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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.

An international 19-laboratory survey was organized to compare assays for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D in plasma. Each participant received two ethanolic standard solutions of each metabolite and eight plasma samples. Each laboratory used its usual procedures. Mean interlaboratory coefficients of variation (CVs) for the eight plasma samples were 35%, 43%, and 52% for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D, respectively. Average CVs for the standard solutions were 27%, 23%, and 25%, respectively. Of the eight plasma samples, five had the same concentration for one of the metabolites. One sample was diluted to 0.6 times its original concentration and three samples were fortified with one or more of the metabolites under investigation. Fourteen of 18 laboratories (78%) could distinguish between the five unchanged samples and the modified ones with their 25-hydroxyvitamin D assay. Nine of 12 (75%) could distinguish the modified samples from the other samples with the 24,25-dihydroxyvitamin D assay. Only eight of 15 (53%) could do this their 1,25-dihydroxyvitamin D assay. Values from different laboratories evidently cannot be intercompared without making an actual comparison of the assay procedures. Furthermore, in case of clinical applications of these assays, each laboratory should establish its own reference values and should continually use an internal reference sample to assess the precision of the procedures.

In order to elucidate if changes in vitamin D metabolism play a role for diabetic bone loss, the serum concentrations of the major vitamin D metabolites were studied in 26 adult male ambulatory insulin-treated diabetics, selected to have normal renal function and a duration of diabetes below 11 years. The patients were studied during usual metabolic control and exhibited wide ranges of hyperglycaemia and glycosuria. The serum concentrations of the major metabolites of vitamin D, 25-hydroxyvitamin D(2 + 3) (25OHD), 24,25-dihydroxyvitamin D(2 + 3) (24,25(OH)2D), and 1,25-dihydroxyvitamin D(2 + 3) (1,25(OH)2D), were measured in diabetics, and in age and sex matched controls. The diabetics had slightly decreased serum levels of 25OHD (42.0 nmol/l versus 55.5 nmol/l in normals, P less than 0.05), markedly decreased serum levels of 24,25(OH)2D (2.98 nmol/l versus 5.91 nmol/l, P less than 0.01), but serum levels of 1,25(OH)2D were virtually normal (64.2 pmol/l versus 68.3 pmol/l, ns). The close correlation between serum concentrations of 25OHD and 24,25(OH)2D observed in the normal subjects, was absent in the diabetics. There were no correlations between the serum levels of any of the vitamin D metabolites and the measured indices of glucose and calcium metabolism. It is concluded that insulin-dependent diabetic patients demonstrate definite alterations in serum levels of vitamin D metabolites, the significance of which remains unknown at present.

25OHD, 1,25,-(OH))2D, and 24,25-(OH)2D were assayed in the serum of 16 patients with the infantile, childhood, or adult form of hypophosphatasia. Except for diminished 1,25-(OH)2D and elevated 24,25-(OH)2D levels in 2 infants (which could be attributed to nonparathyroid hormone-mediated hypercalcemia), the mean circulating level of each vitamin D metabolite was normal in the 3 patient groups. Abnormalities in vitamin D metabolism do not appear to contribute to the pathogenesis of this rare hereditary form of rickets or osteomalacia, which occurs despite normal circulating calcium, inorganic phosphate, and vitamin D metabolite levels.

Using the Haddad modified method, 25-OH-D were measured in the blood of the umbilical cord of 29 infants and in peripheral serum after 6 weeks. 16 infants were given a daily dosage of 1000 I. E., 13 infants 500 I. E. vitamin D against rickets. Further they were fed with an adapted milk containing 400 I. E. vitamin D/1. The mean cord serum values were 13 and 15 ng/ml. After treatment with 1000 I. E., 25-OH-D values around 54 ng/ml were measured after 6 weeks and under 500 I. E. daily, values of 37 ng/ml, respectively. Treatment using a dosage of 500 I. E. vitamin D combined with feeding with vitamin D fortified milk seems adequate, to prevent vitamin D depletion.

A high-performance liquid chromatographic (HPLC) method for estimating plasma 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) is described. The method involves plasma extraction, Lipidex 5000 chromatography and HPLC on straight-phase Zorbax-SIL, collecting the 25-OHD2 + 25-OHD3 fractions. These secosteroids are isomerised to their isotachysterol derivatives and re-run in the same HPLC system, monitoring at 290 nm. 3H-Labelled 25-OHD3 is used as an internal standard. The method was evaluated in terms of reproducibility, and recovery of added secosteroids was quantitative. Values obtained using this method were in close agreement with those values obtained on the same plasma sample using gas chromatography--mass spectrometry.

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.

Plasma levels of 25-hydroxyvitamin D3 (25-OH-D3) and 25-hydroxyvitamin D2 (25-OH-D2) in 758 Japanese healthy subjects (most of them adults) were determined by a high-performance liquid chromatographic method previously reported (6) and the following results were obtained: The mean and standard deviation (M +/- SD) of the assayed values of 25-OH-D (sum of 25-OH-D3 and 25-OH-D2) was 23.8 +/- 10.1 ng/ml. 25-OH-D3 was detected in all the samples and the M +/- SD was 23.0 +/- 10.1 ng/ml. The plasma levels clearly showed the seasonal variation that the levels in summer were significantly higher than those in winter. Moreover, the plasma levels were significantly correlated with the amounts of UV light in solar radiation. These results strongly suggested that 25-OH-D3 in plasma mainly originated from endogenous vitamin D3 formed by photo-conversion of 7-dehydrocholesterol in skin. 25-OH-D2 was detected only in 18.3% of the plasma samples and the M +/- SD in the detected samples was 4.4 +/- 2.9 ng/ml which was much lower than those of 25-OH-D3. The results suggested that few healthy Japanese are taking daily exogenous vitamin D2 from multivitamin preparations or others. The M +/- SD values of 25-OH-D3 plasma levels in men and women were 26.2 +/- 10.4 and 19.3 +/- 8.0 ng/ml, respectively. The formers were significantly higher than the latters. The results were thought to be due to the reason that men might be outdoors for longer periods than women. When age variation of plasma 25-OH-D3 levels was examined, the levels in the twenties were significantly lower than the other generations. This was confirmed to be due to the low values observed in the female twenties group, but the detailed reason is unclear at the present time. When 4 healthy volunteers were orally administered 400 I.U./day of vitamin D2 every day for 8 weeks, maximum levels (average: 11.5 ng/ml) were observed at the 8 weeks and the levels gradually decreased after stopping the administration. The results suggested that the half life of 25-OH-D2 in plasma might be 4-5 weeks.

The authors report on a new case of severe hypercalcaemia induced by prolonged oral treatment with high doses of vitamin D2. (6 mg/day for 9 months). Blood calcium level did not return to normal until 6 months after the drug was discontinued. The plasma concentration of 25 (OH) D was more than three times the normal value and remained very high throughout the observation period. The plasma concentration of 24, 25 (OH)2 D was slightly raised during treatment but became normal after 15 days, while that of 1,25 (OH)2 D, the active form of vitamin D, remained normal throughout, suggesting that the pathogenesis of vitamin D-induced hypercalcaemia is probably complex.

Plasma 25-hydroxyvitamin D and 24, 25-dihydroxy-vitamin D [24,25-(OH)(2)D] concentrations were measured in normal and chronically dialyzed anephric humans and pigs. Measurement of the 24, 25-(OH)(2)D was preceded by three purification steps involving one Sephadex LH-20 column and two high-pressure liquid chromatographic columns. The final high-pressure liquid chromatography step involved resolution of 25-hydroxy-vitamin D(3)-26,23 lactone and 25,26-dihydroxy-vitamin D(2) from 24,25-dihydroxyvitamin D(2) and 24,25-dihydroxyvitamin D(3) [24,25-(OH)(2)D(3)]. The total 25-hydroxyvitamin D [25-hydroxyvitamin D(2) plus 25-hydroxyvitamin D(3) (25-OHD(3))] was 31.7+/-3.6 ng/ml in the plasma of eight anephric human subjects and 40.1+/-3.7 ng/ml in five normal human subjects. Six of the eight anephric patients had undetectable (<0.2 ng/ml) 24,25-(OH)(2)D concentrations. Two of the eight patients had very low (0.51 and 0.41 ng/ml), but detectable, 24,25-dihydroxyvitamin D(2). The normal human volunteers had plasma 24,25-(OH)(2)D concentrations of 2.8+/-0.7 ng/ml. Chronically dialyzed anephric and normal pigs were given intramuscular injections of massive amounts (5 x 10(6) IU) of vitamin D(3) immediately after surgery (day 0) and again on day 7. In anephric pigs, plasma 25-OHD(3) progressively rose from 12+/-4 ng/ml on day 0 to 705+/-62 ng/ml on day 10. The 25-OHD(3) concentrations in normal pigs rose from 8+/-2 ng/ml on day 0 to 439+/-64 ng/ml on day 10. Plasma 25-OHD(3) was higher in anephrics throughout the experiment, and concentrations were significantly higher (P < 0.05) on days 9 and 10. Plasma 24,25-(OH)(2)D(3) concentrations declined progressively in anephric pigs from 3.6+/-0.6 ng/ml on day 0 to 3.2+/-0.7 ng/ml on day 2. During days 4-10, plasma 24,25-(OH)(2)D(3) was not apparent until plasma 25-OHD(3) was >400 ng/ml. In control pigs, plasma 24,25-(OH)(2)D(3) was elevated from 4.3+/-0.6 ng/ml on day 0 to 178+/-2.7 ng/ml on day 3. Plasma 24,25-(OH)(2)D(3) was significantly higher (P < 0.05) in controls on days 1-8. At the end of the experiment (day 10), 24,25-(OH)(2)D(3) concentrations were similar and not significantly different in both groups (87.0+/-18.4 ng/ml in anephric and 110.3+/-32.1 ng/ml in normal pigs). The identity of the 24,25-(OH)(2)D(3) isolated from anephric pig plasma was confirmed by mass spectroscopy. Our data suggest that anephric humans receiving normal dietary levels of vitamin D(3) have little or no ability to produce 24,25-(OH)(2)D. However, we have shown that pigs produce 24,25-(OH)(2)D(3) when plasma 25-OHD(3) is extremely high (>400 ng/ml).

Vitamin D-deficient pigs, rats and chicks were repleted with four daily oral doses of crystalline ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) containing equal concentrations of each. At 24 h after the last dose, the plasma of each species was analysed for vitamin D and 25-hydroxyvitamin D by standard methods. The mean (+/- S.D.) ratios of plasma cholecalciferol to ergocalciferol concentration were 1.5 +/- 0.1 (pig). 1.7 +/- 0.1 (rat) and 6.3 +/- 1.2 (chick). The mean ratios of plasma 25-hydroxycholecalciferol to 25-hydroxyergocalciferol concentration were 4.0 +/- 0.1 (pig), 0.4 +/- 0.02 (rat) and 10.7 +/- 3.4 (chick). The mean plasma cholecalciferol/ergocalciferol ratios for the 24,25-dihydroxy-, 25,26-dihydroxy- and 1,25-dihydroxy-derivatives in the pig were 2.6 +/- 0.6, 5.8 +/- 1.3 and 5.8 +/- 0.8 respectively. This is the first evidence that mammals other than the New World monkey, like birds, discriminate between ergocalciferol and cholecalciferol. These data, therefore, suggest that species discrimination between the different forms of vitamin D is probably a general phenomenon in mammals. Moreover, this is the first indication of a species (rat) that discriminates against a cholecalciferol metabolite in favour of an ergocalciferol metabolite. Species discrimination against particular forms of vitamin D may be important to the choice of experimental animal models for studying the regulation of vitamin D metabolism and may also be an important consideration in dietary vitamin supplementation.

Vitamin D-deficiency rickets continues to be reported in infants fed human milk, and the importance of human milk as a source of vitamin D for infants is controversial. Furthermore, effects of race and of normally consumed maternal vitamin D intake on human-milk vitamin D have not been reported. Milk, serum, and three-day-diet diaries were obtained from 25 mother-infant pairs. Human-milk vitamins D3 and D2 and 25-hydroxyvitamin D3 were lower in blacks vs whites, whereas 25-hydroxyvitamin D2 did not differ. Total-milk vitamin D, but not 25-hydroxyvitamin D, correlated with vitamin D intake. Milk vitamin D2 specifically was correlated with vitamin D intake even after controlling for race. Infant serum 25-hydroxyvitamin D did not correlate with milk vitamin D or 25-hydroxyvitamin D; we speculate that the contribution of vitamin D from human milk in these infants is insignificant relative to the contribution from sunshine exposure.

A study was undertaken in 46 subjects; 21 patients diagnosed as having HRL and 25 volunteers patients. Biochemical and hormonal analyses were performed in the study population, including determination of Ca, P, Mg, Cr in blood and urine, phosphate tubular resorption (PTR), maximum tubular phosphate resorption (MTPR), fasting calcium secretion (FCS), alkaline phosphatase (AP), hydroxyprolinuria (HPR), osteocalcin (BGP), parathormone (PTH), cAMP, and 1-25(OH)2D. The stone formers showed lower calcemia values (p less than or equal to 0.005d), higher phosphaturia, and magnesiuria (p less than or equal to 0.0005), higher FCS (P less than or equal to 0.005) and higher values for PTH (p less than or equal to 0.01) and cAMP (p less than or equal to 0.0025). No significant differences were observed for the other parameters evaluated. Classification of the patient group into 2 subgroups (renal SbR and absorptive SbA) according to FCS values greater or lower that 0.16 mg/dl, the SbR patient group revealed a higher PTH and 1-25(OH)2D values (p less than or equal to 0.05). There appears to be no increase of bone resorption since AP, HPR, and BGP values in our patients fell within normal ranges. The 1-25(OH)2D levels were also normal and, with respect to the control group, were only elevated for the SbR patient group, whose PTH levels were also observed to be elevated. These increments appear to be related and may result in intermediate forms between renal and absorptive hypercalciuria.

The serum concentrations of the major vitamin D metabolites (25-hydroxyvitamin D(2 + 3) (25-OH-D), 1,25-dihydroxyvitamin D(2 + 3) (1,25-(OH)2-D), and 24,25-dihydroxyvitamin D(2 + 3) (24,25-(OH)2-D) were studied in 22 healthy male volunteers before and after one or two treatments with whole body UVB or PUVA in conventional doses. The effect of UVB was investigated in the autumn and early spring, whereas the effect of PUVA was investigated only in the autumn. The pre-treatment values of two metabolites were significantly reduced in the spring compared to the autumn level (25-OH-D: 14.0 ng/ml versus 22.0 ng/ml, P less than 0.02; and 24,25-(OH)2-D: 1.23 ng/ml versus 2.74 ng/ml, P less than 0.01), whereas the serum concentration of 1,25-(OH)2-D was not significantly reduced in the spring. After UVB, a small, but not significant, rise in all metabolites was observed in the spring, whereas virtually no changes were measured after UVB or PUVA in the autumn. We conclude that UVB and PUVA do not lead to harmful concentrations of vitamin D metabolites in the blood of healthy subjects.

This report describes a microassay for 1,25-dihydroxyvitamin D [1,25-(OH)2D] in plasma which does not require high performance liquid chromatography. The assay involves rapid extraction and preliminary purification on a C-18 Sep-Pak cartridge, followed by final purification on a silica Sep-Pak using hexane-isopropanol. Quantitation of 1,25-(OH)2D is achieved using a nonequilibrium assay employing 1,25-(OH)2D receptor from calf thymus. The method is sensitive to 1.5 pg/tube, with B50 occurring at 9 pg/tube and a useful assay range of 1.5-40 pg/tube. The intra- and interassay coefficients of variation are 6.5% and 11.5%, respectively, and the method is linear over a wide range of sample dilutions. In addition, this assay measures both 1,25-(OH)2D2 and 1,25-(OH)2D3 with equal affinity. The importance of using an assay with equal affinity for 1,25-(OH)2D2 and 1,25-(OH)2D3 is demonstrated by the findings that 25-hydroxyvitamin D2 (250HD2) constituted 38.9% of the total 25-OHD found in clinical samples (12.6 +/- 0.7 ng/ml 25-OHD2 vs. 20.1 +/- 0.5 ng/ml 25-OHD3; n = 807). Results of this new assay have been compared to those of the assay of Horst et al. (21), which employs Sephadex LH-20 and high performance liquid chromatography sample purification. The correlation coefficient was r2 = 0.96, and the slope was 1.05. Using this new assay, plasma 1,25-(OH)2D concentrations were as follows: normal adults, 37.4 +/- 2.2 pg/ml (n = 22); chronic renal failure, 10.6 +/- 1.5 pg/ml (n = 7); anephrics, undetectable (n = 10); infant cord blood, 22.9 +/- 4.4 pg/ml (n = 7); and hyperparathyroidism, 68.9 +/- 5.0 pg/ml (n = 13). This assay should be particularly useful in pediatric or other studies in which sample size is limited.

Recent evidence suggests that vitamin D plays an important role in calcium homeostasis during pregnancy and early extrauterine life. Vitamin D is metabolized by successive hydroxylations to 25-hydroxyvitamin D and then to 1,25-dihydroxyvitamin D, the most potent known metabolite of the vitamin. During pregnancy, the concentrations of this metabolite in maternal serum increase in parallel with the increased need to absorb dietary calcium. 1,25-Dihydroxyvitamin D is produced in the fetoplacental unit as well as in the maternal kidneys. Receptors for 1,25-dihydroxyvitamin D appear to be present in the placenta suggesting that the placenta may be a target for vitamin D action. Developmental changes in vitamin D metabolism and action have been documented in the neonate as well as in the mother and fetus. Clinical studies indicate that adequate vitamin D intake is important during pregnancy. Administration of vitamin D or its metabolites appears to be useful in the treatment of neonatal disorders.

The effect of parenteral administration of magnesium was studied in five patients with hypomagnesaemic hypocalcaemia. The initial metabolic state was characterized by a normal level of serum immunoreactive parathyroid hormone (iPTH), and by low or undetectable serum 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25 (OH)2D). A parathyroid response was elicited by the acute intravenous injection of magnesium chloride. In contrast, 1,25(OH)2D did not change up to 24 h after the injection. Intramuscular magnesium sulphate restored serum magnesium and calcium to normal, whereas iPTH was transiently increased. 25OHD remained low and unchanged. 1,25(OH)2D rose very slowly, but the correction of hypocalcemia began before any change in 1,25(OH)2D levels could be demonstrated. Thus, the early correction of hypocalcemia mainly depended on the restoration of an adequate parathyroid function independently of the secretion of 1,25(OH)2D.

1. Protein-binding assays have been used to measure plasma 1,25-dihydroxy-vitamin D [1,25-(OH)2D] as well as 25-hydroxy-vitamin D [25-(OH)D] in rats given 10 mg of phosphorus (P) day(-1) kg(-1) as ethane-1-hydroxy-1,1-diphosphonate (EHDP). 2. In control animals given a normal laboratory chow plasma 25-(OH)D and 1,25-(OH)2D were about 40 nmol/l and 300 pmol/l respectively. 3. EHDP produced a decrease of plasma 1,25-(OH)2D to below 50 pmol/l in 2 days. 4. Both in control and in EHDP-treated rats plasma 1,25-(OH)2D increased when dietary calcium (Ca) was restricted to 0.1%, or dietary P to 0.2%, indicating that the well-known stimulatory effect of Ca or P deprivation was at least partially effective in EHDP-treated rats. 5. In response to an increase of the oral supply of vitamin D3 to 65 nmol/day the plasma level of 25-(OH)D rose in both control and EHDP groups. Plasma 1,25-(OH)2D was not increased above the normal value in control rats. In EHDP-treated rats, however, plasma 1,25-(OH)2D rose to a level equal to that in controls, suggesting that the effect of EHDP on plasma 1,25-(OH)2D can be overcome at high precursor concentration.

Total plasma 25-hydroxyvitamin D (25-OHD) and its components 25-OHD2 and 25-OHD3 were measured during the winter in young and elderly healthy persons and in elderly women patients in a long-stay mental hospital. The long-stay patients had no exposure to sunlight or other ultraviolet irradiation, but their diet alone maintained an average total 25-OHD level of 16 nmol/l (range, 10.3-26.5 nmol/l), with most values within the ranges observed in the other two groups, although the average value was lower. The plasma 25-OHD2 which is derived exclusively from vitamin D2 in the diet was similar in all three groups and the vitamin D intake was also similar in the two elderly groups. 25-OHD3 is obtained from the diet as well as from the skin and in the elderly patients the diet alone maintained equal concentrations of 25-OHD2 and 25-OHD3. These findings taken together suggest that the diet accounted for 70 per cent of the total 25-OHD in the active elderly in winter compared with 50 per cent in the young subjects. In all our subjects, young or old, the diet was the major source of vitamin D.

An assay procedure capable of determining 25(OH)D2 (25-hydroxyvitamin D2), 25(OH)D3 (25-hydroxyvitamin D3) and total 24,25(OH)2D (24,25-dihydroxyvitamin D) in a single 2-5 ml sample of human serum is described. The assay involves methanol/chloroform extraction of serum lipids followed by separation of the mono- and dihydroxyvitamin D metabolites and purification from interfering contaminants by chromatography on Sephadex LH-20 and by high-performance liquid chromatography (HPLC). 25(OH)D2 and 25(OH)D3 are quantified by HPLC using UV detection. Total 24,25(OH)2D is routinely measured by a competitive protein-binding (CPB) assay with human serum as a source of the binding protein. Mean values (+/- SD) in 16 normal human serum samples taken in October-November were: 25(OH)D2 5.1 +/- 2.2 ng/ml (12.8 +/- 5.5 nmol/l); 25(OH)D3 16.6 +/- 4.4 ng/ml (41.5 +/- 11.0 nmol/l); total 25(OH)D 21.7 +/- 4.9 ng/ml (54.3 +/- 12.3 nmol/l); total 24,25(OH)2D 1.6 +/- 0.6 ng/ml (3.8 +/- 1.4 nmol/l) and the ratio 24,25(OH)2D/25(OH)D 0.066 +/- 0.024. The ratio was 0.048 +/- 0.009 in women and 0.081 +/- 0.022 in men (p less than 0.002). Values in three serum samples taken from patients receiving vitamin D3 therapy were: 25(OH)D2 4.1 +/- 2.7 ng/ml (10.3 +/- 6.8 nmol/l); 25(OH)D3 46.6 +/- 20.9 ng/ml (116.5 +/- 52.3 nmol/l); total 24,25(OH)2D 3.4 +/- 1.9 ng/ml (8.2 +/- 4.6 nmol/l); 24,25(OH)2D/25(OH)D 0.075 +/- 0.022.

The skeletal changes in 19 very low birthweight infants (less than 1500 g) were observed from birth to 10 weeks, by means of clinical, biochemical, and radiological techniques. All infants were receiving a supplement of 800 IU vitamin D a day from age 2 weeks. None of the infants showed any specific physical sign of rickets during the period of study. Six infants showed radiological evidence of skeletal demineralisation; 1 of these had severe changes of rickets and 1 had both rickets and fractures. These 6 infants were of shorter gestational periods and lower birthweights than the infants not showing radiological changes. They tended to have more clinical problems and to reach a predetermined volume of feeds (160 ml/kg a day) later than the unaffected infants. Serum alkaline phosphatase values were significantly higher at 5 weeks in the infants with abnormal radiographs than in those without. There were no significant differences between the two groups in relation to serum calcium, inorganic phosphate, 25 hydroxyvitamin D, and immunoreactive parathyroid hormone. The pathogenesis of the skeletal lesions of very low birthweight infants remains unknown.

A comparison of two regimens of vitamin D prophylaxis in elderly institutionalized subjects has been made. Sixty-six subjects received 2.5mg vitamin D2 by mouth in December 1982; a further group of 62 received the same dose in December 1982 and again in June 1983. Blood samples have been analysed for 25-hydroxyvitamin D2 [25(OH)D2] in 10 subjects from each group over a 12-month period. The serum 25(OH)D2 was maintained at normal concentrations for most of the year in the once-a-year dosage group but at the end of 12 months only 60% were fully protected. The twice-per-year regimen offered complete protection maintaining the serum 25(OH)D2 above the threshold associated with osteomalacia. It is concluded that a twice-yearly regimen of vitamin D supplementation is a practical method for prophylaxis in institutionalized elderly people.

Despite great interest in the elevated circulating levels of calcitriol (1,25-[OH]2D) associated with the clinical syndrome of human primary hyperparathyroidism, the relative potencies of known and potential stimuli/suppressors of long-term calcitriol levels have not been evaluated in either clinical or experimentally induced hyperparathyroid states. Based on reports that aparathyroid animals exhibit suppressed plasma calcitriol concentration and that acute administration of parathyroid hormone (PTH) to both humans and experimental animals or to renal slices in vitro results in increased plasma calcitriol concentration/production rate, it might be predicted that a chronic experimental model of either hypercalcemic primary hyperparathyroidism or hypocalcemic secondary hyperparathyroidism would show increased plasma calcitriol concentration. Chronic alterations in plasma calcium concentration have not been implicated as modulating calcitriol levels in any species. Accordingly, we investigated the long-term response of plasma calcitriol concentration in states of sustained experimental primary and secondary hyperparathyroidism. Intact dogs (group I) undergoing continuous intravenous PTH infusion for 12 d developed sustained hypercalcemia and hypophosphatemia, and plasma calcitriol concentration decreased from 23 +/- 3 to 14 +/- 3 pg/ml (P less than 0.01). Subsequent chelator (EGTA)-induced chronic normalization of hypercalcemia during ongoing PTH infusion resulted in a large and sustained increase in plasma calcitriol concentration to supernormal levels, reversible during subsequent cessation of chelator infusion. In additional intact dogs (group II), chronic chelator-induced hypocalcemic secondary hyperparathyroidism resulted in a sustained increase in plasma calcitriol concentration despite hyperphosphatemia. In normal human subjects undergoing a 12-13-d continuous intravenous PTH infusion to result in sustained moderate hypercalcemia (12.0 +/- 0.2 mg/100 ml) and hypophosphatemia, plasma calcitriol concentration decreased significantly (P less than 0.01) as in group I dogs and was followed by reversal to normal levels in a recovery period. The present results provide strong evidence in both humans and dogs that during experimentally induced chronic PTH excess, alterations in plasma calcium concentration dictate the directional response of circulating calcitriol concentrations. The long-term potency of plasma calcium concentration as a modulator of calcitriol metabolism is sufficient to override opposing modulation by plasma phosphorus concentration and PTH.

The levels of vitamin D metabolites were measured in three children with a decreased dietary intake of calcium and vitamin D and sun exposure. All three children had hypocalcemia, hypophosphatemia, and elevated alkaline phosphatase activities. Two children had rickets, aminoaciduria, and elevated immunoreactive parathyroid hormone (iPTH) concentrations. The concentrations of vitamins D2 and D3, 25-hydroxyvitamins D2 and D3 (25-OH-D2 and 25-OH-D3), and 24,25-dihydroxyvitamin D (24,25-[OH]2D) were reduced. Nonetheless, the levels of calcitriol (1,25-[OH]2D) were normal. The combination of hypocalcemia, hypophosphatemia, and increased iPTH concentrations should result in supranormal calcitriol concentrations. Moreover, the ratio of PTH to calcitriol is significantly higher than in normal subjects. Accordingly, in patients with vitamin D deficiency and "normal" calcitriol values, the synthesis of this compound may be reduced. The evaluation of vitamin D deficiency should include the measurement of all metabolites.

Metabolites of vitamin D were measured in plasma from 83 patients with idiopathic infantile hypercalcaemia syndrome who were mentally handicapped but had normal calcium values at the time of the study. No significant difference was detected in the mean plasma concentrations of 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D3, or 25,26-dihydroxyvitamin D3 between patients and age matched controls. The mean plasma concentration of 25-hydroxyvitamin D3 was significantly lower in patients than controls but this may be a secondary phenomenon related to less sunlight exposure. In addition, two hypercalcaemic patients with this syndrome were studied during the first year of life, and were found to have normal concentrations of vitamin D metabolites. These findings do not support a role for abnormal vitamin D metabolism in the pathogenesis of this syndrome.

A survey to assess the vitamin D nutritional state in 119 pregnant women at term and in their newborns was undertaken in Riyadh, Saudi Arabia. Concentrations of 25-hydroxy vitamin D (25-(OH)D) were below 4 ng/ml in 30 of 119 maternal sera, in 11 of which they were undetectable. The median concentration of 25-(OH)D was 5.7 ng/ml, which is comparable to that found in Asian vegetarian women at term in London. Fifty of 119 cord samples had undetectable 25-(OH)D, and a total of 81 samples had 25-(OH)D concentrations of less than 4 ng/ml. Despite the low 25-(OH)D concentrations cord bloods had calcium concentrations higher than those in maternal blood, while serum albumin concentration was similar in maternal and cord samples. Higher socioeconomic background of women, antenatal care, and vitamin D supplementation were associated with significantly higher concentrations of 25-(OH)D. Vitamin D supplementation, however, had no significant effect on 25-(OH)D concentration in cord samples or on the weight of the newborns. This study shows the high prevalence of marginal vitamin D nutrition in women in Saudi Arabia, which may predispose babies to rickets during infancy. In a country endowed with plentiful sunshine, the exclusion of sunshine by thick dark veils and bad housing probably contribute to this marginal state of vitamin D nutrition.

Rickets was diagnosed in an extremely low-birthweight infant 16 weeks after birth. She had a normal plasma concentration of 25-hydroxyvitamin D, a relatively low level of 24,25-dihydroxyvitamin D, and a markedly elevated 1,25-dihydroxyvitamin D level compared with adult standards. The plasma concentrations of the vitamin D metabolites were, however, indistinguishable from those of healthy preterm infants who received a similar diet of human milk and vitamins. The results indicate that rickets was not caused by vitamin D deficiency or by abnormal vitamin D metabolism, but by calcium and/or phosphate deficiency, and that the calcium and phosphorous content of human milk may be inappropriately low for very low-birthweight infants.

We surveyed calcidiol levels (25-OH-D) in a geographically-defined population of 373 women, ages 20-80 yr to test for an association between occult osteomalacia and excess bone loss. Bone mass was measured by photon densitometry and an estimate of vitamin D was determined by measuring dietary and supplemental intake as well as sunlight exposure equivalent. The relationship of smoking practices, alcohol use, exogenous estrogen use, and medications to calcidiol level was assessed. Calcidiol levels were not associated with bone mass levels observed at two different forearm sites. Low levels of calcidiol, indicative of generalized vitamin D deficiency, were not observed in this population though mean estimates of vitamin D intakes from food 119 +/- 148 IU) or food and supplements (319 +/- 463 IU) were less than the Recommended Dietary Allowance (400 IU). Calcidiol was significantly associated with estimates of vitamin D intake from food (r = 0.11), supplement use (r = 0.21), and sunlight equivalent exposure (r = 0.26). Calcidiol levels were negatively related to age (p = 0.0020) and positively related to exogenous estrogen use and premenopausal state independent of age.

The plasma calcemic hormones in mature female reindeer were measured during the major portion of the antler growth cycle, between the months of March and November. Blood samples were collected every 2 months, a total of five samples each from eight reindeer. Plasma levels of 25-hydroxyvitamin D, PTH, and osteocalcin were found to increase progressively from May through July and decline thereafter except 1,25-dihydroxyvitamin D level which peaked in September. No change in plasma total Ca was observed at any time of the year. Plasma estradiol level was elevated in March only. All animals grew and shed antlers between April and February. This suggests that during the antler growth period when the deer undergoes cyclic bone loss, corresponding changes in plasma calcemic hormones occur, which may account for the reported skeletal bone changes seen during the growth of the antler.

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.

Vitamin D appears to influence parathyroid function indirectly through its effects on calcium metabolism rather than by a direct action of its metabolites on the parathyroid glands. In states of both secondary and primary hyperparathyroidism, the quantitative production of 1,25-(OH)2D may be determined by the prevailing concentration of serum 25-(OH)D but there appears to be some constraint that limits the formation of 1,25-0(OH)2D when the provision of its precursor exceeds the physiological. From the absence of this constraint in 'type 2 vitamin D dependency' it is inferred that it may operate through 'self-inhibition' of the renal production of 1,25-(OH)2D. It is shown that the level of serum 25-(OH)D may always exert some influence on the production of 1,25-(OH)2D and that this effect is facilitated by hyperparathyroidism. In developing vitamin D deficiency the reactive secondary hyperparathyroidism may thus function as an adaptive mechanism that sustains the level of serum 1,25-(OH)2D in the face of a diminishing serum 25-(OH)D. Failure of this adaptation and the development of a critical deficiency of 1,25-(OH)2D is regarded as the direct cause of defective mineralisation of bone. This concept would explain the absence of osteomalacia in some patients with very low levels of serum 25-(OH)D and the occurrence of defective osseous mineralisation in hypoparathyroidism.

A sensitive, precise assay for vitamin D in plasma is described. Three to five milliliters of plasma were extracted with methanol:methylene chloride (2:1). The lipid extract was chromatographed on Sephadex LH-20 and then on lipidex-5000 columns. After high pressure liquid chromatography with a reverse phase chromatographic system, vitamin D2 and vitamin D3 were quantitated by ultraviolet absorbance. We used this assay system for monitoring daily changes of vitamin D3 in plasma of two Jersey cows after four intramuscular doses (15 x 10(6) IU) of vitamin D3 administered at weekly intervals. Basal vitamin D in plasma was 3.2 +/- .99 ng/ml with a range of 1.7 to 4.9 ng/ml. Vitamin D3 in plasma remained relatively low (10 to 45 ng/ml) the week after the first vitamin D3 injection. Vitamin D3 was high (130 to 234 ng/ml) after the second, third, and fourth injections. Vitamin D3 decreased steadily to 88 ng/ml by 38 days after the fourth vitamin D3 injection. Phosphorus in plasma increased sharply to a plateau at 9.5 mg/100 ml during the week after the second vitamin D3 injection and returned to normal (4.5 mg/100 ml) at the end of the experiment. Calcium, however, gradually increased to 14.0 mg/100 ml 20 days after the fourth vitamin D3 injection. Both animals remained hypercalcemic (calcium 11.5 mg/100 ml) during the experiment.

Circulating concentrations of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D and 25-hydroxyvitamin D were measured in 21 anephric subjects. 13 subjects had no therapy with vitamin D, dihydrotachysterol or 1 alpha-hydroxyvitamin D3. In 7 subjects of this group 1,25-dihydroxyvitamin D was undetectable (less than 5 pmol/l). In the other 6 patients concentrations ranged from 10 to 43 pmol/l (reference value 111 +/- 33 pmol/l). All subjects taking high doses of vitamin D showed detectable 1,25-dihydroxyvitamin D concentrations in the same range. Dihydrotachysterol therapy caused spuriously high '1,25-dihydroxyvitamin D' values, probably by interference of a metabolite of dihydrotachysterol in our assay. In subjects on vitamin D or dihydrotachysterol therapy 25-hydroxyvitamin D concentrations were significantly elevated (314 +/- 146 nmol/l and 98 +/- 19 nmol/l, respectively; reference value 52 +/- 22 nmol/l). Concentrations of 24,25-dihydroxyvitamin D were only measured in subjects without vitamin D2 intake. In general very low but detectable concentrations were found. One subject on a high dose of vitamin D3 showed a 24,25-dihydroxyvitamin D3 concentration of 10.2 nmol/l (reference value 4.4 +/- 2.9 nmol/l). Our results therefore confirm earlier reports on extrarenal synthesis of 24,25-dihydroxyvitamin D and suggest that there may be extrarenal production of 1,25-dihydroxyvitamin D as well.

A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.

An investigation into plasma calcium concentrations and the biochemical factors which regulate it in vegetarian Asian subjects without clinical or radiological features of osteomalacia revealed the presence of low serum 25-hydroxyvitamin D concentrations and elevated PTH concentrations, even in the presence of calcium levels in the normal range (up to 2.35 mmol/liter). These elevated PTH concentrations, though not as high as those in osteomalacic patients with hypocalcemia, often persisted despite treatment with vitamin D, normalization of 25-hydroxyvitamin D, and an increase in calcium concentrations. In one patient the PTH concentration remained high even when the plasma calcium concentration became supranormal. Therefore, secondary hyperparathyroidism is commonly associated with vegetarianism, and may play an important role in maintaining calcium concentrations within the normal range. Persistent elevation of PTH despite normalization of 25-hydroxyvitamin D also points to autonomous PTH hypersecretion, which may result in osteolysis in the long term, and raises the question of the need for vitamin D supplementation in vegetarians with low dietary intake of vitamin D.

Serum concentrations of 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] of vitamin D2 and D3 origin were determined separately in 10 women before vitamin intake in early pregnancy, and repeated in maternal and cord serum obtained at delivery after 20 to 30 wk of vitamin D2 supplementation in a dose of 400 IU/day. Before supplementation 25-OHD2 and 1,25-(OH)D2D2 were present in just traceable or nondetectable concentrations, but the levels increased in all to a mean +/- 1 SD of 7.3 +/- 3.7 ng/ml and 37.2 +/- 18.1 pg/ml, respectively (p less than 0.0025), by the time of delivery. At delivery the total 25-OHD and 1,25-(OH)2D levels were always lower in the cord than in the maternal serum (30.7 +/- 14.2 versus 20.1 +/- 9.1 ng/ml, and 90.1 +/- 31.2 versus 37.3 +/- 11.6 pg/ml, p less than 0.0025). The paired concentrations of 25-OHD were closely related (r = 0.89, p less than 0.0005), while the association for 1,25-(OH)2D was not statistically significant (r = 0.53, p less than .01). The 25-OHD of D2 and D3 origin accounted for a similar proportion of the total 25-OHD in the maternal and cord serum (ratio of 25-OHD2 to 25-OHD3: 0.40 +/- 0.28 versus 0.45 +/- 0.29, p = NS), as did the respective 1,25-(OH)2D metabolites [ratio of 1,25-(OH)2D2 to 1,25-(OH)2D3: 0.73 +/- 0.35 versus 0.90 +/- 0.50, p = NS].(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D2 was administered orally as a single dose (2 mg) to 19 elderly subjects and 17 young adults. The maximum elevation of serum 25-hydroxyvitamin D was significantly greater in young than in elderly subjects. To evaluate intestinal absorption we also measured serum levels of vitamin D2 5 h after the given dose. A vitamin A absorption test was also performed simultaneously. Small differences between young and old subjects were seen with respect to serum vitamin D2 or vitamin A increments. In both groups the serum level of 25-hydroxyvitamin D was still elevated above initial level 60 days after a dose was given. No side effects, nor any change in serum calcium ion activity were noted during this period. These results speak in favour of the use of intermittent large doses of vitamin D2 as a prophylaxis against vitamin D deficiency in the elderly.

The initial step in cholecalciferol (vitamin D3) metabolism is the photo-conversion of 7-dehydrocholesterol to previtamin D3. This reaction occurs in the epidermis and requires ultraviolet light. We measured the circulating concentration of vitamin D (ergocalciferol and cholecalciferol), 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in 14 normal white, 9 normal black subjects, and in 9 white and 17 black hemodialysis patients. The mean plasma vitamin D in normal white subjects was greater than in normal black subjects, 4.01 +/- 1.02 ng/ml versus 0.96 +/- 0.30 ng/ml, respectively (P less than 0.05). Plasma 25-hydroxyvitamin D in normal blacks was also less than in normal whites, 17.7 +/- 1.5 ng/ml versus 31.3 +/- 3.0 ng/ml, respectively (P less than 0.01). In uremic white subjects, plasma vitamin D, 6.7 +/- 2.6 ng/ml, was similar to normal white subjects. However, vitamin D was not detectable in 12 of 17 uremic black subjects and was depressed in the remainder of the group. Following exposure to a single minimal erythema dose of ultraviolet-B irradiation, the maximal increase in plasma vitamin D was depressed in white dialysis patients as compared to healthy white subjects, 6.3 +/- 1.9 ng/ml versus 21.3 +/- 2.8 ng/ml, respectively (P less than 0.02). 7-Dehydrocholesterol content was similar in epidermis from site-matched skin of fresh cadavers and white hemodialysis patients, 131 +/- 23 ng/mg versus 124 +/- 14 ng/mg skin, respectively. It is concluded that chronic hemodialysis patients exhibit defective photoproduction of cholecalciferol, despite normal epidermal content of substrate, 7-dehydrocholesterol.

Serum calcium, phosphorus, calcitonin, parathyroid hormone, 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D [1,25-(OH)2D] were measured in 6 women and 2 men with medullary carcinoma of the thyroid, 22 normal subjects, 5 patients with chronic renal failure, and 5 patients with primary hyperparathyroidism. Serum 1,25-(OH)2D levels were significantly higher in patients with primary hyperparathyroidism and lower in patients with chronic renal failure than in normal subjects. In patients with medullary carcinoma of the thyroid, the serum calcitonin levels were elevated, but the parathyroid hormone and 1,25-(OH)2D levels were within normal ranges. The serum 25OHD levels were not significantly different in any group. It is concluded that chronic elevation of serum calcitonin has no effect on the serum 1,25-(OH)2D level.

Rapid quantification of serum vitamin D-binding globulin (VDBG) is described. An immunonephelometric method was developed and compared with the conventional immunodiffusion method for VDBG. A correlation coefficient of 0.896 (P less than 0.001) was found with a regression line y = 0.965x + 0.011 (n = 23). The within-assay and day-to-day coefficient of variation of the immunonephelometric method was 6 and 11% at 0.150 g/l (low VDBG concentration), 5 and 7% at 0.230 g/l (normal), and 4 and 8% at 0.570 g/l (high). Levels of VDBG and total 25-hydroxyvitamin D (25(OH)D, 25(OH)D2 + 25(OH)D3) were analysed from 33 serum samples obtained in April-May 1980 from 14-month-old children undergoing vitamin D substitution. 25(OH)D was extracted from 0.1-0.5 ml serum followed by semipreparative silica column chromatography and final quantification by a competitive protein-binding method. The VDBG mean value was 0.308 +/- 0.068 g/l (SD) and 25(OH)D had a mean value of 121 +/- 70 nmol/l (SD). There was no significant correlation between the levels of VDBG and 25(OH)D. The results of this study in general agreed well with other findings.