Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics

Gröer, Christian; Busch, Diana; Patrzyk, Maciej; Beyer, Karl H.; Busemann, A.; Heidecke, Claus–Dieter; Droździk, Marek; Siegmund, Werner; Oswald, Stefan

doi:10.1016/j.jpba.2014.08.016

Cited by 90 publications

(75 citation statements)

References 32 publications

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“…These results were consistent with the recombinant UGT data ( Figure 5, 34, 36). AG formation is primarily mediated by UGT2B7 as confirmed by recombinant UGT and HLM data ( Figure 5, 34, 36). Etio is metabolized by both UGT2B17 (r 2 = 0.7; Figure 5 c ) and UGT2B7 (r 2 = 0.44; Figure 5).…”

Section: Resultssupporting

confidence: 92%

“…For example, there was a good correlation between TG formation and UGT2B17 protein abundance in HLMs (r 2 = 0.87; Figure 5, 34, 36). These results were consistent with the recombinant UGT data ( Figure 5, 34, 36).…”

Section: Resultsmentioning

confidence: 91%

“…( h and i ) These panels show the estimated glucuronidation activity of UGT2Bs toward testosterone, A, and Etio in the human liver and the intestinal microsomes. The scaling was based on UGT2B protein abundance in the liver (36.6, 21.2, and 0.92 pmol/mg36 and the intestine (2.83, 0.0, and 8.87 pmol/mg) for UGT2B7, UGT2B15, and UGT2B17, respectively 34. AG, androsterone glucuronide; EtioG, etiocholanolone glucuronide; TG, testosterone glucuronide…”

Section: Resultsmentioning

confidence: 99%

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Effect of Dose and 5α‐Reductase Inhibition on the Circulating Testosterone Metabolite Profile of Men Administered Oral Testosterone

Basit

Amory

Prasad

2018

Clinical Translational Sci

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Development of an oral testosterone therapy has proven extremely challenging because of extensive and variable first‐pass metabolism. We investigated the in vivo metabolism of testosterone with increasing oral doses of testosterone, both alone and with the co‐administration of dutasteride (5α‐reductase inhibitor) by liquid‐chromatography tandem mass spectrometry (LC‐MS/MS). In eugonadal men prior to dosing, the circulating concentration of testosterone, androstenedione, etiocholanolone‐glucuronide, and androsterone‐glucuronide was 8.6, 20.9, 9.1, and 55.3%, respectively, of the total testosterone‐related species, whereas testosterone‐glucuronide was ∼1%. When testosterone was dosed orally to men with experimental hypogonadism, a proportion of testosterone‐glucuronide increased to 13%. Dutasteride treatment significantly decreased levels of androsterone and its metabolites. This work reveals extensive metabolism of orally dosed testosterone to androsterone glucuronide via androstenedione, with testosterone‐glucuronide appearing to be the second most important metabolite. This information is of importance in the development of an effective oral testosterone therapy and may have implications for testosterone doping research.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Effect of Dose and 5α‐Reductase Inhibition on the Circulating Testosterone Metabolite Profile of Men Administered Oral Testosterone

Basit

Amory

Prasad

2018

Clinical Translational Sci

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“…Our limit of quantitation was 5 fmol/lane, which represents a 6-fold reduction over the 31 fmol/lane limit of quantitation reported by Croom et al (2009) and is comparable to limits of quantitation determined by highly accurate HPLC tandem mass spectrometry methods (Gröer et al, 2014) This increased sensitivity potentially explains, in part, our success in detecting CYP2B6 in all of our liver samples.…”

Section: Discussionsupporting

confidence: 66%

Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation

Pearce

Gaedigk

Twist

et al. 2015

Drug Metabolism and Disposition

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Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

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“…In particular, reaction phenotyping for drug-metabolizing enzymes using correlation approaches is crucially dependent on robust analytical methods used to measure both activity and expression levels in individual samples (Zientek and Youdim, 2015). However, despite recent efforts aimed at developing assays to characterize the abundance and activity of drug-metabolizing enzymes, with considerable success especially in the case of cytochrome P450 enzymes (Walsky and Obach, 2004;Gröer et al, 2014), this level of understanding is still hindered by the lack of standard and consistent methods for quantifying uridine-59-diphospho-glucuronosyltransferase (UGT) expression and function (Guillemette et al, 2014). Correlations of enzyme abundances and activity were previously demonstrated for several cytochrome P450 enzymes (Snawder and Lipscomb, 2000;Olesen and Linnet, 2001) and some UGT enzymes (mainly UGTs 1A1, 1A6, 1A9, and 2B7) (Jones et al, 2012;Sato et al, 2012;Knights et al, 2016), with a variety of substrates and different levels of correlation.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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; R 2 = 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; R 2 = 0.34-0.58; n = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79-0.90, P < 0.0001; R 2 = 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.

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Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics

Cited by 90 publications

References 32 publications

Effect of Dose and 5α‐Reductase Inhibition on the Circulating Testosterone Metabolite Profile of Men Administered Oral Testosterone

Effect of Dose and 5α‐Reductase Inhibition on the Circulating Testosterone Metabolite Profile of Men Administered Oral Testosterone

Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation

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

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