Rejoinder: Arsenic exposure and prevalence of type 2 diabetes: updated findings from the National Health Nutrition and Examination Survey, 2003-2006.

The role of inorganic arsenic exposure in chronic diseases, including type 2 diabetes, is a major public health research question. This has been underscored by recent epidemiologic1-4 and experimental5-8 evidence supporting increased risks at low exposure levels. In this context, it is critical to understand the biology and the technical limitations of biomarkers of inorganic arsenic exposure, usually measured in urine.9-11 Total urine arsenic integrates exposure from multiple sources including inorganic (arsenite, arsenate) and organic (mainly arsenobetaine, arsenosugars and arsenolipids) arsenic compounds and their metabolites (Figure 1). In population-based studies, arsenic speciation in urine is important to differentiate inorganic from organic exposure because organic arsenicals, mostly found in seafood, have little toxicity relative to inorganic arsenic and its metabolites. Despite analytic advances in the measurement of arsenosugars, arsenolipids and their metabolites,9 their determination remains technically challenging in epidemiologic studies. For example, those compounds were not measured in the 2003-2004 National Health Nutrition Examination Survey (NHANES). Moreover, because arsenite, arsenate and methylarsonate—species that directly reflect inorganic arsenic exposure and metabolism (Figure 1)—were measured in NHANES 2003-2004 with high limits of detection,9,12-14 only total arsenic, dimethylarsinate, arsenobetaine and arsenocholine (a minor seafood arsenical) were available for analyses of arsenic and health endpoints. Figure 1 Inorganic and organic arsenic exposure: relevant arsenic compounds in the general population, from source to urine. In NHANES 2003-2004, arsenite, arsenate, methylarsonate (MA) and arsenocholine were below the limit of detection in 96%, 94%, 65% and 98% . To evaluate the association of inorganic arsenic exposure with the prevalence of type 2 diabetes in NHANES 2003-2004,2 we reported two main strategies to remove the contribution of organic arsenicals of marine origin to total urine arsenic. First, we conducted analyses of the association between total urine arsenic and the prevalence of type 2 diabetes adjusted for sociodemographic factors, diabetes risk factors, and two markers of seafood intake (urine arsenobetaine and blood mercury). More importantly, we reported analyses of the association between total urine arsenic and the prevalence of type 2 diabetes adjusted only for sociodemographic and diabetes risk factors but restricted to participants with low arsenobetaine levels (ie participants with unlikely seafood intake, in whom urine arsenic would be derived mainly from inorganic arsenic). The magnitude of the association in this subgroup was similar to the analysis adjusting for urine arsenobetaine and blood mercury, demonstrating that the association between arsenic and type 2 diabetes identified in the whole sample was driven by inorganic arsenic exposure and was not dependent on the statistical method used to control for organic arsenicals of marine origin. In a reanalysis of the association between arsenic exposure and diabetes in NHANES 2003-2004 published in this issue of EPIDEMIOLOGY, Steinmaus et al.15 used total urine arsenic minus urine arsenobetaine and urine arsenocholine (in 13 individuals in whom it was detected) as an “estimate of inorganic arsenic exposure,” in an attempt to remove the contribution of organic arsenicals of marine origin to total urine arsenic. Total urine arsenic minus arsenobetaine and arsenocholine, however, is an inadequate estimate of inorganic arsenic exposure because it does not remove other unmeasured organic arsenicals and their metabolites derived from seafood. Indeed, the correlation between arsenobetaine and total urine arsenic minus arsenobetaine and arsenocholine in NHANES 2003-2004 remained moderately strong (r=0.51, Figure 2). In their Discussion, the authors indicated that after subtracting arsenobetaine and arsenocholine from total arsenic, 24% of the variance in the remaining arsenic was still explained by arsenobetaine “probably due to some dimethylarsinate and some other arsenosugars that are present in the same seafoods that contain arsenobetaine”. 15 The magnitude of the misclassification induced when estimating inorganic arsenic by total urine arsenic minus arsenobetaine and arsenocholine in the NHANES sample can be further appreciated by comparing the distribution of total arsenic minus arsenobetaine and arsenocholine (20th, 50th 80th, 90th and 99th percentiles of 2.7, 6.0, 11.9, 18.4 and 73.6 μg/L, respectively) with the distribution of total urine arsenic among participants with undetectable (<0.4 μg/L) urine arsenobetaine (20th, 50th 80th, 90th and 99th percentiles of 1.9, 3.9, 7.7, 11.8 and 30.4 μg/L, respectively). As a consequence, any analysis that uses total urine arsenic minus arsenobetaine and arsenocholine as an estimate of inorganic arsenic exposure still needs to adjust for markers of seafood intake, such as arsenobetaine itself. Futhermore, the misclassification induced by using total urine arsenic minus arsenobetaine and arsenocholine as an index of inorganic arsenic exposure is aggravated in categorical analyses, e.g. quintiles. Many participants in the highest quintile (≥80th percentile) of total urine arsenic minus arsenobetaine and arsenocholine in NHANES 2003-2004 were exposed to substantial amounts of organic arsenic compounds from seafood, and categorization in this situation could reduce the efficacy of statistical adjustment for seafood intake in multivariable models. Figure 2 Relationship of arsenobetaine with total arsenic and total arsenic minus arsenobetaine (AB) and arsenocholine (AC) in NHANES 2003-2004. Arsenocholine was subtracted only in 13 individuals with detectable levels. Lines represent dose-response relationships . Given the longstanding experience of Steinmaus et al. in investigating the health effects of arsenic, it is difficult to understand why the authors used total urine arsenic minus arsenobetaine and arsenocholine as an “estimate of inorganic arsenic exposure” without controlling for seafood intake in their main analyses and conclusions. Indeed, when controlling for seafood intake, Steinmaus et al. reported findings consistent with out 2008 paper.2 (Table 3 of their manuscript).