Some previous studies have measured tHcy and folate levels among Koreans, although only limited information for reference values for tHcy and folate concentrations has been available because of their small sample size (7) and issues with the representativeness of study subjects (8). Therefore we determined the age-, gender-, and MTHFR C677T genotype-specific reference intervals for plasma tHcy and serum folate concentrations using a large-sized reference sample group in a middle-aged and older Korean population.

Reference individuals, in this study, were participants in the Namwon Study, an ongoing population-based cohort study that includes 10,667 individuals (4,201 men and 6,466 women), aged from 45 to 74 yr, dwelling in the community of Namwon, Korea. The study design and measurements in the Namwon Study were previously described (9). Among the participants, individuals with missing plasma tHcy, serum folate or MTHFR C677T genotype data (n= 52) and with specific conditions that could potentially alter the plasma tHcy or serum folate concentration were excluded to establish reference sample group. The exclusion criteria were as follows: extreme serum folate or plasma tHcy concentration (< 2.5 percentile or > 97.5 percentile by gender), renal insufficiency (glomerular filtration rate, GFR < 60 mL/min per 1.73 m²), hyperglycemia (fasting glucose ≥ 126 mg/dL), thyroid dysfunction (subclinical/overt-hypothyroidism and -hyperthyroidism), hypertension (systolic blood pressure ≥ 140 mmHg or diastolic pressure ≥ 90 mmHg), hyperlipidemia (total cholesterol ≥ 240 mg/dL), severe anemia (hemoglobin < 8 g/dL for men and < 7 g/dL for women), peripheral arterial disease (ankle brachial index < 0.9), body mass index ≥ 30, known coronary heart disease, stroke, and cancer history, and heavy smoker (≥ 20 pack-years or ≥ 20 cigarettes per day), heavy drinker (14 drinks per week for men and ≥ 7 drinks per week for women), current medication for diabetes, hypertension, and hyperlipidemia, using oral contraceptive or thyroid drug. Finally, 3,154 participants (1,029 men and 2,125 women) were used to determine reference values. The study was approved by the institutional review board of Chonnam National University Hospital (IRB No. 1-2007-07-062). Blood was taken from antecubital vein after overnight fasting, at least 12 hr. For measuring tHcy, whole venous blood was collected into EDTA tubes, immediately packed into crushed ice and protected from light. Within 30 min after collection, the plasma was separated by high-speed cold centrifugation, and stored in aliquots at -70℃ until assayed. Whole blood, which was not treated with anticoagulant, was collected into serum separator tubes and held at room temperature for 30 min before centrifugation. Plasma tHcy concentration was estimated with fluorescence polarization immunoassay and serum folate with ion capture assay, both using Abbott AxSYM analyzer (Abbott Park, IL, USA). Genomic DNA was extracted from peripheral blood and genotyping by real-time polymerase chain reaction (PCR) was performed. Our MTHFR genotyping method has been reported previously (10).

All calculations for determining reference values, reference interval (RI) with 90% confidence interval (CI), were based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI) guideline document CA28-A3 (11). Age- and gender-specific percentile curves were constructed by using LMS (lambda, mu, sigma) method with skewness expressed as a Box-Cox transformation. Statistical analyses were done using the GAMLSS package in the statistical program R version 3.0.1 (The R foundation; available from www.r-project.org) and Medcalc Statistical Software version 12.7.3 (Medcalc Software bvba, Ostend, Belgium).

Significant differences of plasma tHcy concentration were observed between the age groups (45-54 yr, 55-64 yr, and 65-74 yr), MTHFR 677 genotypes, and quartiles of serum folate. The RI (90% CI) for plasma tHcy concentration (µM/L) for men (n = 1,029) was 5.03 (4.96-5.12) to 13.80 (13.00-14.85) and for women (n =2,125) was 3.95 (3.90-4.00) to 10.19 (9.87-10.49). No significant difference of serum folate concentration between each age group was found. The RI (90% CI) of serum folate concentration (nM/L) for men was 11.73 (11.48-12.23) to 38.44 (37.69-38.94) and for women was 15.23 (14.98-15.72) to 40.44 (40.19-40.93). MTHFR677 genotype-specific reference values were also significantly different between genotypes in both genders. The highest values for tHcy concentration and the lowest values for folate concentration were observed in the TT genotype (Table 1). Percentile curves of tHcy and folate according to genders and age were presented in Fig. 1.

The present study reported reference values for plasma tHcy and serum folate in middle aged and older Koreans using a large-sized population sample. Age-specific, gender-specific, and MTHFR C677T genotype-specific reference values were also provided. We believe that the present reference values of plasma tHcy and serum folate concentrations were estimated accurately, based on the conservative strategies in sample selection that effectively prevented pre-analytic variation, and the standard guidelines of data analysis. Wide-ranging exclusion criteria were applied to establish the reference sample.

The findings of relative higher concentrations of tHcy and lower concentrations of folate in men, older group and MTHFR 677 TT genotypes are consistent with most previous studies. The median plasma tHcy concentrations (µM/L) in our reference sample (7.7 in men and 6.1 in women) were lower than those of previous reports from Korea (7, 8), other Asian countries (12, 13), European countries (14), the USA and Canada in both of pre- and post-folic acid fortification era (6, 15), even than those of Mexican Americans, an ethnicity with the lowest tHcy level in National Health and Nutrition Examination Survey (6). These differences of tHcy concentrations can be explained by the higher level of serum folate in our population. The median serum folate concentrations in our reference sample (23.7 nM/L in men and 29.0 nM/L in women) were about 2-fold higher than those of the non-fortified populations (5) and similar to those of the fortified USA population (6). Dietary patterns with high intake of vegetables, fruits, and fibers, as well as low meat consumption, have been associated with decreased tHcy and increased folate concentrations (14). In previous reports from nationwide nutrition surveys, fruit and vegetable consumption of Koreans was higher than those of western countries (16, 17). Within the same ethnicity, differences between geographic areas and their characteristics, such as agricultural production, are believed to play a role in maintaining the tHcy concentration. A Chinese study reported that plasma homocysteine concentration in rural areas is lower than in urban areas (12). There have still been inconsistent findings in regional variations (12, 18), although these variations of tHcy concentration are highly suggestive that B vitamin status or intake patterns differ between urban and rural areas (16). All participants of the Namwon study are living in a rural area at a high altitude in Korea. Considering the characteristics of the study design, the impact of the seasonal and geographic variations cannot be ruled out as potential explanations of low tHcy and high folate concentrations in our sample.

In addition, tHcy is released from erythrocytes in a time- and temperature-dependent manner, about 10% increase per hour at room temperature (1). In our study, blood was drawn into EDTA and then immediately stored in an ice bucket. Separation was performed using a 4℃ cold centrifuge within 30 min after the blood draw. It was believed that any artificial increase by tHcy export from blood cell is prevented effectively. Over the past decades, extensive evidence that genetic factors are important determinants for tHcy level has accumulated. Individuals with the MTHFR 677TT genotype, the most common genetic determinant, usually have higher tHcy (up to 2.5 µM/L) than those with the 677CC genotype (1). In our study, plasma tHcy concentrations were significantly higher and serum folate concentrations were significantly lower in the subjects with the TT genotype than in those with the CC or CT genotype (P value< 0.05 by Tukey's post hoc test in both sexes). In addition, the 677TT genotype is associated with high tHcy only in individuals with low folate status (19), however, no folate deficiency (< 7.5 nM/L) was found among our reference individuals, and tHcy concentrations in each MTHFR C677T genotype in our sample were lower than those of other populations with the same genotype (20). Our study has some limitations. First, cobalamin status, an important determinant for tHcy level, was not considered. Second, measurements were not cross-validated with the values being measured by HPLC or stable-isotope dilution method. Third, most participants were recruited in a rural area during the winter season, and impact of these variation has not been fully figured out.

In summary, the reference values of tHcy and folate were lower and higher than those in most previous reports, respectively. These findings were maintained across genders, age groups, MTHFR677 genotypes, and other lifestyle factors.