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.

Keywords: adduct; aflatoxin; albumin; biomarker; dosimetry; mass spectrometry; normalization.

Figure 1

Guatemalan study (A–C), Density plots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. The dark blue tracing represents the empirically observed Guatemalan sample (n = 327); the coral tracing represents the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Regression of HSA levels (mg/mL) vs. albumin-normalized values. (F) Regression of HSA levels vs. raw AFB₁-lys concentrations. (G) Subset of observations from empirical data regression analysis in (D), red arrows denote regression residuals and illustrate the impact of albumin normalization on units of the AFB₁ internal dose. (H) Standardized regression residuals (Z-scored Studentized residuals) vs. serum total HSA concentration. Vertical dashed lines in (A,E,F,H) designate the reference range of HSA.

Figure 1

Guatemalan study (A–C), Density plots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. The dark blue tracing represents the empirically observed Guatemalan sample (n = 327); the coral tracing represents the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Regression of HSA levels (mg/mL) vs. albumin-normalized values. (F) Regression of HSA levels vs. raw AFB₁-lys concentrations. (G) Subset of observations from empirical data regression analysis in (D), red arrows denote regression residuals and illustrate the impact of albumin normalization on units of the AFB₁ internal dose. (H) Standardized regression residuals (Z-scored Studentized residuals) vs. serum total HSA concentration. Vertical dashed lines in (A,E,F,H) designate the reference range of HSA.

Figure 2

Qidong longitudinal study (A–C), Density plots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. Blue and purple tracings represent each of the four years in the empirically observed Chinese cohort (1995, n = 97; 1999, n = 92; 2003, n = 87; 2009, n = 30); coral tracings represent the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Plot of standardized regression residuals from (D) vs. serum total HSA concentration. Vertical dashed lines in (A,E) designate the reference range of HSA.

Figure 2

Qidong longitudinal study (A–C), Density plots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. Blue and purple tracings represent each of the four years in the empirically observed Chinese cohort (1995, n = 97; 1999, n = 92; 2003, n = 87; 2009, n = 30); coral tracings represent the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Plot of standardized regression residuals from (D) vs. serum total HSA concentration. Vertical dashed lines in (A,E) designate the reference range of HSA.

Figure 3

Gallbladder cancer case-control study (A–C), Density and boxplots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. Dark blue and light blue tracings represent persons with gallbladder cancer (Case; Chile n = 23, Shanghai n = 107) and controls, or persons with gallstones (Control; Chile n = 18, Shanghai n = 67), respectively. Coral tracings represent the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Plot of standardized regression residuals from (D) vs. serum total HSA concentration. Vertical dashed lines in (A,E) designate the reference range of HSA.* p < 0.0001.

Figure 3

Gallbladder cancer case-control study (A–C), Density and boxplots of HSA (A), AFB₁-lys (B), and albumin-normalized AFB₁-lys (C) concentrations in the empirically observed and simulated data. Dark blue and light blue tracings represent persons with gallbladder cancer (Case; Chile n = 23, Shanghai n = 107) and controls, or persons with gallstones (Control; Chile n = 18, Shanghai n = 67), respectively. Coral tracings represent the simulated sample (n = 20,000). (D) Regression of AFB₁-lys adducts as raw (pg/mL serum) vs. albumin-normalized values (pg/mg HSA) in empirically observed and simulated datasets. (E) Plot of standardized regression residuals from (D) vs. serum total HSA concentration. Vertical dashed lines in (A,E) designate the reference range of HSA.* p < 0.0001.