The Effect of Magnesium Sulfate on Bleeding Time and Nitric Oxide Production in Preeclamsia
Preeclampsia is a disease regarding with altered vascular reactivity leading to hypertension of the mother and metabolic alterations in the fetus. This study aimed to assess nitric oxide and bleeding time following administration of magnesium sulfate to preeclamtic patients compared to normotensive pregnant women. A total of 112 subjects (56 preeclamtic patients and 56 normotensive pregnant controls) were enrolled in this case-control study. Cases and controls were matched for age, BMI, gestational age, parity and gravidity. Total concentration of nitrite and nitrate (NOx) was measured before and during magnesium sulfate (MgSO4) treatment using a modified Griess-based method. Results: Systolic and diastolic blood pressures were significantly decreased during MgSO4 treatment in preeclamtic patients (p<0.0001). NOx levels were significantly increased in preeclamtic women after MgSO4 administration (33.7±18.5 vs. 50.2±21.6, p<0.0001) but it was not seen in normotensive parturients (52.4±28.9 vs. 57.3±21.7, p = 0.362). The bleeding time was scarcely increased following magnesium sulfate treatment in preeclamptic patients compared to normotensive pregnant women but it was not significant (p = 0.18). In addition, there was only a significantly reverse correlation between NOx levels and systolic or diastolic blood pressure in preeclamtic parturients after MgSO4 treatment (r = -0384; p = 0.003 and r = -0.29; p = 0.03, respectively). This study demonstrates that administrating MgSO4 to preeclamtic patients induced significant changes in NOx production which had a major role in modulating vasculature changes in preeclamsia.
September 28, 2010; Accepted: January 03, 2011;
Published: February 09, 2011
Preeclampsia is a pregnancy-associated multisystem disorder that affects more
than 3-5% of all pregnancies (Khalil et al., 2009).
It remains a leading cause of maternal and naonatal morbidity and mortality
in the world (Friedman et al., 1991). Reduced
placental perfusion in the early stages of pregnancy is a main event in the
development of this condition. Preeclamsia is associated with a complex of coagulation
abnormalities which is due to an increased in platelet function, fibrinolytic
system activation (Dusse et al., 2010) thrombin
formation (Belfort et al., 2006) and accelerated
a hypercoagulable state. Meanwhile, many preeclamptic women have at least some
evidence of an abnormal clotting cascade (Benedetto et
Nitric Oxide (NO) which produce from placenta endothelial cells and platelets,
has a major role in physiological vascular adaptation occurs during normal pregnancy
such as an increasing in hemodynomic parameters in presence of decreased vascular
resistancy (Many et al., 2000; Vatish
et al., 2006). NO induces vasodilatation of placenta (Bachetti
et al., 2004) inhibits platelet aggregation (Jang
et al., 2002) is involved in angiogenesis, acts as a neurotransmitter
and prevents the adhesion of platelets to endothelial cells (Gladwin,
2005; Lowe, 2000). Altered synthesis and/or biological
actions of NO have been related with abnormal blood flow in preeclampsia and
gestational diabetes (Casanello et al., 2007).
Abnormally elevated levels of NO converted metabolites have been reported in
various biological fluids from preeclamptic pregnancies (Von
Mandach et al., 2003). Deficient NO formation has been implicated
in hypertensive disorders of pregnancy.
Magnesium sulfate (MgSO4), which is used as a therapeutic modality
for the prevention of seizures in pregnant women with preeclampsia, has anticoagulant
and antiplatelet effects (Briel et al., 1987).
In addition to its anticonvulsive effects, MgSO4 decreases blood
pressure through a still unknown mechanism (Touyz, 2003;
Souza et al., 2010). MgSO4 has shown
the ability to inhibit arterial thrombus formation in some experimental animal
studies and treatment with magnesium might lower the risk of thromboembolic-related
disorders (Sheu et al., 2003). The results of
studying the effect of MgSO4 infusion on bleeding time in preeclamsia
have been controversial. Kynczl-Leisure showed an increased bleeding time in
the preeclamtic women received MgSO4 (Kynczl-Leisure
and Cibils, 1996) however other studies have reported a decreased in bleeding
time (Ravn et al., 1996) or without significant
affect (Falck et al., 1999; Rukshin
et al., 2001). However, there is no published study has determined
and correlated bleeding time, blood pressure and NO levels in a single sample
of preeclamptic patient through MgSO4 administration. Therefore,
the present study was designed to measure all these variables in normotensive
pregnant and preeclamptic women.
MATERIALS AND METHODS
Study population: A total of 112 pregnant women including 56 preeclamtic
patients and 56 normotensive pregnant women, referred for their prenatal care
at the Department of Obstetrics and Gynecology, Imam Khomeini Hospital, Sari,
North of Iran during Nov. 2008 to May 2009, were recruited in this case control
study. All subjects were on 20-40 weeks of gestation according to a reliable
last normal menstrual period date or sonographic reports. Control patients were
matched with those with preeclampsia for maternal age and gestational age at
the time of blood sampling. Preeclampsia was diagnosed as a new onset of hypertension
(systolic blood pressure (BP) ≥140 mm Hg or diastolic BP≥90 mm Hg) and
proteinuria (>300 mg dL-1) on at least two occasions 6-24 h apart
(ACOG Committee on Practice Bulletins-Obstetrics, 2002).
Women in labor with ruptured membranes, multiple pregnancy, medical complications
including autoimmune disorders, anti-platelet antibodies, diabetes mellitus,
thrombocytopenia, inflammatory conditions and those having a history of coagulopathy
and cases with bleeding time more than 420 seconds were excluded from study.
All subjects gave written or oral informed consent before participating and
the protocol was approved by the Ethics Research Committee of Mazandaran University
of Medical Sciences.
Baseline laboratory data included an initial bleeding time, platelet count
and serum creatinin. All preeclamtic patients received a 4 g bolus of IV MgSO4
diluted in 100 mL of 5% dextrose solution over 20 min, followed by an infusion
of 2 g h-1 for 24 h. Bleeding time was measured by means of a modified
Ivy method (Pagana and Pagana, 2002) with a Surgicatt
device (International Technidyne, Edison, N.J.) before MgSO4 was
given and also 24 h after it infused. To minimize technical variation, all bleeding
time measurements were blindly done by one investigator. The bleeding time was
recorded as mean occasions of every 30 sec until bleeding stopped. Upon hospital
admission, in two occasions, 10 mL blood samples obtained from each parturient.
Blood samples were collected in citrated vials before commencement of the magnesium
bolus and 40 h after the administration of the MgSO4. Blood sampling
was done in control group as the same times as preeclamtic group. Serum aliquots
were immediately frozen at -80°C until assayed. Blood pressure was also
recorded just before sampling.
Measurement of Total Plasma Nitrite Level (NOx): Since NO is unstable and rapidly converted to nitrates and nitrites, it is necessary to determine both total concentrations nitrite and nitrate in samples. In the present study, all nitrate was converted to nitrite using chemical reduction by vanadium chloride. Total concentration of nitrite and nitrate (NOx) was determined in thawed serum supernatant by a modified Griess reaction. Briefly, serum samples were diluted four fold with distilled water and deproteinized by adding 1/20th volume of zinc sulfate (300 g L-1) to give a final concentration of 15 g L-1. After centrifugation at 10000 g for 5 mm at room temperature, 100 μL of supernatant was applied to a microtiter plate well, followed by 100 μL vanadium chloride (400 mg were prepared in 50 mL 1M HCL) and 50 μL of Griess reagent (1 g L-1 sulfanilamide, 25 g L-1 phosphoric acid and 0.1 g L-1 N-1-naphthylethylenediamine). After 10 min of color development at room temperature, the absorbance was measured on a microplate reader at a wavelength of 540 nm. Each sample was assayed in duplicate wells. Calibration curves were made with sodium nitrite in distilled water (linear range 0-100 μmol L-1). The detection limit of the assay is 1.5 μmol.
Statistical analysis: Data are expressed as Mean±SD for variables
with normal distribution. Differences between groups were analyzed using the
independent t-test or χ2-test, appropriately. In addition, differences
into one group were analyzed using the paired-sample t-test. Pearsons
correlation test was used to study associations between NO levels and other
variables. Differences with P-values below 0.05 were considered significant.
Table 1 summarizes the clinical characteristics of the 112
subjects enrolled in the present study. There were no significant differences
in age, gestational age at sampling, Body Mass Index (BMI), parity and gravidity
between preeclamtic patients and normotensive pregnant women (p<0.05). Majority
of study groups were primigravidia. However, women with preeclamsia had higher
creatin in levels and lower platelet count than those in the control group (p<0.05).
||Characteristics of pregnant women with preeclampsia and healthy
|Values are the Mean±SEM or no. (%). P values were determined
by application of Students t-test for continuous variables and by
χ2 test for categorical variables
As expected, higher systolic and diastolic blood pressures were found in women
with preeclampsia compared with the control group (p<0.0001).
The effect of MgSO4 on the bleeding time and nitric oxide levels are shown in (Table 2). Administration of Magnesium sulfate to preeclamtic group leads to significant decreased in bleeding time when compared to healthy pregnant women (<0.0001). However, comparison of bleeding time before and after MgSO4 treatment revealed an increased in coagulation time in preeclamtic patients (Table 3), however, it was not significantly different (p = 0.18). In addition, the based line of serum levels of nitric oxide was significantly lower in preeclamtic group than those of control group (<0.0001). Nonetheless, after magnesium treatment, there was no significant difference in NOx production in preeclamtic patients compared to normotensive controls (p = 0.177). After MgSO4 treatment, the serum levels of NOx was significantly increased in preeclamtic patients (33.75±18.48 vs. 50.78±22.3 μmol mL-1, p<0.0001). However, this high NOx production did not meet statistical significanct difference, in preeclamtic group compared to controls (p = 0.177). On the other hand, NOx levels were significantly different only in peeclamtic women before and after magnesium infusion (p<0.0001 and p = 0.362, respectively).
To address the possibility that any relationships exist between the circulating concentrations of NOx and bleeding time and systolic or diastolic blood pressure, we carried out correlation analysis between these variables. As it was shown in parts A and B of Fig. 1, there is no correlation between NOx levels and systolic or diastolic blood pressure before magnesium sulfate administration in preeclamtic patients (p = 0.18).
|| Effect of magnesium on bleeding time and nitric oxide production
in preeclamtic patients compared to healthy pregnant
|P values were determined by Student T test; P value indicates
a significant difference (p<0.05) between both groups
||Evaluation of laboratory findings in patients with preeclamsia
and healthy controls following magnesium infusion
|P values were determined by Student T test; P value indicates
a significant difference (p<0.05) between both groups
||Correlation between NOx and systolic or diastolic blood pressure
in preeclamtic patients before (A and B parts) and after (C and D parts)
magnesium treatment. There is only a reverse significant correlation between
NOx concentration and systolic or diastolic blood pressure after MgSO4
Nonetheless, after MgSO4 treatment, NOx levels had a significantly reverse correlation to systolic or diastolic BP; p = 0.003 and 0.03, respectively (parts C and D of Fig. 1). We could not find any correlation between NOx concentration and bleeding time, before and after magnesium treatment.
A main finding of this study is that lower plasma NOx levels in parturients with preeclampsia compared with normotensive healthy pregnant women that can be compensated by magnesium administration. In addition, there is only a significantly reverse correlation between NOx levels and blood pressure in preeclamtic patients received magnesium sulfate treatment. Since nitric oxide converts rapidly to its metabolites (nitrite and nitrate) and Griess reaction could only measure nitrite metabolite, in this study we first converted nitrate to nitrite to have more perception about nitric oxide concentration.
Through normal pregnancy, there is several hemodynamic adaptations include
increased cardiac output without increasing in materno-fetal vascular resistancy.
It is postulated that nitric oxide, by dilating of systemic vasculature, has
a major role in the lack of increase in arterial blood pressure in normal pregnant
women (Sladek et al., 1997; Sandrim
et al., 2008). Systolic and diastolic blood pressures of preeclamtic
parturients were significantly after magnesium sulfate administration. These
results are similar to those reported by other investigators, suggesting that
acute administration of magnesium sulfate in women with preeclampsia decreases
systemic vascular resistance and blood pressure and increases cardiac output
(Cotton et al., 1984). Increased No production
following magnesium sulfate infusion in preeclamtic women might be one of the
mechanisms in regulating blood pressure. However, we could not find a significant
difference in serum levels of NO in preeclamsia compared to normotensive pregnant
women. It was suggested that significant decreased in blood pressure after infusing
magnesium sulfate is a synergic mechanism of NO production and other regulatory
mechanisms. This conclusion was according to the results of other study demonstrated
magnesium sulphate acts as a vasodilator by increasing the synthesis of prostacyclin,
as well as inhibiting angiotensin converting enzyme activity (Elsharnouby
and Elsharnouby, 2006).
Endothelial dysfunction and injury most likely result in hypertension, proteinuria
and other systemic manifestations of the preeclamsia. These modifications lead
to ischemic and hypoperfused placentas (Steinberg et
al., 2009). It is also reported that dysfunction of endothelial cells
can contribute to inappropriate vasoconstriction and platelet aggregation, which
are early signs of hypertension and thrombosis (Vane and
It is demonstrated that blocking of NO production in animal models causes microvasculature
changes resemble to preeclamsia (Pandhi and Malhotra, 2002).
The decreased NOx levels in preeclamtic parturients compare to normotensive
pregnant women in our study is also reinforced involvement of nitric oxide in
NO levels are maintained by its production by Nitric Oxide Synthase (NOS),
but several factors could control NO plasma levels, including free radicals
and NOS substrate concentrations. It was demonstrated that decreasing levels
of endothelial NOS (eNOS) but increasing inducible NOS (iNOS) during preeclamsia
may lead to low NO concentration in preeclamtic patients (Ariza
et al., 2009). Here, we showed an increased in the levels of NOx
production in preeclamtic parturients 24 h after MgSO4 administration.
As the levels of NOx is correlated by the eNOS activity, it seems MgSO4
has a profoundly effect on eNOS activity. Recently, Ariza
et al. (2009) showed that magnesium has opposite effects on iNOS
and eNOS in mild preeclamsia (Ariza et al., 2009).
Another postulation that may be made on effect of magnesium on preeclamsia is
due to decreasing free radicals, NOS inhibitors. It was reported that magnesium
gluconate had an anti-radical and cytoprotective effects in vitro (Mak
et al., 2000). We found a significantly reverse correlation between
NOx levels and blood pressure in preeclamtic parturients after receiving MgSO4.
On the other hand, MgSO4 treatment significantly reduced systolic
and diastolic blood pressure in preeclamtic patients. This finding suggects
that the antihypertensive effect of MgSO4 may be mediated, at least
in part, by increasing serum NO levels.
Otherwise, we did not find a significantly positive association between NOx levels and bleeding time in preeclamtic patients. This finding was shown that coagulation is extremely complex process involving many interacting factors other than platlets. However, a scarcely increased bleeding time in patients with preeclamsia treated by MgSO4, was showed an indirect effect of high NO production in coagulation.
There is a limitation to this study that should be mentioned. It is possible that our results may be false positives due to the nitrite/nitrate level does not definitely reflect bioactive amount of NO. It has shown nitrite/nitrate plasma levels are affected by the dietary consumption of nitrite/nitrate. Therefore, to introduce a well-defined correlation between NOx concentration and preeclamsia, eNOS activity should be targeted for further investigation.
In conclusion, this study demonstrates that MgSO4 treatment to preeclamtic pregnant women induced significant changes in NOx production which had a major role in modulating vasculature changes in preeclamsia
ACOG Committee on Practice Bulletins-Obstetrics, 2002. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Obstet. Gynecol., 99: 159-167.
Ariza, A.C., N. Bobadilla, L. Diaz, E. Avila, F. Larrea and A. Halhali, 2009. Placental gene expression of calcitonin gene-related peptide and nitric oxide synthases in preeclampsia: Effects of magnesium sulfate. Magnes Res., 22: 44-49.
Bachetti, T., L. Comini, S. Curello, D. Bastianon and M. Palmieri et al., 2004. Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells. J. Mol. Cell. Cardiol., 37: 939-945.
Belfort, M.A., S.L. Clark and S. Baha, 2006. Cerebral hemodynamics in preeclampsia: Cerebral perfusion and the rationale for an alternative to magnesium sulfate. Obstet. Gynecol. Survey, 61: 655-665.
Benedetto, C., M. Massobrio, E. Bertini, M. Abbondanza, N. Enrieu and C. Tetta, 1989. Reduced serum inhibition of platelet-activating factor activity in preeclampsia. Am. J. Obstet. Gynecol., 160: 100-104.
Briel, R.C., T.H. Lippert and H.P. Zahradnik, 1987. Changes in blood coagulation, thrombocyte function and vascular prostacyclin synthesis induced by magnesium sulphate. Geburtshilfe Frauenheilkd, 47: 332-336.
Casanello, P., C. Escudero and L. Sobrevia, 2007. Equilibrative nucleoside (ENTs) and cationic amino acid (CATs) transporters: Implications in foetal endothelial dysfunction in human pregnancy diseases. Curr. Vasc. Pharmacol., 5: 69-84.
Cotton, D.B., B. Gonek and K.R. Dorman, 1984. Cardiovascular alterations in severe pregnancy-induced hypertension: Acute effects of intravenous magnesium sulphate. Am. J. Obstet. Gynecol., 148: 162-165.
Dusse, L.M., D.R. Rios, M.B. Pinheiro, A.J. Cooper and B.A. Lwaleed, 2010. Pre-eclampsia: Relationship between coagulation, fibrinolysis and inflammation. Clin. Chim. Acta, 412: 17-21.
CrossRef | PubMed |
Elsharnouby, N.M. and M.M. Elsharnouby, 2006. Magnesium sulphate as a technique of hypotensive anaesthesia. Br. J. Anaesth., 96: 727-731.
Falck, G., H. Lundgaard, T. Jareld, S. Skarra, I. Arbo, S. Gunnes and P. Jynge, 1999. Effect of magnesium infusion on bleeding time in healthy male volunteers. Scand. J. Clin. Lab. Invest., 59: 425-430.
Friedman, S.A., R.N. Taylor and J.M. Roberts, 1991. Pathophysiology of preeclampsia. Clin. Perinatol., 18: 661-682.
Direct Link |
Gladwin, M.T., 2005. Nitrite as an intrinsic signaling molecule. Nat. Chem. Biol., 1: 245-246.
Jang, E.K., J.E. Azzam, N.T. Dickinson, M.M. Davidson and R.J. Haslam, 2002. Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside. Br. J. Haematol., 117: 664-675.
Khalil, A.A., D.J. Cooper and K.F. Harrington, 2009. Pulse wave analysis: A preliminary. BJOG: An Int. J. Obstetr. Gynaecol., 116: 268-276.
CrossRef | PubMed |
Kynczl-Leisure, M. and L.A. Cibils, 1996. Increased bleeding time after magnesium sulfate infusion. Am. J. Obstet. Gynecol., 175: 1293-1294.
Lowe, D.T., 2000. Nitric oxide dysfunction in the pathophysiology of preeclampsia. Nitric. Oxide, 4: 441-458.
CrossRef | PubMed |
Mak, T., A.M. Komarov, J.H. Kramer and W.B. Weglicki, 2000. Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity. Cell Mol. Biol., 46: 1337-1344.
Many, A., C.A. Hubel, S.J. Fisher, J.M. Roberts and Y. Zhou, 2000. Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am. J. Pathol., 156: 321-331.
Pagana, K.D. and T.J. Pagana, 2002. Mosbys Manual of Diagnostic and Laboratory Tests. 3rd Edn., Mosby-Year Book, St. Louis. ISBN: 0323039030, pp: 1280.
Pandhi, P., L. Saha and S. Malhotra, 2002. Effect of oral magnesium supplementation on experimental pre-eclampsia induced by prolonged blockade of nitric oxide synthesis in pregnant rats. Indian J. Exp. Biol., 40: 349-351.
Ravn, H.B., H. Vissinger, S.D. Kristensen, A. Wennmalm, K. Thygesen and S.E. Husted, 1996. Magnesium inhibits platelet activity--an infusion study in healthy volunteers. Thromb. Haemost., 75: 939-944.
Rukshin, V., B. Azarbal, P.K. Shah, V.T. Tsang and M. Shechter et al., 2001. Intravenous magnesium in experimental stent thrombosis in swine. Arterioscler. Thromb. Vasc. Biol., 21: 1544-1549.
CrossRef | PubMed |
Sandrim, V.C., A.C. Palei, I.F. Metzger, V.A. Gomes, R.C. Cavalli and J.E. Tanus-Santos, 2008. Nitric oxide formation is inversely related to serum levels of antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluble endogline in preeclampsia. Hypertension, 52: 402-407.
Sheu, J.R., G. Hsiao, M.Y. Shen, Y.M. Lee and M.H. Yen, 2003. Antithrombotic effects of magnesium sulfate in in vivo experiments. Int. J. Hematol., 77: 414-419.
Sladek, S.M., R.R. Magness and K.P. Conrad, 1997. Nitric oxide and pregnancy. Am. J. Physiol., 272: 441-463.
Souza, A.S., M.M. Amorim, I.C. Coutinho, M.M. Lima, C. Noronha Neto and J.N. Figueroa, 2010. Effect of the loading dose of magnesium sulfate (MgSO4) on the parameters of doppler flow velocity in the uterine, umbilical and middle cerebral arteries in severe preeclampsia. Hypertens Pregnancy, 29: 123-134.
CrossRef | PubMed |
Steinberg, G., E.V. Khankin and S.A. Karumanchi, 2009. Angiogenic factors and preeclampsia. Thromb Re., 2: 93-99.
Touyz, R.M., 2003. Role of magnesium in the pathogenesis of hypertension. Mol. Aspects Med., 24: 107-136.
Vane, J.R. and R.M. Botting, 1992. The role of chemical mediators released by the endothelium in the control of the cardiovascular system. Int. J. Tissue React., 14: 55-64.
Direct Link |
Vatish, M., H.S. Randeva and D.K. Grammatopoulos, 2006. Hormonal regulation of placental nitric oxide and pathogenesis of pre-eclampsia. Trends Mol. Med., 12: 223-233.
Von Mandach, U., D. Lauth and R. Huch, 2003. Maternal and fetal nitric oxide production in normal and abnormal pregnancy. J. Matern. Fetal. Neonatal. Med., 13: 22-27.