Plasma ALT activity, an indicator of liver function, was measured using an Alanine Aminotransferase (ALT) Activity Colorimetric Assay Kit (K752, Biovision, Inc, Milpitas, CA, USA) according to the manufacturer’s instructions. We measured the plasma ALT activity for the ETGs and LTGs which were treated with dantrolene for the longest time (11 months). Briefly, 10μl plasma was diluted in a total 100μl reaction mixture, including 86μl ALT Assay Buffer, 2μl OxiRed Probe, 2μl ALT Enzyme Mix, and 10μl ALT Substrate, to analyze the pyruvate transformed from a-ketoglutarate with alanine. A standard curve was generated at the same time, using pyruvate concentrations of 0, 2, 4, 6, 8, and 10 nmol/well. The optical density (OD) at 570 nm was measured at 10 min (A1) then again at 60 min (A2) after incubating the reaction at 37°C. The pyruvate concentration was measured in a linear range of the standard curve. ALT activity was calculated using the formulation: ALTactivity = (A2 - A1)/50 * 10 mU/ml .
Alt activity colorimetric assay kit
Manufactured by Abcam
Sourced in United States, Canada
The ALT Activity Colorimetric Assay Kit measures the activity of alanine aminotransferase (ALT) in biological samples. It uses a coupled enzymatic reaction to produce a colored product, which can be detected colorimetrically.
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3 protocols using alt activity colorimetric assay kit
1
Quantifying Plasma ALT Activity
2
Serum ALT Measurement for Liver Injury
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To assess liver injury, we measured dams’ serum ALT concentrations at baseline (ED 7) and at ED 18, using an ALT Activity Colorimetric Assay Kit (BioVision Inc., San Srancisco, CA). As per the ALT assay protocol, 20 μL of samples or standards was collected in each well along with 100μL of reaction mix (86 μL ALT assay buffer, 10 μL ALT substrate, 2 μL ALT enzyme mix, and 2 μL OxiRed probe). The absorbance was read at 570 nm after 10 minutes (A1) and then again after incubating the reaction at 37°C for 60 min (A2). Because the assay is run kinetically, the difference between A2 and A1 values needed to be obtained. A standard curve generated from this data was used to determine the concentration of pyruvate and activity of ALT in each sample.
3
Liver Metabolic Biomarker Assays
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