Hydrochlorothiazide Effects on Serum Calcium and Immunoreactive Parathyroid Hormone Concentrations

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1 BRIEF COMMUNICATIONS Hydrochlorothiazide Effects on Serum Calcium and Immunoreactive Parathyroid Hormone Concentrations Studies in Normal Subjects ROBERT M. STOTE, M.D., LYNWOOD H. SMITH, M.D., F.A.C.P., DAVID M. WILSON, M.D., F.A.C.P., WILLIAM J. DUBE, M.D., RALPH S. GOLDSMITH, M.D., and CLAUDE D. ARNAUD, M.D., Rochester, Minnesota Nine normal subjects were given 50 mg of hydrochlorothiazide twice daily for 25 days, to investigate the relationships between circulating immunoreactive parathyroid hormone (ipth) and changes in calcium homeostasis induced by this diuretic. Total and ionized plasma calcium concentrations were significantly increased during administration of hydrochlorothiazide and for at least 2 weeks after withdrawal of the drug. There was no clearly definable effect either on protein binding of calcium or on ipth. The normal negative correlation between ionized calcium and ipth appeared to remain intact, and the mechanism of the increase in serum calcium is yet to be elucidated. THE ASSOCIATION between use of benzothiadiazine diuretics and altered calcium metabolism was first reported in 1959 by Lamberg and Kuhlback (1); when they gave chlorothiazide and hydrochlorothiazide to patients with congestive heart failure, the urinary excretion of calcium decreased. Several investigators have confirmed this finding in normal subjects and in patients with various diseases (2-12). In addition to this effect of hydrochlorothiazide From the Mayo Clinic and Mayo Foundation, Rochester, Minn. on urinary calcium excretion, however, serum calcium has been observed to increase with variable frequency in normal subjects (2) and in patients with hypertension (2), idiopathic hypercalcemia (4), or primary hyperparathyroidism (9, 12, 13). An association among long-term administration of thiazides, hypertension, and surgically proved hyperparathyroidism has been reported by Paloyan, Forland, and Pickleman (14). Experimentally, Pickleman and associates (15) produced hypercalcemia and enlargement of the parathyroid glands in normal dogs by prolonged administration of hydrochlorothiazide. Coe, Canterbury, and Reiss (16) studied the effect of giving hydrochlorothiazide for 3 to 12 months to patients with idiopathic hypercalciuria in whom serum immunoreactive parathyroid hormone (ipth) had been measured before hydrochlorothiazide treatment. In patients in whom serum ipth had been increased there was a consistent decrease with therapy, which was thought to be related to the decrease in urinary calcium excretion and improvement in calcium balance. Our study of 9 normal subjects before, during, and after administration of hydrochlorothiazide investigated the relationship between circulating ipth and the changes in calcium homeostasis induced by this diuretic. Annals of Internal Medicine 77: ,

2 Subjects and Methods SUBJECTS Five men and four women, ages 21 to 34 years, were studied. Their medical histories showed no evidence of cardiac, renal, or metabolic disorders, and they were not taking medications, vitamins, or calcium supplements. Fasting blood concentrations of total and ionized calcium, ipth, uric acid, total protein, glucose, creatinine, and potassium were within the normal range. After the patients' diets had been supplemented with 1 g of calcium daily for 3 days, urinary excretion of calcium did not exceed 275 mg/24 hr in the men or 250 mg/24 hr in the women (upper limits of normal) (17). DESIGN The subjects ate ad libitum and were given 50 mg of hydrochlorothiazide twice daily for 25 days. No other medication or supplemental potassium was given. Blood samples were obtained daily for the first 5 days; twice weekly during the second, third, and fourth weeks; and 14 days after the administration of hydrochlorothiazide was discontinued. Blood samples were drawn between 7:30 and 9 AM, with subject in a fasting state, and at least 10 hours after the previous dose of hydrochlorothiazide had been ingested. The samples were collected anaerobically without stasis in Vacutainer tubes evacuated to 1 mm Hg; the tubes were completely filled. The filled tubes were immediately placed in ice and were kept at 4 C until centrifugation. The ph and concentrations of total and ionized calcium, magnesium, phosphorus, and total protein were measured in each sample. The serum ipth level was measured by radioimmunoassay before the study; on days 1, 2, 3, 5, 8, 11, 17, and 25 during hydrochlorothiazide administration; and 2 weeks after discontinuation of administration. Serum potassium levels were measured weekly. To establish the validity of the radioimmunoassay in the presence of hydrochlorothiazide, the following studies were performed. To the hyperparathyroid standard serum normally used in the assay we added hydrochlorothiazide in concentrations of 1.0, 2.0, 10.0, and 50.0 /xg/ml, which' is up to 25 times the maximal concentration found in the serums of patients taking 65 mg of hydrochlorothiazide orally (21). Standard immunoassay curves were not altered by these additions. To provide control for possible effects of thiazide metabolites, two standard immunoassay curves of standard hyperparathyroid serum were done by adding 25% serum from two patients with surgical hypoparathyroidism, one of whom was taking hydrochlorothiazide (100 mg twice daily), to the incubation mixture. The two curves were identical. Statistical analysis to compare the values during the study with those during the control period was done using Dunnett's test (22), a method of analysis especially designed to compare multiple points during a treatment period with a single control period. Comparisons of values during the control period with those during the posttreatment period were performed by hierarchical analysis of variance, in which the differences between individual patients and between replicate mea- ANALYSIS Plasma ionized calcium levels were measured with a flow-through calcium electrode* after withdrawal of plasma by tuberculin syringe through the rubber stopper of the evacuated tube. The coefficient of variation for ionized calcium concentrations in our laboratory is less than 1%. Aliquots of the plasma used to measure the ionized calcium levels were used to measure ph by microelectrode and plasma protein level by refractometryt. Concentrations of serum calcium and magnesium were measured by atomic absorption spectroscopy (18). The coefficient of variation for serum calcium concentrations in our laboratory is less than 1% (normal range, 8.9 to 10.1 mg/100 ml). Serum phosphorus levels were measured by a modification of the Fiske and Subbarow method adapted for use with the Auto- Analyzer (19). The ipth radioimmunoassays were performed by the method of Arnaud, Tsao, and Littledike (20). The intra-assay variability (mean percentage variation) of duplicate ipth measurements assayed at two dilutions is 11.8 ±0.6% (SE), and the inter-assay variability is 12.2 ±0.8% (SE) (20). * Orion-Research, Inc., Cambridge, Mass. t American Optical Corp., Buffalo, N.Y. 588 October 1972 Annals of Internal Medicine Volume 77 Number 4 Figure 1. Mean values (±SE) of serum total calcium, ionized calcium, total protein, and immunoreactive parathyroid hormone (ipth). TZ = hydrochlorothiazide; B.I.D. = twice a day.

3 Table 1. Effect of 50 Mg of Hydrochlorothiazide Given Twice Daily on Nine Subjects Mean Serum Values ± SE Total Ionized Immunoreactive Total Calcium Calcium Parathyroid Hormone Protein mg/100 ml filitre eq/ml g/100 ml Before administration 9.55 ± ± ± ± 0.10 Day ± ± ± ± 0.15 Day ± ± ± ±0.17 Day ± ± ± ± 0.14 Day ± ± ± 0.12 Day ± ± ± ± 0.12 Day ± ± ± ± 0.15 Day ± ± ± ± 0.08 Day ± ± ±0.13 Day ± ± ± ± 0.13 Day 22 Day ± ± ± ± ± ± weeks after drug withdrawn 9.80 ± ± ± ± 0.16 Normal values 8.9 to to38 surements in each patient were considered as separate sources of variation. Results TOTAL SERUM CALCIUM The mean serum calcium concentration increased significantly (P < 0.05) after 1 day of hydrochlorothiazide administration and gradually reached a peak at 11 days. It then decreased toward the control mean value but remained significantly higher (P < 0.001) than the control value even 2 weeks after the medication had been withdrawn (Figure 1; Table 1). Seven of the nine subjects became hypercalcemic while receiving hydrochlorothiazide, and three had an increase of more than 1 mg/100 ml (the greatest was 1.3 mg/100 ml). Regression analysis of total calcium on total protein showed that total calcium was significantly influenced by total protein (P < 0.001). PLASMA IONIZED CALCIUM The mean plasma ionized calcium concentration decreased significantly (P < 0.05) after 24 hours of treatment and then returned to the pretreatment value after 48 hours, despite an increase in total protein levels. It increased during the next 2 weeks, was significantly increased (P < 0.05) at the 17th day, and remained significantly increased (P < 0.001) 2 weeks after cessation of hydrochlorothiazide administration (Figure 1; Table 1). There was no significant correlation between ionized calcium and total protein. TOTAL PROTEIN The total protein concentration increased by an average of 1.06 g/100 ml (15%) at 48 hours, remained slightly increased (average, +6%) throughout the duration of hydrochlorothiazide administration, and returned to control values in the posttreatment period (Figure 1; Table 1). SERUM IMMUNOREACTIVE PARATHYROID HORMONE The mean pretreatment value of ipth was 14.6 /Jitre eq/ml. Throughout the study none of the serum ipth values was significantly different from the control value despite the apparent increase during the first several days of hydrochlorothiazide administration (Figure 1; Table 1). For the entire study there was a significant (P<0.01) regression of ipth on ionized calcium (negative) and serum protein (positive). By multiple regression analysis, concentrations of total protein and of ionized calcium were found to have contributed equally to the effect on concentration of ipth. OTHER MEASUREMENTS No significant changes were noted in the concentrations of serum magnesium and phosphorus. Serum potassium levels were significantly decreased by the second week and remained so while hydrochlorothiazide was being administered. The plasma ph increased only 0.01 in the first 24 hours, when the ionized calcium level had decreased significantly, and the difference from control ph was never greater than Discussion Previous observations that thiazide diuretics may produce hypercalcemia (2, 4, 12) has been con- Stote et a/. Hydrochlorothiazide Effects 589

4 firmed in this study of nine normal subjects. All of them showed an increase in total and ionized plasma calcium levels during administration of hydrochlorothiazide, and seven of the nine developed hypercalcemia. The increased values persisted at least 2 weeks after cessation of administration in eight subjects, and the difference from control value was statistically significant for both total and ionized calcium. The mechanism by which hydrochlorothiazide increases serum calcium levels has not been examined systematically, although the demonstration by Koppel and his associates (23, 24) of a similar response in oliguric patients seems to exclude a renal mechanism. Furthermore, Parfitt (9) found no consistent correlation between increases in serum calcium levels and decreases in urinary excretion of calcium. Duarte and co-workers (12), on the other hand, have suggested that increased calcium binding by plasma proteins might account for the hypercalcemia, since they observed no change in ultrafiltrable calcium in a patient whose total serum calcium level increased from 10.0 to 12.4 mg/100 ml. In this patient, however, the serum protein concentration had increased by only 0.3 g/100 ml at the time of maximal serum calcium, suggesting that hydrochlorothiazide had increased the binding affinity of the proteins for calcium. Our subjects did not have as great an increase in serum calcium levels, but the increase in their serum protein levels was greater during the first week of hydrochlorothiazide administration (0.5 to 1.0 g/100 ml). Although we did not measure ultrafiltrable calcium, the increases in total and ionized calcium levels were generally proportional throughout the study. These data indicate that no gross deviation from normal protein binding (25-28) occurred during hydrochlorothiazide administration. However, in the posttreatment period, when serum protein concentration had returned to the control value, the mean increase in ionized calcium was equal to the mean increase in total calcium. This suggests that, if a deviation from normal protein binding had occurred, it was more likely a decrease than an increase. Pickleman and associates (15) suggested that thiazides may cause increased secretion of parathyroid hormone, from their observation of hypercalcemia and enlarged parathyroid glands in normal dogs that were given up to 200 mg of hydrochlorothiazide daily for 4 to 9 months. We found no significant increase in serum ipth during hydrochlorothiazide administration, but our subjects received less of the drug for a shorter period. We chose to use the standard therapeutic dose in the normal subjects and did observe significant alterations in calcium metabolism throughout the study. The early apparent increase may have been artifactual, since it coincided with the highest values of serum protein. We have reported previously that large quantities of serum proteins may produce minor nonspecific inhibition of the immunoreaction in our radioimmunoassay (21), an effect that we have found with every antiserum. All serum ipth values measured when serum protein was increased may actually represent lower values of serum ipth. If so, it would mean that the serum ipth level was decreased when ionized calcium was increased after the first several days. That this may be correct is indicated by the highly significant multiple regression of serum ipth on ionized calcium (negative) and total protein (positive). Despite these highly significant regression coefficients, there was no significant correlation between ionized calcium and total protein, and the effects of ionized calcium and total protein on serum ipth may have been independent. We cannot state with assurance the mechanisms of the dual effects, but it seems reasonable to consider the protein effect as most likely being an in vitro phenomenon and the ionized calcium effect as an in vivo one. This would require an as yet unidentified mechanism for the observed increase in ionized calcium, with a secondary and appropriate decrease in serum immunoreactive parathyroid hormone. ACKNOWLEDGMENTS: The authors are indebted to the following technologists for invaluable assistance in these studies: Mary Jo Bill, Karen J. Laakso, Judith A. Larsen, Diane J. Brinck, Pamela A. Bonnes, and Mary C. Matthusen. Supported in part by a grant from the Mayo Foundation, Rochester, Minn., and by research grants AM and RR-585, National Institutes of Health, Bethesda, Md. Received 17 March 1972; revision accepted 28 June Requests for reprints should be addressed to Section of Publications, Mayo Clinic, Rochester, Minn References 1. LAMBERG B-A, KUHLBACK B: Effect of chlorothiazide and hydrochlorothiazide on the excretion of calcium in urine. Scand J Clin Lab Invest 11: , SEITZ H, JAWORSKI ZF: Effect of hydrochlorothiazide on serum and urinary calcium and urinary citrate. Can Med Assoc J 90: , NASSIM JR, HIGGINS BA: Control of idiopathic hypercalciuria. Br Med J 1: , YENDT ER, GAGNE RJA, COHANIM M: The effects of thiazides in idiopathic hypercalciuria. Am J Med Sci 251: , DUARTE CG, BLAND JH: Changes in metabolism of calcium, phosphorus and uric acid after oral administration of chlorothiazide. 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