INCREASE IN URINARY THROMBOXANE EXCRETION DURING PREGNANCY AND LABOR W.A. Noort, F.A. de Zwart and M.J.N.C. KeirseI Department of Obstetrics and Gynecology, Leiden University Hospital, The Netherlands ABSTRACT Urinary TXB2 excretion was measured during pregnancy and labor using high pressure liquid chromatography and radioimmunoassay. From the first trimester onwards TXB2 levels in urine of pregnant women (n=60) were significantly (p (0.001) higher than in non-pregnant women (n=12) and they increased, albeit not significantly, with advancing gestation. Labor was associated with a two-fold increase in urinary TXB2 excretion. Levels in established labor were significantly higher than at any other time in pregnancy (p tO.OOl), but the levels in incipient labor showed considerable overlap with these in late pregnancy. Thus urinary TXB2, while not necessarily originating from the pregnant uterus, appears to reflect the uterine activity of labor and may be the expression of a general stimulation of prostanoid production during parturition. INTRODUCTION There is substantial evidence that cycle-oxygenase products, prostaglandins in particular, play a critical role in the onset and progress of human parturition (1, Some of these compounds, especially thromboxane A2 2). (TXA2) and prostacyclin, are further believed to be intimately involved in physiological processes of pregnancy. Moreover, an imbalance in the production of TXA2 and prostacyclin has been put forward as an explanation for several pathological states of pregnancy, such as pregnancy-induced hypertension (3, 4), fetal growth retardation (5), and other states of chronic placental insufficiency Pharmacological suppression of (6). thromboxane production has thus been utilized to improve the outcome of pregnancy in these conditions (7, 8). In vivo assessment of prostanoid production remains fraught with difficulties (9), however, and this applies in particular to TXA2 production in pregnancy. While the placenta is known to contain substantial amounts of lReprint requests to M.J.N.C. Keirse, Department Obstetrics EiGynecology, Leiden University Hospital, P.O.Box 9600, 2300 RC Leiden, The Netherlands
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thromboxane synthase (l), there are virtually no data on TXA2 prOdUCtiOn in vivo during human pregnancy. TXB2 is known to be present in amniotic fluid in amounts that are larger during than before labor (10, 11) but, for obvious reasons, no data are available earlier in pregnancy. TXB2 concentrations measured in plasma and serum mostly reflect the quantity and the biosynthetic capacity of blood platelets in vitro, rather than being an expresof TXA2. Measurements in sion of in vivo prOduCtiOn urine, however, are less likely to be influenced by sample collection and handling. Therefore, we measured urinary TXB2 excretion throughout pregnancy and labor to address three questions in particular: First, is thromboxane production suppressed in normal physiological pregnancies as compared to the non-pregnant state? second, does TXB2 excretion change at the onset of labor? Third, can the excretion of non-metabolized TXB2 provide a useful parameter to study changes in prostanoid production in vivo at and during parturition? MATERIALS AND METHODS Urine was collected from healthy non-pregnant women (540 years), from pregnant women in the first, second and third trimesters of pregnancy and from women during incipient and established labor (each group, n=12). Established labor was defined as the presence of strong and regular contractions with a frequency of at least 3 per 10 min. together with progressive cervical dilatation; otherwise labor was considered to be incipient (in all of these, cervical dilatation was 4 cm or less). Urine samples from women not in labor were early morning samples; during labor samples were collected whenever labor occurred. Urine samples were centrifuged at 4°C immediately after voiding and aliquots were frozen and stored at -20°C until assayed. Extraction and chromatography: Urine samples (4 ml) were spiked with 3,000 cpm JH-TXB2 (New England Nuclear, Boston, Mass.; 180 Ci/mmol), adjusted to pH 4 with citric acid and extracted twice with 10 ml ethyl acetate : petroleum ether (60:4O,v/v). The pooled organic phases were evaporated under nitrogen at 35°C. The dry residue was dissolved in 2 ml H20 with citric acid and further (Waters Ass., extracted using Sep-pak Cl8 CartridgeS Milford, Mass.) (12). The cartridges were pretreated with ethanol (20 ml) and then with water (20 ml) before samples were applied. The cartridges were rinsed with 10 ml acidified (citric acid) ethanol : water (20:8O,v/v) and 5 ml petroleum ether successively. The TXB2 fraction was eluted with 10 ml chloroform : petroleum ether
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(70:3O,v/v). The eluate was dried under nitrogen and dissolved in 0.25 ml water, containing 0.75 % acetic acid (v/v) for high pressure liquid chromatography (HPLC). The HPLC system (Gilson Med. Electronics, Villiers-le-Bel, phase guard column France) was equiped with a reversed (75 x 2.1 mm ID) and a Lichrosorp lo-RP-18 colomn (250 x 4.6 mm ID) (Chrompack, Middelburg, Netherlands) as described by Moonen et al. (13). TXB2 was eluted with an increasing gradient of acetonitrile, from 28 to 100 % (v/v) at a flow rate of 2 ml/min. Fractions (1 ml) containing TXB2 were collected and pooled. Acetonitrile was evaporated under nitrogen at 35°C and the aqueous residue was lyophilized. Acetonitrile, ethyl acetate and chloroform (Merck) were of spectroscopic grade. Radioimmunoassay: The lyophilized residues were suspended in 1 ml 0.05 M Tris-HCl buffer (pH 7.4); 175 pl being used for radiometric determination of recovery after extraction and HPLC. Aliquots of 350 ~1 and 50 ~1 were incubated with 50 ul 3H-TXB2 and 50 u-11antibody in duplicate overnight at 4"~. The antibody (14) was used in a final dilution of 1:11,500. Separation of bound from free ligand was carried out with dextran-coated charcoal. Under these assay conditions the least detectable concentration was 16 pg and 50 % binding in the standard curve corresponded to 60 pg. Intra- and inter-assay coefficients of variation were 6.3 (n=ll) and 11.2 % (n=4) respectively. Recovery of 3H-TXB2 added to urine was 65 f 9.2 % mean & sd; (n=58). TXB2 levels were expressed either in rig/l urine or in rig/g creatinine. Creatinine was determined by a routine method. The Mann-Witney test was used for statistical analysis. Because we could not predict the direction of difference between the non-pregnant and pregnant state, we used a two-tailed test. For testing between different states of pregnancy we used a one-tailed test. Table 1. Urinary excretion of TXB2 during normal pregnancy and labor
TXB2 concentrations (means f sem) rig/l urine rig/g creatinii
non-pregnant 1st trimester 6-13 wk) 2nd trimester 14-25 wk) 3rd trimester 28-42 wk) incipient labor progressive labor
31 51 67 97 80 229
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* f f f f *
5 6 12 17 17 41
18 41 50 87 105 183
f 2 f 6 +- 7 + 16 f 12 f 20
RESULTS TXB2 levels (expressed either in ng TXB2 /l urine or in ng TXB2 /g creatinine) in urine of pregnant women were significantly higher than those in non-pregnant women (p CO.001) (Table 1). The difference with the non-pregnant state was already seen in the first trimester (rig/l urine, p (0.05; rig/g creatinine, p <0.002) with some, albeit non-significant, increase in urinary TXB2 excretion with advancing gestation. TXB2 levels in the third trimester were significantly higher (p (0.025) than in the first trimester of pregnancy, however. TXB2 concentrations in early labor were significantly greater than in the first (p tO.001) and second (p tO.001) trimester of pregnancy, but only when expressed per gram creatinine and not when expressed per litre of urine. There was no difference in TXB2 concentrations between the third trimester of pregnancy, before the onset of labor, and the early stages of labor (incipient labor). In progressive labor with well-established uterine ng TXB, / g CREAT
Fig. 1. Urinary excretion of TXB2 before and during pregnancy and labor (means and 95 % confidence intervals)
,, non pregnant
tridsters of pregnancy
incip. progr. labor
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were significantly concentrations contractions, TXB2 higher than before labor (each trimester of pregnancy, p < 0.001). Fig. 1 shows mean values with 95 % confidence limits for all groups studied, and illustrates the clear pregnancy and distinction between the non-pregnancy, established labor groups. TXB2 concentrations in incipient labor, however, showed considerable overlap with those obtained before the onset of labor (Fig. 1). DISCUSSION When compared to both the non-pregnant state and various pathological states of pregnancy, a relative deficiency in the production of TXA2 over that of other prostanoids is thought to be an important feature of the normal physiology of pregnancy. Our data, however, show that throughout normal pregnancy urinary TXB2 excretion is significantly higher than before pregnancy and that this difference becomes established quite early in the first trimester (Fig. 1). These data seem to be at variance with the recent observations of Ylikorkala et al. (4) who found no statistical difference in urinary TXB2 levels between pregnant and non-pregnant women. But in that study, the non-pregnant group consisted of women who were 6 weeks post-partum and it is not clear whether the data are based on the first voided urine, as in the present study, or on random samples obtained during the day. Although it is known that the human placenta can synthesize TXA2 in early as well as in late pregnancy (I), it cannot be assumed that the increase in urinary TXB2 excretion during pregnancy originates from the pregnant uterus or its contents. For example, urinary TXB2 excretion is known to be influenced by the urinary flow rate, probably by affecting passive reabsorption in the distal nephron (15). In pregnancy, the glomerular filtration rate is known to increase with about 50 %, while renal plasma and blood flow increase with about 25 % (16). Several other physiological adaptations occur in pregnancy (16) and these adaptations may be at least as important for the urinary TXB2 concentration as the contribution, if any, from the reproductive system itself. It should also be realized that 2,3-dinor TXB2 rather than TXB2 itself is the main urinary metabolite of TXB2 (17). It is conceivable, that the increased TXB2 excretion in pregnancy relates not only to an increase in TXA2 production but, to some extent, also to changes in the relative proportions of the various TXA2 metabolites that are excreted in urine. This may well be the reason that, in general, little attention has been paid to urine as a means of assessing
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changes in prostanoid production during pregnancy and labor (18-20) and that, until recently (4), there were no published data on urinary TXB2 excretion in pregnancy. The increase observed in pregnancy as compared to outside explain the increased pregnancy may well partially tendency for thromboembolism in pregnancy. On average, but with wide variations, our data indicate a TXB2 excretion of roughly 70 to 100 ng TXB2 per day in pregnancy. If this were assumed to originate entirely from extra-renal sources and if the excretion or clearance of thromboxane were unaltered by pregnancy, this would mean that daily about 520 to 750 ng TXB2 enters the renal arteries. Indeed, Zipser and Martin (21) found that 13.5 % of 3H-TXB2 infused into the renal artery is excreted as 3H-TXB2 in the urine. Of the 3H-TXB2 infused in the peripheral venous circulation, however, only 2.3 (21) to 2.5 % (17) was excreted unaltered in the urine. Again assuming that all TXB2 is of extra-renal origin and that its excretion is unaffected by pregnancy, this would mean that during pregnancy approximately 3 to 4 ug TXB2 is released in or into the peripheral circulation per day. During labor this would increase more than two-fold, at least if the same assumptions can be upheld. These estimations, however, can only be seen as a rough guideline and they are particularly tenuous because it is known that the kidney itself is, at least in vitro, capable of TXB2 production (22). Whatever the source of the urinary TXB2, it is clear that there is a marked increase with labor. Generally, one would assume that a compound that has not undergone extensive metabolism before being excreted can herald fast and/or acute changes in prostanoid production. Since thromboxane is a poor substrate for the ubiquitous prostaglandin dehydrogenase being 15-hydroxy (23), degraded mainly by the slower processes of fi- and Ooxidation, it was not illogical to expect changes with the onset of labor. Indeed, the onset of labor is known to be associated not only with marked increases in prostaglandin biosynthesis but also with an increase in the production of the other cycle-oxygenase products (9). Nevertheless, we found considerable overlap between values obtained before and after the onset of labor. Only when labor was firmly established with regular contraccervical dilatation did TXB2 tions and progressive excretion reach values that were consistently above those recorded before the onset of labor. Further studies are in progress to examine whether urinary TXB2 excretion can be useful to distinguish true from false preterm labor, a distinction that is often difficult to make in clinical practice.
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ACKNOWLEDGMENTS We are grateful to MS J. Paulusma for the selection of normal pregnancies. The study was supported by grant 28-1118 from the Praeventiefonds, the Netherlands. REFERENCES 1.
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2. Huszar, G., and F. Naftolin. The myometrium and uterine cervix in normal and preterm labor. N. Engl. J. Mea. 311: 571, 1984. 3. Keirse, M.J.N.C., Moonen, P., and G. Klok. Control of prostacyclin synthesis in pregnancy-induced hypertension. Prostaglandins 29: 643, 1985. 4. Ylikorkala, O., Pekonen, F., and L. Viinikka. Renal prostacyclin and thromboxane in normotensive and preeclamptic pregnant women and their infants. J. Clin. Endocrinol. Metab. 63: 1307, 1986. 5. Jogee, M., Myatt, L., and M.G. Elder. Decreased prostacyclin production by placental cells in culture from pregnancies complicated by fetal growth retardation. Br. J. Obstet. Gynaecol. 90: 247, 1983. 6. Ylikorkala, O., and U.-M. MgkilZ. Prostacyclin and thromboxane in gynecology and obstetrics. Am. J. Obstet. Gynecol. 152: 318, 1985. 7. Van Assche, F.A., Spitz, B., Vermylen, J., and H. Deckmijn. Preliminary observations on treatment of pregnancy-induced hypertension with a thromboxane synthetase inhibitor. Am. J. Obstet. Gynecol. 148: 216, 1984. 8. Wallenburg, H.C.S., Makovitz, J.W., Dekker, G.A., and P. Rotmans. Low dose aspirin prevents pregnancy-induced hypertension and preeclampsia in angiotensin sensitive primigravidae. Lancet i: - 1, 1986. 9. Keirse, M.J.N.C. Endogenous prostaglandins in human parturition. In: Human Parturition (M.J.N.C. Keirse, A.B.M. Anderson, and J. Bennebroek Gravenhorst, eds.). Leiden University Press, The Hague, 1979. p.101.
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