No negative impact of reduced leptin secretion on bone metabolism in male decathletes.

This study investigated the effect of intense physical activities that generate high mechanical constraints on bone metabolism and serum leptin concentrations and the potential relationships among bone mineral density (BMD), bone biochemical markers and leptin variation. Thirteen male decathletes (mean age 22.4 +/- 2.9 years), nationally or internationally ranked (15.5 h/week of training), were compared with 13 healthy sedentary subjects (mean age 25.9 +/- 3.3 years). BMD was measured by DEXA and bone turnover was evaluated by specific markers. Leptin and calciotropic hormones levels were analysed in parallel. BMDs were higher in athletes than in controls at total body (13.9%), lumbar spine (17%), femoral neck (25%) and radius (9%), but not at the head. Athletes presented higher concentrations of osteocalcin (59.8%), cross-linked C-telopeptide of type-I collagen (41.1%) and 1,25-dihydroxyvitamin-D (37.1%). Basal leptin concentration was lower in athletes (0.94 +/- 0.54 vs. 5.07 +/- 1.1 ng ml(-1)), and this difference persisted when leptin levels were adjusted for whole body fat mass (WBFM). No difference was observed for bone-specific alkaline phosphatase or intact parathyroid hormone. Serum leptin levels were negatively correlated with various BMD values only when both the groups were pooled (n = 26). This relationship did not persist when leptin levels were adjusted for WBFM. Male athletes, who practise sports generating high mechanical constraints on the body, present a specific bone metabolism that includes high BMD, as well as high bone turnover. The blunted leptin secretion did not seem to have deleterious effect on the process of bone adaptation to high mechanical constraints.

Fibroblast growth factor 23, parathyroid hormone, and 1alpha,25-dihydroxyvitamin D in surgically treated primary hyperparathyroidism.

OBJECTIVE: To determine whether fibroblast growth factor 23 (FGF23) contributes to the hypophosphatemia of primary hyperparathyroldism. PATIENTS AND METHODS: Thirteen adult patients with primary hyperparathyroidism had serum collected before and after parathyroidectomy for analysis of inorganic phosphorus, calcium, 1alpha,25-dihydroxyvitamin D (1alpha,25[OH]2D), parathyroid hormone (PTH), FGF23, creatinine, and bone-specific alkaline phosphatase (BSAP). Patients were recruited between July 24, 2003, and February 11, 2004. RESULTS: Before surgery, patients had elevated serum calcium and PTH concentrations. Serum phosphorus concentrations were in the low-normal range. The FGF23 concentrations were not elevated in patients with primary hyperparathyroidism compared with healthy controls. Within 24 hours of surgery, serum calcium, PTH, 1alpha,25(OH)2D, and BSAP concentrations were lower (P < .002 for all) and phosphorus concentrations were higher (P = .003) than in the preoperative state. The FGF23 concentrations were similar 1 day and 6 weeks after surgery. The FGF23 concentrations did not correlate with serum phosphorus, calcium, PTH, 1alpha,25(OH)2D, creatinine, or BSAP concentrations in the preoperative or postoperative state. CONCLUSION: Parathyroid hormone is the major regulator of serum phosphorus concentrations in patients with primary hyperparathyroidism. Fibroblast growth factor 23 does not appear to play a role in phosphorus homeostasis in patients with surgically treated primary hyperparathyroidism.

An investigation of ethnic differences in bone mineral, hip axis length, calcium metabolism and bone turnover between West African and Caucasian adults living in the United Kingdom.

The aim of the study was to investigate factors relating to calcium and bone metabolism which might explain the low incidence of osteoporotic fracture among Africans. Adult bone mineral status, hip axis length and biochemical indices were investigated in 20 Caucasians (10 male, 10 female) and 19 Gambians (12 male, 7 female) living in the UK. Bone mineral content (BMC), bone mineral density (BMD) and BMC adjusted for bone area, body weight and height (size-adjusted BMC) were measured for the whole-body, lumbar spine, femoral neck, trochanter, radius shaft and radius wrist using dual-energy X-ray absorptiometry. There were no significant differences in whole body or regional BMC; values tended to be lower in the Gambians. Gambian men had higher size-adjusted BMC at the femoral neck (Gambian-British = 21%, 95% CI = 6 to 36%, p < 0.01), associated with a smaller bone area (Gambian-British = -11%, 95% CI = -20 to -2%, p = 0.02). BMD was affected similarly. No other significant differences in BMD or size-adjusted BMC were observed. Gambians had shorter hip axis length (Gambian British, after accounting for sex, = -5%, 95% CI = -9 to -1%, p = 0.02). There were no significant differences in bone turnover (osteocalcin, bone isoenzyme of alkaline phosphatase, urinary deoxypyridinoline) or calciotropic hormone levels (parathyroid hormone, 1,25-dihydroxyvitamin D, calcitonin). Gambian men had lower 25-hydroxyvitamin D concentrations (Gambian = 26.3 SD 12.0 nmol/L, British = 55.5 SD 13.9 nmol/L, p < 0.0001), a difference not seen among the women. Gambian men and women excreted significantly less phosphate and potassium than British subjects by 30-60%; urinary calcium and sodium excretion were similar in the two groups. This study revealed few ethnic differences that could account for the disparity in osteoporotic fracture rates between Africans and Caucasians, with the possible exception of anatomical differences in the hip.

In vivo microautoradiography of [3H]1,24(OH)2D3 (tacalcitol) following topical application to normal rats and in vitro metabolism in human keratinocytes.

This study was conducted to investigate the mechanism of topical absorption of [3H]1,24(OH)2D3 (1,24-dihydroxyvitamin D3; tacalcitol) by applying an ointment containing 4 micrograms2/g [3H]1,24(OH)2D3 to the skin of rats using an occlusion method. Microautoradiography of the skin at the application site 1 h after topical treatment showed a high concentration of radiolabel in the stratum corneum, the epidermis and around the hair follicles. Radiolabel was also seen in the epidermis and hair follicle areas 8 h and 24 h after application. The radiolabel was distributed to a minor extent to the subcutaneous fat layer. Microautoradiography showed two routes of purcutaneous absorption of 1,24(OH)2D3: through the stratum corneum and epidermis into the microvessels, and through hair follicle areas into the bloodstream. After topical application of an ointment containing 4 micrograms/g or 40 micrograms/g [3H]1,24(OH)2D3 to the shaved neck skin of rats, the absorption rate, estimated by excretion in the urine and faeces, was about 30% of the total applied radioactivity. The main excretion route after topical application was in the faeces. Furthermore, 1,24(OH)2D3 added to human adult keratinocytes was not metabolized into other compounds, and only the unchanged compound was detected. These findings strongly suggest that 1,24(OH)2D3 distributed into the epidermis acts on epidermal keratinocytes. Topical application of 1,24(OH)2D3 appears to be a possible approach to the treatment of psoriasis and other skin diseases through its action on the 1,25(OH)2D3 receptor, which reportedly plays a very important role in the regulation of proliferation and differentiation of keratinocytes.

[Synthesis of dihydroxycholecalciferols in maternal and fetal rat organs in the last days of gestation]. / Síntesis de dihidroxicolecalciferoles en órganos maternos y fetales de rata en los últimos días de la gestación.

The in vitro synthesis of 3H1,25(OH)2D3 and 3H24,25-(OH)2D3 in different maternal (kidney and spleen), placental (maternal and fetal sides) and fetal (kidney, intestine, liver and skeleton) tissues and the relative contribution of each of these organs to total production was studied in the last six days of gestation in rats. On day 16, synthesis of both metabolites was higher in fetal tissues than in maternal kidney, and decreased as gestation advanced. On day 16, placental contribution represented more than 50% of the total production of 3H1,25(OH)2D3, while the maternal kidneys and the fetal tissues contributed only 16% and 26%, respectively. On day 18, the synthesis of 3H1,25(OH)2D3 by maternal placenta and fetal tissues was significantly reduced in comparison with that observed on day 16. Between days 16 and 19, the plasma concentrations of 1,25(OH)2D in mothers and fetuses were associated with the magnitude of its in vitro production. Starting on day 19, however, the in vitro production remained at the same level while the plasma concentration increased, suggesting lower utilization or lower catabolism of this metabolite. Similarly, the total synthesis of 3H24,25(OH)2D3 decreased on day 19. Between days 16 and 18, a higher synthesis of 3H24,25(OH)2D3 corresponded with lower plasma concentration of this metabolite suggesting greater utilization. In contrast, between days 19 and 21, the in vitro synthesis and plasma concentration of 24,25(OH)2D increased in parallel fashion. In summary we report the following findings: a) inhibition of the in vitro synthesis of 3H1,25(OH)2D3 and 3H24,25(OH)2D3 on day 19 of gestation in the rat; b) the contribution of each of the different maternal, placental and fetal tissues to the total synthesis of these metabolites in the last six days of gestation; and c) a parallelism between in vitro production and plasma concentration of both metabolites.

Tritium isotope effect in high-pressure liquid chromatography of vitamin D metabolites.

A significant chromatographic isotope effect is reported for 1,25-dihydroxyvitamin D3 in a wide variety of HPLC separation systems. The effect is also observed for 24,25-dihydroxyvitamin D3. Retention times differ from less than 1% up to 4% depending on the separation system and the degree and position of tritium substitution. Such an effect must be corrected for whenever both labeled and unlabeled vitamin D metabolites are used in HPLC cochromatography or assay recovery studies.

Vitamin D supply to the rat fetus and neonate.

The prevention of neonatal rickets by oral supplementation with vitamin D2 (ergocalciferol) has tended to obscure our ignorance of the natural mechanism by which young mammals receive an adequate supply of vitamin D. To investigate the possibility of specific intrauterine transfer and storage of vitamin D in fetal tissues, vitamin D-deficient female rats were given depot injections of 3H- or 14C-labeled vitamin D3 (cholecalciferol) before mating and the 3H-labeled animals were killed at stages during the last third of gestation. Analysis of lipid extracts from whole fetuses revealed a linear increase in the concentration of 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and D3 itself between days 14 and 19 of gestation. During this period the elimination half-time of 3H-labeled molecules in maternal plasma fell from 27.1 to 4.4 d, suggesting that a specific mechanism was transferring vitamin D molecules into the fetuses. The vitamin was stored predominantly as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, with the highest concentrations in fetal muscle. Immediately after birth, pups from 3H- and 14C-labeled mothers were exchanged and later killed after 1-3 wk of suckling. Analysis of total lipid extracts for 3H and 14C content determined the relative contributions of vitamin D supplied before birth via the placenta and after birth in the maternal milk. The vitamin D content of the rat milk was relatively high, between 1.0 and 3.5 micrograms/liter. Nevertheless, the supply of vitamin D in utero, rather than from milk, was the main determinant of vitamin D status in early neonatal life. This is the first indication in a mammal of a specific transfer mechanism that allows the fetus to accumulate vitamin D from the mother during the last third of gestation.

Vitamin D3 metabolites in amniotic fluid in relation with maternal and fetal sera in term pregnancies.

The concentration of 25-hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were determined in amniotic fluid, fetal cord serum and maternal serum in 26 cases of elective cesarean sections at term. All the women had a normal pregnancy and did not get any vitamin D fortified preparations. The samples were collected during December 1982-April 1983, at 37-40 weeks of pregnancy. The respective levels (+/- S.D.) of 25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3 in maternal serum were: 18.03 +/- 10.8 ng/ml, 1.473 +/- 1.562 ng/ml and 36 +/- 21.5 pg/ml; in fetal cord serum: 13.15 +/- 8.3 ng/ml, 0.9 +/- 0.76 ng/ml and 29.2 +/- 18.55 pg/ml and in amniotic fluid: 0.732 +/- 0.508 ng/ml, 0.212 +/- 0.104 ng/ml and 14.3 +/- 10.0 pg/ml. The levels of the three metabolites in maternal and fetal cord serum were not statistically different. There was a statistically significant correlation between maternal and fetal serum levels of 25(OH)D3 and 24,25(OH)2D3 (r = 0.79, p less than 0.01 and r = 0.743, p less than 0.01 respectively). No significant correlation was found in 1,25(OH)2D3 levels between maternal and fetal cord sera. This lack of correlation may well be in agreement with the recent findings of Kouppala, et al. who demonstrated that the fetus contributes to its own pool of 1,25(OH)2D3. A significant difference was found between maternal serum and amniotic fluid levels of the three metabolites. A statistically significant difference was also found between fetal serum levels of 25(OH)D3 and 24,25(OH)2D3 and amniotic fluid levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of active metabolites of vitamin D3 in the callus of fracture repair in chicks.

The levels of the active metabolites of vitamin D were measured in the callus and in the epiphyseal growth plate of chicks given radioactive cholecalciferol during fracture healing. Those levels were correlated with the histological findings. Three groups of chicks were studied: a control group with no fracture, chicks with fractures fixed by Kirschner wire, and chicks with unfixed fractures. A significant increase in the levels of the active metabolites was found in the callus during the first few days after fracture. The levels of 25-hydroxycholecalciferol [25(OH)D3] and of 24,25-dihydroxycholecalciferol [24,25(OH)2D3] were higher when there was no fixation, while those of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were higher after fixation. The concentrations of these metabolites in the proximal epiphysis of the tibia were similar to those found in the callus. Based on these findings it is suggested that the active metabolites of vitamin D are directly involved in the process of fracture repair.

Measurement of 25,26-dihydroxyvitamin D: importance of the configuration of the C-25 hydroxyl group.

25,26-Dihydroxyvitamin D3 [25,26(OH)2D3] is chemically synthesized as two stereoisomers: 25(R),26(OH)2D3 and 25(S),26(OH)2D3. Both the R and S configurations have been claimed to be the natural form. Previous studies, however, have not considered the possibility that the stereochemical configuration of the C-25 hydroxyl group could affect the binding of 25,26(OH)2D3 to the rat serum binding protein used in assays for the measurement of this metabolite. In our study, a 50% displacement of radiolabeled 25-hydroxyvitamin D3 ([3H]25OHD3) from its initial binding is achieved with 325 fmol/mL 25(R),26(OH)2D3 and 850-1000 fmol/mL 25(S),26(OH)2D3--a difference in potency of approximately 3-fold. The potency of the R isomer in displacing [3H]25OHD3 was similar to that of 25OHD3 or 24(R),25(OH)2D3. When 25,26(OH)2D levels were measured in 12 normal subjects with both the R isomer and the S isomer as standards, the results were 625 +/- 360 fmol/mL 25(R),26(OH)2D3 equiv and 1800 +/- 1130 fmol/mL 25(S),26(OH)2D3 equiv. To determine which stereoisomer is biosynthesized, two types of experiments were performed. In the first, radiolabeled 25,26(OH)2D3 was biosynthesized from [3H]25OHD3 by using chick renal mitochondria and compared to [3H]25OHD3 as a ligand in the rat serum binding protein assay, which was used to measure 25OHD3, 25(R),26(OH)2D3, and 25(S),26(OH)2D3. Initial binding of radiolabeled 25OHD3 and 25,26(OH)2D3 to the binding protein was comparable, as was their displacement by the nonradioactive metabolites, indicating that chick renal mitochondria produce primarily 25(R),26(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Losses of 1,25- and 24,25-dihydroxycholecalciferol in the peritoneal fluid of patients treated with continuous ambulatory peritoneal dialysis.

We measured peritoneal losses of the active vitamin D metabolites 1,25(OH)2D3 and 24,25(OH)2D3 in patients receiving continuous ambulatory peritoneal dialysis (CAPD). The serum concentration of 24,25(OH)2D3 was considerably lower than in hemodialysis patients. The serum concentration of 1,25(OH)2D3 was undetectable and rose to levels similar to those in hemodialysis patients only after loading with much higher oral doses of 1-alpha-vitamin D3 than those received by hemodialysis patients. Losses of both metabolites in peritoneal fluid were considerable, averaging approximately 6-8% of the plasma pool per day. These losses lead to low serum levels of these active vitamin D metabolites in CAPD patients, which may be an important factor in exacerbating renal osteodystrophy. Our results indicate the need for increased replacement doses of vitamin D metabolites in CAPD patients.

Vitamin D metabolites in human milk.

The concentrations of unconjugated 25-OHD, 24, 25(OH)2D, and 1,25(OH)2D were measured in human milk by competitive protein-binding radioassays following successive preparative Sephadex LH-20 chromatography and HPLC. The mean (+/- SE) concentration of 25-OHD was 0.37 +/- 0.03 ng/ml, of 24,25(OH)2D was 24.8 +/- 1.9 pg/ml, and of 1,25(OH)2D was 2.2 +/-0.1 pg/ml. The concentration of 25-OHD3 in milk as determined by HPLC and UV detection at 254 nm was 0.27 +/- 0.08 ng/ml. The milk concentrations of vitamin D metabolites did not correlate with the maternal serum 25-OHD levels. The total amounts of unconjugated vitamin D metabolites correspond to the known low bioassayable vitamin D antirachitic activity in human milk.

A modified radioimmunoassay for 1,25-dihydroxycholecalciferol.

A radioimmunoassay for 1,25-dihydroxycholecalciferol which did not cross react with 1,25-dihydroxyergocalciferol is described. IgG fractions were prepared from the serum of rabbits that had been immunized with 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine albumin. Radioligand binding by the IgG fractions was time-, temperature-, and pH-dependent. The IgG fractions had a high affinity for 1,25-dihydroxycholecalciferol but cross reacted with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Vitamin D2 metabolites did not cross react in the assay when amounts up to 9 ng per tube were tested. The determination of 1,25-dihydroxycholecalciferol in human serum required an organic extraction and chromatographic isolation of the metabolite. Radioligand binding was influenced by the presence and concentration of the proteins in the phosphate buffer. The mean concentration of 1,25-dihydroxycholecalciferol in serum from normal adults was 56 (SEM 5.7) ng/L. 1,25-Dihydroxycholecalciferol was not detectable in serum from a nephrectomized subject and the concentration in serum was lower than normal in hypoparathyroid patients. Ingestion of 1,25-dihydroxycholecalciferol by nephrectomized or hypoparathyroid patients restored the concentration of 1,25-dihydroxycholecalciferol in serum to the normal range. The stability of the IgG fraction, the relatively short incubation interval, and the ability to measure 1,25-dihydroxycholecalciferol without interference from 1,25-dihydroxyergocalciferol are unique aspects of this radioimmunoassay.