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 . Depending on the criteria used, the proportion of subjects with impaired folate status in other studies ranges 0–79% [18–21]. 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.  for Spanish adults (205 μg/d in men, 197 μg/d in women) and higher ones that Ford et al.  and Rasmussen et al.  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, 23–25]. 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 [9–11].
In agreement with several other studies [28–32], 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 .
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, 37–39]. 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. .
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 [29–32]. 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  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 1st 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 [41–45] 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].