newsbites mar 04 pv - purina® pro plan® vets · 2017-06-02 · correcting metabolic deficits with...

Nutrition ForumChanging Paradigms in Nutrition

St. Louis, Missouri • October 2–4, 2008

A Supplement to Compendium: Continuing Education for Veterinarians®

Vol. 31, No. 3(B), March 2009

Changing Paradigms in NutritionSt. Louis, Missouri • October 2–4, 2008

A Supplement to Compendium: Continuing Education for Veterinarians™

Vol. 31, No. 3(A), March 2009

Nutrition Forum

Sponsored by an educational grant from Nestlé Purina PetCare Company.

This information has not been peer reviewed and does not necessarily reflect the opinions of, nor constitute or imply endorsement or recommendation by, the Publisher, Editorial Board, or Nestlé Purina PetCare Company. Neither the Publisher nor Nestlé Purina PetCare Company is responsible for any data, opinions, or statements provided herein.

© 2009 Nestlé Purina PetCare CompanyAll rights reserved.

Printed in the United States of America.Nestlé Purina PetCare Company, Checkerboard Square, St. Louis, Missouri 63164

Designed and published by Veterinary Learning Systems780 Township Line Road, Yardley, PA 19067

Cover Images: Radius Images/Jupiterimages, Corbis/Jupiterimages, Masterfile

Supplement to Compendium: Continuing Education for Veterinarians® Vol. 31, No. 3(B), March 2009 3

SCIENTIFIC PROGRAM: CHANGING PARADIGMS IN NUTRITION

Changing Paradigms in Nutrition...............................................................................7Dorothy P. Laflamme

Putting Clinical Trials on Trial .................................................................................10Dorothy Cimino Brown

Where Does Molecular Nutrition Fit Into an Evidence-Based World? ............................14John A. Milner

Metabolism from the Bottom Up: How Molecular Investigation Can Inform Nutritional and Disease Research..............................................................................18Thomas Schermerhorn

Metabonomic Technologies and Their Applications in Physiologic Monitoring, Disease Recognition, and Phenotypic Profiling............................................................23Ziad Ramadam

Can We Personalize Nutrition-Based Preventive Health Care? .....................................27Dennis F. Lawler

Enterococcus faecium SF68 as a Probiotic for Dogs and Cats .....................................31Gail Czarnecki-Maulden

Immunonutrition ....................................................................................................32Ebenezer Satyaraj

Nutrigenomics for Pet Nutrition and Medicine ...........................................................40Jim Kaput and Baitang Ning

RESEARCH ABSTRACTS: ORAL PRESENTATIONS

Feline Mammary Gland Organ Culture Model for Analysis of Pomegranate Juice as a Chemopreventive Agent for Human Breast Cancer........................................49A. Wilson, K.E. Saker, and A.E. Tanner

Correcting Metabolic Deficits with Allogeneic Bone Marrow Stem Cells.........................50P.R. Vulliet, S.M. Halloran, K.M. Tallon, D.L. Bee, L.A. Lyons, Q.R. Rogers, and A.J. Fascetti

Does Glucotoxicity or Lipotoxicity Contribute to the Pathophysiology of Diabetes in Cats?.................................................................................................51E. Zini, M. Osto, M. Franchini, S. Moretti, A. Vögtlin, M. Ackermann, T.A. Lutz, and C.E. Reusch

CONTENTS

4 Proceedings, 2008 Nestlé Purina Nutrition Forum

Effects of Spaying on Adipose and Muscle Gene Expression and Blood Indices in Cats Fed a High- versus a Moderate-Protein Diet ................................52B.M. Vester, K.J. Liu, T.L. Keel, T.K. Graves, and K.S. Swanson

Regulation of Key Metabolic Factors by Triiodothyronine in Cats ..................................53M. Hoenig, Z. Caffall, and D.C. Ferguson

Adiponectin mRNA Expression in Cats .......................................................................54A.L. Lusby, S.A. Kania, J.W. Bartges, and C.A. Kirk

Effect of Dietary Fish Oil on Adiponectin Concentration in Dogs ..................................55M. Mazaki-Tovi, P.A. Schenck, and S.K. Abood

C-reactive Protein in Lean versus Overweight Dogs .....................................................56L.A. Eirmann, L.M. Freeman, D.P. Laflamme, and K.E. Michel

RESEARCH ABSTRACTS: POSTER PRESENTATIONSThe Influence of Physical Activity during a Canine Weight-Loss Program.......................59J.J. Wakshlag, A.M. Struble, M. Panasevich, B. Warren, M. Maley, and F.A. Kallfelz

Using Activity Monitoring to Delineate Time Spent by Pet Dogs in Activities of Differing Intensity: A Preliminary Analysis ...............................................60K.E. Michel, C. Dow, and D.C. Brown

Survey of Diets Designed for Weight Loss in Dogs and Cats..........................................61D.E. Linder and L.M. Freeman

Obesity in Dogs: A Synthesis of Clinical Data ............................................................62S. Serisier, E. Bailhache, C. Gayet, F. Briand, J. Le Bloc’h, V. Leray, T. Magot, K. Ouguerram, and P. Nguyen

Dietary Fiber Type Affects Behavior in Kenneled Dogs .................................................63G. Bosch, B. Beerda, M. Hesta, A.F.B. van der Poel, G.P.J. Janssens, and W.H. Hendriks

Review of Cat Feeding Habits in Spain ......................................................................64V.M. Mariotti, M. Hervera, J. Fatjó, M. Amat, J.L. Ruiz de la Torre, X. Manteca, and M.D. Baucells

Effect of Mineral Supplementation on Hair Growth and Coat Characteristics of Short-Haired Cats .........................................................................65M. Hekman and D.G. Thomas

High Calcium Intake Affects the Apparent Digestibility of Crude Nutrients and Energy in Puppies .............................................................................................66B. Dobenecker, V. Frank, and E. Kienzle

CONTENTS

Energy Requirements of Two Different Dog Breeds for Ideal Growth from Weaning to 6 Months of Age ....................................................................................67B. Dobenecker, V. Frank, and E. Kienzle

Prediction of Metabolizable Energy in Food for Wild Carnivorous Mammals..................68E. Kienzle, M. Clauss, and H. Kleffner

The Effects of Ginkgo biloba Extract on Healthy Geriatric Dogs..................................69A. Pasquini, P. Simonetti, E. Luchetti, C. Gardana, G. Cardini, and G. Re

Evaluation of the Canine Immune Response to Dietary β-Glucans ................................70W. Anderson, E. Satyaraj, and W. Kerr

Composition, Disintegrative Properties, and Labeling Compliance of Commercial Taurine and Carnitine Supplements.........................................................71R.A. Bragg, L.M. Freeman, A.J. Fascetti, and Z. Yu

Effects of Phenobarbital Administration on Serum 25-Hydroxyvitamin D Concentration in Dogs.............................................................................................72P.A. Schenck and S.K. Abood

Usefulness of β-Hydroxybutyrate Measurements for Diagnosing Feline Diabetes Mellitus...........................................................................................73F. Zeugswetter, J. Prokisch, S. Handl, and C. Iben

Renal Pelvic Precipitates in Domestic Cats .................................................................74D. Lawler, Z. Ramadan, J. Lulich, D. Polzin, and R. Evans

Effects of Diacylglycerol Oil in Normal and Lipoprotein Lipase–Deficient Cats...............75C.A. Datz, R.C. Backus, K.L. Fritsche, and J.J. Ramsey

Effects of Linoleic Acid– and γ-Linolenic Acid–Containing Diets on Feline Lipid Metabolism...........................................................................................76L. Trevizan, A.M. Kessler, K. Bigley, W. Anderson, M.K. Waldron, and J.E. Bauer

ROUNDTABLEControversies in Clinical Nutrition............................................................................77


CONTENTS


Paradigm: A philosophical and theoretical framework of a scientificschool or discipline within which theories, laws, and generalizationsand the experiments performed in support of them are formulated.a

For centuries, long before the concept of “nutrients” wasknown, there was a recognition that foods could contribute topoor or good health. From the time of Hippocrates until the19th century, European medical philosophy and practice weredriven by the humoral theory, which held that the body wasfilled with four basic substances, called humors, elements, or tem-peraments, and that all diseases and disabilities resulted from anexcess, deficiency, or imbalance of these humors. Among otherfactors, diet was thought to influence the balance of humors.

The humoral theory of medicine was eventually displacedby scientific advances and the concepts of the scientificmethod. The introduction of experimental medicine and clin-ical trials dates back to the Canon of Medicine, publishedabout 1025 AD, which contributed to a very gradual para-digm shift in medical philosophy. The first “clinical trial” in-volving a nutritional treatment may have been conducted byJames Lind in 1747. Lind carried out a prospective study oftreatments among men affected with scurvy. Among the treat-ments was citrus fruit. It was highly effective at treating thecondition, and this became the basis for the prevention ofscurvy among British sailors. However, it was not until nearly200 years later that basic research identified the true nature ofthis disease as a vitamin C deficiency.

During the 20th century, scientific research made phe-nomenal advances. In the field of nutrition, individual nu-trients were identified for the first time. The concepts ofessential and non-essential nutrients as well as minimum re-quirements were studied. The identification of individual nu-trients was made possible through the use of studies withpurified diets, which were used to identify specific minimumrequirements. The nutritional paradigm of the time was todefine the essential nutrients and the minimum requirementsneeded to prevent recognized deficiency signs. Regarding pet

nutrition, whole-animal studies were conducted, often in-volving reproduction or growth studies, based on the recog-nition that these were nutritionally demanding life stages.Emerging from this research was the concept of “completeand balanced nutrition,” defined as foods or diets that con-tain all the known essential nutrients, both in sufficientamounts to meet the minimum requirements and in properbalance to one another based on known interactions. A foodsufficient for reproduction and growth was considered suffi-cient for all life stages.

Toward the end of the 20th century, another paradigmshift began with the recognition that some individuals mayneed nutrients in excess of the defined minimums and that“optimum” nutrition might support health beyond avoid-ance of recognized signs of nutritional deficiency.1,2 Also, asthe knowledge base about both diseases and nutrients grew,it came to be recognized that some diseases interfere withnormal metabolism, leading to the hypotheses that individ-uals with these disturbances could benefit from nutritionalmodification.

EVIDENCE-BASED MEDICINEMeanwhile, medicine was undergoing a shift with a contin-ued emphasis on scientific evidence. The concept of evidence-based medicine (EBM) has is roots in the mid-19th century,when the scientific method began to be more widely acceptedin medicine. But paradigm shifts can be slow to occur, asmanifested by the Flexner report of 1910, which highly criti-cized medical training in the United States and challengedmedicine to become more science based.3 EBM is defined as“the conscientious, explicit, and judicious use of current bestevidence in making decisions about the care of individual pa-tients,” and the practice of EBM involves integrating individ-ual clinical expertise with the best available external clinicallyrelevant evidence from systematic research (Box 1).4

Several groups have developed schemes to categorize dif-ferent types of clinical evidence and rank them according tovarious criteria.5,6 In the various schemes, rankings are based

Changing Paradigms in NutritionDorothy P. Laflamme, DVM, PhD, DACVN

Nestlé Purina PetCare ResearchFloyd, Virginia

aFrom Merriam-Webster Online Dictionary, 2008.


on the type of study and, in some cases, the quality of thestudy. The key elements that influence quality are summa-rized in Box 1. Although differences exist, the top of the listin each scheme focuses on systematic reviews of multiple ran-domized clinical trials (RCTs). Because RCTs are at the top ofthe evidence pyramid, it seems worthwhile to define whatconstitutes an RCT. Numerous designs might appropriatelybe used for an RCT, depending on the hypothesis and thetreatment to be evaluated. The key elements that should beincluded are shown in Table 1. Not all of these elements areincluded in every clinical trial, potentially decreasing the rel-ative value of the data produced.

It is not unusual for various RCTs to have different—andoften conflicting—outcomes.7 Thus, one must consider thequality of the study design and potential confounding effectsin each study and consider the sum of the available data. Un-fortunately, the luxury of having multiple RCTs rarely, if ever,exists in the veterinary literature. Further, although RCTs mayprovide valuable information, there are limitations to the ques-tions that can be addressed by RCTs and limitations in the rel-evance of these data in the treatment of individual patients.4,5,8,9

In clinical medicine or nutrition, the individual patientmust be treated based on his or her unique needs. However,research is based on studies of populations, or more correctly,on samples of populations. It is not unusual for a treatmentthat has been proven of benefit in controlled clinical trials tobe less effective in an individual patient than anticipatedbased on the study results. One explanation for this is thatclinical trials are conducted under very tightly controlled cir-

cumstances. Often, patients with multiple problems are ex-cluded from such studies. In clinical trials, patients are care-fully monitored, which may alter their compliance withtreatment or have other unrecognized effects. Another expla-nation is that any deviation might be attributable to genetic orepigenetic factors that interact with these pharmaceutical ornutritional factors only in a subset of people or animals. Cer-tainly, this latter factor is being increasingly recognized. Someexamples include sodium-sensitivity hypertension, lipid-sen-sitive cardiovascular disease, and even predisposition to obe-sity.10,11 These individual predispositions can now be studiedusing the research technologies of genomics, transcriptomics,

Box 1. Guidelines for Assessing the Quality of Evidence4–8

• Objective evidence is superior to subjective evidence oropinions.

• Clinical trials provide better evidence regarding clinical care ofpatients.

• Controlled, randomized, blinded studies provide the best data.

• Larger studies are considered more reliable than small studies.

• Longer studies are considered more reliable than short studies.

• Studies in the target species are considered more reliable thanextrapolation from other species.

• In vivo studies are considered more reliable than in vitro studies.

• Peer-reviewed studies are considered more reliable thannonreviewed studies.

• Consistent, repeatable results are more reliable than any singlepiece of evidence.

• Bias, whether intentional or not, limits the reliability of data.

TABLE 1Characteristics of Good-Quality

Randomized Clinical Trials

Element Characteristics

Prospective An ideal clinical trial prospectively evaluatesthe treatment in question, allowing greatercontrol over confounding variablescompared with a retrospective study.

Interventional A clinical trial includes a definedintervention of some type.

Randomized Test subjects must be assigned to receive thetreatment or control in a manner thatminimizes selection bias.

Blinded To avoid inadvertent bias, the test subjectsand investigators should be unaware of thetreatment group assignment until dataanalysis is complete.

Controlled The nontreatment group should receive anappropriate placebo. For nutritional studies,selection of the control diet may introducebias.

Target species The study should be conducted in the speciesto which the results are to apply.

Representative The study subjects should be representative population of the population to which the results are to

apply. The study protocol should identifyappropriate inclusion and exclusion criteria.

Predefined protocol The study should follow a well-definedprotocol that ensures that all study subjectsare treated the same except for theintervention being evaluated.

Outcome measures The specific parameters to be measured,and the schedule for collection should bedefined based on the study hypothesisbefore initiating the study.

Statistical analysis The study should use appropriate statisticalmethods.


CHANGING PARADIGMS IN NUTRITION

metabonomics, and proteomics. This brings us to the mostcurrent paradigm shift: the “-omics” of nutrition research.

THE “-OMICS” OF NUTRITION RESEARCHHow do nutrigenomics, metabonomics, and proteomics fitwith the currently defined world of EBM and evidence-basednutrition? For research questions that cannot be studiedthrough clinical research, the top of the evidence hierarchy islaboratory research that allows for specific causal assump-tions and mechanisms to be tested and for dependent andindependent variables to be isolated through experimentalmethods.8 Certainly, the “-omics” provide different informa-tion than clinical trials and, in some cases, provide informa-tion that cannot be practically evaluated in interventionalstudies. In many cases, nutrigenomics can provide the basisof understanding or the basis for screening nutrients in a waythat will advance our understanding or shorten the discoverytime to identify factors that will promote health or help man-

age disease and set the stage for targeted clinical trials. In ad-dition, these research technologies should eventually allow abetter understanding of differences among individuals in re-sponse to diet, drugs, or other environmental influences.

THE FUTURE OF NUTRITION SCIENCELooking forward, the evidence hierarchy will once again beshifting with a new paradigm in medical and nutrition re-search driven by the new “-omics” technologies. The future ofnutrition science lies in the elucidation of the relationshipsbetween diet and health on both a population and personallevel. The nutritional genomics sciences of metabonomics,proteomics, and transcriptomics, coupled with functional andclinical measures, will one day define nutritional phenotypesand facilitate a better understanding of the interactions amongdiet, disease, and health (Figure 1).12 These tools already arebeing applied to veterinary medicine and nutrition.

REFERENCES1. Combs GF Jr. Considering nutrient needs: adequate vs optimum. Petfood

Industry 1998;40:31-43.

2. Strain JJ. Optimal nutrition: an overview. Proc Nutr Soc 1999;58:395-396.

3. Go VLW, Wong DA, Wang Y, et al. Diet and cancer prevention: evidence-based medicine to genomic medicine. J Nutr 2004;134(suppl):3513S-3516S.

4. Sackett DL, Rosenberg WMC, Muir-Gray JA, et al. Evidence based med-icine: what it is and what it isn’t. Br Med J 1996;312:71-72.

5. Meyers E. Systems for evaluating nutrition research for nutrition careguidelines: do they apply to population dietary guidelines? J Am DietAssoc 2003;103(suppl):34S-41S.

6. Oxman AD. Grading quality of evidence and strength of recommenda-tions. Br Med J 2004;328:1490-1494.

7. Bigby M. Evidence-based medicine in a nutshell. Arch Dermatol1998;134:1609-1618.

8. Block KI, Jonas WB. “Top of the hierarchy” evidence for integrative med-icine: what are the best strategies? Integr Cancer Ther 2006;5:277-281.

9. Coulter ID. Evidence summaries and synthesis: necessary but insuffi-cient approach for determining clinical practice of integrated medicine?Integr Cancer Ther 2006;5:282-286.

10. Logan AG. Dietary sodium intake and its relation to human health: asummary of the evidence. J Am Coll Nutr 2006;25:165-169.

11. Ordovas JM, Shen J. Gene-environment interactions and susceptibilityto metabolic syndrome and other chronic diseases. J Periodontol2008;19:1508-1513.

12. Zeisel SH, Freake CH, Bauman DE, et al. The nutritional phenotype inthe age of metabolomics. J Nutr 2006;135:1613-1616.

Disease

Health

MetabolomeProteomeTranscriptome

Genes DietEnvironment

NutritionalPhenotype

Functional measuresClinical measures

Figure 1. Components of a nutritional phenotype. The nutritionalphenotype defines an individual’s responsiveness to dietary fac-tors as related to health and disease. A nutritional phenotype in-volves the interaction between that individual’s genetic andepigenetic responses to diet, as modified by environmental factors,including physical activity, and as manifested by alterations in clin-ical and functional measures. (Adapted from Zeisel SH, Freake CH,Bauman DE, et al. The nutritional phenotype in the age ofmetabolomics. J Nutr 2006;135:1613-1616.)


Reports of randomized controlled trials (RCTs) are the “goldstandard” by which practitioners make decisions about treat-ment efficacy. As such, more than any other methodology,RCTs may have a powerful and immediate impact on patientcare. The purpose of this presentation is to review the basiccomponents of the methods and results for RCTs and de-scribe the influence of those components on the interpreta-tion of trial results. In addition, this paper reviews how well(or not so well!) these components are reported on in theveterinary nutrition literature.

To make the discussion clearer and more applicable, wewill refer to a (fictitious) specific RCT report as an example(Figure 1). This is a report of the beneficial effects of diet A ondogs with osteoarthritis (OA). The authors conclude thatbased on a double-blind, randomized, controlled clinicaltrial, dogs with OA eating diet A have “significant improve-ments in lameness and mobility.” As we review the basiccomponents of RCTs and describe the influence of thosecomponents on the interpretation of trial results, we will referto this example.

EVALUATION OF METHODSStudy PopulationFor the conclusions of a trial to be useful for practitioners,the study subjects in the trial should be representative of thepatients seen routinely in their practices. This can be deter-mined by evaluating the inclusion and exclusion criteria inthe methods section of the report. Narrow inclusion and ex-clusion criteria confine enrollment in the study to a smallsubset of patients with the disease, which may impose limi-tations on how useful the results are to practitioners. In thisexample, the study concluded that there is a beneficial effectof diet A for dogs with OA; however, the inclusion criteria forthe trial were middle-aged, medium-sized dogs with cox-ofemoral OA and no other underlying conditions. Althoughdiet A appeared to be very beneficial in this subset of dogs

with OA, it is likely that practitioners will see more variableresults in their practices when dogs of varying ages and sizes,dogs with varying joints affected with OA, and dogs with un-derlying diseases consume the diet.

Assignment to Treatment versus Control GroupsThe RCT is the gold standard for evaluating interventions be-cause of its ability to minimize bias. Selection bias occurswhen study subjects with one or more influencing factors ap-pear more frequently in one study group than in another. Forexample, if smaller body size is associated with a better out-come in dogs with OA and if the proportion of smaller studysubjects is greater in the treatment group than in the controlgroup and the treatment and control diets are equally effec-tive, then there would be an observed benefit of the treatmentthat did not really exist.

Using our trial example of medium-sized (10–20 kg) dogsreported in the OA trial, if 50% of the dogs in the diet Agroup weigh 10 kg and only 10% weigh 20 kg and the oppo-site is true in the control group (50% weigh 20 kg and 10%weigh 10 kg), then the diet A group will appear to do muchbetter than the control group, not necessarily because the dietwas more effective but because the dogs in the diet A grouptended to be much smaller, and smaller dogs do better. Ran-domization can take care of this problem by ensuring that a10-kg dog is just as likely to be placed in the diet A group asthe control group. Ideally, with an adequate sample size, ran-domization leads to study groups that are the same with re-spect to all variables except the treatment being studied. Thebeauty of randomization is that not only will known factorsbe evenly distributed between groups but so will factors thatare unsuspected by the investigators because of limitationsof biologic knowledge at the time the trial is initiated.

The randomization code should be concealed from thestudy personnel who will determine which subjects are eligi-

Putting Clinical Trials on TrialDorothy Cimino Brown, MSCE, DVM, DACVS

Associate Professor of Surgery, School of Veterinary MedicineAssociate Scholar, Center for Clinical Epidemiology & Biostatistics, School of Medicine

University of PennsylvaniaPhiladelphia, Pennsylvania


PUTTING CLINICAL TRIALS ON TRIAL

ble to enter into a trial. Whenever a system of assignment isknown, there is the potential for bias. For example, if the firsttwo eligible dogs with OA (i.e., a 10-kg dog and a 20-kg dog)are presented at the same time with different prognoses andaccording to the randomization list, dog 1 is to receive diet Aand dog 2 is to be a control subject, an investigator may, con-sciously or not, enter these dogs into the study in the orderthat would allow the dog with the better potential outcome(the 10-kg dog) to receive the treatment. If a large proportionof study subjects is entered in this way, a serious imbalance inthe treatment groups with respect to factors affecting the out-come under study would result.

In addition, If there is no mention of randomization, thepresence of unequal study groups and a biased result mustbe considered.

Ascertainment of OutcomeObservation bias (by the investigators or the owner of thedog) in ascertainment of outcome can exist in a RCT in thatknowledge of a study subject’s treatment status might, con-sciously or not, influence the identification or reporting ofrelevant events. For example, if investigators know that a dogis receiving diet A, they may be more likely to persistentlyquestion the owner to give some indication of a positive re-sponse on a mobility score than if they know the dog is onthe control diet. In addition, if the dog’s owner knows that hisor her dog is receiving diet A versus a control diet, the owneris likely to overreport an improved mobility score. Con-versely, those who know their dog is in the control group arelikely to overreport no improvement (or perhaps deteriora-tion). This leads to exaggerated estimates of diet A’s benefits.

The key to obtaining an unbiased estimate of effect for atreatment is to subtract out the placebo effect (i.e., the true ef-fect of the treatment is the measured effect in the treatmentgroup minus the measured effect in the control group). If acontrol group is not used, it would be impossible for thepractitioner to tell whether the treatment effect was attribut-able to the actual treatment or merely to placebo effects.

Sample Size ConsiderationsA trial must have a sufficient sample size (i.e., number ofstudy subjects) to have adequate statistical power or the abil-ity to reliably detect the small to moderate but clinically im-portant differences between study groups that are most likelyto occur. A trial undertaken with an insufficient number ofstudy subjects is of little scientific value. In fact, trials with an

inadequate sample size could be scientifically harmful if theirresults are misinterpreted as demonstrating that a treatmenthas no effect when actually the sample size was not sufficientto draw that conclusion.

EVALUATION OF RESULTSTracking Study Subjects through the TrialTo assess the validity and generalizability of the results, onemust be able to follow the flow of study subjects through thetrial.

Number of Animals Assessed for Eligibilityversus Number of Animals RandomizedThose who choose to participate in a clinical trial are verylikely to differ from nonparticipants in ways that may affectthe outcome of the trial. This is particularly true if a large per-centage of those that are offered enrollment in a trial declineand the participants in the trial end up being a select sub-group for reasons not apparent in the inclusion criteria.Whether the subgroup of participants is representative of theentire potential participant pool does not affect the validity ofresults for that subgroup but may affect how relevant those re-sults will be to a more general population. A trial reportshould include the number and characteristics of the studysubjects assessed for eligibility that then did not participate sothat one can assess the presence and extent of differences be-tween participants and nonparticipants. Doing this aids inthe judgment of whether the results among the participantsare representative of the larger population.

Number of Animals That Do Not Comply in Each Study GroupAn RCT requires the active participation and cooperation ofthe study subjects and their owners. After the agreement toparticipate, there may be deviation from the protocol for var-ious reasons, including the development of side effects, for-getting to administer the appropriate diet, or the owner’s

Figure 1. Example RCT studying diet A versus a control diet in dogswith OA.

PopulationDogs with OA

InterventionDiet A

ControlControl diet

OutcomesPeak vertical force

Mobility score


choosing to feed an alternative diet on his or her own initia-tive. To the extent that study subjects in the control group re-ceive therapy or those in the treatment group do not actuallyreceive their assigned regimen, the two study groups will be-come very similar in terms of exposure to the treatment. Thus,noncompliance in any study subject makes the treatment andcomparison groups more alike, which decreases the ability ofthe trial to detect any true differences between groups.

If a null result of a trial is found because a significant num-ber of those enrolled have not complied with the protocol, itis possible that the study is underpowered (i.e., there are notenough compliant subjects in each group) to detect a true dif-ference between the study groups, even if an appropriatenumber was originally randomized. If a positive result of atrial is found in light of a significant number of those en-rolled not complying with the protocol, it is likely that thediet is very effective but may not be practical because there isa lot of noncompliance in its use for some reason. Knowingthat 100% compliance in a trial would be very unusual, if nocompliance information is reported, one needs to considerwhether only compliant study subjects were analyzed for thereport. This can lead to very biased results, which will be dis-cussed later.

Number of Animals Lost To Follow-UpIn addition to the need for uniform ascertainment of outcomebetween groups, complete follow-up of study subjects overthe duration of the trial is required. If the proportion ofnonascertained outcomes is large or differs among or betweenthe study groups, the result could be an under- or overesti-mate of the effect of the diet. Exactly how much is introducedis impossible to determine directly. An indirect approach is tocalculate estimates of diet effect, assuming the most extremesituations. The results of these calculations provide a rangewithin which the true effect of the treatment lies. It is unusualto not lose a single study subject during a trial. If the trial re-port does not address loss to follow-up at all, it is impossibleto gauge how much bias may be associated with the results.

Number of Animals AnalyzedIt is important to know how many of the original animalsrandomized to each study group had outcomes analyzed.Again, this has to do with the bias that is introduced into re-sults because subjects lost to follow-up cannot be includedin the analysis or when noncompliant subjects are not ana-lyzed in the group to which they were randomized.

Baseline DataAn important early step in the evaluation of the results of atrial is to compare the relevant characteristics of the treatmentand control groups to ensure that balance was achieved. Al-though randomization tends to distribute both known and un-known factors evenly among the study groups, if the samplesize is small, randomization may not always result in groupsthat are alike with respect to every factor except the treatmentunder study. Most RCTs reported in veterinary medicine arerelatively small, making it very important for the baseline char-acteristics of the group to be reported. If this information isnot reported, it is impossible to know whether randomizationwas effective in delivering comparable groups for analysis.

Intention to Treat AnalysisA most important thing to assess when evaluating the resultsof a clinical trial is the question of which study subjects wereincluded in the analysis. Some investigators remove from theanalysis subjects who did not comply with the study proto-col; however, the exclusion of any randomized study subjectfrom the analysis may lead to biased results. After study sub-jects are randomized to a study group, their subsequenthealth experience must be assessed and analyzed along withall others in that group, regardless of whether they have com-plied with their assigned regimens. In all circumstances, thecomparison that is optimal to estimate the true benefit to beobtained from the treatment protocol should be analyzed bythe intention to treat (not whether they were actually treated).In other words, after subjects are randomized, they must al-ways be analyzed. In most trials, perfect compliers representsome fraction of the total study population. Although thegoal is to study the actual effect of the treatment, random-ization is done only on the basis of offering the treatment. Topreserve the power of randomization, the data must be ana-lyzed on this basis. Only the entire groups allocated by ran-domization are truly comparable. Subsequent analyses cancertainly be reported based on the subgroup of study subjectsthat actually received their assigned treatment; however, ifthis is reported, it is important to realize that it is impossibleto achieve balance in the distribution of unknown factors thathad originally been achieved through randomization, andthe results of the subgroup of compliers may be biased.

EFFICACY VERSUS EFFECTIVENESSRCTs are designed to test a biologic question on a relativelyhomogeneous population of subjects. These types of trials are


PUTTING CLINICAL TRIALS ON TRIAL

generally called efficacy trials. They test the true biologic ef-fect of a treatment under optimal circumstances. They do nottest the treatment in usual care, which is the effectiveness ofa treatment when widely used in practice. The true effective-ness of a treatment cannot be determined until it is used in aheterogeneous population of thousands of animals.

RECOMMENDED READINGBrown DC. Control of selection bias in parallel-group controlled clinical tri-

als in dogs and cats: 97 trials (2000-2005). JAVMA 2006;229:990-993.

Brown DC. Sources and handling of losses to follow-up in parallel-grouprandomized clinical trials in dogs and cats: 63 trials (2000-2005). Am J VetRes 2007;68:694-698.

Hennekens CH. Epidemiology in Medicine. Philadelphia: Lippincott Williams& Wilkins; 1987.

Moher D, Schulz KF, Altman D. The CONSORT statement: revised recom-mendations for improving the quality of reports of parallel-group ran-domized trials. JAVMA 2001;285:1987-1991.

Piantadosi S. Clinical Trials. A Methodologic Perspective. New York: John Wiley& Sons; 1997.

Ruiz-Canela M. Intention to treat analysis is related to methodological qual-ity. Br Med J 2000;320:1007.


Evidence-based medicine has emerged as “the conscientious,explicit and judicious use of current best evidence in makingdecisions about the care of individual patients.”1 Its applica-tion in veterinary medicine serves to incorporate the best sci-entific principles for issues faced by the profession. Alimitation of this decision process stems from the inevitabil-ity of inadequate time for veterinarians, as well as other prac-titioners, to read and assimilate the mass of scientificinformation available before incorporating these findingsinto standard practice. Although evidence-based decisions aredevoted to assessing the science that leads to practice, thesedecisions have profound implications for management,scheduling, design, and even finance. Clearly, fundamentalto acceptance or rejection of evidence-based guidelines arethe appropriateness and completeness of studies and the ex-perience of the user.2

The use of emerging data invariably raises concerns aboutpossible bias and distortion of information, regardless of thearea being addressed. The temptation is to manipulate theinformation statistically, including post hoc analyses, in thehope of identifying and showcasing findings. This is espe-cially true for the more positive results that surface duringstudies. Sometimes this manipulation results in unsoundclinical conclusions, which may be further compounded bymeta-analyses. Although systematic reviews and associatedmeta-analyses may provide valuable insights into benefit andrisk, they are typically hampered by inadequate informationto make firm conclusions and recommendations. Concernsoften surface about the applicability of designs that are basedon a selection process to yield a maximum response yet areproposed for general real-life situations. Certainly, this ismost concerning when formalized as a guideline.

Today, practices do not always reflect the principles of ev-idence-based practices but rather rely on tradition, recent ex-

perience, education, conversations with friends and col-leagues, and cost.3 Unquestionably, the best evidence needsto be combined with clinical expertise and appreciation ofthe potential pitfalls that may exist with the design, execu-tion, and interpretation of evidence.4 Unfortunately, assessinghealth depends on many factors, including value-of-life judg-ments, which are not always considered in evidence-based re-views. When the response to a treatment is large, the studydesign and analysis may not be as critical, assuming that com-plications can be appropriately assessed. For example, theonly controlled trial of antibacterial therapy in pneumonia hadno placebo group and was largely without statistics.5 However,recent clinical designs reflect the need to magnify small andpossibly real or biologically relevant responses, sometimeswithout consideration of long-term consequences.6

Undeniably, studies should provide fundamentals aboutthe positive benefits, any complications (even under usualcircumstances), and the biologic mechanism of action. Theresponse to foods and food components can bring about arange of responses ranging from mild to profound, possiblyreflecting a host of interactions among nutrients or with ge-nomics. For example, several years of research show the ben-efits of joint supplements such as glucosamine for people andpets, although there is evidence of interactions with drugsand not all of these supplements are equally responsive.7,8

Likewise, omega-3 fatty acids have been recommended foruse in pets for years. Although originally proposed to assistpets with allergic skin disease, new evidence suggests thatomega-3 fatty acids can have profound effects on multipleprocesses that may provide benefits in a number of condi-tions, including heart disease, joint disease, kidney disease,and even cancer.9,10 Again, discrepancies are found acrossstudies, and there are pleas for additional controlled inter-vention studies. Part of the variation in response to foods or

Where Does Molecular Nutrition Fit Into an Evidence-Based World?

John A. MilnerNutritional Science Research Group

Division of Cancer PreventionNational Cancer Institute

Rockville, Maryland


WHERE DOES MOLECULAR NUTRITION FIT INTO AN EVIDENCE-BASED WORLD?

their components may reflect variation in the cellulartargets being modified, possibly reflecting nutrient–genetics interactions. Likewise, nutrient–nutrient anddrug–nutrient interactions may make lead to conclu-sions about the importance of specific foods or theiractive components particularly challenging.

PERSONALIZED NUTRITIONProper dietary habits are fundamental to achieving genetic potential, increasing physical and cognitiveperformance, and decreasing the risk of a variety ofdiseases.11–15 Admittedly, there is considerable confusionabout the proper diet for health promotion because ofthe enormous variability in response reported in thescientific literature. Evidence-based clinical nutritionhas surfaced as an approach to integrate findings andidentify appropriate clinical interventions. The CochraneCollaboration, one of the leaders in this investigativeand recommendation process, has showcased the importanceof diet in health promotion in several recent reviews.16–18 A re-cent review highlights some of the concerns with such an ap-proach when dealing with a topic such as alternativemedicine. In the Cochrane Collaboration’s review, the ma-jority believed there was insufficient evidence to make firmconclusions.19

A recent report from the World Cancer Research Fundhighlights the importance of eating behaviors for reducingcancer risk. This report is also based on an evidence-based re-view.20 Unfortunately, throughout the report are notes aboutinadequate information and concerns about the heterogene-ity in available data. Although statistical analysis allowed forthe development of guidelines in this case, as well as in manyothers, it is clear from the available data that enormous vari-ability occurs across studies and presumably also across in-dividuals. This dearth of information for this and othersystematic reviews raises real concerns about what the mosteffective practices are and when changes are most likely tobring about a positive or protective response.

It is recognized that chronic disease accounts for a signif-icant global burden of illness (~70%) in adults who are 30years of age and older.21 Because pets routinely experiencemany of the same medical complications and conditions ashumans, there is a growing need to develop appropriatestrategies for risk reduction. Paradoxically, despite moremoney, more technology, and unprecedented dedication tomedical research, disturbing trends are apparent in that rates

of chronic disease continue to mount, at least in humans. In-creasingly, individuals are abandoning mainstream medicineand turning to alternative health. At least some of this de-parture stems from scientific evidence that foods and theircomponents have a plethora of biologic effects. Because petsare not often presented with choices, practices based on evi-dence-based recommendations and the overall quality ofdata for making decisions become of paramount impor-tance.15,22

Undeniably, nutrition is a foundational pillar of healthand well-being. Along with reproduction, the adequacy of thefood supply is key to the supreme goal of survival. Animalsand humans are living longer, healthier lives today in part be-cause of improvements in the nutrient supply. Significant re-search is underway to explore advantages that may arise frompatterns of foods, specific foods, or their isolated compo-nents. It is already clear that not all animals or humans re-spond identically to agents, whether they are drugs or foods.23

Integrating contemporary scientific research with Hip-pocrates’ admonition to “let food be thy medicine and med-icine be thy food,” it is clear that understanding basic aspectsof nutrition as it relates to cellular and molecular metabo-lism is not a luxury but rather is fundamental for effective di-etary management practice.

The expanded use of personalized, individualized, and tai-lored approaches is becoming increasingly fundamental tomodern nutrition (Figure 1).24 The recognition that cellularfunction relies on satisfying specific cellular requirements has

DNA

Bioactivefood

components

Needsand

insults

Cellularprocess(es)

Phenotype

Nutrigenetics

Nutritionalepigenetics

Nutritionaltranscriptomics

Proteomics

Metabolomics

Nutrigenomics

DNA

RNA

Protein

Metabolites

Figure 1. The interrelationship between bioactive food components and phe-notype as a function of changes in DNA, RNA, protein, and small-molecular-weight cell components. This interrelationship can be influenced by insults suchas those coming from viruses, bacteria, and oxidative stress.


ushered in the exciting and ever-expanding field of nutrige-nomics. Without adequate intake of bioactive food compo-nents, biochemical processes and molecular metabolismwithin cells are unable to proceed effectively and may lead toa myriad of diseases, including cancer.

Genetic differences, such as polymorphisms or copy num-ber, may affect the digestion, absorption, metabolism, andexcretion of bioactive food components and thus may estab-lish the response to a change in eating behavior24,25 and likelyinfluence the susceptibility to a host of diseases. Seven singlenucleotide polymorphisms (SNPs)—hPRB +331G/A, ARCAG repeat, CYP19 (TTTA)10, CYP1A1 MspI, VDR FOK1,XRCC1 Arg194Trp, and XRCC2 Arg188His—have surfaced assmall but significant risk factors for spontaneous, nonhered-itary breast cancer.21 In addition, meta-analysis of data in theliterature establishes the TGFBR1*6A, HRAS1, GSTPIle105Val, and GSTM1 SNPs as low-penetrance genetic riskfactors for sporadic breast cancer. Some of these same genesalong with the genes for acid phosphatase locus 1, O6-methylguanine DNA-methyltransferase, methylenetetrahy-drofolate reductase, thymidylate synthase, epidermal growthfactor receptor, and matrix metalloproteinase have surfaced asbiomarkers for colorectal cancer risk. It is noteworthy thatnutrition can modify the expression of many of these geneticvulnerabilities and thereby possibly influence the disease re-lationship. Evidence that this is the case comes from diverseobservations about the association between the vitamin D re-ceptor, calcium intake, and colorectal cancer; cytochromeP450 gene expression patterns, meat intake, and colorectalcancer; and DNA repair gene expression, lycopene, andprostate cancer.23,24,27–29 It is conceivable that by providingspecific bioactive food components or eliminating others, cellsand tissue function can be restored and therefore prevent, retard, or reverse genetic limitations.31

Several bioactive food components can modulate the ac-tivity of defective or vulnerable biochemical processes, in-cluding those involved with cellular detoxification, hormonalhomeostasis, cell division, differentiation, apoptosis, angio-genesis, and immunocompetence.23–27 This overall responsecan be influenced by a host of insults, including bacteria,viruses, oxidative stress, and the amount and duration ofphysical activity (Figure 1).22,23,25

The expanding field of epigenetics is devoted to exploringthe layer of biochemical processes and other determinants incells that regulate genetic expression. A host of dietary com-ponents has been found to modify not only DNA methyla-

tion but also influence histone homeostasis.31 At least someof these changes in epigenetic processes appear to be carriedforward for multiple generations, thus complicating the pic-ture about the role of diet in determining disease risk. Re-cently micro-RNAs have also been implicated in thisregulatory process. Although only a few nutrients have beenreported to alter micro-RNAs, this is an emerging area that islikely to be fruitful for understanding the importance of foodcomponents as modifiers of epigenetic processes, cell func-tion, and possibly longer term imprinting.32

A recent review33 examined the extent to which transcrip-tomic methods have lived up to their promise in the contextof nutrition research. The availability of this high-quality plat-form technology, coupled with established standards and sys-tems for data storage and exchange and powerful newmethods of data analysis, mean that these analyses havereached a level of technical maturity at which they can be ex-ploited to their full potential. In the context of nutrition, tran-scriptomic methods have already been widely applied, albeitprimarily in studies using cell lines and animal models. Usingtranscriptomic technologies, a multitude of genes regulated atthe mRNA level that can be influenced by dietary componentshas been identified.24 These findings are providing new andexciting insights into the biologic processes affected by over-and undernutrition. Evidence from what must be considereda dearth of transcriptomic-based nutritional studies in hu-mans suggests that it is feasible to conduct such studies and toobtain meaningful information. On the other hand, gene ex-pression–based biomarker development still poses a majorchallenge in terms of interpretation. A profile or “signature”may provide better information than data about the expres-sion of a single gene.32 Thus, bioinformatic tools will becomeincreasingly important in deciphering information from tran-scriptomics and the “-omic” technologies in general.

CONCLUSIONUnquestionably, it is clear that neither all humans nor all an-imals respond identically to agents, whether they are drugsor dietary components. Within the current ultraspecializationhealth care paradigm, an appreciation of the interconnectednature of the whole body is of paramount importance. Theapplication of molecular approaches to discover fundamen-tal principles about complex interactions among biologicprocesses as influenced by dietary components will ulti-mately identify the best clinical approaches for promotinghealth and wellness. Rather than developing strategies for an


WHERE DOES MOLECULAR NUTRITION FIT INTO AN EVIDENCE-BASED WORLD?

independent collection of isolated organ systems, the wholeorganism should be considered as an interwoven and inter-dependent tapestry of specialized cells working and commu-nicating together in harmony. A systems approach thatsimultaneously evaluates changes in multiple disease risk thatbuilds on nutrigenomics is essential for understanding indi-viduality and promoting long-term health.

REFERENCES1. Sackett DL, Rosenberg WM, Gray JA, et al. Evidence based medicine:

what it is and what it isn’t. Br Med J 1996;312(7023):71-72.

2. Black D. Evidence-free medicine. Clin Med 2002;2(5):474-475.

3. Williams C, Brunskill S, Altman D, et al. Cost-effectiveness of usingprognostic information to select women with breast cancer for adju-vant systemic therapy. Health Technol Assess 2006;10(34):iii-iv, ix-xi, 1-204.

4. Morice AH, Parry-Billings M. Evidence based guidelines—a step too far?Pulm Pharmacol Ther 2006;19(3):230-232.

5. Evans GM, Gaisford WF. Treatment of pneumonia with 2-(p-aminobenzenesulphonamido) pyridine. Lancet 1938;2:14-19.

6. Hampton JR. Guidelines—for the obedience of fools and the guidanceof wise men? Clin Med 2003;3(3):279-284.

7. Aragon CL, Hofmeister EH, Budsberg SC. Systematic review of clinicaltrials of treatments for osteoarthritis in dogs. JAVMA 2007;230(4):514-521.

8. Dahmer S, Schiller RM. Glucosamine. Am Fam Physician 2008;78(4):471-476.

9. Hickman MA. Interventional nutrition for gastrointestinal disease. ClinTech Small Anim Pract 1998;13(4):211-216.

10. Gillies PJ. Preemptive nutrition of pro-inflammatory states: a nutrige-nomic model. Nutr Rev 2007;65(12 pt 2):S217-S220.

11. McGinnis JM, Nestle M. The Surgeon General’s Report on Nutrition andHealth: policy implications and implementation strategies. Am J ClinNutr 1989;49(1):23-28.

12. Milner JA. Incorporating basic nutrition science into health interven-tions for cancer prevention. J Nutr 2003;133(11 suppl 1):3820S-3826S.

13. Ullah MF, Khan MW. Food as medicine: potential therapeutic tenden-cies of plant derived polyphenolic compounds. Asian Pac J Cancer Prev2008;9(2):187-196.

14. Gómez-Pinilla F. Brain foods: the effects of nutrients on brain function.Nat Rev Neurosci 2008;9(7):568-578.

15. Michel KE. Unconventional diets for dogs and cats. Vet Clin North AmSmall Anim Pract 2006;36(6):1269-1281, vi-vii.

16. Stevinson C, Pittler MH, Ernst E. Garlic for treating hypercholes-

terolemia. A meta-analysis of randomized clinical trials. Ann Intern Med2000;133(6):420-429.

17. Goldberg RJ, Katz J. A meta-analysis of the analgesic effects of omega-3polyunsaturated fatty acid supplementation for inflammatory jointpain. Pain 2007;129(1-2):210-223.

18. Millward C, Ferriter M, Calver S, Connell-Jones G. Gluten- and casein-free diets for autistic spectrum disorder. Cochrane Database Syst Rev2008;16(2):CD003498.

19. Committee on the Use of Complementary and Alternative Medicine.Complementary and Alternative Medicine in the United States. Washing-ton, DC: National Academies Press; 2005.

20. World Cancer Research Fund/American Institute for Cancer Research.Food, Nutrition, Physical Activity, and the Prevention of Cancer: A GlobalPerspective. Washington, DC: American Institute of Cancer Research;2007.

21. Strong K, Mathers C, Leeder S. Beaglehole R. Preventing chronic dis-eases: how many lives can we save? Lancet 2005;366:1578-1582.

22. Roudebush P, Schoenherr WD, Delaney SJ. An evidence-based review ofthe use of therapeutic foods, owner education, exercise, and drugs forthe management of obese and overweight pets. JAVMA 2008;233(5):717-725.

23. Milner JA. Nutrition and cancer: essential elements for a roadmap. Can-cer Lett 2008;269(2):189-198.

24. Trujillo E, Davis C, Milner J. Nutrigenomics, proteomics, metabolomics,and the practice of dietetics. J Am Diet Assoc 2006;106(3):403-413.

25. Davis CD, Milner JA. Biomarkers for diet and cancer prevention re-search: potentials and challenges. Acta Pharmacol Sin 2007;28(9):1262-1273.

26. Tempfer CB, Hefler LA, Schneeberger C, Huber JC. How valid is singlenucleotide polymorphism (SNP) diagnosis for the individual risk as-sessment of breast cancer? Gynecol Endocrinol 2006;22(3):155-159.

27. Abrams SA, Griffin IJ, Hawthorne M, et al. Vitamin D receptor Fok1polymorphisms affect calcium absorption, kinetics, and bone mineral-ization rates during puberty. J Bone Miner Res 2005; 20:945-953.

28. Küry S, Buecher B, Robiou-du-Pont S, et al. Combinations of cy-tochrome P450 gene polymorphisms enhancing the risk for sporadiccolorectal cancer related to red meat consumption. Cancer EpidemiolBiomarkers Prev 2007;16:1460-1467.

29. Goodman M, Bostick RM, Ward KC, et al. Lycopene intake and prostatecancer risk: effect modification by plasma antioxidants and the XRCC1genotype. Nutr Cancer 2006;55(1):13-20.

30. Stover PJ, Caudill MA. Genetic and epigenetic contributions to humannutrition and health: managing genome-diet interactions. J Am DietAssoc 2008;108(9):1480-1487.

31. Ross SA. Nutritional genomic approaches to cancer prevention research.Exp Oncol 2007;29(4):250-256.

32. Davis CD, Ross SA. Evidence for dietary regulation of microRNA ex-pression in cancer cells. Nutr Rev 2008;66(8):477-482.

33. Elliott RM. Transcriptomics and micronutrient research. Br J Nutr2008;99(suppl 3):S59-S65.


In clinical medicine, patients presented for treatment poseproblems that require individualized solutions; integrationof existing medical information; and ultimately, a decisionto select the best possible course to achieve success. Despiteour sincerest attempts at logical and rational applications ofevidence-based therapies, the approach needed to effectivelytreat clinical patients is often at odds with the manner bywhich basic research operates, which is conducted under care-fully controlled conditions. Whereas basic research seeksknowledge that can be generally applied to explain funda-mental processes of biology, clinical medicine uses knowl-edge to modify the biology of individual organisms toachieve specific endpoints. Biologic variability is the bane ofresearchers but is an accepted fact of life for clinicians.

As veterinarians, we are aware that research drives ad-vances in animal health and disease management, but manyclinicians look askance at information from basic researchstudies. The lack of appreciation—if that is the right word—of the ability of basic research to directly inform clinical med-icine may be partly because of many clinical veterinarians’unfamiliarity with the process of basic research and partly be-cause of the feeling that basic research is too far away fromclinical application to be helpful in solving the problems ofindividual patients. The purpose of this paper is to highlightsome examples of how basic research can inform and expandour knowledge of disease and drive advances in the diagno-sis, treatment, and management of clinical patients.

DIABETES—A DISORDER IN WHICH BASICRESEARCH MEETS CLINICAL MEDICINEDiabetes is an extraordinarily complex disorder regardless ofthe perspective from which one chooses to view it. The de-

velopment of diabetes is clearly influenced by genetic, meta-bolic, nutritional, and environmental factors in humans, anda similarly complex etiology is suspected in dogs and cats.The hepatic glucose-sensing pathway, which is important fornormal glucose homeostasis and is disordered in diabetics,presents a useful context for a discussion of how research atthe cellular and molecular levels can have dramatic effects onour understanding the abnormal glucose metabolism thatcharacterizes the diabetic state.

The Concept of Hepatic Glucose SensingThe blood glucose concentration is maintained within nar-row limits by strict physiologic controls exerted on the ratesof glucose input or removal from the circulation. Mainte-nance of the glucose concentration requires a system for sens-ing changes in circulating glucose and linking those changesto the appropriate metabolic response. The liver, which is in-volved in both glucose usage and production, is a major reg-ulator of blood glucose and is part of a network ofglucose-sensing tissues that also includes the endocrine pan-creas and specialized gut and brain cells.

A network of interacting hepatocellular proteins serves tosense changes in blood glucose after a meal and initiate theappropriate metabolic response. The “fasted-to-fed” meta-bolic transition in the liver is mediated by nutrients (espe-cially carbohydrates) and hormones (especially insulin andglucagon). In the fasted state, when blood glucose and in-sulin concentrations are low (basal), the insulin:glucagonratio favors hepatic glucose production via gluconeogenesis.After ingestion of a carbohydrate-containing meal and stim-ulation of pancreatic insulin secretion, the combination ofelevated portal glucose concentration and the increased in-

Metabolism from the Bottom Up: How Molecular Investigation Can InformNutritional and Disease Research

Thomas Schermerhorn, VMD, DACVIMAssociate Professor of Small Animal Medicine

College of Veterinary MedicineKansas State University

Manhattan, Kansas


METABOLISM FROM THE BOTTOM UP: HOW MOLECULAR INVESTIGATION CAN INFORM NUTRITIONAL AND DISEASE RESEARCH

sulin:glucagon ratio promotes hepatic glucose uptake viastimulation of glucokinase (GK) activity and simultaneouslyreduces hepatic gluconeogenesis via inhibition of glucose-6-phosphatase (G6Pase) activity. Circulating hormones andnutrients produce their effects on hepatic function by directlyaltering protein interactions and enzymatic activity but exertequally important long-term effects on hepatic glucose me-tabolism by influencing the expression of genes involved inthe glucose-sensing network.

The following examples illustrate how cellular and mo-lecular studies into the expression, function, and regulationof the glucose-sensing proteins have yielded valuable insightsinto normal metabolic function of the liver as well as the roleof the liver in the establishment and propagation of the dia-betic state.

Identification of the Molecular Hepatic Glucose SensorEarly biochemical studies reported an enzyme that phos-phorylated glucose with unusual kinetics in the livers of mostmammals. The peculiar enzyme was eventually identified asGK, a 52 kD protein product of the GCK gene. GK is one offour known mammalian hexokinases (HKs) that use adeno-sine triphosphate to phosphorylate glucose for entry into gly-colysis.1 Subsequent studies determined that the kineticproperties of GK differed substantially from the other HKs.GK has a lower affinity for glucose, its principal substrate, andis less inhibited by its enzymatic product, glucose-6-phosphate, compared with HKs.2 These kinetic features im-part a “sensor” function of the GK enzyme, which respondsto physiologic changes in glucose concentration with a lin-ear increase in enzymatic activity. Gene and protein studieshave showed that the GCK gene has tissue-specific regulationand is almost exclusively found in hepatocytes and pancreaticislet cells. The importance of GK for glycolysis, its apparentsensor function, and its presence in tissues with vital roles inglucose homeostasis provide a plausible mechanism thatlinks changes in blood glucose with enhanced glucose me-tabolism and, ultimately, the appropriate tissue response.1

The major hepatic proteins involved in glucose sensingand their physiologic regulation have since been mapped out.GK activity establishes the rate of hepatic glucose usage, andGK is the de facto hepatic glucose sensor. However, GK func-tion is exquisitely regulated by numerous other proteins thatmodulate the hepatocellular glucose-sensing function, in-cluding, among others, the glucose transporter type 2

(GLUT2), GK regulator protein (GKRP), ketohexokinase(KHK), and G6Pase proteins. Functional coordination ofthese proteins is necessary for normal hepatic glucose me-tabolism and is disordered in diabetic patients.

In health, dietary carbohydrates, especially glucose andfructose, acutely stimulate hepatic GK enzymatic activity.These nutrients act either directly or indirectly (via GKRP) onthe GK protein to enhance its enzymatic activity. Glucosebinds directly to GK as a substrate and stimulates enzymaticactivity. GK activity is also closely regulated by GKRP, whichis localized in the hepatocyte nucleus. GK undergoes a well-described nuclear–cytosolic translocation cycle, and its sub-cellular localization varies with the nutritional state.3 In thefasted state, GK is bound to GKRP and localized within thehepatocyte nucleus, where it is functionally inactive.4 Afterfeeding, GK translocates to the cytosol, where it performs itsenzymatic function.4 The GK–GKRP interaction is inhibitedby glucose and fructose-1-phosphate (derived from dietaryfructose) and enhanced by fructose-6-phosphate, a glycolyticintermediate.1

Nutrients and hormones also exert a regulatory effect ongene expression and therefore alter the long-term hepatic me-tabolism. GCK expression in β cells and the liver is regulatedby separate promoters, which permits differential regulationby nutrients and hormones in those tissues.5 Similar to GKactivity, hepatic GCK expression is highly influenced by nu-tritional status. In contrast to the β-cell promoter, which isnot regulated by insulin or glucagon, transcriptional regula-tion of the hepatic GCK promoter is strongly activated by in-sulin and repressed by glucagon.6

Identification of the genes and proteins involved in glu-cose sensing provided a molecular mechanism to integrateand coordinate signals arising from the digestion of dietarycarbohydrates into a larger body-wide response that servedprevailing metabolic needs. The abnormal glucose sensingthat accompanies the diabetic state5 could now be probed atthe molecular level for gene or protein targets with roles inthe development of diabetes.

Genetic Engineering of the Glucose Sensing MechanismGenetic engineering includes techniques that alter the geneticmaterial within an organism or cell. These techniques arenow standard in most laboratories and have been used ex-tensively to study metabolism and diabetes.

In vitro studies using cell lines with altered gene expres-


sion show that whereas even small reductions in the amountof expressed GK have a dramatic negative impact on glucosesensitivity, GK overexpression generally enhances glucosesensitivity.3 Findings from in vitro experiments are supportedby results of in vivo models. Genetic knockout mouse mod-els that lack the genes encoding either GK or GKRP have beenproduced.7,8 Mice with total lack of GK expression and thosethat have targeted elimination of pancreatic islet GK are dia-betic at birth and die within a few days.7 GK and GKRPknockout models both have abnormal hepatic glucose me-tabolism and exhibit similar phenotypes after glucose chal-lenge. Mice with targeted elimination of hepatic GKexpression are not diabetic, readily develop hyperglycemiaunder stressful conditions, and have mildly impaired glucoseintolerance and insulin secretion.7 Likewise, GKRP knockoutmice are not diabetic but are glucose intolerant and prone todeveloping stress hyperglycemia.9

The transgenic approaches used to modify gene expressionin cell-line and whole-animal models have yielded consistentresults and confirmed the central importance of GK for glu-cose sensing. It is clear that abnormalities in hepatic GK ex-pression impair hepatic glucose metabolism and provide afunctional basis by which GK could be involved in the etiol-ogy and pathogenesis of diabetes.

Molecular Genetics of GCKGenetic influences likely play a major role in the develop-ment of diabetes in humans and animals. The genetic com-ponent appears to be heterogeneous. In a small proportionof cases, mutations in single genes are directly linked withthe diabetic phenotype. The genetic influence in most pa-tients, however, is exerted by the presence of “diabetes” sus-ceptibility genes in the patient’s genome. In human type 2diabetes (the most common form), phenotypic disease re-sults from complex interactions between metabolic, nutri-tional, and environmental factors on a background ofgenetic susceptibility. Before the genomics era, a completeunderstanding of the complex genetics of diabetes wouldhave been nearly impossible to achieve. However, the eluci-dation of the human, canine, and feline genomes and rapidadvances in bioinformatics, mathematics, and statistical sci-ence make it possible to envision that the complex geneticsof diabetes will eventually be uncovered.

After GK was established as the tissue glucose sensor, GCK(which encodes GK) was investigated as a candidate gene fordiabetes mellitus.10,11 The candidate gene approach evaluates

individual genes as possible causes of a particular phenotypeby screening individuals for mutations and polymorphismsin the candidate gene. Studies of GCK proved fruitful, andnearly 200 naturally occurring mutations that are spread outover the entire GCK gene have been described.10 GCK codingmutations cause a monogenetic form of human non–insulin-dependent diabetes known as maturity-onset diabetes in theyoung (MODY) type 2 (there are five subtypes), which accountfor as many as 60% of all MODY cases.10 The single gene mu-tations carried by MODY2 patients usually result in an ab-normal GK protein that may have altered substrate binding orkinetics depending on the type and location of the mutation.

Other studies have postulated a role for GCK variants inthe development of typical human type 2 diabetes (i.e., late-onset diabetes).12,13 Many polymorphisms and other genevariants of diabetic humans are not in the GCK coding region(thus, they do not alter the GK protein) but could alter GCKgene regulation or the efficiency of gene expression. Exam-ples of GCK variants found in diabetic patients include silentbase changes in the coding region14 and base substitutions inupstream promoter regions,13,14 in the 5’-untranslated regionof exon 1a,14 and in introns (including substitutions near theintron–exon junction).14,15

Although GCK mutations are not present in the majorityof humans with type 2 diabetes, extensive genetic analysis re-vealed much about the enzymatic functions of GK. Analysisof various GCK mutations in MODY2 patients provided theopportunity to correlate particular mutations with an enzy-matic phenotype, which permitted amino acids with crucialroles in GK structure and function to be identified. Further-more, recognition of MODY2 (and other MODY phenotypes)as distinct etiologies have important implications for thebroader understanding of diabetes epidemiology and patho-genesis as well as disease prognosis and therapy for affectedpatients.

GK as a Direct Pharmaceutical TargetIncreased hepatocellular glucose flux and improved hepaticglucose metabolism are observed when the amount or activ-ity of GK is increased. Thus, GK is an attractive target for phar-macologic intervention in diabetes. An exciting developmentof the past few years was the discovery of a family of organiccompounds that act as GK activators. Several mechanisms ofGK activation have been described for this group of drugs, in-cluding increasing GK Vmax and enhancing GK affinity for glu-cose.16 GK activators bind directly to GK at a site that is


METABOLISM FROM THE BOTTOM UP: HOW MOLECULAR INVESTIGATION CAN INFORM NUTRITIONAL AND DISEASE RESEARCH

distinct from the catalytic site and exert their action inde-pendent of GKRP (in the liver).16 Studies of various GK acti-vators (GKA1, GKA2, RO-28-1675, compound A) in rodentshave shown enhanced hepatocyte glucose metabolism, re-duced threshold for glucose-stimulated insulin secretionfrom isolated rodent islets, and decreased blood glucose con-centrations after oral glucose administration in various ani-mal diabetes models.16 The dual actions of GK activators toenhance hepatic glucose metabolism and stimulate pancre-atic insulin secretion have great therapeutic potential for di-abetes mellitus.

Hepatic Glucose Sensing Is Integral toDietary Regulation of Hepatic LipogenesisFrom the standpoint of nutritional interventions, targetinggene networks may be more attractive than targeting specificgenes or proteins, such as GK. An example of the complexi-ties of dietary modification of gene expression is discussedbelow, but numerous gene networks have been proposed asdesirable targets for interventions aimed at improving hepaticmetabolism in diabetes.

Hepatic glucose metabolism is intimately linked to he-patic lipid metabolism. A major fate of glycolytic products isto serve as substrate molecules for de novo fatty acid synthe-sis. Hepatic lipid synthesis is under nutritional regulation andresponds to dietary carbohydrate. Similar to crucial glycolyticgenes, such as GCK, transcriptional activation of importantlipogenic genes (e.g., acetyl-CoA carboxylase [ACC] and fattyacid synthase [FAS]) requires both insulin and glucose.

Insulin action on hepatic GCK expression is mediated inpart by a transcriptional activator known as sterol regulatory-element binding protein 1c (SREBP-1c).17 SREBP-1c is a well-established activator of lipogenic genes in the liver, but inrecent years, SREBP-1c has emerged as an intermediate in in-sulin-mediated regulation of gene expression.17 Glucose di-rectly activates GK enzymatic activity but also exerts apermissive effect on GCK gene expression. The carbohydrateresponsive element-binding protein (ChREBP) is anothertranscriptional factor that appears to mediate the direct effectof glucose on gene expression.17

SREBP-1c has been implicated in the development of in-sulin resistance in experimental models.18 The stringent con-trol SREBP-1c exerts over lipogenic gene expression is evidentfrom genetic studies. SREBP-1c knockout mice have impairedexpression of lipogenic genes after carbohydrate challenge.19

Additionally, overexpression of hepatic SREBP-1c induces he-

patic lipidosis in mice.20 Interestingly, the full extent ofSREBP-1c–mediated activation of lipogenic genes requires anintact glucose-sensing mechanism.17 Genetically altered GCKknockout mice do not show complete activation of glycolyticand lipogenic genes in the presence of glucose, even whenSREBP is overexpressed.17 The lack of direct glucose effect ongene expression may be caused by educed ChREBP gene ex-pression that also occurs in these mice.17

The presence of sterol and carbohydrate response elementsthat are targeted by SREBP-1c and ChREBP, respectively, inthe promoter regions of genes important for glycolysis andlipogenesis suggests modulation of gene expression could bea target for pharmacologic or dietary therapy. Genetic analy-sis of these factors may prove useful for probing metabolicabnormalities associated with diabetes. Evidence suggeststhat SREBP-1c polymorphisms in mice produce different he-patic responses to dietary fructose, and some SREBP-1c poly-morphisms are associated with greater induction of lipogenicgene expression by fructose.21

CONCLUSIONBasic research using techniques of biochemistry, cell biology,molecular genetics, genomics, and bioinformatics continuesto provide a progressively detailed understanding of the cel-lular and molecular pathways involved in hepatic glucosesensing. Several excellent examples aptly illustrated howknowledge gleaned from basic research has been used to fur-ther understanding of the etiologic and pathogenic mecha-nisms of diabetes and as a basis for developing noveltherapeutic interventions for this important disorder.

REFERENCES1. Postic C, Shiota M, Magnuson MA. Cell-specific roles of glucokinase in

glucose homeostasis. Recent Prog Horm Res 2001;56:195-217.

2. Pollard-Knight D, Cornish-Bowden A. Mechanism of liver glucokinase.Mol Cell Biochem 1982;44(2):71-80.

3. Fernandez-Novell JM, Castel S, Bellido D, et al. Intracellular distributionof hepatic glucokinase and glucokinase regulatory protein during thefasted to refed transition in rats. FEBS Lett 1999;459(2):211-214.

4. Agius L. The physiological role of glucokinase binding and translocationin hepatocytes. Adv Enzyme Regul 1998;38:303-331.

5. Zelent D, Najafi H, Odili S, et al. Glucokinase and glucose homeosta-sis: proven concepts and new ideas. Biochem Soc Trans 2005;33(pt1):306-310.

6. Nordlie RC, Foster JD, Lange AJ. Regulation of glucose production bythe liver. Annu Rev Nutr 1999;19:379-406.

7. Postic C, Shiota M, Niswender KD, et al. Dual roles for glucokinase inglucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem1999;274(1):305-315.

8. Farrelly D, Brown KS, Tieman A, et al. Mice mutant for glucokinase reg-


ulatory protein exhibit decreased liver glucokinase: a sequestrationmechanism in metabolic regulation. Proc Natl Acad Sci USA 1999;96(25):14511-14516.

9. Grimsby J, Coffey JW, Dvorozniak MT, et al. Characterization of glu-cokinase regulatory protein-deficient mice. J Biol Chem 2000;275(11):7826-7831.

10. Bell GI, Pilkis SJ, Weber IT, Polonsky KS. Glucokinase mutations, in-sulin secretion, and diabetes mellitus. Annu Rev Physiol 1996;58:171-186.

11. Galan M, Vincent O, Roncero I, et al. Effects of novel maturity-onset di-abetes of the young (MODY)-associated mutations on glucokinase ac-tivity and protein stability. Biochem J 2006;393(pt 1):389-396.

12. Marz W, Nauck M, Hoffmann MM, et al. G(-30)A polymorphism in thepancreatic promoter of the glucokinase gene associated with angio-graphic coronary artery disease and type 2 diabetes mellitus. Circulation2004;109(23):2844-2849.

13. Stoffel M, Froguel P, Takeda J, et al. Human glucokinase gene: isola-tion, characterization, and identification of two missense mutationslinked to early-onset non-insulin-dependent (type 2) diabetes mellitus.Proc Natl Acad Sci USA 1992;89(16):7698-7702.

14. Stone LM, Kahn SE, Deeb SS, et al. Glucokinase gene variations inJapanese-Americans with a family history of NIDDM. Diabetes Care1994;17(12):1480-1483.

15. Sun F, Knebelmann B, Pueyo ME, et al. Deletion of the donor splice siteof intron 4 in the glucokinase gene causes maturity-onset diabetes of theyoung. J Clin Invest 1993;92(3):1174-1180.

16. Grimsby J, Sarabu R, Corbett WL, et al. Allosteric activators of glucoki-nase: potential role in diabetes therapy. Science 2003;301(5631):370-373.

17. Dentin R, Pégorier JP, Benhamed F, et al. Hepatic glucokinase is re-quired for the synergistic action of ChREBP and SREBP-1c on glycolyticand lipogenic gene expression. J Biol Chem 2004;279(19):20314-20326.

18. Shimomura I, Hammer RE, Richardson JA, et al. Insulin resistance anddiabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adi-pose tissue: model for congenital generalized lipodystrophy. Genes Dev1998;12(20):3182-3194.

19. Shimano H, Yahagi N, Amemiya-Kudo M, et al. Sterol regulatory ele-ment-binding protein-1 as a key transcription factor for nutritional in-duction of lipogenic enzyme genes. J Biol Chem 1999;274(50):35832-35839.

20. Shimano H, Horton JD, Shimomura I, et al. Isoform 1c of sterol regu-latory element binding protein is less active than isoform 1a in livers oftransgenic mice and in cultured cells. J Clin Invest 1997;99(5):846-854.

21. Nagata R, Nishio Y, Sekine O, et al. Single nucleotide polymorphism (-468 Gly to A) at the promoter region of SREBP-1c associates with ge-netic defect of fructose-induced hepatic lipogenesis. J Biol Chem2004;279(28):29031-29042.


Nutrition researchers are beginning to take advantage ofmetabonomic technologies to identify health biomarkers forhumans1–3 and pets.4 Metabonomics is the comprehensiveanalysis of low-molecular-weight metabolites in biologic flu-ids, along with complex multivariate analysis of the output.5,6

Biomarkers that are identified by these means can be meas-ured quantitatively over time and can be used to monitor themetabolic status of humans and pets. Some biomarkers willultimately be used as diagnostic tools to recommend diets orother practices that reduce the risk for disease.7,8 Therefore, thisprocess of biomarker identification can provide tools that helpencourage consumers to actively participate with their veteri-narians in maintaining their pet companions’ health.9 Finally,metabonomics has the potential to streamline the process offinding foods and ingredients with enhanced nutritional valuefor maintaining health and well-being.

This paper provides an overview of metabonomic tech-nologies and applications in physiologic profiling, diseaserecognition, and dietary interventions. Three examples arepresented, describing studies conducted at Nestlé ResearchCenters in Lausanne, Switzerland, and St. Louis in collabo-ration with Imperial College London. The most recent ana-lytical techniques and statistical analyses that can be used torapidly identify up- or downregulated endogenous metabo-lites in various biofluids (plasma, urine, saliva) include pro-ton nuclear magnetic resonance10,11 (1H NMR) and liquid/gaschromatography mass spectroscopy (LC/GC MS),12 whichwill be emphasized in this discussion.

METABONOMICS ANALYTICS AND DATAANALYSIS METHODSThe aim of metabonomics is to obtain the widest possiblecoverage in terms of understanding the types and numbersof compounds to be recognized. This is achieved by making

use of several complementary analytical methods. 1H NMRand LC/GC MS are the principal analytical platforms used formetabolic profiling. In 1H NMR spectroscopy, all covalentlyattached protons from mobile molecules within a very highdynamic range of concentrations (i.e., from millimolar tonanomolar scales, if one is using modern cryogenic probes)are scanned simultaneously. Because little sample pretreat-ment is necessary in 1H NMR experiments, the process enablesthe simultaneous identification and monitoring of a widerange of low-molecular-weight endogenous metabolites, thusproviding a biochemical fingerprint of an organism.

Mass spectroscopic metabonomic techniques are separa-tion techniques (gas or liquid chromatography) coupled toonline MS detection for the screening of low-molecular-massmetabolites in biologic matrices. The main advantages of MStechniques are high sensitivity and structure-rich information.However, there is a need to preselect analytic conditions sothat the analyte is ionized for MS detection methods. Samplepreparation, such as deproteinization (LC/MS) or derivatiza-tion (GC/MS), is needed in most cases. Furthermore, biologicsamples contain a very wide range of molecular species, in-cluding nonionic, acidic, basic, and amphoteric substances.Therefore, MS analysis should use both positive and negativeionization techniques to ensure the best possible chance ofdetecting the maximum number of metabolites.

Recognizing that no single technique can be expected tomeet all of the field’s diverse challenges, many metabonomicresearch programs use several analytic techniques. Metabo-nomic data are very complex, and it is difficult to make mean-ingful comparisons of large numbers of NMR spectra or MSchromatograms by eye. Multivariate statistical methods areused to compress data into more easily managed forms. Thisprocess may help in visualizing patterns and clustering be-tween samples, which are two of the central issues in metabo-

Metabonomic Technologies and Their Applicationsin Physiologic Monitoring, Disease Recognition,and Phenotypic Profiling

Ziad Ramadan, PhDNestlé Research Center

St. Louis, Missouri


nomic analysis. Multivariate analysis is practically essential inthe fingerprinting approaches but is also helpful in techniquesin which individual metabolites are explicitly quantified.Using both NMR and MS techniques in any metabonomicstudy can be seen as using complementary analytic platformscapable of producing complex metabolic profiles. Both NMRand MS data are coupled with multivariate data analysis andpattern recognition methods for biomarker discoveries.

Many multivariate methods are available for clustering,classifying, and modeling metabonomic data sets. Thesemodeling methods are divided into two categories: unsuper-vised and supervised clustering algorithms. Unsupervisedclustering algorithms require no a priori knowledge aboutthe class of the sample. The most common multivariate un-supervised method used in the evaluation of a metabonomicstudy is principal components analysis (PCA). PCA is alwaysrecommended as a starting point for analyzing multivariatedata; PCA rapidly provides an overview of the informationhidden in the data. PCA enables visualization of biologic datasets based on the inherent similarity or dissimilarity of sam-ples with respect to their biochemical composition.

PCA creates a condensed summary of the data, which canbe analyzed graphically by means of two types of plots, thescores plot and the loadings plot. The scores plot is a sum-mary of the relationship between the observations; it can beused to establish any significant similarities and differencesbetween biologic samples. The loadings plot is a similarsummary of the variables (i.e., spectra, mass:charge ratio list-ings, chromatograms). The loadings plot can be viewed as ameans to interpret the pattern seen in the scores plots be-cause the two are complementary. In this way, PCA may fa-cilitate the simultaneous comparison of a large number ofcomplex objects such as biofluid spectra and may provideinformation on biochemical (metabolite) changes with re-lation to physiologic variations.

Supervised clustering algorithms, on the other hand, re-quire a priori knowledge as to the class of the sample. Themost common multivariate supervised methods used in theevaluation of a metabonomic study is partial least-squaresdiscriminant analysis (PLS-DA) and its variant, the orthogonalpartial least-squares discriminant analysis13 (O-PLS-DA),which adds a data-filtering step. PLS-DA provides a means tofilter out metabolic information that is not correlated to pre-defined class memberships. PLS-DA loadings, similar to PCAloadings, yield information on which spectral signals are as-sociated with the observed clustering, providing a basis for

metabolic interpretation. PLS-DA models can be refined byusing the O-PLS-DA technique, in which the structure noisein the X matrix, unrelated to the Y matrix (class membership),is removed. The O-PLS-DA method provides a predictionsimilar to that of PLS-DA, but the interpretation of the mod-els is improved because the structured noise is modeled sep-arately from the variation common to the X and Y matrices.

METABONOMIC APPLICATIONSCanine StudyA total of 48 Labrador retrievers from seven litters were allot-ted to a paired feeding design.4 The dogs were paired by gen-der and weaning weight within litter and were then assignedrandomly as control-fed (CF) or diet-restricted (DR) pairmates, the latter being given 75% of the amount of the samefood consumed by CF pair mates. Free indoor–outdoor accesswas available, and activity was not restricted. The feeding pro-tocol was initiated at 8 weeks of age. The dogs were housedand fed under the same environmental conditions for life.

An 1H NMR–based metabonomic strategy was used to mon-itor urinary metabolic profiles throughout the lifetimes of CFand DR dogs. Urinary metabolic trajectories were constructedfor each dog, and metabolic variation was found to be pre-dominantly influenced by age. Urinary excretion of creatinineincreased with age, reaching a maximum between 5 and 9 yearsand declining thereafter. Excretion of mixed glycoproteins wasnoted at earlier ages, a likely reflection of growth patterns. Inaddition, consistent metabolic variation related to diet was alsocharacterized, showing that energy-associated metabolites, suchas creatine, 1-methylnicotinamide, lactate, acetate, and succi-nate, were lower in the urine in DR dogs.

Both aging and diet restriction altered activities of gut mi-crobiota, manifested by variation of aromatic metabolitesand aliphatic amine compounds. This analysis allowed themetabolic response to two different physiologic processes(age and food intake) to be monitored throughout the life-time of this population. The results may form part of a strat-egy to monitor and reduce the impact of age-related diseasesin dogs and may provide more general insights into the ex-tension of longevity in higher mammals.

Finally, this study has highlighted the benefits of using ametabonomic strategy for the detection of subtle physiologicchanges and dietary effects on mammalian metabolism. Therole that gut microflora plays in longevity and quality-of-liferesponses to DR is potentially important and merits furtherinvestigation.


METABONOMIC TECHNOLOGIES AND THEIR APPLICATIONS IN PHYSIOLOGIC MONITORING, DISEASE RECOGNITION, AND PHENOTYPIC PROFILING

Feline StudyIn this study, 21 cats were selected based on feline odonto-clastic resorptive lesion (FORL) status, diagnosed by visualexamination, probing the gingival sulcus surrounding thetooth, and full-mouth dental radiography with cats undergeneral anesthesia.14 The severity of the disease was deter-mined and the FORL grade recorded on each affected tooth.Based on the clinical and radiographic examination, 10 cats(eight males; two females) were confirmed to have FORLswith either one lesion at stage 3 or above or two lesions atstage 2 or above. The other 11 cats (six males; five females)were confirmed FORL-free because of the absence of clinicalor radiographic signs of FORL.

1HNMR- and LC/MS-based metabonomic analysis ofsaliva samples obtained from these cats showed clear differ-ences in the metabolic composition of saliva between healthyand FORL-diseased cats. To identify biomarkers, the spectro-scopic data were processed using PLS-DA and validated byleave-one-subject-out cross-validation. The PLS-DA modelpredicted FORL-affected cats with more than 60% accuracy.Affected cats showed increased levels of many organic andamino acids, such as acetate, lactate, propionate, isovalerate,tryptamine, and phenylalanine, suggesting changes in oralmicroflora in the presence of disease.

Human StudyA total of 75 healthy men participated in a study of choco-late consumption. Participants were classified as chocolatedesiring or chocolate indifferent15 based on a preestab-lished score range (score range, 6–30) that reflected eatinghabits. Twenty-two of the subjects were selected for biofluidcollection and data analysis, including 11 subjects at eachend of the score range. Subjects were also matched accord-ing to age and body mass index (BMI) and were selectedaccording to medical evaluation of a confidential healthquestionnaire.

In this study, a new “nutrimetabonomic” approach wasdemonstrated in which spectroscopically generated metabolicphenotypes were correlated with behavioral and psychologicdietary preference, defined as chocolate desiring or chocolateindifferent. Urinary and plasma metabolic phenotypes werecharacterized by differential metabolic biomarkers measuredusing 1H NMR spectroscopy and were included in the post-prandial lipoprotein profile and gut microbial co-metabolism.

The observations demonstrated imprinted differencesin the metabolic activity of gut microbiota of individuals

that appeared to depend on their previous dietary con-sumption habits. These results are supported by reportson the impact of dietary habits on recolonization by thegut microflora16 and imply that dietary influences on mi-crobial activities may be subtle. It is widely thought thatthe gut microbial populations in individuals are stable,but these data suggest that metabolic activity of gut mi-crobes and consequently the nutrients and ingredientsthat are consumed by the host may be more finely modu-lated by diet than previously thought.

This study suggests imprinting of the basal metabolic phe-notype in relation to a behavioral and psychologic dietarypreference that is characterized by chocolate desiring orchocolate indifference. This imprinting is independent of theingested food because chocolate consumption versus placebohad no direct effect. The plasma metabotype within the pref-erence class is mainly characterized by differences in lipopro-tein profiles. In addition, the metabolic phenotypes observedin the urine suggested considerable differences in gut micro-bial metabolic activities that may be of long-term health sig-nificance to the host.

CONCLUSIONThe holistic nature of metabonomic technologies and theirnoninvasive approach help in identification and validationof biomarkers for maintaining the health and well-being ofhumans and pets. A key challenge in metabonomic researchis to devise methods to speed the process of finding foodswith enhanced nutritional value to maintain health statusand delay the onset of homeostasis disruption associatedwith morbidity and mortality. This may result in dietary rec-ommendations based on knowledge of food preferences, nu-tritional requirements, and health status as a step towardpersonalized nutrition for human and pets. The move towardpersonalized nutrition requires more global analysis of thephenotypes of individuals on the health, disease, and dietcontinuum, ascertained through an integrated systems–biol-ogy approach using information from microarray analysis,proteomics, and metabonomics.

REFERENCES1. Rezzi S, Ramadan Z, Fay LB, et al. Nutritional metabonomics: applica-

tions and perspectives. J Proteome Res 2007;6(2):513-525.

2. Kussmann M, Affolter M, Fay LB. Proteomics in nutrition and health.Comb Chem High Throughput Screen 2005;8(8):679-696.

3. Gibney MJ, Walsh M, Brennan L, et al. Metabolomics in human nutri-tion: opportunities and challenges. Am J Clin Nutr 2005;82(3):497-503.

4. Wang Y, Lawler D, Larson B, et al. Metabonomic investigations of aging


and caloric restriction in a life-long dog study. J Proteome Res 2007;6(5):1846-1854.

5. Nicholson JK, Lindon JC, Holmes E. “Metabonomics”: understandingthe metabolic responses of living systems to pathophysiological stimulivia multivariate statistical analysis of biological NMR spectroscopicdata. Xenobiotica 1999;29(11):1181-1189.

6. Nicholson JK, Wilson ID. Opinion: understanding “global” systems bi-ology: metabonomics and the continuum of metabolism. Nat Rev DrugDiscov 2003;2(8):668-676.

7. Lindon JC, Holmes E, Nicholson JK. Metabonomics: systems biologyin pharmaceutical research and development. Curr Opin Mol Ther2004;6(3):265-272.

8. Lindon JC, Holmes E, Bollard ME, et al. Metabonomics technologiesand their applications in physiological monitoring, drug safety assess-ment and disease diagnosis. Biomarkers 2004;9(1):1-31.

9. German JB, Bauman DE, Burrin DG, et al. Metabolomics in the open-ing decade of the 21st century: building the roads to individualizedhealth. J Nutr 2004;134(10):2729-2732.

10. Dunn WB, Ellis DI. Metabolomics: current analytical platforms and

methodologies. Trends Anal Chem 2005;24(4):285-294.

11. Dunn WB, Bailey NJ, Johnson HE. Measuring the metabolome: currentanalytical technologies. Analyst 2005;130(5):606-625.

12. van der G, van der Heijden R, Verheij ER. The role of mass spectrome-try in systems biology: data processing and identification strategies inmetabolomics. In: Ashcroft AE, Brenton G, Monaghan J (eds). Advancesin Mass Spectrometry. Amsterdam: Elsevier Science; 2004, pp 145-164.

13. Trygg J, Wold S. Orthogonal projections to latent structures (OPLS). J Chemom 2002;16(3):119-128.

14. Ramadan Z, Zhang PF, Jacobs DM, et al. An NMR- and MS-basedmetabonomic investigation of saliva metabolic changes in feline odon-toclastic resorptive lesions (FORL)-diseased cats. Metabolomics2007;3(2):113-119.

15. Rezzi S, Ramadan Z, Martin FPJ, et al. Human metabolic phenotypeslink directly to specific dietary preferences in healthy individuals. J Pro-teome Res 2007;6(11):4469-4477.

16. Dumas ME, Barton RH, Toye A, et al. Metabolic profiling reveals a con-tribution of gut microbiota to fatty liver phenotype in insulin-resistantmice. Proc Natl Acad Sci USA 2006;103(33):12511-12516.


Medical formats for approaching aging-related problems arelargely (and necessarily) structured as body systems–associ-ated catalogues of diseases and algorithms for diagnosis andtherapy. Emerging knowledge indicates that this approach,although it is the expected standard in most clinical settings,can limit understanding of the more fundamental place thataging may have in the evolution of the natural biology of hu-mans and animals.1

The term “diseases of aging” may refer to problems thatare mediated intrinsically (and may or may not be influencedextrinsically) to disrupt “normal, healthy aging,” consistentwith the usual understanding. However, a viable alternative isthat some diseases of late life do not represent newly arisendisruptive processes but rather failure of adaptations that evo-lution designed as protective mechanisms. In this view, dis-eases of aging do not disrupt the flow of life events but areintegral parts of it. This (presently) hypothetical view alsoleads to asking research questions in new ways, as suggestedby the following examples.

Hematology and clinical chemistry profiling have beenstandards for veterinary biomedical screening for many years.A recent study of cats revealed that heritability may influencephenotypic expression of many these variables and thus maypotentially also influence interpretations of results2 (Table 1).In a genetic context, changing expression of some variableswith age may represent an additional level of complexity. Ap-propriate questions might be:

• Because interactions between chronology and heritabilitymay lead to outcomes that are not yet explored, what (ifanything) needs to be known about segregating newlyarising intrinsic insults from failures of programmed ef-fects?

• Might this knowledge affect uses of therapeutic agents orinfluence decisions to support rather than mitigate certainaspects of changing systems physiology or morphology?

In another study, cats that died from renal causes in a largecattery had a longer mean life span than cats without renalcauses of death (Table 2).3 Cats that succumbed to nonrenalcauses of death were evaluated across categories of death-causing diseases, with results confirming that the observa-tions did not result from the structure of the colony or otherdisease frequencies.3 Inbreeding coefficients in this colonywere low as well, documenting that the observations alsowere not consequences of the breeding program. The longermean lifespan among cats that died from renal failure, how-ever, does not confirm renal disease as a “default outcome”for this species. Rather, the wide variation in ages at deathand evidence of renal changes from early adulthood suggestthat tubular changes may represent a mechanism for pro-grammed deletion in the face of dysfunction. Appropriatequestions might be:

• What nature of protective mechanisms might influencewhat we view as a disease process?

• What is the nature of the threshold for failure of adapta-tions?

• What is the role of signaled apoptosis in renal tubulardeletion in cats?

The observation that terminal body condition has a smallbut significant component of quantitative heritability (h2 = 0.16)additionally supports the idea of phenotypic programming. Inresponse to the obvious follow-up question about healthy bodycomposition, body composition (dual-energy x-ray absorp-tiometry; DEXA) data from 119 of these cats over their years ofhealthy adult life yielded heritability and principal components(h2 = 0.40, PC2; Table 3) that also documented a role for quan-titative inheritance. The opposing directions of lean–fat com-ponents of body composition (PC2; Table 3) suggestindependent physiologic drivers for fat and lean grams withinindividuals. Given the relationship of body composition tolongevity, this observation also implies programming for aging.3

Can We Personalize Nutrition-Based Preventive Health Care?

Dennis F. Lawler, DVMNestlé Research Center

St. Louis, Missouri


Finally, although nearly 70% of the cats in this databasehad histologic renal changes, no quantitative heritability wasfound for the tubulointerstitial deletion phenotype. The twoprimary interpretations of this observation are that the phe-notype (1) is not heritable and (2) is drifting toward fixationin the population. The latter interpretation remains a possi-bility because only about 130,000 years have passed since di-vergence of Felis silvestris catus from the ancestor, Felis silvestrislybica. These observations further suggest the need for a moreadvanced understanding of the biology of speciation and traitfixation as these processes relate to aging phenotypes andthresholds for failure.

In the lifetime canine diet-restriction (DR) study, high fat

mass (>25%) and declining lean mass each predicted deathmost strongly at 1 year prior, with population decline trajec-tories starting after years 9 and 11 in the control-fed (CF) andDR groups, respectively.4 The correlation of fat to lean gramswithin individuals was negligible (r = –0.08).5 Additionally,increasing insulin resistance (a correlate of fat mass at r =0.67) independently predicted life span and clinical onset ofchronic diseases of late life. Although insulin sensitivity wasconsistently higher among DR dogs, declining sensitivity wasobserved in each group after year 9 (occurring later amongDR dogs).5 This observation suggests the existence of anheretofore uncharacterized threshold that prompts questionsabout its nature and about other detectable associations.

Osteoarthritis (OA) is an important chronic disease of dogs,and DR delayed onset and progression toward joint failure. Lackof bimodal distribution of radiographic hip OA onset indicatesthat early- and late-life onset do not have different causes andthat hip dysplasia OA may occur at any time during life. Bothfindings refute decades of conventional thinking.6 In both feed-ing groups, symptomatic (vs. morphologic) hip OA onset oc-curred only after 84 months of age (but was delayed by DR)even though 23 of the 48 dogs had radiographic OA by 1 yearof age. These observations again suggest that some late-life “dis-eases” might represent failure of protective adaptations. Median“failure” ages for joints or the spine were 10.3 and 13.3 years inthe CF and DR groups, respectively.5 In both groups, 85% ofmortalities occurred after year 9, albeit with a median of 1.8years later among DR dogs, an observation associated with bothlean mass decline and changes in insulin resistance.

Viewed in retrospect, the ninth year represented a pivotalpoint in the life spans of the CF and DR dogs, with groupspecificity such that most events relating to the death trajec-tory occurred later among DR dogs. To further elaborate these

TABLE 1Quantitative Heritability of Hematology

and Clinical Chemistry Variables in a Colony of Adult Domestic Shorthair Cats

Variable Patients (n) Heritability (h2)

Erythrocytes 106/μl 564 0.48Mean cell volume (fl) 564 0.69Packed cell volume (%) 564 0.44Hemoglobin (g/dl) 564 0.41

Albumin (g/dl) 445 0.61Alkaline phosphatase (IU/L) 445 0.78Alanine transferase (IU/L) 445 0.39Calcium (mg/dl) 445 0.39Cholesterol (mg/dl) 445 0.47Chloride (mmol/L) 445 0.28Creatinine (mg/dl) 445 0.26Glucose (mg/dl) 444 0.39Potassium (mEq/L) 530 0.25Sodium (mEq/L) 530 0.16Phosphorus (mg/dl) 530 0.20Triglyceride (mg/dl) 444 0.13Total protein (g/dl) 469 0.38Urea nitrogen (mg/dl) 466 0.23

O2 saturation (%) 629 0.59pCO2 (mm Hg) 629 0.45TCO2 (mEq/L) 629 0.43HCO3

– 629 0.40pO2 (mm Hg) 629 0.40Base excess (mEq/L) 629 0.27pH 629 0.25

Adapted from Lawler DF, Chase K, Teckenbrock R, Lark KG. Heritable componentsof feline hematology, clinical chemistry, and acid-base profiles. J Hered 2006;97(6):549-554.

TABLE 2Renal and Nonrenal Causes of Death among

491 Adult Cats Maintained for life in the Same Cattery, Expressed by Age at Death

Patients Mean AgeCause of Death (n) at Death (mo) P

Renal 187 140

Nonrenal 304 120 <.001

Adapted from Lawler DF, Chase K, Teckenbrock R, Lark KG. Heritable componentsof feline hematology, clinical chemistry, and acid-base profiles. J Hered 2006;97(6):549-554.


CAN WE PERSONALIZE NUTRITION-BASED PREVENTIVE HEALTH CARE?

observations, nuclear magnetic resonance (NMR; metabo-nomics) was used to evaluate small-molecule metabolites inbiologic fluids from the dogs. One potential advantage ofsmall-molecule screening is earlier detection of metabolicchanges that may predict later events in contrast to more tra-ditional methods for clinical chemistry that often reflect onlymore advanced stages of physiologic change. Changes inurine and blood metabolites among CF and DR dogs wereinfluenced by age and by DR.

Urine NMR life trajectories demonstrated that chronologicmetabolite signaling was influenced simply by consuming25% less of the same food on a daily basis. In urine, the pre-dominating signals reflected age, with some influence of feed-ing group and evidence of involvement of gut microbiota(Table 4). Urine signals associated with diet restriction in-cluded downregulation of creatinine, 1-methylnicotinamide,lactate, acetate, and succinate and upregulation of hippurate,phenylacetyl glycine, and 4-hydroxyphenylpropionic acid.The urine signals suggest that DR is associated with a lowerlevel of energy-related metabolism in association with up-regulation of signals from gut microbiota.7

Serum NMR life trajectories, acquired and processed as forurine, demonstrated a shift in the spectrotypes of both groupsfrom year 9 onward. The CF and DR groups both reflectedthe same shift away from glucose-protein predominance tolipid predominance, although the shift was less predominantin the DR group8 (Table 5).

These analyses identified a significant senescence-relatedmilestone and provided longitudinal contribution withineach spectrotype. It is important that this metabolic transi-

TABLE 3Heritability and Principal Component Analyses of Healthy Life Body Composition

Among 119 Adult Cats Maintained for Life in the Same Cattery

PC1 PC2 PC3 PC4 PC5 PC6 PC7

Percent of variance 55.0 24.7 12.2 4.1 2.2 1.3 0.5

Heritability 0.33a 0.40b 0.00 0.21 0.11 0.74b 0.00

Loadings

Bone density 0.30 –0.07 0.85 –0.11 0.29 –0.29 –0.06% fat 0.33 –0.54 –0.08 –0.48 –0.21 0.06 –0.57Body weight 0.37 –0.32 –0.49 –0.23 0.63 –0.25 –0.08BCS 0.38 0.41 –0.01 0.64 0.36 0.39 –0.03Lean grams 0.39 –0.43 –0.06 0.45 –0.49 –0.25 –0.40Bone minerals 0.41 –0.40 0.09 –0.30 –0.17 0.70 0.23Fat grams 0.45 0.30 –0.13 –0.04 –0.29 –0.38 0.68

aP < .05.bP < .01.Adapted from Lawler DF, Chase K, Teckenbrock R, Lark KG. Heritable components of feline hematology, clinical chemistry, and acid-base profiles. J Hered 2006;97(6):549-554.

TABLE 4Effect of Age on Predominant Metabolites

in the Urine of Dogs*

Age (yr)More Predominant

MetabolitesLess Predominant

Metabolites

0.25 N-AcetylglycoproteinDimethylamine

2-Oxobutyrate2-PropanolCreatinineHippurateDimethylglycine3-Hydroxyphenylpropionic

acid

1.5 TaurineHippurate3-Hydroxyphenylpropionic

acidCreatinine

AcetateDimethylamineSuccinateTrimethylamine oxideLactateGlycoproteins1-Methylnicotinamide

9.0 α-KetobutyrateDimethylglycine2-PropanolTrimethylamineLactateAlanineSuccinateCreatinine

Glycoprotein

*Dogs are not separated based on feeding group.


tion was identified before life trajectory changes in body com-position, insulin metabolism, late-life diseases, and mortal-ity. The potential role of gut microbiota in health has beenthe subject of increasing interest in the role of microbiota inhealth-related metabolic signaling, and our data support apossible association.9

SUMMARYThese observations add significantly to the understanding ofhow veterinary scientists and clinicians might go about eval-uating the aging process in dogs and indicate the likelihoodof recognizing individual needs for preventive interventionearlier in life than was previously possible. NMR evaluationof sera and urine from CF and DR dogs revealed a shift to-ward a lipid-predominant explanation for variance in NMRspectrotypes after the ninth year, with the longer-living DR

group expressing the shift less prominently. These data, alongwith a newer view of possible roles of gut microbiota inhealth,9 suggest new possibilities for the design of nutritionalinterventions. However, the evaluation of common feline andcanine late-life changes also indicate that the existence ofmultiple cohorts within certain phenotypes and biologicthresholds for failure provide a level of complexity that mustbe understood as well before effective interventions can bedevised. In any event, this new state of knowledge documentsthat “one size fits all” is no longer true.

REFERENCES1. Makinen V-P, Soininen P, Forsblom C, Ingman MP, et al. 1H-NMR

metabonomics approach to the disease continuum of diabetic compli-cations and premature death. Mol Syst Biol 2008;4:167:1-12.

2. Lawler DF, Chase K, Teckenbrock R, Lark KG. Heritable components offeline hematology, clinical chemistry, and acid-base profiles. J Hered2006;97(6):549-554.

3. Lawler DF, Evans RH, Chase K, et al. The aging feline kidney: a modelmortality antagonist? J Fel Med Surg 2006;8:363-371.

4. Lawler DF, Evans RH, Larson BT, et al. Influence of lifetime food re-striction on causes, time, and predictors of death in dogs. JAVMA2005;226(2):225-231.

5. Larson BT, Lawler DF, Spitznagel EL, Kealy RD. Improved glucose toler-ance with lifetime diet restriction favorably affects disease and survivalin dogs. J Nutr 2003;133:2887-2892.

6. Smith GK, Paster ER, Powers MY, et al. Lifelong diet restriction and ra-diographic evidence of osteoarthritis of the hip joint in dogs. JAVMA2006;229:690-693.

7. Wang Y, Lawler DF, Larson BT, et al. Metabonomics investigation ofaging and dietary intervention: urinary metabolic profiles from a life-long dog diet restriction study of dogs. J Proteome Res 2007;6:1846-1854.

8. Richards SE, Wang Y, Lawler D, et al. Self-modeling curve resolution re-covery of temporal metabolite signal modulation in NMR spectroscopicdatasets: application to a life-long caloric restriction study in dogs. AnalChem 2008;80:4876-4885.

9. Hooper LV, Gordon JI. Commensal host-bacterial relationships in thegut. Science 2001;292:1115-1118.

TABLE 5Predominant Age-Related Metabolites

in the Serum of Dogs*

Predominance Predominance1- to 9-Year Spectrotype 9+-Year Spectrotype

Glucose

Citrate

Lactate

Alamine

Albumin

N-Acetylated glycoproteins

Phosphatidylcholine

Lipoprotein lipids from:TriglyceridesPhospholipidsCholesterolsGlucose

*Dogs are not separated based on feeding group.


The intestinal microbiome—the community of microbiotaresiding in the intestinal tract—consists of a balance of ben-eficial and potentially harmful microbiota. Intestinal micro-biota are essential for establishment of a healthy immunesystem and maintenance of healthy intestinal function.

Probiotics, which are live, beneficial microbiota consumedby animals, may help to restore and maintain microbiota bal-ance during times of stress and may promote the develop-ment of healthy immune function. Specific probioticmicrobiota include bifidobacteria and lactic acid bacteriasuch as Enterococcus faecium SF68 and lactobacilli. Becauseprobiotics are live microorganisms, keeping them stable be-fore consumption is one of the biggest barriers to producingan effective probiotic. Safety is another important factor toconsider when selecting a probiotic. Although numerousstudies have established the benefits of probiotics in humansand livestock, until recently, few studies have evaluated pro-biotic efficacy in dogs and cats.

Numerous studies have proven the efficacy of the probi-otic E. faecium SF68 in humans and livestock. Benefits in hu-mans include a reduction in acute and antibiotic-induceddiarrhea. In published research, E. faecium SF68 has beenshown to reduce mortality, pathogen shedding, and the sever-ity and duration of diarrhea in livestock. Decreased severity ofprotozoal parasitic infections has also been reported; SF68improved immune response to Giardia infection in mice anddecreased shedding of Giardia trophozoites in the feces ofthese mice.

After extensive safety and stability evaluation, a series ofefficacy trials were conducted to determine the benefits of theprobiotic E. faecium SF68 (NCIMB 10415) when fed to dogsand cats. In each study, dogs or cats were fed either a controldiet or the same diet supplemented with SF68.

Puppies fed SF68 had increased fecal bifidobacteria andfecal lactobacilli and better fecal quality than puppies fed thecontrol food. Compared with the control puppies, puppies

fed the probiotic also maintained their vaccination titerslonger and had higher levels of secretary IgA. Similarly, eld-erly beagles fed SF68 for 6 months maintained higher fecalIgA than elderly beagles fed a control food. Kittens fed E. fae-cium SF68 had higher serum IgA compared with kittens fedthe control diet. A decreased incidence and duration of nat-urally occurring diarrhea and improved microbiota balancewere also observed in kittens and adult cats consuming E. fae-cium SF68.

In conclusion, although previous studies have proven thebenefits of probiotics in humans and livestock, new researchhas shown that the probiotic E. faecium SF68 can help pro-mote a stable, healthy microbiota balance and a healthy im-mune system in dogs and cats.

RECOMMENDED READINGAllen SJ, Okoko B, Martinez E, et al. Probiotics for treating infectious diar-

rhoea. Cochrane Database Syst Rev 2004;(2):CD003048.

Benyacoub J, Perez PF, Rochat F, et al. Enterococcus faecium SF68 enhances theimmune response to Giardia intestinalis in mice. J Nutr 2005;135: 1171-1176.

Benyacoub J, Czarnecki-Maulden GL, Cavadini C, et al. Supplementation offood with Enterococcus faecium (SF68) stimulates immune functions inyoung dogs. J Nutr 2003;133:1158-1162.

Czarnecki-Maulden G, Cavadini C, Lawler D, Benyacoub J. Incidence of nat-urally occurring diarrhea in kittens fed Enterococcus faecium SF68. Com-pend Contin Educ Vet 2006;29(suppl 2A):37.

Knorr R, Praplan F, Benyacoub J, Cavadin C. Screening and selection of petprobiotics. Compend Contin Educ Pract Vet 2004;26(suppl 2A):68.

Simmering R, Cavadini C, Rochat F, et al. Development of probiotic strainsfor pet food application. Proc WSAVA; 2001.

Taras D, Vahjen W, Macha M, Simon O. Performance, diarrhea incidence,and occurrence of Escherichia coli virulence genes during long-term ad-ministration of a probiotic Enterococcus faecium strain to sows and piglets.J Anim Sci 2006;84:608-617.

Walker R, Buckley M. Probiotic microbes: the scientific basis. Am Acad Mi-crobiol 2006.

Weese JS. Evaluation of deficiencies in labeling of commercial probiotics.Can Vet J 2003;44(12):982-983.

Weese JS. Microbiologic evaluation of commercial probiotics. JAVMA2002;220(6):794-797.

Weese JS, Arroyo L. Bacteriological evaluation of dog and cat diets that claimto contain probiotics. Can Vet J 2003;44(3):212-216.

Enterococcus faecium SF68 as a Probiotic for Dogs and Cats

Gail Czarnecki-Maulden, PhDNestlé Research Center

St. Louis, Missouri


Nutritional immunology is an emerging field of study thatevolved with the investigation of nutritional deficienciescaused by malnutrition, which are sometimes referred to asnutritionally acquired immune deficiency syndrome (NAIDS).1 Al-though malnutrition still remains a global problem even inthe 21st century because of natural calamities, famine, andwar, many of the detrimental effects of malnutrition can beaddressed by correcting underlying nutritional problems. Un-like immunodeficiency caused by malnutrition, immunode-ficiencies that are age related (life stage) or caused by stress ordietary overindulgence need a more comprehensive strategyand cannot be addressed simply by correcting nutritionalproblems. Although micro- and macronutrient deficienciescaused by changes in nutrient absorption, reduced food in-take, and the like do contribute, these immunodeficienciescan be more difficult to evaluate, understand, and manage.More importantly, practicing clinicians are more likely to seepatients with immunodeficiency that is unrelated to malnu-trition. Immunodeficiency, irrespective of its cause, may se-verely undermine the health of the animal, triggeringdebilitating conditions such as infections, malignancies, andautoimmune diseases.

The immune system is central to a number of physiologicand pathologic processes involved in noninfectious diseasesand aging. In an earlier review,2 I proposed a more integratedview of the immune system to understand the impact of nu-trition on immune health. From this perspective, all tissuesand organs that have a function in protecting the integrity ofthe individual from both external and internal challengeswould be part of the “immune system,” making tissues suchas the skin, coat, and epithelial lining of internal organs comeunder the purview of the “immune system” because of theroles they play in protection. This paper reviews the causes ofimmunodeficiency in otherwise healthy animals, explainswhy what we eat influences the immune system, and pro-poses a framework to understand how nutrition interactswith the immune system.

WHY SHOULD WE ENHANCE IMMUNESTATUS IN A HEALTHY ANIMAL?Age and stress are two very important factors that significantlyimpact the immune status of an animal. The immune re-sponses of neonates and older animals tend to be less vigor-ous than those of young adults, making neonates and olderanimals more susceptible to infection.3 Aging is characterizedby low-level chronic inflammation that contributes to the de-clining ability of the immune system to respond and regulateimmune response.4 This chronic inflammation is believed tobe one of the underlying causes of a variety of diseases such astype II diabetes, cancers, autoimmunity, and declining neu-rologic health along with a greater susceptibility to infection.4

Stress, particularly chronic stress, has been shown to havea significant negative impact on the immune system, both inits ability to respond to challenges as well by chronic in-flammatory conditions caused by stress.5

THE EFFECT OF AGING ON THE IMMUNE SYSTEMImmune Response in NeonatesImmune response varies by age. Immune responses inneonates tend not to be as strong as in adult animals.6 Indogs, for example, responses to mitogenic stimuli are signif-icantly lower in beagle puppies from birth through 4 weeksof age compared with adults.7 Somberg et al8 found that thein vitro lymphocyte proliferation activity of newborn pupswas 50% lower than in adults.

Neonates, although competent in responding to immunechallenges, tend to exhibit a T-helper type 2 (Th2) bias in theirimmune responses.3 A T-helper type 1 (Th1)–biased immuneresponse is characterized by proinflammatory cytokines suchas interferon-γ (IFN-γ), interleukin-6 (IL-6), and tumor necro-sis factor-α (TNF-α) and hence is more effective in preventinginfectious diseases. In contrast, a Th2-biased immune re-sponse is predominated by antiinflammatory cytokines suchas IL-10, IL-4, and transforming growth factor-β (TGF-β) and

ImmunonutritionEbenezer Satyaraj, PhD

Nestlé Research CenterSt. Louis, Missouri


IMMUNONUTRITION

is not as effective in dealing with microbial infections. Thereare several reasons for this Th2 bias, including:

1. Reduced number and function of antigen-presenting cells(APC) because of their reduced capacity to express crucialco-stimulatory molecules CD86 and CD40 and upregu-late major histocompatibility complex (MHC) class IImolecules.9 MHC class II molecules and co-stimulatorymolecules are required to present antigens to CD4+ T cellsto initiate an immune response.

2. Influence of the fetoplacental environment, which tendsto be Th2 dominant because of locally acting cytokinesand hormones3

3. Neonatal B cells, which also function as APC, have alteredsignaling because of lowered MHC class II molecules aswell as lowered accessory signaling molecules. Lack of up-regulation of CD40 and CD40L tends to dampen B-cellresponse as well as their ability to class switch contribut-ing to the Th2 bias.

4. Neonatal Th1 cells undergo apoptosis because they expressunique receptors. In a recent study, Lee et al10 have shownthat although a primary immune response from neonatal Tcells includes a significant Th1 component, the Th1 cellsgenerated have unique characteristics. They tend to havehigh levels of IL-13Rα1, which heterodimerizes with IL-4Rα.As the immune response progresses, because of the lack ofappropriate dendritic cells (DCs), the immune response isdominated by lL-4, which binds the IL-13Rα–IL-4Rα–IL-13Rα–IL-4Rα complex expressed on the Th1 cells and in-duces apoptosis, eliminating the Th1 cells and resulting in aTh2 bias. As the neonate ages, a significant number of ap-propriate DCs start accumulating, especially in the spleen.These DCs produce IL-12, and this IL-12 triggers the down-regulation IL-13Rα 1 on the Th1 cells, rescuing them fromIL-4–induced apoptosis. This study underlines the need forcytokines such as IL-12 to initiate a Th1-biased immune re-sponse.10

Immune Response Changes with AgingAging brings changes in both the humoral and cellular immuneresponses. Mechanisms leading to these changes range from de-fects in the hematopoietic bone marrow to defects in lymphocytemigration, maturation, and function. Chronic involution of thethymus gland is thought to be one of the major contributing fac-tors to loss of immune function with increasing age.11

With age, the immune system loses its plasticity, leading the

organism to be a “low responder.” Immune plasticity is theability of the immune system to remodel itself to promptly re-spond to harmful stimuli, which include oxidative stress, andto return to a quiescent state after the danger passes. One ofthe reasons for this declining immune plasticity is that stressbecomes chronic with age.12 This results in reduced immuneresponse to oxidative stress and a lower cellular capacity inDNA repair, leading to a condition described as “immunose-nescence,” which increases the risk of age-related diseases suchas cancer and infection.13,14 Declining immune plasticity leadsthe cells of the immune system to undergo cell death or necro-sis triggered by oxidative stress.15

In a review article, Rink and Seyfarth16 summarized the de-scribed changes of the immune system in human centenari-ans. Significant differences exist between the immune systemsof elderly individuals compared with those of young individ-uals, although most changes in the elderly individuals werewithin the normal ranges of the appropriate parameter. Theserum levels of the immunoglobulins G, M, and A, as well asthe number of benign monoclonal gammopathies and thenumber of autoantibodies, increased with age. The titers ofzinc, an important mineral needed for the cells of the immunesystem,17 were significantly decreased in the serum of elderlyindividuals. The number of lymphocytes decreased, and thenumber of neutrophils increased with aging. There are manydescriptions about changes of the leukocyte subpopulation inaging, which are not always comparable. However, the num-ber of T cells (CD3) decreases. Within the T cells, the CD8cells decreased more than the CD4 cells, resulting in an in-creased CD4:CD8 ratio. Whereas memory T cells (CD45RO)increased during aging, naive T cells (CD45RA) decreased. Thenumber of B cells (CD19) also decreased, but the number ofnatural killer (NK) cells (CD16, CD56, CD57) increased withaging. Greeley et al18 have described similar changes in the im-mune system of aging dogs.

The life stage of the animal has a significant impact on im-mune status and is one of the important reasons to considernutritional strategies to enhance immune system effectiveness.

STRESSStress has a significant negative impact on the immune sys-tem. Both major and minor stressful events have been shownto have a profound influence on immune responses in bothanimal and human studies. As described earlier, oxidativestress gradually erodes immune plasticity, and one of the hall-marks of chronic stress is the general increase in levels of ox-


idative stress. Research in this area has spawned a new disci-pline called psychoneuroimmunology that has generated a lotof interest among basic researchers as well as clinicians. Usingvaccine responses as a indicator of immune status,19–23 re-searchers have demonstrated that among medical studentstaking examinations, the level of stress had a negative corre-lation to vaccine response (virus-specific antibody and T-cellresponses to hepatitis B virus vaccine), although the degree ofsocial support had a positive correlation to vaccine response.19

Another good example of chronic stress is the stress asso-ciated with caregiving for a spouse with Alzheimer’s disease,which has been shown to be associated with a poorer anti-body response to an influenza virus vaccine compared withcontrol subjects with well spouses.24 Vaccine responsesdemonstrate clinically relevant alterations in an immunologicresponse to challenge under well-controlled conditions andtherefore can be used as a surrogate for responses to an infec-tious challenge. Therefore, individuals who respond poorly tovaccines would likely have greater susceptibility to the infec-tious agent compared with those who have better vaccine re-sponses. Consistent with this argument, adults who showpoorer responses to vaccines also experience higher rates ofclinical illness, as well as longer-lasting infectious episodes.25,26

Therefore, from these vaccine studies, it is hypothesized thatstress puts individuals at greater risk for more severe illness. Insupport of this idea, Cohen et al27 showed that human vol-unteers who were inoculated with five different strains of res-piratory viruses showed a dose-dependent relationshipbetween stress and clinical symptoms observed after infection.

From a mechanistic standpoint, stress appears to delay in-flammation by reducing efficiency of CD62L-mediated im-mune surveillance by phagocytes.28 Stress decreases IFN-γsecretion by lymphocytes and may decrease antigen presen-tation efficiency by downregulating MHC class II moleculeexpression on APCs and delay or impair immune responsesto vaccination.

Stress sets into motion physiologic changes—the fight-or-flight response—that help the organism cope with the stres-sor. However, repeated exposure to stress results in chronicactivation of stress responses, which include activation of thehypothalamic–pituitary–adrenal axis and the sympathetic–adrenal–medullary axis, resulting in the production of glu-cocorticoid (GC) hormones and catecholamine. GC receptorsare expressed by a variety of immune cells, and they bind cor-tisol, interfering with NF-κB (nuclear factor κ light chain en-hancer of activated B cells) function, which in turn regulates

the activity of cytokine-producing immune cells. Severalmodels have been proposed to explain the mechanism of ac-tion of GC hormones in the immune cells.29 GCs impact cy-tokine expression, expression of co-stimulatory molecules,and adhesion molecules and influences immune cell migra-tion, differentiation, proliferation, and effector function.30–32

Adrenergic receptors bind epinephrine and norepinephrineand activate the cyclic adenosine monophosphate (cAMP) re-sponse element binding protein, inducing the transcriptionof genes encoding a variety of immune response genes, in-cluding genes for cytokines. Elevated levels of catecholaminesproduced during stress can modify immune response genes.33

Along with life stage, stress is a key factor that can negativelyimpact the immune status of an animal irrespective of its age.

Age and stress can undermine the immune status in anotherwise healthy animal. Immunodeficiency, regardless ofits cause, may severely undermine the health of the animal,triggering debilitating diseases such as infections, malignan-cies, and autoimmune diseases. Hence, there is a critical needto evaluate immune status and address deviations that, ifmanaged effectively, may significantly enhance the quality oflife for the animal and individual.

HOW CAN DIET INFLUENCE THE IMMUNESYSTEM?The Gut—The Largest Immune OrganBesides being the gateway for nutrient intake, the gut is thelargest immune organ, with more than 65% of all the im-mune cells in the body and more than 90% of all Ig-produc-ing cells.34,35 The intestine in an adult human contains about7 x 1010 Ig-producing cells, considerably more than the bonemarrow (2.5 x 1010).36 The gut is the major compartment forthe humoral immunity in the body, with 90% of the productbeing exported to the lumen as secretory IgA.36 It is estimatedthat a daily total of about 3 g of secretory IgA is pumped intothe lumen of an adult human.37 Thus, a significant part of theimmune system interacts with what we eat.

GALT Plays an Important Role in theDevelopment of the Immune SystemResearch conducted with germ-free animals has documentedthat the immune stimuli from environmental antigens, espe-cially microbiota in the gut, are crucial for development of ahealthy immune system.38 Germ-free animals tend to have veryunderdeveloped immune systems, underscoring the roleplayed by symbiotic microflora and the associated environ-


IMMUNONUTRITION

mental antigens. Gut-associated lymphoid tissue (GALT) isunique in its ability to be exposed to a diverse array of anti-gens from foods (~10–15 kg/yr in humans) and from the morethan 1,000 species of commensal microorganisms (1012 ml perml of colon content, making them the most numerous cells inthe body) and yet remain quiescent until it encounters a threat,such as a pathogen. This is initiated by molecules calledpathogen-associated molecular patterns (PAMPs) expressed by mi-crobial pathogens. PAMPs are highly conserved motifs presentin microorganisms. PAMPs include lipopolysaccharides (LPS)from the gram-negative cell wall, peptidoglycan, lipoteichoicacids from the gram-positive cell wall, the sugar mannose(common in microbial glycolipids and glycoproteins but rarein mammals), bacterial DNA, N-formylmethionine found inbacterial proteins, double-stranded RNA from viruses, and glu-cans from fungal cell walls. PAMPs can also be recognized bya series of soluble pattern-recognition receptors in the bloodthat activate the complement pathway. Therefore, GALT offersunique opportunities for immunomodulation via the diet.

Efficient Antigen Presentation isFundamental for Efficient Immune ResponseAn appropriate innate immune response is a prerequisite fora primary adaptive immune response. It sets the stage that di-rects the course of the adaptive immune response because ofthe co-stimulatory molecules induced on the APCs. In turn,APCs provide important signals in the form of secreted cy-tokines that influence the characteristics of the adaptive im-mune response. An efficient immune response requiresefficient antigen presentation to T lymphocytes. APC func-tion is central to the altered immune response that is charac-teristic of neonatal immune system, the immune response ofan aging immune system, and the immune response duringstress. In all three cases, because of the lack of inflammatorycytokines such as IL-1 and IL-12, APCs responding to an im-mune challenge are not able to efficiently upregulate MHCclass II molecules as well as co-stimulatory molecules such asCD86. Lack of these cytokine signals also modifies the adap-tive immune response, reducing its efficiency and giving it aTh2 bias. The resulting immune response therefore tends toless efficient. Consequently, the approach to addressing thisdeficiency would focus on providing the required signaling tothe APCs.39 The immune receptors of the innate immune sys-tem present in the gut serve this function and are the primarytargets of strategies for immunomodulation via diet. For ex-ample, toll-receptor agonists such as yeast β-glucans,40 yeast

mannans,41 and nucleic acids42 are great examples of the useof PAMPs as immune response modifiers (IRMs). Probioticsinteract with the immune system by virtue of their PAMPsmolecules such as LPS.43 These IRMs primarily initiate a localproinflammatory cytokine secretion that activates local APCsto upregulate MHC class II and co-stimulatory molecules, en-abling APCs to present antigens efficiently to T lymphocytesand initiate an effective immune response. IRMs provided viathe diet enhance APC efficiency. APCs in the gut continuallyprocess and present antigens to T lymphocytes in the GALT.Although the GALT is quiescent to the myriad of antigenicstimuli it receives via the diet, when it encounters a pathogen,it is able to initiate a more efficient immune response.

The enhanced immune activity induced by dietary IRMshas a tendency to spread to the entire immune system. Theconcept of a common mucosal immune system is being pro-posed because of the trafficking of activated lymphocytesfrom induction to effector sites, with significant overlap withthe nonmucosal immune system.44 The beneficial effects ofdietary IRMs on the overall immune system is not fully un-derstood but can perhaps be explained by the circulation oflymphocytes and effector cytokines.

Nutrition Interacts with the Immune System at Multiple LevelsInteraction between nutrition and the immune system takesplace at multiple levels and for simplicity can be consideredin a framework of four stages. Stages I and II are passive be-cause they involve providing the immune system with essen-tial nutrients. Stages III and IV focus on modifying theimmune response using agents such as IRMs that primarilytarget the PAMP receptors in the gut and therefore are moreactive encounters in the immune system and diet interaction.

Stage I: Complete NutritionAt the primary level, the focus revolves around dietary energy,protein, vitamins (vitamins A, C, and E), and minerals (e.g., zinc,magnesium, iron; described in detail in Klaus-Helge and Rink17).Minerals such as calcium and magnesium drive signaling mech-anisms in the immune system and are therefore also importantfor an enhanced immune response. Providing basic nutrition isthe very least we can do for the immune system.

Stage II: Optimizing Macro- and MicronutrientsThe second stage involves optimizing key nutrients that arecritical for the immune cells. The immune system has a need


for certain nutrients, and providing greater amounts of thesekey nutrients better targets the immune cells. A temporary de-ficiency of a key nutrient may negatively impact the immunesystem. For example, during strenuous exercise, muscle cellspreferentially use glutamine as their energy source. As a re-sult, there is a reduction of glutamine levels in circulation.Glutamine is also the preferred energy source for immunecells and after strenuous exercise. Because of low levels of glu-tamine in circulation, immune cells cannot function effi-ciently if challenged, making athletes vulnerable to infectionsimmediately after vigorous bouts of exercise.45

Key ingredients needed for a healthy immune system in-clude higher levels of and higher quality proteins in the diet.At a molecular level, proteins make up the structural compo-nents and mediate key process of the immune system. Recep-tors, cytokines, immunoglobulins, complement components,and bactericidal proteins are all proteins. A source of high-quality protein in the diet is therefore important for a healthyimmune system. Vitamins A, C, and E and minerals such aszinc, magnesium, and iron are critical for the immune system.For this reason, dietary products for companion animals oftenexceed the required levels for dietary energy, proteins, vita-mins, and minerals.

Addressing oxidative stress and subsequent damage to cel-lular DNA is another example of optimizing micro- andmacronutrients. Along with other environmental stressors,aging tends to increase the levels of oxidative damage to cel-lular DNA, including the DNA in immune cells. Cells havethe ability to repair damage in response to injury or stress.However, beyond a point, the damage may be irreparable andmay result in cell death by apoptosis. Oxidative DNA damagecaused by free radicals produced during cellular metabolismis one of the primary causes of cell death.46 Increased apop-tosis may break immune tolerance to self-antigens, resultingin autoimmunity.47 Immunosenescence is characterized by adecreased response to mitogens, decreased cytokine produc-tion, and changes in signal transduction and has been asso-ciated with aging (reviewed in Klaus-Helge and Rink17).Various strategies may help address senescence, tissue dam-age, and apoptosis associated with aging, including:

• Caloric restriction (CR): Apart from increasing the lifespan,48 data from laboratory animals have demonstratedthat CR reduces immunosenescence.49 Recent data from aCR study conducted in Labrador retrievers clearly showsthat CR may help retard immunosenescence.50 A CR diet

is likely to help aging animals maintain a healthier im-mune system.

• Antioxidants: Increased levels of antioxidants such as vi-tamin C,51 vitamin E,17 and carotenoids (β-carotene, α-carotene, lycopene, astaxanthin) may help prevent damagemediated by these free radicals. A number of reports havedocumented the benefits of carotenoids in dogs, particu-larly in older ones.52,53

• Prebiotics that support or help maintain normal gut floraalso fall into this category. Intestinal microflora play animportant role in keeping the immune system primed toprevent colonization by pathogenic microbes. However,under certain conditions, such as after antibiotic therapy,gastrointestinal (GI) infections, stress, or older age, thenormal flora in the GI is perturbed and may lead to achange in the bacterial flora because of an overgrowth ofharmful bacteria (e.g., Clostridium difficile). Prebiotics suchas inulin help animals maintain a healthy commensalpopulation in the gut under stress.54

The first two stages are passive approaches in immunonu-trition. These stages are passive because the focus is on pro-viding dietary energy, protein, vitamins, minerals, andantioxidants and on managing caloric intake to help the im-mune system function optimally. Stages III and IV are differ-ent and involve a more proactive approach to managing theimmune system to obtain the desired outcome.

Stage III: Active Modulation of the Immune SystemIn stage III, the emphasis is on actively interacting with theimmune system in an attempt to modulate its function to-ward a desired goal. Examples include:

• Induction of a Th1 bias and enabling efficient antigenpresentation: A Th1 (proinflammatory) response is im-portant for protection against microbial infections. TheTh1 component of the immune system is boosted by stim-ulating the immune system with probiotic bacteria orPAMP-expressing moieties (e.g., yeast β-glucans). Probi-otics (Enterococcus faecium, Lactobacilli spp, Bifidobacteriaspp) in the diet have been shown to enhance the immunestatus in dogs.55 Milk bioactives from bovine colostrumhave been shown to have immune-enhancing effects inboth human and murine studies and are interesting im-munomodulating ingredients. Colostrum (and whey pro-


IMMUNONUTRITION

tein, which has a very similar composition) contains im-munoglobulins, cytokines, lactoferrin, and lactoperoxi-dase, each of which can influence the immune system.56

Mice fed milk bioactives produced significantly higherserum and intestinal antibodies to several antigens (in-fluenza virus, diphtheria and tetanus toxin, poliomyelitisvaccine, ovalbumin and cholera toxin subunit).57

In another study, mice fed milk bioactives had enhancedresistance to pneumococcal infection.58 In in vitro studiesconducted with human monocytes, Biswas et al59 report thatco-culture with bovine colostrum without antigenic stimu-lus induced a dose-dependent production of IL-12 by CD14+ monocytes but did not induce IFN-γ production. Inter-estingly, in the same study, bovine colostrum differentiallyaffected stimuli-induced IFN-γ production. It enhanced IFN-γ in response to weak antigenic stimulation and inhibitedIFN-γ in response to strong antigenic stimulation. As dis-cussed earlier, IL-12 and IFN-γ are cytokines involved in theTh1 polarization required for a successful immune responsetoward intracellular pathogens, such as bacteria and viruses.

In a clinical study conducted in highly trained cyclists,low-dose bovine colostral protein concentrate supple-mentation favorably modulated immune parameters dur-ing normal training and after an acute period of intenseexercise, which contributed to lowering of the incidenceof upper respiratory illness.60 In a research study con-ducted in adult dogs,a we evaluated the immune-enhanc-ing effect of bovine colostrum. Our results demonstratethat adding bovine colostrum significantly enhanced thedogs’ immune status as measured by their response to acanine distemper virus vaccine as well as by increased levelof GALT activity. Stimulating the immune cells in the gutlikely leads to a cascade of immune cell activation, whichresults in the secretion of cytokines that may reach the restof the immune cells via the circulatory system, resultingin overall activation of the immune system and an increasein the production of IgA in the gut.

• Better managing inflammation to prevent further dam-age: Chronic inflammation is central to the pathophysi-ology of a number of diseases, including cardiovasculardiseases and neurologic diseases (Alzheimer’s disease, im-paired cognition).61 Physiologically, the effects of inflam-mation are mediated by prostaglandins and leukotrienes,which are both endproducts of the arachidonic acid me-

tabolism. A diet rich in DHA (docosahexaenoic acid) andomega-3 fatty acids can control the damaging effects of inflammation by reducing the levels of active prostaglandinsand leukotrienes. This can be an effective strategy in address-ing the effects of chronic inflammation. Reduced inflamma-tion not only improves the quality of life by preventing anumber of cardiovascular and neurologic diseases but alsohelps prevent autoimmunity by reducing exposure of the im-mune system to self-antigens.

Stage IV: Personalized Nutrition: Predictive,Preventive, and Personalized NutritionInteractions between the diet, environment, and genome ul-timately define the individual’s health status and can be crit-ical in influencing chronic disease.62–65 Over the past fewdecades, the science of pharmacogenomics, which deals withthe genetic basis underlying disease susceptibility and vari-able drug response in individuals, has brought about a para-digm shift in the pharmaceutical industry by moving it from“one drug fits all” approach toward personalized therapy.This process has been greatly accelerated by advances in the“-omics” fields, which include single nucleotide polymor-phisms analysis, transcriptomics (cDNA analysis), pro-teomics, and metabolomics. A good example of geneticvariability affecting disease is that breast cancer therapy withthe drug trastuzumab (Herceptin, a humanized monoclonalantibody against the HER2 receptor developed by GenentechInc.) is linked to HER2 overexpression. Individuals expressinglow levels of the HER2 receptor respond poorly to Her-ceptin.66,67 Another example is the influence of genetic vari-ability on cytochrome P450 monooxygenase system enzymes(P450 family of enzymes is important for the metabolism ofmost drugs) and drug toxicity in individual patients.68

The concept of “personalized medicine” is now being ex-plored in nutrition. Although personalized nutrition is still inits infancy, it is practiced in principle, such as in the dietarymanagement of diabetes and in the maintenance of a healthylipid profile to manage the risk of cardiovascular disease. For apractical, personalized diet strategy, there are two basic re-quirements: a clear understanding of the disease pathogenesisand the availability of cheap and reliable disease biomarkers ei-ther to identify disease susceptibility or to diagnose disease.Biomarkers are objectively measured characteristics that are in-dicators of normal biologic processes, pathologic processes, orpharmacologic responses to a therapeutic intervention. The ul-timate goal is to modify physiology through this “personal-

aSatyaraj R, Reynolds A, Nestlé Purina PetCare, St. Louis, MO: Per-sonal communication, 2007.


ized” dietary regimen before the animal enters into the diseasecontinuum, preventing disease or at least delaying the onset ofdisease significantly and thereby enhancing quality of life.

Induction of a local Th2 bias in animals with inflamma-tory bowel disease using dietary means is an example of a tar-geted approach to immunomodulation. Probiotic microbeshave been characterized based on the cytokines responses theyinduce. Certain bacteria induce secretion of antiinflammatorycytokines such as IL-10, TGF-β, and IL-13.69 These probioticagents provide the opportunity to explore probiotic-fortifieddiets that will help animals with inflammatory bowel diseases.Similarly, ingredients rich in TGF-β, such as colostrum andwhey proteins, are being increasingly used to effectively ad-dress localized inflammatory conditions in the gut.

SUMMARYAs research advances in understanding complex physiologicnetworks in health and disease, the role played by the im-mune system and its interaction with diet takes on a wholenew meaning. As our understanding of the relationship be-tween nutrition and the immune system deepens, a vast arrayof diet-based options to address immune needs will becomeavailable. The food we eat and feed our pets can clearly de-liver several other benefits beyond basic nutrition, andtherein lies the promise of immunonutrition.

REFERENCES1. Beisel RW. History of nutritional immunology: introduction and

overview. J Nutr 1992;122:592-596.

2. Satyaraj E. Nutrition and the immune system: review of concepts, strate-gies and techniques. Compend Contin Educ Pract Vet 2005;27:(suppl3A):29-37.

3. Morein B, Abusugra I, Blomqvist G. Immunity in neonates. Vet ImmunolImmunopathol 2002;87(3-4):207-213.

4. Ungvari Z, Buffenstein R, Austad SN, et al. Oxidative stress in vascularsenescence: lessons from successfully aging species. Front Biosci2008;13:5056-5070.

5. Tausk F, Elenkov I, Moynihan J. Psychoneuroimmunology. DermatolTher 2008;21(1):22-31.

6. Toman M, Faldyna M, Knotigova P, et al. Postnatal development ofleukocyte subset composition and activity in dogs. Vet Immunol Im-munopathol 2002;87(3-4):321-326.

7. Gerber JD, Brown AL. Effect of development and aging on the responseof canine lymphocytes to phytohemagglutinin. Infect Immun1974;10(4):695-699.

8. Somberg RL, Tipold A, Hartnett BJ, et al. Postnatal development of Tcells in dogs with X-linked severe combined immunodeficiency. J Im-munol 1996;156(4):1431-1435.

9. Marshall-Clarke S, Reen D, Tasker L, Hassan J. Neonatal immunity: howwell has it grown up? Immunol Today 2000;19(4):150-152.

10. Lee HH, Hoeman CM, Hardaway JC, et al. Delayed maturation of anIL-12-producing dendritic cell subset explains the early Th2 bias in

neonatal immunity. J Exp Med 2008;205(10):2269-2280.

11. Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing.J Pathol 2007;211:144-156.

12. Mocchegiani E, Giacconi R, Cipriano C, et al. MtmRNA gene expres-sion, via IL-6 and glucocorticoids, as potential genetic marker of im-munosenescence: lessons from very old mice and humans. Exp Gerontol2002;37:349-357.

13. Pawelec G, Solana R. Immunosenescence. Immunol Today 1997;18:514-516.

14. Makinodan T. Patterns of age-related immunologic changes. Nutr Rev1995;53(suppl):S27-S34.

15. Mocchegiani E, Santarelli L, Costarelli L, et al. Plasticity of neuroen-docrine-thymus interactions during ontogeny and ageing: role of zincand arginine. Ageing Res Rev 2006;5:281-309.

16. Rink L, Seyfarth M. Characteristics of immunologic test values in theelderly. Gerontol Geriatr 1997;30(3):220-225.

17. Klaus-Helge I, Rink L. Zinc. In Hughes DA, Darlington LG, Bendich A(eds). Diet and the Human Immune Function. New York: Humana Press;2004

18. Greeley EH, Kealy RD, Ballam JM, et al. The influence of age on the ca-nine immune system. Vet Immunol Immunopathol 1996;55(1-3):1-10.

19. Glaser R, Kiecolt-Glaser JK, Bonneau RH, et al. Stress-induced modula-tion of the immune response to recombinant hepatitis B vaccine. Psy-chosom Med 1992;54:22-29.

20. Glaser R, Sheridan J, Malarkey WB, et al. Chronic stress modulates theimmune response to a pneumococcal pneumonia vaccine. PsychosomMed 2000;62:804-807.

21. Morag M, Morag A, Reichenberg A, et al. Psychological variables as pre-dictors of rubella antibody titers and fatigue: a prospective, double blindstudy. J Psychiatr Res 1999;33:389-395.

22. Vedhara K, Cox NK, Wilcock GK, et al. Chronic stress in elderly carersof dementia patients and antibody response to influenza vaccination.Lancet 1999;353:627–631.

23. Jabaaij L, van Hattum J, Vingerhoets JJ, et al. Modulation of immune re-sponse to rDNA hepatitis B vaccination by psychological stress. J Psy-chosom Res 1996;41:129-137.

24. Kiecolt-Glaser JK, Glaser R, Gravenstein S, et al. Chronic stress alters theimmune response to influenza virus vaccine in older adults. Proc NatlAcad Sci USA 1996;93:3043–3047.

25. Burns EA, Goodwin JS. Immunology and infectious disease. In: CasselCK, Leipzig R, Cohen HJ, Larson EB (eds). Geriatric Medicine. New York:Springer-Verlag; 1990, pp 312–329.

26. Patriarca PA. A randomized controlled trial of influenza vaccine in theelderly. JAMA 1994;272:1700-1701.

27. Cohen S, Tyrrell DA, Smith AP. Psychological stress and susceptibility tothe common cold. N Engl J Med 1991;325:606–612.

28. Kehrli ME, Burton JL, Nonnecke BJ, Lee EK. Effects of stress on leuko-cyte trafficking and immune responses: implications for vaccination.Adv Vet Med 1999;41:61-81.

29. Padgett DA, Glaser R. How stress influences the immune response.Trends Immunol 2003;24(8):444-447.

30. Elenkov IJ, Chrousos GP. Stress hormones, proinflammatory and anti-inflammatory cytokines, and autoimmunity. Ann NY Acad Sci2002;966:290–303.

31. Ashwell JD, Lu FW, Vacchio MS. Glucocorticoids in T cell developmentand function. Annu Rev Immunol 1992;18:309-345.

32. Russo-Marie F. Macrophages and the glucocorticoids. J Neuroimmunol1992;40:281-286.

33. Shaywitz AJ, Greenberg ME. CREB: a stimulus-induced transcriptionfactor activated by a diverse array of extracellular signals. Annu RevBiochem 1999;68:821–861.

34. Bengmark S. Gut microenvironment and immune function. Curr OpinClin Nutr Metab Care 1999;2(1):83-85.


IMMUNONUTRITION

35. Brandtzaeg P, Halstensen TS, Kett K, et al. Immunobiology and im-munopathology of human gut mucosa: humoral immunity and in-traepithelial lymphocytes. Gastroenterology 1989;97(6):1562-1584.

36. Brandtzaeg P, Johansen FE. Mucosal B cells: phenotypic characteristics,transcriptional regulation and homing properties. Immunol Rev2005;206:32-63.

37. Conley ME, Delacriox DL. Intravascular and mucosal immunoglobulinA: two separate but related systems of immune defence? Ann Intern Med1987;106:892-899.

38. Cebra JJ. Influences of microbiota on intestinal immune system devel-opment. Am J Clin Nutr 1999;69(suppl):1046S-1051S.

39. Murtaugh MP, Foss DL. Inflammatory cytokines and antigen present-ing cell activation. Vet Immunol Immunopathol 2002;87(3-4):109-121.

40. Decuypere J, Dierick N, Boddez S. The potentials for immunostimula-tory substances (β-1,3/1,6 glucans) in pig nutrition. J Anim Feed Sci1998;7:259-265.

41. Pietrella D, Mazzolla R, Lupo P, et al. Mannoprotein from Cryptococcusneoformans promotes T-helper type 1 anticandidal responses in mice.Infect Immun 2002;70(12):6621-6627.

42. Holen E, Bjørge OA, Jonsson R. Dietary nucleotides and human im-mune cells. II. Modulation of PBMC growth and cytokine secretion. Nu-trition 2006;22(1):90-96.

43. Saavedra JM. Use of probiotics in pediatrics: rationale, mechanisms ofaction, and practical aspects. Nutr Clin Pract 2007;22(3):351-365.

44. Hannant D. Mucosal immunology: overview and potential in the vet-erinary species. Vet Immunol Immunopathol 2002;87(3-4):265-267.

45. Gleeson M, Nieman DC, Pedersen BK. Exercise, nutrition and immunefunction. J Sports Sci 2004;22(1):115-125.

46. Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage:mechanisms, mutation, and disease. FASEB J 2003;17(10):1195-1214.

47. Cline AM, Radic MZ. Apoptosis, subcellular particles, and autoimmu-nity. Clin Immunol 2004;112(2):175-182.

48. Barger JL, Walford RL, Weindruch R. The retardation of aging by caloricrestriction: its significance in the transgenic era. Exp Gerontol.2003;38(11-12):1343-1351.

49. Pahlavani MA. Influence of caloric restriction on aging immune system,J Nutr Health Aging 2004;8(1):38-47.

50. Greeley EH, Spitznagel E, Lawler DF, et al. Modulation of canine im-munosenescence by life-long caloric restriction. Vet Immunol Im-munopathol 2006;111(3-4):287-299.

51. Anderson R, Smit MJ, Joone GK, Van Staden AM. Vitamin C and cellu-lar immune functions. Protection against hypochlorous acid-mediatedinactivation of glyceraldehyde-3-phosphate dehydrogenase and ATPgeneration in human leukocytes as a possible mechanism of ascorbate-mediated immunostimulation. Ann NY Acad Sci 1990;587:34-48.

52. Massimino S, Kearns RJ, Loos KM, et al. Effects of age and dietary beta-carotene on immunological variables in dogs. J Vet Intern Med2003;17(6):835-842.

53. Kim HW, Chew, P, Wong TS, et al. Dietary lutein stimulates immune re-sponse in the canine. Vet Immunol Immunopath 2000;74:315-327.

54. Flickinger EA, Fahey GC Jr. Pet food and feed applications of inulin,oligofructose and other oligosaccharides. Br J Nutr 2002;87(suppl2):S297-S300.

55. Benyacoub J, Czarnecki-Maulden GL, Cavadini C, et al. Supplementa-tion of food with Enterococcus faecium (SF68) stimulates immune func-tions in young dogs. J Nutr 2003;133(4):1158-1162.

56. Artym J, Zimecki M, Paprocka M, Kruzel ML. Orally administered lacto-ferrin restores humoral immune response in immunocompromisedmice. Immunol Lett 2003;89:9-15.

57. Low PP, Rutherfurd KJ, Gill HS, Cross ML. Effect of dietary whey proteinconcentrate on primary and secondary antibody responses in immu-nized BALB/c mice. Int Immunopharmacol 2003;3(3):393-401.

58. Bounous G, Kongshavn PK. Influence of protein type in nutritionallyadequate diets on the development of immunity. In: Friedman M (ed).Absorption and Utilization of Amino Acids. Boca Raton, FL: CRC Press;1989.

59. Biswas P, Vecchi A, Mantegani P, et al. Immunomodulatory effects ofbovine colostrum in human peripheral blood mononuclear cells. NewMicrobiol 2007;30(4):447-454.

60. Shing CM, Peake J, Suzuki K, et al. Effects of bovine colostrum supple-mentation on immune variables in highly trained cyclists. J Appl Phys-iol 2007;102:1113–1122.

61. Casserly I, Topol E. Convergence of atherosclerosis and Alzheimer’s dis-ease: inflammation, cholesterol, and misfolded proteins. Lancet2004;363(9415):1139-1346.

62. Ames BN, Gold LS. The causes and prevention of cancer: the role of en-vironment. Biotherapy 1998;11(2-3):205-220.

63. Ames BN. DNA damage from micronutrient deficiencies is likely to bea major cause of cancer. Mutat Res 2001;475(1-2):7-20.

64. Kaput J, Swartz D, Paisley E, et al. Diet-disease interactions at the molecularlevel: an experimental paradigm. J Nutr 1994;124(8 suppl):1296S-1305S.

65. Milner JA. Molecular targets for bioactive food components. J Nutr2004;134(9):2492S-2498S.

66. Goldenberg MM. Transtuzumab, a recombinant DNA derived human-ized monoclonal antibody, a novel agent for the treatment of metasta-tic breast cancers. Clin Ther 1999;21:309-319.

67. Baselge J, Norton, L, Albanell J, et al. Recombinant humanized anti-HER2 antibody (HERCEPTIN) enhances the antittumor activity of pa-clitaxel and doxorubicin against HER2/neu over expressing humanbreast cancer xenografts. Cancer Res 1998;58:2825-2831.

67. Touw DJ. Clinical implications of genetic polymorphisms and drug in-teractions mediated by cytochrome P450 enzymes. Drug Metab Drug In-teract 1997;14:55-82.

68. Ma D, Forsythe P, Bienenstock J. Live Lactobacillus reuteri is essential forthe inhibitory effect on tumor necrosis factor alpha-induced inter-leukin-8 expression. Infect Immun 2004;72(9):5308-5314.


Nutrigenomics, the study of gene–nutrient interactions, initiallyfocused on maintaining and optimizing human health. Theunique genetic histories and interdependencies of humans andpets make the application of nutrigenomics easier in domesti-cated cats and dogs than in people. Pet animals were bredthrough millennia to produce varieties with defined physicaland behavioral tendencies. Recent sequencing of DNA from se-lected breeds and comparative methodologies have character-ized the genomes of virtually all canine and feline lines.Inbreeding had the unintended consequences of making somebreeds susceptible to specific pathologies that parallel humandisease conditions. Because pets in Western countries and manydeveloping countries rely on humans for food, designing opti-mal nutrition for specific breeds may help prevent or delay theonset of nutrient-influenced conditions.

The sequencing of the human genome stimulated similarefforts in a wide variety of species, including dogs1–4 andcats.1,5–7 Comparative genomics provides invaluable infor-mation not only for genome structure and function but alsofor comparing physiologic and genetic similarities and dif-ferences within8,9 and across species.1,4,10,11 Many dog12,13 andcat6,14 breeds experience pathologies that are remarkably sim-ilar to those in humans.11,15–18 However, in many cases, breedsshow subsets of pathologies that are, in some cases, similar tothose observed in outbred human populations, which maybe a consequence of inbreeding.1

The sequencing and subsequent genome analyses of differ-ent breeds have also provided a resource for nutrigenomics,the study of gene–nutrient interactions that either maintainhealth or initiate or exacerbate chronic diseases.19,20 Althoughthe potential benefits of personalized health care are signifi-cant for humans, public health, and the economy, researchand applications for humans face a diversity of challengesbased on genetic heterogeneity, the complexity of foods, thevariable physiologic mechanisms that produce health or dis-ease states,21,22 and research paradigms that produce popula-

tion rather than individual risk factor data.23 Pet animals, par-ticularly those that are inbred, provide not only a researchopportunity to identify and characterize gene–nutrient inter-actions24 but also a means to develop healthy foods for pre-venting disease and maintaining health in these geneticallydefined lines. However, many pet animals are crossbreeds(i.e., mixed genetic mixtures, or admixtures), which may in-troduce uncertainty in designing healthy foods. The geneticheritage of each crossbreed could be determined through ge-netic testing, which is less problematic than similar testing inhumans. This information could then be used to determinea diet that is most similar to the pet’s ancestral background.This paper reviews the challenges that face nutrigenomics re-search, the advantages of studying gene–nutrient interactionsin pets, and their potential applications.

IMPACT OF GENETIC ARCHITECTURE INDOMESTICATED PET ANIMALSAlthough human populations are outbred and genetically het-erogeneous, pet animals have a more limited genetic architec-ture because of inbreeding. Dogs were derived from wolvesabout 15,000 years ago and experienced artificial selection andhigh-frequency genetic bottlenecks. The result of these prac-tices over the past 200 years is more than 400 distinct breeds,which are defined by their physical features and behaviors.1

The domestic feline was domesticated about 10,000 years agoand underwent similar inbreeding to generate about 68 certi-fied breeds.25 Although selection of pet animal breeds reliedprimarily on genes that exert strong phenotypic traits, envi-ronmental variables, including the diet, are known to exert se-lective pressure on whole chromosomal segments26 as well ason the expression of individual genes.27,28 Any nutrient or setof nutrients that would increase the reproductive success of theanimal would contribute to the creation of breeds.

Inbreeding was done for specific physical or behavioral traits,but a selected gene can produce multiple unintended physio-

Nutrigenomics for Pet Nutrition and MedicineJim Kaput, PhD, and Baitang Ning, PhD

Division of Personalized Nutrition and MedicineFood and Drug Administration

National Center for Toxicological ResearchJefferson, Arizona


NUTRIGENOMICS FOR PET NUTRITION AND MEDICINE

logic processes (called pleiotropy). Alternatively, some disease-causing genes hitchhiked (i.e., genetic hitchhiking) in the traitselection process, resulting in the enrichment of genes for riskloci associated with chronic diseases such as cancer, epilepsy,and metabolic diseases (e.g., type 2 diabetes mellitus).11,14,29

The unique genetic makeup of pet animals is proving valu-able for identifying these disease-causing genes. Classical ge-netic tests that assess the statistical association between oneor more markers and a trait in unrelated cases and controls(i.e., association studies30,31) are more powerful in inbred asopposed to outbred populations because of the defined ge-netic differences between breeds. Characterization of thecausative genes within dog or cat breeds will lead to the de-velopment of genetic tests for genetically admixed pets andideally preventive measures to reduce the incidence or sever-ity of diseases.18,32,33 Such studies can help develop knowl-edge to optimize diets to improve the health of pet animals.In addition, animal gene mapping might also provide infor-mation for humans. Comparative genomic methods haveshown that chromosomal segments in mammals maintain thesame order of genes along the chromosome.4,11,25 These seg-ments may be found on different chromosomes in differentmammals, but the fact that genes and their positions overshort segments are shared means that associations that finda chromosomal segment or a gene within that segment asso-ciated with a disease may help “map” the gene to humanchromosomes.

Epistatic InteractionsGenetic analyses may be confounded by epistatic (gene–gene) and epigenetic (DNA methylation and chromatin re-modeling) interactions. Gene–gene interactions34,35 also alterthe influence of the genetic expression by gene variants.9,36,37

A subset of these interactions may alter expression of genesaffected or involved in nutrient metabolism.38 Proteins andenzymes are interconnected in pathways and networks thatshare metabolites or have direct physical interactions thatalter flux. Hence, the effect of a gene variant is context (orgenome) specific. Among the first examples of epistatic ef-fects were those observed in patients with phenylketonuria, amodel of a monogenic disease. Individuals with the same ge-netic defect may have variable onsets and severities because ofthe inheritance of different chromosomal segments (andtherefore genes) on different chromosomes.39,40 Such gene–gene interactions occur in chronic diseases caused by multi-ple genes and multiple environmental factors. An example is

a polymorphism in ghrelin (GHRL) that interacts with a spe-cific allele of its counterpart receptor (GHSR) to abolish its as-sociation with myocardial infarction (MI) and cardiovasculardisease (CVD).41 These examples confirm the nutrigenomicconcepts in studying gene–gene–phenotype relationshipsthat underlie health and disease processes.

Epistatic interactions may be identified by comparing theeffects of a gene variant among ancestral populations42 or inadmixed populations,43 which may have differing allele fre-quencies. A well-known example is HapK, a haplotype con-sisting of four single nucleotide polymorphisms in theleukotriene 4 hydrolase gene (LTA4H). LTA4H participates inleukotriene and prostaglandin metabolism, which are linkedto dietary fatty acid intake.44 The HapK allele increased thepopulation risk from 1.35 in Europeans (the origin of theHapK allele) for CVD and MI to almost fivefold in AfricanAmericans who carry the same haplotype. This difference ineffect may occur because (1) different environmental factorsalter the influence of LTA4H on MI;45 (2) LTA4H interacts dif-ferently with one or more gene variants in either African orEuropean chromosomal regions, resulting in increased effectof LTA4H activity in African Americans; or (3) a combinationof epistatic and gene–environment interactions is present.42

Inbred mouse experiments have shown large differencesin biologic effect based on epistatic interactions.38,46 Becauseof the similar genetic history of pet animals, epistatic inter-actions should be a significant factor in breeds as well ascrossbreeds.

EpigenesisExpression of genetic information can be altered by mecha-nisms that alter chromosome structure. Chromatin remodel-ing occurs as a consequence of DNA methylation or othermodifications of chromosomal proteins and subsequentchanges in DNA packing.47–53 Chromatin remodeling is partlyregulated by certain chemicals or metabolites derived fromthe diet54–56 and by the energy balance of the cell.57,58

Metabolite flux through the one carbon pathway is a crit-ical regulator of epigenetics because S-adenosylmethionine,the substrate for DNA methyltransferases,59 is a product ofthis pathway.60 Dietary choline, methionine, folate, vitaminB12, vitamin B6, and riboflavin are either substrates or cofac-tors for certain enzymes involved in these pathways. Defi-ciencies in these nutrients alter one-carbon metabolism;impair DNA methylation; and have been linked to an in-creased risk of neural tube defects, cancer, and CVDs.61


COMPLEXITY OF PHYSIOLOGIC ANDPATHOLOGIC PROCESSESSimilar to humans, pet animals are undergoing an increase inthe incidence of obesity with the accompanying metabolicabnormalities, endocrinopathies, orthopedic disorders, car-diorespiratory problems, neoplasia, urogenital dysfunctions,and decreased life span.17 Many of these obesity-related com-plications have higher incidences in specific breeds,1 a directconsequence of the unique genetic history of pet animals andsimilar to the conditions that are “purely” genetic in causa-tion. In an Australian study, the incidence of diabetes melli-tus in cats within breeds was ordered from greatest to lowestas Burmese (2.2%), Abyssinian (1.6%), Chinchilla (1.4%),Siamese (0.6%), Oriental (0%), Devon Rex (0%), and Britishshort hair (0%).62 Crossbreeds had a prevalence of 1.25%, al-though this varied depending on the population studied. Dogbreeds also show a variation in diabetes incidence. In a UKstudy, 13.4% of Labrador retrievers, 4.5% of Samoyeds, andfewer than 1% of boxers had diabetes.15 Although controversyexists about whether classically defined type 2 diabetes oc-curs in dogs,15 conditions associated with or participating intype 2 diabetes mellitus, such as hyperinsulinemia, hyper-glycemia, and glucose intolerance, do occur in dogs.17

Finding discrete aspects but not the entire constellation ofa chronic disease such as diabetes may be explained in part bythe unique genetic history of inbred animals—breeds havedifferent “subphenotypes” of the complex disease because ofthe unique combination of genes inherited during the in-breeding process. We have used type 2 diabetes as an exam-ple of this metabolic and physiologic complexity inhumans.21,22 Five classes of drugs are used to treat type 2 dia-betes mellitus in humans: sulphonylureas and meglitinidesstimulate insulin secretion from the pancreas, biguanidesblock gluconeogenesis in the liver, α-glucosidases block glu-cose uptake in the intestines, and thiazolidinediones increaseinsulin sensitivity in the peripheral tissues. These same drugsare used to treat diabetic dogs and cats,63 suggesting that oneor more of these subphenotypes occur in inbred or mixed-breed pet animals. Each drug class targets different organsand molecular pathways. Individual humans and pet ani-mals63 respond differently to these drugs depending on thepathway that is metabolically abnormal based on lifestyle(e.g., diet and exercise) or inherited gene variants. This com-plexity is reduced in inbred animals, but different breeds arelikely to have different combinations of alleles (gene variants)predisposing them to one or more of the subphenotypes. For

example, inbred mouse strains, which have genetic historiessimilar to those of dogs and cats, have different variations ingenes involved in pathways that produce obesity.21,64 Hence,the genetic differences between breeds or specific genetic ad-mixtures of domestic cats or dogs challenge veterinary med-ical practices relying on animal data generated from the“average” response of dogs or cats rather than how individualbreeds respond. An analogous situation occurs in humanstudies in which most of the nutritional and drug efficacydata are generated by population studies.23

DIETARY INFLUENCES IN CHRONIC DISEASESOF PET ANIMALSGenetic factors may be the most important causes of diseasesaffecting different breeds, but the environment, particularlyunbalanced nutrient intake65 or inactivity, increases the inci-dence or exacerbates the severity of diseases. The gene–envi-ronment connection is now firmly established, although thecontributions and interactions of specific genes and environ-ments are only now being studied in detail. The increase inthe incidence of obesity in humans, which is reported to beabout 35% of the US population, is mirrored by an increasein obesity in pet animals. The incidence of overweight andobesity is 25% to 40% of dogs and cats in certain geograph-ical areas.16–18 The increased incidence and severity of meta-bolic diseases in domesticated pet animals that has occurredover the past 20 years or so is similar to the case in humanswho immigrate and adopt the food and physical activity cul-ture of their new environment.66

Although the clinical definition of obesity in pet animalsis considered a body weight of at least 15% above “ideal” forbody size67 such measures may have greater variance in dogsbecause of the significant differences in body size amongbreeds and crossbreeds. Detailed scoring systems have beendeveloped to assess the degree of obesity, although these sys-tems are not uniformly used.17 Because few epidemiologicstudies have been conducted in pet animals, associations be-tween specific dietary components and obesity (or other dis-eases) are not well known.

IDENTIFYING GENE–NUTRIENT INTERACTIONSINVOLVED IN HEALTH AND DISEASESUSCEPTIBILITYExamining gene–environment interactions in critical targetorgans is not possible in pet animals because of ethical con-cerns. As previously stated, laboratory animals, particularly



inbred strains of mice or rats, may provide information forhumans and pets because of the similarities and known dif-ferences in their physiologies and genetic makeups (i.e., com-parative genomics). However, only a few studies in mice haveused gene expression,68–72 metabolite analyses,73 and com-parative genomics to identify candidate disease genes.68,69,72

Results from such studies could be used to develop ethicalfeeding intervention studies in pets using intermediate-riskfactors (e.g., glucose, lipid, insulin levels) linked to chronicdiseases. Mouse geneticists routinely scan multiple inbredstrains to select responders and nonresponders to dietarychallenges,74–76 which can then be used to identify geneticloci,74,76–78 gene expression,68,72,73 or metabolite73 differencesthat are associated with physiology. Similar noninvasivescreening methods could be used to define groups of breedsthat respond similarly to dietary differences. Comparative nu-trigenomic strategies based on inbred strains (and hencebreeds), defined diets, and analyses of differing physiologicresponses have been proposed.69,79

SUMMARY: THE COMPLEXITY OF NUTRITION, GENETICS, HEALTH, DISEASE,AND PERSONALIZATIONThe significant advances in understanding complex biologicprocess in the past century relied on reductionistic experi-mental strategies (i.e., change one variable while all others arekept constant). Although this strategy was successful for cer-tain phenotypes, understanding complex systems requires an-alytical approaches that incorporate rather than avoidcomplexity. Genes interact with nutrients, and nutrients altergenetic expression; analyzing one and ignoring the other re-sults in incomplete analyses. Therefore, the key challenge forpersonalizing health care for pets and humans is not the com-plexity of the datasets but acquiring those datasets in a man-ner to reduce noise and increase the true signals. This mightbest be accomplished by preselecting phenotypes based onquantitative data or preselecting genotypes that maximize dif-ferences in allele frequencies of candidate genes involved innutrient metabolism or another physiologic trait. Dog and catbreeds would be ideally suited for such research even with theethical limitations that are similar to those in human studies.The genetic histories and resulting architectures of the manydog and cat breeds need to be fully exploited, not only forhuman benefit but also for the benefit of pet animals. Inte-grative whole-system analyses of the datasets and new in vivoand noninvasive visualization methods not only perform

these complex experiments but also develop biologic insightinto the outcomes. The development of nutrigenomics andthe application of this knowledge will provide strategies formaintaining health and improving the medical treatment ofchronic diseases in both humans and pets.

DISCLAIMERThis work includes contributions from and was reviewed bythe Food and Drug Administration. This work has been ap-proved for publication by this agency, but it does not neces-sarily reflect official agency policy.

CONFLICT OF INTERESTThe authors declare there is no conflict of interest.

REFERENCES1. Karlsson E, Lindblad-Toh K. Leader of the pack: gene mapping in dogs

and other model organisms. Nat Rev Genet 2008;9:713-725.

2. Lindblad-Toh K, Wade CM, Mikkelsen TS, et al. Genome sequence, com-parative analysis and haplotype structure of the domestic dog. Nature2005;438(7069):803-819.

3. Parker HG, Kim LV, Sutter NB, et al. Genetic structure of the purebreddomestic dog. Science 2004;304(5674):1160-1164.

4. Switonski M, Szczerbal I, Nowacka J. The dog genome map and its usein mammalian comparative genomics. J Appl Genet 2004;45(2):195-214.

5. O’Brien SJ, Johnson WE. Big cat genomics. Annu Rev Genomics HumGenet 2005;6:407-429.

6. O’Brien SJ, Menotti-Raymond M, Murphy WJ, Yuhki N. The felinegenome project. Annu Rev Genet 2002;36:657-686.

7. Pontius JU, Mullikin JC, Smith DR, et al. Initial sequence and compar-ative analysis of the cat genome. Genome Res 2007;17(11):1675-1689.

8. Serre D, Nadon R, Hudson TJ. Large-scale recombination rate patternsare conserved among human populations. Genome Res 2005;15(11):1547-1552.

9. Togawa K, Moritani M, Yaguchi H, Itakura M. Multidimensionalgenome scans identify the combinations of genetic loci linked to dia-betes-related phenotypes in mice. Hum Mol Genet 2006;15(1):113-128.

10. Bochdanovits Z, Sondervan D, Perillous S, et al. Genome-wide predic-tion of functional gene-gene interactions inferred from patterns of ge-netic differentiation in mice and men. PLoS ONE 2008;3(2):e1593.

11. Paoloni M, Khanna C. Translation of new cancer treatments from petdogs to humans. Nat Rev Cancer 2008;8(2):147-156.

12. Elsinghorst TA. New findings on animal diseases published since 2003.1. Dogs. Vet Q 2004;26(1):18-24.

13. Sutter N, Ostrander E. Dog star rising: the canine genetic system. NatureRev Genet 2004;5:900-910.

14. Rand JS, Fleeman LM, Farrow HA, et al. Canine and feline diabetes mel-litus: nature or nurture? J Nutr 2004;134(8 suppl):2072S-2080S.

15. Catchpole B, Kennedy LJ, Davison LJ, Ollier WE. Canine diabetes mel-litus: from phenotype to genotype. J Small Anim Pract 2008;49(1):4-10.

16. German AJ. The growing problem of obesity in dogs and cats. J Nutr2006;136(7 suppl):1940S-1946S.

17. Gossellin J, Wren JA, Sunderland SJ. Canine obesity: an overview. J VetPharmacol Ther 2007;30(suppl 1):1-10.

18. Laflamme DP. Understanding and managing obesity in dogs and cats.Vet Clin North Am Small Anim Pract 2006;36(6):1283-1295, vii.


19. Kaput J. Diet-disease gene interactions. Nutrition 2004;20(1):26-31.

20. Kaput J, Rodriguez RL. Nutritional genomics: the next frontier in thepostgenomic era. Physiol Genomics 2004;16(2):166-177.

21. Kaput J, Noble J, Hatipoglu B, et al. Application of nutrigenomic con-cepts to type 2 diabetes mellitus. Nutr Metab Cardiovasc Dis2007;17(2):89-103.

22. Kaput J, Perlina A, Hatipoglu B, et al. Nutrigenomics: concepts and ap-plications to pharmacogenomics and clinical medicine. Pharmacoge-nomics 2007;8(4):369-390.

23. Kaput J. Nutrigenomics research for personalized nutrition and medi-cine. Curr Opin Biotechnol 2008;19(2):110-120.

24. Swanson KS, Schook LB, Fahey GC, Jr. Nutritional genomics: implica-tions for companion animals. J Nutr 2003;133(10):3033-3040.

25. O’Brien SJ, Johnson W, Driscoll C, et al. State of cat genomics. TrendsGenet 2008;24(6):268-279.

26. Myles S, Tang K, Somel M, et al. Identification and analysis of genomicregions with large between-population differentiation in humans. AnnHum Genet 2008;72(pt 1):99-110.

27. Ordovas JM, Corella D. Nutritional genomics. Annu Rev Genomics HumGenet 2004;5:71-118.

28. Schuster GU. Nutrients and gene expression. In: Kaput J, Rodriguez RL(eds). Nutritional Genomics. Discovering the Path to Personalized Nutrition.Hoboken, NJ: John Wiley and Sons; 2006:153-176.

29. Fleeman LM, Rand JS. Management of canine diabetes. Vet Clin NorthAm Small Anim Pract 2001;31(5):855-880, vi.

30. Kraft P, Cox DG. Study designs for genome-wide association studies.Adv Genet 2008;60:465-504.

31. Pearson TA, Manolio TA. How to interpret a genome-wide associationstudy. JAMA 2008;299(11):1335-1344.

32. Hoenig M. The cat as a model for human nutrition and disease. CurrOpin Clin Nutr Metab Care 2006;9(5):584-588.

33. Laflamme DP. Nutrition for aging cats and dogs and the importance ofbody condition. Vet Clin North Am Small Anim Pract 2005;35(3):713-742.

34. Carlborg O, Haley CS. Epistasis: too often neglected in complex traitstudies? Nat Rev Genet 2004;5(8):618-625.

35. Moore JH. The ubiquitous nature of epistasis in determining suscepti-bility to common human diseases. Hum Hered 2003;56(1-3):73-82.

36. Cheverud JM, Vaughn TT, Pletscher LS, et al. Genetic architecture of ad-iposity in the cross of LG/J and SM/J inbred mice. Mamm Genome2001;12(1):3-12.

37. Yang RC. Epistasis of quantitative trait loci under different gene actionmodels. Genetics 2004;167(3):1493-505.

38. Chiu S, Diament AL, Fisler JS, Warden CH. Gene-gene epistasis and geneenvironment interactions influence diabetes and obesity. In: Kaput J,Rodriguez RL (eds). Nutritional Genomics. Discovering the Path to Person-alized Nutrition. Hoboken, NJ: John Wiley and Sons; 2006:135-152.

39. Scriver CR. Nutrient-gene interactions: the gene is not the disease andvice versa. Am J Clin Nutr 1988;48(6):1505-1509.

40. Scriver CR. The PAH gene, phenylketonuria, and a paradigm shift. HumMutat 2007;28(9):831-845.

41. Baessler A, Fischer M, Mayer B, et al. Epistatic interaction between hap-lotypes of the ghrelin ligand and receptor genes influence susceptibil-ity to myocardial infarction and coronary artery disease. Hum Mol Genet2007;16(8):887-899.

42. Klos KLE, Kardia SLR, Hixson JE, et al. Linkage analysis of plasma ApoEin three ethnic groups: multiple genes with context-dependent effects.Ann Hum Genet 2005;69(2):157-167.

43. Suarez-Kurtz G, Pena SD. Pharmacogenomics in the Americas: the im-pact of genetic admixture. Curr Drug Targets 2006;7(12):1649-1658.

44. Kelley DS. Modulation of human immune and inflammatory responsesby dietary fatty acids. Nutrition 2001;17(7-8):669-673.

45. Helgadottir A, Manolescu A, Helgason A, et al. A variant of the gene en-

coding leukotriene A4 hydrolase confers ethnicity-specific risk of my-ocardial infarction. Nat Genet 2006;38(1):68-74.

46. Warden CH, Yi N, Fisler J. Epistasis among genes is a universal phe-nomenon in obesity: evidence from rodent models. Nutrition2004;20(1):74-77.

47. Delaval K, Feil R. Epigenetic regulation of mammalian genomic im-printing. Curr Opin Genet Dev 2004;14(2):188-195.

48. Dolinoy DC, Das R, Weidman JR, Jirtle RL. Metastable epialleles, im-printing, and the fetal origins of adult diseases. Pediatr Res 2007;61(5 pt 2):30R-37R.

49. Esteller M. Cancer epigenomics: DNA methylomes and histone-modi-fication maps. Nat Rev Genet 2007;8(4):286-298.

50. Fowler AM, Alarid ET. Dynamic control of nuclear receptor transcrip-tion. Sci STKE 2004;2004(256): pe51.

51. Jiang YH, Bressler J, Beaudet AL. Epigenetics and human disease. AnnuRev Genomics Hum Genet 2004;5:479-510.

52. Jirtle RL, Skinner MK. Environmental epigenomics and disease suscep-tibility. Nat Rev Genet 2007;8(4):253-262.

53. Morgan HD, Santos F, Green K, et al. Epigenetic reprogramming inmammals. Hum Mol Genet 2005;14:R47-R58.

54. Cooney CA. Maternal nutrition: nutrients and control of expression. In:Kaput J, Rodriguez RL (eds). Nutritional Genomics. Discovering the Path toPersonalized Nutrition. Hoboken, NJ: John Wiley and Sons; 2006;219-254.

55. Dolinoy DC, Weidman JR, Waterland RA, Jirtle RL. Maternal genisteinalters coat color and protects Avy mouse offspring from obesity by mod-ifying the fetal epigenome. Environ Health Perspect 2006;114(4):567-572.

56. Fenech M. The genome health clinic and genome health nutrigenomicsconcepts: diagnosis and nutritional treatment of genome and epigenomedamage on an individual basis. Mutagenesis 2005;20(4):255-269.

57. Gallou-Kabani C, Junien C. Nutritional epigenomics of metabolic syn-drome: new perspective against the epidemic. Diabetes 2005;54(7):1899-1906.

58. Picard F, Kurtev M, Chung N, et al. Sirt1 promotes fat mobilization inwhite adipocytes by repressing PPAR-gamma. Nature 2004;429(6993):771-776.

59. Sneider TW, Teague WM, Rogachevsky LM. S-adenosylmethionine:DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cellline. Nucleic Acids Res 1975;2(10):1685-1700.

60. Mason JB. Biomarkers of nutrient exposure and status in one-carbon(methyl) metabolism. J Nutr 2003;133(suppl 3):941S-947S.

61. Stover PJ, Garza C. Bringing individuality to public health recommen-dations. J Nutr 2002;132(8 suppl):2476S-2480S.

62. Lederer R, Rand JS, Jonsson NN, et al. Frequency of feline diabetes mel-litus and breed predisposition in domestic cats in Australia. Vet J 2007,Dec 26. [Epub ahead of print].

63. Nelson RW. Oral medications for treating diabetes mellitus in dogs andcats. J Small Anim Pract 2000;41(11):486-490.

64. Wolff GL. Obesity as a pleiotropic effect of gene action. J Nutr1997;127(9):1897S-1901S.

65. Swanson KS, Mazur MJ, Vashisht K, et al. Genomics and clinical medi-cine: rationale for creating and effectively evaluating animal models.Exp Biol Med (Maywood) 2004;229(9):866-875.

66. Kolonel LN, Altshuler D, Henderson BE. The multiethnic cohort study: ex-ploring genes, lifestyle and cancer risk. Nat Rev Cancer 2004;4(7):519-527.

67. Laflamme DP. Challenges with weight-reduction studies. CompendiumContin Educ Pract Vet 2001;23:45-50.

68. Kaput J, Klein KG, Reyes EJ, et al. Identification of genes contributing tothe obese yellow Avy phenotype: caloric restriction, genotype, diet xgenotype interactions. Physiol Genomics 2004;18(3):316-324.

69. Kaput J, Swartz D, Paisley E, et al. Diet-disease interactions at the mo-lecular level: an experimental paradigm. J Nutr 1994;124(8 suppl):1296S-1305S.



70. Kendziorski CM, Chen M, Yuan M, et al. Statistical methods for ex-pression quantitative trait loci (eQTL) mapping. Biometrics2006;62(1):19-27.

71. Lan H, Stoehr JP, Nadler ST, et al. Dimension reduction for mappingmRNA abundance as quantitative traits. Genetics 2003;164(4):1607-1614.

72. Park EI, Paisley EA, Mangian HJ, et al. Lipid level and type alter stearoylCoA desaturase mRNA abundance differently in mice with distinct sus-ceptibilities to diet-influenced diseases. J Nutr 1997;127(4):566-573.

73. Ferrara CT, Wang P, Neto EC, et al. Genetic networks of liver metabolismrevealed by integration of metabolic and transcriptional profiling. PLoSGenet 2008;4(3):e1000034.

74. Kumar KG, Poole AC, York B, et al. Quantitative trait loci for carbohy-drate and total energy intake on mouse chromosome 17: congenicstrain confirmation and candidate gene analyses (Glo1, Glp1r). Am J

Physiol Regul Integr Comp Physiol 2007;292(1):R207-R216.

75. Paigen B, Morrow A, Brandon C, et al. Variation in susceptibility to ather-osclerosis among inbred strains of mice. Atherosclerosis 1985;57(1):65-73.

76. Smith Richards BK, Belton BN, Poole AC, et al. QTL analysis of self-se-lected macronutrient diet intake: fat, carbohydrate, and total kilocalo-ries. Physiol Genomics 2002;11(3):205-217.

77. Paigen B, Mitchell D, Reue K, et al. Ath-1, a gene determining athero-sclerosis susceptibility and high density lipoprotein levels in mice. ProcNatl Acad Sci USA 1987;84(11):3763-3767.

78. Wang X, Ishimori N, Korstanje R, et al. Identifying novel genes for ath-erosclerosis through mouse-human comparative genetics. Am J HumGenet 2005;77(1).

79. Kaput J. Decoding the pyramid: a systems-biological approach to nu-trigenomics. Ann NY Acad Sci 2005;1055:64-79.

Nutrition ForumResearch Abstracts:Oral Presentations


Feline mammary tumors have been identified as ap-propriate models for human breast cancer (HBC)studies based on similarities in hormone recep-tor–negative and metastatic characteristics. Pome-granate (Punica granatum) fruit juice (PJ) may hindercancer cell proliferation and promote apoptosis inhuman prostate and HBC cell lines. Therefore, theobjective of this study is to develop a feline mam-mary gland organ culture model in which to conductstudies using PJ as the chemopreventive agent.

Mammary glands were removed from adult femalecats obtained from a local animal shelter immediatelyafter euthanasia. The mammary glands were processedand placed in specifically designed culture systems. Ad-ditional tissue samples were procured in liquid nitro-gen or 4% paraformaldehyde for histologic evaluation.Culture systems were incubated in a humidified atmospheric condition with 5% CO2 or 95% air with 5% CO2 with or without hormone treatment. Onculture day 3, the carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), was added to each system in vary-

ing concentrations (3–24 μg/ml). Dimethyl sulfoxideand media-only controls were included. Culture sys-tems were exposed to DMBA for 24 hours, refreshedwith DMBA-free media, and maintained for 15 days.

The cell architecture of fixed-gland (hematoxylinand eosin–stained) and DMBA-exposed tissue wasevaluated for preneoplastic alveolar lesions. POMWonderful® 100% PJ, as the chemopreventive agent,was added to media of each culture system on day 0in five concentrations (1.25% to 20%). DMBA (10μg/mL, optimal lesion-inducing dose) was added onday 3 for 24-hour exposure.

Histologic analysis and interpretation of the cul-tured gland tissue suggested that the PJ concentrationof 5% lowered the frequency of lesions associatedwith carcinogen exposure. Based on results of this invitro study, a viable feline mammary gland organ cul-ture can be maintained, and PJ appears to havechemopreventive abilities in this system. Pomegran-ate and other functional foods may possibly benefitboth feline and human cancer patients.

Feline Mammary Gland Organ Culture Model for Analysis of Pomegranate Juice as a Chemopreventive

Agent for Human Breast CancerA. Wilson, K.E. Saker, and A.E. Tanner

College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina



The objective of this study was to determine if injec-tion of allogeneic lipoprotein lipase (LpL)+/+ ex-pressing bone marrow stromal cells (MSCs) cancorrect metabolic deficiencies in LpL-/- cats.

Bone marrow was harvested from LpL+/+ cats andamplified in culture, and 50 million MSCs were in-jected into LpL-/- cats. Two weeks later, an identicalinjection from the same preparation of MSCs was re-peated. Plasma turbidity, triglycerides, cholesterol,and lipase activity were monitored before and afterthe injection of the MSCs.

Injection of MSCs resulted in a decrease in theplasma lipid profile 3 to 5 days after administration.To confirm that this decrease in plasma lipids re-sulted from circulating lipase activity from the in-jected MSCs, plasma samples were collected and LpLactivity measured. After the first injection, there wasa modest decrease in plasma turbidity and lipids that

correlated with a slight increase in LpL activity. Thesecond injection resulted in a much larger increase inLpL activity and a greater decrease in plasma turbid-ity and lipids. This enhanced response on the secondinjection suggests that there might be an accumula-tion of LpL+/+ cells somewhere in the cat, most likelyin the bone marrow.

Injection of allogeneic MSCs corrects the LpL-/- ge-netic defect and transiently returns the plasma lipidprofile to near normal in a dose-dependent manner.Multiple injections of allogeneic MSCs demonstrateadditivity and increased duration of effect. Behavioralmodifications and additional benefits were also ob-served. Using this dose and injection protocol, thebiphasic duration of therapeutic action appears tolast approximately 3 months. This is a very powerfuland robust animal model for studying cytokineticsand cytotherapeutics.

Correcting Metabolic Deficits with Allogeneic Bone Marrow Stem Cells

P.R Vulliet, S.M. Halloran, K.M. Tallon, D.L. Bee, L.A. Lyons, Q.R. Rogers, and A.J. FascettiDepartments of Veterinary Molecular Biosciences and Public Health and Reproduction,

University of California, Davis, California


The prevalence of diabetes mellitus is increasing incats and humans worldwide. In humans with type 2diabetes, sustained hyperglycemia and hyperlipidemiaexacerbate β-cell dysfunction via islet inflammation.The toxic effects of high glucose and lipid levels on βcells are referred to as “glucotoxicity” and “lipotoxic-ity.” Feline diabetes shares many similarities withhuman type 2 diabetes, including insulin resistancerelated to obesity, decreased β-cell mass, and pancre-atic amyloid deposition. Because of these similarities,we hypothesize that “glucotoxicity” and “lipotoxicity”also contribute to β-cell dysfunction in cats.

Healthy cats were infused with glucose (n = 5) orlipids (n = 6) for 10 days to clamp their blood con-centrations at the approximate level found in un-treated feline diabetes (glucose, 25–30 mmol/L;triglycerides, 3–7 mmol/L). Control cats were infusedwith saline (n = 5) or received no infusion (n = 5).

On day 10, α1-acid glycoprotein levels were deter-mined, an intravenous glucose tolerance test (ivGTT)

was performed, and pancreatic specimens were col-lected. Pancreatic sections were labeled for insulinand myeloperoxidase, and neutrophils were countedin the islets. Cytokine (interleukin [IL]-1β, IL-6,tumor necrosis factor-α) and chemokine (IL-8, mono-cyte chemoattractant protein-1) mRNA were quanti-fied in pancreatic islets. Statistical differences weredetermined with nonparametric tests.

During the ivGTT, hyperglycemic cats had no stim-ulation of insulin secretion; in hyperlipidemic cats,the insulin secretion pattern was normal. Comparedwith control subjects, hyperglycemic and hyperlipi-demic cats had increased α1-acid glycoprotein levels.Islet neutrophils and cytokine or chemokine tran-scripts were not increased in hyperglycemic and hy-perlipidemic cats.

In conclusion, hyperglycemia but not hyperlipi-demia induces β-cell dysfunction in cats. High glucoseand lipid levels cause a systemic inflammatory reac-tion that is not accompanied by islet inflammation.

Does Glucotoxicity or Lipotoxicity Contribute to thePathophysiology of Diabetes in Cats?

E. Zini,a M. Osto,b M. Franchini,c S. Moretti,a A. Vögtlin,c M. Ackermann,c T.A. Lutz,b and C.E. Reuscha

aClinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Switzerland bInstitute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Switzerland

cInstitute of Virology, Vetsuisse Faculty, University of Zurich, Switzerland



Obesity after spaying is a common problem in the do-mestic cat population. The objective of this study wasto evaluate adipose and muscle gene expression andblood metabolites in cats fed a high-protein (HP) ormoderate-protein (MP) diet after ovariohysterectomy.Eight cats were used in a completely randomized de-sign with repeated measures. Cats were spayed at week0 and were fed ad libitum for 24 weeks. Fasting bloodsamples and adipose and skeletal muscle biopsieswere collected at 0, 12, and 24 weeks.

Blood glucose increased (P < .05) linearly over time.Blood nonesterified fatty acids were decreased (P < .05)at weeks 12 and 24 compared with week 0, regardlessof dietary treatment. Blood leptin increased (P < .05)over time and was greater (P < .05) in cats fed the MPdiet. Adipose tissue mRNA abundance of adiponectin,hormone-sensitive lipase, toll-like receptor-4, uncou-

pling protein-2 (UCP2), and vascular endothelialgrowth factor decreased (P < .05) linearly over time, re-gardless of diet. Insulin receptor decreased (P < .05)curvilinearly over time, and glucose transporter-1 wasdetermined to have a curvilinear increase (P < .05) overtime, regardless of dietary treatment. Adipose mRNAinterleukin-6 was higher (P < .05) in cats fed MP, andleptin mRNA was higher (P < .05) in cats fed HP. Skele-tal muscle mRNA glucose transporter-1, hormone-sen-sitive lipase, and UCP2 were decreased (P < .05), andinsulin receptor was determined to have a decreasingcurvilinear effect (P < .05) over time, regardless of di-etary treatment.

This research noted changes in adipose and skele-tal muscle tissue after spaying and that these changescan be influenced by diet, both of which may help ex-plain the metabolic changes observed in spayed cats.

Effects of Spaying on Adipose and Muscle Gene Expression and Blood Indices in Cats Fed a High-

versus a Moderate-Protein DietB.M. Vester,a K.J. Liu,b T.L. Keel,c T.K. Graves,c,d and K.S. Swansona,c,d

aDepartment of Animal Sciences, University of Illinois, Urbana, IllinoisbNatura Manufacturing, Inc., Fremont, Nebraska

cDepartment of Veterinary Clinical Medicine, University of Illinois, Urbana, IllinoisdDivision of Nutritional Sciences, University of Illinois, Urbana, Illinois


Obesity is the result of a positive imbalance betweenenergy intake and energy expenditure, but little isknown about the mechanisms involved in its patho-genesis and the progression to diabetes. Thyroid hor-mone has a profound influence on energy balance.Triiodothyronine (T3) increases the basal metabolic rateand has been proposed to regulate uncoupling proteins(UCPs) that are involved in energy expenditure. T3 alsoregulates lipid metabolism through increased expres-sion of transcription factors such as peroxisome prolif-erator-activated receptors (PPARs) and coactivators suchas the peroxisome-proliferator-activated receptor-γ coac-tivator 1 (PGC-1) family of coactivators.

The effects of a 2-week administration of 75 μg T3

on substrate oxidation, heat production, nonesteri-fied fatty acids (NEFA), and leptin were evaluated ineight lean (three females and five males) and eightobese (five females and three males) age-matchedadult neutered cats. In addition, using real-time re-verse transcriptase–polymerase chain reaction, ex-pression of muscle and adipose tissue UCP2 and

UCP3, deiodinase 1 (D1) and 2 (D2), PPARα and -γ,and PGC-1α were examined.

Compared with lean cats, obese cats had increasedNEFA, leptin, UCP2, and D1 mRNA in muscle andUCP3mRNA levels in fat but lower heat productionand fat PPARs and PGC1. T3 administration increasedthermogenesis and NEFA in lean and obese cats andadipose tissue PPARγ in lean cats. It also increasedmuscle D1 in lean and D2 in obese cats. The increasein muscle D2 was interpreted to be reflective of the re-duced serum total thyroxine concentration after T3

suppression of the pituitary. No effect was seen onleptin or UCP2 and UCP3. This shows that T3 regu-lates thermogenesis but not through changes in un-coupling protein expression. It also indicates thatPPARs have an important role in the pathogenesis ofobesity in cats.

REFERENCE1. Hoenig M, Caffall Z, Ferguson DC. Triiodothyronine differen-

tially regulates key metabolic factors in lean and obese cats.Domest Anim Endocrinol 2007;34:229-237.

Regulation of Key Metabolic Factors by Triiodothyronine in Cats1

M. Hoenig, Z. Caffall, and D.C. FergusonDepartment of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia



Adiponectin is a hormone expressed from adiposetissue in humans, rodents, and dogs. Adiponectin hasantiinflammatory action with beneficial effects oncardiovascular health and insulin sensitivity. With in-creasing fat mass, adiponectin concentrations para-doxically decrease. Adiponectin’s role in metabolismand diabetes mellitus is of interest in feline medicinebecause cats are susceptible to developing type 2 di-abetes with weight gain. This study determined rela-tive amounts of adiponectin mRNA expression fromvarious body tissues and organs in domestic cats.

Two intact male cats and one intact female catwere evaluated postmortem. All cats were estimatedto be young adults and had body condition scores of4 to 5 of 9. Tissues samples from inguinal subcuta-neous adipose, visceral mesenteric adipose, liver fromthe left lateral lobe, skeletal muscle from the gastroc-

nemius, left ventricular cardiac muscle, aorta, stom-ach fundus, duodenum, pancreas, thyroid gland,adrenal gland (cortex and medulla), and renal cortexwere collected and frozen. After RNA extraction,adiponectin mRNA expression of each tissue was de-tected using reverse transcriptase real-time poly-merase chain reaction.

Visceral adipose tissue had the highest level of ex-pression, averaging 12% higher than subcutaneous adi-pose. All other tissues had negligible levels of expressioncompared with adipose samples. This study provides a valuable step for adiponectin research in cats by determining which tissues express this hormone. Catsdiffer from humans by expressing higher levels ofadiponectin in visceral compared with subcutaneousfat. The metabolic impact of this expression pattern isunknown and provides a basis for future research.

Adiponectin mRNA Expression in CatsA.L. Lusby,a S.A. Kania,b J.W. Bartges,a and C.A. Kirka

aDepartment of Small Animal Clinical Sciences, University of Tennessee, Knoxville, TennesseebDepartment of Comparative Medicine, University of Tennessee, Knoxville, Tennessee


Adiponectin is a novel adipokine with antiathero-genic and insulin-sensitizing properties. Circulatingadiponectin concentrations are decreased in obesedogs, similar to what happens in obese humans. Lowadiponectin concentrations in humans have been as-sociated with obesity-related disorders such as insulinresistance, hyperlipidemia, and hepatic steatosis.Polyunsaturated fatty acids (PUFA) are important me-diators in lipid and glucose metabolism; fish oil, arich source of omega-3 PUFA, may have beneficial ef-fects in conditions associated with obesity. Recently,fish oil has been shown to increase adiponectin se-cretion in obese human subjects and rodent models.

The aim of this study was to examine the effect offish oil supplementation on circulating adiponectinconcentrations in healthy dogs. Twenty dogs were ad-ministered a fish oil supplement (220 mg/kg oncedaily) for 30 days. All dogs were fed balanced nutri-tionally complete diets for at least 3 months before

and for the duration of the study. Before and aftersupplementation, dogs were examined and weighed,medical and dietary histories were obtained, bodycondition scores (BCS) were determined (range, 4–6of 9), and body measurements were recorded for cal-culation of percent body fat. Fasted blood samples werecollected for a biochemistry profile and adiponectinand triglyceride determination.

The mean serum adiponectin concentration increasedsignificantly at the end of the supplementation period(pretreatment, 11.4 ± 7.9 μg/ml; post treatment, 13.9 ±9.9 μg/ml). There was no significant difference in serumtriglycerides, body weight, BCS, or percent body fat. Theresults of this pilot study suggest that dietary fish oil sup-plementation is associated with increased circulatingadiponectin concentrations in healthy, non-obese dogs.Additional studies are required to confirm the effect offish oil supplementation on adiponectin concentrationsin dogs of various body conditions.

Effect of Dietary Fish Oil on Adiponectin Concentration in Dogs

M. Mazaki-Tovi,a P.A. Schenck,a and S.K. Abood b

aDepartment of Pathobiology and Diagnostic Investigation, Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, Michigan

bDepartment of Small Animal Clinical Sciences, Michigan State University, East Lansing, Michigan



Proinflammatory adipokines secreted by adipose tis-sue, such as C-reactive protein (CRP), are increased inobese humans and may have adverse health effects,including a role in the development of metabolic syn-drome, insulin resistance, and cardiovascular disease.Tanner et al1 have shown increased CRP in cats fed toan obese state compared with the baseline lean state.However, canine studies2,3 have failed to demonstrateincreased CRP. The purpose of this study was to mea-sure serum CRP in lean and overweight dogs.

Seventy-six healthy adult, client-owned dogs be-tween 2 and 7 years of age and with no recent historyof illness, vaccination, or boarding were enrolled inthe study. Dogs were determined to be in good healthbased on medical and dietary history, physical exam-ination, complete blood count, serum biochemistryprofile, and urinalysis. Body condition score (BCS)was determined by one investigator, and dogs wereassigned to either the lean group (BCS, 4–5 of 9; n =39) or the overweight group (BCS, 7–9 of 9; n = 37).Blood was sampled after an overnight fast, and serumCRP was measured using a validated canine-specific

ELISA. Groups were compared using chi-square andMann-Whitney U tests.

No significant differences between groups were de-tected in age (P = .16) or gender (P = .34). The me-dian CRP concentration was significantly higher in theoverweight group (median, 0.94; range, 0.47–6.22μg/ml) versus the lean group (median, 0.72 μg/ml;range, 0.45–6.04 μg/ml; P = .03), although all valueswere within the expected range for healthy dogs. De-spite wide variation, the higher level of serum CRP inoverweight dogs supports the presence of increasedinflammation. Additional studies to evaluate other in-flammatory mediators, mechanisms, and clinical sig-nificance are warranted.

REFERENCES1. Tanner AE, Martin J, Saker KE. Oxidative stress and inflammatory

state induced by obesity in healthy felines J Anim Physiol AnimNutr 2007;91(3-4):163-166.

2. Veiga APM, Price CA, de Oliveira ST, et al. Association of canineobesity with reduced serum levels of C-reactive protein. J VetDiagn Invest 2008;20:224-228.

3. Yamka RM, Friesen KG, Frantz NZ. Identification of canine mark-ers related to obesity and the effects of weight loss on the mark-ers of interest. Intern J Appl Res Vet Med 2006;4(4):282-292.

C-Reactive Protein in Lean versus Overweight DogsL.A. Eirmann,a,b L.M. Freeman,c D.P. Laflamme,a and K.E. Michel d

aNestlé Purina PetCare, St. Louis, MissouribOradell Animal Hospital, Paramus, New Jersey

cCummings School of Veterinary Medicine, Tufts University, North Grafton, MassachusettsdSchool of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Nutrition ForumResearch Abstracts:

Poster Presentations


Veterinarians often recommend increasing physicalactivity to improve the success of weight-loss pro-grams. Therefore, we chose to examine how physicalactivity influences a traditional weight-loss programover 12 weeks.

All dogs meeting the inclusion criteria underwent adiet and activity evaluation, and their diets wereswitched to Purina OM dry and/or canned formulasduring a 2-week washout period. The weight-loss pro-gram was initiated using the Purina Feeding Guide,which was designed to achieve 2% weight loss everyweek. Dogs were evaluated every 2 weeks to assess theirbody weight, body condition score, dietary history, av-erage daily kilocalorie consumption, and average num-ber of steps daily, registered by collar-mountedpedometers. Dogs were initially stratified into active(>7,500 steps daily) and inactive (<7,500 steps daily)groups based on physical activity patterns during thewashout period. Dogs in the active group were en-couraged to walk an additional 2 miles/day beyondtheir normal activity level, and inactive dogs were notencouraged to increase their physical activity. Signifi-cance was determined using the Wilcoxon signed-ranktest with an α set at 0.05.

Thirteen dogs in the active group and 14 dogs inthe inactive group completed the 12-week dietarytrial. Significant differences between the groups wereobserved in steps/day and kcal/kg0.75/day but not inoverall weight loss during the program (Table 1).

When examining the two groups of dogs, it is evi-dent that active dogs can consume roughly 30% morecalories while achieving the same rate of weight lossas inactive dogs. These findings suggest that physicalactivity plays a modest role in weight reduction andunderscores how physical activity should be takeninto consideration when implementing weight-re-duction protocols.

The Influence of Physical Activity During a Canine Weight-Loss Program

J.J. Wakshlag, A.M. Struble, M. Panasevich, B. Warren, M. Maley, and F.A. KallfelzDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

RESEARCH ABSTRACTS: POSTER PRESENTATIONS

TABLE 1Effect of Physical Activity on

a Weight-Loss Program

Study Group

Parameter Active Inactive P Value

Steps/day 9,702 ± 3,042 5,157 ± 1,654 <.01

kcal/kg0.75/day 57.1 ± 15.6 42.2 ± 10.6 <.01

Weight loss (%) 14.6 ± 6.4 15.5 ± 5.2 .70


Feeding recommendations for pet dogs are based onpredictive equations derived using kenneled labora-tory dogs. Pet dogs, however, are generally housed inconsiderably different environments; in contrast to lab-oratory dogs, pets are likely to have routines that varyfrom day to day and to participate in activities that aremuch more variable and uncontrolled. In our investi-gations of the Actical® activity monitor (AAM; MiniMitter, Bend, OR), an accelerometer-based device, weused standardized activities to established cut-pointsfor discriminating between sedentary, light (walking),and moderate to vigorous (e.g., trotting, jumping) ac-tivities in dogs. The aim of this study was to use thesecut-points to make a preliminary evaluation of thetime pet dogs spent in activities of differing intensity.

Ninety-six clinically normal dogs were included.Owners’ written consent was obtained, and the ab-sence of any planned changes in usual schedule wasconfirmed. Dogs wore an AAM on a neck collar con-tinuously for 2 weeks. The monitor recorded the total

activity counts accumulated over 1-minute intervals,and each dog’s intensity of activity for each minuteof recording was classified using the total counts forthat minute and our preestablished cut-points. Thepercentage of time dogs spent in sedentary, light, ormoderate to vigorous activity each day was calculatedand evaluated over the 14 days of data collection.

The median percentage of time dogs were seden-tary, engaged in light activity, or engaged in moderateto vigorous activity were 87% (range, 54% to 98%),11% (range, 2% to 42%), and 2% (range, 0% to33%), respectively. Based on linear regression analy-sis, for every 1-year increase in age, there was an 0.8%increase in time spent sedentary and 0.6% and 0.2%decreases in time spent in light and moderate to vig-orous activities, respectively.

These are preliminary findings, and the AAM has notyet been cross-validated for activities in a free-living sit-uation. However, the data do suggest that pet dogs aresedentary for a significant portion of their days.

Using Activity Monitoring to Delineate Time Spent by Pet Dogsin Activities of Differing Intensity: A Preliminary Analysis

K.E. Michel, C. Dow, and D.C. BrownSchool of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania


Survey of Diets Designed for Weight Loss in Dogs and CatsD.E. Linder and L.M. Freeman

Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts


TABLE 1Median (Range) Caloric Density of 72 Weight-Loss Diets

Dry Diets (n = 54) Canned Diets (n = 18)

Species kcal/cup kcal/kg kcal/can kcal/kg

Canine 297 (217–395) 3,295 (2,726–3,875) 309 (189–398) 857 (533–1,002)

Feline 320 (235–480) 3,466 (3,018–4,009) 144 (78–172) 941 (744–1,010)

The high prevalence of obesity in companion animalsmakes reduced-calorie diets an important part of aweight-loss program. However, variability in caloricdensity and feeding directions could make it difficultto correctly use these diets. Therefore, the purpose ofthis study was to survey commercially available dietsdesigned for weight loss to determine the range ofcaloric density and feeding directions. The study in-cluded all diets in one region having a weight-man-agement claim and having feeding directions forweight loss. Diets were obtained through mass mer-chandisers, supermarkets, pet specialty stores, and aveterinary hospital. Seventy-two diets (40 dog foods[30 dry and 10 canned] and 32 cat foods [24 dry and8 canned]) were included. An additional 21 diets thathad weight-management claims on the label but no

feeding directions for weight loss were evaluated sep-arately. The results for caloric density of the 72 dietsare shown in Table 1.

According to feeding directions, the median rec-ommended caloric intake was 1.00 × resting energyrequirement (RER) for current weight in dogs andranged from 0.73 to 1.30 × RER. For cats, the medianrecommended caloric intake was 0.95 × RER for cur-rent weight and ranged from 0.67 to 1.38 × RER. Priceper kilocalorie was highly variable among diets. Thesedata suggest that there is a wide variance in recom-mended caloric intake, kilocalories by weight and vol-ume, and price per kilogram across the market ofdiets intended for weight loss. This variability couldcontribute to the challenges of achieving successfulweight loss in pets.


Our laboratory has studied obesity and associatedmetabolic disorders in dogs for almost 10 years. Wehave compiled data from 56 dogs (Exp 1 to 5) thatwere involved in a weight-gain program to make themobese (hyperenergetic high-fat diet) and 14 obesedogs (Exp 6 and 7) that were involved in a weight-loss program (hypoenergetic diet). Body weight (BW)and composition, insulin sensitivity (using the eu -glycemic– hyperinsulinemic glucose clamp technique),and plasma lipids were measured at the beginningand the end of the weight-change period.

Results are shown in Table 1. In the 56 overfed dogs,BW increased by an average of approximately 43% in32 weeks. Obesity led to an average decrease in insulinsensitivity index (IS) of 52%, but neither basal glycemianor insulinemia changed significantly. Obesity also led

to dyslipidemia, with an increase in plasma triglycerides(TG; 74% on average) and nonesterified fatty acids(NEFA; 32% on average). Except in one experiment,plasma total cholesterol (TC) did not change signifi-cantly. Lipoprotein profiles mainly showed an increasein very low-density lipid triglycerides (VLDL-TG) andhigh-density lipid triglycerides (HDL-TG) and a de-crease in high-density lipid cholesterol (HDL-C).

BW loss (Exp 6 and 7) resulted in the inversion ofmetabolic disorders associated with obesity. Clinicalassessment of obesity-related metabolic disorders hadnever been reported before in such a large number ofdogs. In dogs, similar to what happens in humans,overfeeding leads to obesity associated with a de-crease in insulin sensitivity and an increase in plasmaTG and NEFA.

Obesity in Dogs: A Synthesis of Clinical DataS. Serisier, E. Bailhache, C. Gayet, F. Briand, J. Le Bloc’h, V. Leray, T. Magot, K. Ouguerram, and P. Nguyen

Nutrition and Endocrinology, National Veterinary School of Nantes—INSERM U539, University Hospital, Nantes, France

TABLE 1Effect of Diet on Metabolic Parameters

Weight Gain Weight Loss

Parameter Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6 Exp 7

Sample size and gender 7 males 7 females 7 males 11 females 24 females 6 males 7 females

Age (yr) 3.5 ± 2.1 1.8 ± 0.1 8.0 ± 0.8 4.5 ± 0.3 1.1 ± 0.0 5.5 ± 2.1 6.2 ± 0.1

Food allowance Ad libitum 1.6 NRC 1.6 NRC 1.6 NRC 2.1 NRC 75 iBW0.67 0.6 NRC

Food intake (kcal ME/kg0.75) 248 ± 11 182 ± 11 — 225 ± 7 275 ± 13 62 ± 0 73 ± 2

Duration (wk) 28 60 25 22 25 12 11

BW change (%) +43* +45* +39* +44* +47* –28* –24*

Fat mass change (%) +75* — +51* — — –51* –48*

Basal insulin change (%) +140* 0 –19* +33 –7 –17 –21

IS change (%) –47* –54* –56* –58* –46* +61* +43*

TG change (%) +224* +74* +55* +67* — –44* —

NEFA change +70* +47* +25 +46* — –57* –29*

TC change –12 +2 +30* +5 — +21 —

VLDL-TG change (%) +438* — — — — –67 —

HDL-TG change (%) +868* — — — — –80* —

HDL-C change (%) –16* — — — — +17* —

*Significant change (P < .05).— = not determined; iBW = ??????????? ME = ??????????? NRC = ???????????.


Dietary fiber may increase and maintain satiety andprevent feelings of hunger, depending on fiber typeand inclusion level. Prolonged satiety could have fa-vorable effects on behavior in dogs. This study ex-amined whether two diets differing in dietary fiberfermentability and satiety properties (unpublishedresults) would influence the behavior of dogs.

Sixteen healthy adult beagles were housed indi-vidually in indoor pens and fed a low-fermentablefiber (LFF) diet containing 8.5% cellulose or a high-fermentable fiber (HFF) diet containing 8.5% sugarbeet pulp and 2% inulin. Dogs were fed two equalportions at 0830 and 1830 hours according to dailyenergy requirements. Behavior was recorded after thediet had been fed for 4 weeks and analyzed by in-stantaneous scan sampling (2 × 24 hours with 15-min intervals) and focal sampling continuousrecordings (10-min episodes/hr/animal between0900 and 1800). Scores were expressed per clock

hour, and time × diet effects were tested statisticallyusing residual maximum likelihood.

For the scans, a significant (P < .05) time × diet in-teraction was found for the frequency of “lie headrested.” At night and in the morning, HFF-fed dogsrested more than LFF-fed dogs, but they rested lessbetween 1400 and 1700. No significant interactioneffects were found for “lie–sit” or “stand–walk.” Forthe continuous recordings, the main findings were atendency (P < .10) for time × diet for time spent “liehead rested,” with a pattern consistent with that forthe scans. The interaction was significant for “lie–sit,”with HFF-fed dogs having higher values from 1300to 1800. Finally, time spend tail wagging was signifi-cantly higher for LFF-fed dogs just before the eveningmeal, which may indicate a higher level of arousal.In conclusion, fermentability of dietary fiber influ-ences behavior in kenneled dogs, possibly throughits effects on feelings of satiety or hunger.

Dietary Fiber Type Affects Behavior in Kenneled DogsG. Bosch,a B. Beerda,b M. Hesta,c A.F.B. van der Poel,a G.P.J. Janssens,c and W.H. Hendriksa

aAnimal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen, The NetherlandsbAdaptation Physiology Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

cLaboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium



Between 1997 and 2007, a retrospective study was con-ducted to determine (1) the effect of management fac-tors on cats’ feeding habits and (2) the relationshipbetween the method of food and water administration,environment, room availability, physical activity, andbehavioral disorders. Information (sex, age, breed, typeof food, mode of administration, family composition,presence of other cats in the home, housing condi-tions, and behavioral problems) was recorded for 300cats evaluated at the Clinical Behavioral Service of the Veterinary Teaching Hospital of the AutonomousUniversity of Barcelona. Data were analyzed by chi-square test (SPSS 12.0; SPSS, Chicago, IL).

The most common type of food consumed wasmedium- to high-quality dry food (78%) adminis-trated ad libitum. The typical owner is a family ofthree or four members living in a home with littleroom (less than 10 m2) at the cat’s disposal. Indoorcats comprised 77% of the total number. In general,playing represented the cats’ main physical activity. Itwas not possible to quantify the amount of time catsspent being physically active. Eighteen percent of thefamilies surveyed owned more than one cat.

Within this population, there was no provable cor-relation between the method of feeding, type of food,and intraspecies aggression toward other cats livingin the same environment. Unlike dogs, in which alink between administration pattern and aggressive-ness has been observed, the availability of food forcats is not a factor influencing so-called food de-fense–related aggression.

A significant correlation (P = .05) betweenhouse-soiling problems and water availability wasobserved. Cats with a limited access to water moreoften demonstrate problems with urinating inplaces other than the litterbox (P < .05). There wasno direct correlation between overweight andwhether the cat was neutered or not neutered. Thiscan be explained by the fact that appropriate nutri-tion in conjunction with the opportunity for phys-ical activity may counteract the castration-relatedtendency toward obesity. In this regard, a direct link(P = .05) was detected between the number of catsliving in a house and the rate of overweight cats be-cause an interaction and playing between cats stim-ulates physical activity.

Review of Cat Feeding Habits in SpainV.M. Mariotti, M. Hervera, J. Fatjó, M. Amat, J.L. Ruiz de la Torre, X. Manteca, and M.D. Baucells

Animal Nutrition, Management and Welfare Research Group, Department of Animal and Food Science, Autonomous University of Barcelona, Catalonia, Spain


Hair growth in adult short-haired cats shows a strongseasonal pattern, with hair growth rates at maximumin late summer and at minimum in late winter. Re-cent work has indicated that the optimal mineral lev-els for coat quality characteristics during periods ofgrowth may be significantly higher than current rec-ommendations for growth and maintenance. Thisstudy investigated the summer coat growth of catsduring chelated mineral supplementation.

Two groups of eight adult cats (four males and fourfemales), ranging in age from 1.8 to 8.0 years, werehoused at the Centre for Feline Nutrition (latitude40°22’S; longitude 175°31’E) for 16 weeks. The controlgroup was fed a complete and balanced diet containingnonchelated zinc (50 mg/kg on a dry-matter [DM]basis), copper (9.5 mg/kg DM), selenium (0.47 mg/kgDM), and manganese (6.2 mg/kg DM), and the testgroup was fed the same diet supplemented with zinc(150 mg/kg DM), copper (15 mg/kg DM), selenium(0.5 mg/kg DM), and manganese (50 mg/kg DM). Alldiets were fed ad libitum. Hair growth was measured by

clipping a 5 × 5 cm area on the left flank. Photos werethen taken with a digital camera to determine the lengthof hair regrowth over the ensuing 168 hours. Thisprocess was repeated at intervals over the 16-week study.At the beginning and end of the study, a judging panelof five people from a local cat club assessed coat glossi-ness, softness, greasiness, and overall coat condition.

The average rate of hair growth in mineral-supple-mented and control animals was similar at the start ofthe study (178.3 and 173.1 μm/day, respectively) butbecame significantly different (P < .05) by the end ofthe study (91.5 μm/day vs 72.7 μm/day). In addition,the judging panel reported a 48.2% improvement inassessed coat parameters in the supplemented groupcompared with a 36.1% improvement in the controlgroup. This study shows that additional chelated min-eral supplementation improved the growth of the sum-mer coat, and the coat that was grown showedimproved visible and tactile characteristics. However, itremains to be determined whether nonchelated min-eral supplementation would produce the same effects.

Effect of Mineral Supplementation on Hair Growth and Coat Characteristics of Short-Haired Cats

M. Hekman and D.G. ThomasInstitute of Food, Nutrition & Human Health, Massey University, Palmerston North, New Zealand



The aim of this study was to determine the influenceof excess calcium on the digestibility of crude nutri-ents and energy partly as a basis for other work.1

Thirty healthy, growing beagles and 44 foxhound–boxer–Labrador retriever crossbreeds (FBIs) wereused in this study. As a control, one group of everybreed was fed a balanced diet with calcium accord-ing to requirements (normal calcium [NC], ~11 g/kg);the other puppies were fed the threefold amount ofcalcium (high calcium [HC], ~36 g/kg). Digestion tri-als were conducted at the ages of about 12, 18, and24 weeks. Feed and fecal samples were analyzed forcrude nutrients and gross energy (GE; using an adia-batic bomb calorimeter).

Calcium excess significantly impaired the di-gestibility of energy and crude nutrients. There was asignificant interaction between breed and diet. The

impairment of digestibility of energy, protein, and or-ganic matter (Table 1) was significantly higher in theFBIs than in the beagles.

Potential mechanisms of calcium’s effect on di-gestibility are (1) direct inhibition of fat digestibilitycaused by the formation of calcium soaps in the gut,2

(2) reduced pancreatic secretion by an inhibitory ef-fect of calcitonin,3 or (3) a calcium-induced increas-ing intestinal motility.4

REFERENCES1. Dobenecker B. Apparent calcium absorption in growing dogs of

two different sizes. J Nutr 2004;134(8 Suppl):2151S-2153S.2. Meyer H, Zentek J. Ernährung des Hundes. Stuttgart: Parey; 2005.3. Tiscornia OM, Levesque D, Sarles H, et al. Canine exocrine pancre-

atic secretory changes induced by calcium or ethanol plus calciumintraduodenal infusion. Am J Gastroenterol 1976;66(5): 452-459.

4. Kowalewski K, Kolodej A. Effects of calcium infusion on secre-tion and motor activity of totally isolated canine stomach per-fused with homologous blood. Pharmacology 1976;14:537-549.

High Calcium Intake Affects the Apparent Digestibility of Crude Nutrients and Energy in Puppies

B. Dobenecker, V. Frank, and E. KienzleInstitute of Physiology, Physiological Chemistry & Animal Nutrition,

Ludwig-Maximilians University, Munich, Germany

TABLE 1Apparent Digestibility (% aD) of Energy and Macronutrients (Mean ± SD)*

Beagles NC Beagles HC FBI NC FBI HC

% aD energy 89.0a ± 2.4 86.6b ± 3.4 88.3a,b ± 2.6 84.7c ± 3.7% aD protein 88.2a ± 2.5 86.8a ± 2.8 87.3a ± 3.2 84.6b ± 4.1% aD fat 93.7a ± 2.9 89.1a,b ± 5.3 93.3a ± 3.2 87.7b ± 6.6% aD NfE 90.0a ± 3.2 85.2b ± 4.4 88.2a ± 4.4 84.0b ± 4.6% aD organic matter 88.3a ± 2.3 84.7b ± 3.3 87.3a ± 2.5 82.7c ± 3.4

*Letters identify differences between more than two groups; means not sharing a superscript letter are significantly different (two-way analysis of variance; P < .05).NfE = nitrogen-free extract.


Hitherto in growth studies, puppies received eitherfood for free choice or defined amounts of daily en-ergy. From such trials, feeding recommendations weredeveloped.1 The present study was designed the otherway around: Food allowance was adapted to guaran-tee a growth rate as recommended by the NRC.1

Thirty beagles (adult body weight [BW],11–15kg) and 38 foxhound–boxer–Labrador retrievercrossbreeds (FBIs; adult BW, 31–36 kg) were com-pared from 6 to 28 weeks of age. After weaning, thefood rations were adjusted daily according togrowth level, guaranteeing a development consis-tent with the recommended weight curve for the re-spective breed. Digestible energy (DE) in food wasdetermined in digestion trials at the age of 12, 18,and 24 weeks; metabolizable energy (ME) was cal-culated as follows:

ME = DE – 1.25 kcal × g digestible crude protein

All puppies were clinically healthy. The mean en-ergy intake from 6 weeks through 28 weeks of age was

209 ± 27 kcal/kg BW0.75 in the beagles and 226 ± 39kcal/kg BW0.75 in the FBIs (calculated with the actualBW); age had little effect (Table 1). During the study,however, there was a consistent breed difference, witha higher energy intake in the FBIs.

This study suggests that during the major periodof growth, the energy requirement is not a function ofrealized growth. Mature beagles and FBIs show simi-lar differences in energy requirements as the puppiesin this study. This indicates that breed differences inenergy requirements must be taken into account dur-ing growth.

REFERENCES1. National Research Council. Nutrient Requirements of Dogs and

Cats. Washington, DC: National Academies Press; 2006.

2. Kleffner H. [Study of the digestibility of energy and nutrients inwild carni- and omnivorous mammals as a basis for energy pre-dictions in the feed.] Thesis, Veterinary Faculty, Ludwig-Maxim-ilians University, Munich, Germany; 2008. Available athttp://edoc.ub.uni-muenchen.de/9416/1/Kleffner_Helen.pdf; ac-cessed January 2009.

Energy Requirements of Two Different Dog Breeds for IdealGrowth from Weaning to 6 Months of Age

B. Dobenecker, V. Frank, and E. KienzleInstitute of Physiology, Physiological Chemistry & Animal Nutrition,

Ludwig-Maximilians University, Munich, Germany


TABLE 1Metabolizable Energy Requirement (kcal/kg BW0.75) of Beagle and FBI Puppies

6 to 28 Weeks of Age

Weeks 6–10 Weeks 10–14 Weeks 14–18 Weeks 18–22 Weeks 22–26 Weeks 26–28

Beagles 199 ± 31 223 ± 27 216 ± 26 212 ± 19 202 ± 21 193 ± 25

FBIs 227 ± 41 229 ± 42 227 ± 38 228 ± 34 218 ± 38 224 ± 48


Prediction of energy in feed is an important precon-dition for systematic diet calculation. In zoo animals,it may also help to estimate energy requirementsfrom empirical data on feed intake. In this study, theestimated metabolizable energy (ME) by existing pre-dictive equations for dogs and cats were comparedwith the ME derived from experimental data on di-gestibility of energy and protein from the literatureon digestibility in wild carnivores.

Literature was reviewed for data on digestibility ofenergy and nutrients in carnivorous wild mammalsin captivity.1 A total of 111 publications on 48 species(40 publications on 16 Felidae species, 28 publica-tions on six Mustelidae species, 14 publications onnine marine mammal species, 11 publications on fiveUrsidae species, 14 publications on eight Canidaespecies, two publications on two Viverridae species,and two publications on two Hyaenidae species) werereviewed. Two types of diets were used: prepared petfood or unprocessed food such as meat and offal(usually supplemented). The ME was derived fromexperimental data on digestable energy (DE) proteinas follows:

ME = DE – 1.25 kcal × g digestable crude protein

Regressions between nutrients and digestible nutri-ents (Lucas test) were used to estimate true proteinand fat digestibility in the different ration types. Also,regressions between crude fiber in dry matter (DM)and digestibility of energy were calculated.

Lucas test regressions gave true digestibilities forprotein and fat of above 90% for unprocessed food.For processed food, true protein digestibility was

nearly 80%, and true fat digestibility nearly 100%.Predictive equations for ME for dogs2 gave reason-able estimates for all carnivores, including Felidae.Equations for cats gave less accurate predictions. Forunprocessed food, the unmodified Atwater factorswere appropriate:

ME (kcal) = 4 × g protein + 9 × g fat + 4 × g NfE

For processed food, the negative correlation be-tween fiber and energy digestibility was used to makean estimate:

Gross energy (kcal) = 5.7 × g protein + 9.4 × g fat+ (4.1 × [g nitrogen-free extract + g crude fiber])

Apparent energy digestibility (%) = 91.2 – (1.43 ×% crude fiber in DM)

DE (kcal) = gross energy × apparent energy di-gestibility/100

ME (kcal) = DE – 1.04 × g protein

Digestibility of food in wild carnivores appears tobe more similar to digestibility in dogs than in cats.Predictive equations for ME recommended for do-mestic dogs2 provide reasonable estimates for ME inwild carnivores.

REFERENCES1. Kleffner H. [Study of the digestibility of energy and nutrients in

wild carni- and omnivorous mammals as a basis for energy pre-dictions in the feed.] Thesis, Veterinary Faculty, Ludwig-Maxim-ilians University, Munich, Germany; 2008. Available athttp://edoc.ub.uni-muenchen.de/9416/1/Kleffner_Helen.pdf; ac-cessed January 2009.

2. National Research Council. Nutrient Requirements of Dogs andCats. Washington, DC: National Academies Press; 2006.

Prediction of Metabolizable Energy in Food for Wild Carnivorous Mammals

E. Kienzle,a M. Clauss,b and H. Kleffnera

aInstitute of Physiology, Physiological Chemistry & Animal Nutrition, Ludwig-Maximilians University, Munich, Germany

bClinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland


The standardized extract of Ginkgo biloba leaf is one ofthe most frequently used phytomedicines in elderly per-sons. G. biloba contains flavonoids, of which quercetinis recognized as the most important. Flavonoids showpotent antioxidant and free-radical scavenging activities.

In this study, the presence of G. biloba extract addedto a senior dry diet at the end of food production was determined by liquid chromatography–mass spectrometry/ mass spectrometry (LC-MS/MS). Diet A(without G. biloba) and diet B (with G. biloba) were ad-ministered to a group of 11 healthy aging dogs for 1month. The ration was calculated on the basis of idealweight. No other food was administered, but water wasprovided. Absorption and efficacy trials were performedby blood collection of all subjects every 10 days. G.biloba absorption by dogs was investigated by determi-nation of plasma quercetin after meals by LC-MS/MS.Two oxidative stress parameters, derivatives–reactive

oxygen species (d-ROMs) and biologic antioxidant po-tential (BAP) (Diacron International, GR, Italy), werespectrophotometrically assessed in dog plasma. BAPand d-ROMs data were analyzed by analysis of varianceand Tukey-Kramer tests; BAP and quercetin values weretested by correlation test. Differences were consideredsignificant when P <.05. G. biloba absorption was veri-fied by an increase in plasma quercetin values after dietB administration (9.08 ± 6.04 ng/ml).

No significant variation of d-ROMs values oc-curred, but the BAP was considerably increased in thesample on the last day (3,508 μmol/L) in contrast tothe first sampling day (2,537 μmol/L). Plasmatic BAPand quercetin concentrations were correlated (r =0.73). These results confirmed the utility of G. bilobaadministration to healthy geriatric dogs to improvetheir antioxidant potential, which is so importantduring senescence.

The Effects of Ginkgo biloba Extract on Healthy Geriatric DogsA. Pasquini,a P. Simonetti,b E. Luchetti,a C. Gardana,b G. Cardini,a and G. Rec

aVeterinary Clinic Department, University of Pisa, Pisa, ItalybDepartment of Food Science and Microbiology, Division of Human Nutrition, University of Milan, Milan, Italy

cBayer Spa, Milan, Italy



β-glucans are branch-chained polysaccharides foundin a variety of fungi, including yeast (Sacromyces spp).Prior studies suggest that β-glucans may have signif-icant immune-modulating effects. The objective ofthis study was to evaluate the effects of feeding β-glu-cans from yeast on the immune status of adult dogs.The 36-week study included 48 dogs (2–10 years ofage) evenly and randomly allocated to four diets.During an adaptation period of 8 weeks, all dogs werefed a complete and balanced standard diet. Subse-quently, the dogs were fed the standard diet coatedwith the following: diet A, no yeast product; diet B, β-glucan extracted from baker’s yeast; diet C, semipuri-fied β-glucan extracted from brewer’s yeast; and dietD, brewer’s dried whole yeast. Diets B, C, and D werefed to provide 5 mg/kg body weight of β-glucan. At12 and 14 weeks, all dogs were vaccinated with Bor-relia burgdorferi bacterin (LymeVax®; Fort Dodge). Se-rial blood samples were taken to monitor vaccine

response, specifically serum IgG, using a B. burgdorferiantigen ELISA (antigen provided by John F. Ander-son, New Haven, CT). Additional immune parame-ters (total IgG, IgA, IgM) were also measured.

All dogs in the trial had a positive response to theB. burgdorferi bacterin vaccine. After 36 weeks, dogsconsuming diets C and D had significantly (P < .001)higher concentrations of bacterin-specific IgG thandid dogs fed diets A or B. Analysis of covariance in-dicated no significant differences between diets forserum IgG, IgA, and IgM at 36 weeks, demonstratingthat β-glucans modulate the immune system to bet-ter respond to specific immune challenges withoutoverstimulation. As demonstrated using a vaccine-re-sponse model, semipurified yeast β-glucans and β-glucans from brewer’s dried yeast are capable ofpositively and significantly modulating the immuneresponse in dogs when fed as part of a nutritionallycomplete and balanced diet.

Evaluation of the Canine Immune Response to Dietary β-Glucans

W. Anderson, E. Satyaraj, and W. KerrNestlé Purina PetCare, St. Louis, Missouri


Past studies have shown inconsistencies in the ana-lyzed product content of various dietary supplementsas well as labeling violations.1-4 The objective of thisstudy was to test the quality control, disintegrationproperties, and compliance with labeling regulationsfor representative commercial taurine and carnitinesupplements. Eleven commercial taurine and 10 com-mercial carnitine supplements were evaluated. Foreach sample, the amount of taurine or carnitine wasdetermined and compared with the label claim. Allsupplements were evaluated for concentrations ofmercury, arsenic, and selenium, and the disintegra-tion properties of five taurine and eight carnitinetablets were determined in vitro. Labels were alsoevaluated for compliance with Food and Drug Ad-ministration (FDA) regulations.

Ten of 11 taurine and all carnitine products werewithin 10% of the stated label claim. Three of 11 tau-rine and six of 10 carnitine products were within 5%of the stated label claim. No significant contamina-tion of mercury, arsenic, or selenium was found in

any of the samples. During disintegration testing, oneof five taurine tablets and five of eight carnitinetablets did not disintegrate within 45 minutes, anddisintegration times varied widely. All product labelsconformed to FDA regulations. Compared with pre-vious studies, the taurine and carnitine supplementsevaluated in this study more closely adhered to man-ufacturer claims and labeling guidelines. However,disintegration trials suggest high variability in someproducts, possibly limiting their uptake and utiliza-tion by animals that receive them.

REFERENCES1. Russell AS, Aghazadeh-Habashi A, Jamali F. Active ingredient con-

sistency of commercially available glucosamine sulfate products.J Rheumatol 2002;29:2407–2409.

2. Oke S, Aghazadeh-Habash A, Weese JS, et al. Evaluation of glu-cosamine levels in commercial equine oral supplements. EquineVet J 2006;38:93–95.

3. Weese JS. Microbiologic evaluation of commercial probiotics.JAVMA 2002;220:794–797.

4. Weese JS. Evaluation of deficiencies in labeling of commercialprobiotics. Can Vet J 2003;44:982–983.

Composition, Disintegrative Properties, and LabelingCompliance of Commercial Taurine and Carnitine Supplements

R.A. Bragg,a L.M. Freeman,a A.J. Fascetti,b and Z. Yub

aCummings School of Veterinary Medicine, Tufts University, North Grafton, MassachusettsbUniversity of California, Davis, School of Veterinary Medicine, Davis, California



Epilepsy is a common disease in dogs, and pheno-barbital is typically used for therapy. Since the late1960s, decreased serum 25-hydroxyvitamin D (25-OH-D) concentrations and decreased bone mineraldensity have been observed in humans receiving phe-nobarbital. It is unknown whether long-term pheno-barbital therapy in dogs is associated with decreasedconcentrations of 25-OH-D. Thus, the objective ofthis study was to determine if dogs receiving pheno-barbital therapy exhibit changes in serum 25-OH-Dconcentrations.

Adult dogs (n = 227) receiving phenobarbital wererecruited and compared with 35 healthy dogs not re-ceiving phenobarbital. All dogs consumed commer-cially available, nutritionally balanced, complete dogfoods. Serum concentrations of 25-OH-D were sig-nificantly lower in dogs receiving phenobarbital(mean, 118 nmol/L) compared with those not receiv-ing phenobarbital (mean, 137 nmol/L). Significancedepended on the serum concentration of phenobar-bital; dogs with serum phenobarbital concentrationsbelow the therapeutic range (<65 μmol/L; n = 57) had

similar concentrations of 25-OH-D (mean, 130nmol/L) as those not receiving phenobarbital. Dogswith the highest levels of serum phenobarbital (>150μmol/L; n = 13) exhibited the lowest concentrations ofserum 25-OH-D (mean, 83 nmol/L).

Additionally, the duration of phenobarbital ad-ministration had a significant impact on 25-OH-Dconcentrations: Dogs receiving phenobarbital for morethan 1 year had significantly lower 25-OH-D concen-trations (mean, 114 nmol/L; n = 154) than dogs re-ceiving phenobarbital for less than 1 year (mean, 127nmol/L; n = 61). The age or body condition of the dogdid not appear to have a significant effect on serum 25-OH-D concentration. Phenobarbital administration re-sulted in a lower serum concentration of 25-OH-D;however, the mean concentration was still within theaccepted reference range for 25-OH-D. Most completepet foods probably contain sufficient vitamin D tomaintain serum 25-OH-D within an adequate range;however, problems could potentially arise in dogs re-ceiving phenobarbital that are consuming diets with amarginal content of vitamin D.

Effects of Phenobarbital Administration on Serum 25-Hydroxyvitamin D Concentration in Dogs

P.A. Schencka and S.K. Aboodb

aDepartment of Pathobiology and Diagnostic Investigation, Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, Michigan

bDepartment of Small Animal Clinical Sciences, Michigan State University, East Lansing, Michigan


Diabetes mellitus (DM) is a common disease in oldercats. The diagnosis is currently based on glucose andfructosamine measurements. Because of stress-inducedhyperglycemia, elevated glucose concentrations inblood and urine are not specific for DM, and fruc-tosamines are biased by changes in protein concentra-tion and hyperthyroidism. The aim of this study was totest β-hydroxybutyrate (β-OHB) as a diagnostic para-meter to confirm DM in cats. The hypothesis is thatonly cats with DM develop significant ketosis and glu-coneogenesis prevents increases of β-OHB in other dis-eases associated with energy depletion.

Three groups of animals were included in the study:(1) healthy controls (n = 25), (2) cats with DM at firstconsult without insulin pretreatment (n = 31), and (3)anorectic or vomiting cats (n = 30). Venous blood spec-imens were obtained from all animals, and β-OHB wasmeasured on a Hitachi 911 Chemistry Analyzer(Roche). Differences between the groups were ana-

lyzed using the Kruskal-Wallis test, and specificity andsensitivity were calculated with the help of receiver op-erating characteristic curves.

The diabetic cats had significantly higher β-OHB con-centrations than the healthy cats and anorectic or vom-iting cats (P < .001). No difference was found betweenthe nondiabetic groups (P = .0549). None of the non-diabetic cats had a β-OHB value greater than 0.3mmol/L. Using 0.3 mmol/L as a cutoff value to diagnoseDM, the sensitivity and specificity were 74.2% and100%, respectively. In contrast to human patients inwhom rapid ketone production is seen during fasting,no significant ketosis was found in cats with various dis-eases causing energy depletion. If examinations on agreater population corroborate these results, β-OHB canbe used as a specific confirmatory test to diagnose felineDM. The existence of a handheld glucometer that is re-liable for measuring β-OHB in the low range makes thisparameter attractive for its use in private practice.

Usefulness of β-Hydroxybutyrate Measurement for Diagnosing Feline Diabetes Mellitus

F. Zeugswetter,a J. Prokisch,a S. Handl,b and C. Ibenb

aDepartment for Companion Animals and Horses, University of Veterinary Medicine, Vienna, AustriabDepartment for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria



A large colony of adult domestic shorthair cats wasmaintained for nutritional research. Established prac-tices for health maintenance included postmortem eval-uation of all mortalities, at which time renal tissue andrenal precipitates were harvested by standard methodsfor histologic and quantitative mineral evaluation. Pre-cipitates were found in one or both kidneys of 102 of671 (15.2%) cats. As single analytes, calcium oxalate (n= 15) and organic debris (n = 27) occurred most fre-quently. As components of complex precipitates, organiccalcium oxalate (n = 24) and calcium oxalate–calciumapatite (n = 14) occurred most frequently.

Overall, precipitate-affected and precipitate-unaf-fected cats had similar (P > .05) mean ages at death(121 and 124 months, respectively). Among cats thathad renal-associated causes of death, the ages at deathbetween the affected and unaffected cats were similar(121 and 146 months, respectively; P > .05). However,among cats that had nonrenal causes of death, the af-fected and unaffected cats had marginally different

(P = .054) mean ages at death (120 and 104 months,respectively).

Overall, precipitate-affected and precipitate-unaf-fected cats had different (P < .01) mean histologicscores (11 and 9 of 19, respectively). Among cats thathad renal-associated causes of death, affected and un-affected cats had similar (P > .05) mean histologicscores (12 and 13, respectively). Among cats that hadnonrenal causes of death, affected and unaffected catshad different (P < .05) mean histologic scores (9.6and 7.3, respectively).

Relationships between oxalate and organic precip-itates are not yet well understood. The high frequencyof organic debris suggests hemorrhage, but causal fac-tors also are not well understood. The possibility thatsignaling from gut microflora might play a role in fe-line renolith formation needs to be investigated.Among the cats in this study, the presence of renal pre-cipitates was associated with somewhat more histo-logic change but had little apparent additional effect.

Renal Pelvic Precipitates in Domestic CatsD. Lawler,a Z. Ramadan,a J. Lulich,b D. Polzin,b and R. Evansc

aNestlé Research Center, St Louis, MissouribUniversity of Minnesota, College of Veterinary Medicine, St. Paul, Minnesota

cPacific Marine Mammals Center, Laguna Beach, California


A vegetable oil high in diacylglycerol (DAG) has beeninvestigated in humans and animals for treating hy-perlipidemia and related disorders. Two studies wereconducted to evaluate the tolerance and therapeuticpotential of dietary DAG in cats. The first was a two-pan palatability trial of a dry-type commercial dietcoated with triacylglycerol (TAG) oil versus DAG oil(19% metabolizable energy [ME] oil, 48% ME totalfat). Six female (2.8 ± 0.9 years; 3.3 ± 0.2 kg) and twomale (4.7 years; 5.2 ± 0.3 kg) cats were offered bothdiets over a 14-day period. Food intake was the samebetween diets (P = .26), and no adverse effects werefound on body weight, fecal scores, or overall health.

In the second study, 11 males with hypertriglyc-eridemia (1.5 ± 0.4 years; 4.4 ± 0.6 kg) deficient inlipoprotein lipase (LPL) from a heritable mutationwere acclimated to a semipurified diet containing

TAG oil (23% ME fat) for 21 days. After assignmentinto two groups according to baseline triglyceridemia,six cats were fed the adaptation diet and five cats werefed a diet with DAG oil substituted for TAG oil witha similar fatty acid composition for 8 days. The diettreatments were crossed over for 8 more days. Serumfrom jugular venous blood was obtained on days 6,7, 8, 14, 15, and 16.

TAG versus DAG as a dietary fat source did not af-fect serum triglycerides (3,001 ± 1,730 vs 3,282 ±2,311 mg/dL; P = .47), cholesterol (184 ± 51 vs 186 ±60 mg/dL; P = .81), or nonesterified fatty acids (1.39± 1.05 vs 1.36 ± 1.04 mmol/L; P = .62). AlthoughDAG did not lower triglycerides in LPL-deficient catswith hypertriglyceridemia, DAG was well acceptedand tolerated in amounts up to 48% ME in commer-cial and semipurified diets.

Effects of Diacylglycerol Oil in Normal and Lipoprotein Lipase–Deficient Cats

C.A Datz,a R.C. Backus,a K.L. Fritsche,b and J.J. Ramseyc

aDepartment of Veterinary Medicine and Surgery, University of Missouri, Columbia, MissouribDepartment of Microbiology and Molecular Immunology, Division of Animal Sciences, University of Missouri, Columbia, Missouri

cDepartment of Molecular Biosciences, University of California, Davis, California



Conversion of linoleic acid (LA) to arachidonic acid(AA) in cats is limited because of low 6-desaturase.However, dietary γ-linolenic acid (GLA) may bypassthis enzyme step. It may be possible to induce 6-desaturase directly by providing large dietary amountsof LA precursor. Furthermore, an alternative pathwayfor AA may also exist involving LA chain elongationand 8- and 5-desaturation. We hypothesized thatGLA feeding can bypass 6-desaturation for AA syn-thesis. We further hypothesized that feeding cats ahigh-LA diet results in AA accumulation either directlyby inducing Δ6-desaturase or by an alternate route.

To test these hypotheses, fatty acid profiles weredetermined after feeding high-LA (HL), high-GLA(GLA), or adequate-LA (LL, control) diets. Adult fe-male cats (n = 29) were separated into three groupsand fed for 8 weeks. Cats were fed according to theirmetabolic body weight (BW) to maintain body con-dition scores (BCSs) of 5 of 9 with water ad libitum.Daily consumption, weekly BW, and BCS were

recorded. Blood samples were taken after overnightfasting at weeks 0, 2, 4, and 8 to measure plasmatriglycerides (TG), total cholesterol (TC), lipoproteindistribution (LP), and plasma phospholipid fatty acidprofiles. Repeated-measures analyses and Tukey (α =0.05) multiple comparisons were performed.

No significant time effects were observed in foodconsumption, BW, BCS, or metabolic factor afterweek 1. No diet effects were observed for TG, TC, orlipoprotein fractions. Time effects were observed atweeks 2 and 4 with significant increases in these pa-rameters but within normal limits that were likely at-tributable to increased dietary fat content in the testdiets. No evidence of Δ8-desaturase enzyme activitywas found with HL. However, a GLA diet effect wasobserved as early as week 2 with the formation of di-homo-γ-linolenic acid (DGLA; 20:3 8,11,14) and AA asearly as week 2, demonstrating 5-desaturase activity.It is thus possible to use dietary GLA to induce AAsynthesis.

Effects of Linoleic Acid– and γ-Linolenic Acid–Containing Diets on Feline Lipid Metabolism

L. Trevizan,a,b A.M. Kessler,a K. Bigley,b W. Anderson,c M.K. Waldron,c and J.E. Bauerb,d

aLEZO, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, BrazilbComparative Animal Nutrition Laboratory, Texas A&M University, College Station, Texas

cNestlé Purina PetCare, St. Louis, MissouridFaculty of Nutrition, Texas A&M University, College Station, Texas

Nutrition ForumRoundtable

on Controversies inClinical Nutrition


While evidence-based medicine and nutrition are important to patientcare, there are often large gaps in available evidence in veterinarymedicine and nutrition. Further, there are many ways to interpret the available evidence, and different clinicians may take differentapproaches to the same case. The purpose of this roundtable was toexplore ways that different experts approach cases for which evidenceis limited. We presented various types of cases to nutrition experts fromaround the world to understand their clinical approach. While there area number of areas in which the experts agree, there are probably justas many points on which they differ. Here they explain their clinicalapproach to each case and answer questions from the audience inattendance at the 2008 Nestlé Purina Nutrition Forum.

Arleigh J. Reynolds, DVM, PhD,DACVN, roundtable moderator and senior research scientist at the NestléResearch Center, St. Louis, Missouri

Daniel L. Chan, DVM, DACVECC,DACVN, FHEA, MRCVS, lecturer inemergency and critical care and clinical nutritionist at the Royal Veterinary College in London

Richard C. Hill, MA, VetMB, PhD,MRCVS, DACVIM, DACVN, Walthamassociate professor of small animal internal medicine and clinical nutrition at the University of Florida

Stanley Marks, BVSc, PhD, DACVIM(Internal Medicine, Oncology), DACVN,professor of small animal medicine and director of the Companion Animal Gastrointestinal Laboratory at the University of California, Davis, School of Veterinary Medicine

Rebecca Remillard, PhD, DVM,DACVN, clinical nutritionist at AngellAnimal Medical Center in Massachusetts

Julie Churchill, DVM, PhD, assistantclinical professor of companion animalnutrition at the University of Minnesota

Linda Fleeman, BVSc, PhD,MACVSc, senior lecturer of small animal medicine in the Faculty of Veterinary Science at the University of Sydney in Australia

ABOUT THE PARTICIPANTS

Additional questions and comments were provided by audience members attending the2008 Nestlé Purina Nutrition Forum.

The information in this roundtable does not necessarily reflect the opinions of, nor constitute or imply endorsement or recom-mendation by, the Publisher or copyright holder. The Publisher is not responsible for any data, opinions, or statements providedherein. The opinions expressed in this publication are those of the participants and do not necessarily reflect the points of view ofthe company or companies that manufacture and/or market any of the products mentioned.

Dr. Julie Churchill: My initial approach to a case like thisdepends on whether the owners are coming in for weightmanagement counseling or if I’m the first voice to tellthem that their pet is overweight. If I’m that first personinforming them of their pet’s obesity, then I’d spend sometime finding the level of the owners’ understanding thatobesity is a health problem. Otherwise, I would start bytaking a detailed diet history.

If the owners show a readiness for change, then I try toget a more thorough diet history. Ideally, I would quanti-tate the current intake. I tell the owners that if they werecalled away on an emergency and I were to become the petsitter, I’d like to re-create the day for Fiver. So the diet historyshould be as thorough as possible, including the amountand frequency fed of regular food, treats, human foods, andfoods given with medications. Some owners may have ahard time quantifying these amounts. If possible, I havethem measure amounts for a few days before our appoint-ment or after they return home. I try as closely as possibleto determine the current caloric intake. This helps in myapproach to reduce current calorie intake by 25% to 30%. Ialso try to determine how much of the food is coming fromtreats. That tells me a lot about the nutritional balance of thecurrent diet as well as this particular human–animal bondand where my obstacles are going to be.

Dr. Richard Hill: I completely agree that getting a detaileddiet history is essential. I’ve found it very helpful to askthe owners to document their pet’s diet for a week beforewe have an in-depth discussion. Keeping a record involves

them getting information on all the foods they havebecause these pets are often fed a lot of different foods,some of which I’m not familiar with. This record helps mework out how many calories are in each of the foods.

Using the size of the scoop to determine the amount offood fed is inaccurate because cup size varies from client toclient, especially with small animals like cats and dogs. It’sbetter to get clients to weigh the food. So I ask them to go outand buy a small kitchen scale. If they are willing to take thatstep, it gives me a better indication of how willing they areto work with me on trying to figure out the amount to feed.

Dr. Churchill: Similar to what Dr. Hill does, we email ourdiet history form to owners in advance so they have hadsome time to think about it and write it out. We put theonus on them and say that the more accurate informationwe collect, the quicker we can help their pet. We try to cre-ate an environment in which there isn’t any sense of judg-ment. I think I get decent information most of the time.

I also talk about partnering with clients. I ask what theyare able and willing to do in their household, what foodsthey are able and willing to purchase, and whether theyare open to a diet change. When pets are two or more bodycondition scores (BCSs) above healthy lean, I almostalways feel that their nutritional needs are best met withone of the prescription weight-loss products or somethingthat’s formulated specifically to achieve weight loss.

I also pay careful attention to the food I select to makesure that it’s meeting my patients’ dietary protein needs. Thisis not because dietary protein is any more important than


ROUNDTABLE

Moderate obesity

Patient: Fiver, a 5-year-old spayed beagle

BCS: 7.5/9

Weight: 17 kg (37.4 lb)

Ideal weight: Around 12 kg (26.4 lb)

Recent history: Weight gain, lethargy, mild heat and exercise intolerance

Diet: Free-choice, maintenance dry food, about 24% protein/14% fat

Presentation: The patient is panting, and her abdomen is mildly distended. Her heart rate is 120 bpm. Alllaboratory and clinical data, including a thyroid panel, are within normal limits.

case 1

the other nutrients, but in my experience in taking diet his-tories, it’s frequently one of the limiting ones. It’s the onethat’s most likely to be at the low level when we’re feedingand restricting calories. So my general rule is to try to pro-vide a minimum of 1 g of dietary protein per pound of bodyweight per day for dogs and 2 g per pound per day for cats.

In addition to checking patients’ BCSs, I also try to accountfor current muscle conditioning, particularly in patients thatneed weight-loss assistance. This system isn’t quite as refinedas the 9-point BCS scale that we use in our hospital, but itgives me an idea of whether patients have normal lean mus-cle mass or if they have lost lean muscle mass. It can be

deceiving if you have a morbidly obese animal with a 9 of 9BCS; it can be easy to miss loss of lean mass when the patienthas so much excess body fat. So I’m especially attentive to theobese patient’s muscle conditioning.

Once I’ve selected and transitioned the pet to theweight-loss diet, I will schedule checks every 2 weeks untilI am achieving a weight-loss trend. We then switch tomonthly checks, with the target weight loss somewhere inthe range of 1% to 2% of body weight per week.

Dr. Rebecca Remillard: I agree with and follow most ofyour approach. However, I find it interesting to know whythe clients came in. Did they come in because someonetold them the dog was already fat and they’re concerned,or is the dog limping and having some physiologic, clini-cal changes at home that have prompted them to come? Ithink it’s very important to figure out where the caloriesare coming from, and it’s well worth spending the time tofind out what the owners want to get out of the visit and

how vested they are in helping their pet. I could spend 15minutes of a 30-minute appointment trying to figure outwhat has or will motivate the owner to follow through (forinstance, improved skin, better mobility, improved car-diovascular parameters). The success rate is fairly low tobegin with, and if you don’t know where the clients arecoming from, your chances of success diminish greatly.

It’s also important to know whether we’re talking abouta multiple-pet household. Are there cats, dogs, or otherpets in the house? In addition, is the person doing thefeeding the same person who brought the pet in? I can’ttell you how frustrating it is to have a client be all gung-hoonly to find out that she’s not the person responsible forfeeding the dog or the one who thinks the dog isn’t fat. Sonow you’re talking to the wrong person. So I think it’s veryimportant to spend a significant amount of time trying tofigure out where the calories are coming from, who is over-feeding, why, when, where—as much as possible.

Then you’re basically entering into a contract with theclients. What are they willing to give up, what can they giveup, what’s important, and what isn’t? Weight loss is not amystery, and the calculations are simple. It just requiresimplementing behavioral changes in the home. When Iwent to vet school, nobody told me how to change humanbehavior. I never took a course in pet owner psychology.So I don’t really know how to manage the owners. We needhelp in that regard because that’s where I think most of theproblem lies—in the home and the human–animal rela-tionship. Obesity is an owner-induced disease.

My initial estimate for weight loss would be roughlyaround 1% per week. I might even tweak it up to 2% perweek initially. I agree with Dr. Churchill that a designatedweight-loss diet is mandatory if you are to meet the nutri-tional requirements during significant weight loss.

Changing the diet can take 10 days, and then addingcompliance on top of that can take quite a bit of time. Idon’t find many clients who are willing to come back in 2weeks, but they are willing to come back in 30 days. Thefall-off rate is steep. Probably less than 15% of them willcome back on their own accord. They need a reminderphone call for the follow-up visits. I think this is exactlywhere you need specialized nutrition technicians: makingthose follow-up phone calls to ensure that the pets wentthrough the transition period from the original diet totheir new diet, addressing any issues that came up, thenmaking the strategic phone calls to clients for schedulingthe recheck visits. If you’re lucky enough to have a nutri-tion technician, he or she is key to these follow-ups.

Overall, I think that a recheck schedule of roughly


We email our diet history form toowners in advance so they have

had some time to think about itand write it out.

—Julie Churchill, DVM, PhD


ROUNDTABLE

every 30 days works. Our prescription module is elec-tronic, so I can easily check when the person purchasedthe food and how much they purchased and make a quickcalculation to determine when they are due back. Thesemeasures are helpful in keeping owners on track withtheir follow-up schedule.

I usually don’t implement a form of exercise at the sametime. If the pets are exercising at all, I encourage that tocontinue. I also try to get an assessment of what the petsare doing and assign some caloric value to it if possible.However, if they were brought in because they’re havingclinical signs related to mobility or breathing, I’m notgoing to talk about exercise on the first visit.

Dr. Arleigh J. Reynolds: Assuming that you are successful,what do you do once the dogs have lost the weight? Whatis your strategy to prevent them from bouncing back to theirprevious weight?

Dr. Remillard: I have usually found that in a successfulweight-loss program in which you have good communi-cation with the clients and you have established a workingdoctor–client–patient relationship, owners are willing totry any diet you recommend—and that includes stayingon the same diet. Very few clients want to change toanother diet after a long but successful weight-loss pro-gram. It’s probably because they understand how much tofeed and how the product works. They know that the prod-uct is not going to cause any harm because the pet hasbeen on it for a couple months, and they know where andhow to get the product and how to feed it. Familiaritybreeds understanding and compliance.

So I find most of the dogs end up staying on the weight-loss diet. It’s my job to recalculate how many calories theyshould be consuming per day to maintain their optimalbody weight. I’ll often use the Purina weight-loss softwareto do this. At the end of about six visits, if you’ve pluggedthe recheck data into that program, it will give you an esti-mate of the maintenance energy requirement for that petat whatever weight you decide to maintain.

Dr. Churchill: As the dogs begin losing weight, I start plant-ing the seeds early that clients are in it for the long haul. Idon’t want my clients thinking that when they reach thattarget or estimated weight, the work is done. That’s actuallywhen the real work begins. So I start preparing clients early.

I have also found out that very often the amount of calo-ries it takes for dogs to lose weight at that healthy target rateis quite close to what they need to maintain their healthy

lean weight. Just giving owners that information helps themreadjust their mental model of how much food the animalreally needs to remain at its healthy weight. Probably 95%of clients opt to keep their dogs on that prescription diet.When they hear we are switching to a maintenance phase,they find it unpalatable to have to feed their dogs less.

Dr. Francis Kallfelz (Cornell University): If the animal is sig-nificantly overweight, it’s going to take about half a year ormore to get it down to its normal weight. I think it’s impor-tant to impress upon clients the fact that this is going to bea very slow process and that they have to stick with it.

There seems to be a difference of opinion as to whetheryou should start the weight-loss calculation from the ani-mal’s current weight or base it on what the clinician feelsis the animal’s ideal body weight.

Dr. Remillard: I don’t feel comfortable estimating the ani-mal’s optimal body weight. It’s happened too many timeswhere you get down to a target weight that you agreed to inthe first visit, but when I check the BCS at that target weight,I prefer not to stop. I would also prefer not to give clientsa target body weight because they lock onto that number.If you get to that weight and you still haven’t resolved theclinical problem or the pet is still carrying too much bodyfat, you have to try to move the owners off that mark. I’drather they lock onto a number of pounds lost per week oran optimal BCS rather than a final body weight.


Dr. Linda Fleeman: There’s not a lot of hard evidence abouthow to manage diabetes mellitus in dogs and whether onemethod is better than another. Obviously, this dog requiresboth antibiotics to treat the uniary tract infection (UTI) andinsulin therapy. Every single dog with diabetes requiresinsulin, and it’s crucial that the insulin therapy is matchedto meals. When a normal animal eats, the body secretesinsulin to deal with that meal in the postprandial period;when we’re treating a diabetic dog, we’re trying to replicatethat scenario by feeding and administering exogenous

insulin in a specific regimen that’s going to mimic the mostnormal situation. The aim of this method is to control theclinical signs of the disease. We also have to make sure thatwe don’t induce the most serious consequence of treatingdiabetic dogs, which is insulin overdose and hypoglycemia.So the way we manage these patients has to reduce that riskas much as possible.

There’s an enormous amount of day-to-day and injec-tion variability in the way insulin works in dogs, so we donot necessarily match up the time of maximum insulin

Diabetes mellitus and a mild lower urinary tract infection

Patient: Clark, an 8-year-old spayed Labrador retriever

BCS: 3/9

Weight: 23 kg (50.6 lb)

Recent history: Weight loss of about 4 kg (8.8 lb) over the past 3 months, lethargy during the past 2months, and development of polyuria and polydipsia over the past 5 weeks

Diet: Free-choice, maintenance dry food, 24% protein/14% fat

Presentation: The patient is quiet, alert, and responsive. She has muscle wasting, particularly over her temporal and masseter muscles and the lumbar area of her back. She has a dry coat with flaky skin and mildcataracts in both eyes.

Laboratory findings: Her CBC is within normal limits. Her glucose is elevated at 267 mg/dL (14.8mmol/L). Her cholesterol and triglycerides are also elevated. She has +3 glucose, +2 ketones, +2 protein, and+2 white blood cells on dipstick. Her urine specific gravity is 1.025, and a number of cocci and 10 whiteblood cells/high-power field are observed in her sediment. She was negative for metabolic acidosis.

case 2

SUMMARY OF CASE 1Dr. Reynolds: It seems like when we’re dealing with obe-sity, perhaps the single most important message is to dealwith the client and the patient as individuals because ofall the variables that lead to obesity in the first place. Per-haps the single most important component that we takeinto consideration as we formulate our treatment plan isthe diet history of that patient.

We are basically in agreement that we need to use atherapeutic-type diet because we want to ensure that anadequate amount of protein is taken in to try to mini-mize the loss of lean body mass while the animal is

losing weight. But there isn’t a generalized agreementabout how much protein that should be. So maybe weneed more information in that area.

We determined that the amount of exercise should bebased on the requirements of the individual and that thedietary energy requirements should be adjusted to con-sider expenditures from exercise. There is general agree-ment about a rate of 1% to 2% weight loss per week andthe fact that extreme commitment from the owner isrequired. A lack of client commitment is the reason forthe relatively low success rate in these cases.

action with the postprandial period accurately. But that’swhat we’re hoping to do. In my experience, the postprandialperiod with most commercial dog foods lasts for about 5or 6 hours. So I usually choose an insulin that is going tohave a period of maximum insulin action maybe 1 to 6hours after the injection. This allows for a very simple regi-men in which you feed the dog at the same time as givingthe insulin injection.

The biggest thing for this particular owner is that he isused to free-choice feeding. So apart from having to getused to administering insulin injections, this owner alsoneeds to start meal feeding the dog.

We’ve talked about the importance of collecting a diethistory. The thing about treating diabetes is that the ownerhas to do all of the treatment. To get maximum compli-ance, you need to make a contract with him or her aboutwhat each meal is going to comprise. What is the ownergoing to be comfortable with feeding this dog as a mealtwice a day? Is the dog receiving treats? And if the dog doesnormally get treats, then perhaps we can organize a waythat a certain number of treats can be fed at the same timeas the meal or soon afterward or as a reward after theinsulin injection so the owner still has the pleasure oftreating the dog.

Because it’s very important that the meals are matchedto the insulin injections, it’s crucial that those meals arepalatable. That’s part of the deal that I’m making with theowner. What do you think you’re going to be able to feed thatthe dog will eat reliably every time?

If the owner is interested in choosing a diet that is goingto offer some benefits for management of the diabetes, Iwould first focus on the total carbohydrate content. It’svery important that the total carbohydrate content is con-sistent with each meal. But there’s some value to keepingthe total carbohydrate content of each meal fairly lowbecause it’s the carbohydrate content that’s going to beresponsible for most of the postprandial hyperglycemia.So if the owner is prepared to change diets, I would chooseone that provided perhaps less than 30% of calories fromcarbohydrate.

It is important to monitor these animals closely. Iwould obviously monitor glycemia in this dog, as well ascompliance with both the insulin administration and thefeeding to see whether or not the owner is able to con-tinue it. I would also monitor BCS and body weightbecause we want to get this dog to stop losing weight andstart gaining body condition as soon as possible. In addi-tion, I think it is important to monitor fasting triglyceridelevels in diabetic dogs.

Dr. Remillard: My entry into most of these types of casesis usually at the recheck visit when things haven’t gonewell. But even if I was asked at this point in the case, dur-ing the first visit, I’m not sure that I would change the dietjust yet, mostly because we have an underweight dog,some muscle wasting, and a low BCS. In this case, I’m notcertain I would jump right to a moderate-fiber diet, whichwould be the classic answer for a diabetic dog. The dog’sproblems at the first visit were not only diabetes and theUTI, but she was also underweight. I might wait and seehow well the dog does on the current diet with improvedglucose control.

If things are going well in the first couple weeks of reg-ulation, hopefully the dog will regain that weight andmuscle and the triglycerides will come back down to nor-mal. So, from a nutritional standpoint, I wouldn’t be soquick to make another change in the lifestyle of the dogand the owner initially. This is already going to be a trau-matic shift for them. If we can do it on the current dietand correct some or most of these problems, that’s finewith me. I wouldn’t automatically change to a high-fiberdiet unless we needed to do so for glucose control, so I’dwait until the recheck visit.

Dr. Fleeman: If you’ve got a diabetic dog that is eating well,even with ketones in the urine, I would feel more relaxedabout starting long-term management than for any animal


ROUNDTABLE

For this [lean] diabetic dog, Iwouldn’t automatically change toa high-fiber diet unless we neededto do so for glucose control.

—Rebecca Remillard, PhD, DVM, DACVN

that was not eating because anorexia is not a sign of dia-betes. There has to be some sort of concurrent disease goingon in an inappetant or anorectic, thin, diabetic dog. It’s dif-ficult to know how many dogs have pancreatitis, but I thinkabout 30% of diabetes in dogs is caused by chronic pan-creatitis, where there’s been destruction of both exocrineand endocrine function by a chronic disease process. Butwhen you’re talking about why dogs present with diabeticketoacidosis, I would suggest that maybe 80% of them havepancreatitis. Thus, I would always look for pancreatitis inan inappetant diabetic dog.

Pancreatitis is a whole other discussion. But one of myreasons for monitoring triglycerides is that if there is anyrationale at all for dietary fat restriction as a way of prevent-ing pancreatitis or treating chronic pancreatitis in dogs, it’sthat we’re trying to limit hypertriglyceridemia. The theory isthat hypertriglyceridemia is an inciting cause for pancreati-tis, although there’s not a lot of direct evidence to supportthis. This dog’s triglycerides are elevated, which is a typical

finding in dogs with poorly controlled diabetes. Hyper-triglyceridemia does often resolve with insulin therapy, butnot always, and I like to find out if it doesn’t. If a diabeticdog had (1) fasting triglycerides above 500 mg/dL (5.5mmol/L), (2) persistent hypertriglyceridemia over 400mg/dL (4.4 mmol/L) despite good glycemic control, or (3)a history or clinical evidence of concurrent pancreatitis, Iwould definitely institute dietary fat restriction.

Dr. Reynolds: Suppose this animal becomes normo-glycemic but has persistent hyperlipemia. What level offat do you look for in a diet, and are all fats equal? Are

there certain types of fats that you would look for and cer-tain ones you’d like to avoid?

Dr. Fleeman: I’d start by restricting dietary fat to less than30% of calories and then monitoring triglycerides.

Dr. Hill: I have found that 30% of calories as fat is too high.I like to give less than 20% of the calories as fat. That’s lessthan about 25 g per 1,000 calories. I think that starting withthe higher level and seeing how the patient responds is per-fectly reasonable. I don’t think we have any objective datato say what works and what doesn’t. As far as the fat type,fish oil will help reduce the cholesterol.

I’m worried about food aversion in patients when they’resick. It’s really important to control their nausea. I trieddoing an experiment with some pancreatic dogs involvinga new diet, and it was quite effective, but they didn’t wantto ever eat the diet again after they got better. So we stoppedthe study because we asked the funding agency, “Do youreally want to have these dogs never want to eat your dietagain?” So it’s very important to try to control nausea. OftenI will continue with the same diet initially until the animalis feeling better. I’ve seen a lot of patients come into myclinic that have been changed onto a new diet that they’renot eating. If you’ve got a dog that’s already underweightand it’s not eating, you’re going to make its condition worse.

Dr. Lisa Weeth (Red Bank Veterinary Hospital, Tinton Falls,NJ): What would you recommend to owners of thisunderweight dog if their initial gut reaction is to feed it asoften and as much as possible, rather than meal feedingcoinciding with insulin action? How do you handle treatsand supplements and midday meals that the owners maywant to give to try to encourage weight gain?

Dr. Fleeman: I would explain to the owners that the foodthey feed the dog will not be utilized by the dog unlessthere’s also insulin on board. So I would emphasize howimportant it is to match feeding with insulin action. How-ever, there’s often such a big improvement as soon as youinitiate insulin therapy that I wouldn’t worry about howbig this dog’s meals are until the dog was beginning to getoverweight. I wouldn’t tell them to calorie restrict. I let theowners feed big meals if that’s what they want to do for athin dog. It’s very uncomfortable for all of us to feed asmall meal to a hungry dog. However, I would discuss allof the treats that they want to feed the dog. There may besome high-fat, high-carbohydrate, or high-sugar treats thatI would avoid and replace with something else.


Because it’s very important thatthe meals are matched to the

insulin injections, it’s crucial thatthose meals are palatable.

—Linda Fleeman, BVSc, PhD, MACVSc

Dr. Weeth: Would you make any specific recommendationsfor owners who are feeding home-cooked diets? Do youtry to get the owners to convert to a commercial diet?

Dr. Fleeman: What I emphasize is that the total dietary car-bohydrate content needs to be consistent in each mealthat’s fed to a diabetic dog. It’s much easier to do that withcommercial food than it is with a home-cooked diet. How-ever, if they’re prepared to be quite rigorous with the home-cooked diet, then they can do that.

What is also essential, not just for a diabetic dog butfor all dogs, is that the diet should be complete and bal-anced. But I think it’s particularly important for an animalthat has metabolic problems, such as diabetes. I reallypush for the diet to be complete and balanced if it’s goingto be home cooked, so I would prefer it to be formulatedby a nutritionist.

Dr. Reynolds: Suppose this patient was an obese cat thathas been newly diagnosed with diabetes? How would yourtreatment plan differ, and how would it be the same?

Dr. Fleeman: When diabetes is newly diagnosed, weightloss is typically part of the animal’s clinical presentation.Although it’s desirable for an obese animal to lose weight,I’m not comfortable with it losing weight because of apathologic process. So my approach is to try to get somecontrol of the diabetes and actually demonstrate that thiscat will stop losing weight before even thinking aboutimplementing a weight management program.

It’s difficult to have a weight management program indogs or cats without restricting their food intake to meals.It’s advisable to change to a feeding regimen that follows alot of recommendations that we have discussed. Many ofthese animals do well on a carbohydrate-restricted diet, butin my experience, not all of them do. Some animals do bet-ter on a calorie-restricted diet, which is not always a low-car-bohydrate diet. You need to individualize it to the patient.

Dr. Remillard: I don’t always know what to do with anobese cat, much less one that’s diabetic. I know that highprotein is all the rage, but all the therapeutic diets fre-quently considered for diabetic cats have very similar lev-els of protein on a caloric basis (Table 1). We should reallybe talking about readily available carbohydrate (nitrogen-free extract, or NFE) versus fiber levels (and type) in ourdietary options for managing diabetic cats.

I was somewhat comforted by the chapter Dr. ClaudiaKirka published in which she reviewed the data in diabetic

cats fed a low-carbohydrate (NFE) versus high-fiber diet.She said basically that both diets can work. Therefore, Idecide on a case-by-case basis which dietary approach totake. Dr. Kirk concluded that glycemic control was aboutequal on both diet types and that the reversion to noinsulin required occurred on both diet types (although ata three times greater rate using the low-NFE diet). So inpractice for under- or normal-weight diabetic cats, I will

use a low-NFE, calorie-dense diet (Purina Veterinary Diets®

DM Dietetic Management® or Hill’s Prescription Diet®

m/d® Feline), canned or dry, according to the cat’s andowner’s preferences. In obese diabetic cats, I actually pre-fer to use Purina Veterinary Diets® OM Overweight Man-agement®, canned or dry. On a caloric basis, the OM dietcontains more protein, less fat, and more fiber than thetherapeutic diets commonly considered for diabetic catswith a comparable NFE content.

Dr. Deborah Greco (Nestlé Purina PetCare, St. Louis, MO):I’m a little surprised that the conversion rate of cats goinginto remission on high-fiber versus low-carbohydrate dietswas the same. In our study,b 68% went off insulin on alow-carbohydrate diet and 41% went off insulin on a high-fiber diet.

aKirk CA. Feline diabetes mellitus: low carbohydrates versus high fiber? Vet ClinNorth Am Small Anim Pract 2006;36(6):1297-1306.bBennett N, Greco DS, Peterson ME, et al. Comparison of a low carbohydrate–low fiber diet and a moderate carbohydrate–high fiber diet in the managementof feline diabetes mellitus. J Feline Med Surg 2006;8(2):73-84.


ROUNDTABLE

It’s important to control nauseain a sick patient. Often I will continue with the same diet until the animal is feeling better.

—Richard Hill, MA, VetMB, PhD, MRCVS

Dr. Fleeman: I think it’s interesting to read the differentreports about remission rates with different treatmentapproaches to diabetic cats. As far as I’m aware, there arereports of remission rates from 100% in cats that are newlydiagnosed with diabetes and are treated with a long-actinginsulin preparation and a low-carbohydrate diet to awhole range of other remission rates, depending on thepublication. It’s difficult to find two reports where the onlydifference between them is the diet.

At the same time that feeding a low-carbohydrate dietbecame popular, new insulin therapies, including long-act-ing insulins such as glargine, and new ways of monitoringthe animals, including home monitoring of capillary blood

glucose, were introduced. As a result, there are probably anumber of factors contributing to the improved success rateof achieving diabetic remission in cats.

I like the idea of feeding a low-carbohydrate diet to dia-betic cats in combination with long-acting insulin prepara-tions. It is my first choice, but I recognize that people havesuccess with other diets and with other insulin therapies.

Dr. Iveta Becvarova (Virginia-Maryland Regional Collegeof Veterinary Medicine): If a cat has renal proteinuria andhyperlipidemia, you may want to restrict both protein andfat in the diet and boost the carbohydrates, essentially cre-ating a highly digestible carbohydrate diet, which may


Fooda NFE(g/100 kcal ME)

Protein(g/100 kcal ME)

Crude Fiber(g/100 kcal ME)

Fat(g/100 kcal ME)

ME (kcal/g drymatter)

Purina Veterinary Diets® DM DieteticManagement®, canned

1.7 11.9 0.8 5.0 4.6

Purina Veterinary Diets® DM DieteticManagement®, dry

3.4 12.9 0.3 4.0 4.1

Hill’s Prescription Diet® m/d®, dry 3.5 12.2 1.4 5.5 4.2

Hill’s Prescription Diet® m/d®, canned 3.9 13.1 1.5 4.8 4.0

Purina Veterinary Diets® OM OverweightManagement®, canned

5.9 11.4 2.6 3.7 3.7

Royal Canin Veterinary Diet™ Diabetic DS44™, dry

6.4 12.3 1.3 3.2 3.7

Purina Veterinary Diets® OM OverweightManagement®, dry

6.5 16.6 1.8 2.5 3.2

Hill’s Prescription Diet® w/d®, canned 7.6 11.5 3.1 4.8 3.4

Royal Canin Veterinary Diet™ Calorie Con-trol CC 29™ High Fiber, canned

7.9 8.1 1.9 5.2 3.7

Royal Canin Veterinary Diet™ Calorie Con-trol CC 29™ High Fiber, dry

10.6 10.3 4.3 3.1 3.0

Hill’s Prescription Diet® w/d®, dry 10.7 11.1 2.2 2.8 3.5

aFoods are sorted on the basis of NFE.NFE = nitrogen-free extract (a measure of digestible carbohydrate); ME = metabolizable energy.

Veterinary Therapeutic Foods Recommendedfor Management of Diabetic Cats


ROUNDTABLE

require an increased insulin dose. What would be yourapproach to that situation? Would you be more comfort-able increasing the insulin dose?

Dr. Fleeman: Absolutely. In theory, you can always managehyperglycemia by adjusting the insulin dose. If youincrease the dietary carbohydrate, the insulin dose willalso likely need to be increased. But as long as you knowthat and you increase the insulin dose and monitor it tomake sure it’s okay, I am completely comfortable with thatapproach. My recommendation is that the nutritionalrequirements of any concurrent disease should usuallytake precedence in diabetic animals and the insulin ther-apy should be adjusted accordingly.

Dr. Beth Hamper (University of Tennessee): When wouldyou address obesity in a newly diagnosed obese cat?

Dr. Fleeman: My initial priority would be to make surethat the cat was able to maintain weight because I think it’sa terrible metabolic situation for an obese cat to be losingweight before the diabetes is controlled. With a newlydiagnosed diabetic animal, the owner needs to make somany adjustments, and it’s going to be a little bit of timebefore you’re going to achieve glycemic control. So Iwould first focus on all of that.

Once the diabetic control has stabilized, I’d make adjust-ments to the animal’s diet and start with meal feeding. It isoften a straightforward process to introduce calorie restric-tion as you’re making nutritional recommendations for thepurposes of sorting out the diabetes. I give advice aboutcalories and then start a gradual weight-loss program. I dothink weight loss is important.

On the other hand, you also have to watch for weightgain in a lean diabetic animal with good glycemic controlthat is on a long-acting insulin preparation and a fairly high-calorie, low-carbohydrate diet. A lot of these animals gainweight, and you can start to have problems with insulinresistance if you don’t adjust for that. So when you’re man-aging diabetic dogs and cats, you should monitor body con-dition and body weight because you need to stop anychanges before this starts to affect your glycemic control.

Dr. Daniel Chan: In the past few years, there was a bit ofexcitement about the role of diet in controlling diabetes incats. Most of these studies focused on the newly diagnosed

diabetic cat. Now practitioners are changing diets evenbefore the patients arrive at referral institutions. So we’rechanging the population that specialists see. What we’reprobably going to see are those that fail to respond to dietchanges. We have to be cautious because recommenda-tions might be made on a different population that’s seenin general practice. Referral cases now come in where theanimal has had a diet change and has been on two or threedifferent insulin types. It’s a different population thanthose where you change the diet and they no longer needinsulin. We have to remain cognizant of that fact.

SUMMARY OF CASE 2Dr. Reynolds: So we can see that when we take on a dia-betic patient, there are several fairly serious considera-tions. Just as we discussed with obesity, probably thenumber one consideration is individualizing the ther-apy to the patient and ensuring that you have ownercompliance. In this case, it’s probably significantly moreserious and important than in a patient with obesity.

We talked about matching meals with the timing ofinsulin, making sure it is a meal the dog will eat, andmaking sure that the diet is consistent so the responsewill stay the same and can be adjusted to the needs ofthe individual. We also discussed setting goals in termsof what was important to control first for the good ofthe patient—controlling glycemia and trying to main-tain optimal or at least as good as possible BCS andbody weight—and then monitoring other things thatmay be important, such as fasting triglycerides and con-comitant health concerns. If there is fasting triglyc-eridemia, we do want to lower fat intake, but we don’tagree completely on how low we should go and whatthe type of fat would be. We also disagree on the opti-mal protein content and the caloric or fiber distributionof the diet.

It seems like people have had success with varyingregimens of diet and insulin, but the take-home mes-sage is that whatever you’re doing, be consistent. Soeven among some of the clinicians who have handledthese types of diabetes cases more than the rest of us,there isn’t necessarily agreement on the best approach.Various approaches work, but there are some holes in the evidence that we need to fill to better treat theseanimals.


Dr. Stanley Marks: The history of the dog together with theultrasonographic features and SPEC cPL result support adiagnosis of acute pancreatitis. The history and physicalexamination findings also support a diagnosis of acute gas-troenteritis or dietary indiscretion; however, the ultrasono-graphic features are classic for acute pancreatitis. It isimportant to assess this dog’s appetite because that is goingto affect our nutritional approach for this particular patient.In addition, it is valuable to obtain a comprehensive diethistory from the owner before embarking on a specificnutritional recommendation.

The relatively frequent bouts of vomiting in this dogare going to significantly alter how we would try to man-age this patient nutritionally. Historically, we probablywould have used total parenteral nutrition (TPN) becauseof our concerns with exacerbating the pancreatitis with oral or enteral feeding and increasing the risk of

aspiration pneumonia with continued oral feeding. Inaddition, we would have considered the potential forinducing a conditioned food aversion in this dog that feelsvery nauseated.

The historical and current benefits of TPN pertain to ourability to meet this dog’s caloric and protein requirements.Another benefit of TPN is that it’s fairly “clean,” whichmeans that this dog will have limited to no diarrhea in thehospital setting, and if we’ve got a good nursing staff, wecan monitor this patient and make sure our catheters andlines are all maintained appropriately and aseptically. It’sactually quite simple to maintain these patients for themost part, barring metabolic complications.

There has been abundant research in recent years that hasfocused on the role of the intestinal tract in human patientswith acute pancreatitis and rodent models with experimen-tally induced pancreatitis. It has been well documented that

Acute pancreatitis

Patient: Tootsie, a 12-year-old spayed beagle mix

BCS: 4/9

Weight: 6.5 kg (14.3 lb)

Recent history: Acute onset of vomiting following ingestion of pork ribs and potato salad;vomiting continued for 3 days, occurring about two to three times a day; episodes were unas-sociated with meals

Previous treatment: IV fluids and metoclopramide for vomiting

Presentation: The patient is quiet, alert, responsive, and moderately dehydrated (~7%). Her temperature is102.5°F, heart rate is 150 bpm, and respiratory rate is 42 breaths/min. She has moderate pain on cranialabdominal palpation, a slightly tense abdomen, and some diarrhea on the rectal examination. No murmurs orarrhythmias are auscultated.

Laboratory findings: Her chemistry panel shows an elevated ALT (108 IU/L), ALP (1,051 IU/L), and GGT(40 IU/L). She is mildly hyperbilirubinemic (1.5 mg/dL) and hypercholesterolemic (496 mg/dL) and has anincreased blood urea nitrogen (40 mg/dL) and mildly increased creatinine (2.3 mg/dL). Her pancreatic lipaseimmunoreactivity is 602 (high-normal being 200). Urinalysis shows concentrated urine specific gravity (1.041),+2 protein, +1 bilirubin, and a benign sediment. On abdominal radiographs, there is reduced serosal detail inthe cranial abdomen. Abdominal ultrasonography shows a hypoechoic pancreas and hyperechoic surroundingmesentery. A small amount of free abdominal fluid is present in the right anterior quadrant, and the descendingduodenum is thickened.

case 3

there is a significant increased rate of morbidity and septiccomplications in people in whom the intestinal tract is notused from a nutritional standpoint. So the dogma of keep-ing these patients NPO or fasting them to allow “pancreaticrest” is being heavily challenged. There’s no better time thantoday to address this dogma critically.

The evidence that we’re now seeing from our humancounterparts and from rodent models overwhelminglysupports the enteral route for the management of acutepancreatitis. This entails feeding directly into the jejunumwhen feasible if the patient is vomiting intractably. Ourchallenge as veterinarians is that we aren’t usually able toplace a feeding tube or catheter in the jejunum withoutfirst anesthetizing that patient. If we were to try and placea jejunal tube, we would have to put this dog under gen-eral anesthesia and either place the J-tube percutaneouslythrough a gastrostomy tube, which requires practice tofacilitate proper placement and is often associated withrefluxing of the J-tube back into the stomach, or place itsurgically, which does work very well.

If this dog needed to go to surgery, I would want toproactively place a J-tube in that situation. The problem isthat, at least in my experience, most dogs with pancreatitisdon’t need surgical intervention, so the question nowbecomes, “How do we manage this dog that is vomitingtwo or three times a day following stabilization?”

I think all of us would agree with the importance of usingantiemetics in these animals, and we fortunately have at ourdisposal superior drugs today than what we had 10 to 15years ago. I would want to try an effective antiemetic,whether that is a 5-HT3 receptor antagonist, such asondansetron or dolasetron, or an NK1 receptor antagonist,such as maropitant citrate. Even giving both drugs togetherbecause of their additive effects would be a very reasonableapproach for a dog like this. If I could control this dog’s vom-iting adequately and effectively, I would seriously considerthe benefit of feeding the dog a fat-restricted diet containingless than 20% fat calories orally, or consider the value of plac-ing an esophagostomy tube or a percutaneous endoscopicgastrostomy tube, if the dog is anorectic but not vomiting.

So, if I could control this dog’s intractable vomiting withappropriate antiemetic therapy, I would want to do every-thing I could to feed the animal orally or enterally. If thisparticular patient warranted an exploratory laparotomy todebride the pancreas or warranted exploration for a relatedreason, I’d ask our surgeons to place a J-tube proactivelybecause the tube could always be removed at a later time ifnot needed.

What kind of diet would I use? In these particular

patients, we’ve been providing digestible diets that are mod-erately to markedly fat-restricted. In a dog like this withacute pancreatitis, we typically strive to find a commercialdiet that contains less than 20% fat on a caloric basis. Nottoo many commercial diets meet that requirement. It worksextremely well for our canine patients. We use that type ofproduct readily because it’s quite palatable and digestible. IfI had a dog that was a repeat offender and I needed to fur-ther fat-restrict the diet, then I would formulate a home-cooked diet that contains less than 12% to 14% fat calories.

Dr. Chan: It’s very popular to say enteral is better than par-enteral. I think we all agree that enteral is superior, but inwhat population? It’s a very good point that 15 years ago,pancreatic rest was a hot topic, and now we actually havesome evidence that the pathogenesis of pancreatitis is anintracellular process that leads to the autodigestion of thepancreas and not really pancreatic stimulation.

Now, at least for me, the criteria for putting patients onparenteral nutrition are failure to respond to your therapyand the persistence of vomiting. So we do metoclopramideinfusions and try more potent antiemetics like maropitantand ondansetron; if the patient still fails to respond to ther-apy, persisting with enteral nutrition is a mistake becauseyou have already proven that the dog is intolerant of enteralnutrition. If you can get jejunal feeding and the dog toler-ates it, that’s great, but if you place an esophagostomy tubeand the dog continues to vomit, you’re playing Russian


ROUNDTABLE

It’s clear that our paradigm forpancreatitis has changed over the past 10 or 15 years and thatthe individuality of the patienthas to be considered first.

—Arleigh J. Reynolds, DVM, PhD, DACVN

roulette and you’re going to increase morbidity. If the pop-ulation is intolerant of enteral nutrition, then parenteral isstill your best option.

Sometimes we’ll try to get away with using diets withhigher fat content if we can get them through a J-tube with-out any untoward effects. Because I’m not going to feed amaintenance amount of food in the acute phase of treat-ment in this dog, I don’t worry too much about the fat con-tent. Only when the dog goes into a chronic phase ofmanagement would I say lowering the fat content in the dietis appropriate. But in a patient that I want to support via afeeding tube, the diets that I usually use are not low in fat.

Dr. Reynolds: Suppose your patient does not respond toenteral nutrition and you have to go to parenteral nutri-tion. Describe how you would estimate energy and proteinrequirements, and then divide the nonprotein calories.

Dr. Chan: I like to personalize the nutritional plan for eachpatient, so I look at anything in the blood work that will guideme about whether I need to change the protein, fat, or glucoserequirements. The dog that was presented seemed to haveprerenal azotemia as well. There was 5% to 7% dehydration,so with that level of azotemia, I’m not sure I would restrictprotein. We really don’t know the optimal protein levels thatare required to promote homeostasis in a sick animal.

As a starting point, I formulate the solution to containabout 4 to 5 g protein per 100 kcal. If there’s any question

that the animal might be in a catabolic state, I may bumpup the starting protein levels. I do account for the caloriesprovided by the protein in my formulation, and I typicallydivide the remaining nonprotein calories 50/50 betweenlipids and carbohydrates, depending on the case.

If my patient is hypertriglyceridemic, I lower the lipidcontent, but I watch the glucose as well because I am veryconcerned about the complication of hyperglycemia. Weperformed an observational study looking at ICU dogs, andthe rate of septic complications was fourfold if they werehyperglycemic and nondiabetic. There is growing evidenceto say hyperglycemia shouldn’t be ignored anymore.

Dr. Marks: I echo most of Dr. Chan’s sentiments, includingthe protein amount and energy distribution. We determinethe patient’s resting energy requirement (RER) based on cur-rent body weight and begin to feed that patient at 25% to30% of the RER. We monitor the patient closely for meta-bolic complications and will gradually increase the amountof TPN administered to reach RER over a 72-hour period.

We somewhat recently modified our TPN formulationfor our canine patients, predominantly because of the con-cerns with the hyperosmolarity of the older solution. I amnot aware of complications of thrombophlebitis with theinitial formulation; however, despite that, our formulationnow has a lot more lipid in it, at about 52% to 54% fatcalories. We tend to let the ICU folks tweak the electrolytesin the patients’ intravenous fluids, which is easier thanadjusting electrolytes in the TPN. Potassium phosphate isusually added to TPN formulations and piggybacked withpotassium chloride administered in intravenous fluids foranimals that are not hyperkalemic.

I agree about concerns with hyperglycemia, but if we lookat most of the patients that we’ve followed closely, especiallywith glucoses, a lot of them are transiently hyperglycemicand, in fact, over 24 hours or so become euglycemic withoutchanging the rate of TPN administration or administeringany insulin. So whether or not that initial 24-hour hyper-glycemic spike is deleterious in our patients is difficult for usto say based on our canine experience to date.

Dr. Becvarova: Once you decide to feed through a J-tube,the diet is going to be a liquid formulation. There is muchdiscussion about an elemental versus polymeric diet forhumans. Which do you think we should use?

Dr. Marks: This is a really great question, but I don’t havethe answer. If you look at a lot of studies done in peoplereceiving elemental formulations, there may well be some


I like to personalize the nutritionalplan for each patient, so I look at

the blood work to decide whether Ineed to change the protein, fat, or

glucose requirements.—Daniel Chan, DVM, DACVECC, DACVN, FHEA, MRCVS

argument or debate about the benefit of elemental for-mulations because we’re decreasing pancreatic stimula-tion. If you’re feeding distal to the ligament of Treitz inthese patients, then I cannot think of a potential benefit ofgiving an elemental over a polymeric formulation. My oneconcern with elemental formulations is that I might bemore likely to see hyperosmotic diarrhea compared withpolymeric formulations. But I don’t have any good evi-dence or data to show that giving one is superior to theother when feeding in the distal jejunum.

Dr. Chan: Virtually all published veterinary clinical caseseries on using jejunal feeding have used a polymeric diet,and the success rate is quite good. The complications andfears of using a polymeric are probably a little overstated.I’ve always used polymeric diets, and the response hasbeen positive in the majority of these patients. I did notsee worse diarrhea, bloating, or abdominal discomfort. SoI’m comfortable using a polymeric diet.

Dr. Hill: I would like to point out that we have to be care-ful about using human studies because it’s a different pan-creatitis. In humans, it’s alcoholic pancreatitis and bile ductobstruction, which we do not see much of. The averagetime those patients spend in the hospital is more than 3weeks. In fact, there is a distinction in human pancreatitisbetween acute severe and mild edematous pancreatitis. Thecurrent prevailing opinion is that mild pancreatitis willresolve before you need to worry about giving any nutri-ent recommendation.

I don’t know that we’re very good in veterinary medi-cine at distinguishing between acute severe and mild.There have been various attempts at scoring systems,which don’t look very good to me at the present. The bestestimate, even in a recent study, was around 50% accurate.You might as well toss a coin to decide whether you’regoing to give nutritional support.

I’m a bit worried that by pushing nutrition, we are com-mitting people to spend an awful lot of money on patients,some of which would resolve with relatively conservativetreatment that would not involve doing TPN or enteralnutrition. However, if we do not start nutritional support,we run the danger that we are delaying food administration.

So we have to weigh pushing intensive nutritional sup-port (TPN or enteral feeding) versus conservative treatment(antiemetics and oral feeding with liquids) in individualpatients. But it is possible that some of our patients in prac-tice will resolve with relatively conservative treatment thatdoes not involve intensive nutritional support.

Dr. Marks: I completely agree with Dr. Hill’s point. In fact,our debate about what kind of TPN formulations we alluse might be moot if you look at how we have used TPNto date. Several published studies looked at a good numberof dogs, up to 209 dogs in the largest one, and the medianduration of TPN administration was 3.5 days! If it’s takingus 2 or more days to get to the RER, you might as well putwater down that patient’s jugular vein. Our patient selec-tion in the past has been really skewed, and our interven-tion with TPN was associated with a 50% mortality rate in209 dogs studied at UC Davis.c This extraordinarily highmortality rate tells us that we don’t have a clue when to useTPN, at least historically. We’ve picked the wrong patientsat the wrong time and given TPN for 3.5 days.

So we can debate about whether we should be usingthis amount of fat or that amount of fat and this amountof protein, etc. But it’s not making any difference basedon how we have used TPN in this population of dogs. Fol-lowing this discussion, I hope that we’re going to be morecognizant of proactively and aggressively using TPN whenwarranted in appropriate patients.

In this vomiting dog, I’m not worried about what she isgoing to be eating over the next 24 to 48 hours. I’m trying tostabilize her metabolically, at least with intravenous fluid sup-port in the form of crystalloids and colloids, and I’ll considerthe merits of analgesics, antiemetics, and possibly antibioticadministration. And after 48 to 72 hours, I’ll reassess the dogand then decide if I need to intervene with nutritional supportand what route and type would be most appropriate.

Dr. Kathryn Michel (University of Pennsylvania): On ourspectrum of acute pancreatitis, Tootsie looks like a rela-tively stable patient. You’re going to get her in, you’regoing to treat your metabolic issues, you’re going to givesome good antiemetics, maybe some prokinetics, stop hervomiting, and get her stable. But many people are stillfocused on pancreatic rest, and they’re afraid that they’regoing to give some food to this dog and set her back. Sowhen would you start feeding, and what would you offer?

Dr. Marks: I would not offer this dog anything by mouthfor the first 48 hours. I’m using that window because shehas been vomiting intractably, and I’m assuming that I’mnot going to be able to stop it as soon as I start antiemet-ics. So I’m simply not feeding, more because of the vom-iting than my theoretical concern of what I might be doingto this dog’s pancreas.

cReuter JD, Marks SL, Rogers QR, Farver TB. Use of total parenteral nutrition indogs: 209 cases (1988–1995). J Vet Emerg Crit Care 1998;8(3):201-213.


ROUNDTABLE


Hyperthyroidism, moderate lymphoplasmacytic enteritis, hepatic lipidosis, chronic pancreatitis, and Helicobacter infection

Patient: Patches, a 10-year-old castrated domestic shorthaired cat

BCS: 1/9

Weight: 2.4 kg (5.3 lb)

History: Chronic intermittent vomiting for 3 months; significant weight loss of 2 kg (4.4 lb);lethargy, and anorexia; other than being anorectic for 6 weeks at one time, he has no previous medical problems

Presentation: The patient is current on all his vaccines. On physical examination, he has mild elevations in body temperature, pulse, and respiratory rate. He has icteric mucous membranes. A cervical nodule is palpated, but there is nothing remarkable on abdominal palpation.

Laboratory findings: Based on his database, he has a mild, nonregenerative anemia. He has a mildly elevated white blood cell count and slightly low platelets. On his chemistry panel, he has an elevated ALP, ALT,GGT, BUN, and creatinine; his bilirubin and serum T4 are significantly elevated. Urinalysis shows that he isconcentrating his urine at 1.048 and has a +2 bilirubin. His fPLI test is 64 (normal being 0 to 6). Abdominalultrasonography shows hyperechoic areas in the cranial abdomen. An exploratory laparotomy is performed,and biopsy samples are collected from the liver, gastrointestinal tract, pancreas, and mesenteric lymph nodes.

case 4

If after 48 hours, the dog ceases vomiting and is notlooking nauseous, I will offer either a shallow bowl ofwater or some crushed ice cubes. If after another 12 to 24hours, the dog has not vomited and is still on antiemetictherapy, I will offer a low-fat cottage cheese and rice com-bination in small quantities several times a day, depend-ing on whether she is a repeat offender.

Depending on this dog’s response and assuming that thereis still no vomiting and she is improving clinically, I couldtransition her onto one of several commercial formulas. I’mgoing to be more fastidious and more proactive in restrictingdietary fat to less than 20% if this dog is a repeat offender. Ifthat is the case, I will wean her from a cottage cheese and ricecombination onto a commercial fat-restricted diet contain-ing less than 20% fat calories and consider a complete andbalanced home-cooked diet if she is unable to tolerate thecommercial diet.

Dr. Chan: I am not concerned about pancreatic stimula-tion. I haven’t been convinced that if you feed an animaltoo early and it vomits, you go back to square one. So I’m

willing to offer food sooner. I don’t necessarily wait 48hours because if I am able to control the vomiting andabdominal pain, I will offer water and then food, maybeeven in the same day.

SUMMARY OF CASE 3Dr. Reynolds: It’s pretty clear that our paradigm for pan-creatitis has changed over the past 10 or 15 years and,like the other cases we discussed, the individuality ofthe patient has to be considered first and foremost. Theprimary concerns are stabilizing patients and gettingthe serious problems under control before nutritionalmanagement is started.

When it comes to nutritional management, I think theconsensus is that if we can feed the gut, that’s what we’d liketo do. However, deciding on whether or not that is appro-priate is not always easy. We’re also not in complete agree-ment on the route and type of diet used. This is an excitingarea and one that we all see on a fairly regular basis. I lookforward to continuing this discussion in years to come.

ROUNDTABLE

Dr. Marks: This patient’s problems are that he’s icteric,vomiting, emaciated, anorectic, and lethargic; he’s lostweight; and he has a cervical nodule. Like many of thesecats that present with a working diagnosis of chronic pan-creatitis, he has the triad of suspected inflammatory boweldisease, hepatic lipidosis, and pancreatitis, which makeshim a challenging patient to treat.

Dr. Hill: The most important, life-threatening problem for this animal is the hepatic lipidosis, which is almost certainly very severe at this stage, and the blood results corroborate that. The other threatening concern is overfeeding. I think there is a real chance that the animalcould suffer from refeeding syndrome. So we have to becareful of how fast we introduce new food.

Because of the hepatic lipidosis; the moderate lym-phocytic plasmacytic enteritis, which may have preventedvitamin absorption; and possible vitamins B12, E, and Kdeficiencies, I would give this cat a vitamin formula in thefluids.

I would then decide how many calories I want to admin-ister. Unfortunately, we have no data on how many caloriesan animal this thin would be consuming. So it’s an edu-cated guess. Let’s assume an average cat running aroundconsumes about 150 to 250 calories a day. If you scale thatback under the assumption that this is a very thin cat and it’snot doing much, I think 100 calories is a reasonable guessfor what this animal’s energy requirement might be.

I would not want to give all of those calories on the firstday. I would start off probably giving no more than a thirdto a half of that amount, and I would give it slowly overthe first day. I would carefully monitor the patient for signsof refeeding syndrome, looking for signs of hypermagne-semia, hyperphosphatemia, hyperkalemia, and hyperam-monemia. Many of these cats with lipidosis havedeveloped hyperammonemia, which makes me wonderwhether they can handle the high amounts of protein.Nevertheless, I would feed a high-protein diet because theonly data we have on this is from Dr. Vincent Biourge andcolleagues,d who suggested that cats with lipidosis did better on a high-protein diet. So I would choose a high-protein diet. Because I’m giving very few calories, I thinkthat’s okay. I would monitor the patient, and if it doesdevelop hyperammonemia, I would treat that and maybescale back on the amount of protein I give.

Because the cat has an E-tube, I would make sure the

diet was also high in fat. I would be concerned about theamount of fat if the cat had acute severe pancreatitis. If mysurgeons had good evidence that it had pancreatitis, Iwould want them to put in a G-tube. We would put a J-tube through the G-tube into the jejunum so we could givethe patient food directly into the jejunum, eventually with-drawing the tube if we can.

For a convenient mixture that will go down the tube,we take one sachet of Vivonex® Plus (Nestlé HealthCareNutrition, Minneapo-lis), add a little water tomake it into a slurry,and mix it with one canof cat food. You end upwith a mixture thatcontains only 25% ofcalories as fat. If youwant 33% of calories asfat, then you use half asachet of Vivonex Pluswith one can.

Dr. Chan: As a criticalist, I always ask, What’s going to kill mypatient first? In this case, it’s the hepatic lipidosis, so that’sgoing to be my focus. We know that feeding is going to beone of the most important aspects of management. Interms of how aggressively I pursue nutritional interven-tions, I actually tend to be very cautious; the sicker they are,the more conservative I am.

It’s an interesting question regarding estimates of caloricrequirements in the setting of critical illness. We have somedata in this regard using indirect calorimetry, and the con-clusion is that in sick animals, the energy requirement isnot higher than in normal animals. It’s actually lower.

There’s a current concept in critical care medicine inwhich they are talking about hypocaloric feeding andthey’re actually having rather good success with this strat-egy. They are recommending feeding something like two-thirds of calculated energy requirements, and they areseeing a beneficial effect in these patients in terms of

Prevention of recurrent bouts ofpancreatitis in dogs is distinctlydifferent from that in cats.

—Stanley Marks, BVSc, PhD, DACVIM (Internal Medicine, Oncology), DACVN

dBiourge VC, Massat B, Groff JM, Morris JG, Rogers QR. Effects of protein, lipid,or carbohydrate supplementation on hepatic lipid accumulation during rapidweight loss in obese cats. Am J Vet Res 1994;55(10):1406–1415.


reduced complications and even improved hospital out-come. Studies have shown that patients can maintain apositive nitrogen balance and have fewer complicationswhen treated using a hypocaloric nutritional approachcompared with standard regimens.

Dr. Reynolds: For both this case and the previous one,let’s say you have these animals under control andyou’re going to send them home. What can you recom-mend to the owner to try to prevent pancreatitis from com-ing back? Is there anything you can do?

Dr. Chan: It depends on what you believe the underlyingpathology is. We know a little more about chronic caninepancreatitis than chronic feline pancreatitis. Identificationof risk factors has probably been done more in dogs. Socontrolling conditions such as Cushing’s disease, diabetes,and dyslipidemia in dogs might decrease the risk of recur-rence. I don’t think the efficacy of this approach has really

been properly evaluated in dogs, however. In cats, weknow very little of what underlies their propensity to havechronic bouts of pancreatitis. I think very little has beenpublished on this topic, so I don’t know if there are anytherapies that can prevent recurrence of pancreatitis in cats.

Dr. Marks: Prevention of recurrent bouts of pancreatitis indogs is distinctly different from that in cats, at least fromwhat we believe and recognize today. Most of us wouldlikely agree that the single most important nutritional rec-ommendation to prevent a canine repeat offender frompresenting to your practice with pancreatitis is probably

restricting dietary fat and ensuring that the patient doesnot develop hypertriglyceridemia. That’s my philosophycurrently. We need to look at and research what it is aboutcertain canine breeds that increases the inherent risk ofpancreatitis versus other breeds that are so protected. Forinstance, if an overweight 7-year-old spayed Cocker spaniellooks at a pork chop, it’s going to get pancreatitis. On theother hand, if you give a sled dog racing the Iditarod a60% or 70% fat diet, I’m going to bet none of those ani-mals ever develop pancreatitis.

Dr. Fleeman: One thing that might be different betweenthese animals—the working, active dogs versus the petdogs and those that aren’t active—is dyslipidemia. If we’regoing to recommend dietary fat restriction, maybe weshould be monitoring their triglyceride level to see if it’scoming down?

Dr. Marks: I would argue that measuring triglycerides inour patients is unnecessary if the serum is not lipemic. Ifthe serum is clear or not lipemic, I can guesstimate thatthe serum triglyceride concentration is less than 200mg/dL. So we’re going to have to debate whether having aserum triglyceride concentration of 155 mg/dL is deleteri-ous in a patient that’s at risk for pancreatitis. I don’t knowthe answer to the question.

In academia, we have a biased population of animalsthat tend to be sicker and have more complex and severedisease. At least in those animals, I’ve observed an anec-dotal response to severe dietary fat restriction (12% to 16%fat calories) over diets that are moderate in fat content.

Dr. Chan: If a dog has dyslipidemia, would you behappy using fish oil, which is basically fat, to treat thatpatient, knowing that you would increase the fat con-tent of the diet?

Dr. Remillard: Yes, because it is anti-inflammatory. I’mprobably going way out on a limb here, but I am think-ing about the inflammatory mediators of visceral adi-pose tissue. If you look in the abdomen, the kidney andliver have protective capsules, but the pancreas is like apiece of fresh meat bathed in abdominal adipocytes. I think paracrine inflammatory mediators from ab -dominal fat cells may be causing a local effect on thepancreas.

So how do we get these patients that have a problembecause they ate ribs and potato salad? There is some other,preliminary work where they fed normal-weight 20- to 30-


We know that feeding is going tobe one of the most importantaspects of management in apatient with hepatic lipidosis.

—Daniel Chan, DVM, DACVECC, DACVN, FHEA, MRCVS

Although we have learned a lot in the past 10 years,there’s still a lot that we don’t know, and there areholes in our evidence-based nutrition. What is thebiggest hole in clinical nutrition?

Dr. Chan: We’re always looking at the human side tosee what they’re researching. We see that they’re usingomega-3 fatty acid infusions, glutamine, and arginine.I’m interested in whether these therapies will make a difference in our patients.

Dr. Remillard: On the top of my wish list is to movetoward including genomics in our feeding studies. In lightof what has come out of this forum, all the studies thatwe’ve done thus far need to be redefined by genetics,and that’s scary. Most of the remaining questions couldprobably begin to be answered if we incorporated agenetic database or nutrigenomics into our study designs.

Dr. Reynolds: We need to stop looking at entire populationsand gear our investigations more toward individuals—or at least individuals with common backgrounds—andreally define our populations. So I think it’s not so scary.It’s kind of exciting to think that we may be able to do abetter job by focusing our efforts on that task.

our experts weigh in

ROUNDTABLE

year-old people 900 calories of a fast food meal, which istypically high fat (as n-6 and n-9) and protein, and then thenext day gave the same individuals 900 calories of fruit andvegetables.e I can’t imagine eating that many calories asfiber. They drew blood at 0, 1, 2, and 3 hours after each mealand found the circulating inflammatory mediators were sig-nificantly higher after the fast food meal.

We seem to be uncovering this picture of pancreatitis inwhich initiating factors are local in obese animals due toinflammatory mediators from paracrine adipocytes. Thenthe patient eats a high-fat meal that causes an increased cir-culating level of proinflammatory mediators, and that tipsthe balance to where systemic anti-inflammatory and antiox-idant mechanisms are overwhelmed. Local inflammationwill persist as chronic pancreatitis until excessive abdominalfat is lost, and circulating levels will remain low with feedinga low-fat, high-fiber diet.

Dr. Hill: A canned food can be labeled as being low fat eventhough most people here would not regard it as such. So youcannot rely on the accuracy of a canned food label that saysit’s low fat without confirming the actual fat content. I’ve hadmany cases referred to me where the dogs have been on thesediets, and they respond to lower amounts of fat. I have not,however, done what Dr. Fleeman wisely suggested and mon-itored serum fat levels before and shortly after feeding. I havejust noted this result during years of clinical experience. Theremay be a lot of cases in private practices that have respondedbeautifully to that amount of fat. I just don’t see them. I seethe ones that do need greater fat restrictions.

SUMMARY OF CASE 4Dr. Reynolds: One of the big take-home messages fromthis last case is that the diagnosis of pancreatitis in catsis not easy, and we don’t really have a gold standard forit. When we have patients that we believe have pancreati-tis, the initial therapy may be similar to that used forother diseases. The goal is to get them stabilized and takecare of the major problems and then continue to see ifpancreatitis is really the primary problem or if it is sec-ondary to another disease.

We want to be very cautious with overfeeding, par-

ticularly in an animal that has been anorectic for a longtime. As always, we try to start with an enteral route, butif that doesn’t work, we go the parenteral route.

We shouldn’t forget the importance of trace miner-als, particularly in animals that have been anorectic fora while. Also, moderate to elevated protein seems to beof benefit, as does moderate to elevated fat, especially incats. At least in dogs, moderately restricting fat may playa role in prevention of pancreatitis. That concept in catshas not yet been validated.

eMohanty P, Daoud N, Ghanim H, et al. Absence of oxidative stress and inflam-mation following the intake of a 900 calorie meal rich in fruit and fiber. Diabetes2004;53:A405.


newsbites mar 04 pv - purina® pro plan® vets · 2017-06-02 · correcting metabolic deficits with...

Documents