Vitamin D deficiency attenuates endothelial function by reducing antioxidant activity and vascular eNOS expression in the rat microcirculation.

This study examined the effects of vitamin D deficiency on vascular function and tissue oxidative status in the microcirculation; and whether or not these effects can be ameliorated with calcitriol, the active vitamin D metabolite. Three groups (n = 10 each) of male Sprague Dawley rats were fed for 10 weeks with control diet (CR), vitamin D-deficient diet without (DR), or with oral calcitriol supplementation (0.15 µg/kg) for the last four weeks (DSR). After 10 weeks, rats were sacrificed; mesenteric arterial rings were studied using wire myograph. Oxidative stress biomarkers malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were measured in the mesenteric arterial tissue. Vascular protein expression of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Acetylcholine-induced endothelium-dependent relaxation of DR was lower than CR. eNOS expression and SOD activity were lower in mesenteric arterial tissue of DR compared to CR. Calcitriol supplementation to DSR did not ameliorate the above parameters; in fact, augmented endothelium-dependent contraction was observed. Serum calcium was higher in DSR compared to CR and DR. In conclusion, vitamin D deficiency impaired microvascular vasodilation, associated with eNOS downregulation and reduced antioxidant activity. Calcitriol supplementation to vitamin D-deficient rats at the dosage used augmented endothelium-dependent contraction, possibly due to hypercalcaemia.

Alterations in gene expression in vitamin D-deficiency: Down-regulation of liver Cyp7a1 and renal Oat3 in mice.

The vitamin D-deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D-deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α-hydroxylase (Cyp7a1, rate-limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up-regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D-deficiency. When vitamin D-sufficient or D-deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D-deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D-sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)2 D3 /cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D-deficient mice, while 1,25(OH)2 D3 treatment led to hypercalcemia. In conclusion, vitamin D-deficiency resulted in down-regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.

Loss of miR-125b contributes to upregulation of CYP24 in uraemic rats.

AIM: Abnormal upregulation of CYP24 contributes to vitamin D insufficiency and resistance to vitamin D therapy in chronic kidney disease (CKD), because human CYP24 is a key enzyme involved in the inactivation of 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3; calcitriol) and 1,25(OH)2D3. There are multiple mechanisms regulating CYP24 in a variety of types of tissues and diseases. An increasing body of evidence suggests that microRNA-125b (miR-125b) plays an important role in post-transcriptional regulation of CYP24 mRNA. METHODS: We sought to test hypothesis that abnormal elevation of CYP24 in CKD is a consequence of loss of miR-125b in CKD in a uraemia rat model. RESULTS: We found that expression of miR-125b was significantly inhibited in uraemic rats coupled with increased CYP24 at both protein and mRNA levels compared with normal controls. In NRK-52 kidney cells, we further found that miR-125b antagomirs increased CYP24 but miR-125b mimics decreased CYP24, and luciferase assay confirmed that CYP24 is a direct target of miR-125b. Vitamin D status exerted no significant effects on expression of both miR-125b and CYP24 in uraemic rats. CONCLUSION: These results suggest that modulation of miR-125b may be used for treatment of Vitamin D insufficiency in CKD.

Cytochrome P450-mediated metabolism of vitamin D.

The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field.

Expression of vitamin D-metabolizing enzymes in human adipose tissue -- the effect of obesity and diet-induced weight loss.

OBJECTIVE: Low vitamin D (VD) levels are common in obesity. We hypothesized that this may be due to metabolism of VD in adipose tissue (AT). Thus, we studied (1) whether the VD-metabolizing enzymes were expressed differently in AT of lean and obese individuals and in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and (2) whether their expression was influenced by weight loss. METHODS: Samples of SAT and VAT were analyzed for expression of the vitamin-D-25-hydroxylases CYP2R1, CYP2J2, CYP27A1 and CYP3A4, the 25-vitamin-D-1α-hydroxylase CYP27B1, the catabolic vitamin-D-24-hydroxylase CYP24A1, and the vitamin D receptor, using reverse transcriptase-PCR. Moreover, plasma 25-hydroxy-vitamin D (25OHD) level was measured and related to the expression of these enzymes. Samples of SAT and VAT from 20 lean women and 20 obese women, and samples of SAT from 17 obese subjects before and after a 10% weight loss were analyzed. RESULTS: A plasma 25OHD level <50 nmol l(-1) was highly prevalent in both lean (45%) and obese (90%) women (P<0.01). Plasma 25OHD increased by 27% after weight loss in the obese individuals (P<0.05). Expression levels of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 were decreased by 71% (P<0.0001) and 49% (P<0.05), respectively, in SAT of the obese. CYP24A1 did not differ between lean and obese women, but the expression was increased by 79% (P<0.05) after weight loss. CONCLUSION: Obesity is characterized by a decreased expression of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 in SAT, whereas the catabolic CYP24A1 does not differ between lean and obese women. However, the expression of CYP24A1 is increased after weight loss. Accordingly, AT has the capacity to metabolize VD locally, and this can be dynamically altered during obesity and weight loss.

CYP24A1 and kidney disease.

PURPOSE OF REVIEW: Patients with chronic renal disease have elevated serum phosphate levels, elevated fibroblast-like growth factor 23 (FGF-23), and declining vitamin D status. These changes are related and may be responsible for elevated 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) and dysfunctional vitamin D metabolism. This review focuses on the biochemistry and pathophysiology of CYP24A1 and the utility of blocking this enzyme with CYP24A1 inhibitors in chronic kidney disease (CKD) patients. RECENT FINDINGS: CYP24A1 is the cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25-OHD3) and its hormonal form, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], into 24-hydroxylated products targeted for excretion. The CYP24A1-null phenotype is consistent with the catabolic role of CYP24A1. A number of polymorphisms of CYP24A1 have recently been identified. New data from the uremic rat and humans suggest that dysfunctional vitamin D metabolism is due to changes in CYP24A1 expression caused by phosphate and FGF-23 elevations. SUMMARY: Changes in serum phosphate and FGF-23 levels in the CKD patient increase CYP24A1 expression resulting in decreased vitamin D status. Vitamin D deficiency may exacerbate defective calcium and phosphate homeostasis causing renal osteodystrophy and contribute to the other complications of renal disease. These findings argue for increased focus on correcting vitamin D deficiency in CKD patients by blocking CYP24A1 activity.

[The role of vitamin D in the prevention and the additional therapy of cancers]. / A D-nap. A D-vitamin helye a daganatprevencióban és a kiegészíto kezelésben.

The active metabolite of vitamin D apart from a crucial role in maintaining mineral homeostasis and skeletal functions, has antiproliferative, apoptosis and differentiation inducing as well as immunomodulatory effects in cancer. It is well known that with increasing sunshine exposure the incidence of breast, prostate and colorectal cancer is decreasing. A number of in vitro and in vivo experiments documented the effects of vitamin D in the inhibition of the tumorigenesis. In studying the role of vitamin D in cancer, it is imperative to examine the potential pathways that control local tissue levels of vitamin D. The enzyme 24-hydroxylase converts the active vitamin D to inactive metabolite. Extra-renal production of this enzyme is observed and has been increasingly recognized as present in cancer cells. This enzyme is rate limiting for the amount of local vitamin D in cancer tissues and elevated expression is associated with an adverse prognosis. 24-hydroxylase may be a predictive marker of vitamin D efficacy in patients with cancer as an adjunctive therapy. There are many vitamin D analogs with no pronounced hypercalcemizing effects. Some analogs are in phase 1 and 2 clinical test, and they might have a role in the therapy of several types of cancer. At present our main task is to make an effort to decrease the vitamin D deficiency in Hungary. Speer G. The D-day. The role of vitamin D in the prevention and the additional therapy of cancers.

Enzymes involved in the activation and inactivation of vitamin D.

Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1alpha-hydroxylase contains the component CYP27B1, which completes the activation pathway to the hormonal form 1alpha,25-dihydroxyvitamin D(3). A five-step inactivation pathway from 1alpha,25-(OH)(2)D(3) to calcitroic acid is attributed to a single multifunctional CYP, CYP24A1, which is transcriptionally induced in vitamin D target cells by the action of 1alpha,25-(OH)(2)D(3). On the basis of alignments and crystal structures of other CYPs, homology models of vitamin-D-related CYPs have been generated. Two human forms of rickets caused by mutations of CYP2R1 and CYP27B1, as well as mouse knockout models of CYP27A1, CYP27B1 and CYP24A1, are helping us to establish the full in vivo physiological roles of the vitamin-D-related hydroxylases.

Parathyroidectomy reduces 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the hypocalcemic vitamin D-deficient chick.

To test the hypothesis that in the vitamin D-deficient state the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase (25-OHD3-1 alpha-hydroxylase) is modulated by parathyroid hormone and the plasma concentration of phosphate only in the presence of small amounts of 1,25-dihydroxyvitamin D3 (or some other metabolite of vitamin D), we measured the activity of this enzyme 24 h after parathyroidectomy (PTX) in frankly hypocalcemic, vitamin D-deficient chicks that were not supplemented with vitamin D or one of its metabolites. The otherwise predictable complications of PTX in this metabolic setting (hypocalcemia of increasing severity, tetany, moribundity, and death) were prevented by continuous intravenous administration of calcium (as a solution of calcium chloride/calcium gluconate 1:1) through a catheter in the external jugular vein placed at the time of PTX. The findings were as follows: (a) The activity of 25-OHD3-1 alpha-hydroxylase was significantly less in the parathyroidectomized group than in the sham-operated control chicks (P less than 0.001). (b) The reductive effect of PTX on the activity of this enzyme was significantly attenuated when hypophosphatemia was increased in severity by administration of glucose. (c) In the post-PTX state the activity of 25-OHD3-1 alpha-hydroxylase and plasma concentration of phosphate were significantly, inversely related (P less than 0.001). (d) In the sham-operated control group the activity of this enzyme and the plasma concentration of phosphate were not significantly correlated. These findings indicate that in the vitamin D-deficient state, both circulating parathyroid hormone and the plasma concentration of phosphate can significantly modulate the activity of 25-OHD3-1 alpha-hydroxylase in the absence of vitamin D or its metabolites. The findings also suggest that in the vitamin D-deficient state the plasma concentration of phosphate modulates the activity of this enzyme only when the concentration of circulating parathyroid hormone is not increased.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.

Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca(2+)-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl phorbol 13-acetate and 1,25(OH)2D3 activated endogenous PKC, as assessed by inhibition of myristoylated alanine-rich C kinase substrate back-phosphorylation by exogenous PKC. These studies indicate that PKC-alpha, -delta, and/or -epsilon likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.

Vitamin D and adenosine triphosphatase dependent on divalent cations in rat intestinal mucosa.

Intestinal brush borders prepared from vitamin D-deficient rats demonstrate increased susceptibility in vitro to fragmentation by shear forces or to loss of microvillus enzymes on treatment with EDTA. These effects are relatively nonspecific and are also observed in normal rats starved for 48 h. They may underlie prior observations that purport to demonstrate a vitamin D-dependent increase in brush border Ca-dependent ATPase. In addition, however, vitamin D increases ATPase activity dependent on certain divalent cations, including Ca and Zn, in whole-particulate suspensions pelleted by high-speed centrifugation of mucosal homogenates. This action is independent of changes in other microvillus enzymes, i.e. disaccharidases, and tissue distribution and cation specificity studies support the hypothesis that the mucosal whole-particulate ATPase is related to transport of Ca, Zn, and possibly other divalent cations.

Microbiota-Dependent Induction of Colonic Cyp27b1 Is Associated With Colonic Inflammation: Implications of Locally Produced 1,25-Dihydroxyvitamin D3 in Inflammatory Regulation in the Colon.

Our recent studies demonstrated that intestinal epithelial vitamin D receptor (VDR) signaling plays a critical role in regulating colonic inflammation by protecting epithelial barrier integrity. Epithelial VDR is downregulated in colitis, but how mucosal inflammation affects local 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production is unknown. Here we showed that cytochrome P450 27b1 (Cyp27b1), a cytochrome P450 enzyme necessary for 1,25(OH)2D3 biosynthesis, is highly induced in colonic mucosa in inflammatory response. Although VDR is reduced in colon biopsies from patients with ulcerative colitis, Cyp27b1 is markedly upregulated in these samples. Colon mucosal Cyp27b1 was also markedly induced in an experimental colitis mouse model, and this local Cyp27b1 induction and colonic inflammation required the presence of commensal bacteria. Vitamin D deficiency further exaggerated colonic Cyp27b1 induction and aggravated colonic inflammation in mice. In HCT116 cells, lipopolysaccharide or tumor necrosis factor-α treatment induced Cyp27b1 in time- and dose-dependent manners, and the induced Cyp27b1 was enzymatically active. The inflammation-induced upregulation of Cyp27b1 was mediated by nuclear factor κB. Collectively these data suggest that induction of colonic epithelial Cyp27b1, which is expected to increase local production of 1,25(OH)2D3, is a protective mechanism that partially compensates for the downregulation of epithelial VDR during colonic inflammation. Increased local 1,25(OH)2D3 maintains 1,25(OH)2D3-VDR signaling to protect the mucosal barrier and reduce colonic inflammation.

Vitamin D metabolism in peripheral blood mononuclear cells is influenced by chewing "betel nut" (Areca catechu) and vitamin D status.

CONTEXT: Vitamin D deficiency, common in South Asians, is a risk factor for metabolic syndrome, type 2 diabetes, and ischemic heart disease. Vitamin D receptor (VDR) activation depends on activated vitamin D [1,25-dihydroxyvitamin D (1,25(OH)(2)D)] concentration, reflecting opposing actions of 25-hydroxyvitamin D-1alpha-hydroxylase [1-alpha(OH)ase] for formation and 25(OH)D-24-hydroxylase [24(OH)ase] for catabolism. We previously reported that circulating 1,25(OH)(2)D contributed to determination of VDR-protein levels and VDR genotype was a determinant of both VDR mRNA and VDR-protein in South Asians. OBJECTIVE: We hypothesized that chewing betel nut, an addictive habit common throughout South Asian communities, contributes to hypovitaminosis-D by modulating the enzymes regulating circulating 1,25(OH)(2)D concentration. DESIGN: Peripheral blood mononuclear cell (PBMC) 1-alpha(OH)ase and 24(OH)ase mRNA concentrations were measured and examined in relation to cross-sectional data on the vitamin-D axis, diet, smoking, and betel usage, including PBMC VDR-RNA and VDR-protein content in a pilot study of 33 healthy British Bangladeshis. RESULTS: PBMC 24(OH)ase mRNA correlated positively and serum 1,25(OH)(2)D negatively with betel quids per day (r = 0.49, P = 0.006 and r = -0.486, P = 0.006, respectively). Independent determinants for 24(OH)ase included betel quids per day (P < 0.0001) and serum 25-OHD (P = 0.024). Independent determinants for serum 1,25(OH)(2)D were gender, smoking, and betel quids per day. PBMC 1-alpha(OH)ase mRNA correlated inversely with VDR mRNA (r = -0.44; P = 0.013); its independent determinants were serum 1,25(OH)(2)D and VDR TaqI and BsmI polymorphisms (P = 0.03-0.0001). CONCLUSIONS: Betel chewing is a more powerful independent determinant of increased 24(OH)ase expression and of decreased serum calcitriol than serum 25-OHD, supporting the hypothesis that this habit could aggravate the effects of vitamin-D deficiency.

Acute stimulation of creatine kinase activity by vitamin D metabolites in the developing cerebellum.

There is increasing evidence that vitamin D metabolites have a developmental function. We have investigated the influence of the vitamin D status on the activity of creatine kinase in the brain. Normally fed rats show an increase in the specific activity of cerebral and cerebellar creatine kinase during postnatal development. Vitamin-D-depleted rats failed to show this normal increase. Developing cerebellum, but not cerebrum, in both vitamin D-depleted rats and in normally fed animals, responded sequentially to a single injection of a vitamin D metabolite by displaying increased creatine kinase specific activity. In 5-25-day-old rats, 24R,25-dihydroxyvitamin D-3 significantly increased creatine kinase specific activity 24 h after injection. In contrast, 1,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity from 20 days after birth. A similar pattern of sequential responsiveness to vitamin D metabolites, but at an earlier age, was shown in the cerebellum of the rabbit, which is a 'perinatal brain developer' compared to the rat, a 'postnatal brain developer'. Because of the difficulty in obtaining vitamin D-depleted rabbits, studies were carried out in normally fed animals. In these rabbits, 24R,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity between 6 days before birth and 9 days after birth, while 1,25-dihydroxyvitamin D-3 caused an increase in cerebellar creatine kinase specific activity from 8 days after birth. These developmental differences found in creatine kinase basal activity and responsiveness are correlated with differences in cellular growth rates, both in the rabbit and in the rat, suggesting that vitamin D metabolites may be required for optimal cerebellar development.

Intestinal brush border hydrolase topography. Effects of vitamin D-3 and filipin.

Intestinal brush borders were isolated from vitamin D-3-treated and vitamin D-deficient chicks, and protein topography in the paired preparations assessed by the enzymatic release of four marker hydrolases. Exposure of the brush borders to the protease bromelain resulted in soluble levels of alkaline phosphatase, leucine aminopeptidase, maltase, and sucrase activities from preparations of vitamin D-3-treated birds that were 42%, 75%, 64%, and 56%, respectively, of corresponding activities released in preparations from rachitic chicks. Analyses for recovery of enzyme activity revealed that bromelain treatment selectively inactivated 43% of the alkaline phosphatase activity of brush borders obtained from vitamin D-3-replete birds, and preferentially diminished recovered sucrase activity in preparations from vitamin D-deficient chicks. In additional experiments, brush borders isolated from rachitic birds were treated in vitro with the polyene antibiotic filipin or an equivalent volume of vehicle. Subsequent exposure of such preparations to bromelain resulted in little or no differences in levels of marker hydrolase specific activities released from filipin- or vehicle-treated brush borders. However, analyses of membrane-bound specific activities after treatment of brush border preparations with a range of filipin concentrations, revealed a biphasic inhibition of approx. 30% for both maltase and sucrase, relative to vehicle controls, and a smaller effect on alkaline phosphatase and leucine aminopeptidase.

Creatine phosphokinase in long-term dialysis patients.

Of 90 patients undergoing regular dialysis, 42% had elevated levels of creatine phosphokinase (CPK). The BB isoenzyme was not detected, and only one patient had CPK MB. The elevation of CPK MM level did not correlate with values for calcium, phosphorus, calcium times phosphorus product, dry weight, or parathyroid hormone. Elevated levels of the enzyme correlated directly with muscle weakness in male patients and with hypothyroidism (depressed free thyroxin index) and inversely with treatment with vitamin D, supplements. We conclude that high CPK levels in uremia are secondary to skeletal muscle abnormalities and that hypothyroidism and vitamin D, deficiency may contribute to so-called uremic myopathy.

High fat diet-Induced obesity alters vitamin D metabolizing enzyme expression in mice.

Low serum 25(OH)D concentrations have been reported in obese humans. Inadequate sun exposure and impaired hepatic 25-hydroxylation have been suggested as possible reasons for obesity-associated vitamin D deficiency; however, the underlying mechanism has not been elucidated. We investigated the effects of high fat diet-induced obesity on vitamin D status and vitamin D metabolizing enzyme expression. Male C57BL mice (4 weeks old) were fed control diet containing 10% energy from fat (control group) or high fat diet containing 45% energy from fat (obese group) for 18 weeks. There were no differences in serum 25(OH)D concentrations between two groups, while serum 1,25(OH)2 D concentrations were significantly higher in obese mice. Hepatic mRNA levels of 25-hydroxylases (Cyp2r1, Cyp27a1, and Cyp2j3) were lower in the obese group (31, 30, and 48% lower, respectively). Renal 1α-hydroxylase (Cyp27b1) mRNA levels were higher and 24-hydroxylase (Cyp24) mRNA levels were lower in the obese group. Serum 1,25(OH)2 D concentrations correlated positively with renal Cyp27b1 expression levels and negatively with renal Cyp24 expression levels. Serum PTH concentrations were higher in obese mice. In visceral adipose tissue, Cyp27a1, Cyp2j3, and vitamin D receptor mRNA levels were higher in obese mice. Overall, vitamin D metabolizing enzyme expression was influenced by high fat diet-induced obesity, which might partly explain the mechanisms of the altered vitamin D endocrine system associated with obesity. Higher serum PTH and 1,25(OH)2 D concentrations in obese mice suggest abnormal regulation of serum 1,25(OH)2 D concentrations due to hyperparathyroidism, which might have contributed to lower hepatic 25-hydroxylase mRNA levels.

Human 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 subunit maps to a different chromosomal location than that of pseudovitamin D-deficient rickets.

We have cloned part of the human 25-OHD 24-hydroxylase cytochrome P450 (P450cc24) cDNA. The characterized sequence consists of 776 bp of the coding and 720 bp of the 3'-untranslated region interrupted by an intron. In the coding region we found 79.8% similarity in DNA and 87.5% in deduced amino acid sequences between human and rat, with no similarity in the 3'-untranslated region. By Southern blot hybridization of DNA from human-hamster somatic cell hybrids and by in situ immunofluorescence hybridization, we mapped P450cc24 to human chromosome 20q13.1. This location of P450cc24 is different from that of pseudovitamin D-deficient rickets (PDDR), previously assigned to chromosome 12q14 by linkage analysis, thus excluding it as a target of the PDDR mutation. Since it is likely that PDDR is caused by a mutation in the 25-OHD 1 alpha-hydroxylase P450 subunit (P450cc1 alpha) our results do not support the hypothesis that the two cytochromes are encoded by a single gene.

Characteristics of parathyroid hormone-specific cyclic changes of glucose-6-phosphate dehydrogenase activity in the distal convoluted tubule of the guinea pig.

The effect of parathyroid hormone (PTH) on the time course of glucose-6-phosphate dehydrogenase (G6PD) activity in the distal convoluted tubule of a vitamin D-depleted guinea pig was determined using quantitative cytochemistry. G6PD activity decreased to the stable basal level 5 hrs after the initiation of the kidney segment maintenance cultures. The exposure of the tissues to 1 pg/ml of bovine PTH-(1-84) induced a cyclic change of G6PD activity, whereas neither carboxyl-terminal PTH nor other hormones tested showed such activity. After a 16-min exposure to bovine PTH-(1-84), the peak height of each cycle began to decrease until it disappeared at 34 min. The second exposure to this hormone at 46 min reinduced a similar cyclic change with a similar peak, indicating full viability of the cells. When bovine PTH-(1-84) was incubated with an excess amount of anti-bovine PTH antibody, the PTH-induced G6PD activity was completely abolished. Throughout a 14-min exposure to either human PTH-(1-84), human PTH-(1-34) or bovine PTH-(1-84), similar cyclic changes were observed with the constant peak height regardless of the dose (10(-16)-10(-12) M), although the cycle length shortened progressively as the dose was increased. They were equipotent on a molar basis between the concentrations of 10(-16) and 10(-13) M at 6 min of hormone exposure. The present data demonstrate that the cytochemical bioassay of PTH in a vitamin D-depleted animal is based on a dose-dependent difference in the time course of G6PD activity.