Simultaneous Determination of Vitamins D3 (Calcitriol, Cholecalciferol) and K2 (Menaquinone-4 and Menaquinone-7) in Dietary Supplements by UHPLC.

The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market. Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. In this regard, the simultaneous determination of vitamin D3 (calcitriol-CT and cholecalciferol-CHL) and K2 (menaquinone-4-MK-4 and menaquinone-7-MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04-0.05 µg/mL, respectively. The validation results indicated that the method had reasonable linearity (R2 ≥ 0.9990), good recovery (>82%), satisfactory intra-day precision (≤1.9%) and inter-day precision (≤3.5%), and high selectivity and specificity. The validated UHPLC method was demonstrated to be precise, accurate, and robust for the simultaneous determination of vitamins D3 (CT and CHL) and K2 (MK-4 and MK-7) in dietary supplements.

1,25-(OH)2D3/Vitamin D receptor alleviates systemic lupus erythematosus by downregulating Skp2 and upregulating p27.

BACKGROUND: Recent evidence has suggested that the 1,25(OH)2D3/Vitamin D receptor (VDR) acts to suppress the immune response associated with systemic lupus erythematosus (SLE), a serious multisystem autoimmune disease. Hence, the aim of the current study was to investigate the mechanism by which 1,25-(OH)2D3/VDR influences SLE through regulating the Skp2/p27 signaling pathway. METHODS: Initially, the levels of 1,25(OH)2D3, VDR, Skp2, and p27 were measured in collected renal tissues and peripheral blood. Meanwhile, the levels of inflammatory factors, biochemical indicators (BUN, Cr, anti-nRNP IgG, anti-dsDNA IgG) and urinary protein levels were assayed in in VDRinsert and VDR-knockout mice in response to 1,25(OH)2D3 supplement. In addition, the distribution of splenic immune cells was observed in these mice. RESULTS: Among the SLE patients, the levels of 1,25(OH)2D3, VDR and p27 were reduced, while the levels of Skp2 were elevated. In addition, the levels of anti-nRNP IgG and anti-dsDNA IgG were increased, suggesting induction of inflammatory responses. Notably, 1,25(OH)2D3/VDR mice had lower concentrations of BUN and Cr, urinary protein levels, precipitation intensity of the immune complex and complement, as well as the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice. Additionally, 1,25(OH)2D3 or VDR reduced the degree of the inflammatory response while acting to regulate the distribution of splenic immune cells. CONCLUSION: This study indicated that 1,25-(OH)2D3/VDR facilitated the recovery of SLE by downregulating Skp2 and upregulating p27 expression, suggesting the potential of 1,25-(OH)2D3/VDR as a promising target for SLE treatment.

Pentosidine concentration is associated with degenerative lumbar scoliosis in older women: preliminary results.

PURPOSE: Advanced glycation end products (AGEs) have been implicated in the pathogenesis of sarcopenia. The objective of the study was to investigate the prevalence of sarcopenia in degenerative lumbar scoliosis (DLS), and the relationship between biochemical markers including major AGEs, pentosidine, and DLS in older women. METHODS: Our study participants were 20 elderly women with idiopathic DLS (mean age 76.4 years, range 56-88). Nineteen age- and sex-matched volunteers (mean age 74.0 years, range 62-86) served as controls. Spinal and femoral BMD of all participants was measured using dual-energy X-ray absorptiometry. We used a bioelectrical impedance analyzer to analyze body composition, including appendicular skeletal muscle mass index [SMI; appendicular lean mass (kg)/(height (m)]2. SMI < 5.75 was considered diagnostic for sarcopenia. Coronal and sagittal spinal alignments were measured. The following biochemical markers were measured: serum and urinary pentosidine, serum homocysteine, 1,25(OA)2D, and 25(OH)D. The level of each variable was compared between DLS and controls. The relationship between biochemical markers including pentosidine and DLS was examined. RESULTS: Sarcopenia was observed at a high prevalence in participants with DLS: 50% compared with 15.8% of healthy controls. Height, weight, femoral BMI, appendicular lean mass, total lean mass, and SMI all had significantly lower values in the DLS group. Serum pentosidine was significantly higher for the DLS group compared with controls. Correlations with serum pentosidine revealed a significant positive correlation between lumbar scoliosis, pelvic tilt, and pelvic incidence-lumbar lordosis mismatch, and a significantly negative correlation between thoracic kyphosis (P < 0.05). CONCLUSIONS: We found that sarcopenia was involved in DLS, and high serum pentosidine levels are associated with severity of coronal and sagittal malalignment in older women, suggesting that high levels of AGEs are a potential biomarker for the progression of lumbar scoliosis and kyphotic deformity. Further studies are needed to clarify the pathogenesis of DLS.

Evaluation of calcipotriol transdermal permeation through pig, rat and mouse skin using liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method to evaluate the permeation and retention of calcipotriol in excised samples of pig, rat and mouse skin after application of a calcipotriol ointment has been developed and validated. After sample preparation of ointment, skin homogenate and receptor medium by liquid-liquid extraction, chromatography was performed on an Extend-C18 column using isocratic elution. Detection was by electrospray ionization in the negative ion mode using multiple-reaction monitoring of the precursor to product ion transitions of calcipotriol at m/z 411.1 → 393.5, and of lovastatin (internal standard) at m/z 403.2 → 101.2. The assay was linear in all matrices with LLOQs of 1, 0.5 and 40 ng/mL for skin homogenate, receptor medium and ointment samples respectively. In terms of the permeation profiles, it was found that calcipotriol permeated through all skins to only a limited extent over 20 h after application but was efficiently retained in all skins at a level at 20 h of between 40% (pig) and 60% (rat and mouse) of the applied dose. This indicates that calcipotriol ointment has the potential to provide sustained therapeutic benefit in the treatment of psoriasis with minimal systemic side effects.

Maternal vitamin D deficiency affects the morphology and function of glycolytic muscle in adult offspring rats.

BACKGROUND: Fetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals. METHODS: Female rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old. RESULTS: Vitamin D deficiency induced muscle atrophy in the male (M-VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast-twitch skeletal muscles [extensor digitorum longus (EDL)] from the M-VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross-sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M-CTRL; P < 0.05). M-VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF-1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser 473 Akt and Ser 21/9 GSK-3ß. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two-fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow-twitch skeletal muscles (soleus) from the M-VDD group as well as in both EDL and soleus muscles from the female offspring. CONCLUSIONS: These data show that maternal VDD selectively affects the development of type-II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast-twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.

Metabolic changes following 500 µg monthly administration of calcidiol: a study in normal females.

This study was performed to investigate the effect of monthly oral administration of 500 µg of calcidiol (25-hydroxyvitamin D(3)) for 4 months on both serum vitamin D levels and sequential changes of parameters of calcium metabolism; 18 normal women aged 24-72 years were investigated. There was a significant increase of serum 25(OH)D after the first administration; thereafter all values persisted significantly higher compared to the basal value (P < 0.001). Mean 1,25(OH)(2)D serum levels peaked at day 3 and then tended to stabilize following day 30. During the first month, all mean values observed following the initial administration were significantly higher than basal values. The first calcidiol dose produced a significant reduction of serum PTH levels (P < 0.001), which then remained constant over time. Concerning serum calcium and phosphorus, we were not able to demonstrate any significant change during the entire observation period. Considering the single values for both serum ionized and total calcium, the values of Ca(2+) exceeded upper limits of normal on only two occasions. Regarding biochemical markers of bone remodeling, mean changes of serum bone isoenzyme of alkaline phosphatase activity showed a significant trend to decrease, starting at day 30. No significant changes of serum CTX values were noted. Overall, 24-h urinary excretion of calcium did not change, seven values exceeding the threshold of 4 mg/kg body weight. Monthly administration of 500 µg of 25-hydroxyvitamin D(3) may be considered an alternative for vitamin D repletion, without any detrimental effect.

Concentrations of Ca, Mg, P, Prostaglandin E2 in Bones and Parathyroid Hormone; 1,25-dihydroxyvitamin D3; 17-ß-estradiol; Testosterone and Somatotropin in Plasma of Aging Rats Subjected to Physical Training in Cold Water.

Exposure to low temperatures can be considered a stressor, which when applied for a specific time can lead to adaptive reactions. In our study we hypothesized that cold, when applied to the entire body, may be a factor that positively modifies the aging process of bones by improving the mechanisms related to the body's mineral balance. Taking the above into account, the aim of the study was to determine the concentration of calcium (Ca), magnesium (Mg), and phosphorus (P) in bones, and to examine bone density and concentrations of the key hormones for bone metabolism, namely parathyroid hormone (PTH), somatotropin (GH), 1,25-dihydroxyvitamin D3, 17-ß estradiol, testosterone (T) in plasma, and prostaglandin E2 (PGE2) in the bone of aging rats subjected to physical training in cold water. The animals in the experiment were subjected to a series of swimming sessions for nine weeks. Study group animals (male and female respectively) performed swimming training in cold water at 5 ± 2 °C and in water with thermal comfort temperature (36 ± 2 °C). Control animals were kept in a sedentary condition. Immersion in cold water affects bone mineral metabolism in aging rats by changing the concentration of Ca, Mg, and P in the bone, altering bone mineral density and the concentration of key hormones involved in the regulation of bone mineral metabolism. The effect of cold-water immersion may be gender-dependent. In females, it decreases Ca and Mg content in bones while increasing bone density and 17-ß estradiol and 1,25-dihydroxyvitamin D3 levels, and with a longer perspective in aging animals may be positive not only for bone health but also other estrogen-dependent tissues. In males, cold water swimming decreased PTH and PGE2 which resulted in a decrease in phosphorus content in bones (with no effect on bone density), an increase in 1,25-dihydroxyvitamin D3, and increase in T and GH, and may have positive consequences especially in bones and muscle tissue for the prevention of elderly sarcopenia.

Competitive surface-enhanced Raman scattering assay for the 1,25-dihydroxy metabolite of vitamin D3.

This paper describes the development and preliminary testing of a competitive surface-enhanced Raman scattering (SERS) immunoassay for calcitriol, the 1,25-dihydroxy metabolite (1,25-(OH)(2)-D(3)) of vitamin D(3). Deficiencies in 1,25-(OH)(2)-D have been linked to renal disease, while elevations are linked to hypercalcemia. Thus, there has been a sharp increase in the clinical demand for measurements of this metabolite. The work herein extends the many attributes of SERS-based sandwich immunoassays that have been exploited extensively in the detection of large biolytes (e.g., DNA, proteins, viruses, and microorganisms) into a competitive immunoassay for the low level determination of a small biolyte, 1,25-(OH)(2)-D(3) (M(w) = 416 g mol(-1)). The assay uses surface modified gold nanoparticles as SERS labels, and has a dynamic range of 10-200 pg mL(-1) and a limit of detection of 8.4 ± 1.8 pg mL(-1). These analytical performance metrics match those of tests for 1,25-(OH)(2)-D(3) that rely on radio- or enzyme-labels, while using a much smaller sample volume and eliminating the disposal of radioactive wastes. Moreover, the SERS-based data from pooled-patient sera show strong agreement with that from radioimmunoassays. The merits and potential utility of this new assay are briefly discussed.

Vitamin D metabolites and the gut microbiome in older men.

The vitamin D receptor is highly expressed in the gastrointestinal tract where it transacts gene expression. With current limited understanding of the interactions between the gut microbiome and vitamin D, we conduct a cross-sectional analysis of 567 older men quantifying serum vitamin D metabolites using LC-MSMS and defining stool sub-Operational Taxonomic Units from16S ribosomal RNA gene sequencing data. Faith's Phylogenetic Diversity and non-redundant covariate analyses reveal that the serum 1,25(OH)2D level explains 5% of variance in α-diversity. In ß-diversity analyses using unweighted UniFrac, 1,25(OH)2D is the strongest factor assessed, explaining 2% of variance. Random forest analyses identify 12 taxa, 11 in the phylum Firmicutes, eight of which are positively associated with either 1,25(OH)2D and/or the hormone-to-prohormone [1,25(OH)2D/25(OH)D] "activation ratio." Men with higher levels of 1,25(OH)2D and higher activation ratios, but not 25(OH)D itself, are more likely to possess butyrate producing bacteria that are associated with better gut microbial health.

Serum osteocalcin/bone-specific alkaline phosphatase ratio is a predictor for the presence of vertebral fractures in men with type 2 diabetes.

We examined whether or not BMD or bone markers were useful for assessing the risk of vertebral fractures in 248 Japanese men with type 2 diabetes. We analyzed the relationships between bone markers (osteocalcin [OC], bone-specific alkaline phosphatase [BAP], urinary N-terminal cross-linked telopeptide of type-I collagen) or BMD and HbA(1c), urinary C-peptide, insulin-like growth factor-I (IGF-I), parathyroid hormone, 1,25(OH)(2) vitamin D, and the presence of prevalent vertebral fractures. Multiple regression analysis adjusted for age, body height, weight, duration of diabetes, and serum creatinine showed that serum OC and OC/BAP ratio were correlated negatively with HbA(1c) (P < 0.01) and positively with IGF-I (P < 0.01). Multivariate logistic regression analysis adjusted for the above parameters showed that serum OC/BAP ratio was inversely associated with the presence of vertebral fractures (odds ratio = 0.695, P < 0.05). This association was still significant after additional adjustment for lumbar or femoral neck BMD. Our results suggest that poor diabetic control and lower IGF-I level are linked to impaired bone formation and resultant reduction in OC/BAP ratio in men with type 2 diabetes. The OC/BAP ratio could be clinically useful for assessing the risk of vertebral fractures independent of BMD in diabetic men.

Combination of yeast-based in vitro screens with high-performance thin-layer chromatography as a novel tool for the detection of hormonal and dioxin-like compounds.

The combination of classic in vitro bioassays with high-performance thin-layer chromatography (HPTLC) is a promising technique to directly link chemical analysis of contaminants to their potential adverse biological effects. With respect to endocrine disruption, much work is focused on estrogenicity. While a direct combination of HPTLC and the yeast estrogen screen is already developed, it is well accepted that further endocrine effects are relevant for monitoring environmental wellbeing. Here we show that non-estrogenic specific biological endpoints, (partly) related to the endocrine system, can also be addressed by combining respective yeast reporter gene assays with HPTLC to support effect-directed analysis (EDA). These are: androgenicity (YAS), thyroidogenicity (YTS), dioxin-like effects (YDS), effects on the vitamin D (YVS) and the retinoic acid receptor (YRaS). A proof of principle is demonstrated within this study by the characterization of dose-dependent responses to different model compounds for the respective receptors with and without chromatographic development of the HPTLC-plate. Limits of quantification (LOQ) for several model compounds were determined, e.g. 37 pg for testosterone (p-YAS), 0.476 ng for ß-naphthoflavone (p-YDS) and 1.02 ng for calcipotriol hydrate (p-YVS) with chromatographic development. The LOQ for p-YTS and p-YRaS were 10.16 pg for 3,3',5-triiodothyroacetic acid (p-YTS) and 0.41 pg for tamibarotene (p-YRaS), without chromatographic separation. Furthermore, we challenged the developed methodology using environmental samples, demonstrating an elimination efficiency of androgenic activity from municipal wastewater by a wastewater treatment plant between 99.4 and 100%. We anticipate our methodology to substantially broaden the spectrum of specific endpoints combined with HPTLC for an efficient and robust screening of environmental samples to guide a subsequent in-depth EDA.

Pharmacokinetic interaction of calcitriol with 20(S)-protopanaxadiol in mice: Determined by LC/MS analysis.

The physiological and anti-cancer functions of vitamin D3 are accomplished primarily via 1α,25-dihydroxyvitamin D3 (calcitriol), whereas 20(S)-protopanaxadiol (aPPD) is a ginsenoside, which is isolated from Panax ginseng, with potential anti-cancer benefits. In the present study, we report a pharmacokinetic (PK) herb-nutrient interaction between calcitriol and aPPD in mice. A liquid chromatography mass spectrometry (LC/MS) method was developed using 4-phenyl-1,2,4-triazoline-3,5-dione derivatizing agent and we subsequently used the method to quantitate calcitriol in mouse serum. The limit of quantitation was 0.01 ng/ml which is approximately 100 fold lower than the previously reported assay from our laboratory. Calcitriol PK parameters were determined in non-tumor-bearing or C4-2 human prostate tumor-bearing nude mice following oral co-administration of calcitriol either alone or in combination with aPPD. Mice were pretreated with oral aPPD (70 mg/kg) or vehicle control twice daily for seven consecutive days, followed by a single oral dose of 4 µg/kg calcitriol alone or in combination with aPPD. Our PK results demonstrated that co-administration of calcitriol with aPPD (following pre-treatment with vehicle for seven days) resulted in a 35% increase in the area under the curve (AUC0-24 h) and a 41% increase in the maximum serum concentration (Cmax) compared to the calcitriol only group. aPPD therefore significantly increased calcitriol serum exposure. We also saw a reduction in the time required to reach Cmax. In contrast, calcitriol PK in mice co-administered with calcitriol and aPPD as well as those pretreated seven consecutive days with aPPD was no different than that determined for the mice that received vehicle for seven days as pre-treatment. Co-administration of calcitriol with aPPD therefore could increase health benefits of vitamin D3, however any increased risk of hypercalcemia, resulting from this combination approach, requires further investigation. Lastly, we surmise that a cytochrome P450 inhibition-based mechanism may contribute to the observed PK interaction.

Validated Chromatographic Methods for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate in the Presence of Two Dosage Form Additives.

Two chromatographic methods were developed, optimized and validated for simultaneous determination of calcipotriol monohydrate (CPM) and betamethasone dipropionate (BMD) in the presence of two dosage form additives named; butylated hydroxytoluene (BHT) and alpha-tocopherol (TOCO). The proposed methods were accurate, sensitive and specific. The first method based on using aluminum thin-layer chromatographic plates precoated with silica gel GF254 as a stationary phase and chloroform-ethyl acetate-toluene (5:5:3, by volume) as a developing system. This was followed by densitometric measurement of the separated bands at 264 nm. Whereas the second method is RP-HPLC where OnyxMonolithic C18® column was used with a gradient profile using methanol, water and acetic acid at flow rate 2.0 mL min-1. Detection was carried out at 264 nm. The methods were validated according to ICH guidelines. The specificity of the developed methods was investigated by analyzing the pharmaceutical dosage form. The validity of the proposed methods was assessed using the standard addition technique. The obtained results were statistically compared with those obtained by the official methods, showing no significant difference with respect to accuracy and precision at P = 0.05.

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.

Effects of 1,25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model.

Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. OBJECTIVE: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. METHODOLOGY: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. RESULTS: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1ß (IL-1ß) and IL-6 protein expression. CONCLUSIONS: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.

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.

Measurement of vitamin D status: background, clinical use, and methodologies.

Vitamin D and its metabolites are crucial to the overall health and well-being of humans and animals, having important functions in calcium homeostasis and bone metabolism. Exposure of the skin to sunlight may provide adequate levels of vitamin D; however, there are numerous reports of vitamin D insufficiency or deficiency. 25-hydroxyvitamin D (calcidiol, 25(OH)D) is regarded as the best measurement of overall vitamin D status. 1,25-dihydroxyvitamin D (calcitriol, 1,25(OH)2D) is the most biologically active vitamin D metabolite. 25(OH)D has higher affinity for vitamin D binding protein (VDBP) than 1,25-dihydroxyvitamin D; whereas, 1,25-dihydroxyvitamin D has higher affinity for the vitamin D receptor (VDR) than 25-hydroxyvitamin D. HPLC and immunoassays allow the determination of vitamin D status, as measured by 25(OH)D, and 1,25(OH)2D. Recently it has been shown that the vitamin D requirements have been underestimated and that vitamin D2 is much less potent than vitamin D3. Future studies will determine the amount of vitamin D3 necessary for optimal health and well-being.

Regulation of the vitamin D receptor by vitamin D lactam derivatives.

The active metabolite of vitamin D3 , 1α,25-dihydroxyvitamin D3 , acts as a ligand for the vitamin D receptor (VDR) and activates VDR-mediated gene expression. Recently, we characterized 1α,25-dihydroxyvitamin D3 -26,23-lactams (DLAMs), which mimic vitamin D3 metabolites, as noncalcemic VDR ligands that barely activate the receptor. In this study, we present structural insights onto the regulation of VDR function by DLAMs. X-ray crystallographic analysis revealed that DLAMs induced a large conformational change in the loop region between helices H6 and H7 in the VDR ligand-binding domain. Our structural analysis suggests that targeting of the loop region may be a new mode of VDR regulation.

Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light.

Solanum glaucophyllum contains the calciotropic hormone 1, 25-dihydroxy-vitamin D3 (1,25(OH)2D3). The metabolic pathway leading to the formation of 1,25(OH)2D3 in the plant is largely unknown. Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D3 and its metabolites. To investigate the requirement for light, S. glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and high-performance liquid chromatography. HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were detected. The presence of 1,25(OH)2D3 was also evidenced by [3H]1,25(OH)2D3 competitive binding analysis using the chick hormone intestinal receptor. Furthermore, 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were unequivocally identified by mass spectrometry. Incubation of control samples of 7-dehydrocholesterol under the same conditions as S. glaucophyllum cultures did not result in vitamin D3 formation, excluding the influence of light in these experiments. The results suggest that a synthetic route of vitamin D3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro.

The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin D3 in vivo and in vitro.

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.