Dynamics of secretion and metabolism of PTH during hypo- and hypercalcaemia in the dog as determined by the ‘intact’ and ‘whole’ PTH assays

Jose C. Estepa1, Ignacio Lopez1, Arnold J. Felsenfeld2, Ping Gao3, Tom Cantor3, Mariano Rodríguez4 and Escolastico Aguilera-Tejero1,

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



   Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Recent evidence has shown that the assay for ‘intact’ parathyroid hormone (I-PTH) not only reacts with 1–84 PTH but also with large non-1–84 PTH fragments, most of which is probably 7–84 PTH. As a result, an assay specific for 1–84 PTH named ‘whole’ PTH (W-PTH) has been developed. The present study was designed: (i) to determine whether the W-PTH assay reliably measures PTH values in the dog; (ii) to evaluate differences between the W-PTH and I-PTH assays during hypo- and hypercalcaemia; and (iii) to assess the peripheral metabolism of W-PTH and I-PTH.

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-1–84 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



   Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Since the inception of the radioimmunoassay for parathyroid hormone (PTH) in the late 1960s, the measurement of PTH has been complicated by the existence of a number of circulating PTH fragments. The accepted gold standard has been the measurement of presumed intact (1–84) PTH using the ‘intact’ PTH (I-PTH) assay developed in the late 1980s. Until recently, this two-site immunoradiometric assay was thought to be specific for the measurement of 1–84 PTH. However, evidence has now shown that the I-PTH assay reacts not only with 1–84 PTH but also with large non-1–84 C-terminal PTH fragments, most of which is probably the 7–84 PTH fragment [14]. Studies in normal humans have shown that the percentage of non-1–84 PTH fragment relative to 1–84 PTH is ~20–30% [1,3,4], but this ratio is somewhat higher in dialysis patients [15]. Because of the failure of the I-PTH assay to measure only 1–84 PTH, a new assay named the ‘whole’ PTH assay (W-PTH), which only detects biologically active 1–84 PTH, has been developed and validated in the normal population and in both primary and secondary hyperparathyroidism [4].

Values of both 1–84 PTH and C-terminal PTH fragments increase during hypocalcaemia and decrease during hypercalcaemia, but the changes are not proportional. As compared with 1–84 PTH, the increase in C-terminal fragments during hypocalcaemia is less than that of 1–84 PTH [69]. During hypercalcaemia, the decrease in C-terminal fragments is less than that of 1–84 PTH [69]. 1–84 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 7–84 PTH bind to the C-PTH receptor and, even though they do not affect the binding of 1–84 PTH to the PTH/PTHrP receptor, their interaction with the C-PTH receptor was recently reported to inhibit the biological effects of 1–84 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 1–84 PTH and not to large non-1–84 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-1–84 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.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Six healthy mixed-breed dogs, three males and three females, ages 3.6±0.1 years and weighing 22.5±0.2 kg were used in the studies. Dogs were housed in individual cages and twice a day were fed a commercial diet containing 1.2% calcium, 0.9% phosphorus and 1200 IU/kg of vitamin D. Water was provided ad libitum.

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 39–84) 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 1–4 N-terminal region of PTH. The use of this antibody guarantees that only 1–84 PTH is detected. The immunoradiometric assay for I-PTH uses a specific polyclonal antibody directed against PTH (7–34) as a tracer. With this antibody, W-PTH (1–84), the 7–84 PTH fragment and possibly other large, non-1–84 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 8–37 and 13–67 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.



   Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Before the induction of hypocalcaemia and hypercalcaemia, the ionized calcium value was 1.20±0.02 mM. The baseline I-PTH value was greater than that of W-PTH, 36±8 vs 13±3 pg/ml (P=0.01).

The induction of hypocalcaemia progressively increased both I-PTH and W-PTH values (Figure 1AGo). 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 1AGo). 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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Fig. 1.  (A) Values for I-PTH (solid line) and W-PTH (broken line) are shown in the same six dogs in which hypocalcaemia (EDTA) and hypercalcaemia (calcium infusion) were induced. Values which were significantly different (P<0.05) for the same ionized calcium value are marked with an asterisk (*). (B) The W-PTH/ I-PTH (solid line) and W-PTH/non-1–84 PTH ratios (broken line) are shown during the induction of hypocalcaemia during which neither of the two ratios changed.

 
At the baseline ionized calcium concentration (1.20 mM), the W-PTH/I-PTH ratio was 38±4% and the W-PTH/non-1–84 PTH ratio was 65% (Figure 1BGo). Hypocalcaemia did not change these ratios significantly. Even though hypercalcaemia resulted in a decrease in both I-PTH and W-PTH values (Figure 1AGo), the W-PTH/I-PTH ratio increased to 81% by an ionized calcium of 1.30 mM. Moreover, at an ionized calcium of 1.35 mM, this ratio was 158% and exceeded this value at every higher ionized calcium concentration. The W-PTH/I-PTH ratio was also significantly greater (P<0.05) than the baseline ratio at all ionized calcium concentrations >=1.30 mM. Because it was unexpected that, even during hypercalcaemia, the I-PTH value would be less than that of W-PTH, dilution curves of plasma samples were performed to determine the capacity of each assay to detect low PTH values. Two plasma samples, one from a normocalcaemic dog and the other from a hypocalcaemic dog, were diluted with zero standard from the assay kit (Table 1Go). Dilution of plasma samples resulted in a predictable reduction in I-PTH values to <2 pg/ml. In contrast, the same dilution curves showed that the lowest W-PTH value that could be measured accurately was ~5 pg/ml.


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Table 1.  Dilution curve of dog plasma starting with a normal PTH (normocalcaemia) concentration and a high PTH (hypocalcaemia) concentration

 
A comparison of the parallel dilutions between human and dog PTH is shown in Figure 2AGo. Human and dog plasma with similar PTH levels were serially diluted and, as shown in this figure, dilution curves were parallel when assayed with the W-PTH assay. Figure 2BGo shows the correlation between dog PTH values measured with the W-PTH and I-PTH assays. The paired values obtained with the W-PTH and I-PTH assays when measuring the same samples were highly correlated (r2=0.96, P<0.001).



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Fig. 2.  (A) Results of W-PTH measurements are shown when parallel dilutions of human and dog plasma were performed. (B) The correlation between intact and whole PTH is shown for all values.

 
Figure 3AGo shows the effect of sustained hypocalcaemia on the plasma PTH concentration as measured with the W-PTH and I-PTH assays. At the onset of the 90 min hypocalcaemic clamp, the I-PTH concentration decreased slightly but remained elevated at ~150 pg/ml during the remainder of the hypocalcaemic clamp. The W-PTH concentration remained in the range of 60 pg/ml during the entire hypocalcaemic clamp. The W-PTH/I-PTH and W-PTH/non-1–84 PTH ratios did not change during the first 30 min of the hypocalcaemic clamp (Figure 3BGo). However, by 35 min, both ratios were significantly greater (P<0.01) than at the beginning of the hypocalcaemic clamp and remained greater (P<0.05) until the end of the study at 120 min.



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Fig. 3.  (A) Shown are the ionized calcium (solid line), I-PTH (dotted line) and W-PTH (broken line) values during the 90 min hypocalcaemic clamp between 30 and 120 min of the study. At the same ionized calcium value, significant differences (P<0.05) between I-PTH and WPTH are shown by an asterisk (*). (B) The W-PTH/I-PTH (solid line) and W-PTH/non-1–84 PTH ratios (broken line) are shown during the 90 min hypocalcaemic clamp between 30 and 120 min of the study. By 35 min of the hypocalcaemic clamp, both ratios were greater (P<0.01) than that at the start of the hypocalcaemic clamp and remained greater (P<0.05) to the end of the study. *P<0.05 vs the ratios at the start of the hypocalcaemic clamp. For reference, before the induction of hypocalcaemia, the values of W-PTH and I-PTH, W-PTH/I-PTH and W-PTH/non-1–84 PTH ratios, and the ionized calcium value at baseline were 13±3 pg/ml, 36±8 pg/ml, 38±4%, 65±6% and 1.20±0.02 mM, respectively.

 
The clearance of W-PTH and I-PTH from the circulating blood after PTX is shown in Table 2Go. Because PTH values had already decreased to the limits of detection by the W-PTH assay by 5 min, it was not possible to calculate a disappearance curve. At 2.5 min after PTX, the PTH value as a percentage of the pre-PTX value was less for W-PTH than for I-PTH, 25±2 vs 30±3%, P<0.05. By 5 min after PTX, the W-PTH value only decreased to 20±3%, while that of I-PTH decreased to 15±3%. Similarly, the W-PTH/I-PTH ratio before PTX was 39±1% while the ratios at 2.5 and 5 min after PTX were 34±4 and 52±7%, respectively. Between 2.5 and 5 min, it appeared that the decrease of I-PTH was more rapid than that of W-PTH. However, this reversal in the rate of disappearance at 5 min was most probably due to the lack of sensitivity of the W-PTH assay at low PTH values.


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Table 2.  Disappearance rate of W-PTH and I-PTH and the ratio of W-PTH to I-PTH from immediately before parathyroidectomy (0 min) to 2.5 and 5 min after parathyroidectomy

 



   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study was designed to evaluate I-PTH and W-PTH secretion and metabolism in the dog with normal renal function. Our results show that (i) during stimulation of PTH secretion by hypocalcaemia, there is a proportional increase in I-PTH and W-PTH secretion; and (ii) both I-PTH and W-PTH are rapidly metabolized.

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 1–84 PTH and large non-1–84 PTH fragments (subsequently referred to as only non-1–84 PTH) most of which is probably 7–84 PTH [1,3,4]. Conversely, the W-PTH assay detects only 1–84 PTH. In normal humans, the W-PTH/I-PTH ratio is ~70–80%, which means that non-1–84 PTH would be 20–30% [1,3,4]. In our study in normal dogs, this ratio was lower at 38%, which means that non-1–84 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-1–84 PTH would mean that the W-PTH/non-1–84 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 1–34 PTH and thus does not bind to 2–34 PTH, 3–34 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 (1–84). 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 7–84 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 1–84 PTH [13,22]. Of particular interest is that the inhibitory effect of 7–84 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-1–84 PTH/1–84 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-1–84 PTH/1–84 PTH ratio has been ~10% [1,5]. In another study, the 1–84 PTH/non-1–84 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-1–84 PTH may adversely affect the activity of 1–84 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-1–84 PTH produced, non-1–84 PTH as a percentage of 1–84 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-1–84 PTH, which may act to decrease the calcaemic action of 1–84 PTH, should increase during hypocalcaemia. It may be that the non-1–84 PTH/1–84 PTH ratio is the determining factor. However, in our study, the non-1–84 PTH/1–84 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 1–84 PTH [13,14]. It has been shown that as compared with the increase in 1–84 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 1–84 PTH [9]. Thus, it would seem that much more work needs to be done to completely understand the interaction between 1–84 PTH and 7–84 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 1–84 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-1–84 PTH decreased during a hypocalcaemic clamp; (vi) the concentration of non-1–84 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-1–84 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-1–84 PTH to 1–84 PTH did not change during acute hypocalcaemia, non-1–84 PTH alone may not retard the action of 1–84 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.



   Acknowledgments
 
This work was supported by grants BFI2001-1901 and BFI2001-0350 from the Ministerio de Ciencia y Tecnología, the Plan Andaluz de Investigacion (Grupo CTS-179) and the Fundacion Reina Sofia-Cajasur.

Conflict of interest statement. P. G. and T. C. are employed by Scantibodies laboratory.



   Notes
 
Correspondence and offprint requests to: Dr E. Aguilera-Tejero, Department of Medicina y Cirugia Animal, Campus Universitario Rabanales, Ctra. Madrid-Cadiz km 396, 14014 Cordoba, Spain. Email: pv1agtee{at}uco.es Back



   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Brossard JH, Cloutier M, Roy L, Lepage R, Gascon-Barre M, D'Amour P. Accumulation of a non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values. J Clin Endocrinol Metab 1996; 81:3923–3929[Abstract]
  2. Lepage R, Roy L, Brossard J-H et al. A non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 1998; 44:805–809[Abstract/Free Full Text]
  3. Brossard J-H, Lepage R, Cardinal H et al. Influence of glomerular filtration rate on non-(1–84) parathyroid hormone (PTH) detected by intact PTH assays. Clin Chem 2000; 46:697–703[Abstract/Free Full Text]
  4. Gao P, Scheibel S, D'Amour P et al. Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function. J Bone Miner Res 2001; 16:605–614[ISI][Medline]
  5. John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999; 84:4287–4290[Abstract/Free Full Text]
  6. D'Amour P, Palardy J, Bahsali G, Mallette LE, DeLean A, Lepage R. The modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration. J Clin Endocrinol Metab 1992; 74:525–532[Abstract]
  7. Brossard JH, Whittom S, Lepage R, D'Amour P. Carboxylterminal fragments of parathyroid hormone are not secreted preferentially in primary hyperparathyroidism as they are in other causes of hypercalcemia. J Clin Endocrinol Metab 1993; 77:413–419[Abstract]
  8. Cloutier M, Rousseau L, Gascon-Barre M, D'Amour P. Immunological evidences for post-translational control of the parathyroid function by ionized calcium in dogs. Bone Miner 1993; 22:197–207[ISI][Medline]
  9. D'Amour P. Effects of acute and chronic hypercalcemia on parathyroid function and circulating parathyroid hormone molecular forms. Eur J Endocrinol 2002; 146:407–410[ISI][Medline]
  10. Chase LR, Melson GL, Aurbach GD. Pseudohypoparathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone. J Clin Invest 1969; 48:1832–1844[ISI][Medline]
  11. Hruska KA, Moskowitz D, Esbrit P, Civitelli R, Westbrook S, Huskey M. Stimulation of inositol triphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone. J Clin Invest 1987; 79:230–239[ISI][Medline]
  12. Cosman F, Morrow B, Kopal M, Bilezikian JP. Stimulation of inositol triphosphate formation in ROS 17/2.8 cell membranes by guanine nucleotide, calcium, and parathyroid hormone. J Bone Miner Res 1989; 4:413–420[ISI][Medline]
  13. Nguyen-Yamamoto L, Rousseau L, Brossard J-H, Lepage R, D'Amour P. Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentrations in rats by acting on a receptor different from the PTH/PTH-related peptide receptor. Endocrinology 2001; 142:1386–1392[Abstract/Free Full Text]
  14. Divieti P, John MR, Juppner H, Bringhurst FR. Human PTH (7–84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor. Endocrinology 2002; 143:171–176[Abstract/Free Full Text]
  15. Cloutier M, D'Amour P, Gascon Barre M, Aamel L. Low calcium diet in dogs causes a greater increase in parathyroid function measured with an intact than with a carboxylterminal assay. Bone Miner 1990; 9:179–188[ISI][Medline]
  16. 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 1992; 7:1021–1028[ISI][Medline]
  17. D'Amour P, Rousseau L, Rocheleau B, Pomier-Layrargues G, Huet PM. Influence of Ca2+ concentration to the clearance and circulating levels of intact and carboxy-terminal iPTH in pentobarbital-anesthetized dogs. J Bone Min Res 1996; 11:1075–1085[ISI][Medline]
  18. 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 1996; 50 [Suppl 57]:S18–S22[ISI]
  19. 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 1996; 11:1226–1233[ISI][Medline]
  20. Estepa JC, Aguilera-Tejero E, Almaden Y, Rodriguez M, Felsenfeld AJ. Effect of rate of calcium reduction and a hypocalcemic clamp on PTH secretion: a study in dogs. Kidney Int 1999; 55:1724–1733[CrossRef][ISI][Medline]
  21. Lopez I, Aguilera-Tejero E, Felsenfeld AJ, Estepa JC, Rodriguez M. Direct effect of acute metabolic and respiratory acidosis on parathyroid hormone secretion in the dog. J Bone Miner Res 2002; 17:1691–2000[ISI][Medline]
  22. Monier-Faugere M.-C, Geng Z, Mawad H et al. Improved assessment of bone turnover by the PTH-(1–84)/large C-PTH fragments in ESRD patients. Kidney Int 2001; 60:1460–1468[CrossRef][ISI][Medline]
  23. Estepa JC, Aguilera-Tejero E, Lopez I, Almaden Y, Rodriguez M, Felsenfeld AJ. Effect of phosphate on PTH secretion in vivo. J Bone Miner Res 1999; 14:1848–1854[ISI][Medline]
  24. Slatopolsky E, Finch J, Clay P et al. A novel mechanism for skeletal resistance in uremia. Kidney Int 2000; 58:753–761[CrossRef][ISI][Medline]
  25. Yamashita H, Gao P, Noguchi S et al. Role of cyclase activating parathyroid hormone (1–84 PTH) measurements during parathyroid surgery. Ann Surg 2002; 236:105–111[CrossRef][ISI][Medline]
Received for publication: 25. 7.02
Accepted in revised form: 22. 1.03