25-Hydroxyvitamin D variability within-person due to diurnal rhythm and illness

Vitamin D nutrition research requires accurate measures of circulating 25-hydroxyvitamin D [25(OH)D]. Our objectives were to test whether a diurnal fluctuation in blood-spot concentrations of 25(OH)D can be demonstrated statistically in a single individual, and whether such fluctuation is affected by the pre- vs post-dose timing of the blood draw. The subject of this case study was a generally healthy female in her 40s who has taken 5000 IU vitamin D3 supplement at mid-day for over one year. Each blood sample was drawn individually from a finger prick onto filter paper at morning, mid-day or night, on four days (three groups of five individual blood samples per collection day). On Days 1 and 2, the mid-day samples were collected approximately 1 hour after the supplement was taken; on Days 3 and 4, the mid-day samples were collected within an hour prior to supplementation (the classical, daily “trough” value for a drug). There was a significant daily pattern of variation in 25(OH)D concentrations (ANOVA p≤0.02 for 3 of the 4 days): peak mid-day mean 25(OH)D was approximately 20% higher than in the morning, and approximately 13% higher than in the evening. Trough sampling produced no significant difference in 25(OH)D compared to sampling an hour after the dose. An incidental finding was that acute illness during the study was related to acutely lower 25(OH)D at every sampling time in the day (p<0.00001). Conclusions: There was a consistent diurnal variation in 25(OH)D, with the peak at mid-day. There was no difference between trough vs post-dose blood draws. Acute illness may acutely lower serum 25(OH)D levels. Because within-person, within-day variability in 25(OH)D is approximately 20%, sampling time introduces systematic error in vitamin D nutritional assessment that is bigger than random analytical error or choice of assay method.