Changes in lipids and free fatty acid fractions in adult Haemonchus contortus during incubation in vitro

Veterimny Pomsitology, 23 (1987) 96-103 Eisevier Science Publishers B.V., Amsterdam - printed in The Netherlands

95

CHANGES IN LIPIDS AND FREE FATTY ACID FRACI’IONS IN ADULT HAEMONCHUS CONTORTUS DURING INCUBATION IN VITRO

JYOTIKA KAI’UR and M.L. SOOD Department

of Zoology, Punjab Agricultural University, Ludhiuna-141 004 (Indin)

(Accepted for publication

21 January 1986)

ABSTRACT Kapur, J. and Sood, ML., 1987. Changes in lipids and free fatty acid fractions in adult Haemonchus contortus during incubation in vitro. Vet. Paraeitol., 23: 95-103. Adult Haemonchw contortu (Rud., 1803) has been investigated for its ability to utilize lipids with regard to the total lipids, sterois, free fatty acids, acyiglycerole and phospholipids produced during incubation in vitro. AI1 these components exhibit extensive fluctuations, decreasing at some times and increasing at others, thus indicating both biosynthesis and utilization. Aiso, changes in fatty acid components of total lipids have been analyzed by gas liquid chromatography. These observations indicate that H. contortu is capable of utilizing 16:0, 18:0, 18:1,18:2, 20:0, 20:un,, 2O:u%, 21:0, 21:un,, 21:1, 21:~~. 22:un,, 22~3, 22:?, 22:un, and 24~3 fatty acids. At the same time, because of iarge increases in 16:0, 16:0, 18:0, 18:?, 18:1, 18:2, 20:0, 20:un,, 2O:uq, 21:0, 21:un,, 21 :l. and 21:~~ acids, it is postulated that these acids are synthesized by adult H. contortus.

INTRODUCTION

Carbohydrates are the major energy reserve in nematodes (Barrett, 1981). However, the role of lipids cannot be overlooked. Only a few studies have been made with regard to lipid utilization in parasitic helminths. These include studies on Ascaris (Schulte, 1917; Mueller, 1929; Hirsch and BretSchneider, 1937; Greichus and Greichus, 1967) and Hymenokpis diminutu (Over-turf and Dryer, 1968). In Haemonchus contortza, no report is available regarding lipid utilization by the adult worms in vivo or in vitro. Nematodes usually contain lipids in considerable amounts (von Brand, 1979); these are usually utilized for energy production under aerobic conditi0ns.H. contortus is capable of aerobic metabolism (Rogers, 1949; Ward, 1974). Starvation experiments provide the best means of studying endogenous substrate mobilization (Overturf and Dryer, 1968). Therefore, in the present studies in vitro incubations were carried out to investigate the utilization of total lipids and the major fractions in H. contortus. Evidence of individual fatty acid utilization in nematodes is also sparse;

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96

only Ascaris (Greichus and Greichus, 1966, 1967) and Panagrellus rediuiuus (Sivapalan and Jenkins, 1966) have been investigated in this regard. Therefore, in the present studies, changes in individual fatty acids have also been studied during incubation in vitro. MATERIALS AND METHODS

Adults of H. contortus were collected from the abomasa of goats from local abattoirs. The worms were thoroughly washed in physiological saline in order to remove the adhering materials. Incubation medium The incubation medium used in the present investigation! had the following composition: 10.7 g NaCl; 0.2 g KCl; 0.265 gCaC1,*2H,0;0.21 g MgC1,*6H,O; 1.0 g NaHCO,; 0.06 g NaHzP04; 1.0 g glucose; 3.0 g streptopenicillin; 0.01 g nystatin, with the volume made up to 1 1 with double distilled water. Final pH was adjusted to 6.6 f 0.1. This medium, with glucose concentration of 1 g 1-l is the most suitable for H. contortus incubations (Ward, 1974), though the concentration of glucose in sheep blood is 0.6 g 1-l. Batches of live adult worms of both sexes in numbers equivalent to 0.5 g were incubated at 39 f 1°C in 10 ml of the incubation medium for 0, 4, 8, 12, 16, 20 and 24 h. These incubations were carried out in triplicate batches for each period of incubation. After the completion of the respective incubation period, worms were recovered from the media and frozen at -20°C till further processing. Lipids were extracted in chloroform:methanol (2: 1 v/v) by homogenizing the worms. Water-soluble impurities were removed by washing the crude lipid extracts (Folch et al., 1957). Chloroform phases. containing lipids were collected and stored at -20” C until the analyses were completed. Estimation of lipids Total lipid content was determined gravimetrically by drying aliquots of lipid samples. Sterols were estimated by the method of Stadtman (1957), free fatty acids by the method of Lowry and Tinsley (1976), acylglycerols by the method of Van Handel and Zilversmit (1957) and phospholipids by the method of Ames (1966). Fatty acid analysis For fatty acid analyses, aliquots of the lipid samples were dried and converted to the methyl esters by the method of Christie (1973). Esters thus obtained were analyzed by gas liquid chromatography using a stainless

97

steel column, packed with 20% diethyl glycol succinate on 60-80 mesh chromosorb-W. Conditions for the separation were column temperature, 195°C; flow of nitrogen, 60 ml min-‘; hydrogen flow, 40 ml min-‘; air 300 ml min-‘. Peaks were identified by comparing their retention time with those of standard esters, under similar conditions. Results are expressed as mean f S.D. Student’s t-test for unpaired data ~88 used to test the significance of the difference. RESULTS

Results of quantitative changes in total and various classes of lipids of H. contortus during incubation are given in Table I and Fig. 1, respectively. It is evident that with increase in the period of incubation, there was a significant increase in total lipids after 4 (P>O.OOlO.OOl
r

-

STEROLS

C---W

FREE

FATTY

ACIDS

b-gPHOSPHOLIPIOS e----9

TRIACVLGLYC

txoLs

4.5 -

4.0 -

3.5 -

4

6 TIME

Fig.

1.

in vitro.

Quantitative

12

16

PERIOD

change6

20

24

(hr)

in major

fraction6

of lipids of

H. contortus

during

incubation

98

TABLE I Quantitative

changes in the total lipids of Haemonchus

contortus during incubation

in vitro

Time (h) 0 4.419

+0.3

4

8

12

5.457 io.22

3.760 20.41

5.300 kO.31

Results are expressed

16 9.447 to.36

26 8.597 to.35

24 6.508 to.40

as mg g-’ fresh tissue, mean * S.D.

nificant decrease (P >O.OOl O.Ol <0.05) after 20 h. This was followed by a significant decrease (P >O.OOl O.OOl) relative to that in phospholipids (P0.01). Levels of free fatty acids increased during the first 4 h, followed by a decrease in the next 4 h. After 12 h there was a further slight increase followed by a decrease. Thereafter, there was a significant increase (X0.1 >0.05). Results of the analysis of long-chain fatty acids during incubation are given in Table II, from which it is evident that there was no significant difference (P>O.5) in the fatty acid composition of controls and of the worms incubated for 4 h,except n16:0,18:0,18:1, 21:unl acids, which were reported to increase during incubation. At the same time, 15:0, n1’7:0, 17:1, 21:1, 22:?, 22:un, and 24:3 decreased significantly (P O.OOl). There was a complete disappearance of 18:?. After 8 h, a significant decrease (P < O.Ol- < 0.001) was observed in 15:0, 17:1, 20:0, 21:un,, 22:?, 24:3 FA and increase in n16:0, n17:0, 18:0, 18:l and 18:2 acids. On the other hand, 16:un, 21:0, 22:un and 24:?, acids completely disappeared. When the worms were incubated for a total period of 12 h, there was a complete disappearance of 12:1, 21:0, 21:1, 21:un,, 22:un, acids; and ant iso 17:0, 18:0, 18:1, 20:unz, 22:?, 23:3 decreased. This was accounted for by an increase in 14:1, 15:0, n16:0, 16:un,, 17:1, 18:2, 20:0, 20:unl, 22:unz, 24:3 and 24:?, acids. At 16 h of incubation a few acids, which had disappeared after 12 h, reappeared. These included 12:1, 21:1, 22:uq acids, the concentration of 12:l being significantly (P <<
99

for by a corresponding decrease in n16:0, 18:0, 18:l and the complete disappearance of 12:1, 16:un,, ant iso 17:0, 18:?, 18:2,22:un,, 22:unz, 22:3, 24:?1, 24:?,, 24:3 acids. 18:l was reduced to about haIf the value of control worms. 22:? acid increased to about 3-fold and 24:?2 about IS-fold the value of control worms. After one day of incubation 14:0, 15:0, n17:0, 18:?, 18:1, 18:2, 20:un,, 21:un,, 22:3, 22:un, and 24:?* increased. This was accounted for by a corresponding decrease in n16:0, 18:0, 20:un1, 21:1, 21:unz, 22:? and complete disappearance of iso 16:0,17:1,18:un1 and 21:0. Increase in 24:?, acids was highly significant (P<<
It is not surprizing that the nematodes living under anaerobic conditions are not able to utilize lipids (von Brand and Bowman, 1961; Fairbairn et al., 1961; Greichus and Greichus, 1966,1967). On the other hand, SchuIte (1917) and Beames et al. (1968) reported a slight increase in total lipids of Ascaris during starvation. Lipid utilization of H. contortus, as reported in the present studies was expected, since it is capable of aerobic metabolism and a fully functional TCA cycle is present (Ward and Huskisson, 1978). The decrease in lipid content during the initial incubation may be accounted for by the turnover of membranes of lipids excreted/secreted into the medium and by expulsion of eggs. Simultaneous increase in lipid content may be accounted for by lipid biosynthesis. It is weII established that 60-70s of total lipids is located in the reproductive system and eggs of Ascaris. Therefore, it is reasonable to assume that some lipid catabolism could occur in the muscle and/or intestine during incubation, although the overah results of analysis of whole worms would show by lipid synthesis, associated with egg production. Evidence is available to account for lipid utilization in lateral Iine tissues and intestinal waII (Mueller, 1929; Hirsch and Bretschneider, 1937). An increase in lipids of body waII, reproductive system and eggs was reported during starvation of Ascaris (Beames et al., 1968). The same may hold true for H. contortus. Therefore, further studies need to be carried out involving isolated organs. AII the investigations of lipid utilization carried out so, far in nematodes have considered only the total lipids. In the present studies, changes in different fractions have also been studied. Decreases inphospholipids and acylglycerols might be associated with the expulsion of eggs; phospholipids and sterols are also important components of membranes and their loss might be associated with membrane turnover. More pronounced decreases in acylglycerols than in phospholipids were expected, since phospholipids are the structural entities, while acylglycerols are the main lipid energy reserve. Regarding free fatty acids, an increase was expected, being an end product of carbohydrate catabolism along with CQ, propanol, ethanol etc. (Ward, 1974). A decrease is accounted for by their incorporation into eggs and also in excretion/secretion products.

0

Time(h)

4

12:l 0.187f 0.014 0.193i 0.023 14:o 0.691t 0.033 0.800* 0.074 14:l 1.2402 0.232 l.OSl* 0.148 15:o 1.118f 0.199 0.709* 0.097 iao16:0 0.119f 0.019 0.087f 0.037 n16:O 7.463* 0.222 9.56Oi 0.333 16:un, 0.355f 0.149 0.409f 0.109 antiso 0.957f 0.140 0.835* 0.199 17:o n17:O 1.069* 0.151 0.287* 0.116 17:l 0.664i 0.122 0.3OOi 0.208 18:0 11.259i 0.348 13.912* 0.261 18:? 0.037f 0.035 21.587* 0.256 23.287+C0.241 18:l 12.820* 0.178 13.038* 0.210 18:2 20:o 0.435f 0.153 0.376i 0.148 20:un, 0.655* 0.162 0.556* 0.167 3.410* 0.282 3.398i 0.232 2o:un, 21:o 0.653* 0.090 0.607t 0.112 21:uq 7.457* 0.274 8.513* 0.350 21:l 2.328* 0.336 1.469* 0.238 21:un, 0.729i 0.196 0.721i 0.197 1.059f 0.134 0.668f 0.145 22:un, 22:3 2.394+_0.240 2.423i 0.286 22:? 9.292i 0.489 8.126i 0.200 0.162* 0.050 0.097* 0.026 22:un,

Fatty acid* 16

20

0.023 0.499 f 0.019 0.019 1.233* 0.220 1.299f 0.143 0.832f 0.153 0.201 1.830f 0.174 1.097f 0.144 0.988f 0.027 0.059 1.627f 0.237 0.874* 0.130 1.075f 0.135 0.019 0.427* 0.119 0.0415 0.038 0.147f 0.043 0.256 15.41 f 0.399 15.613f 0.277 11.714* 0.204 1.622* 0.318 0.545* 0.196 0.137 0.324* 0.112 1.055* 0.235 -

12

duringincubationin vitro

2.335* 0.222 0.543f 0.146 2.300f 0.282 7.780f 0.414 -

24

0.570f 0.149 1.083i 0.121 0.637* 0.193 0.361* 0.157 2.488* 0.331 0.068* 0.061 1.084* 0.172 1.380f 0.310 16.012i 0.195 0.907* 0.176 16.432* 0.292 12.427t 0.318 7.708* 0.302 0.093i 0.038 2.330* 0.091 26.645* 0.301 19.518i 0.286 28.600* 0.304 10.656i 0.308 14.561f 0.247 16.105* 0.196 32.458* 0.321 11.255* 0.297 13.594t 0.284 0.065* 0.057 0.446i 0.154 2.486* 0.317 0.140f 0.053 0.459* 0.158 7.933* 0.252 0.043* 0.040 4.358f 0.357 0.077f 0.020 3.114f 0.167 0.445f 0.157 4.051* 0.248 3.219f 0.222 6.523f 0.306 1.928f 0.193 2.361t 0.264 2.691* 0.269 7.517* 0.206 10.571* 0.357 4.244* 0.270 2.369i 0.256 1.96 f 0.181 0.996* 0.199 5.058f 0.250 0.716* 0.197 3.269f 0.302 6.285f 0.261 0.417f 0.209 4.016i 0.789 1.195f 0.210 0.916f 0.194 1.386f 0.266 5.02oi 0.199 1.575* 0.323 5.570* 0.246 31.909* 0.328 4.340f 0.360 0.059* 0.026 2.050* 0.247 1.912f 0.183

0.217t 0.973f 0.998* 0.565* 0.098* 13.137f 0.78 *

8

Quantitative changesinthevariousfatty acidsof Haemonchuscontortulr

TABLE11 s 0

1.511 1.639 1.540 4.713

0

f i i f

Time (h)

0.267 0.396 0.338 0.365

4

1.679 1.460 0.431 3.714

*, Relative per cent basis. ?, Unidentified. -, not detected. Un, unsaturated. Results are expressed as mean f S.D.

? 24 :?, 24:?, 2413

Fatty acid*

TABLE II (continued)

f * f i

0.262 0.244 0.220 0.437

8 2.607 t 0.244 1.083 i 0.166 2.067 f 0.144

0.966 1.684 1.236 6.617

12 * * i f

0.100 0.166 0.209 0.194 1.413 f 0.312 1.350 i 0.227 1.361 * 0.263 -

16 -

20

16.796 f 0.351 -

1.691 i 0.161 -

24

102

Only a few studies are available in nematodes which report changes in fatty acid composition during incubation. All the fatty acids remain constant in starving Ascaris (Greichus and Greichus, 1967), except 16:l which decreases slightly in the free fatty acid fraction. However, in Punagrellus rediuiuus (SivapaIan and Jenkins, 1966), Ci2-C& fatty acids decrease, 20:4 does not show any change and 18:O increases slightly. Similarly, H. contortus has been demonstrated to be capable of utilizing most of the fatty acids, viz. 12:1, 14:1, 16:0, 17:0, 18:0, 18:1, 18:2, 24:3 and 20, 21 and 22. At the same time, 15:0,16:0,18,20 and 21 carbon acids increase, indicating their biosynthesis. Since absolute quantitative data were not obtained, it is impossible to determine that there was a net increase or decrease in any particular acid. However, due to great decrease in 16:0, 18:0, 18:1, 18:2, 24:3 and 20, 21, 22 carbon acids, it is reasonable to assume that these acids are actually utilized by the worms during incubation. At the same time, a great increase in the percentage of 15:0, 16:0, 18, 20 and 21 carbon acids leads us to postulate that H. contortus is capable of synthesizing these acids. To confirm this, radio-GLC studies need to be carried out. However, H. contortus is capable of lipid biosynthesis including fatty acids from C14-acetate and glucose, although the individual components of fatty acids have not been analyzed (Kapur and Sood, 1984). ACKNOWLEDGEMENT

J. Kapur is grateful to the authorities of C.S.I.R., New Delhi for a Senior Research Fellowship grant.

REFERENCES Ames, B.N., 1966. Assay of Inorganic Phosphate, Total Phosphate, and Phosphatases. In: E.F. Neufeld and N. Ginsburg (Editors), Methods in Enzymology, Vol. III. Academic Press, New York, 115 pp. Barrett, J., 1981. Biochemistry of Parasitic Helminths. MacMillan, Hong Kong, 308 PP. Beames, C.C., Jr., Jacobsen, N.S. and Harrington, G.W., 1968. Studies on lipid metabolism of Ascaris during starvation. Proc. Okla. Acad. Sci., 47: 40-44. Christie, W.W., 1973. Preparation of volatile derivatives of lipids. In: Lipid Analysis, Pergamon Press, New York, pp. 85-89. Fairbairn, D., Wertheim, G., Harpur, R.P. and Schiller, E.L., 1961. Biochemistry of normal and irradiated strains of Hymenolepis diminuta. Exp. Parasitol., 11: 248263. Folch, J., Lees, M. and Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-609. Greichus, A. and Greichus, Y.A., 1966. Chemical composition and volatile fatty acid production of male Ascaris lumbricoides before and after starvation. Exp. Parasitol., 19: 85-90. Greichus, A. and Greichus, Y.A., 1967. Body-fat composition of male Ascaris lumbricoides before and after starvation. Exp. Parasitol., 21: 47-52.

103 Hirsch, G.C. and Bratschneider, L.H., 1937. Die Arbeitaraume in den Darmzellen von Ascork Die Einwirkung des Hungerns. Die Sekretbildung Cytologia Fujii Jubilaei Volumen, 424-436. Kapur, J. and Sood, M.L., 1984. Haemonchue contortus: lipid biosynthesis from Cl’labelled acetate and glucose. Zbl. Vet. Med. B., 31: 226-230. Lowry, RR. and Tinsley, L.J., 1976. Rapid calorimetric determination of free fatty acids. J. Am. Oil Chem. Sot., 63: 470472. Mueller, J.F., 1929. Studies on the microscopical anatomy and physiology of As~arb lumbricoides and Aecari8 megalocephala. Z. Zelfforsch. Mikrosk. Anat., 8: 361-403. Overturf, M. and Dryer, R.L., 1968. Lipid metabolism in the adult cestode Hymenolepis diminuta. Comp. Biochem. Physiol., 27: 146-176. Rogers, W.P., 1949. On the relative importance of aerobic metabolism in small nematode parasites of the alimentary tract. 2. The utilization of oxygen at low partial pressures by small nematode parasites of the alimentary tract. Aust. J. Sci. Ree., B., 2: 166177. Schulte, H., 1917. Versuch uber Stoffwechselvargange bei Ascari8 lumbricoides. Pflug. Arch. Gesch. Physiol., 166: l-44. Sivapalan, P. and Jenkins, W.R., 1966. Effect of starvation on the long chain fatty acid composition of the free-living nematode Panagrellus rediuiuwr. Nematologica, 12: 99. Stadtman, T.C., 1967. Preparation and Assay of Cholesterol and Ergosterol In: S.P. Colowick and N.O. Kaplan (Editors), Methods in Enzymology, Vol. III. Academic Press, New York, p. 392. Van Handel, E. and Zilversmit, D.B. 1957. Micromethod for direct determination of serum triglycerides. J. Lab. Clin. Med., 60: 162-167. Von Brand, T., 1979. Biochemistry and Physiology of Endoparasitea. Elsevier/North Holland Biomedical Press, N.Y., Oxford, p. 447. Von Brand, T. and Bowman, I.B.R., 1961. Studies on the aerobic and anaerobic metabolism of larval and adult Taenia taeniaeformis. Exp. Parasitol., 11: 276-297. Ward, P.F.V., 1974. The metabolism of glucose by Haemonchus contortus, in vitro. Parasitology, 69: 175-190. Ward, P.F.V. and Huskisson, N.S., 1978. The energy metabolism of adult Haemonchus contortus in vitro. Parasitology, 77: 266-271.