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Positional distribution of fatty acids in fish and other animal lecithins
BIOCHIMICA ET BIOPHYSICAACTA
133
BBA 55Ol 7
POSITIONAL D I S T R I B U T I O N OF FATTY ACIDS IN F I S H AND O T H E R ANIMAL L E C I T H I N S DANIEL B. MENZEL AND H. S. OLCOTT
Institute of Marine Resources, Department of Nutritional Sciences, University of California, Berkeley, Calif. (U.S.A .) (Received October 29th, 1963) SUMMARY Phosphatidyl choline (lecithin) fractions were prepared from tuna, salmon, and menhaden muscle, from egg yolk, and from rat and beef liver. Hydrolysis by snakevenom phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) was used to determine the positional distribution of the fatty acids. The fatty acid composition of the hydrolyzcd fatty acids (fl position) and those remaining with the phosphatidyl moiety (a' position) were separately determined by gas-liquid chromatography of the methyl esters. 36-86 mole % of the fatty acids esterified in the a' position were saturated; 91-99 mole % of the fatty acids in the fl position were unsaturated. Pahnitic and stearic acids were the predominant saturated acids in the a' position. Some similarity was noted in the /5-position distribution of linoleic, arachidonic, eicosapentaenoic, docosaenoic, docosapentaenoic, and docosahexaenoic acids. This observation tends to support the hypothesis of the interchangeability of linoleic and arachidonic acids with "non-essential" polyenoic acids. INTRODUCTION This work was initiated as an extension of studies on the nature of the phospholipids of tuna 1 and menhaden s. It is known that phospholipase A (phosphatide acylhydrolase, EC 3.i.i.4) of snake venom specifically hydrolyzes the/5 position of both synthetic and naturally occurring phospholipids 3-8, and that these fatty acids are primarily unsaturated 4. The fish lecithins are characterized by large amounts of highly unsaturated fatty acidsl, *, hence it appeared of interest to determine the positional distribution of these fatty acids in them. During the course of this investigation BROCKERHOFFet al.9 described the specific distribution of fatty acids in the phospholipids and triglycerides of several marine organisms: cod (Gadus callarias), scallop (Platopectin magellariccus) and lobster (Homarus americanus). The present paper contains our results obtained by similar means on lecithins isolated from albacore tuna (Thunnus alalunga), menhaden (Brevoortia tyrannus), and salmon (Oncorhynchus tshawytscha) muscle, and from beef liver, rat (Long-Evans) liver, and chicken egg yolk.
Biochim. Biophys. Acta, 84 (x964) i33-I39
134
u.B. MENZEL, H. S. OLCOTT EXPERIMENTALPROCEDURE
A. Purification of lecithins Tissues were homogenized in chloroform-methanol (I :I, v/v) solution. Lipids were extracted, and fractionated by column chromatography on silicic acid as described previously a. The lecithin fractions showed only one component with the thin-layer silicic acid chromatography system of VOGEL et al. 1°. The product used in each case contained the bulk of the lecithin fraction, to avoid fractionation with regard to the fatty acid content. Ester ~1, phosphorus ~2 and plasmogen contents aa were determined for each lecithin. These, together with the specific optical rotation in chloroform solution, and the ester/phosphorus ratios are set forth in Table I. During the preparation of the fractions trace amounts of hydroquinone were added as an antioxidant, and all samples were stored under N 2.
B. Liberation of fatty acids by phospholipase A (ref. 4) 30 mg of each lecithin were dissolved in peroxide-free diethyl ether to give a final concentration of 20 /~moles/ml. Lyophilized venom of the eastern diamondback rattlesnake (Crotalus adamanteus) (Ross Allen's Reptile Institute, Silver Spring, Florida) was dissolved in a solution of o.I M Tris buffer, 20 mM CaC12 and 2 mM EDTA (tetrasodium salt) to give a final concentration of 2.0 #g of venom per #l. The buffered enzyme was adjusted to pH 7-4. To each ml of the etheral solution of lecithin, IO ul of the ~.,nzyme solution was added. The mixture was shaken vigorously for 30 sec and allo~ved to stand at room temperature for 2 h. IO-#1 aliquots were taken at fixed time intervals to check the progress of the reaction by chromatographing the aliquot directly in the thin-layer system mentioned above. The reaction was complete by this test at the end of 2 h. The ether solution was evaporated to dryness under N 2 and the residue dissolved in a minimum of solvent (0.5% methanol in chloroform). The resulting solution was transferred to a 12 mm x 155 mm column containing 5 g of silicic acid equilibrated in the same solvent. The free fatty acids liberated during the reaction were eluted from the silicic acid column by an additional 40 ml of solvent. Elution of unreaeted lecithin (trace amounts) was accomplished by the addition of 40 ml of 25% methanol in chloroform to the column. The remaining reaction product, lysolecithin, was eluted from the column with 9 ° m l of 85% methanol in chloroiorm. Each of the fractions isolated showed only a single spot corresponding to an authentic sample when subjected to chromatography on the thin-layer silicic acid system. Approx. 90% of the amount used was recovered.
C. Preparation and analysis of methyl esters of fatty acids Methyl esters of the fatty acids liberated during the reaction were prepared from the first silicic acid chromatography fraction by reaction with 3% (v/v) conc. H~SQ in anhydrous methanol for 2 h at boiling, followed by extraction with light petroleum and washing with water. The fatty acids remaining esterified to the lysolecithin fraction were methanolized in 5 ml of chloroform-methanol (I : I, v/v) to which was added I.O ml of 0.5 N sodium methoxide 14. The reactants were cooled to o ° before reaction, mixed rapidly, allowed
Biochim. Biophys. Acta, 84 (I964) 133-139
POSITION OF FATTY ACIDS IN LECITHINS
135
to stand in the cold for 20 min, then allowed to warm to room temperature, neutralized with 2.5 ml of 3 % HC1 in anhydrous methanol and evaporated to dryness. The residue was suspended in water and extracted with light petroleum. Methyl esters from the various fractions were analyzed by gas-liquid chromatography. An Aerograph Hy-Fi (Wilkens Instrument Co., Walnut Creek, Calif.) flameionization instrument was used. It was equipped with a 366-cm, 3-mm O.D. glass column which had been treated with dichlorodimethylsilane and packed with 1% EGSS-X on ioo-I2o-mesh hexamethyldisilizane-treated Gas-Chrom P (Applied Science Laboratories, State College, Pa.). The separation was conducted at 19°o with N2 (40 ml/min) as the carrier gas. The instrument was calibrated with authentic standards and National Institute of Health standard mixtures of fatty acid methyl esters. RESULTS AND DISCUSSION
The data of Table I illustrate the nature of the preparations. Although all lecithin preparations showed only one spot by thin-layer chromatography and corresponded to the expected infrared absorption spectra, the ester/phosphorus ratios are lower for the tmla and menhaden lecithin samples than can be accounted for by their content of plasmalogen. They probably therefore contained small amounts of non-lecithin lipid material. Analyses for the other lecithin preparations gave the expected results within the limits of precision of the methods used. By using the total fraction from the silica-gel column chromatography, it was hoped to avoid the initial fractionation of lecithins noted by RHODES AND LEA15. TABLEI CHEMICAL AND
Source
PHYSICAL PROPERTIES
Specific rotation ([a] iS)
P content (%)
Ester content (l*equiv. per xoo rag)
3.60 3.94 3.7 ° 3.76 3.59 4.00
2.06 2.22 2.28 2.4 o 2.13 2.61
D
Salmon muscle Tuna muscle Menhaden muscle E g g yolk R a t liver B e e f liver
+4.4 +3.6 +5.0 +6.6 +6.1 +6.0
OF ISOLATED LECITHINS
Ester/P ratio
1.78 1.75 1.92 2.o 4 1.84 2.02
Mole % pIa.~malogen
5-5 8. 7 5.8 1. 5 3.8 3.8
The specific distribution of fatty acids in the lecithins examined is presented in Table II and summarized in Table III. It is apparent from Table III that, regardless of the origin of the lecithin, the fl position is predominantly unsaturated and that the distribution of saturated and unsaturated fatty acids in the fl position is very nearly the same in all samples while the mole per cent of saturated fatty acids in the a' position varies considerably. Egg-yolk, menhaden-muscle and rat-liver leeithins were similar in the degree of saturation of the a' position (80%) while beef liver was of an intermediary value (66%) and salmon and tuna muscle of the lowest degree of saturation (35%). Palmitic acid was the predominant saturated fatty acid in the a' position. Indeed, palmitic and stearic acids represented the highest positional enrichB i o c h i m . B i o p h y s . A c t a , 84 (1964) i 3 3 - 1 3 9
136
D . B . MENZEL, H. S. OLCOTT
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2 Biochim. Biophys. Acta,
84 ( I 9 6 4 ) 1 3 3 - 1 3 9
POSITION OF FATTY ACIDS IN L E C I T H I N S
137
TABLE III RELATIVE DISTRIBUTION OF SATURATED AND UNSATURATED FATTY ACIDS IN THE (2t AND fl POSITIONS OF LECITHIN
Source
Salmon muscle Menhaden muscle
Mole % saturated fatty acids
Mole % unsaturated forty acids
/5
38 9
62 91
Position
a" a"
81
19
/5
7
93
Tuna muscle
a' /5
36 4
64 96
Egg yolk
a' t5
86 6
14 94
R a t liver
a" /5 a' fl
82 3 66 I
18 97 34 99
B e e f liver
ment of any of the fatty acids found. In the fish lecithins, docosahexaenoic acid was the other major fatty acid; however, in the egg-yolk, rat- and beef-liver materials, lower unsaturated fatty acids completed the balance of fatty acids. Only small amounts of arachidonic acid were found in the fish lecithins; oleic, eicosapentaenoic and docosahexaenoic acids were the prevalent fatty acids in fl position. Beef-liver lecithin contained some of the fatty acids normally enconntered in marine organisms. Docosamonoenoic acid was tentatively identified only in the beef sample and was primarily esterified to the fl position. Trace amounts of odd chain length and branch-chained fatty acids were also detected in the beef sample but were not positively identified. These fatty acids are possibly the products of rumen bacteria. The fatty acid composition of the lecithin of egg yolk shows good correlation with that reported by other workers; e.g., in the u' position, palmitic acid 61°/o, stearic acid 25 %, oleic acid io%, compared to palmitic acid 65 % ; stearic acid 33 %, oleic acid 1% (ref. 5), and palmitic acid 55%, stearic acid 36%, and oleic acid 6% (ref. 16); the correlation is also good for the fl position. In the more complex lecithins containing a greater proportion of higher polyenoic acids, two difficulties are of importance : first, the quantitative problem of gas-liquid chromatography, and second, contamination with plasmalogen (compare Table I). The difficulty with quantitative gas chromatography is illustrated by the data in Table II. Particularly in the fatty acid analyses of the fish-lipid fractions, there are discrepancies in the amounts of total compared to the amounts in the a' and fl position. Since careful repetition failed to resolve this difficulty, we are inclined to conclude that it represents an inherent weakness of the methods or equipment. Previous investigators more often than not do not include data for total fatty acids so that direct comparisons are not possible. However DE TOMASet al. x7 have recently published an analysis of phosphatidylethanolamine fatty acids from rats fed diets deficient in essential fatty acids in which similar discrepancies appear. B i o c h i m . B i o p h y s . A c t a , 84 (i964) 1 3 3 - i 3 9
138
D . B . MENZEL, H. S. OLCOTT
RAPPORT AND FRANZLls report that plasmalogens are attacked by phospholipase A and hence the liberated f a t t y acids will also contain the fatty acids esterified to the contaminating plasmalogen. No scheme is currently available to differentiate between the lecithin /3 position and that of plasmalogen. In the plasmalogen-containing lecithins this error would be expected to decrease the apparent recovery of f a t t y acids not associated with the plasmalogen and to increase the apparent recovery of those associated with the plasmalogen. Should the pattern of f a t t y acids of plasmalogen follow that of lecithin, triglycerides and other phospholipids having a highly unsaturated/3 composition ls-22, then one would expect to recover a larger amount of unsaturated and a smaller amount of saturated f a t t y acids than the theoretically calculated amount. With the above reservation in mind, it is of interest to relate the current d a t a with that of others and with the schemes of biosynthesis proposed for phosphatidyl choline. First, regarding the distribution of the polyenoic acids of the "linolenic acid family" vs. those of the "linoleic acid family", the latter being considered "essential f a t t y acids", it is apparent that the primary representative of the linolenic-group, docosahexaenoic acid, is primarily esterified to the/3 position of both the marine and terrestrial lecithins in which it occurs and hence follows the classical distribution of arachidonic and linoleic acid. Linolenic, eicosapentaenoic, docosaenoic, docosatetraenoic and docosapentaenoic acids are also similarly distributed. This distribution is then in agreement with the suggestion of KLENK AND BROCKERHOFF 2a, RICHARDSON et al. ~, and KLENK AND EBERHAGEN25 that the so-called essential f a t t y acids are not absolute requirements in the structure and function of cellular membranes, but that other unsaturated f a t t y acids m a y substitute. The enzymatic studies of LANDS AND MERKL26 would also support such a thesis. Secondly, as was mentioned previously, the most marked positional enrichment of a given fatty acid observed was that of palmitic acid for the a' position. Dietary effects will influence the relative proportion of saturated and unsaturated f a t t y acids esterified to the a' position. RHODES 27, for example, found an increase in the unsaturation of egg-yolk lecithin, when the laying hens were fed cod-liver oil. Yet even in those lecithins having the highest a'-position unsaturation, the emichment of palmitic acid for the a' position was of the same order as that of arachidonic and docosahexaenoic acid for the/3 position (consider salmon and tuna: palmitic acid: ratio of a't/3 = 4-7; arachidonic acid: ratio ilia' =- 0.5-4; docosahexaenoic acid: ratio fl/g' = about 1.5). BROCKERHOr~ et al. 9 also found more palmitic acid esterified to the a' position in cod-muscle and cod-liver, scallop-muscle and lobster-liver lecithin than that found in the g' position of the triglycerides isolated from the same tissue. ACKNOWLEDGEMENTS
We wish to thank J. R. FROINES AND R. D. JENSEN for their technical assistance, and A. GOODBAN of the Western Regional Research Laboratory, U.S.D.A., Albany, Calif., for determining the optical rotation of the samples. This investigation was supported in part b y funds administered by means of a collaborative agreement between the U.S. Fish and Wildlife Service Bureau of Cornmerci al Fisheries and the University of California.
Biochim. Biophys. Acta, 84 (1964) 133-139
POSITION OF FATTY ACIDS IN LECITHINS
139
REFERENCES 1 C. Y. SI-IUSTER, J. FROINES AND H. S. OLCOTT, J. Am. Oil Chemists Soc., 41 (1664) 36. 2 j . FROINES, C. Y. SHUSTER AND H. S. OLCOTT, in preparation. 8 N. H. TATTRIE, J. Lipid Res., i (1959) 60. 4 D. J. HANAHAN, H. BROCKERHOFF AND E. J. BARRON, J. Biol. Chem., 235 (196o) 1917s G. H. DE HAAS, I. MULDER AND L. L. IV[. VAN DEENEN, Biochem. Biophys. Res. Commun., 3 (196o) ~287. 6 G. H. DE HAAS AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 48 (1961) 215. 7 G. V. MARINETTI, J. ERBLAND, K. TEMPLE AND E. STATY, Biochim. Biophys. Acta, 38 (196o) 524 • s G. H. DE HAAS, F. J. M. DAEMEN AND L. L. M. VAN DEENEN, Nature, 196 (1962) 68. 9 H. BROCKERHOFP, R. G. ACKMAN AND R. J. HOYLE, Arch. Biochem. Biophys., ioo (1963) 9. xo W. C. VOGEL, W. M. DOIJAKI AND L. ZIEVE, J . Lipid Res., 3 (1961) 138. n F. SNYDER AND N. STEPHENS, Biochim. Biophys. Acta, 34 (1959) 244. 11 E. J. KING, Biochem. J., 26 (1932) 292. 13 M. M. RAPPORT AND R. E. FRANZL, Biochim. Biophys. Acta, 33 (1957) 319. 14 G. V. MARINETTI, Biochemistry, i (1962) 35 o. t5 D. N. RHODES AND C. H. LEA, in G. I~OPJAK AND E. LE BRETON, Biochemical Problems of Lipids, Interscience, London, 1956, p. 73. te j . C. HAWKE, Chem. Ind. London, (I962) 1761. 17 M. E. DE TOMAS, R. R. BRENNER AND L. O. PELUPFO, Biochim. Biophys. Acta, 7 ° (1963) 472. is M. M. RAPPORT AND R. E. FRANZL, J. Biol. Chem., 225 (I957) 851. 19 M. M. RAPPORT AND R. E. FRANZL, Biochim. Biophys. Acta, 33 (1957) 319. 10 H. DEBUCH, Z. Physiol. Chem., 304 (1956) lO9. ax E. KLENK AND G. KRICKAU, Z. Physiol. Chem., 308 (1957) 98. z* G. M. GRAY, Biochem. J., 7 ° (1958) 425 • 23 E. KLENK AND H. BROCKERHOFF, Z. Physiol. Chem., 31o (1958) 153. 24 T. RICHARDSON, A. L. TAPPEL, L. M. SMITH AND C. R. HOULE, J. Lipid Res., 3 (1962) 344. 2~ E. KLENK AND D. EBERHAGEN, Z. Physiol. Chem., 328 (1962) 189. 26 N. E. IV[. LANDS AND I. MERKL, J. Biol. Chem., 238 (1963) 898. t7 D. N. RHODES, Biochem. J., 68 (1958) 380.
Biochim. Biophys. Aeta, 84 (1964) 133-139
133
BBA 55Ol 7
POSITIONAL D I S T R I B U T I O N OF FATTY ACIDS IN F I S H AND O T H E R ANIMAL L E C I T H I N S DANIEL B. MENZEL AND H. S. OLCOTT
Institute of Marine Resources, Department of Nutritional Sciences, University of California, Berkeley, Calif. (U.S.A .) (Received October 29th, 1963) SUMMARY Phosphatidyl choline (lecithin) fractions were prepared from tuna, salmon, and menhaden muscle, from egg yolk, and from rat and beef liver. Hydrolysis by snakevenom phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) was used to determine the positional distribution of the fatty acids. The fatty acid composition of the hydrolyzcd fatty acids (fl position) and those remaining with the phosphatidyl moiety (a' position) were separately determined by gas-liquid chromatography of the methyl esters. 36-86 mole % of the fatty acids esterified in the a' position were saturated; 91-99 mole % of the fatty acids in the fl position were unsaturated. Pahnitic and stearic acids were the predominant saturated acids in the a' position. Some similarity was noted in the /5-position distribution of linoleic, arachidonic, eicosapentaenoic, docosaenoic, docosapentaenoic, and docosahexaenoic acids. This observation tends to support the hypothesis of the interchangeability of linoleic and arachidonic acids with "non-essential" polyenoic acids. INTRODUCTION This work was initiated as an extension of studies on the nature of the phospholipids of tuna 1 and menhaden s. It is known that phospholipase A (phosphatide acylhydrolase, EC 3.i.i.4) of snake venom specifically hydrolyzes the/5 position of both synthetic and naturally occurring phospholipids 3-8, and that these fatty acids are primarily unsaturated 4. The fish lecithins are characterized by large amounts of highly unsaturated fatty acidsl, *, hence it appeared of interest to determine the positional distribution of these fatty acids in them. During the course of this investigation BROCKERHOFFet al.9 described the specific distribution of fatty acids in the phospholipids and triglycerides of several marine organisms: cod (Gadus callarias), scallop (Platopectin magellariccus) and lobster (Homarus americanus). The present paper contains our results obtained by similar means on lecithins isolated from albacore tuna (Thunnus alalunga), menhaden (Brevoortia tyrannus), and salmon (Oncorhynchus tshawytscha) muscle, and from beef liver, rat (Long-Evans) liver, and chicken egg yolk.
Biochim. Biophys. Acta, 84 (x964) i33-I39
134
u.B. MENZEL, H. S. OLCOTT EXPERIMENTALPROCEDURE
A. Purification of lecithins Tissues were homogenized in chloroform-methanol (I :I, v/v) solution. Lipids were extracted, and fractionated by column chromatography on silicic acid as described previously a. The lecithin fractions showed only one component with the thin-layer silicic acid chromatography system of VOGEL et al. 1°. The product used in each case contained the bulk of the lecithin fraction, to avoid fractionation with regard to the fatty acid content. Ester ~1, phosphorus ~2 and plasmogen contents aa were determined for each lecithin. These, together with the specific optical rotation in chloroform solution, and the ester/phosphorus ratios are set forth in Table I. During the preparation of the fractions trace amounts of hydroquinone were added as an antioxidant, and all samples were stored under N 2.
B. Liberation of fatty acids by phospholipase A (ref. 4) 30 mg of each lecithin were dissolved in peroxide-free diethyl ether to give a final concentration of 20 /~moles/ml. Lyophilized venom of the eastern diamondback rattlesnake (Crotalus adamanteus) (Ross Allen's Reptile Institute, Silver Spring, Florida) was dissolved in a solution of o.I M Tris buffer, 20 mM CaC12 and 2 mM EDTA (tetrasodium salt) to give a final concentration of 2.0 #g of venom per #l. The buffered enzyme was adjusted to pH 7-4. To each ml of the etheral solution of lecithin, IO ul of the ~.,nzyme solution was added. The mixture was shaken vigorously for 30 sec and allo~ved to stand at room temperature for 2 h. IO-#1 aliquots were taken at fixed time intervals to check the progress of the reaction by chromatographing the aliquot directly in the thin-layer system mentioned above. The reaction was complete by this test at the end of 2 h. The ether solution was evaporated to dryness under N 2 and the residue dissolved in a minimum of solvent (0.5% methanol in chloroform). The resulting solution was transferred to a 12 mm x 155 mm column containing 5 g of silicic acid equilibrated in the same solvent. The free fatty acids liberated during the reaction were eluted from the silicic acid column by an additional 40 ml of solvent. Elution of unreaeted lecithin (trace amounts) was accomplished by the addition of 40 ml of 25% methanol in chloroform to the column. The remaining reaction product, lysolecithin, was eluted from the column with 9 ° m l of 85% methanol in chloroiorm. Each of the fractions isolated showed only a single spot corresponding to an authentic sample when subjected to chromatography on the thin-layer silicic acid system. Approx. 90% of the amount used was recovered.
C. Preparation and analysis of methyl esters of fatty acids Methyl esters of the fatty acids liberated during the reaction were prepared from the first silicic acid chromatography fraction by reaction with 3% (v/v) conc. H~SQ in anhydrous methanol for 2 h at boiling, followed by extraction with light petroleum and washing with water. The fatty acids remaining esterified to the lysolecithin fraction were methanolized in 5 ml of chloroform-methanol (I : I, v/v) to which was added I.O ml of 0.5 N sodium methoxide 14. The reactants were cooled to o ° before reaction, mixed rapidly, allowed
Biochim. Biophys. Acta, 84 (I964) 133-139
POSITION OF FATTY ACIDS IN LECITHINS
135
to stand in the cold for 20 min, then allowed to warm to room temperature, neutralized with 2.5 ml of 3 % HC1 in anhydrous methanol and evaporated to dryness. The residue was suspended in water and extracted with light petroleum. Methyl esters from the various fractions were analyzed by gas-liquid chromatography. An Aerograph Hy-Fi (Wilkens Instrument Co., Walnut Creek, Calif.) flameionization instrument was used. It was equipped with a 366-cm, 3-mm O.D. glass column which had been treated with dichlorodimethylsilane and packed with 1% EGSS-X on ioo-I2o-mesh hexamethyldisilizane-treated Gas-Chrom P (Applied Science Laboratories, State College, Pa.). The separation was conducted at 19°o with N2 (40 ml/min) as the carrier gas. The instrument was calibrated with authentic standards and National Institute of Health standard mixtures of fatty acid methyl esters. RESULTS AND DISCUSSION
The data of Table I illustrate the nature of the preparations. Although all lecithin preparations showed only one spot by thin-layer chromatography and corresponded to the expected infrared absorption spectra, the ester/phosphorus ratios are lower for the tmla and menhaden lecithin samples than can be accounted for by their content of plasmalogen. They probably therefore contained small amounts of non-lecithin lipid material. Analyses for the other lecithin preparations gave the expected results within the limits of precision of the methods used. By using the total fraction from the silica-gel column chromatography, it was hoped to avoid the initial fractionation of lecithins noted by RHODES AND LEA15. TABLEI CHEMICAL AND
Source
PHYSICAL PROPERTIES
Specific rotation ([a] iS)
P content (%)
Ester content (l*equiv. per xoo rag)
3.60 3.94 3.7 ° 3.76 3.59 4.00
2.06 2.22 2.28 2.4 o 2.13 2.61
D
Salmon muscle Tuna muscle Menhaden muscle E g g yolk R a t liver B e e f liver
+4.4 +3.6 +5.0 +6.6 +6.1 +6.0
OF ISOLATED LECITHINS
Ester/P ratio
1.78 1.75 1.92 2.o 4 1.84 2.02
Mole % pIa.~malogen
5-5 8. 7 5.8 1. 5 3.8 3.8
The specific distribution of fatty acids in the lecithins examined is presented in Table II and summarized in Table III. It is apparent from Table III that, regardless of the origin of the lecithin, the fl position is predominantly unsaturated and that the distribution of saturated and unsaturated fatty acids in the fl position is very nearly the same in all samples while the mole per cent of saturated fatty acids in the a' position varies considerably. Egg-yolk, menhaden-muscle and rat-liver leeithins were similar in the degree of saturation of the a' position (80%) while beef liver was of an intermediary value (66%) and salmon and tuna muscle of the lowest degree of saturation (35%). Palmitic acid was the predominant saturated fatty acid in the a' position. Indeed, palmitic and stearic acids represented the highest positional enrichB i o c h i m . B i o p h y s . A c t a , 84 (1964) i 3 3 - 1 3 9
136
D . B . MENZEL, H. S. OLCOTT
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2 Biochim. Biophys. Acta,
84 ( I 9 6 4 ) 1 3 3 - 1 3 9
POSITION OF FATTY ACIDS IN L E C I T H I N S
137
TABLE III RELATIVE DISTRIBUTION OF SATURATED AND UNSATURATED FATTY ACIDS IN THE (2t AND fl POSITIONS OF LECITHIN
Source
Salmon muscle Menhaden muscle
Mole % saturated fatty acids
Mole % unsaturated forty acids
/5
38 9
62 91
Position
a" a"
81
19
/5
7
93
Tuna muscle
a' /5
36 4
64 96
Egg yolk
a' t5
86 6
14 94
R a t liver
a" /5 a' fl
82 3 66 I
18 97 34 99
B e e f liver
ment of any of the fatty acids found. In the fish lecithins, docosahexaenoic acid was the other major fatty acid; however, in the egg-yolk, rat- and beef-liver materials, lower unsaturated fatty acids completed the balance of fatty acids. Only small amounts of arachidonic acid were found in the fish lecithins; oleic, eicosapentaenoic and docosahexaenoic acids were the prevalent fatty acids in fl position. Beef-liver lecithin contained some of the fatty acids normally enconntered in marine organisms. Docosamonoenoic acid was tentatively identified only in the beef sample and was primarily esterified to the fl position. Trace amounts of odd chain length and branch-chained fatty acids were also detected in the beef sample but were not positively identified. These fatty acids are possibly the products of rumen bacteria. The fatty acid composition of the lecithin of egg yolk shows good correlation with that reported by other workers; e.g., in the u' position, palmitic acid 61°/o, stearic acid 25 %, oleic acid io%, compared to palmitic acid 65 % ; stearic acid 33 %, oleic acid 1% (ref. 5), and palmitic acid 55%, stearic acid 36%, and oleic acid 6% (ref. 16); the correlation is also good for the fl position. In the more complex lecithins containing a greater proportion of higher polyenoic acids, two difficulties are of importance : first, the quantitative problem of gas-liquid chromatography, and second, contamination with plasmalogen (compare Table I). The difficulty with quantitative gas chromatography is illustrated by the data in Table II. Particularly in the fatty acid analyses of the fish-lipid fractions, there are discrepancies in the amounts of total compared to the amounts in the a' and fl position. Since careful repetition failed to resolve this difficulty, we are inclined to conclude that it represents an inherent weakness of the methods or equipment. Previous investigators more often than not do not include data for total fatty acids so that direct comparisons are not possible. However DE TOMASet al. x7 have recently published an analysis of phosphatidylethanolamine fatty acids from rats fed diets deficient in essential fatty acids in which similar discrepancies appear. B i o c h i m . B i o p h y s . A c t a , 84 (i964) 1 3 3 - i 3 9
138
D . B . MENZEL, H. S. OLCOTT
RAPPORT AND FRANZLls report that plasmalogens are attacked by phospholipase A and hence the liberated f a t t y acids will also contain the fatty acids esterified to the contaminating plasmalogen. No scheme is currently available to differentiate between the lecithin /3 position and that of plasmalogen. In the plasmalogen-containing lecithins this error would be expected to decrease the apparent recovery of f a t t y acids not associated with the plasmalogen and to increase the apparent recovery of those associated with the plasmalogen. Should the pattern of f a t t y acids of plasmalogen follow that of lecithin, triglycerides and other phospholipids having a highly unsaturated/3 composition ls-22, then one would expect to recover a larger amount of unsaturated and a smaller amount of saturated f a t t y acids than the theoretically calculated amount. With the above reservation in mind, it is of interest to relate the current d a t a with that of others and with the schemes of biosynthesis proposed for phosphatidyl choline. First, regarding the distribution of the polyenoic acids of the "linolenic acid family" vs. those of the "linoleic acid family", the latter being considered "essential f a t t y acids", it is apparent that the primary representative of the linolenic-group, docosahexaenoic acid, is primarily esterified to the/3 position of both the marine and terrestrial lecithins in which it occurs and hence follows the classical distribution of arachidonic and linoleic acid. Linolenic, eicosapentaenoic, docosaenoic, docosatetraenoic and docosapentaenoic acids are also similarly distributed. This distribution is then in agreement with the suggestion of KLENK AND BROCKERHOFF 2a, RICHARDSON et al. ~, and KLENK AND EBERHAGEN25 that the so-called essential f a t t y acids are not absolute requirements in the structure and function of cellular membranes, but that other unsaturated f a t t y acids m a y substitute. The enzymatic studies of LANDS AND MERKL26 would also support such a thesis. Secondly, as was mentioned previously, the most marked positional enrichment of a given fatty acid observed was that of palmitic acid for the a' position. Dietary effects will influence the relative proportion of saturated and unsaturated f a t t y acids esterified to the a' position. RHODES 27, for example, found an increase in the unsaturation of egg-yolk lecithin, when the laying hens were fed cod-liver oil. Yet even in those lecithins having the highest a'-position unsaturation, the emichment of palmitic acid for the a' position was of the same order as that of arachidonic and docosahexaenoic acid for the/3 position (consider salmon and tuna: palmitic acid: ratio of a't/3 = 4-7; arachidonic acid: ratio ilia' =- 0.5-4; docosahexaenoic acid: ratio fl/g' = about 1.5). BROCKERHOr~ et al. 9 also found more palmitic acid esterified to the a' position in cod-muscle and cod-liver, scallop-muscle and lobster-liver lecithin than that found in the g' position of the triglycerides isolated from the same tissue. ACKNOWLEDGEMENTS
We wish to thank J. R. FROINES AND R. D. JENSEN for their technical assistance, and A. GOODBAN of the Western Regional Research Laboratory, U.S.D.A., Albany, Calif., for determining the optical rotation of the samples. This investigation was supported in part b y funds administered by means of a collaborative agreement between the U.S. Fish and Wildlife Service Bureau of Cornmerci al Fisheries and the University of California.
Biochim. Biophys. Acta, 84 (1964) 133-139
POSITION OF FATTY ACIDS IN LECITHINS
139
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Biochim. Biophys. Aeta, 84 (1964) 133-139