Serum Transaminase Elevations as Indicators of Hepatic Injury Following the Administration of Drugs

Metabolomics approaches for discovering biomarkers of drug-induced hepatotoxicity and nephrotoxicity.

Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Three forms of human ALT have been identified, ALT1 and 2 and an alternative splice variant of ALT2 (herein called ALT2_2). The standard ALT activity assay does not discriminate between ALT from different organs, or the isoforms measured in the plasma. Here, we show that ALT1 and 2 possess similar enzymatic activity for alanine and pyruvate but with different Km and kcat values, while recombinant ALT2_2 protein does not possess any enzymatic activity. Isolation of organelles from cultured human skeletal muscle cells, showed localisation of ALT2 to the mitochondrial fraction and endoplasmatic reticulum (ER), but not to the cytosol. In human hepatocytes, on the other hand, ALT1 was only localised to the cytosol and ER, with no detection in mitochondria. ALT2 was not detected in cultured human hepatocytes, liver extract or tissue using Western blotting or immunohistochemistry. The islet of Langerhans and cardiomyocytes were other examples of cells with high expression of catalytic ALT2. A clinical method for selective measurement of ALT1 and 2 in human plasma is described, and both ALT1 and 2 were immunoprecipitated from human plasma and structurally detected using Western blotting techniques.

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Detection of the mitochondrial and catalytically active alanine aminotransferase in human tissues and plasma

Metabolic profiling is a technique that can potentially provide more sensitive and specific biomarkers of toxicity than the current clinical measures benefiting preclinical and clinical drug studies. Both nuclear magnetic resonance (NMR) and mass spectrometry (MS) platforms have been used for metabolic profiling studies of drug toxicity. Not only can both techniques provide novel biomarker(s) of toxicity but the combination of both techniques gives a broader range of metabolites evaluated. Changes in metabolic patterns can provide insight into mechanism(s) of toxicity and help to eliminate a potentially toxic new chemical entity earlier in the developmental process. Metabolic profiling offers numerous advantages in toxicological research and screening as sample collection and preparation are relatively simple. Further, sample throughput, reproducibility, and accuracy are high. The area of drug toxicity of therapeutic compounds has already been impacted by metabolic profiling studies and will continue to be impacted as new, more specific biomarker(s) are found. In order for a biomarker or pattern of biomarkers to be accepted, it must be shown that they originate from the target tissue of interest. Metabolic profiling studies are amenable to any biofluid or tissue sample making it possible to link the changes noted in urine for instance as originating from renal injury. Additionally, the ease of sample collection makes it possible to follow a single animal or subject over time in order to determine whether and when the toxicity resolves itself. This review focuses on the advantages of metabolic profiling for drug toxicity studies.

The role of metabolic biomarkers in drug toxicity studies.

Intraperitoneal administration of isoniazid (IN), an antituberculous drug, to mice at a dose of 100 mg/kg body weight induced significant inhibition of serum aspartate aminotransferase (AAT) in several hours. The original activity was not restored readily by in vitro treatment of the sera with pyridoxal phosphate (PLP), in contrast to the rapid activation, by the same treatment, of apoAAT in the sera from control mice. Prolonged incubation with PLP prior to the assay was required to restore most of the lost activity. Serum AAT activity enhanced by experimental and the reversal of the inhibition similarly required prolonged incubation with PLP. The results suggest the possibility that human serum AAT activity measured for clinical diagnosis may be underestimated in cases of IN overdose even if the conditions for in vitro PLP treatment of samples are sufficient for conversion of the apoenzyme to the holoenzyme.

Iwashima A

Papers

Inhibition of serum aspartate aminotransferase induced by isoniazid administration in mice.