Composition and fatty acid content of rat ventral prostate phospholipids

Biochimica et Biophysics Acta 879 (1986) 51-55 Elsevier

BBA 52402

C~m~sition

and fatty acid content of rat ventral prostate phospholipids

J.A. Pulido, N. de1 Hoyo and M.A. PQez-Albarsanz Departamento de Bioquimica y Biologia Molecular, Uniuersidad de Alcald de Henares, Madrid (Spain) (Received

Key words:

Phosphohpid;

21 April 1986)

Fatty acid; Piasmalogen;

(Rat ventral

prostate)

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphors. The prostate also contained small amounts of serine gIycerophospholipids and sphingomyeiin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and steak aldehydes. Ethanolamine pIasm~ogens also contained the vinyl ether of oleic aldehyde.

Introduction The relationship between phospholipids and biomembrane processes has been well documented in several reviews [l-4] and the effect of phospholipids on the functioning of some membrane enzymes is associated with many physiological activities [S-7]. Phospholipids also participate in complex processes both as substrates and chemical mediators [S]. Although phospholipid metabolism has been extensively studied in several animal tissues [9-111, little attention has been directed towards the prostate gland, either under normal or pathological conditions. However, phospholipids play an im-

Abbreviations: PC, choline glycerophospholipids; PE, ethanolamine glycerophospholipids; PS, serine glycerophosphohpids; GLC, gas-liquid chromatography: MS, mass spectrometry. Correspondence address: Dr. M.A. Perez-Albarsanz, Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala de Henares, Madrid, Spain 00052760/86/$03.50

0 1986 Elsevier Science Publishers

portant role in the maintenance of prostate gland structures and in its biochemical functions. Previous data from our laboratory [K-14] indicate that phospholipid biosynthesis in rat ventral prostate is a development-dependent process which is under the control of sex steroid hormones, It has been also shown that arachidonic acid, a precursor of prostaglandins, is a major component of these phospholipids [12]. Properties of membranes can be markedly influenced by the phospholipid fatty acid composition, particularly by their degree of unsaturation [15]. The deficiency of essential fatty acids is known to result in sterility in both male and female rats [16]. The fatty acid composition has been determined for the lipids from testes, ovaries, ovarian follicles and corpora lutea [16-181. However, precise information concerning the fatty acid composition of the various phosphohpid classes present in the prostate gland has not yet been obtained. We report here a detailed analysis of phospholipid composition of the rat ventral prostate, as well as the fatty acid pattern.

B.V. (Biomedical

Division)

52

Materials and Methods Animals and chemicals. Young mature male Wistar rats (65-75 days, 280-340 g body weight) were housed in a temperature-controlled room and maintained on a standard laboratory diet and water ad libitum. The animals were killed by exsanguination under light diethyl ether anaesthesia. The ventral prostate was removed, stripped of connective and adipose tissues, weighed in a Teflon vessel and kept in an ice-water bath until use. All the reagents used were analytical grade. Standard fatty acids were purchased from Carlo Erba (Milan, Italy) and Alltech Associates (Deerfield, IL, U.S.A.). Standard phospholipids were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Phospholipid extraction, separation and quantification. Phospholipids from rat ventral prostate were extracted by the method of Bligh and Dyer [19]. Different classes of phospholipids were separated by thin-layer chromatography on silica. gel G plates (Merck, Darmstadt, F.R.G.) using propionic acid/propanol/chloroform/water (2 : 2 : 1 : 1, by volume) [20]. The phospholipid classes were determined as previously described [12]. Fatty acid methyl esters and dimethyl acetals. Purified phospholipids of ventral prostate were directly treated with BF,/CH,OH to obtain the fatty acid methyl esters and dimethyl acetals. A Pye Unicam Philips gas chromatograph equipped with a flame ionization detector was utilized to assay the products. (2 m glass column (2 mm internal diameter) packed with 20% diethylene glycol succinate (Merck, Darmstadt, F.R.G.) on SO/l00 Chromosorb WHP (Alltech Associates, Deerfield, IL, U.S.A.); with nitrogen carrier gas (flow rate, 30 cm3/min)). The initial temperature of 150°C was maintained for 8 min followed by a linear temperature program from 150 to 178’C at 4 Cdeg/min. Fatty acids were identified by comparing their retention times with those of standards. The identity of fatty acids was confirmed by GLC before and after catalytic hydrogenation with paladium. Quantitation of fatty acids and dimethyl acetals was based on the internal standard, undecanoic acid. Gas chromatography-mass spectrometry. Mass spectra were taken at 70 eV using a Finnigan 4020

(quadrupole) mass spectrometer interfaced to a Finnigan 9610 gas chromatograph equipped with a Carbowax 20M capillary column (30 m x 0.28 mm internal diameter). Statistical treatment of the results. Results were expressed as mean & S.E. The significance of differences between the various groups was determined by Student’s t-test. Results Phospholipids were the most abundant lipid class in the prostate, comprising 38-43% of total lipids. The predominant phospholipid classes were choline glycerophospholipids (PC) (52.3%) and ethanolamine glycerophospholipids (PE) (25.6%), together accounting for 77.9% of total lipid extractable phosphate (Table I). Small amounts of serine glycerophospholipids (PS) (10.6%) and sphingomyelin (4.5%) were also found (Table I). Lysophosphatidylcholine, lysophosphatidylethanolamine and phosphatidylinositol were present only in minor amounts less than 4% (data not shown). The major phospholipid components (PC, PE, PS and sphingomyelin) were characterized as to their fatty acid composition (Table II). Methyl esters of palmitic (43.3%) and oleic (38.3%) acids were the major components in the methanolysates of PC from the prostate. Acid methanolysates of the other three major phospholipid classes in the prostate contained small amounts of the methyl ester of palmitic acid (PE 7.2%, PS 15.1% and sphingomyelin 25.5%). Table II also shows that arachidonic acid was only a major fraction of the

TABLE

I

PHOSPHOLIPID PROSTATE

COMPOSITION

Results are meansfS.E. sphingomyelin.

of six duplicate

Phospholipids

nmol/mg

PC PE PS SM

280 f 16 144k 8 57+ 1 29& 3

’ Percentages

of phospholipid

(TL). b Based on percentage

OF

TL

RAT

VENTRAL

determinations.

SM,

weight% a

mol% b

22.9 f 1.3 11.2kO.6 4.6 + 0.1 2.0*0.2

52.3 26.6 10.6 4.5

classes in relation

of total lipid P.

k + + *

2.1 1.3 0.5 0.2

to total lipids

53

TABLE

II

FATTY

ACID COMPOSITION

OF PC, PE. PS AND SPHINGOMYELIN

IN RAT VENTRAL

PROSTATE

Fatty acids were analysed as methyl esters. A, B and C are dimethyl acetals, as described in Results. (weight%)+S.E. were taken from different samples (n = 6). ( -) = no detected or present at less than 0.2%. Fatty acid 14:o 15:o A

PE

PC 0.9+0.1 0.5 * 0.02 1.0+0.1

16:O 16:l 17:o B C

43.3 2.2 0.2 0.2 _

k * f f

2.9 0.3 0.09 0.03

18:0 18:l 18:2 20:o 20:1+18:3

5.8 38.3 4.3 _ 0.4

k 0.3 * 3.1 + 0.3 * 0.1

PS

0.4 * 0.01 _ 14.7 * 0.2

The mean

SM

0.5 + 0.08 1.7 * 0.07 1.4kO.2

2.7 f 0.02 2.4 f 0.09 _

7.2*0.1 1.6_+0.1 _ 13.2 f 0.7 8.8 f 0.3

15.1*1.9 1.1+0.1 0.7 * 0.1 1.1 kO.1 _

25.5 & 3.7 1.3kO.2 2.4? 0.2

6.6 30.5 4.7 _ 0.9

23.7 ir 1.2 35.4* 2.4 2.1 * 0.2 2.8 * 0.2 1.2+0.2

8.5*1.2 11.9k1.8 1.2kO.2 4.3 * 0.4 _

2.1 f 0.1 5.1*0.5 2.3 k 0.1 2.0 + 0.1 1.7kO.l

5.2 k 0.6 13.9k2.1 _

k 0.6 * 0.4 + 0.1 f 0.05

20:2 20:3+22:0 20:4

0.7*0.1 0.2 * 0.02 1.1*0.1

0.5 f 0.03 1.2kO.l 9.4kO.2

20:5 22:4+24:1

_ 0.6 5 0.07

0.2 f 0.04 _

PE fraction (9.4%). PS contained mainly oleic acid (35.4%), as well as substantial amounts of saturated fatty acids (16 : 0 and 18 : 0). Sphingomyelin fatty acids exhibited a high percentage of 20-, 22- and 24-carbon chain length. However, arachidonic acid was not a major fatty acid in this phospholipid class. Analysis of rat prostate phospholipid fatty acids by GLC showed three unidentified peaks (A, B and C in Table II). These peaks were present at highest levels in the chromatograms of PE. In order to recognize these peaks, GLC was performed before and after catalytic hydrogenation of PE and PC methanolysates (Fig. 1). Peak A showed a similar percentage before and after hydrogenation, demonstrating that this molecular species is saturated. Fig. 1 shows the disappearance of peak C after hydrogenation, whereas peak B increased in a similar proportion. These results demonstrate that peak B is saturated; peak C is unsaturated with the same carbon chain length as peak B. Characterization of unidentified prostate PE acid methanolysates by GLC-MS shows the typical fragmentation of long carbon chain dimethyl

percentages

_

9.8 kO.3 10.8kl.O

acetals [21]. The mass spectra of GLC peaks A and B were dominated by oxygen-cotnaining fragments of m/z 75. Long-chain dimethyl acetals do not give the molecular ion (M)+. However, the identity of dimethyl acetals was established from the (M - 31)+ ion. The mass spectrum of peak A present in the PE chromatogram showed an (M - 31)+ ion at m/z

TABLE

III

ALK-l-ENYL COMPOSITION VENTRAL PROSTATE

OF PE, PC AND PS IN RAT

Results are expressed as nmol/mg total lipids f S.E.M. (n = 6) of the dimethyl acetals obtained by acid-catalyzed methanolysis of the alk-1-enyl group of plasmalogens. The alk-1-enyl group is specified by carbon number and by the number of double bonds (in addition to the double bound in the vinyl linkage). Alk-1-enyl groups

PE

PC

PS

16:0 18:0 18:l

40.0 rt 1.6 34.2 k 2.1 22.8 * 1.0

5.1 f 0.4 l.OkO.5 _

1.5kO.8 1.1 kO.6

Total

97.0

6.1

2.6

54 %

r

40

n

Before

Hydrogenation

0

After

Hydrogenation

30 20

PC

10

I

0

l-l

d-l

I

% 30 20

PE

10

al-

I I

I

14:0

A

16:0 16:l

B

C

I

d-l

1 3

l&O 18:l 18:2 20:0 2O:l 20:2 22:0 20:4 20:5 18:3

20~3

Fig. 1. Fatty acid composition of PC and PE of rat ventral prostate before (w) and after (0) catalytic Fatty acids and dimethyl acetals were designated and analysed as described in Table II.

255 corresponding to l,l-dimethoxyhexadecane (molecular weight 286). The mass spectrum of peak B present in the PE chromatogram showed an (M - 31)+ ion at m/z 283 corresponding to l,l-dimethoxyoctadecane (molecular weight 314). Peak C of PE chromatogram was identified by comparing its relative retention time with those reported in the literature [22]. GLC peak C has only one unsaturation site, corresponding to l,ldimethoxyoctadecene. Dimethyl acetals were the products of acidcatalyzed methanolysis of the alk-1-enyl groups of the plasmalogens (Table III). PE, PC and PS plasmalogens were composed predominantly of the vinyl ethers of palmitic and stearic aldehydes. PE plasmalogens also contained the vinyl ether of oleic aldehyde. Comparing Tables I and III on the basis of a one molar relation between plasmalogens and dimethyl acetals [23-251, we concluded that the PE fraction of rat ventral prostate contained 65-69 mol% plasmalogens. PC and PS fractions contained less than 5 mol% of plasmalogens.

Discussion The results of the present study demonstrate the high PC contents of rat ventral prostate. The

hydrogenation

with paladium.

percentage of PE is half of that for PC, and other phospholipids are found in smaller amounts. These data agree with the percentages reported by Rajalaksmi et al. [26] for testis and male accessory reproductive organs of the hamster. Rat ventral prostate PE contains high amounts of plasmalogens, whereas vinyl ether linkages are detected in PC and PS in only minor proportions. Comparison of the phospholipid composition in rat prostate to that in other rat tissues [27,28] shows that the PC-to-PE ratio is similar in lung, liver, erythrocytes, testis and prostate. In contrast, the PC to PE ratio in brain, heart and kidney is different from that of prostate. Diagne et al. [27] observed the generally high occurrence of ethanolamine plasmalogens in brain, lung and testis, whereas choline plasmalogens were found in very low amounts in all rat tissues investigated with the exception of heart, as reported here. Compared to several other rat tissues, the prostate contains less sphingomyelin and more PS, &en though the proportion of these phospholipids is relatively low. It appears, as previously suggested by Esko and Raetz [ll], that compositional differences are generally greater among tissues in one animal, rather than in a particular tissue in different animal species.

55

Prostate sphingomyelin exhibited a fatty acid profile characterized by a high content of 20-, 22and 24-carbon unsaturated chains in the absence of substantial amounts of arachidonic acid. Evidence for a different profile in sphingomyelin fatty acids isolated from embryonic chick heart and canine myocardial sarcolemma has been presented previously [29,30]. Prostate plasmalogens were composed mainly of the vinyl ethers of palmitic, stearic and oleic aldehydes. A similar type of composition has been reported for rat brain, heart and kidney phospholipids [22,31]. Present results show that the different phospholipid classes of rat ventral prostate contain mainly 16 : 0, 18 : 0, 18 : 1 and 18 : 2, as well as low proportions of long-chain polyunsaturated fatty acids. Fatty acid composition of rat prostatic lipids has been reported previously 1121. However, these studies have only reported the fatty acid analysis of total phospholipids. The data presented in this work indicate that the content of 20 : 4 fatty acid in rat ventral prostate phospholipids is due essentially to the higher 20 : 4 level in PE. It should be emphasized that arachidonic acid in glycerophospholipids behaves as a precursor of prostaglandins, the rate-limiting step for synthesis of prostaglandins being the hydrolytic release of arachidonate from glycerophospholipids [32,33]. Acknowledgements The authors thank Dr. S. Nitz (Lehrstuhl fir Chemisch-Technische Analyse u. Chemische Lebensmitteltechnologie, Technische Universit~t, Miinchen) for performing the mass spectral analyses, and Dr. J. Calderon (Inst. Q&mica Organica, Consejo Superior de Investigaciones Cientificas, Madrid) for catalytic hydrogenation of samples. References 1 Mom?, D.J., Kartenbeck, J. and Franke, W.W. (1979) Biochim. Biophys. Acta 559, 71-152 2 Etemadi, A.H. (1980) Biochim. Biophys. Acta 604,423-475 3 Quinn, P.J. (1981) Prog. Biophys. MOB. Biol. 38, l-104 4 Pagano, R.E. and Se&t, R.G. (1985) Trends Biochem. Sci. 10, 421-425

5 McMurchie, E.J. and Raison, J.K. (1979) Biochim. Biophys. Acta 554, 364-374 6 McMurchie, E.J., Gibson, R.A., Abeywardena, M.Y. and Charnock, J.S. (1983) B&him. Biophys. Acta 727, 163-169 7 McMurchie, E.J., Abeywardena, M.Y., Chamock, J.S. and Gibson, R.A. (1983) B&him. Biophys. Acta 760, 13-24 8 Hanahan, D.J. and Nelson, D.R. (1984) Lipid Res. 25, 1528-1535 9 Horrocks, L.A. (1972) in Ether Lipids (Snyder, F., ed.), pp. 177-272, Academic Press, New York 10 Horrocks, L.A. and Sharma, M. (1982) in New Comprehensive Bi~he~st~ (Hawthorne, J.N. and Ansell, G.B., eds.), Vol. 4, pp. 51-93, Elsevier Biomedical Press, Amsterdam 11 Esko, J.D. and Rae&. C.R.H. (1983) in The Enzymes (Boyer, P.D., ed.), Vol. 16, pp. 207-253, Academic Press, New York 12 Perez-Albarsanz, M.A., Del Hoyo, N., Alcaide, A. and Recio, M.N. (1982) Comp. Biochem. Physiol. 72B, 673-675 13 Del Hoyo, N., Terre, L.A. and Perez-Albarsanz, M.A. (1984) Comp. Biochem. Physiol. 78B, 299-302 14 Recio, M.N., Del Hoyo, N., Carmena, M.J. and Ptrez-Aibarsanz, M.A. (1985) Int. 3. B&hem. 17, 1129-1132 15 Shinitzky, M. and Barenholz, Y. (1978) Biochim. Biophys. Acta 515, 367-394 16 Holman, R.T. and Hofstetter, H.H. (1965) J. Am. Oil Chem. Sot. 42, 540-544 17 Huang. Y.S., Horrobin, D.F.. Manku, M.S., Mitchell, J. and Ryan, M.A. (1984) Nutr. Res. 4, 719-726 I8 Cunnane, SC., Horrobin, D.F. and Manku, M.S. (1984) Proc. Sot. Exp. Biol. Med. 177, 44-446 19 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 91 l-91 7 20 Alonso, F., Garcia Gil, M., Sanchez-Crespo, M. and Mato, J.M. (1982) J. Biol. Chem. 257, 3376-3378 21 Eight Peak Index of Mass Spectra (1983) Vol. 1, part 2, Mass Spectrometry Data Centre, Nottingham 22 Myher, J.J. and Kuksis, A. (1984) Can. J. Biochem. Cell. Biol. 62, 352-362 23 Rapport, M.M. (1984) J. Lipid Res. 25, 1522-1527 24 Naughton, J.M. and Trewhella, M.A. (1984) J. Neurochem. 42, 685-691 25 Gross, R.W. (1985) Biochemistry 24. 1662-1668 26 Rajalakshmi, M., Reddy, P.R.K. and Prasad, M.R.N. (1973) J. Endocrinol. 58, 349-350 27 Diagne, A., Fauvel, J., Record, M., Chap, H. and DousteBlazy, L. (1984) Biochim. Biophys. Acta 793, 221-231 28 Kaya, K., Miura, T. and Kubata, K. (1984) B&him. Biophys. Acta 796, 304-311 29 Wood, R. (1974) Lipids 9,429-439 30 Gross, R.W. (1984) Biochemistry 23, 158-165 31 Gibson, R.A., McMurc~e, E.J., Charnock, J.S. and Kneebone, G.M. (1984) Lipids 19, 942-951 32 Farnsworth, W.E. (1981) in The Prostate (Murphy. G.P., Sandberg, A.A. and Karr, J.P., eds.), part A, pp. 225-230, Alan R. Liss, New York 33 Cavanaugh, A.H., Farnsworth W.E., Greizerstain, H.B. and Wojtowicz, C. (1980) Life Sci. 26, 29-34