Evidence for alteration of the vitamin D-endocrine system in blacks

As compared with values in white subjects, bone mass is known to be increased and urinary calcium to be diminished in black individuals. To evaluate the possibility that these changes are associated with alterations in the vitamin D-endocrine system, an investigation was performed in 12 black subjects, 7 men and 5 women, and 14 white subjects, 8 men and 6 women, ranging in age from 20 to 35 yr. All of them were hospitalized on a metabolic ward and were given a constant daily diet containing 400 mg of calcium, 900 mg of phosphorus, and 110 meq of sodium. Whereas mean serum calcium, ionized calcium, and phosphate were the same in the two groups, mean serum immunoreactive parathyroid hormone (350 +/- 34 vs. 225 +/- 26 pg/ml, P less than 0.01) and mean serum 1,25-dihydroxyvitamin D (1,25(OH)2D) (41 +/- 3 vs. 29 +/- 2 pg/ml, P less than 0.01) were significantly higher, and mean serum 25-hydroxy-vitamin D (25-OHD) was significantly lower in the blacks than in the whites (6 +/- 1 vs. 20 +/- 2 ng/ml, P less than 0.001). Mean urinary sodium and 24-h creatinine clearance were the same in the two groups, whereas mean urinary calcium was significantly lower (101 +/- 14 vs. 166 +/- 13 mg/d, P less than 0.01) and mean urinary cyclic AMP was significantly higher (3.11 +/- 0.47 vs. 1.84 +/- 0.25 nM/dl glomerular filtrate, P less than 0.01) in the blacks. Further, the blacks excreted an intravenous calcium load, 15 mg/kg body weight, as efficiently as the whites (49 +/- 3 vs. 53 +/- 3%, NS). Mean serum Gla protein was lower in blacks than in whites (14 +/- 2 vs. 24 +/- 3 ng/ml, P less than 0.02), and increased significantly in both groups in response to 1,25(OH)2D3, 4 micrograms/d for 4 d. There was a blunted response of urinary calcium to 1,25(OH)2D3 in the blacks, and mean serum calcium did not change. The results indicate that alteration of the vitamin D-endocrine system with enhanced renal tubular reabsorption of calcium and increased circulating 1,25(OH)2D as a result of secondary hyperparathyroidism may contribute to the increased bone mass in blacks. Their low serum 25-OHD is attributed to diminished synthesis of vitamin D in the skin because of increased pigment.     

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++.     

Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D. A role for increased sensitivity to calcium.

Numerous in vitro studies in experimental animals have demonstrated a direct suppressive effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on parathyroid hormone (PTH) synthesis. We therefore sought to determine whether such an effect could be demonstrated in uremic patients undergoing maneuvers designed to avoid changes in serum calcium concentrations. In addition, the response of the parathyroid gland in patients undergoing hypercalcemic suppression (protocol I) and hypocalcemic stimulation (protocol II) before and after 2 wk of intravenous 1,25(OH)2D was evaluated. In those enlisted in protocol I, PTH values fell from 375 +/- 66 to 294 +/- 50 pg (P less than 0.01) after 1,25(OH)2D administration. During hypercalcemic suppression, the "set point" (PTH max + PTH min/2) for PTH suppression by calcium fell from 5.24 +/- 0.14 to 5.06 +/- 0.15 mg/dl (P less than 0.05) with 1,25(OH)2D. A similar decline in PTH levels after giving intravenous 1,25(OH)2D was noted in protocol II patients. During hypocalcemic stimulation, the parathyroid response was attenuated by 1,25(OH)2D. We conclude that intravenous 1,25(OH)2D directly suppresses PTH secretion in uremic patients. This suppression, in part, appears to be due to increased sensitivity of the gland to ambient calcium levels.     

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.     

Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients

Current evidence suggests that administration of 1,25(OH)2D3 to patients with chronic renal insufficiency results in suppression of secondary hyperparathyroidism only if hypercalcemia occurs. However, since the parathyroid glands possess specific receptors for 1,25(OH)2D3 and a calcium binding protein, there is considerable interest in a possible direct effect of 1,25(OH)2D3 on parathyroid hormone (PTH) secretion independent of changes in serum calcium. Recent findings indicate substantial degradation of 1,25(OH)2D3 in the intestine, therefore, it is possible that while oral administration of the vitamin D metabolite increases intestinal calcium absorption, the delivery of 1,25(OH)2D3 to peripheral target organs may be limited. We therefore compared the effects of orally or intravenously administered 1,25(OH)2D3 on the plasma levels of 1,25(OH)2D3 and the effects of these two modes of treatment on PTH secretion. Whereas oral administration of 1,25(OH)2D3 in doses adequate to maintain serum calcium at the upper limits of normal did not alter PTH levels, a marked suppression (70.1 +/- 3.2%) of PTH levels was seen in all 20 patients given intravenous 1,25(OH)2D3. Temporal studies suggested a 20.1 +/- 5.2% decrease in PTH without a significant change in serum calcium with intravenous 1,25(OH)2D3. In five patients the serum calcium was increased by the oral administration of calcium carbonate, the decrement in serum i-PTH was only 25 +/- 6.65% when compared with 73.5 +/- 5.08% (P less than 0.001) obtained by the administration of intravenous 1,25(OH)2D3. Thus, a similar serum calcium achieved by intravenous 1,25(OH)2D3 rather than calcium carbonate has a greater suppressive effect in the release of PTH. These studies indicate that 1,25(OH)2D3 administered intravenously rather than orally may result in a greater delivery of the vitamin D metabolite to peripheral target tissues other than the intestine and allow a greater expression of biological effects of 1,25(OH)2D3 in peripheral tissues. The use of intravenous 1,25(OH)2D3 thus provides a simple and extremely effective way to suppress secondary hyperparathyroidism in dialysis patients.     

Evidence for in vivo upregulation of the intestinal vitamin D receptor during dietary calcium restriction in the rat.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] increases intestinal calcium absorption through events that include binding of 1,25(OH)2D3 to the intracellular vitamin D receptor. In vitro studies using mammalian cell cultures reveal an increase in vitamin D receptor content after exposure to 1,25(OH)2D3. To test the hypothesis that 1,25(OH)2D3 increases enterocyte vitamin D receptor content in vivo, male rats were fed either a normal calcium diet (NCD, 1.2% Ca) or low calcium diet (LCD, 0.002% Ca). After 21 d LCD increased serum 1,25(OH)2D3 levels (27 +/- 3 vs. 181 +/- 17 pg/ml, P less than 0.001) and increased transepithelial mucosal to serosal calcium fluxes (Jms) across duodenum (65 +/- 21 vs. 204 +/- 47 nmol/cm2.h, NCD vs. LCD, P less than 0.01) and jejunum (23 +/- 3 vs. 46 +/- 4, P less than 0.007). No change in serosal to mucosal calcium fluxes (Jsm) were observed. LCD increased 1,25(OH)2D3 receptor number threefold in duodenum (32.9 +/- 6.7 vs. 98.7 +/- 13.7 fmol 1,25(OH)2D3/mg protein) and jejunum (34.1 +/- 9.5 vs. 84.9 +/- 7.7) without a change in the receptor affinity for 1,25(OH)2D3 (Kd is 0.17 +/- 0.06 vs. 0.21 +/- 0.02 nM for NCD and LCD duodenum, respectively). Duodenal polyadenylated vitamin D receptor mRNA determined by Northern blot analysis did not increase appreciably during LCD, suggesting that upregulation in vivo may not be due primarily to increased receptor synthesis. The results of this study indicate that under physiologic conditions as during chronic dietary calcium restriction, increased intestinal vitamin D receptor content accompanies increased calcium active transport. Upregulation of the vitamin D receptor by 1,25(OH)2D3 may result primarily from posttranslational processes that decrease degradation of the receptor with increased receptor synthesis responsible for a negligible portion of the accumulation.     

Effects of active vitamin D3 and parathyroid hormone on the serum osteocalcin in idiopathic hypoparathyroidism and pseudohypoparathyroidism.

Serum osteocalcin was measured in patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism, before or during the treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). Serum osteocalcin and plasma 1,25(OH)2D were decreased in 11 patients with idiopathic hypoparathyroidism before treatment (2.8 +/- 1.27 ng/ml, P less than 0.001 and 14.3 +/- 4.27 pg/ml, P less than 0.001, respectively). In 24 patients with idiopathic hypoparathyroidism during the treatment, serum osteocalcin and plasma 1,25(OH)2D were within the normal range (4.5 +/- 0.74 ng/ml and 25.7 +/- 5.69 pg/ml, respectively). In five patients with pseudohypoparathyroidism before treatment, plasma 1,25(OH)2D was decreased (15.6 +/- 10.6 pg/ml, P less than 0.001) but serum osteocalcin was normal (7.8 +/- 1.66 ng/ml). In nine patients with pseudohypoparathyroidism during the treatment with active vitamin D3, serum osteocalcin and plasma 1,25(OH)2D were normal (6.8 +/- 1.47 ng/ml and 27.2 +/- 6.0 pg/ml, respectively). Serum PTH in pseudohypoparathyroidism was increased before treatment (0.70 +/- 0.34 ng/ml, P less than 0.05) and was normal during the treatment (0.50 +/- 0.13 ng/ml). In idiopathic hypoparathyroidism, the active vitamin D3 increased serum osteocalcin without PTH. In pseudohypoparathyroidism, PTH may increase serum osteocalcin or modulate the effect of active vitamin D3 on serum osteocalcin.     

Demonstration that circulating 1 alpha, 25-dihydroxyvitamin D is loosely regulated in normal children.

The effects of vitamin D, 2.5 mg (100,000 U)/d for 4 d, on serum calcium, serum 25-hydroxyvitamin D (25-OHD) and serum 1 alpha, 25-dihydroxyvitamin D (1 alpha, 25(OH)2D) were compared in 24 normal adults and 12 normal children. The daily dose of vitamin D was 1,500 U/kg body wt in children weighing less than 45 kg. Vitamin D increased mean serum calcium from 9.5 +/- 0.1 to 9.8 +/- 0.1 mg/dl (P less than 0.05), increased mean serum phosphorus from 4.6 +/- 0.1 to 5.0 +/- 0.1 mg/dl (P less than 0.01), increased mean serum 25-OHD from 25 +/- 3 to 34 +/- 4 ng/ml (P less than 0.001), and increased mean serum 1 alpha, 25(OH)2D from 34 +/- 3 to 42 +/- 4 pg/ml (P less than 0.02) in children. In contrast, vitamin D increased mean serum 25-OHD from 18 +/- 2 to 39 +/- 6 ng/ml (P less than 0.001) and did not change mean serum calcium (9.4 +/- 0.1 vs. 9.5 +/- 0.1 mg/dl), mean serum phosphorus (4.0 +/- 0.1 vs. 4.1 +/- 0.1 mg/dl), or mean serum 1 alpha, 25(OH)2D (31 +/- 2 vs. 29 +/- 3 pg/ml) in adults. Mean serum 1 alpha, 25(OH)2D was significantly higher after vitamin D in children than in adults (P less than 0.02). These results provide evidence that circulating 1 alpha, 25(OH)2D is not as tightly regulated in children as it is in adults. This difference in regulation could account in part for the higher values for serum 1 alpha, 25(OH)2D observed in children.     

Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat.

In vivo in the rat 1,25(OH)2D3 decreases and a low calcium increases PTH mRNA levels. We now report the effect of 3 and 8 wk of changes in dietary vitamin D and calcium on PTH mRNA levels. PTH mRNA levels were increased by 3 wk of calcium deficiency (five times), a vitamin D-deficient diet (two times), and combined deficiency (10 times), but not changed by high calcium. Vitamin D-deficient-diet rats' PTH mRNA did not decrease after a single large dose of 1,25(OH)2D3, but did decrease partially after repeated daily doses of 1,25(OH)2D3. Rats after a vitamin D-, calcium-deficient (-D-Ca) diet did not respond to changes in serum calcium at 1 h. Flow cytometry of isolated cells from parathyroid-thyroid tissue separated the smaller parathyroid from the larger thyroid cells and allowed an analysis of parathyroid cell number. In normal vitamin D/normal calcium (NDNCa) rats the parathyroid cells were 24.7 +/- 3.4% (n = 6) of the total cell number, whereas in -D-Ca rats they were 41.8 +/- 6.6% (n = 6) (P less than 0.05). That is, -D-Ca rats had 1.7 times the number of cells, whereas they had 10 times the amount of PTH mRNA, indicating the major contribution (6 times) of increased PTH gene expression per cell. Moreover, a calcium-deficient, more so than a vitamin D-deficient diet, amplifies the expression of the PTH gene, and vitamin D is necessary for an intact response of PTH mRNA to 1,25(OH)2D3 or calcium.     

Plasma 1,25(OH)2D3 response to parathyroid hormone, cyclic adenosine monophosphate, and phosphorus depletion in the spontaneously hypertensive rat

Spontaneously hypertensive rats (SHR) have several abnormalities of calcium metabolism compared with normotensive control Wistar-Kyoto (WKY) rats. Previously the vitamin D metabolite 1,25-dihydroxycholecalciferol (1,25[OH]2D3) was found to be inappropriately low in SHR in view of their ionized hypocalcemia and hyperparathyroidism. We examined the responses of plasma 1,25(OH)2D3 to several known stimuli. Baseline plasma 1,25(OH)2D3 levels tended to be lower in SHR than WKY rats (51.5 +/- 4.3 vs. 82.3 +/- 14.1 pg/ml, P = 0.06). Infusion of a pharmacologic dose of parathyroid hormone (8 U/hr over a period of 17 hours) resulted in a plasma 1,25(OH)2D3 level of 504 +/- 77 pg/ml in SHR vs. 1016 +/- 211 pg/ml in WKY rats (P less than 0.03). Cyclic adenosine monophosphate infusion (1 mumol/hr/100 gm over a period of 17 hours) in thyroparathyroidectomized animals resulted in a 1,25(OH)2D3 level of 121 +/- 24 pg/ml in SHR vs. 557 +/- 26 pg/ml in WKY rats (P less than 0.01). After dietary phosphorus depletion for 3 weeks, SHR also had lower 1,25(OH)2D3 levels than WKY rats (83 +/- 13 vs. 300 +/- 42 pg/ml, P less than 0.001) even though a comparable degree of hypophosphatemia was achieved. Thus, the response of plasma 1,25(OH)2D3 levels to several known stimuli is submaximal in SHR as compared with WKY rats, suggesting defective synthesis or enhanced metabolic clearance of this hormone.     

Oral intake of phosphorus can determine the serum concentration of 1,25-dihydroxyvitamin D by determining its production rate in humans.

Changes in the oral intake of phosphorus could induce the reported changes in the serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) by inducing changes in its production rate (PR) or metabolic clearance rate (MCR), or both. To investigate these possibilities, we employed the constant infusion equilibrium technique to measure the PR and MCR of 1,25-(OH)2D in six healthy men in whom the oral intake of phosphorus was initially maintained at 1,500 mg/70 kg body weight per d for 9 d, then restricted to 500 mg/d (coupled with oral administration of aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. With phosphorus restriction, the serum concentration of 1,25-(OH)2D increased by 80% from a mean of 38 +/- 3 to 68 +/- 6 pg/ml, P less than 0.001; the PR increased from 1.8 +/- 0.2 to 3.8 +/- 0.6 micrograms/d, P less than 0.005; the MCR did not change significantly. The fasting serum concentration of phosphorus decreased from 3.5 +/- 0.2 to 2.6 +/- 0.2 mg/dl, P less than 0.01. With phosphorus supplementation, the serum concentration of 1,25-(OH)2D decreased abruptly, reaching a nadir within 2 to 4 d; after 10 d of supplementation, the mean concentration of 27 +/- 4 pg/ml was lower by 29%, P less than 0.01, than the value measured when phosphorus intake was normal. The PR decreased to 1.3 +/- 0.2 micrograms/d, P less than 0.05; the MCR did not change significantly. The fasting serum concentration of phosphorus increased significantly, but only initially. These data demonstrate that in healthy men, reductions and increases in the oral intake of phosphorus can induce rapidly occurring, large, inverse, and persisting changes in the serum concentration of 1,25-(OH)2D. Changes in the PR of 1,25-(OH)2D account entirely for the phosphorus-induced changes in serum concentration of this hormone.     

Effect of parathyroid hormone activity on gentamicin nephrotoxicity

Dietary calcium (CA++) supplementation attenuates gentamicin nephrotoxicity in rats. It has been proposed that this protective effect results from the ability of Ca++ to interfere with gentamicin binding to renal cell membranes. However, calcium supplementation also suppresses parathyroid hormone (PTH) activity, which may affect gentamicin nephrotoxicity by altering renal brush border phospholipid composition or renal calcium handling. We therefore compared gentamicin nephrotoxicity in PTH-stimulated control rats and parathyroidectomized (PTX) rats. Although their pretreatment serum ionized calcium concentration was significantly higher (1.27 +/- 0.01 vs. 0.88 +/- 0.06 mmol/L; P less than 0.001), PTH-stimulated rats had higher peak renal cortical gentamicin concentrations (543 +/- 20 vs. 395 +/- 49 micrograms/gm; P less than 0.025) and serum creatinine concentrations (3.0 +/- 0.8 vs. 0.9 +/- 0.3 mg/dl; P less than 0.05). Structural injury and depression of renal cortical slice uptake of p-aminohippurate were also less severe in PTX rats. Gentamicin treatment also caused increased urinary Ca++ excretion in control rats (from 2.12 +/- 0.64 mumol/mg creatinine per day [pretreatment] to 16.86 +/- 2.07 mumol/mg creatinine per day; P less than 0.001) but not in PTX rats. Control rats ingesting chow containing a standard Ca++ content (1.2%) resembled PTX rats. These results indicate that PTH stimulation exacerbates gentamicin nephrotoxicity. Increased peak renal cortical gentamicin concentrations in PTH-stimulated rats may be caused by increased gentamicin transport across the brush border as a consequence of PTH-mediated alteration of plasma membrane phospholipid composition, turnover, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dietary calcium restriction on 1,25-dihydroxyvitamin D3 net synthesis by rat proximal tubules

We measured in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by kidney proximal tubules prepared by Percoll density centrifugation from male and female rats. 1,25(OH)2D3 in tubule extracts was determined by a sensitive and specific radioreceptor assay. Ingestion of diets adequate in vitamin D3 and containing either normal calcium (1.2% Ca, NC), reduced calcium (0.6% Ca, RCD) or low calcium (0.002% Ca, LCD) increased 1,25(OH)2D3 net synthesis (for male rats, NC vs. RCD vs. LCD 1.8 +/- 0.1 SEM vs. 9 +/- 2 vs. 17 +/- 2 pmol/mg protein/20 min; P less than 0.05 for all comparisons). At either level of reduced calcium intake, tubules from male rats produced more 1,25(OH)2D3 than tubules from females. Serum 1,25(OH)2D3 and tubule cyclic adenosine monophosphate (cAMP) content rose in parallel with progressive dietary calcium restriction, and males had higher circulating 1,25(OH)2D3 and tubule cAMP content than females at each level of reduced calcium intake. L-Epinephrine (10(-4) mol/L), in vitro, increased tubule accumulation of 1,25(OH)2D3 and cAMP. Yohimbine, and alpha 2-receptor antagonist, blocked this response, whereas prazosin was without effect. Increased 1,25(OH)2D3 net synthesis by tubules from male vs. female rats partly explains the higher serum levels and enhanced mineral conservation demonstrated previously in male rats. Preparation of proximal tubules from vitamin D-replete rats permits studies in vitro of 1,25(OH)2D3 production and regulation under more physiologic conditions in which parathyroid hormone, inorganic phosphorus, and calcium may be varied independently.     

Effect of calcium intake on serum levels of 25-hydroxyvitamin D3

The effects of high calcium intake on vitamin D metabolism were investigated. To the normal diet of 14 healthy men, 2 g calcium were added daily for 6-7 weeks. The mean serum concentration of 25-hydroxyvitamin D3 increased from 73 +/- 7 to 94 +/- 6 nmol l-1 (P less than 0.05, Student's unpaired t-test; P less than 0.01, paired t-test) in the subjects receiving calcium, whereas there was only a minimal increase, from 67 +/- 5 to 71 +/- 4 nmol l-1 in a control group on a normal diet. At the end of the study the difference between the test group and the controls was highly significant (P less than 0.005). The calcium loading caused a statistically significant depression of the serum levels of 1,25-dihydroxyvitamin D. The results obtained are in agreement with previous studies in rats and indicate that calcium intake is of some importance for the serum level of 25-hydroxyvitamin D3. The findings are discussed in relation to our previous finding that there is a relationship between high 25-hydroxyvitamin D3 levels and hypercalciuria in renal-stone formers.     

In vivo and in vitro effects of WR-2721 in experimental hypercalcemia in the rat

The effects of WR-2721 [S-2-(3-aminopropylaminoethyl)phosphorothioic acid] in two in vivo and in vitro models of experimental hypercalcemia in the rat were examined. Chronic WR-2721 administration by osmotic minipump (250 mg/kg/24 hr) reduced serum calcium from 12.0 +/- 0.1 to 9.5 +/- 1.0 mg/dl (P less than .01) in rats receiving 1,25-(OH)2 Vitamin D3. Control rats receiving Vitamin D without WR-2721 had a rise in serum calcium to 13.4 +/- 0.2 mg/dl over the same 5-day period. In an experimental form of humoral hypercalcemia of malignancy, the Walker carcinosarcoma tumor-implanted rat, WR-2721 reduced serum calcium from 13.6 +/- 0.3 to 8.4 +/- 0.6 mg/dl by 5 to 6 days (P less than .001). In vitro bone resorption assays utilizing fetal rat long bones in organ culture showed complementary results. WR-2721 (10(-4) M) blocked bone resorption (assayed as percentage of 45Ca release) induced by both conditioned medium derived from cell lines of Walker carcinosarcoma (7.6 +/- 1.4 vs. 24.0 +/- 1.8%, P less than .01) and by addition of 1,25-(OH)2 Vitamin D3 (10(-8) M) (9.8 +/- 0.8 vs. 17.3 +/- 1.0%, P less than .01). These results suggest that WR-2721 may be effective in controlling clinical hypercalcemia due to excess bone resorption.     

Serum 1,25 dihydroxy vitamin D (1,25(OH)2D3), 25 hydroxy vitamin D (25(OH)D) and parathormone levels in diabetic retinopathy

OBJECTIVES: To investigate whether there is a relationship between serum 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], which is an inhibitor of angiogenesis, concentrations and severity of diabetic retinopathy (DR). DESIGN AND METHODS: Serum 1,25(OH)2D3, 25 hydroxy vitamin D [25(OH)D] and parathormone (PTH) concentrations were measured in diabetic patients (n = 66) and nondiabetic healthy subjects (n = 20). RESULTS: The mean serum 1,25(OH)2D3 concentration in diabetic patients was lower than that in nondiabetics (57.3+/-21.44 vs. 89.4+/-18.01 pmol/L, p<0.001); mean 1,25(OH)2D3 concentrations fell with increasing severity of DR [being 63.4+/-17.26 pmol/L for background DR (BDR), 47.7+/-13.27 pmol/L for preproliferative DR (pre-PDR), and 43.1+/-19.45 pmol/L for proliferative DR (PDR)]. Compared with the control group, serum 25(OH)D concentrations were found to be decreased in diabetic patients (p<0.001).There were negative correlations between 1,25(OH)2D3 and age (r = -0.331, p<0.01) and duration of diabetes (r = -0.255, p<0.05). CONCLUSION: From these findings, it was found that there was an inverse relationship between the severity of the retinopathy, i.e., neovascularization, and serum 1,25(OH)2D3 concentrations, being the lowest in PDR and the highest in diabetic patients without retinopathy (NDR) patients. The measurement of serum 1,25(OH)2D3 concentrations might be helpful to predict severity of DR in patients with diabetes mellitus.     

Pathophysiology of spontaneous hypercalciuria in laboratory rats. Role of deranged vitamin D metabolism.

Recent data suggest a causal role of deranged 1,25(OH)2D metabolism in the syndrome of idiopathic hypercalciuria. To test this hypothesis, we evaluated if vitamin D availability and/or increased serum 1,25(OH)2D were critical for the expression of hypercalciuria in laboratory rats. Ca balance, serum 25OHD3, and 1,25(OH)2D3 were studied in D-deprived (-D) and D-repleted (+D) male progeny (p) born to normocalciuric (NC) and spontaneously hypercalciuric (SH) rats. 7 of the 14 pSH and 2 of 21 pNC had SH, which was defined as urinary Ca greater than two standard deviations above the mean of values for control animals on days 5 and 6 of a low Ca +D diet (1.19 vs. 0.58 mg/d, P less than 0.001). Fasting serum Ca and 25OHD3 were similar to control. Serum 1,25(OH)2D3 was elevated in these nine SH rats (232 vs. 145 pg/ml, P less than 0.005). However, during vitamin D deprivation, their Ca excretion was also increased (1.53 vs. 0.45 mg/d, P less than 0.001), despite comparably reduced serum 1,25(OH)2D3 (102 vs. 106 pg/ml) and undetectable serum 25OHD3. Net intestinal Ca absorption on a low Ca diet was comparable during D repletion (-0.75 vs. -0.82 mg/d) or D deprivation (-0.80 vs. -2.15 mg/d), excluding primary hyperabsorption as the mediator of the hypercalciuria. Mild hypophosphatemia was present in SH on +D (5.8 vs. 6.9 mg/dl, P less than 0.005) and -D diets (6.2 vs. 7.9 mg/dl, P less than 0.005), and was associated with higher rates of cyclic adenosine monophosphate excretion (32.8 vs. 26.9 and 48.5 vs. 41.0 nmol/mg of creatinine, respectively). Spontaneous hypercalciuria is therefore dissociable from increased Ca absorption, serum levels of 25OHD3, or 1,25(OH)2D3. The data are most compatible with the hypothesis of a renal Ca leak which stimulates parathyroid hormone activity and increases serum 1,25(OH)2D3, if provided adequate 25OHD3 as substrate.     

Evidence that calcitonin stimulates 1,25-dihydroxyvitamin D production and intestinal absorption of calcium in vivo.

Although it is well established that parathyroid hormone and phosphate are important regulators of 1,25-dihydroxyvitamin D [1,25(OH)2D] production, it remains unclear whether calcitonin affects vitamin D metabolism in vivo. Experiments were performed in the rat to determine the effect of chronic calcitonin infusion (0.2 U X h-1) on plasma levels of vitamin D metabolites and on calcium metabolism. Thyroparathyroidectomized animals fed a calcium-replete or calcium-free diet were studied for as long as 2 wk before they were killed. In control rats, a calcium-free diet alone for 12 d resulted in an increase in 1,25(OH)2D levels from 24 +/- 5 to 139 +/- 37 pg . ml-1, P = 0.025. The infusion of calcitonin also stimulated 1,25(OH)2D levels compared with controls on a regular diet (80 +/- 17 vs. 38 +/- 6 pg . ml-1, P less than 0.05) and on a calcium-free diet (460 +/- 50 vs. 139 +/- 37 pg . ml-1, P less than 0.001). In addition, calcitonin increased plasma calcium levels in animals on a regular diet by 50%; this effect was most likely due to increased intestinal absorption of calcium, because removal of calcium from the diet markedly blunted this effect. In contrast, calcitonin administration did not significantly affect 25(OH)D plasma levels. Collectively, these data suggest that calcitonin and calcium are independent regulators of 1,25(OH)2D production and that calcitonin stimulates intestinal absorption of calcium, by increasing circulating levels of 1,25(OH)2D.     

Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration, and the level of vitamin D-binding protein (DBP) in 24 normal subjects, 17 pregnant subjects, and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus, the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl, respectively, P less than 0.001), whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml, respectively). In contrast, in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%, P less than 0.001), the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml, P less than 0.001). When data from all three groups were combined, there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels, (b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels, and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions.