Fluorescent pigments by covalent binding of lipid peroxidation by-products to protein and amino acids

The fluorescent products formed on reaction of 12-oxo-cis-9-octadecenoic acid (12-keto-oleic acid) with about 20 different amino acids, polylysine and bovine serum albumin (BSA) were studied. Besides glycine, only the basic amino acids histidine, lysine and arginine gave products with strong fluorescence. N-Acetylation of amino acids greatly reduced the fluorescence of their reaction products. The formation of fluorescent products was inhibited strongly by SH-amino acids such as N-acetyl-cysteine and glutathione. Polyacrylamide gel electrophoresis showed that BSA treated with 12-keto-oleic acid was more acidic than untreated or ricinoleic acid-treated BSA, indicating that basic amino acid residues in BSA were modified by reaction with the keto fatty acid. None of the structural analogs of 12-keto-oleic acid tested–12-oxo-trans-10-octadecenoic acid, 12-oxo-octadecanoic acid, 12-hydroxy-cis-9-octadecenoic acid (ricinoleic acid),cis-9-octadecenoic acid (oleic acid) and linoleic acid—reacted with glycine to give a fluorescent product. The fluorescent products formed on reaction of 12-keto-oleic acid methyl ester with benzyl amine and glycine methyl ester were shown to be 8-(N-substituted-4,5-dihydro-4-oxo-5-hexyl-5-hydroxy-2-pyrrolyl) octanoic acid methyl esters. The fluorescence properties of these compounds were attributed to the chromophobic system NC=CC=O which contains 6π electrons. This investigation contributes to insight of the mechanism of formation of fluorescent pigments, probably by a similar reaction of other compounds of the β,γ-unsaturated carbonyl type.     

Linoleic acid oxidation catalyzed by various amino acids and cupric ions in aqueous media

Model systems were designed to study linoleic acid oxidation in the presence and absence of various amino acids with or without cupric ions. The tested amino acids exhibited a potential prooxidant effect in linoleic acid dispersed in aqueous media. The effectiveness of various amino acids on linoleic acid oxidation decreased in the following order: cysteine > serine > tryptophan > phenylalanine > histidine > alanine. The addition of alanine, serine, phenylalanine, histidine, or tryptophan to linoleic acid showed an autocatalytic chain reaction. With cysteine, there was a linear relation between concentration of hydroperoxides and time during the early stages of oxidation. The prooxidative activity of the tested amino acids in general could be attributed to the presence of the a-amino group in the form H₃-N-R. The apparent difference in the prooxidative activity is mainly due to the functional groups attached in the β-carbon atom in the amino acid molecules. The addition of cupric ions at a concentration of 10^-5M to linoleic acid catalyzed with various a-amino acids showed that these amino acids had no significant effect. Increasing the copper concentration from 10^-5M to 10^-3M had the following effects: a shortening of the induction period of linoleic acid catalyzed by amino acids having an aromatic side chain, no effect on the induction period but an increase in the oxidation rate during the propagation step in the model systems catalyzed by alanine and serine, and in the model system containing cysteine a linear increase in the linoleic acid oxidation from the start of the reaction.     

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.     

Preparation of schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2∶1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15–57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products. Based on presentation at the AOCS Annual Meeting & Expo in Indianapolis, Indiana, April 28–May 1, 1996.     

Synthesis and characterization of poly(aspartic acid) derivatives conjugated with various amino acids

Novel derivatives of poly(aspartic acid) conjugated with various amino acids and their amphiphilic copolymers were synthesized and characterized. Methyl esters of various amino acids (in their hydrochloride form) were synthesized from the reaction of amino acids with methanol in the presence of chlorotrimethylsilane (TMSCl). Aminolysis reaction onto polysuccinimide (PSI) using various amino acid methyl esters in the presence of catalyst and the followed hydrolysis provided the corresponding amino acid-conjugated poly(aspartic acid) derivatives in high reaction yield. Amino acid—conjugated amphiphilic analogs were also prepared by introducing hydrophobic alkylamine along with amino acid using a similar procedure. The chemical structures of copolymers were confirmed by FT-IR and ¹H NMR spectroscopy. The physicochemical properties of amphiphilic copolymers were characterized using dynamic light scattering (DLS), fluorescence spectroscopy and field emission scanning electron microscopy (FE-SEM). In addition, the in vitro cell viability of the copolymers was examined. These polymers have potential applications in the pharmaceutical and cosmetic fields as delivery vehicles for bioactive molecules.     

Surface analysis of PP/EVA blends modified with amino acids for biomedical applications

The copolymers poly(propylene-co-ethylene) (PP/E) and poly(ethylene-co-vinyl acetate) (EVA) and blends of these were modified to develop an artificial matrix which promotes the growth of endothelial cells. Covalent immobilization of amino acids or sequences of adhesion glycoproteins should trigger the formation of an endothelial cell monolayer onto the polymeric surface. Reactive functional groups were generated by saponifying the ester groups of the EVA component. Esterification with oxalylic or malonic dichlorides in the gas phase yielded the required monoesters and gave the best results for further immobilization of amino acids, while reaction with α,ω-dicarboxylic acid dichlorides in solution led to diester formation. Subsequently, various protected amino acids were immobilized via the carbodiimide method. Surface analytical methods like infrared spectroscopy using at tenuated total reflection (IR-ATR), X-ray photoelectron spectroscopy (XPS), and secondary ion mass spectrometry (SIMS) were used to prove the modification steps. The analytical results confirmed covalent side-chain generation in the upper surface region. © 1995 John Wiley & Sons, Inc.     

Reaction of gossypol with amino acids and other amino compounds

The reactions of gossypol with certain amino acids and other amino compounds have been studied spectroscopically with respect to the effect of time and pH in the range from 5.7 to 7.5 at 37 C. The rate of reaction of gossypol with amino acids increases with increase in pH and has been shown to be related to the distance of the amino group from the carboxyl group within the molecule. Reaction products of gossypol with amino acids and other amino compounds were subjected to various purification procedures and analysis to determine combination ratios. In addition to the expected gossypol-to-amino compound ratio of 1:2, dictated by the formation of Schiff base-type bonds with the two aldehyde groups of gossypol, compounds with ratios of 1:3 and 1:4 were isolated. These results indicate that each of the two aldehyde groups of gossypol can react with two amino groups under the conditions studied. Deceased March 9, 1969.     

Fluorescent image analysis of lipid hydroperoxides in fish muscle with 3-perylene diphenylphosphine

A fluorescent image analysis method was developed to evaluate lipid hydroperoxide formation in fish muscle. The lipid hydroperoxides generated in white and dark fish muscles during storage at 5–6°C oxidized 3-perylene diphenylphosphine located in the tissue to yield the fluorescent derivative, 3-perylene diphenylphosphine oxide (3-PeDPPO). 3-PeDPPO thus obtained was determined by digital fluorescent image analysis. The 3-PeDPPO fluorescence intensity of white and dark muscle increased during low-temperature storage (0–24 h) and was clearly correlated with total lipid hydroperoxide levels in muscle extracts, which were determined by using HPLC based on a triphenylphosphine oxidation method (R ²=0.954). These results suggest that 3-PeDPPO fluorescence, coupled with fluorescent image analysis, is a novel tool for direct determination of lipid hydroperoxides in fish muscle without a need for extraction of lipid.     

Adsorption of amino acids on periodic mesoporous organosilicas

In this study, we prepared periodic mesoporous organosilica(PMO) materials from 4,4-bis(triethoxysilyl)biphenyl, 1,4-bis(triethoxysilyl)benzene and bis[3-(trimethoxysilyl)propyl]amine as precursors and investigated on their adsorption behavior of various amino acids as a guest material under various conditions such as different kinds of PMOs as absorbents, various concentrations of amino acids and pH. For amino acids, we tested glycine, L-lysine, and isoleucine. Adsorption of various amino acids on PMOs has been characterized by a UV–Vis spectroscopy using the ninhydrin reaction. The adsorption behavior was strongly dependent on the isoelectric point and hydrophobicity of the PMO as well as the hydrophobicity of an amino acid.     

Antioxidant Effects of Berry Phenolics Incorporated in Oil-in-Water Emulsions with Continuous Phase β-Lactoglobulin

The purpose of this study was to evaluate the effects of berry phenolics, in this case, black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins, in the presence of continuous phase β-lactoglobulin (β-Lg), on the oxidative stability of Brij 35-stabilized corn oil-in-water emulsions. The extent of lipid oxidation in emulsions was measured by determining the formation of lipid hydroperoxides and hexanal, and extent of protein oxidation by monitoring the loss of tryptophan and cysteine residues in the continuous phase β-Lg. Berry phenolics at concentration levels of 20 and 50 μM were able to prevent lipid oxidation with and without β-Lg in the aqueous phase. The results show that a combination of β-Lg and berry phenolics was more efficient in inhibiting hexanal formation than either component alone. Synergistic effects on antioxidant activity toward hexanal were observed only at the 20 μM levels of berry phenolics in combination with continuous phase β-Lg. The berry phenolics were also able to inhibit the oxidation of tryptophan and cysteine residues of β-Lg. The results show that the amino acid residues were oxidized prior to the propagation of lipid oxidation. This suggests that these amino acids were able to inhibit fatty acid scission. The information gained from this study would be useful in protecting emulsion-based food products from oxidative deterioration.     

Rapid complexing of oxoacylglycerols with amino acids peptides and aminophospholipids

We prepared model Schiff bases from 2-[9-oxo]nonanoyl glycerol (2-MAG-ALD) and various amino compounds. 2-MAG-ALD was obtained by pancreatic lipase hydrolysis of trioleoyl glycerol and reductive ozonolysis of the resulting 2-monooleoyl glycerol. The reaction products were purified by thin-layer chromatography. Schiff bases were synthesized in greater than 50% yield by reacting 2-MAG-ALD with twofold molar excess of valine, Nα-acetyl-l-lysine methyl ester and the tripeptides glycyl-glycyl-glycine, glycyl-glycyl-histidine, and glycyl-histidyl-lysine in aqueous methanol and with 1-palmitoyl-2-stearoyl glycerophosphoethanolamine (PE) in chloroform.methanol for 16 h at room temperature. Prior to analysis the bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. Reaction products were analyzed by high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS). Reduced Schiff bases of 2-MAG-ALD with PF and amino acids were analyzed by normal-phase HPLC/ESI/MS and those with peptides by reversed-phase HPLC/ESI/MS. Single adducts were obtained in all cases and both the α-amino group of valine and the ε-amino group of Nα-acetyl-l-lysine methyl ester were reactive. Molecular ions of reaction products were the only detected ions in the negative ionization mode, whereas in the positive ion mode sodiated molecular ions were also detected. The present study suggests that 2-MAG-ALD may form Schiff base adducts with amino compounds in other aqueous media, such as the intestinal lumen and in the hydrophobic environment of cell membranes.     

Preparation and purification of lipid hydroperoxides from arachidonic and γ-linolenic acids

Commercial soybean lipoxygenase may be used under carefully controlled reaction conditions to give high yields of lipid hydroperoxides. Lipid hydroperoxides so derived from γ-linolenic or arachidonic acid may be purified by high pressure liquid chromatography. Thus, commercial lipoxygenase serves as a viable source for 100 mg quantities of lipid hydroperoxides.     

Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1-pyrenylphosphine

A high-performance liquid chromatographic method, using post-column detection with diphenyl-1-pyrenyl-phosphine (DPPP), was developed for the quantitative and qualitative determination of isomeric lipid hydroperoxides (OOH). The OOH eluted from a normal-phase column were passed through a photodiode array detector and then mixed with DPPP solution in a reaction coil heated at 80°C. DPPP oxide formed by the reaction with OOH was determined by monitoring the fluorescence intensity at 380 nm and excitation at 352 nm. The conjugated diene OOH (13-cis, trans- and 9-cis, trans-OOH) and nonconjugated OOH (12-cis-trans- and 10-cis, trans-OOH) from photosensitized oxidation of methyl linoleate were determined in a molar ratio of 31∶29∶19∶21, respectively. However, only the two conjugated hydroperoxides were detected by ultraviolet absorption at 234 nm. Further applications were carried out for the determination of OOH of methyl oleate and methyl linolenate. This method proved to be useful for the determination of the OOH containing both conjugated and nonconjugated diene structures.     

Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their transformation to 4-hydroperoxynonenal

The cytotoxic aldehydes 4-hydroxynonenal, 4-hydroperoxynonenal (4-HPNE), and 4-oxononenal are formed during lipid peroxidation via oxidative transformation of the hydroxy or hydroperoxy precursor fatty acids, respectively. The mechanism of the carbon chain cleavage reaction leading to the aldehyde fragments is not known, but Hock-cleavage of a suitable dihydroperoxide derivative was implicated to account for the fragmentation [Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R. (2001) Two Distinct Pathways of Formation of 4-Hydroxynonenal. Mechanisms of Nonenzymatic Transformation of the 9- and 13-Hydroperoxides of Linoleic Acid to 4-Hydroxyalkenals, J. Biol. Chem. 275, 20831–20838]. Both 8,13- and 10,13-dihydroperoxyoctadecadienoic acids (diHPODE) could serve as precursors in a Hock-cleavage leading to 4-HPNE via two different pathways. Here, we synthesized diastereomeric 9,12-, 10,12-, and 10,13-diHPODE using singlet oxidation of linoleic acid. 8,13-Dihydroperoxyoctadecatrienoic acid was synthesized by vitamin E-controlled autoxidation of γ-linolenic acid followed by reaction with soybean lipoxygenase. The transformation of these potential precursors to 4-HPNE was studied under conditions of autoxidation, hematin-, and acid-catalysis. In contrast to 9- or 13-HPODE, neither of the dihydroperoxides formed 4-HPNE on autoxidation (lipid film, 37°C), regardless of whether the free acid or the methyl ester derivative was used. Acid treatment of 10,13-diHPODE led to the expected formation of 4-HPNE as a significant product, in accord with a Hock-type cleavage reaction. We conclude that, although the suppression of 4-H(P)NE formation from monohydroperoxides by α-tocopherol indicates peroxyl radical reactions in the major route of carbon chain cleavage, the dihydroperoxides previously implicated are not intermediates in the autoxidative transformation of monohydroperoxy fatty acids to 4-HPNE and related aldehydes.     

Thiobarbituric acid-reactive substances from peroxidized lipids

The thiobarbituric acid (TBA) reaction was performed on linoleic acid 13-monohydroperoxide, autoxidized fatty esters, edible fats and oils, rat liver microsomal lipids, and on human erythrocyte ghost lipids in order to determine which substances from peroxidized lipids are TBA-reactive. The reaction was carried out in 2% acetic acid containing butylated hydroxytoluene using two different reaction modes: a one-step mode which involves heating at 100°C, and a two-step mode which involves first treatment at 5°C and subsequent heating at 100°C. Yields of the red 1∶2 malonaldehyde/TBA adduct, as estimated by absorbance, fluorescence intensity and high-performance liquid chromatography, were much higher than the malonaldehyde content as determined by direct chemical analysis. Yields of red pigment obtained by the two-step mode were slightly higher than those obtained by the one-step mode. Pigment yields were dramatically increased by addition oft-butyl hydroperoxide. Red pigment formation from alkenals and alkadienals was similarly enhanced by the two-step mode or by addition oft-butyl hydroperoxide, whereas pigment formation from malonaldehyde was not. It appears likely that a component of the total red pigment formed from the peroxidized lipids was due to aldehyde species other than malonaldehyde.     

Inulin polysaccharide having pendant amino acids: Synthesis and characterization

Inulin polysaccharide was esterified with N-protected α-amino acids (N,N′-di-benzylocarbonyl-L -lysine and N-benzylocarbonyl-glycine) under a mild condition (room temperature) and within short reaction times (6 h). The esterification reactions were conducted in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine as a catalyst. The optimal reaction time (6 h) was determined by monitoring the concentration of free carboxylic acid of the N-protected amino acids during the reaction. The degree of substitution per fructose unit was 0.95 for inulin-lysine and 1.01 for inulin-glycine. The resulting biopolymer was deprotected by catalytic transfer hydrogenation method using 1,4-cyclohexadiene as an effective hydrogen donor. The structures, molecular weight, and thermal properties of the amino acid esters of inulin were determined by Fourier transform infrared spectroscopy, ¹H and ¹³C NMR, UV, viscosity, and dicyclohexylcarbodiimide. This new modified inulin polysaccharide would have the potential as a biomaterial for biomedical applications. © 1998 John Wiley & Sons, Inc. J. Appl. Polym. Sci. 70: 953–963, 1998     

Influence of some aromatic amino acids on the swelling behavior of acrylamide/maleic acid hydrogel

Summary Influence of some aromatic amino acids (histidine, phenylalanine and tryptophan) on the swelling behavior of acrylamide/maleic acid hydrogel (AAm/MA) prepared by γ-radiation was investigated. Swelling tests of AAm/MA hydrogel were made in buffer solutions and amino acid solutions at various pH at 37°C. The pH values are ionization of α-carboxyl groups (pK'₁), α-amino groups (pK'₂) and, isoelectric points (pI) of amino acids. The swelling of AAm/MA hydrogel increased when pH values of solutions were increased. The value of equilibrium swelling of AAm/MA hydrogel was 1035% at pH 10 buffer, while it was 880% at pH 2 buffer. The values of equilibrium swelling of AAm/MA hydrogel in phenylalanine, tryptophan and histidine solutions varied among 1130–1245% at pH 10, while they were among 790–975% at pH 2. The rate constant of swelling, diffusional exponent, network parameter and, diffusion and intrinsic diffusion coefficient were calculated by swelling kinetics. Diffusion of the penetrants into the hydrogel was found to be non-Fickian character. The diffusion coefficients of the hydrogel varied between 3.33×10⁻⁶– 7.71×10⁻⁶ cm ²s⁻¹, while the intrinsic diffusion coefficients waried between 4.03×10⁻⁶– 8.48×10⁻⁶ cm ²s⁻¹. Received: 22 December 1997/Revised version: 3 March 1998/Accepted: 5 March 1998     

Fluorescent products from reaction of peroxidizing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine

Fluorescent chromophores produced by reaction of peroxidizing arachidonic acid or methyl docosahexaenoate with synthetic dipalmityl phosphatidyl ethanolamine were lipid soluble, and those from reaction with phenylalanine were water soluble. In all reaction systems that contained polyunsaturated fatty acid and only one amine compound, the development of fluorescence was linearly related to oxygen absorption for 12–24 hr (p<0.001) and to the amount of thiobarbituric acid reacting materials until the rate of oxygen absorption decreased. The fluorochromes typically had maximum excitation at 360 nm and maximum emission at 430–440 nm, indicating that they were conjugated Schiff bases with the general structure R−N=C−C=C−N−R, where R represents the amino acid phenylalanine or the phospholipid phosphatidyl ethanolamine. The fluorochromes were similar to those extracted from isolated age pigments and tissues of animals that are aged, vitamin E-deficient, or stressed with highly unsaturated lipid diets.     

Modification of lysine amino groups by the lipid peroxidation product 4,5(E)-Epoxy-2(E)-hepteal

The reaction between 4,5(E)-epoxy-2(E)-heptenal (EH) andl-lysine was studied to characterize some of the compounds that may be produced when proteins react with peroxidizing lipids. A mixture of EH and lysine was incubated overnight at room temperature and then fractionated by high-performance liquid chromatography (HPLC). Fractions were freeze-dried and characterized by¹H and¹³C nuclear magnetic resonance (NMR) and mass spectrometry. Four major pyrrole derivatives were obtained, namely 1-(5′-amino-1′-carboxypentyl)-pyrrole (3), 1-(5′-amino-1′-carboxypentyl)-2-(1″-hydroxypropyl)pyrrole (diastereomers 5 and 8), 1-(5′-amino-5′-carboxypentyl)pyrrole (7), and 1-(5′-amino-5′-carboxypentyl)-2-(1″-hydroxypropyl)pyrrole (9). In addition, several lysine complexes were detected. A polymer (1b) that was responsible for the color and the fluorescence produced in the reaction was isolated by gel filtration chromatography from a fraction obtained by HPLC. Formation of pairs of analogs (5 and 3, 9 and 7) with and without a substituent in position 2 of the pyrrole ring suggested that the compounds were produced by the same mechanism, with the formation of the 2-unsubstituted pyrroles corresponding to the loss of the 2-substituent as propanal; propanal was detected by headspace capillary gas chromatography. A reaction mechanism is proposed based on the NMR data obtained when the reaction was monitored in real time in an NMR tube. The results suggest that pyrrolic amino acids 7 and 9 may be present in proteins that have been damaged by peroxidizing lipids.     

Covalent binding of peroxidized phospholipid to protein: III. Reaction of individual phospholipids with different proteins

Various peroxidized phospholipids were reacted with proteins under N₂. In all cases, phospholipid is bound covalently to the proteins whose molecular size is increased. Both the amount of bound phospholipid and the increase in molecular size of the protein depends on the nature of the phospholipid. Ultraviolet (UV) absorption of the proteins is increased in qualitatively similar ways. Their difference spectra, which show a gradual increase in absorption from 400 nm toward shorter wavelength, differ from that of malonaldehyde-protein complexes. The various complexes of proteins and peroxidized phospholipids have similar fluorescence spectra showing two excitation maxima at 310–320 nm and at 340–350 nm, respectively, and emission maximum at ca. 400 nm. This is different from both fluorescence spectra of malonaldehyde-protein complexes and fluorescence spectra reported for proteins after reaction with peroxidized polyunsaturated fatty acids. Amino groups of the proteins are consumed in the reaction with peroxidized phospholipids. Blocking the amino groups decreases the binding of phospholipid considerably. Besides amino groups, other structures of the protein molecule react with the peroxidized phospholipids. The similar features of UV absorption, fluorescence, decrease of amino groups, and covalently bound phospholipid phosphorus of the various complexes suggest that they are formed by common type of reactions. The reactions seem to be different from those generally believed important between peroxidized lipid and protein. Important reacting species are compounds other than malonaldehyde. Preliminary report of this work was presented at the ISF/AOCS World Congress, New York, 1980.