PHOSPHOUFED GXZBATTON [N EMULSTONS Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY GLENN A. CORLISS 1968 Inn—— 7 _ LIBRARY Michigan State UfllVCl It)’ This is to certify that the thesis entitled Phospholipid Oxidation in Emulsions presented by Glenn Arthur Corliss has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science T 7”€@M§m Major! professor Date September 12, 1968 0-169 ABSTRACT PHOSPHOLIPID OXIDATION IN EMULSIONS by Glenn A. Corliss The autoxidation of emulsions consisting of puri- fied phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC), extracted from egg and soybean lipids by silicic acid column chromatography, and 0.1M borate (pH 7.0) buffer was compared by measurement of oxygen uptake, TBA absorbance, ultraviolet absorbance and changes in the percentage composition of unsaturated fatty acids. Egg phospholipids were more reactive than soybean phospholipids and PE from egg and soybean oxi- dized more readily than egg and soybean PC respectively. All phospholipid fractions emulsified in 0.1M borate (pH 7.0)--Tween 20 buffer had comparable activation energies for oxidation. However, those fractions of a given phOSpholipid with the most unsaturation oxidized most rapidly. Although unsaturated fatty acid oxidation occurred in all fractions, phospholipid oxidation in emulsions was only partly a function of fatty acid com- position. The ethanolamine and choline moieties-in- fluenced the induction period for the oxidation of PE and PC respectively. The effect of the nitrogen moiety Glenn A. Corliss on the oxidation of unsaturated fatty acids was investi- gated in model systems with methyl linolenate emulsions. Ethanolamine was observed to exert a strong prooxidative effect in methyl linolenate emulsions compared to choline which had a slight antioxidative effect. Specific changes in the phospholipid molecule dur- ing oxidation were measured by various physico-chemical tests. Of the two major polyunsaturated fatty.acids in egg phospholipids, arachidonic acid disappeared at a more rapid rate during oxidation while the concentration of linoleic acid decreased to a level that was relatively stable. Considerable oxygen was absorbed by PE and PC after arachidonic acid had disappeared completely and linoleic acid had reached its minimum level. Higher TBA and diene and triene conjugation values were observed for PE than for PC. TBA and diene and triene conjugation attained their maximum levels for egg PC when the rate of oxygen consumption began to decrease. These values in— creased with oxygen uptake for egg PE and continued to increase after the rate of oxygen consumption had de- creased. Although diene conjugation values increased as a result of the oxidation of soybean PE, triene con- jugation was low at all stages of oxidation. The concen- tration of the free amino group of egg and soybean PE decreased and a brownish-yellow discoloration developed during oxidation. Glenn A. Corliss Investigations were conducted on the effect of various pro- and antioxidative substances on the autoxi- dation of phospholipid emulsions. The reactivity of egg PE and PC was decreased by the addition of ethanolamine in 1:1 molar ratios of nitrogen moiety and phospholipid, which was in contrast to the prooxidative effect of the ethanolamine in methyl linolenate emulsions. Copper accelerated the oxidation of PE and PC. The antioxidative effect of ethanolamine and glycine was overcome by the addition of cOpper. The induction period for the oxi— dation of PC was less in the presence of ethanolamine plus copper and glycine plus copper than in the presence of COpper alone. The greater reactivity which resulted when c0pper and an amino compound were added together was attributed to the formation of a complex between the metal and the nitrogen moieties. Glycine, lysine, and tryptophan increased the induction period of egg PE while histidine completely inhibited oxidation for the period of study as evaluated by oxygen uptake and TBA determinations. Vari-l ations in the antioxidative effect of glycine, lysine, tryptOphan and histidine on the oxidation of PE were postulated to occur as a result of differences in the activation energy of the antioxidative reaction which depended upon the particular amino acid that was present. Butylated hydroxytoluene (BHT) and sodium tetra, ethyl- enediamine tetraacetate (Na EDTA) completely retarded Glenn A. Corliss oxidation of egg PE as measured by oxygen uptake and the TBA test for the period of study. The ability of Na EDTA, a metal chelating agent, to inhibit phospholipid oxi- dation in emulsions indicated that trace metal impurities could have influenced oxidation. The strong antioxidative effect that was observed for BHT confirmed the work of others and suggested that its presence in lipid samples during their extraction, manipulation, and storage would retard the develOpment of alterations as a result of autoxidation. PHOSPHOLIPID OXIDATION IN EMULSIONS By Ht . 3\\ ' Glenn A: Corliss A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1968 ACKNOWLEDGMENTS Sincere appreciation is extended to Dr. L. R. Dugan, Jr. for his assistance in selecting the author's course of study and his constructive criticism and counsel during the various phases of the research work. The author is grateful to Dr. B. S. Schweigert, Chairman of the Food Science Department, for his interest, advice and encouragement. Appreciation and thanks are extended to the members of the guidance committee: Drs. C. L. Bedford, R. W. Luecke, C. M. Stine and D. E. Ullrey for their critical review of this dissertation. To the Pillsbury Company, Minneapolis, Minnesota, the author is indebted for a Graduate Training Grant which partially supported this study. Lastly, the author is especially grateful to his wife, Nan, and son, Kevin, for their understanding, encouragement and sacrifices during this graduate program. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . LIST OF TABLES . . . . . . . . LIST OF FIGURES . . . LIST OF APPENDICES INTRODUCTION . LITERATURE REVIEW . . . Mechanism of Autoxidation. . . Lipid Oxidation in Aqueous Media . Lipid Autoxidation Tests . . . Phospholipids--As Antioxidants Phospholipids--As Prooxidants . . Metal Catalysis . . Effect of Amino Acids on the Autoxidation of Unsaturated Fatty Acids and Esters Effect of Amino Acids on Phospholipid Oxidation . . . . . Mechanism of Antioxidation . EXPERIMENTAL . . . . . . . . . Materials . . . . . . . . . Sample Preparation . . . Chemicals and Solvents . . . . Methods. . . . Extraction of Egg Phospholipids Silicic Acid Column Chromatography Thin-Layer Chromatography (TLC) . Phosphorus Determination . . Dispersion of Phospholipids in Aqueous Media. Preparation of Methyl Linolenate Dispersions. Introduction of Additives to Phospholipid Dispersions . . . . . . . Oxygen Uptake Measurements. . . Preparation of Methyl Esters for Gas-Liquid Chromatography . . . Gas-Liquid Chromatography (GLC) . Spectrophotometric Determinations as Indices of Phospholipid Oxidation . . iii Page 11 vi ix 10 18 2O 23 28 29 31 31 31 31 32 32 33 37 38 39 H2 MA ”5 M6 “7 Page RESULTS AND DISCUSSION. . . . . . . . . . 51 The Effect of pH on Oxygen Consumption . . . 51 Reactivity of Egg and Soybean Phospholipids. . 53 Activation Energies of PE and PC . . . . 60 The Effect of Fatty Acid Unsaturation on Phospholipid Oxidation . . . . 62 Comparison of Oxidation Rates Obtained by Experimentation and Calculation . . . 68 Effect of the Nitrogen Moiety on Phospholipid Oxidation . . . . 73 Effect of the Nitrogen Moiety on Methyl Linolenate Oxidation . . . 7H Relative Rates of Autoxidation of Polyun- saturated Fatty Acids in Phospholipids. . . 79 Relationship Between Spectrophotometric ; Determinations and Oxygen Uptake. . . . . 8A Influence of the Free Amino Group on Phospholipid Oxidation . . . . . 90 Influence of Added Nitrogen— Containing Compounds on Phospholipid Oxidation. . . . 92 The Effect of Amino Acids on Phospholipid Oxidation . . . . . . . . . 99 Phospholipid Antioxidation . . . . . . . ION General Discussion . . . . . . . . . . 106 SUMMARY AND CONCLUSIONS . . . . . . . . . 115 PROPOSALS FOR FURTHER RESEARCH . . . . . . . 119 BIBLIOGRAPHY . . . . . . . . . . . . . 123 APPENDICES. . . . . . . . . . . . . . 137 iv Table LIST OF TABLES Fatty Acid Composition of Purified Phospholipids . . . Energy of Activation for the Autoxidation of Phospholipids Emulsified in 0.1M Borate (pH 7.0)—Tween 2O Buffer Fatty Acid Composition of Three Egg Phosphatidyl Choline Fractions Separ— ated on a Silicic Acid Column. . . Comparison of the Oxidation Rates of Egg Phospholipids Obtained by Experimen— tation and Calculation . . . . . Comparison of the Oxidation Rates of Soy- bean Phospholipids Obtained by Experi- mentation and Calculation Analysis, By the TBA Test, of the Extent of Oxidation of PE Samples Emulsified in 0.1M Borate (pH 7.0) Buffer After 50 Hours at 25°C . . , . . Page 59 63 65 69 70 102 LIST OF FIGURES Figure l. The Effect of pH on Oxygen Consumption of Phosphatidyl Ethanolamine (PE) and Phosphatidyl Choline (PC) in 0.1M Borate Buffer After 50 Hours. Temper- ature of Reaction: PE-25°C, PC-AO°C 2. The Oxygen Consumption of Egg Phosphatidyl Ethanolamine and Phosphatidyl Choline Emulsified in 0.1M Borate (pH 7.0)- Tween 20 Buffer at 25°C . . . 3. The Oxygen Consumption of Soybean Phos- phatidyl Ethanolamine and Phosphatidyl Choline Emulsified in 0.1M Borate (pH 7. O) -Tween 20 Buffer at “0°C . . A. The Oxygen Consumption of Egg and Soybean Phosphatidyl Ethanolamine Emulsified in 0.1M borate (pH 7. O) -Tween 20 Buffer at 25°C 0 o o o o o o o o o 5. The Oxygen Consumption of Egg and Soybean Phosphatidyl Choline Emulsified in 0.1M Borate (pH 7.0)-Tween 2O Buffer at 40°C 6. Arrhenius Plot of the Autoxidation of Egg and Soybean Phospholipids Relating the Rate of Oxidation to the Reciprocal of Temperature . . . . . . . . . 7. The Effect of Arachidonic Acid Composition on the Oxygen Consumption of Egg Phos— pholipids Emulsified in 0.1M Borate (pH 7.0)-Tween 2O Buffer at AO°C . . 8. The Effect of Arachidonic Acideomposition on the Oxygen Consumption of Egg Phos- pholipids Emulsified in 0.1M Borate (pH 7.0)-Tween 2O Buffer at 25°C vi Page 52 5M 55 56 57 61 66 67 Figure 9. The Effect of Phosphatidyl Ethanolamine (PE) Contamination on the Oxygen Con- sumption of Egg Phosphatidyl Choline (PC). PC was Emulsified in 0.1M Borate (pH 7.0)-Tween 20 Buffer and Oxidized at 40°C . . . . . . . . . . 10. The Effect of Ethanolamine and Choline" Chloride on the Autoxidation of Methyl Linolenate Dispersed in 0.1M Borate (pH 7.0) Buffer at 40°C . . 11. Relationship Between the Disappearance of the Two Major Polyunsaturated Fatty Acids of Egg Phosphatidyl Choline Emulsified in 0.1M Borate (pH 7.0) Buffer and Oxygen Uptake at “0°C. 12. Relationship Between the Disappearance of the Two Major Polyunsaturated Fatty Acids of Egg Phosphatidyl Ethanolamine Emulsified in 0.1M Borate (pH 7.0) Buffer and Oxygen Uptake at 25°C. . l3. Alterations of Egg Phosphatidyl Choline Emulsified in 0.1M Borate (pH 7.0) Buffer During Oxidation at u0°C as Evaluated by Various Physico-Chemical Tests. . . . . . . . . . . 1U. Alterations of Egg Phosphatidyl Ethanola- mine Emulsified in 0.1M Borate (pH 7.0) Buffer During Oxidation at 25°C as Evaluated by Various Physico-Chemical Tests. . . . . . . . . . . 15. Alterations of Soybean Phosphatidyl Ethanolamine Emulsified in 0.1M Borate (pH 7.0) Buffer During Oxidation at 40°C as Evaluated by Various Physico- Chemical Tests. . . . . . . 16. The Effect of Choline Chloride and Ethanolamine on the Autoxidation of Egg PE Dispersed in 0.1M Borate (pH 7.0) Buffer at 25°C . . . . . . vii Page 75 77 80 81 85 86 87 9H Figure 17. 18. 19. 20. The Effect of Ethanolamine and CuSOu on the Autoxidation of Egg PC Dispersed in 0.1M Borate (pH 7.0) Buffer at 40°C . . The Effect of Glycine and CuSOu on the Autoxidation of Egg PC Dispersed in 0.1M Borate (pH 7.0) Buffer at 40°C. The Effect of Various Amino Acids on the Autoxidation of Egg PE Dispersed in 0.1M Borate (pH 7.0) Buffer at 25°C. The Effect of a Metal, An Antioxidant and a Chelating Agent on the Autoxidation of Egg PE Dispersed in 0.1M Borate (pH 7.0) Buffer at 25°C. viii O Page 95 97 100 105 LIST OF APPENDICES Appendix A. Standard Curve for Phosphorus Determi— nation . B. Fatty Acid Composition of Purified Phospholipid Fractions Dissolved in Solvent and Dispersed in Buffer. 0. Standard Curve for Ethanolamine. ix Page 138 140 142 INTRODUCTION Autoxidation of lipid materials is a complicated phenomenon even when it involves only the oxidation of an anhydrous polyunsaturated fatty acid under carefully controlled conditions. Autoxidation of fats in foods is further complicated since fat is present as a diverse mixture of many lipids. The fat is often in a finely divided or emulsified state, intimately associated with a mixture of solid components and a complex aqueous phase containing a variety of both soluble and colloidally dispersed materials. In such a system, numerous reactions, both pro- and antioxidative occur simultaneously. Studies in a variety of food and natural products have shown that the early oxidation reactions are usually most dominant in the phospholipid moieties. The fatty acid composition of phospholipids vary with source but seem to follow a common pattern in that the phoépholipid fatty acids are more unsaturated than those found in associated triglycerides and are-more highly unsaturated at the B—position than at the a'-position. .Since the phospholipids contain phosphorus, a nitrogen containing component such as choline, ethanolamine or serine, and unsaturated fatty acids, they offer a system far more complicated for oxidation than the neutral triglycerides. One of the unexplained factors of basic interest is whether the tendency of phospholipids to oxidize is due simply to the high unsaturation of the fatty acids of the phospholipids or whether there is a different ten— dency and therefore rate of oxidation according to the specific molecular constitution of each individual phospholipid. The purpose of this research was twofold. The major objective was to characterize the role of the nitro— gen moiety during oxidation in order to learn whether the. oxidation of phosphatidyl ethanolamine (PE) and phospha- tidyl choline (PC) is related to the degree of unsatur- ation of the fatty acids or to the nitrogen containing component. Although published reports had indicated that unsaturated fatty acids and the nitrogen moiety are factors affecting the oxidation of phOSpholipids (1, 59, 77, 99), further study was needed in order to determine the relative importance of unsaturation and the nitrogen component on phospholipid oxidation. Another objective was to investigate the influence of various substances on the susceptibility of phospholipids to oxidation. The potential toxic effect of some commercial antioxi- dants has increased interest in the utilization of- antioxidative substances which occur naturally in biological materials. In this study, the effect of amino acids, ethanolamine, choline, copper, butylated hydroxytoluene and sodium tetra, ethylenediamine tetra- acetate on the oxidation of PE and PC was determined. Egg yolks and a commercial soybean phOSphatide preparation were utilized as sources of phospholipids. PE and PC were fractionated by Silicic acid column chromatography and their purity was ascertained by thin—layer chromatography. Purified PE and PC were emulsified in borate (pH 7.0) buffer prior to the measurement of oxidation, since it was felt that oxi- dation under a neutral pH in the presence of moisture could be more readily controlled in terms of oxidation rate and would be more nearly typical of changes occur- ring in a number of natural food systems than oxidation in a dry system. Autoxidation of PE and PC emulsions was measured by oxygen uptake, TBA absorbance, ultraviolet absorbance and by changes in the percentage composition of unsaturated fatty acids. LITERATURE REVIEW Mechanism of Autoxidation The spontaneous reaction between atmospheric oxygen and many types of organic compounds has been termed autoxi- dation. Autoxidation is an autocatalytic reaction in that the rate of reaction increases with time, due to the for— mation of products which themselves catalyze the reaction. Autoxidation may be beneficial or deleterious depending on the conditions and circumstances under which it occurs (115). It is advantageously employed in the production of blown oils and in various oxidation and polymerization pro- ducts in the drying oil industry. Large quantities of organic chemicals are produced by the controlled oxidation of petroleum hydrocarbons (115). Rancidification and other forms of oxidative deterioration of many foods result from autoxidation but are undesirable. Oxidative rancidity in foods has been traced to the lipid portions, in which both the simple triglycerides and the more complex phospholipids may be involved. The common feature of oxidative rancidity is the reactivity of the unsaturated fatty acid moieties in the lipids (28). In practice, the autocatalytic autoxidation of food lipids almost always involves the presence of catalytic entities, such as prooxidants, antioxidants and light. Autoxidation occurs in a series of reactions in- volving initiation, propagation and termination steps. These steps have been formulated as (28): Initiation RH + 02 -—————> R- + oOOH [1] Propagation R- + 02 -—————> ROO- [2] R00- + RH ———> ROOH + R- [3] Termination R- + R- —————> RR [4] R° + ROO° ——> ROOR [5] R00- + R000 ——> ROOR + 02 [6] RH refers to any unsaturated fatty acid in which the H is labile by reason of being on a carbon atom adjacent to a double bond. R- refers to a free radical formed by removal of a labile hydrogen. The over-all oxidation may be expressed as RH + O —————> ROOH up to a certain stage 2 of oxidation. The oxidation process becomes more complex after the development of a quantity of hydroperoxides (ROOH), for the ROOH then decomposes, either through thermal instability or through reaction with other materials, to form more free radicals which then partici- pate further in the chain reactions (28). Several features of the autoxidation of common food fats have been summarized by Lundberg (68). They are: The reaction involves, primarily, unsaturated acyl groups, and the hydroperoxide group formed as a result of autoxidation, appears in an alpha position relative to a double bond. The rates of autoxidation are greatly dependent upon the degree of unsaturation of the fatty acids, increasing in an exponential manner with increasing unsaturation. Derivatives of satu- rated fatty acids autoxidize to form hydro- peroxides of undetermined structures, although under ordinary conditions, the reaction is extremely slow. Various extraneous influences, if present, can affect the rate of oxidation of any given food fat. Prooxidants, including trace metals and biological catalysts, accelerate the rate of oxidation. All forms of light radiation have marked accelerating effects. A multitude of substances, especially various phenolic com- pounds, exert an antioxidant or negative catalytic effect. As with other chemical reactions, an increase in temperature increases the rate of autoxidation. While the major products of fat autoxidation are hydroperoxides, certain secondary products, not derived from peroxides, are formed concurrently. In particular, with unsaturated fatty acids, small amounts of conjugated ketones are produced with the hydroperoxides. The undesirable flavors and odors of autoxidized food materials are not due to the hydroperoxides themselves, but to secondary substances such as aldehydes, ketones, acids and polymeric com- pounds which are derived through various re- actions and to further oxidation of the peroxides and their degradation products. Lipid Oxidation in Aqueous Media Several reactions occur when polyunsaturated fatty acids or their derivatives are diSpersed for extended periods in water in the presence of air. AS early as 1937, Bloor and Snider (8) discovered that unsaturated fatty acids become water-soluble upon autoxidation. Schulte and Schillinger (110), while studying the non- enzymatic oxidation of vitamin A acetate, established that the reaction catalyzed by oleic acid proceeded faster in a two-phase oil-water system than in a homo- geneous nonaqueous phase. In contradiction, Spetsig (114) reported that the autoxidation of methyl linoleate was inhibited by water although, after the chain reaction had started, its course was not affected by the presence of water of pH between 5.1-9.2. Mabrouk and Dugan (70) observed that the oxidation rates of methyl linoleate emulsions increased markedly as the pH of the emulsion was raised from 6.0 to 8.0 while the oxidation rates of linoleic acid emulsions increased gradually from pH 4.5 to 5.5 and then decreased gradually to a minimum at pH 8.0. The oxidation of lipids in water is strongly in- fluenced by the degree of contact between the lipid and the oxygenated aqueous phase. The autoxidation of oil emulsified in water with and without detergents increased with the degree of dispersion of the oil and the effect of the detergents was greatest for oils with the most double bonds (52). Research on the oxidation of carotene in linoleate soap solution has demonstrated that there is a critical middle concentration of linoleate where oxi- dation is minimal. When the linoleate concentration was below this middle concentration, the oxidation of carotene was not coupled to the oxidation of linoleate; however, the rate of autoxidation of both carotene and linoleate increased when the level of either was raised provided the linoleate concentration was above the critical middle concentration (66). Studies on the oxidation of methyl oleate after irradiation with UV light or saturation with oxygen at 50°C have shown that oxidation proceeded slower in water than in the dry state (117). These results were attributed to a higher solubility of oxygen in oil than in water. The addition of a surfactant or improved dispersion in the absence of surfactant increased the rate of oleate oxidation. At one time, it was thought that the presence of water—soluble products in aqueous dispersions of polyun- saturated compounds resulted from the transfer of impuri- ties from the original material to the aqueous phase (107). This possibility was eliminated by the finding that a higher yield of ultraviolet—absorbing water- soluble products were obtained when several successively smaller portions of highly purified starting material were dispersed in water (106). Infra—red spectroscopy of the dry residue from the clear filtered water phase demonstrated the presence of hydroxyl groups and a re- duction in the intensity of double-bond absorption (106). One result of the chemical interaction of linoleic acid and water is the formation of a saturated tetrahydroxy fatty acid (71). The identification of "active oxygen" in the aqueous phase (109) and the finding that the ex- clusion of oxygen from the dispersion prevented the formation of hydroxy compounds (108), strongly indicated that oxidation could occur when unsaturated lipids were dispersed in water. The first major reaction in the aqueous oxidation of linoleic acid is believed to be identical with that which occurs in the autoxidation of dry linoleic acid, namely the formation of hydrOperoxides. Measurement of the UV absorption of the aqueous phase after prolonged dispersion of linoleic acid in water has indicated the presence of conjugated double bonds (105). Hydroperoxides are known to be unstable and to react further with oxygen to yield secondary products such as aldehydes, ketones, dimers and polymers, and epoxides. A characteristic feature of the oxidation in aqueous suspension is that the water favors the formation of such tertiary products as hydrogen peroxide and carboxylic acids by the hydro- lysis of secondary products (105). These products are formed only in minute amounts in the absence of water. 10 Baker and Wilson (4, 5) succeeded in separating the water-soluble products of UV-irradiated linoleic and linolenic acids into volatile and relatively non-volatile fractions, each of which reacted with thiobarbituric acid and peroxidase. The volatile fraction contained hydrogen peroxide and probably malonaldehyde and the non-volatile fraction had at least twelve compounds (approximate chain length, 70-130) containing a-B—unsaturated carbonyl groups. A number of compounds formed as a result of the aqueous oxidation of polyunsaturated acids have been identified by Schauenstein and co-workers (105). Since most of these compounds are short-chain products they are continuously eluted by the aqueous phase and become unavailable for further reaction in the oil phase. As a consequence, the oxidation of polyunsaturated lipids in water provides an Opportunity for the isolation and identification of intermediate products from classical autoxidation. Many of these intermediate products have biological and medical significance in addition to being important in the deterioration of food lipids. Lipid Autoxidation Tests The oxidative degradation of unsaturated fatty acids can be followed by determining the total consumption of oxygen, by determining the amount of a product of lipid oxidation or by measuring the decrease in the con- centration of unsaturated lipids. ll Peroxide Test The quantitative determination of peroxides is the most commonly used analytical method for following the course of autoxidation reactions (115). The peroxide test is based on the assumption that potassium, sodium or hydrogen iodide will liberate iodine in the presence of peroxidized product. The iodine, liberated in a stoichio- metric ratio of two atoms of iodine for each atom of active oxygen, can be quantitated by titration with sodium thiosulfate. Although numerous peroxide methods have been prOposed, results on a given sample, using different methods and different Operators, are often quite erratic. Some investigators attribute this to errors in the methods or procedures, while others are inclined to suSpect the peroxides themselves (65). TBA Test The 2—thiobarbituric acid (TBA) test has been applied to the estimation of rancidity in dairy products, meats, fishery products, cereal and baked products, and fats and oils (16). The pigment produced in this sensitive color reaction is known to be a condensation product of two molecules of TBA and one of malonic dialdehyde. A fea- ture of the TBA test is its comparative specificity for oxidation products of polyunsaturated fatty acids. The TBA test can be applied directly to a lipid-containing material without prior extraction of the fat. The test 12 supposedly gives a more reliable measure of the extent of oxidation than the peroxide determination in cases where substances other than fat are present and cause decomposition of peroxides (67). Ultraviolet Absorption Oxidation of polyunsaturated fatty acids is accompanied by increased ultraviolet absorption due to the formation of conjugated diene and triene hydro- peroxides. The infrared spectra of the mixed hydro- peroxides from autoxidized linoleate, isolated by counter- current distribution, indicated that the hydroperoxides assumed the cis-trans and trans-trans configurations (92). Cis-trans hydrOperoxides predominated at low tem- peratures while trans-trans hydroperoxides were more prevalent at high temperatures and high levels of autoxi- dation. The magnitude of change of ultraviolet absorption is not easily related to degree of oxidation since the effects upon the various unsaturated acids (oleic, linoleic, linolenic, arachidonic, e.g.) are different in quality and magnitude (47). The spectral change for a given substance, however, can be used as a relative mea- sure of oxidation and probably has its best application in the detection of oxidation rather than its measurement (115). The higher the absorption, the greater has been the exposure to oxygen. 13 Oxygen Absorption Tests Manometric respirometers such as those used for biochemical respiration studies have been employed in many lipid laboratories to measure oxidation. In the usual set-up, a lipid sample in a closed glass chamber is agitated in a liquid bath at a constant temperature. The atmosphere in the chamber is either air or oxygen. As oxidation progresses, the amount of oxygen absorbed is indicated by a decrease in pressure which can be determined manometrically (67). Miscellaneous Tests Since there is no single approach to the problem of measuring lipid oxidation,various methods have been developed. Thin-layer chromatography has been used to detect and classify peroxides in complex lipid mixtures (82). Changes in the concentration of unsaturated fatty acids of phospholipids have been evaluated by gas-liquid chromatography (26, 99) and iodine values (59). The disappearance of the free amino group of phosphatidyl ethanolamine during oxidation has been measured by the ninhydrin reaction. Much attention has been directed toward the separation, estimation and identification of the total and volatile carbonyl degradation products from oxidizing fats. The most widely used method of determining carbonyl substances formed by the secondary degradation of hydroperoxides is probably the colorimetric l4 2,4-dinitrophenylhydrazine procedure of Henick §£_al. (45). A comprehensive review of carbonyl determinations is beyond the scope of this review. However, methods for the separation and identification of carbonyl com- pounds have been reviewed by Day (18) and Badings and Wassink (3). All known oxidation tests have limitations, since no single procedure has proved adaptable to all lipid- autoxidizing systems. There is a definite need to de- vise more methods to characterize, objectively, accurately and rapidly, changes which occur during the autoxidation of lipid materials. Phospholipids--As Antioxidants Phosphatides are reported to have two opposing fea- tures when present in food products: participation in the protection of neutral lipid autoxidation and the acquire— ment of a fishy odor and off-flavor by decomposition and/ or change. The debate over whether phospholipids are antioxidants or prooxidants has yet to be completely re- solved. "Lecithin" was among the first of a number of naturally occurring edible products prOposed as an antioxidant in foods (10). Most "lecithin" preparations have been crude fractions obtained from vegetable oils or animal fats on the basis of their solubilities in various solvents and have contained relatively large amounts of phosphatides other than phOSphatidyl choline. 15 The conflicting reports in the literature on the anti- oxygenic activity of "lecithin" in various substrates probably results from the heterogeneity of the "lecithin" fractions utilized as well as the variety of substrates and testing methods employed (14). In 1935 Evans (32) observed that crude vegetable lecithin appreciably lengthened the induction period of vegetable oils. Olcott and Mattill (85) reported that purified "lecithin" was inactive in lard while purified cephalin was a good antioxidant. Swift §t_al, (116) established by peroxide determinations that the anti- oxygenic activity of a-tocopherol in cottonseed oil was increased by the presence of a crude fraction of cotton- seed cephalin. The antioxidant property of the cephalin molecule has been traced to the phosphoric acid group (85). The inactivity of phosphatidyl choline has been attributed to its existence in a zwitterion form involving the free hydroxyl group of the phosphoric acid radical and the nitrogen of the strongly basic choline (83). Since the nitrogen in ethanolamine is a much weaker base than the nitrogen in choline, it was suggested that the phosphoric acid group has a greater tendency to remain free in phosphatidyl ethanolamine (83). The original description of antioxidants and Synergfists (86) differentiated between substances which were effective alone in relatively low concentration such 16 as tocopherols and phenolic compounds and those sub- stances which had little activity by themselves but were effective in combination with the phenolic inhibitors. Citric, ascorbic and phosphoric acids and phospholipids have been classified as synergists. While phospholipids are usually regarded as synergists, certain phosphatides and glyceride acid phosphate esters may have true anti- oxidant activity (22). Privett and Quackenbush (93) observed that wheat germ phosphatides and phosphoric acid prevented the accumulation of peroxides in both fresh and partially oxidized wheat germ oil. This preventive action was attributed to the formation of polymeric substances by a reaction involving peroxides and the phosphoric acid group. Patents proposing the use of phosphoric acid to -retard the development of oxidative rancidity in fats were assigned to the Proctor and Gamble Company as early as 1934. At about the same time, Lea (57) observed that when lard was in contact with a phosphate buffer it oxi- dized more slowly than when it was in contact with other buffers, such as borate and acetate at the same pH. The effectiveness of phosphoric acid in vegetable oils but not in purified methyl esters was established by Olcott and Mattill (86). Golumbic (37) attributed its syner- gistic activity to its ability to regenerate phenolic antioxidants from their quinones. The activity of l7 phosphoric acid has also been ascribed to its ability to chelate heavy metals (31) and to decompose fatty peroxides (94). Studies on the drying of linseed oil indicated that the mechanism of drying inhibition was antioxidant in nature (23). Phosphatides, containing the smallest percentage of nitrogen appeared to have the greatest inhibiting action as evidenced by the increase in drying inhibition for egg and soybean "lecithins" when choline was removed. The abnormally long drying time for a sample of linseed oil was related to the presence of 0.84% phosphatidic acid. Recognition that the phosphate group of phOSpholipids could act as an antioxidant or synergist has encouraged studies to determine if the nitrogen moiety could contrib- ute to the stabilization of unsaturated fatty acids. Choline has been reported to be prooxidative for ethyl oleate (24) and in lard emulsions (63). Urakami and Kameyama (120) found that choline failed to exhibit any antioxygenic action against purified methyl oleate, al- though phosphoryl choline, phosphoryl choline chloride and natural phosphatidic acid were active antioxidants. Olcott and Van der Veen (87) observed that saturated phosphatidyl choline was a more effective synergist for ethoxyquin in menhaden oil than was phosphatidyl ethano- amine. The latter finding was consistent with the l8 observation that tertiary and secondary amines were usually superior to primary amines as synergists for ethoxyquin (84). Phospholipids-~As Prooxidants Even though most research efforts have studied the use of phosphatides as antioxidants or synergists, the prooxidative nature of phospholipids has been under in- vestigation. In general, the antioxygenic mechanisms of phospholipids have been related to the phosphate or nitrogen moieties rather than to the preferential oxi- dation of unsaturated fatty acids. The fatty acid compo— sition of polar and neutral lipids varies with the source but appears to exhibit a common pattern in that the fatty acids of phospholipids are more unsaturated than the fatty acids in associated triglycerides. The tendency of lipids to undergo oxidation is greatly enhanced by the degree of unsaturation. According to Patton (88), the susceptibility of phospholipids to oxidation depends upon whether they are in water or fat. In the aqueous phase of milk, tri- glycerides are relatively stable and the phospholipids are preferentially oxidized. With butter, which repre- sents an aqueous concentrate of phospholipid dispersed in fat, both fat and phospholipid are susceptible, the latter being most readily oxidized. When water is absent, such as in dried milk products, the triglycerides 19 are relatively susceptible to oxidation whereas the phospholipids are more stable and when present with the triglycerides, serve as an antioxidant. In contrast, El—Gharbawi and Dugan (30) reported that the losses of polyunsaturated fatty acids in freeze-dried raw beef were more pronounced in phospholipid than in neutral lipid fractions after storage. Younathan and Watts (126) observed that the tissue lipids remaining after extraction of cooked pork with a mixture of chloroform- methanol were more readily oxidized than the extracted lipid. Lea (60) fractionated egg yolk into crude frac- tions, stored them separately at 37°C and subsequently incorporated the fractions into fresh egg for tasting. In this way the off-flavors that developed were traced to the phospholipid fraction. Experiments on the oxidative deterioration of cod flesh lipids indicated that triglycerides were highly resistant to autoxidation even when hemoglobin, a pro- oxidant was added.‘ Phosphatidyl ethanolamine oxidized rapidly while phosphatidyl choline required a much longer period of time or a prooxidant before it began to absorb appreciable oxygen (99). Lea (59) reported that the phosphatidyl ethanolamine of egg, spread in thin films at 37°C, oxidized at a phenomenal rate while phosphatidyl choline, which constitutes the bulk of egg phosphatide, oxidized at a considerably lower rate. The rapid 2O oxidation of milk phospholipids has been attributed to fractions containing phosphatidyl ethanolamine and phosphatidyl serine (77). Acosta g§_al. (1) observed that phosphatidyl choline fractions oxidized more rapidly than other phospholipid fractions from turkey during early stages of oxidation. However, this anomaly was caused by phosphatidyl serine contamination since phosphatidyl serine was eluted with phosphatidyl choline in the elution scheme. Mattsson and Swartling (77) have summarized those factors known to effect the rate of oxidation of phospho- lipids in aqueous dispersions. They are: (l) the degree of unsaturation of the phospholipid; (2) the nature of its nitrogenous constituent; (3) the presence or absence of oxidation catalysts or repressors; (4) the pH of the diSpersion; and (5) the dispersibility of the phospho- lipid in solution which is related to the electrolytic state of phospholipid polar groups and to the pH. The importance of these factors singly and the manner in which they can interact to influence oxidation requires further study. Metal Catalysis Heavy metals possessing two or more valency states with a suitable oxidation-reduction potential between them (e.g., 00, Cu, Fe, Mn, Ni) generally increase oxi- dative deterioration of lipids by reducing the length 21 of the induction period and by increasing the maximum rate of oxidation. The metal catalyst appears to act by increasing the formation of free radicals since the re- moval of the metal catalyst from the substrate during the initial stages of oxygen uptake reduces the subsequent rate of oxidation (20). Furthermore, the addition of a strong free radical inhibitor to an autoxidizing system suppresses the chain prOpagating steps and prevents further oxidation for a period of time that is pro- portional to the concentration of the added inhibitor (21). Oxidation catalyzed by metals in polar and non-polar systems has been observed to commence at the same time that the catalyst appears in its higher valency state (7). Free radicals are readily formed by the transfer of an electron from a metal ion to a hydroperoxide molecule. Metals can influence the autoxidation of unsaturated lipids in water by reacting in an aqueous phase with water-soluble radicals such as -OH, H02- and hydroperoxides or by reacting at the oil-water interface with radicals, hydrOperoxide and the substrate (49). Many of these radicals which are dissolved in solution can return to the organic phase and increase the rate of oxidation. Many food lipids are present in aqueous emulsions along with hydrated metal ions. These metals, whether purposely added or supplied by metal activated enzymes 22 and their decomposition products, exist in the form of a coordination complex or as the salts of organic and inorganic acids (49). Metal salts dissolved in an aqueous phase in contact with a fat have been shown to have a detectable effect on the rate of autoxidation of the fat at one part in 1000 million (58). At low catalyst concentrations, the rate of oxidation is generally pro— portional to the concentration of the catalyst (49). However, the catalytic effect reaches a constant value at a specific catalyst concentration with the result that further increases in the amount of catalyst are unable to affect the oxidation rate (6). The effectiveness of a chelating agent in reducing the prooxidant nature of metals is related to its concen— tration and the concentration of metals present. Several complexes can be formed from the combination of chelating agent-metal salt-hydroperoxide depending on the relative concentration of each species. These complexes often have different catalytic activity. Chelation may suppress, intensify or have no effect on the catalytic activity of metals depending on the nature of both the chelating agent, the metal and the complex (13). The action of a chelating agent in reducing the catalytic activity of a metal has been attributed to a steric or blocking effect on the formation of a complex between the metal and hydrOperoxides (13). Also electrical conductance 23 determinations have indicated that chelation can modify the oxidation potential of metals (51). Effect of Amino Acids on the Autoxidation of Unsaturated Fatty Acids and Esters Amino acids have been mentioned as being both antioxidants and prooxidants. All amino acids appear to possess a potential antioxidative capacity and this capacity is not dependent on the presence of a primary antioxidant such as the tocopherols (76), although amino acids can behave as synergists in the presence of a pri— mary antioxidant (75). The antioxidative effect of amino acids is only a potential one since they may be pro— oxidative, more or less ineffective, or antioxidative, depending upon the circumstances (76). Whether amino acids act as prooxidants or antioxidants has been shown to be a function of pH, concentration, and the presence or absence of metal ions, nonionic emulsifiers and phos- phate (102). Factors Affecting Oxidation of Lipid—Amino Acid Emulsions The effect of glycine, histidine, and tryptophan on linoleic acid and methyl linoleate dispersed in water was exclusively prooxidative and increased with increasing concentration of amino acid (76). Histidine has been re- ported to catalyze the autoxidation of methyl oleate, a monounsaturated fatty acid ester (39). The threshold 24 concentration of histidine necessary for prooxidative action was observed to be greater than 0.0001M (102). In the presence of 0.1M phosphate buffer at pH 5, 6, and 7, the effect of glycine, histidine, tryptophan, alanine, serine, lysine and proline on methyl linoleate was ex- clusively antioxidative. Cysteine was prooxidative with and without phosphate buffer at pH 5, 6, and 7. However, it acted as an antioxidant at pH 9.5 (76). Nonionic emulsifiers such as Span 20 and Tween 20 repressed the prooxidative action of histidine. Ionic emulsifiers such as potassium myristate, sodium palmitate and sodium dodecylsulfate supported the prooxidative action of histidine (15, 102). The effect of 0.7% Tween 20 on the rate of oxidation of amino acids was slightly retarding in water and in phosphate buffer. At higher concentrations, Tween 20 accelerated oxidation in the presence of phosphate and amino acids at pH 7 and re— tarded oxidation at pH 5 and 6 (76). Trace metals accelerate methyl linoleate oxidation. The effect of ferrous or ferric ions was more than double that of cupric ions. Autoxidation as measured by oxygen uptake was immediate and rapid when ferrous or ferric ions were added with histidine and was greater than that of cupric ions and histidine. The linoleate peroxide content was increased more than three-fold by the addition 2 of 1 ppm of Fe+ and Fe+3, approximately twenty-fold by 25 a 0.01M concentration of histidine and more than sixty- fold by the addition of both histidine and ionic iron (102). Metal Ions Histidine is known to complex with metal ions and oxygen (41). The catalyzing effect of ionic iron and histidine on the autoxidation of emulsified linoleate could result from the formation of an iron-histidine complex. The activity of histidine, when iron is not added, might be due to a complex between histidine and trace metals present as impurities. The catalytic action of high concentrations of amino acids on the autoxidation of methyl linoleate dispersed in water could thus result from an interaction between amino acids and trace metals (102). Linoleic acid (121) distilled in all-glass apparatus and purified methyl linoleate (122) have been reported to contain traces of copper, iron and cobalt. Also it is difficult to obtain amino acids containing less than 1—3 ppm of various trace metals, particularly if the amino acid has been prepared from biological sources (102). Trace metals can be contributed by glass- ware and by the lipid and amino acid samples. Amino Acid Concentration Histidine alone or added with iron has no appre— ciable prooxidative effect at 0.0001M (102). Studies on 26 the chelation of ferrous and ferric ions by histidine established that the ferrous-histidine chelate was un- stable at low histidine concentrations because histidine was not able to compete successfully with the hydroxyl ion. In the presence of oxygen, the chelated Fe+2 was 3 rapidly oxidized to Fe+ and the competition of OH- for Fe+3 was so strong that it prevented the formation of a ferric-histidine chelate at low ratios of histidine (41). 23 The concentration of histidine and metal available for complex formation is probably related to the pH. With increasing pH, the concentration of OH- increases, reducing 2 +3 the effective concentration of Fe+ or Fe At pH values lower than 5.2 it has been reported that histidine was un- available for chelate formation (41). Optimum catalysis would appear to occur when the product of the concentration of available free metal ion and free histidine is maximum, around pH 6.5 (102). Emulsifiers The effect of emulsifiers on the prooxidative and antioxidative action of amino acids is not understood. The reason for using an emulsifier is to decrease the size of lipid particles in an emulsion, thereby increasing surface contact between the lipid and the aqueous phases. However, the prooxidative action of ionic emulsifiers and 27 the antioxidative effect of nonionic emulsifiers in the presence Of histidine suggests that more is involved than the size of the dispersed lipid particles. An investigation by Coleman et_al. (15) Of factors affecting the autoxidation of methyl linoleate in emulsion provides some insight on the possible role Of ionic and nonionic emulsifiers. In an emulsion polar groups such as carbmethoxy, hydroperoxide (initially present or formed in the reaction) and the hydrophilic group of the emulsifier are oriented at the interface toward the water phase. When an ionic emulsifier such as sodium dodecyl- sulfate is utilized, the linoleate boundary will have a negative electrical charge due to the ionic sulfate group. A second charged layer of positive ions (sodium and hydro- gen) exist on the aqueous side of the interfacial boundary. In catalyzed reactions trace metals, present as impuri- ties Or deliberately added, and amino acids, oriented with the amino groups adjacent to metal ions, will be concen- trated at the interface. With dissolved oxygen distri- buted uniformly in each phase and catalysts and reactive groups present at the interface in high concentrations, energy transfer could easily occur. A nonionic emulsi- fier cannot contribute an ionized charge to the lipid— aqueous interface. As a result metal ions and amino acids would be distributed randomly throughout the bulk aqueous phase, reducing the Opportunity for them to react as a complex or singly at the interface. 28 Phosphate The synergistic effect of phosphate on fat oxidation has been reported (12, 29, 37, 50, 78) and has been as- cribed to metal binding, to the regeneration Of primary antioxidants and to the retardation of the oxidation Of primary antioxidants. Since phosphoric acid has been shown to react with fatty peroxides and to prevent their estimation by the usual iodometric methods (94), the phOSphates may inhibit autoxidation by accelerating the decomposition of hydroperoxides. Thus the stronger antioxidant effect Of amino acids in phosphate buffer probably can be attributed to the action of the phosphate group in complexing metal contaminants and in reacting with primary oxidation products, thereby permitting amino acids to function as antioxidants. Effect Of Amino Acids on Phospholipid Oxidation An aqueous dispersion of phospholipids and amino acids might be expected to behave like unsaturated fatty acid esters and amino acids emulsified in phosphate buffer. Hirano (46), on the basis Of manometric studies, reported that amino acids have an antioxidative effect on the autoxidation Of phosphatidyl choline in the absence of nonionic emulsifier and phosphate buffer. Using phosphatidyl choline, methyl linolenate, amino acids, choline chloride and phosphate buffer he demonstrated that the phosphate segment was mainly responsible for the 29 antioxidative effect of amino acids in phosphatidyl choline autoxidation. Mechanism of Antioxidation Antioxidants are considered to function in two ways, either as inhibitors of free radical formation or as de- composers of peroxides. Since the formation of free radicals cannot be entirely suppressed, most research has been concentrated in the area of free radical acceptors or peroxide decomposers. Peroxide decomposers act as catalysts to decompose peroxides initially present as well as those that are formed during further oxidation. An important feature of this decomposition process is that the primary stable products are not free radicals (28). Studies by Bolland and Ten Have (9) led to the pro- posal of a mechanism in which antioxidants acted as hydro— gen donors Or free radical acceptors. From the kinetics of the reaction it appeared that the free radical acceptors (XH2) reacted primarily with R02- and not with R-, as follows: RO2° + XH2‘—————+ ROOH + XH° XH- + XH° —————» X + XH2 Antioxidants that function as free radical inhibitors react with free radicals to form inert products as a termination step in the chain—reaction mechanism. 3O Peroxy radicals probably predominate in this termination step since hydrocarbon free radicals react readily with molecular oxygen (28). The phenomenon Of synergism in antioxidant action occurs when two or more compounds have a more pronounced antioxidant effect together than the sum Of their indi- vidual effects. Synergism has been produced in fat by a combination of two phenolic antioxidants (27, 73) and by the use of an acid with a phenolic antioxidant (12, 37, 50). The increased action Of two phenolic antioxi— dants in reducing autoxidation has been hypothesized to result because one of the two antioxidants has a lower activation energy (due to a lower bond dissociation energy) and a lower steric factor (due to a loss of rotational freedom in the transition state) than the other (123). Acid synergists are assumed to function by deactivating metals present as impurities. Although a free radical acceptor appears to be essential for achieving protection against all prooxidant impurities, it can be supported by a metal-complexing reagent, capable of inactivating part of the trace metal content. Since free radicals are produced in_an electron transfer reaction involving different valency states of the metal, those metal complexing reagents which reduce electron transfer would have metal-inactivating prOp- erties (123). EXPERIMENTAL Materials Sample Preparation Fresh, grade A, large eggs purchased from a local retail market were used as one source of phospholipids. Soybean phospholipid fractions were isolated from Centro— 1ex P, a granular Oil-free phOSphatide preparation supplied by Central Soya, Chicago, Illinois. Chemicals and Reagents Solvents.——All solvents were A.C.S. reagent grade and were used directly from the container unless redistil- lation was specified in the procedure. Lipids.--Methyl esters for GLC standards and methyl linolenate used for model lipid emulsion studies were Ob- tained from the Hormel Institute, Austin, Minnesota. Column Chromatography.—-Silicic acid, employed for column chromatography Of phospholipids, was purchased from Mallinckrodt Chemical Works, St. Louis, Missouri. Thin-Layer Chromatography (TLC).--Si1ica Gel-G, supplied by Brinkman Instruments, Westbury, Long Island, New York, was used for all TLC work. 31 32 Gas-Liquid Chromatography (GLC).--Diethy1ene glycol succinate (DEGS), high temperature stabilized was made by Analabs Inc., Hamden, Connecticut. Chromosorb W, 80/100 mesh size, acid washed, was Obtained from Applied Science Laboratories, State College, Pennsylvania. Cases for the chromatograph came from the Liquid Carbonic Division Of General Dynamics and were supplied through Michigan State University scientific stores. Specific Chemicals.-—Amino acids were ordered from General Biochemicals, Chagrin Falls, Ohio. The various chemicals added to methyl linolenate dispersions to deter- mine the effect Of ethanolamine and choline bases on autoxidation were purchased from Sigma Chemical Company, St. Louis, Missouri. All other chemicals used and not mentioned here were Of analytical reagent grade. Methods Extraction of Egg Phospholipids Egg phospholipids were extracted by the procedure Of Ansell and Hawthorne (2). Yolks from one dozen eggs were homogenized with 500 ml of acetone. The extract was filtered and the solid re-extracted in the same way. The residue was shaken with 300 m1 Of fresh chloroform— methanol (1:1 v/v) and filtered with suction. This extraction was repeated with 300 ml of fresh chloroform- methanol and the extracts were combined and stored under refrigeration until further use. 33 Silicic Acid Column Chromatography Silicic acid (Mallinckrodt) was washed with de- ionized water until no turbidity was Observed in the supernatant after coarse particles had settled to the bottom of the container. The silicic acid was then transferred to a Buchner funnel, washed with methanol, activated by drying for 24 hours at 120°C, and stored in a tightly stoppered bottle. Two types of columns were used for chromatography: (1) a 2.5 cm O.d. x 30 cm column containing a 300 ml reservoir at the tOp Of the column and a coarse sintered glass disc and Teflon stopcock at the bottom of the column; and (2) a 5 step multibore column (33) constructed Of glass sections fused together and having the following dimensions: O.d. (cm) length Of section (cm) 2.80 13.5 2.20 13.5 1.70 13.0 1.50 12.5 1.37 13.5 The 5 step column was designed with a 250 m1 reservoir containing a side arm with Teflon stOpcock to facilitate draining. Glass wool served to prevent the silicic acid from passing through the stOpcock at the bottom Of the column. 34 Better resolution Of individual phospholipids was achieved with the 5 step multibore column than with the conventional column. Assessment of effectiveness of the resolution by the two columns was made on the basis Of TLC of eluted fractions. Less overlap Of components was Observed for fractions collected from the 5 step column. The 5 step column had a larger sample capacity; however, more time and a larger volume of solvents were required to elute phospholipid fractions from the 5 step column than from the conventional column. Activated silicic acid (50 g for the 2.5 x 30 cm column and 65 g for the 5 step column) was slurried in an excess of chloroform in a beaker with a magnetic stirrer. After stirring until the mixture was homogeneous and translucent, it was poured into the column in such a way that no air bubbles were trapped. The silicic acid was allowed to settle and washed respectively with acetone, methanol, and chloroform to determine whether undesirable channeling existed in the column. A 1—2 cm layer Of powdered anhydrous sodium sulfate was then placed on the top of the packed column to absorb any residual moisture in the sample. Column chromatography was performed at room tem- perature under nitrogen and with a fraction collector in a cold room at 2°C in order to minimize oxidation during preparative steps. When working at room temperature 35 nitrogen gas was introduced at the top of the column before the sample was added to condition the column and dry ice was used to create an inert atmosphere in and around the collection tubes. The nitrogen aided in packing the column and in removing some of the small air pockets that were present. Extracts Of egg phospholipids in chloroform- methanol (1:1 v/v) and soybean phospholipids from a granular, Oil-free phosphatide preparation (Centrolex P), dissolved in chloroform, were carefully applied to the top of the column in a ration Of 0.02 g lipid/g silicic acid. Elution was accomplished by various solvent systems and successive 20 ml (2.5 x 30 cm column) or 27 ml frac- tions (5 step column) were collected. Fractions were collected by hand from the conventional column and by a LKB RadiRac fraction collector, distributor type 3402B, from the 5 step column. Neutral lipids were eluted by chloroform and moni— tored by the Salkowski test (55) until a negative reaction was Obtained. Acetone, used as a scavenger for oxidized materials by Nelson and Freeman (80), also removed pig- mented substances from the column. It was usually possi- ble to follow the descent of the pigments visually as a brownish-yellow band. The effluents containing only neutral lipids and pigments were discarded. The third fraction which contained phosphatidyl ethanolamine (PE) 36 was eluted with 15% by volume Of methanol in chloroform. This fraction was followed during the course Of elution with a rapid ninhydrin test (100), consisting of equal volumes of eluate, ninhydrin (4 mg of ninhydrin/ml of 100% butanol), and 2,4—lutidine (20% 2,4-lutidine by volume in 100% butanol). When all Of the PE was removed from the column, as indicated by a negative ninhydrin test, the polarity Of the solvent was raised by increasing the concentration of methanol to 25% by volume in chloroform to elute phos- phatidyl choline. Elution of phosphatidyl choline (PC) was monitored by a molybdate test. The test was per- formed by heating a few drops of the sample in a test tube to dryness, adding a few drOps of concentrated sul- furic acid before heating, cooling, and finally adding molybdate reagent. The molybdate reagent consisted Of: 5 ml Of 60% w/v perchloric acid, 10 m1 of 1N HCl, and 25 ml Of 4% w/v ammonium molybdate (89). The volumes of solvent required to elute each fraction- were variable, being dependent upon the size of the column, the packing of the column, the moisture in the solvents, and the amount Of sample applied to the column. Since the purpose of fractionation by column chromatography was to Obtain sufficient PE and PC for oxidation studies, collection was discontinued after all of the PC was eluted. Fractions were evaporated under nitrogen to 5-10 37 ml and stored at -20°C in capped vials until spotted on TLC plates to check for purity. Tain-Layer Chromato- graphyi(TLC) Thin-layer adsorption chromatography on Silica Gel-G was employed to check the identity and purity of phospho— lipid fractions eluted from the column. RSCO equipment consisting of a plastic mounting board, desiccator, and spotting template were used. Five clean glass plates (20 x 20 cm) were rinsed with ethanol and spread 0.50 mm thick with a Desaga variable thickness (0-2 mm) applicator distributed by Brinkmann Instruments, Westbury, Long Is- land, New York. Fifty g Of Silica Gel-G containing approximately 13% calcium sulfate binder was shaken with 100 ml Of deionized water in a Mfllml glass stoppered Erlenmeyer flask until the mixture was homogeneous. After spreading, the plates were air dried at room temperature for 30 minutes and stored in laboratory drawers. The plates were activated at 100°C for one hour and cooled to room temperature in a desiccator before spotting. Better separation was Obtained when plates were activated immediately before spotting rather than after storage of the activated plates for several days in a desiccator. Phospholipid fractions were spotted on TLC plates with capillary pipettes and the plates were developed in 38 a solvent system consisting Of chloroform: methanol: water (65:25:4 by volume). Samples of lipids methylated for GLC analysis were tested for completeness of methyl- ation by spotting on TLC plates and chromatographing in petroleum ether: ethyl ether: acetic acid (90:10:l by volume). The plate was removed from the chamber and allowed to dry at room temperature after the solvent had ascended at least 10 cm. Lipid component spots were detected by spraying with various indicators, e.g., ninhydrin solution for amino phosphatides (62), molybdic acid for phosphatides (25), and Dragendorf reagent for choline (113). Aqueous sulfuric acid (50%) was sprayed on the plate after first spraying with the other indi- cators. After 1—2 hours in an oven at 110°C, any pre- viously undetected lipids and other impurities as well as phospholipid spots previously Observed were visible as charred spots on the plate. The contents Of vials con- taining more than one component were considered impure and were discarded. Pure fractions of PE were pooled in one container and fractions of PC were grouped in another. The phospholipd samples were flushed with nitrogen and stored at —20°C in capped vials for further study. Phosphorus Determination The phosphorus content Of PE and PC was determined by the method Of Rouser et al. (101). The phosphorus test was performed with equal success on phospholipid fractions 39 dissolved in organic solvents and on phospholipids dis- persed in aqueous media. The importance of the phosphorus determination in these research studies cannot be over- emphasized since it provided a means of quantitating all phOSphOlipid fractions and dispersions. The phosphorus test was performed on aliquots Of a monobasic potassium phosphate standard (Hartman Leddon Company, Philadelphia, Pennsylvania) ranging in concentration from 2—10 ug of phosphorus. The absorbance Of these aliquots was read with a Beckman Model DU spectrOphotometer at 820 mu and a phosphorus standard curve (Appendix A) was Obtained by plotting the absorbance values of the aliquots versus their concentration. Multiplication by the factor, 11.3, calculated from the standard curve, permitted a direct conversion of an absorbance reading to ug of phosphorus in a sample. Rhee and Dugan (97) demonstrated that the method of Rouser a£_al, could be extended to give a linear curve up to 17.44 ug Of phosphorus. However, the concen- tration of most of the phospholipid samples analyzed were diluted to give readings within the 2—8 ug range (.182- .728 absorbance). Dispersion Of Phospholipids in Aqueous Media Phospholipids were emulsified in three ways. In the first method, purified PE and PC from egg and soybean dissolved in chloroform—methanol were pipetted into separate cell homogenizer flasks. After the chloroform- 40 methanol was evaporated from the homogenizer flasks by a stream Of nitrogen, 50 ml of 0.1M borate (pH 7.0) buffer* and 0.1 ml of Tween 20 (polyoxyethylene sorbitan mono- laurate) were added to the dry phospholipid residues which remained in the flasks. The atmosphere within the flasks was purged with nitrogen and emulsification was achieved with a Braun Cell Homogenizer (Bronwill Scientific, Inc., Rochester, New York) at a speed of 4,000 rpm for 1—2 minutes. Carbon dioxide liquified under pressure was employed to keep the'contents of the flask below room temperature while the emulsion was being formed. A con- trol consisting Of equivalent amounts of borate buffer and Tween 20 was prepared in the same manner. No difficulty was encountered in dispersing PC. PE had a much greater tendency to remain together in clumps and to adhere to the sides of the homogenizer flask than phosphatidyl choline, especially if no Tween 20 was present to facilitate the formation of an emulsion. The inability of PE to emulgate in buffer solution was re- ported by Mattsson and Swartling (77). When glass beads were added to the homogenizer flask and the time of agitation was increased beyond 1—2 minutes, the solution turned slightly brown, indicating that degradation of PE had occurred. Consequently neither technique was *Boric acid was dissolved in redistilled deionized water and adjusted to pH 7.0 with 0.1N NaOH. 41 utilized. In order to minimize pipetting errors result- ing in an uneven transfer of PE from the cell homogenizer flask to respirometer vessels, phosphorus determinations were performed on aliquots from individual vessels after the termination of oxygen uptake measurements. PE was Observed to disperse in buffer at about the same time that it began to absorb measurable amounts Of oxygen and was always well emulsified before the oxygen uptake curve reached its maximum level. A second method of emulsifying phospholipids was adopted for studies Of the optimum pH for the oxidation Of PE and PC. Because Of difficulties in dispersing phosphatidyl ethanolamine over a rather wide range of pH, even with the aid of Tween 20, PE and PC, dissolved in chloroform-methanol in amounts corresponding to 40-50 ug phosphorus (101), were introduced directly into respiro- meter flasks. Solvents were removed by nitrogen and 4 m1 of 0.1M borate buffer* was added. The third technique Of emulsifying phospholipids was similar to the second method but was necessitated by an entirely different reason. When an aliquot of Tween 20 (polyoxyethylene sorbitan monolaurate) was methylated and injected into the gas chromatograph, the major fatty acid ester was laurate but palmitate, stearate and oleate *Boric acid was dissolved in redistilled deionized water and adjusted to the desired pH with 0.1N NaOH or 0.1N HCl. 42 were also present. Since their presence would interfere with subsequent GLC determinations of the changes in the fatty acids of PE and PC during oxidation, another means of forming a stable emulsion was needed. Sonication was considered but it has been shown to produce many unknown peaks from PE and PC as evidenced by GLC traces of fatty acid methyl esters (38). A small amount of phosphatidyl serine has been used to disperse PE in a buffer solution (77). Phosphatidyl serine is rapidly oxidized, however (1, 77). The addition Of 10% PC to PE was Observed to reduce the time required to disperse PE. Accordingly, 10% PC was added to each preparation of PE utilized in studies Of phospholipid alteration resulting from phospho- lipid oxidation. A homogeneous emulsion was needed to minimize pipetting errors for ultraviolet absorption spectra, 2-thiobarbituric acid and free amino group determinations. Egg PC was used with egg PE and soybean PC with soybean PE. PE and PC were pipetted directly into respirometer flasks as in the second method of emulsifying phospholipids. Preparation Of Methyl Linolenate Dispersions _ A 0.0133M dispersion of methyl linolenate (Hormel Institute, Austin, Minnesota) in 0.1M borate (pH 7.0) buffer was prepared by agitation for 1 minute with the Braun Cell Homogenizer. Three ml of the dispersion was 43 pipetted into respirometer flasks and 1 m1 of solution was added in the following combinations: 1. 0.5 m1 of 0.08M DL-d-glycerophosphate (sodium salt) and 0.5 m1 of 0.08M ethanolamine. 2. 0.5 ml of 0.08M DL-d—gylcerOphosphate (sodium salt) and 0.5 m1 Of 0.08M choline chloride. 3. 0.5 ml of 0.08M DL-a-glycerophosphate (sodium salt) and 0.5 m1 Of 0.1M borate (pH 7.0) buffer. 4. 1.0 m1 of 0.1M borate (pH 7.0) buffer. All solutions were made in 0.1M borate (pH 7.0) buffer. The final concentration of methyl linolenate and the various additives in the 4 ml Of borate buffer was 0.01M. Introduction of Additives tO Phoapholipid Dispersions The most convenient method of maintaining the borate buffer concentration at 0.1M in the phospholipid emulsions was to make up all solutions in borate buffer. The level of amino acid, choline chloride and ethanolamine in borate solution was such that the introduction Of 1 ml to 3 m1 of phospholipid emulsion resulted in a 1:1 molar ratio.of phospholipid to additive. Sodium tetra, ethylenediamine tetraacetate and CuSOu were pipetted in 1 ml amounts which reduced their initial concentrations four-fold to 0.0025% and l x'lO-uM respectively. One ml of 0.01% BHT dissolved in chloroform was introduced to a respirometer flask immediately after the addition of the phospholipid 44 in chloroform—methanol. Solvents were removed by nitro— gen and 4 ml of 0.1M borate buffer was added. Oxygen Uptake Measurements Oxidation of phosphatidyl ethanolamine, phosphatidyl choline and methyl linolenate was studied by measuring oxygen uptake in a Differential Respirometer (36) (Model GR 14, Gilson Medical Electronics, Middleton, Wisconsin). The volume of borate buffer transferred to each respiro— meter flask was 4 ml, either pipetted in the form Of a lipid emulsion from a cell homogenizer flask or added to PE and PC after these phospholipids had been freed of the organic solvents in which they were introduced to the re— action flasks. The amount Of phospholipid in each reaction flask was 40-50 ug Of phosphorus. The 15 ml capacity respirometer vessel was conical and made of Pyrex glass. Before it was fastened to the flask-holder, a ground joint standard taper 7/15 stOpper was fitted to the vessel's sac and 0.2 m1 Of 5% KOH was added to its center well. The vessels were placed in the water by lowering the flask— holders and allowed to equilibrate 15 minutes before starting measurement. A minimum of 3 duplicates were run against at least 3 controls of borate buffer. Experiments were conducted at temperatures ranging from 15 to 50°C in an atmosphere of air with a shaking rate Of 150 oscill- ations of 5 cm per minute. 45 Preparation of Methyl Esters for Gas-Liquid Chromatography Methylation of the fatty acids in the phospholipids was performed by the low temperature method Of McGinnis and Dugan (79) modified by deletion of the 2 m1 Of sul- furic acid. This modification was based on the work of Zook (127) who found that almost complete methylation Of soybean and egg phospholipids can be accomplished by omitting sulfuric acid and reducing the amount of base to 2-4 g of KOH/15 ml Of methanol. Evidence for this modi- fication was based on TLC results Of methyl ester prepara- tions chromatographed in petroleum ether: diethyl ether: acetic acid, 90:10:l by volume (127). Methyl esters were prepared from phospholipid fractions derived from silicic acid columns and from dis— persions Of phospholipids in aqueous media. Identical samples of PE and PC in solvent and in buffer were methylated and their fatty acid compositions were deter- mined by GLC. Good agreement was Obtained between samples in solvent and samples in buffer (Appendix B). Methylation of phospholipid emulsions during oxidation provided a means Of following changes in the percentage Of specific unsaturated fatty acids in relation to oxygen uptake. Samples Of PE and PC in organic solvent and in borate buffer, usually in volumes Of 1-3 ml, were pipetted into 125 ml Erlenmeyer flasks containing 20 ml of diethyl ether. The flasks were placed in dry ice—acetone baths 46 and stirred by magnetic stirrers. When the contents Of' the flask had cooled to —60°C, 15 m1 of absolute methanol was added and the mixture was stirred for 10 minutes. The temperature was adjusted to -60°C and methanolic—KOH (2 g of KOH dissolved in 15 ml Of absolute methanol) was added. After stirring the mixture for 20 minutes at -60°C, it was allowed to reach room temperature while stirring was continued. The mixture was quantitatively transferred to a 500 ml separatory funnel by rinsing the flask with 150 ml Of water. Methyl esters were extracted once with 30 m1 Of petroleum ether (30°-60°C B.P.) and twice with 15 ml of petroleum ether. All samples were dried by filtration through anhydrous sodium sulfate be- fore being concentrated to a volume of 0.2 ml in graduated 12 ml centrifuge tubes. Gas-Liquid Chromatagraphy (GLC) Gas—liquid partition chromatography was accomplished with a Beckman GC-5 dual column, temperature programmed gas chromatograph. The unit was equipped with thermal conductivity and flame ionization detectors, and a re- corder. All methylated samples were chrOmatographed using the flame ionization detector. Two coiled stainless steel columns (1/8 in. O.d. x 6 ft.) were used for methyl ester separation. Both columns were packed with 20% diethylene glycol succinate 47 (DEGS) and 1% by weight phosphoric acid on acid washed chromosorb w 80/100 mesh as a support phase. Approxi- mately 2.5 g Of column material were packed into each of the columns. The columns were conditioned at 220°C for 24-48 hours before being used. Operating conditions for this study were: column temperature, 184°C; inlet temperature, 230°C; flow rates for air, helium and hydro— gen, 250, 30, and 32 ml/min. respectively; tank pressures for air, helium and hydrogen, 16, 70 and 30 psi respectively. The attenuator settings ranged from 5 x 103 (2.5 x 10-10 amperes, fullscale deflection) to the higher sensitivity 2 (2.5 x 10'11 amperes, fullscale deflection). Of 5 x 10 Fatty acid methyl esters on the chromatogram were identified by direct comparison of major peaks with those Of chromatographic standards (99% pure) passed through the same column under identical conditions. Peak areas were calculated by the triangulation method Of multiplying the peak height by one—half the base (11). §pectrophotometric Deter- minations as Indices Of Phospholipid Oxidation These determinations were based on aliquots contain- ing 5 ug phosphorus as assayed by the procedure Of Rouser et al. (101). The aliquots were made up to a volume of 1.0 ml with 0.1M borate (pH 7.0) buffer. 48 Ultraviolet Absorption Spectra.--Conjugation of oxidized and non-oxidized PE and PC was determined by diluting 1.0 m1 Of emulsified sample with 2 ml Of Fisher certified A.C.S. spectranalyzed methanol. Ultraviolet absorption Spectra were recorded with a Beckman Model DU spectrOphotometer at 232 mu and 268 mu against 1.0 m1 of 0.1M borate (pH 7.0) buffer similarly diluted with methanol. 2-ThiObarbituric Acid (TBA) Test.--Malonaldehyde formation was measured by the TBA test of Placer 22.22: (90) designed to accommodate emulsions Of polyunsaturated ‘fatty acids. In this procedure, the trimethine colored substance, resulting from the reaction Of TBA reagent with the lipid peroxidation product, malonaldehyde, was esti- mated in alkaline solution. The method involved in- cubating 1.0 m1 Of phospholipid emulsion in a stoppered vial for 30 minutes at 37°C. Following incubation, 1.0 m1 of TBA reagent was added and the mixture was heated for 10 minutes in a boiling water bath using glass beads as a condenser. The TBA reagent consisted Of 2 volumes Of a 0.8% TBA solution and 1 volume of 7.0% perchloric acid. The TBA solution was prepared by dissolving TBA in a small amount of 1N NaOH before neutralizing with 7% perchloric acid. After the TBA-emulsion mixture had cooled, 0.7 ml Of 1N NaOH was added and the solution was mixed by shaking. The absorbance of the reaction 49 mixture was measured at 548 mu versus 1.0 ml of borate buffer similarly treated. The original procedure utilized 2.0 ml of pyridine/n—butanol (3/1, v/v) to dissolve any proteins or lipids that might precipitate or become Opalescent as a result Of the reaction. Since the solutions in this study were clear after the addition of 1N NaOH the addition Of pyridine/n-butanol waahamitted. iFree Amino Group Quantitation.--Reaction with ninhydrin has been utilized for the detection and analysis Of lipids contianing free amino groups (19, 61, 72). Ninhydrin reagents are difficult to use since repro— ducible color yields are obtained only after careful control Of all conditions. Also color yields vary with different substances. In contrast, reactions with trinitrobenzene sulfonic acid (TNBS) yield uniform, reproducible and stable color intensities for free amino groups Of a variety of substances, both lipid and non- lipid. The procedure described below is based on the method of Siakotos (111). One ml of phospholipid emul- sion was pipetted into a 12 m1 graduated centrifuge tube followed by 1 ml of neutralized Hyamine hydroxide (Packard Instrument Company, La Grange, Illinois) and 1 ml Of 0.1% TNBS (Nutritional Biochemicals Corporation, Cleveland, Ohio) in distilled water. The Hyamine hydroxide [p-(diisobutylcresoxyethoxyethy1) dimethyl 50 benzyl ammonium hydroxide] was neutralized to pH 8.0 with glacial acetic acid and diluted to 0.5M with methanol. After mixing thoroughly, the samples were incubated at 40°C for 2 hours in a water bath and covered with aluminum foil to exclude light. One ml of methanolic 1N hydrochloric acid (8.33 ml concentrated HCl diluted to 100 ml with absolute methanol) was added to quench the unreacted TNBS color and the volume was made up to 5 ml with methanol. The color intensity was read at 340 mu using a reagent blank as zero. A standard curve for ethanolamine was Constructed by using 2/1 chloroform- methanol aliquots Of 2-aminoethanol (Sigma Chemical Company, St. Louis, Missouri) ranging in concentration from 0.05 to 0.50 uM (Appendix C). Good agreement was Obtained when aliquots Of PE (non—oxidized) were quanti- tated by the phOSphorus determination and the TNBS free amino group determination. RESULTS AND DISCUSSION The Effect ofapH on Oxygen Consumption Preliminary oxygen uptake measurements on phospho— lipids in organic solvents, in aqueous dispersions and in a dry state indicated that the rate of oxidation could be most readily determined under controlled pH in emulsified systems. An additional reason for measuring oxidation under these conditions was that any changes that occurred would be more typical Of changes occurring in a number Of natural food systems. Figure 1 illustrates the influence of pH on the oxygen consumption Of egg phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC). Oxygen consumption after 50 hours was greatest for PC at pH 7.0 and PE at pH 8.0. Mattsson and Swartling (77), working with butter phospho— lipids in phosphate buffers, reported that PE was reluctant to take up oxygen except at pH values in the alkaline range, while PC had a maximum probably below pH 2 and reacted fairly slowly around pH 7.0. Subsequent oxygen uptake determinations were con- ducted at pH 7.0 since it represented the maximum pH for the less reactive PC and a near maximum for PE, and 51 52 .oooe-0d .oommumd "coapomop mo oedemsOOEoe .mpso: om Longs Lehman weapon zfi.o CH Aomv ocflaozo Hzoflumnamond new Ammv OCHEmHocmcpo Hzpflumzdmona mo OOHpQESmOOO cowzxo so me mo poommo OSBII.H mmonm :d . o o e o m e _ _ 4 3 . O O O . . 9.66 . 452182.. 0 O 0 9.5482415 seedlings 3W1dn NBSAXO (ptdnoudsoud alowx zO alow) 53 because it approximated the physiological pH Of most animal and vegetable products. Consequently, studies on the oxidation Of phospholipids could possibly provide information on the mode of autoxidation of foods during processing and storage. Reactivity of Egg and Soybean Phospholipids The relative rate of oxidation Of both PE and PC was temperature dependent, increasing with increasing temperature. PE from egg or from soybean absorbed oxygen in a much more rapid manner than did PC from the same sources (Figures 2, 3). Lea (60) reported that thin films Of egg PE at 37°C oxidized at phenomenal rates. Roubal (99) Observed that PE from the flesh lipids of cod fish exhibited a high rate of oxidation while PC required an added prooxidant before it would react with oxygen. Variations in experimental techniques among indi- vidual researchers studying the oxidation Of phospholipids make it difficult to compare results with regard to the relative reactivity Of phospholipids from different sources. When phospholipids from egg and soybean were compared by the same method of oxidation, egg phospho— lipids were found tO be more reactive. Egg PE and PC oxidized more rapidly than did soybean PE and PC re- spectively (Figures 4, 5). 54 .oomm pm Lehman om cooeequ.e :oV oeoeoo 2H.o ca ooHaHmH:Eo ocflHoEo Hsoflooeomoeo one ocflemfiocmnpo H%Owumzamosa wwo Mo cofipafizmcoo Cowzxo OLBII.N mmDmHm Amuse—t NEE. 8. 8 TA ow Dc at» TLA. _ ON «(1 HH§DOID .:R=h0m o o Ito.” o o H‘s-5,26 JEFdIn—va—WM- . (mdnoudsoud avowx 2O atom 3>IVldn NBOAXO 57 .ooo: um Loemzo om coozenflo.> mow custom Ea.o CH poflmflmazso OCHHOLO Hzpflpmcgmocq cmOOAOm new wwo mo COHOQESmCOO commxo onell.m mmbon A 231V 92:. 00. 8 _ _ wan—0:0 £9.21; g8 8 Jr 0% 8 n_>o_.EImmOIa L J 3 “‘ér (P!d!l°lldSO'-ld aIOW / 20 3low ) 3>|\'LLdn N39AXO 58 A partial explanation for the differences in oxi- dation for egg and soybean PE and PC can be traced to their fatty acid compositions. Lea (60) concluded that a relationship existed between the oxidation rate and the degree Of unsaturation of individual phospholipids. Hawke (40) reported that egg PE contained a higher per- centage Of polyunsaturated fatty acids than egg PC. The fatty acid composition Of the phospholipids used in this study is presented in Table 1. The concentration Of arachidonic acid is markedly higher in egg PE (13.3%) than in egg PC (3.8%). The amount and degree of poly- unsaturated fatty acids are also higher in soybean PE than in soybean PC with nearly twice as much linolenic acid present in soybean PE. The greater reactivity Of egg phospholipids compared to soybean phospholipids cannot be explained solely by the percentage of polyunsaturated fatty acids since the degree of unsaturation is higher for soybean phospholipids. Of perhaps greater significance is the presence Of arachi- donic acid in egg phospholipids. Holman and Elmer (48) reported that the rate of oxidation of methyl arachidonate is about twice that Of ethyl linolenate. These workers concluded that the introduction Of one additional double bond into a fatty acid at least doubled the oxidation rate. Since the fatty acid with the most unsaturation in both egg phospholipids and soybean phospholipids is 59 TABLE l.--Fatty acid composition of purified phospholipids. Egg PC Egg PE Soybean PC Soybean PE _Fatty Acid % % % % C14 trace trace 1.2 4.4 C16 30.2 19.5 21.7 18.8 016:1 1.5 3.6 _- -- 018 16.4 30.0 4.1 1.9 018:1 32.1 16.9 17.0 8.2 018:2 16.0 16.6 50.4 56.0 018:3 trace trace 5.7 10.6 020:“ 3.8 13.3 -- -- polyunsaturates 19.8 29.8 56.1 66.1 PC phosphatidyl choline PE phosphatidyl ethanolamine 60 oxidized before fatty acids with lesser unsaturation, egg phospholipids containing arachidonic acid should be more susceptible to oxidative attack than soybean phospho— lipids with linolenic acid. Activation Energies of PE and PC The rates Of oxygen uptake from 15 to 50°C were utilized to energies for the autoxidation Of oxidation rate for soybean. The measured at 4—5 temperatures and at temperatures ranging determine activation PE and PC from egg and a phospholipid was was Obtained from the straight line portion of the uptake curve after the onset of initiation and other early autoxidation reactions. Thus the rates reflect the oxidizability of the unsatu— rated fatty acids Of PE and PC. expressed as the number of moles Of phospholipid per hour. Oxidation rates were Of 02 absorbed per mole The logarithm of each rate was plotted against the reciprocal Of the absolute temperature to give a straight line (Figure 6). According to the Arrhenius treatment Of kinetic data the slope Of this line is related to the activation energy by the following equations (17): log K Ea 1.. m-[m 2 slope Of log K vs l/T T - T ] .§._,f._1 and 1 2 _ -Ea — 2.303R where 61 do fimoossfloop esp Op cofipmpflxo mo mums opp mafiww Hos mcfiaflfioxamosm .opzpmsmqaou cooanm cam mwo do Cowpmpflxousm on» mo OOHO msflcomssqnu.e mmsoHa \ EGG PE SOYBEAN PE 308 .SOI . 3.282.. S... \ Rafi; . ‘ 8 . a... 1m. 3 O rlml T . . . L _ 4. m m w. m m m m m. v.00. VT 3: IO3 62 Ea = activation energy in K calories per mole log K = logarithm of oxidation rate at T log Kl = logarithm Of oxidation rate at T1 log K2 = logarithm of oxidation rate at T2 T1 = Absolute temperature at which K1 was deter- mined T2 = Absolute temperature at which-K2 was deter- mined R = Gas constant (.001987 K calories per °C per mole). The activation energies presented in Table 2 were found to be quite comparable, which suggests that the mode Of oxidation, at least during the period of steady state oxidation, was the same for the egg and soybean phospho- lipids utilized in this study. The values Obtained were approximately one-half Of the values reported for the autoxidation of methyl linoleate and linoleic acid emul- sions (70) indicating the greater reactivity of the phospholipids. The Effect Of Fatty Acid Unsaturation on Phospholipid Oxidation Silicic acid column chromatography (35, 98) and thin-layer chromatography (69) have been used to Obtain natural phosphatidyl choline with varying degrees of un- saturation. The faster—moving PC subfractions were found to have higher proportions Of stearic and arachidonic 63 TABLE 2.-—Energy of activation for the autoxidation Of phospholipids emulsified in 0.1M borate (pH 7.0)-Tween 20 buffer. Source and Phospholipid K cal./mole Egg PE 11.7 Soybean PE 11.8 Egg P0 12.2 Soybean P0 12.8 64 acids than slower moving fractions. The fatty acid composition Of PC fractions that were initially recovered from the column or that moved the farthest on TLC re- sembled the fatty acid composition of PE (35, 69). When egg PC was chromatographed with chloroform-methanol (25-35% methanol by volume) on a silicic acid column, the first fractions contained a higher degree of un— saturated fatty acids, particularly arachidonic acid, than did later fractions (Table 3). In fact, the per— centage of arachidonic acid in egg PC first recovered from the column was almost identical with the percentage Of arachidonic acid in egg PE (Table l). The oxygen uptake of the high arachidonic acid- containing PC was compared with PC from succeeding frac- tions and with egg PE at 40°C and 25°C (Figures 7, 8). This high arachidonic acid PC was more reactive than typical PC which has approximately 4% of its fatty acid composition as arachidonic acid; however, it was less reactive than PE. The oxygen uptake pattern Of the third PC fraction is not shown in either figure because its induction period was more than double the time scale used for the other phospholipid fractions. The third PC frac- tion containing 1.3% arachidonic acid did not begin to absorb any measurable amount Of oxygen at 40°C until 116 hours had elapsed. 65 TABLE 3.--Fatty acid composition of three egg phosphatidyl choline fractions separated on a silicic acid column. Fatty First Second Third Acid Fraction Fraction Fraction % % % C14 trace trace trace 016 26.5 34.6 34.0 016:1 1.4 1.9 3.3 018 28.8 16.5 11.3 018:1 18.9 28.4 37.7 018:2 12.0 14.4 12.4 018:3 trace trace trace C20:4 12.4 4.2 1.3 66 .ooo: pm sommdp om coo391A0.e mqv mumsop 2H.o ca OOHmHmHSEo mpflmflaozamond mmo mo COHpQESmCOO cowzxo ogu co coflpflmOOEOo Ofiom OHCOOfiLOmLm mo poommo ogell.e mmeHm A 9:0: v us: On Cc on ON 0. . . .F _ 0.04 0.28.52... $3.1m... 8”... d. 92 228.54% statue 8m. .6. 0.04 920910414 o\oN.¢loa 00w Av. O.» CV 0N , (psdnoudsoud 3l0W/ 20 alow ) 3W1dn NBOAXO 67 . 7 .oomm um nomads om coosequ 5 rev oumnon SH.o CH OOHeHmHSEo mpflqflfionmmoca mwo wo :ofiuqumcoo cowzxo one so COHpHmOQEOO Ofiom OHCOOHcompm mo pooemo ozell.w mmDon A 831v msE. 0o. 0N. 00. 2. On mm _ . . . _ . 904 9200—10414 oonAV tom 08 10.. 98 0.209104”? ext—yd. I On— oow anT .. 0.04 220910414 exomfi. I we 09m AT (mdnoudsoud alow / ZO 610w ) axudn Nasxxo 68 The greater reactivity Of PE as compared to the high arachidonic acid-containing PC was characterized by a shorter induction period. The maximum rates of oxidation for the high arachidonic acid PC and PE were measured and found to be almost equal (Table 4, next section). The similarity in rates for the high arachidonic acid PC and PE suggests that the straight line portion of the oxygen uptake curve (representing the maximum rate of oxidation) for a particular phospho- lipid is regulated primarily by the composition Of un- saturated fatty acids. Comparison Of Oxidation Rates Obtained ay Experimentation and Calculation Oxidation rates for egg and soybean phospholipids were compared by two methods to determine quantitatively the influence of fatty acid composition on the reactivity of a given phospholipid. In the first method, maximum rates of oxidation were Obtained from the straight line portion Of each oxygen uptake curve. The values for egg phospholipids are presented in Table 4 and those for soybean phospholipids in Table 5. The second method was based on data published by Holman and Elmer (48). These workers reported the maximum rates of oxidation of various acids and esters. The maximum rate of oxidation Of ethyl linolenate was 2.4 times that of ethyl linoleate, and the rate of oxidation 69 TABLE 4.--Comparison of the oxidation rates of egg phospholipids obtained by experimentation and calculation. Experimental Rates Oxidation Rate (40°C) Ratio of Phospholipid Oxidized mole OZ/mole P. hr Rates Egg PC-—norma1 arachidonic 185 acid .185 T85 = 1 0 Egg PC—-high arachidonic 360 acid .360 T85 = 1 9 Egg PE .383 383 _ - 2 l l 5 Calculated Rates Phospholipid 1 Fatty Oxidation Rate Product Fatty Acid Summa- Ratio Of Oxidized Acid Relative to C18-2 tion Oxidation Rate Rates Egg PC--norma1 arachidonic 34 6 l = f - ' = acid 01822 11.14% (1.0) 114.1 3“ 1.0 1 a = 34.6 Egg PC--high arachidonic 71 5 d .. ' = acid C18z2 12 Op (1 0) - 12 0 3376 2 1 l = 020 A 12.4% (4.8) 59.5 71.5 Egg PE u 80.4 C18:2 16.6/0 (1.0) = 16.6 m- 2.3 1 = C20:4 l3 3% (T 8) 63.8 80.4 NOTE: The bracked numbers (1) and (4.8) are based on Holman and Elmer's data (48). The rate of oxidation of arachidonic acid (C . ) was reported to be 4.8 times 20 4 greater than linoleic acid (C18-2) at 37°C.. ' '70 TABLE 5. --Comparison of the oxidation rates of soybean phospholipids obtained by experimentation and calculation. Experimental Rates Oxidation Rate (NO°C) Ratio of Phospholipid OX1d;ZEd mole OZ/mole P. hr Rates Soybean PC .092 ngg - 1.0 .138 Soybean PE .138 -3§§-- 1.5 Calculated Rates Phospholipid 1 Fatty Oxidation Rate Product Fatty Acid Summa- Ratio of Oxidized Acid Relative to C18-2 tion Oxidation Rate Rates Soybean PC c 50 u; (1 0) a 50 u 6”'1 . 1 0 18:2 ' ' ' 65.1 ° C18z3 5.7% (2.h) - 13.7 6h.l Soybean PE c 56 o (1 0) = 56 o 81'" = 1 3 18:2 ' ' ' 55.1 C1833 10.6% (2.“) 3 25.“ 81.u NOTE: data (H8). times greater than linoleic acid (C18-2) at 37°C The bracketed numbers (1) and (2.“) are based on Holman and Elmer's The rate of oxidation of linolenic acid (C18-3) was reported to be 2.u 71 of methyl arachidonate was U.8 times that of ethyl linoleate. Ethyl oleate in comparison to ethyl linole- ate was highly resistant to oxidation at 37°C, the temperature at which autoxidation was measured. In the second or calculated method, the percentage of each polyunsaturated fatty acid for a given phospho- lipid was multiplied by its rate of oxidation relative to linoleate. The products were totaled to give oxi- dation summation rates for egg phospholipids (Table M) and soybean phospholipids (Table 5). Thus in Table M the oxidation summation rate of 80.“ for egg PE was calculated by adding the product of linoleic acid, 16.6 (derived by multiplying its percentage of 16.6 times its rate of oxidation of l) and the product of arachiodonic acid, 63.8 (derived by multiplying its percentage of 13.3 times its rate of oxidation of “.8, relative to linoleic acid). The oxidation summation rates in themselves repre- sent nothing. However, they assume a measure of signifi- cance when compared to the eXperimental rates by ratios. In the ratio of rates column in Table H the oxi- dation rate of each phospholipid is divided by the oxi- dation rate of egg PC containing the normal quantity of archidonic acid. The ratio of rates for egg PC with a high amount of arachidonic relative to normal PC was 1.9 by the experimental method and 2.1 by the calculated 72 method (Table A). Experimental and calculated rates by ratio were quite similar for egg PE and soybean PE (Table 5). In the case of soybean PE the comparison of the two methods was relative to soybean PC. An interesting aspect of the closeness between the two methods is that it lends further support to the con- clusion that the rate of autoxidation of a phospholipid is regulated specifically by the kind and amount of poly- unsaturated fatty acids it contains. However, compari- sons between egg and soybean phospholipids in the same manner would seem to negate this conclusion. Thus it would appear that if comparisons are to have any validity, they should be made between individual egg phospholipid fractions and between soybean phospholipids. Other workers have tried similar comparisons on the relative oxidative rates of various oils without obtaining any apparent correlation. Sims (112) was unable to find a correlation between the flavor stability of partially hydrogenated soybean oils containing 0.0—8.0% linolenic acid and their oxidative stabilities or content of polyunsaturated glycerides. Pyriadi and Mason (95) reported that the stability of pecan oils was dependent upon the concentration of tocopherols and the degree of unsaturation. The presence of natural prooxidant and antioxidant materials in these oils could account for the lack of correlation between oxidative stability and 73 unsaturation while the absence of these materials in purified phospholipids could explain the relationship between the rate of oxidation of a phospholipid and its concentration of unsaturated fatty acids. The reason the ratio of rates for egg phospholipids was larger than the ratio of rates for soybean phospholipids might be related to the percentage of the most unsaturated fatty acid in each phospholipid (Table l), since it is the fatty acid that is oxidized first and is primarily re- sponsible for the rate of oxidation. Egg PE has 3.5 times more arachidonic acid than egg PC while soybean PE has only 1.9 times more linolenic acid than soybean PC. This rather large amount of arachidonic acid in egg PE could also explain why it had a much higher rate of oxi— dation than the other phospholipids for the temperatures at which the rate was measured (Figure 6). Effect of the Nitrogen Moiety on Phospholipid Oxidation The differences in the amount of time which elapsed before the egg PC fractions in Figures 7 and 8 reached their maximum rate of oxidation can probably be attri- buted to the content of arachidonic acid in these frac- tions. The induction period of the three PC fractions increased as the percentage of arachidonic acid decreased. Since the arachidonic acid concentration of the first egg PC fraction was almost identical with the arachidonic 7“ acid concentration of egg PE, an additional factor, possibly the influence of the nitrogen moiety, could explain the different induction periods for PE and the high arachidonic acid PC. To test this hypothesis egg PC was purposely contaminated with 1% and 5% PE (Figure 9). The rate of oxidation of PC was not altered appre- ciably by the addition of a small amount of PE, which was not unexpected in view of the finding that the straight line portion of the oxygen uptake curve is regulated primarily by the composition of unsaturated fatty acids. However, there was a definite shortening of the induction period with the greatest reduction occurring for the PC with 5% added PE, indicating either that the ethanolamine base has a greater prooxidant effect than choline or that the choline base has a greater antioxidant capability than ethanolamine. Effect of the Nitrogen Moiety on Methyl Linolenate Oxidation The influence of the nitrogen moiety of phospho- lipids on the oxidation of neutral lipids and phoSpho- lipids is readily apparent but not completely understood. Although PE has been regarded as a more effective anti- oxidant than PC (83, 85), the greater susceptibility of PE toward oxidation has also been reported (1, 77, 99). In an effort to investigate the role of the nitrogen moiety during the oxidation of unsaturated fatty acids, 75 .000: um omufiofixo ocm pmmmsn om cmmzeer.~ may mumpon 2H.o Ca ponHmH28o mm: om .Aomv mcHHoco Hzofipmnomono wwo mo coHuQESmcoo cmwzxo opp co cofiumc unempcou Ammo m:HEmHocmcpm Hsoapmcamoca co cameos mgeun.m mmaon Amcafv 2:. 8 9 W 8 o. p _ ..“)...1\ D C mrusrm :htsnyu com .Av. MruJ¢_.+:z,ua 0mm .AT 9a 00m arooa 1v. (D 35 l 4 3 8 (P!d!|°UdS°|4d snow 20 alow ) Human NBOAXO ...._.é Q' 76 model systems were prepared by dispersing methyl linolenate in borate (pH 7.0) buffer. Aliquots of glycerophosphate, choline chloride and ethanolamine were added to approxi- mate the composition of phosphatidic acid, PC and PE. Figure 10 summarizes the effect of these additives on the autoxidation of methyl linolenate. The induction period for the methyl linolenate dispersion was signifi— cantly reduced when glycerophosphate and ethanolamine were added. The effect of glycerophosphate and glycero- phosphate + choline chloride was slightly antioxidative while that of glycerOphosphate + ethanolamine was definitely prooxidative as compared to methyl linolenate without additives. After the onset of oxidation, a brownish—yellow color developed in the methyl linolenate dispersion containing glycerOphosphate + ethanolamine. This color served to distinguish the dispersion containing ethanolamine from the other methyl linolenate treatments presented in Figure 10. The brownish—yellow color resembled the color of aqueous PE dispersions which had undergone oxidation. The readiness with which some phospholipids decompose and become brown has been noted by other investigators. Folch (3“) observed that a PE preparation from brain, although initially white, acquired a tan color after 2 weeks in an evacuated desiccator. Lea (59) reported that browning occurred as a result of the autoxidation 77 000: pm Lowmsn A0. 5 may mpmaon 2H. 0 CH pmmamamfip mpmcoaocHH flagpoE no coaumpaxOpsw onp :0 evapoano mCHHono paw mcHEmHosmspm go poogmo ogell. OH mmDUHm .38... . USE. 422 .fi '\ 1 mzifiozslb + ”.5538..qu .8 be + 5.42302... JED: lol BEBE 96.510 + whdzamozaomugd .5226 + Enzwdz... 4»sz IOII quamoiofioho -x- .5 + ~22”...on REM! lal H.523... Arab! IIOI I 9’ Id 0 a: C5 4 8 o 1 3, O ‘24? (mm mam alowxzo GIOW). axudn Mamxo 78 of PE; however, browning in relation to oxygen absorption was considerably less for PC than for PE. The browning of many food products has been traced to Maillard-type reactions between the amino group of proteins and the aldehydic group of sugars. During the autoxidation of polyunsaturated fatty acids carbonyl compounds are formed. Carbonyl compounds possess an aldehydic group which is capable of reacting with the free amino group of ethanolamine or PE in a Maillard- type reaction, analogous to the reaction between sugars and proteins. In fact, the amino-containing phospholipids of egg yolk have been implicated in the Maillard reaction in dried eggs, although glucose instead of autoxidation products was thought to be the aldehyde mainly responsible for browning (59). Since the only difference between the model system containing ethanolamine and the one containing choline was in the nitrogen moiety, the data suggest that the free amino group exerts a definite prooxidant effect on the autoxidation of unsaturated fatty acids. Further- more, the brownish-yellow color which developed during oxidation was observed in the methyl linolenate dis- persion containing ethanolamine but not in the one con- taining choline. The prooxidant effect of ethanolamine might be related to the participation of its free amino group in the deveIOpment of the brownish-yellow color 79 by a Maillard-type reaction. In contrast, the quaternary nitrogen of choline did not react with the carbonyl group of oxidation products at the temperatures used in this study to produce a brownish-yellow discoloration. Relative Rates of Autoxidation of Polyunsaturated Fatty Acids in Phospholipids El—Gharbawi and Dugan (30) reported that a marked loss of polyunsaturated fatty acids occurred in the phospholipid fractions of freeze—dried raw beef after a short time of storage. The susceptibility of arachi- donic and linoleic acids in egg PE and PC to oxidative attack is illustrated in Figures ll and 12. Oxygen uptake and the percentage of the two unsaturated fatty acids remaining, during the period of oxidation, are plotted as functions of time. The pattern was similar for both phospholipids. Arachidonic acid disappeared at a more rapid rate and was totally absent midway during the period of oxidation. The percentage of linoleic acid decreased to a minimum level that was relatively stable. The observation that arachidonic acid oxidized more rapidly than linoleic acid was predictable from data published by Holman and Elmer (M8). These workers re- ported that the rate of oxidation was 4.8 times greater for methyl arachidonate than for ethyl linoleate. The more rapid disappearance of arachidonic acid compared to 80 (P!d!|°‘«ld9°‘-ld aIOW/ 30 8MN) BWLdn NBSAXO .000: pm campus cowhxo 02m ammmsp Ac.» ma. mpmaon za.o :fi omHMflmHSEm mcHHoco Hmofipmndmoca mwo mo mofiom mppmm oomeSpmm232H0Q LOWmE 030 on» 00 mocmpmmaammfio mzu cmmZumn dflcmCOHpmHmmll.HH mmDon 350.... m2: On 0? on on O. o _ _ . _ _ D I T Q_l_ < QNIu QMru 95% 2856 lol 904 0.9523 Icl 9.: 98 0.28.552 Iol SNINIVWSH OIOV ALLW °/o 81 (P!d!|°‘ld3°qd 3|0W/ zO aION) ENVldn N39AXO _ .oomm pm mxwpaz cowzxo 0cm Lommzn Ao.w ma. opmnop 2H.o CH omflmfimHSEm mcfiEmHocmcpm Hmpfipmcdmocd wwo mo mpfiom 200mm pwmeSpmm55>H0Q acmmE 030 ecu mo mocmpmmaammflo on» cmmzpon QflQmCOHpmHomll.mH mmsuHm .831. m2... 1 N. o. o v N o q _ q d w _ o. I 95% zwo§o lo! 0.04 0.302... IQI 0.04 0.209104% IOI. ow I o 41‘ a 4 u o.» I I I 043.... ALLVd °/o 9NINIVW3L-l OIOV 82 linoleic acid was consistent with the finding that egg PC containing 12.9% arachidonic acid was considerably more reactive than egg PC containing 4.2% arachidonic acid (Figures 7, 8) and supported the conclusion that the rate of oxidation of a phospholipid is regulated by its polyunsaturated fatty acid content. During the 95 hour reaction period for PC and the 13 hour reaction period for PE, the concentration of the other unsaturated fatty acids, 16:1 and 18:1 and the saturated fatty acids, 16:0 and 18:0 remained fairly constant relative to each other. They increased in amount concurrent with the disappearance of arachidonic and linoleic acids. Similar results were reported for the autoxidation of cod PE (99) and human red cells (26). The greater reactivity of PE as compared to PC has been well documented (59, 77, 99). Not only does egg PE oxidize at a more rapid rate, it also has the capacity to react with more oxygen, undoubtedly because it has more unsaturation than egg PC. The curves in Figures 11 and 12 support and in part explain the differ- ences in reactivity for PE and PC. Although the arachi- donic acid concentration is much higher in PE than in PC (Table l), the time required for the total disappear- ance of arachidonic acid was approximately 6 hours at 25°C for PE and about 25 hours at “0°C for PC. The 83 arachidonic acid disappeared so rapidly and selectively in PE that none was present after 6 hours, while almost all of the linoleic acid was still present. However, the disappearance of linoleic acid followed more closely the disappearance of arachidonic acid in PC and reached its minimum for the period of oxidation at about the same time that the percentage of arachidonic acid drOpped to zero. The difference in the disappearance of linoleic acid in PC and PE could have resulted because of a higher rate of reaction at the higher temperature. Also the longer time and higher temperature (40°C) that were re- quired to completely eliminate arachidonic acid from the PC reaction mixture may have allowed products of the oxi- dation of arachidonic acid more opportunity to catalyze the disappearance of linoleic acid. Considerable oxygen was absorbed, especially by PE but also by PC, after arachidonic acid was totally absent and linoleic acid had reached its minimum level. This observation is probably related to the manner in which lipids oxidize. The primary products of the reaction of oxygen with unsaturated lipids are hydrOperoxides, while alcohols, aldehydes and ketones are secondary products formed by degradation of primary hydroperoxides. The latter compounds, particularly if they are unsaturated, are highly susceptible to further oxidation (53). Lillard and Day (64) have reported that unsaturated 84 aldehydes oxidize at a much faster rate than either methyl linoleate or linolenate. Thus, the degradation compounds from the autoxidation of PE and PC would be expected to be highly reactive and as a consequence capable of increasing the total amount of oxygen con- sumed by the reaction mixture. Relationship Between SpectrOphotometric Determinations and Oxygen Uptake Since various degradation compounds are produced by the autoxidation of phospholipids, an attempt was made to correlate oxygen absorption with specific changes which may be evaluated by Spectrophotometric determinations. Figures 13-15 illustrate the relationship between oxygen uptake and the development of alterations in egg PC, egg PE and soybean PE respectively, as measured by the TBA test, ultraviolet absorbance of diene and triene conjug- ation, and changes in the amount of the free amino group of ethanolamine. TBA and diene and triene conjugation followed a similar pattern for egg PC (Figure 13). They attained their maximum levels when the rate of oxygen consumption began to decrease. Tarladgis and Watts (119) measured malonaldehyde production with the TBA test during the oxidation of unsaturated fatty acids. They observed that malonaldehyde production followed oxygen uptake, reaching a peak at the same time that oxygen uptake 85 (mduoudsoud aIOW/ Zo aIOIN) axudn Namxo .mumou HMOHEoflo nooamzna mzofipm> an ooumsam>m mm coo: um COHpmofixo wcfipso powmsn .0. N rd. mumpon EH. 0 ea emacamH250 mcaaoco Haeapmnamosa wmm co mcoaumpmpaa--. MH mmach .38.... mac. 0m on o 4, .1V _ mwr|l . 9! T . ._. Q. RN T QN AXE. 004 On “fleeszmsa Ifil 292828 05.5 lcl ,SEEQZBHQQQ-LT. 9. Bu» 3» lo! 8m 4w EONVBHOSGV 86 (plduoudsoud alowxzo aIOW) axvun Nasxxo .mumou HonEozolo...m \za msoHLm> mp popmsHm>o mo 0com on :oHpmcon wcHLSU Loc..:; He. \ :a. common SH. 0 2H ooHuHmHzEm mcHEmHocwzpm Hthpszamozo awe .Hc 2:3H00.H00H an poomsz>m mm 000: pm coHpmono wcHLSU nogmsn .0.» m0. common :H.o :H oonHmHSEm mcHEmHocmcum HmoHpmcgmocQ cmwn>0m no mcoHpmamoHaII.mH mmDon .950... ME... ON on on} 8 CD -I 9.]. QNI. 9.4.5... 235.0 Ibl emu... 02:24 mum... loII 20.502200 wzm_E. IOI on I . 203402.200 mzma lol emu... 4m... |¢l er . .00. BONVEHOSBV 88 started to decline. These workers concluded that the malonaldehyde precursor was not accumulating as a stable end-product. After reaching the peak, more malonaldehyde precursor was destroyed than produced. The relationship between oxygen uptake and the extent of the ultraviolet absorption (UV) of autoxidized methyl linoleate has been demonstrated (91). At the end of the induction period, the UV absorption of methyl linoleate increased sharply with the formation of stable (conjugated diene) hydro- peroxides (91). Higher TBA and diene and triene conjugation values for egg PE (Figure 14) reflect the more rapid oxidation of PE as compared to PC. These values followed oxygen uptake but continued to increase even though the rate of oxygen consumption has decreased when this study was terminated. Triene conjugation was slightly higher than diene conjugation for egg PE. The greater amount of arachidonic acid in PE as compared to PC (Table 1) un- doubtedly accounted for the higher triene conjugation in the former. TBA absorbance reached a maximum soon after diene and triene conjugation values were at their highest levels and then decreased somewhat for the duration of the autoxi- dation study of soybean PE (Figure 15). Thus an increase in the production of TBA reactables resulted from the oxi- dation of dienes and trienes, although the TBA reactables 89 did not accumulate as stable end—products. Diene conju- gation increased rather rapidly for soybean PE during the early phase of oxidation as measured by oxygen uptake. Only a slight amount of triene conjugation was observed. The large amount of diene conjugation compared to the small amount of triene conjugation is probably related to the fatty acid composition of soybean PE (Table 1). Approximately two-thirds of the fatty acids in soybean PE are linoleic and linolenic acids, which contain 2 and 3 bonds. Considerable diene conjugation of soybean PE could occur by the shift of one double bond in a small percentage of the fatty acids containing 2 or 3 double bonds. Although 10.6% of the fatty acids of soybean PE are linolenic, the autoxidation of soybean PE did not re- sult in the formation of triene conjugation to the extent that occurred by the autoxidation of egg PE and PC, particularly PE. The data indicates that secondary oxi- dation products were produced from the diene conjugates of soybean PE prior to the formation of appreciable triene conjugates. The presence of 4 double bonds in arachidonic acid increased the Opportunity for a shift of two double bonds and probably accounted for the greater triene con- jugation of egg PE and PC compared to soybean PE. The real value of TBA and ultraviolet absorption measurements for the analysis of unsaturated lipids has 9O frequently been questioned. The magnitude of change of TBA and UV readings is not as easily related to the degree of oxidation as the determination of oxygen absorption or the measurement of decreases in specific unsaturated fatty acids. When considered separately, the signifi- cance of TBA and UV measurements is small, since the products they measure do not increase proportionally tojf7 the extent of lipid oxidation. Even though TBA and UV measurements are not always effective indicators of the precise stage of lipid oxidation, they contribute to a more thorough understanding of the complexities of the autoxidation of unsaturated lipids when they are utilized in combination with the quantitative determination of oxygen absorption and with changes in the concentration of specific unsaturated fatty acids. Influence of the Free Amino Group on Phospholipid Oxidation The high reactivity of PE (59, 77, 99) and phos- phatidyl serine (l, 77) in comparison to PC suggests that the free amino group of ethanolamine and serine influences phospholipid oxidation. PC fractions with the same iodine value as PE were reported to oxidize less rapidly (59). The result was similar for PE and a PC fraction containing equivalent amounts of polyunsaturated fatty acids (Figures 7, 8). PE absorbed oxygen more rapidly than the high arachidonic containing PC. 91 In this study, the free amino test was performed on PE to determine if changes in its concentration* might be related to the reactivity of PE. Lea (59) reported that the free amino group of PE disappeared during oxidation in a degree approximately proportional to the amount of oxygen absorbed. The free amino ab- sorbance value of egg PE was significantly reduced during the period of oxidation (Figure 14). The disappearance of the free amino group of soybean PE (Figure 15) was not as pronounced as that for egg PE, even though both phospho- lipids consumed nearly equivalent amounts of oxygen. The disappearance of the free amino group of PE provides support for the contention that a Maillard-type browning reaction can occur as a result of the autoxi- dation of PE. Malonaldehyde and other 2-thiobarbituric- acid-reactive substances, produced during the oxidation of polyunsaturated fatty acids, can react with amino acids and proteins (56). The decrease in the free amino group of PE probably resulted from its interaction with specific autoxidation products of unsaturated fatty acids such as carbonyl compounds (60). Hydroperoxides, epoxides and aldehydes have been mentioned as substances which could contribute to the disappearance of the free *The concentration of ethanolamine can be obtained from a standard curve once the absorbance of the reaction between trinitrobenzene sulfonic acid and PE has been determined (lll). 92 amino group of PE (59). During oxidation, the brownish— yellow color which developed in dispersions of methyl linolenate + ethanolamine, was also observed in PE emulsions. Characteristic features which serve to distinguish the oxidation of PE from the oxidation of PC are the development of the brownish-yellow color, a decrease in the concentration of the free amino group and a shorter induction period. The development of the discoloration and the decrease in the concentration of the free amino group suggest that Maillard-type browning reactions have occurred. The relationship between brown- ing and the shorter induction period of PE is not readily apparent. Maillard-type browning reactions could acceler- ate phospholipid oxidation or else be incidental and occur as a result of the presence of carbonyl and amino groups. Nevertheless, the possibility exists that inter— mediate products from the reaction of free amino and carbonyl groups might accelerate phospholipid oxidation, perhaps by promoting the formation of free radicals or other readily oxidizable substances, and thereby account for the greater reactivity of PE compared to PC. Influence of Added Nitrogen-Containing Compounds on Phospholipid Oxidation Since the nature of the nitrogen moiety is an important factor affecting phospholipid oxidation, experiments were conducted to determine if the addition 93 of ethanolamine and choline would have any influence on the autoxidation of purified phospholipids. Figure 16 illustrates the effect of ethanolamine and choline on the oxidation of PE when both nitrogen moieties were added in a 1:1 molar ratio of nitrogen moiety and phospholipid. Compared to the PE control, the free nitrogen moieties caused a slight reduction in the re- activity of PE. Both ethanolamine and choline (added as choline chloride) were somewhat antioxidative. When ethanolamine was added to PC in a ratio of 1 mole ethanolamine to 1 mole phospholipid the result was similar (Figure 17). Ethanolamine retarded the autoxidation of PC. The antioxidative effect of the added ethanolamine in phospholipid emulsions was in contrast with the pro— oxidative effect of ethanolamine in methyl linolenate emulsions. The added ethanolamine could function in the same way as amino acids which are often prooxidants in neutral lipid emulsions (76) and antioxidants in phospho- lipid emulsions (46). The antioxidative effect of ethanolamine or amino acids in phospholipids, as opposed to neutral lipids, might then be related to the phosphate group in phospholipids which under certain conditions has been reported to have antioxidant activity (22, 93). Furthermore, amino acids are known to act as antioxidants for neutral lipid emulsions in the presence of phosphate buffer (76, 102). Thus, the prooxidative effect which 94 .00mm 00 Emacsp .0.» ma. mpason 2H.o Ca emmpmamau mm was co coapmeaxOBsm asp co menemHocmgpm gem moHEOHgo seaflozo co powwow mneII.wH mmseHm on . .950... . 92.... 04 4 AM . 35.0410 wz...0...0 + up. IIQI wz.s.4n.oz4......m + um InTI Mn. ’7 IT ON 9 . O I Q l 0 rd 0 c I (pgqloquoqd anw/ 2o aIOW) 3xv1dn Nasxxo 95 .000: pm 000050 .o.s 20. oumgon 2H.o ca 00m000mae 00 000 do sowpme Iaxopsm 0:» so 20050 00m mcaemHocmcpm .0 000000 mseuu.sH mmpuHm .2301 . mg... 0m 0v . H .008 20-0.... + ”2.2502425 + on. .6030 2.70. x . + 0.. 2.2302425 + on. on. IOIII I Q n v= 1_ :I (P!d!|°4d904d slow 30 9IOW) ENVidn Namxo 96 the nitrogen moiety of PE has been shown to exert during the oxidation of PE must depend upon the attachment of ethanolamine to the phospholipid molecule. Heavy metals, particularly those possessing two or more valency states with a suitable oxidation-potential between them (e.g., Co, Cu, Fe, Mn, Ni, etc.), generally increase the rate of oxidative deterioration of lipids (49). These metals reduce the length of the induction period and increase the maximum rate of oxidation (49). The presence of l x 10-4 M CuSOu in an aqueous dispersion of egg PC increased the oxidation rate and reduced the length of the induction period (Figure 17). Of interest was the finding that the reactivity of PC was greater for ethanolamine and CuSOu together than for CuSOu alone, despite the fact that ethanolamine exerted an antioxi- dative effect on PC. The results were similar when the amino acid, glycine, was substituted for ethanolamine (Figure 18). Glycine decreased and CuSOu increased the susceptibility of PC to oxidation. When added together, glycine and CuSOu caused an increase in the reactivity of PC. The curves in Figures 17 and 18 indicate that together Cu++ and possibly the amino group of ethanolamine and glycine influence the autoxidation of PC by reducing the induc- tion period and increasing the rate of oxidation. Other workers have observed that lipid oxidation is accelerated by a combined effect of copper and amino 9.7 .000: pm 080030 .o.e :00 mumpon 2H.o 2H emmpmgmae 0a mmm co coHBmUH2003m 020 co somso 0cm mcaosz do pathos mgBII.mH mmson €81. m2... 0... 8 0m 8 0. .0 . H 0 w 3 N 10.. nu M V M . . 3 . I.0~ \I. C ‘ w . . . m. ,0 / I... m W «008 .270. x. + 02.0.50 + on Icl d e0m..o...?0:..+oa 10! w. 02.050 + 8 Iol m on. a 0... I. m” D. ( 98 compounds. COpper—protein complexes were shown to be more effective catalysts for linoleate oxidation than was copper alone (118). Mattsson and Swartling (77) reported that PE which was poorly dispersed in buffer oxidized more readily in the presence of copper even though the copper did not improve its emulsification. Saunders §£_al, (102) studied the effect of histidine and trace metals on the oxidation of linoleate esters. The catalytic action these workers observed was attri- buted to an interaction between histidine and trace metals, since histidine had been shown to complex with metal ions and oxygen (41). The accelerated oxidation illustrated in Figures 17 and 18 could result from an interaction between copper and ethanolamine and copper and glycine. Copper was shown to bind amino acids, peptides and proteins and to exert a strong catalyzing effect on the oxidation of ascorbic acid (103, 104). Compounds containing more than one donor N atom formed‘more than one type of com- plex with copper (103). Those complexes in which copper atoms were firmly bound tended to be the best catalysts (104). Part of the catalytic activity of copper—protein complexes in methyl linoleate oxidation has been as- cribed to the combined effect of copper and protein in forming chelate bridges and in stabilizing the activated complex of linoleate peroxide-cepper-protein (54). On 99 the basis of the data presented, the mechanism(s) by which a complex between copper and an amino compound could enhance lipid oxidation can only be speculated. The complex could conceivably increase the prooxidative effect of copper by changing its orientation at the aqueous-lipid interface. The formation of a complex might make c0pper, oxygen or both more available for oxidation reactions at or near the double bonds of un- saturated fatty acids. A c0pper-amino complex could accelerate the decomposition of hydroperoxides to form new chain-promoting radicals or to paraphrase a mechan- ism previously mentioned (54), the combined effect of copper and an amino compound might promote phospholipid oxidation by stabilizing the active complex of unsatur- ated fatty acid peroxide-copper-amino compound. The Effect of Amino Acids on Phospholipid Oxidation Amino acids have been shown to act as both pro- and antioxidants for the autoxidation of unsaturated fatty acids and esters (76, 102). Amino acids have been re- ported to exert an antioxidative effect on the oxidation of PC in the absence of nonionic emulsifier and phosphate buffers (46). The influence of glycine, lysine, trypto- phan and histidine on the oxidation of egg PE dispersed in borate (pH 7.0) buffer is illustrated in Figure 19. Glycine, lysine and tryptophan increased the induction aggy ‘sfi‘s .0omm pm 000050 .o.e 20. mumpop 2H.o ca campmamac ma wwm co coHpmonOpsm 030 so moHom ocHEm mSOHLm> 00 000000 mgBII.mH mmpon .98.... m2... Om ov Om ON 0. 100 wz==Hm=...m?. IAYI 24Ia0ka>mk.+ ma UaémXJ.+ ma w2n§30.+ mm mm (pIdIIoudsoud slow 30 am) 3xv1dn Naoxxo 101 period for the oxidation of PE. Histidine completely inhibited the oxidation of PE as evaluated by oxygen uptake measurements. Confirmation for the inhibition of PE oxidation by histidine was obtained by TBA determin- ations. The TBA absorbance of PE with histidine was virtually unchanged after 50 hours as compared to the original TBA value of PE before measurements of uptake were initiated (Table 6). In contrast, the TAB absorbance of the PE samples containing the other three amino acids increased considerably. The variability of the antioxidative capacity of different amino acids on linoleic acid and its methyl ester was observed by Marcuse (76). Histidine and tryptophan exerted a relatively strong antioxidative effect, which was especially pronounced under certain conditions such as a low amino acid concentration, a pH of 7 or higher, and the presence of phosphate buffer and/or nonionic emulsifier. A good explanation for the varied antioxidative effect of different amino acids on an unsaturated lipid has not been provided. At higher amino acid concentrations, histidine and tryptophan inverted from an antioxidative to a prooxida— tive effect (76). Such inversion has been demonstrated for phenolic primary antioxidants (l4) and ascorbic acid (74, 124). According to Heimann and Pezold (42, 43, 44), three reactions could be responsible for the final 102 TABLE 6.--Analysis, by the TBA test, of the extent of oxidation of PE samplesa emulsified in 0.1M borate (pH 7.0) buffer after 50 hours at 25°C.b Sample TBA Absorbance PE .534 PE CuSOu .574 PE BHT .009 PE Na EDTA .075 PE Glycine .544 PE Lysine .521 PE Tryptophan .557 PE Histidine .061 aTBA absorbance of PE before oxidation was .060. bDeterminations were made on aliquots containing 5.0 ug phosphorus as assayed by the procedure of Rouser et al. (101). mar—77.“. '. ; 2 103 result in a system containing substances capable of being prooxidative as well antioxidative. They are: (l) the normal oxidation of the substrate; (2) the anti- oxidative reaction; and (3) the prooxidative reaction. In the system that Heimann and Pezold examined, con- taining lard and tocopherol, the prooxidative reaction had a high activation energy and only prevailed when a large amount of tOCOpherol was present. In contrast, only a small amount of tocopherol was needed to induce the antioxidative reaction. The same explanation given by Heimann and Pezold for lard as substrate and tocopherol as a pro— or anti- oxidant could be valid for unsaturated lipids and amino acids. By analogy then, a small concentration of amino acids could protect against lipid oxidation by encouraging the antioxidative reaction, while the prooxidative re- action, having a high activation energy, would be en— hanced by a high amino acid concentration. The vari— ability of the antioxidative capacity of different amino acids when present at the same concentration could be related to their individual activation energies for the prooxidative reaction and the antioxidative reaction. The greater antioxidative effect of histidine in pre- venting the autoxidation of PE (Figure 19) could indi- cate that histidine has a lower activation energy for the antioxidative reaction or a higher activation energy "I-r‘“”—““a-I-r ’ l .- L. 104 for the prooxidative reaction than glycine, lysine and tryptophan. If the antioxidative effect of an amino acid was dependent upon its activation energy for the antioxidative reaction, histidine would have the lowest activation energy for the system illustrated in Figure 19, followed by tryptophan, lysine and glycine respectively. The significance of the antioxidative action of amino acids toward phosphatides is considerable since both exist in continuous interaction with each other in an aqueous system in many food and agricultural products. A better understanding of the prooxidative, antioxidative and synergistic relationships that exist between amino acids and phospholipids can provide information useful in protecting foods from lipid oxidation. Phospholipid Antioxidation Information on the autoxidation of egg PE in the presence of a metal, an antioxidant and a chelating agent is presented in Figure 20. Copper accelerated the oxi- dation of PE as evidenced by an increase in the oxidation rate and a decrease in the induction period. Butylated hydroxytoluene (BHT) and sodium tetra, ethylenediamine tetraacetate (Na EDTA) completely retarded oxidation as measured by oxygen uptake and the TBA test. The TBA absorbance of PE + histidine was less after 50 hours than the value recorded for PE prior to the start of oxygen uptake measurements (Table 6). 105 iriili .00mm 00 000030 Ao.m ma. 000000 EH.o CH 0000000H0 mm wm0 0o 00H000Hx0030 030 so 0:0w0 000000020 0 000 00000000000 :0 .00005 0 00 000000 020--.om 000000 $30.... 92.... 00 f‘lLllli.‘ III‘J I ‘IlIm-IIII'IIII'HI‘IIIIIII o .0. wk<+m044mkmk mz..z<.omzu..>x...w firm... 55.000 ..0300. + ma. I10] 010 «emuoon.. mm .ILQII .0000 2.70. x _ + ma IIDII 10.0. mm IIAYII .100 o. n. IAHI. .AWII 00 (P!d!l04d'5°4d slow 20 EM) BxVIcIn ~39xxo 106 The ability of Na EDTA to retard oxidation suggests that trace metals may play a significant role in lipid oxidation. Uri (121, 122) has reported that lipid materials contain traces of copper, iron and cobalt. By complexing metal impurities, Na EDTA could aid in reducing the formation of free radicals and thereby provide a partial inactivation of oxidation in an autoxidizing system. A free radical acceptor such as BHT is considered to be essential in achieving protection against all pro- oxidant impurities (123). The addition of BHT to organic solvents at the 0.005% level permitted safe storage of egg PE almost indefinitely at —20°C and during preparative work by column and thin—layer chromatography (81, 125). Research on various aspects of phospholipid oxidation has revealed that phosphatides are highly susceptible to autoxidation. A search of the literature will indicate that numerous studies are being conducted to characterize the role(s) of phospholipids in biological systems. Be- cause changes in the chemical, physical and biological properties of phospholipids can arise through degradation, precautions should be taken to prevent oxidation during extraction, manipulation and storage of phospholipids. General Discussion This thesis research, which was designed to investi— gate the autoxidation of phospholipids, had a two-fold ‘2‘“ '-"'-”‘ “m 107 purpose. One purpose was to relate the oxidation of phospholipids to their specific molecular constitution and the other was to examine the influence of various extraneous substances on phospholipid oxidation. Indi- vidual phospholipids have been observed to vary in their susceptibility to oxidation. Several workers (1, 59, 77, 99) have reported that phosphatidyl ethanolamine (PE) oxidizes more rapidly than phosphatidyl choline (PC). Unsaturated fatty acids and the nitrogen moiety are factors known to affect the oxidation of phospholipids. Substances which could be considered extraneous in model systems can act as either pro— or antioxidants in the presence of phospholipids and thus are important in natural food systems where their proximity to phospho- lipids may permit interaction. Oxidation studies were performed on purified phospho- lipids which were emulsified in borate buffer. After the optimum pH for the autoxidation of PE and PC was shown to approximate neutrality, subsequent studies were conducted at pH 7.0. Autoxidation of egg and soybean phospholipids was followed by oxygen uptake determinations, ultraviolet absorption measurements, the TBA test and by changes in the concentration of polyunsaturated fatty acids. Oxygen uptake determinations emphasized that phospho- lipids from different sources can vary in their suscepti- bility to oxidation. Egg phospholipids oxidized more 108 rapidly than soybean phospholipids. The differences that were noted were ascribed to the fatty acid compo- sitions of egg and soybean phospholipids. Arachidonic acid, the most unsaturated fatty acid in egg PE and PC, contains four double bonds. Soybean PE and PC have as their most unsaturated fatty acid, linolenic acid with three double bonds, one less than arachidonic acid. The relationship between the unsaturation of fatty acids and their oxidation rate has been reported by Holman and Elmer (48). These workers measured the oxidation rate of several unsaturated fatty acids and their esters and concluded that an increase of one double bond in an un- saturated fatty acid or ester caused the rate of oxi- dation to increase by a factor of two or more. An-in- crease in the number of double bonds also reduced the length of the induction period. Thus, the higher rate of oxidation and shorter induction period for arachidonic acid compared to linolenic acid could explain why egg phospholipids are more susceptible than soybean phospho- lipids to autoxidation. The greater reactivity of PE compared to PC, ob- served by numerous workers, was confirmed in studies on the oxidation of egg and soybean phospholipids. An examination of the fatty acid composition of these phospholipids verified a finding reported by several lipid investigators. The finding, consistent for both 109 egg and soybean phospholipids, was that PE contained a higher percentage of unsaturated fatty acids than PC. The higher content of polyunsaturated fatty acids, particularly arachidonic acid in egg PE and linolenic acid in soybean PE, provides a partial explanation for the greater reactivity of PE compared to PC. Corollary measurements which were made at inter- vals during the course of phospholipid oxidation were the TBA test, ultraviolet absorption of diene and triene conjugation and changes in the percentage composition of specific unsaturated fatty acids. The results of these tests were consistent with data obtained through oxygen uptake determinations and provided additional information on characteristics of phospholipid oxidation. Higher TBA and diene and triene absorption values for egg PE as compared to egg PC supported the oxygen uptake studies which indicated the greater reactivity of PE. Low triene conjugation values for soybean PE at all stages of oxi- dation suggested that the formation of triene conjugates was more likely to result from a phospholipid containing a A double bonded fatty acid than from one with three double bonds. The susceptibility of the arachidonic acid in egg phospholipids to oxidation was shown by GLC determinations, which permitted a quantitation of the decrease in its composition in relation to oxygen uptake. By comparison, the amount of linoleic acid in egg PE and PC decreased at a considerably slower rate. 110 The separation of egg PC by silicic acid column chromatography into three fractions which differed in their unsaturated fatty acid compositions provided an additional means of illustrating the importance of un- saturated fatty acids on phospholipid oxidation. The induction period increased as the percentage of unsatur- ated fatty acids, particularly arachidonic acid, de— creased. The finding that the first egg PC fraction had a fatty acid composition which approximated the per- centage of arachidonic acid in PE, permitted an exami- nation of the influence of the nitrogen moiety in phospholipid oxidation. The importance of the nitrogen moiety was established by the observation that the in- duction period for PE was significantly less than that for the PC with the high arachidonic acid content. The maximum rate of oxidation for PE and the high arachidonic acid PC, after the end of the induction period, was identical. On the basis of oxygen uptake determinations on PE and the high arachidonic acid PC, it would appear that the maximum rate of oxidation (measured from the straight line portion of the oxygen uptake curve at the end of the induction period) for a phospholipid was regulated by the kind and amount of unsaturated fatty acids. After further examination of the oxygen uptake curves for PE and the high arachidonic acid PC, the following hypothesis was advanced: the nitrogen moiety ill of phospholipids influences the length of the induction period. Confirmation for this hypothesis was obtained by the finding that the length of the induction period of PC was reduced through contamination by minor amounts of PE. The reduction in the induction period appeared to be proportional to the amount of PE added. Experi- ments in model systems of methyl linolenate emulsions demonstrated that ethanolamine was prooxidative while choline was slightly antioxidative. A brownish-yellow color in the methyl linoleate—ethanolamine emulsion, after the onset of oxidation, suggested that a Maillard— type reaction had occurred between the amino group of ethanolamine and oxidation products of methyl linolenate. Since this discolaration, which had also been obserVed in oxidized PE emulsions, indicated that the free amino group of ethanolamine probably was reacting with compounds containing a carbonyl group, a study was initiated to mea- sure the free amino group of PE at various intervals dur- ing autoxidation. Measurements of the free amino group of egg and soybean PE demonstrated that it decreased during oxidation and therefore supported the contention that a Maillard-type browning reaction occurred. The second purpose of this research, which con— sisted of a study on the influence of various extraneous substances on phospholipid oxidation, was not as well defined as the first which related the oxidation of 112 phOSpholipids to their specific molecular consitution. Although the second phase of this research assumed the nature of a general survey and may have raised as many questions as it answered, part of the information that was obtained provided an insight on the effect of various substances on phospholipid oxidation. Substances that were investigated included ethanolamine, choline, amino acids, c0pper, butylated hydroxytoluene (BHT) and the sodium salt of ethylenediamine tetraacetic acid. The gap between the first and second phases was bridged by oxygen uptake studies on PE in the presence of added ethanolamine and choline chloride. The presence of additional ethanolamine, the nitrogen moiety shown pre— viously to accelerate oxidation, had a depressing effect on the autoxidation of PE. Choline chloride also acted as an antioxidant. Ethanolamine and glycine decreased the susceptibility of PC to oxidation as evaluated by oxygen uptake measurements. COpper accelerated the oxi- dation of PC. A greater prooxidative effect was observed for COpper and ethanolamine or c0pper and glycine than for copper alone. Since complexes between metals.and amino compounds are known to occur (41, 103, 118), and to accelerate oxidation (102, 10“, 118), the increased prooxidative effect of copper + ethanolamine and copper + glycine could be attributed to the formation of a complex. 113 Amino acids were observed to exert an antioxidative effect on the oxidation of phospholipids. Considerable differences existed in the amount of protection afforded PE by individual amino acids. Histidine had a strong inhibitory effect as evaluated by oxygen uptake and TBA absorbance measurements. In contrast, glycine, lysine and tryptOphan were less inhibitory than histidine. The differences in the antioxidative effect for each amino acid were thought to relate to a competition between pro- oxidative and antioxidative reactions, since under cer- tain conditions amino acids have been shown to accelerate lipid oxidation. A low activation energy for the anti- oxidative reaction could explain why histidine exerted a stronger antioxidative effect than glycine, lysine and tryptophan. The phenolic antioxidant, butylated hydroxytoluene (BHT), and sodium tetra, ethylenediamine tetraacetate (Na EDTA), a metal chelating agent, completely retarded the oxidation of PE as measured by oxygen uptake and the TBA test. The antioxidative effect of Na EDTA was un- expected even though its ability to chelate metal ions is known. The possibility exists that the effectiveness of Na EDTA in preventing oxidation could be related to a chelation of metal impurities present in phospholipid emulsions. The strong antioxidant effect, observed for BHT, confirmed previous reports of phospholipid anti- oxidation (81, 125) and provided a way to protect llLI phospholipids from oxidative degradation during their extraction, manipulation and storage. A study of the influence of various extraneous substances on phOSpholipid oxidation was undertaken to determine which substances promoted and which sub- stances retarded the oxidation of phospholipid emulsions. Experiments with added nitrogen containing substances provided additional information of the importance of the nitrogen moiety in phospholipid oxidation. Ethanolamine was observed to accelerate the oxidation of methyl linolenate emulsions but to retard the oxidation of phospholipids. The effect of the free amino group in the presence of a metal was definitely prooxidant. These results indicate that phospholipid oxidation is a complicated phenomenon and is influenced by various factors. The objective of the second phase of the thesis was not unrelated to the study of the oxidation of phospholipids in relation to their specific molecular constitution. In both instances, the goal was the same, namely to induce and measure oxidation in order to ob- tain information which could be utilized to reduce the susceptibility of phospholipids to oxidation. SUMMARY AND CONCLUSIONS Phospholipids from egg yolk and a commercial soy- bean phosphatide preparation (Centrolex P) were separated into fractions by silicic acid column chromatography. The purity of individual fractions was ascertained by thin-layer chromatography. Purified phosphatidyl ethanol- amine (PE) and phosphatidyl choline (PC) were emulsified in a Tween 20-borate buffer by a Braun Cell Homogenizer and their oxygen uptakes were followed manometrically with a Gilson Differential Respirometer. Oxygen consumption after 50 hours was greatest for PE at pH 8.0 and PC at pH 7.0. Since uptake was greater for PE than for PC at all temperatures studied, subse- quent oxygen uptake measurements were conducted at pH 7.0. Egg phospholipids were more reactive than soybean phospholipids. The rates of oxygen uptake at different temperatures were utilized to determine activation energies for PE and PC. The activation energies of PE and PC from egg and soybean were comparable; however, the values obtained for PE andPC were approximately one-half of the values reported for the autoxidation of methyl linoleate and linoleic acid emulsions. 115 116 Egg PC was separated into fractions with varying degrees of unsaturation by silicic acid column chromato- graphy. Of the three fractions collected, the PC frac— tion with the highest percentage of arachidonic acid was the most reactive while the fraction with the least amount of arachidonic acid exhibited the lowest rate of oxi- dation. The fatty acid composition of the first PC fraction eluted from the column, containing the highest percentage of arachidonic acid, resembled the fatty acid composition of egg PE. However, this high arachidonic acid PC had a longer induction period than egg PE. When egg PC was purposely contaminated with a small amount of PE the rate of oxidation was not altered appreciably, al- though the induction period was reduced. The addition of ethanolamine to emulsions containing methyl linolenate and a-glycerophosphate significantly reduced the induction period for autoxidation while the addition of choline (as choline chloride) produced a slight antioxidative effect: Various physico-chemical measurements were made of specific changes in the phospholipid molecule during autoxidation. Of the two major polyunsaturated fatty acids in egg phospholipids, arachidonic acid disappeared at a more rapid rate during oxidation while the concen- tration of linoleic acid decreased to a level that was stable. Higher 2-thiobarbituric acid (TBA) and diene and triene conjugation values were observed for PE i 117 than for PC. The concentration of the free amino group of egg and soybean PE decreased during oxidation. The effect of various prooxidative and antioxidative substances on the autoxidation of aqueous emulsions of PE and PC was investigated. Ethanolamine and choline de- creased the reactivity of egg PE. Copper accelerated the oxidation of PE and PC. Ethanolamine and glycine exerted an antioxidative effect on PC; however, when either ethanolamine or glycine was added with copper, PC was observed to oxidize faster than PC with copper alone. Glycine, lysine and tryptophan increased the induction period of egg PE. Histidine, butylated hydroxytoluene and sodium tetra, ethylenediamine tetraacetate completely retarded the autoxidation of PE for the period of study as evaluated by oxygen uptake and TBA determinations. The conclusions reached as a result of this study are summarized below: 1. The activation energies of egg and soybean PE and PC are comparable, suggesting that the mode of oxidation during the period of steady state oxidation is identical. 2. The greater reactivity of egg phospholipids compared to soybean phospholipids is related to the presence of arachidonic acid in egg phospholipids. 118 The rate of phospholipid oxidation during the period of steady state oxidation is regulated by the unsaturated fatty acids. The induction period for a phospholipid is a function of the nitrogen moiety. The ethanolamine moiety of PE has a greater prooxidant effect than the choline moiety of PC. The concentration of the free amino group of PE decreases during autoxidation, and is probably related to the development of a. brownish-yellow discoloration by a Maillard type reaction. The autoxidation of emulsified phospholipids is promoted by copper, retarded by glycine, lysine, tryptophan and histidine and almost completely inhibited by butylated hydroxy- toluene and sodium tetra, ethylenediamine tetraacetate. “£11.— "a. mum .l PROPOSALS FOR FURTHER RESEARCH Nearly every study, and this one was no exception, raises questions which cannot be adequately explained. Moreover, there are always topics, existing in the fringe areas of any study, which seemingly tantalize the re- searcher and attempt quite innocently to make him detour from his original objectives. Some unanswered questions and tantalizing topics which developed as a result of this study are listed below. 1. Silicic acid column chromatography and thin- layer chromatography have been used to obtain phospholipids with varying degrees of un- saturation in their fatty acids. It would be interesting to learn if plasmalogens could be fractionated in an analogous manner. Such a study on the fractionation of plasmalogens could be performed in conjunction with the fractionation of a pure phospholipid to deter— mine the extent of unsaturated and saturated fatty acids in plasmalogens. 2. No attempt was made to relate phospholipid oxidation to the effect of plasmalogens. The plasmalogen content of egg yolk phospholipids 119 120 has been reported to be 1% (2) and the amount of plasmalogen in soybean PE and PC was shown to range from U—7% and 8—9% respectively de— pending upon the method of determination (96). Although the plasmalogen content of egg and soybean phospholipids is low, its effect on 1 oxidation could be considerable since the ‘1 vinyl ether linkage can be readily hydrolyzed in aqueous solution to yield aldehydes. The latter are known to be susceptible to oxi- dation. Measurement of the oxidation of different plasmalogen fractions could provide information to enhance that obtained on the oxidation of various phospholipid fractions. Preliminary experiments were conducted to determine the effect of the enzyme, lipoxidase, on the oxidation of phospholipids. Indications were that lipoxidase retarded the oxidation of PC at 15°C in a borate (pH 7.0) buffer. Studies could be undertaken to explain this phenomenon and to learn if there are conditions in which lipoxidase can accelerate the oxidation of phospholipids. Many intermediates of classical autoxidation can be isolated and identified when oxidation experiments are carried out in aqueous systems. 121 This type of a study would probably require the use of sophisticated instrumentation such as IR, UV, and mass spectrometers, NMB spectro- scOpy, GLC and the polarograph. Product iso- lation and identification could be performed at various temperatures on PE and PC as differ- ent oxidation products are obtained at high temperatures compared to low temperatures. Methodology relative to such a problem has been considered in a review article by Schauenstein (105). Because the free amino group of phosphatidyl ethanolamine has been shown to decrease during oxidation, special attention should be given to products containing ethanolamine and its deriv— atives. Such an emphasis could help to define the exact changes in the free amino group in .phospholipid oxidation and to characterize browning reactions. 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APPENDICES 137 APPENDIX A STANDARD CURVE FOR PHOSPHORUS DETERMINATION 138 139 OH fir— mempw : .msnoanonm m m h L q 1 m m: u m.HH x mocwanoua< n- dI- oom. . ooz. . com. 1 com. . ooo.a eoueqaosqv APPENDIX B FATTY ACID COMPOSITION OF PURIFIED PHOSPHOLIPID FRACTIONS DISSOLVED IN SOLVENT AND DISPERSED IN BUFFER 140 141 Fatty acid composition of purified phospholipid frac- tions dissolved in solvent and dispersed in buffer. j Egg PE Egg PC Fatty Acid In Solvent In Buffer In Solvent In Buffer z x Z z 016 19.8 20.0 33.3 36.3 016:1 1.6 2.1 -- -- 018 34.2 34.8 14.6 14.0 C18:1 14.5 14.3 34.4 33.4 018:3 3.2 3.3 -- -- 020:" 17.6 15.7 2.1 2.0 APPENDIX C STANDARD CURVE FOR ETHANOLAMINE 142 143 v.0 ‘F m.o q- =.o db mafismaocmnpm z: m.o d m.o up .woom. 1.00:. A eoueqaosqv 1com. 1 room. 1 .ooo.H