DUGAN. Introduction. I s o l a t i o n of Fhospholipids

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1 98. PHOSPHOLIPIDS I N REAT H E T H O D S O F i S O 1 A T l O N A N D I N F L U E N C E OM F E A T C H A R A C T E R I S T I C S L. R. DUGAN Introduction The growing r e a l i z a t i o n of t h e influence of minor components on system p r o p e r t i e s and t h e advances i n s o p h i s t i c a t i o n and s e n s i t i v i t y of measurement of molecular moieties has drawn a t t e n t i o n t o a r e a s which long have been neglected i n food systems. One a r e a of t h i s nature i s t h a t of t h e phospholipids i n food systems and e s p e c i a l l y i n foods of animal o r i g i n. The presence of phospholipids has been recognized f o r many years and more r e c e n t l y t h e i r r o l e as s t r u c t u r a l and f u n c t i o n a l components of c e l l s and membranes has been a s u b j e c t of d e t a i l e d i n v e s t i gation. Phospholipids have been used a s e m u l s i f i e r s, a s c r y s t a l modifiers, and a s a n t i o x i d a n t s i n various food a p p l i c a t i o n s. Phospholipids i n muscles have a number of p h y s i o l o g i c a l functions i n t h e l i v e animal but t h e i r importance i n t h e q u a l i t y of f r e s h and processed meats i s s t i l l r e l a t i v e l y ob s cure. I s o l a t i o n of Fhospholipids E x t r a c t i o n A major f a c t o r i n t h e i s o l a t i o n of phospholipids r e s i d e s i n t h e e x t r a c t i o n procedures. The phospholipids a r e bound t o p r o t e i n s i n a manner which d e f i e s e x t r a c t i o n by conventionally used f a t s o l v e n t s which e x t r a c t e s s e n t i a l l y o n l y t h e n e u t r a l l i q u i d s. Most e x t r a c t i o n methods f o r t o t a l l i p i d s follow t h e p r a c t i c e of Folch, e t a l. ( 6 ) or some v a r i a t i o n of t h i s procedure. This involves hoxogenization of t h e t i s s u e w i t h 2:lCHClz: MeOH and washing t h e e x t r a c t by a d d i t i o n t o it of 0.2 i t s volume of water o r appropriate s a l t s o l u t i o n. The r e s u l t i n g mixture s e p a r a t e s i n t o two phases upon standing, The lower phase i s t h e t o t a l l i p i d e x t r a c t. I n t h e washing procedure, t h e system of t i s s u e e x t r a c t p l u s water contains CHC13, MeOH, and water i n t h e proportions 8:4:3 (V:V) a s c r i t i c a l proport i o n s which must be kept constant. Variations on t h i s procedure have been employed f o r f i s h Bligh and Dyer (2) and muscle t i s s u e s Ostrander and Dugan (13). The a l c o h o l e f f e c t i v e l y r e l e a s e s t h e p r o t e i n and t h e chloroform t h e n provides t h e solvent f o r e f f e c t i v e removal of t h e l i p i d from t h e system. Separation Most methods of s e p a r a t i o n of phospholipids, e i t h e r a s a c l a s s or a s i n d i v i d u a l phospholipid components, depend on some means of s e l e c t i v e adsorption. Choudhury and Arnold ( 4 ) were able t o determine t h e n e u t r a l o i l content of vegetable o i l s a s follows: shake f o r 1 0 min. i n a 125 m l e r l e n meyer f l a s k 23 g crude o i l, 25 g s i l i c i c a c i d and 50 m l CHC13. A f t e r f i l t e r i n g through a s i n t e r e d g l a s s funnel under vacuum, t h e s i l i c i c a c i d i s washed with f i v e 50ml lots CHC13. The combined f i l t r a t e and washings a r e f r e e d from CHCIS on a water b a t h and t h e residue heated t o constant weight a t 105OC i n a vacuum oven or under PITz. The phospholipids a r e determined PY washing t h e s i l i c i c a c i d w i t h MeOH, evaporating t h e MeOH and weighing t h e residue. They found phospholipid content t o be 6.25$ i n soybean o i l, 5.83% i n corn o i l and 3.25% i n cottonseed o i l.

2 99. A number of investigators have applied this procedure to determination of phospholipids or to concentrating them prior to separation into individual components. Kuchmak and Dugan (11) reported that phospholipids from pork muscle were well separated in this manner but that some residual neutral fats remained with the phospholipids. Hornstein, et al. (9) made the same observation. They modified the polarity of the solvent and with a mixture of chloroformhexanediethyl ether (2:l:l) were able to effect a quantitative separation of phospholipids. Separation into individual phospholipids has been accomplished by silicic acid column chromatography and by thinlayer chromatography. Column chromatography with silicic acid seldom gives cleancut separation of components. The cephalins, phosphatidyl ethanolamine and phosphatidyl serine, frequently elute together and lecithin overlaps with lysolecithin and occasionally with sphingomyelin. Kuchmak and Dugan (12) used silicic acid and separated the cephalins on a silicic acidsilicate column described by Rouser, et al. (15). Rouser, et al. (16) established a lipid separation scheme based on DEAE Cellulose which permitted elution of lecithin, sphingomyelin, ceramide, cerebroside, lysolecithin, sterol esters and glycerides as a group. Phosphatidyl ethanolamine eluted as a separate fraction and phosphatidyl serine eluted with the gangliosides as shown in Table 1. Table 1. Elution of Lipids from DEAE Cellulose (Acetate Form) Solvent 1. Lecithin, sphingomyelin, ceramide, cerebroside, cholesterol, lysolecithin, sterol esters, and glycerides 2. Phosphatidyl ethanolamine 3, Water soluble nonlipids ( sugars, amino acids, purines, salt) 4. Uncharacterized components and free fatty acids 5. Phosphatidyl serine and ganglioside 6. Cerebroside sulfate, inositol phosphatide and cardiolipin Substance CHC13/MeOH 7/1 CHCl,/MeOH 7/3 MeOH CHC13/glacial CH3COOH 3/1 containing M CH3COOK Glacial CH3COOH CHCL/MeOH 4/1 containing 10 &/liter concentrated aqueous NH40H *From Rouser et al. (16) 7

3 100. They then separated the components of the first fraction from the DEAE Cellulose column on a Silicic AcidSilicateWater column as shown in Table 2, Table 2. Elution from a Silicic AcidSilicateWater Column* Solvent Sub stance CHC13 Cholesterol CHC13/MeOH, 19/1 Ceramide CHC13/MeOH, 4/ % H20 Cerebrosides CHC13/MeOH, 4/ % H20 Lecithin CHCl3/Me0H, 4/ % H20 Sphingomyelin MeOH + 2% H20 *From Rouser, et al. (16) Lysolecithin Hornstein et al. (10) separated beef and pork lipids into four fractions by silicic acid chromatography using successive 300ml portions of CHC13/MeOH 20/1, CHC13/MeOH 1/1, and MeOH. They reported that the first two fractions consisted mainly of neutral lipids, the third frac tion contained the cephalins, and the fourth contained lecithin and sphingomyelin. The range of components in beef and pork lipids are given in Table 3. Table 3, Lipid Lipids of Beef and Pork Phscle (%)* Beef Nonphosphorus 24 Phospholipid Cephalin 4045 Lec i thin 4045 Sphingomyelin 1015 Protein 510 *From Hornstein et, al. (10) Pork

4 101. Hidaka et al. (8) used the procedure of Hornstein et al. to separate the lipids of several muscles of Japanese Black Cattle. of their results are shown in Table 4. (10) Some Table 4. Percentage of Lipid Fractions in Total Lipids from Muscles of Japanese Black Cattle* Total Fraction 1 Fraction 2 Fraction 3 Denatured Muscle (iglyc ( Cephalins ) (Lecithins Proteins Lipid erides ) and Sphingomyelins ) Fore sha'nk Hind shank Tender Loin 100 8% Loin *From Hidaka, et al. (8) Kuchmak and Dugan (11) used the scheme shown in Table 5 for separating the lipids of pork muscle into 6 fractions Table 5. Fraction I I1 I11 IV V VI Scheme for Chromatographic Separation on Silicic Acid* Eluant Volume Components CHC13 2 Nonphospholipids CH3COC% 113 Principally nonphospholipids 10% MeOH in CHC13 1 Cerebrosides (?) 15% MeOH in CHC13 X m l 20% MeOH in CHC13 1 Cephalins 25% MeOH in CHCl3 Xml 35% MeOH in CHC13 4 Lecithins Me OH 2 Sphingomyelins *From Kuchmak and Dugan (11)

5 102. The phospholipids found in various muscles of the hog carcass are shown in Table 6. Table 6. Percentage of Each Phospholipid Type in the Phospholipids of Hog Muscle Tissue* phospholipid Belly Carcass Location Ha Loin ~. Rib Phosphatidyl ethanolamine Phosphatidyl serine Lecithin Sphingomyelin *From Kuchmak and Dugan (11) Lecithin and phosphatidyl ethanolamine are the dominant phospholipids and all phospholipids vary from muscle to muscle in the carcass. A technique applied recently in our laboratory uses a multibore column packed with silicic acid, This has given clean discrete separation of individual phospholipids. The apparent overlap of lecithin, obtained by us and others, with a long tailing of minute quantities of lysolecithin may be explained by the observation of Camejo (3) that lecithin in contact with methanol on certain silicic acid preparations may undergo limited hydrolysis and yield lysolecithin plus some free fatty acid and methyl esters. Phospholipid Effect on Meat Characteristics Composition effects The amount of phospholipid in total lipid of meats is small and the effect on meat characteristics must be derived from the strong contribution of unique characteristics of the phospholipid moieties.. The factors which are unique are the presence of esterified phosphoric acid, a nitrogenous moiety in most phospholipids, the relatively high unsaturation of the fatty acids, and the close association with proteins. The association with protein determines in large measure the difficulty encountered in extracting phospholipids. This binding prevents direct extraction with chloroform and even extends to freezedried meats. Giam and Dugan (7) found a marked difference in the fatty acid composition. of 'free' lipids, those which may be extracted by ordinary fat solvents, and 'bound' lipids, those which require treatment with methanol prior to or concurrent with the use of chloroform as extracting solvent. Representative data for pork, beef, and lamb are shown in Table 7.

6 103. Table 7. F a t t y Acid Composition of "Free" and " bund" Lipids of Freezedried Raw Pork, Lamb, and Beef of t o t a l f a t t y a c i d s ). * (4 Raw Pork Fatty acid Raw Lamb Raw Beef Free Bound Free Bound Free :l :l O :l : : : X 2.o :l Y O Z :1 *From Giam and Dugan ( 7 ) Bound

7 104. The d i f f e r e n c e s noted a r e p a r t i c u l a r l y marked f o r l i n o l e i c and arachidonic a c i d s, both of which a r e found i n e l e v a t e d concentrations i n phospholipids. Kuchmak and Dugan ( 1 2 ) examined t h e f a t t y a c i d composition and t h e d i s t r i b u t i o n of t h e f a t t y a c i d s i n t h e phospholipids from various pork muscles. Marked d i f f e r e n c e s i n composition were noted f o r t h e various carcass l o c a t i o n s. The f a t t y a c i d s of t h e phosphatidylethanolamine f r a c t i o n a r e shown i n Table 8. Table 8. F a t t y Acid Composition of Phosphatidylethanolamine from Porcine Muscles (Mole $)* Fatty Acid 1o:o 12:o 13:O a 14:O b 15:O 16:O 16:l 17:O 18:O 18:l 18:2 18:3 2O:l 21:o 20:4 22:o 22 :1 Yuscle Source Belly Ham Loin Ribs O e From Kuchmak and Dugan ( 1 2 ) E s p e c i a l l y marked variakions were noted i n t h e amounts of s t e a r i c, l i n o l e i c, and arachidonic a c i d s. The arachidonic a c i d content of t h e b e l l y muscle was more t h a n t h r e e times g r e a t e r t h a n t h a t of t h e ham muscle. Allen, e t a l. (1)determined t h e comparative l i p i d composition of t h r e e porcine muscles: t h e diaphragm, L. d o r s i, and Psoas major. I n e v a l u a t i n g t h e d i f f e r e n c e s between composition of n e u t r a l l i p i d s and of t o t a l l i p i d e x t r a c t s, t h e y r e l a t e d d i f f e r e n c e s i n s t e a r i c and o l e i c a c i d s t o an i n f e r r e d cephalin o r l e c i t h i n content. They noted f u r t h e r an apparent g r e a t e r u n s a t u r a t i o n i n nonneutral l i p i d s from psoas major and I

8 105. suggested that lipids fromthis muscle might become rancid more quickly than those of the other two muscles. Peng and Dugan (14)showed that the dark meat of chicken contained proportionally more linoleic acid and arachidonic acid in given phospholipids than were found in the same phospholipids of white meat. Oxidative and other quality effects Some of the quality factors related to phospholipids are only inferred while others may be directly attributed to the phospholipids. Hornstein,.et al. (10) observed strong fishy odors from heating of cephalins from pork or beef and a diminished fishy odor associated with an aroma suggestive of liver when the lecithins of pork or beef were heated. Both total lipid and phospholipid fractions from pork or beef became rancid quickly when exposed to air. They concluded that phospholipids possibly contributed to poor flavors in excessively lean meat. Marked darkening of the phospholipids accompanied the development of rancidity. Younathan and Watts (17) compared the development of rancidity in extracted fat with that in whole tissue from pork. They noted that TM values of the tissues were many factors greater than in extracted fat. When the lipids were examined, it was found that the TBA of neutral lipids was much less than that of a total lipid extract or of the fraction which they referred to as phospho or proteolipids as in Table 9. Table 9. Rancidity in Pork Lipid Fractions* Wt Lipid/g Tissue TBA Values/g Tissue Lipid Fraction Rancid Antioxidant Rancid Antioxidant Sample eated Sample Sample eated Sample g g Total Lipids Neutral fat, Phospho or Proteolipids, *From Younathan and Watts (17) Similar observations were made by Zipser, et al. investigation of lipids from oxidizing mullet. (18) an El Gharbawi and Dugan (5) studied the stability of nitrogenous compounds and lipids during storage of freezedried beef in cans with varying oxygennitrogen ratios. The fatty acid composition of the neutral lipid and the phospholipid fractions changed with oxidation during storage

9 106. according t o t h e oxygen content of t h e can atmosphere. The o x i d a t i o n of t h e t i s s u e l i p i d s appeared t o occur i n two s t a g e s w i t h t h e phospholipids oxidizing f i r s t. Loss of t h e unsaturated f a t t y a c i d s i n t h e phospholipids was more pronounced t h a n i n t h e n e u t r a l f a t. Associated w i t h t h e l i p i d changes were decreased s o l u b l e p r o t e i n and nonprotein nitrogen, decrease of f r e e amino groups and l o s s of rehydration capacity. The r e l a t i o n of composition of phospholipids t o changes i n t h e meat from which t h e s e a r e derived may be d i r e c t e d t o t h e observation of Kuchmak and Dugan ( 1 2 ) t h a t t h e l i n o l e i c a c i d content of l e c i t h i n from pork b e l l y muscle was markedly g r e a t e r t h a n t h e l i n o l e i c content of l e c i t h i n i n t h e o t h e r muscles studied. The l i n o l e i c content of phosphat i d y l ethanolamine from b e l l y muscle i s a l s o g r e a t e r t h a n t h a t i n t h e o t h e r muscles s t u d i e d b u t t h e most dramatic d i f f e r e n c e l i e s i n t h e arachidonic a c i d content of t h i s phospholipid. With t h e higher l e v e l of polyunsaturated f a t t y a c i d s i n t h e phospholipids of b e l l y muscle, it seems reasonable t o s p e c u l a t e t h a t t h e tendency t o s t a l i n g and r a n c i d i t y i n s l i c e d bacon may c o r r e l a t e q u i t e w e l l w i t h t h e s e compositional f a c t o r s. The r o l e of o t h e r f a c t o r s a r e not w e l l e s t a b l i s h e d a s y e t. Among t h e s e a r e t h e f u n c t i o n of t h e nitrogenous moiety as well a s t h a t of t h e phosphoric a c i d p o r t i o n of t h e molecule a s r e g u l a t o r s of o x i d a t i v e processes. The presence of a s u b s t a n t i a l q u a n t i t y of plasmalogen a s s o c i a t e d w i t h t h e comparable d i a c y l phosphoglyceride r a i s e s a number of i n t e r e s t i n g i s s u e s. Our c u r r e n t program i s concerned both with plasmalogens and w i t h o x i d a t i v e mechanisms of t h e phosphoglycerides. References 1. Allen, E., 36 (1967) R. G. Cassens, and R. W. Bray. 26, J. Animal Science, Camejo, German. J. Chromatog, 21, 6 (1966) 4. 30, 5. ElGharbawi, Mohamed I., and L. R. Dugan, Jr. J. Food Sci., 6. Folch, J., M. Lees, and G. H. SloaneStanley. 497 (1957) J. Biol. Chem., ( 1965 ) Giam, Irene, and L, R. Dugan, Jr. J. Food Sci., 2, , (1965) Hidaka, Toshiro, Mioko Endo, and Masaaki Kojima. Memoirs of t h e F a c u l t y of Agr., University of Miyazaki, 4, 2 1 (1965) 9. Hornstein, I., P. F. Crme, and J. R. Ruck. Anal. Chem., 39, 352 (1967)

10 Hornstein, I., P. F. Crove, and M. J. Heimberg. 581 (1961) 11. Kuchmak, M e and L. R. Dugan, Jr. 734 (1963) J. Am. O i l Chemists' SOC., 12e Kuchmak, M. and L. R, Dugan, Jr. 45 (1965) 13. Ostrander, Joyce, and L. R. Dugan, Jr. 39, 178 (1962) 40, J. Am. O i l Chemists' Soc., 42, 14. Peng, C. Y. and L. R e Dugan, J r. 533 (1965) J. Food Sci., 26, J. Am. O i l Chemists' SOC., J. Am. O i l Chemists' SOC., 42, 15 Rouser, G., J. O'Brien, and D. Heller. 38, 14 (1961) 16. Rouser, G., A. J. Barman, G. Kritchevsky, D. Heller, and J. S, O'Brien. J. Am. O i l Chemists' SOC., 38, 544 (1961) 17 Younathan, Margaret T., (1960) 18. Zipser, Marelynn, Jacqueline Dupont, and Betty M. Watts. Sci., 27, 135 (1962) J. Am. O i l Chemists' SOC., and Betty M. Watts. J. Food Sci., 25, 538 J. Food DR. CRAIG: Thank you D r. Dugan f o r a most i n t e r e s t i n g discussion of t h e phospholipids i n meat. We will hold t h e discussion of t h i s paper and t h e other two papers u n t i l t h e end of t h e program. The t i t l e of t h e i s "The I s o l a t i o n and second paper, t o be presented by D r. VanderbJal, Characterization of Pork S k e l e t a l Muscle Sarcolemma." I ' m happy t o present t o you D r. VanderWal t o present t h i s t o p i c t o you.

Classification, functions and structure

Classification, functions and structure Elena Rivneac PhD, Associate Professor Department of Biochemistry and Clinical Biochemistry State University of Medicine and Pharmacy "Nicolae Testemitanu" Lipids