Normal pregnancy is associated with a profound increase in cardiac output and circulating intravascular volume. Despite these changes, blood pressure levels in normal pregnancy decrease from early pregnancy, which occurs as a result of decreased peripheral vascular resistance brought about by marked systemic vasodilation.1 Nitric oxide is a potent vasodilator and is thought to have a major effect on gestational vasodilation.2 Nitric oxide is synthesized from its substrate L-arginine by the enzyme nitric oxide synthase. This enzyme exists in both a constitutive (calcium-dependent) and inducible (calcium-independent) form in endothelial cells, platelets, and placental tissues.3 Increased nitric oxide synthase activity has been observed in villous trophoblast cells, and nitric oxide may be important in the vascular adaptation needed to accommodate increased uteroplacental blood flow as pregnancy advances.4 The endothelial form of nitric oxide synthetase has been localized to the syncytiotrophoblast and villous endothelium in term pregnancies.5,6 The placenta is therefore an important source of nitric oxide during pregnancy.
The early pathogenesis of preeclampsia may be related to the normal spiral arteries failure to adapt by trophoblastic migration,7 leading to reduced placental blood flow. In late pregnancy, failure of trophoblast invasion of the uteroplacental vessels was found in normotensive and hypertensive pregnancies complicated by fetal growth restriction (FGR).8 Subsequently, widespread vascular endothelial cell dysfunction is thought to mediate the vasospasm and manifest as hypertension, which is a central feature of established preeclampsia. Altered release of nitric oxide from endothelial cells in the fetoplacental and peripheral circulations may also influence the pathogenesis of this disease.
Studies on the role of nitric oxide in preeclampsia have yielded conflicting results. Circulating plasma levels of nitric oxide metabolites in the peripheral circulation in preeclampsia have been reported as decreased,9 increased,10,11 and unchanged12,13 compared with normotensive pregnancies. Increased levels of nitric oxide metabolites have been described in the fetoplacental circulation.14 Altered production of nitric oxide may influence the reduced placental blood flow associated with preeclampsia. Placental tissue levels of nitric oxide synthase in preeclamptic pregnancy have been reported as decreased.15–17 Giles et al18 reported that placental nitric oxide synthase activity was reduced in placentae from pregnancies with abnormal umbilical artery flow velocity waveforms. In addition, infusion of a nitric oxide donor improved uterine artery diastolic blood flow in high-risk pregnancies.19 However, levels of nitric oxide in the uteroplacental circulation in either normotensive or preeclamptic pregnancy have not been reported. We hypothesized that levels of nitric oxide metabolism would be altered in the uteroplacental circulation in preeclampsia compared with normotensive pregnancy. To test this hypothesis and to extend the observations made in previous studies we simultaneously measured circulating levels of nitrites, the stable metabolites of nitric oxide, in the uteroplacental, fetoplacental, and peripheral circulation in both normotensive and preeclamptic pregnancies.
Materials and Methods
Sixteen women requiring cesarean delivery for established preeclampsia and 15 age-matched normotensive control women who had elective cesarean for other indications were enrolled in the study over an 8-month period. None of the women were in established labor. Specific exclusion criteria for the study included a history of hypertension, renal disease, cardiac disease, and diabetes mellitus. Preeclampsia was defined strictly according to internationally approved criteria.20 All women gave informed consent, and the study had the approval of the ethics committee of the Rotunda Hospital. Because of the technical difficulties of uterine vein sampling it was not possible to have matched control and preeclamptic subjects. The two groups therefore represent a convenience sample.
After delivery of the infant but before delivery of the placenta, 5 mL of blood was taken from a uterine vein. Simultaneously, 5 mL of blood was taken from an antecubital vein and an umbilical vein with minimum venous stasis. All blood samples were taken into dry plastic syringes and immediately transferred into tubes containing lithium heparin. Samples were centrifuged at 2000 g for 20 minutes at 4C within 2 hours of sampling. Plasma was removed and stored at −70C until assay of nitrite levels.
Nitric oxide is oxidized spontaneously to both nitrite and nitrate. These metabolites can be measured in plasma by determining total nitrite levels using the Greiss reaction after reduction of plasma nitrates using nitrate reductase as previously described.21 Plasma was first deproteinized by adding 200 μL of 10% ZNSO4 followed by 200 μL of NAOH (0.5 N) to 100 μL of plasma. Treated samples were vortexed for 30 seconds and allowed to stand for 15 minutes at room temperature. After centrifugation at 20,000 g for 10 minutes at 4C, 200 μL of supernatant (in duplicate) was removed and added to 100 μL of phosphate buffer (0.1 M, pH 7.5) in brown eppendorf tubes. Nitrate reductase (Aspergillus species) (0.3 U/mL, 10 μL), 15 μM flavine adenine dinucleotide (10 μL), and 90-μM nicotinamide adenine dinucleotide phosphate (10 μL) were added to each tube to a final volume of 330 μL. Tubes were mixed and incubated in a water bath at 37C for 30 minutes. Excess reduced nicotinamide adenine dinucleotide phosphate was oxidized by adding pyruvate (1 mM) and lactate dehydrogenase (32 U/mL) in 10 μL of phosphate buffer to give a final volume of 340 μL. Samples were mixed and incubated at 37C for another 10 minutes. At the end of the incubation period, all tubes were cooled. Suphanilamide (30 μL) (12.5 mM) followed by HCL (6 M) (30 μL) were added, mixed, and each tube was allowed to stand at room temperature for 10 minutes. Napthylene diamine hydrochloride (25 μL) (12.5 mM) was added to 300 μL of the resulting solution in clear plastic tubes. Samples were left to stand for 10 minutes and were read in a spectrophotometer at 548 nm. A range of concentrations of sodium nitrite was prepared, treated as above, and used to convert sample absorbance into nitrite concentration. Interassay and intraassay coefficients of variation were 8.5% and 5.6%, respectively. To check conversion of nitrate to nitrite, known amounts of nitrate were added to control plasma samples; these samples were deproteinized and reduced as above. Recovery of nitrate was 97.3 ± 18.09%.
Data were analyzed using the NCSS statistical system (J. L. Hintze, Kaysville, UT). All data were log transformed to achieve a normal distribution and analyzed using general linear modeling-analysis of variance and paired and unpaired t test. The χ2 test was used to detect any differences between the number of primigravidas in each group. On the basis of previous data,14 the study had sufficient power to detect a significant difference in nitric oxide levels between the preeclampsia and normal pregnancy groups (α = .05, β = .90). As a secondary analysis, the relationships between nitrite levels and gestational age, placental weight, birth weight, and blood pressure were assessed using regression analysis.
The basic clinical characteristics of the preeclamptic and normotensive groups are shown in Table 1. Birth weights of infants of preeclamptic mothers were significantly lower than those of the control group (P < .001). Placental weights were also lower in the preeclamptic group compared with the control group, but this difference was not significant. The gestational age at delivery was significantly lower in the preeclamptic group compared with the control group (P < .001).
With gestation and birth weight as covariates, GLMANOVA showed that preeclampsia had a significant effect on nitric oxide metabolites levels in the peripheral (P < .02), uteroplacental (P < .01), and fetoplacental (P < .001) circulations of pregnant women (Figure 1). Unpaired t test showed a higher level of nitric oxide metabolites in uteroplacental (P < .01), fetoplacental (P < .001), and peripheral circulation (P < .02) of pregnancies complicated by preeclampsia compared with normal pregnancies (Figure 1).
Paired t test showed that there was no significant difference in nitric oxide metabolite levels between the samples taken from the uterine vein compared with fetoplacental or peripheral samples in either the preeclamptic or control groups. There was no significant difference in nitric oxide metabolite levels between primigravidas and multiparous women in the preeclamptic or normotensive groups.
Regression analysis of either the preeclampsia or normal pregnancy group did not show any significant correlation with birth weight, placental weight, blood pressure, or gestational age. The small sample size in each group limited the statistical power to detect significant difference. When the preeclampsia and normal pregnancy groups were combined, regression analysis showed a significant relationship between birth weight and fetoplacental nitric oxide metabolite levels (r = −.544, P < .004) (Figure 2). Nitric oxide metabolite levels in the uteroplacental and peripheral circulations were not significantly related to birth weight. Similarly, gestational age at sampling was significantly correlated with fetoplacental nitric oxide levels (r = −.489, P < .01), but there was no relationship between uteroplacental or peripheral nitric oxide metabolite levels and gestational age (Figure 3).
Blood pressure, placental weight, and maternal age were not related to peripheral, fetoplacental, or uteroplacental nitric oxide metabolite levels.
Preeclampsia is a multisystem disorder involving vasoconstriction and hypertension in the mother and decreased blood flow and growth restriction in the fetus. The primary disturbance appears to be in the uteroplacental circulation. Because normal trophoblastic invasion does not occur, the spiral arteries retain their endothelial lining and underlying smooth muscle. Thus, these vessels still have the capacity to respond to vasoactive agents released by their endothelial cells. Similarly, abnormal placentation can occur in fetal growth restriction in the absence of any maternal features such as hypertension. Preeclampsia is thought to be mediated by widespread vascular endothelial cell dysfunction.22,23 The features of endothelial dysfunction are vasospasm, platelet activation, and leukocyte activation; nitric oxide is known to inhibit all three processes.
In this study, we showed that nitric oxide metabolites are higher in blood taken from the uterine vein of pregnancies complicated by preeclampsia than in blood from normal pregnancies. Little is known about nitric oxide metabolism in the uteroplacental circulation. Studies on nitric oxide synthase in preeclamptic placentae have had conflicting results. Morris et al15 showed that nitric oxide synthase activity is reduced in placental homogenates from pregnancies complicated by preeclampsia. In contrast, a study of placental villous explants showed a difference in nitric oxide metabolite levels between normal and preeclamptic pregnancies.24 Conrad and Davis5 found similar Vmax and Km values for calcium-sensitive nitric oxide synthase activity in placenta from both normotensive and hypertensive pregnancies. Another study showed increased immunostaining for the endothelial form of nitric oxide synthase in stem villous cells of the fetoplacental unit from pregnancies complicated by preeclampsia compared with control pregnancies.25 Some of these discrepancies might be explained by the methodology used to measure nitric oxide synthase levels. Assays of nitric oxide synthase activity (arginine to citrulline conversion) from tissue homogenates rely on oxygen. Low levels of nitric oxide synthase as measured by this method may therefore be caused by reduced availability of intervillous oxygen as a result of placental ischemia rather than by reduced enzyme concentration.5 In contrast, immunostaining gives a measure of the antigen level of nitric oxide synthase enzyme but does not give an indication of the level of functionally active enzyme.2
Measurement of 3H-arginine binding to nitric oxide synthase showed that active levels of the enzyme were present in placentae from normal pregnancies.25 Greater levels of 3H-arginine binding were observed in pregnancies complicated by preeclampsia and FGR than in normal term pregnancy, suggesting that placental nitric oxide synthase activity is indeed higher.26 We did not observe a correlation between placental weight and nitric oxide metabolite levels in either umbilical cord or uterine vein samples. However, because of small sample size and because the placental weights in each group were not significantly different, it is unlikely that this study had sufficient power to detect a significant relationship.
We observed higher levels of nitric oxide metabolites in fetoplacental samples from preeclamptic pregnancies than in normotensive pregnancies. This finding confirms the findings of Lyall et al,14,27 who found increased nitric oxide metabolites in umbilical vein samples from pregnancies complicated by preeclampsia and pregnancies complicated by FGR. We also showed a significant negative correlation between birth weight and nitric oxide metabolite levels in fetoplacental plasma. This correlation was not observed in peripheral or uterine vein samples. Lyall et al27 found a similar correlation in pregnancies complicated by FGR; however, fetoplacental levels in a study of preeclampsia were not correlated significantly with birth weight.14
Studies of umbilical venous endothelial cells have shown similar levels of expression of nitric oxide synthase in normal and preeclamptic pregnancies; however, endothelial nitric oxide synthase has been observed in the terminal vessels of placenta from preeclamptic pregnancies but not in normotensive pregnancies.28 In the growth-restricted fetus, increased production of nitric oxide might compensate for vasoconstriction and decreased blood flow to the fetus.
Infusion of nitric oxide donor in pregnancies with an abnormal Doppler resistance pattern has been associated with improved blood flow to the fetus.19 Other potent vasodilators, such as prostacyclin, did not show this effect.19 Increased compensatory production of nitric oxide by umbilical endothelial cells might therefore improve blood flow to the fetus and improve fetal growth.
We observed higher levels of nitric oxide metabolites in the peripheral circulation of pregnancies complicated by preeclampsia than in normal pregnancies. Increased,10–11 decreased,9 and unchanged levels12,13 have been reported. Most of these studies have been small studies with large interindividual variation in nitric oxide metabolite levels. In a large study of over 400 patients, Nobunaga et al10 reported higher plasma levels of nitric oxide metabolites in preeclampsia compared with normal pregnancy. This increase was not observed in pregnant patients with long-term hypertension, suggesting that the increased nitric oxide levels are specific to preeclampsia and not simply the effect of high blood pressure on the vasculature.10
Reduced platelet nitric oxide synthase may contribute to the vasoconstriction and hypertension in preeclampsia.29 In contrast, platelet cyclic GMP, the second messenger of nitric oxide, is actually higher than in normal pregnancy,30 indicating that platelet production of nitric oxide is higher in preeclampsia. We previously reported that platelet hyperactivity in preeclampsia frequently results in platelet exhaustion and decreased response to platelet agonists in vitro.31 Decreased platelet nitric oxide synthase activity observed in vitro might also be a result of platelet exhaustion.
Endothelial damage in preeclampsia might lead to reduced production of vasodilators such as nitric oxide; however, a recent study has shown that constitutive endothelial nitric oxide synthase could be upregulated by a factor present in plasma from women with preeclampsia. Endothelial cells exposed to 2% plasma from pregnancies complicated by preeclampsia produce greater amounts of nitric oxide metabolites compared with cells treated with plasma from healthy pregnant women.32 Further study showed that this was due to greater gene expression of the endothelial nitric oxide synthase enzyme and that this upregulation occurred at the level of transcription.32 This suggests that although endothelial damage occurs in preeclampsia, a compensatory increase in production of nitric oxide from healthy endothelium takes place. An increase in sheer stress as a result of vasoconstriction can stimulate increased production of nitric oxide in nonpregnant individuals.3 In early pregnancy, nitric oxide and cyclic GMP levels are raised when vascular impedance in the uteroplacental circulation is at its highest before 12 weeks' gestation. A decrease is observed after the sudden reduction of impedance that occurs at the end of the first trimester.33
Vasoconstriction in the peripheral and uteroplacental circulation may also stimulate increased endothelial production of nitric oxide. The increased circulating levels of nitric oxide found in this study might result from increased endothelial and platelet production of nitric oxide.
The patients in the preeclamptic group were not matched for gestational age with the control group. There was a correlation between fetoplacental nitric oxide levels and gestational age. Several authors have studied the effect of gestational age on nitric oxide levels in normal pregnancy.17,26,34 Although some differences have been observed between early and late pregnancy,26 no evidence suggests that fetoplacental nitric oxide levels in the third trimester are related to gestational age in normotensive pregnancy. The correlation between gestational age and nitric oxide levels may be more closely related to the severity of growth restriction than to gestational age.
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