Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography.

1978 ◽  
Vol 24 (2) ◽  
pp. 287-298 ◽  
Author(s):  
G Jones
Keyword(s):  
Vitamin D ◽  
Protein Binding ◽  
High Performance ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Vitamin D Metabolites ◽  
Binding Assays ◽  
Hydroxyvitamin D ◽  
Chemical Assay ◽  
25 Hydroxyvitamin D

Abstract I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.

Simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D in plasma or serum.

1981 ◽  
Vol 27 (10) ◽  
pp. 1757-1760 ◽  
Author(s):  
M J Jongen ◽  
W J van der Vijgh ◽  
H J Willems ◽  
J C Netelenbos ◽  
P Lips
Keyword(s):  
Vitamin D ◽  
Vitamin D Metabolites ◽  
Binding Assays ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Extraction And Purification ◽  
Chromatographic Procedure ◽  
25 Hydroxyvitamin D ◽  
Liquid Chromatographic

Abstract We describe a simultaneous assay for the principal vitamin D metabolites: 25-hydroxyvitamin D, 24-25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D. Special attention has been paid to simplification of the extensive extraction and purification procedures used in previously described simultaneous assays. All three metabolites were isolated with a single extraction step, followed by only one gradient liquid-chromatographic procedure. For final quantitation we used competitive protein binding assays, involving readily available binding proteins and commercially purchased tritiated vitamin D metabolites. Concentrations in the plasma of healthy subjects (mean age, 27 years), sampled during December were 51 (SD 17) nmol/L, 4.1 (SD 1.3) nmol/L, and 124 (SD 26) pmol/L for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, respectively. Intra- and interassay CVs for the three metabolites were 4.4 and 3.9%, 6.7 and 8.0%, and 7.0 and 4.8%, respectively.

Issues of methodology, standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases

2003 ◽  
Vol 40 (5) ◽  
pp. 546-551 ◽  
Author(s):  
Paul Glendenning ◽  
Jane M Noble ◽  
Mario Taranto ◽  
Alexander A Musk ◽  
Marjory McGuiness ◽  
...  
Keyword(s):  
Vitamin D ◽  
Hip Fracture ◽  
Protein Binding ◽  
High Performance ◽  
Binding Assay ◽  
Negative Bias ◽  
Radioactive Label ◽  
Hydroxyvitamin D ◽  
Protein Binding Assay ◽  
25 Hydroxyvitamin D

Background: Deficiency of vitamin D is commonly associated with hip fracture and treatment with vitamin D reduces hip fracture rates. Consequently, the demand for assays to measure 25-hydroxyvitamin D (25-OHD) has increased. The Nichols Advantage chemiluminescence protein-binding assay (CLPBA) for 25-OHD is a first-generation automated immunoassay with decreased turnaround time, reduced manual handling and non-radioactive label. Methods: We compared the CLPBA to the DiaSorin radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) for the measurement of 25-OHD using 161 samples from hip fracture patients and samples before and after institution of ergocalciferol (vitamin D2) therapy. Results: A negative bias for the CLPBA at concentrations below 30 nmol/L and a positive bias at 25-OHD values above 30 nmol/L compared with the RIA resulted in diagnostic discordance for one in three samples when using 30 and 50 nmol/L as decision limits. HPLC analysis confirmed the presence of a negative bias for the CLPBA at low values. Both immunoassays under-estimate 25-hydroxyvitamin D2. Conclusions: The discordance between 25-OHD values may be due to differences in standardization of each assay relative to HPLC. Our results emphasize the need for assay-specific clinical decision limits.

scholarly journals Vitamin D-Mediated Hypercalcemia: Mechanisms, Diagnosis, and Treatment

2016 ◽  
Vol 37 (5) ◽  
pp. 521-547 ◽  
Author(s):  
Peter J. Tebben ◽  
Ravinder J. Singh ◽  
Rajiv Kumar
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Active Form ◽  
Elevated Serum ◽  
Diagnosis And Treatment ◽  
Vitamin D Metabolites ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
Stone Formers ◽  
25 Hydroxyvitamin D

AbstractHypercalcemia occurs in up to 4% of the population in association with malignancy, primary hyperparathyroidism, ingestion of excessive calcium and/or vitamin D, ectopic production of 1,25-dihydroxyvitamin D [1,25(OH)2D], and impaired degradation of 1,25(OH)2D. The ingestion of excessive amounts of vitamin D3 (or vitamin D2) results in hypercalcemia and hypercalciuria due to the formation of supraphysiological amounts of 25-hydroxyvitamin D [25(OH)D] that bind to the vitamin D receptor, albeit with lower affinity than the active form of the vitamin, 1,25(OH)2D, and the formation of 5,6-trans 25(OH)D, which binds to the vitamin D receptor more tightly than 25(OH)D. In patients with granulomatous disease such as sarcoidosis or tuberculosis and tumors such as lymphomas, hypercalcemia occurs as a result of the activity of ectopic 25(OH)D-1-hydroxylase (CYP27B1) expressed in macrophages or tumor cells and the formation of excessive amounts of 1,25(OH)2D. Recent work has identified a novel cause of non-PTH-mediated hypercalcemia that occurs when the degradation of 1,25(OH)2D is impaired as a result of mutations of the 1,25(OH)2D-24-hydroxylase cytochrome P450 (CYP24A1). Patients with biallelic and, in some instances, monoallelic mutations of the CYP24A1 gene have elevated serum calcium concentrations associated with elevated serum 1,25(OH)2D, suppressed PTH concentrations, hypercalciuria, nephrocalcinosis, nephrolithiasis, and on occasion, reduced bone density. Of interest, first-time calcium renal stone formers have elevated 1,25(OH)2D and evidence of impaired 24-hydroxylase-mediated 1,25(OH)2D degradation. We will describe the biochemical processes associated with the synthesis and degradation of various vitamin D metabolites, the clinical features of the vitamin D-mediated hypercalcemia, their biochemical diagnosis, and treatment.

scholarly journals The Role of the Parent Compound Vitamin D with Respect to Metabolism and Function: Why Clinical Dose Intervals Can Affect Clinical Outcomes

2013 ◽  
Vol 98 (12) ◽  
pp. 4619-4628 ◽  
Author(s):  
Bruce W. Hollis ◽  
Carol L. Wagner
Keyword(s):  
Vitamin D ◽  
Clinical Trials ◽  
Parent Compound ◽  
Physiological Role ◽  
Daily Basis ◽  
Biological Action ◽  
Activation Process ◽  
Vitamin D Metabolites ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D

Context: There is no doubt that vitamin D must be activated to the hormonal form 1,25-dihydroxyvitamin D to achieve full biological activity or that many tissues participate in this activation process—be it endocrine or autocrine. We believe that not only is 25-hydroxyvitamin D important to tissue delivery for this activation process, but also that intact vitamin D has a pivotal role in this process. Objective: In this review, evidence on the vitamin D endocrine/autocrine system is presented and discussed in relation to vitamin D-binding protein affinity, circulating half-lives, and enzymatic transformations of vitamin D metabolites, and how these affect biological action in any given tissue. Conclusions: Circulating vitamin D, the parent compound, likely plays an important physiological role with respect to the vitamin D endocrine/autocrine system, as a substrate in many tissues, not originally thought to be important. Based on emerging data from the laboratory, clinical trials, and data on circulating 25-hydroxyvitamin D amassed during many decades, it is likely that for the optimal functioning of these systems, significant vitamin D should be available on a daily basis to ensure stable circulating concentrations, implying that variation in vitamin D dosing schedules could have profound effects on the outcomes of clinical trials because of the short circulating half-life of intact vitamin D.

scholarly journals Vitamin D Metabolism and Profiling in Veterinary Species

Metabolites ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 371 ◽  
Author(s):  
Emma A. Hurst ◽  
Natalie Z. Homer ◽  
Richard J. Mellanby
Keyword(s):  
Vitamin D ◽  
Companion Animals ◽  
Vitamin D Metabolites ◽  
Vitamin D Status ◽  
Vitamin D Metabolism ◽  
Hydroxyvitamin D ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Health And Disease ◽  
25 Hydroxyvitamin D

The demand for vitamin D analysis in veterinary species is increasing with the growing knowledge of the extra-skeletal role vitamin D plays in health and disease. The circulating 25-hydroxyvitamin-D (25(OH)D) metabolite is used to assess vitamin D status, and the benefits of analysing other metabolites in the complex vitamin D pathway are being discovered in humans. Profiling of the vitamin D pathway by liquid chromatography tandem mass spectrometry (LC-MS/MS) facilitates simultaneous analysis of multiple metabolites in a single sample and over wide dynamic ranges, and this method is now considered the gold-standard for quantifying vitamin D metabolites. However, very few studies report using LC-MS/MS for the analysis of vitamin D metabolites in veterinary species. Given the complexity of the vitamin D pathway and the similarities in the roles of vitamin D in health and disease between humans and companion animals, there is a clear need to establish a comprehensive, reliable method for veterinary analysis that is comparable to that used in human clinical practice. In this review, we highlight the differences in vitamin D metabolism between veterinary species and the benefits of measuring vitamin D metabolites beyond 25(OH)D. Finally, we discuss the analytical challenges in profiling vitamin D in veterinary species with a focus on LC-MS/MS methods.

scholarly journals A Comparison of Free and Total 25-hydroxyvitamin D Levels as Functional Indicators of Bone Health in Healthy Children

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A270-A270
Author(s):  
You Joung Heo ◽  
Yun Jeong Lee ◽  
Kyunghoon Lee ◽  
Jae Hyun Kim ◽  
Choong Ho Shin ◽  
...  
Keyword(s):  
Vitamin D ◽  
Bone Health ◽  
Normal Weight ◽  
Healthy Children ◽  
Vitamin D Metabolites ◽  
Dihydroxyvitamin D3 ◽  
Bone Parameters ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
24,25 Dihydroxyvitamin D3

Abstract Abstract Context: The “free hormone” hypothesis suggests that the free 25-hydroxyvitamin D (25OHDFree) level may usefully indicate bone health. Objective: To determine which vitamin D measure is optimally correlated with clinical and bone parameters in healthy children. Design and Participants: A cross-sectional study including 146 healthy children (71 boys, 9.5±1.9 years) at a tertiary medical center. Main Outcome Measures: We used a multiplex liquid chromatography-tandem mass spectrometry-based assay to simultaneously measure vitamin D metabolites. The 25OHDFree level was directly measured (m-25OHDFree) or calculated using genotype-constant or genotype-specific affinity coefficients of vitamin D-binding proteins (con-25OHDFree or spe-25OHDFree). Bone mineral content (BMC) and density (BMD) were assessed via dual-energy X-ray absorptiometry. Results: The concentrations of total 25OHD (25OHDTotal), the three forms of 25OHDFree, and 24,25-dihydroxyvitamin D3 correlated with parathyroid hormone levels (all p<0.01). Serum 25OHDTotal and m-25OHDFree levels reflected age, puberty, season, body mass index (BMI), daylight hours, and vitamin D intake (all p<0.05). The con-25OHDFree level better reflected puberty and daylight hours than did the spe-25OHDFree level (both p<0.01). The association between the 25OHDTotal level and bone parameters varied according to the BMI (interaction p<0.05). In 109 normal-weight children, the con-25OHDFree level correlated with BMC and BMD (both p<0.05), but the 25OHDTotal and 24,25-dihydroxyvitamin D3 levels were associated with BMC (both p<0.05). No association was found in overweight or obese children. Conclusions: In healthy children, total and free 25OHD levels comparably reflected lifestyle factors. In normal-weight children, the con-25OHDFree level reflected BMC and BMD, whereas the 25OHDTotal level was associated with BMC.

Simultaneous determination of 25-hydroxy- and 1,25-dihydroxyvitamin D from a single sample by dual-cartridge extraction.

1984 ◽  
Vol 30 (1) ◽  
pp. 56-61 ◽  
Author(s):  
P C Kao ◽  
D W Heser
Keyword(s):  
High Performance ◽  
Vitamin D Metabolites ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Hydroxylated Metabolites ◽  
Silica Cartridge ◽  
25 Hydroxyvitamin D ◽  
The Mean

Abstract With this dual-cartridge system we extract 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] from a single serum sample by using a nonpolar octadecylsilanol silica cartridge to adsorb the vitamin D metabolites and other nonpolar substances; the polar substances wash through the cartridge. The eluted material is then applied to a second alkylamine cartridge, which adsorbs the relatively polar hydroxylated metabolites; the less-polar substances are washed from the second cartridge. Elution from the second cartridge purifies and also separates 25(OH)D and 1,25(OH)2D with analytical recoveries near 90%. The monohydroxyl metabolites are determined by "high-performance" liquid chromatography (HPLC); the dihydroxyl metabolites are further purified by HPLC and determined by radioreceptor assay according to established procedures. Mean (+/- SD) winter normal values (34 subjects of both sexes; blood drawn in mid-April) were 18 +/- 5 micrograms/L for 25(OH)D and 25 +/- 7 ng/L for 1,25(OH)2D. In nine laboratory volunteers, the mean increase in the serum 25(OH)D3 value 5 h after ingestion of 50 micrograms of 25-hydroxycholecalciferol (Calderol) was 9 (SD 4) micrograms/L.

scholarly journals Controversies in Vitamin D: Summary Statement From an International Conference

2018 ◽  
Vol 104 (2) ◽  
pp. 234-240 ◽  
Author(s):  
Andrea Giustina ◽  
Robert A Adler ◽  
Neil Binkley ◽  
Roger Bouillon ◽  
Peter R Ebeling ◽  
...  
Keyword(s):  
Vitamin D ◽  
Hypovitaminosis D ◽  
Vitamin D Metabolites ◽  
Vitamin D Metabolism ◽  
International Conference ◽  
Hydroxyvitamin D ◽  
Disease States ◽  
25 Hydroxyvitamin D ◽  
Total Body ◽  
Definition Of

Abstract Context Vitamin D is classically recognized as a regulator of calcium and phosphorus metabolism. Recent advances in the measurement of vitamin D metabolites, diagnosis of vitamin D deficiency, and clinical observations have led to an appreciation that along with its role in skeletal metabolism, vitamin D may well have an important role in nonclassical settings. Measurement of the circulating form of vitamin D that best describes total body stores, namely 25-hydroxyvitamin D, can be unreliable despite many sophisticated methodologies that have been proposed and implemented. Likewise, evidence from clinical studies showing a beneficial role of vitamin D in different disease states has been controversial and at times speculative. Moreover, the target concentrations of 25-hydroxyvitamin D to address a number of putative links between vitamin D inadequacy and nonskeletal diseases are further areas of uncertainty. Setting To address these issues, an international conference on “Controversies in Vitamin D” was held in Pisa, Italy, in June 2017. Three main topics were addressed: (i) vitamin D assays and the definition of hypovitaminosis D; (ii) skeletal and extraskeletal effects of vitamin D; (iii) therapeutics of vitamin D. Results This report provides a summary of the deliberations of the expert panels of the conference. Conclusions Despite great advances in our appreciation of vitamin D metabolism, measurements, biological actions on classical and nonclassical tissues, and therapeutics, all of which this report summarizes, much more work remains to be done so that our knowledge base can become even more secure.

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