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The characterization and incorporation of radioactive bases into scallop phospholipids
Comp. Biochem.PhysioL, 1968, Vol. 27, pp. 533 to 541. PergamonPress. Printedin Great Britain
T H E C H A R A C T E R I Z A T I O N AND I N C O R P O R A T I O N OF RADIOACTIVE BASES I N T O SCALLOP P H O S P H O L I P I D S H. S. SHIEH Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada (Received 24 April 1968) Abstract--1. The major phospholipids of the scallop, Placopecten magellanicus
(Gmelin), extractable in methanol-chloroform were shown to be phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine and other unidentified phospholipid components were present only in small amounts. 2. The major fatty acids in scallop phospholipids were 14:0, 16:0, 18:0, 18:1, 20:1, 20:5 and 22:6. Polyunsaturated fatty acids predominated in the various fractions. 3. The in vivo incorporation of choline methyl-Ct4, ethanolamine-l-2-C14 and serine-3-C x4 into scallop phospholipids was demonstrated. INTRODUCTION THE CHEMISTRY, metabolism and function of phospholipids in vertebrates have been intensively studied, and in recent years, invertebrate phospholipids have also received some attention (Lovern, 1956; Froines et al., 1965; de Koning& McMullan 1966a, b). As yet, little is known about the origin of the nitrogen-bearing moiety of phospholipids in marine animals. T h e incorporation in vivo of radioactivity into phospholipid-bound choline was studied by Bilinski (1962) in the lobster (Homarus americanus) following intramuscular administration of C14-1abelled compounds known to be associated with the intermediary metabolism of the methyl group. A study of utilization of choline methyl-C 14 by the crab (Cancer magister) was also reported (Bilinski, 1962). In the present investigation, by injecting the radioactive compounds, choline methyl-C 14, ethanolamine-1-2-C 14, serine-3-C 14and methionine methyl-C 14 into scallops (Placopecten magellanicus) and following the appearance of label in the phospholipids, it was expected to obtain some information concerning the origin of phospholipid bases in this sea animal. MATERIALS AND METHODS Marine Mollusea Sea scallop, Placopecten magellanicus (Gmelin), was obtained from Lunenburg Sea Products Ltd., Lunenburg, Nova Scotia. The live scallops were maintained in a laboratory holding tank supplied with running sea water previously filtered through sand and gravel, and then through fine porosity paper (Stewart & Power, 1963). Injection of radioactive compounds The radioactive compounds listed in Table 4 were administered to scallops by injection at random into the body with a syringe. After a metabolic period of 72 hr, the whole scallop was subjected to lipid extraction.
533
534
H.S.
SmErI
Lipid extraction Lipids were extracted from scallops with chloroform and methanol by the procedure of Bligh & Dyer (1959). Solvents were removed in vacuo and crude lipids were dissolved in a small amount of chloroform. The phospholipids were precipitated by the addition of acetone. Inclusion of small quantities of antioxidant, c~-tocopherol (Witting et al., 1961) or B H T (Wren & Szezepanowska, 1964), dissolved in the solvents protect the lipids from autoxidation during extraction. To avoid lipid oxidation, an atmosphere of nitrogen was provided where possible and samples were stored in ten times their weight of redistilled benzene under a nitrogen atmosphere at 0°C. Thin-layer chromatography Thin-layer plates were prepared from silica gel-G, dried at room temperature and stored in a cabinet. Just before application of the lipid samples, chromatoplates were heated at l l 0 ° C for 30 min. Phospholipids were separated with the solvent system: chloroform-methanol-ammonium hydroxide ( 3 0 : 1 0 : 1 ) . The chromatograms were stained with molybdenum blue stain (Dittmer & Lester, 1964) for the detection of all phospholipid components, with ninhydrin for amino-lipids. Individual phospholipid components were isolated by preparative thin-layer chromatography (Skipaki et al., 1964). The separated bands were visualized under ultraviolet light after spraying with dichlorofluorescein, scraped from the plate, and each sample was eluted from the silica gel by suspending the powder in eluting solvent. Paper chromatography Paper chromatography was performed according to the method of Letters (1966). Gas chromatography Methanolysis of the lipid fatty acids was carried out by the procedure of Metcalfe et al. (1966). The total fatty acids were initially determined by analytical gas-liquid chromatography but some chromatogram peaks were not separated. Complete separation was achieved by first separating the saturated, mono-unsaturated and polyunsaturated fatty acids as their mercuric acetate adducts, and then chromatographing each class separately with gas-liquid chromatography (Goldfine & Bloch, 1961; Davidoff & Korn, 1963; Kom, 1963, 1964; Korn et al., 1965). Fatty acid analyses were made with an F and M model 402 gas chromatograph equipped with a dual-flame ionization detector. Helium, at an outlet flow rate of 70 ml/min, was used as the carrier gas, and all columns were glass 0-shaped with 3 mm i.d. and 6 m m o.d. The following column packings and conditions were used: (1) Diethylene glycol succinate (DEGS), 6% on 80/100 diatoport S; the column (6 ft) was maintained isothermally at 175°C. (2) Organosilicone polyester (EGSS-X), 10% on 100-120 mesh gas chrom P; the column (6 ft) was maintained isothermally at 170°C. (3) Organosilicone polyester (EGSS-Y), 15"5% on 100-120 mesh gas chrom P; the column (6 ft) was maintained isothermally at 165°C. The components were quantitated by the method of Carroll (1961) and the fatty acid methyl esters were identified by comparison of their retention times with those of known standards and marine oil (Ackman et al., 1963 ; Ackman & Burgher, 1965). Hydrolysis of phospholipids Alkaline hydrolysis was carried out by the method of Dawson (1960) and acid hydrolysis of the phospholipids by the technique of Dittmer et al. (1958). Liquid-scintillation counting Samples were prepared and counted in a Packard Tri-carb liquid-scintiUation speetrophotometer by a technique reported previously (Shieh & Spears, 1967).
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
535
RESULTS AND DISCUSSION
Phospholipid composition Analyses of the total phospholipids extracted from scallop revealed the presence of six components. A tracing of a typical chromatograph (Fig. 1) illustrates the type of separation obtained on a silica gel-G plate. T h e spot number, R 1 values and some of the characteristics of the phospholipids from scallop are listed in Table 1. T h e results of deacylation and chromatographic separation of the products of hydrolysis of scallop phospholipids are given in Table 2.
4
A
0
B
0
o °@ cO
FIO. 1. Thin-layer chromatography of scallop phospholipids on silica gel=G, with chloroform-methanol-ammonium hydroxide (30 : 10 : 1) as solvent. Spots stained with I2. Abbreviations: PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SP, scallop phospholipids. TABLE |--CHARACTERISTICS OF SCALLOPPHOSPHOLIPIDS*
Spot
Average R I value
Molybdenum blue stain
Choline stain
Ninhydrin stain
Identity of components
A B C D E F
81 72 53 32 14 5
+ + + + + +
+ + -
+ + +
Undefined Undefined Phosphatidylethanolamine Phosphatidylcholine Undefined Phosphatidylserine
* Chromatograrns of phospholipids on silica gel-G plate with chloroform-methanolammonium hydroxide (30 : 10 : 1) as solvent. Abbreviations: +, positive; _+ faintly positive; - , negative.
Spots (A) and (B). T h e amounts of these two components are very small. On the b a s i s o f thin-layer chromatographic data and their reaction with various detection reagents, these spots could have been either a phosphoinositide or a cardiolipin type of polyglycerol phosphatide.
Glycerylphosphorylcholine Glycerylphosphorylethanolamine Glycerylphosphorylserine
Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine
0"92 0"60 0-25
Standard 0"91 0-60 0"24
Sample
Choline Ethanolamine Serine
Acid hydrolysis product
0"89 0"49 0"13
Standard
0"89 0"50 0.13
Sample
* Hydrolysis products of scallop phospholipids were separated by paper chromatography with phenol-acetic acid-ethanol-water (40 : 5 : 6 : 10) as solvent.
Alkali hydrolysis product
C H R O M A T O G R A P H I C ANALYSIS OF H Y D R O L Y S I S P R O D U C T S OF P H O S P H O L I P I D S FROM S C A L L O P *
Phospholipid
TABLE 2--PAPER
l
¢.~
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
537
Spot (C). This major component of scallop phospholipids has been identified as phosphatidylethanolamine. When separated on silica gel-G plate, stained with ninhydrin and heated at 100°C for a few minutes, this compound appeared purple and had an R t value of 0.53. A standard sample of phosphatidylethanolamine was found to have the same R t and positive reaction with ninhydrin. After deacylation and chromatographic separation, a spot corresponding to glycerylphosphorylethanolamine was detected (Table 2); it had the same R! as that obtained with the deacylated standard sample. Acid hydrolysis of scallop phosphatidylethanolamine released ethanolamine which could be detected with the ninhydrin stain. Spot (D). The other major component of scallop phospholipids has been identified as phosphatidylcholine. This compound gave an orange-brown colour with the Dragendorff reagent and an average R! value of 0.32. A standard sample of phosphatidylcholine was found to have the same R! value and positive reaction with Dragendorff reagent. A choline-containing spot, identified as glycerylphosphorylcholine, was detected in the deacylated phosphatidylcholine of scallop when the phosphomolybdic acid-stannous chloride reagent was used. Further evidence of its identity was obtained by comparing acid hydrolysates of the unknown sample with a standard phosphatidylcholine. Spot (E). This component gave a faintly positive reaction with the choline stain and similar R! value of sphingomyelin. Considering its positive reaction with ninhydrin, it could represent a bigger chunk of phospholipid than sphingomyelin. Spot (F). This component has been identified as phosphatidylserine on the basis of its R! value and positive reaction with ninhydrin as compared with a standard sample. After deacylation, glycerylphosphorylserine was obtained. A serine spot was identified after acid hydrolysis of scallop phosphatidylserine. Phosphatidylcholine and phosphatidylethanolamine have been demonstrated as the major phospholipid components of most marine invertebrates (de Koning & McMullan, 1966a, b). The principal phospholipids from the scallop, Placopecten magellanicus (Gmelin), were also phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine and other unidentified phospholipid components were present only in small amounts. The component fatty acids in scallop phospholipids A typical analysis of the fatty acids of scallop phospholipids is shown in Table 3. The major fatty acids are 14:0, 16:0, 18:0, 18:1, 20:1, 20:5 and 22:6. This agrees in general with the findings of Gruger et al. (1964), who reported the fatty acid composition of scallop lipids. There are only minor differences in the fatty acid pattern of individual phospholipids extracted from scallop. It has been shown that marine animals often contain very high percentages of polyunsaturated acids (Gruger, 1967). As expected, polyunsaturated fatty acids predominate in scallop phospholipids. It is known that fish and some invertebrates can synthesize fatty acids by pathways established for higher animals (Kayama et al., 1963a, b; Kayama & Tsuchiya, 1962). Very little information is available concerning the fatty acid synthesis by marine Mollusca. In the present study, the incorporation of radioactivity into the
538
H.S.
SHIES
fatty acids of scallop phospholipids has not been demonstrated after the injection of acetate-l-C 14 into scallop in vivo. It seems likely that fatty acids in scallop may be of dietary origin. TABLE 3--THE
COMPONENT FATTY ACIDS IN SCALLOP PHOSPHOLIPIDS *
Percentage of total fatty acid methyl esters Total phospholipids
PC
PE
PS
14:0 14:1 16:0 16:1 16:2
6.1 Tr ace 26"3 2.5 0.6
7.0 0 37.1 3.2 0.4
18:0 18:1
6.0 7"8
4.2 Trace 15.8 1"4 0.7 4.6 5.3 7"9 4.1 23.0 1"2 0"3 32.0 Traces
3.6 0 17.5 3.1 0"3 9.1
Fatty acid
20:1 20:4 20:5 22:1 22:5 22:6 Others
6.2 3.1 14.3 2"4 Trace 23.9 Traces
5.8
9"7 4"9 0"3 9.6 3.0 0 19.1 Traces
11.0
6.8 1.7 14"0 1"6 0"4 29.9 Traces
* Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.
Incorporation of radioactive bases into scallop phospholipids The results of the incorporation of radioactive bases into scallop phospholipids are presented in Table 4. When choline methyl-C x4 was injected into scallop, phosphatidyleholine was found to be heavily labelled and no radioactivity was present in phosphatidylserine or phosphatidylethanolamine. When methionine methyl C 14was used as the precursor, phospholipids were not found to be labelled. Two routes for phosphatidylcholine synthesis have been demonstrated in living cells. The first, established by Kennedy et al. (1956, 1957), involves the reaction of n,I3-diglyceride with eytidine diphosphoeholine to give phosphatidylcholine. The second, discovered by Bremer et al. (1960) and Bremer & Greenberg (1961), involves the conversion of phosphatidylethanolamine to phosphatidyleholine via transmethylation reactions. It seems likely that scallop phosphatidylcholine was formed via the first pathway. This agrees with the findings of Bilinski (1962), who reported that choline was utilized for formation of phospholipid choline by lobster (Homarus americanus) and crab (Cancer magister). When ethanolamine-l-2-C 14 was injected into scallop, phosphatidylethanolamine was found to be heavily labelled and no radioactivity was present in phosphatidyleholine or phosphatidylserine. When DL-serine-3-C 14 was used as the precursor, phosphatidylserine was found to be labelled and no radioactivity was
3"1 2"8 7-4 6-5
Precursor injected
Choline-methyl-C 14 Ethanolamine-l-2-C 1' DL-Serine-3-C 14 L-Methionine-methyl-C 1'
10 10 10 10
Amount administered (pc)
0 2"01 x 10' 0 0
(counts/rain)
(counts/rain) 1.84 x 106 0 0 0
Phosphatidylethanolamine
Phosphatidylchol/ne
Incorporation into
~----]NCORPORATION OF RADIOACTIVE BASES I N T O SCALLOP P H O S P H O L I P I D S
Specific activity mc/mM
TABLE
0 0 6"42 x 104 0
(coun~/min)
Phosphatidylserine
0
~D
LO
t~
2
O
0 ~t
0
540
H.S.
SHIEH
present in phosphatidylethanolamine or phosphatidylcholine. This is in contrast to the results obtained with vertebrates. W h e n ethanolamine-l-2-C 14 was incubated with rat liver homogenate, the principal labelled phospholipid derived from ethanolamine was phosphatidylethanolamine. However, when L-serine-3-C x4 was used as the labelled compound, the principal radioactive lipid recovered was also phosphatidylethanolamine, with only about one-fourth to one-third of the radioactivity present as phosphatidylserine (Borkenhagen et al., 1961). T h e possible biosynthesis pathway for scallop phosphatidylethanolamine is one entirely analogous to that involving CDP-choline and a D-2-3-diglyceride where ethanolamine is substituted for choline. Very few studies have been made on the biosynthetic mechanism of serinecontaining phospholipid and how serine is incorporated into the phospholipid molecule of scallop is not known at the present time. REFERENCES ACKMAN R. G. & BURGHERR. D. (1965) Cod liver oil fatty acids as secondary reference standards in the GLC of polyunsaturated fatty acids of animal origin: analysis of a dermal oil of the Atlantic leatherback turtle. 3. Am. Oil Chem. Soc. 42, 38-42. ACKMANR. G., BURGHXaR. D. & JANGAARDP. M. (1963) Systematic identification of fatty acids in the gas-liquid chromatography of fatty acid methyl esters: a preliminary survey of seal oil Can. J. Biochem. Physiol. 44, 1627-1641. BILINSKI E. (1962) Biosynthesis of trimethylammonium compounds in aquatic animals-III. Choline metabolism in marine Crustacea. 3. Fish. Res. Bd Can. 19, 505-510. BLIGH E. G. & DYERW. J. (1959) A rapid method of total lipid extraction and purification. Can.3. Biochem. Physiol. 37, 911-917. BORKENHAGENL. F. & KENNEDYE. P. (1957) The enzymatic synthesis of cytidine diphosphate choline. 3. biol. Chem. 227, 951-962. BORKENHAGENL. F., KENNEDYE. P. & FIELDINGL. (1961) Enzymatic formation and decarboxylation of phosphatidylserine. 3. biol. Chem. 236, PC 28. BREMERJ., FIGARDP. H. & GREENBERGD. M. (1960) The biosynthesis of choline and its relation to phospholipid metabolism. Biochim. biophys. Acta 43, 477-488. BREMERJ. & GREENBERGD. M. (1961) Methyl transferring enzyme system of microsomes in the biosynthesis of lecithin. Biochim. biophys. Acta 46, 205-216. CARROLLK. K. (1961) Quantitative estimation of peak areas in gas-liquid chromatography. Nature, Lond. 191, 377-378. DAVlDOFF F. & KORN E. D. (1963) Fatty acid and phospholipid composition of the cellular slime mold, Dictyostelium discoideum, jY. biol. Chem. 238, 3199-3209. DAWSON R. M. C. (1960) A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem.3. 75, 45-53. DITTMER J. C., FEMINELLAJ. L. & HANAHANn . J. (1958) A study of the quantitative estimation of ethanolamine and serine in phospholipids. 3. biol. Chem. 233, 862-867. DITTMERJ. C. & LESTERR. L. (1964) A simple specific spray for the detection of phospholipids on thin-layer chromatograms. 3. Lipid Res. 5, 126-127. FROINESJ. R., SHUSTERC. Y. & OLCOTTH. S. (1965) Phospholipids of menhaden muscle. 3. Am. Oil Chem. Soc. 42, 887-888. GOLDFII~m H. & BLOCH K. (1961) On the origin of unsaturated fatty acids in Clostridia. 3. biol. Chem. 236, 2596-2601. GRUGERE. H., JR. (1967) Fatty acid composition. In Fish Oils (Edited by STANSBY,M. E.), pp. 11-15. Avi, Westport, Conn.
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
541
GnUGEn E. H., JR., NELSON R. W. A. & STANSBYM. E. (1964) Fatty acid composition of
oils from twenty-one species of marine fish, freshwater fish and shellfish. 07. Am. Oil Chem. Soc. 41, 662-667. KAYAMA M. ~ TSUCHIYAY. (1962) Possible conversion pathway of polyunsaturated acids in fish. Tohoku07. Agric. Res. 13, 229-235. KAYAMA M . , TSUCHIYA Y. & MEAD J. F. (1963a) A model experiment of aquatic food chain with special significance in fatty acid conversion. BuIl.07ap. Soc. Sci. Fish. 29, 452--458. KAYAMAM., TSUCHIYAY., NEV~ZEL J. C., FULCOA. & MEAD J. F. (1963b) The incorporation of linolenic-l-C 14 acid into eicosapentaenoic and docosahexaenoic acids in fish. 07. Am. Oil Chem. Soc. 40, 499-502. KF_~-NEDYE. P. & WEISS S. B. (1956) The function of cytidine coenzymes in the biosynthesis of phospholipids. 07. biol. Chem. 222, 193-214. DE KONING A. J. (1966a) Phospholipids of marine origin--I. The hake. 07. Sd. Fd. Agric. 17, 112-117. DE KONING A. J. (1966b) Phospholipids of marine origin--IV. The abalone. 07. Sci. Fd. Agric. 17, 460--464. DE KONING A. J. & McMULLAN K. B. (1966a) Phospholipids of marine origin--II. The rock lobster. 07. Sci. Fd. Agric. 17, 117-120. DE KONING A. J. & MCMULLAN K. B. (1966b) Phospholipids of marine origin--III. The pilchard. 07. Sci. Fd. Agric. 17, 385-388. KORN E. D. (1963) Fatty acids of Acanthamoeba sp. 07. biol. Chem. 238, 3584--3587. KORN E. D. (1964) The fatty acids of Euglena gracilis. 07. Lipid Res. 5, 352-362. KORN E. D., GREENBLATTC. L. & LEES A. M. (1965) Synthesis of unsaturated fatty acids in the slime mold Physarum polycephalum and the zooflagellates Leishmania tarentolae, Trypanosoma lewis and Crithidia sp. : a comparative study. 07. Lipid Res. 6, 43-50. LETI~S R. (1966) Phospholipids of yeast--II. Extraction, isolation and characterization of yeast phospholipids. Biochim. biophys. Acta 116, 489-499. LOVERN J. A. (1956) The phospholipids of fish. 07. Sci. Fd. Agric. 7, 729-733. MEAD J. F., KAYAMAi . & REISER R. (1960) Biogenesis of polyunsaturated acid in fish. 07. Am. Oil Chem. Soc. 37, 438 A.A,9. METC~X~ L. D., SCHMITZA. A. & I~LKA J. R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38, 514-515. SHISH H. S. & SPEARSD. (1967) Utilization of choline or betaine for phospholipid synthesis in an aerobic marine microbe. Can.07. Biochem. 45, 1255-1261. SKIeSRI V. P., P~maSON R. F. & BARCLAYM. (1964) Quantitative analysis of phospholipids by thin-layer chromatography. Biochem. 07. 90, 374-378. S a ' ~ r ~ T J. E. & PowEn H. E. (1963) A sea water aquarium for marine animal experiments. 07. Fish. Res. Bd Can. 20, 1081-1084. WITrING L. A., HARwY C. C., CENTURY B. & HoRwIa'r M. K. (1961) Dietary alterations of fatty acids of.erythrocytes and mitochondria of brain and liver. 07. Lipid Res. 2, 412418. Wam~ J. J. & Szczse~'~owsg~t D. (1964) Chromatography of lipids in the presence of an antioxidant 4-methyl-2,6-di-tere-butyl phenol 07. Chromat. 14, 405-410.
x8
T H E C H A R A C T E R I Z A T I O N AND I N C O R P O R A T I O N OF RADIOACTIVE BASES I N T O SCALLOP P H O S P H O L I P I D S H. S. SHIEH Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada (Received 24 April 1968) Abstract--1. The major phospholipids of the scallop, Placopecten magellanicus
(Gmelin), extractable in methanol-chloroform were shown to be phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine and other unidentified phospholipid components were present only in small amounts. 2. The major fatty acids in scallop phospholipids were 14:0, 16:0, 18:0, 18:1, 20:1, 20:5 and 22:6. Polyunsaturated fatty acids predominated in the various fractions. 3. The in vivo incorporation of choline methyl-Ct4, ethanolamine-l-2-C14 and serine-3-C x4 into scallop phospholipids was demonstrated. INTRODUCTION THE CHEMISTRY, metabolism and function of phospholipids in vertebrates have been intensively studied, and in recent years, invertebrate phospholipids have also received some attention (Lovern, 1956; Froines et al., 1965; de Koning& McMullan 1966a, b). As yet, little is known about the origin of the nitrogen-bearing moiety of phospholipids in marine animals. T h e incorporation in vivo of radioactivity into phospholipid-bound choline was studied by Bilinski (1962) in the lobster (Homarus americanus) following intramuscular administration of C14-1abelled compounds known to be associated with the intermediary metabolism of the methyl group. A study of utilization of choline methyl-C 14 by the crab (Cancer magister) was also reported (Bilinski, 1962). In the present investigation, by injecting the radioactive compounds, choline methyl-C 14, ethanolamine-1-2-C 14, serine-3-C 14and methionine methyl-C 14 into scallops (Placopecten magellanicus) and following the appearance of label in the phospholipids, it was expected to obtain some information concerning the origin of phospholipid bases in this sea animal. MATERIALS AND METHODS Marine Mollusea Sea scallop, Placopecten magellanicus (Gmelin), was obtained from Lunenburg Sea Products Ltd., Lunenburg, Nova Scotia. The live scallops were maintained in a laboratory holding tank supplied with running sea water previously filtered through sand and gravel, and then through fine porosity paper (Stewart & Power, 1963). Injection of radioactive compounds The radioactive compounds listed in Table 4 were administered to scallops by injection at random into the body with a syringe. After a metabolic period of 72 hr, the whole scallop was subjected to lipid extraction.
533
534
H.S.
SmErI
Lipid extraction Lipids were extracted from scallops with chloroform and methanol by the procedure of Bligh & Dyer (1959). Solvents were removed in vacuo and crude lipids were dissolved in a small amount of chloroform. The phospholipids were precipitated by the addition of acetone. Inclusion of small quantities of antioxidant, c~-tocopherol (Witting et al., 1961) or B H T (Wren & Szezepanowska, 1964), dissolved in the solvents protect the lipids from autoxidation during extraction. To avoid lipid oxidation, an atmosphere of nitrogen was provided where possible and samples were stored in ten times their weight of redistilled benzene under a nitrogen atmosphere at 0°C. Thin-layer chromatography Thin-layer plates were prepared from silica gel-G, dried at room temperature and stored in a cabinet. Just before application of the lipid samples, chromatoplates were heated at l l 0 ° C for 30 min. Phospholipids were separated with the solvent system: chloroform-methanol-ammonium hydroxide ( 3 0 : 1 0 : 1 ) . The chromatograms were stained with molybdenum blue stain (Dittmer & Lester, 1964) for the detection of all phospholipid components, with ninhydrin for amino-lipids. Individual phospholipid components were isolated by preparative thin-layer chromatography (Skipaki et al., 1964). The separated bands were visualized under ultraviolet light after spraying with dichlorofluorescein, scraped from the plate, and each sample was eluted from the silica gel by suspending the powder in eluting solvent. Paper chromatography Paper chromatography was performed according to the method of Letters (1966). Gas chromatography Methanolysis of the lipid fatty acids was carried out by the procedure of Metcalfe et al. (1966). The total fatty acids were initially determined by analytical gas-liquid chromatography but some chromatogram peaks were not separated. Complete separation was achieved by first separating the saturated, mono-unsaturated and polyunsaturated fatty acids as their mercuric acetate adducts, and then chromatographing each class separately with gas-liquid chromatography (Goldfine & Bloch, 1961; Davidoff & Korn, 1963; Kom, 1963, 1964; Korn et al., 1965). Fatty acid analyses were made with an F and M model 402 gas chromatograph equipped with a dual-flame ionization detector. Helium, at an outlet flow rate of 70 ml/min, was used as the carrier gas, and all columns were glass 0-shaped with 3 mm i.d. and 6 m m o.d. The following column packings and conditions were used: (1) Diethylene glycol succinate (DEGS), 6% on 80/100 diatoport S; the column (6 ft) was maintained isothermally at 175°C. (2) Organosilicone polyester (EGSS-X), 10% on 100-120 mesh gas chrom P; the column (6 ft) was maintained isothermally at 170°C. (3) Organosilicone polyester (EGSS-Y), 15"5% on 100-120 mesh gas chrom P; the column (6 ft) was maintained isothermally at 165°C. The components were quantitated by the method of Carroll (1961) and the fatty acid methyl esters were identified by comparison of their retention times with those of known standards and marine oil (Ackman et al., 1963 ; Ackman & Burgher, 1965). Hydrolysis of phospholipids Alkaline hydrolysis was carried out by the method of Dawson (1960) and acid hydrolysis of the phospholipids by the technique of Dittmer et al. (1958). Liquid-scintillation counting Samples were prepared and counted in a Packard Tri-carb liquid-scintiUation speetrophotometer by a technique reported previously (Shieh & Spears, 1967).
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
535
RESULTS AND DISCUSSION
Phospholipid composition Analyses of the total phospholipids extracted from scallop revealed the presence of six components. A tracing of a typical chromatograph (Fig. 1) illustrates the type of separation obtained on a silica gel-G plate. T h e spot number, R 1 values and some of the characteristics of the phospholipids from scallop are listed in Table 1. T h e results of deacylation and chromatographic separation of the products of hydrolysis of scallop phospholipids are given in Table 2.
4
A
0
B
0
o °@ cO
FIO. 1. Thin-layer chromatography of scallop phospholipids on silica gel=G, with chloroform-methanol-ammonium hydroxide (30 : 10 : 1) as solvent. Spots stained with I2. Abbreviations: PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SP, scallop phospholipids. TABLE |--CHARACTERISTICS OF SCALLOPPHOSPHOLIPIDS*
Spot
Average R I value
Molybdenum blue stain
Choline stain
Ninhydrin stain
Identity of components
A B C D E F
81 72 53 32 14 5
+ + + + + +
+ + -
+ + +
Undefined Undefined Phosphatidylethanolamine Phosphatidylcholine Undefined Phosphatidylserine
* Chromatograrns of phospholipids on silica gel-G plate with chloroform-methanolammonium hydroxide (30 : 10 : 1) as solvent. Abbreviations: +, positive; _+ faintly positive; - , negative.
Spots (A) and (B). T h e amounts of these two components are very small. On the b a s i s o f thin-layer chromatographic data and their reaction with various detection reagents, these spots could have been either a phosphoinositide or a cardiolipin type of polyglycerol phosphatide.
Glycerylphosphorylcholine Glycerylphosphorylethanolamine Glycerylphosphorylserine
Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine
0"92 0"60 0-25
Standard 0"91 0-60 0"24
Sample
Choline Ethanolamine Serine
Acid hydrolysis product
0"89 0"49 0"13
Standard
0"89 0"50 0.13
Sample
* Hydrolysis products of scallop phospholipids were separated by paper chromatography with phenol-acetic acid-ethanol-water (40 : 5 : 6 : 10) as solvent.
Alkali hydrolysis product
C H R O M A T O G R A P H I C ANALYSIS OF H Y D R O L Y S I S P R O D U C T S OF P H O S P H O L I P I D S FROM S C A L L O P *
Phospholipid
TABLE 2--PAPER
l
¢.~
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
537
Spot (C). This major component of scallop phospholipids has been identified as phosphatidylethanolamine. When separated on silica gel-G plate, stained with ninhydrin and heated at 100°C for a few minutes, this compound appeared purple and had an R t value of 0.53. A standard sample of phosphatidylethanolamine was found to have the same R t and positive reaction with ninhydrin. After deacylation and chromatographic separation, a spot corresponding to glycerylphosphorylethanolamine was detected (Table 2); it had the same R! as that obtained with the deacylated standard sample. Acid hydrolysis of scallop phosphatidylethanolamine released ethanolamine which could be detected with the ninhydrin stain. Spot (D). The other major component of scallop phospholipids has been identified as phosphatidylcholine. This compound gave an orange-brown colour with the Dragendorff reagent and an average R! value of 0.32. A standard sample of phosphatidylcholine was found to have the same R! value and positive reaction with Dragendorff reagent. A choline-containing spot, identified as glycerylphosphorylcholine, was detected in the deacylated phosphatidylcholine of scallop when the phosphomolybdic acid-stannous chloride reagent was used. Further evidence of its identity was obtained by comparing acid hydrolysates of the unknown sample with a standard phosphatidylcholine. Spot (E). This component gave a faintly positive reaction with the choline stain and similar R! value of sphingomyelin. Considering its positive reaction with ninhydrin, it could represent a bigger chunk of phospholipid than sphingomyelin. Spot (F). This component has been identified as phosphatidylserine on the basis of its R! value and positive reaction with ninhydrin as compared with a standard sample. After deacylation, glycerylphosphorylserine was obtained. A serine spot was identified after acid hydrolysis of scallop phosphatidylserine. Phosphatidylcholine and phosphatidylethanolamine have been demonstrated as the major phospholipid components of most marine invertebrates (de Koning & McMullan, 1966a, b). The principal phospholipids from the scallop, Placopecten magellanicus (Gmelin), were also phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine and other unidentified phospholipid components were present only in small amounts. The component fatty acids in scallop phospholipids A typical analysis of the fatty acids of scallop phospholipids is shown in Table 3. The major fatty acids are 14:0, 16:0, 18:0, 18:1, 20:1, 20:5 and 22:6. This agrees in general with the findings of Gruger et al. (1964), who reported the fatty acid composition of scallop lipids. There are only minor differences in the fatty acid pattern of individual phospholipids extracted from scallop. It has been shown that marine animals often contain very high percentages of polyunsaturated acids (Gruger, 1967). As expected, polyunsaturated fatty acids predominate in scallop phospholipids. It is known that fish and some invertebrates can synthesize fatty acids by pathways established for higher animals (Kayama et al., 1963a, b; Kayama & Tsuchiya, 1962). Very little information is available concerning the fatty acid synthesis by marine Mollusca. In the present study, the incorporation of radioactivity into the
538
H.S.
SHIES
fatty acids of scallop phospholipids has not been demonstrated after the injection of acetate-l-C 14 into scallop in vivo. It seems likely that fatty acids in scallop may be of dietary origin. TABLE 3--THE
COMPONENT FATTY ACIDS IN SCALLOP PHOSPHOLIPIDS *
Percentage of total fatty acid methyl esters Total phospholipids
PC
PE
PS
14:0 14:1 16:0 16:1 16:2
6.1 Tr ace 26"3 2.5 0.6
7.0 0 37.1 3.2 0.4
18:0 18:1
6.0 7"8
4.2 Trace 15.8 1"4 0.7 4.6 5.3 7"9 4.1 23.0 1"2 0"3 32.0 Traces
3.6 0 17.5 3.1 0"3 9.1
Fatty acid
20:1 20:4 20:5 22:1 22:5 22:6 Others
6.2 3.1 14.3 2"4 Trace 23.9 Traces
5.8
9"7 4"9 0"3 9.6 3.0 0 19.1 Traces
11.0
6.8 1.7 14"0 1"6 0"4 29.9 Traces
* Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.
Incorporation of radioactive bases into scallop phospholipids The results of the incorporation of radioactive bases into scallop phospholipids are presented in Table 4. When choline methyl-C x4 was injected into scallop, phosphatidyleholine was found to be heavily labelled and no radioactivity was present in phosphatidylserine or phosphatidylethanolamine. When methionine methyl C 14was used as the precursor, phospholipids were not found to be labelled. Two routes for phosphatidylcholine synthesis have been demonstrated in living cells. The first, established by Kennedy et al. (1956, 1957), involves the reaction of n,I3-diglyceride with eytidine diphosphoeholine to give phosphatidylcholine. The second, discovered by Bremer et al. (1960) and Bremer & Greenberg (1961), involves the conversion of phosphatidylethanolamine to phosphatidyleholine via transmethylation reactions. It seems likely that scallop phosphatidylcholine was formed via the first pathway. This agrees with the findings of Bilinski (1962), who reported that choline was utilized for formation of phospholipid choline by lobster (Homarus americanus) and crab (Cancer magister). When ethanolamine-l-2-C 14 was injected into scallop, phosphatidylethanolamine was found to be heavily labelled and no radioactivity was present in phosphatidyleholine or phosphatidylserine. When DL-serine-3-C 14 was used as the precursor, phosphatidylserine was found to be labelled and no radioactivity was
3"1 2"8 7-4 6-5
Precursor injected
Choline-methyl-C 14 Ethanolamine-l-2-C 1' DL-Serine-3-C 14 L-Methionine-methyl-C 1'
10 10 10 10
Amount administered (pc)
0 2"01 x 10' 0 0
(counts/rain)
(counts/rain) 1.84 x 106 0 0 0
Phosphatidylethanolamine
Phosphatidylchol/ne
Incorporation into
~----]NCORPORATION OF RADIOACTIVE BASES I N T O SCALLOP P H O S P H O L I P I D S
Specific activity mc/mM
TABLE
0 0 6"42 x 104 0
(coun~/min)
Phosphatidylserine
0
~D
LO
t~
2
O
0 ~t
0
540
H.S.
SHIEH
present in phosphatidylethanolamine or phosphatidylcholine. This is in contrast to the results obtained with vertebrates. W h e n ethanolamine-l-2-C 14 was incubated with rat liver homogenate, the principal labelled phospholipid derived from ethanolamine was phosphatidylethanolamine. However, when L-serine-3-C x4 was used as the labelled compound, the principal radioactive lipid recovered was also phosphatidylethanolamine, with only about one-fourth to one-third of the radioactivity present as phosphatidylserine (Borkenhagen et al., 1961). T h e possible biosynthesis pathway for scallop phosphatidylethanolamine is one entirely analogous to that involving CDP-choline and a D-2-3-diglyceride where ethanolamine is substituted for choline. Very few studies have been made on the biosynthetic mechanism of serinecontaining phospholipid and how serine is incorporated into the phospholipid molecule of scallop is not known at the present time. REFERENCES ACKMAN R. G. & BURGHERR. D. (1965) Cod liver oil fatty acids as secondary reference standards in the GLC of polyunsaturated fatty acids of animal origin: analysis of a dermal oil of the Atlantic leatherback turtle. 3. Am. Oil Chem. Soc. 42, 38-42. ACKMANR. G., BURGHXaR. D. & JANGAARDP. M. (1963) Systematic identification of fatty acids in the gas-liquid chromatography of fatty acid methyl esters: a preliminary survey of seal oil Can. J. Biochem. Physiol. 44, 1627-1641. BILINSKI E. (1962) Biosynthesis of trimethylammonium compounds in aquatic animals-III. Choline metabolism in marine Crustacea. 3. Fish. Res. Bd Can. 19, 505-510. BLIGH E. G. & DYERW. J. (1959) A rapid method of total lipid extraction and purification. Can.3. Biochem. Physiol. 37, 911-917. BORKENHAGENL. F. & KENNEDYE. P. (1957) The enzymatic synthesis of cytidine diphosphate choline. 3. biol. Chem. 227, 951-962. BORKENHAGENL. F., KENNEDYE. P. & FIELDINGL. (1961) Enzymatic formation and decarboxylation of phosphatidylserine. 3. biol. Chem. 236, PC 28. BREMERJ., FIGARDP. H. & GREENBERGD. M. (1960) The biosynthesis of choline and its relation to phospholipid metabolism. Biochim. biophys. Acta 43, 477-488. BREMERJ. & GREENBERGD. M. (1961) Methyl transferring enzyme system of microsomes in the biosynthesis of lecithin. Biochim. biophys. Acta 46, 205-216. CARROLLK. K. (1961) Quantitative estimation of peak areas in gas-liquid chromatography. Nature, Lond. 191, 377-378. DAVlDOFF F. & KORN E. D. (1963) Fatty acid and phospholipid composition of the cellular slime mold, Dictyostelium discoideum, jY. biol. Chem. 238, 3199-3209. DAWSON R. M. C. (1960) A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem.3. 75, 45-53. DITTMER J. C., FEMINELLAJ. L. & HANAHANn . J. (1958) A study of the quantitative estimation of ethanolamine and serine in phospholipids. 3. biol. Chem. 233, 862-867. DITTMERJ. C. & LESTERR. L. (1964) A simple specific spray for the detection of phospholipids on thin-layer chromatograms. 3. Lipid Res. 5, 126-127. FROINESJ. R., SHUSTERC. Y. & OLCOTTH. S. (1965) Phospholipids of menhaden muscle. 3. Am. Oil Chem. Soc. 42, 887-888. GOLDFII~m H. & BLOCH K. (1961) On the origin of unsaturated fatty acids in Clostridia. 3. biol. Chem. 236, 2596-2601. GRUGERE. H., JR. (1967) Fatty acid composition. In Fish Oils (Edited by STANSBY,M. E.), pp. 11-15. Avi, Westport, Conn.
INCORPORATION OF RADIOACTIVE BASES INTO SCALLOP PHOSPHOLIPIDS
541
GnUGEn E. H., JR., NELSON R. W. A. & STANSBYM. E. (1964) Fatty acid composition of
oils from twenty-one species of marine fish, freshwater fish and shellfish. 07. Am. Oil Chem. Soc. 41, 662-667. KAYAMA M. ~ TSUCHIYAY. (1962) Possible conversion pathway of polyunsaturated acids in fish. Tohoku07. Agric. Res. 13, 229-235. KAYAMA M . , TSUCHIYA Y. & MEAD J. F. (1963a) A model experiment of aquatic food chain with special significance in fatty acid conversion. BuIl.07ap. Soc. Sci. Fish. 29, 452--458. KAYAMAM., TSUCHIYAY., NEV~ZEL J. C., FULCOA. & MEAD J. F. (1963b) The incorporation of linolenic-l-C 14 acid into eicosapentaenoic and docosahexaenoic acids in fish. 07. Am. Oil Chem. Soc. 40, 499-502. KF_~-NEDYE. P. & WEISS S. B. (1956) The function of cytidine coenzymes in the biosynthesis of phospholipids. 07. biol. Chem. 222, 193-214. DE KONING A. J. (1966a) Phospholipids of marine origin--I. The hake. 07. Sd. Fd. Agric. 17, 112-117. DE KONING A. J. (1966b) Phospholipids of marine origin--IV. The abalone. 07. Sci. Fd. Agric. 17, 460--464. DE KONING A. J. & McMULLAN K. B. (1966a) Phospholipids of marine origin--II. The rock lobster. 07. Sci. Fd. Agric. 17, 117-120. DE KONING A. J. & MCMULLAN K. B. (1966b) Phospholipids of marine origin--III. The pilchard. 07. Sci. Fd. Agric. 17, 385-388. KORN E. D. (1963) Fatty acids of Acanthamoeba sp. 07. biol. Chem. 238, 3584--3587. KORN E. D. (1964) The fatty acids of Euglena gracilis. 07. Lipid Res. 5, 352-362. KORN E. D., GREENBLATTC. L. & LEES A. M. (1965) Synthesis of unsaturated fatty acids in the slime mold Physarum polycephalum and the zooflagellates Leishmania tarentolae, Trypanosoma lewis and Crithidia sp. : a comparative study. 07. Lipid Res. 6, 43-50. LETI~S R. (1966) Phospholipids of yeast--II. Extraction, isolation and characterization of yeast phospholipids. Biochim. biophys. Acta 116, 489-499. LOVERN J. A. (1956) The phospholipids of fish. 07. Sci. Fd. Agric. 7, 729-733. MEAD J. F., KAYAMAi . & REISER R. (1960) Biogenesis of polyunsaturated acid in fish. 07. Am. Oil Chem. Soc. 37, 438 A.A,9. METC~X~ L. D., SCHMITZA. A. & I~LKA J. R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38, 514-515. SHISH H. S. & SPEARSD. (1967) Utilization of choline or betaine for phospholipid synthesis in an aerobic marine microbe. Can.07. Biochem. 45, 1255-1261. SKIeSRI V. P., P~maSON R. F. & BARCLAYM. (1964) Quantitative analysis of phospholipids by thin-layer chromatography. Biochem. 07. 90, 374-378. S a ' ~ r ~ T J. E. & PowEn H. E. (1963) A sea water aquarium for marine animal experiments. 07. Fish. Res. Bd Can. 20, 1081-1084. WITrING L. A., HARwY C. C., CENTURY B. & HoRwIa'r M. K. (1961) Dietary alterations of fatty acids of.erythrocytes and mitochondria of brain and liver. 07. Lipid Res. 2, 412418. Wam~ J. J. & Szczse~'~owsg~t D. (1964) Chromatography of lipids in the presence of an antioxidant 4-methyl-2,6-di-tere-butyl phenol 07. Chromat. 14, 405-410.
x8