Cell specificity and properties of the C-3 epimerization of Vitamin D3 metabolites

It is well documented that Vitamin D3 metabolites and synthetic analogs are metabolized to their epimers of the hydroxyl group at C-3 of the A-ring. We investigated the C-3 epimerization of Vitamin D3 metabolites in various cultured cells and basic properties of the enzyme responsible for the C-3 epimerization. 1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3], 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were metabolized to the respective C-3 epimers in UMR-106 (rat osteosarcoma), MG-63 (human osteosarcoma), Caco-2 (human colon adenocarcinoma), LLC-PK1 (porcine kidney) and HepG2 (human hepatoblastoma)] cells, although the differences existed in the amount of each C-3 epimer formed with different cell types. In terms of maximum velocity (Vmax) and Michaelis constant (Km) values for the C-3 epimerization in microsome fraction of UMR-106 cells, 25(OH)D3 exhibited the highest specificity for the C-3 epimerization among 1alpha,25(OH)2D3, 25(OH)D3 and 24,25(OH)2D3. C-3 epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha --> beta) -hydroxysteroid epimerase (HSE) catalyzed the C-3 epimerization in vitro. Based on these results, the enzyme responsible for the C-3 epimerization of Vitamin D3 are thought to be different from already-known cytochrome P450-related Vitamin D metabolic enzymes and HSE.     

The quantitative determination of vitamin D3 and its metabolites in plasma

A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease.     

Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.     

Vitamin D3 metabolites in rat epididymis: high 24,25-dihydroxy vitamin D3 levels in the cauda region

Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D3 ([3H]25(OH)D3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [3H]-25(OH)D3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D3 and the 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH)2D3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH)2D3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in rat reproductive tissues.     

Effects of phytase and 25-hydroxyvitamin D3 inclusions on the performance, mineral balance and bone parameters of grower-finisher pigs fed low-phosphorus diets

Two experiments, a performance experiment and a mineral balance study, were conducted on grower-finisher pigs (42 to 101 kg live weight) to investigate the effects of Peniophora lycii phytase enzyme and 25-hydroxyvitamin D3 (25-OHD3) on growth performance, carcass characteristics, nutrient retention and excretion, and bone and blood parameters. The two experiments were designed as a 2 × 2 factorial (two levels of phytase and two levels of 25-OHD3). The four diets were T1, low-phosphorous diet; T2, T1 + phytase; T3, T1 + 25-OHD3 and T4, T1 + phytase + 25-OHD3 diet. In all, 25 μg of 25-OHD3 was used to replace 1000 IU of vitamin D3 in diets T3 and T4. Diets were pelleted (70°C) and formulated to contain similar concentrations of energy (13.8 MJ DE/kg), lysine (9.5 g/kg) and digestible phosphorus (P; 1.8 g/kg). Neither the inclusion of phytase nor 25-OHD3 in the diet had any effect on pig performance. There was an interaction between phytase and 25-OHD3 on calcium (Ca) and P retention (P < 0.01) and on the apparent digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.001). Pigs offered phytase diets only, had a higher retention of Ca and P and digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.01) compared with pigs offered unsupplemented diets. However, when the combination of phytase and 25-OHD3 were offered, no effects were detected compared with 25-OHD3 diets only. Pigs fed phytase diets had higher bone ash (P < 0.01), bone P (P < 0.01) and bone Ca (P < 0.05) concentrations compared with pigs offered non-phytase diets. In conclusion, pigs offered phytase diets had a significantly increased bone ash, Ca and P than pigs offered unsupplemented phytase diets. However, there was no advantage to offering a combination of phytase and 25-OHD3 on either bone strength or mineral status compared to offering these feed additives separately.     

The effect of toxic doses of cholecalciferol (Vitamin D3) on the serum zinc levels in rats

Zinc is a trace element important to bone mineralization as well as, in general, nutrition. It is known that cholecalciferol (vitamin D₃) affects bone metabolism. In this study toxic doses of vitamin D₃ were injected subcutaneously (25 μg/d) to rats for 5 wk. It caused a significant increase in serum zinc levels (p<0.02). On the other hand, no significant increase was detected in the other groups. Excessive amounts of vitamin D₃ caused bone breakdown and increased the levels of zinc in blood.     

Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts

A fluorescent Ca2+ indicator, acetoxymethyl Quin2, was used to quantify changes in the cytosolic free calcium concentration ([Ca2+]i) of confluent mouse osteoblasts. 1,25 - Dihydroxycholecalciferol (1,25 - (OH)2D3, 10-100 pM), 25-hydroxycholecalciferol (25-(OH)D3, 10-100 nM), parathyroid hormone (PTH(1-84), 0.1-10 nM), and prostaglandin E2 (PGE2, 10-1000 nM) all induced immediate (t less than 15 s) transient increases in [Ca2+]i, from a basal level of 135 +/- 8 nM to levels of 179-224 nM. These increases rapidly returned to a plateau approximately 10% higher than the basal level. 24,25-Dihydroxycholecalciferol (24,25-(OH)2D2, 0.1-10 nM) induced a rapid increase in [Ca2+]i which remained elevated for 5 min before decreasing. The 1,25-(OH)2D3- and PTH-induced spikes were abolished by the prior addition of EGTA and Ca2+ entry blockers (verapamil, nifedipine, 1 microM) while the responses to 25-(OH)D3, 24,25-(OH)2D3, and PGE2 were unaffected. Addition of 1,25-(OH)2D3 + EGTA or PTH + EGTA caused enhanced Ca efflux. Addition of drugs which interfere with calcium sequestration by the endoplasmic reticulum (ER) (caffeine, 4 mM; 8-(diethyl-amino)-octyl 3,4,5-trimethoxybenzoate HCl, 0.5 mM) or mitochondria (antimycin, 10 microM; oligomycin, 5 microM) showed that 25-(OH)D3 and PGE2 mainly mobilized Ca2+ from ER. 1,25-(OH)2D3 and bovine PTH caused a transient increase in [Ca2+]i, 70% of which resulted from Ca2+ influx from outside the cells and 30% by release from the ER. The [Ca2+]i increase induced by 24,25-(OH)2D3 included a 30% contribution from the ER and 70% from the mitochondria.     

Vitamin A supplementation at pharmacological doses induces nitrosative stress on the hypothalamus of adult Wistar rats

Vitamin A is a micronutrient involved in the regulation of a normal mammalian brain function. In spite of this, it has been demonstrated that vitamin A exerts a wide range of deleterious effects regarding neuronal homeostasis, for instance impairing brain metabolism and suppressing neurogenesis, to cite a few. In addition, vitamin A is a redox active molecule, i.e. it is both anti- and pro-oxidant, depending on its concentration. In the herein presented work, we performed some experiments aiming to investigate the effects of clinically applied doses of vitamin A (1000–9000 IU/kg/day during 28 days) on rat hypothalamic redox state and mitochondrial electron transfer chain (METC) activity, as well as on hypothalamic α-synuclein and D2 receptor (dopamine receptor) contents. Additionally, we quantified caspase-3 activity and tumor necrosis factor-α (TNF-α) levels to assess either neuronal death or an inflammatory state in such brain area. We found that vitamin A supplementation increased free radical production, as well as oxidative and nitrosative stress, in rat hypothalamus. Also, we observed increased complex I-III activity, but decreased complex IV activity in the hypothalamus of vitamin A-treated rats, which may give rise to the increased superoxide anion (O₂⁻) production found here. Other parameters investigated here, i.e. α-synuclein and D2 receptor contents did not change. Even though we did not observe signs of increased cell death or inflammation in the rat hypothalamus, more attention is needed when vitamin A is the choice of treatment in certain pathologies.     

维生素D_3与雄性生殖

维生素D3的经典作用是调节体内钙、磷的代谢平衡和维持骨的健康,有证据显示维生素D3也可调节动物生殖。维生素D代谢酶可合成和降解活性维生素D3及其代谢中间产物,活性维生素D3通过结合至目标组织的维生素D受体(vitamin D receptor,VDR)以发挥其生物学作用。VDR和维生素D代谢酶在雄性生殖系统中表达,表明维生素D3在雄性生殖生物学中起关键作用。本文对维生素D3与雄性生殖的研究进展作一综述,以期为推进维生素D3影响雄性生殖的分子机制和临床治疗男性不育的研究提供理论依据。     

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.     

1,25-Dihydroxyvitamin D3 injections into rat fetuses : effects on fetal plasma calcium, plasma phosphate and mineral content

The in vivo effects of 1,25-(OH)2D3 were assessed using fetuses from normal and thyroparathyroidectomized (TPTX) pregnant rats. 21.5-day old decapitated fetuses from TPTX mothers exhibited lowered basal plasma calcium, elevated basal plasma phosphate and an increased percentage of total ash compared to intact littermates. In decapitated fetuses from normal mothers, neither plasma calcium nor plasma phosphate was changed. Subcutaneous injection of 1 micrograms of 1,25-(OH)2D3/kg of body weight to 19.5-day old fetuses (intact or deprived of their parathyroid glands by decapitation) from TPTX mothers induced a marked rise in plasma calcium levels (2.01 and 3.66 mg/dl, respectively) 48 h later. Little change occurred in fetuses from normal mothers (1.06 mg/dl in decapitated and no change in intacts). A decrease in plasma phosphate levels was observed with the same dose in both decapitated and intact fetuses from TPTX mothers (- 1.39 and - 0.65 mg/dl, respectively), while no modification was found in fetuses from normal females. Therefore, the hypersensitivity of fetuses from TPTX mothers to 1,25-(OH)2D3 was unrelated to the development of the fetal hyperparathyroidism secondary to maternal TPTX. The percentage of ash was unchanged in decapitated fetuses from TPTX mothers and was increased in intact littermates after 1,25-(OH)2D3 treatment. However, these values for total ash may represent alterations in bones and/or soft tissues.     

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.     

维生素D及其受体在帕金森病中的作用

帕金森病(Parkinson's disease,PD)是一种常见的中枢神经系统退行性疾病,引起帕金森病的发病机制至今尚未明确。帕金森病患者及老年人普遍存在维生素D缺乏,这可能是帕金森病的重要发病机制之一。由于维生素D具有免疫调节,抗氧化,调节神经营养因子,降低神经毒性的功能,能同时针对几种导致神经退行性病变因素发挥作用,特别是老年人纠正维生素D缺乏可能会阻止神经元的损失和PD相关的认知功能下降。因此补充维生素D可能成为治疗PD的方法。近年来研究发现,维生素D受体基因多态性与帕金森病的发病有相关性。该文就维生素D及其受体在帕金森病中可能发生的保护作用及其机制作一综述。     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

Effects of vanadate on plasma lipoprotein profiles in experimental diabetic rats

The insulin-like effects of vanadate have been intensively studied in the biological system. Lipids and lipoprotein profiles are altered in diabetes. Rats were made diabetic by a single i.v. injection of streptozotocin (55 mg/kg body weight) in citrate buffer. After the overt of diabetes, the diabetic rats were treated with sodium orthovanadate (0.3 mg/ml) for fifteen days. The altered cholesterol, phospholipids and triglycerides in plasma lipoprotein fractions (HDL, LDL and VLDL) were found to be reverted back to near normal levels in vanadate treated diabetic rats.