Homocysteine and cysteine levels in prepubertal children: Association with waist circumference and lipid profile

Nutrition 29 (2013) 166–171

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Applied nutritional investigation

Homocysteine and cysteine levels in prepubertal children: Association with waist circumference and lipid profile Narjara Pereira da Silva M.S., R.D. a, Fab
ıola Isabel Suano de Souza M.D., Ph.D. a, Aline Ifanger Pendezza M.S., R.D. a, Fernando Luiz Affonso Fonseca Ph.D. b, Sonia Hix Ph.D. c, Allan Chiaratti Oliveira Ph.D. d, Roseli Oselka Saccardo Sarni M.D., Ph.D. a, *, Vania D’Almeida Ph.D. d ˇ

~o Paulo, Brazil Departamento de Pediatria, Faculdade de Medicina do ABC, Santo Andr
e, Sa ~o Paulo, Brazil Curso de Ciencias Farmaceuticas, Faculdade de Medicina do ABC, Santo Andr
e, Sa ~o Paulo, Brazil Disciplina de Bioqu
ımica, Faculdade de Medicina do ABC, Santo Andr
e, Sa d ~o Paulo, Sa ~o Paulo, Sa ~o Paulo, Brazil
rio de Erros Inatos do Metabolismo, Universidade Federal de Sa Laborato a

ˇ

ˇ

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 January 2012 Accepted 30 May 2012

Objective: To evaluate homocysteine and total cysteine levels in prepubertal children and to determine the association between these levels and obesity, increased waist circumference, glucose levels, and lipid profile alterations. Methods: Using a cross-sectional study, 677 prepubertal students 6 to 11 y old were assessed. The weight, height, and waist circumference of the students were measured. Laboratory analyses included triacylglycerols, total cholesterol and its fractions, glucose, vitamin B12, folate, homocysteine, and cysteine. Chi-square tests and logistic regression (forward–stepwise) were used for statistical analysis; the significance level was set at 5%. Results: The median age of the students was 8.9 y (6.5–11.5), and the prevalences of overweight and obesity were 90 in 677 (13.3%) and 81 in 677 (12.0%), respectively. An increase in waist circumference was observed in 180 of 677 children (26.6%). Inadequate levels of low-density lipoprotein cholesterol, triacylglycerols, and high-density lipoprotein cholesterol were found in 95 (14.0%), 129 (19.1%), and 179 (26.4%) of the 677 students, respectively. The median homocysteine and total cysteine plasma levels were 5.6 mmol/L (0.1–11.7) and 365.7 mmol/L (191.5–589.2), respectively. A multivariate analysis showed that children with a waist circumference above the 90th percentile (7.3 mmol/L) were 2.4 times (95% confidence interval 1.4–4.0) more likely to have increased homocysteine levels and that children with increased waist circumferences and those with high low-density lipoprotein cholesterol levels were 2.7 (95% confidence interval 1.6–4.6) and 2.1 (95% confidence interval 1.1–4.0) times more likely, respectively, to have total cysteine levels above the 90th percentile (445.0 mmol/L). Conclusion: The association of abdominal obesity in prepuberty with levels of homocysteine and cysteine found in this study of a prepubertal population could be an early and independent predictor of cardiovascular risk. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Homocysteine Cysteine Waist circumference Obesity Children

Introduction Homocysteine (Hcy) is a metabolic intermediary in the biosynthetic process that converts methionine, derived from dietary protein, into cysteine (Cys) [1]. Studies have shown that an increase in the concentrations of Cys [2] and Hcy [3] are

* Corresponding author. Tel/fax: þ55-11-5571-9589. E-mail address: [email protected] (R. Oselka Saccardo Sarni). 0899-9007/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2012.05.015

associated with an increase in the risk of the development of cardiovascular diseases (CVDs) in adults. Meta-analyses, including prospective studies involving adults without prior CVD, have indicated that an increase of 5 mmol/L in the level of Hcy is associated with a risk increase of 20% in the development of coronary events over a 5-y period [3]. For total Cys, an association with factors related to CV risk, such as dyslipidemia, high blood pressure, and especially for those who are overweight, has been verified [4]. Nevertheless, there is no robust evidence to show its direct relation with

N. Pereira da Silva et al. / Nutrition 29 (2013) 166–171

167

Height was measured using a stadiometer with a precision of 0.1 cm (Leicester Height Measure; Child Growth Foundation, London, UK) according to World Health Organization recommendations. The height measurements were made in triplicate, with the mean of the three values used for the final result. Anthropometric indices (body mass index and height-to-age ratio) as percentiles were calculated using Epi Info 3.4.3 software [18] considering Centers for Disease Control and Prevention growth charts as reference [19]. Overweight was determined by a body mass index greater that the 85th percentile but lower or equal to the 95th percentile in relation to gender and age. Obesity was defined by a body mass index greater than the 95th percentile on the same basis. Waist circumference was measured in the standing position, midway between the lateral lower rib margin and the uppermost point of the iliac crest, and classified according to Freedman et al. [20] who considered values above the 90th percentile to be increased for gender and age. Biochemical analysis

Fig. 1. Description of the selection of the participants.

serious complications associated with CVD [5–7], including mortality [8]. In healthy children, the reference values for Hcy and Cys are unknown, as is whether the most increased concentrations are related to a future risk of CVD [9]. Punctilious studies have described an association of higher concentrations of Hcy in the pediatric age group with obesity and increased abdominal circumference [10], insulin resistance [11], dyslipidemia [12], and systemic arterial hypertension [13]. Until now, similar studies for Cys have not been available. Central fat deposition increases the risk of metabolic complications such as dyslipidemia, hypertension, diabetes, and atherosclerosis [14]. It has been established that waist circumference is more sensitive than other anthropometric measurements in predicting obesity and other complications related to CV risk [15]. The present study aimed to evaluate the Hcy and total Cys levels in prepubertal children and to determine the association between these levels and obesity, increased waist circumference, glucose levels, and lipid profile alterations. Materials and methods Data collection A cross-sectional study was performed in a convenience sampling of 677 prepubertal children 6 to 10 y old. The students were selected from the 911
public school in Santo Andre
, Sa ~o Paulo, Brazil students enrolled in a Santo Andre (Fig. 1). The following exclusion criteria were adopted: parental or guardian nonconsent; the presence of pubertal development as judged by the Tanner criteria (breast stage 2, testicular volume bilaterally 4 mL) [16]; malnutrition and/or stunting; hospitalization or vitamin supplement intake during the 3 mo before data collection; and chronic, infectious, or inflammatory diseases at the time of data collection. The study was previously approved by the Federal ~o Paulo research ethics committee. Informed written consent was University of Sa obtained from the children’s parents or legal guardians. A general questionnaire on clinical and sociodemographic data was obtained from the parents or guardians. Physical examinations were performed by a pediatrician. Anthropometric parameters Body weight, height [17], and waist circumference were measured by dietitians with the same tools. Children were weighed using a digital scale (model E-150/3P; Filizola, S~ ao Paulo, Brazil) that had been calibrated previously and had a capacity of 150 kg, and the weights were recorded to a precision of 0.1 kg.

Blood samples (10 mL) were collected, through peripheral venipuncture, after a 12-h overnight fast in tubes containing ethylenediaminetetraacetic acid as an anticoagulant to quantify the Hcy and Cys plasma levels and in tubes without an anticoagulant to quantify the total levels of cholesterol and its fractions, triacylglycerols, vitamin B12, and folate. The determination of Hcy and Cys was performed by the method described in 1999 by Pfeiffer et al. [21] set up in our laboratory [22]. Briefly a mixture of 50 mL of plasma and 60 mL of a reaction medium containing phosphate buffered saline (pH 7.4), tris(2-carboxyethyl)phosphine (10%), and an internal standard (cystamine 40 mmol/L) was incubated for 30 min at room temperature to reduce and release protein-bound thiols, after which 100 mL of trichloroacetic acid (0.1 g/mL) containing ethylenediaminetetraacetic acid (1 mmol/L) was added for deproteinization. Afterward, the sample was centrifuged for 10 min at 13 000  g. The supernatant (50 mL) was added to an ambar microtube with a reaction medium containing 10 mL of NaOH (1.55 mol/L), 125 mL of borate buffer (0.125 mol/L) containing ethylenediaminetetraacetic acid (4 mmol/L, pH 9.5), and 50 mL of 7-fluorobenzofurazan-4-sulfonic acid ammonium salt (SBDF; 1 g/L in the borate buffer) for derivatization. The sample was then incubated for 60 min at 60 C and transferred to a high-performance liquid chromatographic vial. Highperformance liquid chromatography was carried out on Simadzu, Kyoto, Japan. Equipment with a scanning fluorescence detector (385-nm excitation, 515-nm emission). Separation of the SBDF-derivatized plasma thiols was performed in a Prodigy ODS2 analytical column (150  3.2 mm, 5 mm; Phenomenex, Torrance, CA, USA) with an Alltech ODS C18 5.0-mm guard column (Alltech, Kentucky, OH, USA) using a 10-mL injection volume and acetic acid-acetate buffer (pH 5.5) 0.1 mol/L containing methanol 30 mL/L as mobile phase at a flow rate of 0.7 mL/ min. The intra-assay coefficients of variation for Hcy ranged from 1.1% to 1.8% and the interassay coefficient of variation was 5.6% [22]. Hcy [23] and total Cys plasma levels above the 90th percentile were used as the cutoff value. Serum folate and vitamin B12 serum levels were measured by a solid-phase, competitive chemiluminescence enzyme immunoassay (Immulite, Siemens, München, Deutschland). Total cholesterol and fractions (high-density lipoprotein cholesterol [HDL-C] and low-density lipoprotein cholesterol [LDL-C]) and triacylglycerol levels were analyzed using a colorimetric enzymatic method (Bayer
cnica, MG, Brazil). The Express Plus, München, Deutschland analyzer and Biote cutoff values proposed by the American Academy of Pediatrics were adopted [24]. Glucose levels were analyzed using the glucose oxidase method (Bayer
cnica kit). Glucose levels above 100 mg/dL were Express Plus analyzer and Biote adopted as the cutoff value [25]. Statistical analysis The statistical analysis was performed with the SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). The continuous variables were tested for normality with the Kolmogorov–Smirnov test. In the bivariate analysis, the chi-square test was applied for the qualitative and dichotomous variables. Variables associated with increased levels of Hcy or Cys that had a P value lower than 0.1 were included in the multivariate regression analysis (logistic regression, forward stepwise method), and P < 0.05 was adopted as the level of statistical significance.

Results Among the studied children, 347 of 677 (51.2%) were female and 459 of 677 (67.8%) were Caucasian. The median age was 8.9 y (range 6.5–11.5). In 444 of 677 (65.6%) of the families, the percapita income was less than the minimum wage established in Brazil (US$350.00/mo). Table 1 presents the nutritional status, waist circumference, lipid profile, and the levels of glucose in children with Hcy and

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Table 1 Nutritional status, waist circumference, lipid profile, and serum glucose levels in children according to homocysteine and cysteine levels Cysteiney (mmol/L)

Variable

Homocysteine* (mmol/L) >7.3 (n ¼ 67)

7.3 (n ¼ 610)

Pz

>445.0 (n ¼ 66)

445.0 (n ¼ 608)

Pz

Nutritional status: obesity Waist circumference >90th percentile High LDL-C (mg/dL) Low HDL-C (mg/dL) High triacylglycerols (mg/dL) Glucose serum levels >100 mg/dL

17 29 13 12 12 1

60 143 77 163 114 10

0.001 0.001 0.093 0.064 0.505 0.663

20 33 14 9 19 1

58 140 75 162 108 11

0.001 0.001 0.015 0.012 0.072 0.568

(25.4%) (43.3%) (19.4%) (17.9%) (17.9%) (1.49%)

(9.8%) (23.4%) (12.6%) (26.7%) (18.7%) (1.6%)

(30.3%) (50.0%) (21.2%) (13.6%) (28.8%) (1.5%)

(9.5%) (23.0%) (12.3%) (26,6%) (17.8%) (1.8%)

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol Data are presented as number of subjects (percentage) * Homocysteine 90th percentile: 7.3 mmol/L. y Total cysteine 90th percentile: 445.0 mmol/L. z Level of significance using chi-square test.

total Cys levels below and above the 90th percentile (7.3 and 445.0 mmol/L, respectively). The frequencies of overweight and obesity were 90 (13.3%) and 81 (12.0%) of 677. An increased waist circumference was observed in 180 of 677 children (26.6%). For the lipid profile, 35 (5.2%) and 129 (19.1%) of the 677 children presented increased LDL-C and triacylglycerol values, respectively, and 179 of 677 (26.4%) presented low HDL-C values. Table 2 presents the mean and standard deviation of the biochemical variables studied. The median plasma Hcy and total Cys levels were 5.5 mmol/L (0.1–11.7) and 365.7 mmol/L (191.5–589.2), respectively. Any Hcy and total Cys values above the 90th percentile (7.3 and 445.0 mmol/L, respectively) were considered increased. For children with Hcy levels above and below 7.3 mmol/L, the mean concentrations of vitamin B12 were 689.9  276.2 versus 744.6  326.0 pg/dL (P ¼ 0.003) and of folate were 15.6  5.2 versus 15.0  5.0 ng/dL (P ¼ 0.645), respectively. Among the children whose total Cys levels were above and below 445.0 mmol/L, the mean concentrations of vitamin B12 were 689.9  276.2 versus 744.6  326.0 pg/dL (P ¼ 0.184) and of folate were 15.6  5.2 versus 15.0  5.0 ng/dL (P ¼ 0.395), respectively. Increased Hcy levels (>7.3 mmol/L) were significantly associated with obesity (25.4% versus 9.8%, P ¼ 0.001) and increased waist circumference (43.3% versus 23.4%, P ¼ 0.001; Table 1). Increased total Cys levels were significantly associated with obesity (30.3% versus 9.5%, P ¼ 0.001), increased waist circumference (50.0% versus 23.0%, P ¼ 0.001), high LDL-C levels (21.2% versus 12.3%, P ¼ 0.015), and normal HDL-C levels (13.6% versus 26.6%, P ¼ 0.012; Table 1). Children who were non-overweight/obese and had an increased waist circumference showed a higher frequency of Hcy levels higher than 7.3 mmol/L than children with a normal waist circumference (19.1% versus 9.2%, P < 0.05). Similar results were observed in children with a total Cys level higher than 445.0 mmol/L (28.2% versus 8.6%, P < 0.05; Fig. 2). Table 2 Values of biochemical variables Variable

Mean  SD

Homocysteine (mmol/L) Cysteine (mmol/L) LDL-C (mg/dL) HDL-C (mg/dL) Triacylglycerols (mg/dL) Folate (ng/mL) Vitamin B12 (pg/mL)

5.6 366.4 106.9 43.4 80.3 15.1 737.7

      

1.3 62.4 26.5 9.8 41.3 5.0 320.6

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol

The relation among obesity (P ¼ 0.001), increased waist circumference (P ¼ 0.001), increased LDL-C levels (P ¼ 0.093), normal HDL-C levels (P ¼ 0.064), and Hcy levels higher than 7.3 versus no higher than 7.3 mmol/L were assessed using a multivariate analysis. In this model, an increased waist circumference was associated with a greater than two-fold increase in the odds of Hcy levels being higher than 7.3 mmol/L (Table 3). A multivariate analysis was also conducted for total Cys levels. Obesity (P ¼ 0.001), an increased waist circumference (P ¼ 0.001), high LDL-C levels (P ¼ 0.015), normal HDL-C levels (P ¼ 0.012), and high triacylglycerol levels (P ¼ 0.072) were included in this model. In the same analysis, children with large waist circumferences and high LDL-C levels were 2.7 (95% confidence interval 1.6–4.6) and 2.1 (95% confidence interval 1.1–4.0) times more likely, respectively, to have increased total Cys levels (Table 4). Discussion The present study found a significant association between increased waist circumference and high concentrations of Hcy and total Cys in obese and non-overweight/obese prepubertal children. Obesity in the abdominal area, characterized by deposits of visceral fat, triggers a series of metabolic repercussions through complex mechanisms. The prevalence of b-adrenergic receptors in the abdominal adipocytes explains the high lipolytic activity in this area, allowing the fat deposits that are located there to be more easily mobilized [26]. Measurement of the waist circumference has been emphasized in studies with adults and children [15,27]. An increase in the waist circumference in the pediatric age group has been associated with early markers of atherosclerosis, such as dyslipidemias [28], endothelial dysfunction [29], insulin resistance [30], and hypertension [31]. Some hypotheses may be suggested to explain the observed association between an increased waist circumference and high levels of Hcy found in this study: insulin resistance, inflammation, and oxidative stress [32,33]. Similar results were described by Papandreou et al. [10] in a study in 524 Greek children and adolescents. The investigators found a significant association between Hcy levels higher than 10 mmol/L and an increased waist circumference. The results regarding total Cys that were observed in this study were similar to the results described by Elshorbagy et al. [34]. They reported that women and men who had high concentrations of Cys had waist circumferences that were 9 and 6 cm larger, respectively, than individuals with normal Cys concentrations. Previous studies have emphasized the positive

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WC >= p 90 6.7%

9.6%

WC < p 90

14.3% 38.9%

80.9% 93.3%

90.4%

90.8%

85.7% 61.1%

*

19.1% Hcy >= 7.3

Hcy < 7.3

Hcy > 7.3

Obese

Hcy < 7.3

9.2%

Hcy >= 7,3

Hcy < 7,3

Non-overweight/obese

Overweight

Cysteine 6.7%

9.6% 35.7%

38.5%

71.8% 91.4% 93.3%

90.4% 64.3%

61.5%

*

28.2% 8.6% Cys >= 445

Cys < 445

Cys >= 445

Obese

Cys < 445 Overweight

Cys >= 445

Cys < 445

Non-overweight/obese

Fig. 2. Association between increased WC and levels of homocysteine and total cysteine in non-overweight/obese, overweight, and obese children. * Chi-square significance level or Fisher exact test (P < 0.05). Cys, cysteine; Hcy, homocysteine; p 90, 90th percentile; WC, waist circumference.

association between total plasma Cys levels and obesity [4], body fat mass [35], and metabolic syndrome [36]. The mechanism that explains this association in humans is not yet completely understood. However, some hypotheses may be considered, such as an increase in the synthesis of coenzyme A, the suppression of 3-phosphoglycerate dehydrogenase, the insulin-like effect in adipocytes, and the pro-oxidant action [4,5]. In the present study, children with high levels of LDL-C were twice as likely to have increased cysteine levels. A cohort study Table 3 Adjusted* odds ratio for the effects of increased waist circumference on homocysteine levels Variable

Odds ratio

95% CI

P

Waist circumference >90th percentile

2.4

1.4–4.0

0.001

CI, confidence interval * Adjusted for obesity, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol.

involving 1550 adults found a positive association between total plasma cysteine levels and LDL-C levels [37]. Cys is a major functional and structural component of apolipoprotein B, the protein of LDL-C, which is the most important form of cholesterol transport from the liver to other tissues [38]. The cutoff point adopted for increased Hcy levels was 7.3 mmol/L, which represents the 90th percentile for the sample. Other investigators have used the 90th percentile as a cutoff point for the same purpose, and the values they found varied from 10 to 11.5 mmol/L. In a review, Refsum et al. [38] proposed upper limits of 8 mmol/L in areas with folic acid supplementation and 10 mmol/L in areas without folic acid supplementation for children and adolescents younger than 15 y. Since June 2004, the addition of 150 mg of folic acid/100 g of corn and wheat flour has been mandatory in Brazil [39]. Therefore, the values found in this study may reflect this supplementation effect, because they are lower than the lowest value established in the study by Refsum et al. [8].

N. Pereira da Silva et al. / Nutrition 29 (2013) 166–171

Table 4 Adjusted* odds ratio for the effects of increased waist circumference and high LDL-C levels on total cysteine levels Variable

Odds ratio

95% CI

P

Waist circumference >90th percentile High LDL-C

2.7 2.1

1.6–4.6 1.1–4.0

<0.001 0.025

CI, confidence interval; LDL-C, low-density lipoprotein cholesterol * Adjusted for obesity, high-density lipoprotein cholesterol, triacylglycerols.

and

In this analysis the cutoff point used for total Cys was 445.0

mmol/L, which characterizes the 90th percentile of the population. Studies in adults have used the following values as the upper limits for Cys: 282 mmol/L (95th percentile of sample) [2] and 300 mmol/L (95th percentile of sample) [5,8]. When performing studies in 922 children 7 to 10 y old, Bates et al. [40] adopted 264 mmol/L for boys and 271 mmol/L for girls as the cutoff points for total Cys. In our study, the possibility that high total Cys levels were influenced by the participants’ lifestyle and dietary habits cannot be excluded. There are some limitations to the present study: not assessing genetic polymorphisms in the enzymes involved in the metabolism of Hcy, not using more reliable methods to assess body composition and central adiposity, and not evaluating the dietary intake. There have been no publications assessing the relation between waist circumference and total Cys levels in children. However, recent studies have provided robust evidence of the association between Cys levels and body composition in adults. Regarding Hcy, the findings in this study highlight the importance of its association with central obesity because of insulin resistance, inflammation, and oxidative stress. Conclusion The association of abdominal obesity in prepuberty with levels of Hcy and Cys, found in this study of a prepubertal population, could be an early and independent predictor of CV risk. Therefore, preventive public health measures, such as nutritional education and lifestyle change, should be implemented to contain the rising rates of excessive weight gain and especially of central obesity in children. References [1] Mudd SH. Hypermethioninemias of genetic and non-genetic origin: a review. Am J Med Genet 2011;157:3–32. [2] El-Khairy L, Vollset SE, Refsum H, Ueland PM. Predictors of change in plasma total cysteine: longitudinal findings from the Hordaland Homocysteine Study. Clin Chem 2003;49:113–20. [3] Humphrey LL, Fu R, Rogers K, Freeman M, Helfand M. Homocysteine level and coronary heart disease incidence: a systematic review and metaanalysis. Mayo Clin Proc 2008;83:1203–12. [4] Elshorbagy AK, Kozich V, Smith AD, Refsum H. Cysteine and obesity: consistency of the evidence across epidemiologic, animal and cellular studies. Curr Opin Clin Nutr Metab Care 2012;15:49–57. [5] El-Khairy L, Ueland PM, Refsum H, Graham IM, Vollset SE, European Concerted Action Project. Plasma total cysteine as a risk factor for vascular disease: the European Concerted Action Project. Circulation 2001;103:2544–9. [6] Xiao Y, Zhang Y, Lv X, Su D, Li D, Xia M, et al. Relationship between lipid profiles and plasma total homocysteine, cysteine and the risk of coronary artery disease in coronary angiographic subjects. Lipids Health Dis 2011;10:137. [7] El-Khairy L, Ueland PM, Nyg ard O, Refsum H, Vollset SE. Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma: the Hordaland Homocysteine Study. Am J Clin Nutr 1999;70:1016–24.

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