Assessing the Validity of Normalizing Aflatoxin B1-Lysine Albumin Adduct Biomarker Measurements to Total Serum Albumin Concentration across Multiple Human Population Studies.

Katherine A. McGlynn,Patricia A. Egner,Christian S. Alvarez,María F. Kroker-Lobos,Manuel Ramírez-Zea,Jill Koshiol,Sean M. Burke,Joshua W. Smith,John D. Groopman,Derek K. Ng,Thomas W. Kensler,Jian-Guo Chen,Alvaro Rivera-Andrade

doi:10.3390/toxins14030162

Abstract

The assessment of aflatoxin B1 (AFB1) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB1-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB1 exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB1-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 AFB1 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 AFB1-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB1 internal dose estimates. Using data from AFB1-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 (R2 = 0.04) or raw AFB1-lysine measurements (R2 = 0.0001), and that HSA normalization of AFB1-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), AFB1 exposures (105 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 AFB1-lys adducts—when using LCMS in serum/plasma.

Highlights

Aflatoxin B1 (AFB1 ) is a potent carcinogen that contaminates staple grains worldwide [1] and contributes to the global hepatocellular carcinoma burden [2]
Given the use of human serum albumin (HSA) as the denominator in the normalization of raw aflatoxin B1 (AFB1) -lys levels, total HSA concentrations were, as expected, inversely and significantly associated with HSA-normalized
The effect of HSA “normalization” in HSA-corrected AFB1 -lys adduct values appears to be the result of arithmetic transformation and the addition of technical error, rather than a reduction in biological or technical variance

Summary

Introduction

Aflatoxin B1 (AFB1 ) is a potent carcinogen that contaminates staple grains worldwide [1] and contributes to the global hepatocellular carcinoma burden [2]. Quantitation of the AFB1 -lys adduct biomarker using the current state-of-the-art isotope-dilution liquid chromatography-mass spectrometry (LCMS) analytical method [5] results in data expressed in units of volumetric concentration (e.g., pg AFB1 -lys adduct/mL of serum or plasma). These values are transformed by dividing the measured AFB1 -lys concentration by the concentration of total circulating human serum albumin (HSA), resulting in the quantitation of the AFB1 -lys adduct relative to the total HSA concentration (e.g., pg AFB1 -lys adduct/mg total HSA). HSA precipitation or immunoaffinity purification of digested

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