الفهرس 
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Abstract
Summary
Abstract
No consensus exists on the role of NO production in normotensive and hypertensive pregnant women, because the concentrations of NO metabolites or second messenger cGMP in plasma and urine have been reported to be increased, reduced, or unchanged in preeclamptic subjects when compared with normal pregnant women, in this study we measured L-arginine as a NO precursor aiming to compare the concentration of L-arginine in the blood of pregnant women affected by preeclampsia to healthy pregnant women, using ELISA.
Our study conducted on 2 groups, NP n=25 &PE n=25. L-arginine level was found to be significantly lower in PE than NP as mean L-arginine level (46.5±4.9vs. 85.3±17.7, P=0.001) in PE &NP respectively.
There was inverse correlation between serum L-arginine level and each of systolic blood pressure [rs= -0.79, p< 0.05], diastolic blood pressure [rs=-0.8, p<0.05], mean arterial blood pressure [rs-0.8=, p<0.05].
We suggest that reduced L-arginine levels contribute with the development of preeclampsia. L arginine level was found to be decreased in the serum of pregnant women with sever preeclampsia by 2-2.5 fold compared to the control group. So we recommend measuring serum level of L arginine late in pregnancy as index of severity of PE.
Introduction
Hypertensive pregnancy disorders are the main cause of maternal and prenatal morbidity and Mortality. Preeclampsia (PE) is the most important among these pathologies (khan et al., 2006).
No consensus exists on the role of NO production in normotensive and hypertensive pregnant women, because the concentrations of NO metabolites or second messenger cGMP in plasma and urine have been reported to be increased, reduced, or unchanged in preeclamptic subjects when compared with normal pregnant women, some studies measured L-arginine as a precursor of NO. Evidence suggests that clinical manifestations are caused by endothelial dysfunction. Nitric oxide (NO), which is synthesized from L-arginine in endothelial cells by the endothelial nitric oxide synthase (eNOS), provides a tonic dilator tone and regulates the adhesion of white blood cells and platelet aggregation. Alterations in the L-arginine-NO pathway have been associated with the development of PE (Lopez-Jaramillo et al, 2008).
L-arginine forms nitric oxide (NO) by the enzyme NO synthase (NOS), this means that the Serum level of L-arginine mirror the NO production in the body (Benedetto et al., 2000).
Cells cultured with adequate concentrations of L-arginine only formed NO from NO synthase, cells depleted of L-arginine generated both NO and O2, which then reacted to form peroxynitrite, which impairs the vascular endothelium lead to alteration of vascular reactivity of systemic & local blood flow exacerbating the pathology of preeclampsia(Noris, et al ., 2004)
Kim, et al., suggested that reduced L-arginine levels, contribute to the development of preeclampsia, and decreased placental eNOS expression might constitute a characteristic finding in the preeclamptic placenta (Kim, et al., 2006).
L-arginine is markedly reduced in women affected by preeclampsia compared to NP (about five-fold lower, P<0.01) so, the determination of L-arginine in the blood of pregnant women could potentially constitute an additional marker for the early diagnosis of preeclampsia (Aniello, et al., 2001).
The most important factor influencing endothelial NO production and endothelial dysfunction may the concentration of L-arginine, (Kim, et al., 2006)
Results of the current study show that serum levels of L-arginine which reflect NO production are significantly lower in women with severe preeclampsia than in normotensive pregnant women. So this study suggested that decrease l-arginine concentration lead to endothelial dysfunction, decrease NO production and contribute to the development as well as the severity of the preeclampsia (kim, et al 2006).
In this study we measure the level of L-arginine as a NO precursor to compare the concentration of L-arginine in the blood of pregnant women with preeclampsia to healthy pregnant women.
Patient &method
This study was carried at Benha university Hospital &KafrShokr general hospital in the period from august 2011 to august 2012. The study was conducted on 2 groups, Group I (n=25): including pregnant women with sever PE, Group II (n=25): including pregnant normotensive women as a control group. We measured serum L-arginine level in both groups. We used the (Enzyme-linked immunosorbent assay) ELISA to measure the L-arginine level in the serum of the tested groups.
Preeclampsia is defined according to the Working Group (2000) criteria, as high blood pressure (≥140/90 mmHg after 28 weeks’ gestation) and proteinuria >+2 dipstick.The control pregnant women were monitored at the Department of Obstetrics and Gynecology of our hospital in the 3rd trimester.
Blood samples were drawn from the antecubital vein of each patient upon admission. Both the normal and preeclamptic women were not in labor. We used the serum from each patient using serum separator tube and allow samples to clot for two hours at room temperature before centrifugation for 20 minutes at approximately1000 g. Assayof freshly prepared serum immediately and/or stored samples in aliquot at -20 for later use, using ELISA Kit for Arginine.
Result
All the data were tabulated and statistically analyzed by the aid of software package of (SPSS) program.
-In this study there were no statistically significant differences between women of the two studied groups regarding to Age, G.A. & parity P>0.05, table (1 &2). But there is a highly significant differences regarding to mean WT (KG), mean Height and mean BMI P<0.05, table (1).
-There was a highly significant difference between women of the two studied groups regarding to oedema L.L. as p<0.05, table (3).
-The mean Albuminuria in the PE group is (2.6±0.5) while in the normal group is (0.4±0.5) and this of highly significant differences (P<0.05) table (4).
-There was a highly significant difference between women of the two studied groups regarding to systolic Bp, diastolic BP and mean ABP P<0.05, table (5).
-The mean serum l-arginine was significantly lower among women with severe PE when compared to the normal group P<0. 05, table (6).
-The level of L-arginine in the PE group decreased by2-2.5fold compared to the normal group as P<0.05 and this nearly agree with Aniello, et al (2001) table (6).
-The difference of mean L-arginine in MG to PG is not significant p>0.05, table (7), but the difference of mean L-arginine in cases of +ve oedema LL to cases of -ve oedema LL is highly significant as p value < 0.05, table (8).
-There was no significant correlation between serum l-arginine and each of age (years), gestational age (w), wt (kg), Height, BMI, parity, table (9).
-There was a highly significant inverse correlation between serum l-arginine level and each of systolic blood pressure [rs=-0.79, p< 0.05], diastolic blood pressure [rs=-0.8, p<0.05], mean arterial blood pressure [rs – 0.8=, p<0.05] & Albuminuria[rs=-0.8, p<0.05] table (10).
Receiver operator characteristics (ROC) curve was constructed for estimating the association between serum L-arginine titre and the diagnosis of severe PE. The curve showed a significant association, denoted by the significantly high area under the curve [AUC = 0.999, p<0.001].
A serum l-arginine titre ≤ 52.05 umol/l was significantly associated with diagnosis of severe PE (sensitivity of 88 %, specificity of 96%, PPV 90 %, NPV 96%).
Discussion
The concentrations of NO metabolites or second messenger cGMP in plasma and urine have been reported to be increased (Baksu,et al ., 2005) reduced (Lopez- Jaramillo,et al ., 1996; Schiessl, et al ., 2006) or unchanged (Conrad, et al., 1999) in preeclamptic subjects when compared with controls, Our results show that serum levels of l-arginine which reflect NO production, are significantly lower in women with severe preeclampsia than in normotensive pregnant women.
This study was conducted on 2 groups, group I (n=25): including pregnant women with sever PE and group II (n=25): including pregnant normotensive women as a control. Kim, et al.2006; Noris, et al., 2004; Aniello, et al., 2001, their studies conducted on 2 groups also while Benedetto, et al., 2000 their study conducted on 4 groups, normal control group, pregnancy induced hypertension, mild preeclampsia &sever preeclampsia.
In the current study the mean G.A. was (36.3±2.5 vs. 36.6±2.9 weeks) in PE & NP respectively, also most of the previous studies like us measure l-arginine in 3rd trimester while Khlil, et al.,2013 they measured L-arginine in early pregnancy (11-13)weeks.
Results of the current study show that serum levels of L-arginine which reflect NO production are significantly lower in women with severe preeclampsia than in normotensive pregnant women.So we suggested with Kim, et al., 2006 that decrease l-arginine concentration lead to endothlieal dysfunction, decrease NO production andcontributeto the development as well as the severity of the preeclampsia.
We suggest with other author (Kim, et al. 2006;Noris, et al., 2004 ;Aniello, et al.,2001&Benedetto,et al., 2000) that reduced L-arginine levelscontribute to the development of preeclampsia .
In the current study the mean serum l-arginine was significantly lower among women with severe PE when compared to the normal group (46.5±4.9nmol/ml vs. 85.3±17.7nmol/ml as P=0.001) respectively.
-The level of L-arginine in the PE group decreased by2-2.5fold compared to the normal group as P=0.001 and this nearly agree withAniello, et al., (2001).
-There was a highly significant inversecorrelation between serum l-arginine level and each of systolic blood pressure [rs=-0.79, p< 0.05], diastolic blood pressure [rs=-0.8, p<0.05], mean arterial blood pressure [rs – 0.8=, p<0.05]&Albuminuria[rs=-0.8, p<0.05]
-There was no significant correlation between serum l-arginine and each of age (years), gestational age (w), wt (kg), Height, BMI, parity.
There have been some conflicting results with regard to plasma levels of L-arginine in preeclamptic patients.
According to Pettersson, et al., (1998) plasma arginine levels were identical in preeclamptic and normotensive control women (80±5.8 umol/lvs74±3.8umol/l) respectively.
Powers, et al., (2008) found that L-arginine levelsin both groups NP n=31 &PE n=15, werethe same (36.3 ±17.9 umol/l in both groups).
AlsoMao, et al (2010) they found that L-arginine nearly equally in NP n=30 (78.55 ±3.09umol/l)&PE n=60 (78.58 ±4.76umol/l).
While BENEDETTO, et al(2000) ;Aniello, et al (2001); Noris, et al(2004) ; Kim, et al (2006); Khalil, et al(2013) found that L-arginine levels were decreased in PE group compared to NP.
According to our results, L-arginine levels in preeclamptic women were significantly lower than in the normal pregnant women 46.5±4.9nmol/ml vs.85.3±17.7nmol/ml, respectively, p<0.05.
The current study agree with that of Aniello,et al., (2001)who carried a study on 12 normotensive pregnant women and 12 pregnant women with preeclampsia, 28–35 years at 35–36 weeks of pregnancy. Theyfound thatL-arginine was highly significantly reduced in preeclamptic group by about five-fold compared to the control group (116±33 mg /dl in PE to 610±120 mg/dl in NP, P<0.05).
This study agree with that of Benedtto,et al.,( 2000) who carried a study on four groups of pregnant women. They found that the concentration of L-arginine was lower in patients with severe preeclampsia than the other 3 groups(mean ±SEM, 107.9±7 in the control group no (44) ; 105.5±9.6 Pregnancy-induced hypertension no17) : 102.3±7.8Mild or moderate preeclampsia no(10)&83.6±7.5Severe preeclampsia no(17) ) .
Also agree with that of Kim, et al., (2006) who carried a study on two groups of pregnant women (NP &PE ),G.A. was (28-40) like our study, they found that the concentration of L-arginine in preeclamptic women was significantly lower in the patient with preeclampsia than in the control They measured L-arginine in 166 normal pregnant women &126 PE women, (p value =0.02) & mean L-arginine level 54.2 (range, 39.0-77.5) vs. 85.4 (range, 55.1-117) in PE&NP respectively. Also they found that there was significant difference between two studied groups regarding to systolic and diastolic BP as P<0.05 and there was no significant difference between two studied groups regarding to age as P>0.05.
The current study is in contrast with Pettersson, et al.,( 1998) who carried their study on 12 NP& 12PE women at 32 -39 G. weeks,They found that L-arginine levels did not differ between preeclamptic and normotensive control women (80.7+/-5.8 umol/l and 74.5+/-3.8umol/l respectively).
Also this study is in contrast with Mao, et al., (2010) who carried their study on 30 NP&62PE women. They found that L-arginine levels did not differ between normotensive control women&preeclamptic (78.55± 3.09micromol/l) vs. (78.58± 4.76micromole/l respectively).
Result of the current study is in contrast with khalil, et al., (2013) as they measured L-arginine early in pergnancy (11-13weeks) we measured L-arginine late in (35-36weeks).khalil, et al.,(2013) found that l-arginine decreased in early PE rather than late PE (46.0umol/l vs.51.56 umol/l in early PE& NP respectively ) while Kim,etal.,found that L-arginine markedly decreased in PE women compared to NP(54.2umol/l vs. 85.4umol/l in PE &NP respectively) in late pregnancy,28 -40 weeks, also Benedetto, et al., (2000) found that L-arginine markedly decreased in PE women compared to NP (83.6±7.5 umol/lvs.107±7.0umol/l in PE &NP respectively) in 3rd trimester.
The discrepancies of arginine levels between our study and that of Petterssons, (1998),Powers, et al., (2008)&Mao, et al (2010)may be due to several reasons such as method of assay we used Eliza while they used highy performance liquid chromatography (HPLC).The other reason may comefrom the ethnic difference between Egyptian and other pregnant women (other races) . The different analytical methods of arginine may also have a role in this difference.
Results of this study show that serum levels of L-arginine which reflect NO production are significantly lower in women with severe preeclampsia than in normotensive pregnant women.L-arginineinversely correlated with the severity of disease in preeclampsia.So we suggested that decrease L-arginine concentration lead to endothelial dysfunction, decrease NO production andcontributeto the development as well as the severity of the preeclampsia.
Determination of L-arginine level in the blood of pregnant women may be considered as an index of severity of preeclampsia and as a potential additional marker for early diagnosis.
The observed reduction in the levels of L-arginine in the preeclampsia patients suggests that L-arginine supplementation or its derivatives (NO) in pregnant women may effectively reduce the prevalence of preeclampsia and in the treatment of severe cases of preeclampsia and as prevention in mild and pregnancy –induced hypertension.
Facchinetti, et al.,(2007) evaluate the effects of L-arginine (L-Arg) supplementation on clinical outcomes and blood pressure (BP) changes in patients with gestational hypertension. Their Study demonstrated that L-Arg supplementation effective as a conservative treatment in patients with gestational hypertension. The effect of L-Arg was associated with a significant reduction in systolic and diastolic blood pressures, and the treatment was well tolerated.
According to Ortega, et al., (2011), Supplementation during pregnancy with a medical food containing L-arginine and antioxidant vitamins reduced the incidence of pre-eclampsia in a population at high risk of the condition.
Conclusion &Recommendation
L-arginine level was found to be decreased in the serum of pregnant women with sever preeclampsia.So we recommend measuring serum level of L arginine late in pregnancy as index of severity of PE.
Also we recommend to measure L arginine level in the serum of pregnant women in the second trimester as an additional marker for the early diagnosis of preeclampsia.
We believe that l-arginine contributed in the development of preeclampsia so we recommended using l-arginine in the treatment of severe cases of preeclampsia and as prevention in mild and pregnancy –induced hypertension.
Also we recommend using L-arginine as a food supplement for prevention of PE.