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Laboratory of Nutrition and Clinical Dietetics, Department of Nutrition and Dietetics, and
* Department of Home Economics and Ecology, Harokopio University, Athens, Greece; and
Institute of Child Health, Aghia Sofia Children’s Hospital, Athens, Greece
2To whom correspondence should be addressed. E-mail: tina.papoutsakis{at}hua.gr.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • homocysteine • folate • MTHFR C677T genotype • children • Greece
Heart disease is the leading cause of death in Greece, with a mortality rate that increased between 1980 to 1997 from 218 to 304 per 100,000 (1). However, not too long ago, in the 1960s, Greeks had impressively low cardiovascular disease (CVD)3 rates as described in the Seven Countries Study (2). The increase of CVD in Greek adults and the fact that risk factors established at young ages can set the stage for adult disease necessitate the study of CVD risk factors in young populations (3).
Cigarette smoking, hypertension, an abnormal lipid profile, obesity, diabetes, physical inactivity, an atherogenic diet, family history, age, and male gender are well-known risk factors that explain a substantial proportion but not all of coronary heart disease. Thus, the identification of new risk factors continues to be an area of active research. An increased total homocysteine (tHcy) concentration in the blood has been recognized as such an independent CVD risk factor (4). tHcy is a sulfur-containing amino acid whose concentrations are controlled by 2 metabolic pathways: tHcy remethylation to methionine or tHcy transsulfuration to cysteine, which require folate and vitamins B-12 and B-6 as cofactors (5). Whether homocysteine-lowering efforts will translate into lowering CVD rates remains to be shown by clinical intervention trials. The prospect for prevention supports the need to study tHcy in young populations (6), especially in countries where CVD rates are of concern and tHcy has never been described. This is not to imply that elevation of CVD can be solely attributed to tHcy or the factors that influence it. Most likely, adverse changes in classic risk factors (such as obesity and worsening of blood lipids) are responsible for the majority of the CVD increase observed in Greece (7). However, the possibility that elevated tHcy in childhood contributes to future risk of CVD cannot be discounted.
Elevated tHcy is influenced by nutrition, most notably inadequate status of the B vitamins, folate and vitamins B-12 and B-6, as well as male gender, increasing age, and genetic background (8). The most common genetic defect of tHcy metabolism is the 677C 
T mutation in the gene for 5,10-methylenetetrahydrofolate reductase (MTHFR) (EC 1.7.99.5), where an alanine-to-valine substitution takes place. The 677C T mutation is found in a thermolabile variant of the MTHFR enzyme and results in about half of the usual enzyme activity (9). Adults who are homozygous for valine (or TT genotype) have higher tHcy concentrations under conditions of inadequate folate status (10). Also, folate is the most robust nutritional predictor of tHcy, and thus hyperhomocysteinemia can occur as a consequence of a gene-nutrient interaction (4,8,10). Recent studies suggest that age could modify the contribution of the MTHFR mutation to tHcy concentrations (11), yet few studies have investigated the effects of the MTHFR-folate interaction on tHcy in children (12).
Plasma tHcy concentrations may vary according to geographic location (13), but data for healthy children in Mediterranean countries are minimal (14). To our knowledge, tHcy has not been studied in presumed healthy children living in Greece. In the present study, we measured plasma tHcy in a sample of Greek sixth-grade schoolchildren and we investigated putative associations with folate, vitamin B-12, and other metabolic variables in plasma, as well as dietary intake and the MTHFR C677T polymorphism.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Specimen collection and biochemical analyses.
Overnight fasting (
10 h) venous blood was collected from study participants. The samples were collected in EDTA-containing tubes and protected from light thereafter. Plasma was immediately separated (1800 x g, 15 min) and the buffy coats of nucleated cells were saved for DNA extraction. After being divided into aliquots, plasma samples were shipped in ice under code to the Laboratory of Nutrition and Clinical Dietetics, Harokopio University, Athens. Aliquots were stored at –80°C until assayed. Among other routine blood indices, plasma total cholesterol, HDL cholesterol, triacylglycerol, and glucose were determined in duplicate using commercially available enzymatic colorimetric assays (Sigma Diagnostics) on an automated ACE analyzer (Schiapparelli Biosystems). Within-batch coefficients of variation for the determination of biochemical variables were all below 5%. LDL cholesterol was calculated by the Friedewald equation (15).
Plasma tHcy concentration (total of protein- and nonprotein-bound homocysteine) was measured via reverse-phase HPLC with fluorometric detection. Precolumn derivatization of plasma thiols with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate was implemented prior to HPLC and fluorometric detection. The method of Ubbink et al. (16) as originally described by Araki and Sako (17) was modified to include a novel phosphine reagent: tris(2-carboxyl-ethyl) phosphine (18). Fluorescent intensities were measured with excitation and emission wavelengths set at 245 and 515 nm, respectively. tHcy concentration was determined against a known plasma standard for tHcy (ClinCal-Calibrator, Plasma-Calibrator, Lyophil., for homocysteine from Recipe, Chemicals and Instruments). Also, controls (Clinchek-Control, Plasma Control, Lyophil., for homocysteine, Level I, II from Recipe, Chemicals and Instruments) were used for internal quality assurance of the assay. The intra- and interassay CV were 3 and 4%, respectively. Plasma folate and vitamin B-12 levels were measured using a commercially available RIA kit (Dualcount; solid phase no boil assay; Diagnostic Product) with a sensitivity of 
0.7 nmol/L for folate and 25 pmol/L for vitamin B-12.
Dietary information. In this survey, information was also collected regarding children’s current dietary intake via 2 nonconsecutive 24-h recalls. No child reported taking a vitamin and mineral supplement. Food intake data were analyzed using Nutritionist V software, version 2 (First Databank). The Nutritionist V food database was modified by adding traditional food recipes (19) and local processed food items using nutrient data provided by industry.
DNA isolation and genotyping. Genomic DNA was extracted from leukocyte nuclei by the salting-out method (20). The MTHFR C677T genotype was determined using the method of Frosst et al. (9). Briefly, a 198-bp PCR amplification product was generated using the following set of primers: 5'-TGA AGG AGA AGG TGT CTG CG-3' (forward); 5'-AGG ACG GTG CGG TGA GAG TG (reverse). The PCR conditions were 95°C for 5 min, 60°C for 1 min, and 72°C for 1 min for 1 cycle and subsequently 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min. The PCR products were digested with the restriction endonuclease HinfI (New England Biolabs) and were then electrophoresed on 3% agarose gels, prestained with ethidium bromide to visualize the different genotypes. Digestion of the 677C allele resulted in no cleavage products whereas digestion of the 677T allele produced 2 fragments with sizes 175 and 23 bp.
Statistical analysis. SPSS 11.0 for Windows (SPSS) was used for all statistical analyses. The descriptive presentation of data includes means and standard deviations or number of subjects and respective percentage. Unpaired two-tailed t tests were calculated to detect significant differences between 2 independent groups, and the chi-square test was applied to assess differences in frequencies of measured genotypes. Chi-square tests were also conducted to examine whether the genotype frequencies were in Hardy-Weinberg equilibrium. The normal distribution of the investigated variables was assessed through the Kolmogorov-Smirnov criterion. In all analyses, log-transformed values were used for tHcy, folate, vitamin B-12, triacylglycerol, and dietary intakes of various nutrients, due to their skewed distribution. However, in the tables untransformed means are provided. Pearson’s correlation coefficients were determined to identify significant correlations between continuous variables of interest. ANOVA was used to test for differences between genotypes and for interactions between genotype and vitamin concentrations. For post hoc comparisons of means, overall type I error was controlled using Tukey’s test. Also, the effect of several potential confounders (i.e., age, gender, BMI) was considered, yet adjustment for these variables had no influence on the observed results, and therefore only the unadjusted data are presented. Furthermore, in a recessive model for the T allele, the differences in measured variables in individuals with homozygous mutant genotypes (TT) versus carriers of the wild-type allele (C allele) were explored. Statistical significance was interpreted as values of P < 0.05.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The distribution of plasma tHcy concentrations was positively skewed in this group of children (Fig. 1) as it is in adult populations, whereas tHcy values were appreciably lower than those reported in adults (4). The homocysteine concentrations we present are relatively similar to those reported for healthy children from neighboring Mediterranean countries, namely Italy (21) and Spain (14,22), and higher than those reported in other developed countries (age-appropriate reported values range from 5.0 to 6.8 µmol/L) (13,23–27). tHcy may depend on geographic location and/or ethnicity, reflecting differentiations of nutritional environment and/or genetic profile. Unfavorable socioeconomic characteristics may increase children’s tHcy even when vitamin status is adequate (23). Also, variations in reported tHcy results may be attributed to preanalytic conditions, whether data are presented as medians or arithmetic or geometric means or whether results are presented by gender or not. Some studies have fewer than 30 children in age groups or provide results without categorizing data by age (13). There is no standard definition for hyperhomocysteinemia, but the 95th percentile value has frequently served as a relative cutoff point (26). Consistent with this definition, hyperhomocysteinemia in our sample corresponded to concentrations exceeding 11.5 µmol/L. Osganian et al. (6) reported 95th percentile values of 8.5 µmol/L in US children (ages 13–14 y), and De Laet et al. (28) reported 95th percentile values of 10.2 µmol/L in Belgian children (ages 10–14 y). Even though our sample is not representative of the Greek population, and we did not study a comprehensive pediatric age range, an estimate of what may be deemed as hyperhomocysteinemia in Greek children is clinically relevant because hyperhomocysteinemia has been found to be a potent risk factor for thromboembolism in children (29).
Plasma tHcy was inversely associated with folate and vitamin B-12 concentrations, in agreement with existing research from different countries in adults (30–32) and children (6,12,13,23,24,26,28,33). The repeatability of the inverse relationship between tHcy and B-vitamin status suggests that even though there may be differences in tHcy concentrations between different populations, optimal B-vitamin status is universally important in achieving lower tHcy.
We did not find differences in plasma tHcy concentrations between males and females. Others have reported a lack of gender difference in similar ages, whereas such a difference begins to become important in postpubertal children (5). The absence of gender difference in earlier childhood seems to be related to the process of maturation: as age increases, muscle mass increases, and sex hormones change. We did not account for the influence of puberty stage. Additional research is needed to describe the specific relation between development of gender differences during adolescent years and tHcy. As we show, plasma glucose is inversely related to tHcy (34). Fonseca et al. found that in folate-replete individuals, such as in our sample, insulin sensitivity correlates positively with plasma tHcy concentrations (35). Insulin has recently been shown to directly influence homocysteine metabolism by inhibiting cystathionine ß-synthase expression in liver; a key enzyme involved in the transsulfuration pathway of homocysteine catabolism (36), and it is likely that such a mechanism underlies the above-observed relationships.
We did not find any significant relations between tHcy and plasma lipid concentrations, BMI, or dietary intake. Osganian et al. (6) reported that tHcy was not associated with lipids and was only weakly related to BMI, but De Laet et al. (28) found BMI to relate positively with tHcy across a wide age range of children. The relation of dietary intake and tHcy has not been thoroughly studied in children. In our pediatric sample, we did not detect a relation of reported macro- and/or micronutrient intake with tHcy, folate, and vitamin B-12 in plasma. However, the use of two 24-h dietary recalls may not be sufficient to reveal such associations with micronutrients. Further studies on the diet-tHcy relation in children are warranted because adherence to a Mediterranean eating pattern has been related to lower tHcy concentrations (37) and tHcy may be modifiable through diet, even in children with adequate vitamin status (23).
For the MTHFR polymorphism, we report a frequency distribution of 37.1% for the T allele, similar to the frequency reported for Greek adults (35%) (37,38). Our MTHFR frequency data are in agreement with a higher prevalence of the homozygous mutant genotype TT in Mediterranean countries compared to other countries in Northern Europe (39). Also, our data support the concept of a north-to-south increasing gradient for the frequency of T677 in Europe (39). The relatively high tHcy concentrations observed in our group of Mediterranean children may be related to the increased frequency of the T allele in Southern Europe.
In adults, the TT genotype increases tHcy under conditions of lowered folate status (10), whereas CC adults usually exhibit higher plasma folate concentrations and have 25% lower concentrations of tHcy when compared to TT individuals (40). Such information in children is very limited. Balasa et al. (41) found that the C677T MTHFR polymorphism was an independent determinant of tHcy in 197 healthy U.S. children (aged 6 mo to 16 y), but these investigators did not measure plasma concentrations of B vitamins to examine possible interactions between the genetic profile and nutritional status on tHcy. In our study, TT children had about 16% higher concentrations of tHcy and 15% lower plasma folate concentrations compared to CC children. Moreover, TT status was important in exhibiting increased tHcy only in children who were stratified in the lower folate status group, suggesting that even at young ages the interaction between folate and the genotype influences tHcy concentrations. An interaction between the MTHFR C677T genotype and serum folate on tHcy was previously reported in the study of Tonstad et al. (42) among 92 Norwegian children with familial hypercholesterolemia during cholestyramine treatment. Delvin et al. (12) also reported that in a healthy French Canadian pediatric sample the TT genotype was associated with higher tHcy concentrations only in nutritionally stressed children who were older than 10 y. It is noteworthy that none of the children in our sample qualified as being folate deficient, suggesting that optimal folate status versus avoidance of subclinical or clinical deficiency ought to be the appropriate nutritional goal, especially for individuals carrying the mutant allele. Our study supports the hypothesis that comprehensive interventions to achieve optimal folate status ought to start early to maximize CVD prevention potential.
The cross-sectional study design limits our potential to reveal causal relations. Also, our study included a moderate number of participants and the sample is not representative of the Greek population. Nevertheless, this investigation is the first one to examine simultaneously in a Greek pediatric sample plasma tHcy, folate, and vitamin B-12 concentrations and the MTHFR polymorphism. We showed that, similar to what has been observed in adults, plasma folate is important in determining the contribution of the MTHFR C677T mutation to tHcy concentrations in children. The collection of representative data in young subjects is needed to address important issues such as establishing tHcy reference values in Greek children and investigating further the factors that affect tHcy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: C, cytosine; CVD, cardiovascular disease; MTHFR, methylenetetrahydrofolate reductase; T, thymidine; tHcy, total homocysteine.
Manuscript received 20 September 2004. Initial review completed 10 November 2004. Revision accepted 23 November 2004.
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