Vitamin D Status in Children With Short Stature: Accurate Determination of Serum Vitamin D Components Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Vitamin D is critical for calcium and bone metabolism. Vitamin D insufficiency impairs skeletal mineralization and bone growth rate during childhood, thus affecting height and health. Vitamin D status in children with short stature is sparsely reported. The purpose of the current study was to investigate various vitamin D components by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to better explore vitamin D storage of short-stature children in vivo. Serum circulating levels of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], and 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3, C3-epi] were accurately computed using the LC-MS/MS method. Total 25(OH)D [t-25(OH)D] and ratios of 25(OH)D2/25(OH)D3 and C3-epi/25(OH)D3 were then respectively calculated. Free 25(OH)D [f-25(OH)D] was also measured. 25(OH)D3 and f-25(OH)D levels in short-stature subgroups 2 (school age: 7~12 years old) and 3 (adolescence: 13~18 years old) were significantly lower compared with those of healthy controls. By contrast, C3-epi levels and C3-epi/25(OH)D3 ratios in all the three short-stature subgroups were markedly higher than the corresponding healthy cases. Based on cutoff values developed by Endocrine Society Recommendation (but not suitable for methods 2 and 3), sufficient storage capacities of vitamin D in short-stature subgroups 1, 2, and 3 were 42.8%, 23.8%, and 9.0% as determined by Method 3 [25(OH)D2/3+25(OH)D3], which were lower than those of 57.1%, 28.6%, and 18.2% as determined by Method 1 [25(OH)D2+25(OH)D3+C3-epi] and 45.7%, 28.5%, and 13.6% as determined by Method 2 [25(OH)D2/3+25(OH)D3+C3-epi]. Levels of 25(OH)D2 were found to be weakly negatively correlated with those of 25(OH)D3, and higher 25(OH)D3 levels were positively correlated with higher levels of C3-epi in both short-stature and healthy control cohorts. Furthermore, f-25(OH)D levels were positively associated with 25(OH)D3 and C3-epi levels in children. The current LC-MS/MS technique can not only separate 25(OH)D2 from 25(OH)D3 but also distinguish C3-epi from 25(OH)D3. Measurement of t-25(OH)D [25(OH)D2+25(OH)D3] alone may overestimate vitamin D storage in children, and short-stature children had lower vitamin D levels compared with healthy subjects. Ratios of C3-epi/25(OH)D3 and 25(OH)D2/25(OH)D3 might be alternative markers for vitamin D catabolism/storage in short-stature children. Further studies are needed to explore the relationships and physiological roles of various vitamin D metabolites.

Xu B ，Feng Y ，Gan L ，Zhang Y ，Jiang W ，Feng J ，Yu L ... -  《Frontiers in Endocrinology》

National Estimates of Serum Total 25-Hydroxyvitamin D and Metabolite Concentrations Measured by Liquid Chromatography-Tandem Mass Spectrometry in the US Population during 2007-2010.

The 2007-2010 NHANES provides the first US nationally representative serum 25-hydroxyvitamin D [25(OH)D] concentrations measured by standardized liquid chromatography-tandem mass spectrometry. We describe patterns for total 25(OH)D and individual metabolites in persons aged ≥1 y stratified by race-ethnicity and grouped by demographic, intake, physiologic, and lifestyle variables. We measured 25-hydroxycholecalciferol [25(OH)D3], 25-hydroxyergocalciferol [25(OH)D2], and C3-epimer of 25(OH)D3 [C3-epi-25(OH)D3] in serum samples (n = 15,652) from the 2007-2010 cross-sectional NHANES [total 25(OH)D = 25(OH)D3 + 25(OH)D2]. Concentrations (median, detection rate) of 25(OH)D3 (63.6 nmol/L, 100%) and C3-epi-25(OH)D3 (3.40 nmol/L, 86%) were generally detectable; 25(OH)D2 was detectable in 19% of the population. Total 25(OH)D, 25(OH)D3, and C3-epi-25(OH)D3 displayed similar demographic patterns and were strongly correlated (Spearman's r > 0.70). Concentrations of 25(OH)D2 (90th percentile) were much higher in persons aged ≥60 y (17.3 nmol/L) than in younger age groups (≤4.88 nmol/L). We noted significant race-ethnicity differences in mean total 25(OH)D [non-Hispanic blacks (NHBs), Hispanics, and non-Hispanic whites (NHWs): 46.6, 57.2, and 75.2 nmol/L, respectively] and in the prevalence of total 25(OH)D <30 nmol/L overall (24% of NHBs, 6.4% of Hispanics, and 2.3% of NHWs) as well as stratified by season (winter months: 30% of NHBs, 7.5% of Hispanics, and 3.8% of NHWs; summer months: 17% of NHBs, 4.4% of Hispanics, and 1.6% of NHWs). Persons with higher vitamin D intakes (diet, supplements, or both) and those examined during May-October had significantly higher total 25(OH)D. Significant race-ethnicity interactions in a multiple linear regression model confirmed the necessity of providing race-ethnicity-specific estimates of total 25(OH)D. Race-ethnicity differences in the prevalence of low total 25(OH)D remained strong even after adjustment for season to account for the NHANES design imbalance between season, latitude, and race-ethnicity. The strong correlation between C3-epi-25(OH)D3 and 25(OH)D3 may be because the epimer is a metabolite of 25(OH)D3. The presence of 25(OH)D2 mainly in older persons is likely a result of high-dose prescription vitamin D2.

Schleicher RL ，Sternberg MR ，Looker AC ，Yetley EA ，Lacher DA ，Sempos CT ，Taylor CL ，Durazo-Arvizu RA ，Maw KL ，Chaudhary-Webb M ，Johnson CL ，Pfeiffer CM ... -  《-》

Evaluation of vitamin D storage in patients with chronic kidney disease: Detection of serum vitamin D metabolites using high performance liquid chromatography-tandem mass spectrometry.

Vitamin D (VitD) deficiency is extremely common in chronic kidney disease (CKD). However, the current clinical testing of vitamin D is based on the recommended serum 25-hydroxyvitamin D [25(OH)D]. The levels of VitD components in CKD patients are rarely reported. In this study, we tested various VitD components, and used different methods to evaluate the VitD status of CKD patients in vivo. Totally 173 CKD patients and 111 control individuals were enrolled. Serum levels of 25(OH)D2, 25(OH)D3, C3-epimers (C3-epi) and free 25(OH)D [f-25(OH)D] were measured. The 25(OH)D2/25(OH)D3 ratio, C3-epi/25(OH)D3 ratio, total 25(OH)D [t-25(OH)D], and bioavailable vitamin D (BAVD) were calculated, respectively. The ratios of 25(OH)D2/25(OH)D3, C3-epi/25(OH)D3, and the level of C3-epi in CKD patients were significantly higher than those in the control group (all P < 0.05). The levels of t-25(OH)D, 25(OH)D3, C3-epi, f-25(OH)D and BAVD in patients with CKD stage 5 were significantly lower than those in stages 2, 3, and 4 (all P < 0.05). The calculated VitD storage according to Method 3 [25(OH)D2/3 + 25(OH)D3] was only 32.95 %, which was lower than the results of 53.76 % by Method 1 [25(OH)D2+ 25(OH)D3+C3-epi] and 48.56 % by Method 2 [25(OH)D2/3 + 25(OH)D3+C3-epi]. In addition, the VitD results calculated by three methods were positively correlated with f-25(OH)D and BAVD, while C3-epi levels were also positively correlated with f-25(OH)D and BAVD. Serum levels of t-25(OH)D, 25(OH)D3, C3-epi, f-25(OH)D and BAVD in CKD patients gradually decrease with the progression of CKD stages. Though the results of VitD storage in CKD patients evaluated by different methods are different, simultaneous detection of 25(OH)D2, 25(OH)D3, C3-epi and f-25(OH)D levels and fully estimation of their respective biological activities could accurately evaluate the VitD storage in vivo.

Li YM ，Feng Q ，Jiang WQ ，Wu BT ，Feng JF ... -  《-》

Development and application of a LC-MS/MS assay for simultaneous analysis of 25-hydroxyvitamin-D and 3-epi-25-hydroxyvitamin-D metabolites in canine serum.

Hypovitaminosis D and hypervitaminosis D are well recognised disorders in dogs. Hypovitaminosis D can occur following consumption of a diet inadequately supplemented with vitamin D or as a sequelae of severe intestinal disease. Hypervitaminosis D may occur as a result of consuming proprietary dog foods over-supplemented with vitamin D or through ingestion of vitamin D containing medicinal products or rodenticides. Consequently, there is a clear need to establish a methodology that can accurately quantify vitamin D metabolites across a broad dynamic range in dogs. The existence of C3-epimers of vitamin D metabolites has yet to be elucidated in dogs, yet are known to interfere with the analysis of vitamin D and have unknown biological activity in other species. Here, we describe the development and validation of a sensitive, specific and robust analytical liquid chromatography tandem mass spectrometry (LC-MS/MS) assay capable of separating and accurately measuring 25-hydroxyvitamin-D2/3 (25(OH)D2/3) and 3-epi-25-hydroxyvitamin-D2/3 (3-epi-25(OH)D2/3). We describe a simplified workflow utilising supported liquid extraction (SLE) without derivatization that provides good linearity (mean r > 0.996) and accuracy across a broad dynamic range of 4-500 nmol/L for D3 metabolites and 7.8-500 nmol/L for D2 metabolites. Upon application of this assay to 117 canine serum samples, 25(OH)D3 was detectable in all samples with a median concentration of 82.1 nmol/L (inter-quartile range (IQR) 59.7-101.8 nmol/L). 3-epi-25(OH)D3 could be detected in 87.2 % of the study population, with a median concentration of 5.2 nmol/L (2.4-8.1 nmol/L). However, 3-epi-25(OH)D3 was quantified below the LLOQ in 40.2 % of these samples. 3-epi-25(OH)D3 contributed on average 6.3 % to 25(OH)D3 status (contribution ranges from 0 to 23.8%) and a positive correlation was detected between 25(OH)D3 and 3-epi-25(OH)D3 concentrations. Free 25(OH)D was also measured using an immunoassay with a median concentration of 15.2 pmol/L (12.5-23.2 pmol/L), and this metabolite was also positively correlated to both 3-epi-25(OH)D3 and 25(OH)D3 concentrations. D2 metabolites were not detected in canine serum as expected. Vitamin D metabolite concentrations were variable between individuals, and research into the causes of this variation should include factors such as breed, age, sex and neuter status to determine the impact of genetic and hormonal factors. Given the clinical importance of vitamin D in dogs, and the immense potential for utilising this species as a model for human disease, further elucidation of the vitamin D pathway in this species would provide immense clinical and research benefit.

Hurst EA ，Homer NZ ，Denham SG ，MacFarlane E ，Campbell S ，Boswinkel M ，Mellanby RJ ... -  《-》

Analysis of Vitamin D Components in Serum of Minors by UHPLC-MS/MS.

He YY ，Li YM ，Chen YC ，Wu BT ，Yang YW ，Feng JF ... -  《-》