Human Placental Lipid Metabolism. III. Synthesis and Hydrolysis of Phospholipids ALEX ROBERTSON and HOWARD SPRECHER, Departments of Pediatrics and Physiological Chemistry, The Ohio State University College of Medicine, and The Children's Hospital Research Foundation, Columbus, Ohio

ABSTRACT

Both diacyl GPC (glycerylphosphoryl- choline) and diacyl GPE (glycerylphos- phorylethanolamine) are synthesized in human placental tissue from their respec- tive monoacyl precursors. The origin of the monacyl phosphatides is apparently not the result of placental phosphatide acyl-hydrolase activity. The most likely source is maternal serum. The declining level of 1-acyl GPC in maternal serum is not attributable to lysophosphatide acyl- hydrolase activity and is probably ex- plained by placental utilization for the synthesis of diacyl GPC.

INTRODUCTION

T HE ACTIVITY in placental tissue of acyl GPC acyl-transferase (E.C. 2.3.1.99) (1) has

been previously described. This study reports the synthesis of diacyl GPE from 1-acyl GPE. In placental tissue the monoacyl substrate could arise from phosphatide acyl-hydrolase (E.C. 3.1.1.4) activity. Therefore the hydrolysis of endogenous and exogenous diacyl phosphatides was measured. Another explanation is that acyl GPC may be derived from maternal serum, as was originally suggested by Svanborg and Vikrot (2). The decline in acyl GPC during pregnancy (2) could represent either utilization or hydrolysis by the placenta. The placental activity of lysophosphatide acylhydrolase (E.C. 3.1.1.5) was therefore measured.

MATERIALS AND METHODS

Placental tissue was perfused and prepared as previously described (1). These prepara- tions are referred to as the tissue homogenate.

The 1-acyl GPC and 1-acyl GPE were pre- pared from chicken egg yolk (3). The phos- phorus to ester value of 1-acyl GPC was 0.97 and of 1-acyl GPE, 0.92. Beta 1J~C-diacyl GPC and beta 1-~4C-diacyl GPE were prepared with x~C-oleic acid by the enzymatic action of human erythrocytes on 1-acyl GPC and 1-acyl GPE (4).

The conversion of 1-acyl GPE to diacyl GPE

was measured by incubating the homogenate with 1-acyl GPE, CoA (coenzyme A), ATP (adenosine triphosphate), and 1-14C-oleic acid.

Phosphatide acyl-hydrolase activity was measured by incubating the homogenate with beta 1-~4C-diacyl GPC or beta 1-~4C-diacyl GPE. The hydrolysis of endogenous diacyl phosphatides was measured by determining the decline of diacyl GPC and diacyl GPE phos- phorus values. Lysophosphatide acyl-hydrolase activity was determined by measuring the dis- appearance of added 1-acyl GPC phosphorus. The level of endogenous acyl GPC was too low in this system to measure any hydrolysis.

The lipids from all incubations were ex- tracted and the lipid fractions separated by thin-layer chromatography on silica gel or column chromatography" on silicic acid. All snbstrates used were purified by silicic acid column chromatography and appeared pure on thin-layer chromatography. The a~C was measured by a Nuclear Chicago gas flow- counter to a 5 % accuracy level.

In all instances "phosphate buffer" refers to a 0.1 N, p h i 7 . 4 buffer containing one micro- mole per milliliter of Na deoxycholate. When erythrocytes were used the cells were washed and the hemolysate was used undiluted.

R ES U LTS

Previous studies showed the synthesis of diacyl GPC from 1-acyl GPC (1). Incubations were performed by using 1-acyl GPE as the substrate. When erythrocytes, 14C-oleic acid, and 1-acyl GPE are incubated, 44% of the radioactivity is incorporated into diacyl GPE (Table I) with no incorporation in the diacyl GPC fraction. However when per fused placenta is incubated with ~4C-oleic acid and 1-acyl GPE, 18% of the radioactivity is in- corporated into diacyl GPE and 23% into diacyl GPC. In the absence of 1-acyl GPC or 1-acyl GPE no radioactive diacyl phosphatides were formed by placental tissue. The synthesis of diacyl GPC when 1-acyl GPE was the sub- strate could be explained by the conversion of diacyl GPE to diacyl GPC. However the in- cubation of ~4C-diacyl GPE with placenta

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404 ALEX ROBERTSON AND HOWARD SPRECHER

TABLE I Synthesis of Diacyl GPE and Diacyl GPC

from l-acyl-GPEa

Percentage of C ~4 Incorporated

Erythrocyte Perfused Time hemolysate placenta

(minutes) Diacyl-GPE Diacyl-GPC Diacyl-GPE Diacyl-GPC

0 2 2 2 2 5 4 1 7 6

10 7 1 13 13 15 10 2 17 20 20 13 2 18 19 60 44 4 18 23

aIncubation mixture consists of 1-14C:oleic acid (26 millimicromoles, 116,000 cpm), 400 millimicromoles of 1-acyl-GPE, 0.1 ml of 0.1 NI MgC12, 0.4 rnl of phosphate buffer, 0.1 ml ATP (10 micromoles), 0.2 ml of tissue homogenate, and 0.2 ml CoA (26 millimicromoles). Figures represent the percentage of 14C in each lipid fraction.

y i e l d e d n o ~4C-diacyl G P C . A l s o i n c u b a t i o n o f p e r f u s e d p l a c e n t a w i t h S - A d e n o s y l - l - M e t h i o - n i n e - M e t h y l - ~ 4 C in t h e s y s t e m o f G i b s o n e t al. ( 5 ) g a v e n o ~4C-diacyl G P C w h e r e a s a c r u d e h o m o g e n a t e o f r a t l iver d i d l e a d to ~4C-diacyl G P C . T h e pos s ib i l i t y m u s t be c o n s i d e r e d t h a t t h e s u b s t r a t e , 1 -acy l G P E , m a y be c o n t a m i - n a t e d w i t h 1 -acy l G P C a n d t h a t p l a c e n t a l acy l - G P C a c y l - t r a n s f e r a s e d i f fe rs f r o m t h e e r y t h r o - cy t e e n z y m e in its a f f in i ty f o r 1 -acy l G P E . T h e r e w a s n o e v i d e n c e h o w e v e r f r o m t h e t h i n - l a y e r c h r o m a t o g r a p h y t h a t t h e r e w a s c o n t a m i - n a t i o n o f t h e 1 -acy l G P E s u b s t r a t e .

T h e h y d r o l y s i s o f e n d o g e n o u s d i acy l G P C a n d d i aey l G P E w a s m e a s u r e d , a n d t h e r e s u l t s a r e s h o w n i n T a b l e II. I n t h r e e h o u r s t h e r e is n o a p p r e c i a b l e h y d r o l y s i s o f e i t h e r e n d o g e n o u s s u b s t r a t e . W h e n 1 4 C - d i a c y l p h o s p h a t i d e s a r e i n c u b a t e d w i t h p l a c e n t a l t i s sue , t h e r e is s l i gh t h y d r o l y s i s o f d i acy l G P E in t h r e e h o u r s a n d n o h y d r o l y s i s o f d i acy l G P C ( T a b l e I I I ) . T h e c o n t r o l i n c u b a t i o n w i t h r a t i n t e s t i n e h o m o g - e n a t e s h o w s a l m o s t c o m p l e t e h y d r o l y s i s .

T h e i n c u b a t i o n o f 1 -acy l G P C w i t h p e r f u s e d p l a c e n t a s h o w e d n o s i g n i f i c a n t h y d r o l y s i s ( T a b l e I V ) w h e r e a s h y d r o l y s i s in th i s s y s t e m

TABLE II

Absence of Hydrolysis of Endogenous Diacyl GPC and Diacyl GPE a

Time Endogenous Phospholipid (minutes) Diacyl GPC Diacyl GPE

0 45.1 30.5 60 43.7 29.2

120 41.1 29.2 180 42.4 29.2

aIncubation consists of 1.4 ml of placental homogenate and 0.6 ml of phosphate buffer. Figures represent the average of triplicate incubations expressed as millimicro- moles of phosphorus in total incubation.

TABLE III Hydrolysis of Diacyl Phosphatides a

Incubation with 14C- Diacyl GPC

Percentage of 14C in Lipid Fractions

Fatty Diacyl Acyl Tissue acid phosphatide phosphatide

No enzyme 2 95 3 Rat intestine 86 12 2 Perfused placenta 4 92 4

Incubation with 14C- Diacyl GPE No enzyme 9 89 2 Rat intestine 93 5 2 Perfused placenta 17 82 1

aIncubation mixture consists of 0.2 ml of 14C-diacyl GPC labeled in the 2 position (160 mum, 10,000 clam) or 14C- diacyl GPE labeled in the 2 position (99 mum, 9,400 cpm) sonicated in phosphate buffer, and 0.2 ml of the tissue homogenate. All incubations were stopped at three hours. Figures represent the average percentage of a4C found in the lipid fractions from duplicate incubations.

d o e s o c c u r w i t h r a t i n t e s t i n e h o m o g e n a t e . I n t h e s e e x p e r i m e n t s w h e r e l i t t le or n o h y -

d r o l y s i s o c c u r r e d , t h e p l a c e n t a l h o m o g e n a t e s h o w e d a d e q u a t e p h o s p h a t i d e s y n t h e s i s w h e n c o f a c t o r s a n d s u b s t r a t e w e r e a d d e d ( a s in T a b l e I ) a n d w a s n o t i n a c t i v a t e d b y t h e

m e t h o d o f p r e p a r a t i o n .

DISCUSSION

T h i s w o r k h a s s h o w n t h a t p l a c e n t a l t i s sue c a n c o n v e r t 1 -acy l G P C ( 1 ) a n d 1 -acy l G P E to d i acy l G P C a n d d i acy l G P E r e s p e c t i v e l y . T h e q u e s t i o n a r i ses w h e t h e r o r n o t t he m o n o - acy l s u b s t r a t e is p r o d u c e d in t h e p l a c e n t a l t is- s u e b y p h o s p h a t i d e a c y l - h y d r o l a s e ac t iv i ty . S i nce in t h e s e e x p e r i m e n t s t h e r e is n o h y d r o l - y s i s o f e n d o g e n o u s d i a c y l G P C a n d e n d o g - e n o u s d i acy l G P E , n o h y d r o l y s i s o f e x o g e n o u s d i a c y l G P C a n d o n l y s l i gh t h y d r o l y s i s o f e x o g e n o u s d i acy l G P E , it s e e m s u n l i k e l y t h a t p l a c e n t a l p h o s p h o l i p a s e ac t i v i t y a f fo rd s t h e 1 -acy l s u b s t r a t e f o r s y n t h e s i s o f t h e d i acy l

TABLE IV Absence of Hydrolysis of l=Acyl GPC by Placenta a

Tissue Time

(minutes) No enzyme Placenta Rat intestine

0 2.75 2.48 2.39 10 2.61 2.67 1.95 20 2.39 2.53 1.49 40 2.70 2.72 0.96

a Incubation mixture consists of 0.2 ml of tissue homog- enate, 0.4 ml of 1-acyl GPC solution (4 micromoles), and 0.4 ml of phosphate buffer. At each designated time 0.2 ml were removed and analyzed. Figures represent micro- moles of phosphorus in 1-acyl GPC fraction of total in= cubation and are the average of triplicate incubations.

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HUMAN PLACENTAL LIPIDS METABOLISM. III 405

phosphat ides . These results are different f r o m those of Popjak (6 ) , who injected BeP-labeled phosphol ipids into rabbits and recovered ~2p_ g lycerophosphate and inorganic 32p f rom the placenta. He concluded that this represents placental b r eakdown of phosphol ipids . H o w - ever no tissue incubat ions were repor ted.

The monoacyl substrate may arise f rom the maternal blood, and a decline in mate rna l se rum acyl G P C does occur dur ing p regnancy (2) . This decline cannot be expla ined by placental hydrolysis of acyl G P C since these studies show that this activity is absent in per- fused placenta, The absence of lysophos- phat ide acyl-hydrolase activity differs f r o m the results o f Winkler , w h o showed this activity in rat p lacenta prepara t ions (7 ) . The var ia t ion be tween the results may be expla ined by the action of erythrocytes , which are k n o w n to have lysophosphat ide acyl-hydrolase activity (8 ) , or by species or incubat ion differences. The maternal origin of the monoacy l substrates is fur ther suggested by the work of Eisenberg et al., who have shown that the rat p lacenta removes 1-acyl G P C f rom the materna l circula- t ion and utilizes it to fo rm placental phospho- lipids (9) .

ACKNOWLEDGMENTS

This investigation was supported in part by funds from a Public Health Research Grant, Number tLD 0267502 from the National Institute of Child Health and Human De- velopment and from a Public Health Research Grant, Number AM 0975802 from the National Institute of Arth- ritis and Metabolism, and by a contract with the Ohio Department of Health, Maternal and Child Health Divi- sion. The technical assistance of Mrs. Justina Wilcox and Mrs. Tekla Svanks is acknowledged.

REFERENCES

1. Robertson, A., and H. Sprecher, Pediatrics 38, 1028- 1033 (1966).

2. Svanborg, A., and O. Vikrot, Acta Med. Scand. 178, 615 (1965).

3. Robertson, A., Biochim. et Biophys. Acta 116, 379- 381 (1966).

4. Robertson, A., and W. E. M. Lands, J. Lipid Res. 5, 88-93 (1964).

5. Gibson, K. D., J. D. Wilson and S. Udenfriend, J. Biol. Chem. 236, 673-679 (1961).

6. Popjak, G., Cold Spring Harbor Symposia 19, 200- 208 (1954).

7. Winkler, L., Naturwissenschaften 51, 340 (1964). 8. Mulder, E., J. W. O. Van Den Berg and L. L. M.

Van Deenen, Biochim. et Biophys. Acta 106, 118-127 (1965).

9. Eisenberg, S., Y. Stein and O. Stein, Biochim. et Biophys. Acta 137, 115-120 (1967).

[Received April 7, 1967]

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