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Reply to: ‘Vitamin D deficiency during pregnancy: confronting the issues’

Dear Editor, Thank you for the opportunity to respond to the comments of Karras et al. on our study of a cohort of Danish women in early pregnancy. The authors raise several important questions regarding the analysis method of serum 25(OH)D and the prevalence of vitamin D insufficiency. We measured serum 25(OH)D2+3 by the LC-MS/MS method validated according to the American National Institute of Standards and Technology (NIST). We chose to report the combined 25(OH)D2 + 25(OH)D3 values only. In the vast majority of participants (n = 1319), the level of 25(OH)D2 was below the detection threshold (<6 5 nmol/l). Detectable, but low, levels of D2 were seen in 29 women (2 2%), and for these women, the level of the reported 25(OH)D was the sum of D2 and D3. Vitamin D3 is of animal origin and known to have a higher biological activity than the plant-derived vitamin D2. In Denmark, vitamin D supplementation during pregnancy is recommended at a dose of 10 lg daily, and most products consist of D3. Further dietary vitamin D sources in pregnant Danish women are predominantly of animal origin, especially fish, sea food, low-fat dairy products and eggs. We agree that in other settings with a higher vitamin D2 intake, or specific vitamin D2 supplementation, separate 25(OH)D2 and 25(OH)D3 data could be potentially useful. We did not report on the less active C3and C2-epimers, which in the relatively small (n = 60) study from Karras et al. represented almost 25% of the total 25(OH)D in pregnant women at term and 22% in their offspring. In our study, the C2-epimer was expected to be negligible given the low 25(OH) D2 levels. Our laboratory has separately examined the concentration of 3-epi-25(OH)D in serum from 400 randomly selected patients who needed assessment of serum 25(OH)D. In these samples, none had a content of C3-epimer above the detection limit (1 5 nmol/l), despite full detection rate for the C3-epimer in the set-up (unpublished data). This lack of C3-detection may be due to selection of patients with low total 25(OH)D levels. Engelman recently reported a mean C3-epimer concentration of 0 5 ng/ml (1 25 nmol/l; below our detection limit) in adults with a total level of 25(OH)D below 62 5 nmol/l. In various studies, the C3-epimer represented 3 9–6% of the total 25(OH)D in children and adults, but 11–21% in neonates and infants. It could be hypothesized that the C3-epimer percentage is especially high in late pregnancy and neonates, in keeping with other alterations during pregnancy including the oestrogen-induced rise in vitamin D binding globulin, a twofold increase in 1,25 (OH)2D and a rise in the free fraction of 1,25(OH)2D. In early pregnancy, as in our study, a much lower C3-epimer fraction compared with late pregnancy could be expected. More data during early and late pregnancy including a higher number of pregnant women should be published, before conclusions on a substantially high C3-epimer fraction during late pregnancy eventually can be drawn. Future studies should, furthermore, investigate the clinical significance of the C3-epimer, which is poorly understood especially for the nonskeletal effects of vitamin D. The prevalence of vitamin D deficiency plus insufficiency as defined by 25(OH)D2+3 below 50 nmol/l was relatively low compared with Mediterranean and some other Scandinavian studies. In Danish adults in general, 52% have a 25(OH)D below 50 nmol/l. In two smaller Scandinavian studies of young women, however, only 16–23% had 25(OH)D below 50 nmol/l. Such differences may be explained by random variation (especially in small studies), selection bias, differences in lifestyle/vitamin D supplementation or genetic factors. As mentioned in the paper, our population had a lower proportion of women of non-European origin of 4 8% compared with 9 3% in the background population. Dark-coloured or veiled women in general have a lower 25(OH)D level as reported in Norway. Preliminary analysis of questionnaire data from our cohort gives reason to believe that vitamin D supplementation was widespread, which may in part explain the higher 25(OH)D level in our study. In another recent Danish study, 66% of pregnant women used vitamin D supplementation. In reports from Greece, 25(OH)D levels were much lower during pregnancy in spite of the sunny location; however, vitamin D supplementation was not used at all. Information was lacking regarding smoking status, which was very low in our cohort, and other explanatory variables like BMI and parity. These variables may also explain some of the difference in the prevalence of vitamin D insufficiency. The question of estimating ultraviolet B (UVB) exposure and associating this to serum 25(OH)D levels is complex. Ecological UVB radiation data are a poor indicator of UVB-induced D3 production on the individual level. We believe that data on individual sun-exposure habits (ideally including location, hour of the day, duration, season, clothing, sun bath and sunscreen use) and skin colour are more ideal. However, an ideal sun-exposure data set is difficult to obtain. One major problem is assessing to what extent different sunscreen products inhibit skin penetrance of UVB. Nonetheless, it is clear from the existing literature that a sunny location does not guarantee vitamin D sufficiency. We agree that this knowledge should encourage implementation of vitamin D supplementation to risk groups also in sunny countries, including pregnant women.