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TRANSCRIPT
THE EFFECT OF FATTY ACID CHAIN LENGTH ON ENERGY METABOLISM LN HEALTHY WOMEN
Andrea A. Papamandjaris
School of Dietetics and Human Nutrition McGill University, Montreal
Québec, Canada
A thesis submitted to the Faculty of Graduate Studies and Research in partial fiilfilment of the requirements of the degree of Doctor of Philosophy
@Andrea A. Papamandjaris, 1999
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The effect of fatty acids on energy metabolism has been shown to be dependent on
their acyl structure. In humans, foilowing short term feeding, medium chain triglycerides
(MCT) have been shown to increase the themic effect of food and fat oxidation as
compared to long chain triglycerides (LCT). Short tenn results in animals have been
comparable. In longer term animal studies, MCT vs. LCT have resulted in less weight
gain during overfeeding or refeeding after weight loss. However, observations of the
longer term effects of MCT in humans beyond 7 days are sparse and inconclusive. Hence,
the objective of the thesis was to examine the effects of MCT vs. LCT on total energy
expenditure, its components basal metabolic rate and thermic effea of food, and on
substrate oxidation, including both exogenous and endogenous fat oxidation for a penod
of one week, following one week of prefeeding. Twelve healthy college aged women
were fed eucalonc 14 day diets enriched with either MCT or LCT in a randomired cross
over design, with a two week washout period. Doubly labelled water, respiratory gas
exchange analysis, and 1-I3C labelled myristic, palmitic, and stearic acids were used to
measure total energy expenditure, cornponents of energy expenditure, and endogenous
long chah fatty acid oxidation, respectively. The presence of MCT in the diet significantiy
increased endogenous oxidation of labelled long chah fatty acids following 14 days of
feeding, while the presence of LCT did not. Respiratory gas exchange analysis showed
significantly increased basai metabolic rate on day 7 of MCT vs. LCT feeding, but this
effect of diet was reduced to a trend by day 14. Dietary treatment did not result in
significant differences in total energy expenditure during the second week of feeding.
These results suggest that, d e r two weeks of feeding, MCT continue to affect energy
metabolism through increased endogenous fat oxidation and a suggestion of heightened
basal metabolic rate, but do not affect total energy expenditure as measured using doubly
labelled water. In conclusion, MCT may be useful in the prevention of obesity through
altered adipose tissue utilkation and deposition.
L'effet des acides gras sur le métabolisme énergétique dépend de la structure de
leur chaîne carbonée. Chez les humains, les triglycérides a chahe carbonée moyenne
(MCT) ont diminué la réponse thennogénique a l'alimentation ainsi que I'oxidation des
gras comparé aux triglycérides à chaîne carbonée longue (KT) suite à une alimentation à
court terme. Des résultats comparables ont été observés dans les études a coun terme
chez les animaux. Dans les études à long terme dans les animaux, MCT vs. LCT ont
résulté en une diminution dans le gain de poids soit durant ïhyperalimentation ou soit
durant la réalimentation suivant une perte initiale de poids. Pourtant, les observations sur
les effets des MCT à long terme chez les humains ne permettent pas de tirer de
conclusions. Ainsi, l'objectif de cette thèse fait d'examiner les effets des MCT vs. LCT
sur la dépense énergétique totale, ses composantes le métabolisme de repos et la réponse
thermogénique à l'alimentation, et sur l'oxidation du gras d'origine exogène et endogène
pour une période d'une semaine suivant une semaine d'alimentation. Douze étudiantes
universitaires en bonne santé ont été alimentées pendant 14 jours avec des diètes
isocaloriques riches soit en MCT ou en LCT. L'eau doublement enrichie, I'analyse des
échanges de gaz respiratoires, et les acides myristique. palmitique et stearique e ~ c h i e de
13C ont été utilisés pour mesurer respectivement la dépense énergétique totale, ces
composantes, et l'oxidation du gras d'origine endogène. La présence des MCT dans la
diète a augmenté I'oxidation des gras à chaîne longue d'origine endogène après 14 jours
d'alimentation, mais pas la présence de LCT. L'analyse des échanges de gaz respiratoires
a démontré une élévation dans le métabolisme de repos à la septième journée
d'alimenation des MCT vs. LCT, mais cet effet est presque disparu a la quatorzième
journée. Le traitement alimentaire n'a pas produit de changements dans la dépense
énergétique totale dans la deuxième semaine d'alimentation. Ces résultats suggèrent que,
suivant deux semaines d'alimentation, les MCT continuent d'iduencer le métabolisme
... 111
énergétique au moyen d'une augmentation de I'oxidation des gras d'origine endogène et
une suggestion d'un taux métabolique de base élevé, mais les MCT n'affectent pas la
dépense énergétique totale selon les résultats obtenus par les mesures d'eau doublement
enrichie. En conclusion, les MCT peuvent être utiles pour la prévention de l'obésité à
travers des altérations dans l'utilisation et l'accumulation des réserves de gras.
PREFACE
In this thesis, the effects of medium chain tnglycerides vs. long chah tnglycerides
on total energy expenditure, its components basal metabolic rate and the therxnic effect of
food, total fat oxidation, and exogenous and endogenous oxidation of saturated long chain
meal fats were examined in healthy fernales.
The results are presented in manuscript format, with peninent iiterature reviews on
each chapter of the thesis. Chapter 1 provides an outline and rationale for the project,
including objectives and nul1 hypotheses. Chapter 2 presents an extended literature review
for topics discussed in the body of the thesis. Chapter 3 addresses the effects of MCT vs
LCT feeding on the exogenous and endogenous oxidation of saturated long chah meal
fats using a 13C fatty acid repeated dosing paradigm. Chapter 4 explores the effects of
MCT vs. LCT at the level of components of energy expenditure, basal metabolic rate, and
the thermic effect of food, determined using respiratory gas exchange. In addition, effects
on whole body subnrate oxidation are exarnined. Chapter 5 investigates the effects of
MCT vs. LCT on total energy expenditure, as measured using doubly labelled water,
during the second week of the two week feeding period. Additionally, components of
daily energy expenditure are compared through combination of respiratory gas exchange
and doubly labelled water results.
The thesis ends with a summary and general conclusion drawn from al1 aspects of
the research. Limitations of the thesis and potential directions for continued future
research are dso discussed. This thesis is based on three manuscnpts published or in press
and one manuscript submitted to peer-reviewed joumals.
STATEMENT FROM THESIS OFFICE
According to the regdations of the Faculty of Graduate Studies and Research of
McGill University, the following statement from the Guidelines for Thesis Preparation
(McGill University, October 1 0, 1 997) is included :
Carididotes have ~ h e option of i~dudi~ag, as pari of the thesis, the text of one of more pupers mbmitted or to be mbmittedfor publication, or the clear~'y-duplicated text of oiie or more published papers. These texrs must be bound as a17 integral part of the thesis.
If thzs option is chose~i, comectir~g texts that provide logical bridges between the d@ere!it papers are mu~~datory. me thesis mirst be written in such a wuy thar it is more thmi a rnere collection of martuscripts; in ofher words, remlts of a series of papers mus2 be irltegrated
The thesis rntrst still co~jorm to all other requirements of the 'Gridelines for Thesis Preparatio 7". The fhesis rntls; iirlirde: a Table of Co~itents, an abstract in Etigiish alid Fre»ch. an h~troduction which cleariy States ~ h e rafio~~ale ajd objectives of the stirdy, and a comprehemiw review of the literature, afital conclzrsioii and sirmmary.
Additional muterial must be provided where appropriate (e.g. in appendices) and in nffjcient detail to allow a cIeur and precise judgement to be mude of the importance alid origrn~Iity of the research reported in the thesis.
In the case of manuscripts co-atrthored by the candidaie and others, the candiihte is repired tu make an explicit stuternent in the thesis as to who contributed to such work and to whar extent. Supervisors must attest to the acnrracy of ait doctoral oral defense. Since the task of the examiners is made more dtflctdt in these cases, it is in the candidate e's interest to make perfectly clear the resporsibilities of al1 the uurhors of the co-authored papers.
ADVANCE OF SCHOLARLY KNOWLEDGE
1. Original contributions to knowledge
This thesis examines the effects of medium chah fatty acids (MCFA) contained in
medium chain triglycerides (MCT) vs. long chain fatty acids (LCFA) contained in long
chah triglycendes (KT), respectively, on fat oxidation and energy utilization. The results
from the thesis work have contributed to knowledge in the field of the effects of fw acid
chah length on energy metabolism by:
i) demonstrating validation of assessrnent of endogenous oxidation of meal
delivered fat using repeated doses of 13C labelled fatty acids.
ii) showing that the presence of MCT vs. LCT has the capacity to increase
endogenous oxidation of saturated long chain meal fats following 14 days
of feeding.
iii) demonstrating that it is possible to effect changes in basal metabolic rate
and substrate oxidation at eucaloric Ievels of intake of palatable MCT-
e ~ c h e d North American style diets d e r 7 days.
iv) illustrating that compensatory mechanisms appear to exist which, by day
14, blunt the effects of MCT vs. LCT feeding seen on basal metabolic rate
at day 7.
v) showing that at modest levels of MCT in the diet, total energy expenditure
as meanired by doubly labelled water is not significantly afKected over 14
days of feeding.
vii
vi) establishing that the effects of fatty acid chah length on energy metabolism
are present in women.
viii
2. Research Publications in Referetd Scientific Journals
1 . Papamandjaris MacDougall DE, Jones PJH. Medium Chain Fatty Acid
Metabolism and Energy Expenditure: Obesity Treatment Implications. Life
Sciences l998;M: 1203-1215.
2. White MD, Papamandjaris AA, Jones PM. Enhanced Postprandid Energy
Expenditure with Medium Chain Fatty Acid Feeding 1s Attenuated afler 14 Days
in Premenopausal Women. Americm Jorrnial of Clhical NrctrMon? 1998 (In
press).
3 . Papamandjaris Aq White MD, Jones PM. Components of Total Energy
Expenditure in Healthy Young Women Are Not Anected Mer 14 Day Feeding
with Medium Versus Long Chain Triglycerides. Obesity Research. 1999 (In
press).
4. Papamandjaris AA, Di Buono M. Jones PIH. Fatty Acid Chain-length
Designations are Important to Study Conclusions. Americm Journal of Cli~tical
Nutrition, 1 997;66: 7 10-7 1 1 .
3. Research manuscripts submitted to refetceâ scientific joumds
1. Papamandjaris A4 White Mû, Jones PJH. Increased Endogenous Fat Oxidation
Dunng Medium Chain Versus Long Chain Triglyceride Feeding. Paper to be
submitted to JmrnaI of Nutrition.
CONTRIBUTIONS OF CO-AUTHORS TO MANUSCRlPTS
The candidate was responsible for assisting the s u p e ~ s o r in the development of
the thesis project, including formulation of diets and choice of sarnple population. With
respect to execution of the project, the candidate was responsible for complete
coordination of the dinical trial including preparation of the research facility for ovemight
stay for basai metabolic rate measurement, preparation of kitchen facilities to house 12
subjects, generation of feeding and sampling protocols, and diet testing. The candidate
prepared al1 meals for the subjects assisted by Dr. M. White and Jayne Rop, and was
responsible for sarnple collection during al1 phases of the experimental trial, assisted by Dr.
M. White.
The candidate wrote the manuscript, "Increased Endogenous Fat Oxidation During
Medium Chain Versus Long Chain Triglyceride Feeding" and performed al1 calculations
and statistics presented therein. In preparation for the manuscript, the candidate
conducted al1 analyses in triplicate for each set of 36 breath samples collected per subject
per dietary trial, including sarnple preparation and mass spectrometnc analysis.
With respect to data analysis and presentation of results for the manuscript
"Enhanced Postprandial Energy Expenditure with Medium Chain Fatty Acid Feeding 1s
Attenuated after 14 Days in Premenopausal Women", the candidate reviewed the
calculations made by Dr. White using the respiratory gas exchange data and perfomed
statistical analyses in conjunction with him. For preparation of that manuscript, the
candidate collected and summarized articles and provided extensive editing.
For the samples generated for assessrnent of total energy expenditure using the
DLW method, the candidate was responsible for al1 stages of sample analysis for both
deutenum and "0 samples for al1 subjects for both trials in tnplicate from cryogenic
purification through to mass spectrometric analysis. The candidate performed al1
calcdations and statistics on the collected data, and was responsible for writing the
manuscript, "Components of Total Energy Expenditure in Healthy Young Women Are
Not Affected After 14 Day Feeding with Medium Versus Long Chain Triglycerides".
For the review article, "Medium Chain Fatty Acid Metabolism and Energy
Expenditure: Obesity Treatment Implications", the candidate collected and summarized
many of the articles, created the figures and the table, and wrote the final version of the
manuscript. Ms. D. MacDougall collected anicles for the review paper, and contnbuted
to the writing of the manuscript.
Dr. P.J.H. Jones, the candidate's supervisor, developed the study design in
conjunction with the candidate, and conducted weekly meetings to monitor progress of
the work.
Dr. M.D. White aided in ninning the clinical trial by assisting with meal
preparation and sampie collection. He was responsible for performing the initial analyses
on the respiratory gas exchange data and for writing the initial version of the paper
"Enhanced Postprandial Energy Expenditure with Medium Chain Fatty Acid Feeding 1s
Attenuated afler 14 Days in Premenopausal Women". Dr. White edited the two other
research manuscripts contained in the thesis, Chapters 3 and 5 .
xii
ACKNOWLEDGMENTS
First and foremost, 1 would üke to thank my supervisor, Dr. Peter Jones, for his
guidance and his confidence in me to allow me the opportunity to conduct this research
project. It has been a leaming experience like none other. 1 would also like to
acknowledge my cornmittee members, Dr. K. Ng-Kwai-Hang and Dr. Linda Vfykes, for
their input both into the research as it progressed and into the thesis. Dr. Wykes went
above and beyond the cd1 of duty to serve as a mentor, and is aiso a fiend. The input of
Dr. Matthew White was also appreciated. Dr. Cue was most helpful in my desire to
master statistics. 1 am gratefbl to the twelve women who participated in the study.
The day to day progress of this work would not have been possible without the
help and sympathetic ears of the support staff Lise Grant, Anne Houston, Leslie Ann
LaDuke, Francine Tardif, and Nicole Legault. Never underestimate the power of a simple
gesture.
1 salute my fellow members of the Peter Jones Lab Group: Tim, Tanya, Kirsten,
Jayne, Diane, Fady, Ming, Crystal, Marco, Nimpa, Catherine, Jian Yin& and Mahmoud.
Only we know just what it takes. 1 also would like to thank Tony, Shaila, and al1 my
fellow graduate students, both at Macdonald Campus and downtown, for their good
humour and support.
Throughout this search for the scientific tmth, there has remained but one
absolute: fnendship. There are those who MW me through it dl: Nancy, Susan, April,
Ruth, Gillian, Linda, Kathy, Paul, Chris, Mom, and Marco, the lighthouse keeper. To
them, 1 am unable to express my gratitude, so 1 borrow from a master:
"Thy true fiiendship such wealth brings, That then 1 scom to change my state with kings."
Shakespeare, Sonnet 29
TABLE OF CONTENTS
PREFACE .......................................................... v
STATEMENT FROM THESIS OFFICE ................................ vi
ADVANCE OF SCHOLARLY KNOWLEDGE 1 . Original contributions to knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii 2. Research publications in refereed scientific joumals . . . . . . . . . . . . . . . . . . . ix 3. Research manuscripts submitted to refereed scientific joumals . . . . . . . . . . . xi
CONTRIBUTIONS OF CO-AUTHORS TO MANUSCRIPTS . . . . . . . . . . . . . . . xi
... ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi11
TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
... LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix
LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
CHAPTER 1. RATIONALE AND STATEMENT OF PURPOSE . . . . . . . . . . . . . 1
1 . 1 Project overall objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1 .1 .1 Specific objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.2 Nul1 hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
CEAPTER 2 . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction 9
2.2 Energy expenditure and substrate oxidation assessrnent techniques . . . . . . . . . . 11
2.2.1 Assessment of fat oxidation using stable isotopically labelled fatty
acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.2 Assessment of energy expenditure and substrate utilkation by . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . indirect calorirnetry 13
2.2.3 Total energy expenditure assessment by doubly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . labelled water 15
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4 References 18
Medium chah fatty acid metabolism and energy expenditure: Obesity treatment
implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Abstract 25
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Introduction 26
2.3.3 Digestion and absorption of medium chah triglycerides . . . . . . . 27
2.3.4 Internai transport of medium chah fatty acids . . . . . . . . . . . . . . 27
2.3.5 Oxidative pathways of medium chah fatty acids . . . . . . . . . . . . 28
2.3.6 Ketogenesis and lipogenesis of medium chah fatty acids . . . . . . 31
2.3.7 Macronutnent balance and medium chah fatty acids . . . . . . . . . 32
2.3.8 Oxidation of medium chah fatty acids vs . other fatty acids . . . . 54
2.3.9 Thermogenesis and medium chah fatty acids . . . . . . . . . . . . . . . 35
2.3.10 Energy balance and medium chah fatty acids . . . . . . . . . . . . . . 37
2.3.1 1 Potential for use of medium chain triglycendes for treatment in
obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.3.12 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3.13 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3.14 Figure Legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
CHAPTER 3 . LNCREASED ENDOGENOUS FAT OXIDATION OVER 8 DAYS FOLLOWING ONE WEEK OF PRECEDENT DIET DURING MEDIUM CHAIN VERSUS LONG CHAIN FAT'W ACID FEEDING . . . . . . . . . . . . . . . . . . . . . . . . 57
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Abstract 58
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Introduction 60
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Methods 61
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Subjects and study design 61 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2 Experimentai diets 62
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Protocol 62
3.3.4 Collection, purification, and analysis of carbon dioxide in breath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . samples 63
. . . . . . . . . 3.3.5 Whole body respiratory gas exchange measurements 64
3.3.6 Fatty acid composition of test m a l s . . . . . . . . . . . . . . . . . . . . . 64
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.7 Data Analysis 65
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 .3.8 Statistical Analysis 67
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Results 67
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 Study subjects 67
. . . . . . . . . . . . . . . . . . . . . . . . . . . 3 .4.2 Meal compositional analysis 68
3.4.3 Overall appearance of label in the breath . . . . . . . . . . . . . . . . . . 69
3 -4.5 Dose recovery of cornbined dietary [l -L3C]-myristic, -palmitic, and . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -stearic acids 69
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 .4.6 Net oxidation 70
3.4.7 Percent contribution to fat oxidation . . . . . . . . . . . . . . . . . . . . . 70
3.4.8 Substrate utilization as measured using respiratory gas exchange 71
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Discussion 71
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 References 77
3.7 Figure legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
BRIDGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
CHAPTER 4 . ENHANCED POSTPRANDIAL ENERGY EXPENDITURE WITB MEDIUM C E U N FATTY ACID FEEDING IS A'IïïCNUATED AFTER 14 DAYS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IN PRElMENOPAUSAL WOMEN 91 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Abstracî 92
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Introduction 94
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Subjects and Methods 95
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Results 99
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Discussion 102
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 References 108
4.7 Figure Legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13
CHAPTER 5 . COMPONENTS OF TOTAL ENERGY EXPENDITURE IN HEALTHY YOUNG WOMEN ARE NOT AFFECTED AFTER 14 DAY FEEDING WITH MEDILM VERSUS LONG CHAIN TRIGLYCERIDES . . . . . . . . . . . . . 118
5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
5.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.3.1 Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 5.3.2 Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 5.3.3 Experirnental protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.4 Statistical Andysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
5 .5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 5.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 5.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.8 Figure Legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
. . . . . . . . . . . . . . . CHAPTER 6 OVERALL SUMMARY AND CONCLUSION 146 6.1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 6.2 Figure Legend . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
APPENDIX 1 . THESIS PROTOCOL FIGURE . . . . . . . . . . . . . . . . . . . . . . . . . . 157
APPENDIX II . SUBJECT CEiARACTERISTICS . . . . . . . . . . . . . . . . . . . . . . . . 159
APPENDIX III, FAITY ACID CBALN-LENGTH DESICNATIONS a . . . . . . . . . . . . . . . . . . . . . . . . . . . . IMPORTANT TO STUDY CONCLUSIONS 160
. III 1 Letter to the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
III.2 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
LIST OF TABLES
Table 2.1 Summary of Studies Illustrating the Positive Effect of Medium Chain
Versus Long Chain Triglyceride Consumption on Postprandial
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thennogenesis in Humans 53
. . . . . . . . . . Table 3.1 Sample Menu of Food served During Dietary Intervention 82
Table 3.2 Fatty Acid Profile of Medium Chain Triglyceride and Long Chain
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Triglycende Dietary Treatments 83
Table 3.3 Substrate Oxidation During Medium Chain vs. Long Chain Triglycende
Feeding as Measured Using Respiratory Gas Exchange . . . . . . . . . . . . . 84
. . . . . . . . . . . . . . . . . . . . - . Table 4.1 Fatty Acid Spectrurn of the Two Test Diets 1 12
Table 5.1 Grams of Fatty Acid in Medium Chain and Long Chain Triglyceride Diets
per 1 OOg of Meal Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 40
Table 5.2 Mean and Between Diet Cornparisons of Total Energy Expenditure,
Isotopic Decay Rates, Isotope Dilution Space Ratio, Total Body Water,
and % Body Fat During Medium Chain vs Long Chain Tnglycendes
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Feeding 141
Table 5.3 Mean Basal Metabolic Rate, Thermic Effect of Food, Activity Induced
Energy Expenditure, and Total Energy Expenditure Values by Day and
Diet, and Across Al1 Treatments and Days . . . . . . . . . . . . . . . . . . . . . 142
Appendix II Subject Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
LIST OF FIGURES
Figure 2.1
Figure 2.2
Figure 3.1
Figure 3.2
" .
Figure 3.3
Differential Medium Chain Fatty Acid and Long Chain Fatty Acid
Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Energy Balance and Body Weight Before and M e r Medium Chain Fatty
Acid Feeding at Constant Energy Intake . . . . . . . . . . . . . . . . . . . . . . . . 56
Cornparison of Pre-meal % l 3 C 4 Enrichment in Breath over Baseline for
Medium Chain Triglyceride vs Long Chain Triglyceride Diets from Day 1
to 8 afler Oral Administration of 13C Labelled Mixture of [l-13C]-Myristic
Acid. -Palmitic Acid, and -Stearic Acid at Each Breakfast Meal . . . . . 87
Hourly Dose Recovery of Labelled Mixture of [~-'~C]-Myristic Acid, - Palmitic Acid, and -Stearic Acid as Breath ')CO, on A) Day 7 d e r Fust
Oral Administration of Isotopic Mixture at Breakfast (Time O). B) on Day
14 afier Repeated Oral Administration of Isotopic Mixture at Breakfast
(Time O) Starting on Day 7, Overall Value. C) on Day 14 afler Repeated
Oral Administration of Isotopic Mixture at Breakfast (Time O) Starting on
Day 7, Relative Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Cumulative Dose Recovery Over 5.5 h of Labelled Mixture of [1-"Cl-
Myristic Acid, -Palmitic Acid, and -Stearic Acid as Breath 13C0, (A),
Cumulative Net Oxidation of Myristic Acid, Palmitic Acid, and Stearic
Acid Over 5.5 h (B) and Percent Contribution of Combined Myristic Acid,
Palmitic Acid, and Stearic Acid to Net Postprandial Fat Oxidation (C) on
Both Medium Chain and Long Chain Triglyceride Treatments on Day 7,
Dav 14 Overall and Dav 14 Relative . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Figure 4.1
Figure 4.2
Figun 4.3
Figure 5.1
Figure 5.2
Figun 6.1
Figure AL1
Comparisons on Day 7 and 14 Between Dietary Conditions (Medium
Chain Triglycende or Long Chain Triglyceride) of Basal Metabolic Rate
and Postprandial Energy Expenditure Following a Standardized Breakfast
E ~ c h e d in Either Treatment Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 14
Cornparisuns on Days 7 and 14 Between Dietary Conditions (Medium
Chain Triglyceride or Long Chain Triglycende) of Respiratory Quotient
Together with Fat and Carbohydrate Oxidation in the Pre- and Postprandial
Periods Following a Standardized Breakfast Enriched in Either Treatment
Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
The Comparisons for Medium Chain Triglycende or Long Chain
Triglyceride Diets Between Testing Days 7 or 14 for the Respiratory
Quotient Together with Fat and Carbohydrate Oxidation, in Pre- and
Postprandial Periods Following a Standardized Breakfast Enriched in
Either Treatment Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 16
Total Energy Expenditure (kcauday) by Dietary Treatment for Each
Subject and Overall D i e t q Treatment Average for Medium Chain
Tnglycende vs. Long Chain Triglycende . . . . . . . . . . . . . . . . . . . . . . 144
Partitionhg of Daily Energy Expenditure. Basal Metabolic Rate, Thermic
Effect of Food, and Activity Induced Energy Expenditure Expressed As A
Percentage of Total Energy Expenditure for Medium Chain Triglycende,
. . . . . . . Long Chain Triglyceride, and Combined Dietary Treatments 145
Influence of Medium Chain Triglyceride on Dietary Fat Delivery to
Adipose Tissue Storage and on Subsequent Oxidation of Fat to CO, . 156
Overall Thesis Experimental Protocol . . . . . . . . . . . . . . . . . . . . . . . . . 1 58
LIST OF ABBREVIATIONS
P-OH
AJEE
BMI
BMR
CNRU
CPT
DEE
DLW
EE
FA
IRMS
LCFA
LCT
LF
M A
MCFA
MCT
PA
CO* RGE
RMR
RQ
SA
SNS
TEE
TEF
P-hydroxybutyrate
activity induced energy expenditure
body mass index
basal metabolic rate
clinical nutrition research unit
carnitine palrnitoyl transferase
daily energy expenditure
doubly labelled water
energy expenditure
fatty acids
isotope ratio mass spectrometry
long chah fatty acid(s)
long chain triglycetide(s)
Iow fat
myristic acid
medium chain fatty acid(s)
medium chah triglyceride(s)
pairnitic acid
rate of carbon dioxide production
respiratory gas exchange
resting metabolic rate
respiratory quotient
stearic acid
sympathetic nervous system
total energy expenditure
thermic effect of food
CHAPTER t
RATIONALE AND STATEMENT OF PURPOSE
Dflerences in the metabolic handling of fatty acids (FA) based on acyl structure,
specifically chah length, have been studied since the first half of the century.
Discrimination can occur dunng uptake, distribution and deposition throughout the body,
and in utilization for energy or storage. However, questions remain regarding the effects
of chain length on energy metabolism. specifically with respect to medium chain
tnglycerides (MCT) containing medium chain fatty acids (MCFA) as compared to long
chain triglycerides (LCT) containing long chain fatty acids (LCFA).
Medium chain fatty acids are compnsed of 8 to 12 carbon atoms and are always
saturated (1,2) in contrast to LCFA, which have carbon chain lengths of 14 carbon atoms
or greater and have varying degrees of saturation. They are predominantly consumed in
certain Asian countnes using coconut oil during food preparation, as coconut oii is a
major source of MCFA (3-6). As such, they do not form a major constituent of the typical
Nonh American diet (6,7). As descnbed by Kaunitz et al (8) in the middle of the century,
research up to that point on the differential metabolic eEects of MCFA compared to
LCFA had been relatively sparse. Since that time, there has been increasing evidence,
both animal and human, to support the notion that FA of different chah lengths undergo
disparate metabolic fates and result in different metabolic consequences. However, the
consistency and extent of these dissimilarities remain under explored.
Dif5erences in metabolism between MCT and LCT occur at manifold stages of
metabolism. They are handled dissimilarly during absorption, digestion, and transport
2 9 - 1). The subsequent metabolic disposa1 of MCFA and LCFA towards oxidation vs.
storage is also disparate. In animals, MCFA as compared to LCFA have been show to
affect oxidative processes and energy utilization over the short term following single meal
feeding, including greater and faster oxidation of MCFA vs. LCFA (1 2.1 3). Outcornes of
decreased body weight gain in rats during extended feeding of MCFA vs. LCFA have also
been reported (14- 16).
In humans, similar short term results have been observed with increased themic
effect of food (6,17,18,19) and more extensive and rapid oxidation of MCFA vs. LCFA
(20). In one of the only long term studies to date, no effect of chain length was reponed
on weight loss during an extreme hypocaloric regimen in obese female inpatients over 4
weeks or outpatients over 12 weeks; however, neither components of energy expenditure
nor body composition were measured (21). Thus, the effects of MCFA vs. LCFA on
specific metabolic parameters, such as themiic effect of food, basal metabolic rate, and
total energy expenditure, have not been eiucidated over longer periods. In addition, the
effect of MCFA on endogenous fat oxidation has not been documented.
A systematic exploration of the effects of MCT vs. LCT on energy utilization and
fat oxidation beyond 7 days has not been conduaed. Studies in controlled conditions,
with palatable diets, simultaneously examining effects of fatty acid chah length at dEerent
levels of body energy utilization and fat metabolisrn, from endogenous oxidation to basal
metabolic rate to whole body total energy expenditure, are warranted. Conditions of
weight maintenance or eucaloric intake are also warranted, to ailow energy expenditure,
and not alterations in energy intake, to be the detennining factor in any metabolic
perturbations seen. Specitically, efYects of MCFA on other fats contained within the meal
need to be understood, as does how the presence of MCFA cm affect how these meal fats
are stored and subsequently oxidiied.
Therefore, the objective of the thesis was to examine the effects of MCT compared
to LCT in a clinically controled environment using a combination of metabolic
rneasurement techniques to allow a systematic assessment at different levels of energy
metabolism, and to ailow assessment of endogenous oxidation of saturated meal fats, a
parameter not previously studied.
1.1 Project Overall Objectives
The overall objectives of the thesis were to investigate over the longer term, up to
14 days, the effects of fatty acid chah length, specifically MCT vs. LCT, on human fat
oxidation and energy utilization. In addition, the objective was to study the varying effects
of FA of different chah lengths in wornen, a group whose metabolic responses to MCT
feeding have been understudied.
1.1.1 Specific Objectives
Healthy college age women consumed North Amencan style diets enriched
with either MCT or LCT for a period of two weeks each in a controlled clinical
environment. Based on this protocol, specific objectives were to determine:
a) exogenous and endogenous fat oxidation of the meal fats myristic (MA),
palmitic (PA), and stearic (SA) acids using repeated doses of 1-"C labelled
MA, PA, and SA by determining the excretion of "CO, in the breath using
isotope ratio mass spectrometry.
b) basal metabolic rate (BMR), respiratory quotient (RQ), and fat and
carbohydrate oxidation for 0.5 h prior to consumption of breaWast and the
t h e d c effect of food (TEF), RQ, and fat and carbohydrate oxidation for
5.5 h following consumption of breakfast on day 7 and 14 of feeding using
respiratory gas exchange.
C) total energy expenditure (TEE) during the second week of feeding using
doubly labelled water and isotope ratio mas spectrometry and to combine
TEE with BMR and TEF to derive aciivity induced energy expenditure
(AIEE).
1.1.2 Null Hypotheses
The presence of MCT as cornpared to LCT in the diets of heafthy college
women over a two week feeding period will not influence:
a) endogenous and exogenous oxidation of the saturated meal fats MA,
PA, and SA as determined using 1-13C labelled M& PA, and SA.
b) BMR as measured prior to bre&ast on days 7 and 14 of feeding.
c) TEF as measured following consumption of breakfast on days 7 and 14.
d) AIEE as derived from measurements of BMR, TEF, and TEE.
e) fat or carbohydrate oxidation, or RQ, as detennined using RGE on days
7 and 14 of feeding.
t) TEE as measured using doubly labelled water dunng the second week of
feeding.
1.2 References
Paparnandjaris Di Buono M, Jones PJH. Fatty acid chah-length designations
are important to study conclusions. Am J Clin Nutr 1997;66: 7 10-7 1 1. Letter to the
Editor.
Bach AC, Babyan W. Medium-chah triglycendes: An update. Am J Clin Nutr
1982;36:950-962.
Mendis S. Wissler RW, Bndenstine RT, Podbielski FJ. The effeas of replacing
coconut oil with corn oil on human serurn lipid profiles and platelet derived factors
active in atherogenesis. Nutrition Reports International l989;4O: 773-782.
Das PK. The place of coconut oil in Indian vegetable oils. .4gricultural situation in
India l984;39:3 17-324.
Beegom R, Singh RB. Association of higher saturated fat intake with higher nsk of
hypertension in an urban population of Trivandrum in south India. Int J Cardiol
l997;58:63-70.
Dulloo AG, Fathi M, Mensi N, Girardier L. Twenty-four-hour energy expenditure
and urinary catecholarnines of humans consuming low-to-moderate amount of
medium-chain triglycerides: A dose-response study in a human respiratory
chamber. Eur J Clin Nutr 1996;50: 152- 158.
Chong YH, Ng TK. Effects of palm oil on cardiovascular risk. Med J Malaysia
1991;46:41-50.
Kaunitz H, Slanetz CA, Johnson RE, Babayan VK, Barsky G. Relation of
saturated, medium- and long-chain trigiycerides to growth, appetite, thirst and
weight maintenance requirements. J Nutr 1958;64: 5 13-524.
9. Jensen C, Buist NR. Wilson T. Absorption of individual fatty acids from long chah
or medium chah triglycerides in very small infants. Am J Clin Nutr 1986;43:745-
751.
10. Swift LL, Hill JO, Peters JC, Greene HL. Medium-chah fatty acids: evidence for
incorporation into chylornicron triglycerides in humans. Am J Clin Nutr
lWO;52:834-836.
11. Vallot 4 Bernard 4 Carlier H. Infiuence of the diet on the portal and lymph
transport of decanoic acid in rats. Simultaneous study of its mucosal metabolism.
Comp Biochem Physiol A 1985;A82:693-699.
12. Leyton J, Drury PJ, Crawford MA. Differential oxidation of saturated and
unsaturated fatty acids in vivo in the rat. Br J Nutr l987;57:3 83-393.
- . 1 3 . Johnson RC, Young SK, Cotter R, Lin L, Rowe WB. Medium-chain-triglyceride
O lipid ernulsion: metabolism and tissue distribution. Am J Clin Nutr 1990;52:502-
508.
14. Lavau MM and Hashim SA. Effect of medium chah tnglyceride on lipogenesis and
body fat in the rat. J Nutr 1978; 108:6 13-620.
15. Geliebter 4 Torbay N, Bracco EF, Hashirn SA, Van Itallie TB. Overfeeding with
medium-chah tnglycende diet results in diminished deposition of fat. Am J Clin
Nutr l983;37: 1-4.
16. Crozier G, Bois-Joyeux B, Chanez M, Girard J, Peret J. Metabolic effects induced
by long-term feeding of medium-chah tnglycerides in the rat. Metabolism
1987;36:807-8 14.
17. Sealfi L, Coltorti A, Contaldo F. Postprandial thermogenesis in lean and obese
subjects afier meals supplemented with medium-chain and long-chah tnglycerides.
Am I Clin Nutr W9l;S3:ll3O-lEL
18. Hill JO, Peters JC, Yang D, Sharp T, Kaler M, Abumrad NN, Greene HL.
Thermogenesis in humans during overfeeding with medium-chah triglycendes.
Metabolisrn 1989;38:64l-648.
19. Seaton TB, Welle SL, Warenko MK, Campbell RG. Thermic effect of medium-
chah and long-chah triglycerides in man. Am J Clin Invest l986;44:630-634.
20. Metges CC, Wolfram, G. Medium- and long-chah triglycendes labelled with 13C:
A cornparison of oxidation afler oral or parenteral administration in humans. I
Nutr 1991;121:31-36.
- . 21. Yost TJ, Eckel RH. Hypocaloric feeding in obese women: Metabolic effects of
a medium-chain triglyceride substitution. Am J Clin Nutr l989;49:3 26-330.
2.1 Introduction
Obesity is a disorder which has been linked to increased mortality and is a serious
health concem in North her ica . Reportedly, 3 1.6 of Canadians are obese, as defined by
a body mass index (BMI = Weight in kmeigh t in m)? of 2 27 kglm' (1). The Canadian
and global (2) prevalence of obesity is cause for concem as being ovenveight increases the
probability of developing coronary heart disease, strokes, cancer, diabetes, and digestive
diseases (3). In conjunction with the growing awareness of the national level of obesity,
over half of Canadians surveyed expressed concem about their level and type of fat
consumption (4). Thus, the population appears prepared to accept and apply obesity
interventions related to fat intake. Clearly, appropriate interventions lie in health
education, specifically in the area of nutrition, with the goals of overall health, obesity
prevention, and obesity treatment. However, specific dietary interventions that address
metabolic parameters affecthg body weight and obesity may also play an important role in
the health of the population.
In the context of energy balance and weight regulation, the impact of consumption
of specific types of fat has been somewhat overlooked. Lipid research is showing that the
metabolic utilization of fatty acids (FA), directing ingested fat towards oxidation vs.
storage and afFecting substrate oxidation and energy expenditure, is dependant on FA acyl
structure, including chah length. As maintenance of a reduced or elevated body weight
has been s h o w to be associated with compensatory changes in energy expenditure (5,6),
the effects of different FA on energy expenditure are important in the context of obesity.
in addition, greater oxidation and less deposition of d i e tq fat following ingestion and
greater mobilization of adipose tissue will result in decreased body weight. Thus, an
understanding of the impact of FA acyl structure, specifically chain length, on fat oxidation
and energy utilization is warranteci.
Focus has recently been placed on the metabolic efects of FA of different chah
lengths. A combination of oral and parenteral feeding and 13C tracer studies have
demonstrated greater oxidation of medium chah fatty acids (MCFA) vs. long chain fatty
acids (LCFA) (7,8). Respiratory gas exchange analysis (RGE) has shown the greater
short term effect of MCFA vs. LCFA on the thermic effect of food (TEF) (9-1 2). This
research has been predorninantly conducted over the short term, following single meal
feeding up to periods of 7 days. However, if MCFA are to be implicated in the prevention
or treatment of obesity, their longer term effects on metabolism will have to be
demonstrated. Do the positive effects of MCFA on energy metabolism last beyond 7
days? Only through continued perturbation of energy balance and fat oxidation c m
MCFA affect body weight and adipose deposition.
Stable isotope techniques now permit longer and more specific analysis of the
effects of differing FA on energy metabolism. The doubly labelled water method permits
measurement of total energy expenditure in free living subjects. The advent of the use of
13C labelled FA permits the assessment of both exogenous and endogenous oxidation of
meal delivered FA. These stable isotope techniques can be combined with a more
traditional method of energy expenditure and substrate oxidation assessment, respiratory
gas exchange analysis. Thus, a more complete picture of energy metabolism from
endogenous substrate utilization to whole body energy expenditure can be obtained, and,
as such, the effects of varying the chah length of FA on parameters affecthg fat
metaboiism and energy utilization can be assessed.
2.2 Energy expenditure and substrate oxidation assessment techniques
2.2.1 Assessrnent of Fat Oxidatiori Usir ig Stable Isotopicaily Labe lied Farry
A ci&
Respiratory carbon dioxide (CO,) is the end product of nutrient oxidation in many
biological systems (13). As such, measurement of CO, production offers a means of
assessing substrate oxidation. If nutrients are labelled with carbon isotopes pnor to
undergoing oxidation, the isotopes will appear in the expired CO2 (1 3). Thus, isotopically
labelled tracers contained in nutnents are a means of assessing the rate and extent of
substrate oxidation. An area where such isotopically labelled tracers are widely in use is in
the study of lipid absorption and oxidation (14).
In 1977, following prelirninary experimentation in the 1970's (15), the use of the
non-radioactive isotope, "C, to study lipid metabolism, through the excretion of "CO, in
the breath, was outlined by Schoeller (16). Previously, dating back to the 1950's (15), "C,
a radioactive isotope, was in use in lipid metabolism research, but its harmfûl nature
precluded widespread human use. Based on the relatively large natural abundance of "C
in the environment of 1.1%. initial assessment was made as to the feasibility of measuring
the increase in ratio of '3C02 to 12C02 following consumption of a 13C labelled substrate.
Use of isotope ratio mass spectrometry (IRMS) made the assessment of the presence of
the isotope in the breath possible. Since the initial investigations into the use of "C to
study lipid metabolism, several applications of this methodology have been made (1 7-29).
The natural I3C enrichment of foodstuffs has been examined, in order to determine
both the impact of foodstuff enrichment on loading dose requirements in stable isotope
assessment of lipid oxidation during feeding trials (17) and to exploit the natural presence
of the label in the environment for use as a naturally labelled tracer (18). Equations have
been developed to express Iipid oxidation based on the ratio of 13COz to "CO, in the
breath following administration of tracers labelled with "C (1 9,20). Therefore, fiactional
oxidation of labelled substrate, net oxidation of type of FA labelled, and percent
contribution of labelled species to overall oxidation can be calculated. Correction factors
have been established to account for the sequestering of the label as bicarbonate dunng
stages of metabolism (21,22). Assessrnent of lipid metabolism using "C labelled FA has
been conducted in both normal and diseased adult and child populations (23-26). This
includes lipid absorption in patients with cystic fibrosis, as well as the eRect of FA
differing in both chain length and degree of saturation, on the extent of lipid absorption
and oxidation (27-29).
There are some limitations associated with the assessment of lipid metabolism
using 13C labelled FA. There is variation with respect to the amount of 13C estimated to be
sequestered as bicarbonate in body pools,
(2 1,22,29). Consequently, the correction
with published values ranging from 10V0 to 50%
factor applied and the method and population
used in the determination of the correction factor must be documented. In addition, the
position of the label within the FA chah impacts on the interpretation of the results. If
oniy the carbon in the carboxyl group is labelled, differences observed in oxidation of the
FA based on experimental treatment result from a dflerence in FA transport and cellular
uptake, and not from a difference in metabolic rate in the tricarboxylic acid cycle (30).
The use of the stable isotope I3C has thus permitted great insight into the study of
lipid metabolism. As a tracer, "C offers a non-invasive, safe, and convenient method of
analysis for lipid oxidation. In addition, I3C confers a major advantage in that oxidation of
specific dietary fat components can be assessed, rather than whole body fat oxidation, as is
measured with the standard substrate oxidation measurement made with respiratory gas
exchange analysis.
2.2.2 Assessrnerit of Energy Expertdittire mid Siibstrate Utilization by hidirect
Culorirnet?y
Respiratory gas exchange analysis, or indirect calorimetry, has been used to
estimate metabolic rate or energy expenditure since the turn of the century (3 1,32). The
technique is based on the principle that the heat released by metabolic oxidative processes
during the conversion of nutrients to CO2 and water can be calculated from the
measurement of oxygen consumption and carbon dioxide production (33). As such, it is
referred to as indirect calorimetry because energy or heat production is determined by
measurement of oxygen conmmption and carbon dioxide production rather the by direct
measurement of heat transfer, as is done in direct calorimetry. Standard equations, wch as
de Weir's equation relating 4 consumption, CO2 production, and a measurement or an
estimate of protein utilization, are used to calculate metabolic rate (34.35). SMuly,
measurements of gas exchange can be used to calculate carbohydrate and fat oxidation
(34).
Thus, one of the major advantages of RGE is that in addition to the quantification
of energy expenditure, substrate oxidation at the whoie body level can be determined. In
addition, assessment of pre- and postmeal gas exchange allows for assessment of basal
metabolic rate (BMR) and TEF, respectively, as well as substrate oxidation during these
periods. A ventilated hood system such as a portable metabolic cart allows assessrnent to
be conducted in any clinical setting equipped with a quiet rest area.
There are potential drawbacks to RGE. Specific metabolic situations, such as high
rates of lipogenesis or ketogenesis. or ingestion of large amounts of atypical lipids or
carbo hydrates may require adjustment in the calculations. Such corrections, however, can
be handled individually in each situation (36,37,38). In addition, despite the precision of
24 h energy expenditure measurements using RGE with a coefficient of variation of 2.4%
(39), total daily energy expenditure measurements using RGE may be somewhat
inaccurate (40). The measurement is typically conducted in a respiratory chamber,
disenabling the subject fiom remaining fiee living to perfonn normal daily energy requiring
tasks, and resulting in an underestirnate of typical daily energy expenditure. Therefore,
RGE is best suited for assessrnent of the components of energy expenditure, BMR and
TEF, and substrate oxidation.
2.2.3 Total Energy fipenditure Assessme~ Using Doitbiy Lubelled Water
The doubly labelled water (DLW) method for use in the assessment of free living
energy expenditure was developed in the 1950's by Lifson and colleagues. Lifson et al
(41) demonstrated that the oxygen in body water is in isotopic equilibnum with the
oxygen in respiratory C4. This is due to the rapid action of the enzyme carbonic
anhydrase:
CO2 + H20 H2C0, ,= H' + HCO, (1)
Thus it was concluded that an isotopic label of oxygen introduced into the body water
would be eliminated as water and CO2, whereas an isotopic label of hydrogen would be
lost oniy as water. Therefore, the elirnination rates of oxygen labelled water and hydrogen
labelled water from the body would differ, and this direrence would be proportional to the
rate of carbon dioxide production (rCO,) (42). This hypothesis was first tested using
smail animals (43) to determine r C 0 based on the differential elimination rates of "O and
?H. Frorn the calculation of rCO,, total energy expenditure (TEE) can be calcuiated using
standard cdo~metric equations, such as that of de Weir, and an estimation of the
respiratory quotient (RQ)(32):
TEE (kcdday) = 3.94 1 rCOJRQ + 1. I l rC4 (2)
In 1982, Schoeller and van Santen (42) were the first to apply DLW methodology in the
calculation of TEE of humans.
Since this first human application of the measurement of TEE by isotopic
differential elimination by Schoeller, validation studies have been conducted against
previously existing techniques of TEE assessment. The method has been validated against
respiratov gas exchange (44,45,46) and energyhntake balance methods (42). The
method has been successfùlly applied in several types of metabolic situations to calculate
TEE (47,48,49,5O).
Its popularity as a method of TEE estimation stems from the capacity of the
method to non-invasively measure TEE in free living subjects. Following an oral dose of
water labelled with deutenurn and "O, samples of physiological fluid, such as saliva or
urine, collected at specific time intervals are al1 that are required of the subject in order to
determine TEE. Thus, normal daiiy energy expenditure, including components TEF and
activity induced energy expenditure (AEE), are incorporated into the measurement
without alteration of these values as a result of the method of measurement.
Despite its ease of use and its wide range of applicability, the DLW method is not
without limitations. Factors such as fiactionation and changes in background ennchment
of the isotopes must be addressed (5 1). Estimates of fractionation, the loss of water
different in e ~ c h m e n t fiom body water, have been determined and can be incorporated
into rCO, calculations (52). Changes in background enrichment may also be accounted
for (53); aitemativefy, subjects can be studied in situations where background enrichment
does not fluctuate. In addition, variability in the measurement is also of concem. The
DLW method has an observed intra-individual variation of as much as 12% (54). The
theoretical error is estimated at 6% (54) and biological variation is estirnated at 10%
(square root of 12' - 6'). However, analytical erron can be rninimized through isotope
loading doses of 0.25 and 0.12 gkg total body water for "0 and deutenum labelled water
respectively (55). Error can also be reduced by studying optimal metabolic penods of 5-
28 days in adults (55). Theoretical accuracy is also increased through the choice of
multipoint sarnpling, with sampling clustered at the beginning and end of the measurement
period (56). Biological variation can be minimized through consistent subject activity and
lifestyle. Nonetheless, with weful application addressing the issues discussed above, the
DLW method of TEE assessrnent is a valid and useful technique of measuring TEE in fiee
living subject S.
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2.3 Medium Chain Fatty Acid Metabolism and Energy Expenditure: Obesity
Treatment Implications
Andrea A. Papamandjaris, Diane E. MacDougall, Peter J.H. Jones
(Published in Life Sciences, 1998; 14: 1 203 - 12 1 5 .)
2.3.1 A bstraci
Fatty acids undergo dEerent metabolic fates depending on their chain length and
degree of saturation. The purpose of this review is to examine the metabolic handling of
medium chain fatty acids (MCFA) with specific reference to intermediary metabolism and
postprandial and total energy expenditure. The metabolic discrimination between varying
fatty acids begins in the GI tract, with MCFA being absorbed more efficiently than long
chain fatty acids (LCFA). Subsequently, MCFA are transported in the portal blood
directly to the liver, unlike LCFA which are incorporated into chylomicrons and
transported through lymph. These structure based differences continue through the
processes of fat utilization; MCFA enter the mitochondria independently of the camitine
transport system and undergo preferential oxidation. Variations in ketogenic and
Iipogenic capacity also exist. Such metabolic discrimination is supported by data in
animals and humans showing increases in postprandial energy expenditure after short term
feeding with MCFA. In long terrn MCFA feeding in animals, weight accretion has been
attenuated. These düferences in metabolic handling of MCFA vs. LCFA are considered
with the conclusion that MCFA hold potential as weight loss agents.
2.3.2 Inaochrction
Medium chain fatty acids (MCFA) containing 8-12 cubons are saturated
compared to long chain fatty acids (LCFA) which contain 14 or more carbon atoms and
can possess one or more double bonds. These stnictural differences affect molecular size
and water solubility and can lead to differentiation between MCFA and LCFA during
processes of digestion, absorption, and transport (1). In addition, smail chah length
dependent differences in energy content of triglycendes and constituent fatty acids (FA)
exist (2,3). There has been growing evidence that in addition to the fate of nutrients as
directed by ATP requirements, chain length- and saturation-dependent differentiation in
metabolic disposal of dietary fatty acids occurs, potentially directing ingested fat toward
oxidation vs. storage and thus infiuencing energy expenditure (2,4,5,6). Differences in
MCFA vs. LCFA energy content, uptake, and transport as well as efficiency of energy
transformation may therefore impact on long term energy balance. Data suggest that such
differences have implications in treatment strategies for obesity. Dietary fat substitution of
medium chah tnglycerides (MCT) for long chain triglycerides (KT) has been shown to
influence energy balance and may therefore promote weight reduction (6,7). To address
the question of the effect of MCFA on energy balance, the purpose of this review is to
examine MCFA intermediary metabolism and the effect of MCFA administration on
thermogenesis and total energy expenditure. The potential use of MCT as weight loss
agents is also explored.
2.3.3 Digesfiot~ 4nd Absurpiion o/Medium Chain Thiglycerides
Metabolic discrimination between MCT and LCT commences in the gut. The
smaller rnolecular weight of MCT as compared to LCT facilitates the action of pancreatic
lipase and therefore increases the rate of digestion of MCT (1). Consequently, MCT
undergo ffaster and more complete hydrolysis to MCFA than LCT to LCFA (8,9,10) and
MCFA are absorbed more quickly into the intestinal lumen (1, I l ) . In addition, due to the
longer chah length specificity of acyl-CoA synthetase, MCFA are not significantly
incorporated into tnglycerides and the subsequent chylomicrons as are LCFq and
therefore leave the intestine and enter the blood faster (1 ).
2.3.4 Interd Traqvort of Medium Chair, Fa@ Acids
Followinç digestion and absorption into the intestinal mucosa fkom the lumen,
saturated LCFA are incorporated into chylomicrons and transferred to the circulation
primarily via the thoracic duct, thereby initially bypassing the liver (Figure 2.1) (1 2,13,14).
In contrast, higher concentrations of MCFA and unsaturated LCFA. bound to albumin,
travel directly to the liver in the ponal blood (3,15). For example, 49% of intraduodenally
infused C 10:O was recovered from the rat mucosa in the portal blood systern compared to
7.8%, 6.4%, and 10.6% recovery of C18: 109, Cl 8:2o6, and C20:4o6 respectively (16).
These results are consistent with the fkdings of McDonald et al. (13) who observed that
72%, 58%, 41%, 28%, 58%, and 68% of C 12:O, C14:0, Cl6:O, C l 810, C18:206, and
C 18:303 respectively, bypassed the lymphatic pathway when individuaiiy infiised in the rat
duodenum.
The correlation between increasing FA chah length and incorporation into
chylomicrons is also seen in humans. Results of Swift et al (17) indicate that the mass of
trigiyceride transported in chylomicrons from a formula-fed LCT diet was approximately
five times p a t e r than that from a MCT diet. Chylomicron transport of triglycendes in
the MCT diet, determined by chylomicron tngiyceride concentration, was found to be
increased slightly from day 1 to day 6 of feeding, although the reincorporation of MCFA
as MCT in chylomicrons remained a quantitatively negligible pathway of MCFA
metabolism. MCFA are therefore transported directly to the liver via the portal circulation
unlike LCFA which are preferentially incorporated into chylomicrons as LCT and
transported via lymph.
2.3.5 Oxidarive Pa~hways of Medium Chain Fazty A ci&
Once transponed to the liver, MCFA may follow various catabolic pathways
inciuding beta-oxidation, omega-oxidation, and peroxisornal oxidation. Consequently,
characteristic differences between MCFA and LCFA metabolism also exist following
uptake by hepatic tissues. The fatty acyl synthetase responsible for TG re-esterification is
mon effective with FA of 14 or more carbons (1 8). As a result, few MCFA are recovered
in triglycende (19), phospholipid or cholesterol ester fractions (1 0) and low
concentrations of MCFA are recovered in various tissues (20). The chah length
preference of fatty acyl synthetase exists therefore as a major point of partitioning
dserentiation between MCFA and LCFA.
The majonty of lipids are catabolized by mitochondrial beta-oxidation. LCFA or
their acyl-CoA denvatives, once tnuisfonned into acylcamitine by carnitine palmityl
transferase (CPT) 1, cross the mitochondnal membrane and are regenerated as long chain
acyl-CoA in the mitochondrial matrix by CPT II (1); the concentration of LCFA crossing
the membrane is therefore limited. Conversely, MCFA do not require a shuttle system to
penetrate mitochondria (2 1). Mitochondriai acyl-CoA denved fiom either MCFA or
LCFA then undergo oxidation with production of acetyl-CoA. Berry et al (22) noted that
inhibition of FA entrance into the mitochondna caused a decrease in acetyl-CoA
production, a decrease in CO, production denved fiom the acetyl CoA precursor, and a
decrease in ketone production. Higher concentrations of acetyl-CoA were conducive to
ketone body formation. Inhibition of CPT I more efTectively reduced ketone body levels
associated with LCT consumption compared to MCT consumption (2 1). Christensen et al
(23) found that most C12:O gained access to the mitochondna independently, however
camitine dependent mitochondrial oxidation may provide a minor pathway of C 12:O
metabolism. Interestingly, Rossle et al (24) noted in healthy humans that a MCFA
infusion depressed free camitine levels and increased short chain acylcamitine levels,
relative to LCFA infusion. These results demonstrate the greater necessity of LCFA for a
shuttle, CPT I and II, to enter the mitochondna for oxidation.
An dtemate route for cystolic fat utilkation is peroxisornal oxidation. At high
concentrations of FA in the pefised rat liver, it is estimated that 25% of oxidation occurs
in the peroxisome (25). Unlike mitochondrial beta-oxidation, peroxisomal beta-oxidation
produces H202 and is not coupled to a respiratory chain (26). Brady et al (27) found that
the induction of peroxisomal and mitochondrial beta-oxidation was coordinated.
Regardless of FA substrate, mitochondrial beta-oxidation exceeded (28) and preceded
(29) that occurring in the peroxisome. The chah length specificity of acyl-CoA oxidase,
an enzyme which may be rate limiting for peroxisornal oxidation, is dependent on acyl-
CoA concentrations (1). Below 80 PM, palmitoyl-CoA showed the highest activity in rat
Iiver extract; above that levei, CoA derivatives of C8:O and C 12:0, produced the most
activity, followed by C16:O and C24:O (30). Similarly, Handler et al (25) found that
peroxisomes preferentially oxidized C 12:0 in rat homogenates and perfused rat liven.
This rate decreased with increasing and decreasing chah lengths (25,26). As peroxisomal
beta-oxidation appears to be a significant pathway of fat catabolism and MCFA may have
a high affinity for peroxisomal oxidation, this energetically inefficient pathway may
contribute to enhanced thermogenesis.
Omega-oxidation of fat may also be structure specific. Omega-oxidation
originates in the hepatocyte microsorne, producing water soluble dicarboxylic acid which
can be excreted in the urine. Christensen et al (1 9) found that C 120 and C 10:O have the
greatest affinity for omega-oxidation. Administration of MCT in children and animals has
been associated with dicarboxylic aciduria (3 1,32,33), indicating a possible preference of
MCFA for this oxidative pathway. Christensen et al (1 9) mggested that omega-hydroxy
and dicarboxylic acids formed by FA omega-oxidation in the liver may be then further
metabolized in the liver, accounting for acetate release fiom peroxisomes (34). Normai
adults consuming a 3 day diet with MCT contributing 5 1% of energy, excreted less than
1% of the energy intake in the form of u n n q dicarboxylic and keto acids (35). Thus,
reduced energy accumulation associated with MCT ingestion does not appear to be the
result of urinary excretion of omega oxidation products.
2.3.6 Kelogeitesis and Lipoge~~esis of Medium Chain Fatry A c i h
That MCT ingestion can result in increased ketogenesis in both animals and
humans has been well documented (35,36,37). Several studies have indicated that MCT
metabolism resulted in elevated P-hydroxybutyrate @-OH) compared to LCT metabolism
(4,24,38,39). However, other researchers have failed to notice sirnilar effects of MCT
metabolism (4O,4 1).
Acetyl-CoA derived from MCFA and not directed toward ketone body formation
or oxidation may be resynthesized into longer chain FA and estenfied (7,lO). Thus, LCF4
may result from MCFA ingestion through de novo FA synthesis or through chain
elongation. Christensen et al (23) noted that a significant amount of C 120 was rapidly
converted to C l6:O and lesser amounts of CWO, C 16: 107, C l8:O and C 18: 109 in
isolated hepatocytes derived from rats refed glucose.
Lipogenesis associated with LCT, MCT, and low fat (LF) consumption is
fiequently assessed by measurement of lipogenic enzyme activity in the liver and adipose
tissue. In rat adipose tissue, the consumption of LCT was significantly more effective than
MCT in inhibiting lipogenic enzyme activities (39,42). Chanez et al (40) found in rats that
hepatic glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA
carboxylase and fatty acid synthase activities were 1.7, 2.6, 1.4, 1.5, and 1.4 fold higher on
the MCT diet and 40%, 30%, 55%, 50°h, and 45% lower on the LCT diet compared to
the LF control diet, respectively. Geelen (43) observed that short-tem exposure of
isolated rat hepatocytes to MCFA stimulated fatty acid synthesis, as determined through
increased hepatocellular carboxylase activity. Further to this, in vivo, Geelen et al (44)
determined that in the rat model, a diet of MCT oil vs. corn oil increased hepatic acetyl-
CoA carboxylase and fatty acid synthase. Foufelle et al (45) examined the effect of MCT
consumption on lipogenic enzyme activity and gene expression during the
suckiinglweaning transition period in the rat. During transition, lipogenic enzymes
normally increase with the incorporation of a high carbohydrate rat chow diet. This effect
fails to occur with weaning to a high fat diet, unless the diet contains MCT.
It has been suggested that these discordant lipogenic effects noted with MCT
consumption may be due to carbohydrate content (36,40), polyunsaturated fat content
(40,45) or the thyroid hormone response (36) to dietary nutnents. Consequently, al1
aspects of the diet must be considered for cornparisons of dietary metabolic effects.
2.3.7 Macromltriei~t Balance and Medium Chain Futty Aczds
MCFA metabolisrn rnay be affected by the macronutnent composition of a mixed
meal. Flatt et al (46) found no signifiant difference in postprandial oxidation following
consumption of test meals containing sirnilar amounts of carbohydrate and protein but
differing across LCT, MCT or LF dietary treatments. The authors suggested that
aithough the metabolism of carbohydrate and protein are highiy regulated due to their
lirnited storage ability, fat exhibits a greater degree of metabolic flexibiiity since it can
either be oxidized or readily stored. Flan et ai (46) proposed that the metabolic pathway
of dietary fat is not detennllied only by the composition or the amount of fat ingested;
rather, fat metabolism proceeds in a manner which ensures that carbohydrate, protein, and
energy balance are rnallitained. This theory is supported by B e ~ e t at al (47) who added
50 g of dietary fat to a standard breakfast and did not see an increase in fat oxidation or
energy expenditure dunng the following 24 h in humans. Schutz et al (48) observed a
similar result when a fat supplement of 987 kcai/d did not alter 24 h energy expenditure
and failed to promote the use of fat as a metabolic fiel. Conversely, other workers noted
a significant negative correlation between carbohydrate intake and I3C fatty acid oxidation
rate to CO, (18) or fat intake and carbohydrate oxidation (49). Sato et al (50) found that
total parenteral administration of an emulsion high in MCT resulted in increased glucose
oxidation, cornpared to an emulsion high in LCT. Compared to prediet levels, subjects
consuming 800 kcaVday diets required significantly more glucose to maintain euglycemia
during continuous similar diets high in LCT (41). This result suggested MCT may
stimulate insulin mediated carbohydrate metabolism.
Medium chah triglyceride metabolism may also be influenced by corresponding
consumption of LCT. Johnson et al (20) found that the oxidation rate of "C MCT lipid
emulsion was not significantly reduced when LCT were simultaneously adrninistered,
although there was a trend in this direction. Paust et al (18) observed that sorne patients
oxidized C 18: 109 in a pure LCT emulsion more rapidly than in a mixed MCT/LCT
emulsion. In other patients, no differences in the rate of FA oxidation were detected.
Cotter et al (5 1) also identified competitive interaction between intravenous MCT and
LCT emulsions in beagle dogs. These findings suggest that meal macronutnent
composition must be taken into account if specific alterations of metaboiism attributable to
MCT are to be exploited.
2.3.8 Oxidztion of Medim Chuin Fatiy Acidr Vs Other Fut@ A cids
Structure dependent differences in oxidation of fat as a function of structure have
been show to occur using isotope tracer methodologies and respiratory gas exchange
analysis. The utilkation of [l-14C]octanoate has been shown to exceed [I-"Cloleate by at
Ieast five times in isolated hepatocytes incubated with corresponding fatty acid (52,53).
Similar findings were noted in whole body fatty acid metabolism studies. Leyton et al
(54), analyzing "CO, evolution following oral dosing of various "C FAs to rats, reported
that the oxidation decreased with increasing chain length. Faster oxidation resulted in
lower retention of these LCFA in the carcass and liver. A similar result was reported
where lipid emulsions of "C MCT or "C LCT, when injected into rats, produced a more
complete oxidation of MCT (90%) compared to the oxidation of LCT (45%) after
24 h (20).
Fatty acid chain length seems to affect not ody the quantity of fat oxidized, but the
sequence in which oxidation takes place. In studies with normal children ranging fiom 3
months to 17 years who were adrninistered "C-triolein, '3C-palmitic acid or 13C-
trioctanoin, the appearance of "CO, from 13C-tnoctanoin reached its maximum 2-4 hours
after administration (55). 13CC-t~o~ein oxidation peaked between 4-6 hours whereas
labelled palmitic acid appeared as 13C02 more slowly, gradually increasing over a 6 h
penod. Cumulative excretion of "CO2 over 6 h was 27.6%, 11.3%, and 6.6% for
trioctanoin, triolein, and palmitic acid respectively. Concurrent to these results, a single
bolus intravenous infusion of l3C-triolein or 13C-trioctanoin in newbom Uifants was show
to produce peak 13C0, excretion levels 90 minutes and 45 minutes later respectively (1 8).
Enrichment retumed to baseiine levels 10 hours after triolein and 8 hours d e r trioctanoin
administration.
In human studies using respiratory gas exchange anaiysis, intravenous
administration of MCT or LCT emulsions significantly increased the oxidation of MCFA
over 10 hours, while the oxidation of LCFA remained similar to basal levels (56). Here,
the concurrent rise in energy expenditure due to MCT administration could be entirely
accounted for by energy expended for enhanced fat oxidation. Enhanced oxidation of
MCT compared to LCT has also been shown to occur during exercise (57).
The method of administration of Iipids for the purpose of determining FA
oxidation may have a large bearing when comparing extent of oxidation. For example, the
total oxidation of octanoate exceeds oleate in hurnans (58); this result is sirnilar whether
administered orally or parenterally to humans (S8,5 9). In contrast, "CO2 breath
enrichment of labelled oleate proceeds more rapidly when administered parenterally (58).
Consequently, the length of feeding, the state of activity, and method of administration
must be considered in examining effects of MCT administration on fat oxidation.
2.3.9 27termogenesis and Mednm Chain Farty Acidr
Increases occur in energy expenditure due to meal ingestion. This themiic effect
of food (TEF) can be determined from respiratory gas exchange analysis by comparing
whole body total energy expenditure (TEE) following consumption of a m d to resting
metabolic rate (RMR). Flatt et al (46) cornpared the effect of ingesting an 858 kcal test
meal containing 40 g MCT vs. 40 g LCT over 9 hours. Energy expenditure due to the
consumption of the test meal was similar and equivalent to 1 1.2% and 12.5% of energy
contained in the LCT and MCT meals, respectively. Conversely, Scalfi et al (2) exarnined
the TEF response to consumption of a 1300 kcal test meal containing 30 g of MCT or
LCT in lean subjects. Total energy expenditure of subjects increased and the respiratory
quotient decreased afler the MCT test meal, resulting in a significantly elevated
thermogenic response. Hill et al (7) examined energy balance during 7 days of
overfeeding diets containing 40% MCT or LCT in healthy humans. Body weight, body
composition and RMR did not change significantly during either diet treatment; however,
following ingestion of a 1000 kcal test meal containing MCT, TEF was significantly higher
on both day 1 and day 6 compared to LCT. Seaton et al (4) compared the thermic effect
of meals consisting almost entirely of 48 g of MCT or 45 g of corn oil. The MCT meal
produced a signifiant increase in postprandiai oxygen consumption compared to the LCT
meal, thus resulting in an increased energy expenditure over basal level of 53 kcai and 17
kcalh These changes in energy expenditure were equivalent to 13% and 4% of energy
contained in the MCT and LCT meals, respectively. Dulloo et al (6) saw a 5% increase in
24 h energy expenditure when humans were fed a diet containing 15-30 g MCT. Mascioli
et al (56) noted that enhanced energy expenditure associateci with MCT ingestion
occurred during intravenous administration of MCFA to hospitaiized patients. A summary
of the studies reflecting the positive effect of MCFA vs LCFA on postprandial energy
expenditure can be found in Table 2.1.
2.3.10 Energy Balance und Medium Chain Fatty Acids
Long t e n energy balance studies have examined the effect of MCFA
administration on energy balance, expressed as fluctuations in weight, fat deposition or
energy deposition. In animals studies with rats ingesting diets containing 63% of
metabolizable energy as MCT or LCT, Crozier et al (36) reported that the MCT treatment
resulted in approximately 13% less energy intake and 30% less weight gain than did the
LCT diet. These findings are in agreement with other animal studies conducted by Lavau
and Hashim (42) and Geliebter et al (60) who observed reduced body weight and smaller
fat depots during MCT feeding. Conversely, Wiley and Leveille (39) found that rats fed
an MCT diet ad libitum did not gain less weight compared to rats on sirnilar diets
containing lard and corn oil, but body composition measurements were not made, so that
an effect of MCT in reducing fat gain cannot be ruled out. Geliebter et al (60), however,
stressed the importance of controlling energy intake and physical activity to allow cross
cornparison among studies. Overfeeding rats with an MCT diet (45% energy) via
gastronomy tube for 6 wk reduced weight gain by 20% and fat deposition by 23%
compared to a similar LCT diet (60). A more realistic level of fat consumption by rats
(32% metabolizable energy) showed that energy retention resulting fiom LF and MCT
diet treatments over 45 days was 26% less than that from a LCT diet (40). Sirnilar long
t e n studies in humans are less common, although Yoa and Eckel(41) found that obese
women consuming 800 k d d liquid diets containing 24% of energy as MCT or LCT for
up to 12 weeks did not difFer in either the rate or the arnount of weight lost; however,
body composition was not measured and therefore any changes in total body fat could not
be assessed.
The effect of MCT and LCT on long term energy balance needs to be considered
in the context of possible adaptation to the MCT diet. Crozier et al (36) examineci
progressive adaptation to extended MCT consumption in rats. Initially, elevated ketone
body concentrations associated with consumption of high fat diets were noted, particularly
with the MCT diet. Ketone body concentrations continually declined, and by day 44 were
approximately 50% of the initial levels. In rats receiving 32% metabolirable energy as
MCT or LCT compared to LF controls, Clianez et al (40) observed that plasma ketone
body concentration was initially elevated with MCT consumption, but by day 45 this effect
was no longer significant. This adaptive response may involve decreased ketone
production and /or increased utilization.
In humans, HiIl et al (7) found adaptive affects of MCT consumption to occur
rapidly. Following ingestion of 1000 kcal meals containing LCT, TEF did not change
significantly during a 6 day LCT feeding regime; TEF due to MCT feeding increased
significantly over 6 days (7). Fasting TG levels, which were not different between diet
treatments on day 1 were nearly 3 fold higher on day 6 of the MCT diet treatment vs. the
LCT diet treatment. Hill proposed that increased TEF on day 1 of the MCT diet may be
due to increased ketone body formation. The additional rise in TEF by day 6 of the MCT
diet may have been a result of a shift toward de novo fatty acid synthesis. This possible
explanation is supported by Hill et al (38) who found that, relative to the LCT diet
treatment, the composition of plasma TG on day 6 of the MCT diet contained &ce as
much C 16:O; however, the C16:O content of the MCT diet was 5 fold less than that
contained in the LCT diet. Increases in C 1 8:O and C 1 8 : 1 w9 levels associated with the
MCT diet treatment suggested that chain elongation and desaturation also occurred (3 8).
Possible adaptation was also reported by MacDougall et ai (6 1) who demonstrated that
following an average of 8 and 11 days of feeding, no differences in postprandial energy
expenditure existed between a breakfast nch in MCT vs. LCT.
Results of human feeding studies challenge, therefore, current notions concerning
classical metabolic pathways of ingested food as evidenced by differences in fat oxidation
and TEF of MCFA and LCFA. It has been generally assumed that 5- 10%. 0-3%. and 20-
30% of energy contained in carbohydrate, fat and protein, respectively, will be expended
during the process of thermogenesis (62). However, Hill et al (7) estimated that
obligatory costs of octanoate oxidation were 3.3% if directed toward oxidation, compared
to 6.7% if directed toward ketone formation and 32.3% if directed toward de novo FA
synthesis. Thus, the obligatory thermogenic costs of MCFA ingestion rnay be in excess of
traditional thermogenic costs of fat metabolism based on characteristic long chain
metabolic pathways. This difference bears importance for studying MCT metabolism in
humans using indirect calorimetry; ciassical equations for calculating fat oxidation are
based on values obtained using LCT, and may require modification for trials involving
MCT feeding. With respect to weight control, this increase in themogenesis may affect
energy balance by increasing energy expenditure without altering energy intake and
consequently may induce weight loss if energy intake is aabilized as expendihire increases.
2.3.11 PotentiaI for Use of Medium Chain Tnglyerrides for Treatment in Obesity
The previous discussion illustrates the concept that the metabolism and the
thermogenic effects of MCFA are difTerent as compared to LCFA. The positive effect of
MCFA on postprandial energy expenditure and fat oxidation has a potential application in
body weight regulation. When an organism is in energy balance, the arnount of energy
entering the system equals that being expended. Schutz (63) proposes the model that if
the amount of energy entering the system increases, the amount of energy expended will
also increase as lean tissue mass grows to support the increase in fat tissue. The result is
an elevation in body weight to a level at which a new equilibrium of energy expenditure
and energy intake is achieved. This model expands upon the dynamic equilibrium
hypothesis initially descnbed by Payne and Dugdale (64). A corollary to this is that if the
amount of energy expended increases and the amount of energy intake stays the same, the
body compensates to reach a new energy equilibrium. This is accomplished by decreasing
body weight until a new energy equilibrium is achieved. Such a scenuio is represented in
Figure 2.2. As presented, the source of this energy expenditure increase is MCT feeding
as a substitute for LCT in diet.
The postulated increase in energy expenditure is supported by the research
presented in Table 2.1. Differences in energy expenditure seen between MCT and LCT
feeding may translate into weight loss. For example, Duiioo et al (6) fed 30 g MCT in
addition to a maintenance diet consisting of approximately 15% of energy as protein, 40%
as fat, and 45% as carbohydrate. The difference in energy expenditure over 24 h as a
result of 30 g MCT vs. 30 g LCT was 9370 490 kJ compared to 8899 481, a
difference of 47 1 kJ, or 1 13 kcal. This dflerence cm be translated into the equivalent of
approximately 12.6 g fatlday, or one pound of fat (0.45 kg) over approximately 36 days.
A greater rate of loss due to increased dietary substitution of MCT for LCT above 30 g
andlor continuing the dietary regimen over an extended period may be extremely clinically
important in the treatment of obesity. The relevance of such an increase is supported by
the fat balance theory of Swinbum and Rawssin (65) who state that the major infiuence
on fat oxidation is energy expenditure, with negative energy balance promoting fat
oxidation. Consequently, a negative energy balance created by MCT ingestion may
promote fat oxidation and weight loss in the obese, recognizing that energy intake must be
actively rnaintained at a constant level.
In animal experirnents, decreased weight gain during MCT feeding vs. LCT
feeding has been observed by several researchers. Lasekan et ai (66) demonstrated that
rats receiving an intragastnc or intravenous infusion over 24 h of a 3 : 1 emulsion of MCT
and LCT vs. an LCT ernulsion had one third the weight gain as well as a 13% increase in
energy expenditure. During overfeeding for 6 weeks, Geliebter et al (60) observed that
among rats fed 45% of calories either as MCT or LCT through a ganrostomy tube, the
MCT-fed rats gained 20% less weight and possessed fat depots weighing 23% less than
the LCT-fed rats. Similarly, Lavau and Hashim (42) saw a decrease in body weight and
fat depots in rats fed a 55% by energy MCT diet as compared to a low fat diet, whereas a
55% LCT diet caused an increase in body weight and fat depots. Conversely, W o o and
Girardier (67) saw no effect of carbon-chah length on energy expenditure or on energy
partitionhg duMg two weeks of calorie controlled refeeding in rats with 30% of energy
as fat. However, in a subsequent isocaioric refeeding trial with fat as 50% of energy (69),
MCT fed as coconut oil resulted in higher energy expenditure and less fat deposition as
compared to LCT fed as lard. In generd, these results suggest that MCT feeding is less
weight promoting than LCT in rats.
In humans, there is a relative lack of studies examining the longer term efEect of
MCT keding on weight gain and energy expenditure. Yost and Eckel(41) failed to see a
difference in weight loss between two groups of obese women being fed hypocalonc diets
over 12 weeks containing either 30% of calories as LCT or 24% as MCT and 6% as
LCT; neither energy expenditure nor body composition was measured in this study. Hill
et al (7) saw an increase in postprandial energy expenditure over 6 days of MCT vs LCT
over feeding in healthy young men. These findings suggest that the effect of increased
energy expenditure may be transient.
In addition, the extent of lipogenesis that results after MCT feeding will have to be
detennined over the longer tem. Hill et al (38) saw changes in tnglycerides after 6 days of
MCT vs. LCT feeding that were consistent with the hypothesis that MCT overfeeding may
result in de novo lipogenesis and enhanced FA elongation by the liver. Potential
lipogenesis as a result of MCT feeding is a concern as this effect may negate the positive
effect of MCT on energy expenditure. However, whether significant lipogenesis will
occur in eucaloric feeding remains to be determined.
2.3.12 Cortclusio?r
As presented, ample evidence exists that the pathways and energy costs of MCFA
intemediary metabolism result in a characteristic enhancement of post prandial energy
expenditure. Within a wide variety of circurnstances, MCFA are consistently oxidized to a
greater degree than LCFA. Ease of absorption, hepatic portal transport, carnitine
independent mitochondrial metabolism and a low affinity for esterification may facilitate
the rapid and greater oxidation of MCFA, thus making it a highly available energy
substrate. Understanding the thermogenic effects of MCFA rnay provide valuable insight
into the suitability of MCFA use in various clinical and therapeutic situations.
The capacity of MCFA use as an agent in the treatment of obesity is still to be
determined. Eucalonc and hypocaloric mixed meal feeding paradigms that explore the
long terni effect of MCT on energy expenditure are required to detemine whether dietary
substitution of MCFA for a proporiion of LCFA can result in weight loss or in prevention
of weight gain or regain following slimming. At a constant level of energy intake,
increased dietary MCFA has the potential to be an effective tool in addressing the issue of
obesity .
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during weight recovery d e r low food intake. Metabolism 1995;44:273-279.
TABLE 2.1
Suiriiiiary of Studics Illustrating tlic Positive Eircct of Mcdiuiii Cliairi Vcrsus Long Clrairi Triglyccridc Consuinplion on Postprandial Tlieriiiogenesis in Huiimns
Subjec t s Energy content Macmnutrient Trcatment fat Differcnce in TEFt Reference (kcal) composition (medium va long chain tnglycerides)
7 lean males
IO lean males
6 Iwii i i~dcs G &se males
1300 LCT riical 1270 MCT irieal
100 % fat
15 % protein 45 % carbohydratc 40 % fat
1 5 % proteiii 55 % carboliydrate 30 % fat
15 % proiein 45 % carboliydraie 40 % fat
48 g MCT oil vs 45 g corn oil
MCT oil vs LCT oil
30 g MCT oil vs 30 g corn oil
30 g MCT oil pcr &y/ 10g per incal
53 vs 17 kcal*
day 1 : SM8 vs 5tk8 kcal* day 6: 120* 13 vs 66* 1 O kcal*
Ican: 366.5*8.6 vs 246.8*2Y .6 kcal* obese: 367.1k52.4 vs 222.4k36.8 kcal*
Seaton et al. 1986
Hill et al. 1989
Scalfi et al. lY9 1
DuIbo et al. 19%
'Energy expcnditure was measured for 6 Ikours with the exception of Dulloo et al. (6) where measurement was for 24 hours *indicales signifïcant diffcrcnce (p< 0.05)
2.3. i 4 Figure Legends
Fieure 2.1
Differential MCFA and LCFA Transport. Following absorption fiom the intestine, MCFA
pass directly from the mucosal ce11 into the portal vein and are transported to the liver
attached to alburnin. Conversely, LCFA are incorporated into chyiomicrons which travel
through the lymphatic system and exit at the ieft subclavian vein, circuiating peripherally
en route to the liver.
Figure 3.2
Energy Balance and Body Weight Before and Mer MCFA Feeding at Constant Energy
Intake. At weight maintenance, energy intake equals energy expenditure. An increase in
MCT ingestion at the expense of LCT disturbs the equilibrium by increasing the energy
expenditure. To achieve a new equilibrium, body weight decreases.
ntest ine Mucbsal Cell Circulation
systcm
Left subclavian vein
INCREASED ENDOGENOUS FAT OXIDATION DURING MEDIUM CHAIN
VERSUS LONG CHAIN TRIGLYCEIUDE FEEDING
Andrea A. Papamandjaris, Matthew D. White, Peter J.H. Jones
Paper to be submitted to Journal of Nutrition
School of Dietetics and Hurnan Nutrition,
Facult y of Agncultural and Environmental Sciences,
McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Quebec, H9X 3V9
Corresponding Author:
Peter J.H. Jones, Ph.D.
School of Dietetics and Human Nutrition,
Faculty of Agricultural and Environmental Sciences,
2 1 1 1 1 Lakeshore Road
McGill University, Macdonald Campus,
Ste-Anne-deBellevue, Quebec H9X 3V9
Numerous studies point to differential metabolic handling of medium chah
triglycendes (MCT) vs. long chain triglycendes (LCT), yet the capacity of MCT vs. LCT
to affect oxidation of other meal fats remains unknown. The objective of this study was to
compare the effect of MCT vs. LCT feeding on exogenous and endogenous oxidation of
long chain saturated meal fats. Twelve healthy women were fed weight maintenance diets
providing 15%, 45%, and 40% of energy as protein, carbohydrate, and fat, respectively,
with 80% of this fat compnsed of either a combination of butter and coconut oil (MCT) or
beef tallow (LCT). Following 6 d of feeding, subjects were dosed daily for 8 d with a 10
mgkg bodyweight mixture of 1 -13C labelled-mynstic, -palmitic, and -stearic acids. The
appearance of 13C in expired CO2 was used as an index of fat oxidation. On d 7 and 14,
net CO2 production was assessed using respiratory gas exchange. On d 7, the first day of
isotopic dosing, there was no effect of diet on cumulative dose recovery of dietary "C, net
oxidation, or percent contribution to total fat oxidation. Thus, no difference in exogenous
saturated fat oxidation was observed as a function of diet on d 7. On d 14, greater
cumulative dose recovery (1 6.9 * 2.SW5.S h vs. 9.1 * l.2%/5.S h) (pC0.0 l), net
oxidation (2956 * 4 13 mg vs. 1669 * 224 mg) (p<0.0 l), and percent contribution to total
oxidation (1.63 * 0.23% vs. 0.95 * 0.1 5%) (pC0.0 1) were observed with consumption of
the MCT vs. the LCT diet. Thus, on d 14, the MCT diet resulted in greater combined
exogenous and endogenous fat oxidation. Within the MCT diet, but not the LCT diet,
cumulative dose recovery (pcO.05), net oxidation (p<0.05), and percent contribution to
total oxidation (pc0.05) were significantly increased by d 14 as compared to d 7,
indicating increased endogenous oxidation. The capacity of MCT to increase endogenous
oxidation of long chah saturated meals fats may be helpful in avoiding an increase in
adipose tissue depots over long-term fat consumption.
3.2 Introduction
A growing body of literature suggests metabolic discrimination of fatty acids for
oxidation vs. storage based on chain length. Both human and animal data indicate that
medium chah tnglycendes (MCT), containing medium chah fatty acids (MCFA)
composed of chains of 8-12 carbon atoms, are preferentially oxidized as compared to long
chah triglycerides (LCT) containing long chain fatty acids (LCFA), composed of chains of
14 or greater carbon (1-14). Observations at the whole body level have show that
animals overfed and refed MCT- vs LCT-containing diets had decreased body weight gain
( 1 4 Animal studies of substrate utilization have also show more extensive and rapid
oxidation of MCT in comparison to LCT (5-7). In humans, greater propensity of MCT
for oxidation has also been observed (8-14). Yet it still remains to be determined whether
the presence of MCT in the diet c m alter the oxidation of other meal fatty acids. In
addition, the potential capacity of extended MCT feeding to affect endogenous oxidation,
whether by changing the composition of the adipose tissue pool through altered
endogenous availability, or through induction of enzymes and metabolic processes, has not
been determined. The capability of MCT to increase endogenous fat oxidation could have
implications for reduction of adipose tissue mass. Given that there is some prelirninary
evidence to indicate impaired oxidation of LCT yet normal oxidation of MCT in the obese
(1 S), substitution of MCT for LCT may provide a means to reduce adipose tissue
deposition and increase adipose tissue mobilization.
The goal of this study, therefore, was to examine effects of MCT- vs. LCT-
emiched diets on exogenous and endogenous oxidation of myristic (MA), palmitic (PA),
and stearic (SA) acids over 8 days, following one week of prefeeding. The hypothesis was
that the presence of MCT would affect oxidation of long chah saturated fatty acids
contained in the diet in a marner dflerent from the presence of LCT. Myristic acid, P 4
and SA were chosen as they represent common saturated fatty acids found in the North
Amencan diet. A repeated I3C dosing paradigm was used to study the exogenous and
endogenous components of fat oxidation concurrently.
3.3 Methods
3.3.1 Si< bjects and Sttidy Desipi
Twelve healthy, non-smoking female college students, aged 22.7 * 0.7 y with a
mean height of 1.62 i 0.0 1 m and initial body weight of 56.2 * 1.5 kg (individual subject
data, refer to Appendix II), were recruited from Macdonald Campus, McGill University.
Subjects were screened for normal blood lipid levels (total cholesterol < 5.3 mmoVL,
triglycendes < 1.24 rnmoVL), as well as absence of chronic disease, exercise fiequency,
and regular menstnial history using a questionnaire.
A randomized cross over design was used invoiving two 2 week feeding periods
separated by a 14 day washout. The number of subjects per treatment per diet cycle was
balanced, and subjects were blinded to the diet sequence. Al1 procedures were approved
by the McGill University Ethics Cornmittee and informed, written consent was obtained
Rom all subjects prior to their entrance into the study.
3.3.2 Erperimental Diets
Prepared solid food diets providing 15%, 45%, and 40% of energy as protein,
carbohydrate, and fat, respectively, were fed over each 14 day period in arnounts
calculated to satisfy individual subject energy requirements. Treatment fat, comprised of a
combination of butter and coconut oil for the MCT treatment, or beef tallow for the LCT
treatment, made up 80% of the fat in the diet. Diets (sample menu provided in Table 3.1)
were provided as a two day rotating menu comprised of three meals per day which were
isoenergetic both in total energy and fat. The rotating menu was modified to ensure that
the same meals were fed on pre-test days, day 6 and 13, and on test days, day 7 and 14 of
feeding. Ingestion of foodstuffs and beverages other than those provided was prohibited,
except for water, which was permitted ad libitum. Al1 ingredients were weighed to the
nearest 0.5 g. Energy intake was calculated to maintain energy balance using the Mifflin
equation (1 6) and an activity factor (1 7), and diets were designed to meet the
Recornmended Nutnent Intakes for Canadians (1 8). Al1 meals were prepared and served
at the Mary Emily Clinical Nutrition Research Unit at McGill University.
3.3.3 Protocol
On day 7 and 14 of each diet cycle, following a 12 h fast and overnight stay at the
Unit, subjects were awakened at 7:00 am and a baseline breath sample was then collected.
Basal metabolic rate (BMR) was measured over 30 min using a Deltatrac Metabolic
Monitor (Sensormedics, Anaheim, California). Subjects were then served a hot breakfast,
containing a 10 mg/kg b r e of 1-13C labelled @ PA, and SA (CDN Isotopes,
Quebec, Canada), given at levels proportional to the levels of MA, P 4 and SA in the diet
(Table 3.2). As the three 13C-labelled fatty acid tracers were added to the diet at levels
proportional to that of the tracees, the unlabeiled MA, PA, and SA, the fraction of label
ingested appearing as 13C0, represented oxidation of these specific fatty acids as contained
in the diet. Over 5.5 h foiiowing the breakfast meal, the volume of subjects' expired COz
was measured using the Deltatrac Metabolic Monitor. Breath samples were also collected
at hourly intends during this time. Subjects were then fed lunch, delivered a post-lunch
breath sample, and a 9th breath sarnple was collected pre-dinner, at approximately 18:00
h. From days 8-13, the same dose of isotope mixture was delivered with breakfast, and
breath samples were collected pnor to each meal: breakfast. lunch, and dimer.
Throughout the entire study, subjects kept dianes recording dates of menstruation and
daiiy activities (refer to Appendix 1 for oveniew of Study Protocol) .
3.3.4. Col(eciio)i, pcnfica~ior~, ami a)~alysis of carbm dioxide hi breafh samples
Subjects collected their own breath by exhaling into sealed bags through a sealed
tube equipped with a stopcock. At each collection time, the subjects filled the bags twice,
with only the second exhalation being used as sample. When under the hood of the
metabolic cart, subjects were given the 1.3 cm diameter tube under the hood, and
delivered the sample. A syringe was used to extract 180 rnL of air which was then
bubbled through a giass condenser, 1 .O m in total length, containing 10 rnL 1M NaOH, to
trap the CO2 The samples were then transferred to vials and stored at -20°C until
analysis.
To release the CO2, 2.5 mL of the NaOH - C O solution at room temperature was
injected into vacutainers containing 2.5 mL of phosphoric acid. The vacutainers were
shaken for 90 s to release the CO2. A SIRA isotope ratio mass spectrometer (SIRA 12,
Isomass, Cheshire, U K ) was used to determine the "C enrichment of the samples in delta
per mil (%O) (parts per thousand). The mass spectrometer was corrected for ''0 and
calibrated daily using CO, gas and standards of known isotopic composition. Background
13C abundance, as measured at baseline, either pre-isotope dose on day 7 or day 14, was
subtracted from later samples to determine e~chment due to the oxidation of LnC
labelled fatty acids.
3.3.5 Whole Body Respiratory Gas Exchmge Me~st~rements
On day 7 and 14, day 1 and 8 of isotope delivery, respectively, O2 consumption
and COz production for each subject was measured using a Deltatrac Metabolic Monitor
(refer to Chapter 4, section 4.3 for a full description). Briefly, a transparent ventilated
hood was placed over the subject's head, with a hose co~ecting the hood to the analyzer
system. After a 30 min warm-up, reference gas standards were used to calibrate the
monitor. Respiratory gas exchange (RGE) measurements were collected for 0.5 h pnor to
breakfast following a 12 h fast, and for 5.5 h following conmrnption of the scheduled
breakfast.
3.3.6 Fatty Acid Composiioti of Test Meals
Repücate portions of al1 test meais were homogenized using a commercial blender.
Fatty acid content was determined using gas-liquid chromatography afler lipid extraction
(19) and boron trifluoride methylation (20). The gas chromatograph (Hewlett Packard
5890 Series II, Pa10 Alto, California) was equipped with an autosarnpler and flame
ionization detector. Separation was achieved on an SP 2330 capillary column, 30 m x
0.25 mm x 0.2 Pm. The split ratio was 100: 1. Running conditions were: initial
temperature 1 OO°C, ramp 3 O C /min, final temperature 1 90°C, hold for 25 min. The total
nin time was 57 min. Individual fatty acids were identified against authentic standards
(Sigma Chernical Company, St. Louis, MO) using retention times.
Mean MA, PA, and SA percentages over al1 meals in each dietary cycle were
determined and each expressed as mg per g of the total of these three fatty acids. This
proportion of these individual fatty acids in the diet was used to establish the mixture of 1-
"C labelled PA, and SA delivered to the subjects in a 10 mg/kg body weight dose.
3.3.7 Data Attalyss
Fractional oxidation rates, or hourly dose recovery, of breakfast meal mixture of 1-
13C labelled fatty acids, based on the percentage of the total label ingested appearing in the
breath, were determined using the following equation (12):
hourly dose recovery ( Y i ) (dietary I3C recovery/h)=
mm01 excess 13C/mmol CO, x (mm01 CO, excretedh x 100) x 1.35
mm01 13C administered
where the factor of 1.35 was added to account for bicarbonate retention of "C in body
pools (22,23). Amount of CO, produced ( m o l ) was determined using RGE. The mm01
13C administered was determined as the total number of m o l delivered in the mixture of
the 13C labeiled MA, P 4 and SA. Cumulative fractional oxidation, or cumulative dose
recovery, was determined as the total hourly dose recovery over 5.5 h.
The total amount of combined M& PA and SA in the breakfast meal was
calculated for individuai subjects based on the following formula (2 1):
Breakfast meal fatty acid content (mg) = breakfast meal fat (mg) x (2)
concentration of fany acids in the brealâast meal (% of fatty acids in total fatty
acid spectrum)
Net oxidation rates of combined dietary Mq PA, and SA were calculated as follows (21 ):
Net oxidation (md5.5 h) = breakfast meal fatty acid content (mg) x (3
cumulative dose recovery
Percent contribution of dietary MA, Ph and SA to net postprandial fat oxidation was
calculated as (24):
Percent contribution to total fat oxidation (W5.5 h) = net oxidation (mg)/ (4)
net total fat oxidation (mg) x 100%
where net total fat oxidation was determined using RGE and the equations of de Weir
(25).
For al1 parameters, results were calculated for day 7 and two sets of results were
generated for day 14. E~chment values for day 7 were obtained by s u b ~ i o n of the
day 7 baseline values fiom subsequent breath e~chrnents. Overall enrichment values
were obtained on day 14 by subtraction ofthe subjects' initial baseline as detemiined on
day 7. These enrichrnents were then used to calculate absolute or overall values for hourly
dose recovery, cumulative dose recovery, net oxidation, and percent contribution to fat
oxidation on day 14. Likewise, relative enrichment values were obtained on day 14 by
subtraction of the day 14 baseline value, detemiined prior to breakfast and delivery of
isotope on day 14. These relative enrichments were used to calculate relative values for
hourly dose recovery, cumulative dose recovery, net oxidation, and percent contribution
to fat oxidation on day 14.
3.3.8 Stutistical Aiialyss
A repeated measures ANOVA with factors of diet (MCT or LCT), meal (1 to 24),
and diet by meal interaction was used to compare overall appearance of 13C02 in the
breath over 8 days. Hourly dose recovery over time was assessed using repeated
measures ANOVA with factors of sequence (1 or 2), diet (MCT or LCT), hour (0.5 to
5 . 9 , and diet by hour interaction for day 7, day 14 relative, and day 14 overall.
Cumulative dose recovery, net oxidation, and percent contribution to fat oxidation were
assessed for day 7 and day 14 relative, using repeated measures ANOVA with facton of
sequence (1 or 2), diet (MCT or LCT), day (7 or 14 relative), and diet-by-day interaction.
Similady, cumulative dose recovery, net oxidation, and percent contribution to fat
oxidation were compared between day 7 and day 14 overall, using repeated measures
ANOVA with facton of sequence (1 or 2), diet (MCT or LCT), day (7 or 14 overall), and
diet-by-day interaction. Post-hoc cornparisons were made using contrasts. Al1 values are
expressed as means k SEM.
3.4 Results
3.4.1 Srudy Subjects
During both dietary cycles, subjects were observed to have consumed al1 food
provided. Two subjects' energy intakes were increased based on reported hunger over the
first three days of the first dietary cycle. There were no significant changes in body weight
between start (56.2 1.5 kg) and end (56.7 i 1.4 kg) of the study, either between or
within study groups. Subject diaries indicated regular menstruation, with mean cycle
length of 28.5 + 0.6 days. No abnormaiities in health or large fluctuations in activity
patterns were reported.
3.4.2 Meal Compositior la1 A nalyss
Mean fatty acid composition profiles of al1 meals for each diet are shown Table
3.2. The percentage of MA, PA and SA differed between diets. The relative proportions
of MA, PA, and SA as calculated for the dosing mixture were 28.4 k 0.3%, 54.2 0.2%.
and 17.3 * 0.2% for the MCT diet and 9.0 0.2%. 56.2 * 0.3%, and 34.8 * 0.2% for the
LCT diet, respectively.
3.4.3 OvetaIl Appeurance of Label N t the Breath
Comparkon of pre-meal% %O2 e~chment in breath over basdine over the 8
day isotope delivery period showed a main effect @<0.001) of dietary treatment, with
values of "C in breath on the MCT diet higher than those on the LCT diet (Figure 3.1).
3.4.5 Dose Recaws, of Con1 bined Dieimy [I - J3CJ-h@ristic, -Palmitic, and - Stearic acids
On day 7, the first day of isotope delivery, there was a main effect (p<O.O5) of diet
on hourly dose recovery of breath "CO,, with greater recovery on the MCT diet at 4.5 h
(Figure 3.2A). Similarly, on day 14, there was a main effect (p<0.001) of diet on overall
hourly dose recovery of label in the breath, with increased label recovery on the MCT diet
at 1.5 and 3.5 h (Figure 3.2B). On day 14, there was a main effect (p<0.0 1) of diet on
relative hourly dose recovery, with greater recovery on the LCT diet at 2.5 and 5.5 h
(Figure 3.2C). The diet * hour interaction was not signifiant.
Cumulative dose recovery of "CO, on day 7 on the MCT diet (10.7 * 2.4%/5.5 h)
did not differ from recovery on the LCT diet (6.1 1.8%/ 5.5 h) (Figure 3.3A).
Cornparison of overall cumulative dose recovery between diets over 5.5 h on day 14
showed a main effect (p<0.01) of diet with greater recovery h m the MCT diet (16.9 * 2.5%/S.S h) than from the LCT diet (9.1 i 1.2%/5.5 h) (Figure 3.3A). Within diet
cornparison between day 7 and overdl values for day 14 showed an increase @<O.OS) in
recovery on the MCT diet by day 14 (Figure 3.3A). On day 14, relative cumulative dose
recovery was not different between diet types (MCT: 5.3 I 2.8%/5 -5 h, LCT: 10.6 k
3.6%/5.5 h); however, the interaction t e m of diet by day ap proached sigdcance
(p=0.07) (Figure 3.3 A).
3.46. Net Oxiktio~rl
The combined concentrations of MA, PA, and SA oxidized from the breakfast
meals are shown in Figure 3.3B. The resuits were sirnilar to those found for both between
and within diet comparisons for the cumulative dose recovery of label in the breath.
Between diet cornparison of net oxidation for day 7 showed no significant effect of diet
(MCT: 1836 426 mgl5.5 h vs. LCT: 1135 i 330 rnd5.5 h). On day 14, there was an
effect (pC0.0 1) of diet on overall net oxidation, with greater net oxidation on the MCT
- .. diet (2956 * 413 rnd5.5 h) than on the LCT diet (1669 i 224 rngl5.5 h). Relative net
oxidation on day 14 was not Sected by diet type (MCT: 932 î 497 mg/5.5 h vs. LCT:
1926 * 671 mg/% 5 h); however, as with the cumulative dose recovery data, the interaction
tenn of diet by day approached significance (p=0.08). Within the MCT diet, there was
greater (pC0.05) overall net oxidation on day 14 as compared to day 7.
3.4.7. Percelit cmtributio~rl of MA, SA, ar~d PA to total fat midatrion
Results for percent contribution to fat oxidation (Figure 3.3C) reflect the pattern
observed for between and within diet comparisons for both cumulative dose recovery and
net oxidation. The only significant between diet finding occurred with the overall results
on day 14, with combined MA, PA, and SA fiom the MCT contributing to 1.63
0.23Yd5.5 h oftotal fat oxidation, which dEered (p<O.01) from the 0.95 0.15%/5.5 h
contributed by those fatty acids on the LCT diet. For within diet comparisons, the day 7
contribution on the MCT diet (0.93 i O.l9%/5.S h) was smaller (p<O.OS) than the overd
contribution on the MCT diet on day 14 (1.63 * 0.23%/5.5 h). Again, no difEerences were
observed in between diet cornparisons of either day 7 or relative day 14 values, but the
interaction tem of diet by day approached significance at p=O. 10.
3.1 8. Sitbstrate Uti Iization as Meamred Using Respiratusi Gas Excha~~ge
Full description of data obtained using RGE can be found in Chapter 4, section
4.4. Total fat and carbohydrate oxidation as a function of diet fat following consumption
of breakfast on days 7 and 14 are reported in Table 3.3. Diet fat treatment did not have a
significant effect on total fat or carbohydrate oxidation on either day. At certain
timepoints in the postprandial period on day 7, repeated measures ANOVA showed
greater (pcO.05) rate of fat oxidation on the MCT diet (Chapter 4. Fi y r e 4.2).
3.5 Discussion
Present results indicate that total oxidation of meal-contained saturated long chah
fatty acids can be increased with MCT feeding over a two week penod. Repeated oral
exposure to 1 -"C labelled MA, PA, and SA and assessrnent of breath "CO, enrichment
over a period of 8 days permitted measurement of the endogenous oxidation of the
labelled fatty acids. The combined endogenous oxidation of these saturated LCFA was
aftected by dietary treatment. The acyl structure of the fatty acids within the diet other
than those which were labelled, specifically MCT or LCT, affected the capacity of the
body to retain and subsequently release the label through oxidation. Therefore, rate of
oxidation of fatty acids depends not oniy on their chah length, but also on the number of
carbon in other fatty acids present in the dietary fatty acid mVr.
Delivery of 10°C labelled fatty acids allowed assessrnent of "CO2 in the breath as
the end point of fat oxidation. Day 7 represented the subjects' first exposure to isotope,
therefore, any label that appeared in the breath on that day represented exogenous
oxidation of the labelled P 4 and SA delivered that morning with the breakfast meal.
Cumulative dose recovery did not differ between diets on day 7 despite a greater hourly
recovery of dose at 4.5 h on the MCT diet. Net oxidation and percent contribution to
oxidation data presented similar results with no statistical direrence between diets. These
results indicate that total exogenous oxidation of these fatty acids, following the first
exposure to isotope afler one week of prefeeding, was not significantly affected by dietary
treatment. Other researchers (8 , l l ) reporting increased oxidation of 10°C labelled fatty
acids on MCT diets labelled the MCFA specifically, and therefore were not assessing the
impact of MCFA on oxidation of other fatty acids, as in the present study. In addition, in
those studies (8,ll). the increase in oxidation was observed following initial feeding of
MCT, not following prefeeding. Therefore, it would appear that the presence of MCT in
the diet was not capable of significantly altering the exogenous oxidation of dietary MA,
PA, and SA following 7 days of diet administration.
Examination of exogenous oxidation following two weeks of feeding showed
similar remlts. By day 14 relative, comparable to day 7, cumulative dose recovery, net
oxidation, and percent contribution to total oxidation were not different between dietary
treatments. However, in contrat to day 7, hourly dose recovery was greater at 2.5 and
5.5 h on the LCT diet. Greater hourly dose recovery on the LCT diet by day 14 as
compared to greater hourly dose recovery on the MCT diet on day 7 is supported by
results of the diet'day interaction term for cumulative dose recovery, which approaches
significance. This vanability supports a lack of difference between MCT and LCT diets in
exogenous oxidation of saturated long chain meai fats.
Endogenous oxidation of MA, PA, and SA afler 8 days of repeated isotope
delivery was also assessed. The low percent contribution of the label to total oxidation
and the low cumulative dose recovery detennined on day 7 indicated that most of the label
delivered remained in the body, and was not oxidized postprandially. This is in accord
with other researchers who observed that less than 1% of label was oxidized
postprandially (24) and that breath "C enrichment was low following delivery of label
(12.26). Consequently, label was deposited in the body and available for subsequent
endogenous oxidation. As the same arnount of label had been consumed each day, it may
be hypothesized that the exogenous component of L13C labelled fatty acid oxidation
remained the same over that period. Thus, any increase in overall oxidation of the 1-13C
MA, PA, and SA seen by day 14 as compared to day 7 indicates contribution of label from
endogenous sources. By day 14, following repeated isotope dosing, the overall amounts
of cumulative dose recovery, net oxidation, and percent contribution to oxidation differed
between the two diet s, and were also significantly greater then on day 7 within the MCT
diet. Therefore, the 1-"C labelled MA, P 4 and SA delivered in the MCT diet on
previous days had been deposited within the body, and subsequently released during
endogenous oxidation. Lack of a significant difference between days 14 and 7 within the
LCT diet indicates that endogenous oxidation of stored label did not occur to the same
degree as on the MCT diet. The presence of MCT in the diet thus had the capacity to
influence both the deposition and subsequent mobilization of fatty acids in a manner that
the presence of LCT did not.
Examination of the pattern of label appearance offers insight into what may be
occurring dunng storage and oxidation of the labelled meal fats. Except for the initial rise
and subsequent overall higher level of appearance of label on the MCT diet, the pattern of
label appearance for the two diets is similar. The peaks seen at each day's pre-lunch
breath sampling are most likely the resuit of exogenous oxidation of the label delivered
with the breakfast meal. This rise is similar to that typically seen following ingestion of
I3C labelled fatty acids (1 l,2 1). The imrnediate rise in enrichment in the breath on the
MCT diet not present on the LCT diet may indicate rapid maximal enrichment of a fatty
acid storage pool following the initial dose with isotope. The fatty acid storage site,
having reached capacity on the MCT diet after 1 day of label feeding, began to release the
label, resulting in the rise in label appearance from endogenous oxidation. This pool was
then maintained at maximal enrichment during the following 7 days over which isotope
was delivered. Consequently, fatty acids stored within this pool were constantly available
as substrates for endogenous oxidation. Enrichment of this pool on the LCT diet did not
occur to the same degree, resulting in less endogenous oxidation. Therefore, the presence
of MCT in the diets afTected the use of the labelled fatty acids dinerentiy as compared to
the presence of LCT. On the LCT diet, the label can be hypothesized to have been
deposited in a second adipose pool which was much less metabolically active, and
therefore did not contribute in the same way to the reappearance of label through
endogenous oxidation.
The existence of two storage pools for fat has been postulated previously. In
1960, Hirsch et al (27) hypothesized the existence of two separate metabolic
compartments, one a caloric depot, exchanging siowiy with dietary fat, the other a small
metabolically active cornpartment, which may be in close metabolic relation to dietary
lipids. Such a two pool system has also been proposed by other researchers (28,29).
These pools rnay exist in order to maintain a specific fatty acid pattern within adipose
tissue (28,30,3 1). Thus, a rapidly exchanging pool may be present to act as a sink from
which less desirable fatty acids, with respect to the body's adipose tissue composition and
subsequent lipid synthesis, can be oxidized more readily rather than stored long term. IF.
as indicated by the present results, the presence of MCT within the diet can direct more
fatty acids towards temporary storage in the metabolically active pool, rather than towards
more permanent storage in the inert pool, less fat could be stored during consumption of a
diet containing MCT. Manipulation of dietary fatty acids to include more MCT could be
important in the context of the development and control ofobesity, as it may result in less
adipose tissue deposition.
The substrate utilization results determined with RGE measurements (Chapter 4)
support in part the data obtained presently using the 1-13C labelied fatty acids. As with the
isotopic analyses, the total fat oxidation in the postprandial penod on day 7 was not
different between dietary treatments despite that, at certain time points, the rate of fat
oxidation on the MCT dietary treatment was greater than on the LCT diet. Therefore,
both types of measurements support a nul1 effect of diet on total exogenous oxidation
following one week of prefeeding. However, there is some discrepancy with respect to
day 14. The RGE analysis indicated no effect of diet on total postprandial or basal fat
oxidation, yet the isotopic data show greater overall net oxidation of PA and SA on
day 14 on the MCT diet. Possibly, the RGE method is unable to detect small yet
significant differences in endogenous oxidation that are present and which the isotope
method is capable of measuring. Altematively, oxidation of other fatty acids that were not
labelled may have decreased in compensation for increased oxidation of PA, and SA,
resulting in no overall change in total fat oxidation.
In conclusion, these results demonstrate that MCT can measurably affect
endogenous oxidation of MA, PA, and SA following two weeks of feeding compared to
LCT as detennined using a repeated 13C dosing paradigm. In addition, the results
obtained support the existence of both a rnetabolically active pool and an inert pool of
fatty acid storage. Acyl stmcture, specifically chah length, of dietary fatty acids may
determine in which pool dietary fat is stored, and subsequently how rapidly fatty acids can
undergo endogenous oxidation. Consequently, MCT may have the capacity to increase
endogenous oxidation, and therefore to potentially decrease adipose tissue pool size
dunng extended feeding.
3.6 References
1. Lavau MM and Hashim SA. Effect of medium chah triglyceride on lipogenesis and
body fat in the rat. J Nutr 1978; IO8:613-620.
2. Geliebter A, Torbay N, Bracco EF, Hashim S 4 Van Itallie TB. Overfeeding with
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3. Crozier G, Bois-Joyeux B, Chanez M, Girard J, Peter J. Metabolic effects induced
by long-term feeding of medium-chah triglycerides in the rat. Metabolism
1987;36:807-814.
4. Dulloo AG, Mensi N, Seydoux J, Girardier L. Differential effects of high-fat diets
varying in fatty acid composition on the effeciency of lean and fat tissue deposition -. dunng weight recovery after low food intake. Metabolism 1995;44:273-279.
5. Johnson RC, Young SK, Cotter R, L Lin, Rowe WB. Medium-chain-triglyceride
lipid emulsion: metabolism and tissue distribution. Am J Clin Nutr 1 990; 52: 502-
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6. Souza PF, Williamson DH. Effects of feeding medium-chain triacylglycerols on
matemal lipid metabolism and pup growth in lactating rats. Br J Nutr
1993;69:779-787.
7. Lieber CS, Lefevre 4 Spritz N, Feinrnan L, DeCadi LM. Difference in hepatic
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8. Paust H, Keles T, Park W, Knoblach G. (1990) Fatty acid metabolism in infants.
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al, ed.), pp 1-22. Intercept Ltd., Great Britain.
9. Mascioli Eq Randall S, Porter Kater G, Lopes S, Babayan VK, Blackburn
GL, Bistrian BR Thennogenesis fiom intravenous medium-chah triglycerides.
PEN J Parenter Entera1 Nutr 1991;15:27-3 1.
10. Metges C and Wolfram G. Medium- and long-chah triglycendes labelled with 13C:
A cornparison of oxidiation after oral or parenteral administration in humans. J
Nutr lggl;l21:3 1-36,
1 1. Schoeller DA, Klein PD, Watkins JB, Heim T, Maclean Jr. WC. "C abundances of
nutrients and the effect of variations in ''C isotopic abundances of test meals
- .. formulated for l X O 2 breath tests. Am J Clin Nutr l980;33:2375-3385.
12. Watkins JB, Klein PD, Schoeller DA, Kirschner BS, Park R, Perman IA.
Diagnosis and differentiation of fat malabsorption in children using '-'c-labelled
lipids: Trioctanoin, triolein, and palmitic acid breath tests. Gastroenterology
1982;82:9 11-917.
13. Dernmelrnair H, Sauerwald T, Koletzko B, Richter T. New insights into lipid and
faay acid metabolism via stable isotopes. Eur J Pediatr 1997; 1 56(S 1 ): S70-S74.
14. Jakobs C, Kneer J, Marin D, Boulloche J, Brivet M, Poll-The BT, Saudubray JM.
In vivo stable isotope studies in three patients affected with rnitochondrial fatty
acid disorders: Lirnited diagnostic use of I-"C fatty acid breath test using bolus
technique. Eur J Pediatr 1997; 1 S6(S 1):S78-S82.
15. Binnert C, Pachiaudi C, Beylot M, Hans D, Vandermander J, Chantre P, Riou JP,
Laville M. Influence of human obesity on the metabolic fate of dietary long- and
medium-chah triacylglycerols. Am J Clin Nutr 1998;67:595-60 1.
16. Mitnin MD, St Jeur ST, Hiil L& Scott BJ, Daugherty SA, and Koh YD. A new
predictive equation for resting energy expenditure in healthy individuals. Am I Clin
Nutr 1990;51:241-247.
17. Bell L, Jones PJH, Telch J, Clandinin MT, and Pencharz PB. Prediction of energy
needs for clinical studies. Nutr Res 1985;s : 123- 129.
1 8. Health and Welfare Canada. Ntltrient Recommendariorzs: The report of the
scie~rtfic review cornmittee. Ministry of Supply and Services, Ottawa, Canada,
1990, p203-204.
19. Folch I, Lees M, Sloan S. A simple method for the isolation and purification of the
total lipids from animal tissues. J Biol Chem 1957;226:497-509.
20. Al Makdessi S, Andrieu JL, Bacconin A, Fugier JC Hedier H, Faucon G. Assay of
lipids in dog myocardium using capillary gas chromatography and derivitization
with boron trifluoride and methanol. J Chromatogr 1985;339:25-34.
21. MacDougall DE, Jones PI, Kitts DD, Phang PT. Effect of butter compared with
tallow consumption on postprandial oxidation of myristic and palmitic acids. Am .i
Clin Nutr l996;63 :9 1 8-924.
22. IMng C, Wong WW, Shulman RJ, OBrîan Smith E, Klein PD. ["C]bicarbonate
kinetics in humans: intra- vs. interindividual variations. Am J Physiol
l983;245:Rl9O-R202.
23. Jones AE. The gastroiatestinal handling and metabolic disposal of dietary lipid.
1 996. Doctoral thesis, University of Sout harnpton, United Kingdom.
24. MacDougall DE, Jones PJ, Vogt J, Phang PT, Kitts DD. Utilization of myristic
and palmitic acid in humans fed difFerent dietary fats. Eur J Clin Nutr 1996;26:755-
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25. Weir IB. New methods for calculating metabolic rate with special emphasis to
protein metabolism. J Physiol 1949;226:497-509.
26. Jones PJH, Pencharz PB, Clandinin MT. Whole body oxidation of dietary fatty
acids: Implications for energy utilization. Am J Clin Nutr l985;42:769-777.
27. Hirsch J, Farquhar JW. Ahrens EH, Peterson ML, Stoffel W. Studies of adipose
tissue in man. A microtechnique for sampling and analysis. Am J Clin Nutr
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28. Ostwald R Okey R Shannon A, Tinoco J. Changes in tissue lipids in repsonse to
diet. 1. Fatty acids of subcutaneous, mesenteric and interscapular fat. J Nutr
29. Beynen AC, Hemus RJJ, Hautvast JG. A mathematical relationship between the
fatty acid composition of the diet and that of the adipose tissue in man. Am I Clin
Nutr 1980 33:81-85.
30. Jones P H , Toy BR, Cha MC. Differential fatty acid accretion in heart, liver and
adipose tissues of rats fed beef tallow, fish oil, olive oil and safflower oils at three
levels of energy intake. J Nutr 1995 125: 1 175- 1 182.
3 1. Field CJ, Angel 4 Clandinin MT. Relationship of diet to the fatty acid
composition of human adipose tissue structural and stored lipids. Am J Clin Nutr
l985;4S: 1206-1220.
Table 3.1. Sample menu used during the dietary intervention
- --
Breakfast Lunch Dinner
'Vegetable Omelette1 *Griiled Ham & Cheese Spaghetti with Meat Sauce
Fruit and Fibre Cereal Diet Soda Parmesan Cheese
1% MiIk Grapes * Garlic Toast
Orange Juice *Oatmeal cookie Celery
'Bluebeny muffin Fruit Juice
Raisins *Date Square
I * Denotes food items to which treatment fat was added
Table 3.2. Fatty acid profile and percentage of 1-"C rnyristic, palmitic, and stearic acids delivered in treatment dose on medium chain and long chain triglyceride dieiary treatments
MCT - -
% of total fatty I3C labelled fatty acid (%/g total acid spectmm of C14:0, C16:0, C18:O)
Fatty Acid
C 8:O 3.9
C 10:o 4
C 12:O 17.7
C 14:O 13.3
C 16:O 25.4
C 1 6 1 0 7 1.6
C I8:O 8.1
C 18:l 0 9 19.3
C 18:l 0 7 0.6
C 18:2 06 4.8
LCT
% of total fatty I3c labelled fatty acid (%/g total acid specimni of C14:0, C16:0, C18:O)
Table 3 $3. Substrate oxidation as measured using respiratory gas exchange
Fat (g15.5 h) 20.9 î 1.5 18.3*1.8 19.1i1.3
MCT Parameter
Day 7' Day 14'
Carbohvdrate W 5 . 5 h) 36.0 i 2.4 40.5 & 2.5 1 39.3 * 3.4 38.6 * 2.3
LCT
Day 7' Day 14l
Total oxidation of substrate in the postprandial period as measured over 5 . 5 h following ingestion of scheduled of scheduled breakfast meal.
3.7 Figure Legends
Fiare 3.1
Cornpanson of pre-meal %O "CO, enrichment in breath over baseline for MCT vs. LCT
diets from day 7 to 14 after oral administration of "C labelled mixture of [l-"Cl-M4 -
P 4 and -SA at each breakfâst meal. Values are overall as they are compared to the initial
baseline at day 7. Arrows indicate daily delivery of breakfast isotopic mixture. Letters (A
or B) indicate day of two day rotating menu cycle. Overall whole body oxidation of
labelled fatty acids was greater (p<O.OOI) on the MCT vs. the LCT diet.
Fipure 3.2
Hourly recovery of labelled mixture of [I-'~C]-MA, -PA, and -SA as breath "COz on A)
day 7 after first oral administration of isotopic mixture at breakfast (time O); there was a
main effect (pC0.02) of diet on hourly recovery with greater recovery on the MCT diet at
4.5 h. B) on day 14, after daily oral administration of isotopic mixture at breakfast (time
O) starting on day 7. Values represent overall appearance of label as they were calculated
relative to the day 7 baseline. There was a main effect (p<0.00 1) of diet on hourly
recovery with greater recovery on the MCT diet at 1.5 h and 3.5 h . C) on day 14 after
repeated oral administration of isotopic mixture at breakfast (time O) starting on day 7.
Values represent relative appearance of label as they were calculated relative to the day 14
pre-dose baseline. There was a main effect (p<O.006) of diet on hourly recovery with
greater recovery on the LCT diet at 2.5 h and 5.5 h. Significant dzerence between diets
indicated by * (p<O.OS).
Fi-are 3.3
Cumulative dose recovery over 5.5 h of IabeUed mixture of [ 1 -"C]-h& -P4 and
breath ''CO2 (A), net oxidation of MA, PG, and SA over 5.5 h (B) and percent
contribution of combined P 4 and SA to total fat oxidation (C) on both MCT and
LCT treatments on days 7, day 14 relative to the day 7 baseline (overdl value) and day 14
relative to the day 14 baseline (relative value). Cumulative dose recovery (p<0.007), net
oxidation (p<0.01), and percent contribution to total fat oxidation (pC0.0 1) were different
between dietary treatments for the overall day 14 values. Overall cumulative dose
recovery (p<0.03), net oxidation (p<0.03), and percent contribution to totd fat oxidation
(pC0.02) within the MCT dietary treatment on day 14 were greater as compared to day 7.
Between diet comparisons for each day are indicated by letters, with different letters
indicating significant difference (p<0.05). Significant within diet comparisons are
indicated by * (p<O.OS).
0.0 0.5 1.5 2.5 3.5 4.5 5.5
Hours After Dose
Dny 11 Overrll
Hours After Dose
Day 14 Relative 6 , i
Hours Amr Dose - MCT LCT
Day 7 Day 14 overall Day 14 relative
Days on Diet
Day 7 Day 14 overall Day 14 Relative
Days on Diet
Oay 7 Day 14 ovenll Day 14 relative
I MCT Q LCT QO
BRIDGE
In the previous study, the repeated 13C dosing paradigm demonstrated that the
presence of MCT within the diet increased endogenous oxidation of the combination of
the saturated meal fats, MA, Ph and S 4 by day 14 of feeding in a way that the presence
of predominantly LCT did not. In addition, the pattern of label appearance over the 8 day
isotope administration period indicates that the increased endogenous oxidation may stem
fiom the shunting of FA of different chah lengths into one of two FA storage pools.
However, despite transient increases at certain time points following breakfast meals, there
was no effect of dietary treatment on exogenous oxidation of P 4 and SA.
With the overail thesis objective of a step-wise assessrnent of fat oxidation and
energy utilization, what remained to be determined was whet her the differences detect ed
in endogenous oxidation of the saturated meai fats were influencing pre- or postprandial
whole body substrate oxidation and energy utilization. Would the levels of MCT as
cornpared to LCT present in these palatable Nonh Amencan-style diets measurably affect
BMR, TEF and whole body fat oxidation over 7-14 days, a period longer than previously
studied? Therefore, Our objective was to measure Oz consumption and CO, production
using RGE on days 7 and 14 of feeding to determine the effects of FA chah length on
B m TEF, RQ, and pre- and postprandial fat and carbohydrate oxidation.
CHAPTER 4
ENHAiYCED POSTPRANDIAL ENERGY EXPENDITURE WITE MEDIUM
CHAIN FATTY AClD FEEDING IS AITENUATED AFTER 14 DAYS IN
PREMENOPAUSAL W OMEN
Matthew D. White, Andrea A. Paparnandjaris, Peter J.H. Jones
Americun Journal of Clhical Nutrition, 1998 (in press)
SchooI of Dietetics and Human Nutrition,
Faculty of Agncultural and Environmental Sciences,
McGill University, Macdonald Campus, Ste-Anne-de-Bellevue, Quebec, H9X 3V9
Corresponding Author:
Peter J.H. Jones, Ph.D.
School of Dietetics and Hurnan Nutrition,
Faculty of Agricultural and Environmental Sciences,
21 1 1 1 Lakeshore Road
McGill University, Macdonald Campus,
Ste-Anne-de-Bellevue, Quebec H9X 3 V9
4.1 Abstract
Medium chah triacylglycerides (MCT) have been reported to enhance human
energy expenditure (EE), although few studies involve women and the duration of such
effects is only known for periods of about 7 days. Numerous MCT feeding studies
utilized concentrated MCT-oiis, that unlike mixed-diet feeding of MCT, can cause
gastrointestinal disorders. The objective of this study was to determine whether women
consuMng mixed diets higher in MCT demonstrate changes in EE or substrate oxidation
afier 7 or 14 days relative to consumption of long chah triacylglycerols (LCT) over the
same length period. Twelve non-obese pre-menopausal women were fed eucaloric mixed
diets containhg either butter and coconut oil or beef tallow, during two separate 14 day
feeding periods. Both diets included 40% fat, which included four-fiflhs treatment fat,
45% carbohydrate and 15% protein. On days 7 and 14 of each trial, basal metabolic rates
(BMR, Id/min), in addition to total energy expenditure (TEE, Hlmin) and thermic effect
of feeding (TEF, i.e. TEE-BMR, akJ/min) following a standardized breakfast were
measured by respiratory gas exchange. On day 7, the mean BMR (mean SE) (3.58 * 0.1 1 Idfmin) for the MCT was greater (p=0.0003) than that of 3.43 0.1 1 kJ/min for the
LCT diet. The mean day 7 postprandial TEE (4.36 k 0.04 kJ/rnin) for the MCT diet was
also greater @-0.04) than that (4.23 0.04 kJ/min) for the LCT diet. By day 14, the
postprandial TEE for the MCT was attenuated in cornparison to the LCT diet. No
dserences in the thermie effect of feeding were evident between diets. Present evidence,
fiom the longest controiied MCT feeding study to date, indicates that shon term feeding
of a MCT-enriched relative to a LCT-enriched diet can increase EE, but that this effect
could be transient with continued feeding.
4.2 Introduction
The composition of dietary fat is thought to influence postprandial energy
expenditure (EE) and substrate oxidation rates in rodents (1) and humans (2-6).
Specifically, the fatty acid chah length is thought to be a deteminant of the rate of
postprandial substrate oxidation and total EE (8). Over the postprandial penod, the
thennic effect of food (TEF) and fat oxidation were shown by Hill and colleagues (4) to
be greater when medium chain triacylglycerols (MCT) were ingested relative to long chain
triacylglycerols (KT). In hospital patients receiving total parenteral nutrition, similar
responses were observed (5) when patients showed no metabolic responses to LCT
infusions, but significant TEF and increases in fat oxidation for MCT infusions.
Additionally, a MCT-containing diet was shown to stimulate 2 4 4 EE by 5 percent in
cornparison to a LCT diet (6). These studies suppon the postulate that MCT relative to
LCT ingestion increases postprandial EE possibly due to an enhanced postprandial fat
oxidation.
The evidence supporting that MCT increases postprandial EE and fat oxidation (2-
6) is not without exception. Flatt and colleagues (8) showed that postprandial energy
expenditure, as well as fat and carbohydrate oxidation, were similar following breakfasts
that were low in fat, MCT-enriched or LCT-enriched. Similarly, MacDougall and
colleagues (9) showed no difference for the mean of days 8 and 11 postprandial EE and
substrate oxidation following a brealâast increased in either MCT or LCT. FoUowing
longer term low calorie diets (10,l l), women showed similar weight losses between diets
enriched in either MCT or LCT. The results from studies with feeding of high levels of
MCT suggest that a positive effect on EE and fat oxidation is possible for periods up to
about 7 days (46). It is questionable, however, if longer feeding periods would show
these differences (10,ll). In addition, only 2 studies report MCT vs. LCT feeding in
women (10,l l), since other studies have been generaüy restricted to men (2-6,8).
The purpose of the present study was to determine if farty acid chain length
idiuences EE and substrate oxidation in women being fcd controlled, eucalonc, Norih
Amencan diets that were consumed under supervision in a clinical research unit. Feeding
diets enriched in either MCT or LCT over 2 weeks allowed the subjects' responses to be
compared over a penod at least twice as long as previously reported (4-6).
4.3 Subjects and Methods
Twelve women participated in the study and sarnple size was calculated using the
tables of Machin and Campbell (12). The cntena for selection were that subjects were
normolipidemic, regularly menstmating, non-obese and between 18 and 30 years of age.
The subjects selected were 22.7 a0.7 years of age (mean * SE). They had a mean weight,
height and BMI of 56.2 * 1.5 kg, 1.62 1.20 m and 2 1.5 * 0.8 k W , respectively (refer
to Appendix il for individual subject information). Ail subjects were informed of al1
inherent risks in the study and were instructed that they could withdraw their pariicipation
at any time without prejudice. The protocol was approved by the McGiIl University
Human Ethics Cornmittee and pnor to the study aii subjects signed an informed consent.
The feeding trials were conducted in the Mary Emily Clinical Nutrition Research
Unit (CNRU) of the School of Dietetics and Human Nutrition on the MacDonald Campus
of McGill University in Ste-Anne-de-Bellevue, Canada. The two mixed diets prepared in
the unit included typical North Amencan foods. The composition of the diet included
40% fat, which was comprised of four-Mhs treatment fat, 45% carbohydrate, and 15%
protein. Treatment fats were butter and coconut oil, to increase the proportion of MCT,
or beef tallow to increase the proportion of LCT. Durhg food preparation al1 foods were
weighed to the nearest 0.1 gram. The proportions of fatty acids in each of the MCT and
LCT diets are given in Table 4.1. The eucalonc energy intake was set with the Mifflin
equation to set the basal metabolic rate (13) multiplied by an individualized activity factor
(1 4). The Bell factor, as recommended by the authors (l4), was adjusted for athletically
active subjects using a table adapted fiom Passmore and Dumin (1 5). This gave a mean
activity factor of 1.72 * 0.05. This maintained subjects weights at 56.6 * 0.4 kg for the
MCT- and at 56.5 a 0.4 kg for the LCT-ennched diet. Feeding was closely supe~sed
throughout the trials to ensure al1 food provided was consumed. Subjects showed an
excellent tolerance to these diets and consurned the prescribed food. Over 99 percent of
the meals were eaten in the CNRU and as such on only a few occasions were prepackaged
meals consumed outside the CNRU.
A Deltatrac metabolic monitor (Sensonnedics, Anaheim, Caüfomia, USA) was
employed to determine oxygen consumption and carbon dioxide production, both
expressed at standard temperatun and pressure dry. A transparent ventilated hood was
positioned over the abject's heads with Collins tubing comecting the hood to the
monitor. FoUowing a minimum of 30 minutes warm-up, a reference gas (5% Co,%%
oxygen) was used to calibrate the oxygm and carbon dioxide analysers. Validation of
Deltatrac analysers against a lung model has shown (16) an accuracy of 1.9% for oxygen
consumption and 1.5% for carbon dioxide production. Values fiom the Deltatrac were
collected every minute with a personal computer.
Each diet was fed for a 14-day period, separated by 14 days when the subject
resumed a habitua1 eating pattern. The subject was blinded to the diet type and randornly
placed on either the MCT or LCT diet in the first 14-day period. An equal number of
wbjects began the first 14 day feeding penod on each of the MCT and LCT diets. On
days 6 and 13 of each diet, subjects slept at the CNRU and on the following morning at
approximately 7 a.m., after a 10 to 12 h fast, BMR was measured over 30 min. Following
a standardized breakfast, subject's expired gases were continuously collected with a
ventilated hood over 5.5 h with short washroom breaks permitted. The standardized
breakfast possessed the same composition as the diet and contained one-third of each
subject's predetermined daily energy intake. Throushout the postprandial period the
subject relaxed in a semi-recumbent position and was awake reading or watching
television.
Following ovulation core temperature increases from about 0.5 to 1 O C and for
each degree Celsius increase in body temperature metabolic rate increases by 10 to 12
percent (1 7). This effect has been shown to elevate 24-h energy expenditure by 8 to 16%
(1 8) and post-ovulatory TEF is depressed in cornparison to preovulatory TEF (1 9). To
control for this body temperature effect, we employed a 14 day feeding period followed by
a 14 day washout penod. Since the subjects were regularly menstniating and had close to
28 day cycles, this put them on approximately the same day of their menstrual cycle for
the testing days in each dietary condition.
Replicate portions of the two diets were homogenized using a commercial blender
and relative fatty acid percentages were determined using gas Iiquid chromatography after
iipid extraction (20) and boron trifluoride methylation. The gas chromatograph (Hewlett
Packard 5890 Series LI) was equipped with an autosarnpler and flame ionization detectors.
Separation was achieved on a SP2330, 30 m capillary colurnn, 0.2 mm interna1 diameter,
0.25 pm film thickness. The split ratio was 50: 1. Running conditions were: initial
temperature 80 O C with a ramp of 1 O0C/min until 160" C that was held for 10 min followed
by a ramp at 10°C/min to 220°C that was held for 12 min followed by a final ramp of
10" Clmin until a maximum temperature of 240" C was reached that was held for 5 min.
Individual fatty acids were identified against standards using retention times.
Energy expenditure was expressed as postprandial total energy expenditure (TEE,
Wlmin). The thermic effect of food (TEF, akJ/rnin) was calculated as the difference in
postprandial EE from the EE values measured in basal conditions pnor to breakfast (or the
BMR). Mean postprandial TEE, mean fat @/min) and mean carbohydrate oxidation
(ghin) rates across the entire postprandial period, were calculated as the mean of 30
consecutive min periods. On each diet the integrated TEE (kJ) is given by the culmulative
sum of consectutive 30-min penods following the breakfast. The integrated values for
total wbohydrate (g) and total fat (g) oxidized are also given over the postprandial
period. Respiratory quotient (RQ, unitless) was calcuked as the carbon dioxide
production over oxygen consumption. Weir's fonnula was used to give an estimate of the
fat and carbohydrate oxidation (21). Modifjing Weif s formula to account for the error
due to the oxidation of MCT instead of LCT was estimated at less than 1% (3) and as
such the equation was used unmodified. A constant oxidation of 0.7 g of protein per
kilogram of fat free mass was assumed since an inaccuracy of up to 30% for protein
oxidation wouid have no significant eKect on the substrate oxidation rates (22).
A cross-over repeated measures ANOVA mode1 was employed. The factors
employed, as applicable, were diet (MCTor LCT), day (day 7 or day 14) and hour (0.5 to
5.5 h). A sequence factor for diet was added to control for cross-over effects as
recornmended for this type of repeated measures design (23). Post-hoc comparisons at
selected 30-min intervals were made between treatment conditions using contrasts. For
multiple pst-hoc comparisons the Bonferonni statistic was applied to control for the level
of Type 1 error. To assess body weight, a repeated measures ANOVA (diet and day) was
employed.
4.4 Results
Energy expenditure values on day 7 and 14 are compared as a function of dietary
fat type in Figure 4.1. At time zero, on both measurement days, the mean rate of energy
expenditure in basal conditions is given. The main effect of diet (levels: MCT and LCT)
for BMR was (F46.11, p=0.00 1) and there was no diet by day (levels: day 7 and day 14)
interaction for BMR. On day 7 the BMR of 3 .S8 k 0.1 1 Id/min for the MCT diet was
greater (p=0.003) than that of 3.43 0.11 kllrnin for the LCT diet. On day 14 the BMR
of 3.63 i 0.08 k h i n for the MCT diet also tended to be greater @=0.06) than that of
3.49 * 0.09 idmin for LCT diet.
The MCT relative to the LCT diet gave a greater postprandial TEE on day 7 with
an attenuation of this effect on day 14. The main effect of diet for TEE was significant
(F=30.87, p=0.00 1) and there was a diet by day interaction for TEE (Ft5.22. p=0.02).
Following the standardized breakfast on day 7, post-hoc mean cornparisons showed
postprandial TEE was significantly greater at different time points between 0.5 and 3 h for
the MCT relative to the LCT diet (Fig. 4.1). These postprandial TEE differences are
reflected on day 7 by a mean rate of TEE (4.36 * 0.04 kJ/min) for the MCT diet that was
greater (p=0.04) than the mean rate of TEE (4.23 k 0.04 kJ/min) for the LCT diet. On day
_ . . 14 the postprandial differences in TEE were attenuated, as supported by the significant
O diet by day interaction terni for TEE. This attenuation of the difference between diets was
supported by the day 14 mean rate of TEE for the MCT diet that was 4.38 * 0.03 k.J/min
and this was not different than that of 4.29 * 0.03 k h i n for the LCT diet.
For the MCT diet on day 7, the integrated TEE of 1439 * 34 kJ for the 330 min
postprandial measurement period was not greater than the integrated TEE of 1395 28
kJ/330 min for the LCT diet. On day 14 the integrated TEE postprandial measurement
was 1444 k 25 i d 3 3 0 min for the MCT diet and this was not different than the 14 13 * 27
id330 min for the LCT diet. The main effect of diet and the diet by day interaction for
TEF (Le. TEE-BMR) were both not significant. On day 7 the mean TEF was 0.79 * 0.03
Idlrnin for the MCT and 0.80 * 0.03 Id/min for the LCT. On day 14 the mean TEF for
the MCT diet was 0.75 * 0.04 Wmin and 0.79 * 0.03 k h i n for the LCT diet.
For RQ the main effèct of diet (F=34.72, p=û.0001) and the diet by day interaction
(F=6.98, p4.009) were both significant. No signiticant âiierences for the RQ during the
basal conditions were evident between the two dietary conditions. At different time points
following the standardized breakfast, the RQ on day 7 for the LCT diet was greater than
for the MCT diet (Fig. 4.2). The main effect of diet (F=43.57, p=0.0001) and the diet by
day interaction (F4.69, p=0.004) were both significant for fat oxidation. This main effect
of diet was evident at different time points in the postprandial period on day 7 when the
rate of fat oxidation was greater for the MCT diet than for the LCT diet (Fig. 2). The
main effect of diet (F=18.77, p=O.OO 1) and the diet by day interaction (F=4.23, p=0.04)
for carbohydrate oxidation were also significant. On day 7 for carbohydrate oxidation
there was evidence for a lower rate for the MCT relative to the LCT condition. On day
14, as supponed by the significant diet by day interactions, each of the rates of substrate
oxidation and RQ were sirnilar between conditions.
There was no effect of diet or a diet by day interaction for total fat (g) or total
carbohydrate (g) oxidation. In the postprandial period the total fat oxidation on day 7 was
20.9 * 1.5 g for the MCT diet and 18.3 1.8 g for the LCT diet. By day 14 these total fat
oxidation values were nearly identical at 19.1 I 1.5 g for the MCT diet and at 19.1 * 1.3 g
for the LCT diets. On day 7, carbohydrate oxidation rates were 36.0 2.4 g for the MCT
diet and 39.3 3.4 g for the LCT diet. On day 14 total carbohydrate oxidation was 40.5
2.5 g for the MCT diet and 38.6 2.3 g for the LCT.
There was no main effect of diet, afker correcting the mean rates of fat oxidation
for basal rates of fat oxidation, during the postprandial period on day 7. Mean values
were sirnilar at 0.001 0.002 g/min and -0.007 i 0.002 g/min for the MCT and LCT
diets, respectively. For corrected fat oxidation there was, however, a diet by day
interaction (F=4.34, p=0.04). On day 14 the postprandial fat oxidation rates were -0.007
i 0.002 &min for the MCT diet and -0.005 i 0.002 gfrnin for the LCT diets. For
carbohydrates, corrected for basal rates of oxidation, there was no main effect of diet or
diet by day interaction. The carbohydrate oxidation rates on day 7 were 0.044 1 0.004
g/mh and 0.062 0.004 ghin for the MCT and LCTdiets, respectively. On day 14,
carbohydrate oxidation rates of subjects corrected for basal rates of oxidation were the
same for both conditions at 0.059 0.004 &in.
The RQ and substrate oxidation data were grouped by diet and compared between
days 7 and 14 (Fig. 4.3). No main effect of diet or diet by day interaction for substrate
oxidation rates and RQ were observed for within diet between day comparisons.
4.5 Discussion
The effect of controlling the fatty acid chain length in the fat component of a mixed
diet for these women showed a time dependent effect on both energy expenditure and
substrate oxidation. On day 7, for the MCT- vs. the LCT-containing diet, there were
small and significant increases of BMR and postprandial TEE (Fig. 1). Also on day 7, the
fat oxidation at different time points over the postprandial penod was significantly greater
for the MCT in cornparison to the LCT diet (Fig. 2). This effect of MCT on both of the
postprandial energy expenditure and fat oxidation was attenuated by day 14 of the feeding
trials (Fig. 1 and 2). The B m in contrast, showed a trend for a sustained elevation for
the MCT relative to the LCT diet on day 14 (Fig. 1). The postprandial TEE results on
day 7 are consistent with studies using a mixed, weight maintenance diet, pnor to a single
fat load, as they illustrate an increase in postprandial EE for MCT in cornparison to LCT
treatments (2-5). In addition, for short-term, with a prefeeding of treatment fats for
approximately one week (4,5), there is also an elevated postprandial EE for a MCT
compared to a LCT treatment. The day 14 results are supponed by studies of prolonged
but uncontrolled (out-patient) feeding that are show to have a diminishing effect on
energy balance as the duration of MCT-ennched dietary treatments are extended beyond a
few days to several weeks (1 0,11,24). Specifically, for women fed either MCT-exuiched
vs. LCT-enriched hypocalonc diets (10, I l ) or for subjects fed MCT-ennched vs. non-
- . MCT enriched hypocaloric diets (24), similar weight losses were reported within the
studies irrespective of the treatments. Together the evidence suggests that the positive
effect of MCT on energy balance are diminished with longer feeding trials.
The studies reporting resting or basal metabolic rates of subjects fed MCT and
LCT diets, for periods from 1 to 28 days, show both no effect (4,9) or an increase of
preprandial EE with a MCT relative to either of a LCT infusion (5) or a non-MCT
hypocaloric diet (24). Subjects supplemented with MCT oils during a 28 day hypo~alonc
diet (24). despite a weight loss of 10 kg, maintained their prediet BMR. Concurrently, the
non-MCT oil control group (24) demonstrated the same 10 kg Ioss and the associated
drop in their BMR that is nonnally seen during such weight reductions (25.26). Patients
on total parenteral feeding of MCTand LCT oils (5) showed an elevation in resting
metabolic rate for the MCT infusion on days 1, 3, and 5 of the feeding supporting the view
that MCT increases preprandial EE. Reports of an effect of MCT in the preprandial
period are thus divided with studies showing no effect (4,9) or a positive effect (5,24).
The present results support a positive effect of MCT relative to LCT feeding on BMR
after 7 days, and possibly after 14 days of feeding (Fig. 1); however, this topic clearly
merits fùrther study in order to resolve these discrepancies. If MCT have an effect on
BMR this could have considerable importance in weight maintenance since basai metabolic
processes make up the large part of daily energy expenditure.
Ravussin and Swinbum (27) show that the coefficients of variation for repeated
TEF responses varied between 4 and 48 percent. This large variability of TEF responses
may cast Iight on discrepancies for TEF responses to MCT or LCT feeding. Presently no
- . TEF difference was apparent between MCT and LCT treatments and this was also
O reported previously in similar feeding studies (8,9). Some studies, however, have shown a
greater TEF for MCT relative to LCT treatments (2-5). These discrepancies in TEF
response could be related to existence of a pre- feeding protocol or to the quantity of
MCT utilized. Generally for studies with a prefeeding of the treatment fats (8,9),
including the present study, no TEF difference between MCT- and LCT- diets was
apparent. In contrast, studies (2.3,s) that showed an elevated TEF after a single MCT-
vs. a single LCT-load did not include a prefeeding of treatment fats. In addition, when
marked TEF difFerences between MCT and the LCT regimes were reported (2-5),
commercial MCT oils ranging frorn 75 (5) to 100% (2) of the total fat energy intake were
employed. These quantities of MCT are considerably greater than the quantity of MCT
used presently in the mixed diet (Table 4.1). Together the evidence points towards a TEF
that is most elevated aAer a single fat load including a high proportion of MCT (2-5) and
for a protocol with no pre-feeding of the treatment fats (2, 3, 5). These differences in
protocols for studies comparing TEP responses to MCT and LCT treatments, plus the
highly variable nature of TEF responses (27), suggest it would be difficult to make firm
conclusions about TEF responses to MCT or LCT treatments. These comrnents on TEF
apply equally to substrate oxidation rates corrected for basal rates of oxidation.
A short-tenn effect of MCT feeding on the postprandial fat and carbohydrate
oxidation appeared to be evident during this study but overall the evidence is not
compelling. In comparison to the LCT diet responses on day 7, greater rates of fat
oxidation and lower rates of carbohydrate oxidation were evident for the MCT diet (Fig.
2). There is evidence to suppon that with feeding MCT- vs. LCT-ennched diets, greater
postprandial fat oxidation rates are evident (3-5) although studies results are not without
exception (2,8,9). When considering fat oxidation, it should be mentioned that when
feeding higher levels of MCT (Le. MCT-oils) than those employed in the present rnixed
diet, caution should be exercised when employing indirect calorimetry if the substrate mix
possibly contains ketones (28,29).
The diet and day interaction term was significant for each of the RQ, fat oxidation
and carbohydrate oxidation. The source of these interactions is apparent by the differences
for each of RQ, fat and carbohydrate oxidation on day 7 in the postprandial period that
were diminished by day 14 (Fig. 2), for maintained levels of each variable within each diet
(Fig. 3). Studies of MCT feeding show an improved glycemic control after 5 days, as
evidenced by increased amount of glucose needed for an euglycemic clamp (10,30), and
improved glucose tolerance, as judged by a higher glucose disappearance rate (3 1). With
long term feeding of MCT there is also a higher expression of hepatic lipogenic enzymes
(32,33) and between days 1 and 6 of a MCT diet in humans, a 3-fold increase in total
plasma triacylglycerols was evident (34). These studies (32-34) suggest an increased
hepatic lipogenesis from the medium chah fatty acid (MCFA) rnoieties and this supports a
decreased fat oxidation as MCT feeding is proionged. Together the evidence supports
that with longer term feeding of MCT there is a diminished stimulation of postprandial fat
oxidation and an improved glucose tolerance (8).
The origin of the elevated postprandial energy expenditure for the MCT, in
comparison to the LCT diet, appears attnbutable to the different metabolic fates of
MCTand LCT(7,32). The large majority of MCFA foilowing absorption are weakly
bound to albumin (35) in the portal circulation and are preferentially oxidized to acetyl-
CoA (7). No single mechanism accounting for the elevated postprandial EE by MCT is
evident and several hypotheses have been presented (7). LCT, in contrast to MCT, is
mostly incorporated into chylomicrons and enters the circulation through the lymphatic
system. Long chah tnacylglycerols fi-om chylomicrons are mostly taken up by peripheral
adipose tissue for storage; this is l e s costly energetically than the ketogenesis or
lipogenesis from MCFA (36).
Based on the day 7 increase in EE for the MCT relative to LCT diet, projected to
24 h, this gives about 160 kcal or 6 percent of a typical2500 kcal (- 10, 500 kJ) per day
diet for these subjects. This value is supported by the findings of Dulloo and colleagues (6)
who showed using whole body indirect calorimetry a 5 percent increase in 24-h EE for
MCT vs. LCT feeding. If such an increase in 24-h energy expenditure were maintained
over about 20 days, it would contribute to the loss of calories approximately equivalent to
a pound of fat (0.45 kg). This potential benefit over the longer term, however, a p p a s
questionable since the effect of the MCT diet on EE was diminished by day 14 of the
stud y.
In conclusion, on day 7 of a 14-day feeding trial of moderate amounts of MCT in a
mixed diet, there were increases to the basal metabolic rate, postprandial energy
expenditure, and fat oxidation in non-obese college aged women. These positive effects
of the MCT diet on energy expenditure were decreased when feeding was extended to 11
days, a period longer than previously reported. Evidence supports that dietary MCT, at
the present levels, can increase energy expenditure but this effect could be transient.
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Table 4.1. Fatty acid spectmm of the two test diets. Values are percentage of the total spectrum observed.
Fatty Acid Chain Length
MCT
3 -9
4
17.7
13.3
25.4
1.6
8.1
19.3
0.6
4.8
LCT
4.7 Figure Legends
Figure 4.1
Cornparisons on day 7 and 14 between dietary conditions (MCT or LCT) of basal
metabolic rates and postprandial energy expenditure following a standardized breakfast
enriched in either treatment fat The histograms give the mean rates of the MCT (white
bar) and LCT (black bar) conditions. (Levels of significance are as follows: 5 p<0.05, *
p<0.004; f' pcO.00 1).
Fieure 4.2
Cornparisons on days 7 and 14 between dietary conditions (MCT or LCT) of respiratory
quotient together with fat and carbohydrate oxidation in the pre- and postprandial periods
following a standardized breakfast e ~ c h e d in either treatment fat. For levels of
significance please refer to the legend of Figure 4.1.
Fieure 4.3
The cornparisons for MCTor LCT diets between testing days 7 or 14 for the respiratory
quotient together with fat and carbohydrate oxidation, in pre- and postprandial penods
following a standardized breakfast emiched in either treatment fat. For levels of
significance please refer to the legend of Figure 4.1.
Postpmdial time (hou=) PostprandiaI time @ours)
DAY 14
MCT LCT
BRIDGE
Results from the RGE analysis presented in Chapter 4 demonstrated that up to
periods of 7 days, feeding of MCT vs. LCT had the capacity to positively influence B m
RQ, and rate of fat oxidation at certain tirne points foiIowing consumption of breakfast.
Nonetheless, by day 14, the effect of FA chain length on these parameters was attenuated.
Therefore, it appeared that beyond 7 days, the effect of MCT on acute energy metabolism
was blunted. This methodology, however, was not capable of assessing the effea of MCT
on whole body free-living daily energy expenditure. Were differences seen on day 7 using
the RGE methodology in Chapter 4 and over the two week period using the stable isotope
methodology in Chapter 3 detectable at the whole body level using current techniques of
measurement?
In order to determine the potentiai of these diets enriched with either MCT
or LCT to alter TEE, we used doubly labelled water to meawre TEE during the second
week of each dietary treatment. The effects of MCT on free-living TEE had not been
measured to date. Examination of TEE at the whole body level would cornpiete the
metabolic assessrnent of the effect of MCT on energy utiliition and fat oxidation. In
addition, combination of the RGE measurements with the DLW TEE would allow
partitioning of the components of energy expenditure, BMR, TEF, and AIEE. Therefore,
Our objective was to assess overall TEE and to examine its components, basal metabolic
rate, thermic effect of food, and activity induced energy expenditure.
CI&APTER 5
COMPONENTS OF TOTAL ENERGY EXPENDITURE IN BEALTHY YOUNG
WOMEN ARE NOT AFFECTED AF"IER 14 DAY FEEDING WITEI MEDIUM
VERSUS LONG CHAIN TRIGLYCERIDES
Andrea A. Paparnandjaris, Matthew D. White, Peter J.H. Jones
Obesity Reseurch, 1999 (in press)
School of Dietetics and Human Nutrition,
Faculty of Agricultural and Environmentd Sciences,
McGill University, Macdonald Campus, Ste- Anne-de-Bellewe, Quebec, H9X 3 V9
Comsponding Author:
Peter J.H. Jones, Ph.D.
School of Dietetics and Human Nutrition,
Faculty of Agricultural and Environmental Sciences,
2 1 1 1 1 Lakeshore Road
McGill University, Macdonald Campus,
Ste-Anne-de-Bellevue, Quebec H9X 3 V9
5.1 Abstract
The objective of the study was to examine the effect of consumption of medium
chain triglycerides (MCT) vs. long chain tnglycerides (LCT) on total energy expenditure
(TEE) and its components in young women during the second week of a two week
feeding period. Twelve heaithy lean women (age: 22.7 i 0.7 yrs, BMI: 2 1.5 * 0.8 kgim')
were fed weight maintenance diets containing 15% of energy as yrotein, 45% as
carbohydrate, and 40% as fat, 80% of which was treatment fat, for two weeks in a
randomized cross-over design separated by a two week washout period. Dietary fat was
composed of triglycerides containing either 26% MCFA and 74% LCFq or 2% MCFA
and 98% LCFA. Free-living TEE was measured from day 7 to 14 on each dietary
treatment using doubly labelled water @LW). Basal metabolic rate (BMR) and ihermic
effect of food (TEF) were measured on days 7 and 14 using respiratory gas exchange
analysis (RGE) for 30 min and 330 min, respectively. Activity induced energy expenditure
(AIEE) was derived as the difference between TEE and the sum of BMR and TEF. The
average TEE while consuming the MCT diet (2246 i 98 kcal/day) did not differ fiom that
of the LCT diet (21 86 k 138 kcallday). BMR was significantly higher on the MCT diet on
day 7 (1 2 19 38 kcallday vs 1 179 * 42 kcdday), but not on day 14; there was no effect
of diet on TEF. There were no differences in BMR, TEF, or AlEE between diets when
expressed as percentages of TEE. On average, BMR, TEF, and AIEE represented 54.6%,
8.2%, and 37.2%, respectively, of TEE. Results suggest that between day 7 and day 14
feeding of MCT vs. LCT at these levels TEE is not a£Fected and that increases seen in
energy expenditure following MCT feeding may be of short duration. Thus,
compensatory mechanisms may exist which blunt the effect of MCT on energy
components over the longer tenn.
5.2 Introduction
Totai energy expenditure (TEE) is comprised of three components: basal
metabolic rate (BMR), postprandiai energy expenditure or the thermic effect of food
(TEF), and activity induced energy expenditure (AIEE) (1). An increase in any of these
three components in the presence of a constant energy intake cm lead to an imbalance
between intake and output resulting in weight loss. Clearly, increasing physical activity
will increase AIEE and TEE. However, diet rnay also play a role in altering TEE through
its effect on TEF, Bm or both. Studies have s h o w that dietary fat type, specifically
medium chain tnglycerides (MCT) containing medium chain fatty acids (MCFA) (C8:O - C12:0), can positively influence TEF, and therefore cm modiw TEE (2-9). In shon term
feeding studies in humans (6 h to 7 days), consumption of MCT increased TEF as
compared to long chah tnglycerides (LCT) containing long chain fatty acids (LCT) (>
C12:O) by up to 60 kcaVmeal(3-6). This increase is most likely due to stnicture-
dependent differences in fatty acid metabolic disposal during absorption (6), transportation
(7), and oxidation (8,9). In longer term studies in animais (up to 45 days), rats have
gained less weight during MCT vs. LCT feeding (10,11,12).
In free living subjects, however, data are lacking regarding extended feeding
effects of MCT on energy expenditure. The capacity of MCT to alter the energy balance
equation and modi& TEE over longer periods needs to be determined to evaluate the
potential of MCT for induction of weight loss in the obese or weight maintenance in the
post-obese. The objective of this study, therefore, was to examine the effect on TEE of
MCT- vs. LCT-based eucaloric diets fed to healthy college-age women during the second
week of 14 day feeding periods. The study population was chosen because the effect of
MCT on energy metabolisrn in women has been understudied (13,14) with the majority of
the research on this topic being conducted in men (2-5). Additionally, our goal was to
measure BMR and TEF in order to derive AIEE, and quantifi dl aspects of TEE.
5.3 Methods
5.3. f Sirbjects
Twelve healthy, non-smoking female students aged 19-26 y (Appendix II) were
chosen from a larger sample afler being screened by questionnaire. Plasma lipid analysis
was perfonned to screen for normal levels of total cholesterol(<5.2 mmoV1) and
triglycerides (4 2 4 mmoM). Only those subjects who were free of chronic disease, non-
or moderately exercising, maintaining an approxirnately 28 day menstrual cycle, and had
normal blood lipid parameters were admitted into the study. Subjects were requested to
maintain habitua1 and constant levels of activity throughout the trial. The objective of the
study and its protocol were explained to the subjects prior to obtaining written consent.
Al1 procedures were approved by the McGill University Ethics Cornmittee.
5.3.2 Diets
The study was conducted using a randomked, cross-over design with two 14 day
feeding periods separated by a two week washout period when subjects resumed their
normal diets. Subjects were blinded to diet sequence. There were 6 subjects per dietary
treatment for each 14 day feeding period and al1 subjects received both treatments.
Dunng each penod, subjects consumed prepared solid foods containing 15% of energy as
protein, 45% as carbohydrate, and 40% as fat. Of the total fat consumed, 80% was
provided as either beef tallow, comprising the LCT treatment, resulting in an overall fatty
acid (FA) breakdown of 2% MCFA and 98% LCFq or as a combination of butter and
coconut oil, comprising the MCT treatment, with an overall FA breakdown of 26%
MCFA and 74% LCFA (Table 5.1). Diets provided were designed to meet normal energy
and nutrient needs in accordance with the Recommended Nutrient lntakes for Canadians
(1 5), except for the percentage of fat in the diet (40%) which was chosen to accmnmodate
the level of treatment fat. All meals were prepared and served at the Mary Emily Clinical
Nutrition Research Unit at McGill University and, as such, subjects were outpatients
except for 2 ovemight stays per diet cycle required for BMR measurement. Ingredients
were weighed to the nearest 0.5 g. A two day diet cycle was used to provide variety and
was served as three meals of equal energy content per day. Subjects were required to
consume only food that was provided, with the exception of water, which was permitted
ad libiîum. Consumption of dl food provided was ensured through close supervision of
mealtimes. Energy requirements were determined using the Mifllin equation (1 6)
multiplied by an activity factor of 1.7 for free-living adult students (1 7); further
adjustments were made for increased activity based on a table adapted fiom Passmore and
Dumin (18). The resultant mean activity factor was 1.72 * 0.05. Caloric loads were
matched for both feeding periods. In addition, subjects were instnicted to keep an activity
diary during the first feeding period, and use the diary to dictate activity dunng the second
period.
5.3.3 fiprimerital Proiocol
The doubly labelled water @LW) total energy expenditure measurement penod
began at 07:OO h on day 7 of each dietary trial and lasted through day 14, the final day of
each dietary cycle. On day 7, pnor to consumption of DLW dose, subjects delivered
baseline urine samples after an ovemight fast. Subjects were then given an oral dose of
DLW containing 2.5 g HzMO/kg estimated total body water (TBW) (1 O atom percent
excess (APE) oxygen- 18, normalized for D) and 0.1 g D + N g estimated TBW (99.5
APE) followed by a 50 ml wash of water. Weights of DLW doses were recorded to the
nearest mg, and a 1500 dilution of the dose was prepared as a standard for use during
analyses. Second void morning urine samples were collected on days 8,9, 12, 13, and 14.
Al1 samples were fiozen at -20°C irnmediately following collection.
Both deuterium and oxygen analyses were pcrfonned in trîplicate using a VG
Isogas 903D dual inlet isotope-ratio mass spectrometer; isotopic enrichments were
analysed as diflerences in parts per thousand (1) against Viema standard mean ocean water
(SMOW). For deuterium, 2 pl of water was vacuum distilled over zinc and stored in 9 cm
break-seal tubes until isotopic anaiysis (19). Maximum acceptable precision for deuterium
was 5 Oho at enrichments over 500 O h and 2 960 at enrichments below 200 O h . Oxygen
sarnples were analysed using carbon dioxide equilibration (20). A 1.5 ml sarnple was
injected into a 10 ml vacutainer which was followed by injection of 1.5 ml of CO,.
Samples were then shaken in a 2S°C water bath for 1 h, followed by a 48 h equilibration
period. C"0, was then cryogenically rernoved and stored in 9 cm break seal tubes until
mass spectrometric anaiysis (2 1). Maximum acceptable agreement between replicates for
cM02 was 0.7 %O.
Time-zero dilution spaces and isotope elirnination rates were obtained from the y-
intercept and slope respectively of the semi-Iogarithmic plot of nomalized isotope
enrichment vs. time after dosing. Normalized e~chments were determined using the
following equation, representing ail sample e~chments as a fraction of the initiai dose
given (22):
where d is the enrichment of the sample (d,), pre-dose baseline (41, dose (d3, and tap
water (Q; a is the amount of dose diluted for analysis (g); W is the amount of water used
to dilute the dose (g); A is the amount of dose adrninistered (g); and 18.02 converts g
water into moles. Elimination rates were calculated using enrichment above measured
pre-isotope baseline. Dilution spaces (rnmol), Nd and No for deuterium and oxygen
respectively, were calculated using enrichment above measured baseline for specific diet
cycle. TBW was calculated using the following equation (23):
TBW (mol) = 1(N11.007) + (N,,/1.041)1 2
The rates of CO, production were calculated using the following equation (24):
Rate of CO2 (rC0J production (moVd)
where N is the average dilution space calculated from the time zero dilution spaces of
H,"O and 'H,O; ko and k, are the turnover rates of "O and deuterium, respectively; and
r,, is the water lost by fractionation, estimated as 1 .O5(l .O07 k,, - 1.041 k,,) (24).
Dilution space ratios were calculated as N p o and delta k values were calcuiated
as ko-k,. TEE was calculated using de Weir's equation 12 (25). TBW (mol) was e .
converted to kg by the following equation:
TBW (kg) = (TBW (mol) * 18.021 1 O00
Fat fiee mass (FFM) (kg) was calculated as TBW (kg)/0.732 and % body fat was
calculated using the following equation:
% body fat = Wt ( k d - FFM (kg) x 100 w t (kg)
where Wt is the subject's body weight in kg.
Respiratory gas exchange analysis (RGE) was conducted on day 7 and 14 of each
dietary treatment in order to m e s s BMR and TEF (refer to Chapter 4, section 4.3).
Bnefly, subjects aayed ovemight at the Research Unit on days 6 and 13 and following an
approximate 12 h fast, BMR was measured over 30 min using RGE (Deltatrac Metabolic
monitor, Sensormedics, Anaheim, California, USA). Subjects were then fed their
scheduled breakfast, and their energy expenditure was assessed for 5.5 h, during which
subjects were awake and relaxed, reading or watching television. Shon washroom breaks
were pennitted. Mean postprandial TEE was calculated as the mean of 30 consecutive
min periods. BMR and TEF were expressed in kcaüday, derived fkom the kcal/min RGE
measurement. To determine the daily TEF values, average TEF was multiplied by 330
min, the length of the measurement period during RGE. TEF values were then muitiplied
by a factor of 3, to account for the TEF resulting frorn the three isocaloric meals with
similar macronutrient composition served to the subjects per day. The difference between
TEE and the sum of BMR and TEF was used to denve AIEE. Daily energy expenditure
(DEE) was calculated for both days when RGE testing was conducted (day 7 and 14)
dunng each dietary treatment.
5.4 Statisticai Analysis
Statistical evaluations were made using the paired student's t-test between diet
treatments for al1 variables determined using the DLW method. Repeated measures
ANOVA with main effects of sequence, diet (MCT and LCT), day (day 7 and 14), and the
interaction between diet and day was used to test for differences of 24 h values of TEF,
B a and AIEE. Post-hoc comparisons were made using contrasts. AU tests were
perfomed using the SAS Statistical Package V. 6.12 for Windows. Level of significance
was set at pcO.05. All data are presented as mean astandard error (SE).
5.5 Results
Menstmal diaries indicated cycles of 28.5 * 0.6 days; therefore, based on the
protocol of two 14 day trials separated by a 14 day washout period, subjects were at the
same points in their menstruai cycles at each stage of the treatment phases and menstmal
cycle affected both measurements of TEE equally. There were no statistically significant
changes in body weight during the course of the study (56.2 i 1.5 kg at start vs. 56.7
1.4 kg at end). When questioned, subjects were unable to identify their dietary treatment
fat type dunng either cycle. Cornpliance was assessed by subjects' attendance to meals
and monitoring of consumption of ail food provided. Two subjects' caioric intakes had to
be upwardly adjusted based on reponed hunger over the first three days of the first cycle
One subject was eliminated from al1 calculations as the values for delta k, dilution space
ratio, and TEE for one of the dietaty cycles obtained both during initiai and repeat analysis
indicated contamination of the samples through dilution of isotope.
Table 5.2 lists the mean TBW (kg), % body fat, delta k, dilution space ratios, and
TEE (kcaVday) for 1 1 subjects for both diet treatments, as well the between diet
cornparisons for these parameters. The dinerence in TEE between the MCT and LCT
dietary treatrnents (2246 * 98 vs. 2 186 138 kcalld) was not statistically signifiant, nor
were significant differences present between other parameters tested. Figure 5.1
represents the individual values for TEE for aii 1 1 subjects, as well as the means for both
dietary treatments.
The mean values for 11 subjects for BMR and TEF as determined using RGE are
listed in Table 5.3. There was a sigdcant effect of diet on BMR on day 7 with greater
BMR on the MCT diet (12 19 38 kcallday) as compared to the LCT diet (1 179 i 42
kcallday). No differences were observed in BMR on day 14. #en B m TEF, and
AiEE were expressed as percentages of TEE, there were no longer any dEerences
between diets. Figure 5.2 represents the components of TEE as a percentage of daily
energy expenditure for each diet with day 7 and 14 combined within dietary treatment, and
with both dietary treatments combined.
5.6 Discussion
The objective of our study was to assess the effect of 14 day MCT feeding on TEE
and its components, BMR, TEF, and AIEE by using a combination of both DLW TEE
measurement and RGE. The results indicate that differences in dietary fatty acid
composition did not influence TEE dunng the second week of feeding in any appreciable
fashion, as TEE was not significantly enhanced during the MCT feeding period. The body
composition data (Table 5.2) are consistent with the TEE results in that no decrease in
percent body fat was observed in subjects consuming the MCT diet vs. the LCT diet.
Several aspects of the experimental protocol are relevant in the interpretation of
these results. Specifically, the second week of the two week feeding penod was used for
TEE assessment. This allowed one week of adjustment to the dietary intervention pnor to
measurement and as such was consistent with our objective to determine any longer term
energy metabolism changes on MCT diets. Prefeeding allowed the adipose tissue stores to
begin to incorporate the exogenous fat into endogenous stores pnor to measurement (26).
Therefore, TEE may have been intluenced through changes in endogenous fat oxidation,
&en this change in substrate; however, changes in energy metabolism were not detected.
Interestingly, RGE of BMR on day 7 showed a si@cant dserence between diets (MCT:
12 19 9 38 kcaVday vs LCT: 1 179 * 42 kdday); however, this difference was attenuated
by day 14. Xt appears that compensatory mechanisms may exist that aiter handling of
MCT when their presence in the diet is significantly increased and therefore blunt their
effect on longer term energy expenditure. There is some evidence that following 6 days of
feeding in humans there is a slight increase in chylomicron transpon of MCT, which are
othewise transported mainly in the portal vein (7). Thus, MCT may potentially begin to
be metabolized more like LCT as their presence in the diet continues to be elevated.
Nonetheless, Binnert et al (27) demonstrated impaired oxidation of 30 g of LCT in obese
subjects vs. non-obese controls, yet similar oxidation in obese vs. non-obese subjects of 30
g oil containing 50Y0 MCT. Thus, given that obese subjects rnay have a defect in their
capacity to oxidize LCT, but not MCT, dietary substitution of MCT for LCT may still be
beneficial in the treatment of obesity.
The difference in percentage of MCFA contained in the triglycerides between our
diets was modest, udike the differences between formula diets used by other researchers
(5,27). However, the purpose of these diets was to represent typical North Arnencan
foods and macronutrient percentages that could be incorporated into dietary regimens of
free-living subjects. It rnay be possible to fùrther increase the proportion of dietary MCT,
while maintainhg palatability and ease of preparation, and therefore elicit the changes in
DEE seen over shorter periods (3,4,5) over an extended feeding period. In addition, it
may be desirable to decrease the proportions of Cl2:O and increase the proportions of
C8:O and C 10:O within the diet. While C 12:0, based on its metabolism, is often classifled
as a MCFA (28) (refer to Appendix I), its classification as such is inconsistent within the
literature (29). Increased amounts of caprylic and capric acid in the diets would minirnize
reliance on the potentially questionable metabolism of lauric acid as an MCFA. Diferences
between diets in the percentages of saturated LCT may also have had an impact on our
results. The greater amount of the monounsaturate C 18: l n9 in the LCT diet may have
compensated somewhat for the lack of MCT, as oleate has been reponed to undergo
greater oxidation as compared to C 1 8:O and C 1 8:2n6 (30), possibly similar in oxidation to
MCFA.
Furthemore, the limits of detection of the method employed must also be
considered. The DLW method of measuring TEE may not allow enough accuracy and
precision to detect the small yet vital difference that may occur during longer term MCT
feeding. In their examination of the variability of repeated DLW measurements on fiee-
living individuals, Goran et al (3 1) observed an intra-individual variation of 12%, with an
estimate of intemal precision, or projected theoretical error, of 6%. The authors
concluded that the difference between experimental and theoretical error was due to
biological variation in TEE, which was 10%. In the present study a mean intra-individual
variation of 9% (range, 0.3% to 22.6%) was observed. The multipoint method was
chosen with groups of sarnples collected at the beginning and end of the measurement
penod in order to rninimize error variance in determination of CO, as suggeaed by Cole
and Coward (32), and therefore to rnaximize intemal precision. Therefore, ushg a
theoretical projected error of 6%, error due to biological variation was 7% (square root of
9' - 6'). This level was unexpectedly lower than the 10% determined by Goran et al (3 l),
given that biological variation was intentionally imposed through the dietary treatment.
The biological variation may have been lower than that seen previously based on the short
period of TEE measurement, ody a four week separation between repeat TEE
rneasurements, the use of an activity diary to direct subjects' activity levels during both
measurements, and the consistent Iifestyle of students attending classes.
Our method of DEE assessment was comprehensive. Analysis of DEE is often
made with a respiratory chamber using indirect calorimetry (3 3,34) with physical activity
measured using a radar system (35) and TEF assessed as the difference between the mean
resting energy expenditure (typically measured over 15 h) and basal energy expenditure
(34,35,36). However, it has been suggested that measurement of TEF using a respiratory
chamber is less appropriate than that made with a ventilated hood system with the subject
at rest (33), such as that used in this study. Our TEF measurement duration is in
accordance with the recommendation of Reed and Hill (37) who suggest that TEF be
measured for greater than or equal to 5 h; extrapolation of TEF to 16.5 h is sirnilar to the
15 h measurement used in the respiratory chamber (33). As al1 three meals were of sirnilar
macronutrient breakdown and caloric value, introduction of error into the TEF
measurement through extrapolation from 1 to 3 meals should be minimized (38). We
were then able to derive AiEE as the diEerence between the extrapolated BMR and TEF,
and TEE. This is simijar to the method used by Rising et al (39) who assessed DEE in
Pima Indians. In that study, DLW was used to measure TEE, a 24 h respiratory chamber
measurement was used to assess sedentary energy expenditure (SEDEE), and AIEE was
calculated as TEE - (BMR + 0.1 TEE), where 0.1 TEE was an estimate of TEF as 10 %
of TEE. However, use of the ventilated hood system, as in Our protocol, allows for a
direct measurement of TEF, and therefore only one of the components of DEE, AEE, has
to be estirnated. The present method of combining DLW and RGE, therefore, allows for
direct assessrnent of DEE.
Lack of a significant difference in TEE between dietary treatments and lack of
effect of diet or day on extrapolated 'ïEF and BMR led us to express our measurements as
an overall average of the components for both diets, in addition to between diet
cornparisons. On average, expressed as percentages of TEE, BMR (54.6%), TEF (8.2%),
and AIEE (3 7.2%), correspond to accepted ranges for these parameters in relativeiy
sedentary individuals, where BMR is approximately 50-60% of TEE, TEF is usually
estimated at IO%, and W E is approximately 30% for healthy individuals (40).
In summary, the present results, from the longest feeding trial of this type to date,
assist in examining the feasibility of the use of MCT to increase TEE and therefore alter
energy balance over the long term. At this proportion of MCT in the diet, fed as three
isocaloric meals per day at weight maintenance requirements for a period of two weeks,
TEE was not significantly increased as compared to sirnilar feeding with LCT. In
addition, despite a signifiant difference between diets in RGE BMR on day 7, when
expressed as percentages of TEE, BMR, TEF, and AIEE were not sigiuficantly altered.
Such findings indicate the long term feeding of MCT at this level of intake may not result
in a perturbation of the energy balance equation, and may not result in weight reduction at
a constant energy intake.
Wolfe RR Radioactive and S t d e Isotope Tracers in Biomedicine: Principles
and Prociice of Kinetic Anuijsis. Liss, New York, 1992.
Scalfi L, Coltorti A, Contaldo F. Postprandial thermogenesis in lean and obese
subjects &er meals supplemented with medium-chah and long-chah tnglycerides.
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Themogenesis in humans during overfeedinç with medium-chah triglycendes.
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GL, Bistrian BR Thermogenesis fkom intravenous medium-chah tnglycerides.
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Table 5.1. Gram of fatty acid in MCT and LCT diets per lOOg of meal fat
--
MCT Diet LCT Diet -i
C 8:O 3.9 O
C 109 4.0 0.4
C 120 17.7 1.4
C 14:O 13.3 4.4
C 16:O 25.4 27.3
C 16:1 07 1.6 2.9
C 18:O 8.1 16.9
C 18:l 0 9 19.3 35.9
C 183 07 0.6 0.8
C 182 0 6 4.8 8.0
Table 5.2. Mean and between diet compatisons of TEE, isotopic decay rates, Nd/No, TBW and % body fat for eleven subjects.
MCT LCT MCT - LCT mean (SE) mean (SE) mean (SE)'
% Body ~ a t ' 28.7 (1.54) 28.2 (1.51) 0.5 (0.771)
TEE (kcauday) 2246 (98) 2 186 (1 38) 60 (116)
'Total Body Water ' Calculated fiom TBW and body weight ' Difference in 'w and deuteriurn decay rates
Ratio of deuteriurn to IBO distribution space ' No significant differences were observed between diets
Table 5.3. Mean BMR, TEF, AIEE, and TEE values by day and diet, and across al1 treatments and days, for 11 subjects for whom DLW measures were calculated.
BMR TEF AIEE TEE
kcaVdayt kcaVday t kcaüday ' kcdday
MCT
Day 7 1219 38' 186 î 9 841 108
Day 14 1244 * 29 178 k 22 824 k 100
MCT Average 1232 * 23 182 12 832 -t 72 2246 * 98 @ay 7 & 14)
LCT
- . Day 7 1179*42' 188* 1 1 819* 139
a Day 14 1193 * 33 181 r 21 812* 137
LCT Average 1186 i 26 185 * 12 816 I 95 21861 138
* Detennined by multiplying the kcaVmin value determined using RGE x 1440 (number of minutes in 24 h) ' Determined by multiplying the kcaYrnin value detennined using RGE x 330 (number of minutes of TEF measurement) x 3 (number of meals per day) ' Determined as the difference between TEE and (BMR + TEF)
Determined using doubly labelled water Indicates signifiant difference between diets at p<O.OS
5.8 Figure Legends
Figure 5.1
Total energy expenditure (kcdday) by dietary treatment for each subject (small filled
squares) and overali dietary treatment average for MCT (lined box) (2246 * 98) vs. MCT
(filled box) (2 186 * 1 3 8).
Figure 5.2
Partitioning of daily energy expenditure. BMR, TEF, and AIEE expressed as a percentage
of TEE for MCT, LCT, and combined dietary treatments.
Y'.
CHAPTER 6
OVERALL SUMMARY AND CONCLUSION
The present study examined the effect of North Arnencan diets modestly e ~ c h e d
with either MCT or LCT on comprehensive components of energy metabolism in women
over a 14 day feeding period. An understudied population group was assessed for a
period twice as long as previously studied. The techniques used in the assessrnent of fat
oxidation and energy expenditure were chosen so as to permit measurernent at three levels
of energy utilization: endogenous substrate oxidation, whole body substrate oxidation and
components of energy expenditure, and free living total energy expenditure. This step-
wise approach has yielded a systematic analysis of the effects of MCT- vs. LCT-enriched
diets on energy metabolism. The results offer insight into the way the body discriminates
between FA based on chain length up to 14 days of feeding.
The novel method of the I3C FA repeated dosing paradigm presented in Chapter 3
permitted first tirne examination of endogenous oxidation of saturated LC meal fats
delivered in MCT-enriched diets. The presence of MCT in the diet affected the way meal
fats are stored and utilized, resulting in increased endogenous oxidation when compared to
the presence of LCT. If this capacity of MCT to increase endogenous oxidation could br
exploited, it could have implications for the treatment and prevention of obesity. The
substitution of MCT for LCT in the diet could result in less deposition of FA into adipose
tissue, thereby preventing or combatting obesity. In addition, the abrupt rise in label
appearance, an indication of whole body oxidation, on the MCT diet not evident on the
146
LCT diet supported the existence of two pools of adipose storage, one more rnetaboiically
active and positively iduenced by shorter FA chah lengths, and an inert pool acting as a
sink for longer chain FA. Fatty acids delivered to the active pool rnay undergo more rapid
oxidation as seen by the greater oxidation on the MCT diet (Figure 6.1).
Examination of postprandial substrate utilkation and EE indicated that after 14
days of feeding, the effect of chain length on these parameters was blunted. Day 7
measurernent showed a significant difference in BMR, RQ, and rate of fat oxidation
between dietary treatments as presented in Chapter 4. These differences were attenuated
by day 14; however, a trend towards continued elevated BMR on the MCT diet on day 14
exists. These results indicate that fat metabolism rnay be adapting to chain length of the
dietary fat, and that differences measured with this technique are decreasing. Medium
chah triglycerides rnay be handled in an increasingly similar fashion to LCT. If increased
BMR on the MCT diet is representative of the increased endogenous oxidation measured
in Chapter 3, perhaps the effect is slightly diminished afier an initial peak. Some FA,
despite the influence of the MCT to increase delivery to the active pool, rnay be shunted
towards the inert adipose pool as the body adjusts to the atypical FA source. However,
metabolic requirement for specific adipose composition rnay preclude al1 classes of FA
from being deposited in the inert pool in order to maintain optimal adipose FA
composition (1). Thus, BMR and endogenous oxidation rnay remain somewhat elevated
during continued feeding.
The TEE remlts presented in Chapter 5 showed that, with MCT fed at this level in
a eucaloric diet, alterations seen in energy metabolisrn at the substrate oxidation level were
147
not large andor consistent enough to affect energy expendinire at the whole body level.
However, the DLW method of measuring TEE may not aiiow enough accuracy and
precision to detect the small difference that may occur during extended MCT feeding.
Increases seen in endogenous oxidation and the trend towards maintained increased BMR
on the MCT diet may be altering TEE. Assessrnent of body composition using magnetic
resonance imaging could result in a more precise assessment of TEE changes that result in
a decrease of body fat through increased endogenous oxidation (2,3,4) and therefore may
provide a means to detect small yet vital differences in free living subjects.
Taken together, these results show that metabolic discrimination of FA based on
chain length continues to occur up to 14 days, with MCT resulting in increased
endogenous oxidation as measured using 13C labelled FA. Respiratory gas exchange
results support in pan these findings, with increased BMR on the MCT-e~ched diet on
day 7, and a trend towards increased BMR on day 14. However, these differences do not
affect TEE as measured using DLW when they result from the levels of MCT in the diets
in the present study. Therefore, the levels of MCT in these North Atnencan style diets
alter energy metabolism over 14 days of feeding at the level of endogenous oxidation, but
this difference does not translate into measurable dEerences at the whole body EE level.
The level of MCT in the diet is therefore a limitation of the study.
One objective of the thesis was to examine the effects of North Arnerican style
diets enriched with MCT, triglycerides containhg FA not comrnonly found in this type of
d i e t q regimen. This protocol was followed with the purpose of examining the feasibility
of altering fat oxidation and energy utilization using diets that could potentiaily be
prepared and consumed outside of the clinical setting, and that would not be a major
departure from typical North Amerim foods. As such, the resulting diets did not M e r
widely in FA breakdom. This modest difTerence in MCFA, 26% on the MCT-enriched
diet compared to 2% on the LCT-enriched diet, may be in part responsible for the smail
impact that MCT had on the fat and energy utiiization parameters measured. Greater
amounts of MC?' within the diet may affect exogenous fat oxidation and the thermic eFect
of food, as seen in previous studies (5,6). However, the type of diet required to increase
the proportion of MCT rnay or may not maintain the whole food aspect that was present
in this study, and may require formula preparations. Therefore, the applicability of these
fats outside of the clinic setting and acceptance of use by the subjects would have to be
evaluated.
Another restriction to the study design was the length of dietary treatment. The 14
day dietary treatment period used for this protocol was the longest treatment period to
date examining components of energy expenditure and substrate oxidation. The length
was dictated somewhat by the students' schedules and the schedule of the Clinical
Research Unit; a longer trial would have extended beyond the school yea.. The necessity
of scheduling each dietaty trial at the same point in each subjects' menarual cycle dso
dictated the time frame. A two week dietary trial with a two week washout allowed 28
days between the start of each treatment phase. However, this time fiame did not control
for stage of menstnial cycle within each dietary cycle, between day 7 and day 14. Despite
the extended length of this feeding protocol with respect to previous protocols, even
longer term feeding beyond 14 days, potentially with increased proportions of MCT in the
diet as discussed above, would have permitteci fùrther assessment of the effect of MCT on
components of energy metabolism, specifïcally endogenous oxidation. Additionally,
baseline measurements of energy expenditure components on day 0, which were not
conducted based on protocol restrictions, would have permitted a more comprehensive
evaluation of the effect of diet on energy expenditure.
The sample population is aiso an important factor in the interpretation of the
results. Subjects were chosen to allow assessment of metabolic parameters in a healthy
and nonsbese female population during longer tenn feeding. Thus, results observed
represent normal responses to MCT vs. LCT feeding, and the longer term effects of MCT
on fat metabolism and energy utilization have now been documented in college-age
women. Nonetheless, obese subjects may respond differently than controls to dietary
interventions containing MCT. Binnen et al (7) have shown disparate responses between
obese and controls to oral boluses of LCT, with decreased oxidation of LCT in the obese,
but sirnilar responses to MCT. Therefore, care must be taken in extrapolation of results
fiom one population group to another.
In future studies, the effect of MCT feeding on blood lipids also warrants Further
examination. Despite their saturated nature, MCT may not adversely affect blood lipid
profiles with respect to cardiovascular disease risk (8). Evidence regarding the effects of
C8:O and C 10:O is relatively sparse (9); however, Cater et al (1 0) demonstrated that MCT
oil, containing C8:O and C 10:0, had one-half the potsncy of palmitic acid in raising serum
total and LDL-cholesterol concentrations. With respect to C 12:0, Denke and Gmndy (1 1)
observed that when compared to C16:0, C12:O lowered total and HDL cholesterol yet
compared to C 18: 109, it was hypercholesterolemic. In rats fed MCT vs. corn oil, MCT
increased plasma triacylglycerols and decreased plasma cholesterol(12). Also in rats,
Jones et a1 (13) observed an increase in both triglycerides and plasma total cholesterol
following a coconut oil diet. In contrast, &er 6 days of MCT vs. LCT feeding in humans,
Hiii et a1 (8) saw no effect of MCT feeding on plasma cholesterol, but there was a
significant threefold increase in triglycerides. Matsuo et al (14) saw a decrease in semm
triglycerides following MCT as compared to LCT feeding. Therefore, resultant blood
lipid profiles from MCT feeding need to be further elucidated to determine the potential of
MCT in the context of diets in which they are delivered as hypercholesterolemic and
hypertriglyceridernic agents.
Another area that requires further examination dunng MCT feeding is facultative
thermogenesis. Facultative thermogenesis can anse from events not directly involved in
nutrient processing (6,l S), and may be a mechanism of action of MCT with respect to
their efTect on energy expenditure (15). Results showing increasing unnary excretion of
noradrenaline with augmentation of MCT levels in the diet suggest that the sympathetic
nervous system (SNS) may play a role in increased thermogenesis during MCT feeding
(1 5). Studies combining energy expenditure assessment and SNS activity measurements
will contribute to knowledge of MCT effects on BMR and thermogenesis, both obligatory
and facultative.
Despite the limitations, results obtained have contributed to knowledge in the field
of the effects of fatty acid chah length, specifically MCT as compared to LCT, on fat
oxidation and energy utilization. Future research can in part be directed by results
obtained here, and the limitations of the curtent studies can be addressed in the design and
execution of upcoming studies. Further research examining the existence of the two pools
of FA storage, and their influence on endogenous oxidation, combined with increased
proportions of MCT within experimental diets may offer fùrther insight into the body's
capaciîy to store and oxidize FA of varying chah length.
6.1 References
1. Field CI, Angel A, Clandinin MT. Relationship of diet to the fatty acid
composition of human adipose tissue structural and stored lipids. Am J Clin Nutr
1985 ;42: 1206-1 220.
2. Fuller MF, Fowler PA, McNeill G, Foster MA. Imaging techniques for the
assessrnent of body composition. J Nutr 1994; 1546s- 1 5 50s.
3 . Fowier PA Fuller MF, Glasbey CA, Cameron GG, Foster MA. Validation of the
in vivo measurement of adipose tissue by magnetic resonance imaging of lean and
obese pigs. Am J Clin Nutr lW2;56:7- 13.
4. Ohsum F, Kosuda S. Takayama E, Yanagida S, Nomi M, Kasamatsu H, Kusano -. S, Nakamura H. Imaging techniques for measuring adipose-tissue distribution in
the adbomen: A cornparison between computed tomography and 1 .S tesla
rnagnetic resonance spin-echo imaging. Radiat Med 1998; 16:W- 107.
5 . Seaton TB, Welle SL, Warenko MK, Campbell RG. Thermic effect of medium-
chain and long-chain tnglycerides in man. Am J Clin Invest 1986;44:630-634.
6. Hill JO, Peters JC, Yang D, Sharp T, Kaler M, Abumrad NN, Greene HL.
Thermogenesis in humans during overfeeding with medium-chah tnglycerides.
Met Clin Exp 1989;38:641-648.
7. Bimert C, Pachiaudi C, Beylot M, Hûns D, Vandemander J, Chantre P, Riou JP,
Laville M. Mluence of human obesity on the metabolic fate of dietaiy long- and
medium-chah triacylglycerols. Am J Clin Nutr 1 998;67: 5 95-60 1 .
8. Hill JO, Peters JC, Swift IL, Yang D, Sharp T, Abumrad N, Grene HL. Changes
in blood lipids during six days of overfeeding with medium or long chah
triglycerides. J Lipid Res 1 99O;3 1 :4O7-4 16.
9. Kris-Etherton P, Yu S. Individual fatty acid effects on plasma lipids and
lipoproteins: human studies. Am J Clin Nutr l997;65 : 1 628(S)- l6M(S).
10. Cater NB, Heller HT, Denke MA. Comparison of the effects of medium-chain
triacylglycerols, palm oil, and high oleic acid sudower oil on plasma
triacylglycerol fatty acids and Lipid and lipoprotein concentrations in humans. Am J
Clin Nutr 1997; 65:41-45.
1 1. Denke M, Gnindy S. Comparison of effects of launc acid and palmitic acid on
plasma lipids and lipoproteins. Am J Clin Nutr 1992;56:895-898.
12. Geelen M. Medium-chain fatty acids as short-term regulators of hepatic
lipogenesis. J Nutr l994;302: 14 1-146,
13. Jones PJ, Ridgen JE, Benson AP. Influence of dietary fatty acid composition on
cholesterol synthesis and estenfication in hamsters. Lipids 1 W O ; Z : 8 1 5-820.
14. Matsuo T, Ohiwa M, Taguchi N, Takeuchi H. Effects of medium and long chah
triacylglycerol (structured lipids) on post-ingestive energy expenditure in healthy
young women. (Abstract) FASEB Journal 1999; 13 :A90 1.
15. Dulloo AG, Fathi M, Mensi N, Girardier L. Twenty-four-hour energy expenditure
and urinary catecholamines of hurnans consuming low-to-moderate amount of
medium-chah triglycerides: A dose-response study in a human respiratory
chamber. Eur J Clin Nutr l996;SO: 1 52- 1 58.
6.2 Figure Legend
F&re 6.1
Influence of MCT on dietary fat delivery to adipose tissue storage and on subsequent
oxidation of fat to CO,.
APPENDIX 1
Figure Aï. 1 Overd Thesis Experirnental Protocol
APPENDIX IL Subject Characteristics
Subject Age 69 Weight (kg)' Height (cm) BMI (kgh?)
1 25 45.9 162.0 17.5
2 24 67.9 154.0 28.6
3 23 56.8 163.8 21.2
4 19 58.2 168.0 20.6
5 25 60.5 167.6 21.5
* mean ' standard error ' upon entrance into the study
APPENDIX III
FATTY ACID CHAIN-LENGTH DESIGNATIONS ARE IMPORTANT TO
STllDY CONCLUSIONS
Andrea A. Papamandjaris, Marco Di Buono, Peter J.H. Jones
published in American Journal of Chical Nuîritiof~ 199 7;66: 71 0- 71 1.
. Letter to the Editor
Dear Sir:
Recently, Cater et al (1) compared the effects of feeding diets rich in medium-chah
triacylglycerols, palm oil, and high oleic acid sunfîower oil on blood lipids in humans.
_. They concluded that medium-chain fatty acids (MCFA) have one-half the potency of
pairnitic acid in raising total and LDL-cholesterol concentrations. Their results were based
on specific fatty acid classifications as follows: short chah (C4:O-C6:0), medium chain
(C8:O-C 10:0), long chain (C 12:O-C 1 &O), very long chain (C20:O-C24:O). We believe that
t heir classification of MCFA as exclusively C8:O-C 1 0:O is inconsistent with the consensus
of a substantiai body of literature. For this reason, their conclusion should be restated to
refer specifically to those fatty acids studied, but not necessarily al1 MCFA.
Notably, at present C 12:O is predominantly classified as a MCFA by leading
textbooks (2,3). Several studies examining MCFA also acknowledge classification of
C 12:O as a MCFA (4,5,6). Still, some discrepancy does exist. A recent MCFA review
classified C6:0, C8:0, and C10:O as MCFA and long-chah fatty acids (LCFA) as those
FA with C12:O or greater (7). There are also examples of studies that report results based
on the use of medium-chah tnglyceride (MCT) oil or MCFA, without reporting which FA
were actually part of the MCFA profile studied (8).
As stated by the authors (l), MCFA and long-chah fatty acids @CFA) undergo
different mechanisms of absorption and metabolism. Specificaily, MCFA difise rapidly
into the portal circulation, whereas LCFA are typically reesterified into triacylgIycerols,
and travel tbrough the lymph packaged in chylomicrons. It should be noted that the
enzyme responsible for the reincorporation of fatty acids (FA) into chylomicrons, acyl-
CoA synthetase, is specific for fatty acids with more than 12 carbon atoms (9, 10). This
would warrant the classification of C12:O, lauric acid, as a MCFA according to the
definition provided by the authors.
This inconsistency within the area of lipid research needs to be addressed. Based
upon the stated metabolic rationale, C 12:O should be included in the category of MCFA.
This wiil allow valuable and correct conclusions to be drawn about the different effects of
MCFA vs LCFA. With respect to the study in question, proper classification of C12:O as
a MCFA would allow appropnate interpretations to be made regarding the effects of
MCFA on blood lipids in humans. In addition, diligent reporting of the FA composition of
lipids studied over and above the broad classification of medium- or long-chain will permit
cornparison of specific FA, which may reveal further discrepancies andor similarities of
FA based on chah length.
iU.2 References
1. Cater NB, Heller HJ, Denke MA. Cornparison of the effects of medium-chah
triacylgiycerols, palm oil, and high oleic acid sunflower oil on plasma
triacylglycerol fatty acids and lipid and lipoprotein concentrations in humans. Am J
Clin Nutr l997;65:4 1-45.
2. Lindsheer WG, Vergroesen AI. Lipids. In: Shils ME, ed. Modem nutrition in
health and disease. Philadelphia: Lea and Febiger 1994:42-88.
3. Jaqueline Dupont. Lipids. In: Brown M, ed. Present knowledge in nutrition.
Washington, DC: International Life Sciences Institute-Nutrition Foundation
1990:56-66.
4. Jensen C, Buist Wilson T. Absorption of individual fatty acids fiom long chah
or medium chain triglycendes in very small infants. Am J Clin Nutr 1986;43:745-
751.
5 . Seaton TB, Welle SL, Warenko MK, Campbell RG. Thermic effect of medium-
chain and long-chain triglycerides in man. Am J Clin Nutr 1986;44:630-634.
6. Scalfi L, Coltorti 4 Contaldo F, Postprandial thermogenesis in lean and obese
subjects d e r rneals supplemented with medium-chah and long-chah triglycerides.
Am J Clin Nutr 1991;53:1130-1133.
7. Bach AC, Ingenbleek Y, Frey A. The usefulness of dietary medium-chah
triglycendes in body weight control: fact or fancy? J Lipid Res l996;3 7:708-726.
8. Dulloo AG, Fathi M, Mensi N, Girardier L. Twenty-four-hour energy
expenditure and urînary catecholamines of humans consuming low-to-
moderate arnounts of medium-chah triglycerides: a dose-response study in
a human respiratory charnber. Eu J Clin Nutr l996;5O: 152- 158.
9. Pausî H, Keles T, Park W, Knoblach G. Fatty acid metabolism in infants. In:
Chapman TE, ed. Stable isotopes in paediatric nutntional and metaboiic research.
Andover, Hants, England: Intercept Ltd. 1990: 1-23.
10. Bach AC, Babayan K. Medium-chah triglycerides: an update. Am J Clin Nutr
l982;36:950-962.