The Effect of Ionized Calcium, ph, and Temperature on Bioactive Parathyroid Hormone during and after Open=Heart Operations

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1 The Effect of Ionized Calcium, ph, and Temperature on Bioactive Parathyroid Hormone during and after Open=Heart Operations D. J. Chambers, Ph.D., J. Dunham, Ph.D., M. V. Braimbridge, F.R.C.S., B. Slavin, M.R.C.Path., J. Quiney, M.B., and J. Chayen, D.Sc. ABSTRACT Normal myocardial function is dependent on the metabolic balance of a number of electrolytes and hormones. The calcium ion plays a major role in muscle contraction and is rigorously controlled within narrow limits. Open-heart surgery imposes metabolic disturbances on both electrolytes and hormones, especially ionized calcium. Normally, ionized calcium levels are controlled by parathyroid hormone with a negative feedback from the ionized calcium controlling the system, but the results from this study suggest that during open-heart procedures, ionized calcium does not impose its normal negative feedback on bioactive parathyroid hormone secretion. The low blood ph levels that occurred during the operative conditions of the patients studied and the level of hypothermia imposed on the circulating blood during cardiopulmonary bypass appeared to influence the control of parathyroid hormone secretion, causing high levels of hormone to be secreted during this period. Normal myocardial function is dependent on the ions of calcium, potassium, magnesium, sodium, and inorganic phosphate [ l]. However, during and after cardiac operations, substantial metabolic disturbances are imposed on these electrolytes [2-51 because of changes in blood ph, dilution of blood, and the addition of exogenous electrolytes or citrate from banked blood. Of the electrolytes, the importance of calcium in cardiac muscle contraction has long been recognized [6], and the ionized part of the total circulating calcium is the physiologically active From the Departments of Heart Research (Surgical Cytochemistry), Max Rayne Institute, Cardiothoracic Surgery and Clinical Pathology, St. Thomas Hospital, and the Division of Cellular Biology, The Mathilda and Terence Kennedy Institute of Rheumatology, London, England. Accepted for publication Nov 30, Address reprint requests to Dr. Chambers, Heart Research (Surgical Cytochemistry), Max Rayne Institute, St. Thomas Hospital, London SE1 7EH, England. component [7, 81. The lack of a practical method of measuring ionized calcium has hindered definition of the relative importance of both total and ionized calcium. Recently, however, the use of ion-sensitive electrodes has improved the facility of measuring ionized callcium in body fluids [9, 101, allowing accurate, convenient, and quick measurement of ionized calcium in serial samples of whole blood, plasma, or serum. The level of ionized calcium is best studied in association with parathyroid hormone (PTH) levels, because under normal physiological conditions PTH has a direct effect on the level of ionized calcium in the blood [ll, 121. Previous studies have measured PTH by radioimmunoassay, but this method may give anomalous results, probably because of the fragmentation of the hormone and the recognition of biologically inactive fragments by the immunoassay [13]. A cytochemical bioassay for bioactive PTH has recently been developed [ 141, and this technique has been used to investigate whether changes in plasma ionized calcium during and after openheart procedures correlate with changes in circulating levels of bioactive PTH. Total calcium, potassium, magnesium, sodium, and inorganic phosphate ions, total protein, albumin, and blood ph were also measured. Materials and Methods Twelve patients (10 men and 2 women) ranging in age from 43 to 66 years (mean, 53 years) were studied. The operations performed were 7 coronary artery bypass grafts, 2 aortic valve replacements, 1 mitral valve replacement, 1 multiple valve replacement (aortic and mitral valve replacement and tricuspid valve repair), and 1 aortic aneurysm repair. General anesthesia was induced by thiopentone sodium, Omnopon, and pancuronium bromide and was maintained with a mixture of nitrous oxide and oxygen. 306

2 307 Chambers et al: Effect of Ionized Calcium, ph, and Temperature on Parathyroid Hormone Cardiopulmonary bypass was established with a disposable bubble oxygenator, a priming volume of 2 liters of Hartmann s solution and a systemic flow rate that was initially 2.4 liters per minute per square meter of body surface area. Core cooling reduced the body temperature to around 32 C when the aorta was clamped, at which time the systemic flow rate was reduced to 1.5 L/min/m2 in order to minimize noncoronary collateral flow to the myocardium. Most patients were further cooled to around 25 C which under these conditions tended to produce metabolic acidosis in the patients studied. Cold cardioplegic arrest was induced by an infusion of 1 liter of St. Thomas Hospital cardioplegic solution [15, 161; subsequent infusions were administered if aortic cross-clamp time exceeded 60 minutes. Bypass time ranged from 39 to 177 minutes (mean, 109 f 40 minutes [standard deviation]) and aortic occlusion time ranged from 41 to 149 minutes (mean, 83 f 33 minutes). Arterial blood samples were collected from each patient at the following stages during the operation: 1. When the patient was anesthetized and on the operating table. 2. After the patient s chest was opened. 3. Prior to institution of cardiopulmonary bypass. 4. After 5 minutes of bypass. 5. Approximately halfway through bypass (in some patients with longer bypass times, tyo samples [5A and 5B] were taken). 6. Prior to discontinuation of bypass. 7. Fifteen minutes after end of bypass. 8. During later stages of sewing up the patient s chest. Postoperative samples were then taken hourly for up to eight hours. Levels of ionized calcium and blood ph were measured immediately after sampling; the remaining blood was centrifuged and the plasma frozen for later measurement of sodium, potassium, magnesium, and inorganic phosphate ions, total calcium, total protein, albumin, and bioactive PTH levels. Ionized calcium was measured with a Nova 2 ionized calcium-selective electrode. * This is an automated, computerized, thermostatically controlled flow-through system that uses 350 pl of heparinized whole blood, plasma, or serum and takes 70 seconds for analysis. Blood ph was measured using a Radiometer acid-base analyzer (Model PHM 71). Sodium, potassium, and inorganic phosphate ions, total calcium, total protein, and albumin were measured on a Vickers direct multichannel analyzer, and magnesium was measured by atomic absorption. Bioactive levels of PTH were measured by the cytochemical bioassay technique [ 141. The principle of the technique is to use microdensitometric measurement of the biochemical effect of the hormone acting on its specific target cells in intact guinea pig tissue in vitro. The procedures are described in detail elsewhere [14, 171. The mean index of precision of the assay? standard error of the mean is 0.09? 0.04 (N = 11); the reproducibility of serial samples is? 13%. Statistical analyses were done by Student s f test and by a modification of Gaddum s simplified system of computation for the analysis of biological parallel-line assays [ 181 for individual PTH levels. This method calculates the individual PTH level f 95% confidence limits. Thus, any two samples whose 95% confidence limits did not overlap were taken to be significantly different. Results Estimations of the circulating levels of potassium, magnesium, sodium, inorganic phosphate, total protein, and albumin in samples taken at various stages during and after operation were combined for all 12 patients, except the magnesium estimations, which were done for only 3 patients. These combined results (Fig 1) showed little variation from the normal range in phosphate and magnesium ions. There were slight rises in circulating levels of potassium (up to 4.9 mmol per liter) during the bypass period and a small drop in the sodium levels (down to 130 mmol/l) in the prebypass period. Neither was statistically significant, but these slight fluc- *Nova Biomedical Corp., 1238 Chestnut St, MA Newton,

3 ~~ 308 The Annals of Thoracic Surgery Vol 36 No 3 September 1983 (pg;l:k 72 { T lo M.QY*L RINSE 0 I SAMPLE NUMBERS Fig 2. Total calcium, total calcium correctesd for albumin concentration, (0---0), ionized calcium, ph, and parathyroid hormone (PTH) levels during and after openheart operation. Points represent mean & standard error of the mean. OP B"P D 11 I2 13 I4 15 l6 17 YW WBERS Fig 1. Levels of ionized phosphate, magnesium, sodium, and potassium as well as total protein and albumin during and after open-heart operation. Points represent mean +. standard error of the mean. tuations in the combined results reflected considerable change in 1 or 2 patients. The concentrations of total protein and plasma albumin showed significant decreases from the values found in the initial preoperative sample, both in the prebypass period (sample 3; p < 0.05 and p < 0.02, respectively) and during the bypass period ( p < for both total protein and plasma albumin). These decreases in total protein and plasma albumin (55.9% and 55.6%, respectively) were consistent with dilution in the prebypass period and by the priming volume at the beginning of bypass. The concen- trations of both variables recovered during the postbypass period resulting in a constant level during the postoperative period, although these levels were still significantly lower than the initial preoperative value (p < 0.001). Combined Results for ph, Calcium, and PTH Values The results from all 12 patients (Fig 2) showed a significant drop in ph during the bypass period (p < 0.02 and p < for samples 4 and 5, respectively, compared with samples 1 and 2). There was no change in ionized calcium in the 11 patients in whom this was measured, but there was a significant (p < 0.002; comparison of samples 5 and 1) decrease in total calcium levels to a mean value of 1.95 mmoyl during the bypass period. However, when these results were corrected for the plasma albumin levels [19], the values for total calcium were within or higher

4 309 Chambers et al: Effect of Ionized Calcium, ph, and Temperature on Parathyroid Hormone Plasma Levels of Bioacfive Parathyroid Hormone in Patients Undergoing Open-Heart Operations Lowest Levels of Bioactive PTH (pg/ml)a,b Temperature Pre- Post- Patient during Operation Lowest PH during bypass During Bypass bypass No. ("C) Operation Sample 3 Sample 4 Sample 5A Sample 5B Sample 6 Sample ' ' d ' d lc,e Cff ' T d ' 10.7d 45.2' d d 1.9d 8.2' "Each PTH level, along with its 95% confidence limits, has been calculated by the method of Borth [MI. beach sample number indicates a stage during the operation at which blood was taken (see text for details). The PTH level is significantly higher (95% confidence limits do not overlap) than the previously measured level. dthe PTH level is significantly lower (95% confidence limits do not overlap) than the previously measured level. Temperature rose to 32 C with transient drop in ph to Temperature at 37 C but with transient drop in ph to PTH = parathyroid hormone. than normal levels (see Fig 2). The combined results for the concentration of bioactive PTH in the 11 patients in whom this was measured appeared to be inversely related to the changes in ph. Thus, there was a statistically significant (p < 0.001) rise in PTH levels in the samples taken during bypass and in the sample taken immediately after bypass (samples 4, 5, 6, and 7 compared with all other samples). In some individuals, values of up to 70.0 pg per milliliter were recorded (normal levels up to 15.0 pg/ml 1131). Investigation of the results in individual instances where changes in ph and in circulating levels of bioactive PTH could be directly related (Table) showed that, in general, major increases in PTH levels following a decrease in ph did not occur until the body temperature was above 30 C. Thus, in Patients 1 and 2, whose body temperatures were maintained at or above 30T, highly abnormal levels of over 50 pg/ml of PTH were recorded immediately after the drop in ph. Similarly, high levels were found in Patients 4 and 5, both of whom had been cooled to 25 C. In the forrner a high PTH level occurred only when the temperature rose to 32"C, and in the latter a high PTH level was seen when a transient drop in ph occurred at 37 C. Five of the 8 patients who were cooled to below 30 C had normal or near normal levels of bioactive PTH during the bypass period, even though the ph dropped to ph (mean? standard deviation; N = 8). Higher values were found only when the patient was rewarmed before coming off bypass (see Table). Exceptionally high fluctuating values, not obviously related to ph or temperature, were found in 2 patients (Patients 9 and 11). Effect of Ionized Calcium and of ph at Normal Temperature In 1 patient (Patient 8), the PTH levels did appear to respond to changes both in the concentration of ionized calcium and in ph. This patient had a descending aneurysm, and consequently he was not cooled during the femorofemoral bypass. An initial rise in ionized calcium depressed the PTH level during the prebypass period (Fig 3); the ionized calcium level then decreased, and the bioactive PTH rose to a

5 310 The Annals of Thoracic Surgery Vol 36 No 3 September 1983 tion of ionized calcium rose and then fell while the levels of ionized calcium remained fairly constant. 0.9 oe PRE PRE-.pYPASz- POST.- POST.- OP 'S'lPAS? BYPASS OP L peak within the bypass period. Although the ionized calcium level remained low, the PTH level decreased; however, a continuing fall in ionized calcium and a decrease in blood ph produced a further rise in the level of circulating bioactive PTH. Effect of Exogenous Calcium At various times during the operation, generally at the end of the bypass period when the heart was back in the circulation and again during the postbypass period, various amounts of calcium chloride were injected into the circulation at the discretion of the anesthetist to act as a shortlasting inotropic agent. Under normal conditions, such increases in the circulating concentration of calcium should have suppressed the secretion of PTH. However, there was no obvious correlation between the increased levels of ionized calcium in the circulation caused by addition of exogenous calcium and the concentration of PTH in these patients. Indeed, in some patients PTH levels rose as the concentra- Comment During open-heart operations, metabolic disturbances to major electrolytes, albumin, and total protein, caused by changes in blood ph, blood dilution, and the addition of exogenous electrolytes and banked blood, may influence the level of bioactive PTH. Thus, in this study of the factors affecting the release of bioactive PTH during and after open-heart procedures, it was necessary to establish which components exhibited variations from their normal levels and whether these variations correlated with any changes in the level of bioactive PTH. Circulating concentrations of sodium, potassium, magnesium, and inorganic phosphate appeared to be maintained with a fair degree of consistency during operation and in the early postoperative period (see Fig 1). The reduced circulating level of sodium in the prebypass period may have been caused by the addition of 5% dextrose solution to the circulation of some patients. The increased concentrations of potassium seen during the bypass period (see Fig 1) could have been caused by either the addition of the banked blood (acid-citrate-dextrose blood) or the addition of exogenous potassium by the anesthesiologist during this period. The concentrations of total protein and, specifically, of albumin generally declined in the preoperative period and decreased more markedly during bypass; these concentrations did not become fully normal even seven hours after the end of the operation (see Fig 1). The fact that total protein and albumin showed an almost identical percentage of decrease indicates that these decreases were a result of hernodilution in the preoperative stage and of the priming fluid for the cardiopulmonary bypass. These decreased protein concentrations are in accord with those measured by other workers [2-51 during prolonged cardiac procedures. Previous radioimmunoassay studies of PTH levels in patients undergoing prolonged openheart operations [2, 4, 51 have been inconclusive. Yoshioka and co-workers [4] found that levels of PTH decreased at the start of the by-

6 311 Chambers et al: Effect of Ionized Calcium, ph, and Temperature on Parathyroid Hormone pass period (from approximately 500 pg/ml to approximately 200 pg/ml) but returned to prebypass levels by the end of the bypass period. Similarly, the study by Gray and colleagues [5] showed a decrease in PTH levels during cardiopulmonary bypass, from 80.0 to 53.0 pleq/ml when using an assay that was specific for both the amino terminal and the carboxy terminal of the PTH molecule, and from 1,100 to 700 pg/ml when using an assay that was specific only for the carboxy terminal of the PTH molecule. However, although they found a rapid increase in the levels of the amino and carboxy terminals of the PTH molecule, returning to prebypass levels when their patients were off bypass, the levels shown by the assay specific for the carboxy terminal of the PTH molecule remained depressed for up to five days postoperatively. Gray and colleagues [5] suggest that this demonstrates an increase in the secretion of bioactive PTH during the bypass period. In contrast, Moffitt and associates [2] found a significant increase in levels of PTH at the beginning of the perfusion period (from 25.6 pleq/ml before anesthesia to 48.9 pleq/ml early in the perfusion period) that returned to prebypass levels by the end of the bypass period. The present study was initiated because current radioimmunoassays are unable to distinguish between bioactive PTH and the large amounts of biologically inactive fragments of PTH that have been shown to occur in the circulation [20, 211. The cytochemical bioassay for PTH [ 141 measures only the bioactive hormone, however, and it has been shown that this assay can be used to measure changes in bioactive PTH levels produced by very small physiological alterations in ionized calcium levels [22]. In thii 'study we have shown that there is a considerable rise in the levels of bioactive PTH during the bypass period and immediately after bypass, which seems to agree with the results of Moffitt and associates [2] although the increase in PTH found in their patients was less prolonged. Our results were also in agreement with the suggestion by Gray and colleagues [5] that there is an increase in secretion of the bioactive hormone molecule during the period of cardiopulmonary bypass. The question was, What induced this se- cretion of PTH? Normally, PTH levels respond to circulating levels of ionized calcium [12, 221. Such correlation was indeed found in the only patient who was maintained at 37 C (Patient 8). A perplexing feature of the present study was that under the specific operative conditions at St. Thomas Hospital (see Materials and Methods section), there was no relationship between the circulating levels of PTH and the concentration of either ionized calcium or total calcium when the latter was corrected for plasma albumin concentration. However, the secretion of bioactive PTH appeared to correlate with depression of ph, although this correlation was complex in that it appeared to be temperature dependent. Thus, in 2 patients who had been cooled during cardiopulmonary bypass only to 30 C and whose blood ph dropped to below ph 7.3, there was a brisk rise in the circulating level of PTH (see Table). In the patients who were cooled to less than 30 C, the relationship between a drop in ph and the secretion of PTH was less evident or even absent (see Table). In some of these patients, decreased ph apparently led to increased PTH levels only when the body was rewarmed to 30 C and higher. In the 2 patients in whom there were two episodes of reduced ph (Patients 4 and 5; see Table), PTH levels were unaffected when the episode occurred at 25"C, whereas they were markedly elevated when body temperature had increased to greater than 30 C. Both Yoshioka and co-workers [4] and Moffitt and associates [2] observed a decrease in ionized calcium levels during the perfusion period that they attributed to the respiratory and metabolic alkalosis that persisted in their patients. Moffitt and associates [2] explained the increase they found in PTH on the basis of this alkalosis and decreased ionized calcium levels, whereas Yoshioka and co-workers [4] implicated the decrease they observed in PTH levels as a circulatory insufficiency in the distal organs at the beginning of perfusion. Gray and colleagues [5] also noted a decrease in both ionized and total calcium levels that they attributed to hemodilution. However, the pumppriming solution that they used did not contain any calcium, and no exogenous calcium was given to any of the patients at any time during

7 312 The Annals of Thoracic Surgery Vol 36 No 3 September 1983 operation. Consequently, it was not surprising that a fall was seen in both total and ionized calcium levels. They also associated the observed decrease in PTH levels with hemodilution; however, they suggested that there was an increase in the secretion of bioactive PTH during cardiopulmonary bypass that accounted for the recovery of total and ionized calcium levels and for the rapid recovery of the amino and carboxy terminal levels of the PTH to preoperative levels by the end of the bypass period. In our study, the concentration of ionized calcium remained constant within the normal range throughout the sampling period, although the sampling time interval used (15 to 20 minutes for ionized calcium) may have excluded the possibility of detecting any rapid changes in the level of ionized calcium. However, the levels of bioactive PTH showed a significant rise during the bypass and immediate postbypass period. Under the conditions of these operations, ph seems to have a direct influence on the level of circulating bioactive PTH, increasing this concentration as the ph drops below 7.3, provided the body is not cooled below 30 C. At lower temperatures, any fluctuations in PTH levels, apparently in response to ph, are slight, rising only transiently above normal circulating levels. In contrast, at 30 C or above, the circulating concentrations of bioactive PTH reach values corresponding to those found in primary hyperparathyroidism. The same applies to patients who were cooled below 30 C but whose blood had a low ph when they were warmed to greater than 30 C. The PTH level, suppressed at low body temperature, rises with temperature. In conclusion, it is now possible to measure circulating levels of bioactive parathyroid hormone during the course of prolonged openheart procedures, with the heart protected by cardioplegic arrest. The results indicate that decreased ph of the blood can cause considerable elevation of circulating levels of the hormone if the body temperature is higher than 30 C. References 1. Langer GA: Ion fluxes in cardiac excitation and contraction and their relation to myocardial contractility. Physiol Rev 48:708, Moffitt EA, Tarhan S, Goldsmith RS, et al: Patterns of total and ionized calcium and other electrolytes in plasma during and after cardiac surgery. J Thorac Cardiovasc Surg 65:751, Westhorpe RN, Varghese Z, Petrie A, et al: Changes in ionized calcium and other plasma constituents associated with cardiopulmonary bypass. Br J Anaesth 50:951, Yoshioka K, Tsuchioka H, Abe T, Iyomasa Y: Changes in ionized and total calcium concentrations in serum and urine during open-heart surgery. Biochem Med 20:135, Gray R, Braunstein G, Krutzik S, et al: Calcium homeostasis during coronary bypass surgery. Circulation 62:Suppl 1:57, Ringer S: A further contribution regarding the influence of different constituents of blood on the contraction of the heart. J Physiol (Lond) 4:29, McLean FC, Hastings AB: A biological method for the estimation of calcium ion concentration. J Biol Chem , Robertson WG: Measurement of ionized calcium in body fluids: a review. Ann Clin Biochem 13:540, Moore EW Ionized calcium in normal serum, ultrafiltrates and whole blood determined by ionexchange electrodes. J Clin Invest 49:318, Dobson JV, Taylor MJ: Cation analysis in the clinical laboratory. Lab Equip Digest 18:67, Nordin BEC, Peacock M, Wilkinson R: The relative importance of gut, bone and kidney in the regulation of serum calcium. In Talmage RV, Munson PL (eds): Calcium, Parathyroid Hormone and the Calcitonins. Amsterdam, Elsevier Excerpta Medica, 1972, pp Parsons JA: Parathyroid physiology and the skeleton. In Bourne GH (ed): The Biochemistry and Physiology of Bone. New York, Academic, 1976, VO~ 4, pp Segre GV, Habener JF, Powell D, et al: Parathyroid hormone in human plasma: immunochemical characterization and biological implications. J Clin Invest 51:3163, Chambers DJ, Dunham J, Zanelli JM, et al: A sensitive bioassay of parathyroid hormone in plasma. Clin Endocrinol (Oxf) 9375, Hearse DJ, Stewart DA, Braimbridge MV: Cellular protection during myocardial ischemia: the development and characterization of a procedure for the induction of reversible ischemic arrest. Circulation 54:193, Braimbridge MV, Hearse DJ, Chayen J, et al: Cold cardioplegia versus continuous coronary perfusion: clinical and cytochemical assessment. In Longmore DB (ed): Modern Cardiac Surgery. Lancaster, UK, M.T.P. Press, 1978, pp Chayen J: The cytochemical bioassay of polypep-

8 313 Chambers et al: Effect of Ionized Calcium, ph, and Temperature on Parathyroid Hormone tide hormones. Monographs on Endocrinology. Berlin, Springer, vol 17, Borth R: Simplified mathematics for multiple bioassays. Acta Endocrinol (Copenh) 35:454, Correcting the calcium (editorial). Br Med J 1:598, Dambacher MA, Fischer JA, Hunziker WH, et al: Distribution of circulating immunoreactive components of parathyroid hormone in normal subjects and in patients with primary and secondary hyperparathyroidism: the role of the kidney and of the serum calcium concentration. Clin Sci 57:435, 1979 Segre GV, Niall HD, Habener JF, Potts JT Jr: Metabolism of parathyroid hormone: physiological and clinical significance. Am J Med 56:774, 1974 Chambers DJ, Tully G, Rafferty B, et al: Acute increase and decrease of biologically active circulating human parathyroid hormone (biopth) induced by positive and negative calcium challenges within the physiological range (abstract). Calcif Tiss Int [Suppl] 27:A6, 1979