Alanine transaminase colorimetric activity assay kit

All biochemical parameters were measured using an enzyme-linked immunosorbent assay kit (ELISA). An Alanine Transaminase Colorimetric Activity Assay Kit, Cholesterol Fluorometric Assay Kit, and Glucose Colorimetric Assay Kit were purchased from Cayman Chemical, a TNF-alpha Quantikine ELISA Kit and an Insulin ELISA Kit from R&D Systems, and a Rat Lipopolysaccharides (LPS) ELISA Kit from MyBioSource. Insulin resistance (HOMA-IR) was calculated according to the following formula: fasting serum glucose × fasting serum insulin/22.5.

Hematocrit was measured on 5 μL tail blood using heparinized capillary tubes. Blood glucose was monitored with a TrueResult glucometer and TrueTest strips (Nipro Diagnostics). Body temperature was measured using a Ret-3 rectal thermometer (Braintree Scientific). Mouse activity levels were quantified in an open field test [68 ]: briefly, mice were placed in a white 18" x 18" box and their motions were video recorded for 5 min. Data analysis was performed in the Nikon Imaging Center at UCSF/QB3 using the Tracking tool in NIS-Elements 4.20 to automatically track recorded mice. Tracking options were adjusted to ensure that the tracks accurately followed the mice, were not confused by reflections from the wall of the box, and did not stop prematurely. ALT was measured in plasma using the Alanine Transaminase Colorimetric Activity Assay Kit (Cayman Chemicals). Parasitemia and reticulocyte frequencies were enumerated from thin blood smears stained with Giemsa.

After 10 weeks of infection and therapy regimen, mice were euthanized, and total blood was harvested. Blood was spun at 10 min at 1000–2000 × g using a refrigerated centrifuge to collect plasma and 20 µl of plasma from each mouse in duplicate was used to determine ALT and AST activity using the Alanine Transaminase Colorimetric Activity Assay Kit or Aspartate Aminotransferase Colorimetric Activity Assay Kit according to the manufacturer’s instructions (Cayman Chemical).

After collection of blood in 1.5 mL EDTA tubes, samples were centrifugated at 2000× g for 10 min at 4 °C and the supernatant was collected, snap frozen in liquid nitrogen, and stored at −80 °C. Serum was collected by allowing blood to coagulate in normal Eppendorf vials for 20 min at room temperature with subsequent centrifugation at 2000× g for 10 min at 4 °C. Total homocysteine (tHCy) levels were determined on an Architect system (Abbott, Hoofddorp, The Netherlands) using a homocysteine reagent kit (ABBL482R02, Abbott, Hoofddorp, The Netherlands). Plasma samples were diluted 25-fold to achieve the required 250 µL sample volume. Plasma homocysteine levels in control groups fell below the lower limit of detection (LLOQ, 1 µM). Given that concentrations of tHCy levels in diluted samples of control groups were below plasma tHCy levels before dilution, the concentration could not have exceeded 25 μM. To establish the average tHCy levels in control groups, samples were pooled and reassessed at a 4-fold dilution. Plasma ALAT levels were measured using the alanine transaminase colorimetric activity assay kit (700260, Cayman Chemical, Ann Arbor, MI, USA). Serum hydrogen peroxide levels were determined using an Amplex red H₂O₂ kit, Life Technologies (A22188), Leusden, The Netherlands.

Peripheral blood (50 µL) was collected from retro-orbital sinus of the mice. The sera were collected using a standard protocol by clotting for 30 min at room temperature followed by centrifugation at 2000 g for 15 min. The clear supernatant was collected. The serum alanine transaminase (ALT) enzymatic activity was measured using an Alanine Transaminase Colorimetric Activity Assay Kit (Cayman chemical, #700260) following the manufacturer’s instructions.