Phase 2 biotransformation : the targeted profiling of glycine amino acid conjugation

Abstract

The hepatic biotransformation system plays an important role in drug metabolism and toxicity and has been studied quite extensively. The importance of the very first biotransformation reaction discovered, namely phase 2 glycine conjugation, has however been underestimated and consequently neglected by researchers. Glycine conjugation facilitates the metabolism of potentially toxic aromatic and aliphatic acids, capable of disrupting mitochondrial integrity and energy production. Human exposure to aromatic acids such as benzoic acid and salicylates is continuous and unavoidable. This is due to metabolic interactions with intestinal microbiota and increased consumption of preservatives and medications. Individual glycine conjugation capacity varies significantly among humans however, little is known about in vivo regulatory mechanisms. The glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) gene is highly conserved among humans and polymorphisms with deleterious effects are rare. Thus, characterisation of individual glycine conjugation capacity and its regulatory factors has become increasingly important. Currently, challenge tests using aspirin and sodium benzoate as probe substances are employed for this purpose however, these methods suffer from some substantial drawbacks. Severe adverse reactions to benzoate, aspirin intolerance in adults and fear of Reye’s syndrome in children limit their use. In addition to this, the analytical methods used are susceptible to interferences from other aromatic compounds and have limited sensitivity in biological matrices. No challenge test has been able to provide supplementary information regarding possible causes of impaired glycine conjugation in vivo. What is more, studies have indicated that genetic variations in the GLYAT gene have the potential to modulate enzyme activity. However, no correlations have been made between genetic variation in GLYAT and glycine conjugation in vivo. The goal of this study was to investigate the intricacies of individual glycine conjugation and the factors that regulate it. In the first part of the study, the use of an alternative glycine conjugation probe substance, known as p-aminobenzoic acid (PABA), was investigated. A highly selective and sensitive high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for this purpose. The developed HPLC–MS/MS method could simultaneously quantify the compounds of interest, directly from human urine, with acceptable precision and accuracy. To our knowledge, this is the first HPLC–MS/MS method available for the simultaneous quantification of PABA, benzoic acid and aspirin together with their respective glycine conjugates in human urine. After completion of the method development, the applicability of the method and PABA as an alternative probe was studied during an adapted challenge test, using 10 volunteers. Time dependent quantification of p-aminohippuric acid (PAHA), p-acetamidobenzoic acid (PAABA) and p-acetamidohippuric acid (PAAHA) in the urine of the volunteers supplied useful information regarding the possible regulatory mechanisms of glycine conjugation. Unexpectedly, the potential of PABA as an indicator of Phase 0 biotransformation was also revealed. In the second part of the study, the open reading frame (ORF) of the GLYAT gene was investigated for polymorphisms that could possibly be associated with impaired glycine conjugation. Using 30 unrelated volunteers, with impaired glycine conjugation capacity, the coding regions of the GLYAT gene was investigated with next generation sequencing techniques. Only two non-synonymous variants were detected namely the N156S and S17T. These variants have been associated with increased (N156S) and similar enzyme activity (S17T) compared to the wild-type. These are also the variants with the highest allele frequency across all populations tested. No variants of the GLYAT gene with deleterious effects on enzyme activity were detected. From the results it seemed unlikely that non-synonymous SNPs in the GLYAT gene contributed to the impaired glycine conjugation capacity of these individuals. Finally, the GLYAT gene of the 10 PABA challenge test volunteers was also screened for the two polymorphisms, associated with increased and normal GLYAT activity, using Real-Time PCR assays. All of the individuals were homozygous for the N156S polymorphism associated with increased enzyme activity. Based on the results of this study, the developed analytical method can provide important quantitative data for studies of the glycine conjugation pathway. PABA holds potential as a safer glycine conjugation probe substance, which could contribute significantly to a better understanding of the complex glycine conjugation system. The molecular data supports the notion that the GLYAT ORF is highly conserved among humans due to the importance of glycine conjugation in hepatic metabolism and function. From the available literature and the results of this study it seems that genetic variation in the GLYAT ORF is not the most important contributor to inter-individual variation in glycine conjugation capacity.