Tadashi Kobayashi

Regulation of Type II Renal Na+-dependent Inorganic Phosphate Transporters by 1,25-Dihydroxyvitamin D3 IDENTIFICATION OF A VITAMIN D-RESPONSIVE ELEMENT IN THE HUMAN NAPI-3 GENE

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (Pi) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent Pi transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) to vitamin D-deficient rats increased the initial rate of Pi uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent Pi transporter NaPi-3 gene was increased markedly by 1,25-(OH)2D3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5′-GGGGCAGCAAGGGCA-3′ nucleotides −1977 to −1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates Pi homeostasis is through the modulation of the expression of type II Na+-dependent Pi transporter genes in the juxtamedullary kidney cortex.

In Vitro Metabolism of the Vitamin D Analog, 22-Oxacalcitriol, Using Cultured Osteosarcoma, Hepatoma, and Keratinocyte Cell Lines

Using four cultured cell models representing liver, keratinocyte, and osteoblast, we have demonstrated that the vitamin D analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and side chain truncated metabolites. Four of these metabolic products have been rigorously identified by high pressure liquid chromatography, diode array spectrophotometry, and gas chromatography-mass spectrometry analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as the cleaved molecules, hexanor-1α,20-dihydroxyvitamin D and hexanor-20-oxo-1α-hydroxyvitamin D. Comparison with chemically synthesized standards has revealed the stereochemistry of the biological products. Although differences exist in the amounts of products formed with the different cell types, it is apparent that 22-oxacalcitriol is subject to metabolism by both vitamin D-inducible and noninducible enzymes. Time course studies suggest that the truncated 20-alcohol is derived from a side chain hydroxylated molecule via a hemiacetal intermediate and the 20-oxo derivative is likely formed from the 20-alcohol. Biological activity measurements of the metabolites identified in our studies are consistent with the view that these are catabolites and that the biological activity of 22-oxacalcitriol is due to the parent compound. These results are also consistent with recent findings of others that the biliary excretory form of 22-oxacalcitriol is a glucuronide ester of the truncated 20-alcohol.

A novel high-performance liquid chromatographic assay for vitamin D metabolites using a coulometric electrochemical detector

A new, highly sensitive HPLC assay method using an electrochemical detector (ECD) for multiple assay of vitamin D metabolites is reported. The assay involves extracting lipids from plasma with methylene chloride and methanol, purification on Zorbax SIL column with 5.5% (v/v) iso-propanol in hexane and quantification by HPLC-ECD. A coulometric system, composed of the dual electrode analytical cell and a guard cell, was used for ECD of the eluting compounds. The potentials applied to detectors 1 and 2 in a dual electrode analytical cell were adjusted to +0.20 V and +0.60 V, respectively. This method is sensitive to 20 pg of 25-hydroxyvitamin D3 [25(OH)D3] and of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Calibration curves gave linearity from 20-1000 pg for 25(OH)D3 and 24,25(OH)2D3. The detection limit was approximately 50 pg ml-1 for 25(OH)D3 and 24,25(OH)2D3 in plasma. This sensitivity combined with an overall recovery of 25(OH)D3 (81.5 +/- 2.6%, mean +/- S.E.) allows the measurement of trace amount of 25(OH)D3 with only 20 microliters of plasma. Intra- and interassay RSD values were 5.3 and 9.7% for 25(OH)D3 and 6.3 and 9.7% for 24,25(OH)2D3, respectively. Plasma levels of 25(OH)D3 and 24,25(OH)2D3 in normal adults were 15.9 +/- 2.8 ng ml-1 (n = 10) and 1.4 +/- 0.5 ng ml-1 (n = 10), respectively. This method allows the determination of 25(OH)D2 and 25(OH)D3 for evaluating their nutritional and clinical status. From these results, it is concluded that the proposed HPLC-ECD assay system is useful for the determination of vitamin D metabolites in biological fluids as a highly sensitive physicochemical method.

Saccharated ferric oxide (SFO)-induced osteomalacia : In vitro inhibition by SFO of bone formation and 1,25-dihydroxy-vitamin D production in renal tubules

A 60-year-old man with portal hypertensive gastropathy due to type C liver cirrhosis developed severe bone pains, marked hypophosphatemia with inappropriately increased urinary excretion of phosphate (%TRP; 9.6%), and hyperalkaline phosphatasia, after intravenous administration of saccharated ferric oxide (SFO) at a dose of 80-240 mg/week over a period of more than 5 years. The total iron infused was estimated to be more than 25 g. On a diagnosis of SFO-induced osteomalacia, the infusion of iron was immediately discontinued, and phosphate and vitamin D2 (1000 IU/day) were administered. Serum levels of 25-OHD2 increased after 1 week, whereas levels of 1,25-(OH)2D2 did not increase until 3 months later, accompanied by improvement of renal tubular reabsorption of phosphate and gradual improvement of the bone pains. The patient has been doing well for the last 2 years, with normal serum levels of phosphate, calcium, and alkaline phosphatase, without any supplementation of phosphate, vitamin D, or iron-containing agents. In primary culture of neonatal mouse renal tubules, in which 1,25-(OH)2D3 was produced from 25-OHD3 in response to PTH, SFO significantly inhibited PTH-induced production of 1,25-(OH)2D3 at 30 mumol/L, which is attainable in the urine of patients receiving a therapeutic intravenous dose of SFO. Furthermore, SFO decreased the calcium content and inhibited 45Ca incorporation in cultured fetal mouse parietal bones at 3 mumol/L. Such SFO concentration may be transiently observed in the plasma of patients receiving excessive intravenous doses of SFO for a prolonged period. These in vitro findings together with the clinical observations suggest that SFO, after filtration through the glomerulus and reabsorption in the proximal renal tubules, impaired proximal renal tubular function, such as tubular reabsorption of phosphate and 1 alpha-hydroxylase activity, leading to hypophosphatemic osteomalacia. Furthermore, it is highly likely that SFO in the peripheral blood, when transferrin is saturated with iron, may impair bone formation and aggravate osteomalacia. Although SFO-induced osteomalacia is reversible simply by discontinuation of the agent, excessive and prolonged administration of SFO should be avoided.

THE ROLE OF VITAMIN D METABOLITES IN THE TREATMENT OF OSTEOPOROSIS

The Second Symposium on Osteoporosis was organized in Tokyo, Japan on November 6, 1994, with the purpose of summarizing the current views on the biology of vitamin D, and the applications of vitamin D metabolites in osteoporosis. The Symposium was sponsored by Chugai Pharmaceuticals, Co., and Teijin Ltd., Tokyo, Japan. Dr. Etsuro Ogata (Cancer Research Institute, Tokyo, Japan) introduced the symposium observing that although vitamin D metabolites and analogs are currently being used in Japan and other countries for the treatment of osteoporosis, there is still controversy on their therapeutic role, especially in North America and Europe. Because the molecular aspects of vitamin D action on bone cells constitute the basis for understanding how vitamin D affects bone remodeling, and for the design of analogs with selective actions on bone, the first part of the symposium focused on the biologic action of vitamin D at the cellular level. The second part reviewed the role of vitamin D in the pathogenesis of osteoporosis, and in the last session the most important and controversial issues on the clinical application of active vitamin D analogs in osteoporosis were discussed.

Synthesis and biological character of 1β-hydroxylated vitamin D3 Analogues

Abstract The synthesis of 1,25-dihydroxy-22-oxavitamin D 3 and 1β,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D 3 and the preliminary in vitro biological evaluation are described.

The enzymatic formation of 1α,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 in the liver of fetal rats

We have confirmed the existence of 25-hydroxyvitamin D3 (25-OH-D3)-1 alpha-hydroxylase in the liver of fetal rats, in addition to that in the kidney, by in vitro experiments. The findings are similar to those in the fish liver as reported previously (Takeuchi et al., Life Sci. 32, 275-282, 1991). When [3H]-25-OH-D3 was incubated with liver homogenates of vitamin D-deficient fetal rats, a peak corresponding to [3H]-1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was observed in the profile of high-performance liquid chromatography (HPLC). The formation of the metabolite was confirmed by thermal isomerization into the pre-isomer, binding affinity to the receptor and mass fragmentography. Lineweaver-Burk plot analysis of mitochondrial 25-OH-D3-1 alpha-hydroxylase in the liver gave an apparent Km value of approximately 2 microM of 25-OH-D3 and a Vmax value of 0.2 pmol of 1,25(OH)2D3/30 min/mg protein. These findings suggest that the enzyme in the liver disappeared with the growth of the fetus and became predominant in the kidney of mature rats.

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1alpha,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C18OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1alpha,25-dihydroxyvitamin D [1,25(OH)2D] fractions. The 25-OH-D fraction was purified by a Bond Elut C18 cartridge and 25-OH-D2 and 25-OH-D3 were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)2D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)2D2 and 1,25(OH)2D3 fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.

Effects of dietary supplementation of calcium and vitamin don bone growth in growing male rats

Effects of dietary supplementation of calcium (Ca) and vitamin D(D) on bone growth in growing male rats were investigated. We performed this study using D-deficient rats of 3-month-old. In the experiment 1, the D-deficient rats were fed either low-Ca (0.22% Ca) or high-Ca (1.20% Ca) diets with oral supplementation of different amounts of D3 (0, 0.7, 7 or 70 IU/week) for 28 days. In the dxperiment 2, the D-deficient rats were fed diets containing different concentrations of Ca (0.22, 0.44, 0.88 or 1.20%) with oral D3 supplementation of either low-dose (0.7 IU/week) or relatively high-dose (70 IU/week) for 28 days. After the feeding period, plasma levels of Ca, 1α, 25 (OH)2D3, PTH, bone Gla protein were measured. Bone ash weight, bone mineral density, mechanical bone strength were also measured. In the both experiments, the plasma levels of PTH decreased to the normal levels in response to the increased amounts of dietary Ca intakes as well as D supplementation. In contrast, the bone markers increased to the respective normal levels in response to the increased amounts of dietary Ca intakes as well as D supplementation. In the experiments 1 and 2, a high correlation between the plasma levels of PTH and the bone markers was observed. These results suggest that both dietary Ca and D supplementation may affect bone growth in growing rats by controlling PTH secretion.

Effects of vitamin D3 analogues on parathyroid hormone secretion and calcium metabolism in vitamin D-deficient rats

Abstract22-Oxa-1α, 25-dihydroxyvitamin D3 (OCT) and 2β-(3-Hydroxypropoxy)-1α, 25-dihydroxyvitamin D3 (ED-71) are novel synthetic vitamin D3 analogues. In order to examine their calcemic actions on intestine and bone, we have investigated the effects of OCT and ED-71 on intestinal Ca transport, bone mobilization and plasma parathyroid hormone (PTH) level in vitamin D-deficient rats. The vitamin D-deficient rats were intravenously given either 6.25µg/kg or 0.2µg/kg of 1,25-D3, OCT or ED-71 and theirplasma Ca levels and intestinal Ca transport were measured periodically. At a high dose, 1,25(OH)2D3 and ED-71 showed a strong biphasic stimulation of intestinal Ca transport and bone mobilization, and reduced the plasma PTH levels to the normal level completely. On the other hand, OCT failed to suppress the PTH secretion although it exerted first phase action on the both intestinal Ca transport and bone mobilization in vitamin D-deficient rats. The reason why OCT failed to suppress the PTH secretion even at a high dose, has not yet been clarified, but it may be at least in part due to its weak calcemic action and short half-life in plasma.