Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

FGF23, alpha-Klotho and vitamin D mediated calcium-phosphate metabolism in haemodialysis patients

BackgroundKlotho is a prote˝in that acts as a co-receptor for FGF23. FGF23-Klotho axis has great importance regarding the regulation of mineral metabolism by kidneys. In this study, we analysed FGF23, Klotho, 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D, parathormone, Calcium and Phosphate levels of haemodialysis patients in order to investigate the nature of the mineral metabolism disruption in chronic kidney diseases.MethodsSixty haemodialysis patients and 34 healthy controls were included in the study. Serum iFGF, cFGF, and soluble Klotho were analysed using ELISA kits. Moreover, 1,25-dihydroxyvitamin D3 was determined using LCMS/MS. Calcium, phosphate, iPTH and 25-hydroxyvitamin D were measured using autoanalyzers.ResultsIn haemodialysis patients, iFGF23, cFGF23, iPTH and P levels were significantly higher, and 1,25-dihydroxyvitamin D3, Klotho and Ca levels were significantly lower compared with the control group. There was no significant difference in the 25-hydroxyvitamin D levels.ConclusionsOur study showed that lack of sufficient amounts of Klotho is crucial for mineral metabolism disruptions seen as a complication of chronic kidney diseases. Despite the high levels of the hormone, FGF23 is unable to accomplish its function properly, likely due to deteriorated kidney function in haemodialysis patients.     

Intestinal calcium absorption: Molecular vitamin D mediated mechanisms

Rickets and hyperparathyroidism caused by a defective Vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25-(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min after oral gavage in two strains of VDR-knock out (KO) mice (Leuven and Tokyo KO) and observed a threefold lower area under the curve in both KO-strains. Moreover, we evaluated the expression of intestinal candidate genes, belonging to a new class of calcium channels (TRPV), involved in transcellular calcium transport. The calcium transport protein ECaC2 was more abundantly expressed at mRNA level than ECaC1 in duodenum, but both were considerably reduced (ECaC2 > 90%, ECaC1 > 60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was only significantly decreased in the Tokyo KO, whereas PMCA(1b) expression was normal in both VDR-KOs. In Leuven wild type mice, a high calcium diet inhibited (> 90%), and 1,25(OH)(2)D(3) or low calcium diet induced (sixfold) duodenal ECaC2 expression and, to a lesser degree, ECaC1 and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas both ECaC mRNA expressions remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular ECaC2 or TRPV6) is strongly vitamin D dependent and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D dependent active calcium absorption.     

Effects of 1,25-dihydroxyvitamin D3 on the distribution of androgen and vitamin D receptors in human prostate neonatal epithelial cells

Although many studies have examined the mechanisms of 1,25-dihydroxyvitamin D(3) (calcitriol or 1,25 D) action in different prostate cancer cell lines, little is known regarding the influence of this steroid on the normal prostate. The presence of both VDR and AR in normal prostatic tissues raises the distinct possibility of an important role for this hormone in the normal gland. In order to ascertain the possible role of 1,25 D on both AR and VDR in the normal prostate, the effects of calcitriol and dihydrotestosterone (DHT) on the normal human neonatal prostatic epithelial cell line, 267B-1, were examined. These studies were approached by focusing on how 1,25 D in the presence or absence of DHT affects the distribution of AR and VDR in the cytoplasmic and nuclear compartments of the cells in terms of their protein levels and DNA binding activities. Immunoblot analyses show that 1,25 D increases the AR protein level in both the cytoplasmic and nuclear fractions but not the VDR protein level. On the other hand, the gel shift assays demonstrate that 1,25 D increases both the AR- and VDR-DNA binding activities in the nuclear fraction, whereas there is no increase in DNA binding activities in the cytoplasmic fraction. Addition of DHT along with 1,25 D does not affect the DNA binding activities of both AR and VDR. Overall, these studies suggest that 1,25 D actions on the normal prostate cells may be mediated independently through AR and VDR, respectively.     

Influence of ultraviolet B radiation on vitamin D3 metabolism in vitamin D3-resistant New World primates

We investigated the occurrence of rickets in adolescent tamarins (Saguinus imperator) residing at the Los Angeles Zoo. Compared to tamarins in the same colony without clinical evidence of bone disease (N = 6), rachitic platyrrhines (N = 3) had a decrease in their serum calcium concentration (P < .05). The affected tamarins also had lower serum 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) levels than did nonaffected colony mates, but 2–10-fold higher concentrations than in Old World primates of a comparable developmental stage. New World primates in many different genera are known to exhibit target organ resistance to the active vitamin D₃ metabolite, 1,25-(OH)₂D₃, compensated by maintenance of high circulating concentrations of 1,25-(OH)₂D₃. The relatively low serum 1,25-(OH)₂D₃ concentration in rachitic tamarins and ultraviolet B radiation deficient environment of these primates suggested that bone disease may be linked to a deficiency in substrate for 1,25-(OH)₂D₃, 25 hydroxyvtamin D₃ (25-OHD₃). Chronic exposure of platyrrhines in three different vitamin D resistant genera to an artificial UVB source resulted in 1) a significant increase in the mean serum 25-OHD₃ (P < .001) and 1,25-(OH)₂D₃ (P < .02) level over that encountered in platyrrhines not exposed to UVB; and 2) prevention of rachitic bone disease in irradiated individuals. These data further show that the serum 25-OHD₃ and 1,25-OH₂D₃ levels are positively correlated in vitamin D-resistant platyrrhines (r = 0.64; P= .0014) and suggest that a compromise in cutaneous vitamin D₃ production by means of UVB deprivation may limit necessary 1,25-(OH)₂D₃ production. © 1992 Wiley-Liss, Inc.     

The classic receptor for 1alpha,25-dihydroxy vitamin D3 is required for non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in osteosarcoma cells

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.     

Impact of Vitamin D Supplementation on Inflammatory Markers’ Levels in Obese Patients

In view of research suggesting a possible beneficial impact of vitamin D on systemic inflammatory response, the authors decided to investigate an influence of vitamin D supplementation on serum levels of certain inflammatory markers in obese patients. The current study included such biomarkers as interleukin-6 (IL-6), pituitary adenylate cyclase-activating peptide (PACAP), advanced oxidation protein products (AOPP), C-X3-C Motif Chemokine Ligand 1 (CX3CL1), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO). The measurements were performed with the ELISA method before and after 3-month-long supplementation of 2000 IU of vitamin D orally. The results showed that the therapy did not induce any statistically significant changes in serum levels of MCP-1, IL-6, CX3CL1, and PACAP. The supplementation was related to a significant increase in measurements of NO and AOPP levels, although the correlation analysis between vitamin D concentration after its supplementation and the concentration of the molecular parameters did not show significant relation. In conclusion, our study seems to contradict certain aspects of findings available in the literature regarding the vitamin D’s impact.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Seasonal variation and correlation analysis of vitamin D and parathyroid hormone in Hangzhou,Southeast China

This study aimed to describe the 25‐hydroxyvitamin D (25(OH)D) and parathyroid hormone (PTH) status of Southeast Chinese individuals influenced by season. The secondary aim was to determine the cutoff for sufficient 25(OH)D in a four‐season region. From January 2011 to June 2014, a total of 17 646 individuals were evaluated in our study. The serum levels of PTH were detected simultaneously in 5579 cases. A total of 25(OH)D and intact PTH were measured by the electrochemiluminescent immunoassay. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D and PTH were studied. The mean 25(OH)D concentration in our study was 43.00(30.40) nmol/L. The prevalence of insufficiency (25(OH)D < 50 nmol/L) was 62.87% and that of deficiency (<30 nmol/L) was 28.54%. Mean serum 25(OH)D levels revealed a limited sinusoidal profile throughout the year and were significantly higher in Autumn. On the other hand, PTH levels showed an opposite response to seasonal effects relative to 25(OH)D. Age, BMI and daylight were not significantly correlated with 25(OH)D and serum PTH reached a plateau at higher values of serum 25(OH)D of 42.86 nmol/L. This study demonstrated that Vitamin D insufficiency is highly prevalent in Southeast China. The concentration of 25(OH)D in the male group was generally higher than that in the female group. Seasonal variation was an important aspect of 25(OH)D and PTH concentration. This study revealed that the optimal serum threshold of 25(OH)D for bone health should be between 40 and 50 nmol/L for Southeast Chinese individuals.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

The association between vitamin D receptor expression and prolonged overall survival in breast cancer

In this study, we analyzed vitamin D receptor (VDR) expression and survival in a breast cancer patient cohort of 82 breast cancer patients. Immunohistochemical analysis was possible in 91.5% of the patients (75/82). Staining was evaluated using the semi-quantitative assay according to Remmele and Stegner (immunoreactivity score [IRS]). IRS 0-1 was negative/very low, IRS 2-4 was moderate to high, and IRS 6-12 was high. Statistical analysis was performed by Spearman's correlation test (p<0.05 significant). Overall survival was analyzed using Kaplan-Meier estimations. Only 6 patients had a negative IRS. Moderate IRS values were present in 20 patients. Most of the patients had a high IRS (49). For survival analysis, data were dichotomized (IRS 0-4: negative to moderate and IRS 6-12: high VDR expression). In univariate analysis, VDR expression showed significant differences in progression-free survival (PFS) and overall survival (OS). Patients with high IRS scores showed significantly better PFS and OS than patients with moderate/negative IRS scores for VDR expression. Tumor size was significantly correlated to PFS. When analyzed separately, the three different IRS groups showed significant differences in VDR expression. The present data suggest that VDR expression in breast cancer tissue may be of clinical significance, and the results provide evidence that VDR may be a factor with prognostic relevance.