Homocysteine is an intermediate amino acid in the metabolism of the essential amino acid methionine (Castro et al. Reference Castro, Rivera, Blom, Jakobs and de Almeida2006). Elevated plasma total homocysteine (tHcy) concentration has been related to several adverse conditions and diseases, including adverse pregnancy outcomes (Scholl & Johnson, Reference Scholl and Johnson2000; Daly et al. Reference Daly, Cotter, Molloy and Scott2005), cognitive dysfunction among elderly (Morris, Reference Morris2003) and recently osteoporosis (Gjesdal et al. Reference Gjesdal, Vollset, Ueland, Refsum, Drevon, Gjessing and Tell2006). Plasma tHcy also predicts CVD risk in prospective studies (de Bree et al. Reference de Bree, Verschuren, Kromhout, Kluijtmans and Blom2002; Wald et al. Reference Wald, Law and Morris2002), but intervention trials do not demonstrate risk reduction by tHcy-lowering therapy with high doses of B-vitamins in patients with established CVD (Clarke, Reference Clarke2005; Bonaa et al. Reference Bonaa, Njolstad, Ueland, Schirmer, Tverdal, Steigen, Wang, Nordrehaug, Arnesen and Rasmussen2006; Loscalzo, Reference Loscalzo2006).
Elevated tHcy is observed under a variety of conditions, e.g. deficiency of B-vitamins (Verhoef & de Groot, Reference Verhoef and de Groot2005; Castro et al. Reference Castro, Rivera, Blom, Jakobs and de Almeida2006), smoking (Nygard et al. Reference Nygard, Refsum, Ueland and Vollset1998; Ortega et al. Reference Ortega, Requejo, Lopez-Sobaler, Navia, Mena, Basabe and Andres2004), high coffee consumption (Ranheim & Halvorsen, Reference Ranheim and Halvorsen2005; Verhoef & de Groot, Reference Verhoef and de Groot2005) and impaired renal function (Perna et al. Reference Perna, Ingrosso, Satta, Lombardi, Acanfora and De Santo2004; Castro et al. Reference Castro, Rivera, Blom, Jakobs and de Almeida2006). The concentration of tHcy in plasma can be lowered by enhanced remethylation of homocysteine into methionine or via degradation through the transsulphuration pathway. Folate or betaine donate a methyl group, while vitamin B12, vitamin B6 and riboflavin act as cofactors for enzymes involved in homocysteine metabolism (Finkelstein, Reference Finkelstein1990; Ueland et al. Reference Ueland, Holm and Hustad2005; Castro et al. Reference Castro, Rivera, Blom, Jakobs and de Almeida2006).
Several studies report an association between intake of different nutrients and plasma tHcy concentration (Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001; Verhoef & de Groot, Reference Verhoef and de Groot2005; Castro et al. Reference Castro, Rivera, Blom, Jakobs and de Almeida2006). Many studies, particularly the two largest studies – the Framingham Offspring study (n 1960) and the Dutch study (n 2435) – have found inverse associations between plasma tHcy and intake of folate, vitamin B12, vitamin B6 and riboflavin (de Bree et al. Reference de Bree, Verschuren, Blom and Kromhout2001; Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001). Also, mandatory food fortification with folic acid has increased the level of plasma folate and decreased tHcy concentration in the general US population (Jacques et al. Reference Jacques, Selhub, Bostom, Wilson and Rosenberg1999; Pfeiffer et al. Reference Pfeiffer, Caudill, Gunter, Osterloh and Sampson2005; Ganji & Kafai, Reference Ganji and Kafai2006).
There are contradictory reports on the relation between protein and methionine to plasma tHcy concentration (Verhoef & de Groot, Reference Verhoef and de Groot2005). A few studies that examined the associations with total fat, cholesterol, saturated fat, mono- and polyunsaturated fat (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997) and n-3 fatty acids (Brude et al. Reference Brude, Finstad, Seljeflot, Drevon, Solvoll, Sandstad, Hjermann, Arnesen and Nenseter1999) did not show statistically significant associations with tHcy concentration. Although less studied, dietary choline and betaine have been inversely related to plasma tHcy (Verhoef & de Groot, Reference Verhoef and de Groot2005; Cho et al. Reference Cho, Zeisel, Jacques, Selhub, Dougherty, Colditz and Willett2006).
The alteration in plasma homocysteine concentration may vary substantially depending on the food source (Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001). Previous studies show that tHcy concentration is inversely related to the intake of food groups such as bread (Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001), cereals (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997; Ganji & Kafai, Reference Ganji and Kafai2004), fruits (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001), vegetables (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Brude et al. Reference Brude, Finstad, Seljeflot, Drevon, Solvoll, Sandstad, Hjermann, Arnesen and Nenseter1999; Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001), and to individual food items including cruciferous vegetables (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Ganji & Kafai, Reference Ganji and Kafai2004), peppers (Ganji & Kafai, Reference Ganji and Kafai2004), citrus fruits and juices (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001), cold breakfast cereals (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997; Ganji & Kafai, Reference Ganji and Kafai2004), milk (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997; Ganji & Kafai, Reference Ganji and Kafai2004), yoghurt (Ganji & Kafai, Reference Ganji and Kafai2004) and liver (Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001). Positive associations have been found with caffeine-containing drinks such as coffee (Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001; Verhoef & de Groot, Reference Verhoef and de Groot2005), Coca Cola (Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001) and tea (Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001; Verhoef & de Groot, Reference Verhoef and de Groot2005), while findings on alcohol are conflicting (Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001; Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001). To understand the associations between plasma tHcy concentration and foods with high and low B-vitamin content, we included major food groups and individual food items in the present study. Moreover, we examined to what extent dietary intake of folate, vitamin B12, vitamin B6, riboflavin and other nutrients may alter the plasma concentration of tHcy. We also investigated nutrients other than B-vitamins because our comprehensive dietary data allowed us to quantitatively assess the confounding effect of B-vitamins. Thus, the objective of the present paper was to assess the relationship between nutrients and food intake on plasma concentration of tHcy in middle-aged and older men and women in a large population-based study.
Subjects and methods
Subjects
The second round of the Hordaland Homocysteine Study was conducted as part of the Hordaland Health Study (HUSK), from 1997 to 1999 as a collaboration between the National Health Screening Service (now the Norwegian Institute of Public Health), The University of Bergen and local health services. Of the total sample of 9187 men and women born 1925–7 and 1950–1 who were invited to participate in the Hordaland Health Study, 7074 (77 %) agreed to participate. The participants underwent a brief health examination and donated a non-fasting blood sample. Information on diet and lifestyle was collected via self-administered questionnaires. In total 6140 subjects (87 %) completed a FFQ and 6118 of these also had plasma tHcy values measured. The study protocol was approved by the Regional Committee for Medical Research Ethics and the Norwegian Data Inspectorate. All subjects gave their written consent to participate in the study.
We excluded participants with reported energy intake below the 2·5 percentile (2124 kJ for women 71–74 years and 3899 kJ for women 47–49 years; 3856 kJ for men 71–74 years and 5572 kJ for men 47–49 years) and above the 97·5 percentile (11 098 kJ for women 71–74 years and 12 970 kJ for women 47–49 years; 14 023 kJ for men 71–74 years and 17 590 kJ for men 47–49 years). Thus, 141 men and 187 women were excluded from further analyses, yielding a final number of 5812 participants. Participants with reported seasonal or regular intake of at least one dose of multi- or/and individual vitamin supplement (excluding fish oil and n-3 fatty acids) per day were assigned to the vitamin supplement user group (n 1234).
FFQ
In the present study we used a 169-item FFQ, a slightly modified version of a FFQ previously described in detail (Andersen et al. Reference Andersen, Solvoll, Johansson, Salminen, Aro and Drevon1999). This FFQ aimed to capture the habitual diet during the past year. The FFQ includes frequency alternatives (from once a month to several times per day), the number of units eaten and portion sizes (e.g. slices, glasses, cups, pieces, spoons).
The information from the FFQ is presented as individual food items, food groups (consisting of individual food items) and nutrients. Individual food items correspond to the items listed on the questionnaire, whereas ‘food groups’ include related food items (e.g. the food group ‘bread’ contains ‘bread with low, medium and high quantity of fibre’).
In addition to individual food items, the FFQ also includes nine questions about the most commonly used brands of single- and multi-vitamin supplements on the market. Subjects using at least one dose of vitamin supplement per day seasonally or regularly during the past year were classified as vitamin supplement users.
Daily nutrient intakes were computed from a database and software system developed at the Department of Nutrition, University of Oslo (KBS, version 3.2). The nutrient database is mainly based on the official Norwegian food composition table with an update on folate content from 2001 (Rimestad et al. Reference Rimestad, Borgejordet, Vesterhus, Sygnestveit, Løken, Trygg, Pollestad, Lund-Larsen, Omholt-Jensen and Nordbotten2001). B-vitamin intake in supplements is calculated from information on the contents of vitamin supplements for sale during 1997–9.
Health examination and analytic procedures
A brief examination included measurements of height and weight and drawing of non-fasting blood samples. Tubes with plasma containing EDTA were stored at − 80°C. Plasma tHcy was determined by automated HPLC with fluorescence detection. Intra-assay CV was 3 % (Fiskerstrand et al. Reference Fiskerstrand, Refsum, Kvalheim and Ueland1993). The concentration of plasma folate was measured by a Lactobacillus casei microbiological assay (Molloy & Scott, Reference Molloy and Scott1997) and plasma vitamin B12 concentration by a Lactobacillus leichmannii microbiological assay (Kelleher & Broin, Reference Kelleher and Broin1991). The concentration of serum creatinine was measured with standard alkaline picrate colorimetric assay.
Statistical analysis
Differences between non-users and users of vitamin supplements were assessed by linear regression with adjustment for age and sex for continuous variables and logistic regression with adjustment for age and/or sex for categorical variables. Spearman's partial correlation coefficients were used to estimate associations between dietary and plasma B-vitamins, other nutrients and food groups. Associations between predictor variables (food groups and nutrients) and plasma tHcy concentration were analysed separately for non-users and users of vitamin supplements by multiple regression adjusted for age, sex and energy intake. Quartiles of predictor variables were defined separately for vitamin supplement users and non-users. Results were combined for the two age groups and for men and women, because the observed trends were similar in all the four age–sex groups. The potential effect modification (interaction) of vitamin supplement use on associations between diet and plasma tHcy was assessed by linear regression analysis with product terms of supplement use (0 or 1) and each dietary predictor (with both main effects in the model) adjusted for age, sex and energy intake. We found an interaction effect of vitamin user group on the associations with several food groups and nutrients. Thus, due to the present results and the fact that B-vitamin supplementation may substantially reduce plasma tHcy concentration and therefore mask the effects of dietary components, we restricted the study sample to non-users of vitamin supplements in further in-depth analyses.
Multiple linear regression analysis was used to assess the simultaneous relations between various predictors of plasma tHcy concentration. The predictor variables (food groups, food items and nutrients) were categorized in quartiles, excluding refined bread. Due to low consumption of refined bread (29 % of participants), three non-equal categories were used: ‘non-user’, ‘0–25 g/d’, ‘>25 g/d’, separating users in two groups of similar sizes. Thus, the regression coefficients estimated the mean differences in plasma tHcy concentration between quartiles of the predictor variables. Several models were used: Model I was adjusted for age, sex and energy intake (Table 2); Model II included additional adjustment for smoking and coffee consumption, as well as for BMI and serum creatinine (Tables 4 and 5); and Model III was further adjusted for dietary B-vitamin intake (folate, vitamin B12, vitamin B6, riboflavin) and plasma concentrations of folate and vitamin B12 (Tables 4 and 5). Findings with adjustment for dietary but not plasma B-vitamins are presented in the text only. We used additional adjustment for plasma folate and vitamin B12 because their associations with tHcy were stronger than between tHcy and dietary B-vitamins. In addition, concentrations of folate and vitamin B12 in plasma were relatively weakly correlated with the intake of dietary B-vitamins (Table 3). The continuous predictor variables were categorized in quartiles to use the mean difference of homocysteine per increasing quartile as the main effect measure. All statistical analyses were performed using SAS for Windows version 9.1 (SAS Institute Inc., Cary, NC, USA).
Results
Characteristics of study population
Descriptive statistics of the study population and its dietary habits are shown in Table 1. There was a higher proportion of women in the group of vitamin supplement users compared to non-users. In age- and sex-adjusted analyses, supplement users had a lower proportion of smokers, lower BMI and coffee consumption, and higher intakes of cereals, fruits, vegetables and fish compared to non-users. The former group also had lower concentrations of tHcy, whereas folate and vitamin B12 were higher.
Table 1 Characteristics of the study population and its diet, stratified by vitamin supplement use in the Hordaland Homocysteine Study
* Two-sided P value for the difference between non-users and users of vitamin supplements. Logistic regression for the variables age, sex and smoking adjusted for age and/or sex, linear regression analyses for all other variables (categorized in quartiles) adjusted for age and sex.
† Current smokers versus former and never smokers combined.
‡ Includes intake from food and supplements.
A lower intake than recommended in the Nordic Nutritional Recommendations (NNR) of folate (300 μg/d) was observed among 39 % of vitamin supplement users and among 59 % of non-users; the corresponding proportions were 1 % and 3 % for vitamin B12 (NNR: 2 μg/d), respectively. Less than the recommended intake of vitamin B6 (NNR: 1.2 mg/d for women, 1·6 mg/d for men) was registered among 49 % of women and 57 % of men in the group of non-users of vitamin supplements and 31 % and 38 % in the group of users, respectively. Less than the recommended intake of riboflavin (NNR: 1·3 mg/d for women, 1·7 mg/d for men) was observed among 44 % of the women and 48 % of the men among non-users of vitamin supplements and 22 % of the women and 28 % of the men among supplement users.
Relation between dietary intake and total homocysteine among non-users and users of vitamin supplements
Table 2 shows mean differences in plasma tHcy concentration between quartiles of food group and nutrient intake among non-users and users of vitamin supplements, after adjustment for age, sex and energy intake. While the associations between food groups and nutrients with plasma tHcy were mostly similar between users and non-users of vitamin supplements, there were some marked exceptions. Intake of vegetables was significantly inversely associated with plasma tHcy among non-users only, whereas meat intake was significant only among vitamin supplement users. For these food groups, as well as for eggs, fats, sweets, protein and the nutrient fat, there was a significant effect modification of vitamin supplement use on the associations with tHcy. There was a similar effect for energy intake, which was significantly inversely related to tHcy among non-users only.
Table 2 Mean difference in plasma total homocysteine concentration per increasing quartile of food group, nutrient intake, smoking and coffee consumption among non-users and users of vitamin supplements in the Hordaland Homocysteine Study
* Model I adjusted for age, sex and energy intake.
† Two-sided P value for the effect of food group, nutrient, smoking and coffee consumption.
‡ Two-sided P value for interaction of vitamin user group on the association between predictor variables and total homocysteine concentration, adjusted for age, sex and energy intake.
§ Adjusted for age and sex.
Correlations between dietary and plasma B-vitamins, food groups and other macro- and micro-nutrients
Dietary folate intake was significantly positively correlated with the intake of vegetables, fruits and fibre, as well as with most other vitamins, except vitamin D (Table 3). Dietary vitamin B12 was most strongly associated with fish intake, and also with meat, milk, protein, cholesterol and vitamin B6 and riboflavin. Dietary folate, B6 and riboflavin were positively associated with the intake of complex carbohydrates, and inversely associated with the intake of simple carbohydrates. Fat intake was inversely related to plasma folate and the dietary B-vitamins, except dietary B12.
Table 3 Correlation between dietary intake and plasma B-vitamin concentration, other nutrients and food groups among non-users of vitamin supplements in the Hordaland Homocysteine Study*
* Spearman's partial correlation coefficients adjusted for age, sex and energy intake. All coefficients are significant, except those marked † (P >0·01).
Plasma concentration of folate was moderately correlated with intake of fruits and vegetables, as well as with dietary folate and vitamin B6. Plasma vitamin B12 was significantly associated with fish and milk intake, as well as with dietary vitamin B12 and riboflavin. Plasma tHcy had stronger correlations with plasma folate and vitamin B12 than with dietary intake of folate, vitamin B12, vitamin B6 and riboflavin.
Plasma total homocysteine according to food groups and food items intake
Table 4 shows mean differences in plasma tHcy concentration between quartiles of food group and food item intake with two different adjustment models. In comparison with the data in Table 2 adjusted only for age, sex and energy intake, further adjustment for smoking and coffee consumption made the association between tHcy and cakes stronger and statistically significant, whereas the association with fats became weaker and non-significant. For individual variables, significantly lower plasma tHcy was observed for higher intakes of citrus fruits, orange juice, cruciferous vegetables, spinach/green cabbage, non-processed meat and chicken, while a higher intake of processed meat was associated with higher tHcy concentrations. Further adjustments for BMI and serum creatinine only marginally altered the results, and are therefore not presented.
Table 4 Mean difference in plasma total homocysteine concentration per increasing quartiles of food intake among non-users of vitamin supplements in the Hordaland Homocysteine Study
* Model II adjusted for age, sex, energy intake, smoking and coffee consumption.
† Two-sided P value for the effect of food group and food item.
‡ Model III adjusted for age, sex, smoking, coffee consumption, intake of dietary energy, folate, vitamin B12, vitamin B6 and riboflavin, and plasma folate and vitamin B12.
Further adjustments for dietary B-vitamins generally resulted in weaker associations for all food variables, and the groups of vegetables, non-processed meat, processed meat, chicken, fish and milk were no longer significantly associated with tHcy (data not shown). The groups of fruits and orange juice were still inversely associated, while cakes and sweets were positively associated with tHcy concentration. After additional adjustment for plasma folate and vitamin B12 the associations with fruits, orange juice and cakes became statistically non-significant, while sweets still related positively to tHcy. The inverse association with whole-grain bread and non-processed meat became stronger.
Plasma total homocysteine according to nutrient intake
Differences in plasma concentration of tHcy per increasing quartiles of nutrient intakes are shown in Table 5. After adjustment for smoking and coffee consumption most of the associations became weaker, while for protein, simple carbohydrates, cholesterol, vitamin B12 and vitamin D became stronger. The inverse association between cholesterol intake and plasma tHcy concentration became statistically significant. We observed inverse associations between dietary intake of thiamine, vitamins A (retinol equivalent and β-carotene), E (α-tocopherol), D and C, and the plasma tHcy concentration. Adjustment for serum creatinine and BMI only marginally altered the results, and these data are therefore not presented.
Table 5 Mean difference in plasma total homocysteine concentration per increasing quartiles of nutrient intake among non-users of vitamin supplements in the Hordaland Homocysteine Study
* Model II adjusted for age, sex, energy intake, smoking and coffee consumption.
† Two-sided P value for the effect of nutrient.
‡ Model III adjusted for age, sex, smoking, coffee consumption, intake of energy, dietary folate, vitamin B12, vitamin B6 and riboflavin, and plasma folate and vitamin B12.
§ Not adjusted for energy intake.
∥ Not adjusted for plasma folate and vitamin B12.
After additional adjustment for dietary B-vitamins and plasma concentration of folate and vitamin B12, only complex carbohydrates (inversely), thiamine (inversely) and fat (positively) remained associated with plasma tHcy. Dietary folate and riboflavin (not adjusted for plasma concentrations) remained strongly associated with tHcy even after adjustment for the other dietary B-vitamins. This was not seen for vitamins B12 and B6 after similar adjustment. Vitamin C, retinol equivalent, β-carotene and simple carbohydrates remained significantly associated with tHcy after adjustment for dietary B-vitamins, but not after additional adjustment for plasma folate and vitamin B12.
Discussion
In a large population-based sample of non-users of vitamin supplements, we observed that plasma tHcy is associated with several nutrients and foods. While the intake of complex carbohydrates and protein as well as several B-vitamins were associated with lower tHcy concentrations, the intake of fat and sugar were associated with higher plasma tHcy. Consumption of vegetables, fruits, bread, cereals, fish, non-processed meat, chicken, eggs and milk was associated with lower tHcy concentrations, while intake of sweets, cakes and processed meat was related to higher tHcy concentrations. The present results confirm the importance of lifestyle factors as a major determinant of tHcy.
Limitations and strengths
The current study was conducted in a large population-based sample, using a validated 169-item quantitative FFQ (Andersen et al. Reference Andersen, Nes, Lillegaard, Sandstad, Bjorneboe and Drevon1995, Reference Andersen, Solvoll, Johansson, Salminen, Aro and Drevon1999). This allowed the investigation of dietary predictors of tHcy in multivariate models. A limitation relates to the collection of dietary data using a FFQ, which has inherent potential problems related to inaccuracy and potential misclassification in the estimation of nutrient intake (Willett & Lenart, Reference Willett, Lenart and Willett1998; Flood et al. Reference Flood, Smith, Webb and Mitchell2004): reported associations between dietary non-B-vitamin nutrients and tHcy concentration could be due to confounding effects of dietary B-vitamin intake (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997; Brude et al. Reference Brude, Finstad, Seljeflot, Drevon, Solvoll, Sandstad, Hjermann, Arnesen and Nenseter1999). We therefore repeated the analyses using a model that included adjustment for dietary and plasma folate and vitamin B12 when examining these associations.
Users and non-users of vitamin supplements
In accordance with previous reports (Tucker et al. Reference Tucker, Selhub, Wilson and Rosenberg1996; Nygard et al. Reference Nygard, Refsum, Ueland and Vollset1998; Malinow et al. Reference Malinow, Bostom and Krauss1999; Koehler et al. Reference Koehler, Baumgartner, Garry, Allen, Stabler and Rimm2001), we observed that users and non-users of vitamin supplements differed with respect to lifestyle, dietary habits and plasma vitamin concentrations. Users were more likely to be non-smokers, consume less coffee and generally have a more healthy diet compared to non-users, and to have lower concentrations of plasma tHcy, but higher concentration of folate and vitamin B12 as compared to non-users. There was an effect modification of vitamin supplement use on the association between various nutrients, food groups and plasma tHcy. We are not aware that this has previously been reported.
B-vitamins
The present findings confirm previous studies reporting inverse associations between dietary intake of folate, vitamin B12, vitamin B6 and riboflavin, and plasma concentration of tHcy (de Bree et al. Reference de Bree, Verschuren, Blom and Kromhout2001, Reference de Bree, Verschuren, Kromhout, Kluijtmans and Blom2002; Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001; Verhoef & de Groot, Reference Verhoef and de Groot2005). However, only folate and riboflavin remained significantly related to tHcy after adjusting for other dietary B-vitamins. This does not negate an effect of vitamins B12 and B6 on plasma tHcy concentration, because intake of these four B-vitamins is to a significant extent strongly intercorrelated (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997) due to similar dietary sources. Second, because these B-vitamins are all involved in homocysteine metabolism, they have a complementary lowering effect on tHcy concentration (Bostom et al. Reference Bostom, Jacques, Liaugaudas, Rogers, Rosenberg and Selhub2002; Huerta et al. Reference Huerta, Gonzalez, Vigil, Prada, San Martin, Fernandez, Patterson and Lasheras2004). Adjustment of an individual B-vitamin for other vitamins in a related metabolic cycle could significantly reduce or mask their individual effect. Among the four B-vitamins, our analysis confirmed that the individual effect of folate was stronger than the effects of riboflavin, vitamin B12 and vitamin B6 (de Bree et al. Reference de Bree, Verschuren, Blom and Kromhout2001; Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001).
Other nutrients
We found significant inverse associations between plasma tHcy and dietary intake of vitamins C, E and D, retinol equivalent and β-carotene, in accordance with previous reports (Brude et al. Reference Brude, Finstad, Seljeflot, Drevon, Solvoll, Sandstad, Hjermann, Arnesen and Nenseter1999; Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001). The intake of these vitamins is correlated with dietary B-vitamins, indicating overlapping dietary sources. Nevertheless, after adjustment for dietary B-vitamins, retinol equivalents, β-carotene and vitamin C remained significantly inversely related to plasma tHcy concentration. However, after further adjustments for plasma folate and vitamin B12, these associations were no longer significant.
We observed an inverse association between protein intake and plasma tHcy (Stolzenberg-Solomon et al. Reference Stolzenberg-Solomon, Miller, Maguire, Selhub and Appel1999; Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001). However, there are strong positive correlations between dietary intake of protein and riboflavin, vitamin B12 and vitamin B6, and after adjusting for dietary B-vitamins the association between protein and tHcy became non-significant. As for protein, the association between fibre intake and plasma tHcy was no longer significant after adjustment for the intake of B-vitamins.
The intake of complex carbohydrates was inversely related to tHcy concentration, an association that remained significant after adjusting for dietary and plasma B-vitamins. Complex carbohydrates is a macro-nutrient that is not involved in methionine–homocysteine metabolism, thus the inverse association with tHcy could be due to residual confounding from common dietary sources with B-vitamins. Another possible explanation is related to the construction and limitations of the FFQ. For example, choline and betaine are not included in the Norwegian food composition tables, and we could therefore not examine whether the associations could be due to the dietary content of these factors. Some studies (Sakamoto et al. Reference Sakamoto, Nishimura, Ono and Sakura2002; Zeisel et al. Reference Zeisel, Mar, Howe and Holden2003) as well as the USDA National Nutrient Database for Standard Reference (US Department of Agriculture, 2005) report a high content of these quaternary ammonium compounds in some plant food, and negative associations between intake and plasma concentrations of choline, betaine and plasma tHcy have been reported in previous studies (Olthof et al. Reference Olthof, Brink, Katan and Verhoef2005; Ueland et al. Reference Ueland, Holm and Hustad2005; Cho et al. Reference Cho, Zeisel, Jacques, Selhub, Dougherty, Colditz and Willett2006).
Food groups and food items
In line with previous studies (Vollset et al. Reference Vollset, Refsum, Nygård, Ueland, Carmel and Jacobsen2001; Ganji & Kafai, Reference Ganji and Kafai2004), we found inverse associations between plasma tHcy and dietary intake of citrus fruit, orange juice, cruciferous vegetables, spinach/green cabbage and peppers. Furthermore, we observed, as have others, that plasma tHcy is inversely associated with milk (Shimakawa et al. Reference Shimakawa, Nieto, Malinow, Chambless, Schreiner and Szklo1997; Ganji & Kafai, Reference Ganji and Kafai2004) and fish (Brude et al. Reference Brude, Finstad, Seljeflot, Drevon, Solvoll, Sandstad, Hjermann, Arnesen and Nenseter1999). We also found inverse relations with apples, carrots, onions, lettuce, cucumbers, tomatoes, mushrooms and eggs.
The overall food group of meat was not significantly related to tHcy. However, further examination revealed that non-processed meat and chicken were inversely, while processed meat was positively associated with tHcy. Non-processed meat is a source of protein, vitamin B12, vitamin B6, riboflavin, choline and betaine which all are negatively associated with plasma tHcy (Holm et al. Reference Holm, Ueland, Vollset, Midttun, Blom, Keijzer and den Heijer2005; Cho et al. Reference Cho, Zeisel, Jacques, Selhub, Dougherty, Colditz and Willett2006). Processed meat contains less protein and B-vitamins but more fat than non-processed meat. The nutrient fat is positively related to tHcy, and this may partly explain the positive association with processed meat (Appel et al. Reference Appel, Miller, Jee, Stolzenberg-Solomon, Lin, Erlinger, Nadeau and Selhub2000). Associations between tHcy and different types of fat will be examined in more detail in a forthcoming paper from our group (P Berstad et al., unpublished results).
The intake of sweets and cakes, high in fat and sugar, was associated with high tHcy concentrations, consistent with the present finding that the intake of simple carbohydrates (sugar) was negatively associated with dietary folate, vitamin B12, vitamin B6 and riboflavin.
After additional adjustment for dietary B-vitamins and for plasma folate and vitamin B12, whole-grain bread, eggs, sweets, citrus fruits, carrots, onions and non-processed meat remained significantly associated with plasma tHcy concentration. This could be due to residual confounding, or that these food groups or items contain choline, betaine or methionine (Jacques et al. Reference Jacques, Bostom, Wilson, Rich, Rosenberg and Selhub2001; Verhoef & de Groot, Reference Verhoef and de Groot2005; Cho et al. Reference Cho, Zeisel, Jacques, Selhub, Dougherty, Colditz and Willett2006). A high content of these compounds has been reported in eggs, whole-wheat bread, meat and liver, and a lower content in fruits and vegetables (Zeisel et al. Reference Zeisel, Mar, Howe and Holden2003; US Department of Agriculture, 2005). The relationship between tHcy concentration and dietary choline, betaine and methionine should be further examined.
Because people do not eat individual nutrients and food items in isolation, it is important to examine the effect of the total diet on plasma tHcy concentration. Although in the present study we did not evaluate the effect of the total diet, the present results are in general agreement with studies that have examined associations between dietary patterns and tHcy (Fung et al. Reference Fung, Rimm, Spiegelman, Rifai, Tofler, Willett and Hu2001; Gao et al. Reference Gao, Yao, McCrory, Ma, Li, Roberts and Tucker2003; Lasheras et al. Reference Lasheras, Huerta, Gonzales, Prada, Braga, Fernandez and Patterson2003; Weikert et al. Reference Weikert, Hoffmann, Dierkes, Zyriax, Klipstein-Grobusch, Schulze, Jung, Windler and Boeing2005), namely inverse associations between tHcy and diets rich in fruits, vegetables, fish, meat, milk, whole-grain bread and mushrooms (Gao et al. Reference Gao, Yao, McCrory, Ma, Li, Roberts and Tucker2003; Lasheras et al. Reference Lasheras, Huerta, Gonzales, Prada, Braga, Fernandez and Patterson2003; Weikert et al. Reference Weikert, Hoffmann, Dierkes, Zyriax, Klipstein-Grobusch, Schulze, Jung, Windler and Boeing2005). Dietary patterns high in refined cereals (Gao et al. Reference Gao, Yao, McCrory, Ma, Li, Roberts and Tucker2003), fat (Appel et al. Reference Appel, Miller, Jee, Stolzenberg-Solomon, Lin, Erlinger, Nadeau and Selhub2000; Weikert et al. Reference Weikert, Hoffmann, Dierkes, Zyriax, Klipstein-Grobusch, Schulze, Jung, Windler and Boeing2005) and sugar have been associated with higher tHcy levels (Fung et al. Reference Fung, Rimm, Spiegelman, Rifai, Tofler, Willett and Hu2001).
Implications and conclusion
The present findings suggest that in a Norwegian adult and elderly population not taking vitamin supplements, plasma tHcy concentration may be lowered with a diet rich in complex carbohydrates, protein and B-vitamins. Such a diet includes vegetables, fruits, whole-grain bread and cereals, as well as fish, non-processed meat, chicken and eggs. A high consumption of fat and sugar-rich foods such as sweets, cakes and processed meat may increase tHcy concentration. In conclusion, a diet high in B-vitamins and low in fat and sugar is associated with low plasma tHcy concentration in a population not taking vitamin supplements.
Acknowledgements
This work was supported by a research grant from the Norwegian Research Council, Western Norway Regional Health Authority (project no. 911177), the Foundation to Promote Research into Functional Vitamin B12 Deficiency and Johan Throne Holst Foundation for Nutrition Research. We thank Kari Solvoll for construction of the FFQ, nutrient and food intake assessment. None of the authors have any personal or financial conflicts in this work.
