ABSTRACT
Several genome-wide association studies (GWAS) have revealed that single nucleotide polymorphisms (SNPs) of ABCG2 and SLC22A12 were strongly associated with serum uric acid (SUA), but those of methylene tetrahydrofolate reductase (MTHFR) were not. However, there were several studies indicating the association with MTHFR C677T polymorphism. This study examined the association with the polymorphism, taking into account the genotypes of ABCG2 Q126X and SLC22A12 W258X. Subjects were 5,028 health checkup examinees of Seirei Preventive Health Care Center (3,416 males and 1,612 females) aged 35 to 69 years, who participated in the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study). Hyperuricemia was defined as SUA equal to 7 mg/dL or over. The genotype frequency was 35.9% for CC, 48.1% for CT, and 16.0% for TT, being in Hardy-Weinberg equilibrium (p=0.90). Among 4,425 participants with ABCG2 126QQ and SLC22A12 258WW who were not under medication for hyperuricemia, the mean SUA was 5.6 mg/dL, 5.6 mg/dL, and 5.7 mg/dL, respectively. When 114 participants with ABCG2 126QQ and SLC22A12 258WW under medication for hyperuricemia were included in hyperuricemia cases, the sex-age adjusted odds ratio (OR) of hyperuricemia was not significant; OR=1.00 (95% confidence interval, 0.89–1.24) for CT genotype and OR=0.98 (0.84–1.32) for TT genotype, relative to CC genotype. The present study indicated no association between SUA and MTHFR C677T genotype, after the influences of ABCG2 Q126X and SLC22A12 W258X were removed.
Key Words: Serum uric acid, Urate transporter polymorphisms, MTHFR C677T
INTRODUCTION
It is well known that serum uric acid (SUA) levels are associated with various factors such as sex, age, body mass index (BMI), dietary habit and drinking habit.1-3) In addition, there is evidence that genetic traits influence SUA concentrations; the heritability was estimated to be up to 73%.4) A recent genome-wide association study performed in Japan showed strong associations of SUA with genetic polymorphisms of SLC22A12 coding uric acid transporter 1 (URAT1), SLC2A9 coding glucose transporter 9 (GLUT9), and ABCG2 coding ATP-binding cassette subfamily G member 2 (ABCG2),5) which were also reported to have associations in European ancestry.6) Among the polymorphisms, SLC22A12 W258X,7,8) SLC2A9 R380W and R198C,9) and ABCG2 Q126X and Q141K10,11) were confirmed to have associations with SUA, although the association of ABCG2 Q141K was relatively weak. SLC22A12 258X and ABCG2 126X and 141K are found in a general Japanese population, whereas SLC2A9 R380W and R198C are not because of the extremely rare allele frequency.
Although not detected in the genome-wide association study, methylenetetrahydrofolate reductase (MTHFR) C677T was reported to have associations with SUA.12-14) A recent meta-analysis on the association with six studies (two from Iran, two from China, one from Korea, and one from Japan) demonstrated that the summary odds ratio (OR) was 1.879 (95% confidence interval (CI), 1.596–2.213).15) The present study investigated the association of MTHFR C677T with SUA levels among Japanese health checkup examinees, after taking into account the genotypes of SLC22A12 W258X and ABCG2 Q126X.
MATERIALS AND METHODS
Subjects
Subjects were 5,028 participants of the Shizuoka Study, a part of the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study), aged 35–69 years, whose registered address was the west half of Shizuoka Prefecture including 12 cities (Shizuoka, Fujieda, Yaizu, Makinohara, Kikukawa, Omaezaki, Kakegawa, Shimada, Fukuroi, Iwata, Hamamatsu, and Kosai) and 6 towns (Okabe, Oigawa, Yoshida, Kawane, Kawanehon, and Arai).16) After two individuals without blood samples were excluded, 5,026 participants (3,414 males and 1,612 females) were used for analysis.
Data collection
Written informed consent was obtained from all subjects. The contents of the agreement included 1) permission to use information on lifestyle, disease history, and family history collected with a self-administered questionnaire, 2) permission to use laboratory data obtained through the health checkup, and 3) the donation of blood and urine specimens, as well as the follow-up until 2025 for deaths from any cause, and diagnoses of cancer, cardiovascular, and cerebrovascular diseases.
The self-administered questionnaire used in the Shizuoka Study included questions on employment, eating habits, stress, dental health, and forest-air bathing and walking, as well as questions common to the J-MICC Study.16) The administered questionnaire was examined by a study staff in an isolated room, who asked participants to respond to all unanswered questions except those the participant had intentionally refused to answer.
Venous blood was drawn into a 7 ml of vacuum tube including serum separation, and a 7 ml EDTA-Na added vacuum tube on the day of the health checkup. Eight tubes with 300 μl serum, 8 tubes with 300 μl plasma, and 2 tubes with 300 μl buffy coat were separated. All tubes were stored at –80°C at Nagoya University Graduate School of Medicine.
Genotyping procedure
DNA was extracted from the buffy coat fraction of the 7 ml EDTA-Na added vacuum tube. MTHFR C677T polymorphism was genotyped by a polymerase chain reaction with confronting two-pair primers (PCR-CTPP).17) Each 25 μl reaction tube contained 50–80 ng DNA, 0.12 mM dNTP, 12.5 pmol of each primer, 0.5 U Amplitaq Gold (Perkin-Elmer, Foster City, CA) and 2.5 μl of 10× PCR buffer including 15 mM MgCl2. The PCR-CTPP was conducted with initial denaturation at 95°C for 10 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The primers were F1: 5’-AGC CTC TCC TGA CTG TCA TCC-3’, R1: 5’-TGC GTG ATG ATG AAA TCG G-3’, F2: 5’-GAG AAG GTG TCT GCG GGA GT-3’, and R2: 5’-CAT GTC GGT GCA TGC CTT-3’. The amplified DNA fragments were 128 bp for the C allele, 93 bp for the T allele, and 183 bp for the common band.14)
Statistical analysis
Hyperuricemia was defined as SUA level ≥ 7.0 mg/dL, and hypouricemia as SUA level < 3.0 mg/dL. Hardy-Weinberg equilibrium was examined with a chi-square test. Means between two groups were tested with a t-test. The 95% CI of percentage was calculated based on a binomial distribution. Age- and sex-adjusted OR and 95% CI were estimated using an unconditional logistic model. Two-sided p-values less than 0.05 were considered to be statistically significant. All statistical analyses were performed using SAS Enterprise Guide software version 4.1.
RESULTS
Subject characteristics according to sex are summarized in Table 1. The mean and standard deviation (SD) of age was 50.7±8.6 years in males and 49.2±8.7 years in females. Hyperuricemia was found to be 23.5% (n=801) in males and 1.5% (n=24) in females, while hypouricemia was 0.6% (n=21) and 5.2% (n=84), respectively. The SUA mean was significantly higher in males than in females (6.1±1.2 mg/dL vs 4.4±1.0 mg/dL, p < 0.001).
Table 1.
Characteristics of participants according to sex
| Characteristics | Males (n=3,416) | Females (n=1,612) | |||
|---|---|---|---|---|---|
| N | (%) | N | (%) | ||
| Age (years) | |||||
| 35–40 | 265 | (7.8) | 192 | (12.0) | |
| 40–49 | 984 | (28.8) | 496 | (30.8) | |
| 50–59 | 1,367 | (40.0) | 631 | (39.1) | |
| 60–69 | 800 | (23.4) | 293 | (18.2) | |
| BMI (kg/m2) | |||||
| <18.5 | 92 | (2.7) | 162 | (10.0) | |
| 18.5–24.9 | 2,424 | (71.0) | 1,195 | (74.1) | |
| ≧25 | 900 | (26.3) | 255 | (15.8) | |
| SUA (mg/dL) | |||||
| <3.0 | 21 | (0.6) | 84 | (5.2) | |
| 3.0–4.9 | 524 | (15.4) | 1,074 | (66.6) | |
| 5.0–6.9 | 2,070 | (60.6) | 430 | (26.7) | |
| ≧7.0 | 801 | (23.4) | 24 | (1.5) | |
| Creatinine (mg/dL) | |||||
| <0.5 | 0 | (0.0) | 33 | (2.0) | |
| 0.5–0.7 | 640 | (18.7) | 1,476 | (91.6) | |
| 0.8–1.0 | 2,070 | (60.6) | 100 | (6.2) | |
| 1.0– | 706 | (20.7) | 3 | (0.2) | |
| BUN (mg/dL) | |||||
| <8.0 | 11 | (0.3) | 26 | (1.6) | |
| 8.0–19.9 | 3,173 | (92.9) | 1,520 | (94.3) | |
| 20–22.9 | 232 | (6.8) | 66 | (4.1) | |
| Medication for hyperuricemia | |||||
| Yes | 131 | (3.8) | 0 | (0.0) | |
| No | 3,285 | (96.2) | 1,612 | (100.0) | |
BMI, body mass index; SUA, serum uric acid; BUN, blood urea nitrogen
The genotype frequency of MTHFR C677T among 5,026 subjects was 35.9% for CC, 48.1% for CT, and 16.0% for TT, with 0.40 for the T allele. The distribution was in Hardy-Weinberg equilibrium (P=0.90). Among 5,026 examinees, 4,539 subjects with ABCG2 126QQ and SLC22A12 258WW remained after excluding those with ABCG2 126X allele or SLC22A12 258X allele. From them, 114 subjects under medication for hyperuremia were further removed. The genotypes frequencies of the MTHFR accrding to SUA level among the remaining 4,425 subjects (2,972 males and 1,453 females) are shown in Table 2. In both sexes, the genotype frequencies were similar across the different SUA levels; p=0.31 in males and p=0.83 in females from a 4 by 3 chi-square test. The mean SUA was also similar among the three different genotypes. When those under medication for hyperuremia were added, no differences were observed.
Table 2.
Genotype frequencies (%) of MTHFR C677T according to serum uric acid (SUA)
| SUA (mg/dL) | n | Genotype | ||
|---|---|---|---|---|
| CC | CT | TT | ||
| Total | n=1,573 | n=2,147 | n=705 | |
| Males | n=1,061 | n=1,435 | n=476 | |
| <3.0 | 3 | 0.0 | 100.0 | 0.0 |
| 3.0–4.9 | 367 | 36.8 | 50.4 | 12.8 |
| 5.0–6.9 | 1,902 | 35.5 | 47.6 | 16.8 |
| ≥7.0 | 700 | 35.7 | 48.3 | 15.6 |
| Whole | 2,972 | 35.7 | 48.3 | 16.0 |
| Mean ± SD | 6.2±1.1 | 6.2±1.2 | 6.2±1.0 | |
| Females | n=512 | n=712 | n=229 | |
| <3.0 | 51 | 33.3 | 52.9 | 13.7 |
| 3.0–4.9 | 988 | 34.6 | 50.0 | 15.4 |
| 5.0–6.9 | 392 | 37.2 | 45.7 | 17.1 |
| ≥7.0 | 22 | 31.8 | 54.5 | 13.6 |
| Whole | 1,453 | 35.2 | 49.0 | 15.8 |
| Mean ± SD | 4.5±1.0 | 4.5±1.0 | 4.5±1.0 | |
Adjusted OR and 95% CI of hyperuricemia (SUA ≥ 7.0 mg/dL and/or medication for hyperuricemia) for MTHFR C677T are shown in Table 3. The OR adjusted for sex, age, BMI, and creatinine was 1.00 (95% CI, 0.89–1.24) for CT genotype and 0.98 (95% CI, 0.84–1.32) for TT genotype, relative to CC genotype. When the analysis was conducted for males and females separately, the ORs were not statistically significant.
Table 3.
Adjusted odd ratio (OR) and 95% confidence interval (CI) of hyperuricemia (serum uric acid ≥ 7.0 mg/dL and/or medication for hyperuricemia) for MTHFR C677T among those with ABCG2 126QQ and SLC22A12 258WW
| Genotype | OR | 95% CI | ||
|---|---|---|---|---|
| All subjects | (n = 4,539) | |||
| MTHFR C677T | CC | 1 | (Reference) | |
| CT | 1.00 | 0.89–1.24 | ||
| TT | 0.98 | 0.84–1.32 | ||
| Males | (n = 3,086) | |||
| MTHFR C677T | CC | 1 | (Reference) | |
| CT | 1.00 | 0.88–1.25 | ||
| TT | 0.97 | 0.83–1.35 | ||
| Females | (n= 1,453) | |||
| MTHFR C677T | CC | 1 | (Reference) | |
| CT | 1.02 | 0.32–2.07 | ||
| TT | 0.69 | 0.27–4.08 |
Adjusted for (sex), age, BMI and creatinine
DISCUSSION
In the present study with 4,425 subjects aged 35–69 years with SLC22A12 258WW and ABCG2 126QQ, there were no differences in mean SUA among those with different MTHFR C677T genotypes. The distribution of the genotype was also similar across those with different SUA levels. The findings were inconsistent with those from the previous studies for Japanese12,14,18) and for other ethnic groups.13,19)
There may be several reasons for the inconsistent reports on the association between the MTHFR polymorphism and SUA. The other relatively common genetic traits, such as the polymorphisms of ABCG2 and SLC22A12, would conceal the moderate influence of MTHFR C677T. Few studies have examined the effect after removing the effects of such influential genotypes. This study examined the association among those with SLC22A12 258WW and ABCG2 126QQ. Another reason masking the effect of the MTHFR polymorphism may be the medications for hyperuricemia. In this study, the medications for hyperuricemia were taken into account.
The frequency of T allele varies among different ethnic groups; it was reported in a study in Hawaii that the frequency was 0.41 in Japanese-Americans, 0.36 in Caucasians, 0.41 in Latino, 0.13 in African-American, and 0.22 in Native Hawaiians.20) In the present study, the frequency was 0.40, which was close to the Japanese average (0.391 among 10,854 Japanese).21) The subjects seemed to reflect the Japanese general population.
Although the mechanism of the relationship between the MTHFR polymorphism and SUA is still unknown, there are several studies which assume that the MTHFR polymorphism could affect the mechanisms such as the de novo synthesis of purines via 10-formyl tetrahydrofolate with consequent overproduction of UA by the substrate of the MTHFR reaction.12,21,22) It is evident that the TT genotype of MTHFR is more closely related to the rise of plasma homocysteine in patients with a low folic acid level18,23-26) and high plasma homocysteine is lowered by folic acid fortification.27) A direct relation between plasma homocysteine levels and UA levels was also reported.12,19,28-31) These suggest that in a group with low folic acid intake, the effects of MTHFR gene polymorphism on SUA levels are more likely to be marked, and vice versa. In this study, all of the data were collected in one healthcare center located in Shizuoka Prefecture. Shizuoka is well known for its green tea, which contains folic acid. The yearly amount of expenditure on green tea was the largest in Shizuoka, almost three times higher than the average in Japan in 2011.32) This factor could affect the results of this study.
One of the limitations of our study is the remaining unadjusted potential confounding factors associated with SUA, such as alcohol consumption, physical activity, animal protein intake, folate, and serum vitamin B12 intake, although they seemed to be independent of the MTHFR genotype. A systematic review showed that the ethnicity may affect the relationship between the MTHFR mutation and SUA levels.33)
In conclusion, though some limitations remain, the present study indicated no association between SUA and MTHFR C677T genotype among Japanese, after the influences of ABCG2 Q126X and SLC22A12 W258X were removed.
ACKNOWLEDGMENTS
This study was supported in part by a Grant-in-Aid for Scientific Research on Special Priority Areas of Cancer from the Japanese Ministry of Education, Culture, Sports, Science and Technology. We are grateful to Ms Yoko Mitsuda and Ms Keiko Shibata for their technical assistance.
REFERENCES
- 1).Saag KG, Mikuls TR. Recent advances in the epidemiology of gout. Curr Rheumatol Rep, 2005; 7: 235–241. [DOI] [PubMed]
- 2).Weaver AL. Epidemiology of gout. Cleve Clin J Med, 2008; 75 Suppl 5: S9–S12. [DOI] [PubMed]
- 3).Doherty M. New insights into the epidemiology of gout. Rheumatology, 2009; 48: ii2–ii8. [DOI] [PubMed]
- 4).Whitfield JB, Martin NG. Inheritance and alcohol as factors influencing plasma uric acid levels. Acta Genet Med Gemellol (Roma), 1983; 32: 117–126. [DOI] [PubMed]
- 5).Kamatani Y, Matsuda K, Okada Y Kubo M, Hosono N, Daigo Y, Nakamura Y, Kamatani N. Genome-wide association study of hematological and biochemical traits in a Japanese population. Nat Genet, 2010; 42: 210–216. [DOI] [PubMed]
- 6).Kolz M, Johnson T, Sanna S, Teumer A, Vitart V, Perola M, Mangino M, Albecht E, Wallace C, Farrall M, Johansson A, Nyholt D, Aulcenko Y, Beckmann J, Bergmann, Bochud M, Brown M, Campbell H, Connell J, Dominiczak A, Homuth G, Lamina C, McCarthy M, Meitinger T, Mooser V, Munroe P, Nauck M, Peden J, Prokisch H, Salo P, Salomaa V, Samani N, Schlessinger D, Uda M, Volker U, Waeber G, Waterworth D, Wang-Sattler R, Wright A, Adamki J, Whitfield J, Gyllensten U, Wilson J, Rudan I, Pramtaller P, Watkins J, Doering A, Wichmann H, Spector T, Peltonen L, Volzke H, Nagaraja R, Vollenweider, Caaulfield M, Illig T, Gieger C: Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations. PLoS Genet, 2009; 5: e1000504. [DOI] [PMC free article] [PubMed]
- 7).Ichida K, Hosoyamada M, Hisatome I, Enomoto A, Hikita M, Endou H, Hosoya T. Clinical and molecular analysis of patients with renal hypouricemia in Japan- influence of URAT1 gene on urinary urate excretion. J Am Soc Nephrol, 2004; 15: 164–173. [DOI] [PubMed]
- 8).Hamajima N, Naito M, Hishida A, Okada R, Asai Y, Wakai K. Serum uric acid distribution according to SLC22A12 W258X genotype in a cross-sectional study of a general Japanese population. BMC Med Genet, 12: 33, 2011. [DOI] [PMC free article] [PubMed]
- 9).Matsuo H, Chiba T, Nagamori S Nakamura A, Domoto H, Phetdee K, Wiriyasermkul P, Kikuchi Y, Oda T, Nishiyama J, Nakamura T, Morimoto Y, Kamakura K, Sakurai Y, Nonoyama S, Kanai Y, Shinomiya N. Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemina. Am J Hum Genet, 2008; 83: 744–751. [DOI] [PMC free article] [PubMed]
- 10).Matsuo H, Takada T, Ichida K, Nakamura T, Nakayama A, Ikebuchi Y, Ito K, Kusanagi Y, Chiba T, Tadokoro S, Oikawa Y, Inoue H, Suzuki K, Okada R, Nishiyama J, Domoto H, Watanabe S, Fujita M, Morimoto Y, Naito M, Nishio K, Hishida A, Wakai K, Asai Y, Niwa K, Kamakura K, Nonoyama S, Sakurai Y, Hosoya T, Kanai Y, Suzuki H, Hamajima N, Shinomiya N. Common defects of ABCG2, a high-capacity urate exporter, cause gout: a functional-based genetic analysis in a Japanese population. Sci Transl Med, 2009; 1: 5ra11. [DOI] [PubMed]
- 11).Woodward OM, Kottgen A, Coresh J, Boerwinkle E, Guggino WB, Kottgen M. Identification of a urate transporter, ABCG2, with a common functional polymorohism causing gout. Proc Natl Acad Sci U S A, 2009; 106: 10338–10342. [DOI] [PMC free article] [PubMed]
- 12).Lwin H, Yokoyama T, Yoshiike N, Saito K, Yamamoto A, Date C, Tanaka H. Polymorphism of methylenetrahydrofolate reductase gene (C677T MTHFR) is not a confounding factor of the relationship between serum uric acid level and the prevalence of hypertension in Japanese men. Circ J, 2006; 70: 83–87. [DOI] [PubMed]
- 13).Golbahar J, Aminzadeh MA, Al-Shboul QM, Kassab S, Rezaian GR. Association of methylenetetrahydrofolate reductase (C677T) polymorphism with hyperuricemia. Nutr Metab Cardiovasc Dis, 2007; 17: 462–467. [DOI] [PubMed]
- 14).Itou S, Goto Y, Suzuki K, Kawai S, Naito M, Ito Y, Hamajima N. Significant association between methylentetrahydrofolate reductase 677T allele and hyperuricemia among adult Japanese subjects. Nutr Res, 2009; 29: 710–715. [DOI] [PubMed]
- 15).Wei W, Liu S-Y, Zeng F-F, Ma L, Li K-S, Wang B-Y. Meta-analysis of the association of the C677T polymorphism of the methylenetetrahydrofolate reductase gene with hyperuricemia. Ann Nutr Metab, 2012; 60: 44–51. [DOI] [PubMed]
- 16).Asai Y, Naito M, Suzuki M, Tomoda A, Kuwabara M, Fukuda H, Okamoto A, Oishi S, Ikeda K, Nakamura T, Misu Y, Takase S, Tokumasu S, Nishio K, Ishida Y, Hishida A, Morita E, Kawai S, Okada R, Wakai K, Tamakoshi A, Hamajima N. Baseline data of Shizuoka area in the Japan Multi-institutional Collaborative Cohort Study (J-MICC Study). Nagoya J Med Sci, 2009; 71: 137–144. [PMC free article] [PubMed]
- 17).Hamajima N, Saito T, Matsuo K, Kozaki K, Takahashi T, Tajima K. Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Jpn J Cancer Res, 2000; 91: 865–868. [DOI] [PMC free article] [PubMed]
- 18).Zuo M, Nishio H, Lee MJ, Maejima K, Mimura S, Sumino K. The C677T mutation in the methylene tetrahydrofolate reductase gene increases serum uric acid in elderly men. J Hum Genet, 2000; 45: 257–262. [DOI] [PubMed]
- 19).Houng SH, Lee MJ, Kim KH, Lee SH, Lee YH, Kim BG, Jeong B, Yoon HR, Nishio H, Kim JY. The C677T Mutation in methylene tetrahydrofolate reductase gene:correlation wit uric acid and cardiovascular risk factors in elderly Korean men. J Korean Med Sci, 2004; 19: 209–213. [DOI] [PMC free article] [PubMed]
- 20).Loic Le Marchand, Lynne R.Wilkens, Laurence N. Kolonel, Brian E. Henderson. The MTHFR C677T polymorphism and colorectal cancer: the multiethnic cohort study. Cancer Epidemiol Biomarkers Prev 2005; 14: 1198-12–3. [DOI] [PubMed]
- 21).Iida K, Tomita K, Okada R, Kawai S, Morita E, Hishida S, Naito M, Wakai K, Hamajima N. Applicability of allele/genotype frequency from documented controls for case-control studies on genotypes among Japanese: MTHFR C677T as an example. Asian Pac J Cancer Prev, 2009; 10: 231–236. [PubMed]
- 22).Motti C, Gnasso A, Bernardini S, Massoud R, Pastore A, Rampa P, Federici G, Cortese C. Common mutation in methylentetrahydrofolatereductase. Correlation with homocysteine and other risk factors for vascular disease. Artherosclerosis, 1998; 139: 377–383. [DOI] [PubMed]
- 23).Kawamoto R, Koohara K, Tabara Y, Miki T, Doi T, Tokunaga H, Konishi I. An association of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism and common carotid atherosclerosis. J Hum Genet, 2001; 46: 506–510. [DOI] [PubMed]
- 24).Cristensen B, Frosst P, Lussier-Canon S, Selhub J, Goyette P, Rosenblatt DS, Genet J Jr, Rozen R. Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. Arterioscler Thromb Vasc Biol, 1997; 17: 1662–1666. [DOI] [PubMed]
- 25).Girelli D, Friso S, Trabetti E, Olivieri O, Russo C, Peotto R, Faccini G, Pignatti PF, Mazzuucco A, Corrocher R. Methylenetetrahydrofolate reductase C677T mutation, plasma homocysteine, and folate in subjects from northern Italy with or without angiographically documented severe coronary atherosclerotic disease: evidence for an important genetic-environmental interaction. Blood, 1998; 91: 4158–4163. [PubMed]
- 26).Ma J, Stampfer MJ, Hennekens CH, Frosst P, Selhub J, orsford J, Malinow MR, Willett WC, Rozen R. MEthylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation, 1996; 94: 2410–2416. [DOI] [PubMed]
- 27).Schwartz SM, Siscovick DS, Malinow MR, Rosenddaal FR, Beverly RK, Hes DL, PsatyBM, Longstreth WT Jr, Koepsell TD Raghunathan TE, Reitsma PH. Myocardial infarction in young women in relation to plasma total homocysteine, folate and a common variant in the methylenetetrahydroofolate reductase gene. Circulation 1997; 96: 412–417. [DOI] [PubMed]
- 28).Kang SS, Wong PW, Cook HY, Norusis M, Messer JV. Protein-bound homocysteine. A possible risk factor for coronary artery disease, J Clin Invert, 1986; 7: 1482–1486. [DOI] [PMC free article] [PubMed]
- 29).Jacque PF, Selhub J, Bostom AG, Wilson PW, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Eng J Med, 1999; 340: 1449–1454. [DOI] [PubMed]
- 30).Coull BM, Malinow MR, Beamer N, exton G, Nordt F, de Garmo P. Elevated plasma homocysteine concentration as a possible independent risk factor for stroke. Stroke, 1990; 21: 572–576. [DOI] [PubMed]
- 31).Evers S, Koch HG, Grotemeyer KH, Lange B, Deufel T, Ringelstein EB. Features, symptoms, and neurophysiological findings in stroke associated with hyperhomocysteinenia. Arch Neurol, 1997; 54: 1276–1292. [DOI] [PubMed]
- 32).Ministry of Internal Affair and Communications in Japan. Yearly Amount of Expenditures per Household for City Group, District and City with Prefectural Government (Total Households). Family Income and Expenditure Survey, 2011; Table 11.
- 33).Malinow MR, Levenson J, Giraii P, Nieto FJ, Razavian M, Segond P, Simon A. Role of blood pressure, uric acid, an hemorhelogical parameters on plasma homocysteine concentration. Atherosclerosis, 1995; 114: 175–178. [DOI] [PubMed]