Maternal ingestion of acety lsalicy lie acid inhibits fetal and neonatal prostacyclin and thromboxane in humans

Olavi Ylikorkala, M.D., Ph.D., Ulla-Maija Miikilii, M.D., Ph.D.,t Pekka Kiiiipii, M.D., Ph.D., and Lasse Viinikka, M.D., Ph.D.

Helsinki and Oulu, Finland

Small doses of maternal acetylsalicylic acid have proved to prevent preeclampsia. To study the

mechanism of this action of acetylsalicylic acid, healthy women ingested 100 mg (n = 13) or 500 mg

(n = 14) of acetylsalicylic acid during labor at term. The fetal prostacyclin synthesis, as assessed by the production of 6-ketoprostaglandin F,o (a metabolite of prostacyclin) by the umbilical artery, was reduced

from 21.3 ± 1.6 ng/gm/min of dry weight in the controls (n = 25, mean ± SE) to 7.8 ± 1.1 ng/ml/min

(p < 0.001) in infants of mothers receiving 500 mg of acetylsalicylic acid, but it was unchanged in infants

with mothers receiving 100 mg of acetylsalicylic acid (19.5 ± 2.3 ng/gm/min). Maternal ingestion of 500 mg of acetylsalicylic acid also was accompanied by reduced (p < 0.10) urinary excretion of 6-

ketoprostaglandin F,o in neonates during the first 3 days of life. The fetal platelet thromboxane A2

synthesis, as assessed by the release of thromboxane 6 2 (a metabolite of thromboxane A2) during

spontaneous clotting of the umbilical blood (63.4 ± 4.2 pg/105 platelets, n = 22), was inhibited in infants born to mothers given 100 mg (14.0 ± 3.7 pg/105 platelets, p < 0.001) or 500 mg of acetylsalicylic acid

(6.1 ± 3.5 pg/105 platelets, p < 0.001). The thromboxane 6 2 release by the umbilical artery (1.1 ± 0.1 ngl gm/min, n = 13) also was decreased in infants of mothers receiving 500 mg of acetylsalicylic acid (0.57 ± 0.1 ng/gm/min, n = 7, P < 0.01). Thus a small dose of maternal acetylsalicylic acid (100 mg) inhibits only the fetoplacental thromboxane A2 but leaves prostacyclin production unaffected. (AM J OBSTET

GVNECOL 1986;155:345-9.)

Key words: Pregnancy, acetylsalicylic acid, prostacyclin, thromboxane

Two recent reports have suggested a potential use­fulness of small daily doses of acetylsalicylic acid (as­pirin) in the prevention of acute l and superimposed preeclampsia.2 This effect was assumed to be mainly due to acetylsalicylic acid-induced inhibition in the platelet-activating and vasoconstricting thromboxane A2, I. 2 particularly, because there is a relative overprod­uction of thromboxane A2 in comparison to the vaso­dilating prostacyclin in fetoplacental tissues in pre­eclampsia, as summarized recently." There are, how­ever, no direct data to support this theory, which is further challenged by the fact that acetylsalicylic acid can also inhibit the synthesis of prostacyclin. Thus the hypothesis can hold only in the case that there is a proper dose of acetylsalicylic acid which inhibits throm­boxane A2 synthesis but leaves prostacyclin synthesis

From the Departments of Obstetrics and Pediatrics, University of Helsinki and University of Oulu.

The study was supported by The Academy of Finland and Cancer Research Foundation.

Received for publication December 3, 1985; revised February 19, 1985; accepted March 12, 1986.

Reprint requests: Dr. Olavi Ylikorkala, M.D., Department of Ob­stetrics and Gynaecology, University of Helsinki, Haartmaninkatu 2, 00290 Helsinki 29, Finland.

tDeceased May 16, 1986.

unaffected in fetoplacental tissue. We have studied the concomitant effect of maternal ingestion of various doses of acetylsalicylic acid on the fetal and neonatal prostacyclin and thromboxane A2 •

Material and methods

Subjects. Twenty-seven healthy parturients with term normotensive pregnancies and vaginal deliveries were studied with the approval of the committee of ethics of the University of Gulu after being thoroughly informed about the purpose and course of the inves­tigation (Table I). Participants had not taken any drugs for at least 10 days preceding the investigation. Labor began spontaneously and during it the women ingested in randomized order 100 mg or 500 mg of acetylsalicylic acid with 100 ml of water 28 to 658 minutes before delivery (Table I). The uterine contractions and fetal heartbeat were recorded continuously before and after acetylsalicylic acid intake. Thirty-five comparable women not receiving acetylsalicylic acid were studied as controls. All study subjects gave birth spontaneously to healthy infants with I-minute Apgar scores of ~8.

Measurement of fetal and neonatal prostacyclin. Fe­tal prostacyclin generation was measured from the specimens of umbilical arteries by use of a tissue su-

345

346 Ylikorkala et al. August 1986 Am J Obstet Gynecol

Table I. Clinical data of various study groups (mean ± SE)

No. of women Age (yr)

Range Parity

Nulliparous Parous

Gestational age at delivery (wk) Range

Birth weight (gm) Range

Time from acetylsalicylic acid intake to birth (min) Range

Concentration of salicylate in umbilical cord blood (fJ.molll) Range

Maternal acetylsalicylic acid

JOO mg I 500 mg

13 14 28.5 ± 1.9 27.0 ± 1.0

17 - 37 20 - 33

4 6 9 8

40.2 ± 0.2 40.7 ± 0.2 39 - 42 39 - 42

3700 ± 152 3790 ± 149 3020 - 4500 3080 - 5170 344 ± 71 243 ± 36

35 - 658 28 - 515 34.0 ± 7.4 131.6 ± 32.4*

10 - 86 21 - 486

Controls

35 28.1 ± 0.8

19 - 34

12 23

39.8 ± 0.4 38 - 42

3650 ± 79 2940 - 417

*p < 0.0125 in comparison between acetylsalicylic acid groups.

Table II. Production of 6-ketoprostaglandin F,. and thromboxane B2 by the umbilical artery, fetal platelets' capacity to synthesize thromboxane B2, and neonatal urinary excretion of 6-ketoprostaglandin F,. in infants of mothers with acetylsalicylic acid treatment during labor (mean ± SE)

Umbilical production (nglgmlmin)

6-ketoprostaglandin

n I Control group 25 Maternal ASA 100 mg 13 Maternal ASA 500 mg 14

Flo

Mean±SE n I 21.3 ± 1.6 25 19.5 ± 2.3 13 7.8 ± 1.1*t 7

*p < 0.001 in comparing to the control group. tp < 0.01 in comparing to the control group.

Tx B2

Mean±SE

1.10 ± 0.1 0.83 ± 0.08 0.57 ± O.It

tp < 0.01 in comparing between acetylsalicylic acid group. §p < 0.10 in comparing to the control group.

perfusion method. ' Briefly, umbilical artery samples (10 to 20 mg of dry weight) were placed into plastic chambers and superfused with Eagle's medium (pH 7.4; 37° C; carbon dioxide/oxygen, 5%:95%). After a wash-out period of 2.5 hours, a I-hour fraction was collected and its content of 6-ketoprostaglandin Fl. (a stable hydration product of prostacydin) was measured by radioimmunoassay.' Results are expressed as nano­grams of6-ketoprostaglandin F,. produced per minute per gram of dry weight tissue. The storage of the sam­ple in the frozen state did not affect its capacity to produce prostacydin! The recovery of 6-ketoprostag­landin Fl. added to perfusion buffer was 95.3% ± 3.3% (n = 10, mean ± SD).

Neonatal prostacydin was studied by measuring the concentration of 6-ketoprostaglandin F,. in urine. Urine samples were collected into plastic bags on the first and third day of life. Urine samples (2 ml) were acidified to pH 3.0 with IN hydrochloric acid and passed through an octadecylsilyl silica cartridge (Sep-

Tx B 2 release (Pgll0' platelets)

n I 22 II 11

Mean±SE

63.4 ± 4.2 14.0 ± 3.7* 6.1 ± 3.5*

Neonatal urinary 6-ketoprostaglandin Flo (nglmmol creatinine)

1st day

n I Mean±SE

13 317.7 ± 43.7 6 309.2 ± 31.2

13 204.3 ± 25.1§

3rd day

n I Mean±SE

10 133.3 ± 17.7 5 114.5 ± 18.7

10 79.9 ± 13.3§

Pak C I8 cartridges, Waters Chromatography Division of Milipore, Milford, Massachusetts), prewashed succes­sively with 5 ml of ethanol and 5 ml of petroleum ether. The 6-ketoprostaglandin Fl. absorbed was eluted in 2 ml of ethylacetate, which was evaporated to dryness under a nitrogen stream. The sample was further pu­rified with high performance liquid chromatography (Spectron Physics M-740, Santa Clara, California) with use of a Sherisorb 5 ODS II column (Phase Separa­tions Ltd., Industrial Estate Queensberry, CLWD, United Kingdom) and a mobile phase of water/aceton­itrile/acetic acid (69.95: 30.00: 0.05) with flow rate of 2 ml/min. The fraction containing 6-ketoprostaglandin F,o (5 to 6 ml) was collected, evaporated to dryness, and dissolved in phosphate-buffered saline solution with 0.1 % of gelatin. Two different-size aliquots, both in duplicate, were taken for radioimmunoassay.' The re­covery of 6-ketoprostaglandin F,o added into urine (200 to 400 pg/ml) was 79.6% ± 8.9% (mean ± SD, n = 10) and the interassay variation 10.8% (n = 10) after the

Volume 155 Number 2

Maternal acetylsalicylic acid and fetal prostacyclin and thromboxane 347

500

.... 400

'0 300 E :::L

r: 200 0 -10 ... -100 t: Q) 0 t: 0 0 50 Q)

0 -~ • >-.!:2 iii

20 (/)

~~o 30

•

• 0

0

0

o •

I I I " I 60 100

• •

• •• • • ~

·0 0

o I I I I I

200300 600

Time from ASA intake to birth, min

Fig. 1. Salicylate concentrations in umbilical sera of infants born to mothers given 100 mg (0) or 500 mg (e) of acetylsal­icylic acid (ASA) during labor.

entire procedure. To eliminate the effect of changes of the water content of urine, the results are given as nanograms per millimole of creatinine determined by a routine laboratory method.

Measurement of fetal thromboxane. The vascular production of thromboxane A~ was measured by as­saying the concentrations of thromboxane B~ (a me­tabolite of thromboxane A~) from the superfusate of the umbilical artery. 1 To study the fetal platelets' ca­pacity to produce thromboxane A~, umbilical vein blood samples were collected into dry plastic tubes and al­lowed to clot spontaneously at 370 C for 60 minutes. Serum was separated, and its concentration of throm­boxane B~ was measured by radioimmunoassay." The results are expressed as picograms of thromboxane Bj 105 platelets.

Measurement of salicylate concentration. Serum samples from the mixed umbilical cord blood were col­lected for the salicylate measurement. To this end, fer­ric nitrate was added to serum, and the salicylate con­centration calculated from the light absorbance of the end product at 540 nm. When measured by this method, the therapeutic serum salicylate concentration was between 500 to 2500 J.l.mollL.

Statistical analysis. The t test modified according to the Bonferroni method7 and linear regression analysis were employed for the statistical analysis of the results.

Results

Maternal and fetal findings. The study groups were comparable in regard to the maternal, fetal, and neo­natal findings (Table I). Doses of acetylsalicylic acid did not change uterine contractility or fetal heart rate. The

0

30 0

'" c: 0 Qj

~ ]j ., '" 0 Q. 0. 70 ~ c:

60 ~

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() " ." 30 .2 e u c. 0 20 " . 0 ."

LC • • 0 0 § • §o - Is. Q. • rll I 0 10 Qj 8 00 >< .J< • ... I 10 8 • Qj

'" I So • Gi iii • 5 ... g • - Q. :c • • iii E ::> • Qj

I - 2 IL

• 100 mg 500 mg 100 mg 500 mg

MATERNAL ASA

Fig. 2. Production of 6-ketoprostaglandin Fin by the umbilical artery and release of thromboxane B2 (TxB 2) by the platelets in infants born to the mothers given 100 mg (0) or 500 mg (e) of acetylsalicylic acid (ASA) during labor. The shaded area indicates the normal mean ± I SD.

blood-loss during delivery was <500 ml in each subject. No clinically detectable bleeding tendency or any other adverse effects were seen in newborn infants of moth­ers given acetylsalicylic acid during labor.

Salicylate level. The interval from acetylsalicylic acid intake to birth did not differ significantly between the two acetylsalicylic acid-treated groups (Table I). The salicylate was detectable in umbilical blood in each in­fant, and its concentration was higher after the 500 mg dose than the 100 mg dose of maternal acetylsalicylic acid (Table I and Fig. I). The salicylate level was close to the adult therapeutic range only in one newborn infant (486 J.l.moIlL) whose mother received 500 mg of acetylsalicylic acid 455 minutes before delivery.

Fetal and neonatal prostacydin. Maternal ingestion of 100 mg of acetylsalicylic acid had no effect on the umbilical 6-ketoprostaglandin Fin production (Table II and Fig. 2) or on the neonatal urinary 6-ketoprostag­landin Fin excretion (Table II), whereas the 500 mg dose reduced both fetal and neonatal prostacyclin pro­duction (Table II and Fig. 2). This inhibition was not related to the interval between acetylsalicylic acid intake and delivery or to the salicylate level in the umbilical blood.

Fetal and neonatal thromboxane. Maternal admin­istration of 100 mg of acetylsalicylic acid inhibited the thromboxane A~ synthesis in fetal platelets (Table II and Fig. 2) but not in umbilical artery, whereas the 500 mg dosage was accompanied by reduced thromboxane A2 synthesis by both of them. The acetylsalicylic acid­induced inhibitions in the platelets' thromboxane A2 synthesis were related to the salicylate levels in umbilical blood (Fig. 3), although no statistically significant dif-

348 Ylikorkala et al.

20 r=-0.594 0

" 0 p<0.01

;; a; ;; 15 • Ci 0 ~ 0 0

·0 '" • 0-10

rJj 0

" 0 .... 0; • 0 0 ;; 5 • u. • • • •

, I I I I I • I· I I ·1 , 8 10 20 50 100 200 300400500

Salicylate concentration in umbilical blood, ~mol/I

Fig. 3. Release of thromboxane B, (TxB2J by the fetal platelets in relation to the salicylate concentration in the umbilical blood in infants born to mothers given 100 mg (0) or 500 mg (.) of acetylsalicylic acid during labor.

ference in the mean thromboxane B" releases per 10" platelets was seen between the acetylsalicylic acid

groups.

Comment

We report the concomitant effects of small doses of maternal acetylsalicylic acid both on fetal and neonatal prostacyclin/thromboxane A2 production. Maternal acetylsalicylic acid was transported to the fetus, as shown by the presence of salicylate in fetal circulation and by the inhibition of fetal prostanoid synthesis. Ace­tylsalicylic acid inhibits thromboxane A, synthesis in adult platelets by irreversibly acetylating the platelet cyclooxygenase.' Our data show that 100 mg and 500 mg of acetylsalicylic acid given to mothers also inhibit the fetal platelet thromboxane A2 synthesis in relation to the salicylate level in umbilical blood. The reduction of fetal platelet thromboxane A2 synthesis in the pres­ent work was, however, much less than what occurred in adult platelets after a single dose of acetylsalicylic acid." This does not, however, necessarily indicate a difference in the sensitivity of fetal and adult platelets to the inhibitory action of acetylsalicylic acid. The ex­planation is probably pharmacokinetic. The half-life of acetylsalicylic acid in blood is only 12 to 20 minutes,

and a great deal of orally ingested acetylsalicylic acid is quickly hydrolyzed to salicylate during absorption in the gastrointestinal tract. If'. " Thus it is conceivable that only a minor fraction of maternal acetylsalicylic acid, if

any, enters the fetal circulation as an intact compound. The main metabolite of acetylsalicylic acid, salicylate, is

a much weaker cyclooxygenase inhibitor than acetyl­

salicylic acid itself. This may explain why fetal platelet thromboxane A2 production was only partially inhib­ited even after a 500 mg dose of maternal acetylsalicylic acid. The fetal vascular thromboxane A2 synthesis was even more resistant to the effect of maternal acetylsal-

August 1986 Am J Obstet Gynecol

icylic acid than was fetal platelet thromboxane A2 syn­thesis, which is compatible with the ex vivo findings on the difference between the sensitivity of fetal platelet and vascular cyclooxygenases against cyclooxygenase inhibitors. '2

Maternal acetylsalicylic acid ingestion as a single dose inhibited the fetal and neonatal prostacyclin synthesis. Interestingly, this effect was seen only with the 500 mg but not the 100 mg dose. Thus our data suggest that a small dose of maternal acetylsalicylic acid can direct the fetoplacental prostacyclinlthromboxane A2 balance to the dominance of prostacyclin, because it inhibits thromboxane A2 synthesis more readily than prosta­cyclin. Thus our present findings may reveal the bio­chemical mechanism by which 60 mg or 150 mg of acetylsalicylic acid daily prevented acute' and super­imposed' preeclampsia, respectively. Keeping in mind the irreversible effect of acetylsalicylic acid on the plate­let thromboxane A2 synthesis" and a platelet life-span about 10 days, these doses of acetylsalicylic acid could perhaps have been given only at 5 to 7 days intervals. In this respect the rationale would be much the same as in trials conducted to prevent occlusive arterial dis­ease with acetylsalicylic acid.'3. II Nevertheless, caution is needed. Although we and the othersl. 2. '5 did not

encounter neonatal bleeding or other side effects, such possibility may exist'" even with administration of small doses of acetylsalicylic acid, because in this circum­stance, bleeding-promoting prostacyclin dominates in the fetus.

REFERENCES

J. Wallenburg HCS, Dekker GA, Makovitz jW, Rotmas P. Low-dose aspirin prevents pregnancy-induced hyperten­sion and pre-eclampsia in angiotensin-sensitive primi­gravidae. Lancet 1986; I: 1-3.

2. Beaufils M, Uzan S, Donsimoni R, Colan jc. Prevention of pre-eclampsia by early antiplatelet therapy. Lancet 1985; I :840-2.

3. Ylikorkala 0, Makila U-M. Prostacyclin and thromboxane in gynecology and obstetrics. AM j OSSTET GVNECOL 1985; 152:318-29.

4. Makila UM, Wahlberg L, Viinikka L, Ylikorkala 0. Reg­ulation of prostacyclin and thromboxane production by human umbilical vessels: the effect of estradiol and pro­gesterone in a superfusion model. Prostaglandins Leu­kotrienes Med 1982;8:115-24.

5. Ylikorkala 0, Viinikka L. Measurement of 6-keto-pros­taglandin F,o in human plasma with radioimmunoassay: effect of prostacyclin infusion. Prostaglandins Leuko­trienes Med 1981 ;6:427-36.

6. Viinikka L, Ylikorkala 0. Measurement of thromboxane B2 in human plasma or serum by radioimmunoassay. Pros­taglandins 1980;20:754-66.

7. Wallenstein S, Zucker CL, Fleiss jL. Some statistical meth­ods useful in circulation research. eirc Res 1980;47: 1-9.

8. Roth Gj, Stanford N, Majeras PW. Acetylation of pros­taglandin synthetase by aspirin. Proc Nat! Acad Sci USA 1975;72:3073-6.

9. Viinikka L, Ylikorkala 0. Effect of various doses of ace­tylsalicylic acid in combination with dipyridamole on the

Volume 155 Number 2

balance between prostacyclin and thromboxane in human serum. Br] Pharmacol 1981;72:299-303.

10. Rowland M, Riegelman S. Pharmacokinetics of acetylsal­icylic acid and salicylic acid after intravenous administra­tion in man.] Pharm Sci 1968;57:1313-9.

II. Spenney]G. Acetylsalicylic acid hydrolase of gastric mu­cosa. Am] Physiol 1978;234:E606-1O.

12. Makila UM, Kokkonen E, Viinikka L, Ylikorkala O. Dif­ferential inhibition of fetal vascular prostacyclin and plate­let thromboxane synthesis by nonsteroidal anti-inflam­matory drugs in humans. Prostaglandins 1983;25:39-46.

Maternal acetylsalicylic acid and fetal prostacyclin and thromboxane

13. Mustard ]F, Kinlough-Rathbone RL, Packham MA. As­pirin in the treatment of cardiovascular disease: a review. Am] Med 1983;14:43-9.

14. Fields WS. Aspirin for prevention of stroke: a review. Am ] Med 1983;14:61-8.

15. Lewis RB, Schulman ]D. Influence of acetylsalicylic acid, an inhibitor of prostaglandin synthesis, on the duration of human gestation and labour. Lancet 1973;2: 1159-61.

16. Stuart M], Gross S], Elrad H, Graeber ]E. Effects of ace­tylsalicylic-acid ingestion on maternal and neonatal he­mostasis. N Engl] Med 1982;307:909-12.

Potassium regulation and progesterone-aldosterone

interrelationships in human pregnancy: A prospective study

Mark A. Brown, M.B., B.S., Michael J. Sinosich, M.Sc., Douglas M. Saunders, M.D., and Eileen D. M. Gallery, M.D.

St. Leonards, New South Wales, Australia

Little is known about the intrinsic renal and hormonal regulation of potassium excretion in pregnancy

despite major alterations in many of the potassium regulatory factors. Forty primigravid women on an unrestricted diet were studied during the second and third trimesters and exhibited constant absolute and

fractional potassium excretion despite a significant increase in plasma aldosterone concentration between

these stages. The plasma progesterone level rose significantly between studies but closer analysiS

showed no correlation between individual changes in plasma aldosterone concentration and progesterone between trimesters. In the 14 subjects studied post partum, baseline absolute potassium excretion was not

significantly altered but filtered potassium fell and fractional potassium excretion tended to rise. After dietary sodium manipulation at these stages, absolute potassium excretion, fractional potassium excretion,

and progesterone were unaltered despite significant changes in plasma aldosterone concentration and sodium excretion. These results suggest that potassium excretion is held constant throughout pregnancy

and that renal tubular potassium reabsorption adjusts appropriately to the increased filtered potassium load. Progesterone does not appear to be involved in the acute regulation of potassium or sodium excretion but may have effects on sodium and potassium excretion that are constant, proportional to its

placental production, and unresponsive to endogenous changes in mineralocorticoid production. (AM J OSSTET GYNECOL 1986;155:349-53.)

Key words: Progesterone, aldosterone, potassium, sodium

It is surprising that the normal pregnant woman is not potassium depleted. Not only is there a 50% in­crease in glomerular filtration rate and therefore in filtered sodium and potassium, but urine flow rate, as well as aldosterone, cortisol, and deoxycorticosterone acetate' production are increased from the nonpreg-

From the DepartrnenLI of Renal Medicine and Obstetrics, Royal North Shore Hwpital.

Dr. M. A. Brown is supported by a Postgraduate Medical Research Scholarship of the National Health and Medic{tI Research Council of Australia.

Received for publication December 18, 1985; revised April 14, 1986; accepted April 22, 1986.

Reprint requests: Dr. M. A. Brown, Department of Renal Medicine, Royal North Shore Hospital, St. Leonards, N.S. W. 2065, Aus­tralia.

nant state. In addition, plasma argmme vasopressin content appears to be increased" and there is a mild alkalosis. All of these factors are capable of promoting urinary potassium loss and hypokalemia either inde­pendently or in concert, and mineralocorticoid pro­duction increases further as pregnancy progresses, ag­gravating this potassium-losing tendency. However, there is an overall calculated cumulative retention of approximately 350 mmol of potassium throughout nor­mal pregnancy.'

The mechanisms responsible for maintaining this net positive accumuiation of potassium are unknown al­though an antikaliuretic role has been ascribed to pro­gesterone, I whose production increases throughout pregnancy and parallels that of aldosterone.' However,