Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

Abstract

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes (n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P < 0.0001; R² = 0.30; n = 24]. SIL-based abundance measurements correlated well with enzyme activities, with correlations ranging from moderate for UGTs 1A6, 1A9, and 2B15 (Rs = 0.52-0.59, P < 0.0001; R² = 0.34-0.58; n = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79-0.90, P < 0.0001; R² = 0.69-0.79). QconCAT-based data revealed generally poor correlation with activity, whereas moderate correlations were shown for UGTs 1A1, 1A3, and 2B7. Spurious abundance-activity correlations were identified in the cases of UGT1A4/2B4 and UGT2B7/2B15, which could be explained by correlations of protein expression between these enzymes. Consistent correlation of UGT abundance with catalytic activity, demonstrated by the SIL-based dataset, suggests that quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions.

Fig. 1.

Cross-methodology comparison of UGT abundance levels: box and whiskers plot of abundance measurements (n = 23 or 24 liver samples) of UGT enzymes quantified by SIL standards or QconCAT (A); fold difference (fold error) of matched values (i.e., for each enzyme i) (B); and correlation analysis of individual protein abundance measurements (n = 23–24) using the two methods (C). In (A) and (B), the boxes represent the 25th and 75th percentiles, the whiskers represent the minimum and maximum values and the bars represent the medians. In (A), the (+) sign represents the arithmetic mean. Differences were tested using Mann-Whitney rank order U-test: **P < 0.01; ***P < 0.001. In (B), the shaded area represents values within threefold, illustrating interchangeable abundance data. AFE is the average fold error and AAFE is the absolute average fold error. In (C), Rs is Spearman rank order correlation coefficient; green colored data points represent correlated enzyme levels between methods and gray data points reflect lack of correlation. Supplemental Information contains details of statistical analysis, and Supplemental Table 3 shows differences between generated data. Units of abundance measurements are picomoles per milligram HLM protein. QconCAT, quantitative concatemer standard; SIL, stable isotope-labeled peptide standards.

Fig. 2.

Correlation between individual protein abundance and activity measurements for UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 in the stable isotope-labeled (SIL) quantification dataset (n = 59). Statistically significant, moderate-to-strong correlations are shown in blue color. Substrates: UGT1A1, β-estradiol; UGT1A3, chenodeoxycholic acid (CDCA); UGT1A4, trifluoperazine; UGT1A6, 5-hydroxytryptophol; UGT1A9, propofol; UGT2B7, zidovudine; and UGT2B15, S-oxazepam. Rs, Spearman rank order correlation coefficient; units of abundance measurements (x-axis), picomoles per milligram HLM protein; units of catalytic activity (y-axis), nanomoles (glucuronide) per minute per milligram HLM protein.

Fig. 3.

Correlation between individual protein abundance and activity measurements for UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 in the quantitative concatemer (QconCAT) quantification dataset (n = 23 or 24). Statistically significant, moderate correlations are shown in blue color, and nonsignificant, weak correlations are shown in gray. Substrates: UGT1A1, β-estradiol; UGT1A3, chenodeoxycholic acid (CDCA); UGT1A4, trifluoperazine; UGT1A6, 5-hydroxytryptophol; UGT1A9, propofol; UGT2B7, zidovudine; and UGT2B15, S-oxazepam. Rs, Spearman rank order correlation coefficient; units of abundance measurements (x-axis), picomoles per milligram HLM protein; units of catalytic activity (y-axis), nanomoles (glucuronide) per minute milligram HLM protein.

Fig. 4.

Correlation matrix of individual protein abundance and activity measurements (abundance vs. activity) of UGT enzymes based on the extended dataset obtained using SIL standards (n = 59). Significant correlations are represented in blue (Rs > 0.5, Bonferroni corrected P < 0.01, R² > 0.3). Spurious correlations are shown in red. Supplemental Table 4 shows the statistical analysis used to generate the activity-abundance correlation matrix. AZT, zidovudine; CDCA, chenodeoxycholic acid; EST, β-estradiol; 5HTOL, 5-hydroxytryptophol; OXAZ, S-oxazepam; PRO, propofol; Rs, Spearman rank order correlation coefficient; TFP, trifluoperazine.

Fig. 5.

Correlation matrix of individual protein abundances of UGT enzymes (abundance vs. abundance) using the two proteomic methodologies: SIL standard-based quantification (closed circles, n = 60 livers) and QconCAT-based quantification (open circles, n = 23 or 24 livers). Significant correlations are represented in blue (Rs > 0.5, Bonferroni corrected P < 0.01, R² > 0.3). x- and y-Axes represent abundance levels of UGT enzymes expressed in units of picomoles per milligram HLM protein; Rs, Spearman rank order correlation coefficient. Supplemental Table 6 shows the statistical analysis used to generate the abundance correlation matrix.