Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Phenobarbital suppresses vitamin D3 25-hydroxylase expression: a potential new mechanism for drug-induced osteomalacia.

Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D(3) due to decreased 25-hydroxylation of vitamin D(3) in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D(3) 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.

Tumor-induced osteomalacia: clinical and basic studies.

A patient with classic clinical and biochemical features of tumor-induced osteomalacia (hypophosphatemia, phosphaturia, and undetectable serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D]) was studied before and after resection of a benign extraskeletal chondroma from the plantar surface of the foot. Presurgical laboratory evaluation was notable for normal serum concentrations of calcium, intact parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and osteocalcin, increased serum alkaline phosphate activity, and frankly elevated urinary cyclic adenosine monophosphate (cAMP) and pyridinium cross-link excretion. Quantitative histomorphometry showed severe osteomalacia and deep erosions of the cancellous surface by active osteoclasts. After resection, serum 1,25(OH)2D normalized within 24 h, while renal tubular phosphorus reabsorption and serum phosphorus did not normalized until days 2 and 3, respectively; serum Ca declined slightly, and serum intact PTH, osteocalcin, and urinary pyridinium cross-link excretion increased dramatically. Urinary cAMP excretion declined immediately after resection and then began to increase concomitant with the increase in serum intact PTH. A second bone biopsy taken 3 months after resection demonstrated complete resolution of the osteomalacia, increased mineral apposition rate (1.09 mu/day), resorption surface (9.2%), mineralizing surface (71%), and bone formation rate (0.83 mm3/mm2/day), and marked decrease in cancellous bone volume (13.1%) and trabecular connectivity compared with first biopsy. Tumor extracts did not affect phosphate transport in renal epithelial cell lines or 1 alpha-hydroxylase activity in a myelomonocytic cell line. The patient's course suggests that the normal 1,25(OH)2D and phosphorus metabolism is due to a tumor product that may be acting via stimulation of adenylate activity. Increased bone resorption prior to surgical resection suggests that the tumor may also produce an osteoclast activator. The rise in resorption surface and pyridinium cross-link excretion, increase in serum osteocalcin and bone mineralization, normalization of osteoid width, and fall in cancellous bone volume after resection are consistent with healing of osteomalacia by rapid remodeling.

Clinical usefulness of serum tartrate-resistant acid phosphatase activity determination to evaluate bone turnover.

The study was carried out to evaluate the clinical validity and usefulness of serum tartrate-resistant acid phosphatase (TRAP) activity determined using an improved spectrophotometric assay. Enzyme activity was measured in 84 normal subjects and in 109 patients with common metabolic bone diseases. Mean values of serum TRAP activity in male subjects (n = 19; 10.4 +/- 2.15 U l-1) were not significantly different from those found in female subjects (n = 65; 10.8 +/- 1.8 U l-1). In the latter group mean values were significantly raised in post-menopausal subjects (10.5 +/- 2.0 U l-1; p less than 0.01) compared with mean values in pre-menopausal women (8.45 +/- 1.8 U l-1). We found a significant inverse correlation between serum TRAP activity values and bone mineral density (BMD) measured both at an ultradistal radial point (n = 33, r = -0.506; p less than 0.01), and at the lumbar spine (n = 57, r = -0.261; p less than 0.05). Mean serum TRAP activity values in patients with metabolic bone diseases were: primary hyperparathyroidism, n = 30: 14.2 +/- 4.89 U l-1, p less than 0.001 vs normal subjects; chronic maintenance haemodialysis, n = 19: 17.4 +/- 6.7, p less than 0.001; metastatic cancer, n = 13: 21.2 +/- 6.3, p less than 0.001; post-surgical hypoparathyroidism, n = 10: 9.9 +/- 1.8, NS; involutional osteoporosis, n = 20: 12.5 +/- 2.3 p less than 0.001; Paget's disease, n = 10: 16.8 +/- 3.5, p less than 0.001; osteomalacia, n = 7: 19.5 +/- 3.31, p less than 0.001.(ABSTRACT TRUNCATED AT 250 WORDS)

Timing of screening for osteomalacia in the acutely ill elderly.

Hypocalcaemia and hypophosphataemia either on admission or before discharge from hospital are poor discriminators for the presence of subclinical osteomalacia in the elderly. Reliance has to be placed on the measurement of alkaline phosphatase even though false positive tests are common. Use of paired admission and predischarge data reduces the false positive rate by approximately 50% thereby reducing the requirement for histological confirmation of the diagnosis of osteomalacia. Since the prevalence of osteomalacia is low in this type of hospital population, any measure which reduces the requirement for bone biopsy is of considerable practical value.

Bone disorder following partial and total gastrectomy with reference to bone mineral content.

To study post-gastrectomy metabolic bone disorders, we measured the radial bone mineral content (BMC), serum levels of calcium, inorganic phosphorus, alkaline phosphatase, and 25-hydroxyvitamin D(25-OH-D) in 59 patients with partial- and 39 patients with total gastrectomy. Total gastrectomy patients manifested a higher incidence of decreased BMC levels than did partial gastrectomy patients (56 per cent vs. 25 per cent). Patients subjected to the Billroth II procedure, especially females, manifested abnormally low BMC values. The decline in BMC was age-related; it was pronounced in females. At 10 years postoperatively, many of the partial gastrectomy patients manifested markedly low BMC levels; in totally gastrectomized patients this finding was made at less than 5 years postoperatively. Approximately 30 per cent of our patients showed abnormalities in serum minerals, alkaline phosphatase or 25-OH-D.

Osteomalacia in the elderly: investigation and diagnosis.

Osteomalacia in New Zealand and Australia has previously been thought rare. The medical records of 22 elderly inpatients diagnosed as having osteomalacia on biopsy criteria in Dunedin between January 1980 and December 1983 were reviewed. Risk factors, mode of presentation, month of presentation, biochemistry, bone biopsy, bright line counting and bone scans were assessed. Our findings, and a review of the diagnosis of osteomalacia in the elderly are discussed. We conclude that in the elderly, osteomalacia is not uncommon, and recommend increased attention to risk factors and biochemical screening.

Osteomalacia in the elderly: the value of radio-isotope bone scanning in patients with equivocal biochemistry.

Radio-isotope bone scanning was used to detect osteomalacia in 17 elderly subjects who had equivocal biochemical evidence of this condition. The scan was positive in 10 of the 17 subjects. Bone biopsy confirmed osteomalacia in all 10, but also identified a further two cases. Isotope bone scanning is a practical and relatively non-invasive method of detecting osteomalacia in elderly subjects.

Pathophysiology behind anticonvulsant osteomalacia.

Anticonvulsant drug-induced disorders in mineral and bone metabolism are discussed. 'Anticonvulsant osteomalacia' includes hypocalcaemia, elevated serum alkaline phosphatase, decreased serum 25-hydroxycholecalciferol (25 OHD3), radiological and histological signs of osteomalacia and a lower bone mineral content (BMC) than normal. The pathophysiological mechanism behind anticonvulsant osteomalacia is thought be an induction of the microsomal enzyme system in the liver, leading to a disturbance in the metabolism of vitamin D and a resulting vitamin D deficiency. It has been shown that treatment with vitamin D2 increase BMC whereas serum calcium was unchanged. Treatment with vitamin D3 or 25 OHD3 increases serum calcium whereas BMC was unchanged. These findings suggest that vitamin D2 and D3 are metabolized differently in anticonvulsant treated patients. With the present knowledge, preventive treatment of this relative mild pathological condition is not generally indicated.

Value of plasma calcium, phosphate, and alkaline phosphatase measurements in the diagnosis of histological osteomalacia.

Plasma calcium and phosphate concentrations and alkaline phosphatase activities were examined retrospectively in 50 patients with histologically proven osteomalacia and 50 age- and sex-matched control subjects with normal bone histology. An abnormal plasma alkaline phosphatase activity was more useful than an abnormal plasma calcium or phosphate concentration in distinguishing between normal and osteomalacic subjects, producing a false-negative rate of 14% and a false-positive rate of 8%. False-negative and false-positive rates of 10% and 8% respectively were obtained when the presence of an abnormality in any one of the three biochemical measurements was used as a predictor of histological osteomalacia. When discriminant analysis was applied to plasma calcium, phosphate and alkaline phosphatase together a false-negative rate of 12% and a false-positive rate of 0% was obtained.Sixty-two patients in whom a diagnosis of osteomalacia was suspected were investigated prospectively, using both single biochemical abnormalities and the classification functions derived from the discriminant analysis of all three biochemical measurements to predict the presence or absence of histological osteomalacia. Plasma alkaline phosphatase activity gave false-negative and false-positive rates of 10% and 32% respectively but was a more reliable predictor of abnormal bone histology than were plasma calcium or plasma phosphate concentrations or the presence of an abnormality in any one of the three measurements. Discriminant analysis using plasma calcium, phosphate and alkaline phosphatase together produced a false-negative rate of 16% and a false-positive rate of 10%. We conclude that plasma alkaline phosphatase activity is the best single routine biochemical screening test for osteomalacia, although a high false-positive rate may occur. Direct discriminant analysis of plasma calcium, phosphate and alkaline phosphatase together provides a more sensitive method of detecting histological osteomalacia which should be useful in determining the prevalence of osteomalacia within high-risk populations.

[Remodelling in rickets and osteomalacia (author's transl)].

Based on our experience on more than one hundred cases, bone remodelling in rickets and osteomalacia was investigated. Histomorphometry using tetracycline labelling on nineteen ribs and fibula biopsy specimen revealed a marked increase in number of osteon with osteoid seam, a slight decrease in number of resorption cavity, and extremely depressed tetracycline uptake, of 9.7% on average. The attempt to measure bone formation rate by double tetracycline labelling has failed. Analysing the dynamic behaviour of serum alkaline phosphatase, the hypothesis that the serum alkaline phosphatase was regulated by the three factors, activity of osteoblast, number of osteoblast and elution coefficient from the cell, was presented. Both accretion rate and urinary hydroxyproline excretion showed variable values, from low to high. Although bone remodelling in rickets and osteomalacia was always low at the cellular level, it could be low, normal or high at tissue or organ levels.

[The frequency of adult anticonvulsant osteomalacia in relation to duration of therapy and dosage of anticonvulsants (author's transl)]. / Zur Häufigkeit der Osteopathia antiepileptica beim Erwachsenen in Abhängigkeit von Behandlungsdauer und Medikamentendosis.

Of 837 epileptics over 16 years of age treated with mono- or combined hydantoin therapy 20.3% showed radiographic signs of anticonvulsant osteomalacia. With the exception of the patients with severe disturbances of the skeletal system no positive correlation was found with duration of therapy. The percentage of moderate bone changes was the highest in the patients treated for 1 to 2 years; the percentage of severe bone changes in the group treated over 10 years. The rate of osteomalacia correlated with the total dose of hydantoin, phenobarbital, or primidone; the correlation with the dose per year was even more evident. The risk of osteomalacia rose distinctly with doses over 3000 equivalent units/year. The patients with combined hydantoin-barbiturate treatment showed a higher risk than those treated with phenytoin alone. The rate of osteomalacia was the highest in the patients aged under 20 years and over 50 years. Males showed a relatively higher rate of osteomalacia than females, they were treated however with a higher dose per year. The chemical parameters blood alkaline phosphatase and 25-hydroxycholecalciferol corresponded to the radiographic signs, whereas calcium and anorganic phosphate showed no correlation. Early routine radiologic and chemical control especially of the epileptic patients with high risk of osteomalacia should be routinely performed in future.