The biological consequences of feeding polyunsaturated fatty acids to antioxidant-deficient animals

The addition of polyunsaturated fatty acids (PUFA) to diets deficient in vitamin E and other effective antioxidants results in a variety of symptoms in animals. For example, the feeding of such diets to rats results in muscular dystrophy, testis degeneration, dental depigmentation, brown discoloration of the fat and and uterus and creatinuria Similar diets fed to rabbits and ruminants results in muscular dystrophy. In chickens the symptoms observed are encephalomalacia, lowered egg production, and poor hatchability. The addition of PUFA to diets is known to result in the destruction of vitamin E in the diet or in the tissues of animals as a result of free radicals produced during the autooxidation of the PUFA. However, in several studies, this possible explanation for the development of vitamin E deficiency symptoms has been made untenable. In such studies the more likely explanation for development of symptoms is thein vivo peroxidation of PUFA in the tissues of animals following incorporation of large amounts of PUFA in lipid structures and depletion from the tissues of vitamin E and other biologically effective antioxidants.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. I. Gastrocnemius and quadriceps muscle

The gastrocnemius and quadriceps muscle phospholipids of the antioxidant-deficient rat fed a source of both linoleate and linolenate showed a progressive net increase in arachidonate, a progressive net decrease in all other polyunsaturated fatty acids, and there was a concomitant accumulation of fluorescent pigment of the lipofuscin or ceroid type in the tissue. An increased incorporation of intraperitoneally injected, isotopically labeled acetate into not only arachidonate but also the other higher polyunsaturated fatty acids, was observed. The net loss of the higher polyunsaturated fatty acids from the membrane lipids (presumably via lipid peroxidation) apparently was partially compensated by a homeostatic mechanism which involved conversion of the available precursors, linoleate and linolenate, to the higher polyunsaturated fatty acids. The rates of decrease of the polyunsaturated fatty acids in the muscle phospholipids and accumulation of fluorescent pigment in the tissue were correlated with the rate of production of creatinuria.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. IV. Peroxidation and interconversion of polyunsaturated fatty acids in muscle phospholipids

It has been suggested that the net changes which take place in the composition of the muscle phospholipid fatty acids of the antioxidant-deficient rat represent the balance of two opposing processes. To compensate for (A) the preferential peroxidative destruction of the most highly polyunsaturated fatty acids in the tissue there occurs (B) an increase in the conversion of available precursors to the higher polyunsaturated fatty acids. Analysis of the data in terms of peroxidation kinetics indicated that the onset of creatinuria in one group after 3 weeks and in a second group after 7 weeks on an antioxidant-deficient diet occurred in both cases concomitant with the peroxidative “disappearance” of approximately 125 μg of phospholipid polyunsaturated fatty acid per gram wet weight of tissue or 2% of the total muscle phospholipid fatty acids.     

The relationship between fatty acid peroxidation and α-tocopherol consumption in isolated normal and transformed hepatocytes

The response of normal and transformed rat hepatocytes to oxidative stress was investigated. Isolated normal rat hepatocytes and differentiated hepatoma cells (the Fao cell line was derived from the Reuber H 35 rat hepatoma) in suspension were incubated with the ADP/Fe³⁺ chelate for 30 min at 37°C. Membrane lipid oxidation was assessed by measuring (i) free malondialdehyde (MDA) production by a high-performance liquid chromatography (HPLC) procedure, (ii) membrane fatty acid disappearance as judged by capillary gas chromatography, and (iii) α-tocopherol oxidation as determined by HPLC and electrochemical detection. The addition of iron led to increased MDA production in normal as well as in transformed cells, and to simultaneous consumption of polyunsaturated fatty acids (PUFA) and α-tocopherol. In addition, in Fao cells more α-tocopherol was consumed during lipid peroxidation while less PUFA was oxidized. Lipid peroxidation was lower in tumoral hepatocytes than in normal cells. This could be due to a difference in membrane lipid composition because of a lower PUFA content and a higher α-tocopherol level in Fao cells. During oxidation, Fao cells produced 1.5 to 2 times less MDA than normal cells, while in the tumoral cells the amount of oxidized PUFA having 3 or more double bonds was 7 to 8 times lower. Therefore, measuring MDA alone as an index of lipid peroxidation did not allow for proper comparison of the membrane lipid oxidizability of transformed cellsvs. the membrane lipid oxidizability of normal cells.     

Dolichol: A Component of the Cellular Antioxidant Machinery

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV‐B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid‐bound polyunsaturated fatty acids (PL‐PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α‐tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α‐tocopherol were assessed by high‐pressure liquid chromatography (HPLC), PL‐PUFA by gas–liquid chromatography (GC). UV‐B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α‐tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMG‐CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL‐PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.     

Rat vitamin E status and heart lipid peroxidation: Effect of dietary α-Linolenic acid and marine n−3 fatty acids

Three groups of sixteen male rats each were fed semipurified diets containing 15% by weight of lipid for a period of 4 wk. The diets contained the same amount of polyunsaturated fatty acids (PUFA) (20% of total fatty acids) and saturated fatty acids (19% of total fatty acids). Dietary PUFA were represented exclusively by linoleic acid (18∶2 diet), or 10% linoleic acid and 10% linolenic acid (18∶3 diet), or 10% linoleic acid and 10% long-chain n−3 fatty acids (LCn−3 diet). The overall amount of vitamin E was similar in the three diets,i.e, 140, 133 and 129 mg/kg diet, respectively. Following appropriate extraction, tocopherol levels in heart, liver, brain, adipose tissue (AT) and plasma were measured by high-performance liquid chromatography. The level of vitamin E in the heart decreased with n−3 PUFA diets, most markedly with LCn−3 PUFA. Liver and AT vitamin E contents also decreased with n−3 PUFA diets when expressed as μg/mg total lipids and μg/mg phospholipids, respectively. Total plasma vitamin E was lower in rats fed the LCn−3 diet, but there was no significant difference when expressed as μg/mg total lipids. Brain vitamin E was not affected by the various diets.In vitro cardiac lipid peroxidation was quantified by the thiobarbituric acid reactive substances (TBARS) test. Heart homogenates were incubated at 37°C for 15 and 30 min in both the absence (uninduced) or presence (induced) of a free radical generating system (1 mM xanthine, 0.1 IU per mL xanthine oxidase, 0.2 mM/0.4 mM Fe/ethylenediaminetetraacetic acid). TBARS release was time-independent but significantly higher when LCn−3 fatty acids were fed to rats in either the uninduced or induced system. The study demonstrated that n−3 PUFA diets can influence vitamin E status of rats even in short-term experiments and can change the susceptibility of the heart toin vitro lipid peroxidation.     

Supercritical fluid extraction of bioactive lipids from the microalga Nannochloropsis sp.

Marine microalgae are recognised as an important renewable source of bioactive lipids with a high proportion of polyunsaturated fatty acids (PUFA), which have been shown to be effective in preventing or treating several diseases. For the extraction of oil from microalgae, supercritical CO₂ (ScCO₂) is regarded with interest, being safer than hexane and offering a negligible environmental impact, a short extraction time and a high‐quality final product. Whilst some experimental papers are available on the supercritical fluid extraction (SFE) of oil from microalgae, only limited information exists on the kinetics of the process. In such a contest, a mathematical model able to describe the kinetics of the SFE was applied to the recovery with ScCO₂ of lipids from Nannochloropsis sp., a marine microalga commonly used in aquaculture and characterised by a lipid fraction with a high PUFA content. The aim of this paper was to examine the effect of operating conditions on the kinetics of the SFE, on process yields and on the fatty acid composition of lipid extracts.     

Phospholipid Peroxidation: Lack of Effect of Fatty Acid Pairing

Phospholipids where both fatty acids are polyunsaturated are very rare. Most organisms prefer to couple their polyunsaturated fatty acids (PUFA) with either a saturated (SAT) or a monounsaturated (MUFA) fatty acid. This study examined if these natural couplings are there to protect PUFA from themselves. Specifically, does the coupling of PUFA to SAT or MUFA reduce the potential for increased rates of peroxidation by shrouding these highly peroxidisable fatty acids with less peroxidisable fatty acids? The influence of head group was examined by using the two most common phospholipids found in vertebrate membranes i.e. phosphatidylcholine and phosphatidylethanolamine species. Fatty acid pairings included 16:0/18:2 versus 18:2/18:2 and 16:0/22:6 versus 22:6/22:6. All phospholipids were incorporated into liposomes that were matched for their total PUFA content i.e. 25% PUFA/PUFA or 50% SAT/PUFA with phosphatidylcholine 16:0/16:0 used as the background phospholipid. An iron initiator (Fe²⁺/H₂O₂) was used to induce peroxidation and lipid hydroperoxide production was used to measure peroxidation. The results show that coupling of PUFA together on the same molecule does not increase peroxidation rates and therefore does not support the proposed hypothesis. The lower than expected levels of peroxidation measured for some phospholipid species (e.g. PtdEtn 22:6/22:6) is possibly due to the partitioning of these molecular species into the inner leaflet of the bilayer.     

Modeling the Primary Oxidation in Commercial Fish Oil Preparations

The quality of commercial fish oil products can be difficult to maintain because of the rapid lipid oxidation attributable to the high number of polyunsaturated fatty acids (PUFA), specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). While it is known that oxidation in fish oil is generally the result of a direct interaction with oxygen and fatty acid radicals, there are very few studies that investigate the oxidation kinetics of fish oil supplements. This study uses hydroperoxides, a primary oxidation product, to model the oxidation kinetics of two commercially available fish oil supplements with different EPA and DHA contents. Pseudo first order kinetics were assumed, and rate constants were determined for temperatures between 4 and 60 °C. This data was fit to the Arrhenius model, and activation energies (E _a) were determined for each sample. Both E _a agreed with values found in the literature, with the lower PUFA sample having a lower E _a. The oil with a lower PUFA content fit the first-order kinetics model at temperatures ≥20 °C and ≤40 °C, while the higher PUFA oil demonstrated first-order kinetics at temperatures ≥4 °C and ≤40 °C. When the temperature was raised to 60 °C, the model no longer applied. This indicates that accelerated testing of fish oil should be conducted at temperatures ≤40 °C.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. III. Testes

Production of testicular degeneration in the antioxidant-deficient rat resembles encephalomalacia in the chick in its dependence on essential (ω6) fatty acids and is distinct from the generalized response to all polyunsaturated fatty acids seen in nutritional muscular dystrophy in the rat. The nonessential (ω3) polyunsaturated fatty acids, however, lower the essential fatty acid content of the testicular lipids only slightly, are not themselves incorporated into this tissue to any appreciable degree and thus do not show the inhibitory effect on production of the antioxidant-deficiency sign noted in the studies on encephalomalacia. A direct relationship between the essential fatty acid content of the testes and the rate of testicular degeneration was found, but no effects of biologically available selenium and sulfur amino acids were evident. As the liver and muscle, onset of antioxidant-deficiency is characterized by a decrease in the most highly unsaturated fatty acid in the tissue (22∶5–ω6 in this case) and a net increase in arachidonate.     

Suppression of growth in a leukemic T cell line by n−3 and n−6 polyunsaturated fatty acids

Proliferation in a lekemic T cell line (Jurkat) was suppressed in a dose dependent manner by n−6 and n−3 polyunsaturated fatty acids (PUFA) added to the culture medium. At high concentrations, PUFA have a cytotoxic effect on Jurkat cells. The inhibitory effect of the PUFA was not due to production of prostaglandins, and lipid peroxidation was only partly responsible. In addition to production of peroxides and aldehydes, lipid peroxidation also reduced the plasmalogen levels in these cells. The antioxidant α-tocopherol blocked lipid peroxidation and restored the plasmalogen levels to normal. α-Tocopherol did not totally restore cell proliferation although the MDA-like products in these cultures (supplemented with PUFA) were reduced to control level. Cultures supplemented with n−6 PUFA seemed to respond better to α-tocopherol than n−3 PUFA. This suggests that n−6 PUFA may exert their growth inhibitory effect predominantly via lipid peroxidation while different mechanisms might be operating for the n−3 PUFA.     

The influence of vitamin E and selenium on lipid peroxidation and aldehyde dehydrogenase activity in rat liver and tissue

Malondialdehyde (MDA) production and cytosolic aldehyde dehydrogenase (ALDH) response were examined in rat liver tissues after feeding different levels of dietary vitamin E and/or selenium and polyunsaturated fat for 12–38 wk. MDA production was significantly increased by vitamin E deficiency or by high levels of polyunsaturated fat intake, but not by selenium deficiency. The activity of cytosolic ALDH increased upon increased production of MDA after 12–16 wk of feeding the lipid peroxidation-inducing diets. However, ALDH activity was suppressed after 38 wk of feeding the vitamin E-deficient diet. The results indicate that the hepatic cytosolic ALDH may be involved in the metabolism of MDA during a relatively short-term increase inin vivo lipid peroxidation, but that ALDH activity becomes suppressed after more severein vivo lipid peroxidation has been produced. Hepatic and plasma α-tocopherol levels and lipid peroxidation products were measured for the various dietary groups.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. II. Liver

The hepatic phospholipids of the antioxidant-deficient rat fed a source of both linoleate and linolenate showed a progressive net decrease in eicosapentaenoate, a progressive net increase in arachidonate, and there was a concomitant accumulation of fluorescent pigment of the lipofuscin or ceroid type in the tissue. An increased incorporation of isotopically labeled acetate into both the tetraenoic and pentaplus hexaenoic acid fractions was also noted, indicating that the disappearance of polyunsaturated fatty acids was partially countered by increased synthesis. Comparable results were obtained on diets containing either suboptimum or adequate levels of biologically available selenium. Vesicular dilation of the endoplasmic reticulum was noted in animals fed the tocopherol-deficient diet. In separate experiments using a necrogenic diet containing torula yeast, these subcellular alterations were found to be prevented by tocopherol but not by selenium, although selenium supplementation did prevent macroscopically observable damage.     

Lipid composition of selected margarines

Results of analytical studies on the composition of 10 selected margarines representative of consumeravailable hard and soft types are presented. Paired hard and soft products from the same manufacturer were chosen where possible. All of the margarines were compared on the basis of total fatty acid composition, polyunsaturated to saturated fatty acid ratios, totaltrans and thetrans content of the monoene and diene fractions, location of the double bond in the monoene isomers, per cent conjugation, distribution of the fatty acids at the 2 position of the triglycerides, tocopherol content, and the ratios of α-tocopherol to polyunsaturated fatty acids. As expected the soft margarines contained more polyunsaturated fatty acids than their companion hard types, but all soft margarines did not contain more polyunsaturated fatty acids than all of the hard margarines. The one margarine containing safflower oil had the highest polyunsaturated to saturated ratio. Eight of the ten margarines contained more than 15%trans monoene and nine contained less than 5%trans diene. Positional isomers in the monoene fraction were Δ6 toΔ12 with thecis Δ9 isomer predominating. All of the margarines contained less than 1.9% conjugation. The percentage oftrans monoene at the 2 position was greater for some margarines than that in the total fatty acid. This was attributed to the preferential placement of polyunsaturated fatty acids at the 2 position in the original vegetable oils. The forms of tocopherol found were characteristic of the original vegetable oils. Ratios of α-tocopherol to PUFA varied from 0.1 to 0.5 mg/g. Determination of the relationship of the amount of tocopherol content to either source or hardness is not possible on the basis of our data.     

Plasma and lipoprotein lipid peroxidation in humans on sunflower and rapessed oil diets

The effects of natural mixed diets on lipid peroxidation were investigated in humans. In the first study, 59 subjects were fed a rapeseed oil-based diet rich in monounsaturated fatty acids (MUFA) and a sunflower oil-based diet rich in polyunsaturated fatty acids (PUFA) in a cross-over manner for three and a half weeks. The lipid peroxidation products in plasma were determined by measuring conjugated dienes and malondialdehyde (MDA). In a second study, plasma thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides, and the susceptibility of very low density lipoprotein + low-density lipoprotein (LDL) toin vitro oxidation were measured from subjects fed similar MUFA and PUFA diets for six week diets. No significant differences in plasma MDA or conjugated diene concentrations were found after the rapeseed oil diet or the sunflower oil diet in Study 1. In the second study, a small but significant decrease (P<0.05) in both lipid hydroperoxides and TBARS was observed in the LDL fraction after the sunflower oil diet. Thein vitro oxidation gave opposite results, showing increased oxidation after the sunflower oil diet. Despite a high intake of α-tocopherol during the oil peroids, no increase in plasma α-tocopherol was noticed in either study. The results suggest that moderate changes in the fatty acid composition in the Western-type diet may be adequate to affect lipoprotein susceptibility to oxidationin vitro, but there is considerable disparity with some indices ofin vivo lipid peroxidation.     

An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells1,2

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe²⁺-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe²⁺-induced lipid peroxidation. In separate experiments, 20 μM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37°C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 μM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100-fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co-incubation with α-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content. Presented in part at the Experimental Biology Meetings, Anaheim, California, April 1994. Contribution from the Missouri Agriculture Extention Station, Journal #12,495.     

Distinctive Lipid Composition of the Copepod Limnocalanus macrurus with a High Abundance of Polyunsaturated Fatty Acids

We studied the copepod Limnocalanus macrurus for seasonal variation in the composition of fatty acids, wax esters and sterols in large boreal lakes, where it occurs as a glacial‐relict. Vast wax ester reserves of Limnocalanus were accumulated in a period of only two months, and comprised mono‐ and polyunsaturated fatty acids (PUFA) and saturated fatty alcohols. In winter, the mobilization of wax esters was selective, and the proportion of long‐chain polyunsaturated wax esters declined first. PUFA accounted for >50 % of all fatty acids throughout the year reaching up to ca. 65 % during late summer and fall. Long‐chain PUFA 20:5n‐3 and 22:6n‐3 together comprised 17–40 % of all fatty acids. The rarely reported C24 and C26 very‐long‐chain PUFA (VLC‐PUFA) comprised 6.2 ± 3.4 % of all fatty acids in August and 2.1 ± 1.7 % in September. The VLC‐PUFA are presumably synthesized by Limnocalanus from shorter chain‐length precursors because they were not found in the potential food sources. We hypothesize that these VLC‐PUFA help Limnocalanus to maximize lipid reserves when food is abundant. Sterol content of Limnocalanus, consisting ca. 90 % of cholesterol, did not show great seasonal variation. As a lipid‐rich copepod with high abundance of PUFA, Limnocalanus is excellent quality food for fish. The VLC‐PUFA were also detected in planktivorous fish, suggesting that these compounds can be used as a trophic marker indicating feeding on Limnocalanus.     

Characterization of forced oxidation of sardine oil: Physicochemical data and mathematical modeling

It is of major interest to the food industry to understand the mechanisms and kinetics underlying spontaneous oxidation of marine oils because these polyunsaturated fatty acid (PUFA)-rich oils, the object of several health claims, have been repeatedly recommended for dietary intake. The present study attempts to characterize forced oxidation and hydrolytic breakdown of glycerides and fatty acids in sardine oil. A simple, first-order mathematical model was postulated and successfully fitted to the experimental data. This model confirmed that the rate of decrease in concentration of intact fatty acid moieties is almost directly proportional to the number of double bonds present. Therefore, as expected, the rate of oxidative decay was virtually independent of chain length, with an overall activation energy of ca. 22 kJ mol⁻¹. Additionally, the rate of hydrolysis was correlated with the rate of oxidative decay. With the exception of fatty acids possessing more than four double bonds, PUFA proved to be relatively stable to oxidation for up to 10 h at 50–70°C, and the qualitatively richest pattern of volatiles was obtained when the reaction was performed at the highest temperature (80°C).     

Evaluation of a model system for the selective study of the lipid peroxidation process

The effect of molecular environment on the peroxidation of linoleic acid (LA), a polyunsaturated fatty acid (PUFA), initiated by ferrous ions was investigated in acidic and neutral pH conditions. Mixed nonionic surfactants TWEEN®‐20/LA micelles were established as a model system to obtain a surfactant‐in‐lipid aqueous system at high acidity level. The peroxidation of LA was induced by ferrous ions and the kinetics of the produced conjugated dienes was followed by UV measurements and the ferric thiocynate method. Ferrous ions were oxidized only by the preformed LA hydroperoxides, which under established conditions produced lipid alkoxyl and peroxyl radicals as the sole initiators of propagation. The results revealed the LA peroxidation process remained mainly unaffected within the 2.5<pH<5.5 range, while highly pH sensitive around pH 7. The propagation process prevailed at optimal concentrations of 500 µM of LA and 280 µM TWEEN®‐20, and at the ferrous ion concentration up to 75 µM, irrespective of the buffer used. Practical applications: A simple model system in water, suitable for the selective study of the lipid peroxidation propagation phase induced by ferrous ion is presented here. Fatty acids serve as model compounds susceptible to processes associated with oxidative radical initiated‐modifications of lipids. The obtained results contribute to a better understanding of the oxidative behavior of lipids, particularly those soluble in nonionic surfactant micelles in acidic medium. The oxidative stability of the PUFA in model systems containing TWEEN®‐20 and ferrous ion at low pH could be predicted and controlled by measuring the lipid hydroperoxide formation. The experimental conditions presented may also provide a suitable system for the study of the termination phase of lipid peroxidation.     

Dietary antioxidants in young swine

Young swine obtained by hysterectomy were fed purified diets low in vitamin E and supplemented with d-α-tocopheryl acetate and ethoxyquin (Santoquin^R). It was demonstrated that with very low levels of polyunsaturated fatty acids (PUFA) in the diet, both tocopherol and Santoquin protected the tissues of the pig from increased thiobarbituric acid (TBA) values and from increased hemolysis usually associated with low vitamin E status. When the dietary PUFA were increased to levels over 5%, the supplements of tocopherol and Santoquin protected against increased TBA values of tissue homogenates, but not against increased hemolysis of erythrocytes, even when blood serum showed substantial amounts of tocopherol. Some of the interrelationships of dietary PUFA and α-tocopherol were demonstrated. It was shown that for each 1% of peroxidized corn oil added to the diet above 4%, roughly 100 mg of d-α-tocopheryl acetate was necessary to protect the pigs from erythrocyte hemolysis. The failure to reach a “zero” TBA value in vitamin E-deficient swine tissue homogenates substantiated the theory ofin vivo lipid autoxidation, and the increased TBA values of incubated tissue homogenates demonstratedin vitro lipid autoxidation in tissues not protected by a biological antioxidant. Presented at the AOCS meeting in Toronto, Canada, 1962. Supported by grants from the Monsanto Chemical Co., St. Louis, Mo., and The Hormel Foundation.