A number of endogenous and xenobiotic organic acids are conjugated to glycine, in animals ranging from mosquitoes to humans. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, the recently proposed glycine deportation hypothesis states that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilized as neurotransmitters in the central nervous systems of animals. This hypothesis is based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. In this review it will be argued that the major role of glycine conjugation is to dispose of the end products of phenylpropionate metabolism. Furthermore, glucuronidation, which occurs in the endoplasmic reticulum, would not be ideal for the detoxification of free benzoate, which has been shown to accumulate in the mitochondrial matrix. Glycine conjugation, however, prevents accumulation of benzoic acid in the mitochondrial matrix by forming hippurate, a less lipophilic conjugate that can be more readily transported out of the mitochondria. Finally, it will be explained that the glycine conjugation of benzoate, a commonly used preservative, exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated.
The developed LC-MS/MS assay is to our knowledge the first method available for the simultaneous quantification of PABA and its glycine conjugation metabolites in human urine and provides important quantitative data for studies of this phase 2 biotransformation pathway.
Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism.
Glycine conjugation facilitates the metabolism of toxic aromatic acids, capable of disrupting mitochondrial integrity. Owing to the high exposure to toxic substrates, characterization of individual glycine conjugation capacity, and its regulatory factors has become increasingly important. Aspirin and benzoate have been employed for this purpose; however, adverse reactions, aspirin intolerance, and Reye's syndrome in children are substantial drawbacks. The goal of this study was to investigate p-aminobenzoic acid (PABA) as an alternative glycine conjugation probe. Ten human volunteers participated in a PABA challenge test, and p-aminohippuric acid (PAHA), p-acetamidobenzoic acid, and p-acetamidohippuric acid were quantified in urine. The glycine N-acyltransferase gene of the volunteers was also screened for two polymorphisms associated with normal and increased enzyme activity. All of the individuals were homozygous for increased enzyme activity, but excretion of PAHA varied significantly (16-56%, hippurate ratio). The intricacies of PABA metabolism revealed possible limiting factors and the potential of PABA as an indicator of Phase 0 biotransformation.
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