CYP24A1 Activity Research Articles

In Vivo Contribution of Cyp24a1 Promoter Vitamin D Response Elements.

CYP24A1 is a multifunctional, P450 mitochondrial enzyme that catabolizes the vitamin D hormone (calcitriol, 1,25(OH)2D3), its precursor (calcifediol, 25(OH)D3), and numerous vitamin D metabolites. In the kidney, Cyp24a1 is induced by 1,25(OH)2D3 and fibroblast growth factor 23 (FGF23) and potently suppressed by PTH to control the circulating levels of 1,25(OH)2D3. Cyp24a1 is controlled by a pair of promoter proximal (PRO) vitamin D response elements (VDREs) that are aided by distal, downstream (DS) enhancers. The downstream 1 region of Cyp24a1 (DS1) enhancer is kidney-specific and responsible for PTH and FGF23 actions, and the downstream 2 region of Cyp24a1 enhancer responds to 1,25(OH)2D3 in all tissues. Despite this knowledge, in vivo contributions of the PRO VDREs to basal expression, FGF23 activation, and PTH suppression of Cyp24a1 remain unknown. In this study, we selectively mutated the PRO VDREs in the mouse to address these questions. We found mutation of the VDREs leads to a dramatic loss of VDR occupancy, a reduction of 1,25(OH)D3-induced kidney Cyp24a1 expression, and near elimination of intestinal Cyp24a1 induction. FGF23 induction of Cyp24a1 was reduced but not eliminated and still showed a synergistic increase with 1,25(OH)2D3. PTH suppression of Cyp24a1 was unchanged, despite minor reductions in total for phosphorylated cAMP-response element binding protein occupancy. Finally, VDR recruitment was dramatically reduced across the DS enhancers in the Cyp24a1 locus. Taken together, our data suggest a cooperative relationship between the DS and PRO enhancers in the regulation of Cyp24a1 by 1,25(OH)2D3 and FGF23 and points to the DS1 region as a crucial basal switch for Cyp24a1 activity that further defines the interconnected genomic control in vitamin D catabolism.

Vitamin D Metabolites Before and After Kidney Transplantation in Patients Who Are Anephric

Rationale & ObjectiveKidneys are vital for vitamin D metabolism and disruptions in both production and catabolism occur in chronic kidney disease. Although vitamin D activation occurs in numerous tissues, the kidneys are the most relevant source of circulating active vitamin D. This study investigates extrarenal vitamin D activation and the impact of kidney transplantation on vitamin D metabolism in patients who are anephric. Study DesignCase series. Setting & ParticipantsAdult patients with previous bilateral nephrectomy (anephric) not receiving active vitamin D therapy were evaluated at the time of (N=38) and 1 year after (N=25) kidney transplantation. Liquid chromatography–tandem mass spectrometry was used to measure vitamin D metabolites. Metabolic ratios were expressed CYP24A1 (24,25(OH)2D/25(OH)D) and CYP27B1 (1α,25(OH)2D/25(OH)D) as activities. Differences between evaluation timepoints were evaluated by paired Student’s t-test or Wilcoxon matched-pairs signed-rank test. FindingsAt time of transplantation, 1α,25(OH)2D was detectable in all patients (4 to 36 pg/mL). There was a linear relationship between 25(OH)D and 1α,25(OH)2D-levels (r=0.58, p<0.001) with 25(OH)D explaining 34% of the variation in 1α,25(OH)2D-levels. There were no associations between 1α,25(OH)2D and biointact PTH or FGF23. One year after transplantation, 1α,25(OH)2D levels recovered (+205%) and CYP27B1 activity increased (+352%). Measures of vitamin D catabolism, 24,25(OH)2D and CYP24A1 activity increased 3-5 fold. Also at 12 months after transplantation, 1α,25(OH)2D was positively correlated with PTH (rho=0.603, p=0.04), but not with levels of 25(OH)D or FGF23. LimitationsRetrospective, observational study design with a small cohort size. ConclusionsLow-normal levels of 1α,25(OH)2D was demonstrated in anephric patients, indicating production outside of the kidneys. This extrarenal CYP27B1 activity may be more substrate-driven than hormonally regulated. Kidney transplantation seems to restore kidney CYP27B1 and CYP24A1 activity, as evaluated by vitamin D metabolic ratios, resulting in both increased vitamin D production and catabolism. These findings may have implications for vitamin D supplementation strategies in the setting of kidney failure and transplantation.

The Vitamin D Metabolite Diagnostic Ratio Associates With Phenotypic Traits of Idiopathic Hypercalciuria

IntroductionUnderlying mechanisms for hypercalciuria remain unknown in most cases, hence the designation “idiopathic”. We hypothesized that the Vitamin D-inactivating enzyme CYP24A1 contributes to the pathogenesis of hypercalciuria in kidney stone formers. MethodsWe conducted association analyses between CYP24A1 activity, estimated by the Vitamin D metabolite diagnostic ratio (25(OH) Vitamin D3/total 24,25 (OH)2 Vitamin D ratio; VMDR), and the phenotype of participants in two observational cohorts of kidney stone formers, the Swiss Kidney Stone Cohort and the Bern Kidney Stone Registry. Circulating 25(OH)- and 24,25 (OH)2 Vitamin D were quantified using a validated LC-MS/MS assay. Results974 participants were included in the analysis. We found a positive association of VMDR (and hence negative association of CYP24A1 activity) with total (β 0.009 mmol/L; 95% CI 0.002, 0.016; p = 0.02) and ionized plasma calcium (β 0.005 mmol/L; 95% CI 0.002, 0.008; p < 0.01), absolute and fractional excretion of urinary calcium (β 0.054 mmol/24h; 95% CI 0.010, 0.097; p = 0.02 and β 0.046 %; 95% CI 0.018, 0.074; p < 0.01, respectively). Further, VMDR was associated with an increased likelihood of forming calcium oxalate dihydrate stones (Odds ratio 1.64; 95% CI 1.22, 2.35; p < 0.01) and reduced bone mineral density at the femoral neck (β -0.005 g/cm2; 95% CI -0.010, -0.001; p = 0.04). The described associations became stronger when the analysis was confined to idiopathic calcium stone formers. ConclusionOur study reveals that CYP24A1 activity, estimated by VMDR, is associated with clinical traits previously linked to idiopathic hypercalciuria.

CYP24A1 Activity Associates with Phenotypic Traits in Idiopathic Hypercalciuria

CYP24A1 Activity Associates with Phenotypic Traits in Idiopathic Hypercalciuria

#3325 EXTRARENAL CYP27B1 ACTIVITY CONTRIBUTES TO THE CIRCULATING POOL OF 1,25(OH)2D

Abstract Background and Aims Alterations of vitamin D metabolism are ubiquitous in patients with chronic kidney disease (CKD) and a key feature of the syndrome of CKD-mineral and bone disorder. A central paradigm in vitamin D metabolism is that the kidneys are the only relevant source of activation of vitamin D for systemic effects. However, various cells and tissues exhibit CYP27B1 activity and are capable of producing 1.25(OH)2D, which could contribute to the circulating pool [1]. This longitudinal, observational study challenges the dogma of the kidneys as the only source of 1.25(OH)2D, by investigating vitamin D metabolites in anephric patients free of active vitamin D therapy. Method Anephric patients free of active vitamin D therapy were included at time of kidney transplantation. A subset had a second study visit at 12 months post-transplant. Liquid chromatography–tandem mass spectrometry was used to measure 25(OH)D2/3, 1,25(OH)2D3 and 24,25(OH)2D3. Metabolic ratios of vitamin D metabolites over precursors were used as measures of CYP24A1 (24,25(OH) 2D3/ 25(OH)D3) and CYP27B1(1,25(OH)2D/ 25(OH)D3) activity. Biointact PTH (1-84) was measured by an in-house immunoradiometric assay [2] and FGF23 was measured by ELISA (Kainos Laboratories, Tokyo, Japan). Interleukin 6 (IL6), tumor necrosis factor-Α (TNF- Α) and interferon-Γ (IFN-Γ) were measured using an electrochemiluminescence multiplex immunoassay (Human Proinflammatory 1 V-plex, MSD, Maryland, US). Two independent laboratories performed confirmatory analyses of 1,25(OH)2D. Results All anephric patients (n = 38) had detectable (&amp;gt;2.5 pg/mL) levels of 1,25(OH)2D, with a median value of 13.5 pg/mL (range 3.9 to 35.7 pg/ml). There was a significant correlation between levels of 25(OH)D and 1,25(OH)2D (r = 0.583, p≪0.001), with 25(OH)D-levels explaining 34% of the variation in 1,25(OH)2D (Fig. 1). 1,25(OH)2D levels were not significantly correlated to PTH (rho = 0.279, p = 0.09) or FGF23 (rho = –0.280, p = 0.09), nor to any of the inflammatory markers; IL6 (rho = 0.068, p = 0.69), TNF-Α (rho = –0.264, p = 0.11), IFN-Γ (rho = –0.001, p = 0.99). To validate our findings, anonymized samples of 17 anephric patients and 17 CKD stage 5D with residual renal function were sent to two independent laboratories. The presence of 1,25(OH)2D was confirmed in all anephric patients. Twenty-five anephric patients underwent a successful kidney transplantation and were free of active vitamin D therapy at a study visit 12 months post-transplant. After kidney transplantation, 25(OH)D-level decreased, while levels of 1,25(OH)₂D and 24,25(OH) ₂D substantially increased. The metabolic ratios expressing CYP24A1 and CYP27B1 activity both increased (Fig. 2). At 12 months post-transplant, 1,25(OH)₂D-levels correlated with levels of biointact PTH (rho = 0.603, p = 0.002), but not with levels of FGF23 (rho = –0.262, p = 0.21). Conclusion Extrarenal CYP27B1 activity contributes to the circulating active vitamin D pool and may sustain low-normal levels of active vitamin D, even in anephric individuals. While renal CYP27B1 activity is hormonally regulated, extrarenal CYP27B1 activity seems to be mainly substrate dependent.

Race, Ancestry, and Vitamin D Metabolism: The Multi-Ethnic Study of Atherosclerosis.

A comprehensive characterization of racial/ethnic variations in vitamin D metabolism markers may improve our understanding of differences in bone and mineral homeostasis and the risk of vitamin D-related diseases. Describe racial/ethnic differences in vitamin D metabolism markers and their associations with genetic ancestry. In a cross-sectional study within the Multi-Ethnic Study of Atherosclerosis (MESA), we compared a comprehensive panel of vitamin D metabolism markers across self-reported racial/ethnic groups of Black (N = 1759), White (N = 2507), Chinese (N = 788), and Hispanic (N = 1411). We evaluated associations of proportion African and European ancestry with this panel of markers in Black and Hispanic participants using ancestry informative markers. Latent class analysis evaluated associations between patterns of vitamin D measurements with race/ethnicity. Compared with Black participants, White participants had significantly higher serum concentrations of 25-hydroxyvitamin D and fibroblast growth factor-23; lower concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D; circulating vitamin D metabolite ratios suggesting lower CYP27B1 and higher CYP24A1 activity; higher urinary concentrations of calcium and phosphorus with higher urinary fractional excretion of phosphorus; and differences in vitamin D binding globulin haplotypes. Higher percent European ancestry was associated with higher 25-hydroxyvitamin D and lower parathyroid hormone concentrations among Black and Hispanic participants. Latent classes defined by vitamin D measurements reflected these patterns and differed significantly by race/ethnicity and ancestry. Markers of vitamin D metabolism vary significantly by race/ethnicity, may serve to maintain bone and mineral homeostasis across ranges of 25-hydroxyvitamin D production, and be attributable, at least partly, to genetic ancestry.

Mammary-specific ablation of Cyp24a1 inhibits development, reduces proliferation and increases sensitivity to vitamin D

Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24 A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.

Compromised Activation of Vitamin D After Elective Surgery: A Prospective Pilot Study.

Surgical stress reduces concentrations of most proteins in serum and necessitates a rapid adjustment of hormones dependent on protein binding. Activation of vitamin D by renal 1α-hydroxylation is dependent on protein binding because 1,25-dihydroxyvitamin D (1,25(OH)2D3) is formed after megalin-mediated reabsorption of 25-hydroxyvitamin D (25OHD) bound to vitamin D binding protein (DBP). Postoperative alterations in serum concentrations of DBP and albumin may therefore impair 1,25(OH)2D3 production. Our objective was to determine sex-specific changes in serum concentrations of vitamin D metabolites and sex steroids 2, 6, 24, and 48 hours and 3 weeks postoperatively. Fourteen women and eleven men aged 45 to 77 years without severe comorbidities undergoing unilateral total knee arthroplasty participated in this prospective study in a tertiary center for arthroplasty (trial ID: NCT02336932). The main outcome measures were total and free serum concentrations of 25OHD, 1,25(OH)2D3, 24,25-dihydroxyvitamin-D, DBP, albumin, sex hormone binding globulin (SHBG), calcium, and parathyroid hormone (PTH). Serum albumin and SHBG decreased postoperatively (Δalbumin48h -18% [-22%; -14%]). Unexpectedly, concentrations of DBP and 25OHD remained unaltered, but 1,25(OH)2D3 declined postoperatively. 1,25(OH)2D3 was 3 weeks after surgery -24% (-40%; -8%) lower than preoperative levels, whereas 24,25-dihydroxyvitamin-D remained unchanged in postmenopausal women. The calculated conversion rate of 25OHD to 1,25(OH)2D3 was strongly associated with serum 25-OHD and PTH preoperatively, whereas serum calcium was most predictive postoperatively. In conclusion, surgery had no effect on serum concentrations of DBP, 25OHD, and PTH, whereas production of 1,25(OH)2D3 was markedly reduced. Further studies are needed to determine duration and putative outcome effects of this postoperative 1,25(OH)2D3 deficit in women, which in part may be due to discordance in CYP27B1 and CYP24A1 activity.

Metabolism of 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 by CYP24A1 and biological activity of its 24R-hydroxylated metabolite

Our previous study revealed that the 2α-substituted vitamin D analog 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exhibited a higher osteocalcin promoter transactivation activity in human osteosarcoma cells and a greater effect on bone mineral density in a rat model of osteoporosis than 1α,25(OH)2D3 without increasing blood calcium concentration. Thus, we hypothesized that AH-1 could be a promising therapeutic agent for osteoporosis without any serious side effects. In this study, we compared the CYP24A1-dependent metabolism of AH-1 with that of 1α,25(OH)2D3. The resistance to CYP24A1-dependent metabolism could be an important property of vitamin D analogs in prolonging their biological effects. A kinetic analysis was performed using a membrane fraction prepared from recombinant E. coli expressing human CYP24A1. The kcat/Km (μM−1 min−1) value for AH-1 was 31% of that for 1α,25(OH)2D3, suggesting that AH-1 is not as resistant to CYP24A1-dependent metabolism as the other C2-substituted vitamin D analogs such as eldecalcitol [2β-hydroxypropoxy-1α,25(OH)2D3]. The major metabolite of AH-1 was the 24R-hydroxylated metabolite, which had high vitamin D receptor (VDR) binding affinity and high HL-60 cell differentiation activity similar to AH-1 itself. In contrast, 1α,25(OH)2D3 was metabolized by multistep monooxygenation reactions, which led to the loss of affinity for VDR. Thus, the greater therapeutic effects of AH-1 than those of 1α,25(OH)2D3 in in vivo studies using osteoporosis rat models may be due to 24R-hydroxy-AH-1 whose VDR affinity was 91% of that of AH-1.

Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2 D3 in Affected Patients.

CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3 :24,25-(OH)2 D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2 D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2 D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2 D3 as a contaminant of the 24,25-(OH)2 D3 peak. In normals, the concentration of 24,25-(OH)2 D3 greatly exceeds 25,26-(OH)2 D3 ; however, 25,26-(OH)2 D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2 D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2 D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2 D3 from 25,26-(OH)2 D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2 D3 in IIH patients appears to be multifactorial. © 2017 American Society for Bone and Mineral Research.

Abstract P4-05-02: Conditional inactivation of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) in the mouse mammary epithelium alters mammary gland development

Abstract The biologically active form of vitamin D (1,25(OH)2D) regulates proliferation, differentiation, and apoptosis in diverse cell types. We have previously identified anti-proliferative activities of 1,25(OH)2D in human breast tissue, as well as 1,25(OH)2D target gene expression consistent with reports that elevated vitamin D levels may protect against cancer. In mouse studies, vitamin D signaling modulates normal mammary gland development, including ductal outgrowth and branching, and protects against tumorigenesis. Degradation of 1,25(OH)2D is initiated by the enzyme Cyp24a1 in target tissues, providing critical local control of 1,25(OH)2D bioactivity. In vitro, blockade of Cyp24a1 activity potentiates the anti-proliferative effects of 1,25(OH)2D. However, the extent to which endogenous Cyp24a1 activity within the mammary epithelium regulates local 1,25(OH)2D levels to modulate normal mammary gland development, with possible implications for cancer, has not been investigated. We generated a novel mouse model with conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium (MMTV-Cre x Cyp24a1lox/lox). Ablation of Cyp24a1 activity in the mammary epithelium does not alter either gland or body weight at 4, 6 or 10 weeks of age. Preliminary analyses of mammary gland whole mounts indicate that virgin knockout mice form fewer terminal end buds compared to glands from wild-type littermates at 4 and 6 weeks of age (P&amp;lt;0.05). Moreover, the width of the ducts proximal to the central lymph node of knockout mice was less than that of wild-type mice at 4 and 10 weeks of age (P&amp;lt;0.05 and P&amp;lt;0.01, respectively). In addition, the number of secondary and tertiary branching points is reduced in mammary glands from knockout mice at 6 weeks of age (P&amp;lt;0.05 and P&amp;lt;0.01, respectively). In summary, our findings suggest that Cyp24a1 activity within epithelial cells plays a crucial role to modulate postnatal mammary gland development, presumably by limiting the local accumulation of 1,25(OH)2D. Citation Format: Sheng L, Turner AG, Tarulli GA, Barratt K, Kremer R, Morris HA, Callen DF, Anderson PH. Conditional inactivation of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) in the mouse mammary epithelium alters mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-02.

CYP24A1-deficiency does not affect bone regeneration in distraction osteogenesis

The putative biological activity of 24,25-(OH)2D remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following fracture in chicks. Our laboratory has generated a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25-(OH)2D. We set out to study the role of CYP24A1 and 24,25-(OH)2D in intramembranous bone formation during distraction osteogenesis in wild-type and Cyp24a1-deficient mice. Distraction osteogenesis was applied to mouse tibiae using a miniature external fixator apparatus. Histomorphometric parameters and gene expression differences between the mutant mice and the wild-type controls were measured using micro computed tomography and reverse-transcription quantitative PCR.There were no statistically significant differences between genotypes when bone volume/tissue volume ratios were calculated at mid distraction, end of distraction, mid consolidation, or end of consolidation. We measured reduced expression of the Col10a1 gene in the mutant vs. wild-type mice at mid distraction (0.4±0.1 vs. 1.0±0.2 respectively, p=0.01). Similarly, we measured a significantly lower expression of the osteogenic marker Atf4 in mutant vs. wild-type mice at end of distraction (0.7±0.1 vs. 1.0±0.1 respectively, p=0.01) and at mid consolidation (0.6±0.1 vs. 1.0±0.1 respectively, p=0.0003). These results suggest moderate and restricted differences in chondrogenesis and osteogenesis that did not affect the volume of bone produced following distraction. We conclude that CYP24A1 activity is not essential for intramembranous bone formation.

Dynamics of Vitamin D Metabolism in Maternal-Fetal Dyads.

Metabolites of vitamin D in maternal-neonatal dyads remain relatively unexplored. The goal of this study was to evaluate concentrations of 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3 in maternal-infant pairs at delivery. Serum samples of maternal and infant cord blood were collected on 131 mother-infant pairs at delivery. Vitamin D metabolites were analyzed in triplicate using liquid chromatography-tandem mass spectrometry. Statistical analysis was conducted using the Fisher exact test, Wilcoxon rank sum test, and Spearman correlation coefficients. Mean 25(OH)D3 concentrations in maternal and cord blood were 32.9 and 18.5 ng/mL, respectively; mean maternal and cord 24,25(OH)2D3 were 2.0 versus 1.1 ng/mL, respectively. Absolute concentrations of 3-epi-25(OH)D3 were similar in maternal and cord samples (2.4 vs 2.2 ng/mL), whereas the proportion of the total 25(OH)D as the 3-epimer was 6.5% in maternal samples and 10.5% in cord samples. This suggests that the fetus contributes significantly to 3-epi-25(OH)D3 production. In contrast, the ratio of 25(OH)D3:24,25(OH)2D3 was identical in maternal and cord samples (18.5) suggesting equivalent CYP24A1 activity in mother and fetus. Maternal and cord metabolite levels were highly correlated (r = 0.78, 0.90, 0.89 for 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3, respectively, P = 0.001 for all). Serum concentrations of all metabolites were lower in nonwhite infants compared with white infants. Maternal and cord concentrations of 25(OH)D3 were positively associated with birth weight (r = 0.21, P = 0.02; r = 0.25, P = 0.003, respectively). This data suggests that although maternal and cord concentrations of vitamin D metabolites are highly correlated, regulation of specific vitamin D metabolites in the mother and the neonate may be mediated independently.

Pruning the ricket thicket.

Overexpression of FGF23 results in hypophosphatemic rickets, which is characterized by renal phosphate wasting, inappropriately low circulating levels of the active form of vitamin D, and skeletal abnormalities. The precise mechanisms of how excess FGF23 leads to hypophosphatemic rickets are not clear. In this issue of the JCI, Bai and colleagues demonstrate that deletion or inhibition of CYP24A1, which initiates degradation of the active form of vitamin D, ameliorates skeletal abnormalities in two mouse models of hypophosphatemic rickets. While this work supports an important role for excess CYP24A1 activity in the pathogenesis of FGF23-mediated hypophosphatemic rickets, more work will need to be done before CYP24A1 inhibition can be integrated into the management of patients living with these diseases.

LC-MS/MS for Identifying Patients with CYP24A1 Mutations.

Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%-15% across the analytical measurement range of 0.1-25 ng/mL (0.2-60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7-35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99-467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

Abstract 4655: Plant polyphenols inhibit cellular 24-hydroxylase (CYP24A1) expression and elevate serum 25-hydroxyvitamin D levels

Abstract 1α,25-dihydroxyvitamin D3 (1,25D) is known to regulate various cellular functions, including cell proliferation and differentiation. Both 1,25D and its precursor, 25-hydroxyvitamin D3 [25(OH)D], are catabolized by 24-hydroxylase encoded by the CYP24A1 gene, which is one of the most responsive vitamin D receptor (VDR) target genes, and a is candidate oncogene frequently overexpressed in various malignancies. Therefore, compounds which can inhibit CYP24A1 expression and/or activity, thereby reducing degradation of vitamin D derivatives, may potentiate their anticancer effects. We have previously shown that the plant polyphenols carnosic acid, curcumin and silibinin synergistically enhance differentiation of acute myeloid leukemia (AML) cells induced by near physiologic concentrations of 1,25D. This was associated with the transcription factor Nrf2-assisted elevation of both VDR and the retinoid X receptor alpha (RXRα) protein levels. Here, we demonstrate that despite the latter effect the polyphenols markedly inhibited 1,25D-induced CYP24A1 mRNA expression in AML cell lines but not in osteoblast-like cells. Furthermore, while potentiating 1,25D-induced transactivation of the direct repeat-3 (DR3)-type consensus vitamin D response element (VDRE) in AML cells, as measured by the luciferase (Luc) reporter gene assay, the polyphenols strongly inhibited 1,25D-dependent activation of the CYP24-Luc reporter construct containing an early (-298 bp) promoter region with two critical DR3-type VDREs (VDRE1 and VDRE2). In contrast, the polyphenols failed to inhibit and even enhanced 1,25D-induced transactivation of CYP24-Luc transfected into osteoblastic cells. Inactivating mutations of the VDRE1 or VDRE2 sequence in this construct did not abolish the inhibitory effect of polyphenols. However, overexpression of Nrf2 resulted in reduced CYP24-Luc activation by 1,25D, while dominant-negative Nrf2 had an opposite effect. Importantly, feeding of healthy Balb/c mice with standard rodent chow supplemented with carnosic acid-rich rosemary extract resulted in a marked dose- and time-dependent increase in serum levels of 25(OH)D, as compared to control animals. This was associated with a significant decrease in CYP24A1 mRNA levels in the kidneys of these mice. Collectively, the above data suggest that by downregulating CYP24A1 in a tissue-dependent manner, plant polyphenols may potentiate therapeutic and preventive effects of vitamin D derivatives. These findings may also contribute to our understanding of the beneficial effects of healthy diets. (Supported by the Israel Science Foundation grant 635/11 to M.D. and Y.S. and by the American Institute for Cancer Research grant #10A049 to G.P.S. and M.D.). Citation Format: Ehud Sharony, Ze'ev Barvish, Victoria Novik, Marina Khanin, Merav Cohen-Lahav, Doron Amichay, Yoav Sharoni, George P. Studzinski, Michael Danilenko. Plant polyphenols inhibit cellular 24-hydroxylase (CYP24A1) expression and elevate serum 25-hydroxyvitamin D levels. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2015-4655

CYP24A1 Mutations in a Cohort of Hypercalcemic Patients: Evidence for a Recessive Trait.

Loss-of-function mutations of CYP24A1 (which encodes the 25-OH-D3-24-hydroxylase) have recently been reported to cause hypercalcemia. The aims of this study were: 1) to evaluate the frequency of CYP24A1 mutations in patients with medical history of hypercalcemia; 2) to show the clinical utility of a simultaneous assay of serum 25-hydroxyvitamin D3 (25-OH-D3) and 24,25-dihydroxyvitamin D3 (24,25-[OH]2D3) by liquid chromatography tandem mass spectrometry (LC-MS/MS); and 3) to investigate biochemical parameters in heterozygous gene carriers with CYP24A1 mutations. We screened for CYP24A1 mutations in 72 patients with serum calcium levels > 2.6 mmol/L and PTH levels < 20 pg/mL and recruited 24 relatives after genetic counseling for subsequent investigations. Vitamin D metabolite concentrations were assessed in a subset of patients by LC-MS/MS and results expressed as a ratio (R) of 25-OH-D3:24,25-(OH)2D3. Twenty-five patients with hypercalcemia (35%) harbored CYP24A1 variations. Twenty (28%) had biallelic variations, mostly found in subjects with nephrocalcinosis or renal stones (19/20). Five patients, all neonates, were heterozygous, without renal disease. We describe 15 new variations leading to loss-of-function according to pathogenicity prediction programs, and we functionally characterized 5 of them in vitro. A dramatic increase of R, usually >80, was found in patients harboring biallelic mutations providing evidence in vivo for the loss of CYP24A1 activity. In contrast, R value remains <25 in patients without CYP24A1 mutations. Subjects carrying one mutant allele, hypercalcemic individuals, as well as gene-carrier relatives, had a detectable 24,25-(OH)2D3 level and R < 25, indicating normal 24-hydroxylase activity. CYP24A1 biallelic mutations are frequently found in patients presenting with hypercalcemia, low PTH, and renal disease. We confirm the accuracy and effectiveness of a novel blood test estimating the ratio between relevant vitamin D metabolites as a useful screening tool for CYP24A1 mutations. Haploinsufficiency is not associated with CYP24A1 deficiency.

Plasma 3‐epi‐25‐hydroxyvitamin D3 in Very Low Birth Weight Preterm Infants in Canada During the First Five Weeks After Birth

Vitamin D status during infancy is important for skeletal growth and development. Response to vitamin D3 supplementation is complicated by the presence of 3‐epi‐25‐hydroxyvitamin D3 (3‐epi‐25‐OH‐D3). We undertook a small prospective study to simultaneously examine changes in 25‐OH‐D3, 3‐epi‐25‐OH‐D3 and 24,25‐dihydroxyvitamin D3 (24,25‐(OH)2D3) in preterm infants over the first five weeks after birth. Nineteen infants from Manitoba, born before 32 weeks of gestation and &lt;1500 g, were included in the study. Plasma samples were obtained at the end of the first (wk1) and fifth (wk5) week after birth.Vitamin D metabolites were analyzed in triplicate using LC‐MS/MS. Differences over time were tested using ANOVA and Bonferroni post hoc test, p&lt;0.05. Mean gestational age at birth was 29.0 ± 1.5 weeks and birth weight 1144.5 ± 244.4 g. Total vitamin D intake increased over time (Table). Increases were observed in vitamin D status based on plasma 25‐OH‐D3 concentration over the first 5 weeks (wk1: 18.1 ± 7.1 vs wk5: 25.3 ± 10.6 ng/ml, p=0.02) as well as in 3‐epi‐25‐OH‐D3 (wk1: 5.1 ± 4.2 vs wk5: 20.0 ± 15.9 ng/ml, p&lt;0.0001). Over the same time‐period 24,25‐(OH)2D3 (wk1: 0.9 ± 0.5 vs wk5: 0.7 ± 0.5 ng/ml, p=0.13) was not significantly changed.This work showed that 3‐epi‐25‐OH‐D3 increased over the first 5 weeks in parallel with increases of 25‐OH‐D3 while 24,25‐(OH)2D3 was relatively stable suggesting the CYP24A1 activity does not readily mature during 5 weeks postnatally in prematurely born infants. Vitamin D intake from all sources (IU/day) Mean SD Min Max Week 1 202.8a 115.9 4.5 400.7 Week 2 189.3a 118.0 54.9 442.8 Week 3 250.8a 155.7 65.0 551.9 Week 4 366.2b 163.6 42.5 626.3 Week 5 489.3c 186.9 93.1 736.7 Week 6 515.9c 192.5 177.7 779.6 Means with different superscript letters are different, p&lt;0.05

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

CYP11A1 can hydroxylate vitamin D3 at carbons 17, 20, 22, and 23, producing a range of secosteroids which are biologically active with respect to their ability to inhibit proliferation and stimulate differentiation of various cell types, including cancer cells. As 1α-hydroxylation of the primary metabolite of CYP11A1 action, 20S-hydroxyvitamin D3 [20(OH)D3], greatly influences its properties, we examined the ability of both human and mouse CYP27B1 to 1α-hydroxylate six secosteroids generated by CYP11A1. Based on their kcat/Km values, all CYP11A1-derived metabolites are poor substrates for CYP27B1 from both species compared with 25-hydroxyvitamin D3. No hydroxylation of metabolites with a 17α-hydroxyl group was observed. 17α,20-Dihydroxyvitamin D3 acted as an inhibitor on human CYP27B1 but not the mouse enzyme. We also tested CYP27B1 activity on 20,24-, 20,25-, and 20,26-dihydroxyvitamin D3, which are products of CYP24A1 or CYP27A1 activity on 20(OH)D3. All three compounds were metabolized with higher catalytic efficiency (kcat/Km) by both mouse and human CYP27B1 than 25-hydroxyvitamin D3. CYP27B1 action on these new dihydroxy derivatives was confirmed to be 1α-hydroxylation by mass spectrometry and nuclear magnetic resonance analyses. Both 1,20,25- and 1,20,26- trihydroxyvitamin D3 were tested for their ability to inhibit melanoma (SKMEL-188) colony formation, and were significantly more active than 20(OH)D3. This study shows that CYP11A1-derived secosteroids are 1α-hydroxylated by both human and mouse CYP27B1 with low catalytic efficiency, and that the presence of a 17α-hydroxyl group completely blocks 1α-hydroxylation. In contrast, the secondary metabolites produced by subsequent hydroxylation of 20(OH)D3 at C24, C25, or C26 are very good substrates for CYP27B1.

Enhanced induction of Cyp24a1 by FGF23 but low serum 24,25-dihydroxyvitamin D in CKD: implications for therapy

In normal people, induction by FGF23 of renal Cyp24a1, the enzyme that degrades 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, helps to protect from the disorders caused by excessive 1,25-dihydroxyvitamin D. Dai and co-workers report, however, that in human and mouse kidney disease, high FGF23 concurs with low rather than high serum 24,25-dihydroxyvitamin D, a biomarker of Cyp24a1 activity. Their characterization of the underlying mechanisms provides new understanding of how kidney disease impairs the health benefits of vitamin D-FGF23/klotho interactions.