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Keywords:

  • humoral hypercalcemia of malignancy;
  • primary hyperparathyroidism;
  • PTH;
  • PTH-related protein;
  • vitamin D

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Osteoblast activity and plasma 1,25(OH)2vitamin D are increased in HPT but suppressed in HHM. To model HPT and HHM, we directly compared multiday continuous infusions of PTH versus PTHrP in humans. Continuous infusion of both PTH and PTHrP results in marked and prolonged suppression of bone formation; renal 1,25(OH)2D synthesis was stimulated effectively by PTH but poorly by PTHrP.

Introduction: PTH and PTH-related protein (PTHrP) cause primary hyperparathyroidism (HPT) and humoral hypercalcemia of malignancy (HHM), respectively. Whereas HHM and HPT resemble one another in many respects, osteoblastic bone formation and plasma 1,25(OH)2vitamin D are increased in HPT but reduced in HHM.

Materials and Methods: We performed 2- to 4-day continuous infusions of escalating doses of PTH and PTHrP in 61 healthy young adults, comparing the effects on serum calcium and phosphorus, renal calcium and phosphorus handling, 1,25(OH)2vitamin D, endogenous PTH(1-84) concentrations, and plasma IGF-1 and markers of bone turnover.

Results: PTH and PTHrP induced comparable effects on renal calcium and phosphorus handling, and both stimulated IGF-1 and bone resorption similarly. Surprisingly, PTH was consistently more calcemic, reflecting a selectively greater increase in renal 1,25(OH)2 vitamin D production by PTH. Equally surprisingly, continuous infusion of both peptides markedly, continuously, and equivalently suppressed bone formation.

Conclusions: PTHrP and PTH produce markedly different effects on 1,25(OH)2vitamin D homeostasis in humans, leading to different calcemic responses. Moreover, both peptides produce profound suppression of bone formation over multiple days, contrasting with events in HPT, but mimicking HHM. These findings underscore the facts that the mechanisms underlying the anabolic skeletal response to PTH and PTHrP in humans is poorly understood, as are the signal transduction mechanisms that link the renal PTH receptor to 1,25(OH)2vitamin D synthesis. These studies emphasize that much remains to be learned regarding the normal regulation of vitamin D metabolism and bone formation in response to PTH and PTHrP in humans.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

CONTINUOUS SECRETION OF PTH in primary hyperparathyroidism (HPT) causes hypercalcemia, phosphaturia, and hypophosphatemia; enhanced renal tubular calcium reabsorption; and an increase in circulating concentrations of 1,25(OH)2D, with resultant increases in intestinal calcium absorption. (1–3) This increase in intestinal calcium absorption, coupled with the enhanced renal tubular reabsorption of calcium, are the proximal causes of the hypercalcemia observed in HPT. (1–3) At the level of bone, patients with HPT, whose skeletons are exposed to continuous presence of PTH, display increases in both osteoblastic bone formation and osteoclastic bone resorption. (1–5) The increases in bone formation and resorption are closely coupled, such that net loss of skeletal mass in hyperparathyroidism, if it occurs at all, requires years to become manifest. (1–5) In contrast, intermittent (once per day) injection of pharmacologic doses of PTH causes a rapid increase in bone mass. (4, 6–10) Whereas both bone formation and resorption are increased by intermittent injections of PTH, the marked increase in bone mass with PTH treatment reflects a net increase in osteoblastic bone formation over osteoclastic bone resorption. (4, 6–10)

PTH-related protein (PTHrP) was discovered through its role as the quintessential skeletal catabolic agent: continuous secretion of PTHrP is the cause of humoral hypercalcemia of malignancy (HHM). (11–17) Patients with HHM resemble those with HPT in that both groups display increases in serum calcium, reductions in renal phosphorus absorption, increases in renal calcium reabsorption, and increases in osteoclastic bone resorption. (11–17) In contrast with those with HPT, patients with HHM display marked reductions in circulating 1,25(OH)2D. (13, 14, 16) In addition, osteoblast-osteoclast activities are completely uncoupled in HHM, with marked reductions in osteoblast activity, in contrast to the tight coupling that occurs in HPT. (16, 17) The reasons for these differences are not known. Interestingly, PTHrP stimulates 1,25(OH)2D production and also stimulates osteoblastic activity in rodents. (18–20) Thus, rodents are a poor model of the human situation, and the differences in HHM and HPT therefore must be explored in humans.

Surprisingly, given its skeletal catabolic role in HHM, intermittent injections of PTHrP cause a marked anabolic response in humans with postmenopausal osteoporosis, (21, 22) just as occurs with intermittent injections of PTH. (4, 6–10) Unlike the response that occurs to intermittent PTH that stimulates bone resorption, (4, 6–10) intermittent injections of PTHrP are not associated with activation of osteoclastic bone resorption or the development of hypercalcemia. (21, 22) Recently, Miao et al. have shown that PTHrP is produced by osteoblasts and other bone cells and is required for maintenance of adult skeletal mass: conditional deletion of PTHrP in osteoblasts(23) and haploinsufficiency(24) of PTHrP in mice results in osteoporosis.

Thus, the explanation for the differences in 1,25(OH)2D metabolism and osteoblast activities in HHM compared with HPT remain elusive despite almost 20 years of study. We have previously reported that continuous infusions of human PTH {hPTH(1-34)} and human PTHrP {PTHrP(1-36)} at a single dose of 8 pmol/kg/h over 46 h were identical in their effects on serum phosphorus, their renal phosphaturic response, and in stimulating renal tubular calcium reabsorption. (25, 26) However, PTH and PTHrP differed mildly but statistically significantly in their effects on serum calcium and 1,25(OH)2D: PTHrP appeared statistically less calcemic, but these differences were quantitatively small. PTHrP produced markedly lower increments in 1,25(OH)2D concentrations, and these differences were also statistically significant.

With respect to bone turnover, several investigators have examined the effects of a continuous infusion of PTH over a few hours for up to 18 h. In these studies, markers of bone resorption increase beginning at 5–12 h. (27–31) Surprisingly, despite the well-documented stimulatory effect of PTH on bone formation in HPT, markers of bone formation, such as osteocalcin and alkaline phosphatase, decline in every study of chronic PTH infusion. (27–31) We had interpreted this to mean that there must be a temporal lag in the anabolic response to PTH but that bone formation markers would eventually increase over time, as occurs in HPT. Data exploring the effects of PTHrP on bone turnover in humans are very limited. Fraher et al. (31) reported that infusion of PTHrP(1-34) for 12 h led to an increase in bone resorption as measured using hydroxyproline. In that same study, osteocalcin was measured as a marker of bone formation. As has been observed with PTH infusions, osteocalcin concentrations in blood declined over 12 h of PTHrP(1-34) infusion. (31)

Thus, the goals of this study were 3-fold. First, we wanted to unequivocally confirm or refute our earlier observations suggesting that PTH may be more potent than PTHrP in inducing hypercalcemia and in stimulating 1,25(OH)2D in humans using a robust dose-response protocol. Second, we wanted to determine how long the anticipated anabolic response to PTH would require to appear using a novel, multiday infusion protocol. Third, we wanted to explore the potency and time-course of the skeletal anabolic and resorptive effects of PTHrP and to directly compare these with those of PTH.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Peptide production and characterization

hPTHrP(1-36) and hPTH(1-34) were prepared and characterized as described in detail previously. (26, 32, 33) These peptides are referred to hereafter as “PTH” and “PTHrP.” Briefly, peptides were prepared using solid phase synthesis, purity documented by analytical reverse-phase HPLC, and peptide structure and content confirmed using MALDI-TOF mass spectrometry and amino acid analysis. Peptides were sterilely aliquoted into vials and lyophilized, and peptide content was confirmed using amino acid analysis. Bioactivity was examined using the SaOS-2 adenylyl cyclase assay described in detail previously. (34) Pyrogenicity and sterility were assayed using standard techniques. (26, 32, 33) Use of the peptides was approved by the Food and Drug Administration under Investigational New Drugs (INDs) 49175 and 60979.

Study subjects

Sixty-one healthy young adult volunteers between the ages of 24 and 35 years were studied. All were in excellent health, and none were on medications other than oral contraceptives. Of these subjects, 55 were studied using a 2-day PTH or PTHrP protocol, and 6 were studied using a 4-day protocol. The demographics of the subjects in the 2-day protocol are shown in Table 1. For the 2-day protocol, 14 subjects who received PTH or PTHrP at a single dose, 8 pmol/kg/h, have been reported previously(26) and are included for comparison with the 39 new subjects receiving the same (n = 2 at 8 pmol/kg/h PTH) and higher doses of PTH and PTHrP. Demographics for the 4-day study subjects were similar (ages 24–34 years; mean, 26.7 years; 2 males, 4 females). All studies were approved by the University of Pittsburgh Institutional Review Board, and all subjects provided informed written consent.

Table Table 1.. Study Subjects Demographics
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Study design

Subjects were studied between February 2001 and March 2003. They were admitted to the Inpatient General Clinical Research Center at the University of Pittsburgh School of Medicine between 7:00 a.m. and 8:00 a.m. on the first day of the study. Subjects were asked to fast from midnight on the evening before admission until 4:00 p.m. on the day of the study, except for water. Meals (400 mg calcium, 800 mg phosphorus, 2 g sodium, 15% fat, 50% carbohydrate, and 35% protein/day) were provided at 5:00 p.m. on day 1 of the study, 7:00 a.m., 12:00 p.m., and 5:00 p.m. on day 2 of the study, and 7:00 a.m. on the third day after completion of the study. Baseline vital signs, serum chemistries, and hematology studies were performed, and two intravenous lines were inserted in opposite arms: one for continuous infusion of peptide and the other for blood sampling. Beginning between 8:00 a.m. and 9:00 a.m., subjects were infused continuously with either PTH or PTHrP for 46 h at a rate of ∼2-4 ml/h. (25, 26, 33, 35) Before infusion, peptides were resuspended in sterile bacteriostatic saline and mixed with 1 ml of the study subject's whole blood and sterile saline, as described previously. (25, 26, 33, 35) We have shown that PTHrP is stable under these conditions. (35) Subjects were assigned in a matched fashion (Table 1) to receive progressively escalating doses of PTH or PTHrP at 12, 16, 22, 28… pmol/kg/h in groups of five until a serum calcium of 12.0 mg/dl, a predetermined safety cut-off, occurred. Ten subjects were studied at the highest tolerable dose. In the case of PTH, three of four subjects receiving the 16 pmol/kg/h dose achieved a serum calcium value >12.0 mg/dl at or after 29 h, and their infusions were accordingly discontinued. Ten subjects were therefore studied at the next lower dose of PTH, 12 pmol/kg/h. In the case of PTHrP, 5 subjects were studied at 12, 16, and 22 pmol/kg/h and 10 subjects at 28 pmol/kg/h. In the 28 pmol/kg/h group, 4 of 10 developed a serum calcium value >12.0 mg/dl at or after 29 h, and their infusions were therefore terminated, and the dose escalation was discontinued. Blood and urine were obtained at the time-points shown in the figures for serum and urine chemistries. Blood draws ranged between 4 and 28 ml and totaled ∼390 ml over the 2 days of the study. Whereas all time-points for all subjects were analyzed for calcium, phosphorus, and creatinine, because of limited sample availability and cost considerations, only selected samples were analyzed for PTH(1-84), vitamin D, and bone markers.

In addition to these 55 subjects, an additional 6 subjects were infused with PTHrP(1-36) at 8 pmol/kg/h for 96 h in a previous pilot study and had archival samples available for this study.

Plasma and urine assays and calculations

Serum total calcium, ionized calcium, phosphorus, creatinine, and urine calcium creatinine and phosphorus were measured using standard automated chemistries in the University of Pittsburgh Medical Center Clinical Chemistry Laboratory. The fractional excretion of calcium and tubular maximum for phosphorus were calculated as described previously. (25, 26, 33, 35) Plasma 1,25(OH)2D was measured using a previously described assay. (36) PTH(1-84) was measured by ICMA (Quest Laboratories, San Juan Capistrano, CA, USA). hPTH(1-34) was measured using a kit from Immutopics (Minneapolis, MN, USA). The PTH(1-34) two-site assay recognizes both endogenous PTH(1-84) and infused PTH(1-34) and thus cannot reliably be used as an exclusive measure of infused PTH(1-34). Therefore, to approximate an estimate of “infused” PTH(1-34) concentrations achieved during PTH(1-34) infusions {i.e., PTH(1-34) infused without interference from endogenous PTH(1-84)}, the value from the PTH(1-84) assay was subtracted from this value to yield “net” or “free” PTH(1-34), as detailed in Fig. 4. PTHrP(1-36) was measured on protease-protected plasma samples using a two-site immunoradiometric assay as described previously. (37) Plasma osteocalcin was measured by radioimmunoassay (RIA) as described previously. (38) The amino-terminal telopeptides of procollagen 1 (PINP), the serum amino-terminal telopeptide of collagen-1 (sNTx), and carboxy-terminal telopeptide of collagen 1 (CTx) were measured using commercial kits from Orion Diagnostics RIA (Espoo, Finland), Osteomark ELISA (Ostex International, Seattle, WA, USA), and Crosslaps ELISA (Nordic Bioscience Diagnostics, Herlev, Denmark), respectively. IGF-1 was assayed using a double antibody technique after extraction of insulin-like growth factor binding proteins (IGFBPs; ALPCO, Windham, NH, USA) as previously published. (39)

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Figure FIG. 4.. Immunoassay of PTH(1-84), PTH(1-34), and PTHrP(1-36) in plasma at time 0 and at 35 h. To determine steady-state circulating concentrations of PTHrP during the infusions, PTH and PTHrP were measured at the 0- and 35-h time-points. Endogenous PTH(1-84) (stippled bars), measured using the Quest two-site immunochemiluminometric assay (ICMA), decreased after infusion of both PTH and PTHrP from ∼4 to 0.4 pM. hPTH(1-34) (hatched bars), measured using the Immutopics two-site ELISA, reflects total PTH(1-34) immunoreactivity, including components of endogenous, intact PTH(1-84); endogenously generated amino-terminal PTH fragments; and exogenously infused PTH(1-34). To develop an approximate idea as to the concentrations of PTH(1-34) resulting from the PTH(1-34) infusion, we subtracted the “endogenous PTH(1-84)” from “total PTH(1-34) immunoreactivity” to yield “net” or “infused PTH(1-34)” (open white bars). PTHrP (black bars) was measured using a two-site immunoradiometric assay on samples collected in protease inhibitor-containing tubes. (37, 61) We have previously shown that PTHrP(1-36) achieves steady-state levels within 1 h. (35) PTHrP(1-36) values, shown as the black bars, were increased over baseline in all groups and increased in a dose-related manner. In contrast, whether assessed as PTH(1-84), as “total” PTH(1-34), or as “net” PTH(1-34), values were lower than the PTHrP values in PTHrP-infused subjects. These studies make it clear that no matter how one approximates PTH concentrations, PTHrP concentrations are always higher in PTHrP-infused subjects, and the relative failure of PTHrP to stimulate 1,25(OH)2D is not likely caused by lower concentrations of PTHrP than PTH achieved during the infusion. (Inset) Adenylyl cyclase activity in human SaOS-2 cells in response to PTH and PTHrP. To confirm that the PTH and PTHrP preparations were fully active, they were directly compared in vitro for bioactivity using a standard osteoblast-like cell bioassay. As seen, the peptides were comparable in potency in vitro, with PTHrP being slightly more active than PTH. These findings, together with the structural data, indicate that the differences in 1,25(OH)2D between PTH and PTHrP cannot be the result of biologically inactive PTHrP peptide.20

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Statistics

Descriptive statistics were computed to describe the sample in terms of demographic and clinical characteristics. Frequency counts and percentages were computed for categorical variables (e.g., sex), and means and SDs were calculated to summarize measures of central tendency and dispersion, respectively, for continuous variables (e.g., weight). Although frequency matched on key characteristics, groups were compared on baseline characteristics using contingency table analysis with χ2 test statistics for categorical descriptors and using factorial ANOVA on continuous characteristics to identify possible covariates for the analysis of endpoint data. Endpoints considered for efficacy analyses included the observed values of serum calcium, ionized calcium, fractional excretion of calcium (FECa), serum phosphorous, tubular maximum for phosphorus (TmP/GFR), circulating concentrations of 1,25(OH)2D, and the percentage change relative to baseline values for the biomarkers of CTx, NTx, P1NP, serum osteocalcin, and IGF-1. Circulating plasma levels of peptide were also examined. Because some subjects had their peptide infusions discontinued because of reaching the predetermined safety cut-off of 12.0 mg/dl for serum calcium, the analysis of endpoints included repeated measurements up to 29 h for rapid turnover parameters such as serum calcium and 35 h for longer turnover parameters such as 1,25(OH)2D and bone turnover markers. To compare response profiles on endpoints between peptide groups among doses over time, repeated-measures analysis using mixed effects modeling was used using SAS PROC MIXED (version 8.2; SAS Institute, Cary, NC, USA). If significant differences were found between peptide and/or dose groups over time, groups were compared using linear contrasts at individual time-points. If only significant time effects were observed, linear contrasts of mean changes relative to baseline for subsequent measurements were examined. Data from the 96-h study of PTHrP at 8 pmol/kg/h were also analyzed using mixed effects modeling methods to examine the change in serum levels of calcium, CTx, and PINP over time. For these analyses, observed values were used for the analysis of serum calcium, and percentage change values relative to baseline values were analyzed for serum CTx and PINP. The level of significance was set at 0.05 for nondirectional hypothesis testing.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The effect of the multiple doses of PTH and PTHrP on serum calcium are shown in Fig. 1. Note that the data for the 8 pmol/kg/h doses of PTH and PTHrP have been reported previously. (26) As is clear from Fig. 1, both PTH and PTHrP induced quantitatively and statistically significant increases in serum calcium compared with baseline. As is also apparent, PTH consistently yielded greater increases in serum calcium than PTHrP. This is apparent from the observations that the PTH group as a whole was statistically significantly more hypercalcemic (p = 0.0016) than the PTHrP group as a whole, that more subjects receiving PTH at the highest dose (3 of 4 for PTH versus 4 of 10 for PTHrP) required termination of their infusions because of crossing the serum calcium safety threshold of 12 mg/dl, and because the maximally tolerated dose was lower for PTH (12 pmol/kg/h) than for PTHrP (28 pmol/kg/h). These findings indicate that PTH is clearly more potent in causing hypercalcemia than PTHrP. Importantly, the differences in serum calcium between the PTH and PTHrP groups was most apparent on day 2, a result that takes on significance with respect to the 1,25(OH)2D data below. The results in ionized serum calcium (data not shown) mirror those for total serum calcium.

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Figure FIG. 1.. Serum calcium in subjects receiving hPTH(1-34) or hPTHrP(1-36). (Left) PTH or PTHrP was infused continuously in the doses indicated for 46 h, and serum calcium was measured at the times indicated. The doses of PTH and PTHrP used, and the symbols used for each, are indicated in the two insets. The arrow indicates the time (∼29 h) at which the infusions were terminated in subjects in the highest does of PTH or PTHrP as a result of reaching or surpassing the predetermined safety limit (serum calcium = 12.0 mg/dl). Representative SE bars are shown for one PTH and one PTHrP group. The subjects receiving PTH and PTHrP at 8 pmol/kg/h have been reported previously(26) and are included in Figs. 1–3 for comparison. There was a statistically significant increase in serum calcium in both the PTH and PTHrP groups over time compared with baseline (p > 0.0001), and there was a statistically significant difference between the PTH group as a whole compared with the PTHrP group (p = 0.0016), but there was no difference within the PTH or PTHrP groups. Note that there was little difference between the PTH and PTHrP groups during the first 24 h and that the differences emerged on the second day of the study. (Right) Endogenous PTH(1-84). Induction of hypercalcemia by PTH or PTHrP resulted in suppression of secretion of endogenous PTH(1-84).20

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Figure 1 also shows the effects of the increase in serum calcium resulting from PTH or PTHrP infusion on circulating concentrations of endogenous PTH(1-84). PTH(1-84) was measured on those receiving the highest and lowest doses of PTH and PTHrP. As expected, endogenous PTH was rapidly and nearly completely suppressed by the infusion of both peptides.

Figure 2 shows the effects of the two peptides on serum phosphorus and the tubular maximum for phosphorus (TmP/GFR) in the eight groups of subjects. Meals are indicated by asterisks. Both peptides induced an early and statistically significant decline in serum phosphorus and TmP/GFR. This was transiently interrupted by the first meal in the study. There were no differences between PTH or PTHrP at any dose, nor were there differences between individual doses of PTH or PTHrP. The latter finding suggests that the inhibition of phosphate reabsorption is already maximal at the lowest dose of either peptide, 8 pmol/kg/h, and cannot be inhibited further.

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Figure FIG. 2.. (Left) Serum phosphorus and (right) tubular maximum for phosphorus (TmP/GFR). See Fig. 1 for details of symbols, error bars, and study groups. *Times of meals. Infusion of PTH or PTHrP results in a statistically significant decline from baseline in serum phosphorus and TmP/GFR (p < 0.0001). There was no difference between the PTH or PTHrP groups in the serum phosphorus or TmP/GFR response. Note that there was an increase in serum phosphorus after the first meal late on day 1 of the study.20

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The fractional calcium excretion (FECa) is shown in Fig. 3. As can be seen, there is a statistically significant increase in FECa over the course of the study, indistinguishable between the PTH and PTHrP groups or within each peptide group. More importantly, FECa in both the PTH and PTHrP groups was markedly lower than the 6.5% FECa value we observed earlier in subjects receiving a “calcium clamp” in which serum calcium was “clamped” at 10.3 mg/dl, the same serum calcium achieved in that study as well as this study at 14–24 h. (25) These findings collectively indicate that PTH and PTHrP are potent and equivalent anticalciuric agents in humans. Moreover, they indicate that, as with the phosphaturic response, both peptides achieve maximal stimulation of calcium reabsorption at the lowest doses of PTHrP and PTH tested.

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Figure FIG. 3.. (Left) Fractional calcium excretion (FECa). See Fig. 1 for details of symbols, error bars, and study groups. FECa in the subjects receiving PTH and PTHrP is compared with that detailed previously (gray bar) in a group of nine normal subjects who received a “calcium clamp” in which the calcium was raised to 10.3 mg/dl for 8 h, leading to suppression of endogenous PTH(1-84). (25) Note that both PTH and PTHrP lead to significant (p < 0.001) increases in FECa as the serum calcium and filtered load of calcium increase but that there is no difference between the PTH and PTHrP groups. Importantly, both groups display FECa values that are ∼50% (p = 0.0024) of that which occurs in normal individuals “calcium clamped” to a serum calcium of 10.3 mg/dl who have similar calcium filtered loads. (25) These findings indicate that PTH and PTHrP stimulate renal tubular calcium reabsorption to a comparable degree. (Right) Plasma 1,25(OH)2D. Note that the 8 pmol/kg/h data have been reported previously(26) and are included for comparison. Plasma 1,25(OH)2D values rose in a dose-related fashion in subjects receiving PTH, and there was a significant increase in the subjects receiving the all three doses of PTH compared with those receiving PTHrP (p < 0.05). In contrast, there was a relatively modest increase in plasma 1,25(OH)2D in the subjects receiving the two lower doses of PTHrP. The 1,25(OH)2D elevations were comparable in the subjects receiving the middle PTH dose, 12 pmol/kg/h, and the highest PTHrP dose, 28 pmol/kg/h. These findings indicate that PTHrP is a significantly weaker agonist than PTH for renal 1,25(OH)2D in the human kidney.20

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Figure 3 also shows the effects of the two peptides on circulating concentrations of 1,25(OH)2D. In contrast to their quantitatively similar or identical effects on serum calcium, TmP and FECa, PTH and PTHrP display very different profiles with respect to 1,25(OH)2D (p < 0.05). The 8 pmol/kg doses of PTH and PTHrP reported previously(26) are included here for reference. As is apparent from Fig. 3, escalating doses of PTH produced progressively higher concentrations of 1,25(OH)2D. In contrast, the two lowest doses of PTHrP produced almost no increment in 1,25(OH)2D, and the highest dose, 28 pmol/kg/h, whereas statistically significant compared with baseline (p = 0.003), produced a smaller response than the 16 pmol/kg/h dose of PTH. Perhaps most illustrative, whereas the 12 pmol/kg/h dose of PTH produced a robust 1,25(OH)2D response, the identical dose of PTHrP produced a strikingly smaller response (p < 0.05). These observations make it clear that PTH and PTHrP have very different actions on 1,25(OH)2D metabolism in the human kidney.

One explanation for the apparent weaker effect of PTHrP on 1,25(OH)2D could be that the PTHrP preparation was not fully potent. To address this concern, we compared the batches of PTH and PTHrP used in this study in the SaOS-2 osteosarcoma adenylyl cyclase bioassay. As shown in Fig. 4, inset, PTHrP was fully potent and indeed appeared to be slightly more potent than PTH in this bioassay.

Another possibility for the lower apparent efficacy of PTHrP could be that PTHrP may be more subject to degradation than PTH, either during the infusion process or after entry into the circulation. To address this possibility, we measured circulating PTH and PTHrP levels in subjects at baseline and 35 h of the infusion. We previously showed that PTHrP reaches steady state by 60 minutes, (35) so both PTH and PTHrP would have achieved steady-state concentrations and have been stable for many hours by this time-point. As seen in Fig. 4, PTHrP levels gradually increase as the dose of PTHrP was increased, and PTHrP concentrations were higher at every PTHrP dose than those of PTH. Thus, the apparent weaker effect of PTHrP than PTH on 1,25(OH)2D cannot likely be explained by lower bioactivity of PTHrP or lower circulating concentrations or PTHrP.

The effects of PTH and PTHrP on bone resorption have not been compared previously. To study their potency as resorptive agents when administered continuously, we measured CTx and the serum levels of NTx on selected groups of subjects during the study. As anticipated, both peptides induced rapid activation of bone resorption as assessed using CTx (Fig. 5), and the effects were comparable for PTH and PTHrP. As for TmP and FECa, there was no apparent dose response for either peptide, suggesting that bone resorption may have been maximally activated at the lowest doses used. Similar results were observed for serum NTx (data not shown), where NTx increased for both peptides (p < 0.0001), but no difference was observed between the two peptides.

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Figure FIG. 5.. (Left) Plasma concentrations of the bone resorption marker, CTx. See Fig. 1 for details of symbols, error bars, and study groups. The mean baseline concentration in all subjects was 0.82 ± 0.07 (SE) ng/ml. Bone resorption, as measured by plasma CTX, increased early in the infusion as has been reported by others during briefer studies. (26–30) There was no difference between the PTH and PTHrP groups, and there was no apparent dose response. (Right) Plasma PINP concentrations. The mean baseline concentration in all subjects was 54.3 ± 4.6 (SE) μg/liter. PINP, a marker of bone formation, progressively declines throughout the 48 h of the study, indicating that bone formation progressively declines over 48 h in response to continuous infusion of either PTH or PTHrP. There was no difference between PTH and PTHrP and no dose response within the two groups.20

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Although HPT is associated with increases in bone formation in humans, in this study, bone formation or osteoblast activity was markedly suppressed by both PTH and PTHrP. This was best shown by measurement of PINP in serum (Fig. 5). Both PTH and PTHrP caused rapid and sustained (48 h) suppression of bone formation as assessed by PINP. There was no dose response for either peptide and no difference between PTH and PTHrP.

Circulating osteocalcin is widely used as a marker of bone formation. Figure 6 shows the biphasic effects of PTH and PTHrP on osteocalcin in this study. As reported previously in studies lasting 6–18 h, (27–31) PTH caused a rapid decline in osteocalcin at 14 h. PTHrP, as also reported previously in a 12-h study, (31) caused a comparable decline in osteocalcin at the 14-h time-point. Both decrements were statistically significant, and there was no difference between the PTH and PTHrP groups. Osteocalcin began to increase after 14 h and rose in a statistically significant and quantitatively comparable manner for both peptides for the remainder of the 48 h.

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Figure FIG. 6.. (Left) Plasma osteocalcin concentrations. See Fig. 1 for details of symbols, error bars, and study groups. The mean baseline concentration in all subjects was 7.47 ± 0.46 ng/ml. As reported previously, (27–31) osteocalcin concentrations declined during the initial 14 h of the infusion, interpreted to indicate an initial decline in bone formation. Osteocalcin concentrations increased over the ensuing 34 h, consistent either with activation of bone formation or bone resorption (Fig. 5). The PINP data in Fig. 5 make the latter interpretation more likely. (Right) Plasma IGF-1 concentrations in response to PTH and PTHrP. PTH and PTHrP produced rapid and equivalent increases in circulating IGF-1.20

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Studies in vitro and in a variety of mouse genetic models suggest that IGF-1 may mediate some or most of the anabolic actions of PTH and PTHrP on osteoblasts. (40, 41) Surprisingly, no data have been reported describing IGF-1 responses to either PTH or PTHrP in humans. As shown in Fig. 6, both PTH and PTHrP induced rapid and marked increments in IGF-1. Again, there was no apparent dose response to either peptide, and the two peptides induced comparable responses.

We hypothesized that more prolonged infusions of PTH or PTHrP might ultimately stimulate bone formation, as occurs in humans with HPT. To determine when this might occur, we examined plasma CTx and PINP concentrations in a cohort of six subjects who were continuously infused with PTHrP for 4 days at a dose of 8 pmol/kg/h. As shown in Fig. 7, 4 days of continuous infusion of PTHrP at this dose caused mild hypercalcemia in the 10.0-11.0 mg/dl range and stimulated bone resorption for the 4 days of the study. As seen in the 2-day studies described in Fig. 5, an additional 2 days of continuous PTHrP infusion yielded continued suppression of bone formation as assessed using PINP.

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Figure FIG. 7.. Serum calcium, plasma CTx, and plasma PINP in response to a continuous 4-day infusion of 8 pmol/kg/h PTHrP. Error bars indicate SE. Note that even after 4 days of infusion, bone formation remained suppressed in response to PTHrP.20

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

To our knowledge, this is the first study to compare multiple doses of PTH in healthy human volunteers over a multiday period, the first to examine multiple doses of PTHrP over a multiday period, and the first to compare these two peptides in a direct fashion in multiple doses on 1,25(OH)2D over a multiday period. It is also the first to examine the effects of multiday infusions of either peptide on skeletal turnover in humans, and the first to report the effects of either PTH or PTHrP on IGF-1 production in humans. The findings include a number of surprising observations, among them a striking differential in potency as agonists for 1,25(OH)2D production in humans, a prolonged dissociation of bone formation and resorption in response to both peptides in humans, and a surprisingly prolonged suppression of markers of bone formation by both peptides. These results require that the physiology and pathophysiology of the effects of PTH and PTHrP on the skeleton and the kidney in HPT and HHM be re-evaluated.

One important observation is that PTH and PTHrP seem to differentially regulate 1,25(OH)2D synthesis in the human kidney (Fig. 3). These differences cannot be explained by differences in potency of the two synthetic peptides, because the PTHrP preparation used was fully active in vitro using a standard adenylyl cyclase assay. Moreover, in prior studies using this human model, we reported that hPTH(1-34) and hPTHrP(1-36) cause indistinguishable responses in nephrogenous cAMP excretion. (33) The differences cannot be caused by failure of delivery of hPTHrP into the circulation, for again, we have reported identical nephrogenous cAMP responses in humans using these techniques in the past. (33) Moreover, in this study, using modern hPTH(1-34) and hPTHrP(1-36) two-site assays, we showed that PTHrP concentrations were at least as high in the PTHrP-infused subjects as PTH in the PTH-infused subjects.

Thus, the discordant 1,25(OH)2D responses are surprising. PTH(1-34) and PTHrP(1-36) have been clearly documented to bind to the PTH-1 receptor with equivalent affinity and have long been believed to signal through intracellular signaling pathways with equivalent potency. (42) Moreover, in rodent studies, PTH and PTHrP both produce similar increments in circulating 1,25(OH)2D. (18–20) On the other hand, human tumors obtained from patients with HHM in which 1,25(OH)2D is suppressed lead to paradoxical increases in 1,25(OH)2D when transferred to immunodeficient rats or mice. (18–20) These observations collectively suggest two important interpretations. First, whereas hPTH(1-34) and hPTHrP(1-36) activate adenylyl cyclase, intracellular calcium, and phospholipase pathways with equivalent potency, the precise pathways that link the PTH-1 receptor to mitochondrial 1-α-hydroxylase in the human kidney are unknown. These observations suggest that additional signaling pathways may be important in 1-α-hydroxylase regulation and may be subject to differential regulation by PTH and PTHrP. For example, recent studies have shown that MAP kinase, PI3 kinase, phospholipase D (PLD), wnt/low-density lipoprotein-related protein (LRP), and other pathways are downstream of the PTH-1 receptor in a number of cells and tissues. (43–46) To our knowledge, however, a direct comparison of the efficacy of PTH and PTHrP in activating these pathways in renal tubular cells has not been reported. In addition, differential regulation of the PTH-1 receptor has been documented to occur through variations in the local PTH-1 receptor microenvironment. For example, the sodium-hydrogen exchange regulatory proteins (NHERF) may be present in cell type-specific patterns and may result in cell type-specific signaling by the PTH-1 receptor. (47) Second, the observation that PTHrP appears to be a weaker agonist of 1-α-hydroxylase in humans stands in contrast to rodent data and underscores the importance of studying 1,25(OH)2D regulation in human, compared with rodent, renal tubular cells.

These studies are consistent with, but do not fully explain, the longstanding observation that 1,25(OH)2D concentrations are reduced in humans with HHM, in contrast to the elevations in 1,25(OH)2D observed in patients with HPT. (13, 14, 16) Thus, the increase in 1,25(OH)2D in response to PTH is consonant with events in HPT. In contrast, 1,25(OH)2D concentrations are actually reduced in HHM, a finding not observed in this study. We hypothesize that more prolonged exposure of the kidney to PTHrP, as occurs in HHM, may lead to the ultimate desensitization or downregulation of the PTHrP response, with an ultimate decline in 1,25(OH)2D.

The discordant calcemic response to the two peptides was unanticipated. The similarly discordant 1,25(OH)2D concentrations are likely the explanation for the more marked hypercalcemia in the PTH-infused subjects. This interpretation is supported by (1) the observation that neither bone resorption nor renal calcium handling differed between the groups, leaving differential intestinal calcium absorption, directed by differential 1,25(OH)2D concentrations, as the most likely explanation; and (2) the concordant temporal profiles of the two events—serum calcium only differed between the two groups on day 2 of the study and 1,25(OH)2D, also, was only different between the groups in the second day.

In contrast to the differences observed in 1,25(OH)2D metabolism, there were no differences between the peptides on fractional calcium excretion or TmP/GFR. These findings are consistent with the well-documented phosphaturic and the anticalciuric effects of PTH in HPT(1–3, 26) and of PTHrP in HHM. (26, 48) The enhanced tubular reabsorption of calcium supports a renal tubular anticalciuric role for PTHrP in the physiology of lactation(49) and in the pathophysiology of HHM. (11, 12, 26)

A second surprising observation in this study was that, whereas bone resorption promptly increased in response to both PTH and PTHrP, as anticipated from the reports of others using PTH for 5–18 h, (27–31) markers of bone formation rapidly declined and remained suppressed for as long as 4 days. Whereas a number of studies have documented that markers of bone formation such as osteocalcin decline during the initial few hours of a PTH or PTHrP infusion, (27–31) one might have reasonably anticipated that they would increase over time, as occurs with chronic exposure to PTH in HPT. Thus, it is surprising that a 2-day continuous infusion of PTH and 4-day continuous infusion of PTHrP mimic the equally profound suppression of bone formation observed in HHM and contrast markedly with skeletal events observed in HPT. These observations prompt a number of important questions. For example, “How long might it take for continuous infusion of PTH to stimulate bone formation in humans?” The answer to this is certainly unknown. “Will continuous infusion of PTH ever stimulate bone formation in humans?” Also, given these observations, “Is the pulsatile secretion of PTH, or its diurnal variation, reported in normal individuals and those with HPT, (50–53) required to elicit a skeletal anabolic response?” Because bone formation and bone resorption are completely uncoupled in this study, closely resembling the uncoupled skeletal histology in HHM, “Is continuous (i.e., nonpulsatile) secretion of PTHrP by HHM-associated tumors, tumors we have shown to be incapable of pulsatile secretion of PTHrP, (53) the explanation for the marked suppression of bone formation, observed in HHM?” The answers will require longer-term infusion studies with PTH and PTHrP.

The biphasic pattern of the osteocalcin response was unexpected. Osteocalcin peptides are secreted by osteoblasts during bone formation and released by osteoclasts as they resorb bone. Thus, the initial decline in osteocalcin observed in this study, as well as by Fraher et al., Cosman et al., Leder et al., and others, (27–31) presumably reflects a generalized suppression of bone formation and complements the PINP results at these early time-points. In contrast, the reversal of osteocalcin concentrations after 14 h contrasts with the continued and progressive suppression in formation as measured by PINP but parallels the increase in circulating CTx, a marker of bone resorption. We interpret this to indicate that, at the early time-points, osteocalcin is an accurate measure of bone formation, but at later time-points, more accurately reflects bone resorption. More broadly, these findings suggest that osteocalcin, which is widely used as a marker of bone formation, may well serve this purpose in steady-state conditions of high bone turnover or when bone resorption is not present. On the other hand, it's fidelity as a marker of bone formation under nonsteady states is open to question.

Another important question prompted by these studies relates to the teleology of the suppressed bone formation response to PTH and PTHrP. Whereas the acute and presumed transient suppression by PTH of bone formation markers in vivo(27–31) and bone differentiation markers in osteoblasts models in vitro(54–57) has been apparent for years, the physiology underlying this observations is obscure. One might hypothesize that acute suppression of formation by PTH may serve to accentuate the net resorptive response to PTH in settings of acute hypocalcemia. This seems an unsatisfactory explanation, however, because suppression of osteoblast activity would have no immediate effect on the ongoing process of osteoid mineralization in vivo. Some have suggested that dedifferentiation of osteoblasts is a necessary prerequisite for their proliferative response to PTH. This may explain events in cultured osteoblasts but is not a likely explanation for the rapid and sustained decline in formation markers in vivo, because not all, or even most, osteoblasts display a proliferative response to PTH in vivo. (50–52) A third possibility relates to the potential requirement for normal pulsatile secretion of PTH to elicit a formation and osteoblast differentiation response. Again, these questions can be addressed in future studies using variations on the techniques described herein.

The bone formation in response to PTH and PTHrP is believed to be mediated, at least in part, by skeletal IGF-1. The evidence for this comes from studies using isolated osteoblasts(58, 59) and in PTH treatment in mouse genetic models deficient for IGF-1 or its receptor. (40, 41) Despite this well-documented requirement in vitro and in rodents, circulating IGF-1 has never been examined in humans infused with PTH or PTHrP. Here, we showed that both PTH and PTHrP induce elevations in circulating IGF-1 in humans. Although IGFBP3 was not measured, given the rapidity of the increase in IGF-1, an acute increase in IGFBP-3 seems unlikely to be the cause of the increase in IGF-1. The increase in IGF-1 is significant for several reasons. First, the increases are rapid. Second, the increments in IGF-1 are quantitatively large and comparable with the increases observed in children and adults treated with growth hormone. (60) Third, because osteoblastic bone formation depends on, and is stimulated by, IGF-1, it is surprising that bone formation was suppressed rather than stimulated. These studies do not identify the source of IGF-1 in plasma, but the liver seems a reasonable candidate: hepatocytes have receptors for PTH/PTHrP, produce IGF-1, and have long been known to display intracellular signaling responses to PTH and PTHrP. Alternately, the osteoblast or other skeletal cells could be a source.

One limitation in these studies is that it used two amino-terminal peptides of PTH and PTHrP. Both peptides are secreted as larger precursors in HPT and HHM, respectively, and both are metabolized to families of smaller peptides. Thus, events observed using infusions of these amino-terminal peptides may not faithfully reflect events in HPT and HHM in all respects. Unfortunately, the physiologic carboxy-terminal peptides of PTH and PTHrP are not well defined, their physiological effects are also imprecisely defined, and infusion of these peptides into humans is therefore untenable. Nonetheless, both amino-terminal peptides are potent agonists of bone formation when administered intermittently in humans, so their failure to do so when administered continuously intravenously is not likely to reflect confounding effects of non-amino-terminal PTH or PTHrP peptides. A second potential weakness is that the circulating concentrations of PTH(1-34) and PTHrP(1-36) achieved during the infusion may not accurately reflect events in HPT and HHM, respectively. For HHM, we reported using a two-site assay with a mean PTHrP concentration of ∼20 pM, values similar to those reported here during the high-dose PTHrP infusion. For HPT, the circulating concentrations of authentic amino-terminal PTH peptides are not well defined.

These studies show that much remains to be learned regarding both normal and pathological control of mineral and skeletal homeostasis in humans in response to PTH and PTHrP. Specifically, these studies, in addition to the large body of evidence indicating that intermittently administered PTH and PTHrP are skeletal anabolic agents, (6–10, 21–24) point to a need to understand more completely the cellular basis of bone formation induced by PTH and PTHrP in humans. Conversely, the uncoupled suppression of bone formation, and the failure to stimulate 1,25(OH)2D, mimic skeletal events in HHM. These studies show that chronic infusion of PTH is not an ideal model for the skeletal events that occur in HPT. Furthermore, they underscore the need to more carefully evaluate the signaling mechanisms that couple the shared PTH/PTHrP receptor to renal 1-α-hydroxylase in the human kidney. Finally, these studies provide a human experimental model in which these questions—longer-term administration and pulsatile versus intermittent administration—can be evaluated in the regulation of coupling of bone formation to resorption and the regulation of 1,25(OH)2D biosynthesis in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank the staff of the University of Pittsburgh GCRC for support of this study, Jim Elliot and Myron Crawford for peptide synthesis and composition analysis, and the staff of the University of Pittsburgh General Clinical Research Center for help with these studies. This study was supported by NIH Grants DK 51081 and RR 00056 and the University of Pittsburgh General Clinical Research Center.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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