Metabolism is dynamic. Acetaminophen (APAP) overdose is a classic example of non-steady state stress that initiates a dynamic cascade of responses to toxic events occurring to varying severity depending on dose. Many metabolic pathways can protect against APAP toxicity, ranging from phase I and phase II conjugation pathways to superior bioenergetic metabolism. Therefore, the goal of this research proposal is to developed a nonsteady-state challenge test using stable-labeled nutrients that directly measures a multitude of biological functions. The quantification of multiple flux rates and using them characterize a biological system is """"""""fluxomics"""""""", and herein four biomedical engineering technologies will be combined to achieve this: (1) tissue engineering of NMR-compatible bioartificial liver, (2) in vivo 13C NMR spectroscopy, (3) metabolic flux modeling, and multivariate statistical analysis to identify biomarkers. These are state-of- the-art in vivo methods will be used to verify the existing paradigm of the mechanism(s) of APAP drug injury. A considerable contribution to metabolomics will be made by the added fluxomic dataset acquired from real experimental in vivo 13C NMR spectral time courses. This new biotechnology is important for understanding and developing therapies for liver disease. In order to simultaneously determine multiple flux rates and thus perform fluxomics, a recently develop NMR-compatible human bioartificial liver will be established to track the course of u-13C-glucose. A multitude of metabolites will be resolved using a narrow- bore 600 MHz NMR spectrometer. The mentors are an in vivo NMR spectroscopist/tissue engineer (Dr. Macdonald), in a collaboration with a world-expert hepatologist on APAP toxicity (Dr. Watkins), creating a translational team for the training of the graduate student. There are two specific aims: (1) In the first year electrostatically encapsulated rat and human hepatocytes will be cultured for 3 days in a recently established NMR-compatible bioartificial liver, and on day 2 of culture 13C NMR studies of u-13C-glucose metabolism will will be obtained for 4 hr of infusion and then switched to 12C-glucose to determine flux rates of mutliple biochemicals from the decay curve, while the uptake curve will be used to identify the cascade of toxic events by comparison to control;and (2) the same study described in (1) will be performed followed by a APAP challenge on day 2 of culture. Three APAP doses will tested, 1,10, and 20 mM dissolved in the culture media, and the the fluxome and metabolome analyzed to determine the sub-lethal effects of APAP toxicity. At the end of the experiment, the cells will be extracted and analyzed by high resolution 1D 1H and 2D 1H-{13C} HSQC NMR spectroscopy and confocal microscopy for determining viability.
| Jeffries, Rex E; Gamcsik, Michael P; Keshari, Kayvan R et al. (2013) Effect of oxygen concentration on viability and metabolism in a fluidized-bed bioartificial liver using ³¹P and ¹³C NMR spectroscopy. Tissue Eng Part C Methods 19:93-100 |
| Jeffries, Rex E; Macdonald, Jeffrey M (2012) New advances in MR-compatible bioartificial liver. NMR Biomed 25:427-42 |
