Hexokinase glucose 6 phosphate dehydrogenase assay

Participants were classified in the following way (“study group”): no intervention [participants from Monica10], lifestyle counseling [group B from Inter99], lifestyle and group counseling [group A from Inter99]). Education was classified as no education beyond basic or education including students. Physical activity during leisure time was divided into sedentary, light, or moderate/vigorous. Smoking habits were divided into never smokers, ex-smokers, occasional smokers, current smokers <15 g/day including occasional smokers, 15-<25 g/day, or ≥25 g of tobacco/day. BMI was divided into the following groups: <18.5, ≥18.5–25, ≥25–30, or ≥30 kg/m². Alcohol consumption was classified as consumption of 0, >0–7, >7–14, or >14 standard drinks per week.
The lipid profile was determined by enzymatic colorimetric methods (Roche, Mannheim, Germany) [16] (link), [18] (link)–[20] . Fasting plasma glucose was measured by the hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostics, former Boehringer Mannheim, Germany) [16] (link), [19] (link), [21] (link).

Fasting blood plasma samples were drawn during the health examination, which were analyzed for lipids (total cholesterol, HDL-cholesterol, and triglycerides) and glucose, insulin, and glycated haemoglobin (HbA1c). Lipids were measured using colorimetric slide methods with Vitros 4600/5600 Ortho Clinical Diagnostics (OCD), and non-HDL cholesterol was calculated by using the equation: Total cholesterol − HDL-cholesterol. Glucose was assessed by the hexokinase/glucose-6-phosphate dehydrogenase assay (Roche Diagnostics, former Boehringer Mannheim, Germany), insulin was assessed by the flouroimmunoassay technique, AutoDelfia (Perkin Elmer-Wallac, Finland), and HbA1c was assessed by the HPLC method (TOSOH, Minato, Japan). The homeostasis model of insulin resistance, HOMA-IR, was calculated using the formula: ((fasting plasma insulin [pmol/L]* fasting plasma glucose [pmol/L]/22.5) × 0.144) [29 (link)]. Based on the concept that blood glucose and insulin concentrations are related by the feedback of glucose on beta-cells to increase insulin secretion, this model is used as a surrogate measure of insulin sensitivity [29 (link)].

The same grains as used for RNA-seq were used for endosperm starch quantification, as described in [68 (link)]. Briefly, three to five dissected endosperms of known fresh weight were used for each sample. These were homogenised in 0.7 M perchloric acid. Insoluble material was collected by centrifugation, washed three times in 80% ethanol, then resuspended in water. Starch was digested using α-amylase/amyloglucosidase (Roche, Basel), and the released glucose was assayed using the hexokinase/glucose-6-phosphate dehydrogenase assay (Roche).
For curve fitting to the data, the nls() function with the SSlogis model in R (version 4.2.0) was used. This was plotted with the ‘growthcurve’ package (https://github.com/briandconnelly/growthcurve) The parameters of the model were extracted with the coef() function.