25-hydroxyvitamin D3 In Serum Research Articles

Vitamin D(3) auto-/paracrine system in rat brain relating to vitamin D(3) status in experimental type 2 diabetes mellitus

Growing evidence suggests that vitamin D3 (D3, cholecalciferol) deficiency and impaired signaling of the hormonally active form of D3, 1α,25(OH)2D3 (1,25D3), through its cellular receptor (VDR) can be significant risk factors for the development of numerous multifactorial diseases, including diabetes. Our investigation was aimed at researching the D3 status in relation to the state of the D3 auto-/paracrine system in the brain and clarifying the effectiveness of the therapeutic use of D3 as a neuroprotective agent in experimental type 2 diabetes mellitus (T2DM). T2DM was induced in male Wistar rats by a combination of a high fat diet and a low dose of streptozotocin (25 mg/kg BW). Diabetic animals were treated with or without cholecalciferol (1,000 IU/kg BW, 30 days). The content of 25-hydroxyvitamin D3 (25D3) in blood serum and brain tissue was determined by ELISA. Analysis of mRNA expression of CYP24A1 and CYP27B1 genes was performed by RT-PCR. Protein levels of VDR, vitamin D3 binding protein (VDBP), CYP27B1 and CYP24A1 were investigated by Western blotting. A significant T2DM-associated decrease in the content of 25D3 in the blood serum was revealed, which correlated with a reduced content of this metabolite in the brain tissue. Impaired D3 status in animals with T2DM was accompanied by an increase in the levels of mRNA and protein of both 25D3 lα-hydroxylase (CYP27B1) and 1,25-hydroxyvitamin D3-24-hydroxylase (CYP24A1), which, respectively, provide local formation and degradation in the nervous tissue of the hormonally active form of D3 – 1,25D3. At the same time, a significant T2DM-induced down-regulation of the brain content of VDBP was shown. In addition, diabetes caused a slight increase in the protein expression of the VDR, through which the auto-/paracrine effects of 1,25D3 are realized in the brain. We have established a complete or partial corrective effect of cholecalciferol on D3 status, its bioavailability in the CNS and the level of protein expression of CYP27B1 and CYP24A1 in the brain of rats with T2DM. Abnormal D3 status in animals with T2DM was accompanied by compensatory changes in the expression of key components of the auto-/paracrine vitamin D3 system. Cholecalciferol was demonstrated to be partially effective in counteracting the impairments caused by T2DM. Keywords: 25-hydroxyvitamin D3, brain, type 2 diabetes, vitamin D3, vitamin D3 auto-/paracrine system

Characterization and Application of Membrane-emulsified HLB Microspheres for Quantification of 25-Hydroxy Vitamin D2 and 25-Hydroxy Vitamin D3 in Serum by UPLC-MS/MS

Characterization and Application of Membrane-emulsified HLB Microspheres for Quantification of 25-Hydroxy Vitamin D2 and 25-Hydroxy Vitamin D3 in Serum by UPLC-MS/MS

A Novel UHPLC-MS/MS Method for the Measurement of 25-Hydroxyvitamin D3 in Canine Serum and Its Application to Healthy Dogs.

Several studies have shown the importance of vitamin D3 supplementation in small animals. In dogs, a low vitamin D3 status is associated not only with bone metabolism but also with different kinds of disorders, such as congestive heart failure, gastrointestinal diseases, chronic kidney diseases, and some types of cancer. However, it is crucial to maintain balance and monitor the introduction of this essential nutrient through the diet because over-supplementation can result in toxicity. Due to the clinical importance of assessing the vitamin D3 status in small animal patients, a quick, simple, and highly performing analytical method for its measurement is needed. In this study, we describe the development of a novel liquid chromatography-tandem mass spectrometry method for 25-hydroxyvitamin D3 quantification in canine serum. The approach was successfully validated following current European guidelines, proving excellent linearity (R2 always ≥0.996), accuracy (always within ±13%) and precision (always <10%). The application of the validated approach to samples collected from 40 healthy dogs made possible the definition of a reliable reference interval for 25-hydroxyvitamin D3, the main biomarker of vitamin D3. In addition, variations below 5% in the results obtained quantifying the same samples using a water-based calibration curve demonstrated that a surrogate matrix may be used without affecting data accuracy. Thanks to its simplicity, the proposed technique represents a useful tool for supporting clinical routine and investigating correlations between serum concentrations of this metabolite and multiple diseases. Additionally, it could enable the monitoring of supplementation in small animal patients in veterinary clinical practice.

EVALUATION OF A NOVEL LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY BASED METHOD FOR VITAMIN D ANALYSIS IN BLOOD

Objective: To evaluate a novel liquid chromatography tandem mass spectrometry based method for serum 25 hydroxy vitamin D (D2 and D3 metabolites) analysis.&#x0D; Study Design: Cross sectional validation study.&#x0D; Place and Duration of Study: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Pakistan, from Mar 2019 to Mar 2020.&#x0D; Methodology: Samples were extracted and 25 OH vitamin-D was separated by means of chromatography and finally quantified via mass spectrometer. A quadrupole- tandem mass spectrometer with Electron spray Ionization coupled to high performance liquid chromatography was adopted for detection and quantitation of 25-hydroxyvitamin D2 and D3 in serum.&#x0D; Results: Limit of detection (LOD) was at the level of 2.49 ng/ml and limit of quantitation (LOQ) was estimated to be 3.9 ng/ml for both the metabolites. The correlation coefficient was 0.99. For D3 observed recovery was 98% and 97.5% respectively while for D2 the recovery was calculated to be 97% and 98%. Percentage relative standard deviation (RSD) was 0.8% and 1.3% respectively. This method has an advantage of less matrix effects, minimal cross-reactivity with 24, 25 hydroxy vit D and 25, 26 di-hydroxy vitamin D metabolite than the routinely used antibody-based assays.&#x0D; Conclusion: This LC-MS/MS methodology is sensitive, specific and can quantitate Vitamin D2 and D3 quite efficiently. This method may be employed for vitamin D analysis in clinical laboratories.

Long-Term Bioavailability of Single Doses of Intramuscular Vitamin D2

Objective: We sought to determine the long-term bioavailability of single doses of intramuscular (IM) vita-min D2 (D2) in healthy adults.Methods: Forty healthy volunteers with hypovitaminosis D received a single dose of 200,000, 400,000, or 600,000 IU intramuscular D2 or no treatment. Levels of 25-hydroxyvitamin D2 (25[OH]D2) and 25-hydroxyvitamin D3 (25[OH]D3) in serum were measured by liquid chromatography-tandem mass spectrometry. Vitamin D binding protein (DBP) and intact parathyroid hormone (iPTH), bone turnover markers (BTMs), and serum and urinary calcium were also measured.Results: After a single dose of D2 injection, the level of 25(OH)D2 increased slowly and reached a plateau at 8 weeks. The plateau remained stable for 12 weeks. The mean increase in 25(OH)D2 was 6.8, 9.6, or 15.6 ng/mL after injection of 200,000 IU, 400,000 IU, or 600,000 IU D2. Although endogenous 25(OH)D3 levels were reduced by IM D2, the total 25(OH)D levels increased by 5.0, 7.0, or 10.3 ng/mL in average after injection of 200,000 IU, 400,000 IU, or 600,000 IU D2. The iPTH levels were also decreased by IM D2. However, levels of serum calcium, BTMs, and DBP and urinary calcium were not altered by IM D2.Conclusion: A single dose of 200,000 IU, 400,000 IU, or 600,000 IU IM D2 raises total 25-hydroxyvitamin D levels by 5.0, 7.0, or 10.3 ng/mL on average for at least 12 weeks and reduces iPTH and endogenous 25(OH)D3 levels without affecting levels of serum calcium, BTMs, DBP, and urinary calcium.

Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice

Factors that can modify the bioavailability of orally administered vitamin D are not yet widely known. Ergosterol is a common fungal sterol found in food which has a chemical structure comparable to that of vitamin D. This study aimed to investigate the effect of ergosterol on vitamin D metabolism. Therefore, 36 male wild type-mice were randomly subdivided into three groups (n = 12) and received a diet containing 25 μg vitamin D3 and either 0 mg (control), 2 mg or 7 mg ergosterol per kg diet for 6 weeks. To elucidate the impact of ergosterol on hepatic hydroxylation of vitamin D, human hepatoma cells (HepG2) were treated with different concentrations of ergosterol. Concentrations of vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in cells, livers and kidneys of mice and additionally 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in serum were quantified by LC-MS/MS. The concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in serum was analyzed by commercially-available enzyme immuno assay. The concentrations of cholesterol and triglycerides were analyzed in livers of mice by photometric assays.Analyses revealed that mice receiving 7 mg/kg ergosterol with their diet had 1.3-, 1.7- and 1.5-times higher concentrations of vitamin D3 in serum, liver and kidney, respectively, than control mice (P < 0.05), whereas no significant effects were observed in mice fed 2 mg/kg ergosterol. The hydroxylation of vitamin D remained unaffected by dietary ergosterol, since the concentration of 25-hydroxyvitamin D3 in serum and tissues and the concentrations of 1,25(OH)2D3 and 24,25(OH)2D3 in serum were not different between the three groups of mice. The lipid concentrations in liver were also not affected by dietary ergosterol. Data from the cell culture studies showed that ergosterol did not influence the conversion of vitamin D3 to 25(OH)D3. To conclude, ergosterol appears to be a modulator of vitamin D3 concentrations in the body of mice, without modulating the hydroxylation of vitamin D3 in liver.

Calcioic acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D3 and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D3

Calcitroic acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3) proceeds via analogous steps culminating in calcioic acid; however this C23-truncated acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2D3 was found to restore normal fracture repair in Cyp24a1−/− mice devoid of 24,25-(OH)2D3. We set out to study the multi-step catabolism of D3 metabolites in vivo using LC–MS/MS methods in vehicle or 24,25-(OH)2D3-treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2D3 (7 ng/mL) and 25−OH-D3-26,23-lactone (4 ng/mL). We also detected 24-oxo-25−OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1−/− mice. In 24,25-(OH)2D3-treated Cyp24a1+/- mice, serum 24,25-(OH)2D3 rose to 200 ng/mL while 25-OH-D3-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2D3 Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)2D3-treated Cyp24a1+/- mice. In 24,25-(OH)2D3-treated Cyp24a1−/− mice, serum 24,25-(OH)2D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2D3 dose. Serum 1,25-(OH)2D3 levels were suppressed in 24,25-(OH)2D3-treated Cyp24a1+/- and Cyp24a1−/− mice. Circulating 1,24,25-(OH)3D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2D3. While undetectable in vehicle-treated Cyp24a1−/− mice, 1,24,25-(OH)3D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2D3-treated nulls suggesting conversion of 24,25-(OH)2D3 to 1,24,25-(OH)3D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.

Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography: An economical and validated method with bovine serum albumin

A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples’ preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 – 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87–108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

Rapid Quantification of 25-Hydroxyvitamin D3 in Human Serum by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

LC-MS/MS is widely utilized today for quantification of vitamin D in biological fluids. Mass spectrometric assays for vitamin D require very careful method optimization for precise and interference-free, accurate analyses however. Here, we explore chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) as a rapid alternative for quantitative measurement of 25-hydroxyvitamin D3 in human serum, and compare it to results from LC-MS/MS. The method implemented an automated imaging step of each MALDI spot, to locate areas of high intensity, avoid sweet spot phenomena, and thus improve precision. There was no statistically significant difference in vitamin D quantification between the MALDI-MS/MS and LC-MS/MS: mean ± standard deviation for MALDI-MS-29.4 ± 10.3ng/mL-versus LC-MS/MS-30.3 ± 11.2ng/mL (P = 0.128)-for the sum of the 25-hydroxyvitamin D epimers. The MALDI-based assay avoided time-consuming chromatographic separation steps and was thus much faster than the LC-MS/MS assay. It also consumed less sample, required no organic solvents, and was readily automated. In this proof-of-concept study, MALDI-MS readily demonstrated its potential for mass spectrometric quantification of vitamin D compounds in biological fluids. Graphical Abstract ᅟ.

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum.

We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease. Graphical Abstract ᅟ.

Tissue content of vitamin D3 and 25-hydroxy vitamin D3 in minipigs after cutaneous synthesis, supplementation and deprivation of vitamin D3

Information regarding the endogenous storages of vitamin D3 after cutaneous vitamin D synthesis compared to oral vitamin D3 supplementation is sparse. Furthermore it is not known whether vitamin D3 can be stored for later use during periods of shortages of vitamin D3. To investigate the endogenous storages of vitamin D3 two studies were carried out in Göttingen minipigs. In study 1 one group of minipigs (n=2) was daily exposed to UV light corresponding to 10–20min of midday sun and another group (n=2) of pigs were fed up to 60μg vitamin D3/day corresponding to 3.7–4.4μg/kg body weight.Study 1 demonstrated that daily UV-exposure of minipigs stimulated the cutaneous synthesis of vitamin D3 and resulted in increasing serum vitamin D3 and 25-hydroxy vitamin D3, but also carcasses containing vitamin D3 and 25-hydroxy vitamin D3. The vitamin D3 content in adipose tissue from the UV-exposed minipigs was 150–260ng/g and the content was 90–150ng/g in the orally supplemented minipigs.In study 2, minipigs were UV-exposed daily for 49days. Subsequently, one group (n=2) was fed a vitamin D-free diet and another group (n=2) was dosed daily with 13C-labeled vitamin D3. The concentrations of vitamin D3 and 25-hydroxy vitamin D3 in serum and skin- and subcutaneous adipose tissue biopsies were repeatedly monitored. Vitamin D3 and 25-hydroxy vitamin D3 were eliminated from the skin and the adipose tissue after UV-exposure was ceased. Supplementation of 13C-vitamin D3 did not seem to affect the decline in the endogenous vitamin D3 in the adipose tissue formed during UV-exposure.

Quantitation of 25-hydroxyvitamin D2 and D3 in serum and plasma by LCMS/MS.

We developed and validated an analytical method for quantifying 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in serum and plasma. Samples, pretreated with zinc sulfate and methanol, were extracted with hexane. Separation was achieved via UHPLC and 25OHD quantification was accomplished by a triple quadrupole mass spectrometer. Imprecision was 3.6-15.1%CV and bias 88.0-126.0%. Extraction efficiency was 76.5-110.5%, whereas the matrix effect ranged from -46.7 to -32.0%. The method was applied to authentic specimens. The results showed no significant difference between serum and plasma; strong correlation with paired values from an external laboratory; and analyte stability for 15 days. This method provides reliable and accurate measurement of 25OHD for use in clinical practice.

Relationship between serum 25-hydroxy vitamin D3 concentration and obesity childhood

Objective To examine the relationship between the concentration of 25-hydroxy vitamin D3[25-(OH)D3] in the serum and the body mass, the severity of obesity, body mass index(BMI), blood lipid, and their predicting role in obesity children. Methods The study recruited 244 subjects, who see the doctor in Wuxi Maternal and Child Health Hospital, Childhood Nutrition Outpatient from Jul.2011 to Feb.2013.The intake dose of vitamin D each day was investigated, and weight, height, BMI, concentration of 25-(OH)D3 in serum, and microelement were also mea-sured.In addition, lipid metabolism of 38 cases with obesity over 3 years old was determined. Results 1.The serum 25-(OH)D3 concentration of obese children was (68.31±23.06) nmol/L.The concentration of 25-(OH)D3 was lowest in the group of obese children over 36 months of age[(55.03±15.18) nmol/L].2.The concentration of 25-(OH)D3 in the group of obese and overweight children was far lower than that of the children in the normal group(F=4.739, P<0.05).3.The concentrations of 25-(OH)D3 in the severely obese children was significantly lower than that of the mild and moderate obesity children(F=9.711, P<0.05).4.There were significantly inverse associations of serum 25-(OH)D3 with weight, weight and height percentage, BMI(r=-0.365, -0.237, -0.175, all P<0.001).5.There were significantly inverse associations between the concentration of 25-(OH)D3 in serum with weight, triglyceride in obese children more than 3 years old(r=0.476, -0.324, all P<0.05). Conclusions The decreasing level of 25-hydroxy vitamin D3 in the serum was associated with obesity.The cause of it might be the increase of the obese adipose tissue, vitamin D getting trapped in fat cells, and all these factors can lead to a less serum vitamin D levels.The vitamin D consumption of obese children is higher than that of normal children, and should supply more vitamin D to reach normal 25-(OH) D3 level. Key words: Serum 25-hydroxy vitamin D3 concentration; Child; Obesity

Vitamin D status of county hospital patients assessed by the DiaSorin LIAISON ® 25-hydroxyvitamin D assay

Background Vitamin D is an essential nutrient, and there is a growing appreciation of its clinical significance. In addition, there has recently been a discussion as to the best method of measuring this analyte in serum: immunoassay, HPLC or LC–MS/MS. Due to the increased interest in vitamin D, there has been an exponential increase in the number of clinicians testing their patients and therefore also in the volume of this test run in clinical laboratories. Methods Vitamin D levels were determined by chemiluminescence immunoassay in a reference laboratory and a subset by LC–MS/MS at San Francisco General Hospital. Results We developed a robust and rapid LC–MS/MS assay to detect 25-hydroxyvitamin D3 and D2 in serum for use in the routine clinical laboratory. Additionally, we determined that 71% of patients served by San Francisco General Hospital have insufficient serum vitamin D levels (≤ 29 ng/ml) and that these levels are significantly associated with parathyroid hormone levels, total calcium concentration, age and ethnicity. Conclusions The high degree of vitamin D insufficiency at San Francisco General Hospital may be reflective of the status of the patients served by this county hospital; largely an underserved, multi-ethnic urban population.

Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method

BackgroundMeasurement of vitamin D molecules are important in the management of patients with bone disease. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 25OHD3 and 25OHD2 in human serum and compared it to the traditionally used DiaSorin radioimmunoassay (RIA). MethodsSerum samples (200 µl) were treated with acetonitrile and centrifuged to remove protein. An online solid-phase extraction procedure was used. Calibration solutions (5–100 ng/ml) of 25OHD2 and 25OHD3 were prepared using 4% albumin in phosphate-buffered saline. Chromatography: C18 column, isocratic ethanol/water (83/17, v/v). Mass spectrometry system: atmospheric pressure chemical ionization in the positive ion mode. Transitions: 25OHD3, m/z 401.4→383.4; 25OHD2, m/z 413.4→395.4; and the internal standard hexadeuterated-25OHD3, m/z 407.7→389.7. ResultsDetection limits were 0.49 ng/ml (25OHD3) and 1.86 ng/ml (25OHD2). Intra- and inter-assay coefficients of variation (CV) were <7% and <11%, respectively, for 25OHD3 and <9% and <16%, respectively, for 25OHD2. Recovery averaged (SD) 99% (2%) for 25OHD3 and 95% (0.8%) for 25OHD2. In a method comparison of 551 specimens from the National Health and Nutrition Examination Survey, the LC-MS/MS method gave values that were on average 13% higher (95%CI: 11–15%) than RIA results. ConclusionsThis high throughput candidate reference method requires minimal sample preparation and is suitable for routine use for analysis of vitamin D status.

Candidate reference method for the quantification of circulating 25-hydroxyvitamin D3 by liquid chromatography-tandem mass spectrometry.

Quantification of 25-hydroxyvitamin D3 (25-hydroxycholecalciferol) in serum is the best-established indicator of vitamin D status (1). Vitamin D3 (cholecalciferol) is absorbed from the diet, and, given sufficient ultraviolet irradiation, nutritionally adequate amounts of vitamin D3 are formed in the skin from its precursor, 7-dehydrocholesterol. In the liver, vitamin D3 undergoes hydroxylation to 25-hydroxyvitamin D3, which is further metabolized in the kidney to form the active metabolite 1,25-dihydroxyvitamin D3. Vitamin D2 (ergocalciferol) is derived solely from plant sources; relevant serum concentrations of 25-hydroxyvitamin D2 are observed only after ingestion of vitamin D2 drug preparations, and biological equivalence to vitamin D3 has never been demonstrated conclusively. The prevalence of hypovitaminosis D has been recognized as substantial (2)(3), even in regions with high sun exposure (4), contributing not only to osteoporosis (5)(6) but possibly to a loss of muscle strength in aging as well (7). Various assays are used for the quantification of circulating 25-hydroxyvitamin D3 that incorporate either vitamin D-binding globulin or anti-vitamin D antibodies for analyte recognition (8)(9)(10). Fully automated tests have become available during recent years (11). Several HPLC methods with ultraviolet detection have been described as well (12)(13), but their routine use is limited by complex sample preparation requirements. A key problem for the quantification of circulating 25-hydroxyvitamin D3 is the strong binding of the molecule to vitamin D-binding globulin. Precipitation of serum constituents by use of organic solvents or acids can lead to variable coprecipitation of the analyte. Release of 25-hydroxyvitamin D3 from its bonds to the binding protein is technically challenging in automated assays in particular. In light of these analytical problems, a reference method for the quantification of 25-hydroxyvitamin D …

Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum

We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 mumol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antibody raised against 1,25-dihydroxyvitamin D3-3-hemisuccinate conjugated to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection limit of the EIA was 4.4 micrograms/L; recovery 102%; on-plate CV 11%; within-run CV including extraction 12%, and between-run CV 15%. There was no clinically important cross-reactivity with other vitamin D metabolites, and results obtained by the EIA were compared with results obtained by a previously described RIA.

Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. Implications for metabolic bone disease.

Serum vitamin D metabolites and PTH were measured in seven subjects with a history of previous partial gastrectomy (PGX) and metabolic bone disease. The elimination t1/2 of [3H]25-hydroxyvitamin D3 ([3H]25OHD3) in serum was assessed after an iv pulse dose of 5 microCi [26,27-3H]25OHD3. Median serum 25OHD3 was 37.5 (27.5-101.3) nmol/L, [normal range (NR) 10.8-58.5 nmol/L], mean serum 1,25-dihydroxyvitamin D [1, 25-(OH)2D3] was raised at 175 +/- 72 pmol/L, (NR 48-120 pmol/L) and mean PTH was also high, 67 +/- 27 ng/L, (NR 10-60 ng/L). Serum t1/2 [3H]25OHD3 ranged from 10.9-21.2 days. A strong negative correlation existed between t1/2 [3H]25OHD3 and serum 1,25-(OH)2D3 [Spearman's rank correlation coefficient (r = -0.82, P = 0.002)] and PTH [Spearman's rank correlation coefficient (r = -0.81, P = 0.001)]. Four subjects who had high initial PTH concentrations (60-115 ng/L) and elevated 1,25-(OH)2D levels (162-300 pmol/L) were reassessed after calcium supplementation to suppress secondary hyperparathyroidism (2 degrees HPT). In this subgroup, after-treatment PTH fell from 82 +/- 24 to 52 +/- 24 ng/L (mean +/- SD), not significant; 1,25-(OH)2D fell from 210 +/- 61 to 116 +/- 28 pmol/L, P = 0.015; and t1/2 [3H]25OHD3 increased from 13.2 +/- 1.9 to 18.9 +/- 3.1 days, P = 0.012. Patients with PGX and evidence of 2 degrees HPT with elevated 1,25-(OH)2D have a reduced t1/2 [3H]25OHD3, and this may explain the increased susceptibility of the subjects to osteomalacia. Calcium supplementation suppresses 2 degrees HPT, increases t1/2 [3H]25OHD3 and may protect against PGX osteoporosis and osteomalacia.

Determination of 25-hydroxyvitamin D3 in human serum by fluorescence labelling and high-performance liquid chromatography

A method is described for the determination of 25-hydroxyvitamin D3 in human blood serum. The problems of sensitivity and selectivity encountered with previous techniques were avoided by the formation of a highly fluorescent Diels-Alder adduct following solid-phase extraction of the vitamin. After excess of reagent had been eliminated, quantification was achieved by high-performance liquid chromatography. The recovery of the vitamin from serum was 76.4 +/- 1.76%. The precision of the method was determined, and the relative standard deviations were 8.38% at a concentration of 47.0 x 10(-9) mol dm-3, 6.74% at a concentration of 99.8 x 10(-9) mol dm-3 and 3.79% at a concentration of 146.8 x 10(-9) mol dm-3. The detection limit for the adduct was 2.93 x 10(-14) mol injected, for a signal-to-noise ratio of 3:1, and serum concentrations of 0.25 x 10(-9) mol dm-3 could easily be quantified. No interference from endogenous or exogenous substances was observed.

Three commercial kits and one liquid-chromatographic method evaluated for determining 25-hydroxyvitamin D3 in serum.

The accuracy of three commercial kit methods and one liquid-chromatographic (HPLC) method for determining 25-hydroxyvitamin D3 in serum was evaluated by using isotope dilution-mass spectrometry as a reference technique. The kit methods were based on radioimmunoassay and radioreceptor assay. Serum samples were analyzed from male and female volunteers, some of whom had been exposed to ultraviolet B light or given vitamin D3 orally. Results obtained with the three commercial kits were less accurate than those by HPLC. The agreement between the HPLC method and the comparison method was relatively good: r = 0.99; slope = 1.23; intercept = -2.9 micrograms/L. We conclude that methods based on HPLC should be preferred in routine clinical work and that the accuracy of the three commercial kits tested is not sufficient for their intended use.