1,720,977 research outputs found
Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin
No full textIntroduction: Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway.
Areas covered: This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies.
Expert opinion: Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknow
The simultaneous detection and quantification of p-aminobenzoic acid and its phase 2 biotransformation metabolites in human urine using LC-MS/MS
No full textBackground: p-Aminobenzoic acid (PABA) can be used as a probe substance to investigate glycine conjugation, a reaction of phase 2 biotransformation. Methodology/Results: An LC–MS/MS method for simultaneous quantification of PABA and its metabolites from human urine was developed and validated. The metabolites can be quantified with acceptable precision and accuracy directly from human urine samples after ingestion of 550 mg PABA. Conclusion: 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 pathwa
Unbalanced biotransformation metabolism and oxidative stress status: implications for deficient fatty acid oxidation
Get PDFThe concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endo-genous, as in the case of inborn errors of me-tabolism, and lead to some of the same symp-toms as seen in xenobiotic intoxication. Bio-transformation of both exogenous and endo-genous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. We propose a novel model, to describe the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status, with resulting pathological consequences. We further used deficient fatty acid oxidation to verify the proposed model, as deficient fatty acid oxidation is associated with the accumulation of characteristic metabolites. These accumulating metabolites undergo both Phase I and Phase II biotransformation reactions, with resulting depletion of biotransformation substrates and co-factors. Depletion of these important biomolecules is capable of disturbing the balance between Phase I and Phase II reactions, and disturbance of this balance will increase oxidative stress status. The value of the proposed model is illustrated by its application to a clinical case investigated in our laboratory. In this case the possibility of deficient fatty acid oxidation only became evident once the critical balance between Phase I and Phase II biotransformation was restored with oral replenishment of biotransformation substrates. In addition to bio-chemical improvement, there was also significant clinical improvement. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. This in turn may reduce the chances of, or delay the onset of certain disease states
Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites
No full textAn imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolite
Data on the optimisation of a solid phase extraction method for fractionating estrogen metabolites from small urine volumes
Get PDFCertain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysi
The use of p-aminobenzoic acid as a probe substance for the targeted profiling of glycine conjugation
No full textGlycine 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 biotransformatio
A new perspective on the importance of glycine conjugation in the metabolism of aromatic acids
No full textA 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 underestimatedThe
National Research Foundation for a PhD bursary awarded to
C.P.S. Badenhorst, and an MSc bursary awarded to C. Nortje.
The National Research Foundation also awarded a Thuthuka
research grant [TTK20110803000023154] to R. van der Sluis
Clinically proven mtDNA mutations are not common in those with chronic fatigue syndrome
Get PDFBackground
Chronic Fatigue Syndrome (CFS) is a prevalent debilitating condition that affects approximately 250,000 people in the UK. There is growing interest in the role of mitochondrial function and mitochondrial DNA (mtDNA) variation in CFS. It is now known that fatigue is common and often severe in patients with mitochondrial disease irrespective of their age, gender or mtDNA genotype. More recently, it has been suggested that some CFS patients harbour clinically proven mtDNA mutations.
Methods
MtDNA sequencing of 93 CFS patients from the United Kingdom (UK) and South Africa (RSA) was performed using an Ion Torrent Personal Genome Machine. The sequence data was examined for any evidence of clinically proven mutations, currently; more than 200 clinically proven mtDNA mutations point mutations have been identified.
Results
We report the complete mtDNA sequence of 93 CFS patients from the UK and RSA, without finding evidence of clinically proven mtDNA mutations. This finding demonstrates that clinically proven mtDNA mutations are not a common element in the aetiology of disease in CFS patients. That is patients having a clinically proven mtDNA mutation and subsequently being misdiagnosed with CFS are likely to be rare.
Conclusion
The work supports the assertion that CFS should not be considered to fall within the spectrum of mtDNA disease. However, the current study cannot exclude a role for nuclear genes with a mitochondrial function, nor a role of mtDNA population variants in susceptibility to disease. This study highlights the need for more to be done to understand the pathophysiology of CF
New insights into the catalytic mechanism of human glycine N‐acyltransferase
No full textEven 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 mechanis
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