Dietary habits in a population may be a determining factor of health, and inadequate intakes of certain nutrients have been associated with increased risk for chronic disease, such as cardiovascular disease, diabetes, and cancer. More recently, folate has drawn attention as a key nutrient involved in health maintenance and chronic disease prevention 1 . Folate acts as a coenzyme in several single-carbon transfers leading to the biosynthesis of nucleic acids, certain neurotrasmitters, phospholipids, and hormones. It is involved in homocysteine metabolism, donating a methyl group during homocysteine remethylation into methionine, and inadequate folate status has been characterized as a major cause of hyperhomocysteinaemia 2 . Moreover, folate deficiency is an established risk factor for the development of certain types of cancer, cardiovascular disease 3 4 , developmental defects (e.g. neural tube defects), and neurological or psychiatric disorders 5 6 7 . Although much of the adverse effects of impaired folate status is explained through its interplay with homocysteine levels, there is evidence suggesting direct health-beneficial effects of folate, including its favourable effects on vascular endothelial function and redox cellular status 1 .

From a public health perspective, assessing the folate status in a population and examining its dietary or other correlates, is of great importance. This would allow the identification of groups at risk for folate deficiency that would mostly benefit from public health interventions to increase dietary folate intake.

Crete (Greece), once known for the low cardiovascular mortality among adult men in early 1950's, has now increasing rates of cardiovascular disease, a trend that appears to be related to dietary and lifestyle changes that have been taking place during the last decades 8 9 . The traditional Cretan diet – a variant of the Mediterranean diet – has been gradually abandoned, and current Cretans consume higher amounts of saturated fat, meat, and cheese, and lower amounts of bread, fruits, vegetables, legumes, and fibre 9 10 11 . Among other consequences, such dietary changes are expected to result in decreased intake of dietary folate and thus, impaired folate status. To date, however, no data are available with regard to the folate status of the adult population in Crete.

In this study, we report on the folate status of a group of apparently healthy Cretan adults, personnel of the University of Crete and the University Hospital of Heraklion (Crete), which were examined as part of a health prevention programme by the Preventive Medicine and Nutrition Clinic. We describe the distribution of serum folate concentrations by age and sex, and investigate associations with nutrient intakes, food consumption, and lifestyle habits, namely tobacco, coffee, and alcohol consumption. Our findings document certain patterns of dietary intake and lifestyle habits associated with folate levels, with potential implications for the development of future health intervention studies.

The mean ± standard deviation (SD) age was 39 ± 14 years for men and 39 ± 13 years for women (Table 1). Approximately 86% of the subjects lived in urban areas and current smokers were the 38% of men and 31% of women (p > 0.05).

The distribution of serum folate concentrations and dietary folate intakes is presented for all subjects by gender and age in Table 2. The geometric mean of serum folate was 15.6 nmol/l (95% CI, 14.6–16.8 nmol/l) in men and 19.2 nmol (17.9–20.7 nmol/l) in women (p < 0.001). In both men and women age was positively associated with serum folate. The prevalence of sub-optimal serum folate (≤7 nmol/l) was 6.8% in men and 2.1% in women (p < 0.001). Men had higher mean intake of dietary folate than women (294 vs. 247 μg/d, p = 0.003). However, the energy-adjusted intake of folate was higher in women than men (161 vs. 144 μg/1000 kcal/d, p > 0.05). Eighty-seven percent of women and 76% of men (p = 0.013) consumed folate <400 μg/day. Dietary intake of folate increased by age, especially in men (p for trend < 0.01).

Associations between age and sex-specific quartiles of serum folate and mean levels of total homocysteine and vitamin B₁₂ are presented in Table 3. Serum folate showed an inverse linear association with total homocysteine (p for trend < 0.001), and subjects at the highest folate quartile had 18.5% lower mean total homocysteine concentrations compared to those at the lowest quartile. No significant association was observed between serum folate and vitamin B₁₂ levels.

The relations between serum folate and the use of tobacco, coffee, and alcohol are shown in Table 4. The age- and gender-adjusted geometric mean serum folate was ≈2 nmol/l lower in current smokers than in non- or ex-smokers (p < 0.05). Similarly, serum folate levels were inversely related to the quantity of tobacco (p for trend < 0.05), and individuals who smoked ≥20 cigs/day had 21.2% lower mean folate concentrations compared to those who smoked <10 cigs/day. Increasing coffee intake was also related to decreased serum folate (p for trend < 0.05), and subjects consuming >200 g/day had 14.8% lower mean serum folate levels compared to non-consumers. However, the associations between tobacco or coffee consumption and serum folate disappeared after adjusting for intakes of nutrients, especially dietary folate, vitamins A and C (multivariate-adjusted). No significant relationship was observed between alcohol intake and serum folate.

The associations between nutrient intakes and serum folate are shown in Table 5. In multivariate analysis, intake of mono-unsaturated fatty acids (MUFA) – both as raw quantity and as percentage of energy intake – was positively associated with serum folate. Intakes of dietary fibre (r = 0.20), calcium (r = 0.14), and magnesium (r = 0.10) were also positively related to serum folate, independently of age, gender, energy intake, consumption of tobacco, coffee, and alcohol. Serum folate concentrations demonstrated favourable associations with dietary intakes of several vitamins, namely folate (r = 0.14), vitamins A (r = 0.23), E (r = 0.15), C (r = 0.21), B₁ (r = 0.13), and B₆ (r = 0.12).

We further assessed food consumption by the study participants according to their serum folate status (Table 6). Multivariate logistic regression analysis showed that increased consumption of potatoes, legumes, fruits and/or vegetables were associated with decreased risk for low serum folate (determined as folate concentrations <1^st quartile, age- and gender-specific). Specifically, individuals consuming fruits and/or vegetables ≥360 g/day had 79% lower risk for low serum folate, compared to those with no consumption. In contrast, increased intake of cereals and meat (especially red meat) was inversely related to serum folate status.

Folate has recently emerged as a key nutrient to optimising health, and inadequate folate status has been identified as risk factor for cardiovascular disease, various types of cancers, and neurocognitive disorders 1 17 . The present study described the distribution of serum folate concentrations in a healthy adult population in Crete, Greece and examined associations with dietary intakes and lifestyle habits, namely tobacco, coffee, and alcohol consumption. The relationship between serum folate and vitamin B₁₂ and total homocysteine was also examined.

The results of the study should be viewed in light of some limitations. The present data used a cross-sectional design that implies that no conclusions can be drawn on the causal effect of the determinants of serum folate levels. The dietary survey was performed using one single 24-h dietary record and may thus be subject to both systematic and random bias, one source being the lack of inclusion of weekends. The regular use of vitamin supplements was not recorded and this precluded further analyses of the serum folate status. Also, the participation rate was relatively low (<20%) and selection bias cannot be excluded. Lastly, the unique sample of Hospital/Medical School personnel is both strength (as it decreases potential for confounding) and limitation (as the sample is not representative of the general Greek adult population) of the survey.

Despite these limitations, there are several findings in this study that are worth to be discussed. In this sample of apparently healthy Cretan adults, sub-optimal folate levels were found in 6.8% of men and 2.1% of women, using serum concentrations ≤7 nmol/l as a cut-off value 16 . Depending on the criteria used, the proportion of subjects with impaired folate status in other studies ranges 0–79% 18 19 20 21 . Differences in serum folate concentrations and cut-off values may be due to analytical methods, race-ethnicity differences and genetic backgrounds 1 22 . The mean daily intake of folate by the study subjects (294 μg/d in men, 247 μg/d in women) also stands between lower figures that have been reported by Plannells et al. 19 for Spanish adults (205 μg/d in men, 197 μg/d in women) and higher ones that Ford et al. 21 and Rasmussen et al. 23 have described for US men (343 μg/d) and Dutch women (283 μg/d in the 20–35-year-old group), respectively. In contrast to the low prevalence of sub-optimal folate concentrations, >75% of the study participants did not meet the RDI for folate (400 μg/day). Such a discrepancy between serum folate levels and dietary folate intake has been reported in several other surveys, including those where dietary folate intake was assessed by 3-day dietary records or food-frequency questionnaires, and is explained by insufficient capturing of important sources of dietary folate by dietary records 20 23 24 25 . As already discussed, regular use of vitamin supplements was not recorded in our survey, which may have resulted in underestimation of dietary folate intake. Finally, some researchers argue that total homocysteine is a more sensitive indicator of folate status than is serum (or erythrocyte) concentration of the vitamin 20 26 . Similarly to the results of other studies 19 20 , men had higher mean intakes of dietary folate than women, but when expressed by energy intake, women had higher intake of folate than men. This might – at least in part – explain the higher serum folate concentrations in women, although other factors (e.g. hormonal, bioavailabilty of dietary folate etc.) may be responsible for this discrepancy.

In both men and women, subject age was positively related to serum folate levels. One possible explanation for this association is the higher intake of folate and folate-rich foods (especially fruits and vegetables) with increasing age among adults (data not shown). A similar trend has been reported in other studies and is generally attributed to the preference for more "healthy" or "traditional" diets among older adults 19 20 27 . With regard to the adult population in Crete, recent data indicate the gradual abandonment of the traditional Cretan diet in favour of more "westernised" diets, with consumption of higher amounts of saturated fat, meat, and cheese, and lower amounts of fruits, vegetables, legumes, and fibre 9 10 11 .

In agreement with several other studies 28 29 30 31 32 , serum folate was inversely related to total homocysteine concentrations. Hyperhomocysteinaemia is a well-established risk factor for atherothrombotic disease, Alzheimer's disease and other neuropsychiatric disorders, osteoporosis and hip fractures 33 34 . This overlap of medical disorders that are linked both to increased homocysteine and inadequate folate status has led to the suggestion that the disease-promoting effects of low folate are mediated through increased homocysteine levels 1 4 7 32 35 . Yet, recent data indicate that folate may act independently of homocysteine, especially through its direct effects on vascular endothelium and cellular redox status 1 .

Another finding was the inverse relationship between tobacco consumption and serum folate concentrations. Smokers had significantly lower serum folate levels than non-smokers, and serum folate concentrations decreased by increasing consumption of cigarettes. A negative association between smoking and serum folate has been reported in other studies 19 28 29 32 36 , and is generally attributed to the different nutritional status of smokers. More specifically, smokers tend to consume lower amounts of several vitamins, fruits and vegetables, resulting in decreased intake of dietary folate 12 37 38 39 . Similarly, in our study, the association between smoking and serum folate abolished its statistical significance after controlling for intakes of dietary folate, vitamins and C. It should be noted, however, that smoking may have more direct anti-folate effects, producing local vitamin deficiency in individual tissues as demonstrated by Piyathilake et al. 40 .

In the present study, serum folate concentrations were inversely related to coffee consumption. Such a relationship has only been shown indirectly, in studies examining serum total homocysteine levels in association with coffee intake. In some of these studies, the positive association between coffee consumption and homocysteine was lost after accounting for serum folate and/or dietary folate intake, indicating a negative association between coffee intake and folate status 29 30 31 32 . As in the case of tobacco consumption, the association between serum folate and coffee was confounded by differences in nutrient intakes.

The results from the dietary survey demonstrated significant associations between serum folate and intakes of several important nutrients, including MUFA, fibre, calcium, magnesium, folate, and vitamins A, E, C, B₁, and B₆, independently of other confounders. A relationship between dietary folate intake and serum folate concentrations has been reported in some 18 19 23 but not all 20 studies. The favourable associations between serum folate and dietary micronutrients were reflected by differences in consumption of various foods. Individuals with higher intakes of potatoes, legumes, fruits and/or vegetables – all these foods considered major sources of folate 19 20 – had significantly decreased risk for low serum folate (below the 1^st quartile), compared to those with no consumption. Conversely, higher intakes of cereals and meat products were related to decreased serum folate concentrations. These findings are in accordance with those reported by both cross-sectional 41 42 43 44 45 and diet-intervention studies 46 47 , which suggest a positive association between folate status and a dietary pattern characterized by high consumption of fruits, vegetables, legumes and low consumption of refined cereals and meat. It should be noted however, that the lower intakes of cereals and meat among the study subjects with lower serum folate levels might be simply due to substitution by other foods, namely fruits, vegetables, and legumes. Finally, an alternative explanation for the associations between serum folate levels and nutrients and foods, might be that serum folate serves as an overall nutritional biomarker and indicator of diet and health quality 48 49 .

Conclusively, in this cross-sectional study of healthy Cretan adults we found various demographic, lifestyle and dietary factors associated with serum folate concentrations. Considering the importance of folate in health maintenance and prevention of chronic disease, it is important to increase the public's awareness on modifiable lifestyle habits and especially diet, associated with improved folate status. In view of the high percentage of adults with inadequate intake of dietary folate, our results also emphasize the need to undertake large-scale epidemiological studies within the general Greek adult population, in order to assess the prevalence of impaired folate status, and further examine associations between dietary patterns, serum folate, and chronic disease risk.

The author(s) declare that they have no competing interests.

CH performed blood sampling, collected data, and reviewed the manuscript. GB collected data and drafted the manuscript. ML collected data and carried out the statistical analysis. In JS' laboratory biochemical measurement of serum folate, vitamin B₁₂ and homocysteine was performed. JS also reviewed the manuscript. AK conceived of the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.