Published online before print June 18, 2007, doi: 10.1161/HYPERTENSIONAHA.107.091223
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(Hypertension. 2007;50:396.)
© 2007 American Heart Association, Inc.
Original Articles |
Homocysteine and Nitric Oxide Are Related to Blood Pressure and Vascular Function in Small-for-Gestational-Age Children
From the Division of Nephrology (M.C.P.F., E.M.S.H., R.S.), Division of Genetics (V.D., F.G.d.S.), and Department of Physiology (A.L.S.), School of Medicine, Federal University of São Paulo, São Paulo, Brazil; and the Department of Pharmacology (Z.B.F.), University of São Paulo, Biomedical Sciences Institute, São Paulo, Brazil.
Correspondence to Maria C.P. Franco, Division of Nephrology, Federal University of São Paulo, R. Botucatu, 740 São Paulo, São Paulo, Brazil 04023-900. E-mail mdcfranco{at}nefro.epm.br
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Abstract |
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Leptin, homocysteine (Hcy), and C-reactive protein are risk factors potentially useful in predicting future cardiac events. These plasma biomarkers may participate in the regulation of cardiovascular function through an NO-dependent mechanism. Our purpose was to investigate whether alterations in C-reactive protein, Hcy, leptin, and NO are present in small-for-gestational-age children and to determine whether the levels of these plasma biomarkers are associated with birth weight, vascular function, and blood pressure. Concentrations of leptin, Hcy, C-reactive protein, and NO were measured in 69 children (36 boys and 33 girls; ages 8 to 13 years). Leptin (means difference: 1.4 ng/mL; 95% CI: 0.4 to 2.4) and Hcy (means difference: 0.9 µmol/L; 95% CI: 0.3 to 1.5) levels were significantly elevated in children born small for gestational age compared with those with appropriate birth weight. Nevertheless, NO (means difference: 342.9 µmol; 95% CI: 124.2 to 561.6) concentration was significantly reduced in small birth weight children, and the levels of C-reactive protein remained unchanged. There was a significant association between the circulating levels of both NO and Hcy with vascular function, as well as with blood pressure levels, in our population. Because both Hcy and NO are associated with a risk of cardiovascular disease, it is possible that part of the association of low birth weight with elevated risk for vascular and metabolic disease in later life is mediated by perturbation in pathways for these biomarkers.
Key Words: homocysteine • NO • endothelium function • blood pressure • children
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Introduction |
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There is accumulating evidence to suggest that small-for-gestational-age (SGA) children have long-term adult health consequences including obesity, type 2 diabetes, hypertension, and cardiovascular disease (CVD).1–3 Mortality from vascular disease approximately doubles from the highest to the lowest extremes of birth weight.1,2 The occurrence of cardiovascular damage can be the result of exposure to multiple risk factors and is usually preceded by the development of asymptomatic structural and functional abnormalities at the vascular level. It has already been reported that a higher prevalence of endothelial dysfunction and arterial stiffness exists in SGA infants4 and children.5,6
Several plasma biomarkers have been investigated to determine their use as tools for predicting the risk of CVD including high-sensitivity C-reactive protein (hs-CRP), homocysteine (Hcy), and leptin. hs-CRP is a marker of systemic inflammation and has been shown to be an independent risk factor for CVD.7 Plasma Hcy, a sulfhydryl-containing amino acid formed during the metabolism of methionine, is associated with endothelial damage and hypertension.8 Finally, leptin is a circulating hormone that regulates adipose tissue and, along with several metabolic effects, is recognized to have many vascular effects.9 Evaluation of these risk factors in SGA children may provide insights into mechanisms involved in the development of disease in later life. On the other hand, many studies have focused on the cardioprotective effects of NO and have shown that hypertension and other circulatory disorders may be associated with insufficient NO production and availability.10,11 In addition, it has been reported that NO concentration is significantly associated with leptin, Hcy, and hs-CRP in pathological processes such as hypertension, obesity, diabetes, and coronary artery disease.9,12,13
To the best of our knowledge, no studies have been done to evaluate simultaneously circulating levels of NO, Hcy, hs-CRP, and leptin in children born SGA. Therefore, the purpose of this study was to investigate whether alterations in hs-CRP, Hcy, leptin, and NO are present in 8- to 13-year-old SGA children and to determine whether the levels of these plasma biomarkers are associated with birth weight, vascular function, and blood pressure (BP). In addition, we evaluated classical risk factors for CVD, ie, body mass index, blood lipids, serum uric acid, and insulin sensitivity, and investigated their relationship with leptin, NO, Hcy, and hs-CRP in SGA children.
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Methods |
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A total of 113 children aged 8 to 13 years were recruited and evaluated between November 2004 and July 2005. Participants were selected among 289 children screened in an anthropometric census performed in 5 shantytowns by the Nutritional Rehabilitation Center of the Federal University of São Paulo. Initially, 18 shantytowns were identified within 15 km of the Federal University of São Paulo, which is located in the southern region of São Paulo. The region was then subdivided into 3 geographic areas, and a secondary sample of 5 shantytowns was randomly selected, taking into account this stratification by region. We have reported previously the demographic, anthropometric, and biochemical characteristics of this cohort.5 Personal and family medical histories were obtained by a questionnaire completed during an interview with parents or guardians. Exclusion criteria were the presence of renal disease, acute or chronic infections, chronic illness, positive family history or clinical signs of CVD, or endocrinopathy. Twenty four of the 113 selected children were excluded because they had birth weight (BW) within the range 2501 to 2999 g. In addition to these, 9 children did not have adequate amount of blood or urine for evaluation of the biomarkers, 7 children were excluded because of family history of hypertension, 3 had laboratory tests indicative of diabetes mellitus, and 1 child had renal disease. Sixty-nine children remained eligible for this study. During enrollment, the weight, height, serum lipids, insulin, uric acid, and BP levels were measured with described methods.5 NO-mediated vascular function was measured by assessing brachial artery flow-mediated dilation (FMD) with a noninvasive ultrasound technique as described previously.5
Children were divided into 2 groups: the AGA group was composed of 34 children, who were born at term with appropriate birth weight (BW: 
3.0 kg), and the SGA group was composed of 35 children, who were born at term with small birth weight for gestational age (BW: 
2.5 kg). The anthropometric indicators used to assess child nutritional status were height for age, weight for age, and body mass index for age, which were expressed as z scores. These indicators were calculated using the National Center for Health Statistics reference.14 A Quantitative Insulin Sensitivity Check (QUICK) index was used as a measure of insulin sensitivity. Details of BW data validity and repeatability of brachial artery ultrasound measurements have been published previously.5 The study was approved by the ethics committee of the Federal University of São Paulo, and informed consent was obtained from one of the parents of the children enrolled in the study.
Assessment of Leptin, Hcy, hs-CRP, and NO
Blood samples were centrifuged, and aliquots of heparin plasma were stored at –80°C until assay. The first morning urine samples (2 mL) were collected before any other test; they were kept refrigerated during the collection period, after which they were immediately stored at –80°C until assay. Plasma leptin levels were determined by a radioimmunoassay (Human Leptin RIA kit, Linco Research, Inc). Total plasma Hcy levels were determined according to the methodology described by Pfweiffer et al15 using high-pressure liquid chromatography with fluorometric detection and isocratic elution, with a specific substrate of the thiol group, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate. hs-CRP was measured in plasma samples by an immunoturbidimetric assay with the Roche/Hitachi 912 analyzer. Urine NO was determined by the chemiluminescence method with the Model 280 Nitric Oxide Analyzer (NOA, Sievers Instruments Inc), a high-sensitive detector for measuring NO, based on a gas-phase chemiluminescent reaction between NO and ozone. Although NO is the primary source for circulating NO metabolites, its levels could be influenced by high nitrate diets.9 However, it has been shown that urinary nitrates reach maximal levels 4 to 6 hours after an oral load and return to baseline after 12 hours.9 Thus, it seems likely that, after overnight fasting, the influences of dietary nitrates are eliminated.
Statistical Analysis
Data were analyzed with the statistical program SPSS 11.0 for Windows (SPSS Inc). All of the continuous variables were examined for normality with the Kolmogorov-Smirnov test. Because plasma hs-CRP levels were not normally distributed, a logarithmic transformation was applied before testing with parametric tests. Categorical variables were compared with the 
2 test. Student t test was used to compare mean values of continuous variables between 2 groups. Pearson’s and partial correlation coefficients, as well as stepwise forward multiple linear regression analysis of the overall cohort, were used to investigate associations between biomarker parameters (leptin, Hcy, hs-CRP, and NO) and the other variables. Values of continuous variables are expressed as the mean±SEM. Statistical tests were 2 tailed, and the significance level was set at P<0.05.
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Results |
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The characteristics of SGA and AGA children are shown in Table 1 and 2
. As expected, boys and girls from the SGA group showed values significantly lower for birth weight compared with AGA children. There were no significant differences in gestational age, anthropometric evaluation, and laboratory parameters among girls or boys from both groups, though higher levels of uric acid were observed in the SGA children. No difference in heart rate was found between the 2 groups of children. In contrast to AGA children, mean systolic BP levels were markedly elevated in both boys and girls from the SGA group. No significant differences were detected in blood flow and brachial artery diameter during baseline conditions between children groups. Nevertheless, mean FMD was significantly lower in boys and girls of the SGA group than in the AGA group.
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hs-CRP, Hcy, Leptin, and NO in Relation to Birth Weight
Plasma hs-CRP levels did not differ between AGA and SGA children (Table 3), and no correlation was found between this inflammatory marker and BW or anthropometric parameters investigated in the present study (Tables 4 and 5). As shown in Table 3, the plasma concentrations of Hcy and leptin were significantly higher in the SGA group compared with the AGA group. No significant ethnic (P=0.607) or gender (P=0.654) differences impacted mean Hcy levels in the study group; however, we observed a significant sexual dimorphism for leptin levels, with girls tending to have higher levels than boys (5.5±0.4 ng/mL versus 3.1±0.3 ng/mL, respectively; P<0.001). No difference was noted between ethnicities (P=0.995) for this marker. On the other hand, mean urinary NO levels were lower in the SGA group than in AGA children (Table 3). No statistical differences were found in gender (P=0.512) or race (P=0.085) in relation to NO levels. For the entire cohort, leptin, Hcy, and NO concentrations correlated significantly with BW. After controlling for gender and race, the association between leptin and BW was reduced and was no longer significant, whereas this adjustment did not modify the associations between Hcy or NO with BW (Table 4).
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Other Factors Associated With Leptin, Hcy, and NO Levels
Because obesity is expected to contribute to increased leptin levels, we investigated the association among leptin, body mass index z score (BMIZ), and weight-for-age z score (WAZ). For the entire cohort, plasmatic leptin levels were positively correlated with BMIZ and WAZ (Table 5). After controlling for race and BW, these associations remained significant (BMIZ r=0.364 and WAZ r=0.321; P=0.002 and P=0.007, respectively). Another important result is that leptin concentration was significantly correlated with insulin levels (r=0.425; P<0.001), QUICK index (r=–0.425; P<0.001), and triglycerides (r=0.335; P=0.011) in our population. Adjustment for race, gender, BW, and BMIZ did modify these associations (insulin: r=0.422, P=0.001; QUICK index: r=–0.470; P<0.001; and triglycerides: r=0.386; P=0.001).
After adjustment for BW, race, and gender, Hcy and age were found to be significantly correlated (r=0.314; P= 0.007). On the other hand, urinary NO was associated with uric acid levels in a model adjusting for gender and race (r=–0.286; P=0.031). Both Hcy and NO levels were not correlated with any other demographic, anthropometric, or biochemical parameters investigated in the present study.
Influence of Biomarkers on BP Levels and Vascular Function
Systolic BP Levels
For the entire cohort, significant correlations were observed between systolic BP levels and urinary NO concentrations (Table 5 and Figure 1A) and with plasmatic Hcy (Table 5 and Figure 1B). No relationship was found between systolic BP levels and other biomarkers evaluated in this study (Table 5). Subsequently, multiple regression analyses were carried out. In a model testing BW, age, gender, race, BMIZ, blood lipids, uric acid, Hcy, and NO as independent variables, only Hcy (ß=3.294; SE=1.195; P=0.008) and BW (ß=–0.0085; SE=0.004; P=0.021) showed a significant association with systolic BP levels. Separate analyses of SGA children yielded findings that supported these associations. According to the regression analysis, only Hcy was positively associated with systolic BP in SGA children (ß=5.026; SE=1.333; P=0.001). Similar analyses were performed in AGA children, and none of the covariates reached statistical significance.
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Vascular Function
In all of the children, urinary NO concentration significantly correlated with FMD (Table 5 and Figure 2), and this relationship was not significantly affected in a model adjusting for BW, race, and gender (r=0.312; P=0.016). On the other hand, the correlation between FMD and plasmatic Hcy level was significant only when adjusting for race and gender (r=–0.220; P=0.050). To establish that these correlations were not merely casual, we included BW, gender, race, age, BMIZ, uric acid, blood lipids, QUICK index, NO, and Hcy as covariates in a multiple regression model. This analysis showed that BW (ß=0.0056; SE=0.003; P=0.042) and NO (ß=0.0068; t=0.003; P=0.018) were independent determinant factors affecting vascular function in our population.
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Discussion |
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Elevated levels of C-reactive protein, Hcy, and leptin have been associated with the increased likelihood of cardiovascular events, including hypertension and endothelial dysfunction.7–9 Although substantial evidence of cardiovascular morbidity among SGA children has been accumulated in the past decade,4–6 no studies have simultaneously examined the levels of these biomarkers in SGA children. The results of this study showed that SGA children have elevated circulating levels of both leptin and Hcy associated with the presence of reduced urinary NO concentrations. In addition, our study demonstrates for the first time that there is a significant association between circulating levels of both NO and Hcy with vascular function, as well as with BP levels in 8- to 13-year-old SGA children. Because both Hcy and NO are associated with the risk of CVD, it is possible that part of the association of low birth weight with elevated risk for vascular and metabolic disease in later life is mediated by perturbation of pathways of these biomarkers.
In this study, we did not find any significant difference in the concentration of hs-CRP between SGA and AGA children. Also, there was no evidence of a significant relationship between hs-CRP with both systolic BP and vascular function in these children. Previous studies in children failed to find significant correlations between CRP and birth weight.16,17 Recently, Sattar et al18 evaluated 1663 individuals and demonstrated that low birth weight contributes to elevated CRP concentrations in adult life. The negative findings of the present study could be a result of the limitation in sample size, though we had 85% of statistical power to detect a difference of 15% in the mean values of hs-CRP between the groups. Larger study populations would be required to detect smaller associations between hs-CRP and birth weight or other clinical parameters.
On the other hand, we found leptin concentration to be higher in SGA than in AGA children. This adverse effect on leptin production observed among SGA children provides an important example of the consequences of reduced fetal growth on hormonal functions of adipose tissue. For the entire cohort, circulating levels of this hormone were significantly correlated to birth weight, WAZ, and BMIZ. Moreover, we found differences in leptin levels according to gender, being higher in girls than in boys. Our findings agree with results described by Pulzer et al,19 who also found elevated leptin concentrations in SGA children. Our results are also comparable to those found in SGA children with catch-up growth who presented enhancements in leptin levels.20 Moreover, there is evidence that adults with a history of low birth weight have higher circulating leptin concentrations.21 However, there is conflicting information regarding the levels of leptin in SGA children. Other studies reported lower leptin levels in SGA children.22,23 We do not have an explanation for this discrepancy; it is possible that diet composition, metabolic control, predisposition to obesity, and the presence of catch-up growth could influence leptin levels in these studies regarding the SGA population.
Another interesting result of the present study is the fact that higher Hcy levels were found in SGA children, though the values are considered as within the reference interval. We also found that Hcy was inversely correlated with birth weight, even after adjustments for gender and race. There are few reports addressing the relationship between Hcy and birth weight. It has been described that elevated levels of Hcy in both maternal and amniotic fluids can be associated with the birth size of SGA newborns.24,25 Moreover, Engel et al26 hypothesized that variants in the folate metabolism pathway affect the accumulation of Hcy and can be linked to adverse birth outcomes. These authors suggested the possibility of a direct or indirect role for the SHMT1 (1420T) variant in SGA births. Although little information is available regarding the link between Hcy and fetal growth retardation, high levels of Hcy during childhood may contribute to the accelerated CVD process in SGA children.
Although circulating levels of Hcy have shown an increase in SGA children, urinary NO concentrations were decreased in these children. Previous studies have reported reduction in NOS activity in the umbilical artery endothelium,27 and fetoplacental NO synthesis is decreased during pregnancies complicated by fetal growth restriction.28,29 On the other hand, other authors have found that concentrations of plasma nitrates and nitrites are higher in the fetal circulation of growth-restriction pregnancies in comparison with normal pregnancies.30,31 The reasons for these apparent discrepant results are currently not known, but our study is the first to investigate NO levels in SGA children, suggesting that alterations to NO pathways could contribute to adult disease. The finding of an inverse correlation between NO and serum uric acid levels is also relevant. It has been demonstrated that uric acid is a mediator of endothelial dysfunction and impairs NO release in human vascular cells.32 Because SGA children have high levels of uric acid, it is not inconceivable that impairment of NO synthesis observed in our study may be influenced by this route. On the other hand, the availability of NO could also be disturbed by elevated oxidative stress, and this is consistent with a previous report showing that both isoprostanes and thiobarbituric acid reactive substance levels are higher in SGA than in AGA children (unpublished observation). Still, the mechanisms that determine impaired NO release in SGA children are highly speculative, as discussed above, and are beyond the scope of the present study.
Finally, the most striking result of the present study is the fact that Hcy and NO levels are significantly correlated with both BP and vascular function. We found that systolic BP levels were correlated positively with Hcy and inversely with NO levels in all of the children. In a multiple regression analysis, which included several risk factors, only birth weight and Hcy emerged as significantly important factors associated with systolic BP. In separate analyses, we found that systolic BP correlated with Hcy only in SGA children. This was both statistically significant and independent of other risk factors, including lipid profile, uric acid, and NO. Our data suggest that Hcy could contribute to the development of hypertension in SGA children. However, the mechanism by which Hcy alters systolic BP is unclear. Its direct action on the arterial wall by a variety of mechanisms has been demonstrated, including endothelial dysfunction, smooth muscle cell proliferation, collagen synthesis, and deterioration of elastic material of the arterial wall, all of which increase arterial stiffness.8 Interestingly, a previous study already demonstrated that SGA children show a trend toward increased carotid stiffness.6 In light of these findings, we observed that heart rate was similar between SGA and AGA children; therefore, high BP cannot be attributed to increased cardiac output, but rather must be because of increased systemic vascular resistance. Elevated Hcy levels may be associated with functional abnormalities in the small arteries and/or arterioles, leading to increased stiffness of these arteries and contributing to delays in the recovery of systemic vascular resistance. Therefore, we believe that elevated circulating levels of Hcy observed in SGA children could contribute to arterial stiffness, leading to an early increase in systolic BP.
The relationship found between vascular function and both Hcy and NO levels is also relevant. It is well established that SGA children show impaired endothelial function4–6; however, the mechanisms involved in this endothelial damage have yet to be elucidated. It has been postulated that the impairment in the vasodilatory function during infancy and later increased risk of diabetes and CVD in adults occur on the basis of genetically determined insulin-resistant endothelium, incapable of normal NO formation and vasodilatory capacity.33 We reported previously that uric acid correlated inversely with FMD, demonstrating that increased uric acid levels, even within the physiological range, were closely linked with impaired endothelial function in SGA children. However, this association became negligible when known vascular risk factors were simultaneously considered in a multivariate analysis.5 In the present study, our interest was focused on the association of multiple biomarkers with endothelial function in SGA children. In univariate analyses, we observed that FMD was significantly correlated with Hcy and NO levels but not with leptin or hs-CRP. Not surprisingly, when we performed a multivariate analysis incorporating conventional risk factors, the changes in birth weight and NO were significantly correlated with FMD, reinforcing the evidence that birth weight was a significant predictor of vascular dysfunction in the present cohort. Consistent with current results that indicated both impairment of NO production and a negative correlation between NO and birth weight, our results confirm that NO is a mediator of endothelial dysfunction in our population. The underlying mechanism linking fetal life factors to vascular function remains unclear. However, our study provides the first evidence that NO is involved in the vascular programming observed in 8- to 13-year-old SGA children.
A limitation of our study is that the analyses are based on measurements of plasma biomarkers at a single time point. The results should be interpreted with caution, because the study was carried out in a relatively limited number of children living in shantytowns, with a higher prevalence of underweight and stunting than in populations of developed countries. Larger prospective studies are required to confirm these findings.
In summary, our results show that hs-CRP concentration was not affected, whereas circulating levels of both leptin and Hcy were significantly elevated, and NO was decreased in SGA children. We demonstrated that Hcy plays an important role in BP, whereas NO plays a role in endothelial dysfunction in SGA children. Although the underlying mechanisms that link restricted fetal growth with cardiovascular or metabolic diseases have not been recognized, the present findings further support the hypothesis that SGA children show several cardiovascular and metabolic abnormalities, often referred as syndrome X, that are already evident in the first decade of life.
Perspectives
To date, there is no convincing evidence that metabolic parameters of cardiovascular risk should be monitored in SGA children. Our findings suggest that alterations, even within a reference range, might be helpful to identify any evidence of cardiovascular or metabolic disease processes in these children. A close monitoring of metabolic risk factors, such as leptin, Hcy, and NO, should be evaluated in SGA children and their effects assessed in the late development of diseases.
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Acknowledgments |
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Sources of Funding
Financial support for this study was provided by Fundação de Amparo a Pesquisa do Estado de São Paulo No. 04/10342-7 and No. 04/01672-3.
Disclosures
None.
Received March 22, 2007; first decision April 13, 2007; accepted May 20, 2007.
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