Diacyl, alkylacyl and alkenylacyl phospholipids of the nematode Turbatrix aceti

Comp. Biochem. Physiol. Vol. 69B, pp. 115 to 120

0305-0491/81/050115-06S02.00/0 Copyright © 1981 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

DIACYL, ALKYLACYL AND ALKENYLACYL PHOSPHOLIPIDS OF THE NEMATODE T U R B A T R I X ACETI* D. J. CHITWOODand L R. KRUSBERG Department of Botany, University of Maryland, College Park, MD 20742, U.S.A. (Received 9 July 1980)

Abstract--1. Phospholipid comprised 10.2% of the dry weight of Turbatrix aceti and largely consisted of choline and ethanolamine phosphoglycerides. 2. Choline phosphoglyceride contained a larger proportion of polyunsaturated fatty acid than ethanolamine phosphoglyceride. 3. Fatty acids esterified at the 1-position were generally shorter and more saturated than those esterified at the 2-position. 4. Ethanolamine phosphoglyceride contained proportionally more alkenylacyl and alkylacyl phosphoglyceride than choline phosphoglyceride. 5. The most abundant alkenyl and alkyl groups were octadecyl compounds. 6. Fatty acid compositions of alkenylacyl and alkylacyl phosphoglycerides were similar to those of the 2-positions of the corresponding diacyl compounds, but differences did occur.

INTRODUCTION Although the phospholipids of animal-parasitic nematodes have been studied in some detail, little information is available regarding these compounds in free-living nematodes. Only in Pana#reUus redioivus and Turbatrix aceti have the phospholipid or polar lipid content, composition, and fatty acid composition been investigated (Sivapalan & Jenkins, 1966; Fletcher & Krusberg, 1973). The presence of ethercontaining phospholipids, i.e. alkylacyl and alkenylacyl (or plasmalogen) phosphoglycerides in free-living nematodes has yet to be ascertained. In addition, positional analysis of phospholipid fatty acids from any nematode has yet to be performed, nor has there been comparison in any helminth of the acyl, alkyl and alkenyl group compositions of specific diacyl, alkylacyl and alkenylacyl phosphoglycerides. The purpose of this investigation was to quantify the phospholipid composition and investigate the positional distribution of fatty acyl, alkyl and alkenyl groups in phosphoglycerides from T. aceti. MATERIALS AND METHODS

Lipid standards and lipolytic enzymes

Phospholipid, fatty acid methyl ester, fatty alcohol, hexadecanal, and alkylglycerol standards were purchased from Applied Science Division, State College, Pennsylvania; Supelco, Inc., Bellefonte, Pennsylvania; and Avanti Biochemicals, Inc., Birmingham, Alabama. Additional fatty aldehydes were prepared from fatty alcohols by use of the * Scientific Article No. A2807, Contribution No. 5857 of the Maryland Agricultural Experiment Station. This investigation was supported in part by Regional Research Project NE-101, Cooperative State Research Service, U.S. Department of Agriculture.

prodedure of Mahadevan et al. (1966). Phospholipase C from Bacillus cereus and porcine pancreatic lipase were obtained from Calbiochem, San Diego, California and Sigma Chemical Co., St Louis, Missouri, respectively. Culture methods and lipid extraction T. aceti was propagated at 28°C in a liquid medium

containing liver extract, yeast extract, soy peptone and acetic acid (Cole & Krusberg, 1968). Nematodes from 21day-old cultures were collected as previously described (Fletcher & Krusberg, 1973), and the method of Folch et al. (1957) was used to extract lipids from lyophilized nematodes. Phospholipid isolation, identification and quantification

Total lipid was fractionated into neutral lipid, glycolipid and phospholipid on a column of activated silicic acid (Unisil, Clarkson Chemical Co., Williamsport, Pennsylvania) by the method of Rouser et al. (1967). Phospholipids were tentatively identified by their behaviour during thinlayer chromatography (TLC) on Adsorbosil 5 (Applied Science Division) with a solvent of chloroform:methanol: water 65:25:4 (v/v per voi) (TLC system I) (Rouser et al., 1967), n-butanol:acetic acid:water 3:1:1 (TLC system II) (Rouser et al., 1967) and, on plates prepared with 0.4 M boric acid, chloroform:methanol:water:28% NH4OH 70:30:3:2 (TLC system III) (Poorthuis et aL, 1976). Phospholipids were recovered from Adsorbosii by elution with chloroform :methanol 1:1, chloroform: methanol 1:4, and methanol. The compounds were characterized further by the spraying of chromatograms with several reagents specific for various compounds, including sodium hypochloritebenzidine spray for detection of compounds containing secondary amino groups (Bischel & Austin, 1963), ninhydrin for free amino groups (Christie, 1973), Dragendorff reagent for choline (Skidmore & Entenman, 1962), copperammonium molybdate for phosphorus (Goswami & Frey, 1971), ammoniacal silver nitrate for inositol (Skidmore & Entenman, 1962), and 2, 4-dinitrophenylhydrazine for plasmalogens (Marinetti, 19621 In addition, serine phosphogly115

116

D.J. CHITWOODand L. R. KRUSBERG

ceride was identified by TLC of acid hydrolysis products on TLC plates coated with Cellulosepulver MN300 (Macherey, Nagel & Co., Duren, W. Germany) with a solvent of 77~ ethanol (Dittmer & Wells, 1969; Block et al., 1958). Sphingomyelin was further characterized by spectrophotometric analysis (Lauter & Trams, 1962). Phospholipids were quantified by separation with TLC systems I and III and subsequent phosphorus determination of the purified compounds by a modified Bartlett procedure (Dittmer & Wells, 1969).

Table 2. Photodensitometric analysis of classes of glyceride acetates derived from choline phosphoglyceride (CP) and ethanolamine phosphoglyceride (EP) from Turbatrix aceti

Structural analysis of choline and ethanolamine phosphoglycerides Choline and ethanolamine phosphoglycerides were initially isolated with TLC system I. Choline phosphoglyceride was further purified by DEAE-cellulose column chromatography by the method of Rouser et al. (1967). The phosphorylcholine and phosphorylethanolamine moieties of the compounds were then removed by phospholipase C hydrolysis (Blank et al., 1975), and the glycerides produced were acetylated with pyridine/acetic anhydride (Kuksis & Marai, 1967). The resultant 1,2-diacyl-, 1,3-diacyl-, I-alkyl-2-acyl- and 1-alkenyl-2-acylglycerolacetates were separated by TLC on silica gel G (E. Merck, Darmstadt, W. Germany) as described by Wood & Snyder (1969) and quantified by photodensitometric analysis of similar plates after the method of Privett et al. (1973) with a Chromaflex K-495000 densitometer (Kontes Glass Co., Vineland, New Jersey). The small amounts of 1,3-diacylglycerolacetates present, which were formed by acyl migration in 1,2-diacyl compounds, were discarded. Positional distribution of fatty acids in 1,2-diacylglycerol acetates was determined by pancreatic lipase hydrolysis after the method of Luddy et al. (1964). Hydrolysis products were separated by TLC on silica gel G with a solvent of hexane:diethyl ether:acetic acid 50:50:1 (TLC system IV)(Renkonen, 1966). Alkylglycerols and free fatty acids were derived from alkylacylglycerol acetates by saponification after the method of Blank et al. (1975) and subsequent separation of the products with TLC system IV. Aldehydes and acyl groups in alkenylacylglycerol acetates were separated by HCl-reaction TLC as described by Schmid &Mangold (1966).

Results are expressed as relative percentage of total glyceride acetate.

Acyl, alkyl and alkenyl group analysis Fatty acid methyl esters were prepared from lipids by transesterification with BC13-methanol(Supelco, Inc.) and purified by TLC on silica gel G with benzene as the developing solvent (Morrison & Smith, 1964). Methyl esters were identified and quantified as previously described (Krusberg, 1967, 1972). Alkyl groups were analyzed by preparation of 2,3-O-isopropylidene-l-O-(alkyl) glycerols by the method of Hanahan et al. (1963). The isopropylidene glyceryl ethers were characterized by gas-liquid chromatography (GLC) with columns containing 3Yo SE-30 (methyl silicone) and 15yo EGS (ethylene glycol succinate polymer) coated onto 100/120 mesh Gas-Chrom Q

Table 1. Percentage of total phospholipid phosphorus in phospholipids from Turbatrix aceti Phospholipid Choline phosphoglyceride Ethanolamine phosphoglyceride Sphingomyelin Diphosphatidylglycerol Serine phosphoglyceride Inositol phosphoglyceride Lysophosphatidylcholine Phosphatidic acid

45.1 40.4 5.3 4.4 3.4 1.4 Trace Trace

Glyceride acetate 1,2-diacylglycerolacetate 1,3-diacylglycerolacetate l-alkenyl-2-acylglycerol acetate l-alkyl-2-acylglycerolacetate

CP

EP

85.6 3.6 7.0 3.8

44.1 2.3 43.2 10.5

(Applied Science Division). Column temperatures were 210° and 195°C, respectively. Fatty aldehydes were identified by GLC with the same columns as used for fatty acid methyl esters but at a lower temperature. Results presented are the means of at least three separate experiments with different harvests of nematodes.

RESULTS Phospholipid content and composition

Lipid comprised 19.8~ of the dry weight of T. aceti and consisted of 44.2~o neutral lipid, 4.5~ glycolipid and 51.4~o phospholipid. Choline and ethanolamine phosphoglycerides were the most abundant phospholipids (Table 1). These two phospholipids contained alkenylacyl and alkylacyl phosphoglycerides as well as diacyl compounds (Table 2). Ethanolamine phosphoglyceride consisted of greater amounts of these ether lipids than choline phosphoglyceride. Phospholipid fatty acids

The fatty acid compositions of total phospholipid, choline and ethanolamine phosphoglycerides, and diphosphatidylglycerol are shown in Table 3. Ethanolamine phosphoglyceride contained greater percentages of 18:0 and 20:0 acids than choline phosphoglyceride, which contained larger proportions of 20:3, 20:4 and 20:5 acids. The fatty acid composition of diphosphatidylglycerol was distinctive in that 20:3 and 20:4to 3 acids comprised 84~o of the total fatty acid. Positional distribution of fatty acids

Iso-15:0, 16:0, iso-17:0, 18:0, 20:0, 18:1 and 20:1 fatty acids were more abundant at the 1-position of diacyl choline phosphoglyceride, whereas 18:2, 18:3, 20:3, 20:4 and 20:5 acids were more abundant at the 2-position (Table 4). Similarly, the 1-position of diacyl ethanolamine phosphoglyceride contained more iso-17:0, 18:0, 20:0 and 20:1 acids than the 2-position, which contained more iso-15:0, 16:1, 18:1, 20:2, 20:3, 20:4 and 20:5 acids (Table 5). The fatty acid compositions of alkenylacyl and alkylacyl choline phosphoglycerides (Table 4) were similar to that of the 2-position of their diacyl analogue; however, the ether-containing phosphoglycerides contained larger proportions of 16:0 and 18:l acids and smaller amounts of 18:2 acid. In addition, the alkenylacyl compound contained a moderate amount of iso-15:0 acid. The alkylacyl compound was characterized by presence of larger proportions of 16:0, 16:l and 18:1 acids and lesser percentages of

Phospholipids of Turbatrix aceti Table 3. Fatty acid composition of total phospholipid (PL), choline phosphoglyceride (CP), ethanolamine phosphoglyceride (EP) and diphosphatidyiglycerol (DPG) from Turba-

trix aceti Fatty acid

PL

CP

EP

DPG

iso-14:O: 14:0 iso-15:0 15:0 iso-16:0 16:0 16:1 iso-17:0 17:0 iso-18:0 18:0 18:1 18:2 18: 3 20:0 20:1 20:2 20:3 20:4~o6 20:4co 3 20:5

0.01 0.15 3.7 Ti" 0.11 1.3 0.13 2.0 0.16 0.26 19.0 26.0 11.3 0.42 0.90 1.8 1.7 9.3 4.5 6.5 10.9

0.03 0.10 3.2 T 0.06 1.9 0.28 3.3 0.10 0.18 9.3 26.2 12.1 0.30 0.50 2.7 1.9 9.6 6.3 5.8 16.1

0.03 0.18 6.1 T 0.20 1.1 0.10 2.1 0.04 0.53 22.1 29.8 13.2 1.2 1.8 1.5 2.0 4.8 3.1 3.6 6.6

--:~ 0.26 0.66 0.05 0.01 0.79 0.06 0.23 0.05 0.01 2.8 3.8 4.8 0.02 0.10 0.15 0.59 53.7 0.01 30.9 1.0

117

20:3, 20:4 and 20:5 acids than either the alkenylacyl analogue or the 2-position of the diacyl compound. The fatty acid compositions of alkenylacyl and alkylacyl ethanolamine phosphoglyceride (Table 5) also resembled that of the 2-position of the diacyl analogue; however, iso-15:0, iso-17:0, 18:1, 20:1 a n d

20:2 fatty acids were more abundant and 18:2, 20:3, 20:4 and 20:5 acids less abundant in the ethercontaining phospholipids. Also, the alkenylacyl compound contained greater percentages of iso-15:0, 18:2 and 20:5 acids and smaller proportions of 16:0, 18:0 and 18:1 acids than the alkylacyl compound. Alkenyl and alkyl group composition

The fatty aldehyde compositions of choline and ethanolamine plasmalogens (Table 6) were similar to each other and also to the alkyl group compositions of alkylacyl choline and ethanolamine phosphoglycerides (Table 7). At least 80% of the total alkenyl or alkyl groups was the 18:0 compound. DISCUSSION

* Fatty acids are represented as follows: the first number represents the number of carbon atoms; the second, the number of double bonds. 1"Trace amounts detected. ~tNot detected. Results are expressed as relative percentage of total fatty acid.

The phospholipid content of T. aceti (10.2% of dry weight) was higher than that reported by Sivapalan & Jenkins (1966) for Panagrellus redivivus (7.9%). In the only study of glycolipid content in a nematode, Trichinella spiralis was found to contain 0.8% glycolipid (Castro & Fairbairn, 1969), a value close to the 0.9% found for T. aceti. The major phospholipids of T. aceti were choline and ethanolamine phosphoglycerides, which are also the major phospholipids of P. redivivus (Sivapalan &

Table 4. Fatty acid composition of the 1- and 2-positions of 1,2-diacyiglycerol acetate, the 2-position of l-alkenyl-2-acylglycerol acetate, and the 2-position of 1-alkyl-2-acylglycerol acetate, derived from choline phosphoglyceride from Turbatrix aceti Fatty acid

iso-14: 0 14:0

iso-15:0 15:0 iso-16:0 16:0 16:1 lso-17:0 17:0

iso-18:0 18:0 18 : 1 18:2 18: 3 20:0 20:1 20:2 20:3 20:4¢o ~ 20:4co3 20:5

Diacyl 1-position

Diacyl 2-position

Alkenylacyl 2-position

Alkylacyl 2-position

0.02 0.18 5.5 T* 0.14 6.3 0.56 6.9 0.18 0.25 20.8 35.9 8.2 -0.59 4.4 1.4 5.2 0.85 2.7 --

0.04 0.17 0.26 0.07 0.03 1.2 0.40 0.41 0.10 0.39 0.87 22.5 18.1 0.82 -0.72 1.9 14.2 8.8 7.7 21.4

--t 0.14 1.9 0.05 0.09 4.2 0.72 0.99 0.14 0.34 1.6 29.8 7.4 0.09 0.02 0.53 2.1 11.5 8.9 8.4 21.1

-0.40 0.20 0.03 0.09 6.8 1.4 0.55 0.07 0.23 3.6 50.6 7.1 0.42 -1.2 2.1 4.5 4.1 6.3 10.5

* Trace amounts detected. 1"Not detected. Results are expressed as relative percentage of total fatty acid at each position of each specific glyceride acetate.

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D.J. CHITWOODand L. R. KRUSBERG Table 5. Fatty acid composition of the 1- and 2-positions of 1,2-diacylglycerot acetate, the 2-position of l-alkenyl-2-acylglycerolacetate, and the 2-position of 1-alkyl-2-acylglycerol acetate, derived from ethanolamine phosphoglyceride from Turbatrix aceti Fatty acid

iso- 14: 0 14:0 iso-15:0 15:0 iso-16:0 16:0 16:1 iso- 17:0 17:0 iso-18:0 18:0 18 : 1 18:2 18: 3 20:0 20:1 20: 2 20:3 20:4o96 20:4~o3 20:5

Diacyl l-position

Diacyl 2-position

Alkenylacyl 2-position

Alkylacyt 2-position

0.05 0.22 0.40 0,03 0.02 2.4 0.15 1.9 0.08 0.25 80,7 8,5 0.90 -1.6 1.8 0.25 0.45 0.08 0,31 ---

0.03 0.18 1.9 0.03 0.09 2.0 0.74 0.87 0.01 0.31 0,85 26.6 27.0 0.62 -0.10 0.76 9.6 6.6 7.1 14,6

0,0 t 0.33 14.4 T* 0.34 0.41 0.46 3.2 T 0.21 0,21 50.5 17.6 0.22 0.09 1.5 3.8 1.7 1.4 1.3 2.4

0.05 0.33 5.6 --t 0.18 3.5 0.79 2,5 0.05 0.27 1,6 66.0 7.8 0.20 -1.4 3.9 2.1 0.79 2.3 0,71

* Trace amounts detected. l" Not detected. Results are expressed as relative percentage of total fatty acid at each position of each specific glyceride acetate.

Jenkins, 1966) and the animal-parasitic nematodes Ancylostoma caninum (Khuller et al., 1977), Ascaris lumbricoides (Beames, 1964; Subrahmanyam & Venkatesan, 1968), Dirofilaria immitis (Hutchison et al., 1976), Litomosoides carinii (Subrahmanyam, 1967), Setaria cervi (Ansari et al., 1973) and T. spiralis (Castro & Fairbairn, 1969). Where quantified, the relative proportions of specific phosphoglycerides vary among these genera. Ethanolamine phosphoglyceride consisted of 46.4% diacyl, 43.2% alkenylacyl and 10.5% alkylacyl phosphogtyceride, whereas 89,2% of the choline phosphoTable 6. Fatty aldehyde composition of alkenylacylglycerol acetates derived from choline phosphoglyceride (CP) and ethanolamine phosphoglyceride (EP) from Turbatrix aceti Fatty aldehyde

CP

EP

14:0 15:0 16:0 17:0 18:0 18:1 I9:0 20:0 20:1 20:2

--t -0.02 0.64 89.8 -0.32 0.69 8.3 0.25

0,0t T* 0.02 0.16 88.0 T 0.54 1.2 9.8 0.23

* Trace amounts detected. t Not detected. Results arc expressed as relative percentage of total aldehyde from each glyceride acetate.

glyceride was in the diacyl form, Similarly, the ethercontaining phospholipids of mammalian tissues are also predominantly ethanolamine phosphoglycerides (Horrocks, t972). The only additional quantitative investigations of ether lipids in nematodes have revealed that phospholipid from A. lumbricoides and L carinii contains approximately 20% plasmalogen and 10% alkylacyl phospholipid (Subrahmanyam & Venkatesan, 1968; Subrahmanyam, 1967). Total phospholipid or polar lipid fatty acid compositions have been determined in T. aceti, P. redivivus, 7". spiralis and the plant-parasitic nematodes Meloidogyne arenaria, M. incognita and Globodera solanacearum; each genus examined thus far possesses a characteristic such distribution (Sivapalan & Jenkins, 1966; Table 7. Alkyl group composition of alkylacylglycerol acetates derived from choline phosphoglyceride (CP) and ethanolamine phosphoglyceride (EP) from Turbatrix aceti Alkyl group

CP

EP

16:0

0.94 .--* 81.8 6.2 0.67 10.3 --

0.42 0.01 92.8 0.38 0.71 5.5 0.17

17:0

18:0 19:0 20:0 20:t 20:2

* Not detected. Alkyl groups were analyzed as alkyl isopropylidene glyceryl ethers. Results are expressed as relative percentage of total alkyl group from each glyceride acetate.

Phospholipids of Turbatrix aceti Castro & Fairbairn, 1969; Krusberg et aL, 1973; Orcutt et al., 1978). The fatty acid composition of phospholipid from T. acet/reported herein is similar to that of polar lipid reported previously (Fletcher and Krusberg, 1973), with the exception that in the previous study iso-18:0 acid comprised 11.5% of the polar lipid fatty acid, whereas our present investigation has produced a value of 0.3%. We believe that purification of methyl esters with an alumina column in the previous study resulted in contamination of the methyl esters by dimethylaeetals produced from plasmalogen alkenyl moieties during transesterification. The earlier results can be replicated by GLC of the mixture of TLC-purified methyl esters and dimethylaeetals obtained after transesterification. Polyunsaturated fatty acid from T. aceti was more abundant in choline than in ethanolamine phosphoglyceride. However, the reverse situation is true in the liver fluke Fasciola hepatica (Oldenborg et al., 1975) and in most other organisms (Strickland, 1973). As in the rat tapeworm Hymenolepis diminuta (Ginger & Fairbairn, 1966), diphosphatidylglycerol from T. aceti contained a distinctive abundance of polyunsaturated fatty acids. The 1-positions of choline and ethanolamine phosphoglyeerides contained fatty acids which were predominantly shorter and more saturated than those at the 2-position. Association of saturated acids with the 1-position and unsaturated acids with the 2-position occurs in most organisms (Holub & Kulds, 1978). Since 18:1 acid from T. aceti consists of equal parts oleic (o?) and vaccenic (co7) acids (Krusberg, 1972), investigation of the positional distribution of these acids would be interesting The fatty acid compositions of alkenylacyl and alkylacyl phosphoglyeerides from T. aceti resembled those of the 2-positions of the corresponding diacyl compounds. However, fatty acid esterified to the ether lipids contained a smaller percentage of polyunsaturated acid, with the possible exception of choline plasmalogen. Similar comparisons have not been frequently performed, but ether-containing phosphoglycerides have tended to contain larger percentages of polyunsaturated acids than the 2-positions of their diacyl analogues (Holub & Kuksis, 1978). Choline plasmalogen from T. aceti contained larger proportions of iso-15:0, 20:3, 20:4 and 20:5 acids and a smaller percentage of 18:1 acid than its alkylacyl analogue. Ethanolamine plasmalogen contained larger percentages of iso-15:0 and 18:2 acids but lesser proportions of 16:0, 18:0 and 18:1 acids than its alkylacyl analogue. Some difference in fatty acid composition between alkenylacyl and alkylacyl phosphoglycerides has been described elsewhere (Hotrocks, 1972; Curstedt, 1977), but no common pattern is apparent. The alkenyl and alkyl group compositions of choline and ethanolamine phosphoglyeerides were composed largely of the 18:0 compounds. Among animalparasitic nematodes, alkenyl groups from A. lumbricoides and D. immitis are predominantly unsaturated (Beames, 1964; Hutchison et a£, 1976). The only other nematode in which the alkyl group composition has been investigated is L. carinii, in which only 18:0 alkyl groups were identified (Subrahmanyam, 1967). In summary, variation in fatty acid composition of

119

specific phosphoglycerides occurs in T. aceti, whereas aikenyl and alkyl group compositions vary little. In addition, large differences exist among nematode genera in phospholipid composition and in phospholipid acyl, alkenyl and alkyl group content and composition. Presence of such differences at the level of species remains to be determined. No common patterns in composition of phospholipids or phospholipid constituents among genera living in the same general habitat are yet apparent.

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