Firhaad Ismail

Deranged Bone Mineral Metabolism in Chronic Alcoholism

Chronic alcoholic subjects may suffer from osteopenia with either osteomalacia or osteoporosis as the main histologic finding. The reasons may be multifactorial, including nutrition, direct effects of alcohol on bone, and deranged liver function. Seventeen asymptomatic subjects with chronic alcoholism were studied. Serum PTH (carboxyl and midmolecule fragments), 25 hydroxyvitamin D [25(OH)D], 1-25 dihydroxyvitamin D [1-25(OH)2D], and ionized calcium were measured in each subject. In addition to these tests, we employed a sensitive technique of dual photon absorptiometry to measure vertebral bone density and a radioimmunoassay of serum bone gla protein (BGP) to estimate osteoblast function. Our results show that subjects suffering from chronic alcoholism had significantly lower serum 25(OH)D and higher ionized calcium, BGP, PTH (midmolecule) and 1,25(OH)2D while four patients had bone density values below the fracture threshold (0.96 g/cm2). These findings demonstrate that asymptomatic patients with chronic alcoholism have deranged bone mineral metabolism including abnormal BGP and some subjects may even have abnormal dual photon absorptiometry measurements. These particular subjects may be at risk in the future for developing osteopenia and consequent vertebral compression fractures.

1,25 dihydroxyvitamin D3 modifies cyclosporine-induced bone loss.

SummaryWe have previously shown that cyclosporin A (CsA) produces high bone remodeling with resorption exceeding formation and loss of bone volume in the rat. This may have important clinical implications where CsA is widely used in organ transplantation. 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is a bone mineralizing hormone which also has immune modifying properties. Consequently, we studied the effect of combined CsA and 1,25(OH)2D3 administration over 28 days in four groups of rats. Group A received vehicle (n=10), group B CsA (15 mg/kg) (n=10) alone, group C 1,25(OH)2D3 plus CsA (n=15), and group D 1,25(OH)2D3 alone (20 ng/100 g) (n=15). Rats were bled periodically at day 0, 7, 14, and 28 and Ca, parathyroid hormone (PTH), 1,25(OH)2D, osteocalcin (bone Gla-protein, BGP), BUN, and creatinine were measured. Rats were sacrificed on day 28 and bones were examined histomorphometrically. Compared to controls, CsA resulted in significant elevation of BGP and a transient increase in 1,25(OH)2D with excess bone remodeling and loss of bone volume. 1,25(OH)2D3 administration produced hypercalcemia, a significant rise in BGP, with suppression of PTH and increased osteoid volume. Combined therapy prevented the loss of bone volume probably due to increased osteoid tissue and enhanced osteoblast activity. Renal dysfunction, a side-affect of CsA, was not a factor. In conclusion, 1,25(OH)2D3 combined with CsA restores bone volume which is accompanied by increases in serum calcium and BGP.

The effect of chronic caffeine administration on serum markers of bone mineral metabolism and bone histomorphometry in the rat.

SummaryCaffeine has been cited as a risk factor for osteoporosis in humans. In rats, caffeine increases calcium absorption and excretion and raises parathyroid hormone (iPTH) levels. This study investigated the effect of chronic caffeine administration on bone histomorphometry and serum markers of bone mineral metabolism. Twenty-seven male Sprague Dawley rats weighing approximately 300 g were divided into three groups: Group A (n=8) served as controls, Group B (n=9) received 2.5 mg/100 g caffeine in their drinking water, and Group C (n=10) received 10 mg/100 g body weight caffeine in their drinking water. Animals were bled serially for the 8 week study period: Ionized calcium was measured from tail vein blood and serum iPTH and osteocalcin (BGP) from orbital sinus blood. All three groups received two doses of tetracycline for bone histomorphometry which was performed on a right tibial section from each animal. Ionized calcium was not different among the three groups at any time point. No alteration in serum iPTH levels was demonstrated except for day 56 when the high-dose group (C) showed a raised level (mean=59.1, SE=±8.9 pg/ml (P<0.05). By week 8 Group C showed a failure to gain weight compared with Group A. Group C mean weight=384.0±6.6 g, Group A 427.4±10.8 g (P<0.005). Serum BGP was significantly increased in Group C compared with control (P<0.001). No differences in bone histomorphometry were observed among the three groups. We conclude that although chronic caffeine administration in the rat may slightly increase bone turnover as evidenced by the BGP increase, bone histomorphometry was not altered. This makes the role of caffeine by itself doubtful as an etiological factor for osteoporosis.

Serum bone gla protein (BGP) and other markers of bone mineral metabolism in postmenopausal osteoporosis

Bone gla protein, the vitamin K-dependent protein synthesized by osteoblasts and measured in blood by radioimmunoassay, has been used as an index of the rate of bone turnover. The relationship of bone gla protein with other markers of bone mineral metabolism was determined in 31 untreated postmenopausal women with the osteoporotic syndrome. In addition to serum osteocalcin (BGP) we measured parathyroid hormone (PTH) (carboxyl and mid-molecule fragments), 25(OH)D, alkaline phosphatase, estradiol (E2), estrone (E1), dietary calcium intake, 24 hour urinary calcium excretion, and bone mineral density by CT scan of the lumbar vertebrae. Significant osteopenia was present on CT in untreated postmenopausal osteoporotic women (bone density in 18 out of 31 was below the critical value of 60 mg/cm3). Serum BGP correlated positively with CT scan (r+0.647,P<0.001). CT and age were negatively correlated (r−0.661,P<0.001) while CT and E2 showed a positive correlation (r+0.554,P<0.01). Unexpectedly, BGP and age revealed a significant negative correlation (r−0.421,P<0.05). These findings suggest a state of low bone turnover in this group with untreated postmenopausal osteoporosis.

A high-molecular-weight cysteine endopeptidase from rat skeletal muscle.

A cytosolic enzyme of high molecular weight (about 500 000), which attacks native or denatured proteins (inter alia, casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radioactively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn2+ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42 degrees C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn2+, but with marked aminopeptidase activity and the properties of hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 6B of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine released by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.

The effect of high-dose salmon calcitonin on bone mineral metabolism in the normal rat.

SummaryThe paucity of information on the effect of long-term high-dose salmon calcitonin administration on normal bone mineral metabolism and histology prompted an investigation of the influence of high-dose synthetic calcitonin in the rat. Serum ionized calcium, osteocalcin or BGP (bone gla protein), and immunoreactive PTH were measured serially during calcitonin administration and bone histomorphometry analyzed at 6 weeks (after sacrifice). Daily injections of salmon calcitonin, 0.4 IU/100 g (group B) and 2 IU/100 g (group C), resulted in significant hypocalcemia at 4 hours for both experimental groups (P<0.004). Serium iPTH was significantly higher over the study period for both groups administered calcitonin. Serum BGP levels were significantly lower than controls during the study in group C (P<0.002) and to a lesser extent in group B (P<0.05). In group C, bone histomorphometry revealed increased resorption (onteoclast count), decreased trabecular bone volume, and decreased double-labeled tetracycline surface (bone formation). In group B an increase in osteoclast count but no alteration in bone formation was observed. To assess the role of PTH in the above findings, high-dose calcitonin was administered to parathyroidectomized rats. All of the above changes in bone histomorphometry were not observed in this group of animals. In conclusion, high doses of calcitonin promote hypocalcemia, secondary hyperparathyroidism, and osteoclastosis in the normal rat in a dose-dependent manner with very high-dose calcitonin impairing bone formation.

The response of circulating parameters of bone mineral metabolism to ethanol- and EDTA-induced hypocalcemia in the rat

The mechanism of the acute hypocalcemia that follows acute ethanol administration has not been established. Measurements of parathyroid hormone (PTH) performed during this hypocalcemia reveal conflicting results. We compared the response of ionized calcium (Ca2+), immunoreactive PTH and bone Gla protein (BGP) after ethanol- and EDTA-induced hypocalcemia. 103 male Sprague Dawley rats each weighing approximately 300 g received ethanol and 100 rats of similar weight received EDTA. In each of these studies the animals were divided into experimental and control groups. The ethanol-treated rats received ethanol, 2 g/kg body weight, by ip injection and the EDTA-treated rats received 100 mg EDTA/kg body weight by im injection. Controls received normal saline by the corresponding route of administration. Rats were sacrificed at 0, 30, 60, 90, 180 and 360 min for the measurement of the above parameters. In both experimental groups Ca2+ levels were significantly reduced to the same degree by 30 min with return to control values by 360 min. There was no significant difference in immunoreactive PTH, and BGP between control and ethanol-treated groups. In the EDTA-treated rats, however, PTH values were significantly increased at 30 (P less than 0.005) and BGP at 60 and 90 minutes (P less than 0.005) vs. control. Therefore acute ethanol administration appears to blunt the PTH response to hypocalcemia. A direct inhibitory effect of ethanol on osteoblast function ie BGP production cannot be excluded. In addition, PTH may stimulate BGP.

Hyperstosis induced by the bisphosphanate (2-PEBP) in the oophoretomized rat

SummaryTo prevent the high turnover bone remodeling associated with acute estrogen deficiency, the bisphosphonate [2-(2-pyridinyl) ethylidene-BP] (2-PEBP) was administered to oophorectomized (OX) rats. Three groups of 15 rats each (250 g) were studied. Group (Gp) A was sham operated, Gp B was OX, and Gp C received 2-PEBP (1.72 mg/kg/day) intraperitoneally for 3 days commencing 4 days postoophorectomy. Oophorectomy was confirmed with serum estradiol measurements. Blood samples were collected on days −7, 0, 7, 14, 21, and 28 for ionized calcium (Ca2+), PTH, and serum bone gla protein (BGP). Rats received tetracycline for bone histomorphometric labeling. All results were compared to Gp A. Body weight increased significantly in Gps B and C (P<0.005 by day 28). There was no significant difference in Ca2+, and PTH levels in Gps B and C were similar to Gp A. BGP levels were significantly higher on day 28 in Gp B (P<0.05). In Gp C, BGP levels were significantly decreased on days 7,21, and 28 (P<0.03). Gp B revealed increased bone turnover without loss of bone volume (BV/TV). BV/TV was significantly increased in Gp C despite a decrease in parameters of bone formation and normal osteoclast number. In conclusion, 2-PEBP in the OX rat inhibited bone resorption more than formation with resultant hyperostosis. Serum BGP appeared to be a good marker of the changes observed on bone histomorphometry.

Alteration in osteoblast activity and nutritional vitamin-D deficiency in non-hypercalcemic malignancy

SummaryThe biochemical parameters of bone mineral metabolism in patients with nonhypercalcemic malignancy have not been extensively investigated. Therefore, a group of 29 such patients with different types of malignancy was studied. Ten patients received corticosteroids. In the entire group, serum ionized calcium (Ca2+), bone gla protein (BGP), 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D (1,25(OH)2D) were all lower than in age-matched controls, and carboxy-terminal parathyroid hormone (CPTH) was higher. Although both corticosteroid- and noncorticosteroid-treated patients had decreased BGP values, the corticosteroid-treated patients had lower BGP levels than those not on steroids (4.24±0.70 SE vs. 11.50±2.20 ng/ml;P<0.005). Patients on corticosteroids had lower 1,25(OH)2D values than controls (18.81 ±2.71 vs. 27.83±1.17 pg/ml;P<0.01), whereas those not on corticosteroids had normal 1,25(OH)2D values. These results suggest that patients with nonhydpercalcemic malignancy have nutritional vitamin-D deficiency and secondary hyperparathyroidism with perhaps corticosteroid-induced suppression of serum 1,25(OH)2D and BGP. The decreased levels of serum BGP in the nonsteroid-treated patients suggest, in addition, a defect in osteoblast function.