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Division of Endocrinology, University of Pittsburgh School of Medicine (M.J.H., M.B.T., A.G.-O., A.F.S.), and Graduate School of Public Health (S.M.S.), Pittsburgh, Pennsylvania 15213; and Department of Pediatrics, Medical University of South Carolina (B.W.H.), Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Mara J. Horwitz, M.D., Division of Endocrinology, Falk 580, University of Pittsburgh School of Medicine, 3601 Fifth Avenue, Pittsburgh, Pennsylvania 15213. E-mail: horwitz+{at}pitt.edu.
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Abstract |
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In two groups (seven subjects each) of healthy young (25- to 35-yr-old) normal volunteers, PTH and PTHrP infused at 8 pmol/kg·h displayed similar calcemic effects, although PTH was slightly more potent in this regard. Both peptides also displayed similar phosphaturic effects. In addition, both peptides had similar effects on renal tubular calcium handling, yielding fractional calcium excretion values of approximately 3.5%, some 50% below the values (6.5%) observed in subjects rendered similarly hypercalcemic by the infusion of calcium. In contrast to these several quantitatively similar effects of PTH and PTHrP, PTH tended to be selectively more effective than PTHrP in stimulating renal production of 1,25-(OH)2D.
These studies indicate that renal tubular calcium reabsorption is likely to contribute to hypercalcemia in patients with HHM. In addition, PTH may be selectively more effective than PTHrP in stimulating 1,25-(OH)2D production, in contrast to its phosphaturic, calcemic effects and its effects to stimulate nephrogenous cAMP excretion and renal tubular calcium reabsorption.
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Introduction |
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To date, there have been no head to head studies directly comparing the calcemic and renal calcium reabsorptive potencies of PTH and PTHrP or their effects on 1,25-(OH)2D under such sustained, steady state conditions in normal human volunteers. The current study therefore was designed to answer the following questions. 1) Why are plasma 1,25-(OH)2D concentrations elevated in HPT, but reduced in HHM? 2) What are the relative potencies of PTH and PTHrP in driving renal calcium reabsorption in humans in vivo? 3) What exactly are the relative potencies of PTHrP and PTH in inducing hypercalcemia in vivo in humans?
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Materials and Methods |
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Human (h) PTH-(1–34) (hereafter referred to as PTH) and hPTHrP-(1–36) (hereafter referred to as PTHrP) were synthesized using solid phase synthesis as described previously in detail (11, 20, 22, 23, 24). Both peptides were dissolved in 10 mM acetic acid, sterile-filtered, aliquoted into sterile vials, and lyophilized (11, 20, 22, 23, 24). Vials were assayed for peptide content using amino acid analysis as well as for sterility and pyrogenicity as described previously (11, 20, 22, 23, 24) and were stored for future use at -80 C. Before infusion, peptides were resuspended in sterile bacteriostatic saline and then mixed with 2 ml of the study subject’s plasma in 120 ml sterile saline, as described previously (11, 20, 22, 23, 24). Infusions were administered as described below. We have previously shown that PTHrP is stable under these conditions (20). The use of PTHrP was approved by the FDA (IND 49,175), as was the use of PTH (IND 60,979).
Study subjects
Fourteen healthy subjects between the ages of 25 and 35 yr (mean age ± SD, 28.8 ± 3.0 yr) were recruited from the university community. Baseline demographics are shown in Table 1
. Five were female, and nine were male. All were in excellent health, and none was talking medications of any type, except for oral contraceptives. Exclusion criteria were cardiac, kidney, liver, pulmonary, cardiac, hematological, or other disorders. The research protocol was approved in advance by the University of Pittsburgh School of Medicine institutional review board and the General Clinical Research Center advisory committee. Each subject provided informed signed consent.
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Subjects were admitted to the General Clinical Research Center at the University of Pittsburgh School of Medicine at 0700 h on the first day of the study and remained there for 48 h. Meals were provided at 1700 h on d 1 of the study; at 0700, 1200, and 1700 h on d 2 of the study; and at 0700 h after completion of the study on the third day. The diet was a 400 mg/d calcium, 800 mg/d phosphorus, 2 g/d sodium, 15% protein, 55% carbohydrate, and 30% fat diet. Caloric intake was 30 kcal/kg·d. Subjects fasted from the night before admission until dinner on the first day. Ad libitum access to ionized water was permitted. Before admission, subjects were assigned to receive either PTHrP (n = 7) or PTH (n = 7). The peptides were suspended as described above and in detail previously (11, 20, 21, 22, 23, 24) in saline and infused at a rate of 8 pmol/kg·h (11, 20, 21), beginning at 0800 h on the first day of the study. This time point is considered 0 h in the figures. The primary end points were serum calcium, fractional calcium excretion, and plasma 1,25-(OH)2D, with serum phosphorus, tubular maximum for phosphorus/glomerular filtration rate (TmP/GFR) and endogenous PTH-(1–84) as secondary end points. Blood and urine samples were obtained for these measures at the times indicated in the figures.
Measurements and calculations
Blood was analyzed for serum total and ionized calcium, creatinine, phosphorus, endogenous PTH-(1–84), and plasma 1,25-(OH)2D. Urine was also obtained for calcium, phosphorus, and creatinine determinations at the time points indicated in the figures. Routine chemistries, including ionized serum calcium, were measured at the University of Pittsburgh Medical Center clinical laboratory, and fractional calcium excretion (FECa) and TmP/GFR were calculated as previously described (11, 20, 23, 24). FECa data from PTH- and PTHrP-infused subjects were compared both to one another as well as to our prior data for FECa derived from subjects infused with iv calcium during a calcium clamp (11). Plasma 25-hydroxyvitamin D and 1,25-(OH)2D were measured using previously reported methods (25). PTH-(1–84) was measured using an immunochemiluminometric assay (Quest Diagnostics, Inc., San Juan Capistrano, CA).
Statistical analysis
Descriptive statistics were computed to describe the groups and to summarize the measures of central tendency and dispersion. To assess imbalances that may have occurred during assignment, groups were compared using t and 
2 statistics. The effects of the groups (PTH, PTHrP) over time on serum calcium, FECa, plasma 1,25-(OH)2D, serum phosphorous, TmP/GFR, and endogenous PTH-(1–84) were analyzed using repeated measures analysis with a mixed modeling approach (26). The PROC MIXED procedure in SAS (version 8.2, SAS Institute, Inc., Cary, NC) was employed for fitting these mixed models. Both linear and nonlinear trends over time were evaluated. The level of statistical significance was set at 0.05 (two-tailed). If significant differences were identified for a particular effect, a Bonferroni adjustment was applied to the post hoc comparisons that followed.
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Results |
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The effects of the two peptides on serum total calcium are shown in Fig. 1. The mean serum calcium concentration was approximately 2.32 mmol/liter at the start of the study and rose to approximately 2.87 mmol/liter for PTH and 2.57 for PTHrP. The increment in serum calcium observed for PTHrP was similar to that observed in our previous studies in which PTHrP was infused at the same rate, 8 pmol/kg·h (11). The increase in serum calcium in response to PTH was mildly, but significantly, greater over time than that for PTHrP, demonstrating a quadratic change over time that varied by group (P < 0.001).
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. Again, the effects of the two peptides are similar to the changes in total serum calcium at the dose administered, rising from approximately 1.12 mmol/liter at baseline to 1.35 and 1.47 mmol/liter at the conclusion of the study for PTHrP and PTH, respectively. Statistically significant differences were again observed between the response profiles for ionized serum calcium, suggesting a greater curvilinear increase for PTH than for PTHrP (P = 0.012, linear time by group interaction; P = 0.009, quadratic time effect).
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As shown in Fig. 3, endogenous PTH-(1–84) declined with peptide infusion. Although the baseline values were different (P = 0.005), the values during the second day of the study were comparably and significantly suppressed (P < 0.001). Similar effects were found when controlling for baseline differences in plasma PTH-(1–84) in the analysis. The differences in baseline PTH presumably reflect the differences in prestudy calcium intake. It is important to note that although the two groups differed at baseline, comparable values were observed between groups during the period between 7 and 48 h while subjects were hypercalcemic. This is important to the interpretation of the FECa data shown below.
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As is clear from the data presented in Fig. 4, plasma 1,25-(OH)2D concentrations rose in a curvilinear fashion in both groups (P < 0.001), and significant differences were found between these response profiles for the two groups (P = 0.0495, linear time by group interaction). In the patients infused with PTH, 1,25-(OH)2D increased from approximately 72 to 168 pmol/liter, and this increase from baseline was statistically significant at 7, 23, 35, and 48 h (P < 0.001). In contrast, in the PTHrP group, the change in plasma 1,25-(OH)2D was smaller, rising from 72 to 120 pmol/liter, and this difference was significantly different from baseline at only 23 and 35 h (P < 0.0125). Plasma 25-hydroxyvitamin D concentrations were comparable in the two groups at baseline (PTH group, 67.1 ± 11.3 nmol/liter; PTHrP group, 74.6 ± 7.6 nmol/liter; P = NS).
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As is shown in Fig. 5, serum phosphorus concentrations decreased significantly over time (P < 0.001 for linear time effect). Serum phosphorous response profiles were not statistically significantly different between the two peptides (P = NS). There was a significant transient rise in serum phosphorus in both groups from 11–14 h into the study corresponding to dinner on the first day of the study (P < 0.001). Compared with baseline values, a significant decrease in serum phosphorus was observed in both groups at 11, 23, 29, 35, 38, and 48 h (P < 0.005).
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) declined from approximately 1.13 to approximately 0.80–0.97 mmol/liter at 46 h, showing a similar curvilinear response profile for both groups (P = 0.008 for quadratic time effect). These differences from baseline were significant at 7, 11, 23, 29, and 35 h (P < 0.005), but there were no statistically significant differences between the two response profiles for the two peptides (P = NS).
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As shown in Fig. 7, there was a gradual linear increase in FECa in both groups during the study, from approximately 1–2% at baseline to approximately 3.5% during d 2 of the study (P < 0.001), as we had previously reported for PTHrP alone (11). Importantly, however, there was no statistical or physiological difference between the two groups with regard to the linear change in FECa over time (P = NS).
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No subject in the PTH or PTHrP group developed a change in serum creatinine or blood pressure, and no subject reported significant adverse effects, such as nausea, vomiting, flushing, diarrhea, constipation, or headache.
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Discussion |
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These observations are of interest for a number of reasons. As described in the introduction, HHM and HPT share a number of features, but differ with respect to coupling of osteoclast-osteoblast function and renal production of 1,25-(OH)2D. The pathophysiological reasons for these discrepancies remain unclear despite the identification of PTHrP more than 15 yr ago (4, 5, 6, 7). In addition to these issues, the quantitative significance of the contribution of renal tubular calcium reabsorption to hypercalcemia in humans with HHM remains undefined (4, 5, 6, 7, 11, 12, 13, 16, 17, 18, 19, 20, 21, 27, 28). A related question concerns the relative potencies of PTH and PTHrP in stimulating renal calcium reabsorption. We demonstrated recently that continuous infusion of PTHrP under steady state conditions for 46 h did indeed stimulate renal calcium reabsorption in normal healthy volunteers (11). One goal of the current study was to directly address the relative potency of PTH vs. PTHrP in stimulating renal calcium reabsorption using a head to head comparison of the two peptides.
The techniques employed were adapted from standard diabetes insulin and glucose clamp techniques and extended from our previously described calcium and PTHrP clamp studies (11, 23). In those studies we demonstrated that PTHrP was stable under the infusion conditions employed, and that continuous infusion of PTHrP over 46 h resulted in a gradual, but progressive, increase in serum calcium to concentrations that were unequivocally (but mildly) hypercalcemic. We also determined the degree of fractional calcium excretion (6.5 ± 0.1%) that results from mild hypercalcemia (total serum calcium, 2.57 mmol/liter; ionized serum calcium, 1.27 mmol/liter) induced by calcium infusion alone in the setting of suppressed endogenous PTH-(1–84). What was lacking in these prior studies was a direct comparison of PTHrP with PTH. Such studies could not be performed at the time because of a lack of commercial availability of PTH. To overcome this obstacle, for the current study we synthesized and received FDA permission to use hPTH-(1–34).
These studies indicate that PTHrP and PTH have similar potencies in humans with respect to regulation of both serum calcium and FECa. There may be subtle differences in the abilities of the two peptides to elevate serum calcium, with PTH appearing more effective in statistical terms, but these differences were quantitatively small. The differences in serum calcium may be due to the slightly higher plasma 1,25-(OH)2D concentrations in the subjects infused with PTH, as discussed below.
In addition to this similarity of effects on serum calcium and renal calcium handling, it is important to note that the FECa values observed in the PTH and PTHrP groups (peak values of 
3.5%) were some 50% below that (6.5 ± 0.1%) observed in normal healthy human volunteers in whom serum total and ionized calcium had been clamped to 2.57 and 1.27 mmol/liter, respectively (11). Importantly, the filtered loads of calcium in the PTH and PTHrP groups were similar and were comparable to those in the calcium clamp group reported previously (11). These values were obtained under conditions in which endogenous PTH was markedly suppressed. Thus, the FECa changes observed are due to exogenously infused PTH or PTHrP. These findings indicate that not only did PTHrP and PTH have similar effects on FECa, but the peptides stimulated renal (presumably distal) tubular calcium reabsorption with equal potency. This is an important finding in terms of the pathophysiology of the HHM syndrome, as we and others had believed for years that the hypercalcemia observed in the syndrome was due principally or exclusively to the markedly increased osteoclastic bone resorption observed in patients and animal models of HHM (4, 14, 27, 28). One can now reasonably conclude that renal calcium reabsorption driven by PTHrP probably contributes to the hypercalcemia of HHM. These results may also influence thinking regarding the management of HHM. It has long been standard therapy for HHM to administer saline iv in large volumes. This is intended to expand the GFR, increasing the filtered load of calcium, and, by increasing lumenal sodium concentrations, interfere with proximal tubular calcium reabsorption. There is now an additional rationale for this practice: to overcome the PTHrP-mediated renal tubular calcium reabsorption.
The effects of the two peptides on serum phosphorus concentrations and renal phosphorus handling were also equivalent. This was anticipated, as earlier studies from our group as well as others observed this in shorter-term studies (11, 20, 21).
PTH and PTHrP appeared to produce different quantitative effects on plasma 1,25-(OH)2D concentrations, and these differences were statistically significant (P = 0.0495). As these differences were borderline significant, they should be confirmed in studies using larger numbers of subjects. The greater increase in plasma 1,25-(OH)2D, particularly on the second day of the study, may have contributed to the slightly higher serum total and ionized calcium concentrations observed in the PTH group through direct effects on intestinal calcium absorption, effects on the renal tubule to enhance calcium reabsorption, or direct effects on the skeleton to augment the resorptive response to PTH. We were surprised by these differences, because we and the Fraher group (20, 21) had previously shown that both PTH and PTHrP stimulate 1,25-(OH)2D increases in humans over periods of 6–12 h. In our studies, PTH and PTHrP produced qualitatively and quantitatively similar effects over the first 6 h using the same doses of PTH and PTHrP employed herein (20), but these studies only lasted for 6 h. In the Fraher studies (21, 29), PTHrP was less potent than PTH over a 12-h period, but this was true for all of the effects of PTHrP on serum calcium, renal phosphorus handling, and urinary cAMP as well. In those studies the researchers employed PTHrP-(1–34) instead of the PTHrP-(1–36) used in our studies, and it may be that PTHrP-(1–34) is less effective or more rapidly cleared than PTHrP-(1–36). Those results are different from those described herein, as PTH and PTHrP appeared to be equivalent in calcemic, renal calcium handling, phosphaturic, and PTH-suppressive effects in the current study, and we had previously shown that the effects of PTHrP-(1–36) are identical to those of PTH-(1–34) in humans on nephrogenous cAMP excretion (20). Thus, these current studies suggest that PTHrP-(1–36) may be selectively less effective than hPTH-(1–34) in stimulating plasma 1,25-(OH)2D increments in humans. The interpretation of these findings in light of the repeated documentation that plasma 1,25-(OH)2D concentrations are low in HHM but high in HPT remains uncertain (1, 2, 3, 4, 5, 6, 7). PTHrP may indeed be less effective than PTH in driving 1,25-(OH)2D production, but this alone would not explain a decline in 1,25-(OH)2D in humans with HHM.
These studies have at least one significant limitation. The biologically active circulating forms of PTH are not known, but probably do not include the hPTH-(1–34) employed in the current study. As for PTHrP, although there is good reason to believe that the PTHrP-(1–36) employed herein is an authentic secretory form of PTHrP (20, 30), other secretory forms of PTHrP comprising the amino terminus, the mid region, and the carboxyl terminus exist (30), and they could conceivably have additional or alternative effects on serum calcium and renal calcium handling. As these peptides have not been fully characterized and are not available for study in humans, these must remain open questions.
In summary, these studies demonstrate that multiday, steady state PTH and PTHrP infusion studies can be performed safely and effectively in humans and can provide important total body pathophysiological and physiological information not obtainable from laboratory animals, from human tissue in vitro, or from brief injection/infusion studies in vivo. They also demonstrate that PTHrP and PTH have comparable effects over the long term on renal tubular handling. They further suggest that PTH may be more effective than PTHrP in driving increases in plasma 1,25-(OH)2D in humans.
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Acknowledgments |
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Footnotes |
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Abbreviations: 1,25-(OH)2D, 1,25-Dihydroxyvitamin D; FECa, fractional calcium excretion; h, human; HHM, humoral hypercalcemia of malignancy; HPT, hyperparathyroidism; PTHrP, PTH-related protein; TmP/GFR, tubular maximum for phosphorus/glomerular filtration rate.
Received May 17, 2002.
Accepted January 2, 2003.
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, Subjects infused with PTH; 
, subjects infused with PTHrP. The bars indicated the SD. The shaded bar indicates the time of infusion of PTH or PTHrP. Both peptides were infused at a rate of 8 pmol/kg·h for 46 h.
