Bruce Lobaugh

Abnormal regulation of renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the X-linked hypophosphatemic mouse.

Abnormal vitamin D metabolism has been suspected in patients with X-linked hypophosphatemic rickets (XLH) and X-linked hypophosphatemic mice (Hyp-mice), the murine homologue of the human disease. We compared 25(OH)D-1 alpha-hydroxylase activity in the Hyp-mouse kidney to that in normal and phosphate-depleted mouse kidney. Weanling normal and Hyp-mice were fed a 0.6% phosphorus diet; phosphate-depleted mice received a 0.02% phosphorus diet. At 8-10 wk of age the serum phosphorus values in Hyp (3.35 +/- 0.12 mg/dl) and phosphate-depleted mice (3.83 +/- 0.56) were not significantly different. Despite the similar magnitude of phosphate depletion, however, the maximum levels of 25(OH)D-1 alpha-hydroxylase activity were disparate: phosphate-depleted mouse kidney had profoundly increased activity compared to normal (17.04 +/- .104 vs. 4.96 +/- 0.23 fmol 1,25(OH)2D3 produced/mg kidney per min) while Hyp-mouse kidney had a fourfold lesser increment (8.18 +/- 0.62). These data indicate that phosphate depletion is a potent stimulus of 25(OH)D-1 alpha-hydroxylase activity in the (C57BL6J) mouse. Moreover, the results show that abnormal regulation of 25(OH)D-1 alpha-hydroxylase activity is manifest in the Hyp-mouse.

Abnormal parathyroid hormone stimulation of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the hypophosphatemic mouse. Evidence for a generalized defect of vitamin D metabolism.

Abnormal regulation of vitamin D metabolism is a feature of X-linked hypophosphatemic rickets in man and of the murine homologue of the disease in the hypophosphatemic (Hyp)-mouse. We previously reported that mutant mice have abnormally low renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity for the prevailing degree of hypophosphatemia. To further characterize this defect, we examined whether Hyp-mouse renal 1 alpha-hydroxylase activity responds normally to other stimulatory and inhibitory controls of enzyme function. We studied stimulation by parathyroid hormone (PTH) using: (a) a calcium-deficient (0.02% Ca) diet to raise endogenous PTH; or (b) 24-h continuous infusion of 0.25 IU/h bovine PTH via osmotic minipump. In both cases enzyme activity of identically treated normal mice increased to greater levels than those attained by Hyp-mice. The relative inability of PTH to stimulate 1 alpha-hydroxylase activity is not a function of the hypophosphatemia in the Hyp-mouse since PTH-infused, phosphate-depleted normal mice sustained a level of enzyme activity greater than that of normal and Hyp-mice. In further studies we investigated inhibition of enzyme activity by using: (a) a calcium-loaded (1.2% Ca) diet to suppress endogenous PTH; or (b) 24-h continuous infusion of 0.2 ng/h 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 1 alpha-hydroxylase activity of normal and Hyp-mice was significantly reduced to similar absolute levels following maintenance on the calcium-loaded diet. Further, infusion of 1,25(OH)2D3 caused a comparable reduction of 1 alpha-hydroxylase activity in normal, Hyp-, and phosphate-depleted normal mice. These observations indicate that the inhibitory control of 1 alpha-hydroxylase by reduced levels of PTH or increased 1,25(OH)2D3 concentrations is intact in the mutants. However, the inability of PTH and hypophosphatemia to stimulate enzyme activity in a manner analogous to that in normal and phosphate-depleted mice indicates that a generalized defect of 1 alpha-hydroxylase regulation is manifest in Hyp-mice.

Effects of Ocular Carteolol and Timolol on Plasma High-density Lipoprotein Cholesterol Level

Fifty-eight healthy, normolipidemic adult men participated in a prospective, masked, randomized crossover study designed to compare the effects of two topical nonselective beta-adrenergic antagonists, carteolol and timolol, on plasma high-density lipoprotein cholesterol levels. Two eight-week treatment periods were separated by an eight-week drug-free period. Carteolol 1.0% or timolol 0.5% was used, one drop twice daily, in both eyes without nasolacrimal occlusion. Fresh plasma was assayed for levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and apolipoproteins A-I and B-100. With indistinguishable effects on intraocular pressure, carteolol and timolol induced different (P = .013) decrements in high-density lipoprotein cholesterol levels. Carteolol treatment decreased high-density lipoprotein cholesterol levels by 3.3% (-0.04 mmol/l) and raised the ratio of total to high-density lipoprotein cholesterol levels by 4.0% (0.15 unit); timolol treatment decreased high-density lipoprotein cholesterol levels by 8.0% (-0.10 mmol/l) and raised the ratio of total to high-density lipoprotein cholesterol levels by 10.0% (0.37 unit). There was no differential drug effect on the other lipid variables measured. Ocular nonselective beta-adrenergic antagonist therapy can produce clinically relevant decrements in high-density lipoprotein cholesterol levels in healthy men.

Calcitonin stimulation of renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity in hypophosphatemic mice. Evidence that the regulation of calcitriol production is not universally abnormal in X-linked hypophosphatemia.

Hypophosphatemia (Hyp) mice have defective regulation of 25(OH)D-1 alpha-hydroxylase activity in response to hypophosphatemia, hypocalcemia, and parathyroid hormone (PTH) administration. However, recent observations support the existence of anatomically distinct, independently regulated renal 1 alpha-hydroxylase systems in mammalian proximal convoluted and straight tubules. To more completely define the extent of the 1 alpha-hydroxylase regulatory defect in Hyp-mice, we compared enzyme maximum velocity in normal and mutants after infusion of calcitonin. Upon stimulation, renal 1 alpha-hydroxylase activity increased to similar levels in normal and Hyp-mouse renal homogenates. Moreover, time-course and dose-dependence studies revealed similar patterns of response in the animal models. Subsequently, we examined whether PTH and calcitonin stimulatory effects on enzyme activity are mediated through different mechanisms. In both animal models administration of PTH and calcitonin increased enzyme activity to levels greater than those obtained after maximal stimulation by either hormone alone, consistent with additive effects. These observations indicate that a calcitonin-sensitive component of 1 alpha-hydroxylase is not compromised in the X-linked hypophosphatemic syndrome.

Uremic Tumoral Calcinosis: Preliminary Observations Suggesting an Association With Aberrant Vitamin D Homeostasis

Periarticular tumoral calcification is a unique form of soft tissue calcification that occurs infrequently in patients with end-stage renal disease. The mechanism underlying such massive periarticular calcifications is unknown. The radiographic similarity between uremic tumoral calcifications and those found in hereditary tumoral calcinosis, a disorder of calcitriol and phosphorus homeostasis, caused us to examine whether abnormalities in vitamin D metabolism were associated with uremic calcinosis as well. We examined two uremic subjects with massive periarticular tumoral calcifications and found that they had inappropriately high serum calcitriol levels for the degree of renal function, hyperparathyroidism, and hyperphosphatemia. The source of calcitriol could not be identified in one subject, but likely was derived from granulomatous tissue in the other. In the subject with marrow granulomas, we found that calcitonin administration further stimulated calcitriol production. Although epidemiological studies are needed to confirm this preliminary association between calcitriol and uremic tumoral calcinosis, our observations suggest that normal serum calcitriol levels in association with hyperphosphatemia may be a contributing factor in the development of this rare disorder.

Combination insulin and sulfonylurea therapy in insulin-requiring type 2 diabetes mellitus

PURPOSE To determine the effect(s) on glucose control, insulin dose, and circulating insulin levels of the addition of a sulfonylurea (glipizide) to the treatment regimen of patients with insulin-requiring type 2 diabetes mellitus. PATIENTS AND METHODS Thirty seven patients with type 2 diabetes mellitus taking insulin for at least 1 year prior to study and treated with > or = 40 U of insulin per day were recruited for a randomized, double-blind, placebo-controlled, crossover trial. Patients were treated with 3 months of insulin + placebo (I + P) and 3 months of insulin + glipizide (I + G), with an intermediate 1 month washout period using insulin therapy alone. Adjustments were made initially to the maximum dose of glipizide (40 mg/day), followed by insulin dose adjustments. Twenty-nine of the 37 patients demonstrated a significant C-peptide response to Ensure and were selected for analysis. RESULTS The fasting plasma glucose in the I + G arm was 6.8 (121.8 mg/dl) vs. 8.7 mmol/L (156.0 mg/dl) in the I + P arm, P < 0.001. Mean plasma glucose over 24 hours was 9.8 (176.9 mg/dl) for I + G vs. 11.3 mmol/L (203.8 mg/dl) for I + P, P < 0.001. Glycated hemoglobin was significantly different (9.8 I + G vs. 11.4% I + P, P < 0.008). The total daily insulin dose required was significantly lower with I + G (69.1 vs. 87.3 U, P < 0.0005). However, there were no significant differences in free insulin levels. CONCLUSION The addition of a sulfonylurea (glipizide) to insulin therapy in patients with insulin-requiring type 2 diabetes mellitus taking large doses of insulin results in a rapid and substantial improvement in glucose control despite a significant reduction in insulin dose. Therefore, this form of combination therapy should be considered for patients with the above characteristics whose diet and exercise programs are correct but whose response to insulin therapy is inadequate.

Abnormalities of Vitamin D Metabolism and Action in the Vitamin D Resistant Rachitic and Osteomalacic Diseases

The burgeoning science of vitamin D metabolism and action has brought new perspective to disorders of calcium and bone metabolism in man. Rapid scientific advances have been paralleled by the development of new insights into the pathogenesis of, and new therapies for, human diseases. It is not surprising that the growing body of vitamin D knowledge has been applied to the vitamin D resistant rachitic diseases. Although “vitamin D resistance” may in many instances be a sobriquet imprecisely applied to these diseases, further definition of the disorders must be realized in order to affirm the validity of the newly born scientific assumptions concerning vitamin D.

Vitamin D receptor alleles do not correlate with bone mineral density in premenopausal Caucasian women from the Southeastern United States

Genetic factors are important in determining peak bone density. Recent studies indicate that polymorphisms of the vitamin D receptor (VDR) may account for much of the genetic contribution to bone density, and VDR genotype may be useful to predict the risk of developing osteoporosis. However, the association between VDR genotype and bone mineral density (BMD) has not been observed in all populations. We determined VDR genotype in 69 healthy premenopausal Caucasion women from the southeastern United States and measured BMD at the lumbar spine (anterior-posterior [AP] and lateral views) and proximal femur. We found no association between VDR genotype and BMD at any site. Our results indicate that in this population, VDR genotype does not predict peak bone density and should not be used to predict the risk of developing osteoporosis.

Abnormal parathyroid function in the X-linked hypophosphatemic mouse

SummaryEmploying a cytochemical bioassay, we compared parathyroid function in normal and X-linked hypophosphatemic (Hyp) mice. Under basal conditionsHyp mice manifested hypocalcemia and, in accord, had a plasma bioactive parathyroid hormone concentration (3.04±0.14 pg/ml) significantly greater than that of normals (2.16±0.14 pg/ml). We confirmed the validity of the bioassay by demonstrating that the plasma collected from both mouse models diluted parallel to the assay standard curve. Moreover, after parathyroidectomy, normal andHyp mice had plasma bioactive parathyroid hormone levels approximately 90% less than those obtained under basal conditions and indistinguishable from one another. In further studies we observed that dietary calcium and/or vitamin D deprivation in both animal models resulted in a comparable decline of the plasma calcium concentration. However, the concordant increase of the circulating bioactive parathyroid hormone level was greater in the normal mice. thus, the bioactive parathyroid hormone concentration obtained in response to a low calcium challenge in normals was significantly greater than that inHyp mice In contrast, in response to dietary calcium loading, the plasma bioactive parathyroid hormone levels did not decrease significantly from basal values in either animal model. These data illustrate that the bioactive parathyroid hormone concentration in both normal andHyp mice is inversely correlated with the plasma calcium. However, while theHyp mice maintain an elevated plasma parathyroid hormone concentration under basal conditions (in response to a decreased plasma calcium), the parathyroid activity of the mutants after a more severe hypocalcemic challenge is attenuated, resulting in a significantly different model of linear correlation. Thus, these data indicate thatHyp mice manifest abnormal regulation of parathyroid function.

Measurement of 25-hydroxyvitamin D-1α-hydroxylase activity in mammalian kidney

Publisher Summary This chapter examines the measurement of 25-hydroxyvitamin D 1α-hydroxylase activity in mammalian kidney, and describes the method that increases the sensitivity of the assay, permits accurate estimation of reaction product recovery, and limits the purification procedures necessary to adequately prepare the 1,25-dihydroxyvitamin D 3 for quantification. The renal 25-hydroxyvitamin D 1α-hydroxylase enzyme is a mitochondrial mixed-function oxidase consisting of three proteins. Two of these, a nonheme iron protein, ferredoxin, and ferredoxin reductase function in the transfer of electrons from NADPH to the terminal component cytochrome P -450. The 1α-hydroxylation is supported by succinate, malate, or almost any other Krebs cycle substrate, and is inhibited by carbon monoxide–oxygen mixtures, glutethimide and metyrapone. In the assay method, unlabeled 25-hydroxyvitamin D is added to crude renal homogenates at a concentration sufficient to both saturate the inhibitory factor present in mammalian systems and provide optimal substrate for 1α-hydroxylation. Production of 1,25-dihydroxyvitamin D is quantified by a competitive binding protein radioassay of the purified reaction product, a method which confers great sensitivity to the measurement. The high-performance liquid chromatography step is crucial for accurate assay of lα-hydroxylase activity. Examination of a representative sample HPLC elution profile reveals a substantial amount of 25-hydroxyvitamin D 3 still present in the sample following Sephadex LH-20 chromatography. Since measurement of the 1,25-dihydroxyvitamin D 3 biosynthesized by the renal preparation is achieved by either (1) competitive binding protein radioassay of the unlabeled metabolite, as one shall describe, or (2) determination by scintillation counting of the amount of 25-hydroxy[ 3 H]vitamin D 3 converted to 1,25-dihydroxy[ 3 H]vitamin D 3 , as performed by others, accurate quantification depends upon a priori removal of the 25-hydroxyvitamin D 3 substrate. This can only be achieved by using prohibitively longer LH-20 columns or by the inclusion of further purification steps following LH-20 chromatography.