Journal of lnternal Medicine 1990; 2 2 8 ; 367-371
The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women J. GRAM, J. BOLLERSLEV*, H. K. NIELSENt, H. F. L A R S E N t & L. MOSEKILDES Prom the Department of Clinical Immunology and the * Departmeit of Medical Endocrinology. Odense
University Hospital, the t Department o/ Medical Endocrinology. Arhus AmtssHgehus, and the $ Department of Clinical Chemistry. Svendborg Sygehus. Denmark
Abstract. Gram J, Bollerslev J, Nielsen HK, Larsen HF. Mosekilde L (Department of Clinical Immunology and Department of Medical Endocrinology, Odense University Hospital, Department of Medical Endocrinology, Arhus Amtssygehus. and Department of Clinical Chemistry, Svendborg Sygehus, Denmark). The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women. Journal of lnternal Medicine 1990; 228: 367-371. Carbonic anhydrase localized in bone resorptive cells generates the protons necessary for bone resorption. Inhibition of the enzyme is a potential mechanism for decreasing bone resorption. Eight healthy post-menopausal women received oral acetazolamide 2 50 mg twice daily for 28 d. Bone resorption, evaluated by serum acid phosphatase activity and the renal excretion of hydroxyproline. was unaltered, as was bone formation estimated by serum levels of alkaline phosphatase and osteocalcin. The fasting renal excretion of calcium was increased, whereas serum ionized calcium was unchanged. The maximal renal reabsorption of phosphate decreased, but it was not an effect of PTH as it decreased significantly during the treatment period. In conclusion, no significant effect on biochemical markers of bone remodelling could be detected during the study period. The observed changes in calcium and phosphate metabolism may be secondary to the renal effect of acetazolamide. Keywords ; acetazolamide. bone resorption, carbonic anhydrase.
Introduction Carbonic anhydrase (CA) catalyses the reversible hydration of CO, to H,CO,, and is a widespread enzyme in biological systems. Three soluble isoenzymes (CA 1-111) are known in man. CA I1 is the only isoenzyme found in bones, where it has been localized exclusively in osteoclasts [1,21. Several studies have indicated that CA plays a role in bone resorption. The active metabolite of vitamin D, (1.25(OH),D3), a potent stimulator of bone resorption, increases the CA activity of bones when added to mouse calvarian culture systems [3]. The CA inhibitor acetazolamide antagonizes both the calcium-releasing effect of 1,25(OH),D, [3, 41 and the spontaneous release of calcium from bones [3, 51. Furthermore, in vivo studies have shown that partial
prevention of denervation induced bone loss in rats when acetazolamide was incorporated in the diet [61. The mechanism of action of CA in osteoclasts has been postulated to be facilitation of bone resorption by generation of protons. These are actively transported across the membrane of the ruffled border into the site of resorption [7]. This hypothesis is consistent with the very acidic extracellular fluid found in the ruffled border area of osteoclasts [S], and the inability to maintain this environment on exposure to acetazolamide [9]. Inhibition of CA II may therefore be a potential mechanism for decreasing bone resorption in vivo, and it may be useful in the treatment of metabolic bone disorders characterized by increased bone
367
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resorption. The aim of the present study was to describe the effect of short-term CA inhibition on bone and calcium homeostasis in normal postmenopausal women.
Methods Eight volunteer women aged 48-62 years (median 5 1 years) participated in the study. All subjects were post-menopausal (median 3.5 years after menopause, range 1-10 years) and received no concomitant medication. They were all healthy with no history or clinical signs of endocrine or renal disorders. The study was conducted according to the Declaration of Helsinki II, and was approved by the Regional Ethical Committee. All subjects were treated with oral acetazolamide, 250 mg twice daily for 28 d, as a sustained release preparation (Glaupax Retard'@,Ercopharm). Blood samples were collected after an overnight fast 2 weeks before and on the day of initiation of acetazolamide administration, and also 1 , 2 , 3 , 4 and 6 weeks later. Fasting urine samples were obtained at the same time. Changes in the metabolic component of blood acid-base equilibrium were evaluated by plasma standard bicarbonate (P-HCO,). Bone formation was .evaluated by serum osteocalcin (S-OC) and alkaline phosphatase (S-AF'). Bone resorption was evaluated by serum acid phosphatase (S-ACP) and the fasting renal excretion of hydroxyproline mol-' excreted creatinine (U-OHP/U-Cr). Calcium and phosphate metabolism was evaluated by serum ionized calcium (S-Ca2+),serum intact parathyroid hormone [S-PTH (1-84)], serum phosphate (S-Ph) and the fasting renal excretion of calcium (U-Ca/U-Cr) and phosphate (U-Ph/U-Cr) mol-' excreted creatinine. Serum osteocalcin was determined by radioimmunoassay using a modification of the method of Price and Nishimoto [11, 121. Urine hydroxyproline was measured by the method described by Pedenphant [131. Serum ionized calcium was measured by a calcium ion-selective electrode (ICA 1, Radiometer) employing an incorporated correction for pH [14]. S-PTH (1-84) was determined with a commercial kit (Allegro Intact PTH (IRMA), Nichols Institute) [15]. AU other analyses were performed using standard laboratory methods. The maximum theoretical renal reabsorption capacity of phosphate (TmE'/GFR) was determined according to Bijvoet [16].
Statistical methods
All measurements are given as median values. Pre-, intra- and post-treatment values were compared using Friedman's test for one-way analysis of variance. In the case of significant variance, pretreatment values were compared with intra- and post-treatment values using Wilcoxon's paired rank sum test. The level of statistical significance was set at P < 0.05.
Results All subjects completed the study. Reversible and tolerable peripheral paraesthesia was present in all participants as the only recorded side-effect. The symptoms were aggravated by cold during the whole treatment period. There was no difference between the two measurements before acetazolamide treatment, and therefore the results from day 0 are used as initial values. Treatment with acetazolamide induced a significant change in P-HCO, (P < 0.00001, Friedman test) which decreased by 22 % (median value) (P < 0.01) during the 6rst week (Fig. 1). During the last 2 weeks of treatment P-HCO, increased, but remained 15% lower than the pretreatment value (P < 0.02). The biochemical markers of bone formation, S-AP and S-OC, did not change significantly during the study period. The markers of bone resorption, S-ACP and U-OHP/U-Cr, also remained unaltered. Serum ionized calcium (S-CaZ+)did not change significantly. There was a significant alteration in UCa/U-Cr (P < 0.008), with a 50% increase in the second treatment week ( P < 0.02). It later normalized and 2 weeks after the treatment period it was insignificantly reduced compared with the initial value. S-Ph showed a moderate but significant decrease after 1 and 3 weeks (8%,P < 0.05 and 4%, P < 0.02, respectively), but U-Ph/U-Cr did not change significantly. Consequently, TmP/GFR did change significantly (P < 0.008), with a decrease in the first and third treatment week (13%. P < 0.01 and 20% P < 0.05, respectively). S-iPTH (1-84) changed (P < 0.0006),and was significantlyreduced in the third week (18 %. P < 0.05). returning to the initial value after the treatment period.
CARBONIC ANHYDRASE INHIBITION
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The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women J. GRAM, J. BOLLERSLEV*, H. K. NIELSENt, H. F. L A R S E N t & L. MOSEKILDES Prom the Department of Clinical Immunology and the * Departmeit of Medical Endocrinology. Odense
University Hospital, the t Department o/ Medical Endocrinology. Arhus AmtssHgehus, and the $ Department of Clinical Chemistry. Svendborg Sygehus. Denmark
Abstract. Gram J, Bollerslev J, Nielsen HK, Larsen HF. Mosekilde L (Department of Clinical Immunology and Department of Medical Endocrinology, Odense University Hospital, Department of Medical Endocrinology, Arhus Amtssygehus. and Department of Clinical Chemistry, Svendborg Sygehus, Denmark). The effect of carbonic anhydrase inhibition on calcium and bone homeostasis in healthy postmenopausal women. Journal of lnternal Medicine 1990; 228: 367-371. Carbonic anhydrase localized in bone resorptive cells generates the protons necessary for bone resorption. Inhibition of the enzyme is a potential mechanism for decreasing bone resorption. Eight healthy post-menopausal women received oral acetazolamide 2 50 mg twice daily for 28 d. Bone resorption, evaluated by serum acid phosphatase activity and the renal excretion of hydroxyproline. was unaltered, as was bone formation estimated by serum levels of alkaline phosphatase and osteocalcin. The fasting renal excretion of calcium was increased, whereas serum ionized calcium was unchanged. The maximal renal reabsorption of phosphate decreased, but it was not an effect of PTH as it decreased significantly during the treatment period. In conclusion, no significant effect on biochemical markers of bone remodelling could be detected during the study period. The observed changes in calcium and phosphate metabolism may be secondary to the renal effect of acetazolamide. Keywords ; acetazolamide. bone resorption, carbonic anhydrase.
Introduction Carbonic anhydrase (CA) catalyses the reversible hydration of CO, to H,CO,, and is a widespread enzyme in biological systems. Three soluble isoenzymes (CA 1-111) are known in man. CA I1 is the only isoenzyme found in bones, where it has been localized exclusively in osteoclasts [1,21. Several studies have indicated that CA plays a role in bone resorption. The active metabolite of vitamin D, (1.25(OH),D3), a potent stimulator of bone resorption, increases the CA activity of bones when added to mouse calvarian culture systems [3]. The CA inhibitor acetazolamide antagonizes both the calcium-releasing effect of 1,25(OH),D, [3, 41 and the spontaneous release of calcium from bones [3, 51. Furthermore, in vivo studies have shown that partial
prevention of denervation induced bone loss in rats when acetazolamide was incorporated in the diet [61. The mechanism of action of CA in osteoclasts has been postulated to be facilitation of bone resorption by generation of protons. These are actively transported across the membrane of the ruffled border into the site of resorption [7]. This hypothesis is consistent with the very acidic extracellular fluid found in the ruffled border area of osteoclasts [S], and the inability to maintain this environment on exposure to acetazolamide [9]. Inhibition of CA II may therefore be a potential mechanism for decreasing bone resorption in vivo, and it may be useful in the treatment of metabolic bone disorders characterized by increased bone
367
368
J. G R A M et al.
resorption. The aim of the present study was to describe the effect of short-term CA inhibition on bone and calcium homeostasis in normal postmenopausal women.
Methods Eight volunteer women aged 48-62 years (median 5 1 years) participated in the study. All subjects were post-menopausal (median 3.5 years after menopause, range 1-10 years) and received no concomitant medication. They were all healthy with no history or clinical signs of endocrine or renal disorders. The study was conducted according to the Declaration of Helsinki II, and was approved by the Regional Ethical Committee. All subjects were treated with oral acetazolamide, 250 mg twice daily for 28 d, as a sustained release preparation (Glaupax Retard'@,Ercopharm). Blood samples were collected after an overnight fast 2 weeks before and on the day of initiation of acetazolamide administration, and also 1 , 2 , 3 , 4 and 6 weeks later. Fasting urine samples were obtained at the same time. Changes in the metabolic component of blood acid-base equilibrium were evaluated by plasma standard bicarbonate (P-HCO,). Bone formation was .evaluated by serum osteocalcin (S-OC) and alkaline phosphatase (S-AF'). Bone resorption was evaluated by serum acid phosphatase (S-ACP) and the fasting renal excretion of hydroxyproline mol-' excreted creatinine (U-OHP/U-Cr). Calcium and phosphate metabolism was evaluated by serum ionized calcium (S-Ca2+),serum intact parathyroid hormone [S-PTH (1-84)], serum phosphate (S-Ph) and the fasting renal excretion of calcium (U-Ca/U-Cr) and phosphate (U-Ph/U-Cr) mol-' excreted creatinine. Serum osteocalcin was determined by radioimmunoassay using a modification of the method of Price and Nishimoto [11, 121. Urine hydroxyproline was measured by the method described by Pedenphant [131. Serum ionized calcium was measured by a calcium ion-selective electrode (ICA 1, Radiometer) employing an incorporated correction for pH [14]. S-PTH (1-84) was determined with a commercial kit (Allegro Intact PTH (IRMA), Nichols Institute) [15]. AU other analyses were performed using standard laboratory methods. The maximum theoretical renal reabsorption capacity of phosphate (TmE'/GFR) was determined according to Bijvoet [16].
Statistical methods
All measurements are given as median values. Pre-, intra- and post-treatment values were compared using Friedman's test for one-way analysis of variance. In the case of significant variance, pretreatment values were compared with intra- and post-treatment values using Wilcoxon's paired rank sum test. The level of statistical significance was set at P < 0.05.
Results All subjects completed the study. Reversible and tolerable peripheral paraesthesia was present in all participants as the only recorded side-effect. The symptoms were aggravated by cold during the whole treatment period. There was no difference between the two measurements before acetazolamide treatment, and therefore the results from day 0 are used as initial values. Treatment with acetazolamide induced a significant change in P-HCO, (P < 0.00001, Friedman test) which decreased by 22 % (median value) (P < 0.01) during the 6rst week (Fig. 1). During the last 2 weeks of treatment P-HCO, increased, but remained 15% lower than the pretreatment value (P < 0.02). The biochemical markers of bone formation, S-AP and S-OC, did not change significantly during the study period. The markers of bone resorption, S-ACP and U-OHP/U-Cr, also remained unaltered. Serum ionized calcium (S-CaZ+)did not change significantly. There was a significant alteration in UCa/U-Cr (P < 0.008), with a 50% increase in the second treatment week ( P < 0.02). It later normalized and 2 weeks after the treatment period it was insignificantly reduced compared with the initial value. S-Ph showed a moderate but significant decrease after 1 and 3 weeks (8%,P < 0.05 and 4%, P < 0.02, respectively), but U-Ph/U-Cr did not change significantly. Consequently, TmP/GFR did change significantly (P < 0.008), with a decrease in the first and third treatment week (13%. P < 0.01 and 20% P < 0.05, respectively). S-iPTH (1-84) changed (P < 0.0006),and was significantlyreduced in the third week (18 %. P < 0.05). returning to the initial value after the treatment period.
CARBONIC ANHYDRASE INHIBITION
-
EE
1.28.
-
+
NS
, ' - -
OI
1.24. E
d2
369
1.20.
t 3 0.60-
P
