The objective of this controlled feeding study was to investigate the relationship between well-defined measures of adiposity and serum folate, vitamin B12, homocyteine, and methylmalomic acid in postmenopausal women. Additional precision was gained by adjusting for breast cancer risk factors (family history) and estrogen exposure (parity and age of menarche) in the models. Serum folate concentrations were inversely related to adiposity as measured by BMI, percent total fat, and both central and peripheral fat mass. Adiposity was not related to serum vitamin B12 levels, nor to indicators of folate/B12 metabolism, methylmalonic acid, or homocysteine.
This type of study is important because it offers potential mechanistic insight into the obesity-cancer association. Folate and vitamin B12 are critical for the maintenance of DNA stability, which is, in turn, required for normal cell function. Depletion of folate and/or vitamin B12 result in alterations of one-carbon metabolism, which may lead to increased cancer susceptibility by mechanisms such as uracil misincorporation, DNA strand breaks, DNA repair abnormalities, and aberrations in DNA methylation (Choi et al. 2000
; Bailey et al. 2001
It is possible that serum folate concentration is not the appropriate index of folate status. Herbert (Herbert 1962
) demonstrated that serum folate levels fell into the deficient range (<3 ng/ml) within 22 days of ingesting a folate deficient diet, while red cell folate levels fell only at 3–4 months, as folate replete red cells senesced. From these data, he concluded that serum folate levels reflected recent intake, while red cell folate concentration was a measure of folate storage. However, serum and red cell folate concentrations are closely correlated (r = 0.5, P<0.0001) (Ettinger 1987
). Thus, serum folate concentrations have been taken as an index that reflects steady-state folate equilibration and is routinely measured in clinical laboratories (Galloway et al. 2003
Because all the women in our study consumed the same diet, the lower serum folate concentrations found in women with higher BMI and fat mass may reflect a perturbation of whole body steady state folate concentrations with increased fat mass. Although our study was conducted within the control (0 g) segment of the alcohol trial, like all crossover studies, there may be residual treatment effects and for these reasons the design included a two to five week washout period. Because of these washout periods between each treatment group, and because biologic samples were collected and tested in the final (eight week) of each dietary period, carryover effects, if any, should have been minimized. There was also a similar washout period prior to enrolling in the study. Further, as described in the statistical analysis section, we specifically tested for carryover effects. The addition of alcohol group assignment order did not affect the precision of our estimates. We have also previously reported that the moderate doses of alcohol in this study did not significantly affect serum folate concentrations (Laufer et al. 2004
Three possibilities could explain lower steady state serum folate concentrations with increased fat mass: increased cellular uptake, increased intracellular retention, and/or increased renal excretion (Villanueva et al. 1998
). Folate enters the cell by at least two independent transport systems. The first system is folate receptor (FR) mediated (Rothberg et al. 1990
). FRs have limited distribution in normal tissues, but are highly expressed in some tumors. The second system, used by most normal tissues, is the reduced folate carrier (RFC), a 12-pass transmembrane protein (Ferguson et al. 1999
) with high affinity for reduced folates (eg, 5-MeTHF). Cell-specific folate homeostasis mediated by the RFC is regulated by multiple promoters and non-coding exons (Whetstine et al. 2002
) as well as dietary folate (Liu et al. 2005
), such that each cell type has the flexibility to regulate its intracellular folate concentration according to immediate need.
Serum folate taken up by the cell is demethylated by vitamin B12 dependent methionine synthase and conjugated with variable numbers of glutamate residues by folylpoly-γ-glutamate synthase (conjugase) (Lowe et al. 1993
). The methyl group is transferred to homocysteine with the formation of methionine, an essential amino acid. Methionine is adenosylated to S-adenosyl methionine (SAM) and donates its methyl group to a wide range of macromolecules including DNA (Townsend et al. 2004
) and estrogens (Goodman et al. 2001
). Demethylated tetrahydrofolate (THF) accepts and transfers activated one-carbon units to multiple macromolecules, including uracil in generation of thymine, required for DNA synthesis and repair (Prasannan et al. 2003
). Substituted THF is reduced to 5-MeTHF by the enzyme, methyltetrahydrofolate reductase (MTHFR) and re-enters the serum pool. Polymorphisms of MTHFR and other genes involved in folate metabolism can perturb intracellular and serum folate. We are in the process of genotyping single nucleotide polymorphisms (SNPs) for genes in the folate metabolic pathway for all subjects in the current study.
We have recently reported significant elevation of circulating estrogen metabolites with increased fat mass in overweight and obese women in this study population (Mahabir et al. 2006
), and this suggests a third system for the observed folate reductions. Although the women did not take HRT, it is established that postmenopausal women aromatize adrenal androsteinedione to estrogen metabolites in adipose tissue (Nevton et al. 1986
; Szymczak et al. 1998
). Thus, in our overweight and obese subjects, peripheral conversion in the more abundant adipose tissue is a likely source for the elevated estrogen metabolites. For many years, it has been known that estrogen supplements adversely affect folate status (Krumdieck et al. 1975
; Shojania et al. 1975
). Oral contraceptive intake reduced serum folate availability sufficiently to produce cervical megaloblastosis (Butterworth et al. 1992
) and to potentiate platelet hyperactivity (Durand et al. 1997
). Estrogen inhibits glutamate conjugation activity thereby increasing the “free” folate pool that can leave the cell and is readily filtered by the kidney (Krumdieck et al. 1975
). Estrogen also increases folate utilization by its requirement for methylation via catechol-O-methyltransferase (COMT) prior to urinary excretion (Goodman et al. 2001
). Thus, lower serum folate concentrations observed in obese subjects cold be explained by increased utilization and urinary excretion secondary to increased adipose tissue-derived estrogen concentrations.
It must be noted that serum homocysteine concentration was not associated with obesity in this well nourished study population. Since homocysteine methylation to methionine requires adequate 5-methylTHF, a reduction in serum folate concentration could be expected to compromise this pathway. However, serum folate concentrations, even in the obese subjects were within the normal range, and homocysteine can be remethylated through the independent betaine homocysteine methyltransferase that derives its methyl group from choline (Finkelstein et al. 1988
Although we used a cross-sectional design with only modest numbers of subjects, the strengths of this study include a homogenous study population (e.g., women who were smoking or taking HRT were excluded) and measurement stability, which resulted from use of a carefully controlled diet and use of fasting blood samples for analysis. The small sample size of our study may have limited our power to detect weaker associations. The few studies to date (described in the introduction section) of adiposity and serum folate levels used only BMI, and made adjustments for dietary folate intake assessed by questionnaires, which have known limitations. The DEXA scans employed in our study are considered a reference method for body composition analysis (Panotopoulos et al. 2001
In summary, while our data suggest that increased fat mass is associated with reduction in serum folate concentrations, an accepted measure of folate availability, because of the cross sectional design of our study, causality cannot be inferred from our findings. It is not possible to determine from these data whether these lower serum folate levels are physiologically significant, as the mean of the obese group (>14 ng/ml) was still well within the normal range (5–16 ng/ml). With obesity at epidemic proportions and increasing with each new survey, further investigations into the interactions between folate metabolism and obesity appear warranted.