Inter‐individual variability and differential tissue abundance of mitochondrial amidoxime reducing component 1 (mARC1) enzyme in human

Abstract

Mitochondrial amidoxime-reducing component (mARC) enzymes are the recently discovered molybdenum-containing enzymes that metabolize a multitude of endobiotic and xenobiotics, e.g., N-hydroxylated nucleobases, N-oxides, N-hydroxy compounds. While two isoforms of mARC enzymes (mARC1 and mARC2) have been identified in humans, little is currently known about their inter-individual variability and differential tissue abundance. To fill this knowledge gap, the specific aims of this study were to i) develop a novel microflow liquid-chromatography tandem mass spectrometry (µLC-MS/MS) method for mARC1 quantification in human tissues, ii) quantify mARC1 abundance in five human tissues (liver, intestine, kidney, lung, and, heart), and iii) quantify inter-individual variability in mARC1 abundance by determining the effect of age and sex. Tissue samples were digested by trypsin using an optimized protocol and peptide separation was performed on an Ionkey column using an M-class microflow LC system coupled to Xevo TQ-XS MS instrument (Waters). Surrogate peptides of mARC1 were selected and quantified in multiple reaction monitoring (MRM) mode. The best two surrogate peptides of mARC1 for the targeted analysis were: DLLPIK and VGDPVYLLGQ. Using recombinant protein as a calibrator, the absolute levels of mARC1 in the liver was 41 ± 4 pmol/mg liver protein, which is comparable to the microsomal abundance of major cytochrome P450 enzymes, CYP3A4, CYP2E1, and CYP2C9. The average mARC1 abundance in the human kidney was 8-fold lower than the liver, but the abundance was independent of age in both the organs. In particular, mARC1 abundance in the liver was 35 ± 8, 41 ± 11, 39 ± 13, 42 ± 11, and 41 ± 11 pmol/mg liver protein in infants (0 to 1 year), early childhood (1 to 6 years), middle childhood (6 to 12 years), adolescence (12 to 18 years), and adults (>18 years). Similarly, the abundance was not associated with sex, i.e., mARC1 abundance in men and women was 39 ± 11 and 35 ± 10 pmol/mg protein, respectively. mARC1 was also expressed in other tissues, but its abundance (mean) in the human intestine, lung, and heart was 6-, 3-, and 28-fold lower than the liver. In summary, mARC1 abundance in humans is higher in the liver than in other studied tissues. In addition, the enzyme did not show age or sex-dependent changes in the abundance, which signifies its important physiological role. The mARC1 abundance data presented here can be used to develop physiologically-based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.