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Annals of Clinical & Laboratory Science 32:257-263 (2002)
© 2002 Association of Clinical Scientists

Nitric Oxide Production Increases during Normal Pregnancy and Decreases in Preeclampsia

Jong Weon Choi1, Moon Whan Im2 and Soo Hwan Pai1
1 Department of Clinical Pathology and 2 Department of Obstetrics, Inha University College of Medicine, Inchon, Korea

Address correspondence to Soo Hwan Pai, M.D., Department of Clinical Pathology, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon, 400-103, Korea; tel 82 32 890 2502; fax 82 32 890 2529; e-mail shpaimd{at}inha.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
To investigate the changes in nitric oxide (NO) production during and after normal pregnancy and in pregnancies complicated by preeclampsia, we measured serum nitrates and nitrites (NOx) concentrations and serum iron markers in 347 subjects. Serum NOx concentrations were determined after reduction of nitrates to nitrites using the Griess reaction. Serum iron and serum ferritin were assayed using an automatic chemical analyzer and a chemiluminescence method. Serum NOx concentrations were significantly higher in the first trimester (117.3 ± 31.4 µM) than in nonpregnant women (23.8 ± 7.1 µM). High NOx concentrations persisted throughout normal pregnancy, irrespective of serum ferritin concentrations, and returned to nonpregnant levels by 9–12 wk postpartum. Mean NOx concentrations in preeclamptic women were 43.1 ± 12.7 µM, which were significantly lower than those in the gestation age-matched normal pregnant women (249.7 ± 51.3 µM). In summary, NO production increases with advancing gestation during normal pregnancy and decreases in preeclampsia, regardless of serum ferritin concentrations. Elevated NOx concentrations during pregnancy return to normal within 12 wk after delivery.

(received 27 December 2001; accepted 31 December 2001)

Keywords: nitric oxide, pregnancy, preeclampsia, ferritin, iron


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In normal pregnancy, profound physiologic changes occur in the maternal cardiovascular system, including increases in blood flow through uterine blood vessels, altered responses to vasopressor agents, and reduced peripheral resistance and blood pressure [1]. The mechanisms underlying these adaptations of normal pregnancy are currently unclear; however, these cardiovascular changes ultimately ensure the adequate delivery of oxygen and nutrients to the fetus. Preeclampsia is associated with increased vascular reactivity and vasoconstriction. Preeclampsia is characterized by placental abnormalities and maternal vascular endothelial dysfunction, and is the leading cause of maternal death and a major contributor of maternal and perinatal morbidity. The mechanisms responsible for the pathogenesis of preeclampsia have not been elucidated [2,3].

Nitric oxide (NO) is a biological mediator synthesized from L-arginine by a family of NO synthases. NO is produced in many different cells and is involved in the regulation of physiological and pathological processes, such as inflammation and metabolism [4]. Depending on cell type, NO is produced in an enzymatic reaction catalyzed by one of the three isoforms of NO synthase (NOS): neuronal NOS, endothelial NOS, and inducible NOS [5]. Because NO is highly labile, measurement of the relatively stable metabolites, nitrate and nitrite (NOx), is employed as an index of NO production and as a marker of NOS enzyme activity [6].

Some investigators demonstrated that endothelium-derived NO plays a role in the regulation of vascular resistance during normal pregnancy and preeclampsia [7–9]; however, most studies have been based mainly on measurement of NOx concentrations without assessment of serum ferritin levels in pregnant women. In previous work, we reported that iron depletion induces NO production in healthy human adolescents [10]. On the other hand, iron deficiency anemia is commonly encountered in pregnant women, as gestation progresses. Hence, in the present study we investigated the changes in NO production in pregnancy and in preeclampsia by comparing serum ferritin concentrations with serum NOx levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
NOx and iron markers were measured in serum specimens from 347 women, age 21 – 43 yr, including 167 pregnant women, 80 postpartum women, 52 pregnant women with preeclampsia, and 48 nonpregnant women (Table 1Go). Gestational age was determined by sonographic examination and the date of the last menstrual period. The pregnant women were divided into 4 groups by gestational age; the first trimester (1.0 – 12.0 wk, n = 47), the second trimester (12.1 – 24.0 wk, n = 45), early third trimester (24.1 – 32.0 wk, n = 34) and late third trimester (32.1 – 40.0 wk, n = 41). Postpartum women were divided into 3 groups: 1–4 wk postpartum (n = 25), 5–8 wk postpartum (n = 27), and 9–12 wk postpartum (n = 28). A control group was comprised of age-matched healthy women (n = 48), without history of pregnancy or recent disease.


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  		Table 1. Clinical features and laboratory data (mean ± SD) of the subjects included in this study
 
This study was approved by the Committee on Ethics of the Inha University, and all subjects gave informed, written consent. Women (n = 4) who smoked were excluded, as were women (n = 3) with underlying chronic illness such as renal disease or diabetes mellitus. We also excluded patients (n = 2) with acute or chronic infection because infections may give rise to inflammation-induced NOx elevation. All criteria for subject selection and subject exclusion were declared prior to the measurement of serum NOx concentrations.

Body weight, blood pressure, and 24-hr urine protein concentrations were evaluated in all subjects. The diagnosis of preeclampsia was made by strict criteria as described previously [11]: onset of hypertension during late gestation with systolic and diastolic blood pressure >140/90 mmHg on at least two occasions and urinary protein excretion greater than 300 mg/24 hr. These subjects were normotensive during the first trimester and had no history of chronic hypertension. Systemic NO production was assessed in preeclamptic women on admission to the hospital (gestational age, 25.4 – 32.0 wk, mean = 29.3 wk) before drug administration.

After the subjects had fasted >12 hr, venous blood was drawn into an evacuated serum separator tube on the initial visit, before iron supplementation. Serum was prepared from whole venous blood, immediately separated by centrifugation, and frozen at -70°C until assayed. NOx concentrations were measured by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent nitrate reductase assay [12] in serum of women on a reduced nitrate and nitrite diet. After serum nitrate (NO3-) was converted to nitrite (NO2-) by NADPH-dependent nitrate reductase (incubated with glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADPH in 14 mmol/L sodium phosphate buffer, pH 7.4), the total concentration of nitrite was determined by spectrophotometry at 540 nm.

We measured NOx directly in serum without deproteinization and without dilution since there were no significant differences in NOx concentrations between deproteinized and non-deproteinized sera or between diluted and non-diluted sera, based on 25 specimens selected randomly from the subjects. To avoid dietary effects on serum NOx concentrations, the subjects were given a list of foods potentially rich in nitrate and were requested to abstain from these foods before sample collection after an overnight fast. Specifically, cured meat, fish, cheese, herbal or black teas, beer, wine, and malt beverages were excluded from the diet [13].

Because iron depletion may elevate serum NO concentrations [10,14], we measured iron markers in the pregnant women and the controls. Serum iron was assayed using an automated chemical analyzer (Hitachi 747 analyzer; Hitachi Corp., Tokyo, Japan), and serum ferritin was measured by a chemiluminescence method (ACS 180; Chiron, Inc, MA).

Data analyses were performed using the SAS statistical software (version 6.12; SAS Institute Inc, Cary, NC). The Mann-Whitney U test was used to test the difference of values. Confidence intervals (central 95th percentile) were computed by nonparametric statistics; p values <0.01 were statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Clinical characteristics of the subjects included in this study are summarized in Table 1Go. At the time of study, there were no significant differences in blood pressure, urine protein concentrations, or pregnancy parity among the 3 groups of normal pregnant women. Women with preeclampsia demonstrated marked hypertension and proteinuria.

Data for serum NOx concentrations and iron markers of the subjects are shown in Table 2Go. There were no significant differences in serum ferritin concentrations between the first trimester of pregnancy and nonpregnant women. However, NOx concentrations averaged 117.3 (SD ± 31.4) µM during the first trimester, which was significantly above the values in nonpregnant women (23.8 ± 7.1 µM, p < 0.01). NOx concentrations continued to increase throughout pregnancy, attaining peak levels after 32 wk of pregnancy (249.7 ± 51.3 µM). These levels were 2 to 10 times higher than during the first trimester or in nonpregnant women. Elevated NOx concentration declined abruptly to the mean level of 183.1 ± 40.6 µM at 1 – 4 wk postpartum and returned to nonpregnant levels by 9 – 12 wk after delivery.


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  		Table 2. Serum NOx concentrations and iron markers during and after normal pregnancy and in preeclampsia
 
Mean NOx concentration in preeclamptic women was 43.1 ± 12.7 µM, which was significantly higher than in nonpregnant women (23.8 ± 7.1 µM, p <0.01), but significantly lower than the gestational age-matched normal pregnant women (185.8 ± 42.2 µM, p <0.01). On the other hand, there were no significant differences in serum iron markers between preeclamptic women and gestational age-matched normal pregnant women (Table 2Go).

To compare the serum NOx concentrations in the subjects with no differences in serum concentrations of iron markers, we selected the nonpregnant controls (n = 17) with serum ferritin concentrations <50 µg/L and the pregnant women in the third trimester (n = 21), who had serum ferritin concentrations >30 µg/L. There were no significant differences in serum iron and serum ferritin concentrations between these groups; however, the serum NOx concentration in women in third trimester was still significantly higher than those in nonpregnant women (p <0.01) (Table 3Go).


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  		Table 3. Serum NOx concentrations of nonpregnant controls with serum ferritin concentrations <50 µg/L and pregnant women with serum ferritn concentration >30 µg/L.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we investigated the changes in NO production during and after pregnancy and in pregnancies complicated by preeclampsia. We found that NO production increases with gestational age during normal pregnancy and decreases in preeclampsia, and that elevated NOx concentrations return to nonpregnant level 12 wk after delivery. Our data are in general agreement with the results of Jo et al [15] and Shaamash et al [16], who showed that serum NO production was increased in normal pregnancy, especially in the second trimester, and peaked in the third trimester of pregnancy. However, the changes in NO production during normal pregnancy have varied in different studies. Hata et al [17] reported that maternal circulating nitrite level decreased with advancing gestation. Brown et al [18] and Smarason et al [19] found that there were no changes in NO production during normal pregnancy compared to the nonpregnant state.

These observations suggest that the status of NO biosynthesis in women during normal pregnancy remains still controversial. These discrepancies may derive from methodological shortcomings for measuring plasma NOx concentrations. Dietary intake of nitrate can affect the blood level of NOx. Many of the studies relied on the measurement of NOx in the plasma; however, the plasma level is influenced by the clearance, as well as the production, of NO metabolites [13].

We measured the major metabolites (nitrates and nitrites) of NO as an index of NO production in the serum of women subjected to a reduced nitrate and nitrite diet. Ni et al [14] reported that plasma NOx concentrations increased markedly in the iron deficiency anemic group of an animal model. Mabbott and Sternberg [20] found that NO production correlates directly with the development of anemia, and systemic inhibition of NO synthesis leads to a significant increase of hemoglobin in animal experiments. In our previous work, we confirmed that iron deficiency anemia increases NO production in humans, and elevated NOx concentrations in iron deficiency anemia return to normal with iron supplementation [10]. Therefore, in the present study, we measured serum iron markers to evaluate precisely the NOx concentrations during pregnancy, because iron deficiency is frequently observed in this period. NOx concentrations were nearly 5-fold higher in the first trimester of pregnancy than in nonpregnant women, although there were no significant differences in serum ferritin concentrations between the 2 groups.

To exclude the impact of iron deficiency on serum NOx concentrations, we reanalyzed the NOx concentrations in only the subjects who had serum ferritin concentrations similar to those of nonpregnant women. Serum NOx concentrations were slightly decreased but still significantly higher in the pregnant women of the third trimester than those of nonpregnant women, even after we selected the pregnant women in the third trimester, who showed no differences in serum ferritin concentrations compared to nonpregnant women. These results suggest that increased NO production during normal pregnancy may be derived from other causes than the depletion of iron. On the other hand, we investigated the NOx level during puerperium. The NOx concentrations decreased abruptly after delivery and declined to the levels similar to nonpregnant controls within 9 – 12 wk after delivery. It thus appears that elevated NO concentration during pregnancy returns to normal around 12 wk postpartum.

The role of NO in preeclampsia is still uncertain. Lyall et al [21] found that there was no significant difference in maternal serum nitrite concentrations between a control group and a preeclamptic group. Cameron et al [22] demonstrated that the plasma or urinary nitrate (or nitrite) level was increased during preeclampsia compared to normal gestation. Contrary to these results, in our study, NOx concentrations in preeclamptic women were significantly lower than those in the gestational age-matched normal pregnant women, who showed no differences from preeclamptic women in serum ferritin levels. Our data are in accordance with the previous reports, where the levels of NO products were significantly reduced during pregnancy in preeclampsia [23] and placental NO synthase activity was significantly reduced in preeclampsia [24]. Markedly decreased NO concentrations in preeclamptic women, who showed no significant differences in iron markers from the gestational age-matched pregnant women, suggest that NO biosynthesis is decreased in preeclampsia regardless of serum ferritin levels. Because NO is a potent relaxant of vascular smooth muscle, these results suggest that reduced NO production may have an adverse effect on placental hemodynamic function in preeclampsia, and could be involved in the pathogenesis of this important obstetric complication.

In conclusion, NO biosynthesis increases with advancing gestation during normal pregnancy and decreases in preeclampsia, irrespective of serum ferritin concentrations. Elevated NO concentrations during pregnancy return to normal by 12 wk after delivery.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by a Science Research Center grant from Korea Science and Engineering Foundation to the Nitric Oxide Radical Toxicology Research Center (NORTRC).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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  11. National High Blood Pressure Education Program Working Group. Report on high blood pressure in pregnancy. Am J Obstet Gynecol 1990;163:1689–1712.
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