Konelab 20xt clinical chemistry analyzer

MMTs were performed at baseline and at 18 months after a 10-hour overnight fast. Blood was sampled at 0, 30, 60, 90 and 120 min. Participants were asked to stop the use of metformin, when used, 24 hours before the study visit. Participants consumed a 250ml liquid drink (Fortisip; 18.4 g carbohydrate per 100 ml) over a period of 2–5 min. Plasma glucose (mmol/l) was determined using the enzymatic glucose hexokinase method (Konelab 20 XT Clinical Chemistry analyzer, Thermo Fisher Scientific, Vantaa, Finland). C-peptide (nmol/l) and plasma insulin (pmol/l) were determined using chemiluminometric immunoassay (Liaison Insulin and Liaison C-peptid, DiaSorin S.p.A, Saluggia, Italy). HbA_1c (mmol/mol) was measured by ion-exchange high performance liquid chromatography on a TOSOH G8 analyser (Tosoh Bioscience, Inc, CA, USA). Triglycerides, total and high density lipoprotein (HDL) cholesterol were enzymatically assessed. More details on sample handling and biomarker determination are described elsewhere [6 (link)].

The oxidoreductase activity of purified GluD and GluE proteins was assayed using Konelab 20XT Clinical Chemistry Analyzer (Thermo Scientific). The reaction mixture contained 50 mM Tris buffer, 400 μM NAD+ or NADH, a substrate in different concentrations and purified proteins in a final concentration of 3.6 mg l^-1. The pH 8 was used with NAD+ and L-idonate and pH 7 with NADH and 2-keto-L-gulonate and 5-keto-D-gluconate. The reaction was started by addition of the purified protein and the formation/consumption of NADH was followed at 340 nm. The kinetic parameters were determined using the IC50 tool kit¹. L-Idonate and 2-keto-L-gulonate were ordered as custom synthesized by Omicron Biochemicals Inc, USA while 5-keto-D-gluconate was ordered from Sigma-Aldrich.

The oxidoreductase activity of purified GluC was assayed using Konelab 20XT Clinical Chemistry Analyzer (Thermo Scientific). The reaction mixture contained 50 mM Tris buffer, 400 μM NAD⁺ or NADH, a substrate in different concentrations and purified GluC in a final concentration of 3.6 mg l⁻¹. The pH 8 was used with NAD⁺ and L-idonate (Omicron Biochemicals Inc, USA) and pH 7 with NADH and 2-keto-L-gulonate (Omicron Biochemicals Inc, USA). The reaction was started by addition of the purified GluC and the formation/consumption of NADH was followed at 340 nm. For the HPLC analysis, a similar reaction mixture but 10 mM NAD⁺/NADH and L-idonate/2-keto-L-gulonate were used.