Studies of the biochemical basis of steroid sulphatase deficiency—III. Phospholipid composition of microsomes from normal and steroid sulphatase deficient placentas

J. sreroid Biochetu. Vol. 18, No. 3, pp. 309-312, 1983

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STUDIES OF THE BIOCHEMICAL BASIS OF STEROID SULPHATASE DEFICIENCY-III. PHOSPHOLIPID COMPOSITION OF MICROSOMES FROM NORMAL AND STEROID SULPHATASE DEFICIENT PLACENTAS JOSEPHW. A. MCKEE and JOHN T. FRANCE* Postgraduate School of Obstetrics and Gynaecology, University of Auckland, National Women’s Hospital, Auckland 3, New Zealand (Received 28 May 1982) SUMMARY The phospholipid composition has been determined for placental microsomes from 11 normal and eight pregnancies complicated by steroid sulphatase deficiency. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal placental microsomes, comprising respectively 41.6 & 4.6% (mean + SD), 30 + 5.7% and 22.5 + 4.9% of the total phospholipid content. There was no correlation between the steroid sulphatase activity of the microsomes and the content of any of the three phospholipids. Though their contents were significantly decreased, (P < 0.001) phosphatidylcholine, phosphatidylethanolamine and sphingomyelin similarly constituted the major portion of the total phospholipids in sulphatase deficient microsomes, representing 36 k 4.2%, 34 k 6.1% and 22.4 +_6.7% respectively. Only the percentage of phosphatidylcholine was significantly different (P < 0.02) from normal microsomes. The results show that the decreased phospholipid content of steroid sulphatase deficient placental microsomes reflects a lower content of all major classes of phospholipids, particularly phosphatidylcholine.

INTRODUCTION A number of steroid metabolizing enzymes from various tissues [l, 23, including the placenta [3,4], have been found to require phospholipids for activity. Preliminary findings from this laboratory suggested that the placental steroid sulphatase enzyme could also be a phospholipid dependent enzyme [S]. We suggested that a disordered enzyme-membrane structure in the endoplasmic reticulum, possibly a defect in phospholipid composition, could be involved in the biochemical mechanism of steroid sulphatase deficiency [S]. Recently, we demonstrated that microsomes from sulphatase deficient placentas have a decreased phospholipid content compared with microsomes from normal placentas [6]. The present study was designed to determine whether the decreased phospholipid content of placental microsomes lacking steroid sulphatase activity reflects the absence of, or reduced concentration of one specific or several phospholipids. The phospholipid compositions of steroid sulphatase deficient and normal placental microsomes have been determined and compared.

grade quality. Reference phospholipids were obtained from Sigma Chemical Co., St Louis MO, U.S.A. and were stored in chloroform or chloroform: methanol solution at -20°C in a dessicator. Preparation

of placental microsomes

Placental microsomes were prepared from both normal and steroid sulphatase deficient placentas according to the procedure described by McNaught and France [5]. Measurement of steroid sulphatase actioity Microsomal steroid sulphatase activity was determined with tritium labelled dehydroepiandrosterone sulphate (DHAS) as substrate by the method of Burstein and Dorfman as modified by McNaught and France[S]. Determinations were carried out in duplicate. For placental microsomes, our range of normal levels of activity of the enzyme is 0.99-1.69 nmol DHAS hydrolysed/min/mg protein with a mean (f SD) of 1.26 + 0.22 nmol DHAS hydrolysed/min/mg protein (n = 11) [6]. The limit of sensitivity of the assay is 2 pmol DHAS hydrolysed/min/mg protein. Separation and quantitation of major phospholipids

MATERIALS AND Organic

solvents

METHODS

and reagents

*To whom correspondence should be addressed

Lipids were extracted from placental microsomes by the method of Bligh and Dyer[7] but with use of chloroform-methanol (1: 1, v/v) as the initial extracting solvent. Duplicate aliquots of the chloroform phase were removed for determination of the total

were of analytical

and requests for reprints 309

phospholipid content. The remainder of the extract was taken for separation of the phospholipids into their individual classes by thin-layer chromatography on silica gel G (Merck, Darmstadt. F.G.R.) using chloroform~methanol-water: acetic acid (85:15:3:10, v/v) as the solvent system. The phosphol~pid bands were detected on the thin layer plate by exposure to iodine vapour and identified by comparison with standards. Each band was scraped individually into a Pyrex test-tube and after addition of a drop of water, the phospholipid was extracted into chloroformmethanol (I: I. v;v). The amount of pllospt~olipjd was determined From analysis of duplicate aliquots of the extract. Control areas of the thin layer plate, free from phospholipids were similarly eluted and assayed to provide method blanks. The method of Bartlett as modified by Marinetti[S] was used to measure phospl~olipids as lipid phosphorus. Protein content was determined by the revised Lowry method of Schacterle and Pollard[9].

Phosphatidylcholine. phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal human placental microsomes. These three phospholipids together constituted over 90”,, of the phospholipids. having respective mean (+ SD) percent contents of 41.6 F 4.6”,,, 30.1 + 5.7’!;, and 22.5 + 4.9”,,. Phosphatidylinositol and phisphatidylserine were analysed together because of poor chromatographic separation but constituted a minor proportion of the total phospholipids (2.6 + 2.6”~. Other minor phospholipid bands were pooled for analysis and made up to less than 0.5’:; of the total phosphoiipid content. No correlation was found between the content of each of the major phospholipid classes and the steroid sulphatase activity of the normal preparations. The correlation coefficient expressing the relationship between enzyme activity and phospholipid content was for phosphatidylchoiine, I’ = 0.49; for phosphatidylethanolamine, r = 0.44: and for sphingomyelin, r = 0.13.

The total lipid phosphorus content of six of the sulphatase deficient preparations have been reported previously [6]. The values of the two additional preparations. 0.76 and 0.74 pmoiP/mg protein respectively, confirm the earlier finding of a tower phospholipid content in microsomes deficient in steroid sulphatase activity. The content of phosphatidylinositol/phosphatidylserine and of other minor phosphoiipids did not differ from levels observed in normal microsomes but the major phospholipids, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were all present in much lower amounts. The decrease was most marked with phosphatidylcholine. The phospholipid composition expressed as percentages of the total phospholipid was: phosphatidylcholine. 36.0 f 4.2”,,; phosp~~atidyl-ethanolamine, 34.4 + 6. I”,,; sphingomyelin, 22.4 +- 6.7”,: and phosphatidylinositol/phosphatidylserine. 3.6 i 2.9”,,. The proportions of phosphatidylethanolamine and sphingomyelin were

RESULTS

The levels of steroid sulphatase activity of the 11 normal placentas and six of the eight enzyme deficient placentas investigated were reported previously [6]. Two further placentas from pregnancies with abnormally low oestriol levels were confirmed to be deficient in steroid sulphatase when their microsomes were found to have no detectable activity of the enzyme. These placentas were also included in the study. The phospholipid composition of the microsomal preparations of the 11 normal and 8 sulphatase deficient placentas are listed in Table 1. The content of each phospholipid class is described in terms of ,nmolP/mg protein and as a percentage of the total phosphofpid in the initial chloroform/methanol extract. Recovery of phosphohpids from the thin layer plate averaged 97.6 2 5.4”i:, of the total phospholipid. Table 1. The phosphotipid

composition of normal and sulphatase deficient (and range) for the individllal phospholipids Normal

microsomes

(jcmolP/mg protein) Total

phosphoiipid

1.53 i: 0.23 (1.31--1.9X) 0.64 + 0.15 (0.45-0.99) 0.46 Ifr 0.10 (0.24,“0.60) 0.35 i: 0.10 (0.17-0.51 t 0.04 I: 0.04 (tr -0.10) 0.01 i: 0.01 (tr-0.04)

Phosphatidy~choline Phosphatidylethanolatnine Sphingomyelin Phosphatid~linositol + phosphatid~lserine Minor phospholipids

Difference significant.

from

values

for normal

microsomes:

(n =

Ii)

(I’,)of total phospholipid) 100 41.6 * 4.6 (32.8-50.0) 30.1 f 5.7 (18.3-37.6) 22.5 & 4.9 (12629.3) 2.6 f 2.6

( < 1.0 7.7) 0.4 f 0.8 (< 1.0 2.7) * Highly

significant

placental microsomes. are listed Sulphatase

deficient

(jtmolP.!mg protein) 0.78 ri: 0.09* (0.7~~.~0.9~) 0.28 & 0.05” (0.22.0.371 0.27 f 0.07* (0.20-0.421 0.1 s * 0.05’ (0.07 0.23) 0.03 & 0.02: (tr O.Oh) 0.03 i 0.03: (tr- 0.03) at P < O.OOl. $ Significant

Mean

content

microsomes

+ SD

(~1= 8)

(‘I,, of total phospholipid) IO0 36.0 k 4.21 (28. I-40.1) 34.4 2 6.lf (27.0-42.9) 22.4 * 6.7$ (9628.1) 3.6 + 2.9:

( < 1.o-8.0) 1.9 + 1.71

( < l.Ok4.5) at P < 0.02. 1 Not

Phospholipid composition of placental microsomes comparable in sulphatase deficient and normal microsomes. However, phosphatidylcholine represented a significantly lower proportion of the total phospholipid in affected microsomes than in normal microsomes (P < 0.02, Mann-Whitney non-parametric test, students t-test). DISCUSSION

Lipids comprise l-2% of the wet weight of human placental tissue, with phospholipids constituting about 6&64% of the total lipid content and cholesterol, cholesterol esters, triglycerides, and non-esterified fatty acids making up the remainder [lo, 111. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are the major phospholipids of term placental whole tissue [12], first trimester placentas [13] and placentas associated with prematurity and babies small for gestational age [14]. Phosphatidylserine, phosphatidylinositol, lysolecithin and cardiolipin are minor phospholipid constituents. The present study has shown that the phospholipid composition of microsomes prepared from term placentas is similar to that of the whole tissue. Microsomes from steroid sulphatase deficient placentas have a lower phospholipid content relative to protein than normal placental microsomes [6]. This difference could represent an elevation in protein concentration rather than a decrease in phospholipid. However, the former appears unlikely since when expressed relative to protein content, the activities of the enzymes 17/?-hydroxysteroidoxidoreductase, A4,5 isomerase, aromatase [ 151 and 3/l-hydroxysteroid dehydrogenase [16] are similar in sulphatase deficient and normal placental microsomes. The present study indicates that the lower phospholipid content of sulphatase deficient microsomes reflects a general lowering in content of all major phospholipids. The relative proportions of the individual phospholipids were found to be similar in normal and sulphatase deficient placental microsomes except for phosphatidylcholine which constituted a significantly lower percentage of total phospholipid in the affected microsomes. Several enzymes involved in steroid metabolism have been found to require lipids, in particular phospholipids., for activity [l-4]. McNaught and France [S] have shown that phosphatidylcholine can stimulate the steroid sulphatase enzyme activity in solubilized enzyme preparations from normal and sulphatase deficient placental microsomes. It would not be surprising, therefore, if the absence of the enzyme activity in microsomes deficient in sulphatase activity was related to the lower content of phosphatidylcholine. On the other hand, the steroid sulphatase activity of normal placental microsomes shows no correlation with total phospholipid [6] or, as we have found in this study, with the individual content of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin. Furthermore, activities of other steroid metabolising enzymes located in the microsomes

311

appear unaffected by the presence of decreased phospholipid, at least in preparations from fresh, unfrozen placentas [15 161. These observations, nevertheless, do not rule out the possibility of an association between lack of enzyme activity and phospholipid content in steroid sulphatase deficiency. Only a minimal threshold level of phospholipid may be required in the microsomes for normal sulphatase activity. It is also possible that the steroid sulphatase enzyme depends for its function on a minor phospholipid component or on a specific phospholipid with a specific fatty acid composition present as a minor constituent. It seems likely that a distortion in membrane structure occurs in sulphatase deficient microsomes as a result of the reduced phospholipids:protein ratio. The membrane appears to be less stable than in normal microsomes for the process of freezing and thawing has a significantly more marked effect on lowering 3b-hydroxysteroid dehydrogenase-isomerase activity

C161. In conclusion, we have shown that microsomes deficient in steroid sulphatase have subnormal levels of each of the major phospholipids particularly phosphatidylcholine. Whether the absence of activity of the enzyme is a consequence of the disorder in phospholipid metabolism remains to be proven. Acknowledgements-J. W. A. McKee is a MRC(NZ) Training Fellow in Reproductive Endocrinology. We thank the obstetricians, nurses and laboratory staff who assisted in the supply of placental tissue for the study.

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