Effects of clofibric acid on porcine hepatocytes: a new model for the study of peroxisomal metabolism

Data on interspecies peroxisomal metabolism are limited almost entirely to rodents. Species differences are likely and the application of rodent data to man is questionable. An alternative to rodent models is desirable. Porcine tissue offers such an alternative and was explored. Porcine hepatocyte organelles were separated by isopycnic sucrose gradient centrifugation from livers of 6-month-old Yorkshire pigs. The presence of a peroxisomal palmityl-CoA oxidizing pathway, peroxisomal superoxide dismutase (SOD), and a peroxisomal NAD:aldehyde dehydrogenase (ALDH) with high K[subscript] m for acetaldehyde was demonstrated. Peroxisomal palmitate oxidizing capacity was found to be equal to that of the surviving mitochondria. The high K[subscript] misozyme of ALDH was mainly located in the mitochondria (54%), with a significant portion in the peroxisomes (32%). Remaining activity is distributed among the microsome (8.3%) and cytosol (4.6%). The low K[subscript] m isozyme was confined almost exclusively to the mitochondria. ALDH may exist in the peroxisome as a detoxification mechanism and contribute to shorter half-lives of reactive aldehydes in the cell. SOD was distributed among the peroxisomes (10%), mitochondria (20%), and cytosol (70%). SOD may scavenge reactive species of oxygen produced through peroxisomal [beta]-oxidation. A protocol for the isolation and growth of viable porcine hepatocytes is reported. The effects of clofibric acid on isolated porcine hepatocytes was investigated. Activity of selected enzymes from intact tissue were compared to isolated cells not exposed to the drug. Catalase activity was lower in isolated cells, but NAD:glutamate dehydrogenase, peroxisomal [beta]-oxidation, mitochondrial [beta]-oxidation, aldehyde dehydrogenase, and NADPH:cytochrome c reductase were similar. Isolated hepatocytes were exposed to clofibric acid concentrations ranging from 0 to 3.0 mM. Catalase, mitochondrial [beta]-oxidation, and peroxisomal [beta]-oxidation were not affected by treatments. Treatments did result in a 40% increase in protein content, a 900% increase in NADPH:cytochrome c reductase, a 400% increase in both isozymes of aldehyde dehydrogenase, and a 40% induction of superoxide dismutase activity. Responses were significantly quadratic. Porcine hepatocyte peroxisomes appear to differ significantly from those of rodents. Our data support the hypothesis that phylogenetically higher animals are different from rodents in peroxisome metabolism. Interspecies differences in metabolism is discussed

Recent advances in biomedical applications of accelerator mass spectrometry

After publication of our article, it was noted that we inadvertently failed to include the complete list of authors. The full list, including co-authors, has now been added and the Authors' contributions and Competing interests sections modified accordingly

Statistical Analysis of Variation in the Human Plasma Proteome

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery

Meeting Report--NASA Radiation Biomarker Workshop

A summary is provided of presentations and discussions from the NASA Radiation Biomarker Workshop held September 27-28, 2007, at NASA Ames Research Center in Mountain View, California. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including for long-duration space travel. Topics discussed include the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose responses, multivariate analysis of gene expression, biomarkers in biodefense, biomarkers in radiation oncology, biomarkers and triage following large-scale radiological incidents, integrated and multiple biomarker approaches, advances in whole-genome tiling arrays, advances in mass-spectrometry proteomics, radiation biodosimetry for estimation of cancer risk in a rat skin model, and confounding factors. Summary conclusions are provided at the end of the report

Effects of clofibric acid on porcine hepatocytes: a new model for the study of peroxisomal metabolism

Data on interspecies peroxisomal metabolism are limited almost entirely to rodents. Species differences are likely and the application of rodent data to man is questionable. An alternative to rodent models is desirable. Porcine tissue offers such an alternative and was explored. Porcine hepatocyte organelles were separated by isopycnic sucrose gradient centrifugation from livers of 6-month-old Yorkshire pigs. The presence of a peroxisomal palmityl-CoA oxidizing pathway, peroxisomal superoxide dismutase (SOD), and a peroxisomal NAD:aldehyde dehydrogenase (ALDH) with high K[subscript] m for acetaldehyde was demonstrated. Peroxisomal palmitate oxidizing capacity was found to be equal to that of the surviving mitochondria. The high K[subscript] misozyme of ALDH was mainly located in the mitochondria (54%), with a significant portion in the peroxisomes (32%). Remaining activity is distributed among the microsome (8.3%) and cytosol (4.6%). The low K[subscript] m isozyme was confined almost exclusively to the mitochondria. ALDH may exist in the peroxisome as a detoxification mechanism and contribute to shorter half-lives of reactive aldehydes in the cell. SOD was distributed among the peroxisomes (10%), mitochondria (20%), and cytosol (70%). SOD may scavenge reactive species of oxygen produced through peroxisomal [beta]-oxidation. A protocol for the isolation and growth of viable porcine hepatocytes is reported. The effects of clofibric acid on isolated porcine hepatocytes was investigated. Activity of selected enzymes from intact tissue were compared to isolated cells not exposed to the drug. Catalase activity was lower in isolated cells, but NAD:glutamate dehydrogenase, peroxisomal [beta]-oxidation, mitochondrial [beta]-oxidation, aldehyde dehydrogenase, and NADPH:cytochrome c reductase were similar. Isolated hepatocytes were exposed to clofibric acid concentrations ranging from 0 to 3.0 mM. Catalase, mitochondrial [beta]-oxidation, and peroxisomal [beta]-oxidation were not affected by treatments. Treatments did result in a 40% increase in protein content, a 900% increase in NADPH:cytochrome c reductase, a 400% increase in both isozymes of aldehyde dehydrogenase, and a 40% induction of superoxide dismutase activity. Responses were significantly quadratic. Porcine hepatocyte peroxisomes appear to differ significantly from those of rodents. Our data support the hypothesis that phylogenetically higher animals are different from rodents in peroxisome metabolism. Interspecies differences in metabolism is discussed.</p

Includes a Commentary by the Institute’s Health Review Committee HEALTH

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