Vitamin D: Metabolism

Nephrology, 2006

The renal proximal tubule exhibits a very extensive apical endocytic apparatus that is involved in the reabsorption of molecules filtered in the glomeruli. Several key receptors appear to be involved in this function, which serves not only to conserve protein but also to reabsorb different vitamins in complex with their binding proteins. Recent research has established megalin as probably the most important receptor in this endocytosis process. Cubilin is another receptor identified in the proximal tubule endocytic apparatus. Because cubilin lacks transmembrane or cytoplasmic domains required for endocytosis, this receptor associates with megalin to recycle and internalize its ligands. Recent studies have shown that vitamin D-binding protein (DBP)/25-(OH)D3 complex is one of the megalin/cubilin ligands. Megalin knockout mice develop vitamin D deficiency and bone disease owing to an inability of the proximal tubules to capture the DBP/25-(OH)D3 complexes from the glomerular filtrate. In the same way, kidney-specific megalin knockout mice have severe plasma vitamin D deficiency, hypocalcaemia and serious bone disease, like the complete megalin knockout mice. Anti-cubilin antibodies inhibit cellular uptake of DBP/25-(OH)D3 by up to 70%. Anti-megalin antibodies produced a similar reduction in DBP/25-(OH)D3 endocytosis. When both antibodies were applied, impairment of DBP/25-(OH)D3 was only slightly more impaired (around 80%), suggesting that cubilin and megalin function through the same endocytic pathway. Specific forms of renal Fanconi syndrome are associated with endocytic pathway dysfunction with disruption of megalinmediated uptake DBP/25-(OH)D3 complex, producing metabolic bone disease in affected individuals as a prominent clinical finding.

Nephrology Dialysis Transplantation, 2010

Archives of Biochemistry and Biophysics, 1983

It has been shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)z-D3) and dietary Ca modulate renal metabolism of 25-hydroxyvitamin D3 (25-OH-Da) to 1,25-(OH)2-D3 and 24,25-dihydroxyvitamin D3 (24,25-(OH),-D,) in the rat. However, it is not known if 1,25-(OH)z-D3 and Ca act directly on the kidney to modulate 25-OH-D3 metabolism or indirectly through other mechanisms, such as the modulation of parathyroid hormone secretion. Therefore, we have used isolated renal cortical slices from the rat to study the effect of 1,25-(OH)z-D3 and Ca in vitro on renal 25-OH-D3 metabolism.

Biochemistry, 1979

Frontiers in Endocrinology

Vitamin D has a long-established role in bone health. In the last two decades, there has been a dramatic resurgence in research interest in vitamin D due to studies that have shown its possible benefits for non-skeletal health. Underpinning the renewed interest in vitamin D was the identification of the vital role of intracrine or localized, tissue-specific, conversion of inactive pro-hormone 25-hydroxyvitamin D [25(OH)D] to active 1,25-dihydroxyvitamin D [1,25(OH) 2 D]. This intracrine mechanism is the likely driving force behind vitamin D action resulting in positive effects on human health. To fully capture the effect of this localized, tissue-specific conversion to 1,25(OH) 2 D, adequate 25(OH)D would be required. As such, low serum concentrations of 25(OH)D would compromise intracrine generation of 1,25(OH) 2 D within target tissues. Consistent with this is the observation that all adverse human health consequences of vitamin D deficiency are associated with a low serum 25(OH)D level and not with low 1,25(OH) 2 D concentrations. Thus, clinical investigators have sought to define what concentration of serum 25(OH)D constitutes adequate vitamin D status. However, since 25(OH)D is transported in serum bound primarily to vitamin D binding protein (DBP) and secondarily to albumin, is the total 25(OH)D (bound plus free) or the unbound free 25(OH)D the crucial determinant of the non-classical actions of vitamin D? While DBP-bound-25(OH)D is important for renal handling of 25(OH)D and endocrine synthesis of 1,25(OH) 2 D, how does DBP impact extra-renal synthesis of 1,25(OH) 2 D and subsequent 1,25(OH) 2 D actions? Are their pathophysiological contexts where total 25(OH)D and free 25(OH)D would diverge in value as a marker of vitamin D status? This review aims to introduce and discuss the concept of free 25(OH)D, the molecular biology and biochemistry of vitamin D and DBP that provides the context for free 25(OH)D, and surveys in vitro, animal, and human studies taking free 25(OH)D into consideration.

Biochemistry, 1981

actually involved in the two very different processes. Detailed structural work will have to be performed with aldehyde dehydrogenase to determine the groups whose pKs were identified.

Journal of Biological Chemistry, 1981

We isolated 5,6-trans-25-hydroxyvitamin D3 from the plasma of vitamin D-deficient rats which had received 5,6-cis-vitamin D3 orally. The relative amounts of 5,6-tmmd5-hydroxyvitamin D3 and 5,6-cis-25-hydroxyvitamin D3 in plasma were 1:s. Control experiments in which 5,6-cis-25-hydroxyvitamin D3 was added to plasma or water showed that less than 0.5% of the added 25-hydroxyvitamin DS was converted to 5,6trans-25-hydroxyvitamin D3 during the isolation procedure (p < 0.001, control versu8 experimental). We synthesized 5,6-tr~m~-25-hydroxyvitamin D3 from 25hydroxyvitamin D3 and demonstrated that authentic 5,6-tnuzs-25-hydroxyvitamin D3 co-eluted with the isolated putative 5,6-trans-25-hydroxyvitamin D3 on a high performance liquid chromatography system. In addition, the isolated metabolite demonstrated mass spectral, ultraviolet absorption, and protein-binding properties similar to those of synthetic 5,6-tmns-25hydroxyvitamin D3. As 5,6-tmm-25-hydroxyvitamin D3 binds more efficiently to intestinal cytosol-binding protein than 25-hydroxyvitamin D3, this observation could explain, in part, how vitamin D3 and 25-hydroxyvitamin D3 are effective in large doses when administered to anephric patients or patients with advanced renal failure or hypoparathyroidism. This observation may also hold true for other analogs of vitamin D. Further, our observation provides strong evidence for the existence of 5,6-cis-tnuts isomerization in vivo. Whether this process is enzymatic or nonenzymatic is unknown, and the site of formation of the 5,6-tnurs metabolite has not been determined. Vitamin D3 plays an important role in normal calcium and phosphorus physiology (1). D :

Proceedings of the National Academy of Sciences, 2003

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.

Archives of Biochemistry and Biophysics, 2006

During the past two and half decades the elucidation of the metabolic pathways of 25OHD 3 and its active metabolite 1 ,25(OH) 2 D 3 progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identiWcation of the end products of 25OHD 3 metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD 3 and 1 ,25(OH) 2 D 3 are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1 ,25(OH) 2 D 3 metabolism through C-24 oxidation pathway is 1-OH-23-COOH-24,25,26,27-tetranor D 3 or calcitroic acid; the metabolism of 25OHD 3 into 23-COOH-24,25,26,27-tetranor D 3 has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D 3 as a water soluble metabolite of 24R,25(OH) 2 D 3 produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D 3 was also assumed as another end product of 25OHD 3 metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We Wrst studied the metabolism of 25OHD 3 at low substrate concentration (3 £10 ¡10 M) using [1,2-3 H]25OHD 3 as the substrate in the perfused rat kidneys isolated from both normal and vitamin D 3 intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity suYcient for its structure identiWcation by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD 3 (5 £ 10 ¡6 M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D 3 and 24-COOH-25,26,27-trinor D 3 in a ratio of 4:1. The same metabolite X containing both acids in the same ratio of 4:1 was also produced when 24R,25(OH) 2 D 3 was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D 3. Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D 3. In summary, for the Wrst time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 25OHD 3 metabolism in rat kidney through C-24 oxidation pathway.

The Journal of Steroid Biochemistry and Molecular Biology, 2005

The Vitamin D-activating enzyme 25-hydroxyvitamin D-1␣-hydroxylase (1␣-hydroxylase) is now known to be expressed in a much wider range of tissues that previously thought, suggesting a role for 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), which is more in keeping with a cytokine than a hormone. In this capacity, the function of 1␣-hydroxylase in tumors is far from clear. Studies from several groups including ours have shown altered expression of 1␣-hydroxylase in different types of neoplasm including breast, prostate and colon cancers. However, functional analysis of Vitamin D metabolism in cancer is complicated by the heterogenous composition of tumors. Immunohistochemical analysis of breast tumors has shown that 1␣-hydroxylase is expressed by both epithelial cells and by tumor-infiltrating macrophages, suggesting an immunomodulatory component to 1,25(OH) 2 D 3 production in some types of cancer. The demonstration of 1␣-hydroxylase activity in tumors and their equivalent normal tissues has implications for both the treatment and prevention of cancers. For example, in tumors chemotherapy options may include the use of non-1␣-hydroxylated Vitamin D analogs to increase local concentrations of active metabolites without systemic side-effects. The role of 1␣-hydroxylase in protection against cancer is likely to be more complicated and may involve anti-tumor immune responses. (M. Hewison). significant hypercalcemic side-effects . As a consequence there has been a concerted effort to improve the specificity of 1,25(OH) 2 D 3 therapy through the generation of synthetic analogs or deltanoids that retain the anti-proliferative properties of the hormone while minimising calciotropic sideeffects . A more rational approach to this strategy has been facilitated by recent studies which have recognized that 1,25(OH) 2 D 3 signaling via nuclear Vitamin D receptors (VDR) is likely to be subject to gene and tissue-specific 'tuning' by virtue of their interaction with accessory proteins .