Hexokinase

Fasting blood samples were drawn and plasma samples were used to determine the concentration of glucose by spectrophotometry, using hexokinase as the primary enzyme (Roche Diagnostics, Japan). Diabetes mellitus was defined by fasting plasma glucose concentration of ≥ 7.0 mmol/l and/or HbA1c ≥48 mmol/mol and/or the use of glucose-lowering agents. Total cholesterol, triglycerides, and HDL cholesterol were determined by colorimetric spectrophotometry. LDL cholesterol was calculated according to the Friedewald formula [25] (link). Glycated hemoglobin (HbA1c) was measured by high-performance liquid chromatography technology (TOSOH, Tokyo, Japan). The hs-CRP concentration was measured in heparin plasma by a particle enhanced immunoturbidimetric assay. Human CRP agglutinates with latex particles were coated with monoclonal anti-CRP antibodies. The aggregates were determined turbidimetrically with a Cobas 702c analyzer (Roche Diagnostics, Mannheim, Germany). In 622 participants, the CRP-value was below the detection limit (<0.3  mg/L) and replaced by a value of 0.15  mg/L. D-dimer concentrations were quantified by commercially available ELISA kits. Fibrinogen levels were determined with the immunoprecipitation test.

Samples were autoclaved for 20 min at 120 °C to solubilise the starch. Starch was digested to glucose using α-amylase and amyloglucosidase (both from Aspergillus oryzae, manufactured by Megazyme Ltd., Co. Wicklow, Ireland) and the glucose measured enzymatically as described in Rösti et al. (2006) (link). Coupling enzymes in the glucose assay, hexokinase and (NADP-dependant) glucose-6-phosphate dehydrogenase were manufactured by Roche Diagnostics (Basel, Switzerland). The reaction was monitored at 340 nm on either a spectrophotometer or a microtitre plate reader. Initial absorbance was recorded before the reaction was started by the addition of (NADP-dependant) glucose-6-phosphate dehydrogenase. The reaction was monitored until steady absorbance was reached. The difference between initial absorbance and final absorbance was used to calculate glucose in the samples. The mass of starch (g) was calculated as moles of glucose ×162. Undigested controls were included in the analysis to adjust for glucose in the pellet not from starch.

Whole blood samples were centrifuged at 1650 g for 15 min; then, samples were pipetted into polyethylene cryovials and stored at −80 °C. Laboratory results, at all time-points, for all subjects, were performed in batch, within the same assay. Serum insulin and C-peptide were measured using a specific insulin electrochemiluminescence immunoassay (ECLIA) (Roche, Cat. No. 12017547) for the Elecsys® analyzer (Roche Diagnostics, Mannheim, Germany), with a coefficient of variation of 1.5%. Serum total cholesterol (total-cholesterol and triglycerides (TG) concentrations were measured enzymatically with Roche kits and calibrators on a Cobas Mira analyzer, as was plasma glucose (Roche Hexokinase Cat. No. 11447513216). Coefficients of variation were 2.8% for total-cholesterol, 4.4% for TG and 0.5% for plasma glucose. HDL-cholesterol was measured in the supernatant after precipitation of apolipoprotein B containing lipoproteins with phosphotungstate/magnesium chloride solution [17 (link)] with a coefficient of variation of 3.6%. LDL-cholesterol was calculated using the Friedewald equation: (total – cholesterol − HDL-cholesterol − (TG/2.18)) [18 (link)].

Participants completed a structured questionnaire with records on demographic, socioeconomic, and health-related behavior. Height measurement was performed without shoes with SECA 217 stadiometer to the nearest 0.1 cm. Weight was measured without shoes and in light clothing with SECA 877 scales to the nearest 0.1 kg. Body mass index (BMI) was determined by dividing measured body weight (kg) by height squared (m²). Fasting blood samples were drawn, and plasma samples were used to determine the concentration of glucose by spectrophotometry, using hexokinase as the primary enzyme (Roche Diagnostics, Tokyo, Japan). In this study, we defined individuals suffering from T2D according to whether they self-reported as such, had increased fasting glucose (≥7 mmol/L), or used glucose-lowering medication. Blood samples were drawn from all participants in a fasted state (>8 h of fasting). Serum TG, total cholesterol (TC), HDL-cholesterol (HDL-C), glucose, and LDL-cholesterol (LDL-C) concentrations were measured/calculated from plasma samples, while whole blood was used to determine hemoglobin A1C (HbA1c) concentrations as described previously, using an in-house assay [36 (link)]. The continuous measurements were log₁₀-transformed prior to association testing.