**High Plasma Homocysteine Increases Risk of Metabolic Syndrome in 6 to 8 Year Old Children in Rural Nepal**

**Mohsin Yakub <sup>1</sup> , Kerry J. Schulze 1,\*, Subarna K. Khatry 2 , Christine P. Stewart <sup>3</sup> , Parul Christian <sup>1</sup> and Keith P. West, Jr. 1**


*Received: 18 December 2013; in revised form: 21 March 2014 / Accepted: 2 April 2014 / Published: 21 April 2014* 

**Abstract:** Little attention has been given to the association of plasma homocysteine (Hcy) and metabolic syndrome (MetS) in children. We have evaluated the risk of MetS with plasma Hcy in a cohort of 6 to 8 year old rural Nepalese children, born to mothers who had participated in an antenatal micronutrient supplementation trial. We assessed Hcy in plasma from a random selection of *n* = 1000 children and determined the relationship of HOHYDWHG +F\ ! ȝPRO/ WR 0HW6 defined as the presence of any three of the following: abdominal adiposity (waist circumference WK SHUFHQWLOH RI WKH VWXG\ population), high plasma glucose ( WK SHUFHQWLOH KLJK V\VWROLF RU GLDVWROLF EORRG pressure (WK SHUFHQWLOH RI UHIHUHQFH SRSXODWLRQ WULJO\FHULGH PPRO/ DQG KLJK density lipoprotein < 0.9 mmol/L.) and its components. There was an increased risk of low high-density lipoproteins (HDL), [odds ratios (OR) = 1.77, 95% confidence intervals (CI) = 1.08–2.88; *p* = 0.020], high blood pressure [OR = 1.60, 95% CI = 1.10–2.46; *p* = 0.015] and high body mass index (BMI) [OR = 1.98, 95% CI = 1.33–2.96; *p* = 0.001] with elevated Hcy. We observed an increased risk of MetS (OR = 1.75, 95% CI = 1.06– 2.90; *p* = 0.029) with elevated Hcy in age and gender-adjusted logistic regression models. High plasma Hcy is associated with increased risk of MetS and may have implications for chronic disease later in life.

**Keywords:** metabolic syndrome; homocysteine; Nepal

#### **1. Introduction**

Metabolic syndrome (MetS) is a complex disorder comprising abdominal adiposity, high-blood pressure (BP), plasma glucose (PG), dyslipidemia [high-plasma triglycerides (TG) and/or low concentrations of high-density lipoproteins (HDL)]. Insulin resistance and cardiovascular disease (CVD) have gained attention as major manifestations of the syndrome. MetS has been considered an illness of adulthood; however an increase in the prevalence of insulin resistance and MetS has been reported among children recently [1,2]. According to a systematic review by Friend *et al*., the median prevalence of MetS was 3.3% and 22% among Far East (India, South Korea and China) non-obese and obese children, respectively [3]. Given global trends toward increased adiposity, obesity and diabetes, deaths due to outcomes related to MetS, such as coronary heart disease (CHD) and type-2 diabetes, are expected to rise across the age spectrum [4].

In low income societies, the incidence in MetS among children has also been associated with a pattern of intrauterine conditions leading to low birth weight (<2500 g, LBW), rapid postnatal weight gain and less frequent breastfeeding during early life [5,6]. Given a high prevalence of LBW, rural Asian populations experiencing the nutrition transition to diets higher in fat and processed foods [7] may, therefore, be at particular risk of developing MetS. In the terai of southern Nepal, longitudinal studies have shown the incidence of LBW to be 43% [8] and the prevalence of MetS to be 11.7% at 6–8 years of age [9], with a lower risk among children whose mothers were provided antenatal folic acid supplements [9]. The research described here examines homocysteine (Hcy) as a risk factor for the MetS that was observed in this rural setting.

Homocysteine, an intermediary product of methionine metabolism, is elevated in folate and vitamin B12 deficiencies [10]. Hcy has also been accepted widely as an independent risk factor for CVD such as CHD and stroke [11,12]. Moreover, Hcy has been shown to be a thrombogenic and atherogenic substrate that potentiates atherosclerotic phenomena that may lead to adverse cardiometabolic events [13].

Despite its link with CVD, whether Hcy is more broadly associated with MetS is not well established. It is known that atherosclerosis begins in childhood and CVD can occur if children and adolescents have earlier exposure to risk factors such as elevated Hcy and components of MetS. However, to the best of our knowledge, no study has yet been done to assess the relationship of Hcy to MetS among young children living in rural South Asia. Detection of risk factors for MetS early in life in South Asian populations may suggest means to attenuate the progression of MetS and possible development of type-2 diabetes and CVD later in life [14,15].

Our hypothesis in the present study was that MetS and its components such as excess adiposity, hypertension, dyslipidemia and high glucose are associated with increased plasma Hcy concentrations in children. We addressed this question by determining the prevalence of hyperhomocysteinemia and evaluating its associations with aspects of MetS in a cohort of 6–8 year old children in rural Nepal.

### **2. Experimental Section**

Children born to mothers who participated in a 5-arm trial of antenatal (to 3 months postpartum) micronutrient supplementation (1999–2001) in the rural District of Sarlahi, Nepal [8], were revisited and consented in 2006–2008 at 6–8 years of age as part of a large cohort follow-up study [9]. Procedures and primary outcomes of the original trial [8,16] and follow-up assessment [9] have been previously reported. Briefly, from 2006 to 2008, 3524 of 4130 children who were born to mothers enrolled in the micronutrient supplementation trial [8,16] were revisited in their homes to reassess vital status, height (measured by stadiometer; Harpenden, Croswell, UK), weight (measured by electronic scale; Model 881, Seca, Cambridge, MD, USA) and BP, and a 10 mL venous blood sample was collected in a heparinized tube in 3305 children, two-thirds of whom reported being fasted. For BP, the mean of last three values out of four measurements collected at one minute intervals using an automated oscillometric device (BpTRU™ BPM-300 Medical Devices Ltd., Coquitlam, BC, Canada) was taken. Standard test kits (DCA 2000 analyzer MN, Bayer HealthCare LLC, Elkhart, IN, USA) were used to estimate glycosylated hemoglobin (HbA1c) from whole blood. Plasma was separated in a field laboratory and total cholesterol, HDL-cholesterol, TG and glucose concentrations were measured using a Cholestech LDX analyzer (Cholestech Corp., Haywood, CA, USA). Low density lipoprotein (LDL) cholesterol was estimated using Friedewald's formula [17]. Frozen plasma was shipped to Johns Hopkins University Bloomberg School of Public Health in liquid nitrogen for further analysis, including plasma insulin, in fasted children only, by ultrasensitive sandwich immunoassay (ALPCO Diagnostic, Salem, NH, USA). Data from these laboratory analyses have been reported previously [9].

Based on criteria of having multiple aliquots of plasma and complete data collected from the initial trial and follow up activity, 2130 children were identified as eligible for further biomarker analysis, forming the sampling frame for this analysis. This group of children was similar to the 1394 children without sufficient plasma aliquots or complete data on a range of personal and household characteristics [9]. From the 2130 children we randomly selected 1000 children (511 boys and 489 girls), balanced across maternal supplementation groups (*n* = 200 children per group evenly distributed over the duration of the field activity), for a subsequent study of micronutrient status. Hcy was measured in this subset of samples by chemiluminescent immunoassay (Immulite 1000, Siemens Diagnostics, Los Angeles, CA, USA), along with other measures of inflammation and micronutrient status [18]. Out of the 1000 participating children, 30.8% children had cholesterol, 7.2% had HDL, 6.0% had TG, and 3.2% had glucose concentrations that were below the detectable limits for the Cholestech assay. For all those who had values below the detectable limits we used the minimum detectable value of that biomarker as an estimate of biomarker concentration, allowing us to retain complete data in our analyses, but thereby overestimating the actual concentrations of those analytes for those children. LDL was not calculated when other lipid data were out of range. Also, 324 children had not adhered to fasting instructions, in whom insulin was not measured and therefore HOMA not determined. Data for lipids and glucose were analyzed from both fasted and non-fasted participants and included in the final analysis as we have reported previously [9].

Ethical clearance of the study was obtained from Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health (protocol H.22.06.05.26.A2, 20 September 2006) and the Ethics

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Review Committee at Institute of Medicine at Katmandu, Nepal. The study was conducted according to the principles of the Declaration of Helsinki.
