Synthesis of (6-3H)-1alpha-hydroxyvitamin D3 and its metabolism in vivo to (3H)-1alpha,25-dihydroxyvitamin D3.

[6-3H]-1alpha-Hydroxyvitamin D3 was chemically synthesized and its full biological activity and radiochemical purity were demonstrated. With the use of this preparation it has been possible to demonstrate in vivo that in rats the [6-3H]-1Alpha-hydroxyvitamin D3 is converted to [6-3H]-1alpha,25-dihydroxyvitamin D3, the natural hormone. In fact, in the intestine and bone of rats given 32 picomoles of [6-3H]-1alpha-hydroxyvitamin D3 each day for 6 days, more than 80 percent of the lipid-soluble radioactivity exists as [6-3H]-1alpha,25-dihydroxyvitamin D3, a finding that suggests that much of the biological effectiveness of 1alpha-hydroxyvitamin D3 is due to its conversion to 1alpha,25-dihydroxyvitamin D3.

Evidence for extra-renal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in pregnancy.

The kidneys are thought to be the only organs capable of 1 alpha-hydroxylation of vitamin D and its metabolites. We have examined the in vivo conversion of 3H-(25,26)-25-hydroxyvitamin D3(25OHD3) to 3H-(25,26)-1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in vitamin D-deficient, pregnant and nonpregnant rats. As expected, nephrectomy of nonpregnant, vitamin D-deficient rats prevented the conversion of 25OHD3 to 1 alpha,25(OH)2D3. In contrast, nephrectomy of pregnant, vitamin D-deficient rats reduced but did not abolish the formation of 1 alpha,25(OH)2D3 from its precursor. The identity of the radioactive metabolite formed from 3H-25OHD3 which circulated in nephrectomized, pregnant rats was established as 1 alpha,25(OH)2D3 by comigration with synthetic 1 alpha,25(OH)2D3 on high-pressure liquid chromatography. The simultaneous absence of 1 alpha,25(OH)2D3 in the fetal kidneys indicated that the site of 1 alpha-hydroxylation after nephrectomy of the pregnant rat was probably extra-renal in origin. Two sites of 1 alpha-hydroxylation of 25OHD3, one renal and the other extra-renal, either fetoplacental or maternal, may exist in the pregnant, vitamin D-deficient rat.

Stimulation of 24,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3.

Vitamin D-deficient rats produce [(3)H]1,25-dihydroxyvitamin D(3) from [(3)H]25-hydroxyvitamin D(3) regardless of dietary content of calcium or phosphate. A daily dose of 130 picomoles of 1,25-dihydroxyvitamin D(3) for a period of 5 days reduces production of [(3)H]1,25-dihydroxyvitamin D(3) to essentially zero and stimulates production of [(3)H]24,25-dihydroxyvitamin D(3). A daily dose of 325 picomoles of 25-hydroxyvitamin D(3) has a similar but less dramatic effect. On the other hand, 650 picomoles daily of 24,25-dihydroxyvitamin D(3) given to vitamin D-deficient rats had no effect. Thus it appears that 1,25-dihydroxyvitamin D(3) is an important factor in the regulation of kidney metabolism of 25-hydroxyvitamin D(3).

25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol: conversion impaired by systemic metabolic acidosis.

An acute systemic acidosis in vitamin D depleted rats that was induced by ammonium chlroide feeding resulted in defective biological hydroxylation of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol. Impaired enzymatic hydroxylation occurred despite the presence of either hypophosphatemia or hypocalcemia. The data suggest that acidosis interfers with the adaptive enzymatic control of 25-hydroxycholecalciferol metabolism in the vitamin D depleted state.

Local action of phosphate depletion and insulin-like growth factor 1 on in vitro production of 1,25-dihydroxyvitamin D by cultured mammalian kidney cells.

The hormonal form of vitamin D, 1,25(OH)2D, is synthesized mostly in proximal renal tubular cells. Experimental and clinical studies suggest that the growth hormone may be involved in growth-related fluctuations of plasma 1,25(OH)2D and in the increase of 1,25(OH)2D induced by in vivo phosphate deprivation, an action possibly mediated by insulin-like growth factor 1 (IGF 1). We tested the effects of phosphate depletion and IGF 1 addition on 1,25(OH)2D3 production in cultured kidney cells: opossum kidney (OK) cells, LLC-PK 1, and rabbit's proximal tubular cells. Confluent cell monolayers were preincubated in various phosphate concentrations, in the presence and absence of IGF 1. Then, 5 nM of [3H]25 (OH)D3 or 2 microM of 25 (OH)D3 were added to the medium and the cells were incubated for a further 120 min. The amount of biosynthesized 1,25(OH)2D3 in lipid extracts was determined after two different straight phase high performance liquid chromatographies. The experiment showed the following: (a) LLC-PK 1 and rabbit's cells expressed a detectable ability to synthesize 1,25(OH)2D3, while OK cells did not. (b) Partial or total phosphate deprivation increased the amount of 1,25(OH)2D3 produced, respectively in LLC-PK 1 and in rabbit's cells. (c) IGF 1 (25 ng/ml) increased 1,25(OH)2D3 production in rabbit's cells, particularly in phosphate-free medium (1.6-fold), and in LLC-PK 1 cells, in partial phosphate depletion (2.75-fold in 1 mM phosphate, P = 0.015, n = 5, and 3.2-fold in 0.5 mM phosphate, P = 0.043, n = 4). Our findings demonstrate a local action of phosphate depletion and of IGF 1 on 1,25-dihydroxyvitamin D3 production.

The ionic control of 1,25-dihydroxyvitamin D3 production in isolated chick renal tubules.

Isolated renal tubules prepared from vitamin D-deficient chicks catalyze the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 (250HD3) in vitro. The effect of calcium and phosphate on the rate of synthesis of the product, 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3), was studied at two levels: the long-term effects of various dietary calcium and phosphate contents on the ability of the tubules to produce 1, 25 (OH)2D3, and the acute effects of different calcium and phosphate concentrations in the incubation medium on the rate of synthesis of 1,25(OH)2D3 by the tubules. Manipulation of dietary calcium and phosphate sufficient to produce marked changes in the concentration of calcium and phosphate in the serum led to altered rates of 1,25(OH)2D3 synthesis by the isolated renal tubules. The renal tubules isolated from chicks raised on a vitamin D-deficient diet containing 0.43% calcium and 0.3% P as inorganic phosphate showed the highest rate of synthesis of 1,25(OH)2D3. Diets containing more or less of either calcium or phosphate produced chicks whose renal tubules had a slower rate of 1,25(OH)2D3 production. The calcium, phosphate, and hydrogen ion content of the incubation medium were manipulated to determine the possible factors concerned with the immediate regulation of 1,25(OH)2D3 production. A calcium concentration of 0.5-1.0 mM was necessary for optimal enzymatic activity. Concentrations of calcium greater than this optimal concentration inhibited 1,25(OH)2D3 production if phosphate was also present, and this inhibition was more pronounced as the phosphate concentration was increased. The stimulation of 1,25(OH)2D3 production by calcium was less at pH 6.7 than at 7.4. Raising the phosphate concentration from 0 to 6 mM in the absence of calcium also stimulated the rate of synthesis of 1,25(OH)2D3. This stimulatory effect was blocked by 4 mM calcium. However, at 1-2 mM calciu, phosphate had a biphasic influence on 1,25(OH)2D3 production; extracellular concentrations of phosphate from 0.6 to 1.2 mM resulted in less 1,25(OH)2D3 production than higher or lower phosphate concentrations. This biphasic effect was seen both at pH 7.4 and 6.8.

The ionic control of 1,25-dihydroxyvitamin D3 synthesis in isolated chick renal mitochondria. The role of calcium as influenced by inorganic phosphate and hydrogen-ion.

Isolated kidney mitochondria prepared from Vitamin D-deficient chicks catalyze the conversion of 25-hydroxyvitamin D3 to 1,25 dihydroxyvitamin D3. It wasfound that changes in the concentrations of Ca-2plus, HPO4-2minus, and Hplus altered synthesis in an interrelated fashion. Increasing the Ca-2plus concentration from 10-6 to 10-5 M caused a four- to fivefold increase in 1 alpha-hydroxylase activity when the medium pH was between 6.5 and 7.0. increasing the [Ca2+] to 10-4 M caused to furhter stimulation. At higher pH values, Ca-2plus had little effect upon 1 alpha-hydroxylase activity. In the absence of calcium [Ca2+] less than or equal to 10-7 M), a change in pH from 6.5 to 7.1 had no effect upon 1 alpha-hydroxylase activity in the presence of 10-5 M calcium, increasing the medium pH had a biphasic effect. An increase in pH from 6.5 to 6.9 caused a 1.5-fold increase in 1 alpha-hydroxylase activity, but a further increase of the pH to 7.1 caused a profound decrease in rate of hydroxylation to approximately 20% of the peak value. Neither 10-5 M LaC13 nor 10 mug/ml of oligomycin altered the effects of Ca2+ upon hydroxylate activity. However, the effect of calcium was blocked by 2.5 times 10-5 M ruthenium red, 0.83 mug/ml of antimycin A, and 500 muM dinitrophenol. The clcium ionophore, A23187, decreased but did not prevent the stimulatory effect of calcium. These data are consistent with the concept that the [Ca2+ in the mitochondrial matrix space is of importance in regulating the 1 alpha-hydroxylase. Phosphate exerted a biphasic effect on 1,25(OH)2D3 production with maximal stimulation (approximately twofold) at 1-3 mM. Calcium enhanced the stimulation by phosphate at all concentrations studied. The presence of potassium modified the interrelated effects of calcium and phosphate in two ways: 10-3 M calcium blocked the stimulation by phosphate; and in the presence of phosphate, 10-3 M calcium resulted in less 1,25(OH)2D3 production by production by isolated mitochondria are qualitatively similar to the effects of these ions on 1,25(OH)2D3 production yb isolated renal tubules.

Stimulation of 1,25-dihydroxycholecalciferol production by prolactin and related peptides in intact renal cell preparations in vitro.

Ovine prolactin stimulated the 1 alpha-hydroxylase activity in isolated renal tubules and especially in primary kidney cell cultures, both prepared from vitamin D-deficient chicks. In primary chick kidney cell cultures, treated for 48 h with 1,25-dihydroxycholecalciferol (to induce the 24-hydroxylase activity) ovine prolactin, after a 1 h incubation period, stimulated the 1 alpha-hydroxylase activity without affecting the 24-hydroxylase activity. Similar results were obtained with related peptide hormones such as human growth hormone, chicken growth hormone, and human placental lactogen. These observations are discussed in relation to the possible role of these peptide hormones as modulators of 1,25-dihydroxycholecalciferol production in physiological situations of calcium stress, such as pregnancy, lactation, growth in mammals and egg laying in birds.

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12).

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria. The role of potassium.

In previous studies it was found that change in the concentrations of Ca2+, H+, and HPO2-4 in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)2D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K+ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)2D-3 synthesis by isolated chick mitochondria; that the magnitnitude of this K+-dependent stimulation is enhaced by optimal concentrations of calcium (pCa = 5) and phosphate (pPi = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na+ concentration; and that the stimulatory effect of K+ is not blocked by ruthenium red, and inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH) 2D-3 synthesis. It was also found that valinomycin, a K+-specific ionophore, enhanced the sensitivity of the mitochondrial 1 alpha-hydroxylase activity to K+. In the presence of valinomycin, an increase of pK+ to 3 was sufficient to cause a significant stimulation of 1,25(OH)2D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulated the activity of the 1 alpha-hydroxylase.

Vitamin D metabolism in man. Effect of corticosteroids.

This study evaluated the effects of acute intravenous calcium supplementation on vitamin D metabolism in nine patients maintained on long-term prednisone therapy for chronic obstructive lung disease. Vitamin D turnover studies, employing 1,2-(3)H,4(14)C vitamin D3, and coincident measurement of serum 25-hydroxycholecalciferol (25-OHCC) concentrations demonstration (P less than .05) and half-time (P less than .05) in the prednisone-treated patients when compared with the mean 25-OHCC concentration and half-time found in these patients prior to calcium infusion. However, there was no significant difference in the amounts of 24,25-(OH)2CC) or 1,25-dihydroxycholecalciferol (1,25-(OH)2CC) appearing after calcium infusion. The results of the study showed that the intravenous administration of calcium to corticosteroid-treated patients accelerated the disappearance of 25-OHCC from the plasma of these patients without parallel increases in the production of 1,25-(OH)2CC or 24-25-(OH)2CC.

The regulation of 24,25-dihydroxyvitamin D3 production in cultures of monkey kidney cells.

Primary kidney cell cultures from normal rhesus monkeys were used to study the regulation of 24,25-dihydroxyvitamin D3 production. Kidney cells were grown to confluency in the modified National Cancer Institute Medium NCTC 135 with 2% fetal calf serum and then maintained in a serum-free medium (2% bovine serum albumin) for five additional days prior to a study of regulation. Morphologically, 80% of the cultured cells were epithelial in nautre. These cells produced 24,25-dihydroxy-[3H]vitamin D3 from 25-hydroxy-[3H]-vitamin D3. The identity of the 24,25-dihydroxy-[3H]vitamin D3 was established by Sephadex LH-20 chromatography, by co-chromatography with authentic 24,25-dihydroxyvitamin D3 on high pressure liquid columns, and by periodate sensitivity. Bovine parathyroid hormone at a level of 3 U/ml or human 1-34 parathyroid hormone at 0.05 U/ml for five days suppressed 24,25-dihydroxyvitamin D3 production. Nonradioactive 1,25-dihydroxyvitamin D3 (13 pmol/ml) added once every two days for eight days led to a 2-fold increase in production of 24,25-dihydroxyvitamin D3. Exposure of renal cells to 3 mM instead of 1 mM calcium led to a marked increase in 24,25-dihydroxyvitamin D3 production. These results suggest that renal cell cultures may be an important new approach to a study of regulation of the vitamin D renal hydroxylases.

Cells isolated from embryonic intestine synthesize 1,25-dihydroxyvitamin-D3 and 24,25-dihydroxyvitamin-D3 in culture.

Cells isolated from embryonic chick intestine in culture convert 25-hydroxyvitamin-D3 to a number of more polar metabolites. Two of these metabolites have been identified with chemical and chromatographic methods as 24,25-dihydroxyvitamin-D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). The enzymes conform to substrate saturation kinetics. The apparent Km for substrate for the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 is 70 nM and 29 nM, respectively.

Recurring hypocalcemia of bovine parturient paresis is associated with failure to produce 1,25-dihydroxyvitamin D.

Parturient paresis (milk fever) is a hypocalcemic disorder caused by the onset of lactation in the dairy cow. In most cows a complete recovery follows a single iv calcium treatment to correct the acute hypocalcemia. However, about 20% of cows treated for parturient paresis experience recurring episodes of hypocalcemia (relapses) requiring further treatment. Analysis of plasma from 8 nonrelapsing parturient paretic and 11 relapsing parturient paretic cows revealed differences in plasma 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations before and during the development of hypocalcemia. In nonrelapsing cows, plasma 1,25-(OH)2D increased to 4- to 5-fold as plasma calcium concentrations declined during the first stage of parturient paresis. In relapsing cows, decreases in plasma calcium concentrations during the first stage of parturient paresis were accompanied by just a 2- to 2.5-fold increase in plasma 1,25-(OH)2D. Plasma 1,25-(OH)2D eventually increased 4- to 5-fold in the relapsing cows, but this response was delayed 24-48 h compared with the response in the nonrelapsing cows. Plasma PTH concentration profiles were similar in relapsing and nonrelapsing cows, suggesting that renal 25-hydroxyvitamin D 1 alpha-hydroxylase was temporarily refractory to stimulation by PTH in the relapsing cows. In both groups of cows recovery from parturient paresis began about 12-24 h after plasma 1,25-(OH)2D concentrations had increased 4- to 5-fold. These data imply that lack of production of 1,25-(OH)2D is an important factor in predisposing the cow to relapses of parturient paresis and is critical for recovery from the hypocalcemia associated with the onset of lactation.

Synthesis of 1,25-dihydroxyvitamin D in the nephrectomized pregnant rat.

Pregnant rats were maintained on diets either adequate or deficient in vitamin D. On the 20th day of gestation, animals were either nephrectomized bilaterally or sham operated. Immediately therafter, four groups of nephrectomized or sham-operated pregnant rats received iv [26,27-3H]25-hydroxyvitamin D3 ([26,27-3H]25OHD3), while two groups received [1,2-3H,4-14C]D3. The animals were sacrificed 10-24 h later. The distribution of the radiolabeled metabolites of vitamin D3 was determined in extracts of maternal plasma, maternal intestinal tract, placentae, and fetuses after Sephadex LH-20 column chromatography. Both vitamin D3 and 25OHD3 crossed the placenta and entered the fetus. In anephric animals receiving [26,27-3H]-25OHD3, 24,25-dihydroxyvitamin D and a polar peak eluting in the position of 1,25-dihydroxyvitamin D [1,25(OH)2D] and 25,26-dihydroxyvitamin D were identified in extracts of maternal plasma and intestinal tracts and of placentae and fetuses. The identities of 24,25-dihydroxyvitamin D and 1,25 (OH)2D were confirmed by high pressure liquid chromatography. In rats receiving [1,2-3H,4-14C]D3, approximately 50% of the polar metabolite consisted of 1,25(OH)2D. We conclude that the anephric pregnant rat is able to synthesize 1,25(OH)2D, that the fetal portion of the feto-placental unit is the most likely site of production of this hormone, and that this metabolite of vitamin D is able to cross the placenta from the fetus to the mother.

Insulin permits parathyroid hormone stimulation of 1,25-dihydroxyvitamin D3 production in cultured kidney cells.

Primary cultures of chick kidney cells in serum free medium respond to PTH with increased production of 1,25(OH)2D3 only when exposed to insulin. The response of 1,25(OH)2D3 is maximal at 5 ng bPTH (1-34) per ml and decreases at higher hormone concentrations. Increased 1,25(OH)2D3 synthesis is not evident after 30 minutes exposure to bPTH and is maximal at 4-6 hours of treatment. Insulin does not increase the cyclic AMP response to PTH suggesting that whatever permissive role it is playing occurs beyond the generation of cyclic AMP.

In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of 1alpha,25-dihydroxyvitamin D3 synthesis in rats.

Teh stimulatory effect of parathyroid hormone (PTH) on renal 1alpha-hydroxylation of 25-hydroxyvitamin D3 (25-OH-D3) was studied in thyro-parathyroidectomized (TPTX), vitamin D-deficient rats into which bovine PTH, theophylline, cAMP or dibutyryl cAMP (dbcAMP) was constantly infused. The accumulation in plasma of 1alpha,25-dihydroxy-vitamin D3 [1alpha,25-(OH)2-D3], produced from 25-OH-D3, was enhanced by infusion of either cAMP (0.9 MUMOL/H) OR DBCAMP (1 mumol/h) to a level similar to the maximum obtained by PTH (i95--7.5 U/h) infusion. A submaximal dose (1 U/h) of PTH caused a similar extent of stimulation, when infused with theophylline. When either 2 mumol/h of cAMP or 7.5 U/h of bovine PTH was infused starting 18 h after TPTX, the accumulation of 1alpha,25-(OH)2-D3 in plasma was similarly restored withing 6 h to the level found in the sham-operated animals. These results strongly support the concept that cAMP plays an important intermediary role in the stimulation of 1alpha,25-(OH)2-D3 production induced by both exogenous and endogenous PTH.

Conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in renal slices from the rat.

Isolated renal cortical slices were used to study the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25](OH2)D3] by the rat kidney. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was linear with time (30-90 min) and tissue weight (40-250 mg). Production of 1,25-(OH)2D3 was greatest (134 +/- 17 pg/mg tissue.h) in animals fed a low calcium, vitamin D-deficient diet. The greatest 24,25-(OH)2D3 production (106 +/- 17 pg/mg tissue.h) was seen in animals fed a high calcium, vitamin D-replete diet, 1,25-(OH)2D3 production was reduced to 23% of maximum by the addition of 1.2% calcium or 0.8% strontium to the vitamin D-deficient, low calcium diet. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was greatly reduced in renal cortical slices that had been heated before incubation. Slices of renal medulla produced only small amounts of 1,25-(OH)2D3 compared to slices of renal cortex. These studies provide direct evidence for the production of 1,25-(OH)2D3 and 24,25-(OH)2D3 by the mammalian renal cortex. They also demonstrate that this production may be modulated by dietary calcium, strontium, and vitamin D.

Differential effects of parathyroid hormone on the renal 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 production of young and adult rats.

In young rats, PTH markedly stimulates the renal conversion of 25-hydroxyvitamin D3 (25OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active form of vitamin D3. With increasing age, serum 1,25-(OH)2D3 decreases while serum PTH increases. Therefore, the effect of PTH on the renal metabolism of 25OHD3 to 24,25-(OH)2D3 or 1,25-(OH)2D3 was compared in young and adult rats. Rats were housed in the dark and fed a low Ca, vitamin D-deficient diet for 4-6 weeks, and thyroparathyroidectomy was performed. Renal 25OHD3 metabolism was measured in vitro by incubating renal cortical slices with tritiated 25OHD3 and quantifying tritiated metabolites by high pressure liquid chromatography. When young (2 months old) thyroparathyroidectomized (TPTX) rats were repleted with PTH by ip injection, 1,25-(OH)2D3 production increased 61%, and 24,25-(OH)2D3 production decreased to 40%. When adult (13 months old) TPTX rats were repleted with PTH, there was no increase in 1,25-(OH)2D3, but 24,25-(OH)2D3 production decreased to 43%. When PTH was added in vitro by incubating renal slices from young TPTX rats for 4 h, 1,25-(OH)2D3 production increased 68%, and 24,25-(OH)2D3 production decreased to 71%. In slices from adult rats, 24,25-(OH)2D3 production was decreased significantly to 71%, and 1,25-(OH)2D3 production was unaffected by PTH. The PTH-stimulated increase in the cAMP content of renal slices from adult rats was 75% that of slices from young rats. These studies demonstrate that PTH modulates renal 24,25-(OH)2D3 production in the adult. However, PTH does not modulate renal 1,25-(OH)2D3 production in the adult under the same conditions that produce a PTH effect in the young animal.

Modulation of renal production of 24,25- and 1,25-dihydroxyvitamin D3 in young and adult rats by dietary calcium, phosphorus, and 1,25-dihydroxyvitamin D3.

The purpose of this study was to examine the effects of dietary calcium (Ca), phosphorus (P), and vitamin D3 metabolites on the renal metabolism of 25-hydroxyvitamin D3 (25OHD3) to either 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] in the rat. The regulation of 25OHD3 metabolism was studied in both young and adult rats, since previous studies have suggested a change in the renal metabolism of 25OHD3 with age. Renal 25OHD3 metabolism was measured in vitro by incubating renal cortical slices with tritiated 25OHD3 and quantifying tritiated metabolites by high pressure liquid chromatography. The apparent Michaelis constant for the conversion of 25OHD3 to 1,25-(OH)2D3 in this system was 1.16 microM. Experiments were conducted in rats fed a vitamin D-deficient diet containing either 0.02% Ca (low Ca) or 1.20% Ca (high Ca) for 4 weeks. Young rats (4 weeks old) fed the low Ca diet demonstrated a 2.8-fold increase in 1,25-(OH)2D3 production, but no change in 24,25-(OH)2D3 production compared to young rats fed the high Ca diet. Adult rats (12 months old) fed the low Ca diet showed no change in 1,25-(OH)2D3 production, but exhibited a decrease in 24,25-(OH)2D3 production compared to adult rats fed the high Ca diet. Repletion of the young rats fed the low Ca diet with 1,25(OH)2D3 resulted in a marked decrease in 1,25-(OH)2D3 production and an increase in 24,25-(OH)2D3 production. Repletion of the adult rat resulted in no change in 1,25-(OH)2D3 production, but a significant increase in 24,25-(OH)2D3 production. When young rats were fed diets containing various levels of Ca and P, it was found that 1,25-(OH)2D3 production was inversely correlated with plasma Ca over the range 4--13 mg/dl. Since the plasma Ca level of the adult rat was 11-12 mg/dl regardless of diet, this high concentration may explain the lack of 1,25-(OH)2D3 production observed in the adult.