800

LIPID OXIDATION

Health implications of lipid oxidation

Could eating food containinglipid autoxidized productsfonned during preparation and

storage be harmful 10 your health?Can anything be done to prevent orminimize lipid peroxidatlon withinyour body?

The answer to both questions ispotentially yes-potentially becauseresearchers do nor know the extent ofoxidation nor the quantity of oxidationproducts needed to produce a harmfuleffect.

Lipid oxidation is a normal biolog-ical process by which we obtain ener-gy from fat. Deleterious lipid oxida-tion occurring in foods generally isreferred to as autoxidation; the corre-sponding deleterious lipid oxidationoccurring in the body generally iscalled peroxidanon.

Various aspects of lipid oxidationhave been studied for many years. Howto identify lipid oxidation products infoods and in biological systems. how-ever, continues to be investigated.what the results mean and the mecha-nisms involved also are unresolved.

There is tremendous interest in thetopic, according to David Mills,chairperson for the AOCS Health and

"Based on our sensory research,untrained testers are less sensitive torancidity than trained sensory pan-elists. Therefore, consumers may beeating oxidized food. Certainly whenrancidity is noticeable, people avoidconsuming the product. Just think howmuch food we may eat that's slightlyor moderately oxidized but we eat itanyway. I'd like researchers to answerhow much we can eat before weshould be concerned," Warner said,adding. "I don't believe anyone hasset levels of when these oxidationproducts become harmful."

Because the body has many protec-uvc mechanisms, it is questionablewhether moderately oxidized productsare harmful in the body when eaten,according to Edwin N. Frankel of theDepartment of Food Science andTechnology. University of Californiaat Davis. For example. fish oil cap-sules may increase the levels of highlyunsaturated fatty acids circulating inthe body. Some authorities recom-mend taking additional vitamin E

INFORM. Vol. 4. no. 7 (July 1993)

Tleis artide on lipid tnidolionwas wrinen by Barbara FilchHQJUffQIUI. semor ~dilorlwril~rfor INFORM.

Nutrition Division: "Oxidation, partic-ularly with its possible health andnutrition implications, is probably oneof the most important areas of currentlipid research. There are weightyproblems, such as chronic diseasesand the process of aging. that havebeen found to be influenced by lipidoxidation."

Oxidized foodsMany believe that oxidized foods donot present a real risk because peoplewill throw out food that smells ortastes rancid without consuming it.

Kathleen Warner. research foodtechnologist at the U.S. Departmentof Agriculture's National Center forAgricultural Utilization Research.however. wonders if people could beconsuming foods that may be slightlyoxidized. [continued orrpoge803)

LIPID OXIDATION

BOO

[continued from poge 800) metals and metal compounds, lightand heat. Protection against lipid oxi-dation should be taken as early as pos-sible, Lingner! noted. Oxidation canoccur in raw materials and during pro-cessing. One way to limit oxidation is

along with these fish oil health supple-ments, but a definite need remains tobe established.

The food industry is interested pri-marily for economic reasons in how toprevent and measure oxidarion ,according 10 David B. Min, professorat Ohio Slate University; "Flavorchanges in food are due mainly tolipid oxidation. Off-flavors meanproducts will be less acceptable toconsumers,"

Lipid oxidation also means II lossof essential fatly acids, and highlyoxidized foods may have a negativeeffect on health. "The question is howwe can minimize lipid oxidation,"Min said, noting this needs to includeinformation about the role of the vari-ous antioxidants in preventing oxida-non.

In a talk given at a Lipidforumseminar in October 1992 in Denmark,Hans Lingnen of SIK (The SwedishInstitute for Food Research) noted thatoxidative processes are complex innature and affect many aspects of foodquality. "Flavor deterioration is oneimportant effect, but impaired colorand texture are also common conse-quences of lipid oxidation, as are loss-es in nutritional value due, forinstance, to vitamin reactions," hesaid.

The main targets of oxygen attackare unsaturated fatty acids, but othercompounds such as sterols,carotenoids and aroma compoundsalso may react. "In addition to theundesirable sensory deterioration ofautoxidized foods, questions havebeen raised about the safety of oxida-tion products in food. Mutageniceffects are reported for cholesteroloxidation products, but the possibleactions of hydroperoxides, epoxidesand certain carbonyl compounds alsoare being debated," Lingnen said.

According to Lingnert, the reactionbetween oxygen and lipids does nottake place spontaneously. "It needs tobe initiated either by the formation offree radicals from the lipids or by theformation of active oxygen speciesthat are able 10 react directly with thelipid molecule." The main prooxida-rive factors in foods are enzymes,

"Lipid oxidation remainsa major problem in thefood industry and animportant cause of

quality deterioration."

to limit the oxygen and light availableduring food processing and packag-ing. "Inactivation of enzymes is cru-cial to the oxidative stability of manyfood products." Lingnert added. Low-temperature storage is another way. Athird is the use of antioxidants.

"Despite considerable progress inthe understanding of lipid oxidation infoods during the last 20 years, ourknowledge is still far from complete.Lipid oxidation remains a major prob-lem in the food industry and an impor-tant cause of quality deterioration,"Lingnert said.

A number of products formed arebelieved to be or shown to be cytotox-ic, carcinogenic or atherogenic,according to industry consultant Ger-hard Maerker. who, however, added,"At the U.S. Department of Agricul-ture's Eastern Regional Research Cen-ter (ERRC), we have done workdemonstrating that carcinogenic prod-ucts fonned-the epoxides of cboles-terot=do not survive exposure to sim-ulated gastric juice. So one would notexpect them to survive ingestion in thehuman stomach. However, the productformed from these epoxides is shownto be cytotoxic."

Other work at ERRC has demon-strated that irradiation of food con-taining cholesterol generales certaincholesterol oxidation products whichexist normally but are not known to betoxic. Thus. these compounds poten-tially could be used as a marker toindicate past irradiation of foods. "The

issue of finding a marker for irradia-tion is not trivial," Maerker, said,explaining, "People have looked forsuch compounds for the past 35 years.This indicates the changes to foodswhen irradiated are very minute."

Deep-fat fryingPeople like the taste of some oxidizedlipids. according to Roben G. Ack-man, Research Professor at the Cana-dian Institute of Fisheries Technology,Department of Food Science andTechnology, Technical University ofNova Scotia, who noted that desirableflavors often are oxidation products ora result of the oxidation process.

Fried snack products are a goodexample of this, according to EdwardG. Perkins, professor of food chem-istry and nutritional science at theUniversity of Illinois. "American con-sumers like a little hexanal, which isprimarily an oxidation product," hesaid.

Warner noted that USDAresearchers have found that the desir-able fried food flavor typical of potatochips is related to the oxidation oflinoleic acid and the development ofbreakdown products such as 2,4·deca-diena!.

Lipid oxidation occurs very rapidlyin deep-fat frying because of the hightemperature, Min said. adding that30% of the frying fat is oxidized after72 hours. Studies have shown that ratsfed highly oxidized frying fat experi-enced decreased growth and increasedliver weight and fatty tissue deposits.

Citing studies at the University ofIllinois in which rats were fed fat fromcommercial deep-frying operations,Perkins confirmed similar results."There are indeed nutritional and toxi-cological effects in rats," he said,pointing to reduced growth, a changein liver enzyme levels and enlargedlivers. Previous studies showed thatwhen foods were fried in these fats,the foods absorbed some of the fryingoil, and the absorbed fat contained thesame concentration of oxidation prod-ucts as the bulk of the frying medium.

"When the fat gets bad enough tocause excess smoking and colordevelopment. it is thrown out. Gener-ally speaking, I think food operatorsthrow it away before it is harmful,"

INFORM. Vol. 4. no. 7 (July 1993)

804

LIPID OXIDATION

FIgure 2. HPlC MpWaIUon of ehoIe*ferol oxld.Uoo products In JHlflCllke mi. end lhe.. me extl"Kt to which authentic 7.utocholeat8101 was added

Perkins said. adding, '" think we'veidentified the lipid oxidation productsfrom heated Iars. and most of thehigher molecular weight productsfonned from heating fats are probablyexcreted, based on rat studies."

Of more concern, he said, may becompounds such as cyclic monomersof fatty acids which are formed bothduring processing of the fats and indeepfrying. "l thtnk the primarycon-cern should be the cyclic fatty acidsin the fats and their health effects,"he said. "These have already beenshown 10 cause toxicological effectsat levels as low as 0.1 % of the diet inrat studies.

"What is needed is to have some-one fund studies to synthesize largequantities of lipid oxidation productsand do definitive studies with rats. Italso would be interesting to do a studyof the effects of exposure of personswho operate the fryers all day andmay inhale volatile fat oxidation prod-ucts. No one yet has done a classicalchronic toxicity test with some ofthese heated fat compounds. You can'tbuy them. You have to generate themin a laboratory so you are workingwith only small amounts."

At the University of Illinois,researchers are studying these effectsin tissue cultures. J.-L. Sebedio's labo-ratory at INRA in Dijon, France. is theonly other group in the world studyingthe nutritional and toxicological

INFORM, Vol. 4, no, 7 (July 1993)

effects of cyclic fatty acids, accordingto Perkins.

David Firestone of the U,S. Foodand Drug Administration, meanwhile,believes there needs to be more regu-lation of fats and oils used for deep-fatfrying, In his talk at the 1993 AOCSAnnual Meeting and Exposition, Fire-stone noted that although many stud-ies have shown that improper use offrying fats causes fat degradation andreduced quality of the fried food. thereare no general worldwide regulationsfor the control of frying fats.

Diological systemsThere is evidence that, in the body,lipid oxidation can cause inflamma-tion and playa role in such diseases asarthritis, atherosclerosis, heart diseaseand breast and colon cancer.

Free radicals of polyunsaturatedfatty acids can form in living systems,and oxygen can react with the radicalsto fonn peroxy radicals and hydroper-oxides. These compounds can chainreact to cause membrane damage. Forinstance, in cardiovascular disease.arterial clogging apparently beginswhen low-density lipoprotein (LDL)is oxidized in a reaction induced byfree radicals.

Karen Schaich, assistant professorin the Food Science Department atRutgers University, noted that recom-mendations to increase the proportionof unsaturated fats and decrease total

fat intake in the diet may have beenmade prematurely from observationsthat the material found in plaques con-tained cholesterol oxides and saturatedfats.

"The belief was that atherosclerosiswas caused by cholesterol and saturat-ed fats and thus these should bedecreased in the diet. However, wenow know that neither cholesterol norsaturated fats per se causes atheroscle-rosis. Instead, the original lesions arethought to arise through oxidativeevents in the blood vessel walls.When lesions are formed, they put outchemical markers that are like anten-nas that attract mast cells. This actiontriggers a series of events, such asdeposition of cholesterol. What wemissed was that the initial trigger wasoxidation," Schaich said. "By increas-ing PUFA (polyunsaturated fatty acid)consumption, we may be aggravatingour susceptibility to cardiovasculardisease. We've been treating thesymptom, not the disease."

According to an article in the April1993 issue of Food Technology. thelate John E. Kinsella and co-authorsE.N. Frankel, B. Gennan and J. Kan-ner noted that each cell is exposed tonumerous free radical attacks. Unlesscontrolled, these agents can damagetissue. The pcroxidauon of tissuelipids potentially can disrupt mem-brane-related functions, alter plateletfunctions. modify macrophage func-tions, alter the arachidonic acid cas-cade. cause protein polymerization,promote atherogenesis by modifyingLDL and cause DNA mutation. How-ever, the body has an array of protec-tive mechanisms to control peroxide-tion and to quench, eliminate or inac-tivate free radical generators. Theseinclude enzymes (superoxide dismu-lase. catalase. glutathione peroxidase).antioxidants and complementaryagents such as ascorbic acid, ~-carotene and retinoids. Natural antiox-idants. primarily provided by diet,include vitamins E and C.

In the March/April 1990 issue ofFood & Nutrition News. Paul B.Addis. professor in the Depanment ofFood Science and Nutrition, Universi-

(conlinu~d on fXJgt8(6)

806

Lab Division of the Department ofHealth for New Mexico.

Because cancer tumors have verylow tolerance to lipid oxidation prod-ucts. the latter might be used [or thera-peutic purposes. Mills said, citingstudies by Michel Begin showingPUFAs are capable of killing cancercells but nor healthy cells in vitro. Inarticles published in AnticancerResearch (Vol. 6, pp. 291-296. 1986)and the Journal of (he National Can-cer Institute (Vol. 77. pp. 1053--1062.1986). Begin related this phenomenon10 the oxidation products of PUFAs.

"Healthy cells have a protectivemechanism. However. if the cellsalready have been altered, you may beable 10 use the toxicity of the lipidoxidation products to your advantage10kill the cancer cells," Mills said.

LIPID OXIDATION

Agu ... 3. Cholesterol oxld.llon products

(cominned from pagt' 804)

ty of Minnesota. SI. Paul. cited evi-dence that lipid oxidation productsmay accelerate all three phases ofchronic heart disease: initiation. pro-gression and termination. "Evidenceexists. strong in some cases. thatdietary lipid oxidation products areinvolved in arterial injury, ethcroscte-rotic plaque formation and thrombo-sis/spasm and therefore may be moredeleterious than was once assumed,"he noted.

Lipid oxidation products involvedmay include cholesterol oxides. ferryacid hydroperoxidcs. malonaldehydeand other secondary breakdown prod-ucts. and may be obtained from thediet or formed in vivo, Addis notedthat cholesterol oxides possessangiorcxic properties although purecholesterol does not. There also havebeen studies indicating that consum-ing rancid oils directly acceleratesfoam cell formation. the hallmark ofatherogenesis.

"In the context of cholesterol oxi-dation. it appears that much of the rex-icity of oxidized lipoproteins can beattributed 10 the cholesterol oxide con-tent:' according 10 Alex Sevanian ofthe University of Southern Califor-nia's Institute of Toxicology. Schoolof Pharmacy. Sevanian and H. Hodisand colleagues have done rabbit stud-ies that show cholesterol oxide con-

tent goes up in hypercholesterolemiabut can be reversed by the antioxidantprobucol.

Addis' studies on cholesterol oxideabsorption indicate Ihal cholesteroloxidation products found in the dieteventually end up in plasma Hpopro-reins. "The main sources of choles-terol oxidation products in the humandiet are probably not fresh. freshlycooked or cured meat products andfresh dairy products. but rather frenchfried potatoes (if fried in animal fat).powdered eggs and other dehydratedfoods," Addis wrote, adding, "Muchfurther quantitative work is needed toidentify foods which supply lipid oxt-dation products to the diet of humans.Research is needed to eliminate' lipidoxidation products from frying oils,dehydrated foods and powderedeggs."

Future research. he concluded,should emphasize protecting lipidsfrom oxidation-"an attainable goal atreasonable cost"-rather than attempt-ing to remove cholesterol fromfoods-"a very expensive process."

lrontcany. however. someresearchers have found that lipid oxi-dation products might help 10destroy cancer cells in the body,according 10 Mills. professor andchairperson of the Department ofHealth Studies at the University ofWaterloo. who this month becomesthe Bureau Chief of the Scientific

The role of antioxidantsAntioxidants in the diet are seen as away to minimize lipid oxidarion.Researchers. for instance, are studyingthe role jl-carotene and vitamins E andC might play in inhibiting LDL-oxida-uon.

"Vitamin E is seen by some as a'cure-all,' although it is nor that sim-ple," noted Ackman, who cited a trendtoward more use of natural antioxi-dants because consumers are per-ceived as more willing 10 accept "nat-ural" vs. synthetic additives.

Antioxidants, in fact, have beenshown to provide protective mecha-nisms. Primary antioxidants such asvitamin E donate hydrogen to inacti-vate initial free radical peroxy orhydroxy radicals. Thus. dietary vita-min E helps offer protection. as doesvitamin C which spares and mayregenerate vitamin E. Carotenoidsalso have been shown to be effectivefree radical quenchers in membranes.according to Frankel and COlleagues atthe University of California at Davis.

"We can show beneficial effects ofantioxidants which are far greater thanthe possible adverse effects. Consum-ing fruits and vegetables and redwinc-bcverages containing fruits andvegetables-is a very healthy way ofcounteracting the effects of lipid oxi-dation." Frankel said.

Franke! and colleagues, forinstance. have shown that the nonetco-

tNFORM.VOl.4. no. 7 (July 1993)

807

hotic phenolic components of redwine may reduce the oxidation ofhuman LDL. Their study found that inin vitro studies red wine's phenolicsubstances effectively inhibited cop-per-catalyzed oxidation of LDL(Lancer. Feb. 20, 1993),

AI Ohio State University. Min andresearchers continue to look at p-carotene's role in food and body sys-tems. Thomas Webb at Ohio StateUniversity, for instance. has a Nadon-a1 Cancer Institute grant to study theeffects of p-carolcne on mammarycancer in rats. Min. meanwhile, isstudying the possible health benefitsof jH:arotene and soluble fiber.

In fact, many researchers through-out the world are looking at phyto-chemicals from plants 10 possibly ame-liorate the effects of lipid peroxidativereactions. Research efforts on "design-er food" products include possiblymixing compounds found in fruits.vegetables and other plant sources toperform as effective free radicalquenchers and protective antioxidantsin foods [INFORM 4:344 (I993)J.

An article in The Wall Street Jour-nal of April 13. 1993. said that stud-ies indicating possible benefits ofantioxidants have boosted their sales,particularly of vitamin E. "VitaminE's growing popularity comes as morestudies suggest that antioxidants lowerrisk of various diseases by blockingcell damage from highly reactive oxy-gen-containing chemicals called freeradicals." the article noted.

The article explained that the argu-ment for taking vitamin E supple-ments in addition to eating vitamin-rich foods is stronger than that formost other vitamins because the maindietary sources are vegetable oils,nuts and other fatty foods. Wilh con-sumers told to cut back on fat intake,it is difficult to get large quantities ofvitamin E in the diet. According tothe Vitamin E Research & Informa-tion Service, a not-for-profit organi-zation established by Henkel KGaA,one would have to drink two quarts ofvegetable oil or eat more than fivepounds of wheat germ to obtain 400international units (IUs) of vitamin E,the amount in a typical supplementcapsule. When used as antioxidants infoods. vitamins C and E are used only

A1dehydes. _--_.- -".---'- ------~-.- --

Acids//

//

/__• Pot~" ,k', ,

",",--

figure 4. Composlt ...... of lhit .. ...,.1 products of tIM oxkSfilonprocesses common In fets end oils

in small, pharmacologic amounts.The U.S. recommended daily

allowance (RDA) for vitamin E is 30IUs. However. studies indicate higherdoses may be needed 10 achieve dis-ease-prevention benefits.

lshwarlal Jialal and Scott Grundyat The University of Texas Southwest-ern Medical Center, for instance, haveshown that megadoses of vitamin Emay slow the development ofatherosclerosis. The results of theirstudy, published in the June 1992issue of the Journal of LipidResearch, noted that the oxidation rateof LDL was reduced by half in volun-teers given daily doses of 800 IUs ofa-tocopherol (vitamin E).

However. Jialal and Grundystopped short of recommending mega-doses of either vitamin C or E withoutclinical studies to establish benefits. Infact. researchers generally agree onthe need for further research on therole of antioxidants in the protectionagainst oxidation of the PUFAs ofLDL. Recent work by M. Stamford ofBrigham and Women's Hospital. forinstance, suggests thai vitamin E sup-plementation above laO IUs a dayserves no useful purpose.

Addis personally eats plenty offruits and vegetables and lakes someamounts of supplemental antioxidants.especially if traveling. "I am con-vinced there should be more use ofantioxidants in foods." Addis said.Benefits include retarding lipid oxide-lion in the food. so consumers are lessexposed to secondary lipid oxidation

products. Also, they help maintainvitamin levels in the foods; vitaminsnot spent in the foods to extend shelflife will be absorbed in !he body.

(continll~don pag~808)

Instructor:Dr. Konrad Grob

of Kantonales Laboratory.Switzerland

October 14

GC LC Course on

Fats & Oils Analysis

At Department of Food Scienceand Nutrition

University of Minnesota

•Co-sponsored by (he American Soci-ety of Testing Materials and the Min-

nesota Chromatography ForumContact Dr. Behroze S. Mistry

for detailsPhone: 612-625-4738

Fax: 612-625-5272

fof Informotlon circle t235

INFORM. Vol. 4. no. 7 (Jlly 1993)

808

LIPID OXIDATION

(CQntilllledfrom page 807)

"It is important to have a variety ofantioxidants in your diet. from suchsources as whole grain cereals. fruitsand vegetables," Addis said.

Agreeing with Addis, Schaichbelieves the official recommendationfor vitamin E intake will be increased."Our dietary recommendations forantioxidants are probably too low,"Schaich said.

Measuring lipid oxidationAnother crucial aspect of lipid oxida-tion concerns methodology.

"The biggest confusion is causedby unreliable methods," according toFrankel. "The effect of lipid oxidationin biological systems is confusedbecause researchers are using suchtests us TBA (thiobarbituric acidassay. used to measure the decayproducts of lipid hydroperoxides), Themajor drawback is it measures manythings besides lipid oxidation and thusthe impact of liquid oxidation may beexaggerated. "

Agreeing, Mills noted that measur-ing for the many various compoundsis tricky. "Current methods aren't ade-quate. and there is a lot of confusionover what is really being measuredbecause the methods are so nonspecif-ic," Mills said. "I think there is a lot ofenergy wasted trying to measure thesecompounds. In fact, Ibelieve this willslow down the interpretation."

Several sessions at the 1993 AOCSAnnual Meeting and Exposition heldin April focused on methods for mea-suring the oxidative Status of lipidsand lipid stability. Research continueson various methods. including front-face rutiometric fluorometry, poten-tiometry. high-performance liquidChromatography, spectrophotometry,nuclear magnetic resonance spec-troscopy. headspace gas chromatogra-phy of volatile oxidation products,chemiluminescence assays and con-ductometric determination. The latterhas resulted in a conductometricdetennination method which the JapanOil Chemists- Society has adopted asan official method for testing autoxi-dation of fats and oils. as an alternateto the active oxygen method (AOM).

To evaluate oil stability, different

measuring methods arc used after asample is oxidized under standardizedconditions to a given endpoint. Frankelis critical of the use of AOM in thisapplication because the mechanism ofoxidation is different at the high tern-

"Our dietaryrecommendationsfor antioxidants

are probablytoo low."

we do. we have a long way to gobefore we can approach preventivemedicine." he said.

Frankel and colleagues have devel-oped a static heads pace gas chro-matography mcthod to determinehuman LDL oxidation. "This simple.rapid and sensitive method for oxida-tive susceptibility plays a useful rolein analyzing prooxidam/amioxldamstatus of biological samples," Frankeland co-workers wrote in the Decem-ber 1992 issue of Lipids.

Because this method measures spe-cific volatile aldehydes, it not onlyprovides infonnation on the degree ofhydropcroxide decomposition but alsocan distinguish between oxidationproducts of n-6 PUFA (pentane, hex-anal) and n-S PUFA (propanal).

Lipid oxidation "is a very, vcrycomplex reaction," Frankel said. not-ing that hundreds of compounds maybe formed. "To sort them all out is amonumental task ...

Schaich also sees the need for bet-ter methodology. "When one looks fordamage in a cell, it is very difficult todifferentiate between oxygen radicalsand lipid oxidation. A few peoplehave tried to measure lipid oxidationconcurrently with a loss of cell func-tion and they haven't always found acorrelation. One of the reasons is thatthe TBA test is used extensively as ameasure of lipid oxidation products inbiological systems. TBA measures adownstream product in small quanti-ties from only some lipids and thus isa very limited test. The TBA lest isnOI going to show the early free radi-cals from lipids. Most other testsrequire the extraction of lipid from thetissues. These are tedious and time-consuming," she said.

Frankel, however, noted that hisheadspace gas chromatographic testmeasuring hexanal and other volatileoxidation products can be carried outdirectly with biological tissues andfoods without extraction of lipids.

Lipid extraction allows researchersto look for conjugated dienes, perox-ide values and carbonyl products. "Byputting those all together, you can geta picture of the extent of lipid oxida-tion. But, because lipid oxidation is adynamic process. if you pick anypoint. you won't get any accurate pic-

INFORM. Vol. 4. no. 7 (July 1993)

perature used, vs. at lower tempera-tures. "The mechanism of oxidationchanges drastically at IOO·C andabove, nnd the results of the AOM testdo not predict well the future stabilityor quality of oils under normal storageconditions," he said, adding, "Similar-ly, in the evaluation of antioxidants,the AOM test, like other high-tempera-ture stability tests, gives unreliable andmislcading results that do not translatewell to actual antioxidant performanceat normal storage conditions."

Meanwhile, lipid peroxidation inbiological tissues usually is investigat-ed by measuring major peroxldauonproducts, lipid hydroperoxides andconjugated dienes and minor productssuch as malcnaldehyde, hexanal, fluo-rescent carbonyl-amine products andvolatile hydrocarbons. Most studiesfor lipid oxidation in biological sys-tems have been done in vitro.

However, there are two kinds of invivo studies, according to Frankel. Oneis to give the animal oxidative stressand to measure the toxicological effectafter the animal is killed. The secondanalyzes exhaled breath to measurevolatile hydrocarbons such as pentaneand ethane that form when unsaturatedlipid hydroperoxides decompose. AlTappel described the tener-e-a gaschromatographic method-e-in the bookLipid Peroxides in Biology andMedicine, published in 1982.

Frankel believes researchers need10 accept more valid methods for ana-lyzing lipid oxidation. "We are deal-ing with an extremely complicatedseries of reactions. We don't knowwhich events are significant. Unless

810

LIPID OXIDATION

rure. For that. you have to do a finger-print of the whole process. If you doextracts and only have small amountsof tissue. you have 10 have many ani-mals available to provide adequateamounts of tissues." For these rea-sons, Schaich said, "The definitivestudies required to clarify the role oflipid oxidation in pathological pro-cesses really haven't been done."

Currently working on chemilumi-nescence assays, Schaich hopes thismethodology may eventually provesuccessful in determining lipid per-oxidation. Her first goal is 10 try 10develop an alternative method 10 theTBA test for food quality. A subse-quem goal is to apply such methodol-ogy to determine lipid oxidation inliving tissue.

What about plant sterols?Although much research has beendone on cholesterol oxides, not muchis known about plant sterol oxidation,Perkins and others pointed out.

"As oil chemists, we should not justbe concerned with lipid oxidation prod-ucts but plant sterol oxidation in gener-al," according to Michael Eskin, pro-fessor in the Department of Foods andNutrition at the University of Manitobaand chairperson of the AGeS LipidOxidationjQuality Division. "We needto look at the health implications of theconsumption of lipid oxidized productsincluding sterols.

"A few of the oxidized productsfrom plant sterols have been recog-nized but they have not been charac-terized," Eskin said, pointing to theneed to remedy this. "I think we'veonly identifie4.a small number ofthem. There hasn't been much empha-sis on plant sterols, so there is notmuch of a database."

Maerker agreed that the mecha-nisms of plant sterol oxidation stillneed to be understood. "Frying oilscontain substantial amounts of plantsterols. Chemically these compoundsare very similar to cholesterol, butthey exist in vegetable oils in muchhigher concentrations than cholesterollevels in meat and poultry:' Maerkersaid, noting that some oils are higherin plant sterols than others. "Plantsterols can range from 500 to 800 mil-ligrams per 100 grams. where alJ

meats and poultry contain approxi-mately 75-85 milligrams of choles-terol per 100 grams. If a large amountof oxides of these plant sterols arefonned, what is their effect?"

He added. "It is known that plantsterol content drops dramatically dur-ing frying operations. It is expectedthat the principal products fonned are

"Essentially nothingis knownabout the

health effects ofplant sterol products."

"Further research is needed on ana-lytical procedures. the various lipidoxidation products and the question ofabsorption-which lipid oxidationproducts are absorbed in the blood-stream and which are not," Addis said."For example. although researchshows cholesterol oxides are readilyabsorbed into the bloodstream, wedon't have enough infonnation on themechanism involved.

"The kinetics of absorption is verycomplicated," Addis said. noting hehopes to do more research on thisaspect.

In the meantime. however, Addis isconvinced food companies and con-sumers can take steps to cut down onexposure to lipid oxidation. "l don'tthink we have 10 do 30 more years ofresearch before we tell people theyshouldn't consume oxidized products.It is prudent to tell people to use high-er and broader levels of antioxidantsnow. Use them so we don't have prob-lems with lipid oxidation, or toss ourthe products before they are con-sumed.

"Even I cannot detect oxidizedcompounds at low levels. There mayneed 10 be concem at these low levels.People would be beuer off if theydidn't consume oxidized foods."

Meanwhile, Eskin cautions thatsuch risks should not be blown out ofproportion. "Nothing in life is abso-lutely safe. It is unreasonable toexpect no risk. Just think what wouldhappen if we applied the same stan-dards to driving a car? The problem is,when you improve your methods ofdetections to such a degree that youcan detect a needle-or one-half orone-quarter of a needle-in ahaystack, what you find could be wayout of proportion to the actual risk.So, one has to be cautious concemingthe actual significance of what hasbeen found. If there is a risk, we needto determine what is an acceptablerisk."

It is evident lipid oxidation willremain a topic of interest to AOCSmembers. In fact. the Health andNutrition Division and Lipid Oxida-tion/Quality Division already aregearing up to organize sessions onthe health implications of lipid oxi-dation at future AOCS meetings.

INFORM. Vol. 4, no. 7 (.kAy 1993)

plant sterol compounds very similarchemically to the oxidation productsof cholesterol. But essentially nothingis known about me health effects ofplant sterol products. This is a veryvalid concern."

Eskin agreed. "We don't want tomake things more difficult for indus-try, but we want to do whatever wecan to minimize oxidized productsand improve shelf life of products. Ifoxidation is occurring, we can take theappropriate measures to counteract it.We should minimize the damage."

Analytical techniques have not yetbeen developed to measure plantsterol oxidation products, althoughtechniques used for measuring choles-terol oxides possibly could be used."However, there are no standards forplant sterol oxidation products asthere are for cholesterol oxidationproducts," Maerker pointed out.

Further research neededThus, improving methodology willcontinue to be a crucial researchassignment in this field, both fordetecting lipid oxidation in the foodchain and in living tissue.

At the October 1992 Lipidforumlipid oxidation seminar in Denmark.Gunhild Helmer of the Technical Uni-versity of Denmark noted that moreresearch is necessary to understandthe complicated process of the diges-tive absorption of hydroperoxides andvolatile and nonvolatile secondaryoxidation products.

Evaluation of oxidized lipids in foodsAs is well known. flavor and aromaare two of the most important partsof human existence. Responses 10flavor or aroma stimuli can be eitherpositive or negative. and we areexposed 10 a wide variety of tastesand aromas each day. While ourresponse 10 many foods and aromasis the result of lifelong conditioning,there are some aromas, e-g., vanilla.that have very broad appeal, and oth-ers, e.g .. mercaprans. that areunpleasant 10 most humans.

When lipid-derived flavor compo-nents are studied, the problem ofacceptance or rejection of individualflavor components remains. [0 addi-tion. the level of the component inthe food product must be considered.For example. mildly oxidized fattyacids are important 10 the perceptionof good beef flavor. yet 100 much orcertain flavor components, e.g .. hex-anal, will ruin the overall flavor.

The early work concerning the Ila-vor or oxidized lipids in roods wasseverely limited by the lack or devel-opment or instruments and proce-dures fer studying flavor compo-nents. Derivatives, such as 2,4 dim-trophenylhydrazones. were valuablein the identification or individualcompounds, but were not very usefulfor the quantitation necessary todecide whether a component wasimportant to the overall flavor. Therewere other tests, such as the benzi-dine or anisidine tests which werereasonably quantitative. but their use-fulness was limited primarily to themeasurement of unpleasant aldehy-des in rats and oils.

The TBA (thiobarbituric acid) reac-tion, on the other hand. has been usedto measure lipid oxidation products in avariety or rood systems. The outstand-ing advances in gas chromatography(GC). mass spectrometry (MS) andrelated computerized instrumentationhave helped immensely in the under-standing or both the qualitative andquantitative aspects or flavor; First theTBA reaction will be discussed thenthe use or GC and MS for evaluatingFlavor will be reviewed brieny.

This article is based on a pU~rrlalio" "lltStru-fMrual tmd ~nsory analysis of oxidiz«1 lipidJ ;"foodJR by Glen A. Jacobson. a ",ind ,.~arc"fellow of Campbell Soup Compa",. tIIId 110M' aconsultant from his harM at JJJ Windsor AlIt'.•Haddonfield. New Jersey, during Ihe .4.0CSAnnua/ Muting and Exposition held joiNI, .. ,ilJrIh, Japan Oil Chemisls' Society f,om April25-29, 199), in Ana/teim. CaJi{onria.

TBA reaction, flavor studiesThe TBA reaction has been knownsince 1916. In the 1940s, the TBAreaction was applied to biological sys-tems such as liver tissue. Next camerood systems, primarily because or theability or TBA 10 detect lipid oxlda-lion products at very low levels. Ini-tially, many workers believed that thered chromogen that formed in theTBA test was the sole reaction prod-

uct or malondialdehyde and TBA asshown in Scheme I (I).

As the TBA test was used in manylaboratories for a variety or applica-tions, it soon became apparent thatthere were many pitfalls 10 avoid.One or the first considerations wasthaI TBA reacts with a number oraldehydes to give color in the yellowand orange region or the spectrum(450-510 nm) (Figure I). It is inter-

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INFORM. Vol. 4. no. 7 (July 1993)

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812

LIPID OXIDATION

estlng 10 note that in the lest shown,saturated aldehydes and monoenalsgave yellow color, while the dienalTBA peak occurs in the 530 omregion. with appreciable absorptionin the yellow (450) om region. Also,some absorption occurred for theblank. which we found to result fromtraces of iron. When a source of fer-ric ion was added, about four timesas much yellow color was developedduring the TBA reaction with beef fat(Figure 2). It was later found thaicopper as well as iron affects thereaction.

A high degree of purity of TBAitself, glassware. acid. water andother entities used in the lest isimperative. Several workers haveshown that many aldehydes besidesmalondialdehyde will react withTBA to influence test results; somereactants, such as the dienals, form ared chromogen. In many cases, thelevel of the reaction products of thesealdehydes is more related to off-Ila-vors in foods than malondialdehydeitself, Reaction time and temperaturealso can be very important in rate ofcolor development and the proportionof the chromogen developed. Tern-peratures from ambient to 100°Chave been used.

Applying the TBA test to foodsoften involves a very complex system.Meat products are a good example ofthis. Several sources of rnalondialde-hyde could be present and can affectthe level of red color developed. Somenon lipid sources of malondialdehydeare nucleic acids/nucJeotides, sugars,bile pigments and protein.

Despite numerous pitfalls of theTBA test, many laboratories use it,and good correlations can be devel-oped between the TBA test and Ila-vcr scores. One of the ways to mini-mize artifacts is to steam distill thevolatiles from the food sample andreact the distillate with TBA. Thisprocedure is widely used, but iscumbersome and slow if severalsamples must be analyzed. RecentlyPikul and co-workers (2) modifiedWille's aqueous extraction methodand applied it to chicken and othermeats. The modified method datacorrelated very well with that of the

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tNFORM. VOl. 4. no. 7 (JIjy 1993)

81.

LIPID OXIDATION

standard distillation procedure. Theextraction procedure significantlyreduces the analysis time. whichmakes the test morc attractive forroutine use. It also appears 10 beamenable 10 partially automatedanalysis.

The TBA test has been applied tomany food products and ingredients.including dairy products. fats andoils, bakery products, and particular-ly meal. fish and poultry. The datashown in Figure 3 obtained withcracker meal arc typical for bakeryproducts and highlight how formula-tion and processing effects can befollowed by the TBA reaction. Whenbaked powdered cracker meal wasadded back to the unbaked ingredi-ents at an 18% level. the TBA valueincreased dramatically. When a highbaking temperature (3200F) wasused. the cracker meal was border-line in flavor acceptability after thefirst six or seven weeks. An accept-able flavor score was found in thesesamples when the TBA number wasbelow 1.5. Although routine lastingof this type of ingredient is stillneeded for quality assurance. theTBA test could help offset the sub-jectivity of Flavor scoring with limit-ed or untrained personnel. The TBAreaction was used 10 show the rela-tive oxidation rates of different partsof fish as they were held frozen (3).The data shown in Figure 4 defini-tively reflect the relative susceptibil-ity to oxidation of different parts offrozen fish tissue.

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GC, MS evaluationSeveral years ago. SI. Angelo andco-workers (4) at the U.S. Depart-ment of Agriculture's SouthernRegional Research Center demon-strated the importance of hex anal tothe warmed-over flavor of cookedbeef. More recently. Hwang and co-workers (5). working with frozencooked beef. also found that thepresence of hexanal decreased themealy flavor note and increased thewarmed-over. cardboard and oxi-dized Flavor notes. The correlation

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(conlimlet/ 011 po8~ 816)Figure 4. The reaction I1Itea 01 lipid oxidatIon in lerml 01 change of TBA molar IIlIlue lorautumn mackerel atored at ·15 Ind -30°C

INFORM. VOl. 4, 00.7 (July 1993)

817

• CHime

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AgullI 6. Comparllon 01 peroxide values and 10111uc volatiles 01 vagetable oils during slol'1lge .1 we

The correlation coefficient betweenbexanel level and degree of oxidationof linoleic acid was found 10 be 0.99.Young and Hovis (10) also used arapid GC headspace technique formeasuring the off flavor in Taw androasted peanuts. The peaks identifiedin the headspace chromatographderived from heating ground peanutsin sealed vials for 30 minutes (Figure10) reflect flavor defects used to gradepeanuts. Threshold levels also weredeveloped for the chromatographicpeaks through use of trained panelsand established descriptors. For exam-ple. the musty flavor of peak 14 (N·methyl pyrrole) was found (0 bedetectable by most trained panelists atabout one-third full scale. The ubiqui-tous hexanal, peak 16, was describedas "beany" in peanuts.

130

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INFORM, Vol. 4, no. 7 (JlAy 1993)

B1B

[continued from fHJg~8/7)

LIPID OXIDATION

8

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Flavor score predicted by bexenal-pentanal multiple regll$$ion

Figure 8. COrrelations olactuallnd calculated flavor scores lor aged vegetable alia

ReferencesI. Janero. n.R .. Free Radic. Bioi .

Med. 9:515 (1990).2. Pikul, J .. D.E. Leszcynski and

EA. Kummerow, 1. Agr. FoodChem. 37: 1309 (\989).

II has been found that many foodscontain a number of volatile compo-nents which impact total flavor. Usinga stream splitter at the detector to sortvolatiles by their aromas, "thresholds"in terms of chromatographic responsecan be estimated. In this way, beuerunderstanding can be developed ofwhich peaks and which chromatogramsreflect the right response and balanceof pleasant and unpleasant volatiles.

Presented here have been a num-ber of examples of how to solveproblems brought on by lipid oxida-tion. The TBA reaction is relativelysimple. has some potential forautomation and correlates with fla-vor scores in many foods. GC andMS help to determine which flavorcomponents are responsible for agood or poor flavor. and offer guid-ance in what to do to achieve thedesired flavor.

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Figure 9. Amount 01 oxidiJ:ed linoleic seld (A) and the Increased amount 01 t).hexanal (8) during storage 01 broWn rice at 5 and we

o

INFORM, Vol. 4. no. 7 (July 1993)

819

• ~~ 0

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Figure 10. Headspace chromatogram by lhe minimum analysis lime system, showing thepeak/llavor reLaUonlhlpl 01 a raw peanut sample. Descriptive lenns we,.. d.rI....d ullng •tr.lned flavor panel, Average threshokll.vrel. are approximately full·scale lor musty etter-lasta, one·half lel'alor frully and one·thlrd eeere lor musty uevcrs.

3. Ke. P,J .. D.M. Nash and R.G. 7. SIQcik, S.M., J.1. Gray, A.M.Ackman, J. Am. Oil Cnem. Soc. Booren and O.J. Buckley. 1.54:417(1977). Food. Sci. 56:597 (1991).

4. 51. Angelo. A.J .. J,R. vercclfoui. 8. Warner. K., C.D. Evans. G.R.M.G. Legendre. C.H. vineu. J.w. List. H.R. Dupuy, J.1. WadsworthKuan. C. James and H.P. Dupuy. and G.E. Goheen, 1. Am. Oil1. Food Sci. 52: 1163 (1987). Chern. Soc. 55:252 (1978).

5. S. Y. Hwang, J.A. Bowers and 9. Shin. M.G., S.H. Yoon. J.S. RheeD.H. Kropf. 1. Food Sci. 55:26 and T.-W. Kwon. 1. Food Sci.(1990). 51:460(1986).

6. Shahidi, E. J. Yun. L.J. Rubin and 10. Young, CT. and A.R. Hovis.D.E Wood. 1. lnst . Can. Sci. IbM. 55:279 (1990). •Technol, Aliment, 20: 104 (1987).