- Email: [email protected]
Effect of rate of calcium reduction and a hypocalcemic clamp on parathyroid hormone secretion: A study in dogs
Kidney International, Vol. 55 (1999), pp. 1724–1733
Effect of rate of calcium reduction and a hypocalcemic clamp on parathyroid hormone secretion: A study in dogs JOSE C. ESTEPA, ESCOLASTICO AGUILERA-TEJERO, YOLANDA ALMADEN, MARIANO RODRIGUEZ, and ARNOLD J. FELSENFELD Departamentos de Nefrologı´a y Unidad de Investigacio´n del Hospital Universitario Reina Sofı´a; Departamento de Patologı´a Clı´nica Veterinaria, Universidad de Co´rdoba, Co´rdoba, Spain; and the Department of Medicine, West Los Angeles VA Medical Center and UCLA, Los Angeles, California, USA
Effect of rate of calcium reduction and a hypocalcemic clamp on PTH secretion: A study in dogs. Background. The parathyroid hormone (PTH) calcium curve is used to evaluate parathyroid function in clinical studies. However, unanswered questions remain about whether PTH secretion is affected by the rate of calcium reduction and how the maximal PTH response to hypocalcemia is best determined. We performed studies in normal dogs to determine whether (a) the rate of calcium reduction affected the PTH response to hypocalcemia and (b) the reduction in PTH values during a hypocalcemic clamp from the peak PTH value observed during the nadir of hypocalcemia was due to a depletion of stored PTH. Methods. Fast (30 min) and slow (120 min) ethylenediaminetetraacetic acid (EDTA) infusions were used to induce similar reductions in ionized calcium. In the fast EDTA infusion group, serum calcium was maintained at the hypocalcemic 30-minute value for an additional 90 minutes (hypocalcemic clamp). To determine whether the reduction in PTH values during the hypocalcemic clamp represented depletion of PTH stores, three subgroups were studied. Serum calcium was rapidly reduced from established hypocalcemic levels in the fast-infusion group at 30 and 60 minutes (after 30 min of a hypocalcemic clamp) and in the slow-infusion group at 120 minutes. Results. At the end of the fast and slow EDTA infusions, serum ionized calcium values were not different (0.84 6 0.02 vs. 0.82 6 0.03 mm), but PTH values were greater in the fastinfusion group (246 6 19 vs. 194 6 13 pg/ml, P , 0.05). During the hypocalcemic clamp, PTH rapidly decreased (P , 0.05) to values of approximately 60% of the peak PTH value obtained at 30 minutes. A rapid reduction in serum calcium from established hypocalcemic levels at 30 minutes did not stimulate PTH further, but also PTH values did not decrease as they did when a hypocalcemic clamp was started at 30 minutes. At 60 minutes, the reduction in serum calcium increased (P , 0.05) PTH to peak values similar to those before the hypocalcemic clamp. The reduction in serum calcium at 120 minutes in the slow
Key words: hypocalcemia, parathyroid hormone, EDTA, PTH-calcium curve, hyperparathyroidism. Received for publication April 17, 1998 and in revised form October 30, 1998 Accepted for publication December 15, 1998
1999 by the International Society of Nephrology
EDTA infusion group increased PTH values from 224 6 11 to 302 6 30 pg/ml (P , 0.05). Conclusions. These results suggest that (a) the reduction in PTH values during the hypocalcemic clamp may not represent a depletion of PTH stores. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH-calcium curve, and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium.
The parathyroid hormone (PTH) calcium curve has been used to evaluate parathyroid function in normal volunteers [1–6] and in patients with primary [7–9] and secondary hyperparathyroidism [10–27]. In studies performed in normal volunteers and in dialysis patients on continuous ambulatory peritoneal disease, the standard method of inducing hypocalcemia is the infusion of a calcium-chelating agent, either ethylenediaminetetraacetic acid (EDTA) or citrate. However, for convenience in hemodialysis patients, a reduction in the dialysate calcium concentration is generally used to induce hypocalcemia. Furthermore, the use of a low-calcium dialysate concentration generally produces a more rapid reduction in serum calcium than the usual two-hour infusion of a chelating agent. In a previous study in normal volunteers, the effect on PTH stimulation of a 60and 120-minute reduction in ionized calcium of similar magnitude was similar [1]. However, the effect of a more rapid reduction in serum calcium (30 min) on parathyroid function as compared with a 120-minute calcium reduction has not been evaluated in a controlled setting. In addition to the possible effect of the rate of calcium reduction, the PTH calcium curve has been shown to be modified by the level of serum calcium. In a recent study, we showed that the PTH response to hypocalcemia was less when hypocalcemia was induced from hypercalcemia, which had resulted after the serum calcium had been clamped at a hypercalcemic level for 90 minutes
1724
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
than when hypocalcemia was induced from a normal serum calcium [28]. These results suggested that the PTH secretory response was affected by changes in the steadystate calcium concentration. Whether or not a hypocalcemic clamp affects the PTH response during the recovery from hypocalcemia has not been evaluated previously. Another important issue was addressed after our study was started. When maximal PTH levels are attained during the induction of hypocalcemia, the initiation of a hypocalcemic clamp results in a reduction of PTH values [29, 30]. Whether this decrease in PTH represents an adaptation to the stabilization of the serum calcium or the deceleration in the rate of calcium reduction, as has been previously suggested [2, 13, 31], or the depletion of PTH stores [26] is an important issue. In a recent study, based on the concept of depletion of PTH stores, Madsen et al elected to use the PTH value obtained during the hypocalcemic clamp as the maximal PTH value, even though it was lower than the peak PTH value [26]. The use of this lower PTH value as the maximal PTH not only intimates a reduced secretory capacity for the parathyroid gland, but also affects the determination of the set point and slope of the PTH calcium curve. The goal of this study was to evaluate whether (a) differences in maximal PTH were observed for a slow (120 min) as compared with a fast (30 min) reduction in serum calcium of similar magnitude, (b) the PTH values observed during the recovery from hypocalcemia were different when hypocalcemia was progressively induced during 120 minutes or after a rapid 30-minute reduction in serum calcium followed by a 90-minute hypocalcemic clamp, and (c) the reduction in PTH values during a hypocalcemic clamp was due to a depletion of PTH stores.
METHODS Healthy mongrel dogs, ages two to five years, with a mean weight of 23 6 2 kg were studied. The dogs were housed individually and observed during the two-week period prior to the studies. Dogs had free access to food and water, and the diet contained 1200 IU/kg of vitamin D; the dietary calcium and phosphorus content was 1.2 and 0.9%, respectively. After an overnight fast, the dogs were anesthetized with intramuscular ketamine (10 mg/kg) and intravenous sodium thiopental (15 mg/kg/hr). The left jugular and cephalic veins were then cannulated. The jugular catheter was used for blood sampling and the administration of the anesthetic. Disodium EDTA was used to lower the serum calcium, and during the recovery from hypocalcemia, calcium chloride was used to increase the serum calcium; both EDTA and calcium chloride were administered via the cephalic vein.
1725
The experimental protocol consisted of three phases, which are shown graphically in Figure 1A. In the first phase, the effect of hypocalcemia on PTH stimulation was evaluated for a fast, linear induction (30 min) of hypocalcemia followed by a 90-minute hypocalcemic clamp. These results were compared with those obtained during a slow, linear induction (120 min) of hypocalcemia of a similar magnitude. In the second phase, a calcium infusion was started in both groups after the 120-minute EDTA infusion, and PTH values were measured during the recovery from hypocalcemia. The third phase was designed to evaluate whether the depletion of PTH stores was the cause of the reduction in PTH levels during the hypocalcemic clamp. To evaluate this possibility, three subgroups of dogs had either (a) a fast induction of hypocalcemia (30 min), (b) a fast induction of hypocalcemia (30 min) followed by a 30-minute hypocalcemic clamp, or (c) a slow induction of hypocalcemia (120 min), and then underwent an accelerated reduction in serum calcium from an already hypocalcemic level; this accelerated reduction in serum calcium was performed to determine whether PTH could be further stimulated. After baseline blood sampling, sodium EDTA (1 g/kg), dissolved in one liter of 5% dextrose and water, was administered with an infusion pump during either a slow (120 min) or fast (30 min) induction of hypocalcemia. The desired reduction in ionized calcium in both groups was 0.4 mm. As shown in Figure 1B, to achieve a linear decrease in serum calcium during the slow infusion, the initial rate of EDTA administration (25 ml/hr) was progressively increased during the 120-minute infusion. During the fast EDTA infusion (30 min), the initial rate of EDTA infusion was 75 ml/hr, and this was progressively increased during the infusion (Fig. 1B). When the nadir of the serum calcium was achieved at 30 minutes, the serum calcium was clamped at this hypocalcemic value by reducing the rate of EDTA infusion. At 120 minutes, the EDTA infusion was stopped in both groups, and calcium chloride was infused for 60 minutes at a progressively increasing rate until serum ionized calcium was greater than 1.50 mm. In the third phase of the study, the goal was to reduce the serum calcium from hypocalcemic levels to still lower values and to increase the rate of reduction. In the subgroup studied at 30 minutes, the rate of EDTA infusion was rapidly increased between 30 and 36 minutes (Fig. 1B), and blood samples for ionized calcium and PTH were obtained at 33 and 36 minutes. In the subgroup studied at 60 minutes, the rate of EDTA infusion during the first 30 minutes was the same as other dogs in the fast-infusion group, and at 30 minutes, a hypocalcemic clamp was initiated as described previously. At 60 minutes, the rate of the EDTA infusion was rapidly increased (Fig. 1B), and blood for ionized calcium and PTH was obtained at 62, 64, 66, and 68 minutes. In the subgroup
1726
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 1. (A) Shown is the schematic diagram of the study design with the number (N) of dogs in each segment. The solid line represents the fast EDTA infusion group (a) and the hypocalcemic clamp and, after 120 minutes, the infusion of calcium chloride (f). The broken line represents the slow EDTA infusion group (b) and, after 120 minutes, the infusion of calcium chloride (g). The dotted line represents the rapid lowering of serum calcium from already hypocalcemic levels, which was performed at 30 (c), 60 (d), and 120 (e) minutes. (B) A schematic diagram of the infusions of EDTA and calcium chloride. The solid line represents the rate of EDTA infusion in the fast EDTA-infusion group (a) and during the hypocalcemic clamp (a) and, after 120 minutes, the rate of calcium chloride infusion (g). The broken line represents the rate of EDTA infusion in the slow EDTA infusion group (b) and, after 120 minutes, the rate of calcium chloride infusion (f). The rate of EDTA infusion used in the fast EDTA infusion group to lower serum calcium rapidly from existing hypocalcemic levels at 30 (c) and 60 (d) minutes is shown as a solid line. The rate of EDTA infusion used in the slow EDTA infusion group to lower serum calcium rapidly from existing hypocalcemic levels at 120 minutes (e) is shown as a broken line.
receiving the slow EDTA infusion, the rate of EDTA infusion was rapidly increased at 120 minutes (Fig. 1B), and blood samples for ionized calcium and PTH were obtained at 123 and 126 minutes. During the fast infusion of EDTA and during the hypocalcemic clamp, heparinized blood samples were obtained at five-minute intervals. During the slow infusion of EDTA, blood samples were obtained at 10-minute intervals, except for those obtained at 5-minute intervals during first and last 10 minutes of the 120-minute infusion. During the calcium infusion, which was started at 120 minutes, blood samples were obtained at 10-minute intervals. During the studies in which an accelerated lowering of serum calcium from already hypocalcemic levels was performed, blood samples were obtained every two or three minutes. Ionized calcium was measured with a calcium-selective electrode (Ciba-Corning Diagnostics, S.A., Madrid, Spain),
and the measurement was performed immediately after the sample was obtained. Intact PTH was measured with an IRMA assay (Allegro; Nichols, San Juan Capistrano, CA, USA); the use of this assay for measurement of PTH in the dog has been validated previously [31–35]. The PTH values for a single dog were always measured in one assay. Statistics For the comparison of two samples, the paired or unpaired Student t-test was used. For the comparison of three or more samples, one-way or repeated-measures analysis of variance was used, depending on whether data were unpaired or paired. If the analysis of variance test showed a statistical difference, a post hoc test, the Fisher LSD, was performed to determine intergroup differences; for the post hoc test, differences were at the P , 0.05 level. Multivariate analysis of variance was
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
1727
Fig. 3. The PTH values at the end of the slow (j; 120 min) and fast (h; 30 min) EDTA infusions. In the slow-infusion group, PTH values at 120, 115, and 105 minutes were less than PTH values at 30 minutes in the fast-infusion group. These differences in PTH values were observed despite no difference in ionized calcium for all three comparisons. *P , 0.05 vs. 30 minutes.
Fig. 2. (A) The ionized calcium concentration during the fast (d; 30 min) induction of hypocalcemia (EDTA), followed by a 90-minute hypocalcemic clamp, and subsequently during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a solid line. The ionized calcium concentration during the slow (s; 120 min) induction of hypocalcemia (EDTA) and during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a broken line. (B) The PTH concentration during the fast (d; 30 min) induction of hypocalcemia (EDTA), followed by a 90-minute hypocalcemic clamp, and subsequently during the 60 minute recovery from hypocalcemia (calcium chloride infusion) is shown as a solid line. The PTH concentration during the slow (s; 120 min) induction of hypocalcemia (EDTA) and during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a broken line. aP , 0.05 vs. fast at 30 min (ANOVA); b P , 0.05 vs. slow at the same time (MANOVA); cP , 0.05 vs. slow from 55 to 120 min (ANOVA).
used to compare mean values affected by more than one variable. A P value of less than 0.05 was considered significant. Results are shown as the mean 6 se. RESULTS As shown in Figure 2A, the EDTA infusion resulted in a linear decrease in serum calcium in both the fast and slow groups. In the fast group, the nadir of hypocalcemia (0.84 6 0.02 mm) was at 30 minutes, after which serum calcium was clamped at this hypocalcemic level for an additional 90 minutes. In the slow group, the nadir of hypocalcemia (0.82 6 0.03 mm) was at 120 minutes. In
both groups, a calcium infusion was started at 120 minutes and resulted in a similar rate of recovery from hypocalcemia and an increase to similar hypercalcemic values. The PTH response to hypocalcemia is shown in Figure 2B. In the fast group, a rapid increase in PTH that corresponded with the magnitude of hypocalcemia was observed during the first 30 minutes. During the hypocalcemic calcium clamp, PTH values decreased and from 40 to 120 minutes were less (P , 0.05) than the PTH value at 30 minutes. Moreover, PTH values at 75, 85, 95, and 105 minutes obtained during the hypocalcemic clamp were less (P , 0.05) than those at the same time intervals during the slow EDTA infusion even though the clamped serum calcium values at 75, 85, and 95 minutes were less (P , 0.05) or at 105 minutes, not different than calcium values in the slow group. During the recovery from hypocalcemia (calcium infusion), PTH values were similar in the two groups. As shown in Figure 3, the PTH value at 30 minutes in the fast group was greater than PTH values at 105, 115, and 120 minutes in the slow group. These differences in PTH were observed even though the 30-minute serum calcium value (0.84 6 0.02 mm) was not different than those at 105 (0.88 6 0.02 mm), 115 (0.85 6 0.02 mm), and 120 (0.82 6 0.03 mm) minutes. Moreover, when PTH values were matched for serum calcium during the induction of hypocalcemia (Fig. 4), PTH values in the fast group tended to be greater for similar serum calcium values and were greater for a similar serum calcium at 5 versus 15 minutes and 30 versus 115 minutes (fast versus slow). Whether or not a rapid reduction in serum calcium from hypocalcemic levels further stimulated PTH was
1728
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 4. PTH values matched for the serum calcium concentration in the fast (d) and slow (s) EDTA infusion groups. For similar serum calcium values at 5 (fast) versus 15 (slow) minutes and at 30 (fast) versus 115 (slow) minutes, the PTH values were significantly greater in the fast group (*P , 0.05; **P , 0.01).
evaluated. As shown in Figure 5A, an accelerated reduction in serum calcium between 30 and 36 minutes did not further increase PTH (264 6 26 vs. 279 6 27 pg/ml, analysis of variance, P 5 0.61). When serum calcium was reduced to its nadir at 30 minutes and a hypocalcemic clamp was maintained for the next 30 minutes before a further reduction in serum calcium was initiated between 60 and 68 minutes, PTH values at 66 and 68 minutes were greater than at 60 minutes (ANOVA, P , 0.001; Fig. 5B). Moreover, the PTH value at 30 minutes, obtained at the nadir of hypocalcemia before the hypocalcemic clamp, and PTH values at 66 and 68 minutes were similar to and greater than those at 45, 50, 55, and 60 minutes during the hypocalcemic clamp (ANOVA, P , 0.001). The slow infusion of EDTA resulted in a linear decline in serum calcium during 120 minutes (Fig. 5C). Between 120 and 126 minutes, the rate of serum calcium decrease was accelerated, and the PTH value at 126 minutes (302 6 30 pg/ml) was greater (ANOVA, P 5 0.03) than at 120 minutes (224 6 11 pg/ml) and all other PTH values from 0 to 120 minutes (ANOVA, P , 0.001).
Whether or not the depletion of PTH stores was the cause of the reduction in PTH values during a hypocalcemic clamp was evaluated (Fig. 6). A group of dogs in which serum calcium was reduced between 0 and 30 minutes was subjected to an accelerated rate of calcium reduction between 30 and 36 minutes (Fig. 6A). These dogs were compared with another group in which the calcium reduction was similar during the first 30 minutes, but after which a hypocalcemic clamp was started. In the group with the accelerated reduction in serum calcium, PTH values were not different than those at 30 minutes. However, during the hypocalcemic clamp, PTH values decreased and at 40, 45, and 50 minutes were less (P , 0.05) than those at 30 minutes. As shown in Figure 6B, the PTH values in the group with an accelerated calcium reduction were greater at similar times than those in the group with the hypocalcemic clamp, even though the 30-minute PTH values were similar. In Figure 7, the PTH response to the fast EDTA infusion is shown as curve 1. Shown as open squares are PTH values obtained during the hypocalcemic clamp between 30 and 90 minutes; this time range was used because it is the same as used by Madsen et al [26]. During the hypocalcemic clamp, the two highest PTH values were at 35 and 40 minutes. To construct curve 2, we used as the maximal PTH, the mean PTH (163 pg/ml) of the 12 PTH values during the hypocalcemic clamp. To complete the curve, the first two PTH values during the induction of hypocalcemia were used, and the connection to the mean PTH value for the hypocalcemic clamp was extrapolated. The five highest PTH values were excluded because these values exceeded the maximal PTH value for curve 2. Finally, curve 3 had the same maximal PTH as curve 2, and as was done by Madsen et al [26], the other PTH values were from the recovery from hypocalcemia. As shown in Figure 7, the three PTH-calcium curves are entirely different. DISCUSSION In this study in normal dogs, we showed that for a similar magnitude of hypocalcemia at the end of the slow and fast EDTA infusion, the PTH response was less in the slow than the fast group. Moreover, in the slowinfusion group, PTH values appeared to reach a plateau and, despite a continual reduction in serum calcium, did not increase further. We also showed that during a hypocalcemic clamp, PTH values decreased from maximal levels. However, this reduction in PTH values during the hypocalcemic clamp did not appear to represent a depletion of PTH stores because PTH could again be stimulated to maximal PTH levels when the serum calcium was further reduced below the level of the hypocalcemic clamp. The reason for comparing the PTH response to hypo-
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
1729
Fig. 5. (A) The ionized calcium (d) and PTH (s) values in dogs in which a fast EDTA infusion (30 min) was performed, and from 30 to 36 minutes, ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). The rapid lowering of ionized calcium from 30 to 36 minutes did not further increase PTH levels. (B) The ionized calcium and PTH values in dogs in which a fast EDTA infusion (30 min) was performed; this was followed by a 30-minute hypocalcemic clamp, after which ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). During the hypocalcemic clamp, PTH values decreased from the 30-minute PTH value (P , 0.05, a vs. b). During the rapid reduction of ionized calcium initiated at the end of the hypocalcemic clamp at 60 minutes, PTH values increased to levels similar to those observed at 30 minutes and were greater than PTH values during the hypocalcemic clamp (P , 0.05, a vs. b). (C) The ionized calcium and PTH values in dogs in which a slow EDTA infusion (120 min) was performed, after which ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). The rapid reduction of ionized calcium resulted in an increase in the PTH value to a level that was greater than all the PTH values during the slow EDTA infusion (P , 0.05, a vs. other PTH values).
1730
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH b Fig. 6. (A) The ionized calcium (d) and PTH (s) values obtained when a fast (30 min) EDTA infusion was performed, followed by a hypocalcemic clamp versus a fast (30 min) EDTA infusion followed by a more rapid lowering of the ionized calcium. During the hypocalcemic clamp, PTH values at 40, 45, and 50 minutes were lower than the peak PTH value at 30 minutes (P , 0.05, a vs. 30-min PTH value). In the group in which ionized calcium was rapidly lowered from 30 to 36 minutes, PTH values did not change from the 30-minute value. (B) The PTH values for similar time intervals in the two groups shown in (A). Symbols are: ( ) accelerated; ( ) clamp. At 30 minutes, the PTH values were not different. When the PTH value at 35 minutes during the hypocalcemic was compared with the PTH value obtained at 33 minutes during the rapid lowering of serum calcium, the latter PTH value was greater. Similarly, when the hypocalcemic clamped PTH value at 35 minutes was compared with the PTH value obtained at 36 minutes during the rapid lowering of serum calcium, the latter PTH value was greater. A similar result was observed when the 36-minute PTH obtained during the rapid lowering of serum calcium was compared with the 40-minute hypocalcemic clamped PTH value. These results suggest that the reduction in PTH values during the hypocalcemic clamp was not due to depletion of PTH stores. *P 5 NS; **P , 0.02; †P , 0.01.
calcemia during a fast and slow induction of hypocalcemia was to determine whether differences in the rate of calcium reduction affected parathyroid function. In a previous clinical study, Brent et al reported that the PTH response to hypocalcemia in normal volunteers was similar whether the calcium reduction was during 60 or 120 minutes [1]. In a study in dogs, we showed that the PTH response to hypocalcemia was similar whether the calcium reduction was during 30 or 60 minutes [31]. Furthermore, we showed that the maximal PTH response
was similar whether the calcium reduction was linear or nonlinear; the latter was designed to simulate the gradient-induced reduction in serum calcium (low calcium dialysis) often used to test parathyroid function in hemodialysis patients [10–12, 14–16, 23–25, 27]. However, others have shown that short, rapid reductions in serum calcium produce a greater PTH response than long, slow reductions in serum calcium [2]. Furthermore, in a preliminary report in hemodialysis patients, it was shown that a low calcium dialysis for approximately 60 minutes induced a greater maximal PTH response than a three-hour EDTA infusion even though the magnitude of hypocalcemia was similar (abstract; de Precigout et al, J Am Soc Nephrol 7:1789, 1996). Our current study in dogs also supports the concept that the rate of calcium reduction might affect the magnitude of the PTH response to hypocalcemia. In this study, the maximal PTH response to hypocalcemia was greater at the end of the EDTA infusion in the fast than the slow group despite a similar reduction in calcium. Moreover, in the slow group, PTH values did not change between 55 and 120 minutes even though serum calcium decreased by more than 0.20 mm during this time. An accelerated reduction in calcium at 120 minutes further increased PTH values. Thus, our results suggest that because of the slow rate of calcium reduction, the maximal PTH secretory capacity was not achieved. Because in clinical studies nonuniform protocols of calcium reduction are used to stimulate PTH secretion maximally, it is possible that differences in the rate of calcium reduction may lead to different conclusions regarding the dynamics of PTH secretion. In a recent study, we observed that the PTH response to hypocalcemia was different when the reduction in serum calcium was started after a 90-minute hypercalce-
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 7. The PTH-calcium curve (curve 1) was drawn from values obtained during the fast (30 min) EDTA infusion. When, as suggested by Madsen et al, PTH values obtained during the hypocalcemic clamp (open squares) were used as the maximal PTH (mean of the 12 values), the PTH-calcium curve (curve 2) was distinctly different than curve 1 [26]. Finally, as was done in [26], the PTH value from the hypocalcemic clamp was used as the maximal PTH, and the other PTH values were obtained during the recovery from hypocalcemia, the resulting PTHcalcium curve (curve 3) is markedly different than curve 1.
mic clamp than from the normal state [28]. As a result, we decided to evaluate the converse, that is, whether or not the PTH response during the recovery from hypocalcemia was different after a hypocalcemic clamp than after a linear reduction in serum calcium. Our results show that during the recovery from hypocalcemia, PTH values for similar serum calcium values were not different between the two groups. Several of our findings support the conclusion that a deceleration in the rate of calcium reduction rather than a depletion of PTH stores was the cause of the decrease in PTH levels during the hypocalcemic clamp. After the 30-minute reduction in serum calcium, a subsequent, more rapid reduction in serum calcium did not further increase PTH levels, but PTH levels did not decrease as was observed during the hypocalcemic clamp. Moreover, PTH values during the six-minute accelerated reduction in serum calcium from 30 to 36 minutes were greater
1731
than those at similar times during the hypocalcemic clamp. Thus, if the depletion of PTH stores was responsible for the reduction in PTH levels, lower PTH values would have been expected during the accelerated reduction in serum calcium from 30 to 36 minutes. Other observations also suggest that depletion of PTH stores was not the cause of the reduction in PTH levels during the hypocalcemic clamp. The PTH values during the hypocalcemic clamp were lower than the 30-minute PTH value, and a reduction in calcium after 30 minutes of the hypocalcemic clamp resulted in an increase in PTH. If depletion of PTH stores was responsible for the lower PTH values during the hypocalcemic clamp, a further reduction in calcium would not have been able to increase PTH secretion. Moreover, the fact that during the slow EDTA infusion at the nadir of hypocalcemia at 120 minutes, PTH values could be stimulated by a more rapid reduction in serum calcium also indicates that PTH depletion was not present. Thus, our results suggest that depletion of PTH stores was not the cause of the reduction in PTH levels during the hypocalcemic clamp. Rather, we believe that the reduction in PTH levels was more likely due to the deceleration in the rate of calcium reduction [2, 13, 31]. As such, it may be related to the phenomenon of hysteresis in which after a directional change in serum calcium, the PTH level for the same serum calcium concentration is less during the recovery than the induction of hypocalcemia [2, 13, 31]. In a previous study, we showed that the PTH response to two consecutive 30-minute reductions in serum calcium separated by a 30-minute recovery period was similar [31]. Although unlikely, it could be argued that the 30-minute recovery period provided sufficient time for reutilization of degraded PTH. In our current study, it is possible that a decrease in the metabolic clearance rate of PTH contributed to the differences in circulating PTH levels. In a recent study in dogs, anesthesia with pentobarbital was shown to decrease the hepatic clearance of intact PTH and increase circulating PTH levels [36]. However, in this study, this effect should have been similar in all of the dogs. An important issue is whether hypocalcemia changed the metabolic clearance rate of PTH. At least for our initial 0.4 mm reduction in ionized calcium, most previous studies have shown that this degree of hypocalcemia did not change the metabolic clearance rate of PTH even in anesthetized animals [36–38], and some studies have even reported an enhanced metabolic clearance [39]. Either result would not increase circulating PTH levels. Studies of parathyroidectomized animals have shown that even more severe hypocalcemia did not change the metabolic clearance rate of PTH [37, 40]. However, it is possible that in our study the accelerated reduction in serum calcium could have produced hemodynamic instability, which decreased the metabolic clearance rate of PTH. Although we cannot
1732
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
eliminate this possibility, several results suggest that this explanation is not likely. First, in the group of dogs in which the accelerated reduction in serum calcium was performed during the hypocalcemic clamp, PTH values increased when serum calcium decreased from 0.87 to 0.71 mm. This range of serum calcium has been evaluated in previous studies and was not associated with changes in the metabolic clearance rate of PTH [36, 37]. Second, in dogs in which the accelerated reduction in calcium was performed in the fast- and slow-infusion groups, the PTH response was different for similar reductions in calcium and a similar final serum calcium. In the fast group, PTH values did not increase, although they did increase in the slow group. If hemodynamic instability decreased the metabolic clearance rate of PTH during marked hypocalcemia, it would have been expected to have a similar effect in both groups. Third, the venous blood pH, measured during the determination of ionized calcium, was not different before and after the rapid lowering of ionized calcium (7.36 6 0.02 vs. 7.34 6 0.02, respectively). Fourth, although blood pressure was not measured, these dogs displayed no outward signs of distress, and all recovered without incident. Based on the concept of depletion of preformed PTH stores, Madsen et al recently advocated that the PTH value obtained during the hypocalcemic clamp should be used as the maximal PTH rather than the peak PTH value obtained at the nadir of hypocalcemia [26]. This concept of depletion of preformed PTH stores also implies that in response to hypocalcemia, all parathyroid cells secrete PTH simultaneously. However, the in vitro studies of Sun et al suggest another possibility should be considered [41, 42]. During a reduction in the calcium concentration in these studies, only a certain percentage of parathyroid cells secreted PTH at any one time, and secretion by other parathyroid cells was held in reserve for a subsequent rechallenge to a reduced calcium concentration [41, 42]. In such a schema, recruitment of previously quiescent cells would prevent depletion of PTH stores. Our results in dogs also suggest that depletion of preformed PTH stores may not be a factor. In previous clinical studies, it has been shown that rapid changes in the rate of calcium reduction affect the magnitude of the PTH response to hypocalcemia [2, 43]. In this study in dogs, PTH values decreased by approximately 40% during the hypocalcemic clamp but could be further stimulated during a further reduction in the serum calcium concentration. Such a result would also support the concept that the reduction from peak PTH values does not represent a depletion of PTH stores. In addition to its physiological significance regarding hormone secretion and synthesis, an accurate definition of the maximal PTH level is also important because, as has been shown in Figure 7, it affects the calculation of the set point and the slope of the PTH calcium curve. More-
over, the calculation of the set point and slope would also be affected by the use of PTH values obtained during the recovery from hypocalcemia, which are lower for the same serum calcium concentration than those obtained during the induction of hypocalcemia [3, 13, 31]. Thus, additional clinical studies are needed to resolve these important issues. In conclusion, our results suggest that (a) the reduction in PTH values during a hypocalcemic clamp may not represent a depletion of PTH stores, but rather may be a response to the stabilization of the serum calcium. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH calcium curve; and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium. ACKNOWLEDGMENTS This work was supported by grants #PB97–0090 and #PM95–0184 from the Direccio´n General Ensen˜anza Superior, a grant from the Fundacio´n Reina Sofı´a-Cajasur, and a grant from the Baxter Extramural Grant Program. The authors also wish to acknowledge the technical support of E.M. Saneamientos Cordoba, S.A. Reprint requests to Dr. Mariano Rodriguez, Unidad de Investigacion, Hospital Universitario Reina Sofia, Avda Menendez Pidal S/N, 14004 Cordoba, Spain. E-mail: [email protected]
REFERENCES 1. Brent GA, Leboff MS, Seely EW, Conlin PR, Brown EM: Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans. J Clin Endocrinol Metab 67:944–950, 1988 2. Grant FD, Conlin PR, Brown EM: Rate and concentration dependence of parathyroid hormone dynamics during stepwise changes in serum ionized calcium in normal humans. J Clin Endocrinol Metab 71:370–378, 1990 3. Conlin PR, Fajtova VT, Mortensen RM, Leboff MS, Brown EM: Hysteresis in the relationship between serum ionized calcium and intact parathyroid hormone during recovery from induced hyper- and hypocalcemia in normal humans. J Clin Endocrinol Metab 69:593–599, 1989 4. El-Hajj Fuleihan G, Gundberg CM, Gleason R, Brown EM, Stromski ME, Grant FD, Conlin PR: Racial differences in parathyroid hormone dynamics. J Clin Endocrinol Metab 79:1642–1647, 1994 5. Schwarz P, Sorensen HA, Transbol I, McNair P: Induced hypocalcemia controlled by a citrate clamp technique and the intact parathyroid hormone response obtained. Scand J Clin Lab Invest 50:891–897, 1990 6. Schwarz P, Sorensen HA, Transbol I, McNair P: Regulation of acute parathyroid hormone release in normal humans: Combined calcium and citrate clamp. Am J Physiol 263:E195–E198, 1992 7. Gardin JP, Patron P, Fouqueray B, Prigent A, Paillard M: Maximal PTH secretory rate and set point for calcium in normal subjects and patients with primary hyperparathyroidism. Miner Electrolyte Metab 14:221–228, 1988 8. Paillard M, Gardin JP, Borensztein P, Prigent A: Determinants of parathormone secretion in primary hyperparathyroidism. Horm Res 32:89–92, 1989 9. Schwarz P, Sorensen HA, Momsen G, McNair P, Transbol
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
I: Normal pattern of parathyroid hormone response to calcium lowering in primary hyperparathyroidism, a citrate clamp study. Clin Endocrinol (Oxf) 37:344–348, 1992 Dunlay R, Rodriguez M, Felsenfeld A, Llach F: Direct inhibitory effect of calcitriol on parathyroid function (sigmoidal curve) in dialysis patients. Kidney Int 36:1093–1098, 1989 Cunningham J, Altmann P, Gleed JH, Butter KC, Marsh FP, O’Riordan JLH: Effect of direction and rate of change of calcium on parathyroid hormone secretion in uraemia. Nephrol Dial Transplant 4:339–344, 1989 Rodriguez M, Felsenfeld AJ, Williams C, Pederson JA, Llach F: The effect of long-term intravenous calcitriol administration on parathyroid function in hemodialysis patients. J Am Soc Nephrol 2:1014–1020, 1991 Felsenfeld AJ, Ross D, Rodriguez M: Hysteresis of the parathyroid hormone response to hypocalcemia in hemodialysis patients with low turnover aluminum bone disease. J Am Soc Nephrol 2:1136–1143, 1991 Felsenfeld AJ, Rodriguez M, Dunlay R, Llach F: A comparison of parathyroid-gland function in haemodialysis patients with different forms of renal osteodystrophy. Nephrol Dial Transplant 6:244– 251, 1991 Malberti F, Surian M, Cosci P: Effect of chronic intravenous calcitriol on parathyroid function and set point of calcium in dialysis patients with refractory secondary hyperparathyroidism. Nephrol Dial Transplant 7:822–828, 1992 Kwan JTC, Almond MK, Beer JC, Noonan K, Evans SJW, Cunningham J: “Pulse” oral calcitriol in uraemic patients: Rapid modification of parathyroid response to calcium. Nephrol Dial Transplant 7:829–834, 1992 Ramirez JA, Goodman WG, Gornbein J, Menezes C, Moulton L, Segre GV, Salusky IB: Direct in vivo comparison of calciumregulated parathyroid hormone secretion in normal volunteers and patients with secondary hyperparathyroidism. J Clin Endocrinol Metab 76:1489–1494, 1993 Combe C, Aparicio M: Phosphorus and protein restriction and parathyroid function in chronic renal failure. Kidney Int 46:1381– 1386, 1994 Messa P, Vallone C, Mioni G, Geatti O, Turrin D, Passoni N, Cruciatti A: Direct in vivo assessment of parathyroid hormonecalcium relationship curve in renal patients. Kidney Int 46:1713– 1720, 1994 Ramirez JA, Goodman WG, Belin TR, Gales B, Segre GV, Salusky IB: Calcitriol therapy and calcium-regulated PTH secretion in patients with secondary hyperparathyroidism. Am J Physiol 267:E961–E967, 1994 Sanchez CP, Goodman WG, Ramirez JA, Gales B, Belin TR, Segre GV, Salusky IB: Calcium-regulated parathyroid hormone secretion in adynamic renal osteodystrophy. Kidney Int 48:838–843, 1995 Goodman WG, Belin T, Gales B, Juppner H, Segre GV, Salusky IB: Calcium-regulated parathyroid hormone release in patients with mild or advanced secondary hyperparathyroidism. Kidney Int 48:1553–1558, 1995 Caravaca F, Cubero JJ, Jimenez F, Lopez JM, Aparicio A, Cid MC, Pizarro JL, Liso J, Santos I: Effect of the mode of calcitriol administration of PTH-ionized calcium relationship in uraemic patients with secondary hyperparathyroidism. Nephrol Dial Transplant 10:665–670, 1995 Felsenfeld AJ, Jara A, Pahl M, Bover J, Rodriguez M: Differences in the dynamics of PTH secretion in hemodialysis patients with marked secondary hyperparathyroidism. J Am Soc Nephrol 6:1371–1378, 1995 Pahl M, Jara A, Bover J, Rodriguez M, Felsenfeld AJ: The
26. 27.
28.
29. 30.
31.
32. 33. 34. 35.
36.
37. 38. 39.
40.
41. 42. 43.
1733
set point of calcium and the reduction of parathyroid hormone in hemodialysis patients. Kidney Int 49:226–231, 1996 Madsen JC, Rasmussen AQ, Ladefoged SD, Schwarz P: Parathyroid hormone secretion in renal failure. Kidney Int 50:1700– 1705, 1996 Rodriguez M, Caravaca F, Fernandez E, Borrego MJ, Lorenzo V, Cubero J, Martin-Malo A, Betriu A, Rodriguez AP, Felsenfeld AJ: Evidence for both abnormal set point of calcium and adaptation to serum calcium in hemodialysis patients with hyperparathyroidism. J Bone Miner Res 12:347–355, 1997 Sanchez J, Aguilera-Tejero E, Estepa JC, Almaden Y, Rodriguez M, Felsenfeld AJ: A reduced PTH response to hypocalcemia after a short period of hypercalcemia: A study in dogs. Kidney Int 50(Suppl 57):S18–S22, 1996 Fox J, Heath H III: The “calcium clamp”: Effect of constant hypocalcemia on parathyroid hormone secretion. Am J Physiol 240:E649–E655, 1981 Schwarz P, Sorensen HA, McNair P, Transbol I: Cica-clamp technique, a method for quantifying parathyroid hormone secretion: A sequential citrate and calcium clamp study. Eur J Clin Invest 23:546–553, 1993 Aguilera-Tejero E, Sanchez J, Almaden Y, Mayer-Valor R, Rodriguez M, Felsenfeld AJ: Hysteresis of the PTH-calcium curve during hypocalcemia in the dog: Effect of the rate and linearity of calcium decrease and sequential episodes of hypocalcemia. J Bone Miner Res 11:1226–1233, 1996 Torrance AG, 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 Torrance AG, Nachreiner R: Intact parathyroid hormone assay and total calcium concentration in the diagnosis of disorders of calcium metabolism in dogs. J Vet Intern Med 3:86–89, 1989 Cloutier M, Gascon-Barre M, D’Amour P: Chronic adaptation of dog parathyroid function to a low-calcium-high-sodium-vitamin D-deficient diet. J Bone Miner Res 7:1021–1028, 1992 Cloutier M, Brossard JH, Gascon-Barre M, D’Amour P: Lack of involution of hyperplastic parathyroid glands in dogs: Adaptation via a decrease in the calcium stimulation set point and a change in secretion profile. J Bone Miner Res 9:621–629, 1994 D’Amour P, Rousseau L, Rocheleau B, Pomier-Layrargues G, Huet PM: Influence of Ca21 concentration to the clearance and circulating levels of intact and carboxy-terminal iPTH in pentobarbital-anesthetized dogs. J Bone Miner Res 11:1075–1085, 1996 Fox J, Scott M, Nissenson RA, Heath H III: Effect of plasma calcium concentration on the metabolic clearance rate of parathyroid hormone in the dog. J Lab Clin Med 102:70–77, 1983 Daugaard H, Egfjord M, Olgaard K: Influence of calcium on the metabolism of intact parathyroid hormone by isolated perfused rat kidney and liver. Endocrinology 126:1813–1820, 1990 Canterbury JM, Bricker LA, Levey GS, Kozlovskis PL, Ruiz E, Zull JE, Reiss E: Metabolism of bovine parathyroid hormone: Immunological characteristics of fragments generated by liver perfusion. J Clin Invest 55:1245–1253, 1975 Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, Potts JT Jr: Peripheral metabolism of [35S]parathyroid hormone in vivo: Influence of alterations in calcium availability and parathyroid status. J Endocrinol 122:237–245, 1989 Sun F, Ritchie CK, Hassager C, Maercklein P, Fitzpatrick LA: Heterogenous response to calcium by individual parathyroid cells. J Clin Invest 91:595–601, 1993 Sun F, Maercklein P, Fitzpatrick LA: Paracrine interactions among parathyroid cells: Effect of cell density on cell secretion. J Bone Miner Res 9:971–976, 1994 Schmitt CP, Schaefer F, Bruch A, Veldhuis JD, Schmidt-Gayk H, Stein G, Ritz E, Mehls O: Control of pulsatile and tonic parathyroid hormone secretion by ionized calcium. J Clin Endocrinol Metab 81:4236– 4243, 1996
Effect of rate of calcium reduction and a hypocalcemic clamp on parathyroid hormone secretion: A study in dogs JOSE C. ESTEPA, ESCOLASTICO AGUILERA-TEJERO, YOLANDA ALMADEN, MARIANO RODRIGUEZ, and ARNOLD J. FELSENFELD Departamentos de Nefrologı´a y Unidad de Investigacio´n del Hospital Universitario Reina Sofı´a; Departamento de Patologı´a Clı´nica Veterinaria, Universidad de Co´rdoba, Co´rdoba, Spain; and the Department of Medicine, West Los Angeles VA Medical Center and UCLA, Los Angeles, California, USA
Effect of rate of calcium reduction and a hypocalcemic clamp on PTH secretion: A study in dogs. Background. The parathyroid hormone (PTH) calcium curve is used to evaluate parathyroid function in clinical studies. However, unanswered questions remain about whether PTH secretion is affected by the rate of calcium reduction and how the maximal PTH response to hypocalcemia is best determined. We performed studies in normal dogs to determine whether (a) the rate of calcium reduction affected the PTH response to hypocalcemia and (b) the reduction in PTH values during a hypocalcemic clamp from the peak PTH value observed during the nadir of hypocalcemia was due to a depletion of stored PTH. Methods. Fast (30 min) and slow (120 min) ethylenediaminetetraacetic acid (EDTA) infusions were used to induce similar reductions in ionized calcium. In the fast EDTA infusion group, serum calcium was maintained at the hypocalcemic 30-minute value for an additional 90 minutes (hypocalcemic clamp). To determine whether the reduction in PTH values during the hypocalcemic clamp represented depletion of PTH stores, three subgroups were studied. Serum calcium was rapidly reduced from established hypocalcemic levels in the fast-infusion group at 30 and 60 minutes (after 30 min of a hypocalcemic clamp) and in the slow-infusion group at 120 minutes. Results. At the end of the fast and slow EDTA infusions, serum ionized calcium values were not different (0.84 6 0.02 vs. 0.82 6 0.03 mm), but PTH values were greater in the fastinfusion group (246 6 19 vs. 194 6 13 pg/ml, P , 0.05). During the hypocalcemic clamp, PTH rapidly decreased (P , 0.05) to values of approximately 60% of the peak PTH value obtained at 30 minutes. A rapid reduction in serum calcium from established hypocalcemic levels at 30 minutes did not stimulate PTH further, but also PTH values did not decrease as they did when a hypocalcemic clamp was started at 30 minutes. At 60 minutes, the reduction in serum calcium increased (P , 0.05) PTH to peak values similar to those before the hypocalcemic clamp. The reduction in serum calcium at 120 minutes in the slow
Key words: hypocalcemia, parathyroid hormone, EDTA, PTH-calcium curve, hyperparathyroidism. Received for publication April 17, 1998 and in revised form October 30, 1998 Accepted for publication December 15, 1998
1999 by the International Society of Nephrology
EDTA infusion group increased PTH values from 224 6 11 to 302 6 30 pg/ml (P , 0.05). Conclusions. These results suggest that (a) the reduction in PTH values during the hypocalcemic clamp may not represent a depletion of PTH stores. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH-calcium curve, and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium.
The parathyroid hormone (PTH) calcium curve has been used to evaluate parathyroid function in normal volunteers [1–6] and in patients with primary [7–9] and secondary hyperparathyroidism [10–27]. In studies performed in normal volunteers and in dialysis patients on continuous ambulatory peritoneal disease, the standard method of inducing hypocalcemia is the infusion of a calcium-chelating agent, either ethylenediaminetetraacetic acid (EDTA) or citrate. However, for convenience in hemodialysis patients, a reduction in the dialysate calcium concentration is generally used to induce hypocalcemia. Furthermore, the use of a low-calcium dialysate concentration generally produces a more rapid reduction in serum calcium than the usual two-hour infusion of a chelating agent. In a previous study in normal volunteers, the effect on PTH stimulation of a 60and 120-minute reduction in ionized calcium of similar magnitude was similar [1]. However, the effect of a more rapid reduction in serum calcium (30 min) on parathyroid function as compared with a 120-minute calcium reduction has not been evaluated in a controlled setting. In addition to the possible effect of the rate of calcium reduction, the PTH calcium curve has been shown to be modified by the level of serum calcium. In a recent study, we showed that the PTH response to hypocalcemia was less when hypocalcemia was induced from hypercalcemia, which had resulted after the serum calcium had been clamped at a hypercalcemic level for 90 minutes
1724
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
than when hypocalcemia was induced from a normal serum calcium [28]. These results suggested that the PTH secretory response was affected by changes in the steadystate calcium concentration. Whether or not a hypocalcemic clamp affects the PTH response during the recovery from hypocalcemia has not been evaluated previously. Another important issue was addressed after our study was started. When maximal PTH levels are attained during the induction of hypocalcemia, the initiation of a hypocalcemic clamp results in a reduction of PTH values [29, 30]. Whether this decrease in PTH represents an adaptation to the stabilization of the serum calcium or the deceleration in the rate of calcium reduction, as has been previously suggested [2, 13, 31], or the depletion of PTH stores [26] is an important issue. In a recent study, based on the concept of depletion of PTH stores, Madsen et al elected to use the PTH value obtained during the hypocalcemic clamp as the maximal PTH value, even though it was lower than the peak PTH value [26]. The use of this lower PTH value as the maximal PTH not only intimates a reduced secretory capacity for the parathyroid gland, but also affects the determination of the set point and slope of the PTH calcium curve. The goal of this study was to evaluate whether (a) differences in maximal PTH were observed for a slow (120 min) as compared with a fast (30 min) reduction in serum calcium of similar magnitude, (b) the PTH values observed during the recovery from hypocalcemia were different when hypocalcemia was progressively induced during 120 minutes or after a rapid 30-minute reduction in serum calcium followed by a 90-minute hypocalcemic clamp, and (c) the reduction in PTH values during a hypocalcemic clamp was due to a depletion of PTH stores.
METHODS Healthy mongrel dogs, ages two to five years, with a mean weight of 23 6 2 kg were studied. The dogs were housed individually and observed during the two-week period prior to the studies. Dogs had free access to food and water, and the diet contained 1200 IU/kg of vitamin D; the dietary calcium and phosphorus content was 1.2 and 0.9%, respectively. After an overnight fast, the dogs were anesthetized with intramuscular ketamine (10 mg/kg) and intravenous sodium thiopental (15 mg/kg/hr). The left jugular and cephalic veins were then cannulated. The jugular catheter was used for blood sampling and the administration of the anesthetic. Disodium EDTA was used to lower the serum calcium, and during the recovery from hypocalcemia, calcium chloride was used to increase the serum calcium; both EDTA and calcium chloride were administered via the cephalic vein.
1725
The experimental protocol consisted of three phases, which are shown graphically in Figure 1A. In the first phase, the effect of hypocalcemia on PTH stimulation was evaluated for a fast, linear induction (30 min) of hypocalcemia followed by a 90-minute hypocalcemic clamp. These results were compared with those obtained during a slow, linear induction (120 min) of hypocalcemia of a similar magnitude. In the second phase, a calcium infusion was started in both groups after the 120-minute EDTA infusion, and PTH values were measured during the recovery from hypocalcemia. The third phase was designed to evaluate whether the depletion of PTH stores was the cause of the reduction in PTH levels during the hypocalcemic clamp. To evaluate this possibility, three subgroups of dogs had either (a) a fast induction of hypocalcemia (30 min), (b) a fast induction of hypocalcemia (30 min) followed by a 30-minute hypocalcemic clamp, or (c) a slow induction of hypocalcemia (120 min), and then underwent an accelerated reduction in serum calcium from an already hypocalcemic level; this accelerated reduction in serum calcium was performed to determine whether PTH could be further stimulated. After baseline blood sampling, sodium EDTA (1 g/kg), dissolved in one liter of 5% dextrose and water, was administered with an infusion pump during either a slow (120 min) or fast (30 min) induction of hypocalcemia. The desired reduction in ionized calcium in both groups was 0.4 mm. As shown in Figure 1B, to achieve a linear decrease in serum calcium during the slow infusion, the initial rate of EDTA administration (25 ml/hr) was progressively increased during the 120-minute infusion. During the fast EDTA infusion (30 min), the initial rate of EDTA infusion was 75 ml/hr, and this was progressively increased during the infusion (Fig. 1B). When the nadir of the serum calcium was achieved at 30 minutes, the serum calcium was clamped at this hypocalcemic value by reducing the rate of EDTA infusion. At 120 minutes, the EDTA infusion was stopped in both groups, and calcium chloride was infused for 60 minutes at a progressively increasing rate until serum ionized calcium was greater than 1.50 mm. In the third phase of the study, the goal was to reduce the serum calcium from hypocalcemic levels to still lower values and to increase the rate of reduction. In the subgroup studied at 30 minutes, the rate of EDTA infusion was rapidly increased between 30 and 36 minutes (Fig. 1B), and blood samples for ionized calcium and PTH were obtained at 33 and 36 minutes. In the subgroup studied at 60 minutes, the rate of EDTA infusion during the first 30 minutes was the same as other dogs in the fast-infusion group, and at 30 minutes, a hypocalcemic clamp was initiated as described previously. At 60 minutes, the rate of the EDTA infusion was rapidly increased (Fig. 1B), and blood for ionized calcium and PTH was obtained at 62, 64, 66, and 68 minutes. In the subgroup
1726
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 1. (A) Shown is the schematic diagram of the study design with the number (N) of dogs in each segment. The solid line represents the fast EDTA infusion group (a) and the hypocalcemic clamp and, after 120 minutes, the infusion of calcium chloride (f). The broken line represents the slow EDTA infusion group (b) and, after 120 minutes, the infusion of calcium chloride (g). The dotted line represents the rapid lowering of serum calcium from already hypocalcemic levels, which was performed at 30 (c), 60 (d), and 120 (e) minutes. (B) A schematic diagram of the infusions of EDTA and calcium chloride. The solid line represents the rate of EDTA infusion in the fast EDTA-infusion group (a) and during the hypocalcemic clamp (a) and, after 120 minutes, the rate of calcium chloride infusion (g). The broken line represents the rate of EDTA infusion in the slow EDTA infusion group (b) and, after 120 minutes, the rate of calcium chloride infusion (f). The rate of EDTA infusion used in the fast EDTA infusion group to lower serum calcium rapidly from existing hypocalcemic levels at 30 (c) and 60 (d) minutes is shown as a solid line. The rate of EDTA infusion used in the slow EDTA infusion group to lower serum calcium rapidly from existing hypocalcemic levels at 120 minutes (e) is shown as a broken line.
receiving the slow EDTA infusion, the rate of EDTA infusion was rapidly increased at 120 minutes (Fig. 1B), and blood samples for ionized calcium and PTH were obtained at 123 and 126 minutes. During the fast infusion of EDTA and during the hypocalcemic clamp, heparinized blood samples were obtained at five-minute intervals. During the slow infusion of EDTA, blood samples were obtained at 10-minute intervals, except for those obtained at 5-minute intervals during first and last 10 minutes of the 120-minute infusion. During the calcium infusion, which was started at 120 minutes, blood samples were obtained at 10-minute intervals. During the studies in which an accelerated lowering of serum calcium from already hypocalcemic levels was performed, blood samples were obtained every two or three minutes. Ionized calcium was measured with a calcium-selective electrode (Ciba-Corning Diagnostics, S.A., Madrid, Spain),
and the measurement was performed immediately after the sample was obtained. Intact PTH was measured with an IRMA assay (Allegro; Nichols, San Juan Capistrano, CA, USA); the use of this assay for measurement of PTH in the dog has been validated previously [31–35]. The PTH values for a single dog were always measured in one assay. Statistics For the comparison of two samples, the paired or unpaired Student t-test was used. For the comparison of three or more samples, one-way or repeated-measures analysis of variance was used, depending on whether data were unpaired or paired. If the analysis of variance test showed a statistical difference, a post hoc test, the Fisher LSD, was performed to determine intergroup differences; for the post hoc test, differences were at the P , 0.05 level. Multivariate analysis of variance was
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
1727
Fig. 3. The PTH values at the end of the slow (j; 120 min) and fast (h; 30 min) EDTA infusions. In the slow-infusion group, PTH values at 120, 115, and 105 minutes were less than PTH values at 30 minutes in the fast-infusion group. These differences in PTH values were observed despite no difference in ionized calcium for all three comparisons. *P , 0.05 vs. 30 minutes.
Fig. 2. (A) The ionized calcium concentration during the fast (d; 30 min) induction of hypocalcemia (EDTA), followed by a 90-minute hypocalcemic clamp, and subsequently during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a solid line. The ionized calcium concentration during the slow (s; 120 min) induction of hypocalcemia (EDTA) and during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a broken line. (B) The PTH concentration during the fast (d; 30 min) induction of hypocalcemia (EDTA), followed by a 90-minute hypocalcemic clamp, and subsequently during the 60 minute recovery from hypocalcemia (calcium chloride infusion) is shown as a solid line. The PTH concentration during the slow (s; 120 min) induction of hypocalcemia (EDTA) and during the 60-minute recovery from hypocalcemia (calcium chloride infusion) is shown as a broken line. aP , 0.05 vs. fast at 30 min (ANOVA); b P , 0.05 vs. slow at the same time (MANOVA); cP , 0.05 vs. slow from 55 to 120 min (ANOVA).
used to compare mean values affected by more than one variable. A P value of less than 0.05 was considered significant. Results are shown as the mean 6 se. RESULTS As shown in Figure 2A, the EDTA infusion resulted in a linear decrease in serum calcium in both the fast and slow groups. In the fast group, the nadir of hypocalcemia (0.84 6 0.02 mm) was at 30 minutes, after which serum calcium was clamped at this hypocalcemic level for an additional 90 minutes. In the slow group, the nadir of hypocalcemia (0.82 6 0.03 mm) was at 120 minutes. In
both groups, a calcium infusion was started at 120 minutes and resulted in a similar rate of recovery from hypocalcemia and an increase to similar hypercalcemic values. The PTH response to hypocalcemia is shown in Figure 2B. In the fast group, a rapid increase in PTH that corresponded with the magnitude of hypocalcemia was observed during the first 30 minutes. During the hypocalcemic calcium clamp, PTH values decreased and from 40 to 120 minutes were less (P , 0.05) than the PTH value at 30 minutes. Moreover, PTH values at 75, 85, 95, and 105 minutes obtained during the hypocalcemic clamp were less (P , 0.05) than those at the same time intervals during the slow EDTA infusion even though the clamped serum calcium values at 75, 85, and 95 minutes were less (P , 0.05) or at 105 minutes, not different than calcium values in the slow group. During the recovery from hypocalcemia (calcium infusion), PTH values were similar in the two groups. As shown in Figure 3, the PTH value at 30 minutes in the fast group was greater than PTH values at 105, 115, and 120 minutes in the slow group. These differences in PTH were observed even though the 30-minute serum calcium value (0.84 6 0.02 mm) was not different than those at 105 (0.88 6 0.02 mm), 115 (0.85 6 0.02 mm), and 120 (0.82 6 0.03 mm) minutes. Moreover, when PTH values were matched for serum calcium during the induction of hypocalcemia (Fig. 4), PTH values in the fast group tended to be greater for similar serum calcium values and were greater for a similar serum calcium at 5 versus 15 minutes and 30 versus 115 minutes (fast versus slow). Whether or not a rapid reduction in serum calcium from hypocalcemic levels further stimulated PTH was
1728
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 4. PTH values matched for the serum calcium concentration in the fast (d) and slow (s) EDTA infusion groups. For similar serum calcium values at 5 (fast) versus 15 (slow) minutes and at 30 (fast) versus 115 (slow) minutes, the PTH values were significantly greater in the fast group (*P , 0.05; **P , 0.01).
evaluated. As shown in Figure 5A, an accelerated reduction in serum calcium between 30 and 36 minutes did not further increase PTH (264 6 26 vs. 279 6 27 pg/ml, analysis of variance, P 5 0.61). When serum calcium was reduced to its nadir at 30 minutes and a hypocalcemic clamp was maintained for the next 30 minutes before a further reduction in serum calcium was initiated between 60 and 68 minutes, PTH values at 66 and 68 minutes were greater than at 60 minutes (ANOVA, P , 0.001; Fig. 5B). Moreover, the PTH value at 30 minutes, obtained at the nadir of hypocalcemia before the hypocalcemic clamp, and PTH values at 66 and 68 minutes were similar to and greater than those at 45, 50, 55, and 60 minutes during the hypocalcemic clamp (ANOVA, P , 0.001). The slow infusion of EDTA resulted in a linear decline in serum calcium during 120 minutes (Fig. 5C). Between 120 and 126 minutes, the rate of serum calcium decrease was accelerated, and the PTH value at 126 minutes (302 6 30 pg/ml) was greater (ANOVA, P 5 0.03) than at 120 minutes (224 6 11 pg/ml) and all other PTH values from 0 to 120 minutes (ANOVA, P , 0.001).
Whether or not the depletion of PTH stores was the cause of the reduction in PTH values during a hypocalcemic clamp was evaluated (Fig. 6). A group of dogs in which serum calcium was reduced between 0 and 30 minutes was subjected to an accelerated rate of calcium reduction between 30 and 36 minutes (Fig. 6A). These dogs were compared with another group in which the calcium reduction was similar during the first 30 minutes, but after which a hypocalcemic clamp was started. In the group with the accelerated reduction in serum calcium, PTH values were not different than those at 30 minutes. However, during the hypocalcemic clamp, PTH values decreased and at 40, 45, and 50 minutes were less (P , 0.05) than those at 30 minutes. As shown in Figure 6B, the PTH values in the group with an accelerated calcium reduction were greater at similar times than those in the group with the hypocalcemic clamp, even though the 30-minute PTH values were similar. In Figure 7, the PTH response to the fast EDTA infusion is shown as curve 1. Shown as open squares are PTH values obtained during the hypocalcemic clamp between 30 and 90 minutes; this time range was used because it is the same as used by Madsen et al [26]. During the hypocalcemic clamp, the two highest PTH values were at 35 and 40 minutes. To construct curve 2, we used as the maximal PTH, the mean PTH (163 pg/ml) of the 12 PTH values during the hypocalcemic clamp. To complete the curve, the first two PTH values during the induction of hypocalcemia were used, and the connection to the mean PTH value for the hypocalcemic clamp was extrapolated. The five highest PTH values were excluded because these values exceeded the maximal PTH value for curve 2. Finally, curve 3 had the same maximal PTH as curve 2, and as was done by Madsen et al [26], the other PTH values were from the recovery from hypocalcemia. As shown in Figure 7, the three PTH-calcium curves are entirely different. DISCUSSION In this study in normal dogs, we showed that for a similar magnitude of hypocalcemia at the end of the slow and fast EDTA infusion, the PTH response was less in the slow than the fast group. Moreover, in the slowinfusion group, PTH values appeared to reach a plateau and, despite a continual reduction in serum calcium, did not increase further. We also showed that during a hypocalcemic clamp, PTH values decreased from maximal levels. However, this reduction in PTH values during the hypocalcemic clamp did not appear to represent a depletion of PTH stores because PTH could again be stimulated to maximal PTH levels when the serum calcium was further reduced below the level of the hypocalcemic clamp. The reason for comparing the PTH response to hypo-
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
1729
Fig. 5. (A) The ionized calcium (d) and PTH (s) values in dogs in which a fast EDTA infusion (30 min) was performed, and from 30 to 36 minutes, ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). The rapid lowering of ionized calcium from 30 to 36 minutes did not further increase PTH levels. (B) The ionized calcium and PTH values in dogs in which a fast EDTA infusion (30 min) was performed; this was followed by a 30-minute hypocalcemic clamp, after which ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). During the hypocalcemic clamp, PTH values decreased from the 30-minute PTH value (P , 0.05, a vs. b). During the rapid reduction of ionized calcium initiated at the end of the hypocalcemic clamp at 60 minutes, PTH values increased to levels similar to those observed at 30 minutes and were greater than PTH values during the hypocalcemic clamp (P , 0.05, a vs. b). (C) The ionized calcium and PTH values in dogs in which a slow EDTA infusion (120 min) was performed, after which ionized calcium was rapidly lowered by increasing the rate of EDTA infusion (Fig. 1). The rapid reduction of ionized calcium resulted in an increase in the PTH value to a level that was greater than all the PTH values during the slow EDTA infusion (P , 0.05, a vs. other PTH values).
1730
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH b Fig. 6. (A) The ionized calcium (d) and PTH (s) values obtained when a fast (30 min) EDTA infusion was performed, followed by a hypocalcemic clamp versus a fast (30 min) EDTA infusion followed by a more rapid lowering of the ionized calcium. During the hypocalcemic clamp, PTH values at 40, 45, and 50 minutes were lower than the peak PTH value at 30 minutes (P , 0.05, a vs. 30-min PTH value). In the group in which ionized calcium was rapidly lowered from 30 to 36 minutes, PTH values did not change from the 30-minute value. (B) The PTH values for similar time intervals in the two groups shown in (A). Symbols are: ( ) accelerated; ( ) clamp. At 30 minutes, the PTH values were not different. When the PTH value at 35 minutes during the hypocalcemic was compared with the PTH value obtained at 33 minutes during the rapid lowering of serum calcium, the latter PTH value was greater. Similarly, when the hypocalcemic clamped PTH value at 35 minutes was compared with the PTH value obtained at 36 minutes during the rapid lowering of serum calcium, the latter PTH value was greater. A similar result was observed when the 36-minute PTH obtained during the rapid lowering of serum calcium was compared with the 40-minute hypocalcemic clamped PTH value. These results suggest that the reduction in PTH values during the hypocalcemic clamp was not due to depletion of PTH stores. *P 5 NS; **P , 0.02; †P , 0.01.
calcemia during a fast and slow induction of hypocalcemia was to determine whether differences in the rate of calcium reduction affected parathyroid function. In a previous clinical study, Brent et al reported that the PTH response to hypocalcemia in normal volunteers was similar whether the calcium reduction was during 60 or 120 minutes [1]. In a study in dogs, we showed that the PTH response to hypocalcemia was similar whether the calcium reduction was during 30 or 60 minutes [31]. Furthermore, we showed that the maximal PTH response
was similar whether the calcium reduction was linear or nonlinear; the latter was designed to simulate the gradient-induced reduction in serum calcium (low calcium dialysis) often used to test parathyroid function in hemodialysis patients [10–12, 14–16, 23–25, 27]. However, others have shown that short, rapid reductions in serum calcium produce a greater PTH response than long, slow reductions in serum calcium [2]. Furthermore, in a preliminary report in hemodialysis patients, it was shown that a low calcium dialysis for approximately 60 minutes induced a greater maximal PTH response than a three-hour EDTA infusion even though the magnitude of hypocalcemia was similar (abstract; de Precigout et al, J Am Soc Nephrol 7:1789, 1996). Our current study in dogs also supports the concept that the rate of calcium reduction might affect the magnitude of the PTH response to hypocalcemia. In this study, the maximal PTH response to hypocalcemia was greater at the end of the EDTA infusion in the fast than the slow group despite a similar reduction in calcium. Moreover, in the slow group, PTH values did not change between 55 and 120 minutes even though serum calcium decreased by more than 0.20 mm during this time. An accelerated reduction in calcium at 120 minutes further increased PTH values. Thus, our results suggest that because of the slow rate of calcium reduction, the maximal PTH secretory capacity was not achieved. Because in clinical studies nonuniform protocols of calcium reduction are used to stimulate PTH secretion maximally, it is possible that differences in the rate of calcium reduction may lead to different conclusions regarding the dynamics of PTH secretion. In a recent study, we observed that the PTH response to hypocalcemia was different when the reduction in serum calcium was started after a 90-minute hypercalce-
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
Fig. 7. The PTH-calcium curve (curve 1) was drawn from values obtained during the fast (30 min) EDTA infusion. When, as suggested by Madsen et al, PTH values obtained during the hypocalcemic clamp (open squares) were used as the maximal PTH (mean of the 12 values), the PTH-calcium curve (curve 2) was distinctly different than curve 1 [26]. Finally, as was done in [26], the PTH value from the hypocalcemic clamp was used as the maximal PTH, and the other PTH values were obtained during the recovery from hypocalcemia, the resulting PTHcalcium curve (curve 3) is markedly different than curve 1.
mic clamp than from the normal state [28]. As a result, we decided to evaluate the converse, that is, whether or not the PTH response during the recovery from hypocalcemia was different after a hypocalcemic clamp than after a linear reduction in serum calcium. Our results show that during the recovery from hypocalcemia, PTH values for similar serum calcium values were not different between the two groups. Several of our findings support the conclusion that a deceleration in the rate of calcium reduction rather than a depletion of PTH stores was the cause of the decrease in PTH levels during the hypocalcemic clamp. After the 30-minute reduction in serum calcium, a subsequent, more rapid reduction in serum calcium did not further increase PTH levels, but PTH levels did not decrease as was observed during the hypocalcemic clamp. Moreover, PTH values during the six-minute accelerated reduction in serum calcium from 30 to 36 minutes were greater
1731
than those at similar times during the hypocalcemic clamp. Thus, if the depletion of PTH stores was responsible for the reduction in PTH levels, lower PTH values would have been expected during the accelerated reduction in serum calcium from 30 to 36 minutes. Other observations also suggest that depletion of PTH stores was not the cause of the reduction in PTH levels during the hypocalcemic clamp. The PTH values during the hypocalcemic clamp were lower than the 30-minute PTH value, and a reduction in calcium after 30 minutes of the hypocalcemic clamp resulted in an increase in PTH. If depletion of PTH stores was responsible for the lower PTH values during the hypocalcemic clamp, a further reduction in calcium would not have been able to increase PTH secretion. Moreover, the fact that during the slow EDTA infusion at the nadir of hypocalcemia at 120 minutes, PTH values could be stimulated by a more rapid reduction in serum calcium also indicates that PTH depletion was not present. Thus, our results suggest that depletion of PTH stores was not the cause of the reduction in PTH levels during the hypocalcemic clamp. Rather, we believe that the reduction in PTH levels was more likely due to the deceleration in the rate of calcium reduction [2, 13, 31]. As such, it may be related to the phenomenon of hysteresis in which after a directional change in serum calcium, the PTH level for the same serum calcium concentration is less during the recovery than the induction of hypocalcemia [2, 13, 31]. In a previous study, we showed that the PTH response to two consecutive 30-minute reductions in serum calcium separated by a 30-minute recovery period was similar [31]. Although unlikely, it could be argued that the 30-minute recovery period provided sufficient time for reutilization of degraded PTH. In our current study, it is possible that a decrease in the metabolic clearance rate of PTH contributed to the differences in circulating PTH levels. In a recent study in dogs, anesthesia with pentobarbital was shown to decrease the hepatic clearance of intact PTH and increase circulating PTH levels [36]. However, in this study, this effect should have been similar in all of the dogs. An important issue is whether hypocalcemia changed the metabolic clearance rate of PTH. At least for our initial 0.4 mm reduction in ionized calcium, most previous studies have shown that this degree of hypocalcemia did not change the metabolic clearance rate of PTH even in anesthetized animals [36–38], and some studies have even reported an enhanced metabolic clearance [39]. Either result would not increase circulating PTH levels. Studies of parathyroidectomized animals have shown that even more severe hypocalcemia did not change the metabolic clearance rate of PTH [37, 40]. However, it is possible that in our study the accelerated reduction in serum calcium could have produced hemodynamic instability, which decreased the metabolic clearance rate of PTH. Although we cannot
1732
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
eliminate this possibility, several results suggest that this explanation is not likely. First, in the group of dogs in which the accelerated reduction in serum calcium was performed during the hypocalcemic clamp, PTH values increased when serum calcium decreased from 0.87 to 0.71 mm. This range of serum calcium has been evaluated in previous studies and was not associated with changes in the metabolic clearance rate of PTH [36, 37]. Second, in dogs in which the accelerated reduction in calcium was performed in the fast- and slow-infusion groups, the PTH response was different for similar reductions in calcium and a similar final serum calcium. In the fast group, PTH values did not increase, although they did increase in the slow group. If hemodynamic instability decreased the metabolic clearance rate of PTH during marked hypocalcemia, it would have been expected to have a similar effect in both groups. Third, the venous blood pH, measured during the determination of ionized calcium, was not different before and after the rapid lowering of ionized calcium (7.36 6 0.02 vs. 7.34 6 0.02, respectively). Fourth, although blood pressure was not measured, these dogs displayed no outward signs of distress, and all recovered without incident. Based on the concept of depletion of preformed PTH stores, Madsen et al recently advocated that the PTH value obtained during the hypocalcemic clamp should be used as the maximal PTH rather than the peak PTH value obtained at the nadir of hypocalcemia [26]. This concept of depletion of preformed PTH stores also implies that in response to hypocalcemia, all parathyroid cells secrete PTH simultaneously. However, the in vitro studies of Sun et al suggest another possibility should be considered [41, 42]. During a reduction in the calcium concentration in these studies, only a certain percentage of parathyroid cells secreted PTH at any one time, and secretion by other parathyroid cells was held in reserve for a subsequent rechallenge to a reduced calcium concentration [41, 42]. In such a schema, recruitment of previously quiescent cells would prevent depletion of PTH stores. Our results in dogs also suggest that depletion of preformed PTH stores may not be a factor. In previous clinical studies, it has been shown that rapid changes in the rate of calcium reduction affect the magnitude of the PTH response to hypocalcemia [2, 43]. In this study in dogs, PTH values decreased by approximately 40% during the hypocalcemic clamp but could be further stimulated during a further reduction in the serum calcium concentration. Such a result would also support the concept that the reduction from peak PTH values does not represent a depletion of PTH stores. In addition to its physiological significance regarding hormone secretion and synthesis, an accurate definition of the maximal PTH level is also important because, as has been shown in Figure 7, it affects the calculation of the set point and the slope of the PTH calcium curve. More-
over, the calculation of the set point and slope would also be affected by the use of PTH values obtained during the recovery from hypocalcemia, which are lower for the same serum calcium concentration than those obtained during the induction of hypocalcemia [3, 13, 31]. Thus, additional clinical studies are needed to resolve these important issues. In conclusion, our results suggest that (a) the reduction in PTH values during a hypocalcemic clamp may not represent a depletion of PTH stores, but rather may be a response to the stabilization of the serum calcium. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH calcium curve; and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium. ACKNOWLEDGMENTS This work was supported by grants #PB97–0090 and #PM95–0184 from the Direccio´n General Ensen˜anza Superior, a grant from the Fundacio´n Reina Sofı´a-Cajasur, and a grant from the Baxter Extramural Grant Program. The authors also wish to acknowledge the technical support of E.M. Saneamientos Cordoba, S.A. Reprint requests to Dr. Mariano Rodriguez, Unidad de Investigacion, Hospital Universitario Reina Sofia, Avda Menendez Pidal S/N, 14004 Cordoba, Spain. E-mail: [email protected]
REFERENCES 1. Brent GA, Leboff MS, Seely EW, Conlin PR, Brown EM: Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans. J Clin Endocrinol Metab 67:944–950, 1988 2. Grant FD, Conlin PR, Brown EM: Rate and concentration dependence of parathyroid hormone dynamics during stepwise changes in serum ionized calcium in normal humans. J Clin Endocrinol Metab 71:370–378, 1990 3. Conlin PR, Fajtova VT, Mortensen RM, Leboff MS, Brown EM: Hysteresis in the relationship between serum ionized calcium and intact parathyroid hormone during recovery from induced hyper- and hypocalcemia in normal humans. J Clin Endocrinol Metab 69:593–599, 1989 4. El-Hajj Fuleihan G, Gundberg CM, Gleason R, Brown EM, Stromski ME, Grant FD, Conlin PR: Racial differences in parathyroid hormone dynamics. J Clin Endocrinol Metab 79:1642–1647, 1994 5. Schwarz P, Sorensen HA, Transbol I, McNair P: Induced hypocalcemia controlled by a citrate clamp technique and the intact parathyroid hormone response obtained. Scand J Clin Lab Invest 50:891–897, 1990 6. Schwarz P, Sorensen HA, Transbol I, McNair P: Regulation of acute parathyroid hormone release in normal humans: Combined calcium and citrate clamp. Am J Physiol 263:E195–E198, 1992 7. Gardin JP, Patron P, Fouqueray B, Prigent A, Paillard M: Maximal PTH secretory rate and set point for calcium in normal subjects and patients with primary hyperparathyroidism. Miner Electrolyte Metab 14:221–228, 1988 8. Paillard M, Gardin JP, Borensztein P, Prigent A: Determinants of parathormone secretion in primary hyperparathyroidism. Horm Res 32:89–92, 1989 9. Schwarz P, Sorensen HA, Momsen G, McNair P, Transbol
Estepa et al: Calcium reduction, hypocalcemic clamp, and PTH
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
I: Normal pattern of parathyroid hormone response to calcium lowering in primary hyperparathyroidism, a citrate clamp study. Clin Endocrinol (Oxf) 37:344–348, 1992 Dunlay R, Rodriguez M, Felsenfeld A, Llach F: Direct inhibitory effect of calcitriol on parathyroid function (sigmoidal curve) in dialysis patients. Kidney Int 36:1093–1098, 1989 Cunningham J, Altmann P, Gleed JH, Butter KC, Marsh FP, O’Riordan JLH: Effect of direction and rate of change of calcium on parathyroid hormone secretion in uraemia. Nephrol Dial Transplant 4:339–344, 1989 Rodriguez M, Felsenfeld AJ, Williams C, Pederson JA, Llach F: The effect of long-term intravenous calcitriol administration on parathyroid function in hemodialysis patients. J Am Soc Nephrol 2:1014–1020, 1991 Felsenfeld AJ, Ross D, Rodriguez M: Hysteresis of the parathyroid hormone response to hypocalcemia in hemodialysis patients with low turnover aluminum bone disease. J Am Soc Nephrol 2:1136–1143, 1991 Felsenfeld AJ, Rodriguez M, Dunlay R, Llach F: A comparison of parathyroid-gland function in haemodialysis patients with different forms of renal osteodystrophy. Nephrol Dial Transplant 6:244– 251, 1991 Malberti F, Surian M, Cosci P: Effect of chronic intravenous calcitriol on parathyroid function and set point of calcium in dialysis patients with refractory secondary hyperparathyroidism. Nephrol Dial Transplant 7:822–828, 1992 Kwan JTC, Almond MK, Beer JC, Noonan K, Evans SJW, Cunningham J: “Pulse” oral calcitriol in uraemic patients: Rapid modification of parathyroid response to calcium. Nephrol Dial Transplant 7:829–834, 1992 Ramirez JA, Goodman WG, Gornbein J, Menezes C, Moulton L, Segre GV, Salusky IB: Direct in vivo comparison of calciumregulated parathyroid hormone secretion in normal volunteers and patients with secondary hyperparathyroidism. J Clin Endocrinol Metab 76:1489–1494, 1993 Combe C, Aparicio M: Phosphorus and protein restriction and parathyroid function in chronic renal failure. Kidney Int 46:1381– 1386, 1994 Messa P, Vallone C, Mioni G, Geatti O, Turrin D, Passoni N, Cruciatti A: Direct in vivo assessment of parathyroid hormonecalcium relationship curve in renal patients. Kidney Int 46:1713– 1720, 1994 Ramirez JA, Goodman WG, Belin TR, Gales B, Segre GV, Salusky IB: Calcitriol therapy and calcium-regulated PTH secretion in patients with secondary hyperparathyroidism. Am J Physiol 267:E961–E967, 1994 Sanchez CP, Goodman WG, Ramirez JA, Gales B, Belin TR, Segre GV, Salusky IB: Calcium-regulated parathyroid hormone secretion in adynamic renal osteodystrophy. Kidney Int 48:838–843, 1995 Goodman WG, Belin T, Gales B, Juppner H, Segre GV, Salusky IB: Calcium-regulated parathyroid hormone release in patients with mild or advanced secondary hyperparathyroidism. Kidney Int 48:1553–1558, 1995 Caravaca F, Cubero JJ, Jimenez F, Lopez JM, Aparicio A, Cid MC, Pizarro JL, Liso J, Santos I: Effect of the mode of calcitriol administration of PTH-ionized calcium relationship in uraemic patients with secondary hyperparathyroidism. Nephrol Dial Transplant 10:665–670, 1995 Felsenfeld AJ, Jara A, Pahl M, Bover J, Rodriguez M: Differences in the dynamics of PTH secretion in hemodialysis patients with marked secondary hyperparathyroidism. J Am Soc Nephrol 6:1371–1378, 1995 Pahl M, Jara A, Bover J, Rodriguez M, Felsenfeld AJ: The
26. 27.
28.
29. 30.
31.
32. 33. 34. 35.
36.
37. 38. 39.
40.
41. 42. 43.
1733
set point of calcium and the reduction of parathyroid hormone in hemodialysis patients. Kidney Int 49:226–231, 1996 Madsen JC, Rasmussen AQ, Ladefoged SD, Schwarz P: Parathyroid hormone secretion in renal failure. Kidney Int 50:1700– 1705, 1996 Rodriguez M, Caravaca F, Fernandez E, Borrego MJ, Lorenzo V, Cubero J, Martin-Malo A, Betriu A, Rodriguez AP, Felsenfeld AJ: Evidence for both abnormal set point of calcium and adaptation to serum calcium in hemodialysis patients with hyperparathyroidism. J Bone Miner Res 12:347–355, 1997 Sanchez J, Aguilera-Tejero E, Estepa JC, Almaden Y, Rodriguez M, Felsenfeld AJ: A reduced PTH response to hypocalcemia after a short period of hypercalcemia: A study in dogs. Kidney Int 50(Suppl 57):S18–S22, 1996 Fox J, Heath H III: The “calcium clamp”: Effect of constant hypocalcemia on parathyroid hormone secretion. Am J Physiol 240:E649–E655, 1981 Schwarz P, Sorensen HA, McNair P, Transbol I: Cica-clamp technique, a method for quantifying parathyroid hormone secretion: A sequential citrate and calcium clamp study. Eur J Clin Invest 23:546–553, 1993 Aguilera-Tejero E, Sanchez J, Almaden Y, Mayer-Valor R, Rodriguez M, Felsenfeld AJ: Hysteresis of the PTH-calcium curve during hypocalcemia in the dog: Effect of the rate and linearity of calcium decrease and sequential episodes of hypocalcemia. J Bone Miner Res 11:1226–1233, 1996 Torrance AG, 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 Torrance AG, Nachreiner R: Intact parathyroid hormone assay and total calcium concentration in the diagnosis of disorders of calcium metabolism in dogs. J Vet Intern Med 3:86–89, 1989 Cloutier M, Gascon-Barre M, D’Amour P: Chronic adaptation of dog parathyroid function to a low-calcium-high-sodium-vitamin D-deficient diet. J Bone Miner Res 7:1021–1028, 1992 Cloutier M, Brossard JH, Gascon-Barre M, D’Amour P: Lack of involution of hyperplastic parathyroid glands in dogs: Adaptation via a decrease in the calcium stimulation set point and a change in secretion profile. J Bone Miner Res 9:621–629, 1994 D’Amour P, Rousseau L, Rocheleau B, Pomier-Layrargues G, Huet PM: Influence of Ca21 concentration to the clearance and circulating levels of intact and carboxy-terminal iPTH in pentobarbital-anesthetized dogs. J Bone Miner Res 11:1075–1085, 1996 Fox J, Scott M, Nissenson RA, Heath H III: Effect of plasma calcium concentration on the metabolic clearance rate of parathyroid hormone in the dog. J Lab Clin Med 102:70–77, 1983 Daugaard H, Egfjord M, Olgaard K: Influence of calcium on the metabolism of intact parathyroid hormone by isolated perfused rat kidney and liver. Endocrinology 126:1813–1820, 1990 Canterbury JM, Bricker LA, Levey GS, Kozlovskis PL, Ruiz E, Zull JE, Reiss E: Metabolism of bovine parathyroid hormone: Immunological characteristics of fragments generated by liver perfusion. J Clin Invest 55:1245–1253, 1975 Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, Potts JT Jr: Peripheral metabolism of [35S]parathyroid hormone in vivo: Influence of alterations in calcium availability and parathyroid status. J Endocrinol 122:237–245, 1989 Sun F, Ritchie CK, Hassager C, Maercklein P, Fitzpatrick LA: Heterogenous response to calcium by individual parathyroid cells. J Clin Invest 91:595–601, 1993 Sun F, Maercklein P, Fitzpatrick LA: Paracrine interactions among parathyroid cells: Effect of cell density on cell secretion. J Bone Miner Res 9:971–976, 1994 Schmitt CP, Schaefer F, Bruch A, Veldhuis JD, Schmidt-Gayk H, Stein G, Ritz E, Mehls O: Control of pulsatile and tonic parathyroid hormone secretion by ionized calcium. J Clin Endocrinol Metab 81:4236– 4243, 1996