Genetic variants in the vitamin D pathway and their association with vitamin D metabolite levels: Detailed studies of an inner-city pediatric population suggest a modest but significant effect in early childhood.

OBJECTIVES: In a large cohort of healthy infants and toddlers 6-36 months of age (n = 776), we have been exploring the potential role of genetic variation in predisposition to vitamin D insufficiency. The genes encoding the key cytochrome P450 hydroxylases (CYP2R1, CYP24A1, and CYP27B1) harbour recurrent mutations of uncertain effect. This study was undertaken to look for biochemically relevant associations of these variants with inter-individual differences in vitamin D metabolism in an at-risk pediatric population. METHODS: Genotyping for CYP2R1-CT (c.-1127 C>T, rs10741657), CYP24A1-AG (c.-686A>G, rs111622401), and CYP27B1-CA (c.-1261 C>A, rs10877012) mutations were performed using SNaPshot assay, followed by Sanger sequencing confirmation. Vitamin D metabolites and vitamin D binding protein (DBP) were measured by established methods. RESULTS: In a multivariate regression model, with corrections for co-variates, subjects with the homozygous CYP2R1-TT variant had significantly higher concentrations of 25(OH)D, free 25(OH)D, and 24,25(OH)2D levels. In subjects with the CYP24A1-AG mutation, concentrations of 25(OH)D were significantly higher. CONCLUSIONS: The CYP2R1-TT and CYP24A1-AG variants have measurable effects on the vitamin D pathway. It seems unlikely that they will be clinically relevant in isolation, but they may be members of the large pool of infrequent mutations contributing to different risks for the vitamin D deficiency phenotype.

Circulating vitamin D and breast cancer risk: an international pooling project of 17 cohorts.

Laboratory and animal research support a protective role for vitamin D in breast carcinogenesis, but epidemiologic studies have been inconclusive. To examine comprehensively the relationship of circulating 25-hydroxyvitamin D [25(OH)D] to subsequent breast cancer incidence, we harmonized and pooled participant-level data from 10 U.S. and 7 European prospective cohorts. Included were 10,484 invasive breast cancer cases and 12,953 matched controls. Median age (interdecile range) was 57 (42-68) years at blood collection and 63 (49-75) years at breast cancer diagnosis. Prediagnostic circulating 25(OH)D was either newly measured using a widely accepted immunoassay and laboratory or, if previously measured by the cohort, calibrated to this assay to permit using a common metric. Study-specific relative risks (RRs) for season-standardized 25(OH)D concentrations were estimated by conditional logistic regression and combined by random-effects models. Circulating 25(OH)D increased from a median of 22.6 nmol/L in consortium-wide decile 1 to 93.2 nmol/L in decile 10. Breast cancer risk in each decile was not statistically significantly different from risk in decile 5 in models adjusted for breast cancer risk factors, and no trend was apparent (P-trend = 0.64). Compared to women with sufficient 25(OH)D based on Institute of Medicine guidelines (50- < 62.5 nmol/L), RRs were not statistically significantly different at either low concentrations (< 20 nmol/L, 3% of controls) or high concentrations (100- < 125 nmol/L, 3% of controls; ≥ 125 nmol/L, 0.7% of controls). RR per 25 nmol/L increase in 25(OH)D was 0.99 [95% confidence intervaI (CI) 0.95-1.03]. Associations remained null across subgroups, including those defined by body mass index, physical activity, latitude, and season of blood collection. Although none of the associations by tumor characteristics reached statistical significance, suggestive inverse associations were seen for distant and triple negative tumors. Circulating 25(OH)D, comparably measured in 17 international cohorts and season-standardized, was not related to subsequent incidence of invasive breast cancer over a broad range in vitamin D status.

Correction: Vitamin D4 in Mushrooms.

[This corrects the article DOI: 10.1371/journal.pone.0040702.].

Role of glucuronidated 25-hydroxyvitamin D on colon gene expression in mice.

25-Hydroxyvitamin D3-3ß-glucuronic acid (25OHD-Gluc) is produced in the liver and is a constituent of human blood and bile. Bacterial glucuronidases (GUS) in mammalian digestive microbiota cleave glucuronide conjugates, such as 25OHD-Gluc, and release the free aglycone (i.e., 25OHD) inside the intestinal lumen. We hypothesized that 25OHD-Gluc would elicit a VDR-dependent mRNA response in the colon after cleavage by gut microbiota. The activity of 25OHD-Gluc was investigated by measuring expression of cytochrome P450 24A1 (Cyp24) mRNA both in vitro and in vivo. In cell culture, Caco2 cells responded to 25OHD-Gluc, whereas HT29 cells did not. When coincubated with GUS, both cell lines elicited a robust response as indicated by a 5 Ct (32-fold) increase in Cyp24 mRNA. In vitamin D-sufficient mice, we found that both oral and subcutaneous administration of 1 nmol 25OHD-Gluc induced expression of Cyp24 mRNA in the colon whereas 25OHD did not. In contrast, 25OHD, but not 25OHD-Gluc, was active in the duodenum. When the jejunum was surgically ligated to block flow of digesta to the colon, neither oral nor subcutaneous administration of 2 nmol 25OHD-Gluc was able to induce expression of Cyp24 in the colon. Our findings suggest that 25OHD-Gluc, a vitamin D metabolite found in bile, induces VDR-mediated responses in the colon by crossing the apical membrane of the colon epithelium.NEW & NOTEWORTHY We found that 25OHD-Gluc, an endogenously produced metabolite, is delivered to the colon via bile to induce vitamin D-mediated responses in the colon.

Oral 25-Hydroxycholecalciferol Acts as an Agonist in the Duodenum of Mice and as Modeled in Cultured Human HT-29 and Caco2 Cells.

BACKGROUND: 25-Hydroxycholecalciferol [25(OH)D] is the predominant circulating metabolite of vitamin D and serves as the precursor for 1α,25-dihydroxycholecalciferol [1,25(OH)2D], the hormonally active form. The presence of 1α-hydroxylase (1α-OHase) in the intestine suggests that 1,25(OH)2D can be produced from 25(OH)D, but the effects of oral 25(OH)D on the intestine have not been determined. OBJECTIVES: We investigated the acute intestinal response to orally consumed 25(OH)D in mice by assessing mRNA induction of cytochrome p450 family 24 subfamily A member 1 (Cyp24), a vitamin D-dependent gene. The mechanism of action then was determined through in vitro analyses with Caco2 and HT-29 cells. METHODS: Adult male C57BL6 mice were given a single oral dose of 40, 80, 200, or 400 ng 25(OH)D (n = 4 per dose) or vehicle (n = 3), and then killed 4 h later to evaluate the duodenal expression of Cyp24 mRNA by qPCR and RNA in situ hybridization. The 25(OH)D-mediated response was also evaluated with Caco2 and HT-29 cells by inhibition assay and dose-response analysis. A cytochrome p450 family 27 subfamily B member 1 (CYP27B1) knockdown of HT-29 was created to compare the dose-response parameters with wild-type HT-29 cells. RESULTS: Oral 25(OH)D induced expression of Cyp24 mRNA in the duodenum of mice with 80 ng 25(OH)D by 3.3 ± 0.8 ΔΔCt compared with controls (P < 0.05). In vitro, both Caco2 and HT-29 cells responded to 25(OH)D treatment with 200-fold and 175-fold greater effective concentration at 50% maximal response than 1,25(OH)2D, yet inhibition of 1α-OHase and knockdown of CYP27B1 had no effect on the responses. CONCLUSIONS: In mice, orally consumed 25(OH)D elicits a vitamin D-mediated response in the duodenum. In vitro assessments suggest that the response from 25(OH)D does not require activation by 1α-OHase and that 25(OH)D within the intestinal lumen acts as a vitamin D receptor agonist.

Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, ß-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.

Circulating Vitamin D and Colorectal Cancer Risk: An International Pooling Project of 17 Cohorts.

BACKGROUND: Experimental and epidemiological studies suggest a protective role for vitamin D in colorectal carcinogenesis, but evidence is inconclusive. Circulating 25-hydroxyvitamin D (25(OH)D) concentrations that minimize risk are unknown. Current Institute of Medicine (IOM) vitamin D guidance is based solely on bone health. METHODS: We pooled participant-level data from 17 cohorts, comprising 5706 colorectal cancer case participants and 7107 control participants with a wide range of circulating 25(OH)D concentrations. For 30.1% of participants, 25(OH)D was newly measured. Previously measured 25(OH)D was calibrated to the same assay to permit estimating risk by absolute concentrations. Study-specific relative risks (RRs) for prediagnostic season-standardized 25(OH)D concentrations were calculated using conditional logistic regression and pooled using random effects models. RESULTS: Compared with the lower range of sufficiency for bone health (50-<62.5 nmol/L), deficient 25(OH)D (<30 nmol/L) was associated with 31% higher colorectal cancer risk (RR = 1.31, 95% confidence interval [CI] = 1.05 to 1.62); 25(OH)D above sufficiency (75-<87.5 and 87.5-<100 nmol/L) was associated with 19% (RR = 0.81, 95% CI = 0.67 to 0.99) and 27% (RR = 0.73, 95% CI = 0.59 to 0.91) lower risk, respectively. At 25(OH)D of 100 nmol/L or greater, risk did not continue to decline and was not statistically significantly reduced (RR = 0.91, 95% CI = 0.67 to 1.24, 3.5% of control participants). Associations were minimally affected when adjusting for body mass index, physical activity, or other risk factors. For each 25 nmol/L increment in circulating 25(OH)D, colorectal cancer risk was 19% lower in women (RR = 0.81, 95% CI = 0.75 to 0.87) and 7% lower in men (RR = 0.93, 95% CI = 0.86 to 1.00) (two-sided Pheterogeneity by sex = .008). Associations were inverse in all subgroups, including colorectal subsite, geographic region, and season of blood collection. CONCLUSIONS: Higher circulating 25(OH)D was related to a statistically significant, substantially lower colorectal cancer risk in women and non-statistically significant lower risk in men. Optimal 25(OH)D concentrations for colorectal cancer risk reduction, 75-100 nmol/L, appear higher than current IOM recommendations.

Association of 25-Hydroxyvitamin D with Liver Cancer Incidence and Chronic Liver Disease Mortality in Finnish Male Smokers of the ATBC Study.

Background: Although circulating 25-hydroxyvitamin D [25(OH)D] concentrations were linked to liver cancer and chronic liver disease (CLD) in laboratory studies, few epidemiologic studies have addressed the associations.Methods: Within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, we measured 25(OH)D in baseline serum of 202 incident liver cancer cases and 225 CLD deaths that occurred during nearly 25 years of follow-up, and 427 controls. ORs and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. We examined predetermined clinically defined cut-points, and season-specific and season-standardized quartiles.Results: Low serum 25(OH)D concentrations were associated with higher risk of liver cancer (<25 nmol/L vs. ≥50 nmol/L: 1.98; 95% CI, 1.22-3.20; Ptrend across categories = 0.003) and CLD mortality (1.93; 95% CI, 1.23-3.03; Ptrend = 0.006) in models adjusted for age and date of blood draw. After additional adjustment for body mass index, diabetes, smoking, and other potential confounders, the association remained statistically significant for liver cancer (1.91; 95% CI, 1.16-3.15; Ptrend = 0.008), but was somewhat attenuated for CLD mortality (1.67; 95% CI, 1.02-2.75; Ptrend = 0.05). Associations were similar for analyses using season-specific and season-standardized quartiles, and after excluding participants with diabetes, or hepatitis B or C.Conclusions: Our results suggest a possible preventive role for vitamin D against liver cancer and CLD, although the importance of the liver for vitamin D metabolism and the lack of information about underlying liver disease makes reverse causality a concern.Impact: Future studies are needed to evaluate associations of vitamin D with liver cancer and liver disease in other populations, particularly those with a different constellation of risk factors. Cancer Epidemiol Biomarkers Prev; 27(9); 1075-82. ©2018 AACR.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.

Simplified 25-Hydroxyvitamin D Standardization and Optimization in Dried Blood Spots by LC-MS/MS.

Previous studies have assessed vitamin D status based on the 25-hydroxyvitamin D [25(OH)D] concentration measured in samples from dried blood spots (DBSs). In 40 individuals participating in a clinical study, we compared 25(OH)D levels measured from DBSs and in serum using an LC-MS/MS reference procedure in collaboration with the Vitamin D Standardization Program. The main objective was to simplify and optimize current methods to produce an assay that can be used as a screening tool for 25(OH)D concentration assessment without derivatization. The DBS 25(OH)D levels, compared to serum concentrations, were found to have 101% accuracy overall, and the correlation coefficient (r) was 0.83 (P < 0.0001), with a significant linear relationship. Free 25(OH)D and vitamin D binding protein (VDBP) were assessed in the serum samples for potential correlations to the DBS calculations: the levels of free 25(OH)D had moderate to strong correlation to DBS and serum concentrations, with r values of 0.67 (P < 0.0001) and 0.76 (P < 0.0001), respectively. VDBP and hematocrit had no significant correlation to either DBS or serum sample types, with r values <0.1. In conclusion, the use of two DBSs and an increase in DBS sample size improved overall sample representation without the need for derivatization, and produced an accurate and robust method that can be used to screen 25(OH)D levels.

Vitamin D mitigates the adverse effects of obesity on breast cancer in mice.

Obesity is an established risk factor for postmenopausal breast cancer (BCa), insulin resistance, and vitamin D deficiency, and all contribute to increased synthesis of mammary estrogens, the drivers of estrogen receptor-positive (ER+) BCa growth. As both dietary vitamin D and calcitriol treatments inhibit breast estrogen synthesis and signaling, we hypothesized that vitamin D would be especially beneficial in mitigating the adverse effects of obesity on ER+BCa. To assess whether obesity exerted adverse effects on BCa growth and whether vitamin D compounds could reduce these unfavorable effects, we employed a diet-induced obesity (DIO) model in ovariectomized C57BL/6 mice. Breast tumor cells originally from syngeneic Mmtv-Wnt1 transgenic mice were then implanted into the mammary fat pads of lean and obese mice. DIO accelerated the initiation and progression of the mammary tumors. Treatments with either calcitriol or dietary vitamin D reduced the adverse effects of obesity causing a delay in tumor appearance and inhibiting continued tumor growth. Beneficial actions of treatments with vitamin D or calcitriol on BCa and surrounding adipose tissue included repressed Esr1, aromatase, and Cox2 expression; decreased tumor-derived estrogen and PGE2; reduced expression of leptin receptors; and increased adiponectin receptors. We demonstrate that vitamin D treatments decreased insulin resistance, reduced leptin, and increased adiponectin signaling and also regulated the LKB1/AMPK pathway contributing to an overall decrease in local estrogen synthesis in the obese mice. We conclude that calcitriol and dietary vitamin D, acting by multiple interrelated pathways, mitigate obesity-enhanced BCa growth in a postmenopausal setting.

Androgens Attenuate Vitamin D Production Induced by UVB Irradiation of the Skin of Male Mice by an Enzymatic Mechanism.

Cutaneous exposure to UVB irradiation is an important source of vitamin D. Here, we examined sex-specific differences in cutaneous vitamin D production in mice. Both male and female mice on a vitamin D-deficient diet manifested vitamin D deficiency, with mineral abnormalities, secondary hyperparathyroidism, and osteomalacia. UVB irradiation significantly increased vitamin D levels in the skin of female mice and normalized serum 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 levels, as well as mineral and skeletal abnormalities. However, in male mice, the vitamin D response to UVB was attenuated and mineral and skeletal abnormalities were not normalized. The vitamin D precursor, 7-dehydrocholesterol (7DHC), was significantly lower in the skin of male than female mice. This reduction was due to local androgen action in the skin as demonstrated by castration studies and skin-specific androgen receptor deletion in male mice, both of which reversed the male phenotype. Local androgen regulation in the skin of the CYP11A1 gene, which encodes a crucial enzyme that metabolizes cholesterol, 7DHC, and vitamin D, appeared to contribute to the gender differences in UVB-induced vitamin D production and to its reversal of vitamin D deficiency. Sex-specific, enzymatically regulated differences in cutaneous production of vitamin D may therefore be of importance to ensure vitamin D sufficiency.

Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D.

The anticancer actions of vitamin D and its hormonally active form, calcitriol, have been extensively documented in clinical and preclinical studies. However, the mechanisms underlying these actions have not been completely elucidated. Here, we examined the effect of dietary vitamin D and calcitriol on mouse breast tumor-initiating cells (TICs, also known as cancer stem cells). We focused on MMTV-Wnt1 mammary tumors, for which markers for isolating TICs have previously been validated. We confirmed that these tumors expressed functional vitamin D receptors and estrogen receptors (ER) and exhibited calcitriol-induced molecular responses including ER downregulation. Following orthotopic implantation of MMTV-Wnt1 mammary tumor cells into mice, calcitriol injections or a vitamin D-supplemented diet caused a striking delay in tumor appearance and growth, whereas a vitamin D-deficient diet accelerated tumor appearance and growth. Calcitriol inhibited TIC tumor spheroid formation in a dose-dependent manner in primary cultures and inhibited TIC self-renewal in secondary passages. A combination of calcitriol and ionizing radiation inhibited spheroid formation more than either treatment alone. Further, calcitriol significantly decreased TIC frequency as evaluated by in vivo limiting dilution analyses. Calcitriol inhibition of TIC spheroid formation could be overcome by the overexpression of ß-catenin, suggesting that the inhibition of Wnt/ß-catenin pathway is an important mechanism mediating the TIC inhibitory activity of calcitriol in this tumor model. Our findings indicate that vitamin D compounds target breast TICs reducing tumor-initiating activity. Our data also suggest that combining vitamin D compounds with standard therapies may enhance anticancer activity and improve therapeutic outcomes.

Vitamin D-binding protein and pancreatic cancer: a nested case-control study.

BACKGROUND: Vitamin D-binding protein (DBP) is the primary carrier of 25-hydroxyvitamin D [25(OH)D] in the circulation. One prospective study in male smokers found a protective association between DBP and pancreatic cancer, particularly among men with higher 25(OH)D concentrations. OBJECTIVE: The objective was to examine the association between DBP and pancreatic cancer risk in an American population. DESIGN: We conducted a nested case-control study in the Prostate, Lung, Colorectal, and Ovarian Cancer screening trial cohort of men and women aged 55-74 y at baseline. Between 1993 and 2010, 295 incident pancreatic adenocarcinoma cases were reported (follow-up to 15.1 y). Two controls (n = 590) were matched to each case by age, race, sex, and month of blood draw. We calculated smoking- and diabetes-adjusted ORs and 95% CIs with the use of conditional logistic regression. RESULTS: DBP concentration was not significantly associated with pancreatic cancer overall [highest (≥7149.4 nmol/L) vs. lowest (<3670.4 nmol/L) quintile; OR: 1.75; 95% CI: 0.91, 3.37; P-trend = 0.25]. For serum 25(OH)D compared with the referent (50 to <75 nmol/L), individuals in the highest group had a significantly higher risk (≥100 nmol/L; OR: 3.23; 95% CI: 1.24, 8.44), whereas those in the lowest group had no significant association (<25 nmol/L; OR: 2.50; 95% CI: 0.92, 6.81). Further adjustment for DBP did not alter this association. CONCLUSION: Our results do not support the hypothesis that serum DBP or 25(OH)D plays a protective role in pancreatic cancer. This trial was registered at clinicaltrials.gov as NCT00339495.

Targeted delivery of 1,25-dihydroxyvitamin D3 to colon tissue and identification of a major 1,25-dihydroxyvitamin D3 glycoside from Solanumglaucophyllum plant leaves.

Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333µg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000µg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a ß-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and the 1,3-diglycoside is one of the major forms of 1,25D3 found in the leaf. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Serum 25-hydroxyvitamin D, vitamin D binding protein and risk of colorectal cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

The potential role of vitamin D in cancer prevention has generated substantial interest, and laboratory experiments indicate several anti-cancer properties for vitamin D compounds. Prospective studies of circulating 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, suggest an inverse association with colorectal cancer risk, but with some inconsistencies. Furthermore, the direct or indirect impact of the key transport protein, vitamin D binding protein (DBP), has not been examined. We conducted a prospective study of serum 25(OH)D and DBP concentrations and colorectal cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, based on 476 colorectal cancer cases and 476 controls, matched on age, sex, race and date of serum collection. All subjects underwent sigmoidoscopic screening at baseline and once during follow-up. Conditional logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Circulating 25(OH)D was inversely associated with colorectal cancer (OR = 0.60, 95% CI 0.38-0.94 for highest versus lowest quintile, p trend 0.01). Adjusting for recognized colorectal cancer risk factors and accounting for seasonal vitamin D variation did not alter the findings. Neither circulating DBP nor the 25(OH)D:DBP molar ratio, a proxy for free circulating 25(OH)D, was associated with risk (OR = 0.82, 95% CI 0.54-1.26, and OR = 0.79, 95% CI 0.52-1.21, respectively), and DBP did not modify the 25(OH)D association. The current study eliminated confounding by colorectal cancer screening behavior, and supports an association between higher vitamin D status and substantially lower colorectal cancer risk, but does not indicate a direct or modifying role for DBP.

Combination of oral vitamin D3 with photodynamic therapy enhances tumor cell death in a murine model of cutaneous squamous cell carcinoma.

Photodynamic therapy (PDT), in which 5-ALA (a precursor for protoporphyrin IX, PpIX) is administered prior to exposure to light, is a nonscarring treatment for skin cancers. However, for deep tumors, ALA-PDT is not always effective due to inadequate production of PpIX. We previously developed and reported a combination approach in which the active form of vitamin D3 (calcitriol) is given systemically prior to PDT to improve PpIX accumulation and to enhance PDT-induced tumor cell death; calcitriol, however, poses a risk of hypercalcemia. Here, we tested a possible strategy to circumvent the problem of hypercalcemia by substituting natural dietary vitamin D3 (cholecalciferol; D3 ) for calcitriol. Oral D3 supplementation (10 days of a 10-fold elevated D3 diet) enhanced PpIX levels 3- to 4-fold, and PDT-mediated cell death 20-fold, in subcutaneous A431 tumors. PpIX levels and cell viability in normal tissues were not affected. Hydroxylated metabolic forms of D3 were only modestly elevated in serum, indicating minimal hypercalcemic risk. These results show that brief oral administration of cholecalciferol can serve as a safe neoadjuvant to ALA-PDT. We suggest a clinical study, using oral vitamin D3 prior to PDT, should be considered to evaluate this promising new approach to treating human skin cancer.

Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

Serum 25(OH) vitamin D concentration changes after Roux-en-Y gastric bypass surgery.

OBJECTIVE: To describe serum 25(OH)D changes after Roux-en-Y gastric bypass (RYGB) and to determine if fat mass (FM) loss and vitamin D intake are associated with changes in serum levels. DESIGN AND METHODS: The relationship between serum 25(OH)D and 1) FM, 2) weight, 3) % excess weight loss (EWL), and 4) BMI was investigated after controlling for potential confounders using a mixed effects linear model in 20 women before and up to 1-year post-RYGB. Subcutaneous (SAT) and visceral adipose tissue (VAT) vitamin D concentrations at time of RYGB were also evaluated. RESULTS: Weight and FM decreased 1-year after surgery by 45 ± 1 kg and 37 ± 1 kg, respectively while 25(OH)D increased by 10 ± 2 ng mL(-1) . Weight, FM, BMI, and %EWL changes were associated with 25(OH)D change. VAT had an average 21% more vitamin D per gram than SAT and concentrations were highly correlated. CONCLUSIONS: Although weight loss may lead to increased serum 25(OH)D after RYGB, low levels remain a concern in some patients 1-year postsurgery. Additional research is needed to clarify the relationship between adipose storage of vitamin D and serum 25(OH)D in obesity, and how that relationship might change after surgery. This could lead to improved clinical management of vitamin D in this ever-growing clinical population.

Circulating levels of 25-hydroxyvitamin D and risk of breast cancer: a nested case-control study.

INTRODUCTION: Experimental evidence suggests a protective role for circulating 25-hydroxyvitamin D (25(OH)D) in breast cancer development, but the results of epidemiological studies have been inconsistent. METHODS: We conducted a case-control study nested within two prospective cohorts, the New York University Women's Health Study and the Northern Sweden Mammary Screening Cohort. Blood samples were collected at enrollment, and women were followed up for breast cancer ascertainment. In total, 1,585 incident breast cancer cases were individually-matched to 2,940 controls. Of these subjects, 678 cases and 1,208 controls contributed two repeat blood samples, at least one year apart. Circulating levels of 25(OH)D were measured, and multivariate odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. RESULTS: No association was observed between circulating levels of 25(OH)D and overall breast cancer risk (multivariate-adjusted model OR = 0.94, 95% CI = 0.76-1.16 for the highest vs. lowest quintile, ptrend = 0.30). The temporal reliability of 25(OH)D measured in repeat blood samples was high (intraclass correlation coefficients for season-adjusted 25(OH)D > 0.70). An inverse association between 25(OH)D levels and breast cancer risk was observed among women who were ≤ 45 years of age (ORQ5-Q1 = 0.48, 95% CI = 0.30-0.79, ptrend = 0.01) or premenopausal at enrollment (ORQ5-Q1 = 0.67, 95% CI = 0.48-0.92, ptrend = 0.03). CONCLUSIONS: Circulating 25(OH)D levels were not associated with breast cancer risk overall, although we could not exclude the possibility of a protective effect in younger women. Recommendations regarding vitamin D supplementation should be based on considerations other than breast cancer prevention.