WWW mmas m :22, a: wacmm mam E) m mm: mm LIBRARY“: Michigan State University ( ,, ABSTRACT QUANTITATIVE ANALYSIS FOR D, a-TOCOPHEROL (VITAMIN E) IN PORCINE MUSCLE By Patrick Reginald Hill The objectives of the present study were to develop a reliable method for the measurement of D, a—tocopherol (vitamin E) in porcine muscle, and to compare levels in muscle classified as being normal or as pale, soft and exudative. D, a-tocopherol levels were measured in 18 samples of porcine muscle. A 20.0g sample of powdered muscle, previously prepared and stored at ~20°C, and 20.0g of anhydrous sodium sulfate were ground together in a mortar with enough acetone to produce a paste. The paste was transferred to a 43 mm Soxhlet thimble and the total lipid was extracted by hot acetone in a Soxhlet extraction apparatus, followed by saponification in the presence of hot alkali and pyrogallol. The non-saponifiable material was ether extracted and D, a—tocopherol was separated by thin-layer chromatography on alumina chromatographic sheets, using a solvent system of benzene:diethyl ether (60:40 v/v) to which was added 2 ml of glacial acetic acid per 100 ml. The tocoph— erol Spots were develOped with 0.004% ethanolic 2', 7'—dichlorofluorescein, detected under ultra-violet light, removed, and measured colorimetrically at 534 nm using 4, 7-diphenyl-1, lO-phenanthroline (bathophenanthroline). Replicate concentrations of pure D, a-tocopherol yielded a recovery factor of 85% which was applied in correcting all subsequent experimental data. Using this method, the D, a-toc0pherol content of porcine muscle for 18 samples analyzed in duplicate ranged from 0.91 to 1.78 ug/g muscle, while the lipid content varied from 6.5 to 10.0%. The average weight of lipid extract was 1.6g. This gave a mean value of 8.0% lipid, containing an average concentration of D, a-tocopherol of 1.22 ug/g muscle or 15.09 ug/g lipid. Results obtained in the present study indicate that the method is reproducible and hence suitable as an indicator of the D, u—tocopherol level in muscle. Results from measurement of levels of D, a—tocopherol in a separate study of normal versus pale, soft and exudative porcine muscle were inconclusive in demonstrating any differences. Speculation that the tocopherol may have been destroyed during prolonged freezer storage is supported by reports in the literature. QUANTITATIVE ANALYSIS FOR D, a-TOCOPHEROL (VITAMIN E) IN PORCINE MUSCLE By Patrick Reginald Hill A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1971 .’ y/ ,/ V I (7 '/ / ACKNOWLEDGEMENTS The author expresses his sincere appreciation to his major professor, Dr. A. M. Pearson, for the excellent research facilities provided and his assistance during the preparation of this manuscript. The author thanks Drs. R. A. Merkel and D. E. Ullrey for serving as members of his guidance committee. Sincere appreciation is extended to the author's colleagues in the laboratory for their advice and encouragement throughout the research program. Special thanks are offered to Michael Tate for his assistance with some of the analytical work and to Joice Adams for typing the manuscript. The author is deeply indebted to his parents for their continuous support and encouragement throughout his college career. ii TABLE OF CONTENTS I INTRODUCTION II REVIEW OF LITERATURE Biological Role of D, a-Tocopherol (Vitamin E) Relationship Between Vitamin E and Other Antioxidants Measurement of D, a—TocOpherol in Animal Tissues Preparation and Extraction of the Sample Removal of Fat Removal of Interfering Substances Separation of Tocopherols Measurement of Toc0pherols Pale, Soft and Exudative Pork Incidence and Importance of PSE Muscle Characteristics of PSE Muscle III EXPERIMENTAL MATERIALS AND METHODS Experimental Animals Sampling D, a-Tocopherol (Vitamin E) Assay Preparation of Carbonyl—free Solvents Preparation of Muscle Tissue Extraction of Muscle Lipid Saponification of Muscle Lipid Extract Ether Extraction of Non—saponifiable Fraction Thin-layer Chromatography of Non—saponifiable Fraction Colorimetric Determination of D, a—Toc0pher01 IV RESULTS AND DISCUSSION Development of a Standard Curve Establishment of TLC Recovery Factor D, a—Tocopherol Content of Porcine Muscle iii PAGE l4 14 14 15 15 16 16 17 18 19 20 22 22 24 26 TABLE OF CONTENTS (con't) IV RESULTS AND DISCUSSION (con't) Methodology of Tocopherol Analysis Extraction of Muscle Lipid Saponification of Muscle Lipid Extract Thin-layer Chromatography of Non-saponifiable Fraction Colorimetric Determination of D, a-Tocopherol D, a-Tocopherol in Normal and PSE Porcine Muscle V SUMMARY BIBLIOGRAPHY iv PAGE 28 28 29 30 32 32 34 35 TABLE II III LIST OF TABLES D, o-Toc0pherol Standard Curve Data Data Showing Recovery of D, a—Tocopherol by TLC D, a-Tocopherol Analysis of 20.03 Porcine Muscle (wet weight) PAGE 23 25 27 INTRODUCTION The problem of the biological role of vitamin E in animals is still unsolved. There are currently two schools of thought concerning its function in living tissue. Tappel (1962; 1965) believes that the vitamin functions in viva solely as a physiological lipid antioxidant. The other theory is that vitamin E may function as a catalytic agent in intermediary metabolism, acting at a specific metabolic site in energy metabolism (Schwarz, 1961; 1969). Vitamin E deficient rations have been reported by Michel (1969) to result in muscle degenerative changes among some commercial swine herds in Michigan. HistOpathological observation of porcine muscles by Weatherspoon (1969) has revealed some degeneration in both normal and pale, soft and exudative (PSE) muscle. However, no relationship has been established regarding the role of D, a-toc0pherol in the development of pale, soft and exudative muscle. In order to study this relationship, a suitable method for its measurement in porcine muscle is necessary. Several complications in the methodology of tocopherol assay procedures found in the literature necessitated the development of a procedure suitable for assaying D, a-tocopherol in porcine muscle. Consequently, the present study was undertaken to develop a reliable method for the measurement of D, a—tocopherol (vitamin E) in porcine muscle, and to compare levels in muscle classified as being normal or as pale, soft and exudative. LITERATURE REVIEW Biological Role of D, a-Toc0pherol (Vitamin E) There are two basic concepts of the biological role of a—tocopherol (vitamin E). One is that the vitamin functions in the animal solely as an antioxidant, being particularly involved in the protection of unsaturated tissue lipids against peroxidation (Tappel, 1962; 1965). The other View considers that vitamin E has a metabolic role, which is Specific or general, but which is not directly concerned with its properties as an antioxidant in;zivg_(8chwarz, 1961; 1969; Green and Bunyan, 1969). Although early attempts to show a reversible reaction between a—toc0pherol and its known oxidation product a—toc0pherquuinone were unsuccessful, and vitamin E deficiency could not be correlated with any decrease in cellular oxidation processes, the function of vitamin E seems to be related to the oxidation-reduction properties inherent in its structure (Green and Bunyan, 1969). Pennock.gt_§13 (1964) indicated that there appears to be eight naturally occurring forms of vitamin E. Bieri (1970) stated that the considerably greater biological activity of a—tocopherol compared with its 8, y, and 6 homologs in the tocol series (saturated side chain), and those in the tocotrienol series (unsaturated side chain) is explained by the fact that for optimal utilization and retention at the cellular level, the saturated side chain and three methyl groups on the chroman ring (as in a-tocopherol) are required. For a functional group, which is necessary for biochemical reactivity, the presence of a reactive hydrogen is critical. Green and Bunyan (1969) in a critical review of the biological antioxidant theory concluded that all direct attempts to find an increase in lipid peroxidation in the tissues of vitamin E deficient animals have failed, and in general, the onset of the disease cannot be correlated with tissue peroxidation. They stated the evidence all showed that dietary stresses leading to increased tissue peroxidation, whether produced by increased unsaturated fat or by toxic agents, in fact do not result in increased catabolism of a—tocopherol, secondary antioxidants (such as ascorbic acid or sulfhydryl compounds), vitamin A or other so- called peroxidizable intracellular substances. Although there is a close relationship between vitamin E and the metabolism of unsaturated fat, they suggested that the relationship is complex and cannot be interpreted solely in terms of the peroxidizability of the fat and an antioxidant function for vitamin E. They feel that the concentration of lipid peroxides in the tissues is independent of dietary lipid or vitamin E, except in the case of adipose tissue. Bunyan g£_§1,(l968) suggested that the peroxides accumulating in adipose tissue are largely exogenous and that vitamin E may act by preventing their absorption by the gut. Relationship Between Vitamin E and Other Antioxidants Vitamin E, the major biological antioxidant, shows complex re— lationships with other antioxidants and reducing compounds (Tappel, 1962). In a review of these relationships, Tappel (1970) stated that the major biological function of vitamin E in inhibition of lipid peroxidation is radical chain-breaking. He added that as an ancillary function of ubiquinol, small amounts can react as a chain—breaking lipid antioxidant, and can provide, like vitamin C, important homosynergism with vitamin E. He further stated that sulfhydryl compounds, mainly glutathione, sulfhydryl proteins, cysteine, and also methionine apparently react in small amounts as free radical scavengers and as peroxide decomposers. Tappel and Caldwell (1967) also reported that seleno—amino acids are powerful catalysts of sulfhydryl-disulfide exchange, and that they react as free radical scavengers and as peroxide decomposers as well. Green and Bunyan (1969) stated that the observed dietary relation- ships between selenium, vitamin E, antioxidants and sulfur amino acids, varying as they do from species to species, invite speculation as to their nature. These researchers stress it is essential to recognize that vitamin E and related compounds may operate at more than one level and in more than one way. As an example, they suggest some of the observed synergistic effects may arise from protective or antioxidant interrelationships in the gastrointestinal tract, especially when they are manifested only in the presence of vitamin E. Schwarz (1962; 1965) stated that vitamin E and selenium are separate biochemical entities and that animals probably require both, in the sense that there are separate selenium and vitamin E deficiency diseases, although they may overlap. Bieri (1964) has shown that selenium may exert a synergistic action with synthetic antioxidants as well as with vitamin E. Scott (1970) demonstrated that selenium is an essential nutrient required in metabolic processes, that are not protected by vitamin E. The evidence from his research strongly indicated that one of the important functions of vitamin E is concerned with protection of traces of selenium in the animal body. Measurement of D, a—Tocopherol (Vitamin E) in Animal Tissues a-tocopherol occurs naturally in animal tissues in the non- saponifiable portion of the lipid fraction, together with sterols, non-a-tocopherols, vitamin A, vitamin K, and other naturally occurring antioxidants (Mervyn and Morton, 1959; Bieri, 1969). Separation of a-tocopherol from its associated contaminants has been extensively investigated. However, data for animal tissues obtained with most of the older methods is unreliable since it has been shown that the results were either too high or too low (Edwin e£_al,,l960). Variation in the data is due to the fact that various workers have utilized different techniques in the several stages of purification for tocopherol analysis, while others have utilized techniques resulting in the measurement of non-toc0pherol reducing Substances, which are normally present in much greater amounts than toc0pherol itself (Edwin Efihilr, 1960). The primary difference in procedures occurs in the initial extraction and semi-purification of the tocopherols from other lipids. Once the tocopherols are obtained relatively free of lipid, thin—layer chromatography and colorimetric assay can be used in almost all applications (Bieri, 1969). The sequence of analysis for vitamin E in general consists of: (1) preparation and extraction of the sample, (2) removal of fat, (3) removal of interfering substances, (4) separation of the tocopherols, and (5) measurement of the tocopherols (Bunnell, 1967). Preparation and Extraction of the Sample Hot ethanolic extraction of the lipid fraction in an all-glass apparatus is based on either the method of Quaife and Harris (1948), that used by Bunnell (1955) or that of Pudelkiewicz §£_al,(1960). Hot acetone extraction in a Soxhlet apparatus was used by Edwin e£_§l, (1960) and is particularly well suited to animal tissue. Chloroform extraction (Bolliger and Bolliger-Quaife, 1955) is useful for soft tissues which are easily ground, such as liver. Diethyl ether extraction (Draper and Csallany, 1958) has been used primarily for animal tissues. Removal of Fat Removal of saponifiable fat from the extract of a sample being assayed for tocopherols is usually necessary for several reasons (Bieri, 1969). The presence of appreciable quantities of lipid adversely affects some purification and all separation procedures, such as column, thin— layer, paper and gas—liquid chromatography. In addition, excessive amounts of lipid inhibit the development of color if the Emmerie— Engel colorimetric reaction, which measures total reducing substances, is used (Bunnell, 1967). Saponification has been routinely used to remove lipid substances. Bunnell (1967) and Bieri (1969) stated that the sensitivity of tocopherol to oxidation in the presence of hot alkali requires the use of either an N2 atmosphere or the addition of an antioxidant such as pyrogallol or sodium ascorbate, during saponification. In the case of pyrogallol, washing the solvent extract is usually necessary to remove the antioxidant in order to avoid interference during later analysis (Bunnell, 1967). Removal of Interfering Substances Most analytical procedures require additional purification steps before separation and determination of the tocopherols can be accomplished. Vitamin A, carotenoids, sterols and a considerable quantity of unknown reducing substances usually must be removed (Bunnell, 1967). Column chromatography using floridin (Fuller's earth) has been widely used in vitamin E analysis to remove vitamin A, carotenoids, and some sterols (Pudelkiewicz et_al,, 1960; Edwin et_al,, 1960). However, Diplock gt_§1, (1966) stated that silicic acid- celite columns (Draper e£;§13, 1964) and floridin columns (Diplock.e£_alf, 1960) give low recoveries of vitamin E when used with animal tissues. Sterols have been removed from the non-saponifiable extract by freezing—out of a methanolic solution (Edwin 35 31,, 1960). This step is important if subsequent analysis is by paper or gas-liquid chromatography (Bunnell, 1967; Edwin e£_a1,, 1960). Herting and Drury (1967) reported satisfactory results for thin—layer chromatography of the non-saponifiable lipids of animal tissue on using Eastman alumina chromagram sheets, even when used without performing any purification steps. Alumina (Bieri, 1961) and secondary magnesium phOSphate (Bro- Rasmussen and Hjarde, 1957) column chromatography have also been report- ed by Bunnell (1967) as suitable methods of purification prior to separation of tocopherols by paper or thin-layer chromatography. Bieri (1969) stated, however, that he has satisfactorily separated a-tocopherol from the non-saponifiable fraction of a variety of animal tissues, and also from other tocopherols using a small mixed alumina-zinc carbonate—celite column. He cautioned that careful attention to the prOperties of the adsorbents was necessary. Separation of Toc0pherols The primary methods for separation of the various toc0pherols from each other and from any remaining contaminants are paper and thin—layer chromatography (Bunnell, 1967). Bieri (1969) stated that there have been no significant improvements in paper chromatographic techniques for vitamin E since the comprehensive study of the vitamin E Panel of the British Society of Analytical Chemists (1959). The assay proposed by this group is a two-dimensional paper chromatography system entailing both adsorption and reversed— phase partition chromatography in which the tocopherols are separated into five zones. Although good separation of the tocopherols from other non-saponifiable components can be achieved by two—dimensional paper chromatography, Bieri (1969) stated that this method is considerably slower than thin—layer chromatography. The majority of thin-layer methods for tocopherols utilize plates with silica gels G, GF , and HF 254 254’ aluminum oxide, or magnesium phosphate as the stationary phase (Bunnell, 1967). Bieri (1969) obtained 90 - 96% recovery of a-tocopherol using standard grade silica gel with calcium sulfate binder. He also used glass-fiber sheets impregnated with silica gel and obtained satisfactory separations of a,.y- and 6—tocopherols. But he advised against the use of plastic sheets coated with silica gel, because the thinness of the coating (100 u) greatly restricted the amount of lipid that could be applied and produced rather indistinct separations. Herting and Drury (1967) used both one and two—dimensional thin- layer chromatography on plastic precoated alumina sheets to separate tocopherols in a variety of samples, including rat liver and adipose tissue. They achieved recoveries of approximately 90% for a, y, and 5—tocopherols. They obtained resolutions superior to those on glass plates coated with alumina and their results compared favorably with those obtained by column chromatography on magnesium phosphate. Ames (1971) in the report to the Association of Official Analytical Chemists on a collaborative study of the determination of Vitamin E in foods and feeds, recommended the use of precoated alumina chromagram sheets (Type X-6062 alumina adsorbent without fluorescent indicator, for AOAC vitamin E assay - Eastman Kodak Co.). This method was recently adopted by the AOAC (1971) as the official method of vitamin E determination. Bieri (1969) indicated that of all the reagents used to identify tocopherols under ultra—violet light after thin—layer chromatography, sodium fluorescein was the most sensitive. He reported that as little as 0.2 ug of tocopherol could be detected with this method. The application of gas—liquid chromatography to the analysis of tocopherols in animal tissues is limited due to the problem of interfering substances (Bieri, 1969). The primary difficulty is in achieving a preliminary purification so that interfering substances, primarily cholesterol, do not mask the smaller amounts of tocopherol (Bieri Et_al,, 1963; Nair gt El}: 1963) Measurement of Tocopherols The method of Emmerie and Engel (1939) is the most widely used, and one of the most precise and sensitive methods for measurement of 10 tocopherols (Bunnell, 1967). The method is based on the fact that tocopherol reduces ferric to ferrous iron in the presence of 2, 2'-bipyridine and produces a red complex. The red color is then readily measured in a colorimeter or spectrophotometer at 520 nm (Emmerie and Engel, 1939). Tsen (1961) substituted 4, 7-dipheny1- 1, 10-phenanthroline (bathophenanthroline) for the 2, 2'-bipyridine used in the original Emmerie - Engel reaction. By so doing, the sensitivity of the original method was increased by two and one half fold. Duggan (1959) reported the use of a simple and extremely sensitive Spectrofluorometric method for the determination of free tocopherols and tocopherol esters in tissues. He indicated that vitamin A and other fat-soluble reducing substances did not interfere and that phenolic estrogens were about the only class of compounds in tissues that might cause interference in the determination. Pale, Soft and Exudative Pork Ludvigsen (1953) first described a condition in Danish Landrace swine, which he called "muscle degeneration disease". The muscle was pale, soft and watery in appearance and had a very low pH (5.3 — 5.5) at 45 minutes post mortem, whereas, the pH of normal muscle was much higher (6.8 — 7.0). He noted that the condition was especially prevalent in the longissimus muscle, but that occasionally it was found in all muscles of a given animal. Incidence and Importance of PSE Muscle Other researchers have reported the same condition in pigs ll (Wismer - Pedersen, 1959; Briskey 3£_§13, 1959a; 1959b; Briskey and Wismer-Pedersen, 1961a; 1961b; Bendall and Wismer-Pedersen, 1962; McLoughlin and Goldspink, 1963; Briskey, 1964). This condition has been reported to occur in 18% of some 15,000 pigs surveyed in the United States (Forrest §£_313, 1963) and in 35 - 40% of all pigs slaughtered in Denmark (Clausen and Thomsen, 1960). Lower incidences of PSE muscle have been reported in pigs from other countries, including England, Norway, Hungary, Canada (Briskey, 1964) and Ireland (McLoughlin, 1965). Higher incidences have been encountered in pigs in Yugoslavia and the Netherlands (Briskey, 1964). Briskey (1964) indicated that several factors affect the incidence of the PSE condition, including geographical origin, season, temperature, weight, sex, lean-to—fat ratio and most importantly the breed. He reported that Landrace, Hampshire and Poland China pigs had a higher incidence of PSE muscle than Yorkshire, Berkshire and Chester White pigs. Weight loss, due to a lowered water holding capacity of PSE muscle, causes the condition to be of economic importance to the pork industry (Briskey, 1964). The effects of this condition in pork at the consumer level are not fully known. Characteristics of PSE Muscle PSE muscle is characterized by a rapid rate of post-mortem glycolysis, resulting in a rapid drop in pH soon after death (Ludvigsen, 1953; Wismer-Pedersen, 1959; Briskey and Wismer-Pedersen, 1961a; McLoughlin, 1963; Briskey e£_a13, 1960; Briskey, 1964). The accumulation of lactic acid while the muscles are still at a high temperature 12 (>35°C) accounts for the low pH. However, the underlying cause of the rapid glycolytic rate which produces the PSE condition is still not fully established (Briskey, 1964). Cassens (1966) suggested that the combination of a high temperature and a low pH soon after death might be responsible for the visual loss of color and the drastic lowering of water-binding capacity as reported by Wismer—Pedersen and Briskey (1961b) and Bendall and Wismer-Pedersen (1962). A rapid decrease in labile phOSphate compounds concurrently with a rapid drop in pH in PSE pigs was reported by Briskey and Wismer- Pedersen (1961a). Bendall §£_§13 (1963) have shown that the onset of rigor mortis occurs only after the ATP concentration declines to about 30% of the initial level. Briskey et_§1, (1962) and Bendall 33 31, (1963) have found muscles that ultimately become PSE showed rapid development of rigor mortis. Weatherspoon (1969) stated that differences existed between various muscles and breeds of pigs in their potential to utilize oxygen from myoglobin. He found that in general, red fiber area was larger among low quality (PSE) pigs than in those with normal muscle quality. He further stated that low quality pigs had less capillaries per sq mm of muscle and also the efficiency of the capillary bed was further aggravated by fiber hypertrophy in these muscles. On histopathological and histochemical observation of some muscles of both normal and PSE pigs, Weatherspoon (1969) noted changes similar to those observed in vitamin E and/or selenium deficiency. Michel et_§l, 13 (1969) reported that field cases show a deficiency of these nutrients in some commercial swine herds in Michigan. Hence, Weatherspoon (1969) suggested the need for further study concerning the possibility of a relationship between these nutrients and ultimate muscle quality. EXPERIMENTAL MATERIALS AND METHODS Experimental Animals The muscle used in the present study was taken from the longissimus muscle of hogs slaughtered in the Michigan State University Abbatoir. The samples used in the study of D, a-toc0pherol levels in normal versus PSE muscle originated from pigs raised on farms where there was a history of the PSE condition. Samples were taken from the longissimus muscle and were classified by Weatherspoon (1969) on the basis of a combined evaluation of 45 minutes post-mortem pH and 24 hour transmission values. Sampling Samples of longissimus muscle were immediately frozen in liquid N2 and stored at -20°C. The muscle samples were powdered in a —20°C room as described by Borchert §£_§13 (1965) and Koch (1969). Shattered pieces of the frozen muscle samples (stored at -20°C following liquid N2 freezing) were placed in a Waring Blendor jar with chipped dry ice, pulverized for approximately 30 seconds and then sifted. The coarse material, which remained on the sieve (16 mesh), was discarded. The powdered samples were placed in sterile polyethylene bags and 12 hours were allowed to elapse for CO sublimation before sealing 2 the containers. The powdered samples were then stored at -20°C until used for analysis. 14 15 D, a-Tocopherol (Vitamin E) Assay Preparation of Carbonyl—free Solvents Because of the ease of oxidation of the hydroxyl group of tocOpherols, a primary chemical property from the analytical vieWpoint, it was deemed necessary to prepare carbonyl—free solvents. This was accomplished by refluxing a derivative of hydrazine (2, 4-dinitro— phenylhydrazine-DNPH) in the presence of an acid catalyst (Schwartz _g£flal., 1961). Trichloroacetic acid (TCA) was used for this purpose. Solvents used in the various parts of the assay were analytical grade (Baker or Merck) reagents (A. C. S. specifications), and were prepurified in glass prior to use. Acetone: (B. P. 56.2°C) It was redistilled. Ethyl alcohol: (B. P. 78.4°C) 5 g DNPH + 1 g TCA per liter of ethanol were refluxed for 1 hour, then distilled. The distillate, which was slightly yellow in color, was redistilled to remove any traces of color. Benzene: (B. P. 80.1°C) 5 g DNPH + 1 g TCA per liter of benzene were refluxed 1 hour, then distilled. A drying tube packed with Drierite (anhydrous CaS04—8 mesh — W. A. Hammond Drierite Co.) was attached to the delivery tube of the distillation apparatus to remove any moisture. Diethyl ether: (B. P. 34.6°C) The ether was treated to remove peroxides prior to distillation by the method of Eik Nes (1968). A 100 m1 portion of 20% (w/v) FeSO in 0.7N H SO was 4 2 4 added to 1 liter of ether in a 2000 m1 separatory funnel and the l6 mixture was shaken for a few minutes. The green to brownish- green layer, depending on the peroxide content, was discarded. The ether was then rinsed repeatedly with 100 m1 portions of distilled water until neutral to litmus paper. A mixture of S g DNPH + 1 g TCA per liter of ether was refluxed 1 hour and then distilled, Preparation of Muscle Tissue A modification of the method of Edwin et 31' (1960) was used to prepare duplicate samples of muscle tissue for lipid extraction. A 20 g sample of powdered muscle, previously prepared and stored at -20°C, and 20 g anhydrous sodium sulfate were ground together in a mortar with enough acetone to produce a paste. Muscle Lipid Extraction Total lipid was extracted using a Soxhlet apparatus (Vari Heat— 6 place Soxhlet Extraction Apparatus - Precision Scientific Co.). The paste was transferred to a 43 mm Soxhlet thimble. The excess acetone was filtered through and the residue extracted for 3 1/2 hours with 150 ml of acetone. Following extraction the flasks were cooled and flushed with N2. The acetone was evaporated under vacuum using a rotary vacuum evaporator (Bfichi Rotavapor - R — Rinco Instrument Co.) while warming the solution in a temperature-controlled water—bath. In preliminary work the acetone was evaporated under a steady stream of N while heating in a water—bath, 2 but this proved too time consuming and was abandoned. The total lipid residue weight was derived by weighing the flasks before extraction and after acetone evaporation. l7 Saponification of Muscle Lipid Extract Saponification of the weighed lipid extract (minimum of 1.0 g) and recovery of the non-saponifiable material were carried out as described by the Analytical Methods Committee (1959) of the British Society for Analytical Chemistry, except for modifications in the equipment. The saponification equipment assembly utilized in the present study allowed six samples to be handled simultaneously. It consisted of a large Magni Whirl Water-Bath (Blue M Co.) into which the six 250 ml Soxhlet extraction flasks attached to their respective condensers (Liebig — West Modification - 500 mm) were immersed during saponification. A 4 m1 portion of pyrogallol (5% (w/v) in E+OH), prepared fresh daily, was added per gram of lipid extract to each flask and the contents allowed to boil. When the mixture boiled, the condensers with the flasks still attached were removed from the water-bath (100°C). 1 m1 KOH solution (160 g/100 ml HéO) per gram of lipid extract was added using a 5 m1 measuring pipette, which fitted snugly into the Open end and extended about half of the length of the condenser, thus, allowing a measured quantity of KOH to be carefully dropped directly into the flask. The flasks were reimmersed in the boiling water-bath and heated under vigorous reflux for 5 minutes with occasional shaking. The flasks were then raised from the water-bath and 20 ml of deionized distilled water (room temperature) were added through the top of the condenser. The flasks were then removed, stoppered and cooled. 18 Ether Extraction of the Non—saponifiable Fraction Immediately following saponification, the non-saponifiable fraction was extracted with diethyl ether three times, using 25 ml for each extraction. If the phases failed to separate quickly and sharply, 1 or 2 ml of ethanol were usually added to break the emulsions. The combined ether extracts were washed with 20 m1 portions of deionized distilled water (room temperature) until neutral to phenolphthalein indicator solution (1 g phenolphthalein in 100 m1 absolute alcohol), avoiding vigorous shaking at all times. Three or four washes were usually necessary. The ether solution was then flushed with nitrogen. The ether was evaporated using a rotary vacuum evaporator as previously employed in the case of acetone. The residue was dried, if necessary, by adding a little absolute ethanol and benzene and reevaporating. The dry residue from each flask was dissolved in 5 ml of benzene and transferred carefully to 12 m1 centrifuge tubes (w/stop. grad. — Corning 8144) flushed with a steady stream of N and steppered. An 2 additional 5 ml of benzene, in portions of 2,2 and 1 ml, were used to wash down the sides of flasks and tubes with the aid of a Pasteur pipette. The solutions of non-saponifiable material were reevaporated 2 to 3 times, washing down the sides of the tubes with benzene as needed. Finally, the contents of each tube were adjusted to 1 ml, the tubes flushed with N2 and stoppered. The samples were stored at 5°C until used for thin-layer chromatography. l9 Thin—Layer Chromatography of Non—saponifiable Fraction One dimensional thin-layer chromatography on 20 x 20 cm precoated alumina sheets (Eastman Alumina Chromagram Sheet - adsorbent coated on flexible, 200 u thick polyethylene terephthalate - #6062 and #X6062 without fluorescent indicator - Eastman Kodak Co.) was employed to separate D, a—toc0pherol (Vitamin E) from other tocopherols and non- saponifiable lipids. The method used by Herting and Drury (1967) to estimate D, a—tocopherol in various biological materials from both plant and animal sources, including rat liver and adipose tissue, was applied to porcine muscle. 2 The plates were activated in an oven at 100°C for 30 minutes. 40 pl aliquots of non—saponifiable lipid from porcine longissimus muscle were spotted on the plates using a repeating dispenser (Hamilton Co.) and an Application Box — Model DB (Brinkmann Instruments, Inc.) for main- taining a N atmosphere. After inserting the plates in the box, air and 2 traces of 02 were removed by flushing with N were equipped with nipples so that continuous passage of a small stream 2. The two sides of the box of N2 could be accomplished during application of samples. The plates were developed in a Brinkmann—Desaga Controlled Atmosphere Developing Tank (Brinkmann Instruments 00.), fitted with a special cover equipped with gas inlet and outlet connections. A stainless steel plate holder fitted with two glass plates for Supporting the flexible polyethylene precoated plates was employed. The holder was inserted into the tilted tank so that one edge was immersed in the solvent, while the other supporting the alumina Sheet was above the solvent level. 20 The tank, which was lined on three sides with chromatography paper to improve solvent saturation, was flushed with N2 for 15 minutes. After waiting another 20 minutes so that the atmosphere would become saturated with solvent vapor, the tank was brought back into a horizontal position so that the solvent came into contact with the layer to initiate separation. The solvent system utilized was benzene : diethyl ether (60:40 v/v) to which was added 2 ml glacial acetic acid per 100 ml. The solvent front travelled 15 cm. After chromatography the plates were dried in the dark for 5 minutes and sprayed lightly with a 0.004% solution of 2',7'—dichlorofluorescein (Eastman Organic Chemicals) in ethanol. When the tocopherol Spots were completely dry (about 5 minutes), they were located with a 2537 A° short wave light. The spots were circled with pencil (allowing a safety margin of ca 5 mm around each Spot), cut out with scissors, and carefully scraped into 12 ml centrifuge tubes (w/stop. grad. Corning 8144) in the dark. Colorimetric Determination of D,gu-Toc0pherol The method of Tsen (1961), adapted for measurement of small amounts of tocopherol by Herting and Drury (1969), was used with minor modifications. The modifications were that the tocopherol Spot was scraped off into a 12 ml centrifuge tube rather than merely cutting out the spot and placing it in a vial. Also, it was necessary to include two centrifugation steps. After scraping each a-toc0pherol Spot into a 12 m1 centrifuge 3 tube, 2 m1 of an ethanolic solution of bathophenanthroline (3 X 10— M— G. Frederick Smith Chemical Co.) were added to each centrifuge tube. 21 An unused but sprayed area of the TLC Sheet was used to prepare a blank in the same manner as for the tocopherol spots. The centrifuge tubes were stOppered and Shaken on a Vortex Shaker for 15 seconds. After standing in the dark for 15 minutes, they were again shaken for 15 seconds, then centrifuged for 10 minutes at 3000 rpm in a refrigerated centrifuge at 4°C (International Refrigerated Centrifuge - Model PR-6). After centrifugation, 1 m1 aliquots were transferred to clean 12 m1 centrifuge tubes for the colorimetric reaction. A 0.3 m1 portion of 3 an ethanolic FeCl solution (2 X 10- M - J. T. Baker Chemical Co.), 3 prepared fresh daily, was added to each sample with mixing on a Vortex Shaker. Exactly 15 seconds after the last drop of the FeCl was added, 3 0.3 ml of an ethanolic solution of H PO (0.172 M — 86% — J. T. Baker 3 4 Chemical Co.) was added with mixing to complex the residual ferric ion. The resulting salmon—pink color was stable in a normally lighted room for at least 90 minutes (Herting and Drury, 1969). The samples were centrifuged for 10 minutes at 3000 rpm in a refrigerated centrifuge at 4°C as described previously. This step removed any material that could cause a precipitate to form in the cuvettes during spectrophotometric measurement. Following centrifugation a portion of the solution was transferred to a 1.6 ml capacity, 1 cm light path microcell (Arthur H. Thomas Co.). The absorbance was measured in a Beckman DU-2 SpectrOphotometer at 534 nm (Beckman Instrument Co.). RESULTS AND DISCUSSION Development of a Standard Curve A standard curve was prepared for pure D, a—tocopherol (M. W. 430.72) isolated from vegetable oil and obtained from the Eastman Kodak Company. Standards were prepared in ethanol at concentrations from 0.7 to 7.2 ug/ml, which covers the range of sensitivity for the colorimetric reaction. Table 1 shows the mean absorbance values for duplicate samples at different concentrations of D, a—tocopherol read at 534 nm on a Beckman DU—2 Spectrophotometer. A linear relationship was found to exist between the concentration of D, a- tocopherol and absorbance values which conforms with Beer's Law and is in agreement with the results of Tsen (1961). Tsen (1961) also indicated that by using 4, 7—diphenyl-1, lO-phenanthroline (bathophenanthroline) as the colorimetric reagent instead of 2, 2'—bipyridine as in the original Emmerie-Engel (1939) method, the sensitivity could be increased by two and one half fold. On the basis of Beer's Law, therefore, a factor of 10.2 was derived from the standard curve for pure D, a-tocopherol measurement at 534 nm. An equation which included correction factors of 25 for thin-layer chromatography and 3.2 for the colorimetric reaction, was then derived for calculating the concentration of D, a—tocopherol from the absorbances of various samples as follows: C = 10.2 X 3.2 X 25 X A II where C pg D, a-tocopherol and A absorbance of sample 22 23 Table l D, a-Tocopherol Standard Curve Data D, a-tocopherol Absorbance Readings at 534 nm ug/ml on Beckman DU-2 Spectrophotometer 0.7 0.071 1.4 0.139 2.2 0.216 2.9 0.282 3.6 0.349 4.3 0.423 5.0 0.498 5.8 0.571 6.5 0.642 7.2 0.703 24 A standard was analyzed daily with each set of tocopherol extracts from muscle. Results were compared to the standard curve in order to make corrections for day to day variation. Establishment of TLC Recovery Factor Herting and Drury (1967) reported that recovery of D, a—tocopherol from precoated alumina chromatographic sheets was 90.0% on the basis of 39 trials using levels ranging from 3.2 to 32.4 pg. They further stated that when ethanol was used as the eluting solvent, recoveries declined appreciably for amounts exceeding the maximum value cited above. In view of this, several thin-layer chromatography recovery experiments were performed using pure D, a—tocopherol on precoated alumina chromagram sheets (Eastman Kodak Co.). In each experiment duplicate samples of 5.33, 10.66, and 21.31 pg of D, a-tocopherol were Spotted on alumina chromatOgraphic Sheets. The samples were subjected to one-dimensional thin—layer chromatography as described earlier herein. The absorbance data in Table 11 shows the means of duplicate samples. Percentage recovery data for 4 to 5 replicates at each concentration of D, a- tocopherol are reported. The mean percentage recovery for 5 replicates of 5.33 pg D, a—toc0pherol was 85.5%, while that for 4 replicates of 10.66 and 21.31 pg of D, a—tocopherol was 79.3 and 85.4%, respectively. The overall mean percentage recovery for all replicates for all three concentrations was 83.6%. These results compare favorably with those of other workers. Ames (1971) reported a range from 75 to 85% recovery using 10 pg D, a—tocopherol on Eastman alumina chromagram sheets in a collaborative Table 11 Data Showing Recovery of D, a—Tocopherol by TLC 25 l D, a-toc0pherol “8 5.33 Replicate Mean 10.66 Replicate Mean 21.31 Replicate Mean Overall Mean Replicate No #wNH male—I J-‘(valnI ..————- Mean Absorbance at 534 nm 0.137 0.142 0.141 0.136 0.143 0.140 0.247 0.248 0.270 0.271 0.259 0.507 0.599 0.528 0.598 0.558 Percentage Recovery 83.6 86.7 86.4 83.3 87.3 85.5 75.5 76.0 82.7 83.0 79.3 77.7 91.6 80.9 91.5 85.4 83.6 lEastman Alumina Chromatographic Sheet #X6062 l—I-n 26 study among six laboratories. In the present study recoveries were over 80% for 10 of 13 replicates. If the three replicates below 80% were ignored the mean percentage recovery for the 10 remaining was approximately 85%. On the basis of the results achieved, a recovery factor of 85% was employed to correct all subsequent experimental data. D, a-Tocopherol Content of Porcine Muscle The data for 18 samples of porcine muscle analyzed for D, a- tocopherol are given in Table 111. Absorbance values reported are the means of duplicate samples. The D, a—tocopherol content of porcine muscle ranged from 0.91 to 1.78 pg/g of muscle, while the lipid content varied from 6.5 to 10.0%. For the 18 samples of porcine muscle analyzed, the mean weight of lipid extract was 1.6 g, which gave a mean value of 8.0% lipid containing an average concentration of D, a—tocopherol of 1.22 pg/g muscle or 15.09 pg/g lipid. There is a limited amount of data in the literature on the tocopherol content of foods. A paucity of information exists regarding the toc0pherol content of porcine muscle. A recent compilation of the technical literature on the vitamin E content of foods and feeds (Dicks, 1965) lists 455 references, of which only 38 give data on the individual toc0pherols. All the others give either total tocopherol content, or amounts of a— and non-a-tocopherols (Slover et_al,, 1967). Most of the data reported on porcine muscle is for total tocopherol, however, a few investigators have determined D, a-tocopherol in porcine muscle. Harris g£_§l, (1950) in a study of the vitamin E content of foods, Amw\oOH u pouomm mpm>oommv UHH cH mommOH pom monomuuoom < N mm M N.m N N.OH n AmHomda w om\wnv SOHumuucmoaoo Houmnmoooula .QH mo.mH NN.H qo.H Nu.om mmo.o mo.w o.H sum: wo.mH oo.H om.o mm.mH NNo.o o.m ¢.H wH oq.mH mm.H wH.H oo.m~ ¢N0.0 o.m w.H NH no.mH mq.H mN.H om.m~ Hmo.o m.m m.H 0H oo.mH om.H mo.H o¢.o~ mmo.o o.w o.H mH o~.oH om.H OH.H mo.- “No.0 o.m o.H «H N¢.NH mo.H om.o mm.NH NNo.o m.m n.H mH ow.mH 0H.H «m.o mn.wH mwo.c o.w o.H NH n“ wo.qH mo.H om.o mm.mH «No.0 m.n m.H HH om.nH mm.H Hm.H mH.om mmo.o 0.0H o.~ 0H Hm.mH mq.H n~.H om.mm Hmo.o m.w m.H a oo.mH o~.H No.H 0¢.0N mmo.o o.w o.H m no.0H mm.H mm.H mm.om mmo.o m.a m.H u o~.oH om.H 0H.H mo.NN nmo.o o.w o.H o mm.qH om.o mm.o mm.oH ONan m.o m.H m om.NH om.o Nw.o mm.oH o~o.o m.n m.H q mo.¢H Hm.o wn.o om.mH mHo.o m.o m.H m mm.mH OH.H qm.o mn.wH mmo.o 0.x ¢.H N qw.mH Ho.H ow.o «H.nH HNo.o m.m m.H H meHH w\w: oHomsa w\w1 mHowsa w\wn AwHomsa wo~\wnv as «mm um va uomuuxm .oz Houmsmooouls .Q HouanOUOula .Q Houwsmooouls .9 H .sUcoo mommapomnm vaHH N meHH .uz oHQEMm wmuooppoo vmuumuuoo mo Ho>mH Houmnmoooula .Q Austma umBV oHowsz oaHouom m o.o~ mo mehHms< Hononm000Hls .Q HHH oHnma 28 analyzed one sample of pork chOps from a store. These authors reported an a-tocopherol content of 6.3 pg/g in a sample containing 24% fat. They used the difference between total and y- plus G—tocopherol values as a measure of a—tocopherol content, and estimated non-a-tocopherol to be less than 0.8 pg/g. These data are misleading for the D, a-toc0pherol content of porcine muscle, because of the method of measurement and the high lipid content of the sample. Edwin 25 El: (1960) used hot acetone extraction in a Soxhlet followed by saponification, sterol precipitation, floridin chromato- graphy and paper chromatography to determine the a-tocopherol content of porcine muscle. Data reported by these investigators compares favorably with that obtained in the present study. They reported values for a-tocopherol in porcine muscle to be 1.28 pg/g of muscle for three samples analyzed, and 1.23 pg/g of muscle for a fourth sample. Since complete initial lipid extraction is critical, they compared methods of extraction of the lipids using acetone, ethanol or ether in a Soxhlet, and hot acetone or ethanol vapors in an apparatus similar to that of Quaife and Dju (1949). From their results they concluded that acetone was the best solvent for tocopherol extraction. The results Shown in Table 111 indicate that the method utilized in the present investigation is reproducible. However, it is essential that great care be taken throughout the entire analytical procedure to prevent the destruction of D, a-tocopherol by oxidation. Methodology of Toc0pherol Analysis .Mpscle Lipid Extraction Total lipid extraction is critical in obtaining complete extraction 29 of D, a-toc0pherol from muscle. Edwin pp E£.(1960) stated that in their experience lipid extraction is complete in 2 hours using hot acetone in a Soxhlet extraction apparatus. In experiments with animal tissue, they extracted for 3 hours. In the present study, this period was extended to 3 1/2 hours to insure prOper extraction. Edwin E£_a1. (1960) indicated that it was unnecessary to add any antioxidant material, such as pyrogallol, during the initial lipid extraction Since it is actually deleterious. Paper chromatographic analysis showed that non-saponifiable reducing impurities are formed by prolonged heating of such phenols. Without the use of any antioxidants, Edwin g£_ai3 (1960) recovered over 90% of the toc0pherols added to muscle samples subjected to hot acetone extraction prior to chromatographic separation of the unsaponifiable fraction, including the tocopherols. Consequently, no antioxidant was included during initial lipid extraction in the present study. Saponification of Muscle Lipid Extract Bieri (1969) and Edwin 3E a}: (1960), however, indicated the need for an antioxidant during saponification because of the sensitivity of tocopherol to oxidation in the presence of hot alkali. This observation was verified during preliminary investigations in the present study. The addition of insufficient pyrogallol or improper saturation of the atmosphere in the flasks resulted in the destruction of the tocopherol as evidenced by poor recoveries on alumina thin- layer sheets. At the same time recoveries of pure D, a-toc0pherol not subjected to saponification were high. The problem of improper saturation of the flasks with vapors of ethanolic pyrogallol was solved 30 by adding the KOH through the top of the condensers directly into the flasks. This problem was, however, related to incomplete or improper saponification. Bieri (1969) stressed the importance of complete saponification of the total lipid material in order to attain better separation of the individual tocopherols by thin-layer chromatography. The same difficulty was experienced during preliminary work, and it was found that a saponification time of 5 minutes was satisfactory for quantities of lipid normally extracted in this study. When working with the larger quantities of lipid extracts, it was necessary to use 50 rather than 25 ml portions of ether to extract the non— saponifiable material. This was usually done when the phases failed to separate cleanly, even after addition of 1 to 2 ml of ethanol. Thin-Layer Chromatography of Non—saponifiable Fraction Sample sizes of 10, 20, 30 and 40 p1 of non—saponifiable material in benzene, which had been previously evaporated to 1 ml under N were 2, employed in preliminary work on alumina thin—layer chromatography. Along with varying the concentration of the sample applied to the alumina sheet, several different proportions of benzene and diethyl ether were tried in order to develop a suitable solvent system. It was decided on the basis of experience, that 40 p1 aliquots of non-saponifiable material provided a Suitable sample size in view of the apparent concentration in the extracts. Also, after prolonged effort it was found that benzene: diethyl ether (60 : 40 v/v) provided a suitable solvent system for separation of D, a-tocopherol from the other contaminants in the non- saponifiable fraction of porcine muscle lipid. In addition, 2 ml of glacial acetic acid per 100 ml were added to the solvent system to reduce trailing. It was necessary to allow the solvent system to advance 15 cm for prOper Separation. Herting and Drury (1967), using a solvent 31 system of benzene: diethyl ether (50:50 v/v) for one—dimensional alumina thin-layer chromatography in a conventional or sandwich chamber, reported that they were able to successfully separate D, a—toc0pherol from other contaminants of the non—saponifiable fraction in a variety of biological materials, including rat liver and adipose tissue. However, this solvent mixture gave unsatisfactory results in the present study. 0n using alumina Sheets and the same solvent system, it was found that a conventional chamber gave better separation of D, a-tocopherol from its contaminants in the non-saponifiable fraction than either a sandwich chamber or the Brinkmann-Desaga Controlled—Atmosphere DevelOping Tank. AS evidenced from colorimetric data, there was no noticeable destruction of tocopherol due to the use of the conventional chamber as compared to the controlled-atmosphere tank. The RF values of D, a-toc0pherol under the conditions used in this study varied from about 0.5 to 0.7. Satisfactory results were not obtained by simply cutting out the toc0pherol spot from the alumina sheet and eluting. Scraping of the spot from the sheet was required for proper elution. In view of this, it was necessary to centrifuge the samples in order to provide clear solutions for the colorimetric measurement. Preparation of the blank was critical in order to achieve satisfactory colorimetric data, since net extinction for the toc0pherol solution was low. For the blank, it was essential to use approximately the same area of the sprayed but unused alumina sheet, in order to get meaningful colorimetric data for D, a-tocopherol. 32 Colorimetric Determination of D, a—Tocopherol It was necessary to increase the quantity of bathophenanthroline from 1.4 m1, as Suggested by Herting and Drury (1969), to 2.0 ml in order to have an adequate quantity of solution to remove a 1.0 ml aliquot for the colorimetric reaction. Also, it was essential to centrifuge the samples after the colorimetric reaction prior to reading on the Spectrophotometer in order to remove any precipitates that might be formed in the cuvettes during measurement. Precipitates were encountered in preliminary work and produced erratic data. It was thought that the cause of this problem was probably the spray reagent used for visualization. Therefore, spraying of the alumina sheets after chromatography was reduced to a minimum. If Spraying was too light, however, this presented problems of visualization under ultra-violet light due to the low concentrations of D, a—tocopherol in the samples. Consequently, the centrifugation step was included. D, a-Tocopherol in Normal and PSE Porcine Muscle As indicated earlier herein, samples used in the study of D, a- tocopherol levels in normal versus PSE muscle were classified by Weatherspoon (1969) on the basis of a combined evaluation of 45 minutes post—mortem pH and 24 hours transmission values. Analysis was carried out on 4 samples from normal and 5 samples from PSE muscle from Yorkshire pigs. However, D, a-tocopherol was not detectable in any of these samples. Since D, a-tocopherol has been found in porcine muscle in the present study, it is believed that prolonged freezer storage (ca 2 years), and malfunction of the freezer upon two occasions may have caused oxidation of the tocopherol. This Speculation is supported by Zaehringer EE.E13 (1963) 33 who found significant decreases in the levels of toc0pherols during frozen storage of pork. Bunnell (1965) also reported losses of tocopherol in foods in frozen storage at —12° C. He suggested that low storage temperatures do not prevent the oxidation of tocopherols, since formation of hydroperoxides, which is one of the first degradation steps in the oxidation of fatty acids (Frankel, 1962), is not Suppressed by frozen storage. The present study was inconclusive in demonstrating any differences in the D, a—tocopherol content of normal versus pale, soft and exudative porcine muscle. The probable destruction of the tocopherol in samples of both quality groups during freezer storage precluded any comparison upon analysis for D, a-tocopherol. There is a need for such a comparison in order to investigate any possible connection between D, a—toc0pherol content and ultimate muscle quality. In any such study, considerable care will have to be taken to perform complete analyses for D, a-tocopherol as soon after the muscles are frozen in liquid N as possible. This 2 would prevent destruction of tocopherol during freezer storage as experienced in the present study. If tocopherol analysis could not be performed immediately, consideration should be given to storing the frozen samples for short periods of time prior to analysis in bags sealed under N2 as an extra precaution. SUMMARY D, a-tocoPherol was measured in porcine muscle. The method involved extraction of the muscle lipid, followed by saponification, ether extraction of the non-saponifiable fraction, separation of the D, a-tocopherol from the remainder of the non—saponifiable material and colorimetric measurement. A recovery factor of 85% for D, a-tocopherol from thin—layer alumina chromatographic Sheets was determined. The D, a-tocopherol content of porcine muscle for 18 samples analyzed in duplicate ranged from 0.91 to 1.78 pg/g muscle, while the lipid content varied from 6.5 to 10.0%. The average weight of lipid extract was 1.6 g which gave a mean value of 8.0% lipid, containing an average concentration of D, a-tocopherol of 1.22 pg/g muscle or 15.09 pg/g lipid. Results indicate that the method is reproducible, and hence suitable as an indicator of the D, a-tocopherol level in muscle. Analysis of 4 normal and 5 PSE muscle samples showed that D, a-tocopherol was absent. 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