Mechanistic Aspects and Novel Biomarkers of Responder and Non-Responder Phenotypes in Galactosamine-Induced Hepatitis
Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, United Kingdom.Journal of Proteome Research (Impact Factor: 4.25). 10/2009; 8(11):5175-87. DOI: 10.1021/pr9005266
The amino sugar galactosamine (galN) induces alterations in the hepatic uridine nucleotide pool and has been widely used as a model of human viral hepatitis. Histopathological and clinical chemistry analyses of a cohort of rats following administration of galN revealed extreme interindividual variability in the extent of the toxic response which enabled classification of 'responder' and 'non-responder' phenotypes. An integrative metabolic profiling approach was applied to characterize biomarkers of exposure to galN in urine, serum, feces and liver from responders and non-responders. The presence of N-acetylglucosamine and galN in the urine correlated with the occurrence and extent of toxic response. Conversely, the novel identification of galN-pyrazines in the feces of non-responders and their virtual absence in the feces of responders suggests an alternative means of distribution and metabolism of galN in non-responders. The absence of the UDP-hexosamines in the liver of non-responders further supports differential metabolism of galN and suggests an ability of non-responders to avoid UDP-glucose depletion. An observed disturbance of gut microbial derived metabolites in the urine and feces of non-responders may suggest a role of the microflora in reducing the effective dose of galN. This systems level metabonomic approach has provided new mechanistic insights into differential response to galN and is widely applicable to the study of interindividual variation in metabolism for any xenobiotic intervention.
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ABSTRACT: Spectroscopic profiling of biological samples is an integral part of metabolically driven top-down systems biology and can be used for identifying biomarkers of toxicity and disease. However, optimal biomarker information recovery and resonance assignment still pose significant challenges in NMR-based complex mixture analysis. The reduced signal overlap as achieved when projecting two-dimensional (2D) J-resolved (JRES) NMR spectra can be exploited to mitigate this problem and, here, full-resolution (1)H JRES projections have been evaluated as a tool for metabolic screening and biomarker identification. We show that the recoverable information content in JRES projections is intrinsically different from that in the conventional one-dimensional (1D) and Carr-Purcell-Meiboom-Gill (CPMG) spectra, because of the combined result of reduction of the over-representation of highly split multiplet peaks and relaxation editing. Principal component and correlation analyses of full-resolution JRES spectral data demonstrated that peak alignment is necessary. The application of statistical total correlation spectroscopy (STOCSY) to JRES projections improved the identification of previously overlapped small molecule resonances in JRES (1)H NMR spectra, compared to conventional 1D and CPMG spectra. These approaches are demonstrated using a galactosamine-induced hepatotoxicity study in rats and show that JRES projections have a useful and complementary role to standard one-dimensional experiments in complex mixture analysis for improved biomarker identification.
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ABSTRACT: As with many clinical studies, trials using probiotics have shown clearly that some patients benefit from the treatment while others do not. For example if treatment with probiotics leads to 36% cure rate of diarrhea, why did the other 64% not have the same result? The issue is important for human and indeed experimental animal studies for two main reasons: (i) Would changing the design of the study result in more subjects responding to treatment? (ii) If a subject does not respond what are the mechanistic reasons? In order to tackle the issue of responders and non-responders to therapy, a workshop was held by the International Scientific Association for Probiotics and Prebiotics (ISAPP). The outcome was four recommendations. 1. Clearly define the end goal: this could be supporting a health claim or having the highest clinical effect and impact. 2. Design the study to maximize the chance of a positive response by identifying precise parameters and defining the level of response that will be tested. 3. Base the selection of the intervention on scientific investigations: which strain(s) and/or product formulation should be used and why. 4. Carefully select the study cohort: use biological or genetic markers when available to stratify the patient population before enrollment and decide at what point intervention will provide the best outcome (for example, in acute phase of disease, or during remission, with or without use of pharmaceutical agents). By following these recommendations and selecting an appropriate primary outcome, it is hoped that clinical data will emerge in the future that expands our knowledge of which probiotics benefits which subjects and by what mechanism.
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ABSTRACT: We have developed an ultra performance liquid chromatography-mass spectrometry (UPLC-MS(E)) method to measure bile acids (BAs) reproducibly and reliably in biological fluids and have applied this approach for indications of hepatic damage in experimental toxicity studies. BAs were extracted from serum using methanol, and an Acquity HSS column coupled to a Q-ToF mass spectrometer was used to separate and identify 25 individual BAs within 5 min. Employing a gradient elution of water and acetonitrile over 21 min enabled the detection of a wide range of endogenous metabolites, including the BAs. The utilization of MS(E) allowed for characteristic fragmentation information to be obtained in a single analytical run, easily distinguishing glycine and taurine BA conjugates. The proportions of these conjugates were altered markedly in an experimental toxic state induced by galactosamine exposure in rats. Principally, taurine-conjugated BAs were greatly elevated ( approximately 50-fold from control levels), and were highly correlated to liver damage severity as assessed by histopathological scoring (r = 0.83), indicating their potential as a sensitive measure of hepatic damage. The UPLC-MS approach to BA analysis offers a sensitive and reproducible tool that will be of great value in exploring both markers and mechanisms of hepatotoxicity and can readily be extended to clinical studies of liver damage.
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