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The anesthetic isoflurane decreases ionized calcium and increases parathyroid hormone and osteocalcin in cynomolgus monkeys
Bone Vol. 23, No. 5 November 1998:479 – 484
The Anesthetic Isoflurane Decreases Ionized Calcium and Increases Parathyroid Hormone and Osteocalcin in Cynomolgus Monkeys C. E. HOTCHKISS, R. BROMMAGE, M. DU, and C. P. JEROME Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
(EDTA) infusion in animals anesthetized with isoflurane lowered ionized calcium but did not cause the expected increase in PTH. This alerted us to possible effects of the anesthetic on calcium and bone metabolism. We hypothesized that anesthesia had increased PTH by release of preformed hormone, and that additional stimulation of the parathyroids could not increase secretion further. Whereas general anesthesia with a combination of agents has been shown to lower ionized calcium and increase PTH in humans,37 there is no information on the effects of specific anesthetics on serum PTH in humans or nonhuman primates. However, such an effect should not be surprising, because several anesthetic regimens have been shown to decrease serum ionized calcium in humans and animals,2,16,31,32,54 and an increase in PTH following methoxyflurane anesthesia has been reported in rats.48 This study was designed to measure the effects of commonly used anesthetics on ionized calcium and PTH concentrations in cynomolgus monkeys.
The effects of anesthetics on calcium metabolism in cynomolgus monkeys were studied. Eight adult female cynomolgus monkeys were used in a crossover design. Blood was collected from each of the monkeys at four timepoints: (1) while conscious; (2) following induction of anesthesia with ketamine, ketamine and atropine, isoflurane, or no anesthetic; (3) at 30 min; and (4) 120 min thereafter. Four experiments were performed with a 1 week washout period between sessions, such that each monkey received each treatment. Potassium was lower in anesthetized monkeys than in those that remained conscious. Cortisol, although high, did not differ among anesthetic treatments. Ketamine and ketamine/ atropine did not consistently affect ionized calcium or parathyroid hormone (PTH) concentrations. Isoflurane decreased ionized calcium (0.05 mmol/L), and increased PTH and osteocalcin twofold. The serum inorganic fluoride concentration was higher in monkeys anesthetized with isoflurane than with ketamine/atropine, which may partially account for the decrease in ionized calcium with isoflurane. The increases in PTH and osteocalcin are presumably secondary to the decrease in ionized calcium. (Bone 23:479 – 484; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Materials and Methods Animals All procedures in monkeys were approved by the Bowman Gray School of Medicine Animal Care and Use Committee. Penhoused, ovariectomized, natural-habitat-bred, adult, female Indonesian cynomolgus monkeys, weighing 2.5–3.5 kg, fed a purified diet containing 0.3% calcium and 0.3% phosphorus, were used for these studies. On a caloric basis, this dietary calcium content is approximately equivalent to the amount consumed by a calcium-supplemented human (1500 mg/day).29
Key Words: Anesthesia; Parathyroid hormone; Calcium; Osteocalcin; Nonhuman primate; Fluoride. Introduction Cynomolgus monkeys have been used successfully as a model for the study of osteoporosis and the effects of antiosteoporotic agents. During such studies, the monkeys are anesthetized for the majority of experimental procedures. Although the effects of anesthesia on cardiopulmonary parameters are generally well known, the effects of anesthetics on serum markers of skeletal relevance, such as parathyroid hormone (PTH) and bone turnover markers, are often not considered. Unexpected results were obtained during the validation of an intact PTH assay in isoflurane-anesthetized monkeys. Whereas intravenous calcium infusion in monkeys sedated with ketamine caused a decrease in serum PTH, intravenous ethylene-diamine tetraacetic acid
Validation of PTH Assay Intact PTH was measured in duplicate using an ELISA kit (Active I-PTH, Diagnostic Systems Laboratories, Webster, TX), which had not been previously validated for use in monkeys. The coefficient of variation for six replicates of monkey serum was 6.3%, with a mean of 31 pg/mL, close to the mean value for normal humans of 33 pg/mL using this assay. Decreasing volumes of monkey serum led to a linear decrease in calculated PTH concentration, as expected (R2 5 0.98). To confirm the physiologic relevance of the PTH measured, infusions of calcium (to decrease PTH secretion) and of citrate (to chelate calcium and increase PTH secretion) were performed. Two monkeys were used for each infusion. Monkeys were sedated with 30 mg of ketamine for infusions and blood collections. Calcium gluconate
Address for correspondence and reprints: Dr. Charlotte E. Hotchkiss, Section on Comparative Medicine, Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
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Table 1. Experimental treatments Treatment
Description
Awake Isoflurane
Monkeys did not receive any anesthetic. Anesthesia was induced with atropine (0.07 mg/kg, im), followed 10 min later with ketamine (20 mg/kg, im). The monkeys were intubated, and administered 100% O2 for 5 min by inhalation, followed by 5% isoflurane for 5 min and then maintenance with 2% isoflurane. Anesthesia was induced with atropine and ketamine, as indicated above, to distinguish effects of isoflurane from those of atropine. Additional ketamine was given for later blood collections as needed. Monkeys were sedated with ketamine (30 mg/monkey, im). This is the normal procedure used for blood collection in our facility. The monkeys were allowed to recover between blood collections; additional ketamine was given for later collections.
K&A (ketamine and atropine) Ketamine
(3 mg/kg elemental calcium) was infused intravenously over a 10 min period, and blood was collected prior to infusion, and at 15, 60, and 120 min following the start of infusion. Citrate (1 mmol/kg in 100 mL normal saline) was infused over a 2 h period, with blood collections at the same timepoints. The doses were selected based on published studies in other species.46,50,53 Anesthesia and Sample Collection Monkeys were trained to enter a catch box so that blood samples could be obtained without sedation. Each monkey underwent four trials in a crossover design, such that each monkey received each treatment in random order (Table 1). Blood was collected from the femoral vein or artery of each monkey at four timepoints: at baseline prior to anesthesia; at “time 0” following induction of anesthesia (approximately 3 min after ketamine injection, or at time of isoflurane reduction to 2%); and at 30 and 120 min after “time 0.” Monkeys were allowed a 1 week recovery period between trials. Biochemical Analyses Ionized calcium samples were maintained anaerobically until analysis. An AVL 988-4 electrolyte analyzer (AVL Scientific Corp., Roswell, GA) was used to measure ionized calcium, and also provided data for sodium, potassium, and blood pH. Serum osteocalcin and cortisol were measured using ELISA (Mid-tact human osteocalcin EIA kit, Biomedical Technologies and Active Cortisol EIA, Diagnostic Systems Laboratories, respectively). For all ELISA assays, all samples from a single monkey were evaluated on the same plate. Osteocalcin was measured in the awake and isoflurane groups, and at baseline and 30 min in the ketamine/atropine group. The remaining samples were not analyzed due to limitations of sample volume. Blood for fluoride analysis was collected in plastic syringes, and serum was separated in plastic microcentrifuge tubes. Fluoride was measured in 120 min samples from monkeys anesthetized with isoflurane using a fluoride ion-selective electrode according to the method of Kissa.34 Samples from monkeys at 120 min of anesthesia with ketamine and atropine, were used for comparison, rather than samples from conscious monkeys, because those were collected in glass Vacutainers, that can leach fluoride.6
ments and the individual monkeys were included in the model as random effects. The baseline values for each parameter were used as covariates. A Bonferroni correction was used to judge the significance of planned multiple comparisons. A paired t-test was used to compare osteocalcin at 30 min vs. baseline in ketamineand atropine-treated monkeys. Pearson correlation coefficients between variables were also determined using SAS. In all cases, a p , 0.05 was considered significant. Results Validation of PTH Assay A 10 min infusion of calcium caused an increase in serum ionized calcium and a decrease in serum PTH at 15 min (Figure 1A). These values tended to return to baseline over time. A 2 h citrate infusion caused a continuing decrease in ionized calcium, and an initial increase in PTH that declined toward baseline values (Figure 1B). Anesthesia Effects Anesthesia had no effect on serum sodium levels, and all three anesthetics decreased serum potassium equally (Figure 2A). Cortisol did not differ among groups overall, but decreased with time in all groups (Figure 2B). Ionized calcium, both at blood pH (data not shown) and adjusted to pH 7.4 (Figure 3), was significantly decreased in monkeys anesthetized with isoflurane compared with all other groups, and continued to decrease as the animals were maintained on the anesthetic. Isoflurane caused a significant increase in serum PTH compared with the other three groups (Figure 4). Ionized calcium and PTH were significantly inversely correlated when monkeys were awake (r 5 20.56, p 5 0.005) or treated with ketamine (r 5 20.66, p 5 0.006). Isoflurane caused a significant increase in osteocalcin compared with awake monkeys (Figure 5). Osteocalcin was unchanged in monkeys treated with ketamine and atropine at 30 min compared with baseline (20.0 6 2.8 vs. 17.1 6 1.4 ng/mL). Mean serum fluoride concentrations were significantly higher at 120 min in monkeys anesthetized with isoflurane compared with those anesthetized with ketamine and atropine (3.7 6 0.3 mmol/L vs. 2.4 6 0.2 mmol/L; p 5 0.009 by paired t-test).
Data Analysis Data were analyzed using the SAS procedure, “mixed” (SAS Institute, Cary, NC). The model examined fixed effects of treatment (anesthetic) and time after induction. The order of treat-
Discussion Isoflurane anesthesia had a striking effect on ionized calcium, PTH, and osteocalcin in cynomolgus monkeys. These effects
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Figure 1. The effect of calcium (A) and citrate (B) infusions on ionized calcium and PTH in cynomolgus monkeys (each point represents mean for two monkeys). Hatched bar represents time of infusion.
have not previously been reported in nonhuman primates and, to our knowledge, the increase in osteocalcin following inhalant anesthesia has not been reported in any species. The effects of certain anesthetics on calcium metabolism have been examined in other species. The reported effects of barbiturate anesthesia are not consistent. In rats, inaktin has been shown to lower total calcium without affecting PTH,26 whereas pentobarbital elevated PTH without affecting calcium,48 and sodium thiopental had no effect.26 In humans, thiopental decreased ionized calcium a small but statistically significant amount.2 In rabbits, pentobarbital did not affect total calcium.5 In dogs, pentobarbital decreased ionized calcium and increased serum PTH by both increasing production and decreasing hepatic clearance.12 The dissociative anesthetic ketamine did not affect calcium or parathyroid hormone in rats,48 and in this study there was no effect in monkeys. Urethane caused a decrease in serum calcium in rabbits5,9 and rats.42 Ether had no effect in rats,48 but was reported to decrease total calcium in humans.25 Suxamethonium and succinylcholine have been associated with decreased ionized calcium in humans;31,32,54 however, the patients in those studies received multiple anesthetic agents, including halothane. In regard to halogenated anesthetics, a decrease in calcium was seen with enflurane in humans,16 and halothane in rabbits5 and horses.21 This decrease was not seen with methoxyflurane in rats, although PTH was increased.48 In humans, enflurane increased renal phosphorus clearance, which may be interpreted as parathyroid gland activity.16 In dogs, the hypocalcemic response to citrate infusion was altered by halothane, isoflurane, and
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Figure 2. The effect of anesthesia on serum potassium (A) and cortisol (B) in cynomolgus monkeys (mean 6 SEM, N 5 8). All three anesthetic regimens significantly decreased potassium (p , 0.001). Baseline cortisol values were 116 6 9 mg/dL (awake), 102 6 8 mg/dL (isoflurane), 104 6 5 mg/dL (K&A), and 110 6 5 mg/dL (ketamine). K&A 5 ketamine and atropine.
enflurane.11,28 In the current study, both a decrease in calcium and an increase in PTH were seen with isoflurane anesthesia in monkeys. Some of the contradictory results reported may be due to the different assays used for PTH, especially in different species. A radiometric assay (Incstar Corp.) has been validated for use in vervet monkeys,19 and partially validated for cynomolgus monkeys.39 On the other hand, we used an IRMA kit that gave very
Figure 3. The effect of anesthesia on ionized calcium corrected to pH 7.4 (mean 6 SEM), in cynomolgus monkeys (N 5 8). Isoflurane significantly decreased ionized calcium (p , 0.001).
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Figure 4. The effect of anesthesia on serum intact PTH (mean 6 SEM) in cynomolgus monkeys (N 5 8). Baseline values were 50 6 8 pg/mL (awake), 52 6 10 pg/mL (isoflurane), 61 6 9 pg/mL (K&A), and 50 6 13 pg/mL (ketamine). Isoflurane significantly increased PTH (p , 0.001).
low values for certain monkey sera, which increased when the serum was heated, suggesting the presence of an inhibitor in some monkeys’ sera (unpublished results). Validation of the PTH ELISA used in this study for cynomolgus macaques provides an additional degree of confidence in the results obtained. Although changes in calcium metabolism were seen only with isoflurane, a decrease in potassium was observed with all anesthetics. Hypokalemia is a result of immobilization, as it is seen with many anesthetics, and even with curare in rabbits,5 and may be secondary to alkalosis, as potassium moves into cells to replace H1. Because the change in potassium was not specific to isoflurane, it was not the cause of the changes in calcium metabolism. In an experiment of this sort, calcium metabolism could be affected indirectly. Conscious venipuncture is stressful to monkeys, causing elevations in serum cortisol.1,15,45 Anesthesia is also physiologically stressful, and ketamine may increase cortisol45 or blunt the cortisol rise due to restraint.1 Corticosteroids, in turn, decrease serum osteocalcin,4,10,41,43 and can increase PTH in vivo10 and in vitro.3 Thus, the high cortisol concentrations in
Figure 5. The effect of isoflurane anesthesia on serum osteocalcin (mean 6 SEM) in cynomolgus monkeys (N 5 8). Baseline values were 12 6 3 ng/mL (awake) and 9 6 2 ng/mL (isoflurane). Isoflurane significantly increased osteocalcin (p , 0.01).
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these monkeys could conceivably be related to some of the changes in calcium metabolism seen here. However, it is unlikely that cortisol is responsible for the changes seen in calcium, PTH, and osteocalcin with isoflurane, because there is no difference in cortisol among the three anesthetized groups. The increase in PTH is most likely secondary to the decrease in ionized calcium. The patterns of calcium and PTH following citrate infusion and isoflurane anesthesia are similar. In both, PTH rises severalfold initially, but decreases toward baseline over time, despite continued decreases in ionized calcium. This response to hypocalcemia has been demonstrated previously, and may be due to an initial rapid release of preformed, stored hormone, followed by a sustained increase in hormone synthesis.20,49 The similarity in patterns suggests that isoflurane decreases serum ionized calcium, which in turn stimulates PTH secretion. Had the PTH secretion come first, one would expect an increase in ionized calcium. Osteocalcin was elevated by isoflurane. Such an increase could be caused by increased production by osteoblasts, an increase in the percentage of osteocalcin released into the bloodstream rather than incorporated into bone, a release of osteocalcin from bone surfaces, or decreased clearance. Whereas PTH administered long term raises osteocalcin,14,23,27 acute infusions of PTH decrease it.23,27,30,47 Conversely, osteocalcin does not affect PTH secretion in vitro.23 However, decreases in ionized calcium as a result of EDTA52 or citrate24 infusion have been reported to increase osteocalcin, as have calcium-deficient diets.18,35 This provides further evidence that the primary effect of isoflurane is to decrease ionized calcium, and that the increases in PTH and osteocalcin are secondary. Further studies are needed to determine the mechanism for an osteocalcin increase in response to decreased calcium. Whereas the increases in PTH and osteocalcin appear to be secondary to the decrease in ionized calcium, the mechanism of this decrease in response to isoflurane is not clear. Because the decrease in calcium does not occur with ketamine or most barbiturates, it is not a result of the anesthetic state or immobilization. The decrease could be effected by any one or combination of three mechanisms. First, there could be direct action of isoflurane on peripheral tissues such as bone, kidney, or muscle increasing uptake of calcium into such tissues, or increased calcium excretion. Halothane, a similar halogenated inhalant anesthetic, increases the uptake of calcium by guinea pig ventricular plasma membranes in vitro.44 Such an increase in intracellular calcium could result in a decrease in blood calcium. Enflurane causes a nonsignificant increase in urinary calcium excretion in humans.16 Alternately, calcium could be chelated by isoflurane, as occurs with citrate or EDTA. Addition of isoflurane to calciumcontaining solutions lowers calcium, but only at concentrations more than tenfold higher than serum concentrations achieved during anesthesia (data not shown). A third possibility would be that inorganic fluoride released from isoflurane during biotransformation is responsible for the decrease in ionized calcium. Fluoride could reduce ionized calcium by precipitation of CaF2 in kidney or other soft tissues, increasing excretion of calcium, or by increasing calcium deposition in bone. Toxic doses of fluoride increase renal tissue calcium 14- to 19-fold,38 and in some studies therapeutic doses of fluoride acutely increase urinary calcium.7,51 Chronically, fluoride increases bone formation by increasing the osteoblast perimeter,8,33 resulting in increased uptake of calcium into bone, as evidenced by decreased serum and urine calcium.13,17 Fluoride has been shown to decrease ionized calcium acutely,22,36,51 and serum fluoride levels following isoflurane anesthesia were significantly higher than those following ketamine
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and atropine anesthesia. In addition, halothane, which contains fewer fluoride residues, caused smaller elevations in serum fluoride than isoflurane,40 and did not prevent EGTA-induced PTH secretion in monkeys.19 However, it is unlikely that the increase of 1.2 mmol/L serum fluoride could entirely account for the rapid drop in ionized calcium of 85 mmol/L. In conclusion, it is likely that the decrease in ionized calcium and associated increases in PTH and osteocalcin following isoflurane anesthesia are due to a combination of factors. Additional experiments are needed to identify the mechanisms that control the uptake of calcium by bone or soft tissue in the presence of isoflurane. Finally, caution must be used when interpreting data obtained from anesthetized subjects.
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Acknowledgments: The authors thank Kathy Kaplan for her assistance with statistics. This article was supported in part by the Forsyth County United Way and by a Venture Grant from the Bowman Gray School of Medicine of Wake Forest University.
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Bone Vol. 23, No. 5 November 1998:479 – 484 48. Schultz, V. L., Boass, A., Garner, S. C., and Toverud, S. U. Several anesthetics, but not diethyl ether, cause marked elevation of serum parathyroid hormone concentration in rats. J Bone Miner Res 10:1298 –1302; 1995. 49. Schwartz, P., Sørensen, H. A., Transbøl, I., and McNair, P. Regulation of acute parathyroid hormone release in normal humans: Combined calcium and citrate clamp study. Am J Physiol 263:E195–E198; 1992. 50. Schwarz, P., Sørenson, H. A., Transbøl, I., and McNair, P. Induced hypocalcaemia controlled by a citrate clamp technique, and the intact parathyroid hormone response obtained. Scand J Clin Lab Invest 50:891– 897; 1990. 51. Stamp, T. C. B., Jenkins, M. V., Loveridge, N., Sphier, P. W., Katakity, M., and MacArthur, S. E. Fluoride therapy in osteoporosis: Acute effects on parathyroid and mineral homeostasis. Clin Sci 75:143–146; 1988. 52. Thomas, S., Movsowitz, C., Epstein, S., Jowell, P., and Ismail, F. The response of circulating parameters of bone mineral metabolism to ethanol- and EDTAinduced hypocalcemia in the rat. Bone Miner 8:1– 6; 1990. 53. Torrance, A. G. and Nachreiner, R. Human-parathormone assay for use in dogs: Validation, sample handling studies, and parathyroid function testing. Am J Vet Res 50:1123–1127; 1989. 54. Wandrup, J., Kancir, C., and Hyltoft Petersen, P. Ionized calcium and acid-base status in arterial and venous whole blood during general anesthesia. Scand J Clin Lab Invest 48:115–122; 1988.
Date Received: February 17, 1998 Date Revised: June 12, 1998 Date Accepted: July 16, 1998
The Anesthetic Isoflurane Decreases Ionized Calcium and Increases Parathyroid Hormone and Osteocalcin in Cynomolgus Monkeys C. E. HOTCHKISS, R. BROMMAGE, M. DU, and C. P. JEROME Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
(EDTA) infusion in animals anesthetized with isoflurane lowered ionized calcium but did not cause the expected increase in PTH. This alerted us to possible effects of the anesthetic on calcium and bone metabolism. We hypothesized that anesthesia had increased PTH by release of preformed hormone, and that additional stimulation of the parathyroids could not increase secretion further. Whereas general anesthesia with a combination of agents has been shown to lower ionized calcium and increase PTH in humans,37 there is no information on the effects of specific anesthetics on serum PTH in humans or nonhuman primates. However, such an effect should not be surprising, because several anesthetic regimens have been shown to decrease serum ionized calcium in humans and animals,2,16,31,32,54 and an increase in PTH following methoxyflurane anesthesia has been reported in rats.48 This study was designed to measure the effects of commonly used anesthetics on ionized calcium and PTH concentrations in cynomolgus monkeys.
The effects of anesthetics on calcium metabolism in cynomolgus monkeys were studied. Eight adult female cynomolgus monkeys were used in a crossover design. Blood was collected from each of the monkeys at four timepoints: (1) while conscious; (2) following induction of anesthesia with ketamine, ketamine and atropine, isoflurane, or no anesthetic; (3) at 30 min; and (4) 120 min thereafter. Four experiments were performed with a 1 week washout period between sessions, such that each monkey received each treatment. Potassium was lower in anesthetized monkeys than in those that remained conscious. Cortisol, although high, did not differ among anesthetic treatments. Ketamine and ketamine/ atropine did not consistently affect ionized calcium or parathyroid hormone (PTH) concentrations. Isoflurane decreased ionized calcium (0.05 mmol/L), and increased PTH and osteocalcin twofold. The serum inorganic fluoride concentration was higher in monkeys anesthetized with isoflurane than with ketamine/atropine, which may partially account for the decrease in ionized calcium with isoflurane. The increases in PTH and osteocalcin are presumably secondary to the decrease in ionized calcium. (Bone 23:479 – 484; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Materials and Methods Animals All procedures in monkeys were approved by the Bowman Gray School of Medicine Animal Care and Use Committee. Penhoused, ovariectomized, natural-habitat-bred, adult, female Indonesian cynomolgus monkeys, weighing 2.5–3.5 kg, fed a purified diet containing 0.3% calcium and 0.3% phosphorus, were used for these studies. On a caloric basis, this dietary calcium content is approximately equivalent to the amount consumed by a calcium-supplemented human (1500 mg/day).29
Key Words: Anesthesia; Parathyroid hormone; Calcium; Osteocalcin; Nonhuman primate; Fluoride. Introduction Cynomolgus monkeys have been used successfully as a model for the study of osteoporosis and the effects of antiosteoporotic agents. During such studies, the monkeys are anesthetized for the majority of experimental procedures. Although the effects of anesthesia on cardiopulmonary parameters are generally well known, the effects of anesthetics on serum markers of skeletal relevance, such as parathyroid hormone (PTH) and bone turnover markers, are often not considered. Unexpected results were obtained during the validation of an intact PTH assay in isoflurane-anesthetized monkeys. Whereas intravenous calcium infusion in monkeys sedated with ketamine caused a decrease in serum PTH, intravenous ethylene-diamine tetraacetic acid
Validation of PTH Assay Intact PTH was measured in duplicate using an ELISA kit (Active I-PTH, Diagnostic Systems Laboratories, Webster, TX), which had not been previously validated for use in monkeys. The coefficient of variation for six replicates of monkey serum was 6.3%, with a mean of 31 pg/mL, close to the mean value for normal humans of 33 pg/mL using this assay. Decreasing volumes of monkey serum led to a linear decrease in calculated PTH concentration, as expected (R2 5 0.98). To confirm the physiologic relevance of the PTH measured, infusions of calcium (to decrease PTH secretion) and of citrate (to chelate calcium and increase PTH secretion) were performed. Two monkeys were used for each infusion. Monkeys were sedated with 30 mg of ketamine for infusions and blood collections. Calcium gluconate
Address for correspondence and reprints: Dr. Charlotte E. Hotchkiss, Section on Comparative Medicine, Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
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Table 1. Experimental treatments Treatment
Description
Awake Isoflurane
Monkeys did not receive any anesthetic. Anesthesia was induced with atropine (0.07 mg/kg, im), followed 10 min later with ketamine (20 mg/kg, im). The monkeys were intubated, and administered 100% O2 for 5 min by inhalation, followed by 5% isoflurane for 5 min and then maintenance with 2% isoflurane. Anesthesia was induced with atropine and ketamine, as indicated above, to distinguish effects of isoflurane from those of atropine. Additional ketamine was given for later blood collections as needed. Monkeys were sedated with ketamine (30 mg/monkey, im). This is the normal procedure used for blood collection in our facility. The monkeys were allowed to recover between blood collections; additional ketamine was given for later collections.
K&A (ketamine and atropine) Ketamine
(3 mg/kg elemental calcium) was infused intravenously over a 10 min period, and blood was collected prior to infusion, and at 15, 60, and 120 min following the start of infusion. Citrate (1 mmol/kg in 100 mL normal saline) was infused over a 2 h period, with blood collections at the same timepoints. The doses were selected based on published studies in other species.46,50,53 Anesthesia and Sample Collection Monkeys were trained to enter a catch box so that blood samples could be obtained without sedation. Each monkey underwent four trials in a crossover design, such that each monkey received each treatment in random order (Table 1). Blood was collected from the femoral vein or artery of each monkey at four timepoints: at baseline prior to anesthesia; at “time 0” following induction of anesthesia (approximately 3 min after ketamine injection, or at time of isoflurane reduction to 2%); and at 30 and 120 min after “time 0.” Monkeys were allowed a 1 week recovery period between trials. Biochemical Analyses Ionized calcium samples were maintained anaerobically until analysis. An AVL 988-4 electrolyte analyzer (AVL Scientific Corp., Roswell, GA) was used to measure ionized calcium, and also provided data for sodium, potassium, and blood pH. Serum osteocalcin and cortisol were measured using ELISA (Mid-tact human osteocalcin EIA kit, Biomedical Technologies and Active Cortisol EIA, Diagnostic Systems Laboratories, respectively). For all ELISA assays, all samples from a single monkey were evaluated on the same plate. Osteocalcin was measured in the awake and isoflurane groups, and at baseline and 30 min in the ketamine/atropine group. The remaining samples were not analyzed due to limitations of sample volume. Blood for fluoride analysis was collected in plastic syringes, and serum was separated in plastic microcentrifuge tubes. Fluoride was measured in 120 min samples from monkeys anesthetized with isoflurane using a fluoride ion-selective electrode according to the method of Kissa.34 Samples from monkeys at 120 min of anesthesia with ketamine and atropine, were used for comparison, rather than samples from conscious monkeys, because those were collected in glass Vacutainers, that can leach fluoride.6
ments and the individual monkeys were included in the model as random effects. The baseline values for each parameter were used as covariates. A Bonferroni correction was used to judge the significance of planned multiple comparisons. A paired t-test was used to compare osteocalcin at 30 min vs. baseline in ketamineand atropine-treated monkeys. Pearson correlation coefficients between variables were also determined using SAS. In all cases, a p , 0.05 was considered significant. Results Validation of PTH Assay A 10 min infusion of calcium caused an increase in serum ionized calcium and a decrease in serum PTH at 15 min (Figure 1A). These values tended to return to baseline over time. A 2 h citrate infusion caused a continuing decrease in ionized calcium, and an initial increase in PTH that declined toward baseline values (Figure 1B). Anesthesia Effects Anesthesia had no effect on serum sodium levels, and all three anesthetics decreased serum potassium equally (Figure 2A). Cortisol did not differ among groups overall, but decreased with time in all groups (Figure 2B). Ionized calcium, both at blood pH (data not shown) and adjusted to pH 7.4 (Figure 3), was significantly decreased in monkeys anesthetized with isoflurane compared with all other groups, and continued to decrease as the animals were maintained on the anesthetic. Isoflurane caused a significant increase in serum PTH compared with the other three groups (Figure 4). Ionized calcium and PTH were significantly inversely correlated when monkeys were awake (r 5 20.56, p 5 0.005) or treated with ketamine (r 5 20.66, p 5 0.006). Isoflurane caused a significant increase in osteocalcin compared with awake monkeys (Figure 5). Osteocalcin was unchanged in monkeys treated with ketamine and atropine at 30 min compared with baseline (20.0 6 2.8 vs. 17.1 6 1.4 ng/mL). Mean serum fluoride concentrations were significantly higher at 120 min in monkeys anesthetized with isoflurane compared with those anesthetized with ketamine and atropine (3.7 6 0.3 mmol/L vs. 2.4 6 0.2 mmol/L; p 5 0.009 by paired t-test).
Data Analysis Data were analyzed using the SAS procedure, “mixed” (SAS Institute, Cary, NC). The model examined fixed effects of treatment (anesthetic) and time after induction. The order of treat-
Discussion Isoflurane anesthesia had a striking effect on ionized calcium, PTH, and osteocalcin in cynomolgus monkeys. These effects
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Figure 1. The effect of calcium (A) and citrate (B) infusions on ionized calcium and PTH in cynomolgus monkeys (each point represents mean for two monkeys). Hatched bar represents time of infusion.
have not previously been reported in nonhuman primates and, to our knowledge, the increase in osteocalcin following inhalant anesthesia has not been reported in any species. The effects of certain anesthetics on calcium metabolism have been examined in other species. The reported effects of barbiturate anesthesia are not consistent. In rats, inaktin has been shown to lower total calcium without affecting PTH,26 whereas pentobarbital elevated PTH without affecting calcium,48 and sodium thiopental had no effect.26 In humans, thiopental decreased ionized calcium a small but statistically significant amount.2 In rabbits, pentobarbital did not affect total calcium.5 In dogs, pentobarbital decreased ionized calcium and increased serum PTH by both increasing production and decreasing hepatic clearance.12 The dissociative anesthetic ketamine did not affect calcium or parathyroid hormone in rats,48 and in this study there was no effect in monkeys. Urethane caused a decrease in serum calcium in rabbits5,9 and rats.42 Ether had no effect in rats,48 but was reported to decrease total calcium in humans.25 Suxamethonium and succinylcholine have been associated with decreased ionized calcium in humans;31,32,54 however, the patients in those studies received multiple anesthetic agents, including halothane. In regard to halogenated anesthetics, a decrease in calcium was seen with enflurane in humans,16 and halothane in rabbits5 and horses.21 This decrease was not seen with methoxyflurane in rats, although PTH was increased.48 In humans, enflurane increased renal phosphorus clearance, which may be interpreted as parathyroid gland activity.16 In dogs, the hypocalcemic response to citrate infusion was altered by halothane, isoflurane, and
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Figure 2. The effect of anesthesia on serum potassium (A) and cortisol (B) in cynomolgus monkeys (mean 6 SEM, N 5 8). All three anesthetic regimens significantly decreased potassium (p , 0.001). Baseline cortisol values were 116 6 9 mg/dL (awake), 102 6 8 mg/dL (isoflurane), 104 6 5 mg/dL (K&A), and 110 6 5 mg/dL (ketamine). K&A 5 ketamine and atropine.
enflurane.11,28 In the current study, both a decrease in calcium and an increase in PTH were seen with isoflurane anesthesia in monkeys. Some of the contradictory results reported may be due to the different assays used for PTH, especially in different species. A radiometric assay (Incstar Corp.) has been validated for use in vervet monkeys,19 and partially validated for cynomolgus monkeys.39 On the other hand, we used an IRMA kit that gave very
Figure 3. The effect of anesthesia on ionized calcium corrected to pH 7.4 (mean 6 SEM), in cynomolgus monkeys (N 5 8). Isoflurane significantly decreased ionized calcium (p , 0.001).
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Figure 4. The effect of anesthesia on serum intact PTH (mean 6 SEM) in cynomolgus monkeys (N 5 8). Baseline values were 50 6 8 pg/mL (awake), 52 6 10 pg/mL (isoflurane), 61 6 9 pg/mL (K&A), and 50 6 13 pg/mL (ketamine). Isoflurane significantly increased PTH (p , 0.001).
low values for certain monkey sera, which increased when the serum was heated, suggesting the presence of an inhibitor in some monkeys’ sera (unpublished results). Validation of the PTH ELISA used in this study for cynomolgus macaques provides an additional degree of confidence in the results obtained. Although changes in calcium metabolism were seen only with isoflurane, a decrease in potassium was observed with all anesthetics. Hypokalemia is a result of immobilization, as it is seen with many anesthetics, and even with curare in rabbits,5 and may be secondary to alkalosis, as potassium moves into cells to replace H1. Because the change in potassium was not specific to isoflurane, it was not the cause of the changes in calcium metabolism. In an experiment of this sort, calcium metabolism could be affected indirectly. Conscious venipuncture is stressful to monkeys, causing elevations in serum cortisol.1,15,45 Anesthesia is also physiologically stressful, and ketamine may increase cortisol45 or blunt the cortisol rise due to restraint.1 Corticosteroids, in turn, decrease serum osteocalcin,4,10,41,43 and can increase PTH in vivo10 and in vitro.3 Thus, the high cortisol concentrations in
Figure 5. The effect of isoflurane anesthesia on serum osteocalcin (mean 6 SEM) in cynomolgus monkeys (N 5 8). Baseline values were 12 6 3 ng/mL (awake) and 9 6 2 ng/mL (isoflurane). Isoflurane significantly increased osteocalcin (p , 0.01).
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these monkeys could conceivably be related to some of the changes in calcium metabolism seen here. However, it is unlikely that cortisol is responsible for the changes seen in calcium, PTH, and osteocalcin with isoflurane, because there is no difference in cortisol among the three anesthetized groups. The increase in PTH is most likely secondary to the decrease in ionized calcium. The patterns of calcium and PTH following citrate infusion and isoflurane anesthesia are similar. In both, PTH rises severalfold initially, but decreases toward baseline over time, despite continued decreases in ionized calcium. This response to hypocalcemia has been demonstrated previously, and may be due to an initial rapid release of preformed, stored hormone, followed by a sustained increase in hormone synthesis.20,49 The similarity in patterns suggests that isoflurane decreases serum ionized calcium, which in turn stimulates PTH secretion. Had the PTH secretion come first, one would expect an increase in ionized calcium. Osteocalcin was elevated by isoflurane. Such an increase could be caused by increased production by osteoblasts, an increase in the percentage of osteocalcin released into the bloodstream rather than incorporated into bone, a release of osteocalcin from bone surfaces, or decreased clearance. Whereas PTH administered long term raises osteocalcin,14,23,27 acute infusions of PTH decrease it.23,27,30,47 Conversely, osteocalcin does not affect PTH secretion in vitro.23 However, decreases in ionized calcium as a result of EDTA52 or citrate24 infusion have been reported to increase osteocalcin, as have calcium-deficient diets.18,35 This provides further evidence that the primary effect of isoflurane is to decrease ionized calcium, and that the increases in PTH and osteocalcin are secondary. Further studies are needed to determine the mechanism for an osteocalcin increase in response to decreased calcium. Whereas the increases in PTH and osteocalcin appear to be secondary to the decrease in ionized calcium, the mechanism of this decrease in response to isoflurane is not clear. Because the decrease in calcium does not occur with ketamine or most barbiturates, it is not a result of the anesthetic state or immobilization. The decrease could be effected by any one or combination of three mechanisms. First, there could be direct action of isoflurane on peripheral tissues such as bone, kidney, or muscle increasing uptake of calcium into such tissues, or increased calcium excretion. Halothane, a similar halogenated inhalant anesthetic, increases the uptake of calcium by guinea pig ventricular plasma membranes in vitro.44 Such an increase in intracellular calcium could result in a decrease in blood calcium. Enflurane causes a nonsignificant increase in urinary calcium excretion in humans.16 Alternately, calcium could be chelated by isoflurane, as occurs with citrate or EDTA. Addition of isoflurane to calciumcontaining solutions lowers calcium, but only at concentrations more than tenfold higher than serum concentrations achieved during anesthesia (data not shown). A third possibility would be that inorganic fluoride released from isoflurane during biotransformation is responsible for the decrease in ionized calcium. Fluoride could reduce ionized calcium by precipitation of CaF2 in kidney or other soft tissues, increasing excretion of calcium, or by increasing calcium deposition in bone. Toxic doses of fluoride increase renal tissue calcium 14- to 19-fold,38 and in some studies therapeutic doses of fluoride acutely increase urinary calcium.7,51 Chronically, fluoride increases bone formation by increasing the osteoblast perimeter,8,33 resulting in increased uptake of calcium into bone, as evidenced by decreased serum and urine calcium.13,17 Fluoride has been shown to decrease ionized calcium acutely,22,36,51 and serum fluoride levels following isoflurane anesthesia were significantly higher than those following ketamine
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and atropine anesthesia. In addition, halothane, which contains fewer fluoride residues, caused smaller elevations in serum fluoride than isoflurane,40 and did not prevent EGTA-induced PTH secretion in monkeys.19 However, it is unlikely that the increase of 1.2 mmol/L serum fluoride could entirely account for the rapid drop in ionized calcium of 85 mmol/L. In conclusion, it is likely that the decrease in ionized calcium and associated increases in PTH and osteocalcin following isoflurane anesthesia are due to a combination of factors. Additional experiments are needed to identify the mechanisms that control the uptake of calcium by bone or soft tissue in the presence of isoflurane. Finally, caution must be used when interpreting data obtained from anesthetized subjects.
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17.
18.
19.
20. 21.
Acknowledgments: The authors thank Kathy Kaplan for her assistance with statistics. This article was supported in part by the Forsyth County United Way and by a Venture Grant from the Bowman Gray School of Medicine of Wake Forest University.
22.
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Date Received: February 17, 1998 Date Revised: June 12, 1998 Date Accepted: July 16, 1998