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Effect of Folic Acid and Vitamin B12 Supplementation on Homocysteine Level

in association with MTHFR C677T Polymorphisms in Overweight Female

Adults _{1 1 2 2}

  † ₁ Fidelia Fidelia , Antonius Suwanto , Felicia Kartawidjajaputra , Susana Susana ₂ Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta Bioanalytical Laboratory Nutrifood Research Center, PT Nutrifood Indonesia, Jakarta A R T I C L E I N F O A B S T R A C T

  Objective: The aim of this study was to evaluate the effect of folic acid and vitamin B12 supplementation on Hcy Article history: level based on the MTHFR C677T polymorphisms in Indonesian overweight females.

  Received: February 2015 Accepted: June 2015

UNCORRECTED PROOF

  Methods: The study involved 110 female adults (20±1.3 years) with a body mass index (BMI) above 22.9 (mean: Available online:

August 2015 25.3±2.3). DNA was extracted from saliva in order to identify the SNP of MTHFRC677T using PCR-RFLP. 15 of 110

subjects were chosen to represent all MTHFR genotypes (5 CC, 6 CT and 4 TT) and assigned two different treatments: control (placebo), and supplement (500µg folic acid and 1 mg vitamin B12) for 4 weeks. Hcy levels

  Keywords: were measured using direct immunochemical assay.

  Cardiovascular disease Folate

FOR REVIEWER

  Result: This study population (n=110) showed that 677CC allele had the highest frequency (67%), followed by Homocysteine 677CT (29%) and 677TT (4%). The results demonstrated no significant differences of Hcy level among all Methylenetetrahydrofolate subjects before treatment, but Hcy level was slightly higher in T allele subjects. The folate and vitamin B12 reductase C677T treatment significantly reduced the level of Hcy in supplement group (p = 0.038), but not in placebo group. The Vitamin B12 _† difference between placebo and treatment was observed to be the highest in TT, followed by CT and CC subjects.

  Corresponding author: Conclusion: This study showed that subjects who had T allele, tended to have a higher level of Hcy. Bioanalytical Laboratory Supplementation of folate and vitamin B12 was proven to lower the level of Hcy, and TT subjects were proven to Nutrifood Research Center, be the most responsive. PT Nutrifood Indonesia, Jakarta E-mail: felicia@nutrifood.co.id

1. Introduction

  and harmful use of alcohol, which are responsible for about 80% of atherosclerotic heart disease, are Cardiovascular disease (CVD) is known as the able to be modified by lifestyle or pharmaceutical leading cause of death and disability worldwide, intervention. Both of modifiable and non- including Indonesia. In 2008, an estimated of 17.3 modifiable factors can lead to intermediate risk million people died from CVDs and by 2030, it is factors, such as hypertension, hyperlipidemia, predicted to be risen over 23 million people. glucose intolerance and overweight or obesity, Atherosclerotic heart disease, or widely known as which can lead to chronic disease development coronary heart disease (CHD) is one of CVD [4]. group occupied the first place of cause of death in 80-90% of people dying from CHD have one or Indonesia and the number keeps rising over the more major risk factors that are influenced by years [1-3]. lifestyle. Among the numerous factors above,

  There are many factors associated with overweight is one to be highlighted especially increased risk of CVD. Some of risk factors are because the incidence is increasing each year. classified as non-modifiable, such as age, sex and World Health Organization stated that the genetics; whereas behavioral risk factors, such as prevalence of overweight in Indonesian women is higher than men and also expected to increase higher in women over 10 years [5].

  The actual risk factors of CVD (blood pressure, blood lipid profiles, BMI, blood glucose) can be measured, but they do not accurately predict the cardiovascular events. Therefore, several studies over years focused on the identification of novel risk factors. To date, homocysteine (Hcy), a non- protein-forming sulfur amino acid that is synthesized as by-product of methionine metabolism, was promoted to be a novel risk factor for the development of CVD [6, 7]

  An elevated Hcy level in the plasma or serum, a condition called hyperhomocysteinemia (HHcy), has been reported to be a significant and independent risk factor for CVD as it can cause multi-disease manifestations for cardiovascular pathogenesis [8]. The concentration of Hcy is determined by both genetic and non-genetic factors. An increase in Hcy level could be influenced by genetic variation that involved in methionine pathway. The most widely accepted polymorphism is the 677C>T mutation in the MTHFR gene (rs1801133). Evidence suggested that plasma Hcy concentrations were significantly higher in the subject with the 677TT genotype than those with CC or CT genotype [9] and the homozygosity for the MTHFR 677TT polymorphism was associated with an increased risk of atherosclerotic heart disease [10]. Besides, Hcy level could also be influenced by nutrition that is involved in the methionine pathway, including folic acid and vitamin B12. Both supplements were commonly used in trials and studies which were aimed to lower Hcy levels [7, 11]

  To date, most studies have assessed the effects of B- vitamins and the correlation of MTHFR C677T polymorphisms on Hcy levels mainly on middle-aged or elderly individuals, with the relation on certain specific diseases. Some studies also reported that differences in gender, age, nutrition status, and BMI were also involved in the prediction of Hcy concentration [12-14]. Accordingly, we decided to focus on certain subjects with specific gender, age, BMI in order to see the relation the genetic and nutrition factor effect only. In this research, we chose Indonesian overweight female adults that considered not developing CVD disease. Therefore, the purpose of this study was to evaluate the effect of folic acid and vitamin B12 before and after supplementation in Indonesian overweight female adult on Hcy level based on carrier status for the MTHFR C677T polymorphisms.

  2. Materials and Methods

  2.1 Sample Collection

  This study included 110 female participants. All participants were Indonesian people who lived in Jakarta and surroundings, aged 18-30 years old, classified with BMI (Body Mass Index) ≥ 22.9 (kg/m2), and had been identified as non-smokers, non-alcohol drinker, non-pregnancy. Saliva was obtained from all participants as the source of DNA. For intervention research, blood samples were collected from fasted subjects (n=16) for determination of biochemical parameter. Hcy concentrations were measured using ADVIA Centaur® System (Siemens, US).

  2.2 DNA Isolation

  Genomic DNA from saliva was isolated using phenol-chloroform method. Saliva was transferred into micro centrifuge tube, added with Tris EDTA 1x and sodium acetate 0.2M. SDS 10% and Proteinase-K (20mg/ml) were added and the sample was incubated at 56oC for 30 minutes. Samples were added with phenol and chloroform, mixed vigorously and centrifuged at 13800 g for 2 minutes. Supernatant was transferred into new micro centrifuge tube and was added with ethanol absolute, mixed vigorously. The sample was incubated at -40oC for 5 minutes and centrifuged 13800g for 2 minutes. Supernatant was discarded and the pellet was added with ethanol 70%, and centrifuged at 13800 x g for 2 minutes. The pellet was centrifuged again without the supernatant, at 13800 x g for 1 minute. Pellet was added with ddH2O and incubated 56oC for 15 minutes. The DNA concentration was measured using NanoDrop 2000 (ThermoScientific).

UNCORRECTED PROOF FOR REVIEWER

  2.3 MTHFR C677T Mutation Analysis.

  The MTHFR C677T (rs1801133) gene mutation was identified by PCR-RFLP methods. Briefly, about 50 to 80 ng DNA samples were amplified in a final volume of 25 µL containing 1x Go Taq Green mastermix buffer, 0.4 µM of each primer. The following sense and antisense primer to amplify SNP rs1801133 were 5’- GCCCAGCCACTCACTGTTTTA-3’ and 5’- AGGACGGTGCGGTGAGAGTG-3’ [15]. The PCR reactions were carried out as follows initial denaturation at 95ᴼC for 5 minutes, denaturation at 95ᴼC for 30 seconds, annealing at 62ᴼC for 30

  A

  UNCORRE FOR R

  Visualization of PCR-ampli gene for MTHFR and the patterns for C677T MTHFR g shown in Fig. 1. The amplified was digested with HinfI, result bands for the wild genotype (6 and 341 bp bands for heteroz 77, 165, and 176 bp band (677TT). Due to the separatio agarose gel, 165 and 176 bp b single band.

  THFR polymorphisms her Hcy values in those A

  easured before the pare the effect of the statistical analysis, Hcy h 677CC MTHFR was ed to be an outlier. The h SNP were 10.77, 11.67, ely (Fig. 3). The results nt difference regarding

  olymorphisms genotype

  0.67, 0.29, and 0.04 for respectively (Fig. 2).

  study involved 110 mean age: 20±1.3 years; h the distribution of agreement with the ratio (p > 0.05). The

  THFR C677T fragment 677CC, MTHFR 677CT,

  tion of MTHFR C677T rker 100bp DNA Ladder N, no template control; ment gene (400bp). (b)

  plified fragment in the he different genotype R gene mutation were ified product of 418 bp sulting in 77 and 341 bp e (677CC); 77, 165, 176, rozygous (677CT); and ands for homozygous ation limitation of the p band were seen as a

  Hcy levels were meas intervention study to compar mutant allele (T). For the stat level of 1 participant with 6 excluded as it was considered t means of Hcy level of each SN and 11.83 umol/L, respectively demonstrated no significant d Hcy level between 677CT MTH but there were slightly higher who had T allele.

  Figure 2. MTHFR 677 poly frequency (n=110).

  The pre-intervention st overweight female adults (mea mean BMI: 25.3±2.3) with t MTHFR genotypes is in ag expected Hardy-Weinberg ra genotype frequencies were 0.6 C/C, C/T, and T/T carriers, res

  Figure 1. (a) PCR visualizatio fragment gene. Lane M, marke (New England Biolabs); lane N, lane 1-3, MTHFR C677T fragme Digestion visualization for MTH gene. Lane 1-3: MTHFR 677C MTHFR 677TT.

  3. Result

  seconds, extension at 72ᴼC for 45 secon extension at 72ᴼC for 5 minutes, total nu cycles: 35. The DNA fragments were visua a 2.5% agarose gel followed by staini ethidium bromide. Analysis was perfo least twice to confirm the genotype.

  Hcy level in igned at P- pproved by nity Service med written participants

  (500µg folic orally and fter 12 hours on-additive e serum is 1 hour after as measured munoassay, logy using s, US). Both nation were n studies in oratorium. using SPSS requency of tically with are test (χ²) tion of the tests for ata. The 2- and Mann son between of folic acid

  conds, final l number of isualized on aining with erformed at epresent all ubjects with bjects with treatments:

  RECTED PRO R REVIEWER

  This study was reviewed and appr Institute of Research and Community Atma Jaya Catholic University. Informed consent was obtained from all par following the contents of the study.

  2.7 Ethics statement

  Statistical analysis was performed usi (version 21.0, IBM, USA). Genotype freq MTHFR C677T was analyzed statistica Hardy-Weinberg predictions. Chi-square was used to examine the distribution different genotypes. Shapiro Wilk t normality were performed for all data. tailed nonparametric Kruskall Wallis an Whitney U-test was used for comparison groups in order to evaluate the effect of f and vitamin B12 supplementation on Hcy each genotype. Significance was assigne value of <0.05.

  2.6 Statistical Analysis

  All blood samples were collected after of fasting by venipuncture into a non containing tube. Blood clots and the s separated by centrifugation within 1 ho blood collection. Total serum Hcy was m with the principle of competitive immu direct, chemiluminescent technology ADVIA Centaur® HCY assay (Siemens, U of blood collection and Hcy determinati performed before and after intervention s coorporation with Prodia Clinical Laborat

  2.5 Homocysteine Determination

  16 of 110 subjects were chosen to repr the possible MTHFR genotypes (6 subje 677CC, 6 subjects with 677CT, 4 subje 677TT) and assigned two different tre control (placebos) and supplements (500 acid and 1 mg vitamin B12) ora consecutively for 4 weeks.

  2.4 Intervention Study

PROOF ER

  UNCORRE FOR R Figure 3. Hcy levels before intervention study > 0.05).

  B

  T) and flow-mediated e girls, compared with tes that the correlation and FMD is apparently with a history of CVD, ild status) were widely ndent risk factor [9 und that heterozygotes concentrations of Hcy -type genotype (CC). this result might be y in MTHFR gene and

  T genotype had lowest plied that the presence mon than the T allele. study, Suryandari [17] distribution of this population. As result, ion study.

  [16]. sidered to be in Hardy- which follow the uency and genotypic- d random mating. As a genotype had highest

  Therefore, PCR-RFLP is d to any other available ization, allele-specific ligonucleotide ligation

  can be applied for 677T polymorphisms. ould differentiate each ing at the restriction g single nucleotide ased on endonuclease d is a relatively simple,

  As shown in Fig.3, we found (TT) had slightly higher con levels than people with wild Although not significant, th occurred due to deficiency in

  ). Although d on the BMI the higher intima-media-thickness (IMT) dilatation (FMD) on obese g the lean girls, which indicates of Hcy elevation with IMT and mediated by BMI. In adults wi elevated Hcy levels (even mild considered to be an independe

  only find 4 allele. The d with a subjects for to abnormal ds on the e mean Hcy male adults

  d after intervention; B. Δ Hcy levels during intervention

  The population was conside Weinberg proportions, w assumptions of allelic-frequen frequency equilibrium and ra whole population, the CC ge proportion (67%) while TT g proportion (4%) which implie of C allele was more common Compared to the previous stu also reported the same d genotype in Indonesian pop

  Figure 4 shows the result of reduction level before and after 4-weeks interventi in each treatment. Admission of 500µg f and 1 mg vitamin B12 did reduce the leve in subject who received the supp compared to subjects who received the (P-value : 0.038).

  PCR- RFLP method can determining MTHFR C677 Proven in this study, we coul genotype clearly by looking patterns. For genotyping polymorphisms (SNPs) based cleavage, this classic method is inexpensive, and reliable. The a good method compared to methods such as hybridiza PCR, primer extension, oligo and endonuclease cleavage [16

   Discussion

  µg folic acid groups (P- ce between d to be the enotype TT te genotype e CC (0.02 4.

  tion of Hcy ntion study µg folic acid level of Hcy upplements the placebo were found

  RECTED PRO R REVIEWER tudy (P-value

  The Hcy classification depends references by Ingrosso et al. [18]. The m level of all genotypes in overweight fema was in the mild status (10-16 µmol/L). A we were not able to compare it based on status, but previous study also shown th

  over 110 subjects collected, we could onl required subjects with homozygous T al intervention study then conducted disproportionate number as we use 6 sub CC and CT genotype which can lead to a distribution.

  Figure 4 A. Homocysteine level before and af *P-value: 0.038.

  A

  However, no statistical differences we for Hcy levels after admission of 500 µg f and 1 mg vitamin B12 in all genotype gr value <0.05) (Fig 5). The difference placebo and treatment was observed to highest in subjects with homozygote geno (3.02 µmol/L), followed by heterozygote g CT (2.27 µmol/L) and wild genotype C µmol/L), respectively.

PROOF ER

  UNCORRE FOR R Figure 5. Δ Hcy levels during intervention stu

  as mentioned previously. A meta study reported that people with the TT g have approximately 20% higher Hcy le could be vary depending on age, sex, B other environmental factor [20, 21]

  In the present study, we found t supplementation of folate and vitamin about 4 weeks give significant reduction level, while the placebo giving non-si effect. This result indicated that Hcy l dependent on the folate and vitamin B and admission of each individual. Limit this study was because there was no meas in both folate and vitamin B12 status subject, but some study also suggested th and vitamin B12 deficiencies could trig elevation [22]. Still, a further investigatio the correlation of MTHFR gene, B supplementation, and Hcy levels as bi with the relationship to CVD develop needed.

  Hcy, such as other HFR gene which leads ystinuria (e.g. A1298C, polymorphisms other Hcy metabolism (e.g.

  RECTED PRO R REVIEWER study based on MTHFR C677T polymorphisms.

  eta-analysis T genotype y levels but x, BMI, and d that the in B12 for tion of Hcy significant y level was n B12 status imitation in easurement us on each d that folate trigger Hcy ation about

  ents were higher than or the 677CC subjects. cance result could be presence of many kinds

PROOF ER

  ] that found supplement 677T allele, , Anna et al. T genotype hen treated e Hcy level after those vitamin treatment the initial values, except for t However, the non-significan possibly explained by the pres of factors that control Hc polymorphisms in the MTHF to mild HHcy and homocysti C599T, G482A, A983G), po genes that contributes in Hc A2756G in MTR, A66G in MT at exon 8 CBS gene); renal sta vitamin B12 status.

  T allele and wever, there lementation t in subject CT and CC ed by the

  5. Conclusion

  In summary, Hcy levels are critical functions, the genetic not be changed, and nutritio with overweight BMI statu status of Hcy levels which ten subjects who had T allele. Adm vitamin B12 during 4-weeks w the level of Hcy level with su 677TT MTHFR genotype responsive ones. Moreover, several limitations, this resear the pre-liminary study focusin

  , B-vitamin s biomarker elopment is s result of xpression of ase in the all e lack of

  When we stratified the 4-weeks r intervention study according to the expre T allele, we found no significant increase genotype. This result implied the association between the presence of T a the high Hcy levels in subjects. Howev was a trend that the effect of supplem given was observed to be the highest in with heterozygotes (TT), followed by CT subjects. This trend was supported previous study done by Liu et al. [20] th the Hcy-lowering effect of the folate sup was significant in subjects with the 677 but not in 677C carriers. Interestingly, An [23] also reported the decrease of TT g was greater but more significant when with supplement, yet, surprisingly the H

  MTRR, 680bp insertion l status, and folate and in this study were the length of intervention, ochemical parameter cused more on the

  , which targeting on derate characteristics e with no severe CVD he possible explanation und in this study. an be used as the er research. are influenced by two etic factor which may itional factor. Subjects atus have mild-range tended to be higher on

  Admission of folate and s was proven to lower subject possessed the e as the the most r, although there are earch could be used as using of Hcy elevation

  Several limitations exist in number of population, the len and the lack of bioch measurement. As we focus prevention stage of CVD, w population with modera (overweight BMI, adult age w incidences), this could be the p why no significance foun Moreover, this result can preliminary study for further r

  

UNCORRECTED PROOF

FOR REVIEWER

  15. Frederiksen J, Juul K, Grande P, Jensen GB, Schroeder TV, Tybjaerg.Hansen A, Nordestgaard BG. Methylenetetrahydrofolate reductase polymorphism (C677T), hyperhomocysteinemia, and risk of ischemic cardiovascular disease and venous thromboembolism: prospective and case. control studies from the Copenhagen City Heart Study. BLOOD. 2004;104(10):3046.51.

  Homocysteine metabolism, hyperhomocysteinaemia and vascular disease: An overview. J Inherit Metab Dis.

  2006;29:3.20.

  12. Kuzelicki NK, Milek M, Mlinaric.Rascan I. MTHFR and TYMS genotypes influence TPMT activity and its differential modulation in males and females. Clin Biochem. 2010;43: 37.

  42.

  13. Liu CS, Chen CH, Chiang HC, Kuo CL, Huang CS, Cheng WL, Wei YH, Chen HW. B.group vitamin, MTHFR C677T polymorphism and carotid intima.media thickness in clinically healthy subjects. Eur J Clin Nutr. 2007;61:996.03.

  14. Rekha S, Patel ML, Pooja G, Pushpalata S, Natu SM, Pradeep Y. Correlation of plasma homocysteine levels with BMI and insulin resistance, among obese, overweight and non.obese infertile women. Int J Sci Res Pub. 2012;2(5):1.6.

  16. Zhang R, Zhu Z, Zhu H, Nguyen T, Yao F, Xia K, Liang D, Liu C. SNP cutter: a comprehensive tool for SNP PCR.RFLP assay design. Nucl Acid Res. 2005;33:W489.92.

  Elevated plasma homocysteine is positively associated with age independent of C677T mutation of the methylenetetrahydrofolate reductase gene in selected Egyptian subjects. Int J Med Sci. 2004;1(3):181.92.

  17. Suryandari DA, Yurnadi, Wiweko B, Yunaini L. Genotype distribution of methylenetetrahydrofolate reductase A1298C and C677T gene in Indonesian infertile men. Med J Indones. 2012;21(1):23.27.

  18. Ingrosso D, Cimmino A, Perna AF, Masella L, Santo NGD, Bonis MLD, Vacca M, D’Esposito M, D’Urso M, Galleti P, Zappia V. Folate treatment and unbalanced methylation and changes of allelic expression induced by hyperhomocysteinaemia in patients with uraemia. Lancet. 2003;361: 1693.99.

  19. Zhu W, Huang X, Li M, Neubauer H. Elevated plasma homocysteine in obese schoolchildren with early atherosclerosis. Eur J Pediatr. 2006; 165: 326.31.

  20. Liu CS, Chen CH, Chiang HC, Kuo CL, Huang CS, Cheng WL, Wei YH, Chen HW. B.group vitamin, MTHFR C677T polymorphism and carotid intima.media thickness in clinically healthy subjects. Eur J Clin Nutr. 2007;61:996.03.

  21. Abaci A, Akelma AZ, Ozdemir O, Hizli S, Razi CH, Akin KO. Relation of total homocysteine level with metabolic and anthropometric variables in obese children and adolescents. Turk J Med Sci. 2012;42(1):9.76.

  22. Malinowska A, Chmurzynska A. Polymorphism of genes encoding homocysteine metabolism.related enzymes and risk for cardiovascular disease. Nutr J. 2009; 29:685.95.

  11. Castro R, Rivera I, Blom HJ, Jakobs C, Almekia IT.

  10. El.Sammak M, Kandil M, El.Hifni S, Hosni R, Ragab M.

  on overweight female adult in Indonesian population towards CVD prevention.

  3. Hermansyah, Citrakesumasari, Aminuddin. Physical activity and mental health toward coronary heart disease on outpatients in RSUP dr. Wahidin Sudirohusodo and Labuang Baji hospital Makassar. Media Gizi Masyarakat Indones. 2012;1(2):79.83.

  Acknoledgement

  This work was supported by PT Nutrifood Indonesia

  Conflict of Interest

  The authors report no conflicts of interest

  References 1. WHO resources page. World Health Organization Web site. http://www.who.int/cardiovascular_diseases/publications/atlas _cvd/en/. Accesed January 11, 2013.

  2. Dantas AP et al. Vascular aging: facts and factors. Frontiers in Vascular Pathology. 2012;3(325):1.2.

  4. Ordovas JM. Genetic interactions with diet influence the risk of cardiovascular disease. Am J Clin Nutr. 2006;83(2):443S.

  9. Brown NM et al. Detection of 677CT/1298AC “double variant” chromosomes: implications for interpretation of MTHFR genotyping results. Genec Med. 2005;7(4):1.5.

  46S.

  5. WHO resources page. World Health Organization Web site. http://www.who.int/chp/chronic_disease_report/media/impact/ indonesia.pdf. Accessed January 11, 2013.

  6. Jakubowski H. The pathophysiological hypothesis of homocysteine thiolactone.mediated vascular disease. J Physiol Pharmacol. 2008;59:155.67.

  7. Shishehbor MH, Oliveira LP, Lauer MS, et al. Emerging cardiovascular risk factors that account for a significant portion of attributable mortality risk in chronic kidney disease. Am J Cardiol. 2008;101:1741.46.

  8. Huang T, Yuan G, Zhang Z, Zou Z, Li D. Cardiovascular pathogenesis in hyperhomocysteinemia. Asia Pac J Clin Nutr.

  2008;17(1):8.16.

  23. Anna P, Sandro DA, Stefania C, Rosalba R, et al. Effects of folic acid before and after vitamin B12 on plasma homocysteine concentration in hemodialysis patients with known MTHFR genotypes. Clin Chem. 2006; 52(1):145. 48.