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

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.

Isoform-specific alanine aminotransferase measurement can distinguish hepatic from extrahepatic injury in humans.

Serum alanine aminotransferase (ALT) is used as a clinical marker to detect hepatic damage and hepatoxicity. Two isoforms of ALT have been identified, ALT1 and ALT2, which have identical enzymatic capacities and are detected simultaneously in human serum/plasma using classical clinical chemical assays. Differences exist in the expression patterns of the ALT1 and ALT2 proteins in different organs which suggest that changes in the proportion of ALT1 and ALT2 in plasma may arise and reflect damage to different human organs. However, this has not been previously studied due to the lack of a selective methodology that can quantify both ALT1 and ALT2 isoforms in the total ALT activity normally measured in clinical samples. To the best of our knowledge, our current study reveals for the first time, that under 3 different conditions of liver damage (non-alcoholic fatty liver disease, hepatitis C and during liver surgery) the leakage of ALT1 activity into plasma greatly exceeds that of ALT2, and that the measurement of ALT1 during liver damage is equal to the measurement of total ALT activity. By contrast, during skeletal muscle injury, induced in volunteers by physical exertion, the leakage of ALT2 exceeds that of ALT1 and the proportion of circulating ALT isoforms changes accordingly. The ALT isoform changes occurring in plasma reflect previously demonstrated relative contents of ALT1 and ALT2 activities in human liver and skeletal muscle. These data suggest that assessing the percentage contribution of ALT1 and ALT2 activities to total ALT activity in plasma may distinguish hepatic from extrahepatic injury using the same standard analytical platform.