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

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.

Optimization of Aflatoxin B1-Lysine Analysis for Public Health Exposure Studies

“…As an alternative, Dried Blood Spots have shown promise since measured values were normalized by the amount of HSA, not serum volume [ 20 ]. There is recent evidence that suggests that aflatoxin serum adducts should be normalized by serum volume and not by HSA [ 18 ]. This means that unless the volume of serum or blood is carefully applied to a DBS card prior to desiccation and shipping, it may not easily compatible with this analysis.…”

“…Historically, the amount of HSA in the serum has been quantified to normalize the reported AFB 1 -Lys concentration in units of pg/mg albumin. A recent analysis of a large number of samples from many countries suggested that this normalization step may not be necessary [ 18 ]. Of the various analytical approaches reported, the most reliable data come from liquid chromatography–tandem mass spectrometry methods [ 17 ].…”

Child Aflatoxin Exposure is Associated with Poor Child Growth Outcomes: A Prospective Cohort Study in Rural Malawi

“…AFB 1 -lysine adduct concentration presented in this study was not expressed relative to albumin. It has been reported elsewhere that normalizing AFB 1 -lysine adduct concentration to serum albumin is not necessary when the former was measured by IDMS [ 24 ]. We did human serum albumin (HSA) normalization in plasma samples of mothers ( n = 98) collected at baseline and 36 weeks of gestation in this current study to see if normalization would make a difference.…”

Assessing the Impacts of Preanalytical Field Sampling Challenges on the Reliability of Serum Aflatoxin B1-Lysine Measurements by Use of LC-MS/MS

“…We hypothesize that the proteolytic digestion in our AFB1-lys methodology likely addresses any potential interference from the presence of fibrin, and that the presence of the heparin additive itself does not interfere with proteolysis. However, the comparability of heparinized plasma albumin measurements with serum is also needed since an albumin concentration measurement is used by convention to normalize for the circulating albumin concentration from which the AFB1-lys analyte is proteolytically generated [25]. It has been documented that heparin can form insoluble precipitates with the bromocresol green reagent used in the colorimetric determination of albumin and can yield erroneously low results [26,27].…”

“…The use of bromocresol purple [26] or additives with bromocresol green [27] has been shown to restore accuracy to heparinized plasma albumin measurements but we have not tested these approaches. The need for normalizing AFB1-lys measurements itself has been called into question [25], and so we believe further evaluation of heparinized plasma for AFB1-lys measurements is worth consideration.…”