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Background The circulating concentration of 1α,25-dihydroxyvitamin D [1α,25(OH)2D] is very low, and the presence of multiple isomers may lead to inaccurate quantitation if not separated prior to analysis. Antibody-based immunoextraction procedures are sometimes used to remove structurally related isomers of 1α,25(OH)2D prior to an LC-MS/MS analysis. However, immunoextraction increases sample preparation time and cost. In addition, some dihydroxyvitamin D metabolites are not completely removed by immunoextraction. Method We developed an HPLC method using a phenyl-hexyl column to investigate interfering isomers of 1α,25(OH)2D. Result Using this method, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization product of 1α,25(OH)2D was found to be present as 2 epimers, which were separated chromatographically with an area ratio of 2:1. PTAD derivatized metabolite of 25-hydroxyvitamin D3 [i.e., 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3)] eluted out between 6R and 6S epimers of derivatized 1α,25(OH)2D3. If not chromatographically resolved, 4β,25(OH)2D can affect 1α,25(OH)2D quantitation. In a method comparison study, it was found that the presence of 4β,25(OH)2D produced positive bias up to 127% on 1α,25(OH)2D3 quantitation. Conclusion The LC-MS/MS method we developed without an immunoextraction procedure was able to resolve the major interference peak from 1α,25(OH)2D and achieved reliable quantitation of 1α,25(OH)2D.

The vitamin D hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2D3), and related compounds derived from vitamin D3 or lumisterol as a result of metabolism via the enzyme CYP11A1, have been shown, when applied 24 hours before or immediately after UV irradiation, to protect human skin cells and skin from DNA damage due to UV exposure, by reducing both cyclobutane pyrimidine dimers (CPD) and oxidative damage in the form of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdG). We now report that knockdown of either the vitamin D receptor or the endoplasmic reticulum protein ERp57 by small, interfering RNA (siRNA) abolished the reductions in UV‐induced DNA damage with 20‐hydroxyvitamin D3 or 24‐hydroxylumisterol3, as previously shown for 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced oxygen consumption rates in UV‐exposed and sham‐exposed human keratinocytes and reduced phosphorylation of cyclic AMP response binding element protein (CREB). Both these actions have been shown to inhibit skin carcinogenesis after chronic UV exposure, consistent with the anticarcinogenic activity of 1,25(OH)2D3. The requirement for a vitamin D receptor for the photoprotective actions of 1,25(OH)2D3 and of naturally occurring CYP11A1‐derived vitamin D–related compounds may explain why mice lacking the vitamin D receptor in skin are more susceptible to UV‐induced skin cancers, whereas mice lacking the 1α‐hydroxylase and thus unable to make 1,25(OH)2D3 are not more susceptible. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Objective The objective is to develop a LC-MS/MS method to quantify 1α,25-dihydroxyvitamin D without using antibody-based extraction procedure. Methods Vitamin D is a lipid-soluble micronutrient that regulates calcium and phosphate absorption. 25-Hydroxyvitamin D is the primary form in circulation and 1α,25-dihydroxyvitamin D (DHVD) is the biologically active form. DHVD assay is challenging because of very low concentrations (picomolar) and numerous isomers or epimers present in serum. Solid phase extraction and Cookson-type triazolinedione derivatization of vitamin D increases the analytical sensitivity of DHVD assay. However, presence of isomers and epimers remain to be a challenge. One approach is adding antibody-based purification steps prior to PTAD derivatization. However, antibody-based extraction increases sample preparation time, test turn-around time, and the cost per reportable. In this study, we developed a liquid chromatography method to separate DHVD peak from major interference without using affinity purfication. Specimens were added acetonitrile to precipitate serum proteins. After centrifugation and solid phase extraction, analytes were derivatized with PTAD reagent at ambient temperature. Analytical column was a Kinetex Phenyl-hexyl column, 2.1X100 mm, 1.7 µm particle size. AB Sciex 5500 QTrap mass spectrometer was operated in triple quadrupole mode. MRM transitions and ion source parameters were optimized according to previous report. Quantitation was performed using MultiQuant software with a six-point calibration curve. Results Previous reports showed that 4β,25-dihydroxyvitamin D3 and 3-epi-1α,25-dihydroxyvitamin D3 may be coeluted with 1α,25-dihydroxyvitamin D3. In a method comparison study, we found that this interference could result in a 70% positive bias if using a short LC separation method on a C18 column. On the phenyl-hexyl column, we found that PTAD derivatized 1α,25-dihydroxyvitamin D3 has two epimer peaks and the peak area ratio was 1 to 2. Neat 4β,25-dihydroxyvitamin D3 has only one peak following derivatization. We did a mixing study and found that 4β,25-dihydroxyvitamin D3 peak was eluted between the two epimer peaks of 1α,25-dihydroxyvitamin D3. This is a direct evidence proving 4β,25-dihydroxyvitamin D3 causes positive bias in DHVD3 quantitation. DHVD3 assay based on the dominant epimer peak is the optimal strategy to quantify DHVD3 accurately in serum. The results were within 15% of expected values. We observed similar interference for DHVD2 quantitation, which was likely due to the presence of 4β,25-dihydroxyvitamin D2. Our LC separation method was 9 minute, which is slightly long and but acceptable as a clinical method. The analysis time can be reduced by using a multiplex LC system. Conclusions We developed a LC-MS/MS method for the quantitation of DHVD in serum without using immunoextraction procedure. Results demonstrated that 4β,25-dihydroxyvitamin D3 is a major interference. DHVD assay based on the dominant epimer peak is an optimal method to quantify DHVD3 and DHVD2 levels.

RATIONALE: Vitamin D deficiency is common in patients with asthma and COPD. Low 25-hydroxyvitamin D (25[OH]D) levels may represent a cause or a consequence of these conditions. OBJECTIVE: To determine whether vitamin D metabolism is altered in asthma or COPD. METHODS: We conducted a longitudinal study in 186 adults to determine whether the 25(OH)D response to six oral doses of 3 mg vitamin D3, administered over one year, differed between those with asthma or COPD vs. controls. Serum concentrations of vitamin D3, 25(OH)D3 and 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) were determined pre- and post-supplementation in 93 adults with asthma, COPD or neither condition, and metabolite-to-parent compound molar ratios were compared between groups to estimate hydroxylase activity. Additionally, we analyzed fourteen datasets to compare expression of 1α,25[OH]2D3-inducible gene expression signatures in clinical samples taken from adults with asthma or COPD vs. controls. MEASUREMENTS AND MAIN RESULTS: The mean post-supplementation 25(OH)D increase in participants with asthma (20.9 nmol/L) and COPD (21.5 nmol/L) was lower than in controls (39.8 nmol/L; P=0.001). Compared with controls, patients with asthma and COPD had lower molar ratios of 25(OH)D3-to-vitamin D3 and higher molar ratios of 1α,25(OH)2D3-to-25(OH)D3 both pre- and post-supplementation (P≤0.005). Inter-group differences in 1α,25[OH]2D3-inducible gene expression signatures were modest and variable where statistically significant. CONCLUSIONS: Attenuation of the 25(OH)D response to vitamin D supplementation in asthma and COPD associated with reduced molar ratios of 25(OH)D3-to-vitamin D3 and increased molar ratios of 1α,25(OH)2D3-to-25(OH)D3 in serum, suggesting that vitamin D metabolism is dysregulated in these conditions.

In recent years the apparent impact of vitamin D deficiency on human health has gained increased awareness. Consequently, the development of appropriate assays to measure the status of medicinally most relevant vitamin D metabolites in human blood, serum or relevant tissue is continuously being improved. Particularly, assaying of 1α,25-dihydroxyvitamin D3, in turn considered as the most active metabolite, is mainly indicated in disorders leading to calcaemia or those resulting from an impaired 1α-hydroxylation of 25-hydroxyvitamin D3. Thus, in some competitive protein binding and ELISA assays, biotin-linked 1α,25-dihydroxyvitamin D3 (1α,25-dihydroxyvitamin D3 LC-biotin) is employed for measurement of actual calicitriol concentration. A new efficient synthesis of 1α,25-dihydroxyvitamin D3 LC-biotin is described, starting with readily available vitamin D2, and combining a classical approach to access 1α,25-dihydroxyvitamin D3, appropriate OH-protective group transformations, and a C-3-O-alkylation, suitable to connect the biotin-linker in a reliable, selective and high yielding strategy. The developed methodology is applicable to the synthesis of a wide variety of C-3-OH-linked vitamin D3 and D2 derivatives.

Background Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH) 2 D quantification. An accurate method is required for the determination of the 1,25(OH) 2 D status. Methods We designed a serum/plasma 1,25(OH) 2 D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH) 2 D 3 concentrations were determined in 232 healthy Japanese individuals. Results The intra- and inter-assay CVs for 1,25(OH) 2 D 3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH) 2 D 3 was 7.1 pg/ml. Rheumatoid factor (RF) at concentrations below 517 IU/ml did not affect serum 1,25(OH) 2 D analysis. No significant differences were observed for various blood collection tubes, repeated freeze–thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r = 0.786), but serum 1,25(OH) 2 D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH) 2 D 3 concentrations by LC-MS/MS were 18.7–53.9 pg/ml. Conclusion A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH) 2 D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH) 2 D concentrations in the clinical setting.

Background: 1α,25-Dihydroxyvitamin D [1,25(OH)2 vitD] is the bioactive form of vitamin D. Due to the very low concentrations of 1,25(OH)2 vitD in the blood and its lipophilic character, measurement of this parameter is analytically challenging. Requiring preceding manual extraction steps before analysis, previous assays have been laborious. Methods: In the presented study, we evaluated the performance of two immunoassays from DiaSorin and from Immunodiagnostic Systems (IDS) which combine fully automated extraction and measurement of 1,25(OH)2 vitD. Imprecision and linearity were verified according to Clinical and Laboratory Standards Institute EP15-A3 and EP6-A guidelines, respectively. Ninety-three patient serum samples sent to our institute for determination of 1,25(OH)2 vitD, as well as 20 Vitamin D External Quality Assessment Scheme (DEQAS) samples, were used to evaluate correlation and agreement of 1,25(OH)2 vitD measurements between the two immunoassays and with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results: Total imprecision was 5.2% or less for the DiaSorin test but reached 20.1% for the IDS iSYS test. 1,25(OH)2 vitD concentrations measured with the DiaSorin assay showed a strong correlation with 1,25(OH)2 vitD levels measured by LC-MS/MS and a good agreement with method specific means of DEQAS samples. By contrast, the IDS iSYS test overestimated 1,25(OH)2 vitD concentrations in human serum, particularly at higher concentrations. Conclusions: Due to its high sensitivity, low imprecision, broad measurement range, and good agreement with 1,25(OH)2 vitD concentrations measured by LC-MS/MS, the DiaSorin test is a valuable analytical option for the determination of 1,25(OH)2 vitD.

An efficient convergent synthesis of nonadeuterated 1α,25-dihydroxyvitamin D2 (1) by Pd(0)-catalyzed coupling between the boronate ester (upper fragment) and the enol triflate (A-ring fragment) is described. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Fibroblast growth factor 23 (FGF23), which is primarily produced by osteocytes in bone, regulates renal phosphate excretion and 1α,25-dihydroxyvitamin D [1,25(OH)2D3] metabolism. Patients with chronic kidney disease (CKD) have increased levels of circulating serum FGF23, but the direct effect on circulating FGF23 levels in renal insufficiency is still unclear. To identify the major regulator of FGF23 synthesis in renal insufficiency, we compared the effect of parathyroid hormone (PTH) and 1,25(OH)2D3 on FGF23 synthesis in the calvariae of normal rats with that of uremic rats in vitro. 1,25(OH)2D3 treatment significantly increased the FGF23 concentration in the medium from both groups, but the degree of increase in the uremic group was markedly higher than in the control group. A significant increase in FGF23 mRNA expression occurred as early as 4 h after treatment and reached the maximum within 8 h in the uremic group, whereas in the normal group a significant increase in FGF23 mRNA expression was observed only at 8 h. In addition, the expression of vitamin D receptor (VDR) mRNA in the calvariae of uremic rats was markedly higher than in normal rats. However, in neither group did PTH treatment affect the medium FGF23 concentration or the FGF23 mRNA levels. These results suggest that FGF23 synthesis in bone is regulated by 1,25(OH)2D3 directly, not by PTH, and that increased VDR mRNA expression induced the relatively swift and strong response in the uremic group.

Biologically active metabolites of vitamin D that have been successfully developed for the clinical market are described. Their properties that resulted in their success in the clinic are also provided. Precursors of the metabolically active 1α,25-dihydroxyvitamin D have been prepared and successfully marketed not only for renal failure patients but also for a variety of patients having metabolic bone disorders. Finally, successful analogs of 1α,25-dihydroxyvitamin D in use in the clinic worldwide are presented including properties that have contributed to their success.

The objective of this study was to present analysis of 1α,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques.Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra- and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing.This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, RThe study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.

Convergent syntheses of three new analogues of 1α,25-dihydroxyvitamin D 3 with α-hydroxyalkyl substituents at C12 ( 4a – c ) are described. The A-ring and triene system of each analogue were assembled by a tandem Pd-catalysed intramolecular cyclization and Suzuki–Miyaura coupling process. The stereoselective introduction of substituents at C12 was achieved by Johnson–Claisen rearrangement on allylic alcohol 15 as the key step. This article is part of a Special Issue entitled ‘Vitamin D Workshop’.

A sensitive radioimmunoassay (RIA) for 1 alpha,25-dihydroxyvitamin D [1 alpha,25(OH)2-D] with a double antibody (DAB) separation technique to separate free from bound antigen has been developed. The hormone was extracted from 1 ml serum or plasma by Extrelut columns and normal phase high performance liquid chromatography and quantitated in the DAB-RIA. The detection limit of the assay was 3.75 ng/l. The intraassay variation coefficients were 15.9% and 10.5% for samples with 1 alpha,25(OH)2D3 concentrations of 54 ng/l and 130 ng/l, respectively. The interassay variation coefficients were 18.0% and 16.7% for these two concentrations. Mean (and SD) values for 1,25(OH)2D in serum of 40 healthy subjects and 38 patients with chronic renal failure who did not receive 1,25(OH)2D3 were 62.8 ng/ml (22.2) and 12.4 ng/ml (9.8), respectively. The mean value for 7 patients with primary hyperparathyroidism was 66.5 ng/ml (35.8) before surgery. These results compared well with those of an established charcoal-based RIA. Compared to charcoal-based RIAs, the DAB-RIA is faster and requires less laborious assay procedures.

During a 20-year collaboration the laboratories of UGent and KU Leuven have developed different series of Vitamin D analogs characterized by structural modifications in the central CD-ring system. Modifications have first involved the introduction of substituents at C11 and the epimerization at C14, and subsequently more drastic changes consisting in both ring deletion and enlargement relative to the natural CD-ring system. Lately, the focus has shifted towards the synthesis of analogs featuring a symmetrical CD-ring core. As an illustration two different series are presented.

The synthesis of 14-epi-19-nor-22-oxa-1α,25(OH)2D3 5 was started from diol 8 via Fall's method, oxidation, epimerization, protection, coupling with the 19-nor-A-ring 7, and then deprotection of the hydroxyl functions.

Stereochemistry and Biological Activity of 1α,23,25-Trihydroxy-24-Oxo-Vitamin D 3 , a Natural Metabolite of 1α,25-Dihydroxyvitamin D 3 † 413

Current concepts concerning the mechanisms, diagnosis and means of treatment of a number of the major causes of hypercalcemia and hypocalcemia are reviewed. In particular, the role of abnormalities in metabolism of vitamin D including (1) excessive hepatic production of 25-hydroxyvitamin D (vitamin D intoxication), (2) increased production of 1 alpha, 25-dihydroxyvitamin D (hyperparathyroidism and sarcoidosis), (3) impaired production of 1 alpha, 25-dihydroxyvitamin D (hypoparathyroidism, renal failure, vitamin-D-dependent rickets type I, pseudohypoparathyroidism) and (4) resistance to 1 alpha, 25-dihydroxyvitamin D; the use of vitamin D and its metabolites therapeutically is discussed.

A new improved preparation of key intermediates ( 9b, 11 ) for the synthesis of 1α, 25-dihydroxyvitamin D is described.

PREVIOUS studies have indicated that in pseudohypoparathyroidism there is a defect in the renal production of 1α,25-dihydroxyvitamin D. The mean concentration of the hormone in the circulation is abnormally low.1 , 2 We have reported in short-term studies that impaired intestinal absorption of calcium, hypocalcemia and secondary hyperparathyroidism can be corrected by small doses of 14α,25-dihydroxyvitamin D3.1 , 3 We now describe some complications of the long-term use of 1α,25-dihydroxyvitamin D3 in pseudohypoparathyroidism. We find that increases in serum 1α,25-dihydroxyvitamin D3 may occur after its prolonged administration, and that these increases in hormone value are associated with hypercalcemia. The increased . . .

Decrease in serum calcium level leading to hypocalcemia during the first week of life is a frequent finding in premature neonates. Eight premature neonates presenting with such an episode of hypocalcemia in the course of their first 4 days of life were studied. They were fed with a phosphate-enriched human milk and given vitamin D3 (2,100 IU/day per os). We have evaluated the effect of a 24-hour pharmacologic calcium infusion on the circulating levels of calcium, inorganic phosphate, magnesium, 25-hydroxycalciferol (25-OHD), 1α,25-dihydroxycalciferol [1,25(OH)2D] and immunoreactive parathyroid hormone (iPTH). After the infusion, circulating iPTH and Pi levels dropped significantly (p < 0.025 and p < 0.005 respectively) whereas serum Ca and 25-OHD (p < 0.005) increased. Mg and 1,25(OH)2D serum levels remained unchanged. Our data show that an increased calcium supply sustained for 24 h induces an appropriate response in iPTH secretion. Effects on circulating levels of 1,25(OH)2D were variable and probably reflected individual differences in half life of 1,25(OH)2D or in set points in the feedback mechanisms involved in the control of 1,25(OH)2D synthesis.

The preparation of high-affinity and high-specificity monoclonal antibodies to 1 alpha,25-dihydroxyvitamin D is described. Monoclonal antibodies were derived from Balb-c mice immunised with either 1 alpha-hydroxy-25,26,27-trinor-24-cholecalcioic acid or with 1 alpha-hydroxy-26,27-dinor-cholecalciferol-25-oxime, and spleen cells were hybridised with mouse myeloma cells. From six fusions nine monoclonal antibodies (MAb's) were selected from 676 antibody-secreting hybrids. Antibodies varied widely in their ability to bind 1 alpha,25-dihydroxyvitamin D3 (50% displacement of radioligand ranged from 25 - 900 pg); two had particularly useful characteristics for 1 alpha,25-dihydroxyvitamin D assay. MAb 5F2 has high affinity (Ka = 1.39 X 10(10) M-1) and does not discriminate between 1 alpha,25-dihydroxyvitamin D2 and D3, thus enabling the two forms to be measured together. MAb 1G7 is highly specific, having no cross-reactivity with 25-hydroxy-, 24,25-dihydroxy- or 25,26-dihydroxyvitamin D at concentrations found in normal human serum; this MAb has the potential to eliminate the lengthy extraction procedures involved in currently available assays for 1 alpha,25-dihydroxyvitamin D.

In order to investigate possible modulation of vitamin D metabolism by prolactin, circulating 1α,25-dihydroxyvitamin D (1α,25-(OH) 2 D) was measured by radioreceptor assay in chicks given injections of prolactin for five days. At a dose of 100 μg/day, the lactogenic hormone elicited a two-fold increase in plasma 1α,25-(OH) 2 D. This effect may explain the known action of prolactin in producing hypercalcemia and could be physiologically important in birds. The laying hen represents a physiologic state in which calcuim absorption is known to be stimulated and prolactin has been reported to be elevated. Assay of serum 1α,25-(OH) 2 D in the laying hen demonstrates a nine-fold enhancement over non-laying controls. Since this marked increase during egg laying is at least partially mimicked by injecting prolactin, a possible causative relationship between elevated prolactin and 1α,25-(OH) 2 D is suggested.