Alt activity kit

ALT and AST levels were measured using an ALT activity kit and AST activity assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

The concentrations of total cholesterol and triglycerides were determined in whole plasma samples of each mouse with aid of the enzymatic colorimetric assays as described above. The distribution of cholesterol and triglycerides over the different lipoproteins was analysed by fractionation of plasma pools containing equal volumes of all mice in each treatment group using a Superose 6 column (3.2 × 300 mm, Smart-system, Pharmacia). In addition, plasma pools containing samples from 2 mice from per treatment group (total n = 4/5 of pooled samples) were run in an untargeted LC–MS/MS lipidomics analysis as set up by Schoeman et al.^{33 (link)}. The metabolite panel consisted of > 100 different species, including essential fatty acids, bile acids, isoprostanes, sphingoid mediators, and lysophosphatidic acids. Integrated peak sizes of each individual metabolite were expressed relative to those of the control group. Plasma alanine aminotransferase (ALT) levels were measured with the ALT activity kit from Sigma according to the protocol of the supplier.

Alanine aminotransferase activity (ALT) in liver tissue lysate was measured using an ALT activity kit (MAK052; Sigma-Millipore, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, from each mouse 40 mg of collected liver tissue were homogenized in 300 µl of ALT Assay Buffer using a Power Gen 125 tissue homogenizer (Thermo Fisher Scientific, Hampton, NH, USA). Homogenized liver samples were centrifuged at 15,000g for 15 min at 4 °C. Supernatant containing ALT from each liver sample was diluted 1:100 for ALT activity determination. 20 µl of the diluted samples was pipetted in duplicates onto a 96-well microplate. Wells were treated with ALT Master Mix solution. Changes in colorimetric intensity was measured at 570 nm over 30 min (5 min reading intervals, 37 °C) using a Synergy™ H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT, USA).
The amount of generated pyruvate, a measure of ALT activity, was determined using a standard curve. ALT activity was calculated using Eq. (1). $$\text{ALT}=\frac{(nmolofALT\times DilutionFactor)}{\left(T_{\mathit{final}},-,T_{\mathit{initial}}\right)\times \left(V,o,l,u,m,e,o,f,S,a,m,p,l,e\right)}$$
ALT activity is reported as nmole/min/ml = milliunit/mL (mU/ml), where one milliunit (mU) of ALT is defined as the amount of enzyme that generates 1.0 nmole of pyruvate per minute at 37 °C.