Subjects with Norwegian (in the following quoted as Norwegians) and other ethnic background were recruited primarily among medical students and nurse students in Oslo. In addition, subjects with Sri Lankan Tamil background (in the following quoted as Tamils) were recruited through an organisation for Tamils in Oslo (Tamil Resource and Counselling Centre). The participants recruited through medical or nursing school attended pre-test baseline examination within the period 14–17 February, and the participants recruited through the Tamil Resource and Counselling Centre attended within the period 11–14 March. The four-week intervention was thus completed in mid-April by the latest, i.e. prior to Easter holidays when sunlight exposure would be expected to increase. Those who already took a vitamin D supplement regularly, defined as once a week or more, or had been travelling to sunny areas or used a tanning bed during the previous three months, were defined as ineligible to participate. A total of 74 subjects of the 143 subjects who had agreed to participate (51.7%), were randomised to receive multivitamin tablets or fish oil capsules .
The participants each received a daily supplement of 10 μg (400 IU) vitamin D3 from one multivitamin tablet of type Vitaplex ABCD (Cederroth AS, Revetal, Norway), or one fish oil capsule, specially manufactured for this study by Peter Möller (now Möller’s, Oslo, Norway). Vitamin D3 content of the supplements was determined by an independent laboratory (AS Vitas, Oslo, Norway). Mean content was 9.79 μg per multivitamin tablet (SD 1.51), and 9.99 μg per fish oil capsule (SD 0.23), respectively. In addition to vitamin D3, the multivitamin tablets were declared by the manufacturer to contain 750 μg vitamin A, 1.5 mg vitamin B1, 1.7 mg vitamin B2, 1.5 mg vitamin B6, 20 mg niacin, 5 mg pantothenic acid, and 30 mg vitamin C. The fish oil capsules contained 660 μg vitamin A, 3.7 mg vitamin E, and 0.5 g n-3 polyunsaturated fatty acids, as analysed by the manufacturer. Retinol concentration in the fish oil capsules was standardised to be similar to the multivitamin tablets. Supplements were handed out to each participant at baseline, along with a self-administered compliance form. Participants were encouraged to maintain their usual diet during the study period. The tablets or capsules were ingested daily for 4 weeks, which is considered to be a sufficient duration in order to reach equilibrium (plateau concentration) in s-25(OH)D .
A venous serum sample was drawn on day 1, prior to the intervention, and on day 29, the day subsequent to ingesting the final tablet or capsule. The participants completed a self-administered three-page questionnaire at baseline. During completion of the questionnaire, they had the possibility to ask for assistance (i.e. clarification of questions, or language issues) from one of the project leaders. The questionnaire included questions about usual intake of vitamin D-containing foods, supplement use, clothing and sun exposure habits, self-reported height and weight, date of birth, education, and ethnic background. At follow-up, all participants’ height and weight were measured with an electronic height- and weight-measuring device.
Serum sample handling and analyses
Blood samples were centrifuged (10 min, 2000 g at 10 °C) within 30 minutes after blood collection and sera were immediately frozen and kept at −70 °C until analyzed. Pre- and post-intervention serum from all participants were analysed blinded, in one batch at the Hormone Laboratory (Department of Endocrinology, Oslo University Hospital Aker, Oslo, Norway).
S-25(OH)D was measured by radioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA). This assay measures both 25(OH)D3 and 25(OH)D2. The intra- and interassay coefficients of variation (CV) were 6% and 14-15%, respectively. The detection limit was 6 nmol/l.
Intact PTH (iPTH) were measured by chemiluminoimmunometric assay (DPC, Los Angeles, CA, USA). The intra- and interassay CVs for iPTH were 4% and 10%, respectively.
S-1,25(OH)2D was measured by competitive RIA (DiaSorin, Stillwater, MN, USA). Prior to the 1,25(OH)2D determination, serum lipids and interfering vitamin D metabolites were removed by chromatography on a C18OH column. Cross reaction with 25(OH)D after chromatography is noted to be 0.002%. The intra- and interassay CVs for the s-1,25(OH)2D assay were 7 and 14%, respectively. The limit of detection was 12 pmol/l.
S-TRACP was measured by enzyme activity assessment after immune extraction (Suomen Bioanalytiikka Oy, Oulu, Finland). This assay measures the active isoform 5b derived from osteoclasts. The intra- and interassay CVs for s-TRACP were 5–12 and 8–14% respectively.
The data were analysed in SPSS and Stata. According to normality plots, the dependent variables: Δ25(OH)D, Δ PTH, Δ1,25(OH)2D and ΔTRACP did not deviate substantially from a normal distribution, and non-parametric tests yielded similar results. Therefore, means and distributions are presented for continuous variables, and percentages are presented for categorical variables. Pre- and post-intervention concentration of biochemical parameters were compared by paired samples t tests. Crude differences between the two intervention groups were tested using t test. Potential predictors of change in biochemical parameters were analysed using linear regression.