Journal of the American College of Nutrition

ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: http://www.tandfonline.com/loi/uacn20

Influence of the C677T Polymorphism of the

MTHFR Gene on Oxidative Stress in Women With
Overweight or Obesity: Response to a Dietary
Folate Intervention

Marina Ramalho Ribeiro, Raquel Patrícia Ataíde Lima, Jéssica Vanessa de

Carvalho Lisboa, Thamires Ribeiro Chaves, Rafaella Cristhine Pordeus Luna,
Rayner Anderson Ferreira do Nascimento, Yohanna de Oliveira, Darlene
Camati Persuhn, Alexandre Sérgio da Silva, Maria da Conceição Rodrigues
Gonçalves, Flávia Emília Leite de Lima Ferreira, Roberto Teixeira Lima,
Alcides da Silva Diniz, Alessio Tony Cavalcanti de Almeida, Ronei Marcos de
Moraes, Eliseu Verly Junior & Maria José de Carvalho Costa

To cite this article: Marina Ramalho Ribeiro, Raquel Patrícia Ataíde Lima, Jéssica Vanessa de
Carvalho Lisboa, Thamires Ribeiro Chaves, Rafaella Cristhine Pordeus Luna, Rayner Anderson
Ferreira do Nascimento, Yohanna de Oliveira, Darlene Camati Persuhn, Alexandre Sérgio da
Silva, Maria da Conceição Rodrigues Gonçalves, Flávia Emília Leite de Lima Ferreira, Roberto
Teixeira Lima, Alcides da Silva Diniz, Alessio Tony Cavalcanti de Almeida, Ronei Marcos de
Moraes, Eliseu Verly Junior & Maria José de Carvalho Costa (2018): Influence of the C677T
Polymorphism of the MTHFR Gene on Oxidative Stress in Women With Overweight or Obesity:
Response to a Dietary Folate Intervention, Journal of the American College of Nutrition, DOI:
10.1080/07315724.2018.1460224

To link to this article: https://doi.org/10.1080/07315724.2018.1460224

Published online: 27 Apr 2018. Submit your article to this journal

View related articles View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=uacn20
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
https://doi.org/10.1080/07315724.2018.1460224

Influence of the C677T Polymorphism of the MTHFR Gene on Oxidative Stress

in Women With Overweight or Obesity: Response to a Dietary Folate Intervention
Marina Ramalho Ribeiro, MDa, Raquel Patr
ıcia Ata
ıde Lima, PhDa, J
essica Vanessa de Carvalho Lisboa, MDa,
Thamires Ribeiro Chaves, MDa, Rafaella Cristhine Pordeus Luna , PhDa, Rayner Anderson Ferreira do Nascimento, MDb
, Yohanna de Oliveira, MDa, Darlene Camati Persuhn , PhDc, Alexandre S
ergio da Silva , PhDc,
Maria da Conceiç ~ao Rodrigues Gonçalves , PhDc, Fl
avia Em
ılia Leite de Lima Ferreira, PhDc,
Roberto Teixeira Lima, PhDc, Alcides da Silva Diniz, PhDc, Alessio Tony Cavalcanti de Almeida, PhDd,
Ronei Marcos de Moraes, PhDe, Eliseu Verly Junior, PhDf, and Maria Jos
e de Carvalho Costa , PhDc
a
Graduate Program in Nutrition Sciences, (Centro de Ci^encias da Sa
ude–CCS), Federal University of Paraiba (Universidade Federal da Para
ıba–UFPB),
Jo~ao Pessoa, Brazil; bGraduate Program in Molecular and Human Biology, Center of Exact and Natural Sciences (Centro de Ci^encias Exatas e da
Natureza–CCEN), Federal University of Paraiba (Universidade Federal da Para
ıba–UFPB), Jo~ao Pessoa, Brazil; cDepartment of Nutrition, Federal
University of Paraiba (Universidade Federal da Para
ıba–UFPB), Jo~ao Pessoa, Brazil; dDepartment of Economics, Center of Applied Social Sciences
(Centro de Ci^encias Sociais Aplicadas–CCSA), Federal University of Paraiba (Universidade Federal da Para
ıba–UFPB), Jo~ao Pessoa, Brazil; eGraduate
Program in Health Decision Models, Center of Exact and Natural Sciences (Centro de Ci^encias Exatas e da Natureza–CCEN), Federal University of Paraiba
(Universidade Federal da Para
ıba–UFPB), Jo~ao Pessoa, Brazil; fDepartment of Epidemiology, Institute of Social Medicine, State University of Rio de
Janeiro (Universidade Estadual do Rio de Janeiro–UERJ), Rio de Janeiro, Brazil

ABSTRACT ARTICLE HISTORY

The C677T polymorphism of the methylenetetrahydrofolate reductase gene (MTHFR) is related to folate Received 14 July 2017
metabolism and can alter the levels of biochemical markers. Accepted 27 March 2018
Objective: Investigate the influence of the MTHFR C677T polymorphism on the effects of a dietary folate KEYWORDS
intervention on oxidative stress in women with overweight or obesity. Dietary intake; folate; MTHFR
Methods: Forty-eight adult women with overweight or obesity were subjected to a 24-hour dietary recall, C677T polymorphism;
anthropometric measurements, biochemical analysis, and genotyping of the MTHFR C677T polymorphism. obesity; oxidative stress
They were allocated by convenience sampling to 2 groups, which received 300 g of folate-rich vegetables
containing 191 mg/d (Group 1) (n D 24) or 95 mg/d (Group 2) (n D 24) of folate for 8 weeks.
Results: The dietary intervention increased the serum folic acid levels in the 2 analyzed groups. The
intervention with 191 mg/d of folate led to relevant results in terms of homocysteine levels (p D 0.0005)
and total antioxidant capacity (p D 0.0261); the effect was larger among carriers of the TT genotype.
Conclusions: The study demonstrated the beneficial effect of folate intake in terms of a TAC elevation for
the CC and TT genotypes of the MTHFR C677T polymorphism, an increase in folic acid levels for all
genotypes, and a reduction in the Hcy levels for the TT genotype in response to an intervention consisting
of an intake of 191 mg/d of folate supplied by vegetables.

Introduction
participates in DNA repair and maintenance (8), thus having pos-
Oxidative stress arises from an imbalance between the antioxi- itive effects on oxidative stress through its protective action
dative defense system and the production of reactive oxidative against cell damage (9), certainly due to the increase the stability
species (ROS) (1), resulting in an elevated risk for chronic and of the MTHFR enzyme, thus reducing the difference in enzyme
acute inflammation and several diseases (2,3). Individuals with activity among the polymorphism genotypes (2).
overweight or obesity tend to exhibit increased oxidative stress The methylenetetrahydrofolate reductase (MTHFR) enzyme
markers because excessive fat deposition is associated with an participates in the metabolism of homocysteine (Hcy) and
increase in inflammatory biomarkers and cell damage (4,5). folate, catalyzing the reduction of 5,10-methylenetetrahydrofo-
The consumption of an antioxidant-rich diet is important for late to 5-methylenetetrahydrofolate, which converts Hcy to
controlling and repairing the damage caused by oxidative stress methionine, thus providing a methyl group for various biologi-
because, in addition to exerting beneficial effects on the diseases cal reactions, including DNA methylation (10).
associated with this process, this consumption prevents the occur- The MTHFR C677T polymorphism is the most common
rence of endothelial oxidative injury and contributes to gene regu- mutation in the Hcy metabolism pathway. This mutation
lation (6). Folate is considered an important nutrient in the occurs when the nitrogenous base cytosine (C) is replaced by
regulation of the inflammatory response (7), and it also thymine (T) at nucleotide 677 of the MTHFR gene, where by

CONTACT Rafaella Cristhine Pordeus Luna, PhD rafaellacpluna@gmail.com P
os-graduaç~ao em Ci^encias da Nutriç~ao, Centro de Ci^encias da Sa
ude, NIESN–
ucleo Interdisciplinar de Estudos em Sa
ude e Nutriç~ao/Universidade Federal da Para
ıba, Castelo Branco, Jo~ao Pessoa, PB 58059–900, Brazil.
N

© 2018 American College of Nutrition

2 M. R. RIBEIRO ET AL.

alanine is converted to valine by the MTHFR enzyme (11). This regarding the study aims, in compliance with ethical guidelines;
polymorphism results in the expression of a thermolabile the women agreed to participate by signing an informed con-
enzyme (12); thus, this enzyme has less specific activity in het- sent form. This study was approved by the research ethics com-
erozygous (CT) and abnormal homozygous (TT) carriers (13). mittee of the Center of Health Sciences (Centro de Ci^encias da
Several studies have demonstrated a relationship between Sa
ude–CCS), Federal University of Paraiba (Universidade Fed-
the MTHFR C677T polymorphism and abnormal levels of oxi- eral da Para
ıba–UFPB), ruling number 0569/15. The study was
dative stress markers in individuals with b-thalassemia or pso- also registered at ClinicalTrials.gov with the following ID:
riasis, women with pre-eclampsia, and adolescents at high risk NCT02953522.
of cardiovascular disease due to overweight and dyslipidemia
(2,12,14,15). The presence of gene polymorphisms can lead to
Experimental protocol
variations in the response to dietary interventions. Nutrige-
netics research has demonstrated the relevance of studies of After the eligibility criteria were assessed, the women were
dietary interventions to assess this variability in response and instructed to maintain their body weight, dietary habits,
its relationship with genetic factors (16). and physical activity level at the same values that were
Previous studies have demonstrated the effect of certain die- recorded at baseline (23). In addition, they received an indi-
tary interventions in carriers of the MTHFR C677T polymor- vidualized dietary plan 1 week before the onset of the die-
phism (17,18,19,20,21); however, none of these studies tary intervention (plateau week). All medical treatments of
investigated the effect of folate on oxidative stress in this popu- the participants remained unchanged throughout the study
lation. Thus, in view of the existing relationships between folate period.
and oxidative stress, it is expected that women with overweight The calculation for recommended energy intake was based
or obesity with MTHFR C677T polymorphism may obtain an on the formulas included in the Dietary Reference Intake (DRI)
improvement in the markers of oxidative stress in relation to (24) for weight maintenance. Macronutrient recommendations
the consumption of a dietary intervention with folate. were based on those of the American Heart Association (AHA)
Therefore, the aim of the present study was to investigate the (25). The nutrients in the recommended diet were calculated
influence of the MTHFR C677T polymorphism on the effect of and analyzed using the system of equivalences modified and
a folate-containing diet on oxidative stress in women with over- validated for use in Brazil by Costa (26), and the diet comprised
weight or obesity, to provide a basis for individualized dietary the following: carbohydrates, 45–65% (recommended: 55%);
recommendations based on individual nutritional and genetic protein, 10–35% (recommended: 15%); and total fat, 25–35%
profile. (recommended: 30%).
In study week 2, the participants were randomly allocated to
2 groups, using STATA Software version 14.0 for Windows:
Materials and methods Group 1 (G1) received 300 g of vegetables and legumes con-
taining 191 mg/d of folate, and Group 2 (G2) received 300 g of
Study design
vegetables and legumes containing 95 mg/d of folate. These
The present interventional, double-blind study is related to a folate levels were achieved through the following foods: lentils,
population-based study titled “Second Cycle of Diagnosis and soybeans, corn, peas, carrots, zucchini, lettuce, chard, beets,
Intervention on the Diet, Nutrition and Most Prevalent Non- broccoli, cauliflower, tomatoes, and cucumbers.
communicable Diseases in the Population of Jo~ao Pessoa, Para- Each group comprised 24 participants, with 8 individuals
iba” (II Ciclo de Diagn
ostico e Intervenç~ao da Situaç~ao per possible genotype (CC, CT, or TT). In addition to receiving
Alimentar, Nutricional e das Doenças n~ao Transmiss
ıveis mais folate-containing vegetables and legumes for 8 weeks, the par-
Prevalentes da Populaç~ao do Munic
ıpio de Jo~ao Pessoa/PB—II ticipants were instructed to consume folate-fortified and natu-
DISANDNT/JP), conducted from May 2015 to May 2016. The rally folate-rich foods to achieve a usual intake of 400 mg of
participants were selected from a sample of adults with over- folate, corresponding to its estimated average requirement
weight or obesity identified based on the presence of MTHFR (EAR) (27).
C677T polymorphism. Some information on II DISANDNT/JP
was recently published (22).
Nutritional assessment
The inclusion criteria were female gender, an age of 20 to
59 years, overweight or obesity, variable socioeconomic status, Body weight and height were measured in triplicate; the mean
and preserved cognitive status; participants were not restricted of the 3 measurements was used for analysis. Body mass index
regarding the use of medications other than those listed in the (BMI) was calculated by dividing the body weight (kg) by the
exclusion criteria. The exclusion criteria were alcoholism; square of the height (meters); the cutoff points recommended
smoking; neuropsychiatric disorders; use of a medication by the World Health Organization (WHO) were used (28).
known to interfere with the metabolism of folic acid (in the Waist circumference (WC) was used to define abdominal obe-
past 3 months), multivitamin or mineral supplements, anorexi- sity, with a cutoff point of 88 cm according to the AHA rec-
genic agents, or anabolic steroids; chronic disease affecting the ommendations (29).
endocrine or metabolic systems; and pregnancy or plans to The participants’ usual dietary intake was assessed based
become pregnant during the study period. on two 24-hour dietary recalls (R24hs), one weekend and
Following screening, 48 female adults were selected by con- one weekday, performed with a 15-day interval before the
venience sampling and given appropriate information onset of the intervention. The aim of this procedure was to
 JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3

establish the participants’ dietary habits as a basis for the Amplification was performed under the following condi-
menu definition and intake comparison at the end of the tions: initial denaturation at 94 C for 10 minutes; 35 cycles of
study. A third R24h was performed after the plateau week denaturation (94 C for 30 seconds), annealing (61 C for 30 sec-
to investigate adherence to the nutritional instructions that onds), and extension (72 C for 30 seconds); and an additional
were given. final extension for 10 minutes. The 198 base pair (bp) product
A fourth R24h was performed at the end of the 8-week inter- was digested with HinfI, which recognizes and cleaves the poly-
vention to analyze the participants’ usual caloric and nutrient morphic T allele, giving rise to 2 fragments (175 bp and 23 bp);
intake to establish whether they had adhered to the recommen- in contrast, the C allele remains uncleaved at 198 bp. The geno-
dations and whether changes in their global and folate dietary types were analyzed using electrophoresis with 12% polyacryl-
intake had occurred after the intervention. amide gels and staining with 0.5% silver nitrate.
For the R24h, an album was provided with illustrations of
foods in portion sizes used at home; the illustrations were based
on the weight of the average intake of the corresponding foods Statistical analysis
validated for this population, thus minimizing the possible
The sample characteristics were evaluated using descriptive sta-
biases of this method (30,31).
tistics. The data were tested for normality using the Shapiro–
The foods were analyzed using the nutrition software Die-
Wilk test. The effects of the intervention were compared among
twin (Fator Digital ©2017). The Multiple Source Method
the 3 possible genotypes using analysis of variance (ANOVA).
(MSM) was used to calculate the participants’ usual intake. The
Regarding their values before and after the dietary intervention,
MSM is a statistical technique proposed by the European Pro-
variables with a normal distribution were analyzed using Stu-
spective Investigation Into Cancer and Nutrition (EPIC) (32);
dent’s t-test. The correlation data between the change in the
the method performs two regression models, one for positive
parameter levels studied according to the genetic profile were
data of daily intake and another for consumption. It is applica-
analyzed from the Pearson correlation. All statistical analyses
ble to nutrients and food intake, including food eaten episodi-
were performed using STATA 13. For all analyses, a p value <
cally. The MSM is suitable to estimate the habitual
0.05 was considered significant.
consumption of the individual from repeated measurements in
a given period of time (32).
Results
Biochemical analysis
The general characteristics of the sample are described in
Biochemical analyses were performed at 2 time points; the first Table 1. A comparison of the usual folate intake before and
was during the period of data collection of II DISANDNT/JP, after the intervention among the 3 possible genotypes of the
and the second was after the end of the nutritional intervention. MTHFR C677T polymorphism showed that most participants
The following measurements were performed at both times: had increased the intake of this nutrient at the end of the inter-
levels of total cholesterol (TC) and its fractions (low-density vention, except CT carriers in G2. Dietary intervention did not
lipoprotein [LDL-C] and high-density lipoprotein [HDL-C]), result in increased intake of other nutrients with anti-inflam-
triglycerides, malondialdehyde (MDA), total antioxidant capac- matory and antioxidant function (Table 2). Regarding the mac-
ity (TAC), Hcy, folic acid, and vitamin B12. Blood samples ronutrients, the recommended consumption of carbohydrate
were collected at the participants’ homes after a 12-hour fast, was 55%, protein, 15%; and lipids, 30%.
using sterile vacuum tubes with and without anticoagulant. The According to analysis of variance (ANOVA) and the post
blood collection complied with the norms for the use of perfo- hoc Bonferroni test, the anthropometric and biochemical varia-
rating/cutting instruments. bles before and after intervention did not indicate any differ-
The lipid profile was analyzed using the turbidimetry ence in most cases, with the exception of LDL-C for CC and
method with the Labmax 240 Premium (Labtest) automatic CT (p D 0.047) and folate intake for CC and TT (p D 0.015)
biochemical analyzer. Antioxidant activity was assessed based and CT and TT (p D 0.032) before the intervention, and folic
on MDA and TAC according to a previously used protocol acid for CT and TT (p D 0.030) after the intervention in G1
(33,34) Hcy levels were measured using high-performance liq- (Table 3), and folic acid after the intervention between CC and
uid chromatography (HPLC) (35). Serum folic acid and vita- TT in G2 (p D 0.019) (Table 4).
min B12 levels were measured using the chemiluminescence An analysis with independent t-test of each genotype
and electrochemiluminescence immunoassay methods, respec- showed a significant elevation of TAC in genotypes CC (G1
tively (22). and G2) and TT (G1 but not G2), as well as a reduction in the
Hcy levels in the genotype CT and TT carriers in G1 and TT in
G2. In both groups, folate intake increased the serum folic acid
Genotyping
concentration, demonstrating the impact of the intervention
DNA samples were extracted from peripheral blood leukocytes (Table 3 and Table 4).
using a previously described method (36). The MTHFR C677T According to Pearson’s correlation test, we observed correla-
polymorphism was analyzed using polymerase chain reaction tions between CT genotype and homocysteine values in G1
(PCR) with the following primers: 50 -TGA AGG AGA AGG (r D 0.8754, p D 0.0044) and G2 (r D 0.9729, p D 0.0000); cor-
TGT CTG CGG GA-30 (sense) and 50 -AGG ACG GTG CGG relation between the TT genotype and homocysteine values
TGA GAG TG-30 (antisense) (37). (r D 0.9456, p D 0.0004) in G2; and correlation between the
4 M. R. RIBEIRO ET AL.

Table 1. General characteristics of adult subjects from both groups before the intervention.1

G1 (24) G2 (24) p Value

Age (years) 44.88 § 12.47 44.33 § 9.26 0.88

Body weight (kg) 74.65 § 8.65 77.90 § 15.24 0.81
Height (m) 1.59 § 0.06 1.58 § 0.08 0.56
BMI (kg/m2) 29.88 § 3.01 30.47 § 4.82 0.62
Overweight 27.92 § 1.04 28.16 § 1.21 0.12
Obesity 33.12 § 2.29 34.31 § 6.12 0.39
WC (cm) 91.63 § 7.96 91.78 § 12.02 0.96
Total cholesterol (mg/dL) 200.17 § 36.44 202.21 § 38.55 0.84
LDL-C (mg/dL) 123.12 § 35.97 120.98 § 41.71 0.81
HDL-C (mg/dL) 41.46 § 9.20 41.25 § 8.81 0.93
TG (mg/dL) 148.79 § 69.82 164.75 § 86.40 0.47
TAC (%) 39.63 § 14.49 41.95 § 12.99 0.58
MDA (mmol/L) 3.23 § 0.78 3.19 § 1.18 0.91
Homocysteine (mmol/L) 9.64 § 2.44 11.65 § 5.99 0.14
Folic acid (ng/mL) 14.32 § 4.46 13.84 § 4.13 0.69
Vitamin B12 (pg/mL) 253.15 § 103.58 304.67 § 133.05 0.12
Folate (mg) 158.00 § 36.00 148.00 § 41.00 0.33
Lifestyle n % n % p Value
Menopause and postmenopause (50 years old) 9 37.5 11 45.8 0.22
Overweight or obesity C 1 morbidity 9 37.5 5 20.8 0.33
Overweight or obesity C 2 or more morbidities 5 20.8 4 16.7 0.42
Use of medication 8 33.3 8 33.3 —
Anti-inflammatory — — — —

Values are expressed as the mean § standard deviation (SD) and n (%); significance is at p < 0.05.
1

CT genotype and folic acid values in G2 (r D 0.9571, p D Discussion

0.0002) (Table 3 and Table 4).
The dietary intervention consisting of different amounts of
Differences between the groups for each genotype before
folate resulted in a significant clinical improvement in folic acid
and after intervention showed significance for folic acid in CC
levels. The intake of 191 mg/d and 90 mg/d of folate derived
after intervention, according to independent t-test results
from vegetables and legumes over 8 weeks led to relevant
(Table 5).

Table 2. Nutrition intake before and after folate intervention according to genotype.
Before intervention After intervention

CC (8) CT (8) TT (8) CC (8) CT (8) TT (8)

Group 1 (191 mg)

Vitamin A (mg) 1120.41 § 1153.12 485.84 § 297.61 1994.85 § 2498.84 2049.21 § 5136.95 291.11§144.47 1176.53 § 2155.32
Vitamin C (mg) 107.95 § 70.02 94.67 § 71.61 99.03 § 85.84 233.73 § 221.77 71.60 § 59.87 167.97 § 314.87
Vitamin E (mg) 11.69 § 10.10 10.19 § 10.66 34.07 § 52.60 4.44 § 3.4 10.79 § 8.28 6.98 § 5.20
Selenium (g) 0.14 § 0.22 0.04 § 0.07 0.07 § 0.09 0.07 § 0.13 0.11 § 0.19 0.03 § 0.07
Zinc (mg) 10.09 § 3.91 7.96 § 5.69 23.89 § 45.57 9.14 § 4.47 7.79 § 4.20 9.56 § 2.26
Betacarotene (mg) 197.60 § 188.16 120.66 § 119.31 1579.84 § 2170.22 327.71 § 578.94 88.91 § 31.27 1146.44 § 1982.25
Polyunsaturated fat (g) 13.30 § 5.42 13.22 § 4.81 14.65 § 3.67 5.26 § 1.88 9.39 § 5.93 7.32 § 3.31
Monounsaturated fat (g) 13.99 § 4.41 13.20 § 4.28 13.13 § 4.55 10.25 §2.15 12.12 § 6.28 13.03 § 4.99
Oleic (g) 11.07 § 4.15 8.64 § 4.57 8.41 § 3.90 7.63 § 3.12 7.53 § 4.15 7.96 § 4.89
Linoleic (g) 6.82 § 5.37 5.72 § 6.14 3.00 § 2.88 4.39 § 2.23 6.29 § 3.83 6.3 § 3.10
Linolenic (g) 0.63 § 0.41 0.66 § 0.58 1.62 § 2.44 0.44 § 0.26 0.61 § 0.38 0.86 § 0.38
Saturated fat (g) 14.83 § 3.66 13.91 § 4.41 14.83 § 6.80 15.85 § 9.59 13.17 § 7.75 14.21 § 4.33
DHA (g) 0.00 § 0.00 0.13 § 0.32 0.01 § 0.01 0.03 § 0.03 0.01 § 0.01 0.03 § 0.03
Glycemic index 116.51 § 41.23 120.52 § 61.59 141.75 § 66.87 121.29 § 61.38 81.62 § 31.62 109.21 § 59.90
Group 2 (90 mg)
Vitamin A (mg) 736.86 § 821.30 242.68 § 144.58 233.68 § 115.28 1564.42 § 2528 1384.69 § 3046 2178.36 § 5088.48
Vitamin C (mg) 524.80 § 619.02 405.88 § 319.90 341.23 § 156.15 353.44 § 413.62 123.66 § 125.81 78.50 § 67.94
Vitamin E (mg) 5.97 § 1.98 9.28 § 5.79 9.02 § 5.33 4.38 § 3.77 6.39 § 5.02 8.30 § 8.09
Selenium (g) 0.04 § 0.05 0.06 § 0.75 0.06 § 0.16 0.08 § 0.10 0.14 § 0.19 0.10 § 0.14
Zinc (mg) 8.40 § 0.75 9.05 § 0.71 8.90 § 0.57 9.09 § 3.22 7.57 § 2.70 9.47 § 6.21
Betacarotene (mg) 291.70 § 454.17 268.92 § 247.43 162.31 118.79 336.39 § 515.21 208.38 § 172.08 84.56 § 51.16
Polyunsaturated fat (g) 31.98 § 31.88 18.54 § 7.15 22.70 § 18.12 6.17 § 3.17 9.68 § 5.44 7.55 § 4.79
Monounsaturated fat (g) 11.53 § 7.44 15.54 § 4.50 14.36 § 2.56 9.57 § 4.30 15.12 § 6.89 11.31 § 5.18
Oleic (g) 7.20 § 3.78 10.94 § 2.91 9.67 § 1.74 5.68 § 3.42 7.40 § 4.75 7.57 § 2.14
Linoleic (g) 6.18 § 6.74 7.59 § 4.20 6.28 § 3.13 3.35 § 2.87 8.58 § 5.50 4.59 § 2.29
Linolenic (g) 0.64 § 0.78 0.71 § 0.39 0.72 § 0.41 0.74 § 0.64 0.83 § 0.62 0.46 § 0.25
Saturated fat (g) 14.78 § 6.46 18.35 § 3.63 17.25 § 3.70 12.57 § 6.56 15.01 § 5.18 13.24 § 3.81
DHA (g) 0.08 § 0.21 0.00 § 0.00 0.00 § 0.01 0.00 § 0.01 0.02 § 0.02 0.11 § 0.25
Glycemic index 109.19 § 61.01 96.29 § 45.94 120.32 § 54.25 88.45 § 25.48 124.52 § 109.46 97.04 § 38.41
1
All values are expressed as the mean § SD. A t-test was used for to evaluate differences in each genotype before and after the intervention.
DHA: docosahexaenoic acid.
 JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5

Table 3. Effect of the intervention with 191 mg/d of folate (G1) according to genotype.1

Before intervention After intervention

CC (8) CT (8) TT (8) CC (8) CT (8) TT (8)

Body weight (kg) 74.26 § 7.57 71.74 § 4.36 77.95 § 12.12 70.15 § 6.82 71.49 § 6.63 77.31 § 12.12
Height (m) 1.60 § 0.07 1.57 § 0.05 1.58 § 0.07 — — —
BMI (kg/m2) 29.04 § 2.84 29.57 § 2.36 31.01 § 3.73 28.75 § 5.94 28.93 § 3.16 30.77 § 3.92
WC (cm) 91.00 § 9.97 88.75 § 6.04 95.13 § 6.96 86.32 § 7.62 85.81 § 4.28 94.31 § 7.56
TC (mg/dL) 204.88 § 25.55 170.13 § 34.46 225.50 § 27.06 202.88 § 22.66 188.25 § 55.74 229.13 § 31.49
LDL-C (mg/dL) 124.23 § 18.33a 90.80 § 28.34b 154.33 § 28.35 123.58 § 18.81 115.03 § 51.85 153.59 § 34.89
HDL-C (mg/dL) 45.88 § 12.53 37.75 § 7.25 40.75 § 5.49 42.88 § 11.81 44.13 § 12.37 44.13 § 6.75
TG (mg/dL) 158.13 § 97.14 136.13 § 53.99 152.13 § 58.04 174.75 § 52.43 148.50 § 71.40 157.06 § 78.32
TAC (%) 32.25 § 5.92a 44.62 § 21.63a 42.00 § 9.47a 49.12 § 11.28b 45.50 § 11.65a 56.37 § 13.32b
MDA (mmol/L) 3.28 § 0.79 3.02 § 0.62 3.42 § 0.96 3.86 § 0.24 3.42 § 0.17 4.18 § 0.93
Hcy (mmol/L) 8.97 § 1.53a 9.91 § 3.71a 10.05 § 1.67a 7.60 § 1.79a 8.01 § 2.27b,£ 7.11 § 0.78b
Folic acid (mg/mL) 12.94 § 5.29a 13.48 § 1.62a 16.52 § 5.12a 22.21 § 7.27b 18.11 § 3.10b 25.48 § 4.36b
Vitamin B12 (mg/mL) 251.81 § 113.13 261.25 § 139.79 246.37 § 52.71 237.50 § 52.69 258.00§113.03 228.62 § 78.15
Folate (mg) 123.22 § 32.73a 129.53 § 41.18a 180.43 § 34.54b 337.95 § 73.07c 372.35 § 46.00c 363.64 § 68.32c
1
All values are expressed as the mean § SD. BMI: body mass index; WC: waist circumference; TC: total cholesterol; LDL-C: low-density lipoprotein; HDL-C: high-density
lipoprotein; TG: triglycerides; TAC: total antioxidant capacity; MDA: malondialdehyde; Hcy: homocysteine. A
t-test was used for to evaluate differences in each genotype before and after the intervention: a,b,cMDifferent letters mean statistical difference. Analysis of variance
(ANOVA) was used to evaluate differences between genotypes before and after the intervention. Post hoc Bonferroni test was used for multiple comparisons: a,bDiffer-
ent letters mean statistical difference only for LDL-C and folate before intervention.
£
Significant Pearson’s correlation test between the CT genotype and homocysteine values.

results; this intervention was able to elevate the TAC levels in Thus, consistent with published data, TT carriers
women with overweight or obesity carrying the CC and TT respond well to dietary treatment with additional folate
genotypes and the folic acid levels of all the analyzed genotypes, (17). However, compared to the other genotypes, TT car-
and it also reduced the Hcy levels in carriers of the TT riers need a larger amount of this nutrient (39) to achieve
genotype. benefits that improve oxidative stress parameters. It should
Despite having lower enzyme activity because the thermola- be stressed that the total sum of folate consumed through
bile characteristics of the enzyme differ from those of the unmu- the usual diet and the intervention folate was close to the
tated MTHFR (38), genotype TT carriers who consumed EAR, especially in G1.
191 mg/d of folate exhibited an elevation in TAC and a reduction Other studies have also reported a better response to dietary
in Hcy. This effect was not observed for the women who con- interventions among carriers of the 677T allele, probably due
sumed a lower amount of folate, although the lower intake also to better adherence to healthy dietary habits among carriers of
induced an elevation in folic acid concentration. These findings this allele (18). This better response may also be explained by
demonstrate that higher folic concentrations tend to increase the physiological effects of the MTHFR C677T polymorphism,
the stability of the MTHFR enzyme, thus reducing the difference which make carriers of this allele more responsive to a certain
in enzyme activity among the polymorphism genotypes (2). dietary treatment (21). However, none of the previous studies

Table 4. Effect of the intervention with 95 mg/d of folate (G2) according to genotype.1
Before intervention After intervention

CC (8) CT (8) TT (8) CC (8) CT (8) TT (8)

Body weight (kg) 77.42 § 18.57 84.65 § 16.19 71.61 § 7.65 75.10 § 18.56 83.94 § 17.79 70.13 § 7.50
Height (m) 1.59 § 0.06 1.58 § 0.10 1.56 § 0.07 — — —
BMI (kg/m2) 30.72 § 7.76 30.88 § 2.93 29.80 § 2.57 29.95 § 7.69 33.58 § 6.67 28.96 § 2.49
WC (cm) 92.34 § 14.02 94.13 § 14.19 88.87 § 7.75 87.63 § 13.48 92.13 § 14.67 87.19 § 7.85
TC (mg/dL) 192.50 § 37.51 194.75 § 35.79 219.38 § 41.08 185.00 § 42.72 209.63 § 46.11 215.00 § 51.28
LDL-C (mg/dL) 112.63 § 39.67 104.88 § 43.25 145.43 § 34.84 111.15 § 34.03 131.45 § 38.77 136.38 § 36.62
HDL-C (mg/dL) 38.38 § 7.48 43.75 § 11.93 41.63 § 6.35 42.63 § 5.32 48.63 § 7.69 45.13 § 13.79
TG (mg/dL) 178.88 § 75.05 153.75 § 100.60 161.63 § 91.66 156.13 § 63.06 147.75 § 70.11 167.50 § 72.74
TAC (%) 34.87 § 7.72a 47.12 § 11.87a 43.87 § 16.19a 57.00 § 15.93b 46.50 § 11.50a 48.37 § 12.24a
MDA (mmol/L) 2.93 § 0.72 2.91 § 0.88 3.56 § 0.87 3.78 § 0.68 3.37 § 0.56 3.46 § 0.57
Hcy (mmol/L) 9.13 § 2.39a 10.98 § 6.11a 14.85 § 7.43a 7.83 § 2.52a 9.86 § 4.43a,£ 14.91 § 10.40b,£
Folic acid (mg/mL) 13.18 § 4.09a 13.32 § 3.98a 15.00 § 4.60a 14.88 § 3.79a 16.40 § 4.96b,£ 22.62 § 6.30b
Vitamin B12 (mg/mL) 268.62 § 92.63 345.50 § 179.14 299.87 § 118.65 271.00 § 92.16 355.26 § 202.59 273.62 § 83.60
Folate (mg) 137.45 § 14.43a 161.82 § 50.44 153.45 § 53.84a 297.97 § 65.25b 248.59 § 85.27 316.26 § 63.07b
1
All values are expressed as the mean § SD. BMI: body mass index; WC: waist circumference; TC: total cholesterol; LDL-C: low-density lipoprotein; HDL-C: high-density
lipoprotein; TG: triglycerides; TAC: total antioxidant capacity; MDA: malondialdehyde; Hcy: homocysteine. A
t-test was used for to evaluate differences in each genotype before and after the intervention: a,bDifferent letters mean statistical difference.
Analysis of variance (ANOVA) was used to evaluate differences between genotypes before and after the intervention. Post hoc Bonferroni test was used for multiple
comparisons.
£
Significant Pearson’s correlation test between CT genotype and folic acid values, and correlation between the TT genotype and homocysteine values.
6 M. R. RIBEIRO ET AL.

Table 5. Oxidative stress markers before and after the folate intervention according to group and genotype.1

Before intervention After intervention

CC (8) CT (8) TT (8) CC (8) CT (8) TT (8)

TAC G1 32.25 § 5.92 44.62 § 21.63 42.00 § 9.47 49.12 § 11.28 45.50 § 11.64 56.37 § 13.32
TAC G2 34.87 § 7.71 47.12 § 11.87 43.87 § 16.19 57.00 § 15.93 46.50 § 11.50 48.37 § 12.23
p Value 0.4580 0.7787 0.7815 0.2730 0.8653 0.2314
MDA G1 3.27 § 0.78 2.97 § 0.60 3.42 § 0.96 3.85 § 0.24 3.53 § 0.35 4.18 § 0.92
MDA G2 2.92 § 0.73 2.91 § 0.87 3.74 § 1.67 3.78 § 0.68 3.37 § 0.56 3.26 § 1.12
p Value 0.3704 0.8702 0.6540 0.7915 0.4965 0.0934
Hcy G1 8.97 § 1.53 9.91 § 3.71 10.05 § 1.67 7.60 § 1.79 8.01 § 2.27 7.11 § 0.77
Hcy G2 13.61 § 13.10 10.98 § 6.12 19.33 § 12.76 7.83 § 2.51 9.86 § 4.43 14.91 § 10.40
p Value 0.3368 0.6792 0.0608 0.8310 0.3126 0.0529
Folic acid G1 12.94 § 5.30 13.48 § 1.62 16.52 § 5.12 22.21 § 7.27 18.11 § 3.10 25.48 § 4.36
Folic acid G2 13.18 § 4.09 13.32 § 3.97 15.00 § 4.60 14.88 § 3.79 16.40 § 4.96 22.62 § 6.30
p Value 0.9215 0.9143 0.5429 0.0241 0.4212 0.3093

TAC: total antioxidant capacity; MDA: malondialdehyde;


Hcy: homocysteine.
1
G1: 191 mg/d of folate; G2: 95 mg/d of folate. p < 0.05; independent t-test.

assessed the influence of this polymorphism on oxidative stress Although the present study adds relevant data to the lit-
while evaluating folate intake. erature, one limitation is the sample size and the use of
For the CC genotype carriers, TAC increased in both groups BMI to classify nutritional status, because BMI does not
after the dietary intervention; however, the folic acid levels measure body fat directly (50,51). However, significant asso-
increased only in G1. Surprisingly, the group without an increase ciations and the existence of a dietary response under the
in folic acid levels exhibited an elevation in MDA levels. influence of the MTHFR C677T polymorphism were
Compared to carriers of the 677C allele, carriers of the 677T observed nonetheless.
allele exhibited a greater tendency toward lower TAC levels,
which may be due to the influence of this polymorphism on
Hcy concentrations (40,41). Previous studies have demon-
strated a relationship between hyperhomocysteinemia and pro- Conclusions
oxidant conditions, especially for genotype TT carriers, as In summary, the present study is the first to demonstrate the
hyperhomocysteinemia results in greater generation of ROS beneficial effects of folate intake in terms of a TAC elevation
(2,14), thus contributing to the development of oxidative stress. for the CC and TT genotypes of the MTHFR C677T polymor-
However, to the best of our knowledge, this was the first phism, an increase in folic acid levels for all genotypes, and a
study to show that carriers of the 677T allele exhibited higher reduction in Hcy levels for the TT genotype in response to an
TAC than 677C carriers. This finding might be associated with intervention consisting of an intake of 191 mg/d of folate sup-
the normal Hcy levels exhibited by the risk allele carriers, as plied by vegetables. The data reported here indicate the impor-
suggested by the previously reported negative correlation tance of folate intake by individuals carrying this
between Hcy concentration and TAC for genotype TT (2). polymorphism to improve the levels of oxidative stress
Although the concentrations were already adequate, a markers. The results showed that the higher levels of folate con-
reduction in Hcy levels occurred in genotype TT carriers in G1 sumption, which were close to the recommended values, bene-
after the dietary intervention with folate, which corroborates ficially affected the TAC and Hcy levels, thus further
the results of previous studies (42,43,44). These findings indi- emphasizing the need for individualized dietary instructions,
cate that carriers of the 677T allele have a lower risk of hyper- especially for carriers of the TT genotype.
homocysteinemia when they consume adequate amounts of
folate (12,45).
The results of the present study show that the investigated
anthropometric, biochemical, and dietary intake parameters Acknowledgments
were similar for all genotypes of the MTHFR C677T polymor- We thank the project coordinator, Dr. Maria Jos
e de Carvalho Costa, and
phism before the intervention, except for the LDL-C and folate the funding agencies that enabled the project (National Council of Scien-
in G1. Most of the consulted literature reports that carriers of tific and Technological Development [Conselho Nacional de Pesquisas–
the 677T allele exhibit higher LDL-C levels (40,46,47), which CNPq], Ministry of Health and Para
ıba Research Support Foundation
[Fundaç~ao de Apoio a Pesquisa do Estado da Para
ıba–FAPESQ, PB]), and
agrees with the results of the present study when considering
our research collaborators (Graduate Program in Nutrition Sciences, Fed-
the notable elevation in the lipid profile values in these eral University of Para
ıba).
individuals.
These findings might be due to an alteration in the lipid
metabolism pathway resulting from a reduction in enzyme
activity caused by the polymorphism of interest (40). Thus, Funding
modulation of the lipid profile is a factor that should be taken This work was supported by the Para
ıba Research Support Foundation
into account along with the complexity of oxidative stress (48) (Fundaç~ao de Apoio a Pesquisa do Estado da Para
ıba - FAPESQ, PB -
in individuals with overweight or obesity (49). EFP_00008187).
 JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7

ORCID adhesion protein 1, preliminary report. J Eur Acad Dermatol Vene-

reol. 2014;28(9):1192–98.
Rafaella Cristhine Pordeus Luna http://orcid.org/0000-0003-2477-8258 16. Petkeviciene J, Smalinskiene A, Luksiene DI, Jureniene K, Ramazaus-
Darlene Camati Persuhn http://orcid.org/0000-0001-5291-5454 kiene V, Klumbiene J, Lesauskaite V. Associations between apolipo-
Alexandre S
ergio da Silva http://orcid.org/0000-0003-3576-9023 protein E genotype, diet, body mass index, and serum lipids in
Maria da Conceiç~ao Rodrigues Gonçalves http://orcid.org/0000-0001- Lithuanian adult population. PloS One. 2012;7(7):e41525.
7223-8470 17. Hung J, Yang TL, Urrutia TF, Li R, Perry CA, Hata H, Cogger EA, Mor-
Maria Jos
e de Carvalho Costa http://orcid.org/0000-0001-5441-8654 iarty DJ, Caudill MA. Additional food folate derived exclusively from nat-
ural sources improves folate status in young women with the MTHFR
677 CC or TT genotype. J Nutr Biochem. 2006;17(11):728–34.
References 18. Pitsavos C, Panagiotakos D, Trichopoulou A, Chrysohoou C, Dedous-
sis G, Chloptsios Y, Choumerianou D, Stefanadis C. Interaction
1. Marchi E, Baldassari F, Bononi A, Wieckowski MR, Pinton P. Oxida- between Mediterranean diet and methylenetetrahydrofolate reductase
tive stress in cardiovascular diseases and obesity: role of p66Shc and C677T mutation on oxidized low density lipoprotein concentrations:
protein kinase C. Oxid Med Cell Longev. 2013. doi:10.1155/2013/ The ATTICA study. Nutr Metab Cardiovasc Dis. 2006;16(2):91–99.
564961. 19. Di Renzo L, Marsella LT, Sarlo F, Soldati L, Gratteri S, Abenavoli L,
2. Abd-Elmawla MA, Rizk SM, Youssry I, Shaheen AA. Impact of De Lorenzo A. C677T gene polymorphism of MTHFR and metabolic
genetic polymorphism of methylenetetrahydrofolate reductase C677T syndrome: response to dietary intervention. J Transl Med. 2014.
on development of hyperhomocysteinemia and related oxidative doi:10.1186/s12967-014-0329-4.
changes in Egyptian b-thalassemia major patients. PloS One. 20. Fukuda N, Hamajima N, Wakai K, Suzuki K. A cross-sectional study
2016;11:5. to find out the relationship of methylenetetrahydrofolate reductase
3. Biswas SK. Does the interdependence between oxidative stress and (MTHFR) C677T genotype with plasma levels of folate and total
inflammation explain the antioxidant paradox? Oxid Med Cell Lon- homocysteine by daily folate intake in Japanese. J Nutr Sci Vitaminol.
gev. 2016. doi:10.1155/2016/5698931. 2014;60(40):231–38.
4. Huang CJ, Mcallister MJ, Slusher AL, Webb HE, Mock JT, Acevedo 21. Massa NM, Silva AS, de Oliveira CV, Costa MJ, Persuhn DC, Barbosa
EO. Obesity-related oxidative stress: the impact of physical activity CVS, Gonçalves MCR. Supplementation with watermelon extract
and diet manipulation. Sports Med Open. 2015;1:32. reduces total cholesterol and LDL cholesterol in adults with dyslipide-
5. Farinha JB, Steckling FM, Stefanello ST, Cardoso MS, Nunes LS, Bar- mia under the influence of the MTHFR C677T polymorphism. J Am
celos RP, Duarte T, Kretzmann NA, Mota CB, Bresciani G. Response Coll Nutr. 2016;35(6):514–20.
of oxidative stress and inflammatory biomarkers to a 12-week aerobic 22. LIMA RPA, Nascimento RAF, Luna RCP, Persuhn DC, Silva AS,
exercise training in women with metabolic syndrome. Sports Med Gonçalves MCR, Almeida ATC, Moraes RM, Verly Junior E, Fouil-
Open. 2015;1:3. loux-Meugnier E. Effect of a diet containing folate and hazelnut oil
6. Mangge H, Becker K, Fuchs D, Gostner JM. Antioxidants, inflamma- capsule on the methylation level of the ADRB3 gene, lipid profile and
tion and cardiovascular disease. World J Cardiol. 2014;6(6):462–77. oxidative stress in overweight or obese women. Clin Epigenetics.
7. Bonaa KH, Njølstad I, Ueland PM, Schirmer H, Tyerdal A, Steigen T, 2017. doi:10.1186/s13148-017-0407-6.
Wang H, Nordrehaug JE, Arnesen E, Rasmussen K. Homocysteine 23. Switzeny OJ, M€ ullner E, Wagner KH, Brath H, Aum€ uller E, Hasl-
lowering and cardiovascular events after acute myocardial infarction. berger AG. Vitamin and antioxidant rich diet increases MLH1 pro-
N Engl J Med. 2006;354(15):1578–88. moter DNA methylation in DMT2 subjects. Clin Epigenetics.
8. Lee JY, Shin C, Baik I. Longitudinal associations between micronutri- 2012;4:1.
ent consumption and leukocyte telomere length. J Hum Nutr Diet. 24. National Academy of Sciences. Dietary reference intakes: folate, other
2016. doi:10.1111/jhn.12403. B vitamins and choline. Washington (DC): National Academies Press;
9. Quaranta N, De Ceglie V, D’elia A. Endothelial dysfunction in idio- 1998.
pathic sudden sensorineural hearing loss: a review. Audiol Res. 25. American Heart Association (AHA): Dietary Guidelines for American.
2016;6:1. 2010. https://www.heart.org/idc/groups/heartpublic/@wcm/@adv/docu
10. Wang L, Shangguan S, Chang S, Yu X, Wang Z, Lu X, Wu L, Zhang T. ments/downloadable/ucm_312853.pdf. Accessed April 1, 2016.
Determining the association between methylenetetrahydrofolate 26. Costa MJCC. Nutriç~ao cl
ınica: Uso do sistema de equivalentes na
reductase (MTHFR) gene polymorphisms and genomic DNA methyl- pr
atica dietoter
apica. 2rd ed. Jo~ao Pessoa (Brazil): Editora da UFPB;
ation level: a meta-analysis. Birth Defects Res A Clin Mol Teratol. 2013.
2016;106(8):667–74. 27. Otten JJ, Jennifer PH, Linda DM. Dietary reference intakes: The essen-
11. Li WX, Lv WW, Dai SX, Pan ML, Huang JF. Joint associations of tial guide to nutrient requirements. 2006. https://www.nal.usda.gov/
folate, homocysteine and MTHFR, MTR and MTRR gene polymor- sites/default/files/fnic_uploads//DRIEssentialGuideNutReq.pdf.
phisms with dyslipidemia in a Chinese hypertensive population: a Accessed November 1, 2016.
cross-sectional study. Lipids Health Dis. 2015;14:101. 28. WHO (World Health Organization). Obesity: preventing and manag-
12. Morais CC, Alves MC, Augusto EM, Abdalla DS, Horst MA, Comi- ing the global epidemic. Report of a WHO consultation. World Health
netti C. The MTHFR C677T polymorphism is related to plasma con- Organization. 2000;894:11–253.
centration of oxidized low-density lipoprotein in adolescents with 29. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin
cardiovascular risk factors. J Nutrigenet Nutrigenomics. 2015;8 BA, Gordon DJ, Krauss RM, Savage PJ, Smith SC. Diagnosis and man-
(3):105–13. agement of the metabolic syndrome: An american heart association/
13. Hobbs CA, Sherman SL, Yi P, Hopkins SE, Torfs CP, Hine J, Pogribna national heart, lung, and blood institute scientific statement: Executive
M, Rozen R, James J. Polymorphisms in genes involved in folate summary. Crit Pathw Cardiol. 2005;4(4):198–203.
metabolism as maternal risk factors for down syndrome. Am J Hum 30. Asciutti LSR. Manual de porç~ oes m
edia em tamanho real baseado no
Genet. 2000;67(3):623–30. programa dietsys para estudo de base populacional. Jo~ao Pessoa (Bra-
14. Rahimi Z, Malek-Khosravi S, Rahimi Z, Jalilvand F, Parsian A. zil): Editora da UFPB; 2005.
MTHFR C677T and eNOS G894T variants in preeclamptic women: 31. Lima FEL, Latorre MRDO, Costa MJC, Fisberg RM. Diet and cancer
contribution to lipid peroxidation and oxidative stress. Clin Biochem. in northeast Brazil: evaluation of eating habits and food group con-
2013;46(1):143–47. sumption in relation to breast cancer. Cad Saude Publica. 2008;24
15. Asefi M, Vaisi-Raygani A, Khodarahmi R, Nemati H, Rahimi Z, Vaisi- (4):820–28.
Raygani H, Tavilani H, Pourmotabbed T. Methylentetrahydrofolatere- 32. MSM. The Multiple Source Method. Department of Epidemiology of
ductase (rs1801133) polymorphism and psoriasis: contribution to oxi- the German Institute of Human Nutrition Potsdam. https://msm.dife.
dative stress, lipid peroxidation and correlation with vascular de/tps/msm. Accessed November 17, 2014.
8 M. R. RIBEIRO ET AL.

33. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides peroxides in diet responsiveness of plasma homocysteine in healthy women. J
animal tissues by thiobarbituric acid reaction. Anal. Biochem. Nutr. 2001. 131(10):2643–47.
1979;95:351–58. 43. Chmurzynska A, Malinowska AM, Twardowska-Rajewska J, Gawecki
34. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical J. Elderly women: homocysteine reduction by short-term folic acid
method to evaluate antioxidant activity. LWT Food Sci. Technol. supplementation resulting in increased glucose concentrations and
1995;28:25–30. affecting lipid metabolism (C677T MTHFR polymorphism). Nutri-
35. Kuo K, Still R, Cale S, McDowell I. Standardization (external and tion. 2013. 29(6):841–44.
internal) of HPLC assay for plasma homocysteine. Clin Chem. 44. Zappacosta B, Mastroiacovo P, Persichilli S, Pounis G, Ruggeri S, Min-
1997;43(9):1653–55. ucci A, Carnovale E, Andria G, Ricci R, Scala I. Homocysteine lowering
36. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for by folate-rich diet or pharmacological supplementations in subjects with
extracting DNA from human nucleated cells. Nucleic Acids Res. moderate hyperhomocysteinemia. Nutrients. 2013. 5(5):1531–43.
1988;16:3. 45. Gil-Prieto R, Hern
andez V, Cano B, Oya M, Gil A. Plasma homocyste-
37. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews ine in adolescents depends on the interaction between methylenetetra-
RG, Boers GJH, Heijer M, Kluijtmans LAJ, Van Den Heuve LP. A hydrofolate reductase genotype, lipids and folate: a
candidate genetic risk factor for vascular disease: a common seroepidemiological study. Nutr Metab. 2009;6(1):39.
mutation in methyleneethahydrofolate reductase. Nat Genet. 46. Zhang L, Yin RX, Liu WY, Miao L, Wu D, Aung LHH, Hu XJ, Cao XL,
1995;10(1):111–13. Wu JZ, Pan SL. Association of methylenetetrahydrofolate reductase
38. de Arruda IT, Persuhn DC, de Oliveira NF. The MTHFR C677T poly- C677T polymorphism and serum lipid levels in the Guangxi Bai Ku
morphism and global DNA methylation in oral epithelial cells. Genet Yao and Han populations. Lipids Health Dis. 2010. doi:10.1186/1476-
Mol Biol. 2013. 36(4):490–93. 511X-9-123.
39. Ashfield-Watt PA, Pullin CH, Whiting JM, Clark ZE, Moat SJ, New- 47. Jiang S, Zhao R, Pan M, Venners SA, Zhong G, Hsu Y. Associations of
combe RG, Burr ML, Lewis MJ, Powers HJ, McDowell IF. Methylene- MTHFR and MTRR polymorphisms with serum lipid levels in Chi-
tetrahydrofolate reductase 677C ! T genotype modulates nese hypertensive patients. Clin Appl Thromb Hemost. 2014;20
homocysteine responses to a folate-rich diet or a low-dose folic acid (4):400–10.
supplement: A randomized controlled trial. Am J Clin Nutr. 2002. 76 48. Tanko Y, Jimoh A, Ahmed A, Mohammed A, Ayo JO. Resveratrol
(1):180–86. protects rabbits against cholesterol diet-induced hyperlipidaemia.
40. Husemoen LL, Skaaby T, Jørgensen T, Thuesen BH, Fenger M, Niger J Physiol Sci. 2016;31(1):71–5.
Grarup N, Sandholt CH, Hansen T, Pedersen O, Linneberg A. 49. Yin RX, Wu DF, Miao L, Aung LH, Cao X, Yan T, Long XJ, Liu WY,
MTHFR C677T genotype and cardiovascular risk in a general popula- Zhang L, Li M. Several genetic polymorphisms interact with over-
tion without mandatory folic acid fortification. Eur J Nutr. 2014. 53 weight/obesity to influence serum lipid levels. Cardiovasc Diabetol.
(7):1549–59. 2012. doi:10.1186/1475-2840-11-123.
41. Chen YY, Wang BN, Yu XP. Correlation between the 677C>T poly- 50. De Lorenzo A, Bianchi A, Maroni P, Iannarelli A, Di Daniele N, Iaco-
morphism in the methylene tetrahydrofolate reductase gene and pino L, Di Renzo L. Adiposity rather than BMI determines metabolic
serum homocysteine levels in coronary heart disease. Genet Mol Res. risk. Int J Cardiol. 2013;166:111–17.
2016;15:1. 51. Oliveros E, Somers VK, Sochor O, Goel K, Lopez-Jimenez F. The con-
42. Silaste ML, Rantala M, S€ampi M, Alfthan G, Aro A, Kes€aniemi YA. cept of normal weight obesity. Prog Cardiovasc Dis. 2014;56(4):426–
Polymorphisms of key enzymes in homocysteine metabolism affect 33. doi:10.1016/j.pcad.2013.10.003.