Basu et al. capacity (Hsueh et al. 2009). However, as can be seen in Table 4, we find that full methyla- tion to DMA was more strongly related to urinary creatinine than to lycopene. The relationship between creatinine and arsenic methylation has been reported previ- ously as an aside, without noting that it is the most important predictor nor being able to compare it with multiple nutritional factors as we have done here. In the past, urinary crea- tinine has usually been used to normalize for urine concentration, to compensate for varia- tion in diuresis. However, problems with doing so have been identified (Barr et al. 2005), in particular for studies of arsenic (Nermell et al. 2008; Steinmaus et al. 2009). Gamble et al. reported a strong correlation between urinary creatinine and DMA% (p < 0.001) (Gamble et al. 2005), and Nermell et al. (2008) noted that urinary creatinine was correlated mainly with DMA. One recent study found that low urinary creatinine was associated with arsenic- induced skin lesions (Pilsner et al. 2009). It is noteworthy that the formation of creatinine from methylation of guanidinoacetate accounts for approximately 75% of all folate-dependent transmethylation reactions (Barr et al. 2005; Mudd and Poole 1975). Another new finding was that animal fat in the diet was by far the strongest predictor of MMA%, with a high intake of animal fat being associated with a high MMA%. Some investigators have thought that a high MMA% would be due to an increase in the first step of methylation from InAs% to MMA. However, because 85% of InAs, on average, is methy- lated, at least to MMA, and the large majority ends up as DMA, increases in MMA% are more likely due to a reduction in the second step of methylation from MMA to DMA. We do not know why a high intake of animal fat would reduce the second step of methylation from MMA to DMA. One possibility is that animal fat intake is related to some unknown confounding factor. The only published study we could find that mentioned fat intake and methylation was a study of 66 adults who were kept on a fixed diet (Poirier et al. 2001). The investigators reported that during the experimental period of the study, when dietary fat intake was higher than during the usual diet, blood S-adenosylmethionine levels were inversely correlated with fat intake. The posi- tive association between dietary animal fat and MMA% is a new finding not reported elsewhere. In a study on dietary intake and arsenic methylation based on a U.S. popula- tion (n = 30), Steinmaus et al. (2005) did not find any relationship between fat intake and MMA% in urine. A possible reason why our findings were different from the U.S. study could be attributed to the different levels and patterns of nutrition between a U.S. popula- tion and this rural Indian population. 1312 The inverse association between serum folate and selenium and MMA% is supported by findings from other epidemiological studies. Using data from a cross-sectional survey in a comparable Bangladeshi population (n = 300), Gamble et al. reported that serum folate was inversely associated with InAs% (Spearman r = –0.12; p < 0.05) and MMA% (r = –0.12; p = 0.04) and was positively associated with serum homocysteine (r = 0.21; p < 0.05) and urinary DMA% (r = 0.21; p < 0.001) (Gamble et al. 2005). Gamble and colleagues reported univariate correlation estimates of associations and did not report results of multivariate asso- ciations after adjustment of possible confound- ing variables. On the same population, Gamble et al. (2005) conducted a follow-up double- blind randomized controlled trial (n = 200) that compared 12-week administrations of folate versus placebo and found that after 12 weeks of folic acid supplementation, the increase in the proportion of total urinary arsenic excreted as DMA in the folate group (72% at baseline and 79% after supplementation; p < 0.0001) was greater than that in the placebo group, as was the reduction in the proportions of total uri- nary arsenic excreted as MMA (13% and 10%, respectively; p < 0.0001) and as InAs (15% and 11%, respectively; p < 0.001). However, in this study, the intervention group and the placebo group were exposed to arsenic during the time of the trial (Gamble et al. 2006). Because this study was conducted among individuals con- comitantly exposed to high concentrations of InAs, it cannot be inferred that folate supple- mentation would benefit individuals who were no longer exposed to arsenic in drinking water. Studies have consistently reported higher MMA% in males than in females (National Research Council 1999). In the univari- ate comparison, the difference in MMA% between men and women was 1.9 (Table 1). This difference was not much less than for animal fat (2.3, Table 3). The difference between males and females may be explained in part by differences in animal fat intake and by differences in urinary creatinine. Associations that might have been expected but were largely absent include homocysteine, vitamin B12 (cobalamin), and methionine. Homocysteine was strongly related to MMA% in univariate analysis, but the association became weaker in multivariate modeling when folate was added (Table 4). Heck et al. 2007 reported a relationship between cobalamin and increased MMA to InAs ratio, but we found no evidence of it in this study (MMA% for the lowest tertile of vitamin B12 = 8.0 and for the highest tertile MMA% = 8.1; p = 0.75). Data for this study came from a cross- sectional survey. Information on the dietary variables was collected at the same time as blood was sampled for estimation of serum micronutrients, and urine was collected for estimation of urinary InAs%, MMA%, and DMA%. This provides a snapshot of inter- relationships between diet, micronutrients, and arsenic methylation variables. Usually, the main limitation of cross-sectional studies per- tains to inferring long-term outcomes from data obtained at one point in time. However, this limitation does not apply here, because it is reasonable to believe that arsenic methylation patterns would relate to current nutritional factors. In addition, it has been found that arsenic methylation efficiency in individuals is relatively stable over time (Concha et al. 2002). Another limitation is that because we did not have measures of urinary specific gravity, it was not possible to adjust for urine concentration. Our findings and those of others indicate that one should not use urinary creatinine to adjust for urine concentration in studies of arsenic, especially those involving speciation. However, it should be noted that the parameters of inter- est in this study were InAs%, MMA%, and DMA%. The proportion of arsenic in these various forms is unlikely to be related to urine concentration, even though the concentrations in absolute terms would be. Conclusions This study revealed that urinary creatinine in the highest versus lowest tertile had a stron- ger association with arsenic methylation to DMA than did similar contrasts for any of the dietary or micronutrient factors assessed. This finding provides further evidence that urinary creatinine should not be used to adjust urine concentrations for diuresis effects. Further studies are needed to investigate the determi- nants of urine creatinine concentrations in arsenic-exposed populations, because such fac- tors are also likely to influence arsenic methy- lation. The new findings that plasma lycopene had an inverse relationship with InAs% and that animal fat intake had a positive relation- ship with MMA% are new and warrant fur- ther assessment in other studies. Ref Rencs Aposhian HV. 1997. Enzymatic methylation of arsenic species and other new approaches to arsenic toxicity. Annu Rev Pharmacol Toxicol 37:397–419. Barr DB, Wilder LC, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL. 2005. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ Health Perspect 113:192–200. Chung JS, Haque R, Guha Mazumder DN, Moore LE, Ghosh N, Samanta S, et al. 2006. Blood concentrations of methio- nine, selenium, beta-carotene, and other micronutrients in a case-control study of arsenic-induced skin lesions in West Bengal, India. Environ Res 101(2):230–237. Concha G, Vogler G, Nermell B, Vahter M. 2002. Intra-individual variation in the metabolism of inorganic arsenic. Int Arch Occup Environ Health 75(8):576–580. Cullen W, Reimer K. 1989. Arsenic speciation in the environ- ment. Chem Rev 89:713–764. Drobna Z, Waters SB, Walton FS, LeCluyse EL, Thomas DJ, Styblo M. 2004. Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes. Toxicol Appl Pharmacol 201(2):166–177. ovlume 119 | number 9 | September 2011 • Environmental Health Perspectives