1 Department of Medicina y Cirugia Animal, Universidad de Cordoba, Campus Rabanales, Ctra Madrid-Cadiz km 396, 14014 Cordoba, 4 Department of Nefrologia y Unidad de Investigacion, Hospital Universitario Reina Sofia, Avda Menendez Pidal s/n, 14004 Cordoba, Spain, 2 Department of Medicine, West Los Angeles VA Medical Center and UCLA, Los Angeles and 3 Department of R&D and Diagnostics, Scantibodies Laboratory Inc., Santee, CA, USA
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Abstract |
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Methods. In normal dogs, hypocalcaemia was induced by EDTA infusion and was followed with a 90 min hypocalcaemic clamp. Hypercalcaemia was induced with a calcium infusion.
Results. I-PTH and W-PTH values increased from 36±8 and 13±3 pg/ml (P=0.01) at baseline to a maximum of 158±40 and 62±15 pg/ml (P=0.02 vs I-PTH) during hypocalcaemia. The W-PTH/I-PTH ratio, 38±4% at baseline, did not change during the induction of hypocalcaemia, but sustained hypocalcaemia increased (P<0.05) this ratio. During hypercalcaemia, maximal suppression for I-PTH was 2.0±0.5 and only 5.7±0.6 pg/ml for W-PTH, due to a decreased sensitivity of the W-PTH assay at values <5 pg/ml. The disappearance rate of PTH was determined in five additional dogs which underwent a parathyroidectomy (PTX). At 2.5 min after PTX, W-PTH was metabolized more rapidly, with a value of 25±2% of the pre-PTX value vs 30±3% for I-PTH (P<0.05).
Conclusions. (i) The W-PTH/I-PTH ratio is less in the normal dog than in the normal human, suggesting that the percentage of non-184 PTH measured with the I-PTH assay is greater in normal dogs than in normal humans; (ii) the lack of change in the W-PTH/I-PTH ratio during acute hypocalcaemia is different from the situation observed in humans; and (iii) the dog appears to be a good model to study I-PTH and W-PTH assays during hypocalcaemia.
Keywords: calcium; hypercalcaemia; hypocalcaemia; intact PTH; parathyroid hormone; whole PTH
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Introduction |
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Values of both 184 PTH and C-terminal PTH fragments increase during hypocalcaemia and decrease during hypercalcaemia, but the changes are not proportional. As compared with 184 PTH, the increase in C-terminal fragments during hypocalcaemia is less than that of 184 PTH [69]. During hypercalcaemia, the decrease in C-terminal fragments is less than that of 184 PTH [69]. 184 PTH binds to the PTH/PTHrP receptor and activates G-protein-coupled pathways to generate second messengers such as cAMP, diacylglycerol and inositol triphosphate to produce its biological effects [1012]. C-terminal fragments including 784 PTH bind to the C-PTH receptor and, even though they do not affect the binding of 184 PTH to the PTH/PTHrP receptor, their interaction with the C-PTH receptor was recently reported to inhibit the biological effects of 184 PTH [13,14].
In previous studies, the dog has been used to study the PTH response to hypocalcaemia and hypercalcaemia [520]. In these studies, the I-PTH assay was shown to reflect the expected PTH secretion, with hypocalcaemia markedly stimulating and hypercalcaemia suppressing I-PTH values. However, the degree to which the hypocalcaemia-induced increase in PTH values is due to 184 PTH and not to large non-184 PTH fragments is not known in the dog. Also whether the W-PTH assay, which was designed for use in humans, cross-reacts with dog PTH has not been studied in detail. Human and dog PTH are almost identical in their amino acid sequence. The only amino acid differences in the first 40 amino acids are at positions 7 and 16. Moreover, the first amino acid of the N-terminal end (serine), which is essential for antibody binding in the W-PTH assay [4], is the same in dog and human PTH. Thus, it would be expected that the W-PTH assay would recognize dog PTH.
Our goal was to correlate the response of the W-PTH and I-PTH assays to hypocalcaemia and hypercalcaemia in the normal dog and to evaluate the production of large non-184 PTH fragments during these conditions. Furthermore, in order to determine whether peripheral metabolism contributes to differences between I-PTH and W-PTH, the rate of disappearance of W-PTH and I-PTH was measured after parathyroidectomy.
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Methods |
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After an overnight fast, dogs (n=6) were premedicated with intramuscular ketamine (10 mg/kg) and then anaesthetized with intravenous sodium thiopental (15 mg/kg/h), after which the left jugular and cephalic veins were cannulated. The jugular catheter was used for blood sampling and anaesthetic administration, and the cephalic vein for EDTA administration during the induction of hypocalcaemia. One week later, the same protocol was used in the same six dogs for the induction of hypercalcaemia, except that the cephalic vein was used for the administration of calcium chloride (CaCl2).
Induction of hypocalcaemia and the hypocalcemic clamp
Three heparinized blood samples were obtained at 5 min intervals to establish baseline ionized calcium and PTH values. As previously described [19,20], hypocalcaemia was induced during 30 min with an EDTA infusion. To achieve a linear reduction in the ionized calcium concentration during the 30 min, the rate of the EDTA infusion was increased progressively from 75 mg/kg/h to a final rate of 210 mg/kg/h. During the induction of hypocalcaemia, blood for ionized calcium and PTH was drawn every 5 min. Between 30 and 120 min, the ionized calcium concentration was clamped at the same hypocalcaemic level achieved at the end of the 30 min induction phase. To maintain the same degree of hypocalcaemia during the hypocalcaemic clamp, the rate of EDTA infused was reduced from 210 to 70 mg/kg/h between 30 and 45 min, maintained at 50 mg/kg/h between 45 and 90 min, and progressively increased to 80 mg/kg/h between 90 and 120 min. Heparinized blood samples for ionized calcium and PTH were obtained every 10 min between 30 and 120 min.
Induction of hypercalcaemia
Before the infusion of CaCl2, three heparinized blood samples were obtained at 5 min intervals to establish baseline ionized calcium and PTH values. CaCl2 was dissolved in 5% dextrose and water and infused at a constant rate of 0.66 mg/kg/h during 30 min. Heparinized blood samples were obtained every 5 min.
Studies of PTH metabolism
Five additional dogs, three males and two females, ages 3.1±0.2 years and weighing 24.0±0.5 kg were used to evaluate PTH metabolism. Dogs were again premedicated with intramuscular ketamine and anaesthetized with intravenous thiopental before the placement of the intravenous catheters described in the original protocol. Because of the need to perform a parathyroidectomy (PTX), these dogs were intubated, and deep anaesthesia, which provided hypnosis, analgesia and muscle relaxation, was used. Anaesthesia was maintained during the study by the intermittent intravenous administration of midazolam (0.2 mg/kg), fentanyl (2 µg/kg) and pancuronium bromide (0.1 mg/kg). Because of dead space during mechanical ventilation, an FiO2 of 27% was used to maintain a normal PaO2. The thyroid and parathyroid glands were surgically exposed and sutures were placed loosely around the thyroid veins before baseline values were established. Three heparinized blood samples were obtained at 5 min intervals to establish baseline ionized calcium and PTH values. To induce hypocalcaemia, dogs received an EDTA infusion during 30 min using the same protocol described previously. Removal of the parathyroid glands (time 0) was performed at 30 min, at which time all dogs had a plasma calcium below 0.9 mM. The pre-placed sutures were tightened in <10 s and then the thyroid and parathyroid tissue was removed. The thyroparathyroidectomy was completed in <5 min and, after each surgery, four parathyroid glands were identified in the resected tissue. To determine the disappearance rate of PTH after PTX, blood samples were obtained every 2.5 min for 10 min. The disappearance rate was based on a monoexponential model in which Ct=C0xe-kt, where Ct is the plasma concentration of PTH at a given time (t), C0 is the plasma concentration of PTH at time zero, and k is the constant which determines the rate of disappearance [21].
Laboratory measurements
Ionized calcium was measured with a calcium selective electrode (Bayer Diagnostics, Barcelona, Spain) and the measurements were performed immediately after the sample was obtained. PTH was measured using the Duo PTH Kit (Scantibodies Laboratory Inc., Santee, CA). The kit contains two immunoradiometric assays. Both assays share a polyclonal antibody (anti-PTH 3984) coated on to the surface of polystyrene beads as a solid phase. The immunoradiometric assay for W-PTH utilizes a tracer antibody directed against the 14 N-terminal region of PTH. The use of this antibody guarantees that only 184 PTH is detected. The immunoradiometric assay for I-PTH uses a specific polyclonal antibody directed against PTH (734) as a tracer. With this antibody, W-PTH (184), the 784 PTH fragment and possibly other large, non-184 PTH fragments are detected. In humans, normal values for the W-PTH (Scantibodies Laboratory, Santee, CA) and I-PTH (Nichols, San Juan Capistrano, CA) assays are 837 and 1367 pg/ml, respectively [4].
Statistical analysis
ANOVA was used to compare changes in W-PTH, I-PTH and the W-PTH/I-PTH ratio during the induction of hypo- and hypercalcaemia. When differences (P<0.05) were detected by ANOVA, PTH values at different serum calcium concentrations were compared with PTH values at baseline calcium using paired t-tests. Comparisons between W-PTH and I-PTH values were performed by the paired t-test. Data are expressed as the mean±SE.
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Results |
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The induction of hypocalcaemia progressively increased both I-PTH and W-PTH values (Figure 1A). At an ionized calcium of 0.85 mM, the I-PTH concentration was greater than that of W-PTH, 158±40 vs 62±15 pg/ml (P=0.02). At all measured ionized calcium values between 0.85 and 1.15 mM, the concentration of I-PTH was greater (P<0.05) than that of W-PTH. The induction of hypercalcaemia decreased both I-PTH and W-PTH values (Figure 1A
). The lowest value for both assays was observed at an ionized calcium concentration of 1.55 mM (I-PTH, 2.0±0.5 pg/ml vs W-PTH, 5.7±0.6 pg/ml, P=0.14).
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Discussion |
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During hypo- and normocalcaemia, W-PTH values in dog plasma were consistently lower than I-PTH values. This result was as expected because the assay for I-PTH detects both 184 PTH and large non-184 PTH fragments (subsequently referred to as only non-184 PTH) most of which is probably 784 PTH [1,3,4]. Conversely, the W-PTH assay detects only 184 PTH. In normal humans, the W-PTH/I-PTH ratio is 7080%, which means that non-184 PTH would be 2030% [1,3,4]. In our study in normal dogs, this ratio was lower at 38%, which means that non-184 PTH would be 62%, a value higher than in normal humans and even higher than in dialysis patients [5]. Also, the relatively large amount of non-184 PTH would mean that the W-PTH/non-184 PTH ratio, recently advocated to indicate bone activity [22], would be low in the normal dog. In most studies, the basal I-PTH concentration has been shown to be similar in normal humans and dogs [15,1721,23]. Thus, our study would suggest that W-PTH values are lower in dogs than in humans.
Because the W-PTH exceeded the I-PTH value during hypercalcaemia, we were concerned about the affinity of the tracer antibody for W-PTH. As a result, we performed dilution curves with plasma from a hypocalcaemic dog with high PTH values and from a normocalcaemic dog with normal PTH values. Both the W-PTH and I-PTH assays measured the diluted PTH values, as expected, until values of 5 pg/ml were approached with the W-PTH assay. Thus, as shown by the stimulatory response to hypocalcaemia, the precision of the dilution curves for W-PTH and I-PTH in hypo- and normocalcaemic dogs, the parallels of the human and dog dilution curves, the high correlation between dog PTH values measured with the I-PTH and W-PTH assays and the post-PTX fall in PTH values, the W-PTH assay provided consistent results in the normal dog except at values <5 pg/ml.
The reason for the lack of sensitivity at low PTH values is unclear. The antibody for W-PTH is designed to bind the first four amino acids of human 134 PTH and thus does not bind to 234 PTH, 334 PTH, etc. The first amino acid in human PTH is serine, as it is in dog PTH. The only amino acid differences between the human and dog in the first 40 amino acids are at positions 7 and 16. The slight difference in the C-terminus between dog and human PTH potentially could reduce assay sensitivity, but should not change the detection specificity of dog PTH (184). Thus, the precise reason for the decreased sensitivity of the W-PTH assay at low PTH concentrations in the dog is not known.
Recent studies have shown that the 784 PTH fragment detected by the I-PTH assay and other C-terminal PTH fragments not measured by the I-PTH assay decrease the calcaemic action of 184 PTH [13,22]. Of particular interest is that the inhibitory effect of 784 PTH and other C-terminal PTH fragments was attributed to their interaction with the C-PTH receptor [13,14]. In a study in dialysis patients with a wide range of pre-dialysis serum calcium values, hypocalcaemia decreased the non-184 PTH/184 PTH ratio [24]. In studies in normal humans and dialysis patients in whom hypocalcaemia was induced with a chelating agent, the decrease in the non-184 PTH/184 PTH ratio has been 10% [1,5]. In another study, the 184 PTH/non-184 PTH ratio correlated with bone activity, with a high ratio indicative of high bone turnover and a low ratio indicative of adynamic bone [22]. Again this is a suggestion that non-184 PTH may adversely affect the activity of 184 PTH. In our study, the induction of hypocalcaemia did not change the W-PTH/I-PTH ratio as the total amount of both W-PTH and I-PTH increased. Thus, during hypocalcaemia, even though there was a marked increase in the overall amount of non-184 PTH produced, non-184 PTH as a percentage of 184 PTH was essentially unchanged. If during hypocalcaemia the primary goal of enhanced PTH secretion is to restore the serum calcium concentration to normal, then it becomes important to understand why non-184 PTH, which may act to decrease the calcaemic action of 184 PTH, should increase during hypocalcaemia. It may be that the non-184 PTH/184 PTH ratio is the determining factor. However, in our study, the non-184 PTH/184 PTH ratio did not change. Another consideration is that smaller C-terminal PTH fragments, which are not detected by the I-PTH assay, also bind to the C-PTH receptor and these likewise have been shown to decrease the calcaemic action of 184 PTH [13,14]. It has been shown that as compared with the increase in 184 PTH during the induction of hypocalcaemia, the increase in all C-terminal PTH fragments (small, medium and large) is proportionally less [68]. D'Amour has suggested recently that it is the total amount of C-terminal PTH fragments which determines the biological activity of 184 PTH [9]. Thus, it would seem that much more work needs to be done to completely understand the interaction between 184 PTH and 784 PTH and the other C-terminal PTH fragments. Our results suggest that at least during hypocalcaemia, the dog model could be used to gain a better understanding of the complex interactions cited above.
PTH concentrations during the hypocalcaemic clamp were studied to determine whether sustained hypocalcaemia changed the W-PTH/I-PTH ratio. The I-PTH concentration decreased slightly during the initial phase of the hypocalcaemic clamp, while W-PTH values remained at maximal levels during this time, resulting in an unchanged W-PTH/I-PTH ratio. However, after 35 min of the hypocalcaemic clamp, the W-PTH concentration increased slightly, resulting in an increase in the W-PTH/I-PTH ratio. It could be argued that our hypocalcaemic clamp was imperfect because the ionized calcium concentration at the start was greater than that at the end. However, eliminating the first and last two ionized calcium values results in a desirable hypocalcaemic clamp which still shows a significant and sustained increase in the W-PTH/I-PTH ratio after 30 min of the hypocalcaemic clamp. These results suggest that the parathyroid gland may respond to sustained hypocalcaemia by increasing the production of 184 PTH. It is possible that this phase might represent the release of newly synthesized PTH in contrast to the release of previously stored hormone.
The second goal of our study was to evaluate whether there was a difference in the rates at which I-PTH and W-PTH were metabolized. However, because of the inability to measure low values of W-PTH, it was only possible to measure W-PTH values accurately at 2.5 min after PTX, at which time the percentage reduction in W-PTH was greater than that of I-PTH. While the metabolism study was incomplete due to the decreased sensitivity of the W-PTH assay at low PTH values, the data available at 2.5 min after PTX do indicate that the peripheral metabolism of both W-PTH and I-PTH was rapid. In our study in dogs, the rapidity and magnitude of the PTH reduction in both PTH moieties following PTX as well as the small but significantly greater reduction in W-PTH than in I-PTH after PTX were similar to those recently reported in humans with primary hyperparathyroidism [25]. The high PTH values and normal renal function in our dogs might be expected to present post-PTX conditions similar to those in patients with primary hyperparathyroidism.
In summary, our results show that (i) the W-PTH/I-PTH ratio was less in the normal dog than in studies of the normal human; (ii) acute hypocalcaemia did not increase the W-PTH/I-PTH ratio; (iii) both the W-PTH and I-PTH assays appeared to reflect accurately changes in PTH values in the hypocalcaemic dog; (iv) there was decreased sensitivity of the W-PTH assay in the hypercalcaemic dog; (v) the percentage of non-184 PTH decreased during a hypocalcaemic clamp; (vi) the concentration of non-184 PTH markedly decreased during hypercalcaemia; and (vii) PTH as measured by the W-PTH and I-PTH assays was rapidly metabolized after PTX. In conclusion: (i) levels of non-184 PTH detected by the I-PTH assay appear to be greater in the normal dog than in the normal human; (ii) because the ratio of non-184 PTH to 184 PTH did not change during acute hypocalcaemia, non-184 PTH alone may not retard the action of 184 PTH in hypocalcaemia; and (iii) the dog appears to be a good model to study differences in PTH measurements between the I-PTH and W-PTH assays during hypocalcaemia.
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Acknowledgments |
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Conflict of interest statement. P. G. and T. C. are employed by Scantibodies laboratory.
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Notes |
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References |
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