Nutrient-Gene Expression

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1 Nutrient-Gene Expression Polymorphisms of Key Enzymes in Homocysteine Metabolism Affect Diet Responsiveness of Plasma Homocysteine in Healthy Women 1 Marja-Leena Silaste, 2 Maire Rantala, Maritta Sämpi, Georg Alfthan,* Antti Aro* and Y. Antero Kesäniemi Department of Internal Medicine, University of Oulu, Kajaanintie 50, FIN Oulu, Finland and Biocenter Oulu, University of Oulu, Kajaanintie 52, FIN Oulu, Finland; and *National Public Health Institute (KTL), Mannerheimintie 166, FIN-00300, Helsinki, Finland ABSTRACT High plasma total homocysteine (thcy), low dietary intake of folate and other B vitamins, and genetic polymorphisms related to metabolism of homocysteine may interactively contribute to the risk of cardiovascular disease. We investigated whether known mutations in genes regulating homocysteine metabolism affect the responsiveness of serum folate and plasma thcy to high intake of natural folate from food. Healthy females (n 37) aged y volunteered to participate in a crossover dietary intervention with two 5-wk diet periods (low and high folate diets). Concentrations of serum and RBC folate, serum vitamin B-12 and plasma thcy were measured at baseline and at the end of each diet period. The prevalences of C677T transition of methylenetetrahydrofolate reductase (MTHFR) gene, 844ins68 of cystathionine -synthase (CBS) gene and A2756G mutation of methionine synthase (MS) gene were determined. Compared with the low folate diet, the high folate diet increased the serum folate concentration by 85% (P 0.001), 77% (P 0.001) and 55% (P 0.05) in the subjects with the genotypes C/C (n 19), C/T (n 13) and T/T (n 5), respectively, of the MTHFR gene. Also, the plasma thcy of the subjects with the genotypes C/C, C/T and T/T was decreased by 11% (P 0.001), 15% (P 0.01) and 18% (P 0.05), respectively, during the high folate diet period. The subjects carrying the G2756 allele of the MS gene (n 15) had a more extensive reduction (P 0.05) of plasma thcy during the high folate diet period than the subjects with the genotype A/A (n 22). The 844ins68 of CBS gene did not affect plasma thcy concentrations or diet responsiveness. In conclusion, diet responsiveness of plasma homocysteine may be genetically regulated. J. Nutr. 131: , KEY WORDS: homocysteine folate diet methylenetetrahydrofolate reductase methionine synthase humans An elevated concentration of plasma total homocysteine (thcy) 3 has been recognized as a risk factor for vascular diseases (1). Both nutritional and genetic factors affect plasma thcy levels. Among the nutritional factors, supplementation with folic acid alone (2,3) and in combination with other B vitamins (4) has been shown to reduce the plasma thcy concentration. Also, a high intake of natural folate from food decreases plasma thcy (5 8). The genetic factors affecting plasma thcy levels include mutations in the genes of the key enzymes participating in homocysteine metabolism. The three key enzymes are cystathionine -synthase (CBS), methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS). CBS is the key enzyme in the transsulfuration pathway, whereas MTHFR and MS play important roles in the remethylation pathway. Severe mutations, such as T833C and 1 Supported in part by the Research Council for Health of the Academy of Finland and the Finnish Foundation for Cardiovascular Research. 2 To whom correspondence should be addressed. Marja-Leena.Silaste.oulu.fi. 3 Abbreviations used: BMI, body mass index; CBS, cystathionine -synthase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; PCR, polymerase chain reaction; thcy, total homocysteine. G919A in the CBS gene, are rare, affecting 1% of the general population (9,10). However, a 68-bp insertion (844ins68) of the CBS gene is fairly prevalent; it is present in the heterozygous state in 12% of the U.S. population (11). This insertion of the CBS gene may be associated with low plasma thcy concentrations (12,13). In the MS gene, the A2756G transition is highly prevalent (14). The presence of the G2756 allele in the MS gene has been reported to be associated with a lowered fasting concentration of plasma thcy (14,15). An important genetic determinant of the plasma thcy concentration is a common polymorphism of the MTHFR gene (16). This defect results from a C to T mutation at the nucleotide position 677 in DNA (17), which reduces the basal activity of the enzyme by 50% (18). Homozygosity for the T677 allele of the MTHFR gene is associated with elevated plasma thcy concentrations (16,19). Although some studies have recently focused on the influence of natural folate on the plasma thcy concentration (5 8), the effects of gene mutations on the responsiveness of serum folate and plasma thcy to increased dietary folate have not been determined. Previously, we studied the influence of a high dietary intake of natural /01 $ American Society for Nutritional Sciences. Manuscript received 28 February Initial review completed 14 May Revision accepted 17 July

2 2644 SILASTE ET AL. folate on the concentrations of serum folate and plasma thcy in a controlled crossover diet intervention (20). In the current study, we determined the role of the gene polymorphisms of MTHFR, CBS and MS in the diet responsiveness of serum folate and plasma thcy. SUBJECTS AND METHODS Subjects. The study subjects were healthy female volunteers who were working at the University Hospital of Oulu. Of the 86 women screened, 37 were eligible for the study. The subjects were eligible for inclusion if they fulfilled the following criteria: 1) no gastrointestinal, renal or hepatic disease; 2) normal blood glucose and lipid concentrations; 3) body mass index (BMI) between 20 and 29 kg/m 2 ; 4) no alcoholism; 5) not a current smoker; 6) no use of supplemental vitamins and/or minerals for at least 6 mo before the onset of the study; and 7) no food allergy. Pregnant and lactating women were excluded from the study. Six subjects were using oral contraceptives, and three subjects were using postmenopausal estrogen/progestin supplementation. The study was carried out in accordance with the instructions of the Declaration of Helsinki. Informed consent was obtained from each participant. The study was approved by the Ethical Committee of the Faculty of Medicine, University of Oulu. Study design. This crossover study consisted of a 2-wk baseline period and two 5-wk study periods (low folate diet and high folate diet) with a 3-wk washout period in between. During the baseline and washout periods, the subjects consumed their habitual diets. At the end of the baseline period, the subjects were randomly assigned to two groups. Half of the subjects (n 18) first consumed a low folate diet followed by a high folate diet, whereas the other half (n 19) consumed the diets in the reverse order. Diets. Both diets were designed on the basis of the regular hospital meals (a 5-wk menu) and contained conventional foods and beverages. A basic day s menu included breakfast, lunch, afternoon snack, dinner and evening snack. Breakfast was based on bread and/or breakfast cereals and low fat milk products. For lunch and dinner, warm dishes of meat, poultry or fish together with potatoes, pasta or rice were served. Lunch and dinner also included a dessert. The afternoon snack contained a low fat cake and coffee or tea, and the evening snack consisted of bread and cheese. The foods were prepared, packaged and delivered to the subjects by the hospital kitchen (University Hospital of Oulu, Oulu, Finland). On working days, the lunches and dinners were served at the hospital cafeteria. The participants could also take the packaged dinner home. On weekends, the subjects were able to eat the lunches and dinners at the hospital cafeteria or take the packaged weekend meals home on Friday. Other foodstuffs, such us bread, milk and fruit, were delivered to the subjects twice a week. Both study diets were energy balanced and designed to be low in dietary cholesterol ( 200 mg/d) and saturated fat ( 10% of total energy intake). In both diets, the quantity and quality of dietary fat was controlled by using low fat meat and dairy products, low fat cooking methods, and vegetable oil and margarine. The low folate diet contained one serving of both fresh vegetables and fresh fruit or fruit juice per day. The intake of dietary folate with the low folate diet was designed to be 200 g/d. The intakes of vitamins B-6 and B-12 as well as other nutrients during the low folate diet period were designed to meet the Nordic nutrition recommendations (21). Dietary folate was increased in the high folate diet by increasing the consumption of fresh vegetables, citrus fruits and berries. At breakfasts during the high folate diet period, the subjects ate 30 g of fresh pepper and a piece of fruit, e.g., orange or kiwi, or 125 ml of juice in addition to whole-grain bread, breakfast cereals and low fat milk products. The high folate lunch included g of salad from carrots, cauliflower or cabbage, for example, and 100gof steamed vegetables, e.g., broccoli, peas, carrots or cauliflower in addition to the basic diet. Fresh berries, e.g., strawberries or black currants, were served as dessert. At dinner, approximately the same amounts of fresh and steamed vegetables as at lunch were consumed. The dessert at dinner consisted of either fresh berries or a piece of fruit, e.g., orange or kiwi. At both lunch and dinner, 100 ml of orange juice was consumed. In the evening, the subjects ate 30 g of red pepper and 125 ml of orange or pineapple juice together with whole grain bread and low fat cheese. The high intake of vegetables, citrus fruit and berries was designed to result in an average of 600 g folate/d. Dietary intakes of other nutrients during the high folate diet period were designed to meet the Nordic nutrition recommendations (21). The same experienced dietician (M.-L.S.) interviewed all of the participants concerning their eating and exercise habits and determined an isocaloric energy intake level for each. During the intervention, the participants weighed themselves daily before lunch, and their dietary energy intake was adjusted, when necessary, to maintain their body weight unchanged during the study. Alcohol consumption was determined at baseline by interviewing the subjects who were advised to restrict their use of alcohol to less than four drinks per week during the study. According to the subjects reports, the amount of alcohol consumed was negligible, and alcohol was therefore not included in the calculations of diets. Laboratory methods. At baseline, overnight fasting blood samples were drawn for the clinical chemistry tests and for the measurements of plasma thcy and serum and RBC folate and serum vitamin B-12 concentrations, which were also measured at the end of both study periods. The concentrations of plasma thcy and serum folate and vitamin B-12, but not RBC folate, were also measured at the end of the washout period. Concentrations of serum and red blood cell folate and serum B-12 were determined using the Quantaphase II B-12 and Folate Radioassays (Bio-Rad Laboratories, Espoo, Finland). For folate, the intra- and interassay CV were %, depending on the folate concentration. For vitamin B-12, the intra- and interassay CV were 3.3%. The plasma thcy concentration was analyzed by the immunofluorometric IMX method (Abbott Laboratories, Chicago, IL) (22). The interassay CV was 3.2%. The accuracy was ascertained by using a Nordic quality assurance system, in which the mean bias for 7 sera was 3.5% (23). Genomic DNA was isolated from peripheral leukocytes isolated from anticoagulated blood (EDTA) by using a salting-out method according to Miller and co-workers (24). The analysis of the C677T polymorphism in the MTHFR gene was investigated by polymerase chain reaction (PCR) of a DNA fragment followed by restriction enzyme digestion with Hinf I (18). The presence or absence of the 844ins68 of the CBS gene was tested using PCR amplification and digestion with the restriction enzyme Bsr I (11). The A2756G mutation of the MS gene was detected using PCR amplification and Hae III restriction analysis (25). All of the enzymes used were provided by Finnzymes (Espoo, Finland). The fragments of all three gene polymorphisms were visualized on a UV transilluminator after electrophoresis on a 3% low melting point agarose gel (3:1 NuSieve, Bio- Whittaker Molecular Applications, Rockland, ME) containing nucleic acid gel stain (GelStar, BioWhittaker Molecular Applications). Dietary analyses. Four-day diet records were completed during the baseline diet period after the first visit to our laboratory. The dietary intake during the study was analyzed from duplicate portions collected every day. The analyses were done in the Agricultural Research Center of Finland (Jokioinen, Finland). The intakes of folate and vitamins B-6 and B-12 during the study diets were calculated from the study menus using the Nutrica computer program (Social Insurance Institution, Helsinki, Finland) based on the Finnish nutrient database. Statistical analyses. Because the concentrations of serum and RBC folate and plasma thcy were not normally distributed, logarithmic corrections were used in all statistical analyses. The changes in the concentrations of serum and RBC folate, serum vitamin B-12 and plasma thcy between the low and high folate diets were calculated by subtracting the values of the low folate diet from those of the high folate diet. Student s t test for paired samples was used to determine the differences in the concentrations of serum and RBC folate, serum vitamin B-12 and plasma thcy between the diets. The diet-induced changes in the serum folate and plasma thcy concentrations and the effect of the genotypes of MTHFR, CBS and MS were tested with respect to the intraindividual variation during the diet intervention. This was done by using a layered design in the

3 GENE POLYMORPHISMS AND PLASMA HOMOCYSTEINE 2645 FIGURE 1 Body mass index (BMI) of women at baseline and after consumption of low and high folate diets. Values are mean SD, n 37. form of repeated measurements across time (ANOVA of repeated measurements). The top layer in the model was the between-subject layer, in which the effect of having a certain genotype was tested with respect to the interindividual variation. The bottom layer was the within-subject layer, in which the repeated measures for the diet periods (the baseline, low folate diet and high folate diet) were tested with respect to the variation from one dietary period to another. In addition, the differences in the concentrations of serum folate and plasma thcy between the different genotypes of each gene were detected using either Student s t test for independent samples (CBS and MS genes) or one-way ANOVA (MTHFR gene). After one-way ANOVA, Scheffé s test was used as the post-hoc test. The differences were considered significant at the 5% level. The SPSS software 9.0 (SPSS, Chicago, IL) was used in the statistical analyses. The values are expressed as means SD, unless otherwise stated. RESULTS The age of the subjects ranged from 22 to 57 y and their BMI at the baseline ranged from 20 to 29 kg/m 2. The BMI of the subjects was unchanged during the study (Fig. 1). The dietary intake of nutrients at baseline and during the low and high folate diet periods is presented in Table 1. The high folate diet resulted in significant increases in the serum folate TABLE 1 Nutrient intakes of women at baseline and after consumption of low and high folate diets1 Baseline Low folate diet High folate diet Carbohydrate,2 E% Protein, E% Total fat, E% Saturated fatty acids, E% Monounsaturated fatty acids, E% Polyunsaturated fatty acids, E% Dietary fiber, g Calcium, mg Potassium, mg Iron, mg Folate,3 g Vitamin B-6,3 mg Vitamin B-12,3 g The intakes at the baseline were calculated from the 4-d food records, and the intakes during the low and high folate diet periods were analyzed from the duplicate portions. The intakes of folate and vitamins B-6 and B-12 were calculated. 2 E%, percentage of total energy intake. 3 Calculated. concentration (78%) (P 0.001) and in the RBC folate concentration (14%) (P 0.001) compared with the low folate diet. The plasma thcy concentration decreased by 13% (P 0.001) when the women consumed the high folate diet. The serum concentration of vitamin B-12 was lower (P 0.05) at baseline (328 nmol/l) than during the low and high folate diet periods (366 and 351 nmol/l, respectively). There were no differences in the concentrations of serum vitamin B-12 between the low and high folate diet periods. The frequencies of the C/C, C/T and T/T genotypes of the MTHFR gene among the subjects were 51, 35 and 14%, respectively. The serum folate concentrations at baseline did not differ among the subjects with different genotypes of the MTHFR gene (Table 2). The basal thcy concentration tended to be higher (P 0.251) in the subjects homozygous for the T677 allele in the MTHFR gene than in the subjects with the genotypes C/T and C/C (Table 2). When the subjects consumed the low folate diet, the serum folate and plasma thcy concentrations remained at the baseline level. When they consumed the high folate diet, serum folate increased by 85% (P 0.001), 77% (P 0.001) and 55% (P 0.05) in the subjects with the genotypes C/C, C/T and T/T, respectively, compared with the low folate diet. Also, the plasma thcy concentration decreased by 11% (P 0.001), 15% (P 0.01) and 18% (P 0.05) in the subjects with the genotypes C/C, C/T and T/T, respectively, when they consumed the high folate diet. When tested by ANOVA of repeated measurements across time, the dietary changes affected the serum folate (P 0.001) and plasma thcy (P 0.001). However, no MTHFR genotype effect was found in the diet-induced changes in the serum folate and plasma thcy concentrations. Thirty-one subjects (84%) did not have the 68-bp insertion in the CBS gene, whereas six subjects (16%) were heterozygous for the 844ins68 of the CBS gene. There were no differences in the plasma thcy concentrations of the subjects related to the 844ins68 of the CBS gene (Table 3). The subjects carrying the CBS 844ins68 of the CBS gene had a larger decrease (P 0.05) of plasma thcy due to the high folate diet TABLE 2 Concentrations of serum folate and plasma total homocysteine (thcy) of women according to their C677T genotypes of the MTHFR gene at baseline and after consumption of low and high folate diets1,2 C/C, (n 19) C/T, (n 13) T/T, (n 5) Serum folate, nmol/l Baseline Low folate diet High folate diet Percentage change from low to high folate diet 85 51*** 77 50*** 55 33* Plasma total homocysteine, mol/l Baseline Low folate diet High folate diet Percentage change from low to high folate diet 11 9*** 15 18** 18 9* 1 Values are means SD. *P 0.05; ** P 0.01; *** P 0.001: significant change within genotype; (Student s t test for paired samples). 2 MTHFR, methylenetetrahydrofolate reductase.

4 2646 SILASTE ET AL. TABLE 3 Plasma total homocysteine (thcy) concentration of women according to their 844ins68 genotypes of the CBS gene at baseline and after consumption of low and high folate diets1,2 844ins68 /, (n 31) 844ins68 /, (n 6) Plasma total homocysteine, mol/l Baseline Low folate diet High folate diet Percentage change from low to high folate diet 12 12** 22 16* Percentage change from baseline to high folate diet 13 14** Values are means SD. * P 0.05, ** P 0.001: significant change within genotype (Student s t test for paired samples). P 0.05: Significant difference between genotypes (Student s t test for independent samples). 2 CBS, cystathionine -synthase. than the subjects without the insertion (Table 3). There was no CBS genotype effect in the diet-induced changes of the serum folate and plasma thcy concentrations. The genotype frequencies of the A/A (wild-type), A/G and G/G genotypes of the A2756G mutation of the MS gene were 59, 38 and 3%, respectively. Because there was only one subject homozygous for the G2756 allele, the genotypes A/G and G/G were combined for statistical analyses. The MS genotype affected the plasma thcy concentration during the study (P 0.05). The subjects with the allele G2756 tended (P 0.110) to have lower plasma thcy concentration than the subjects with a genotype A/A (Table 4). The subjects with the allele G2756 also had a lower plasma thcy (P 0.01) during the high folate diet period and a more extensive decrease in plasma thcy (P 0.05) due to the high folate diet than the subjects without the mutation. DISCUSSION In the present study, the diet high in natural folate resulted in similar concentrations of plasma thcy among the subjects with the different genotypes of MTHFR gene. The subjects homozygous for the T677 allele (genotype T/T) of the MTHFR gene tended to have the most extensive reduction ( 18%) in the plasma thcy concentration during the diet high in natural folate. This finding is in line with a previous study (26) in which the subjects homozygous for the T677 allele had a larger decrease in plasma thcy after folic acid supplementation than the subjects heterozygous for the mutation or with a wild type (genotype C/C) of the gene. According to several previous studies (16,19,27 30), subjects homozygous for the T677 allele in the MTHFR gene have a higher fasting plasma thcy concentration and a lower serum folate concentration before vitamin supplementation than the subjects with the genotypes C/T and C/C. In the present study, the differences between the basal concentrations of plasma thcy in the different genotypes were not significant. However, the subjects homozygous for the T677 allele tended to have higher mean plasma thcy concentrations at baseline than the subjects with the genotypes C/T or C/C. The basal concentrations of plasma thcy in the present study were quite low (mean 8.2 mol/l) compared with the suggested optimal level ( 10 mol/l) (31). Also, none of the subjects had a plasma thcy concentration 15 mol/l, which is the limit for mild hyperhomocysteinemia. The small number of subjects and the lack of subjects with hyperhomocysteinemia in the present study may explain in part the lack of differences between the basal concentrations of plasma thcy of the subjects with different genotypes of the MTHFR gene. In our study, the serum folate concentrations of the subjects with the genotypes C/C, C/T and T/T of the MTHFR were similar at baseline. The dietary intake of folate at baseline was not low, which may explain in part the lack of difference in the initial serum folate concentration among the MTHFR genotypes. In a previous study (19), subjects homozygous for the T677 allele of the MTHFR gene had a higher plasma thcy concentration when plasma folate was in the lower range but not when plasma folate was high. These researchers (19) suggested that homozygous subjects have a higher folate requirement for the regulation of plasma thcy. In our study, the subjects homozygous for the T677 allele showed a good response of plasma thcy to a high intake of natural folate, even though they tended to have a lower response of serum folate than the subjects with the genotypes C/T or C/C. Thus, our study does not support the theory that the subjects homozygous for the T677 allele need more folate to control the plasma thcy concentration. On the other hand, we used natural folate from food, which might affect the serum folate and plasma thcy concentrations differently than folic acid. Also, we used a whole-diet approach, and other dietary components, such as vitamins B-6 and B-12, may have contributed to the increase in the serum folate concentration. However, the calculated intake of these vitamins did not differ between the diet periods, and the plasma concentration of vitamin B-12 did not change during the study. Therefore, effects of vitamins B-6 and B-12 on plasma thcy are unlikely in the current study. In addition to the MTHFR gene polymorphism, we detected the 68-bp insertion of the CBS gene and the A2756G transition of the MS gene. Both the presence of the 68-bp insertion of the CBS gene and the G2756 allele of the MS gene are associated with lower fasting levels of plasma thcy (14). Whether these gene mutations are associated with the diet responsiveness has not been determined. In the present study, we found no differences between the basal thcy levels of the different genotypes of either the CBS or the MS genes. The more extensive decrease in the plasma thcy concentration of the subjects with the 68-bp insertion of the CBS gene is explained by the higher plasma thcy TABLE 4 Plasma total homocysteine (thcy) concentration of women according to their A2756G genotypes of the MS gene at baseline and after consumption of low and high folate diets1,2 A/A, (n 22) A/G and G/G, (n 15) Plasma total homocysteine, mol/l Baseline Low folate diet High folate diet Percentage change from low to high folate diet 9 11* 19 14* 1 Values are means SD. *P 0.001: significant change within genotype (Student s t test for paired samples). P 0.05, P 0.01: significant difference between genotypes (Student s t test for independent samples). 2 MS, methionine synthase.

5 GENE POLYMORPHISMS AND PLASMA HOMOCYSTEINE 2647 of those subjects during the low folate diet period than at the baseline. However, the G2756 allele of the MS gene was associated with a lower plasma thcy and a larger decrease in plasma thcy during the low folate diet period compared with the subjects with the genotype A/A. In the present study, the duration of the diet periods was 5 wk and the length of the washout period was 3 wk. The washout period was quite short compared with the previous study (2) testing the effects of a low dose folic acid supplementation on the plasma thcy concentration. In that study (2), an 8-wk washout period was not sufficient for blood folate and plasma thcy concentrations to return to the baseline concentrations. In the present study, the subjects who initially consumed the high folate diet tended to have a higher (P 0.10) serum folate concentration during the low folate diet period than at baseline. In addition, they tended to have a lower (P 0.07) plasma thcy concentration during the low folate diet period than at baseline. The differences and the carryover effect were small and may have occurred by chance. However, the relatively short washout period may have masked some of the effects of the high folate diet in the present study. The number of subjects in the present study was quite small. Therefore, the results should be considered as preliminary data. However, this study suggests that the diet response of plasma homocysteine may be genetically regulated. It was of particular interest that the plasma thcy of the subjects homozygous for the T677 allele of the MTHFR gene responded well to a high intake of natural folate, resulting in similar plasma thcy levels as in the genotypes C/T and C/C. Also, the presence of the G2756 allele of the MS gene was associated with a more extensive decrease in plasma thcy and a lower plasma thcy concentration during the high folate diet period. 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