Kirsten G. Dowling

Vitamin D deficiency in Europe: pandemic?

Background: Vitamin D deficiency has been described as being pandemic, but serum 25-hydroxyvitamin D [25(OH)D] distribution data for the European Union are of very variable quality. The NIH-led international Vitamin D Standardization Program (VDSP) has developed protocols for standardizing existing 25(OH)D values from national health/nutrition surveys. Objective: This study applied VDSP protocols to serum 25(OH)D data from representative childhood/teenage and adult/older adult European populations, representing a sizable geographical footprint, to better quantify the prevalence of vitamin D deficiency in Europe. Design: The VDSP protocols were applied in 14 population studies [reanalysis of subsets of serum 25(OH)D in 11 studies and complete analysis of all samples from 3 studies that had not previously measured it] by using certified liquid chromatography–tandem mass spectrometry on biobanked sera. These data were combined with standardized serum 25(OH)D data from 4 previously standardized studies (for a total n = 55,844). Prevalence estimates of vitamin D deficiency [using various serum 25(OH)D thresholds] were generated on the basis of standardized 25(OH)D data. Results: An overall pooled estimate, irrespective of age group, ethnic mix, and latitude of study populations, showed that 13.0% of the 55,844 European individuals had serum 25(OH)D concentrations <30 nmol/L on average in the year, with 17.7% and 8.3% in those sampled during the extended winter (October–March) and summer (April–November) periods, respectively. According to an alternate suggested definition of vitamin D deficiency (<50 nmol/L), the prevalence was 40.4%. Dark-skinned ethnic subgroups had much higher (3- to 71-fold) prevalence of serum 25(OH)D <30 nmol/L than did white populations. Conclusions: Vitamin D deficiency is evident throughout the European population at prevalence rates that are concerning and that require action from a public health perspective. What direction these strategies take will depend on European policy but should aim to ensure vitamin D intakes that are protective against vitamin D deficiency in the majority of the European population.

Vitamin D and mortality: Individual participant data meta-analysis of standardized 25-hydroxyvitamin D in 26916 individuals from a European consortium

Background Vitamin D deficiency may be a risk factor for mortality but previous meta-analyses lacked standardization of laboratory methods for 25-hydroxyvitamin D (25[OH]D) concentrations and used aggregate data instead of individual participant data (IPD). We therefore performed an IPD meta-analysis on the association between standardized serum 25(OH)D and mortality. Methods In a European consortium of eight prospective studies, including seven general population cohorts, we used the Vitamin D Standardization Program (VDSP) protocols to standardize 25(OH)D data. Meta-analyses using a one step procedure on IPD were performed to study associations of 25(OH)D with all-cause mortality as the primary outcome, and with cardiovascular and cancer mortality as secondary outcomes. This meta-analysis is registered at ClinicalTrials.gov, number NCT02438488. Findings We analysed 26916 study participants (median age 61.6 years, 58% females) with a median 25(OH)D concentration of 53.8 nmol/L. During a median follow-up time of 10.5 years, 6802 persons died. Compared to participants with 25(OH)D concentrations of 75 to 99.99 nmol/L, the adjusted hazard ratios (with 95% confidence interval) for mortality in the 25(OH)D groups with 40 to 49.99, 30 to 39.99, and <30 nmol/L were 1.15 (1.00–1.29), 1.33 (1.16–1.51), and 1.67 (1.44–1.89), respectively. We observed similar results for cardiovascular mortality, but there was no significant linear association between 25(OH)D and cancer mortality. There was also no significantly increased mortality risk at high 25(OH)D levels up to 125 nmol/L. Interpretation In the first IPD meta-analysis using standardized measurements of 25(OH)D we observed an association between low 25(OH)D and increased risk of all-cause mortality. It is of public health interest to evaluate whether treatment of vitamin D deficiency prevents premature deaths.

Standardizing serum 25-hydroxyvitamin D data from four Nordic population samples using the Vitamin D Standardization Program protocols: Shedding new light on vitamin D status in Nordic individuals

Abstract Knowledge about the distributions of serum 25-hydroxyvitamin D (25(OH)D) concentrations in representative population samples is critical for the quantification of vitamin D deficiency as well as for setting dietary reference values and food-based strategies for its prevention. Such data for the European Union are of variable quality making it difficult to estimate the prevalence of vitamin D deficiency across member states. As a consequence of the widespread, method-related differences in measurements of serum 25(OH)D concentrations, the Vitamin D Standardization Program (VDSP) developed protocols for standardizing existing serum 25(OH)D data from national surveys around the world. The objective of the present work was to apply the VDSP protocols to existing serum 25(OH)D data from a Danish, a Norwegian, and a Finnish population-based health survey and from a Danish randomized controlled trial. A specifically-selected subset (n 100–150) of bio-banked serum samples from each of the studies were reanalyzed for 25(OH)D by LC-MS/MS and a calibration equation developed between old and new 25(OH)D data, and this equation was applied to the entire data-sets from each study. Compared to estimates based on the original serum 25(OH)D data, the percentage vitamin D deficiency (< 30 nmol/L) decreased by 21.5% in the Danish health survey but by only 1.4% in the Norwegian health survey; but was relatively unchanged (0% and 0.2%) in the Finish survey or Danish RCT, respectively, following VDSP standardization. In conclusion, standardization of serum 25(OH)D concentrations is absolutely necessary in order to compare serum 25(OH)D concentrations across different study populations, which is needed to quantify and prevent vitamin D deficiency.Knowledge about the distributions of serum 25-hydroxyvitamin D (25(OH)D) concentrations in representative population samples is critical for the quantification of vitamin D deficiency as well as for setting dietary reference values and food-based strategies for its prevention. Such data for the European Union are of variable quality making it difficult to estimate the prevalence of vitamin D deficiency across member states. As a consequence of the widespread, method-related differences in measurements of serum 25(OH)D concentrations, the Vitamin D Standardization Program (VDSP) developed protocols for standardizing existing serum 25(OH)D data from national surveys around the world. The objective of the present work was to apply the VDSP protocols to existing serum 25(OH)D data from a Danish, a Norwegian, and a Finnish population-based health survey and from a Danish randomized controlled trial. A specifically-selected subset (n 100-150) of bio-banked serum samples from each of the studies were reanalyzed for 25(OH)D by LC-MS/MS and a calibration equation developed between old and new 25(OH)D data, and this equation was applied to the entire data-sets from each study. Compared to estimates based on the original serum 25(OH)D data, the percentage vitamin D deficiency (< 30 nmol/L) decreased by 21.5% in the Danish health survey but by only 1.4% in the Norwegian health survey; but was relatively unchanged (0% and 0.2%) in the Finish survey or Danish RCT, respectively, following VDSP standardization. In conclusion, standardization of serum 25(OH)D concentrations is absolutely necessary in order to compare serum 25(OH)D concentrations across different study populations, which is needed to quantify and prevent vitamin D deficiency.

The positive impact of general vitamin D food fortification policy on vitamin D status in a representative adult Finnish population: evidence from an 11-y follow-up based on standardized 25-hydroxyvitamin D data

Background: A systematic vitamin D fortification of fluid milk products and fat spreads was started in 2003 in Finland to improve vitamin D status. Objective: We investigated the effects of the vitamin D fortification policy on vitamin D status in Finland between 2000 and 2011.Design: Serum 25-hydroxyvitamin D [S-25(OH)D] concentrations of a nationally representative sample comprising 6134 and 4051 adults aged ≥30 y from the Health 2000 and Health 2011 surveys, respectively, were standardized according to the Vitamin D Standardization Program with the use of liquid chromatography-tandem mass spectrometry. Linear and logistic regression models were used to assess the change in S-25(OH)D concentrations.Results: Between 2000 and 2011, the mean S-25(OH)D increased from 48 nmol/L (95% CI: 47, 48 nmol/L) to 65 nmol/L (95% CI: 65, 66 nmol/L) (P < 0.001). The prevalence of vitamin D supplement users increased from 11% to 41% (P < 0.001). When analyzing the effect of fortification of fluid milk products, we focused on supplement nonusers. The mean increase in S-25(OH)D in daily fluid milk consumers (n = 1017) among supplement nonusers was 20 nmol/L (95% CI: 19, 21 nmol/L), which was 6 nmol/L higher than nonconsumers (n = 229) (14 nmol/L; 95% CI: 12, 16 nmol/L) (P < 0.001). In total, 91% of nonusers who consumed fluid milk products, fat spreads, and fish based on Finnish nutrition recommendations reached S-25(OH)D concentrations >50 nmol/L in 2011.Conclusions: The vitamin D status of the Finnish adult population has improved considerably during the time period studied. The increase is mainly explained by food fortification, especially of fluid milk products, and augmented vitamin D supplement use. Other factors, such as the difference in the ultraviolet radiation index between 2000 and 2011, may partly explain the results. When consuming vitamin D sources based on the nutritional recommendations, vitamin D status is sufficient [S-25(OH)D ≥50 nmol/L], and supplementation is generally not needed.

Improved accuracy of an tandem liquid chromatography-mass spectrometry method measuring 24R,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150μL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.

Prevalence of vitamin D deficiency and insufficiency among schoolchildren in Greece: The role of sex, degree of urbanisation and seasonality

The current study was aiming to report the prevalence of suboptimal vitamin D status among schoolchildren in Greece and investigate the role of sex, urbanisation and seasonality on vitamin D status. A sample of 2386 schoolchildren (9-13 years old) from four distinct prefectures was examined. The prevalence of 25-hydroxyvitamin D (25(OH)D) concentration <30 and <50 nmol/l (vitamin D deficiency and insufficiency respectively) was 5·2 and 52·5 %, respectively. Girls had a higher prevalence of 25(OH)D<30 (7·2 v. 3·2 %) and 50 nmol/l (57·0 v. 48·0 %) than boys (P<0·001). The highest prevalence rates of 25(OH)D<30 and 50 nmol/l (9·1 and 73·1 %, respectively) were observed during spring (April to June), whereas the lowest (1·5 and 31·9 %, respectively) during autumn (October to December). The prevalence of 25(OH)D<50 nmol/l was higher in urban/semi-urban than rural regions, particularly during spring months (74·6 v. 47·2 %; P<0·001). Female sex, urban/semi-urban region of residence and spring months were found to increase the likelihood of vitamin D deficiency and insufficiency, with the highest OR observed for spring months (7·47; 95 % CI 3·23, 17·3 and 5·14; 95 % CI 3·84, 6·89 for 25(OH)D<30 and 50 nmol/l respectively). In conclusion, despite the southerly latitude, the prevalence of low vitamin D status among primary schoolchildren in Greece is comparable to or exceeds the prevalence reported among children and adolescents on a European level. Sub-populations at highest risk are girls in urban/semi-urban areas during spring months, thus indicating the need for effective initiatives to support adequate vitamin D status in these population groups.

General Steps to Standardize the Laboratory Measurement of Serum Total 25-Hydroxyvitamin D.

The Vitamin D Standardization Program (VDSP) has collaborated with numerous groups and agencies to assemble a set of tools, i.e., a reference measurement system, that can be used to establish the traceability of 25-hydroxyvitamin D [25(OH)D] assays to relevant reference measurement procedures and reference materials. This is done with the goal of verifying end-user laboratory performance using precise statistical criteria to determine whether a specific assay is standardized. The purpose of this paper was to outline a set of steps that routine clinical and research laboratories can use to standardize their 25(OH)D assays using these tools. These steps apply to laboratories using commercially developed immunoassay measurement systems as well as in-house assays, usually based on high HPLC or LC tandem MS measurement systems. The steps are (1) initial calibration, (2) initial assessment of accuracy and bias, (3) assessment of total percent CV and mean bias, (4) use of trueness controls, and (5) participation in accuracy-based performance testing and/or external quality assessment schemes. The goal of each laboratory assay is to have a total CV of ≤10% and mean bias of ≤5%. Rigorous and less rigorous but low-cost options for meeting these statistical criteria are provided. Research laboratories who infrequently measure 25(OH)D are advised to repeat steps 1-4 for every measurement cycle. For users of commercial immunoassays who have relatively little control over standardization, we present an option for using trueness controls to develop a master equation that can be used to standardize results to the reference methods.

Vitamin D-biofortified beef: A comparison of cholecalciferol with synthetic versus UVB-mushroom-derived ergosterol as feed source

This study investigates dietary fortification of heifer feeds with cholecalciferol and ergocalciferol sources and effects on beef total vitamin D activity, vitamer, respective 25-hydroxymetabolite contents, and meat quality. Thirty heifers were allocated to one of three dietary treatments [(1) basal diet + 4000 IU of vitamin D3 (Vit D3); (2) basal diet + 4000 IU of vitamin D2 (Vit D2); and (3) basal diet + 4000 IU of vitamin D2-enriched mushrooms (Mushroom D2)] for a 30 day pre-slaughter period. Supplementation of heifer diets with Vit D3 yielded higher (p < 0.001) Longissimus thoracis (LT) total vitamin D activity (by 38-56%; p < 0.05) and serum 25-OH-D concentration (by 20-36%; p < 0.05), compared to that from Vit D2 and Mushroom D2 supplemented animals. Irrespective of vitamin D source, carcass characteristics, sensory and meat quality parameter were unaffected (p > 0.05) by the dietary treatments. In conclusion, vitamin D3 biofortification of cattle diets is the most efficacious way to enhance total beef vitamin D activity.

Interlaboratory Comparison for the Determination of 24,25-Dihydroxyvitamin D3 in Human Serum Using Liquid Chromatography with Tandem Mass Spectrometry.

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratory methods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (-1.6% mean bias), three laboratories at 10-15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a method calculation error and another laboratory modified and improved their LC-MS/MS method.

The use of synthetic and natural vitamin D sources in pig diets to improve meat quality and vitamin D content

This study investigated the effects of synthetic and natural sources of vitamin D biofortification in pig diets on pork vitamin D activity and pork quality. One hundred and twenty pigs (60 male, 60 female) were assigned to one of four dietary treatments for a 55 d feeding period. The dietary treatments were (1)50 μg vitamin D₃/kg of feed; (2)50 μg of 25-hydroxvitamin D₃/kg of feed (25-OH-D₃); (3)50 μg vitamin D₂/kg of feed; (4)50 μg vitamin D₂-enriched mushrooms/kg of feed (Mushroom D₂). The pigs offered the 25-OH-D₃ diet exhibited the highest (P < 0.001) serum total 25-hydroxyvitamin D concentration and subsequently exhibited the highest (P < 0.05) Longissimus thoracis (LT) total vitamin D activity. Mushroom D2 and 25-OH-D3 supplementation increased pork antioxidant status. The vitamin D₂-enriched mushrooms improved (P < 0.05) pig performance, carcass weight and LT colour. In conclusion, 25-OH-D₃ is the most successful source for increasing pork vitamin D activity, while Mushroom D2 may be a new avenue to improve animal performance and pork quality.