Blood Sampling and Laboratory Assays
Non-fasting venous blood was drawn at baseline and at the follow-up after 16 weeks. Blood for serum was collected in serum-separator gel tubes and centrifuged after 30 min to 2 h, and blood for plasma was collected in EDTA tubes and centrifuged within 30 min at room temperature at the study site. Serum and plasma were separated and frozen in several aliquots at −20°C the same day and within 1–2 weeks frozen at −80°C until they were analyzed. After the completion of the study, all serum samples from baseline and follow-up were analyzed in one batch at the Fürst Medical Laboratory (http://www.furst.no), which is accredited by the International Organization for Standardization and is part of the vitamin D External Quality Assessment Scheme (DEQAS).
Serum 25-hydroxyvitamin D (s-25(OH)D) was measured using high-pressure liquid chromatography tandem mass spectrometry, with Waters Acquity UPLC and Waters triple quadrupole mass spectrometer instruments. Both 25(OH)D2 and 25(OH)D3 levels were measured and the sum of the two was used for analysis (termed 25(OH)D, even though 25(OH)D2 was negligible). The within-batch coefficient of variation for s-25(OH)D3 was 4.8% within high concentrations and 7.2% within low concentrations.
HbA1c was analyzed on a cation exchange column chromatograph using an automated high-pressure liquid chromatography instrument (HLC-723 G7, Tosoh Corporation, Tokyo, Japan). The reference upper normal for HbA1c is <6.1%. The total coefficient of variation was 2% at HbA1c levels around 6.1%.
Fructosamine was measured using a colorimetric enzymatic method (ADVIA 2400 Siemens). The interassay coefficient of variation (CV) was 2.3% and reference upper normal was 285 μmol/L.
Total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were measured using an enzymatic method (ADVIA 2400 Siemens). The interassay coefficients of variation were 1.3% (total cholesterol), 1.6% (LDL-cholesterol), 1.8% (HDL-cholesterol), and 3.8% (triglycerides).
Body weight was measured with a Bosogramm 3000 Scale (loading capacity 150 kg) to the nearest 100 g with participants in indoor clothing without shoes. Height was measured to the nearest centimeter with a rigid meter standard. The same devices were used both at baseline and at follow-up. The participants completed an interviewer-administered questionnaire at baseline and follow-up. Information about age, ethnicity, education, and duration of residence in Norway was collected at baseline.