Assessing the Validity of Normalizing Aflatoxin B-1-Lysine Albumin Adduct Biomarker Measurements to Total Serum Albumin Concentration across Multiple Human Population Studies

The assessment of aflatoxin B-1 (AFB(1)) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB(1)-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB(1) exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB(1)-lysine adduct concentrations (e.g., pg/mL serum or plasma) to the total circulating HSA concentration (e.g., pg/mg HSA). It is hypothesized that this practice corrects for technical error, between-person variance in HSA synthesis or AFB(1) metabolism, and other factors. However, the validity of this hypothesis has been largely unexamined by empirical analysis. The objective of this work was to test the concept that HSA normalization of AFB(1)-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB(1) internal dose estimates. Using data from AFB(1)-lysine and HSA measurements in 763 subjects, in combination with regression and Monte Carlo simulation techniques, we found that HSA accounts for essentially none of the between-person variance in HSA-normalized (R-2 = 0.04) or raw AFB(1)-lysine measurements (R-2 = 0.0001), and that HSA normalization of AFB(1)-lysine levels with empirical HSA values does not reduce measurement error any better than does the use of simulated data (n = 20,000). These findings were robust across diverse populations (Guatemala, China, Chile), AFB(1) exposures (10(5) range), HSA assays (dye-binding and immunoassay), and disease states (healthy, gallstones, and gallbladder cancer). HSA normalization results in arithmetic transformation with the addition of technical error from the measurement of HSA. Combined with the added analysis time, cost, and sample consumption, these results suggest that it may be prudent to abandon the practice of normalizing adducts to HSA concentration when measuring any HSA adducts-not only AFB(1)-lys adducts-when using LCMS in serum/plasma.