Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects.

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4-27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

Serum and synovial fluid vitamin D metabolites and rheumatoid arthritis.

Vitamin D-deficiency has been linked to inflammatory diseases including rheumatoid arthritis (RA). Studies to date have focused on the impact of serum 25-hydroxyvitamin D3 (25(OH)D3), an inactive form of vitamin D, on RA disease activity and progression. However, anti-inflammatory actions of vitamin D are likely to be mediated at sites of RA disease, namely the inflamed joint, and may involve other vitamin D metabolites notably the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the current study serum and synovial fluid samples from n = 20 patients with persistent RA and n = 7 patients with reactive arthritis (ReA) were analysed for multiple vitamin D metabolites. Serum data for RA and ReA patients were compared to healthy controls (HC). There was no significant difference between RA or ReA patients relative to HC for 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 or 25(OH)D2. However, 3-epi-25(OH)D3 was significantly lower in RA and ReA patients compared to HC (p < 0.05). All vitamin D metabolites, apart from 25(OH)D2, were lower in SF compared to serum, and SF 1,25(OH)2D3 was unquantifiable in 13/20 RA and 4/7 ReA samples. SF 25(OH)D3, 3-epi-25(OH)D3 and DBP correlated inversely with swollen joint score, and serum 25(OH)D2 and SF DBP correlated directly with C-reactive protein levels. These data indicate that serum 25(OH)D3 provides only limited insight into the role of vitamin D in RA. Alternative serum metabolites such as 3-epi-25(OH)2D3, and SF metabolites, notably lack of SF 1,25(OH)2D3, may be more closely linked to RA disease severity and progress.

Selection of High-Quality Spermatozoa May Be Promoted by Activated Vitamin D in the Woman.

Context: The vitamin D receptor (VDR) and enzymes involved in activation (CYP2R1, CYP27B1) and inactivation (CYP24A1) of vitamin D are expressed in ovary, testes, and spermatozoa. Objective: Determine responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in spermatozoa from normal and infertile men, and identify the site of exposure and how 1,25(OH)2D3 influences sperm function. Design: Spermatozoa expressing VDR, CYP2R1, CYP27B1, and CYP24A1 were analyzed in normal and infertile men. 25-Hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D3], and 1,25(OH)2D3 were measured in serum, seminal fluid, cervical secretions, and ovarian follicular fluid. 1,25(OH)2D3 was tested on human spermatozoa. Setting: Tertiary center for fertility. Participants: Protein expression in spermatozoa and semen quality were assessed in 230 infertile and 114 healthy men. Vitamin D metabolites were measured in fluids from 245 men and 13 women, while 74 oocytes and 17 semen donors were used for sperm-function tests. Main Outcome Measures: VDR and CYP24A1 expressions in spermatozoa, fluid concentrations of 25-OHD, 24,25(OH)2D3, and 1,25(OH)2D3, and 1,25(OH)2D3-induced effects on intracellular calcium concentration ([Ca2+]i) and sperm-oocyte binding in vitro. Results: VDR and CYP24A1 were expressed in a >2-fold higher fraction of spermatozoa from normal than infertile men (P < 0.01). Concentrations of 25-OHD, 24,25(OH)2D, and 1,25(OH)2D3 were undetectable in seminal fluid but high in ovarian follicular fluid. Follicular concentrations of 1,25(OH)2D3 induced a modest increase in [Ca2+]i and sperm-oocyte binding in vitro (P < 0.05). Conclusion: Presence of VDR and CYP24A1 mainly in spermatozoa of higher quality supports that 1,25(OH)2D3 available in the female reproductive tract may promote selection of the best gametes for fertilization.

25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4nmol/L), 24R,25(OH)2D3 (14.4nmol/L) and 24S,25(OH)2D3 (57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.

CONTEXT: The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D]. OBJECTIVE: We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 µL of serum with a 5-minute run time. DESIGN: The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter. SETTING: We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models. RESULTS: The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia. CONCLUSIONS: The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation.

24,25-Dihydroxyvitamin D (24,25VD) is a major catabolite of 25-hydroxyvitamin D (25VD) metabolism, and may be physiologically active. Our objectives were to: (1) characterize the response of serum 24,25VD(3) to vitamin D(3) (VD(3)) supplementation; (2) test the hypothesis that a higher 24,25VD(3) to 25VD(3) ratio (24,25:25VD(3)) predicts 25VD(3) response. Serum samples (n=160) from wk 2 and wk 6 of a placebo-controlled, randomized clinical trial of VD(3) (28,000IU/wk) were analyzed for serum 24,25VD(3) and 25VD(3) by mass spectrometry. Serum 24,25VD(3) was highly correlated with 25VD(3) in placebo- and VD(3)-treated subjects at each time point (p<0.0001). At wk 2, the 24,25:25VD(3) ratio was lower with VD(3) than with placebo (p=0.035). From wk 2 to wk 6, the 24,25:25VD(3) ratio increased with the VD(3) supplement (p<0.001) but not with placebo, such that at wk 6 this ratio did not significantly differ between groups. After correcting for potential confounders, we found that 24,25:25VD(3) at wk 2 was inversely correlated to the 25VD(3) increment by wk 6 in the supplemented group (r=-0.32, p=0.02) but not the controls. There is a strong correlation between 24,25VD(3) and 25VD(3) that is only modestly affected by VD(3) supplementation. This indicates that the catabolism of 25VD(3) to 24,25VD(3) rises with increasing 25VD(3). Furthermore, the initial ratio of serum 24,25VD(3) to 25VD(3) predicted the increase in 25VD(3). The 24,25:25VD(3) ratio may therefore have clinical utility as a marker for VD(3) catabolism and a predictor of serum 25VD(3) response to VD(3) supplementation.

Medición de 25-hidroxivitamina D sérica: comparación de dos inmunoensayos / Measurement of 25-hydroxyvitamin D in serum: comparison of two immunoassays

La concentración sérica de 25-hidroxivitamina D (25-OHD) es utilizada como indicador del estado nutricional de Vitamina D (VD). El método más utilizado para medirla es el RIA. El desarrollo reciente de métodos automatizados no radiactivos facilitaría la práctica diaria de laboratorio y el diagnóstico de necesidad de suplementación. Objetivos: Comparar los datos de 25-OHD obtenidos usando un RIA y un método de quimioluminiscencia (QLIA) automatizado disponible en nuestro medio. Materiales y métodos: Concentraciones de 25-OHD se midieron en suero de 45 pacientes: 8 hombres y 37 mujeres; 18 no suplementados y 27 suplementados con VD (n=5 con VD2 y n=22 con VD3). Las mediciones de 25-OHD se realizaron con un RIA y un QLIA automatizado (LIAISON), ambos DiaSorin. Se calcularon los coeficientes de variación intraensayo (CV intra) e interensayo (CV inter) para ambos métodos. Análisis estadístico: la comparación entre métodos se realizó con los programas Analyse-it y Med Calc Se consideró significativa una p<0.05. Resultados: Los CV% intra e inter fueron: para RIA menores de 10,6 y 19,9 vs QLIA menores de 8,0 y 13.2, respectivamente. En la población total y en el subgrupo no suplementado con VD los datos de RIA vs QLIA fueron: coeficiente de correlación de Pearson (0,9259 vs 0,9412), Bias%: (6.1 vs 2.7), coeficiente de concordancia (0,9244 vs 0,9329). Conclusiones: 1) Ambas metodologías son adecuadas para mediciones de 25OHD, especialmente en casos no medicados con VD, 2) La tendencia hacia un mayor bias% observado en pacientes suplementados con VD no parecería ser atribuible a variabilidad metodológica, y sugeriría que la VD exógena o alguno de sus metabolitos interactuaría en forma diferente en la medición de 25-OHD por cada una de las metodologías utilizadas. Mayor número de casos es necesario a fin de confirmar esta hipótesis.

Serum concentration of 25-hydroxyvitamin D (25OHD) is used as an indicator of nutritional status of Vitamin D (VD). The methodolgy more frequently used for its measurement is RIA. The recent development of automated non-radioactive methodologies would help the laboratory daily practice to diagnose the need for supplementation. Objectives: To compare the data of 25-OHD obtained using a RIA and an automated chemiluminescence method (CLIA) automated available in our country. Materials and methods: Concentrations of 25-OHD were measured in serum of 45 patients: 8 men and 37 women, 18 unsupplemented and 27 supplemented with VD (n=5 with VD2 and n=22 with VD3). For 25-OHD measurements we used a RIA and a QLIA under an automated platform (LIAISON), both DiaSorin. We calculated intra-assay (intra) and interassay (inter) coefficients of variation (CV%) for both methods. Statistical analysis: comparison between methods was conducted with Analyse-it and Med Calc softwares; p <0.05 was considered significant. Results: The intra and inter CV% were below 19.9 and 10.6 for RIA vs 8.0 and 13.2 for CLIA, respectively. In the overall population and in the subgroup never supplemented with VD, data for RIA vs CLIA were: Pearson correlation coefficient (0.9259 vs 0.9412), Bias% (6.1 vs. 2.7), concordance coefficient (0.9244 vs 0.9329). Conclusions: 1) Both methods are suitable for measurements of 25OHD, particularly in cases not medicated with VD, 2) The trend toward greater bias% observed in patients supplemented with VD does not appear to be attributable to methodological variability, and suggests that exogenous VD or its metabolites interact differently in the measurement of 25-OHD by each of the methodologies used. A higher number of cases is needed to confirm this hypothesis.

Peripheral blood regulatory T cell measurements correlate with serum vitamin D levels in patients with multiple sclerosis.

Vitamin D has been associated with a decreased risk of multiple sclerosis (MS). In this study, serum 1, 25-dihydroxyvitamin D (1, 25-(OH)2 vitD) and 25-hydroxyvitamin D (25-OH vitD), regulatory T cell percentages and naïve and memory T helper cell subsets were measured in 26 patients with multiple sclerosis, 21 who were not on treatment with disease modifying therapy. These studies showed an inverse correlation between 25-OH vitD levels and Treg cell percentages and a direct correlation between Treg cell percentages and 1, 25-(OH)2 vitD:25-OH vitD ratios. In addition, 25-OH vitD levels correlated directly and 1, 25-(OH)2 vitD:25-OH vitD ratios correlated inversely with CXCR3+ naïve T helper cell percentages and CXCR3+naïve:CXCR3+ memory T helper cell ratios. All together, these data demonstrate that vitamin D measurements can reflect measures of immune status among patients with MS.

Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D3 catalyzed by hydroxysteroid dehydrogenase.

Studies on the C-3 epimerization in (24R)-24,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] were performed using hydroxysteroid dehydrogenases (HSDs). 3-Epi-24R,25(OH)(2)D(3) was formed from 24R,25(OH)(2)D(3) by the catalysis of 3alpha- or beta-HSD. These HSDs also catalyzed the C-3 epimerization in 3-epi-24R,25(OH)(2)D(3) to form 24R,25(OH)(2)D(3). 24R,25(OH)(2)D(3) and its C-3 epimer were separated by inclusion high-performance liquid chromatography using gamma-cyclodextrin (gamma-CD) as the mobile phase additive or a gamma-CD bonded chiral column. The product derived from the intermediate during the C-3 epimerization was isolated from the incubation specimens and identified as (7Z)-(24R)-24,25-dihydroxy-9,10-secocholesta-4,7,10(19)-trien-3-one by several instrumental analyses including (1)H-nuclear magnetic resonance spectrometry. The occurrence of this compound strongly proves that the formation of the C-3 epimer by HSD involves a dehydrogenation process. The present study suggests that HSDs may catalyze the C-3 epimerization of vitamin D compounds and modulate their concentrations and biological activities in animals and humans.

Determination of vitamin D3 metabolites: state-of-the-art and trends.

The steps involved in the methods for the determination of vitamin D3 metabolites (namely, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3) mainly in clinical samples are critically reviewed. Sample pretreatment (e.g. deproteinization, saponification, liquid liquid and liquid solid extraction, etc.) as a function of both type of sample and detection system, quantitation based on protein saturation and liquid as well as gas chromatography are discussed. The chemical principles on which the methods are based and the derivatization procedures, which facilitate separation and/or detection, are also commented upon. Finally, the future prospects of the research on methods for the determination of these metabolites are outlined.

Specificity of polyclonal antibodies raised against a novel 24,25-dihydroxyvitamin D3-bovine serum albumin conjugant linked through the C-11alpha or C-3 position.

Novel hapten-carrier conjugants were prepared by coupling 11 alpha-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D3 or (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] 3-hemiglutarate with bovine serum albumin (BSA), to obtain an antibody with high specificity and affinity for use in 24,25(OH)2D3 immunoassay. The polyclonal antibodies showing high titre were each elicited in three or four rabbits against these two conjugants; the antibodies obtained from the former and the latter conjugants were expressed as Ab11 and Ab3, respectively. These had a much higher affinity for 24,25(OH)2D3 than that of the vitamin D binding protein (DBP). Specificity of the antibodies was investigated by crossreactivities with 11 related compounds in a radioimmunoassay (RIA) system. The Abll well discriminated the 1 alpha-hydroxylated metabolites such as 1,24,25(OH)3D3 (< or = 0.69%) and 1,25(OH)2D3 (< or = 0.25%), but significantly crossreacted with some side chain modified compounds such as (24S)-24,25-dihydroxyvitamin D3 [24S,25(OH)2D3] (> or = 67%), 25(OH)D3 (> or = 14%) and 25,26(OH)2D3 (> or = 23%). On the other hand, the Ab3 showed only negligible crossreactivities with the compounds having a different side chain structure such as 24S,25(OH)2D3 (< or = 3.0%), 25(OH)D3 (< 0.3%) and 25,26(OH)2D3 (< or = 0.53%). A significant crossreaction was found only with 1,24,25(OH)3D3 (> or = 68%). These results demonstrated that the Ab3 are promising for developing an immunoassay system which is much more specific and sensitive than conventional competitive protein binding assays based on DBP.

Gas chromatography/mass spectrometric analysis of 24R,25-dihydroxyvitamin D3 using 24R,25-dihydroxy[6,19,19-2H]vitamin D3 as internal standard.

Gas chromatography/mass spectrometric (GC/MS) analysis of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3, 1a) in rat serum is examined using 6,19,19-trideuterated derivative (24,25-(OH)2[6,19,19-2H]D3, 1c) as an internal standard. Pyro- and isopyro-24,25-(OH)2D3 (2a and 3a) are synthesized and the structures are determined unambiguously by their spectral data including nuclear overhauser enhancement study of the NMR spectra. The two peaks which appear on the total ion chromatogram of 24,25-cyclic n-butyl boronate-3-trimethylsilyl derivative of 24,25-(OH)2D3 are identified to be those of the pyro and isopyro isomers (2a and 3a) by direct comparison with the synthetic standards. Analysis of the GC/MS spectrum of 24,25-(OH)2[6,19,19-2H]D3 (1c) indicated that the CH3(19) is preferentially eliminated in the fragmentation giving rise to a base peak (M-TMSOH-Me)+. Thus the molecular ion peak, which is about 20% of the base peak, rather than the base peak is used for GC/MS assay of 24,25-(OH)2D3 in serum.

Calcium regulating activity of 26,27-dimethyl analog of 24R,25-dihydroxyvitamin D3.

To determine the possibility that methyl substitution in 26- and 27-positions of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] alters activities of the original compound, the effects of 24,25(OH)2D3 on calcium (Ca) regulating activity were compared with those of its methyl analog [24,25(OH)2(CH3)2D3] in addition to 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 24,25(OH)2D3 at 10(-6) M and 24,25(OH)2(CH3)2D3 at 10(-7) M and above significantly stimulated both bone resorption in neonatal mouse calvaria cultures and formation of osteoclast-like multinucleated cells (MNC) in mouse bone marrow cultures. A stimulative effect of 1,25(OH)2D3 on bone resorption and MNC formation was recognized in very low concentrations (10(-11) M and above). Although a potency of 24,25(OH)2(CH3)2D3 in stimulating bone calcium (Ca) mobilization and intestinal Ca transport was higher than that of 24,25(OH)2D3, the potencies of both compounds were similar to that of 1,25(OH)2D3 unlike in vitro experiments. As 1,24R,25-trihydroxy-26,27-dimethylvitamin D3 showed almost the same effect as 24,25(OH)2(CH3)2D3, the dihydroxy form is suggested to be hydroxylated at 1 alpha position and converted to trihydroxy form in vitamin D-deficient rats. From these results, methyl substitution in 26- and 27-position of 24,25(OH)2D3 was found to elevate Ca regulating activity of the original compound. In addition, it is suggested that the basis for a similarity in potency between 1,25(OH)2D3 and 24,25(OH)2D3 or its dimethyl analog in vitamin D-deficient rats is likely the result of 1 alpha-hydroxylation.

The relationship between endogenous oestradiol and vitamin D3 metabolites in serum and follicular fluid during ovarian stimulation for in-vitro fertilization and embryo transfer.

The present study was undertaken to examine the effect of circulating oestradiol on serum levels of 25-hydroxyvitamin D3 (25-OHD3), 24,25-dihydroxyvitamin D3[24,25-(OH)2D3], and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] during gonadotrophin-induced ovarian stimulation in 10 healthy women undergoing in-vitro fertilization and embryo transfer (IVF). The presence of these metabolites in the follicular fluid was also investigated. Plasma oestradiol increased from 25 +/- 3.2 (mean +/- SE) pg/ml before initiation of treatment to 2563 +/- 328 pg/ml on the day of injection of human chorionic gonadotrophin (HCG) and 1641 +/- 299 pg/ml on the day of ovum retrieval (P < 0.01). Serum levels of 1,25-(OH)2D3 increased from 32.0 +/- 1.9 (mean +/- SE) pg/ml to 46.6 +/- 8.1 and 48.5 +/- 7.7 pg/ml (P < 0.05) on the day of HCG and ovum retrieval, respectively. No changes in blood levels of 25-OHD3 and 24,25-(OH)2D3 were found. The presence of vitamin D metabolites in follicular fluid is documented herein for the first time. All three metabolites were present in the follicular fluid but were significantly lower than in the concurrent serum (P < 0.01). A highly significant correlation was found between serum and follicular fluid levels: r = 0.787, P < 0.001 for 1,25-(OH)2D3; r = 0.738, P < 0.01 for 25-OHD3; and r = 0.751, P < 0.01 for 24,25-(OH)2D3. Our results suggest that raised levels of circulating oestradiol during gonadotrophin-induced ovarian stimulation are associated with a significant increase of serum 1,25-(OH)2D3.

Differential metabolism of 25-hydroxyvitamin D3 by cultured synovial fluid macrophages and fibroblast-like cells from patients with arthritis.

Differential metabolism of 25-hydroxyvitamin D3 (25(OH)D3) has been shown for macrophages and fibroblast-like cells (possibly synoviocytes) cultured for two to 50 days after isolation from the synovial fluid of 12 patients with various forms of inflammatory arthritis. Macrophages synthesised the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the synthesis of which was increased by bacterial lipopolysaccharide, a known macrophage activating factor. In contrast, fibroblast-like cells formed 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3), synthesis of which was stimulated by 1,25(OH)2D3 and inhibited by lipopolysaccharide. The synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 by macrophages and fibroblast-like cells respectively was inhibited by ketoconazole, indicating that both hydroxylases are dependent on cytochrome P-450. Mean (SEM) synovial fluid and serum 25(OH)D3 concentrations were 16.7 (1.7) and 22.2 (2.6) ng/ml and those of 1,25(OH)2D3 were 29.4 (4.8) and 43.3 (4.0) pg/ml respectively. In most cases concentrations were lower in synovial fluid than in paired serum samples, but in two patients 1,25(OH)2D3 concentrations were greater in synovial fluid than in serum, suggesting local synthesis within the affected joints.

Analysis of vitamin D and its metabolites using thermospray liquid chromatography/mass spectrometry.

A new method is described for the analysis of vitamin D and its metabolites utilizing thermospray (TSP) mass spectrometry as an on-line detector for high performance liquid chromatography. Ionization conditions were optimized for use with isocratic reversed phase chromatography. TSP mass spectrometry was employed in series with a UV absorbance detector to facilitate comparisons between the two methods of detection. Positive ion TSP mass spectra were recorded for vitamin D2, vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). The spectra contained protonated molecular ions, ammonium adduct ions and fragment ions due to the loss of one or more molecules of water. A comparison of quantitative precision was made by determining UV absorbance and TSP standard curves for vitamin D3 using two different methods: (1) External standard method with post-column (post UV detector) addition of ammonium acetate. (2) As (1) but using the method of internal standards with a closely eluting internal standard (vitamin D2). In each case the quantitative precision (correlation coefficient) for UV absorbance detection was superior owing to intrinsic instability of the TSP ion beam. A stable isotopically labelled internal standard was employed in the development of an assay for 1,25(OH)2D3. The assay was used to quantify in vitro enzymic conversion of 25(OH)D3 to 1,25(OH)2D3 in guinea pig and sheep renal mitochondrial incubations. TSP LC/MS was also applied to analysis of an extract of human blood plasma in which D3 and each of its principal metabolites were identified in a single analysis.

Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites.

The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.

25-Hydroxyvitamin D-24-hydroxylase in phytohemagglutinin-stimulated lymphocytes: intermediate bioresponse to 1,25-dihydroxyvitamin D3 of cells from parents of patients with vitamin D-dependent rickets type II.

A method for assay of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10(-8) mol/L and (1.3 +/- 0.4) x 10(-9) mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10(-8), 10(-7), and 10(-6) mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated the presence of an intracellular receptor-effector system for 1,25-(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24-hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease.