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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Objective  To verify if neurokinin B plasma level is increased in pre-eclampsia and IUGR. Also, to ascertain if there is a correlation between neurokinin B plasma level and nitric oxide production.

Design  A total of 90 pregnant women were studied. Thirty had a gestation complicated by pre-eclampsia and 30 by isolated IUGR; the other 30 were controls. In all patients, neurokinin B plasma level and nitric oxide metabolites (nitrites/nitrates) level were measured.

Setting  University, General Hospital, Messina, Italy.

Methods  Neurokinin B blood samples were taken at 33.5 weeks of gestation and at term. Samples for nitric oxide breakdown products were taken at delivery from the antecubital vein and then from the umbilical vein.

Results  Neurokinin B plasma levels in the pre-eclamptic and IUGR groups were significantly higher than controls. Nitric oxide metabolites levels in pre-eclamptic and IUGR patients were also higher than controls. Regression analysis showed a significant correlation among neurokinin B plasma values and nitric oxide metabolite levels either in pre-eclampsia, in IUGR or in the control group.

Conclusion  Neurokinin B could be involved in pregnancy haemodynamic adaptation via nitric oxide production. In pregnancies complicated with pre-eclampsia and IUGR, increased neurokinin B plasma level, correlated well with increased nitric oxide metabolite level, which may be a compensatory mechanism to improve blood flow to the uteroplacental unit.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Recently, pre-eclampsia has been associated with an increased plasma level of a peptide neurotransmitter neurokinin B.1 The mammalian tachykinins: substance P, neurokinin A and neurokinin B are widely distributed throughout the central and peripheral nervous system; their effects are mediated via specific receptors, designated NK1, NK2 and NK3, respectively. These receptors have been found in a number of locations throughout the human body and their biological actions are involved in pain transmission, neurogenic inflammation, smooth muscle contraction and activation of the immune system.2

Previous reports1,3 have stated that there is an increased plasma neurokinin B level throughout normal pregnancy, which seems to be correlated with the placental weight. Page et al.1 have demonstrated that neurokinin B expression is confined to the outer syncytiotrophoblast of the placenta and that significant concentration of neurokinin B can be detected as early as the ninth week of gestation.

It has been shown in animal studies that NK3 activation by neurokinin B causes nitric oxide release from the endothelium.4,5 Recent in vitro reports have been shown that nitric oxide acts as a vasodilator in uterine arteries6 and placental vessels.7,8 It is therefore reasonable to suggest that neurokinin B could be involved in normal pregnancy haemodynamic adaptation via nitric oxide production.

Failure of trophoblastic invasion of the uteroplacental vessels was found in hypertensive and normotensive pregnancies complicated by fetal growth restriction.9 Furthermore, higher levels of nitric oxide metabolites in the peripheral circulation complicated by pre-eclampsia compared with that in normal pregnancies have been reported.10–12

The aim of our study is (a) to verify whether neurokinin B plasma level is increased in pre-eclampsia and isolated IUGR; (b) whether there is a correlation between placental neurokinin B and nitric oxide production.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Sixty women admitted to the Department of Gynaecology and Obstetrics of Messina University were enrolled in the study over a two-year period. Thirty patients had a diagnosis of pre-eclampsia in accordance with Davey and McGillivray classification13: pre-eclampsia was defined as the association of gestational hypertension (diastolic blood pressure ≥90 mmHg on at least two consecutive occasions after the 20th week of gestation) with significant proteinuria (>300 mg/L in a 24-hour urinary collection). The other 30 were defined as isolated IUGR: the patients were non-smokers and had no clinical evidence of hypertension, diabetes, genetic disorders, infections or autoimmune disease. The fetal growth restriction was probably related to insufficient uteroplacental perfusion; in fact, all the patients showed an impaired umbilical artery Doppler velocimetry, with a Pulsatility index > 2SD. The definition of IUGR was based on a fetal weight below the 10th centile, according to the charts currently in use at our Department.14 All the patients in the pre-eclampsia (gestational age at delivery: 34.3 [2.1]) and in the IUGR group (gestational age at delivery: 33.9 [1.9]) required caesarean delivery.

Thirty outpatients, non-smokers, normotensive pregnant women, with an appropriate for gestational age fetus and an uneventful pregnancy were selected as a control group. These patients underwent an elective caesarean section for other indications (the most frequent was a previous caesarean section) at term. Neurokinin B blood samples were taken from an antecubital vein at 33.5 (1.8) weeks of gestation to verify whether neurokinin B plasma values were in the normal range, according to a previous study,3 and then at term. Furthermore, samples for nitric oxide breakdown products were taken at delivery from the antecubital vein and then from the umbilical vein. All these samples were centrifuged at 3.500 ×g for 15 minutes within 2 hours of sampling; plasma was then stored at −70°C.

Neurokinin B extraction from plasma was performed as described for Met-enkephalin, but using octadecasilyl-silica cartridges, Sep-Pack Vac/1 cm3, C18 (Millipore, Milford, Massachusetts, USA); neurokinin B eluted between 30% and 50% acetonitrile in aqueous 0.1% trifluoroacetic acid (v/v). Both extracts were reconstituted in 500 μL of neurokinin B radio-immunoassay buffer (kit RIK 7357; Peninsula Labs, Belmont, California), and assays were carried out in duplicate according to manufacturer's protocols. Results were corrected by reference to extracted standards (CV% intraassay 4.9; CV% interassay 12.7 at the level 1.1 pmol/L).

Nitric oxide production was assessed by monitoring plasma levels of nitrites and nitrates, the two stable oxidation products of nitric oxide metabolism by chemiluminescence detection. Blood samples were centrifuged at 2500 rpm for 20 minutes at 10°C. The supernatant was removed and stored at −70°C. The nitric oxide assay was standardised by a calibration curve using known concentration of nitrate (0.01–100 μmol/L) obtained from sodium nitrate. For each measurement, a 4-μL sample was placed in a reducing vessel with 5 mL of 0.1 mol of vanadium II chloride per liter, 1 mol of hydrochloric acid per liter and 100 μL of antifoaming agent at 90°C; each standard was analysed three times and each plasma sample was analysed at least five times. The mean value was used for all subsequent analysis.

Data are given as the mean (SE). Significance testing was performed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post-test. The relation between neurokinin B plasma concentrations and nitric oxide metabolite levels was assessed by linear regression analysis. A P value of 0.05 or less was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Neurokinin B mean plasma values in the pre-eclamptic group (3.95 [0.52]) and isolated IUGR group (3.53 [0.47]) were significantly higher than the control group at corresponding gestational age (33.5 [1.8] weeks) (1.68 [0.39]) and at term (1.85 [0.41]) (Fig. 1A). There was no significant difference in the control group between neurokinin B plasma values at 33.5 weeks and at term (P > 0.05).

image

Figure 1. (A) Neurokinin B plasma level in the study groups (mean [SE]). (B) Peripheral circulating nitrites/nitrates level in the study groups. (C) Fetoplacental nitrites/nitrates level in the study groups.

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There were no statistically significant differences between the pre-eclamptic group and the isolated IUGR group mean values.

In the pre-eclampsia group, among clinical parameters, only systolic blood pressure was strongly correlated with neurokinin B plasma values (r= 0.719, P= 0.029); but not diastolic blood pressure (r= 0.027, P= 0.94), nor proteinuria (r= 0.058, P= 0.88). Regression analysis of neurokinin B plasma values in the three study groups did not show any significant correlation with birthweight (controls: r= 0.38, P= 0.2; pre-eclampsia: r= 0.277, P= 0.54; IUGR: r= 0.251, P= 0.431), placental weight (controls: r= 0.57, P= 0.18; pre-eclampsia: r= 0.30, P= 0.4; IUGR: r= 0.28, P= 0.36) or pregnancy length (controls: r= 0.22, P= 0.34; pre-eclampsia: r= 0.282, P= 0.46; IUGR: r= 0.367, P= 0.241).

Nitric oxide metabolites (nitrites/nitrates) levels in the peripheral circulation of pregnancies complicated with pre-eclampsia and isolated IUGR were significantly higher than those in normal pregnancies (Fig. 1B). We obtained similar results in umbilical venous assays from the pre-eclamptic group and isolated IUGR group when compared with the control group (Fig. 1C).

There was no significant difference in nitric oxide metabolites levels between the samples taken from fetoplacental or peripheral vessels in either the pathological groups or the control group.

Regression analysis showed a significant relationship for neurokinin B plasma levels and fetoplacental and peripheral nitric oxide metabolites levels in the control group (Fig. 2A), in the isolated IUGR group (Fig. 2B) and in the pre-eclamptic group (Fig. 2C). Furthermore, there was a strong correlation between the nitrite/nitrate levels in peripheral plasma and in umbilical venous plasma in the pre-eclampsia group (r= 0.55, P= 0.001) and in the IUGR group (r= 0.80, P < 0.0001); instead, there was no correlation in the control group (r= 0.49, P= 0.07). In a multiple regression analysis, using placental weight as covariate, the correlations were still significant in the pre-eclampsia and in the IUGR groups.

image

Figure 2. The relationship between nitrites/nitrates and neurokinin B plasma levels in the peripheral and fetoplacental circulation in the control (A), isolated IUGR (B) and pre-eclampsia (C) groups.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Our results are in accordance with the only previous report about neurokinin B plasma behaviour in pre-eclamptic pregnancies.1 It was a small series of eight patients, but our results in 30 pregnancies complicated with pre-eclampsia corroborate these preceding findings. Furthermore, for the first time in the literature, we have found a similar increase in isolated intrauterine growth restriction. Nevertheless, in the pre-eclampsia group, a strong positive correlation exists only between neurokinin B plasma values and systolic blood pressure.

Neurokinin B is the only tachykinin expressed by the placenta1 and its concentration increases throughout pregnancy.1,3 Neurokinin B acts predominantly through the NK3 receptor, whose activation is mediated by the production of nitric oxide4,5 and a recent in vitro study has shown how NK3 receptors may determine the activation of nitric oxide synthase.15 Page et al.16 suggested that NK3 receptors may play a role in local nitric oxide release and our work is in agreement with this. Little is known about nitric oxide metabolism in pregnancy, but previous reports have shown that human trophoblast cells are able to express the inducible nitric oxide synthase (iNOS)8 and that in normal pregnancy there is an up-regulation of endothelial nitric oxide synthase (eNOS) in the uterine artery.6 Thus, it is reasonable to suggest that nitric oxide, dilating uteroplacental vessels, plays an important role in placental growth and function.8 Furthermore, nitric oxide is important in suppressing myometrial contractility throughout pregnancy,17 probably via activation of Ca2+-activated K+ channels.18

We have demonstrated that there is a significant correlation between the neurokinin B plasma values of 30 normotensive pregnant women and nitric oxide metabolite plasma levels, which suggests that neurokinin B plays an important role in normal pregnancy haemodynamic adaptation. A recent report has shown that neurokinin B secreted from the placenta passes into both maternal and fetal circulation,19 and may then affect nitric oxide release in umbilical vein. Sakamoto et al.19 showed that in uncomplicated pregnancies, neurokinin B concentrations in the umbilical vein and artery were significantly higher than in the peripheral circulation. The authors hypothesised an active transport from the mother to the fetus via the placenta or a preferential secretion from the placenta into the fetal circulation.19

Furthermore, we have evaluated nitric oxide metabolite plasma concentrations in pre-eclampsia and isolated IUGR, and we have found, in peripheral and fetoplacental circulation, a statistically significant increase over the control group. Similar data have been reported also by other authors.10–12,20 We would argue that no statistical difference between pre-eclampsia and isolated IUGR groups should be expected. Both pathological events have the same pathogenesis: an abnormal placentation and impaired uteroplacental changes.9 We agree with authors who suggest that nitric oxide increases in such pathological pregnancies. This may be a compensatory mechanism by healthy endothelium, to improve blood flow to the fetus in damaged placentae.10–12,20 Also in the pathological pregnancies we have studied, we have shown a significant correlation between neurokinin B and nitric oxide metabolite plasma increases. The correlation was particularly strong in pre-eclamptic and IUGR groups, but there was no statistical difference on comparison with the control group.

In conclusion, our data might help to define the role of neurokinin B production during normal and pathological pregnancies. Neurokinin B and its receptor NK3 seem to be involved in pregnancy nitric oxide release. Nitric oxide is a potent vasodilator agent which plays an important role in normal pregnancy haemodynamic adaptation and in suppression of myometrial contractility .In pathological pregnancies complicated by pre-eclampsia or IUGR, in which abnormal placentation has occurred, it seems that placental damage induces neurokinin B production, with the aim of increasing nitric oxide release and therefore improving blood flow in the uteroplacental circulation.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Accepted 2 April 2004