1,25-Dihydroxyvitamin D in biological fluids: a simplified and sensitive assay.

A competitive binding assay for 1,25-dihydroxyvitamin D [1,25-(OH2D] in plasma has been developed in which intestinal cytosol preparations from rachitic chicks are used as the binding protein. A new method of extraction and two new chromatographic procedures are used for this assay. The method is sensitive to as little as 10 picograms of 1,25-(OH)2D, and triplicate assays can be done on 5 milliliters of plasma. This assay shows that in the plasma of normal adult subjects there is a 1,25-(OH)2D concentration of 29 +/- 2 picograms per milliliter, while none can be detected in the plasma of nephrectomized subjects and end-stage renal failure patients.

Normal intrauterine development of the fetus of a woman receiving extraordinarily high doses of 1,25-dihydroxyvitamin D3.

We monitored the course of pregnancy in a 28-yr-old patient with hereditary insensitivity to 1,25-dihydroxyvitamin D (1,25-(OH)2D). During pregnancy, she received 1,24-(OH)2D3 in doses of 17--36 micrograms/day (usual dose for hypocalcemia, 0.25--1.0 microgram/day). We separately measured 25-hydroxyvitamin D3 (25OHD3), 25OHD2, 1,25-(OH)2D3, and 1,25-(OH)2D2 to assess contributions to total 1.25-(OH)2D from endogenous biosynthesis (1 alpha-hydroxylation of 25OHD2 or 25OHD3) vs. contributions from exogenously administered 1.25-(OH)2D3. Serum concentrations of 1,25-(OH)2D were extraordinarily high throughout gestation. The evaluations of total 1,25-(OH)2D in maternal plasma reflected large amounts from both 1 alpha-hydroxylation of 25OHD and oral intake of 1,25-(OH)2D3. At parturition, the calcium concentration was 9.6 mg/dl in maternal serum and 10.2 mg/dl in cord serum. The concentration of total 1,25-(OH)2D in cord serum was high (470 pg/ml; normal mean for placental venous serum, 19 pg/ml); most derived from transplacental passage of maternal 1,25-(OH)2D3 and not from fetoplacental biosynthesis. The child manifested mild hypercalcemia in the first 2 days of life, indicating that the 1,25-(OH)2D3 measured in cord serum was active in vivo. The child showed no somatic features of the syndrome of elfin facies and supravalvular aortic stenosis. We conclude that an extraordinarily high concentration of 1.25-(OH)2D in maternal serum throughout gestation was not apparently toxic to the fetoplacental unit, though the maternal metabolite entered the fetal circulation.

A bioassay capable of measuring 1 picogram of 1,25-dihydroxyvitamin D3.

Fetal rat bones in organ culture constitute a sensitive system for assay of the vitamin D metabolite, 1 alpha,25-dihydroxyvitamin D3. Significant bone resorption is obtained with as little as 2 pg 1,25-dihydroxyvitamin D3 after 48 h of culture and with 1 pg after 64 h of culture. In the current study, organ cultures of fetal rat bone are used as a bioassay for 1,25-dihydroxyvitamin D i normal human plasma, which was prepared for assay by extraction with dichloromethane, chromatography on Sephadex LH-20, and purification on silicic acid by high pressure liquid chromatography. The concentration of 1 alpha,25-dihydroxyvitamin D3 in normal adult human plasma was 24.8 +/- 2.0 pg/ml (n = 19) by this assay.

Absence of seasonal variation in serum concentrations of 1,25-dihydroxyvitamin D despite a rise in 25-hydroxyvitamin D in summer.

The serum concentrations of the vitamin D metabolites 25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D (calcitriol) have been measured in normal subjects whose ages varied from 18 months to 35 yr. Samples were obtained in all months of the year in order to assess the effects of season on serum concentration. During the months of April to September, 25OHD3 levels are higher than in the winter months. No seasonal variation in the 25OHD2 or calcitriol serum concentration was observed. Age-related differences in 25OHD2 and D3 concentrations did not exist. The levels of calcitriol are higher in adolescence and increase from 35 +/- 19 pg/ml (SD) at 1.5-10 yr of age to 54 +/- 21 pg/ml at 10-20 yr of age. In young adults, the levels fall again to 28 +/- 16 pg/ml. Accordingly, despite a seasonal variation in the precursor of calcitriol, the levels of this most active metabolite of vitamin D do not change in relation to sunlight exposure. This lack of seasonal variation is further evidence of the tight feedback regulation of calcitriol.

A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D.

Typical features of hereditary vitamin D-dependent (pseudovitamin D-deficient) rickets were observed beginning at ages 20 and 5 months in a brother and sister. Both had calcium malabsorption correctable with high doses of 25-hydroxyvitamin D. During periods of hypocalcemia they both manifested secondary hyperparathyroidism with hypophosphatemia and high serum concentrations of endogenously produced 1,25-dihydroxyvitamin D. In each, normalization of serum calcium concentration and resolution of osteomalacia were obtained with continuous administration of high doses of ergocalciferol or high doses of 1,25-dihydroxycholecalciferol. Chemical features of vitamin D deficiency were corrected in the presence of high circulating concentrations of 1,25-dihydroxyvitamin D2, produced endogenously, or of 1,25-dihydroxyvitamin D3, administered by mouth. Serum concentrations of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D were normal in five first degree relatives. We conclude that in these five first degree relatives. We conclude that in these siblings, rickets and osteomalacia resulted from a hereditary decreased sensitivity to 1,25-dihydroxyvitamin D at the intestine and perhaps other vitamin D target tissues.

Long-term influence of calcitriol (1,25-dihydroxyvitamin D) and supplemental phosphate in X-linked hypophosphatemic rickets.

Ten patients with hypophosphatemic rickets (eight with X-linked familial form) were treated with vitamin D2 (10,000 to 75,000 units per day) and oral phosphate (1.5 to 3.6 gm) for a total of 438 treatment months. Therapy was then changed to calcitriol (17 to 34 ng/kg/day) and the same phosphate dose. Patients served as their own controls, and significant biochemical changes noted were an increase in immunoreactive parathyroid hormone from 29 +/- 9 (SD) microliters Eq/ml (pre-phosphate) to 62 +/- 34 on vitamin D2 plus PO4, then decreasing to 40 +/- 20 on a regimen of 1,25-dihydroxyvitamin D (1,25(OH)2D) plus PO4; serum PO4 rose from 2.44 +/- 0.45 (SD) mg/100 ml to 3.06 +/- 0.79 and then to 3.43 +/- 0.76; alkaline phosphatase activity decreased from 677 +/- 298 (SD) IU/liter to 457 +/- 183 to 290 +/- 176. Serum calcium and creatinine levels were unchanged. Both urinary calcium excretion and calcium-creatinine ratio decreased after therapy with 1,25(OH)2D. Urinary phosphate excretion was higher after calcitriol administration. Serum 1,25(OH)2D levels were low in these vitamin D2-treated patients, and an inverse relationship between serum 25(OH)D and 1,25(OH)2D was found. Improved bone mineralization was evident from serial assessment by photon absorptiometry, and radial bone mineral content rose from 75.3% +/- 2.2% of expected to 82.2% +/- 1.4% (P less than .005). Stature was improved when assessed by standard deviation for chronologic age but did not reach statistical significance. Long-term 1,25(OH)2D plus phosphate therapy appears to be more efficacious than vitamin D2 in this form of rickets, particularly in improving phosphate homeostasis.

Vitamin D metabolites in renal insufficiency and other vitamin D disorders of children.

Serum vitamin D metabolites were measured in relation to factors responsible for the synthesis of 1,25(OH)2D in children with renal disease and other disorders of vitamin D metabolism. Severe renal failure (CCr less than 15 ml/min/1.73 m2) was associated with a reduction in 1,25(OH)2D to 25% of mean normal levels, a very high PTH/1,25(OH)2D ratio of 75 (normal = 0.74), and a 60% reduction of serum 24,25(OH)2D. At CCr values between 15 and 50 ml/min 1.73 m2, 1,25(OH)2D was 50% of normal, despite a PTH/1,25(OH)2D ratio of 7 and a normal 24,25(OH)2D value. Above clearance values of 75 ml/min/1.73 m2, 1,25(OH)2D and the ratio were normal. Oral 1,25(OH)2D therapy in severe chronic renal insufficiency caused a fall in this ratio from 75 to 8. High PTH/1,25(OH)2D ratios were found in hypophosphatemic rickets and vitamin D-deficiency rickets, indicating reduced synthesis of 1,25(OH)2D relative to PTH. Low ratios were found in hypoparathyroidism and sarcoidosis, possibly indicating independence of PTH stimulation in 1,25(OH)2D production. The ratios of calcium/1,25(OH)2D and phosphate/1,25(OH)2D also provided useful information in each of these conditions, indicating that the use of such series of ratios in selected patients may promote a better understanding of pathogenic mechanisms and of the response to therapy.

Rickets of prematurity. Supranormal levels of serum 1,25-dihydroxyvitamin D.

Rickets, hypocalcemia, hypophosphatemia, and hyperparathyroidism were found in a low-birth-weight premature infant. The concentration of plasma calcitriol (1,25-dihydroxyvitamin D) was 145 pg/mL. With additional exogenous calcitriol (37.5 ng/kg/24 hr) given for eight weeks, the biochemical abnormalities were corrected and healing of rickets was evident. Three months later, while receiving only 400 IU of ergocalciferal daily, the patient had normal levels of serum calcium, phosphate, and alkaline phosphatase with a serum calcitriol concentration of 36 pg/mL. These observations suggest that rickets of prematurity may involve a malabsorption of calcium and phosphorus with an elevated calcitriol level needed to overcome this inadequate absorption. Additional doses of calcitriol may be of benefit in these infants, although it must be given carefully. Furthermore, the role of phosphate supplementation in these infants requires consideration.

Determination of vitamin D and its metabolites in plasma from normal and anephric man.

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.

Supranormal 25-hydroxyvitamin D and subnormal 1,25-dihydroxyvitamin D: their role in X-linked hypophosphatemic rickets.

Serum 25-hydroxyvitamin D (25-OH-D) and 1,25-dihydroxyvitamin D (1,25-(OH)2D) and bone mineral content by the photon-absorption technique were determined in eight patients with X-linked hypophosphatemic rickets treated for at least 24 months with oral sodium phosphate and high-dosage ergocalciferol (vitamin D2). Mean 25-OH-D2 level was 129.5 +/- 67.5 ng/mL (mean +/- SD); the level of 25-OH-D3 was 10.5 +/- 5.8 ng/mL. These values were significantly higher than in normal subjects (total 25-OH-D mean of 27 +/- 10 ng/mL). Serum 1,25-(OH)2D was 16.9 +/- 8.5 pg/mL (mean +/- SD) in the eight patients, significantly lower than 47 +/- 16 pg/mL in 27 age-matched controls. Values indicative of significant demineralization were found in seven of the eight phsophate-treated patients, who had no radiologic evidence of rickets. These results suggest that any theory of the pathogenesis of this disorder must account for inappropriate renal vitamin D metabolism and for renal hyperphosphaturia. The failure of high-dosage oral phosphate and ergocalciferol to fully correct demineralization may suggest a role for calcitriol (1,25-(OH)2D3) as a therapeutic agent.

Vitamin D metabolite levels in oncogenic osteomalacia.

Measurements of 1,25 (OH)2D3 and other metabolites of vitamin D in a patient with oncogenic osteomalacia confirm the selective, reversible deficiency of 1,25 (OH)2D3 in this syndrome, and indicate the rapidity of normalization (within days) of the hormone level and associated hypophosphatemia after resection of the tumor. In this patient, the tumor was an intranasal hemangiopericytoma.

Serum 1,25-dihydroxyvitamin D levels in normal subjects and in patients with hereditary rickets or bone disease.

The serum concentration of 1,25-dihydroxylvitamin D (1,25-[OH]2D) in normal children and in children with inherited diseases of bone was compared by use of a competitive binding assay. Observed values were: in 12 normal children and adolescents, 37.1 +/- 1.9 pg per milliliter (mean +/- S.D.); in 14 patients with X-linked hypophosphatemic rickets treated with vitamin D2 and phosphate supplements, 15.6 +/- 7.8 (P less than 0.01 versus control); in six patients with autosomal recessive vitamin D dependency treated with vitamin D2, 9.5 +/- 2.9 (P less than 0.01 versus control); and in four untreated patients with autosomal dominant hypophosphatemic (non-rachitic) bone disease, 30.2 +/- 6.3 (not significantly different from the controls). The difference in bone disease between X-linked hypophosphatemia (severe) and hypophosphatemic bone disease (mild) at comparable low serum levels of phosphate implies that 1,25-(OH)2D and phosphate may have independent roles in the pathogenesis of defective bone mineralization.

Circulating vitamin D metabolite concentrations in childhood renal diseases.

Vitamin D metabolites were measured in children, untreated with glucocorticoids, who had renal disease. Two groups were defined in relation to endogenous creatinine clearance values: those with impaired clearance , 0 to 48 ml/min per 1.73 m2; and those with unimpaired clearance, 75 to 150 ml/min per 1.73 m2. Serum 1.25(OH)2D was 16 +/- (SD) 12 pg/ml in impaired patients (N=24) and 48 +/- 16 pg/ml in unimpaired patients (N=18). The latter level is not different from healthy childhood controls (43 +/- 12 pg/ml; N=194). Serum samples of 25(OH)D2 and D3 were comparable in each group and not different from control values of 33.2 +/- 10.3 ng/ml. Serum 24,25(OH)2D was 0.6 +/- (SD) 0.14 ng/ml in patients with a clearance of less than 13 ml/min per 1.73 m2, 1.39 +/- 0.54 ng/ml in those with a clearance of 18 to 48 ml/min per 1.73 m2, and 152 +/- 0.91 ng/ml in patients without an impairment of clearance. Only patients with the lowest clearance had values different from control values of 1.70 +/- 0.57 ng/ml. In our study we suggest that a significant reduction in 24,25(OH)2D and 1,25(OH)2D are found at low clearance values in children with tubulointerstitial disease. Our study further suggests that a reduction in renal tubular mass is important in accounting for these changes in vitamin D metabolite values.

Serum 1,25-dihydroxyvitamin D levels in normal children and in vitamin D disorders.

Using a precise assay for 1,25-dihydroxyvitamin D in serum, the levels in 103 children, aged 13 months to 16 years, were found to be 43 +/- 2 pg/mL (mean +/- SE). This value is higher than reported values in adults and in neonates. Age-related changes in 1,25-dihydroxyvitamin D levels during childhood were also evident. Older children have significantly higher levels than children less than 11 years, possibly indicating changes with puberty and the adolescent growth spurt. The values were significantly reduced in childhood uremia (13 +/- 5 pg/mL), in hypoparathyroidism (16 +/- 1 pg/mL), and in children with Fanconi's syndrome. Treatment with oral calcitriol in these three groups of patients led to increased serum levels.

Reduction of serum-1, 25-dihydroxyvitamin-D3 in children receiving glucocorticoids.

Serum-1,25-dihydroxyvitamin-D3 (1,25-[OH]2D3) was subnormal in children receiving long-term glucocorticoid treatment for various glomerular diseases, including nephrotic syndrome. In children with chronic glomerulonephritis not treated with glucocorticoids who had similar serum-creatinine with glucocorticoids who had similar serum-creatinine concentrations, serum-1,25-dihydroxyvitamin-D3 concentrations resembled those in healthy controls, indicating that glomerular renal disease per se does not account for reduced serum-1,25(OH)2DE concentrations in steroid-treated patients. The reduction in concentration of this most active vitamin-D metabolite correlated with the dose of steroid administered and with reduction in forearm bone mineral content measured by the photon absorption technique. Reduced serum-1,25-(OH)2D3 concentration may be important in the pathogenesis of steroid-induced osteopenia.

Vitamin-D-dependent rickets type II. Resistance of target organs to 1,25-dihydroxyvitamin D.

Studies were done to determine the cause for hypocalcemia, secondary hyperparathyroidism, osteomalacia and osteitis fibrosa cystica in a 22-year-old black woman. The patient had normal serum 25-hydroxyvitamin D (14 ng per milliliter) and markedly elevated serum 1,25-dihydroxyvitamin D (137 pg per milliliter). Vitamin D3, 4000 units per day for four weeks, increased the serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D to as high as 29 and 297 pg per milliliter, respectively, and corrected the hypocalcemia and secondary hyperparathyroidism. The results suggest that the disorder results from impaired end-organ response to 1,25-dihydroxyvitamin D. We propose that the entity be called vitamin-D-dependent rickets Type II.