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Obstetrics & Gynecology 1999;93:180-183
© 1999 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Increased Platelet-activating Factor–acetylhydrolase Activity in the Umbilical Venous Plasma of Growth-restricted Fetuses

AKIHIRO OHSHIGE, MD, TOSHIHIRO YOSHIMURA, MD, TAKAHIRO MAEDA, MD, MASAHARU ITO, MD and HITOSHI OKAMURA, MD

From the Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, Kumamoto, and the Department of Obstetrics and Gynecology, Ehime University School of Medicine, Ehime, Japan.

Address reprint requests to: Toshihiro Yoshimura, MD Department of Obstetrics and Gynecology Kumamoto University School of Medicine Honjo 1-1-1, Kumamoto City Kumamoto 860-8556 Japan E-mail: yoshimur{at}kaiju.medic.kumamoto-u.ac.jp


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To determine whether platelet-activating factor–acetylhydrolase activity in fetal plasma obtained at birth from umbilical vessels is different from that in maternal plasma, and (2) to compare platelet-activating factor–acetylhydrolase activity in cord plasma from fetuses with fetal growth restriction (FGR) and those with appropriate growth for gestational age (AGA).

Methods: Platelet-activating factor–acetylhydrolase activity was measured in the plasma of 22 nonpregnant healthy women, 16 pregnant women at term during labor, 28 fetuses exhibiting AGA, and seven fetuses with FGR.

Results: Plasma platelet-activating factor–acetylhydrolase activity in normotensive pregnant women at 37–41 weeks’ gestation was 28.1 ± 16.6 nmol/mL per minute, which was not statistically different from the activity in nonpregnant women (30.8 ± 11.1 nmol/mL per minute). Platelet-activating factor–acetylhydrolase activity in venous cord plasma from AGA fetuses was significantly (P < .01) lower than that in maternal plasma (6.3 ± 2.6 nmol/mL per minute), and there was no difference between the activities found in arterial and venous cord samples. In FGR fetuses, venous cord platelet-activating factor–acetylhydrolase activity was significantly (P < .01) higher (12.1 ± 1.4 nmol/mL per minute), than the activity seen in AGA fetuses, and when the data from AGA and FGR fetuses were considered together, there was a negative correlation between cord plasma platelet-activating factor–acetylhydrolase activity and neonatal body weight (r = .46, P = .006).

Conclusion: Platelet-activating factor hydrolysis is significantly lower in fetuses than adults. Further, the comparatively high platelet-activating factor–acetylhydrolase activity in FGR fetuses suggests the existence of a compensatory mechanism to maintain microcirculation within the placenta.

Asymmetric fetal growth restriction (FGR), the most frequently occurring form of FGR, is caused by placental insufficiency. This condition results in diminished glucose transfer, hepatic storage, and fetal abdominal circumference (which reflects the smaller liver size). In such fetuses, placental vascular resistance is increased, as evidenced by Doppler velocimetric measurements of the umbilical artery.1

Platelet-activating factor induces platelet aggregation at concentrations in the range of 10-9 to 10-8 M.2 It also operates through cell surface receptors to exert various other effects, including arterial vasoconstriction, alterations in vascular permeability, and increased leukocyte adhesion.3 Circulating levels of platelet-activating factor are too low to be detected by established methods4; therefore, they must be assessed indirectly by analyzing the activity of platelet-activating factor–acetylhydrolase. This circulating enzyme rapidly metabolizes biologically active platelet-activating factor to the inactive derivative, lysoplateletactivating factor. It has been suggested that lower platelet-activating factor–acetylhydrolase activity results in higher plasma platelet-activating factor levels,5 and ultimately, higher platelet-activating factor levels should increase platelet activation and vasoconstriction.

The purpose of this study was to determine whether, at birth, platelet-activating factor–acetylhydrolase activity in fetal umbilical plasma is different from that in the maternal plasma, and to compare platelet-activating factor–acetylhydrolase activities in fetuses with FGR and in those exhibiting appropriate growth for gestational age (AGA).


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
The protocol for these studies was approved by the Institutional Review Board of the Kumamoto University School of Medicine. Umbilical venous plasma was obtained from 28 singleton AGA fetuses at vaginal or cesarean delivery after 37 to 41 weeks’ gestation. Plasma from the umbilical artery was also obtained from 14 of the AGA infants. In addition, umbilical artery venous plasma was obtained from seven singleton FGR infants at delivery after 36 to 40 weeks’ gestation. Based on the standards for Japanese infants, the FGR infants were all below the tenth percentile in weight for gestational age. None of the FGR or AGA (control) infants had been exposed to exogenous glucocorticoids in utero, and none exhibited 1- or 5-minute Apgar scores of less than 7. During labor, plasma was obtained from the mothers of 14 AGA infants and two FGR infants; for control purposes, plasma was also obtained from 22 nonpregnant healthy woman. Patients with pregnancy-induced hypertension were not included in this study. The clinical characteristics of the study participants are shown in Table 1Go.


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  		Table 1. Clinical Characteristics of Women and Infants Participating in the Study
 
Blood samples were collected into tubes containing ethylenediaminetetraacetic acid (1 mg/mL). The plasma from each sample was separated by centrifugation at 4 C and then stored at -35 C until analyzed. Platelet-activating factor–acetylhydrolase activity was assayed according to the method of Maki et al.6 [3H]Acetyl-platelet-activating factor (1-0-hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphocholine; 10 Ci/mmol; New England Nuclear, Boston, MA) and nonradiolabeled platelet-activating factor (Sigma Chemical Co, St. Louis, MO) were suspended in a 0.1% solution of bovine serum albumin (fatty acid free, Sigma Chemical Co, St. Louis, MO). Immediately before assay, plasma samples were diluted 25-fold with 0.25 M sucrose. Selected quantities of the diluted plasma were then added to the assay mixture, which also contained 0.3 mL Tris-HCl (50 mM, pH 7.4), bovine serum albumin (2.0 mg/mL), and radiolabeled platelet-activating factor substrate (0.05 mM) for a final volume of 0.5 mL. The assay mixture was allowed to react for 20 minutes at 37 C, at which time, the reaction was terminated by the addition of 2 mL of a 1:2 chloroform/methanol mixture. The assay mixture was then centrifuged for 5 minutes at 4 C (600 g). After centrifugation, an aliquot of the supernatant (0.4 mL) was removed and mixed with 4 mL of scintillation fluid (Dojin Chemical Institute, Kumamoto, Japan). The water-soluble [3H]acetate hydrolyzed from [3H]acetyl-platelet-activating factor was assayed by liquid scintillation spectroscopy; platelet-activating factor–acetylhydrolase activity was expressed as nmol/mL of plasma per minute. A standard plasma sample from a human control was also analyzed with each assay group; throughout the course of the study, no significant variation in platelet-activating factor–acetylhydrolase activity was noted in the standard plasma samples. The intra- and interassay coefficients of variation were 2.5% and 4.4%, respectively.

Data are presented as the mean ± standard deviation. Paired and unpaired t tests were used for statistical evaluation of the results. The correlation coefficient was calculated by simple linear regression using least-squares minimization and equal weighting of the data points. The Fisher exact test was done to compare the two groups. Probability values (P) less than .05 were considered significant.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
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The mean platelet-activating factor–acetylhydrolase activity measured in the plasma of parturients at term was 28.1 ± 16.6 nmol/mL per minute, which was not statistically different from the activity in plasma from nonpregnant women (30.8 ± 11.1 nmol/mL per minute). In contrast, platelet-activating factor–acetylhydrolase activity in the umbilical venous plasma of AGA infants was significantly (P < .01) lower than that in maternal plasma (6.3 ± 2.6 nmol/mL per minute). There were no differences in platelet-activating factor–acetylhydrolase activities between the simultaneously drawn venous and arterial cord samples (8.5 ± 3.2 and 8.6 ± 4.1 nmol/mL per minute, respectively). The venous cord platelet-activating factor–acetylhydrolase activity was 12.1 ± 1.4 nmol/mL per minute in FGR infants. Although this value was still significantly (P < .01) lower than the activity in maternal plasma, it was significantly (P < .01) higher than that in the plasma of AGA infants (Figure 1Go). Indeed, when the data from AGA and FGR infants were plotted together, there was a significant negative correlation between umbilical venous plasma platelet-activating factor–acetylhydrolase activity and neonatal body weight (Figure 2Go; n = 35; r = .46; P = .006).



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  		Figure 1. Platelet-activating factor (PAF)–acetylhydrolase activity measured in plasma samples from the umbilical veins of fetuses with fetal growth restriction (FGR) or fetuses exhibiting appropriate growth for gestational age (AGA).

 


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  		Figure 2. Correlation between the plasma platelet-activating factor (PAF)–acetylhydrolase activity and neonatal birth weight. shaded circles = fetal growth-restricted fetuses; solid circles = fetuses with appropriate growth for gestational age.

 

   Discussion
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Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we assayed platelet-activating factor–acetylhydrolase activity in umbilical plasma obtained from AGA and FGR infants and from maternal plasma. We found no difference in the activities measured in plasma samples from pregnant and nonpregnant women, although previous reports found that platelet-activating factor–acetylhydrolase activity is decreased during pregnancy in both humans7 and rabbits.6 Nevertheless, our finding is not surprising, because the activity of platelet-activating factor–acetylhydrolase is known to increase as gestation progresses,6 and in the present study, samples of maternal plasma were obtained at term during labor. Consistent with the study by Kobayashi et al,7 the platelet-activating factor–acetylhydrolase activities in both AGA and FGR infants were markedly lower than the activities measured in the mothers. The physiologic significance of the decreased platelet-activating factor–acetylhydrolase activity, under the conditions of low perfusion pressure and low vascular resistance, which are characteristic of the fetal-placental circulation,8 remains unknown.

Placental vascular resistance is estimated by Doppler velocimetry of the pulsatile blood flow through the umbilical artery. Using this method, it has been shown that when placental vascular resistance is increased, umbilical arterial flow, which normally decreases during fetal cardiac diastole, is decreased even more than usual. This condition results in increased systolic-diastolic blood flow ratio,1 and several studies have demonstrated an association between increased systolic-diastolic ratios and fetal growth restriction and adverse perinatal outcome. Because platelet-activating factor–acetylhydrolase activity in the circulation of FGR fetuses is significantly higher than normal, the clinical importance of the simultaneous increase in fetal platelet-activating factor–acetylhydrolase activity and placental vascular resistance merits contemplation.

There has been considerable debate as to whether platelet-activating factor exerts a vasodilator or vasoconstrictor effect on resistance vessels.9 Dillon et al10 convincingly showed that platelet-activating factor elicited arteriolar constriction in hamster cheek pouch and that the constriction was largely mediated by thromboxane A2.3 Endothelial cells,11 smooth muscle cells,11 and neutrophils12 all have the ability to generate thromboxane A2 in response to challenge by platelet-activating factor. Moreover, hypoxia induces flowing neutrophils to adhere to umbilical vein endothelium13 as well as to cultured endothelial cells,14 which means that under conditions of chronic hypoxemia, as in FGR fetuses, there is increased stimulus-induced synthesis and release of platelet-activation factor from human umbilical vein endothelial cells.15 We speculate that in FGR fetuses, increased catabolism of platelet-activating factor by platelet-activating factor–acetylhydrolase might be a compensatory mechanism for decreasing arteriolar constriction and maintaining placental microcirculation.

In human plasma, 70% of platelet-activating factor–acetylhydrolase activity is associated with low density lipoprotein (LDL) and 30% with high density lipoprotein (HDL).16,17 Circulating cholesterol, particularly LDL, is an important substrate for fetal adrenal steroidogenesis, and in pregnancies complicated by FGR, maternal estrogen levels are usually below normal. Levels of total cholesterol and LDL cholesterol in fetal plasma were found to be inversely related to the concentration of dehydroepiandrosterone sulfate,18 which suggests that the rate of plasma cholesterol utilization for steroidogenesis might exert a significant effect on circulating fetal cholesterol levels. Although we did not measure maternal urinary estrogen secretion or fetal plasma LDL, increased cord plasma LDL might have contributed to the higher platelet-activating factor–acetylhydrolase activity seen in FGR fetuses. The specific functions of platelet-activating factor in FGR, however, remain a subject for further study.


   Footnotes
 
PII S0029-7844(98)00407-4

Received April 20, 1998. Received in revised form July 31, 1998. Accepted August 7, 1998.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Gaziano EP, Knox H, Ferrera B, Brandt DG, Calvin SE, Knox GE. Is it time to reassess the risk for the growth-retarded fetus with normal Doppler velocimetry of the umbilical artery? Am J Obstet Gynecol 1994;170:1734–41.[Medline]

2. Kloprogge E, Akkerman JWN. Binding kinetics of PAF-acether (I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets. J Biochem 1984;223:901–9.

3. Duran WN, Dillon PK. Acute microcirculatory effects of platelet activating factor. J Lipid Mediator 1990;2 Suppl:S215–7.

4. Ostermann G, Till U, Theilmann K. Cooperative effects of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-Acether) and exogenous arachidonic acid in stimulation of human blood platelets. Thromb Res 1984;30:409–18.

5. Frenkel RA, Muguruma K, Johnston JM. The biochemical role of platelet-activating factor in reproduction. Prog Lipid Res 1996;35: 155–68.[Medline]

6. Maki N, Hoffman DR, Johnston JM. Platelet-activating factor acetylhydrolase activity in maternal, fetal, and newborn rabbit plasma during pregnancy and lactation. Proc Natl Acad Sci U S A 1988;85:728–32.[Abstract/Free Full Text]

7. Kobayashi F, Sagawa N, Ihara Y, Kitagawa K, Yano J, Mori T. Platelet-activating factor-acetylhydrolase activity in maternal and umbilical venous plasma obtained from normotensive and hypertensive pregnancies. Obstet Gynecol 1994;84:360–4.[Abstract/Free Full Text]

8. Yoshimura T, Magness RR, Rosenfeld RR. Angiotensin II and {alpha}-agonist. I. Responses of ovine fetoplacental vasculature. Am J Physiol 1990;259:H464–72.

9. Vandongen R. Platelet activating factor and the circulation. J Hypertens 1991;9:771–8.[Medline]

10. Dillon PK, Ritter AB, Duran WN. Vasoconstrictor effects of platelet-activating factor in the hamster cheek pouch microcirculation: Dose-related relations and pathways of action. Circ Res 1988;62: 722–31.[Abstract/Free Full Text]

11. Takayasu-Okishio M, Terashita Z, Kondo K. Endothelin-1 and platelet activating factor stimulate thromboxane A2 biosynthesis in rat vascular smooth muscle cells. Biochem Pharmacol 1990;40: 2713–7.[Medline]

12. Salzman PM, Salmon JA, Moncada S. Prostacyclin and thromboxane A2 synthesis by rabbit pulmonary artery. J Pharmacol Exp Ther 1980;215:240–7.[Abstract/Free Full Text]

13. Arnould T, Michiels C, Janssens D, Delaive E, Remacle J. Hypoxia induces PMS adherence to umbilical vein endothelium. Cardiovasc Res 1995;30:1009–16.[Medline]

14. Rainger GE, Fisher A, Shearman C, Nash GB. Adhesion of flowing neutrophils to cultured endothelial cells after hypoxia and reoxygenation in vitro. Am J Physiol 1995;269:H1398–406.

15. Caplan MS, Adler L, Kelly A, Hsueh W. Hypoxia increases stimulus-induced PAF production and release from human umbilical vein endothelial cells. Biochem Biophys Acta 1992;1128:205–10.[Medline]

16. Stafforini DM, McIntyre TM, Carter ME, Prescott SM. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J Biol Chem 1987;262:4215–22.[Abstract/Free Full Text]

17. Ohshige A, Ito M, Koyama H, Maeda T, Yoshimura T, Okamura H. Effects of estrogen and progesterone on plasma platelet-activating factor-acetylhydrolase activity and low-density lipoprotein cholesterol concentration in men. Artery 1996;22:115–24.[Medline]

18. Parker CR Jr, Simpson ER, Bilheimer DW, Leveno K, Carr BR, MacDonald PC. Inverse relationship between LDL-cholesterol and dehydroisoandrosterone sulfate in human fetal plasma. Science 1980;208:512–4.[Abstract/Free Full Text]




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