Extrarenal synthesis of 1,25-dihydroxyvitamin D: sensitivity to glucocorticoid treatment

The response of circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] to challenge with vitamin D treatment both before and after 7-10 days of prednisone therapy (25 mg/day) was investigated in five anephric subjects, six patients with chronic renal failure (CRF), two patients with vitamin D intoxication and four patients with hypoparathyroidism. In anephric subjects serum 25-hydroxyvitamin D [25-(OH)D] rose from 58 +/- 48 (SD) to 377 +/- 221 (SD) nmol/l after administration of 150 micrograms of 25-(OH)D3 for 1 month. Serum 1,25-(OH)2D, which was barely detectable in only two out of five patients under basal conditions, rose to 30 +/- 21 pmol/l after 2 weeks of therapy with 25-(OH)D3, but fell to 10 +/- 5 pmol/l during prednisone treatment. In CRF patients circulating 1,25-(OH)2D rose from 37 +/- 24 to 58 +/- 24 pmol/l during 25-(OH)D3 therapy, but fell to 41 +/- 31 pmol/l during prednisone treatment. In two patients with rheumatoid arthritis, hypercalcaemia due to vitamin D intoxication was associated with raised levels of 1,25-(OH)2D (288 and 317 pmol/l). Administration of prednisone resulted in suppression of 1,25-(OH)2D levels (132 and 96 pmol/l respectively) and reduction of serum calcium to within the normal range. In the hypoparathyroid patients prednisone therapy did not affect circulating 25-(OH)D levels but serum 1,25-(OH)2D fell from 192 +/- 42 to 117 +/- 23 pmol/l and serum calcium from 2.41 +/- 0.21 to 2.20 +/- 0.05 mmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic 1,25-dihydroxyvitamin D3 administration in the rat reduces the serum concentration of 25-hydroxyvitamin D by increasing metabolic clearance rate.

Administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can lower the serum concentration of 25-hydroxyvitamin (25-OH-D). To determine if 1,25(OH)2D3 lowers serum 25-OH-D by increasing clearance or reducing production, we directly measured the metabolic clearance rate (MCR) of 25-OH-D in rats chronically infused with 1,25(OH)2D3. Chronic 1,25(OH)2D3 administration (0 to 75 pmol/d) reduced, in a time- and dose-dependent fashion, the serum concentrations of 25-OH-D3 and 24,25(OH)2D3 from 18 +/- 2 to 9 +/- 1 ng/ml and from 4.8 +/- 0.7 to 1.3 +/- 0.3 ng/ml, respectively, and increased sevenfold the in vitro conversion of 25-OH-D to 24,25(OH)2D3 by kidney homogenates. The reduction in serum 25-OH-D3 was completely accounted for by an increase in MCR. No change in production occurred. The influence of 1,25(OH)2D3 on serum 25-OH-D3 and 24,25(OH)2D3 was shown not to be dependent on induction of hypercalcemia. These data suggest that chronic 1,25(OH)2D3 administration lowers serum 25-OH-D by increasing the metabolic clearance of 25-OH-D3 and not by decreasing its production.     

Size at birth, adult intestinal calcium absorption and 1,25(OH)(2) vitamin D

BACKGROUND: Adult bone mineral status is modified by early environmental influences, but the mechanism of this phenomenon is unknown. Intestinal calcium absorption and vitamin D metabolism are integrally involved in bone metabolism and may be programmed during early life. AIM: To examine the early-life influences on calcium absorption and its control in 322 post-menopausal female twins. METHODS: Intestinal calcium absorption was assessed by the stable strontium (Sr) method. Serum PTH, 25(OH) and 1,25(OH)(2) vitamin D were measured and recalled birth weight recorded. RESULTS: Fractional intestinal Sr absorption (alpha Sr) was correlated with serum 1,25(OH)(2) vitamin D (p<0.001), but not with 25(OH) vitamin D. Birth weight was inversely associated with serum 1,25(OH)(2) vitamin D (p=0.04), the association being independent of serum calcium, phosphate, creatinine and PTH. Birth weight was inversely correlated with alpha Sr (p=0.03), this association being independent of age, season, customary calcium intake and serum 25(OH) vitamin D; however, when serum 1,25(OH)(2) vitamin D was added into the model, the association became non-significant, suggesting that the association was partially mediated via serum 1,25(OH)(2) vitamin D. DISCUSSION: We found a significant inverse association between birth weight and intestinal calcium absorption that is partially explained by an association between serum 1,25(OH)(2) vitamin D and birth weight. This suggests a mechanism whereby the intra-uterine environment might affect adult skeletal status.     

Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts.

1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. In conclusion: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.     

A sensitive and simplified radioimmunoassay for 1,25-dihydroxyvitamin D3

A sensitive radioimmunoassay system for 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] with an improved extraction procedure has been developed. Following one-step extraction and prepurification of 1,25(OH)2D3 by 'Extrelut-1' minicolumns final purification was achieved by high-performance liquid chromatography (HPLC) using a radial compression separation system equipped with a mu Porasil cartridge. The HPLC method applied allows the purification of 4 extracts/h. Recovery of 1,25(OH)2[3H]D3 after HPLC was 77 +/- 2.6% (mean +/- SD, n = 51). Since the recovery of 1,25(OH)2[3H]D3 was very reproducible, addition of labelled steroid to each single serum sample for monitoring recovery was omitted. The sensitivity of the assay was 0.8 pg/tube resulting in a detection limit of 3 ng/l, when 1 ml of serum was extracted. Intra-assay and inter-assay coefficients of variation were 12% and 16.8%, respectively. Serum 1,25(OH)2D3 concentration in 30 normal subjects (mean age: 25 yr) was 55 +/- 12 ng/l (mean +/- SD). In 55 elderly patients (mean age: 77 yr) the 1,25(OH)2D3 serum level was 32 +/- 12 ng/l (mean +/- SD) and in three patients with chronic renal failure on 1,25(OH)2D3 therapy 146 +/- 67 ng/l (mean +/- SD). Patients with chronic renal failure had reduced 1,25(OH)2D3 serum levels (mean 5.4 ng/l, range less than 3-11 ng/l, n = 10). In one patient with renal failure, following kidney transplantation the serum 1,25(OH)2D3 and creatinine levels were monitored from the 4th to the 12th post-surgical day: a highly significant negative correlation (r = 0.85) was found.     

Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man

Previous in vitro studies in rachitic rat liver suggested that 1,25-dihydroxyvitamin D inhibits the hepatic production of 25-hydroxyvitamin D (25-OHD). An investigation therefore was carried out in eight normal subjects to determine whether concomitant administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD, from 26.3 +/- 2.9 to 66.7 +/- 12.6 ng/ml (P less than 0.01). In contrast, 1,25(OH)2D3, 2 micrograms/d for 4 d, completely prevented an increase in serum 25-OHD in response to the same dose of vitamin D in the same individuals (25.1 +/- 2.2 vs. 27.4 +/- 5.3 ng/ml, NS). In a post-control study in seven of the normal subjects, vitamin D again significantly increased mean serum 25-OHD, from 18.2 +/- 3.1 to 42.8 +/- 4.7 ng/ml (P less than 0.001). In each of the three studies, mean serum calcium, phosphorus, and creatinine did not change and remained within the normal range. Whereas mean urinary calcium did not change in response to vitamin D alone during the 4 d of the two control studies, it increased significantly in the study in which vitamin D and 1,25(OH)2D3 were given together. A dose-response inhibition of the response of serum 25-OHD to vitamin D by 1,25(OH)2D3 was demonstrated in two of the normal subjects. The results provide evidence that 1,25(OH)2D3 inhibits the hepatic synthesis of its precursor 25-OHD in man.     

25-羟维生素D、脂蛋白相关磷脂酶A2与糖尿病视网膜病变的相关性:单中心回顾性研究

  目的  探讨25-羟维生素D[25-hydroxy vitamin D, 25(OH)D]、脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2, LP-PLA2)与糖尿病视网膜病变(diabetic retinopathy, DR)的相关性。  方法  回顾性收集并分析2014年5月至2017年1月沧州市中心医院内分泌科收治的2型糖尿病患者的临床资料,根据眼底摄片结果,分为糖尿病不伴视网膜病变组(no DR, NDR)组,背景期糖尿病伴视网膜病变(background DR, BDR)组和增殖期糖尿病伴视网膜病变(proliferative DR, PDR)组,选取同期于本院进行体检的健康人作为对照组。比较4组相关生化指标水平,采用Pearson相关分析及多元Logistic回归分析DR的相关及独立危险因素。  结果  共340例符合纳入和排除标准的2型糖尿病患者入选本研究,其中NDR组125例、BDR组118例、PDR组97例, 对照组100例。4组间的性别、年龄、体质量指数、收缩压、舒张压、总胆固醇、甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、空腹血糖差异无统计学意义(P均＞0.05);NDR组、BDR组、PDR组的病程逐渐增长(P=0.003),NDR组、BDR组、PDR组糖化血红蛋白A1C(glycated hemoglobin A1c, HbA1c)、糖化白蛋白(glycated albumin, GA)、血清胱抑素C(serum cystatin C, Cys-C)、LP-PLA2均显著高于健康对照组, 25(OH)D显著低于健康对照组(P均＜0.05);NDR、BDR、PDR组间两两比较,HbA1c、GA、Cys-C、LP-PLA2、25(OH)D差异亦有统计学意义(P均＜0.05)。Pearson相关分析显示,病程、HbA1c、GA、Cys-C及LP-PLA2与DR呈正相关(P均=0.000),25(OH)D与DR呈负相关(P=0.000)。多元Logistic回归分析显示,病程、HbA1c、Cys-C、LP-PLA2、25(OH)D与DR及其严重程度均独立相关(P均＜0.05)。  结论  25(OH)D 、LP-PLA2水平与DR的发生、发展密切相关,25(OH)D是其保护因素,而LP-PLA2是其危险因素。     

Vitamin D and vitamin-D-binding protein kinetics in patients treated with continuous ambulatory peritoneal dialysis (CAPD)

Serum and dialysate levels of 25-hydroxycholecalciferol (25-OHD3), 1,25 dihydroxycholecalciferol (1,25-(OH)2D3), and vitamin-D-binding protein (DBP) were measured in 14 patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Serum levels of 25-OHD3 and DBP were within normal range (29.1 +/- 22.9 nmol/L and 5.9 +/- 1.1 mumol/L, respectively). Serum levels of 1,25-(OH)2D3 were subnormal in all (less than 16 pmol/L) but one. In 5 patients, dialysate concentrations of 25-OHD3 were 2.3 +/- 0.9 nmol/L, the rest had levels less than 1.0 nmol/L. Small quantities of 1,25-(OH)2D3 were found in the dialysate effluents. DBP could be detected in the dialysate in all patients (0.24 +/- 0.06 mumol/L). Mass transfer (MT) of 25-OHD3 and DBP were respectively -10.4 +/- 8.3 nmol/24 h and -1.46 +/- 0.46 0.46 mumol/24 h. Peritoneal clearances of 25-OHD3 and DBP were low (0.40 +/- 0.37 mL/min and 0.18 +/- 0.06 mL/min, respectively. We conclude that CAPD leads to losses of 25-OHD3 and DBP. However, the peritoneal loss of DBP is well compensated and does not result in serum deficiency. Serum 25-OHD3 levels did not correlate with time on CAPD.     

Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat.

This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.     

Influence of calcium or 1,25-dihydroxyvitamin D3 supplementation on the hepatic microsomal and in vivo metabolism of vitamin D3 in vitamin D-depleted rats.

Hypocalcemic vitamin D (D)-depleted rats were supplemented with calcium or 1,25(OH)2D3, and the metabolism of D3 to 25(OH)D3 was studied. Infusion with 7 or 65 pmol 1,25(OH)2D3 X 24 h-1 led to normal or slight hypercalcemia associated with physiological and supraphysiological plasma concentrations of the hormone while calcium supplementation normalized plasma calcium despite 1,25(OH)2D3 concentrations as low as those observed in hypocalcemic controls. Constant administrations of [14C]D3 during the supplementation regimens uncovered a stimulation of the in vivo 25(OH)D3 production by calcium supplementation; this was further confirmed in vitro by an increase in the hepatic microsomal D3-25 hydroxylase. The group supplemented with pharmacological doses of the hormone displayed lower circulating concentrations of both D3 and 25(OH)D3 while the in vitro 25(OH)D3 production remained unaffected by 1,25(OH)2D3. Investigation of the kinetics of intravenous 25(OH)[3H]D3 revealed similar elimination constants in all groups. The data indicate that calcium supplementation of hypocalcemic D-depleted rats results in an increased transformation of D3 into 25(OH)D3 while supplementation with 1,25(OH)2D3 does not affect the in vitro D3-25 hydroxylase but seems to influence the in vivo handling of the vitamin by accelerating its metabolism.     

Assessment of vitamin D status in male osteoporosis

BACKGROUND: Clinical assessment of vitamin D status often relies on measuring total circulating 25-hydroxyvitamin D3 (25OHD3), but much of each vitamin D metabolite is bound to plasma vitamin D-binding protein (DBP), such that the percentage of free vitamin is very low. We hypothesized that measurement of free rather than total 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 25OHD3 may provide better assessment of vitamin D status. We therefore aimed to assess vitamin D status in men with idiopathic osteoporosis, in whom possible secondary causes of osteoporosis had been excluded, and to determine the extent of change in biologically active "free" vitamin D caused by variation in plasma DBP concentrations. METHODS: We measured 1,25(OH)2D3 and 25OHD3 in plasma samples from 56 men with idiopathic osteoporosis [mean (SD) age, 59.6 (13.6) years; range, 21-86 years] and 114 male controls [62.4 (10.4) years; range, 44-82 years]. RESULTS: Mean total plasma 25OHD3 in the 56 men with osteoporosis and the 114 controls was 44.7 (21) and 43.3 (17) nmol/L, respectively; total plasma 1,25(OH)2D3 measured in randomly selected men with osteoporosis (n = 50) and controls (n = 50) was 90 (37) and 103 (39) pmol/L, respectively. Mean plasma DBP was significantly higher (P <0.001) in men with osteoporosis [224 (62) mg/L; n = 56] than in the controls [143 (34) mg/L; n = 114], but calculated free plasma 25OHD3 and 1,25(OH)2D3 were significantly lower in the osteoporotic men than in controls [6.1 (3.1) vs 9.1 (4.4) pmol/L (P <0.00001) and 77 (37) vs 142 (58) fmol/L (P <0.00001), respectively]. CONCLUSIONS: Measurement of total vitamin D metabolites alone, although providing a crude assessment of vitamin D status, may not give an accurate indication of the free (biologically active) form of the vitamin. The ratio of total 25OHD3 and 1,25(OH)2D3 to plasma DBP, rather than total circulating vitamin D metabolites, may provide a more useful index of biological activity. Further studies are required to substantiate this hypothesis.     

Prevalence of 25(OH) Vitamin D Insufficiency and Deficiency in Pediatric Patients on Chronic Dialysis

♦ Background: 25(OH) Vitamin D [25(OH)D] is the major circulating form of vitamin D and the parameter used to reflect vitamin D status. Patients with chronic kidney disease (CKD) are likely to have low levels of 25(OH)D, and recent observations have linked suboptimal vitamin D status with adverse cardiovascular outcomes, inflammation, insulin resistance, and the rate of progression of renal insufficiency. Little is known about the magnitude of vitamin D deficiency in pediatric patients with stage 5 CKD on chronic dialysis.♦ Objectives: The aim of the present cross-sectional study was to assess the prevalence of abnormal vitamin D status in children on chronic dialysis.♦ Methods: Serum 25(OH)D, 1,25(OH)2 vitamin D [1,25(OH)2D], calcium, phosphorus, and parathyroid hormone (PTH) were evaluated in 59 pediatric patients on chronic dialysis. Weekly renal Kt/V and creatinine clearance (CCr) were evaluated as parameters reflecting residual renal function. In these patients, serum 25(OH)D concentrations less than 10 ng/mL were considered deficiency and concentrations of 10 - 30 ng/mL were considered insufficiency.♦ Results: Of the 59 pediatric patients (mean age: 14.4 ± 5.1 years), 51 (86.4%) were on peritoneal dialysis (PD), and 8 (13.6%) were on hemodialysis. Vitamin D deficiency was found in 32.2% of the patients (n = 19), and vitamin D insufficiency, in 50.8% (n = 30). Patients with serum 25(OH)D concentrations less than 30 ng/mL were older than those with normal 25(OH)D concentrations (15.4 ± 4.5 years vs 9.2 ± 5.1 years, p = 0.000). Patients with 25(OH) D concentrations less than 30 ng/mL had higher PTH levels than did those with normal 25(OH)D concentrations (349.5 ± 318.3 pg/mL vs 142.5 ± 116.9 pg/mL, p = 0.001). In the univariate analysis, there was no correlation between serum 25(OH)D and serum 1,25(OH)2D (r = 0.242, p = 0.064), calcium (r = 0.108, p = 0.415), phosphorus (r = -0.050, p = 0.706), or body mass index (r = -0.046, p = 0.729). In PD patients, serum 25(OH)D was positively correlated with weekly renal Kt/V (r = 0.385, p = 0.005) and CCr (r = 0.443, p = 0.001). In addition, serum 25(OH)D and serum albumin were positively correlated (r = 0.297, p = 0.035) in the PD patients.♦ Conclusions: The present study found a high prevalence of 25(OH)D deficiency and insufficiency in children on chronic dialysis. Serum 25(OH)D was associated with residual renal function in children on PD. Further studies to evaluate the consequences of vitamin D deficiency and the impact of therapeutic interventions are needed in pediatric CKD patients.Key words: Chronic kidney disease, 25(OH) vitamin D deficiency, chronic dialysis, residual renal functionVitamin D plays a central role in skeletal development and has a protective effect against hypertension, cardiovascular morbidity, diabetes mellitus, and cancer (1,2). Vitamin D is sourced from the diet or synthesized in the skin by ultraviolet B sunlight, metabolized in the liver to 25(OH) vitamin D [25(OH)D (calcidiol)], and then in the kidney to the biologically active 1,25(OH)2 vitamin D form [1,25(OH)2D (calcitriol)] under the control of parathyroid hormone (PTH) and fibroblast growth factor (FGF) 23 (3,4). In a dual effect, FGF23 reduces circulating 1,25(OH)2D by suppressing production of Cyp27b1 and stimulating Cyp24 catabolism of 1,25(OH)2D (5). Although 1,25(OH)2D is considered the biologically active form of vitamin D, 25(OH)D is the major circulating form, which is used as the parameter reflecting vitamin D status (4-6). It is known that 25(OH)D activates the vitamin D receptor and circulates in human plasma at approximately 1000 times the concentration of 1,25(OH)2D (7). In addition, because many tissues—including colon, prostate, skin, macrophages, and parathyroid—are recognized to express 1-α-hydroxylase, normal concentrations of 25(OH)D are important for local synthesis of 1,25(OH)2D in those tissues (8,9).Vitamin D inadequacy results from reduced sun exposure and dietary deficiency, and it is believed to be an epidemic of worldwide proportions in all age groups (10). Currently, for children, severe vitamin D deficiency is defined as a serum 25(OH)D concentration of 5 ng/mL or less, which is associated with an increased risk of rickets and myopathy (11). It has been recommended that a serum 25(OH)D concentration of 15 ng/mL or less be considered a state of vitamin D deficiency. Vitamin D insufficiency is usually defined as a 25(OH)D concentration of 15 - 20 ng/mL, which is associated with osteomalacia (11-13). Although a serum concentration of 25(OH)D greater than 20 ng/mL is considered indicative of vitamin D sufficiency in children, adult data indicate that a level of 32 ng/mL is desirable (11).Patients with chronic kidney disease (CKD), including dialysis patients, are likely to have low levels of 25(OH) D (14-21). The prevalence of vitamin D insufficiency or deficiency in pediatric CKD patients before dialysis ranges from 60% to 82.1% (15,17). The prevalence of 25(OH)D deficiency is known to be higher in patients on peritoneal dialysis (PD) than in those on hemodialysis (HD) because vitamin D binding protein is lost in peritoneal effluent (20,21). Other studies have shown that the prevalence of 25(OH)D deficiency or insufficiency at the time of renal transplant in adults is 88% (22). In CKD patients, a serum 25(OH)D concentration of less than 30 ng/mL is usually used as the definition of vitamin D deficiency or insufficiency (14-17). In the present study, a serum 25(OH)D concentration of less than 10 ng/mL was considered to represent deficiency and a concentration of 10 - 30 ng/mL was considered to represent insufficiency.Low levels of serum 25(OH)D, the substrate for the active hormone 1,25(OH)₂D, may exacerbate secondary hyperparathyroidism in patients with early CKD (16). Recent observations have linked a suboptimal 25(OH) D status to adverse cardiovascular outcomes and also to the rate of progression of renal insufficiency in CKD (14,23,24). Nonrenal synthesis of 1,25(OH)₂D has been described, suggesting that supplementation with cholecalciferol or ergocalciferol in addition to 1,25(OH)₂D may have beneficial effects in CKD patients (15,21). The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend measuring serum 25(OH)D in children with CKD stages 2 - 5 and 5D once annually and supplementing with ergocalciferol or cholecalciferol if serum 25(OH)D is less than 30 ng/mL (25).Most previous studies of vitamin D status in patients on dialysis were performed in adults, and few reports have looked at serum 25(OH)D in pediatric patients on dialysis. In the present study, we evaluated the prevalence and severity of abnormal vitamin D status in children on chronic dialysis, and the relationship between serum 25(OH)D and other parameters of mineral metabolism, nutrition, and residual renal function (RRF).     

Increased renal catabolism of 1,25-dihydroxyvitamin D3 in murine X-linked hypophosphatemic rickets.

The hypophosphatemic (Hyp) mouse, a murine homologue of human X-linked hypophosphatemic rickets, is characterized by renal defects in brush border membrane phosphate transport and vitamin D3 metabolism. The present study was undertaken to examine whether elevated renal 25-hydroxyvitamin D3-24-hydroxylase activity in Hyp mice is associated with increased degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by side chain oxidation. Metabolites of 1,25(OH)2D3 were separated by HPLC on Zorbax SIL and identified by comparison with standards authenticated by mass spectrometry. Production of 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, and 24-oxo-1,23,25-trihydroxyvitamin D3 was twofold greater in mitochondria from mutant Hyp/Y mice than from normal +/Y littermates. Enzyme activities, estimated by the sum of the three products synthesized per milligram mitochondrial protein under initial rate conditions, were used to estimate kinetic parameters. The apparent Vmax was significantly greater for mitochondria from Hyp/Y mice than from +/Y mice (0.607 +/- 0.064 vs. 0.290 +/- 0.011 pmol/mg per protein per min, mean +/- SEM, P less than 0.001), whereas the apparent Michaelis-Menten constant (Km) was similar in both genotypes (23 +/- 2 vs. 17 +/- 5 nM). The Km for 1,25(OH)2D3 was approximately 10-fold lower than that for 25-hydroxyvitamin D3 [25(OH)D3], indicating that 1,25(OH)2D3 is perhaps the preferred substrate under physiological conditions. In both genotypes, apparent Vmax for 25(OH)D3 was fourfold greater than that for 1,25(OH)2D3, suggesting that side chain oxidation of 25(OH)D3 may operate at pharmacological concentrations of substrate. The present results demonstrate that Hyp mice exhibit increased renal catabolism of 1,25(OH)2D3 and suggest that elevated degradation of vitamin D3 hormone may contribute significantly to the clinical phenotype in this disorder.     

Seasonal and age-related differences in serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway

Abstract

Objective. The aim of this study was to investigate the seasonal and age-related variation of vitamin D and PTH serum concentrations in a large general patient population in Western Norway. Design. A retrospective study was conducted at the Hormone laboratory, Haukeland University Hospital, Bergen, Norway. All analyses of 25-hydroxyvitamin D (25(OH)D) (n = 8325), 1,25-dihydroxyvitamin D (1,25(OH)₂D) (n = 4509) and PTH (n = 4203) requested from private practitioners from 2005 to 2008 were included. All three analytes were available in 1551 subjects. Subjects. Mean age of the study population was 49.8 years and 70.9% of the samples were from women. Results. The highest concentrations of 25(OH)D and 1,25(OH)₂D were observed in July–September. In April 43% of the studied population had 25(OH)D concentrations below 50 nmol/L. There was a positive correlation between 25(OH)D and 1,25(OH)₂D (p < 0.001). The levels of 25(OH)D and PTH were negatively correlated (p < 0.001) while 1,25(OH)₂D and PTH showed a weak positive correlation (p = 0.015). We observed higher concentrations of 25(OH)D (p = 0.003) and lower 1,25(OH)₂D levels (p < 0.001) in the older age groups. PTH increased throughout the whole age span (p < 0.001). Conclusion. We observed a seasonal variation in 25(OH)D and 1,25(OH)₂D with low serum concentrations during winter/early spring while PTH showed an inverse pattern. Higher levels of PTH in winter and the elderly may reflect an impaired vitamin D status that may affect calcium homeostasis and bone health.     

2型糖尿病视网膜病变患者血清25-羟维生素D水平及其发生和严重程度的影响因素分析

目的 探讨2型糖尿病视网膜病变(diabetic retinopathy,DR)患者血清25-羟维生素D[25-hy-droxy vitamin D,25-(OH)D]水平及其发生和严重程度的影响因素.方法 选取2型糖尿病(type 2 diabetes mellitus,T2DM)300例,根据疾病不同阶段分为无DR...     

基于UPLC-MS/MS 建立早产儿血清25-(OH)D3 和3-epi-25-(OH)D3 检测新方法及临床初步应用

目的 建立血清25- 羟维生素D3[25-hydroxyvitamin D3, 25-(OH)D3] 及3-epi-25- 羟维生素D3[3-epi-25-hydroxyvitaminD3, 3-epi-25-(OH)D3] 超高效液相色谱- 串联质谱检测新方法（ultra-performance liquid chromatography-tandem massspectrometry,UPLC-MS/MS）,并在早产儿中开展初步应用。方法 采用液- 液萃取法提取化合物,五氟苯基丙基（pentafluorophenylpropyl,PFPP）色谱柱分离25-(OH)D3 与3-epi-25-(OH)D3;从最低定量限、线性关系、精密度与准确度等四个方面对新建方法进行验证,并对134 例早产儿血清样本进行检测分析。结果 25-(OH)D3 及3-epi-25-(OH)D3 的最低定量限分别为6.05±0.78 nmol/L,1.48±0.20 nmol/L;线性范围3.78 ～ 480.00 nmol/L,1.00 ～ 128.00 nmol/L;相关系数r2 为0.992 2,0.992 8;25-(OH)D3 与3-epi-25-(OH)D3 低、中、高浓度质控品的日内精密度和日间精密度均小于15.00%;回收率109.92%,102.25%,98.76%;97.75%, 95.25%,99.80%。134 例早产儿血清25-(OH)D3 平均浓度22.35±13.28nmol/L,3-epi-25-(OH)D3 平均浓度5.43±4.35 nmol/L;3-epi-25-(OH)D3 占总25-(OH)D3 的平均比例为19.96%±12.08%,范围0.00% ～ 60.62%。以25-(OH)D3 计算,维生素D（Vitamin D, Vit D）缺乏率、不足率、充足率分别为78.36%,19.40%,2.24%;以总25-(OH)D3 计算,Vit D 缺乏率、不足率、充足率分别为65.67%,26.12%,8.21%;二者Vit D 缺乏率及充足率的比较, 差异均有统计学意义（χ2=5.351,1.719,4.823,均P ＜ 0.05）。结论 本研究建立的UPLC-MS/MS检测法性能良好且能精准检测早产儿血清25-(OH)D3 浓度,在早产儿Vit D 评估方面具有较好的应用价值。     

Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.

We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.     

Evaluation of a 1,25-dihydroxyvitamin D enzyme immunoassay

BACKGROUND: Radioactive reagents are used in most assays for measurement of 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. We evaluated a 1,25(OH)(2)D enzyme immunoassay (EIA) from IDS Ltd. that uses solid-phase immunoextraction and colorimetric detection and compared results to those of the thymus radioreceptor assay (RRA) for 1,25(OH)(2)D. METHODS: We collected serum samples (n = 145) representing an even distribution (0-200 pmol/L) of 1,25(OH)(2)D concentrations and Vitamin D External Quality Assessment Scheme (DEQAS) proficiency survey samples from 2004 surveys (n = 15) and stored them at -20 degrees C. We analyzed all samples with both EIA and RRA methods. We calculated imprecision using 5 QC samples in quadruplicate in each run (n = 6), including both pooled patient material used for QC with the RRA and QC material included in the EIA reagent set. We evaluated calibration stability by analyzing calibrators from different lots on the same plate and determining if calculated sample values drifted significantly. RESULTS: Deming linear regression between IDS EIA and RRA methods yielded slope 1.25 (95% CI 1.13-1.37), y-intercept -3 (95% CI -18 to 12), R(2) = 0.74. DEQAS proficiency survey samples for 2004 were all within 30% of the all-methods-trimmed mean. Imprecision CVs were 12%-16% within-run and 15%-20% between-run. CONCLUSIONS: We find no evidence of inferiority to the classic calf-thymus receptor assay for 1,25(OH)(2)D and no disadvantage in the results generated by the IDS EIA using samples from the major proficiency survey for 1,25(OH)(2)D. According to the product insert, however, the IDS EIA underestimates 1,25(OH)(2)D(2) compared with the D(3) form.     

Differential effects of 19-nor-1,25-dihydroxyvitamin D(2) and 1,25-dihydroxyvitamin D(3) on intestinal calcium and phosphate transport

19-Nor-1,25-dihydroxyvitamin D(2) (19-norD(2)) a less calcemic and phosphatemic analog of 1,25-dihydroxyvitamin D (1,25[OH](2)D(3)), is approved for the treatment of secondary hyperparathyroidism in patients with kidney failure. We have previously demonstrated that 19-norD(2) is less active than 1,25(OH)(2)D(3) in stimulating bone resorption. In this study, we compared the potencies of 19-norD(2) and 1,25(OH)(2)D(3) in stimulating net calcium and phosphate absorption in the intestine. Mineral balance was assessed in normal rats during the last 4 days of a 14-day treatment with various daily doses of 19-norD(2) or 1,25(OH)(2)D(3). Calcium absorption increased from 16.5% +/- 7.8% in vehicle-treated rats to 27.5% +/- 7.2% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 21.6% +/- 3.9%, 26.2% +/- 5.5%, and 27.4% +/- 5.1% in rats treated with 10, 50, and 100 ng/day 19-norD(2), respectively. Thus comparable stimulation of calcium transport was attained with 10 ng 1,25(OH)(2)D(3) and 100 ng 19-norD(2). Similar results were obtained for phosphate absorption, with an increase from 28.2% +/- 5.5% in vehicle-treated rats to 40.2% +/- 4.7% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 32.9% +/- 2.2%, 36.2% +/- 4.5%, and 36.8% +/- 3.8% in rats given 10, 50, and 100 ng/day 19-norD(2), respectively. Vitamin D compounds are believed to increase calcium absorption by inducing a calcium channel (epithelial calcium transporter or calcium transporter-1 [CaT1]) on the luminal membrane, a calcium-binding protein (Calbindin D9k) in the cytosol, and a calcium pump (plasma membrane calcium adenosine triphosphatase-1 [PMCA1]) on the basolateral membrane. Northern-blot analysis of intestinal ribonucleic acid of vitamin D-deficient rats given seven daily injections of vehicle or 100 ng 1,25(OH)(2)D(3) or 19-norD(2) revealed that 19-norD(2) was less potent than 1,25(OH)(2)D(3) in stimulating expression of CaT1, Calbindin D9k and PMCA1. In summary, the reduced calcemic and phosphatemic activities of 19-norD(2) can be attributed to lower potency in stimulating intestinal calcium and phosphate absorption.     

Transport of vitamin D sterols in human plasma: effect of excess vitamin D, 25 hydroxyvitamin D and 1,25 dihydroxyvitamin D

Abstract. Vitamin D and its more active metabolites, 25 hydroxyvitamin D (25-OH-D) and 1,25-dihydroxy-vitamin D (1,25-(OH)₂-D), are transported in human plasma on a specific binding protein (DBP), which has been shown to have an α-globulin electrophoretic mobility. Since the concentration of DBP in normal human plasma is approximately 5 μmol/l, whereas that of all the vitamin D metabolites is less than 0·2 μmol/l, DBP is less than 3% saturated under physiological conditions. We have studied the transport of the above-mentioned metabolites in human plasma in vitro at normal and saturating concentrations. Human plasma was incubated with increasing amounts of vitamin D metabolites together with their radiolabelled tracers. Ultracentrifugation was used to isolate plasma lipoproteins (density, d < 1·21 g/ml) and agarose gel electrophoresis of lipoprotein-free plasma (d > 1·21 g/ml) to separate DBP (α globulin) from albumin. The recovery of the tracer in plasma proteins was always more than 80%. At physiological concentrations [³H]25-OH-D bound almost exclusively to DBP (98%), [³H]vitamin D or [¹⁴C]vitamin D bound both to DBP and to lipoproteins (40%), and [³H]1,25-(OH)₂-D bound to DBP (62%), to lipoproteins (15%) and also to albumin (23%). When the concentration of vitamin D metabolites was increased, DBP became saturated. The binding capacity of DBP was similar for all three sterols, about 5 μmol/l plasma, or one mole of sterol per mole of protein, but the saturating concentration was different for the three sterols (vitamin D > 1,25-(OH)₂-D > 25-OH-D). 25-OH-D had the greatest affinity for DBP, and it completely displaced both vitamin D and 1,25(OH)₂-D from DBP at higher concentrations. All sterols bound to both plasma lipoproteins and albumin: vitamin D preferentially to lipoproteins and both 25-OH-D and 1,25-(OH)₂-D to albumin. A similar binding pattern for vitamin D in plasma was observed previously by us in a child with vitamin D toxicity. The increased binding of vitamin D to lipoproteins and especially to albumin may help explain the pathogenesis of toxicity in hypervitaminosis D, where the plasma levels of the more active metabolites are insufficient to account for the clinical signs.