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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.

Background & Aims There is a demand for more sensitive, specific and predictive biomarkers for drug-induced liver injury ( DILI) than the gold standard used today, alanine aminotransferase ( ALT). The aim of this study was to qualify novel DILI biomarkers (keratin-18 markers M65/M30, micro RNA-122, glutamate dehydrogenase and alpha-foetoprotein) in human DILI. Methods Levels of the novel biomarkers were measured by enzyme-linked immunosorbent assay or real-time quantitative reverse-transcription PCR ( qRT-PCR) in two human DILI cohorts: a human volunteer study with acetaminophen and a human immunodeficiency virus (HIV)/tuberculosis (TB) study. Results In the acetaminophen study, serum M65 and micro RNA-122 levels were significantly increased at an earlier time point than ALT. Furthermore, the maximal elevation of M65 and micro RNA-122 exceeded the increase in ALT. In the HIV/ TB study, all the analysed novel biomarkers increased after 1 week of treatment. In contrast to ALT, the novel biomarkers remained stable in a human cohort with exercise-induced muscular injury. Conclusions M65 and micro RNA-122 are potential biomarkers of DILI superior to ALT with respect to sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Abstract: Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements. [Copyright &y& Elsevier]