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ORIGINAL RESEARCH |
From the Department of Gynaecology and Obstetrics, University of Turin, Turin, and the Department of Gynaecology, Obstetrics and Pediatrics Sciences, University of Modena, Modena and Reggio Emilia, Italy.
Address reprint requests to: Chiara Benedetto, MD, PhD Department of Obstetrics and Gynecology University of Turin Via Baiardi 43 Turin 10126 Italy E-mail: chbened{at}tin.it
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Abstract |
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Methods: Four groups of pregnant women were included: 17 patients with pregnancy-induced hypertension, ten with mild or moderate preeclampsia, 17 with severe preeclampsia, and 44 normotensive women matched for weeks of gestation at blood sampling with the cases. Plasma levels of L-citrulline and L-arginine were measured by using high-performance liquid chromatography.
Results: The mean plasma levels of L-citrulline and the ratio of L-citrulline to L-arginine, which reflects NO production, were higher in women with severe preeclampsia than in controls, patients with pregnancy-induced hypertension, and patients with mild or moderate preeclampsia.
Conclusion: Nitric oxide production is enhanced in severe preeclampsia, possibly as a compensatory phenomenon for the increased synthesis and release of vasoconstrictors and platelet-aggregating agents.
Nitric oxide (NO) is released by the vascular endothelium and controls blood pressure (BP) by direct vasodilatory action and by blunting the responsiveness to vasoconstrictors. Moreover, it is produced in the platelets, where it inhibits aggregation through the second messenger cyclic guanosine monophosphate (cGMP).1 Because of such properties, it has been hypothesized that derangement of the NO pathway might be involved in the pathogenesis of preeclampsia.2
Animal experiments have shown that administration of NO-synthetase inhibitors to rats produces signs similar to those of preeclampsia, such as sustained hypertension, proteinuria, thrombocytopenia, and fetal growth restriction (FGR).3–6 Furthermore, these effects are reversed by administration of L-arginine substrate of NO; or sodium nitroprusside, an NO donor that increases the endogenous production of NO.5,7–9
Nitric oxide is extremely labile and cannot be measured directly because it rapidly undergoes oxidation to the inorganic end-products nitrite and nitrate.1 In humans, indirect evaluation of NO production through measurement of nitrite, nitrate, and cGMP levels in plasma and urine samples during pregnancy has produced conflicting results.10–19 NO production was found to be higher, lower, or unchanged in preeclamptic women compared with normotensive pregnant women. Such discrepancies could be due to different assays; patient heterogeneity; or confounding factors, such as dietary intake, infections, cigarette smoking, alcohol consumption, atmospheric pollution, and exercise.1 Another possible explanation is that measurement of NO metabolites (nitrite and nitrate) or second messenger (cGMP) is not a reliable method to assess NO production and metabolism.
L-Citrulline is the stoichiometric metabolite resulting from the conversion of L-arginine to NO. Measurement of L-citrulline may be the most specific and reliable method to assess NO synthetase activity in vivo. Such conversion has proven to be consistent with the evaluation of mRNA of NO synthetase, both in platelet preparation2 and placental tissue.20 It has been observed that L-citrulline production after L-arginine loading in preeclamptic patients is smaller than in normotensive pregnant women.21
To verify the hypothesized derangement of the NO pathway in preeclampsia, we measured plasma levels of L-citrulline and L-arginine in normotensive pregnant women and in patients with pregnancy-induced hypertension or preeclampsia.
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Materials and Methods |
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All patients with pregnancy-induced hypertension or preeclampsia were seen consecutively over a 6-month period and were selected from women referred to the antenatal clinics and obstetric units of the Departments of Gynecology and Obstetrics, University Hospitals of Turin and Modena, Italy.
Blood samples were obtained at the time of diagnosis, and controls were matched for gestational age at the time of sampling. Only one blood sample was taken from each patient. No patient was in labor at the time of blood sampling. The study was approved by the Local Ethical Committee. Informed consent was obtained from each patient before inclusion in the study.
We assigned patients to the pregnancy-induced hypertension group or the preeclampsia group by using the criteria reported by Davey and MacGillivray.22 According to these criteria, pregnancy-induced hypertension was diagnosed when two consecutive measurements (obtained 4 hours or more apart) of diastolic BP equal 90 mmHg or greater were found after the 20th week of pregnancy in a previously normotensive woman. Preeclampsia was diagnosed when two consecutive measurements of diastolic BP 90 mmHg or greater and proteinuria 300 mg or greater in one 24-hour urine collection were found after the 20th week of pregnancy in a previously normotensive and nonproteinuric woman. Severe preeclampsia was diagnosed in the presence of at least two of the following findings: diastolic BP 110 mmHg or greater, proteinuria more than 1 g/24 hours, fetal growth restriction, birthweight less than the tenth centile, or delivery before 34 weeks. According to the criteria currently in use in our departments, preeclampsia was considered to be severe also in the presence of the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, disseminated intravascular coagulation, or intrauterine fetal death, even if diastolic BP was less than 110 mmHg. The assignment of patients to each group was reevaluated at the end of pregnancy to confirm the diagnosis.
Blood was obtained by venipuncture and centrifuged; serum was stored at -80C until assay. One mL of serum was added to 30 mg of sulfosalicylic acid powder (Sigma Chemical Co., St. Louis, MO), vortexed, and centrifuged at 2000g for 20 minutes at 4C. A fraction of clear supernatant was mixed with an equal volume of ophtaldialdeide for 1 minute. This fluorescent derivative was then injected into a high-performance liquid chromatography apparatus (Waters Inc., Millipore, MN) equipped with an RP C-18 column. Flow rate was adjusted to 1 mL/min. The mobile phase was constituted by a mixture of 0.012 M phosphate buffer with acetonitrile and methanol (91:4:5), pH 5.9. Citrulline and arginine have a retention time of 6.8 and 10.5 minutes, respectively.
A pool of serum from pregnant women served as external standard and was tested over 6 months. Citrulline levels were 32.7 µmol/L and arginine levels were 97.1 µmol/L. Interassay coefficients of variation were 5.4% and 5.8%, respectively.
Statistical analysis was performed by using the Statistical Package for Social Science software (SPSS Inc., Chicago, IL), and data were analyzed for significant differences by using the Student–Newman-Keuls test. Differences were considered significant if P < .05. The linear regression equation was applied to correlate the plasma levels of L-citrulline and L-arginine with BP values, proteinuria, and birth weight.
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Results |
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. Mean (± SE) levels of L-citrulline and L-arginine in each group of pregnant women are shown in Table 2. No differences were observed in mean gestational age at the time of blood sampling (controls, 30 ± 0.9 weeks; pregnancy-induced hypertension group, 31 ± 1.3 weeks; mild or moderate preeclampsia group, 30 ± 1.6 weeks; severe preeclampsia group, 28 ± 1.4 weeks).
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Discussion |
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Our results show that plasma levels of L-citrulline and the L-citrulline/ L-arginine ratio values, which reflect NO production, are significantly higher in women with severe preeclampsia than in normotensive pregnant women, patients with pregnancy-induced hypertension, or patients with mild or moderate preeclampsia. Because the decrease in L-arginine plasma levels mirrors the increase in L-citrulline, we can exclude the possibility that L-citrulline may be derived from other sources, such as the urea cycle. It should also be considered that L-citrulline could be reconverted to L-arginine, although the reduced production of L-citrulline that occurs with L-arginine loading in less severe cases of preeclampsia limits such a possibility.21 Of note, however, a trend toward reduced L-arginine levels in preeclamptic patients was recently reported (Jerath V, Awoniyi L, Leslie KK. L-Arginine and nitric oxide levels in pregnancy: Possible correlation with preterm labor and preeclampsia [abstract]. J Soc Gynecol Investig 1999;6(Suppl 1):120). These observations suggest that NO production is enhanced in the most severe forms of preeclampsia, and they do not support the data of Seligman et al,11 who found a reduction in nitrite plasma levels in preeclampsia and proposed that diminished NO synthesis may play a key role in the pathogenesis of the disease.
To reconcile the above-reported discrepancies, a stepwise hypothesis was made23: in preeclamptic women, reduced endothelial and platelet activity of NO occur early in pregnancy, representing one among several predisposing factors; vascular resistance increases in uteroplacental circulation and platelet aggregability is enhanced because of targeted reduction of NO release; and an increased NO production seems to be stimulated when overt preeclampsia develops. Such a finding could be interpreted as a compensatory phenomenon, although it does not necessarily represent improvement in the clinical condition. Indeed, the abundance of NO in the presence of superoxide produces peroxynitrite, which further impairs vascular function.20,24 Another possibility is that the NO pathway is upregulated by other metabolic systems, activated by hypoxia or endothelium damage, that are involved in the control of the maternal and fetoplacental circulation. For instance, hypoxia complicating preeclampsia may directly induce the expression and activity of endothelial NO synthetase in placental tissues, as is the case in cerebral endothelial cells during focal ischemia.25 Furthermore, placental corticotropinreleasing hormone, levels of which are increased in the plasma of preeclamptic patients compared with controls,26 has been demonstrated to stimulate NO synthesis.27 For these reasons, the enhanced NO production in preeclampsia may be regarded as a secondary mechanism to maintain BP and peripheral perfusion within normal ranges, probably through interactions with other vasodilatory systems.
A more interesting hypothesis may be formulated if one considers the metabolic processing of NO after synthesis and release. Nitric oxide rapidly interacts with oxygen and is oxidized to the biologically inactive nitrite and nitrate.1 Under physiologic conditions, this is the main pathway of NO inactivation. However, NO may also interact with superoxide to form peroxynitrate, which is a major cytotoxic agent produced during inflammation, sepsis, and ischemic injuries.1 It has been suggested that the vascular endothelium can regulate the effects of NO by generating superoxide. An increase in superoxide production28 and the vascular formation of peroxynitrite have been demonstrated in preeclampsia29; moreover, the generation of superoxide and peroxynitrite may be enhanced when neutrophils and macrophages are activated, as in the HELLP syndrome.30 Therefore, it is likely that in the most severe forms of preeclampsia, the higher NO production, together with the increased levels of superoxide, may generate greater amounts of peroxynitrite, potentially leading to alterations in vascular reactivity and in systemic and local blood flow, thus exacerbating the pathologic features of the disease.
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Footnotes |
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Received November 1, 1999. Received in revised form March 8, 2000. Accepted March 16, 2000.
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2. Boccardo P, Soregaroli M, Aiello S, Noris M, Donadelli R, Lojacono A, et al. Systemic and fetal-maternal nitric oxide synthesis in normal pregnancy and preeclampsia. Br J Obstet Gynaecol 1996; 103:879–86.[Medline]
3. Yallampalli C, Garfield RE. Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia. Am J Obstet Gynecol 1993;169:1316–20.[Medline]
4. Molnar M, Suto T, Toth T, Hertelendy F. Prolonged blockade of nitric oxide synthesis in gravid rats produces sustained hypertension, proteinuria, thrombocytopenia, and intrauterine growth retardation. Am J Obstet Gynecol 1994;170:1458–66.[Medline]
5. Diket AL, Pierce MR, Munshi UK, Voelker CA, Eloby-Childress S, Greenberg SS, et al. Nitric oxide inhibition causes intrauterine growth retardation and hind-limb disruption in rats. Am J Obstet Gynecol 1994;171:1243–50.[Medline]
6. Ahokas RA, Lubarsky SL, Park GC, Friedman SA, Sibai BM. Chronic nitric oxide synthesis inhibition does not prevent pregnancy vasodilation in the rat. Hypertens Preg 1998;17:55–68.
7. Buhimschi I, Yallampalli C, Ckwalisz K, Grafield RE. Preeclampsia-like conditions produced by nitric oxide inhibition: Effects of L-Arginine, D-Arginine, and steroid hormones. Hum Reprod 1995; 10:2723–30.
8. Helmbrecht GD, Farhat MY, Lochbaum L, Brown HE, Yadgarove KT, Eglinton GS, et al. L-Arginine reverses the adverse pregnancy changes induced by nitric oxide synthase inhibition in the rat. Am J Obstet Gynecol 1996;175:800–5.[Medline]
9. Chwalisz K, Buhimischi I, Garfield RE. Role of nitric oxide in obstetrics. Prenat Neonat Med 1996;1:292–328.
10. Cameron IT, van Papendorp CL, Palmer RMJ, Smith SK, Moncada S. Relationship between nitric oxide synthesis and increase in systolic blood pressure in women with hypertension in pregnancy. Hypertens Preg 1993;12:85–92.
11. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944–8.[Medline]
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13. Brown MA, Tibben E, Zammit VC, Cario GM, Carlton MA. Nitric oxide excretion in normal and hypertensive pregnancy. Am J Obstet Gynecol 1996;175:1013–7.[Medline]
14. Silver RK, Kupfermink MJ, Russel TL, Adler L, Mullen TA, Caplan MS. Evaluation of nitric oxide as a mediator of severe preeclampsia. Am J Obstet Gynecol 1996;175:1013–7.
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18. Di Iorio R, Marinoni E, Emiliani S, Villaccio B, Cosmi EV. Nitric oxide in preeclampsia: Lack of evidence for decreased production. Eur J Obstet Gynecol Reprod Biol 1998;76:65–70.[Medline]
19. Facchinetti F, De Martis S, Neri I, Caputo AS, Volpe A. Effects of transdermal glyceryltrinitrate on 24-hour blood pressure changes in patients with gestational hypertension. Prenat Neonat Med 1997;2:22–8.
20. Myatt L, Rosenfield RB, Eis AL, Brockman DE, Greer I, Lyall F. Nitrotyrosine residues in placenta. Evidence of peroxynitrite formation and action. Hypertension 1996;28:488–93.
21. Facchinetti F. L-arginine infusion reduces blood pressure in preeclamptic women through nitric oxide release. J Soc Gynecol Investig 1999;6:202–7.[Medline]
22. Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Clin Exp Hypertens 1986; B5(1):97–133.
23. Neri I, Mazza V, Galassi C, Volpe A, Facchinetti F. Effects of L-arginine infusion on uteroplacental circulation in growth-retarded fetuses. Acta Obstet Gynecol Scand 1996;75:208–12.[Medline]
24. Davidge ST. Oxidative stress and altered endothelial cell function in preeclampsia. Semin Reprod Endocrinol 1998;16:65–73.[Medline]
25. Zhang ZG, Chopp M, Zaloga C, Pollock JS, Forstermann U. Cerebral endothelial nitric oxide synthase expression after focal cerebral ischemia in rats. Stroke 1993;24:2016–22.
26. Petraglia F, Florio P, Benedetto C, Gallo C, Woods RJ, Genazzani AR, et al. High levels of corticotropin-releasing factor (CRF) are inversely correlated with low levels of maternal CRF-binding protein in pregnant women with pregnancy-induced hypertension. J Clin Endocrinol Metab 1996;81:852–6.[Abstract]
27. Clifton VL, Read MA, Leitch IM, Giles WG, Boura ALA, Robinson P, et al. Corticotropin-releasing hormone-induced vasodilation in the human fetal placental circulation: Involvement of the nitric oxide-cyclic guanosine 3', 5'-monophosphate-mediated pathway. J Clin Endocrinol Metab 1995;80:2888–93.
28. Tsukimori MI, Maeda H, Ishida K, Nagata A, Koyanagi T, Nakano H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. Obstet Gynecol 1993;81:536–40.
29. Roggensack AM, Zhang Y, Davidge ST. Evidence for peroxynitrite formation in the vasculature of women with preeclampsia. Hypertension 1999;33:83–9.
30. Walsh SW. Lipid peroxidation in pregnancy. Hypertens Preg 1994;13:1–32.
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