Effects of parathyroidectomy on plasma iPTH, (1-84)PTH and (7-84)PTH levels in patients with stage 5 chronic kidney disease

Objective Currently, parathyroid hormone (PTH) is mainly measured by the second generation intact PTH (iPTH) assay which detects both full-length (1-84)PTH and (7-84)PTH fragments. The third generation whole PTH (wPTH) assay however has turned out to be specific for (1-84)PTH. The aim of this study is to investigate the features of plasma iPTH, (1-84)PTH, (7-84)PTH levels in patients with stage 5 chronic kidney disease (CKD), and evaluate the effects of parathyroidectomy (PTX) on above markers in severe secondary hyperparathyroidism (SHPT) patients. Methods A cross-sectional study including 90 controls and 233 stage 5 CKD patients, and a prospective follow-up study in 31 severe SHPT patients were conducted. Plasma iPTH and (1-84)PTH levels were measured by the second and third generation assay, respectively. Circulating (7-84)PTH level was calculated by subtracting the (1-84)PTH value from the iPTH value. Results Plasma levels of iPTH, (1-84)PTH, (7-84)PTH were higher (P＜0.01), and (1-84)PTH/iPTH was lower (P＜0.01) in stage 5 CKD patients than in controls. For severe SHPT patients with PTX (n=74), plasma iPTH, (1-84)PTH and (7-84)PTH concentrations were significantly increased compared to non-PTX group (n=159) (P＜0.01), and the increase of (7-84)PTH level was more striking than (1-84)PTH. Meanwhile, the value of (1-84)PTH/iPTH was decreased (P＜0.01). Plasma iPTH level was strongly correlated with (1-84)PTH level (r=0.980, P＜0.01) in stage 5 CKD patients. Also, both iPTH and (1-84)PTH levels were positively correlated with serum alkaline phosphatase, dialysis vintage and serum phosphorus (P＜0.01). After PTX (median interval of follow-up: 7.1 months), plasma iPTH, (1-84)PTH, (7-84)PTH concentrations were decreased (by 92.9%, 89.7%, 95.8%, P＜0.01, respectively) greatly and (1-84)PTH/iPTH was increased (P＜0.01). Conclusions In stage 5 CKD patients, plasma iPTH, (1-84)PTH, (7-84)PTH levels are greatly increased while (1-84)PTH/iPTH is decreased, and PTX can significantly improve abnormality of above markers in severe SHPT patients. The second generation PTH assay overestimates the severity of SHPT, and the accurate measurement of (1-84)PTH by the third assays is more conducive to diagnosis and treatment of CKD and SHPT patients.     

Enhancement of Lumbar Fusion and Alleviation of Adjacent Segment Disc Degeneration by Intermittent PTH(1‐34) in Ovariectomized Rats

Osteoporosis, which is prevalent in postmenopausal or aged populations, is thought to be a contributing factor to adjacent segment disc degeneration (ASDD), and the incidence and extent of ASDD may be augmented by osteopenia. Parathyroid hormone (PTH) (1‐34) has already been shown to be beneficial in osteoporosis, lumbar fusion and matrix homeostasis of intervertebral discs. However, whether PTH(1‐34) has a reversing or retarding effect on ASDD in osteopenia has not been confirmed. In the present study, we evaluated the effects of intermittent PTH(1‐34) on ASDD in an ovariectomized (OVX) rat model. One hundred 3‐month‐old female Sprague‐Dawley rats underwent L₄–L₅ posterolateral lumbar fusion (PLF) with spinous‐process wire fixation 4 weeks after OVX surgery. Control groups were established accordingly. PTH(1‐34) was intermittently administered immediately after PLF surgery and lasted for 8 weeks using the following groups (n = 20) (V = vehicle): Sham+V, OVX+V, Sham+PLF+V, OVX+PLF+V, OVX+PLF+PTH. The fused segments showed clear evidence of eliminated motion on the fusion‐segment based on manual palpation. Greater new bone formation in histology was observed in PTH‐treated animals compared to the control group. The extent of ASDD was significantly increased by ovariotomy. Intermittent PTH(1‐34) significantly alleviated ASDD by preserving disc height, microvessel density, relative area of vascular buds, endplate thickness and the relative area of endplate calcification. Moreover, protein expression results showed that PTH(1‐34) not only inhibited matrix degradation by decreasing MMP‐13, ADAMTS‐4 and Col‐I, but also promote matrix synthesis by increasing Col‐II and Aggrecan. In conclusion, PTH(1‐34), which effectively improves lumbar fusion and alleviates ASDD in ovariectomized rats, may be a potential candidate to ameliorate the prognosis of lumbar fusion in osteopenia. © 2015 American Society for Bone and Mineral Research.     

Effects of parathyroidectomy on plasma PTH fragments and heart rate variability in stage 5 chronic kidney disease patients

IntroductionCirculating intact parathyroid hormone (iPTH) levels include full-length (1-84) PTH and long C-PTH fragments, but primarily (7-84) PTH, which have been reported to have antagonistic effects on the bones and kidneys. However, their effects on the cardiovascular system remain unclear. In this study, the relationships between the plasma PTH fragments levels and heart rate variability (HRV) in stage 5 chronic kidney disease (CKD5) patients are explored. Furthermore, the effects of parathyroidectomy (PTX) on the above indices are investigated.MethodsIn this cross-sectional study, 164 healthy controls and 354 CKD5 patients, including 208 secondary hyperparathyroidism (SHPT) subgroup with PTX, were enrolled. Circulating (7-84) PTH levels were calculated by subtracting plasma (1-84) PTH levels from iPTH levels. The HRV parameters were measured using a 24-hour Holter.ResultsThe baseline levels of plasma iPTH, (1-84) PTH, and (7-84) PTH in the CKD5 patients were 930.40 (160.65, 1792.50) pg/mL, 448.60 (99.62, 850.45) pg/mL, and 468.20 (54.22, 922.55) pg/mL, respectively. In the CKD5 patients, plasma (1-84) PTH levels were independently correlated with the standard deviation of the normal-to-normal R-R intervals (SDNN) and the standard deviation of the five-minute average of the normal R-R intervals (SDANN). With a median follow up time of 6.50 months after PTX in the SHPT patients (n = 30), improved SDNN and SDANN markers were related with decreased (1-84) PTH levels. Furthermore, an improved SDNN was related with decreased (7-84) PTH levels.ConclusionsThe CKD5 patients’ baseline (1-84) PTH levels were correlated with the SDNN and SDANN. After PTX, an improved SDNN was related with decreased (1-84) PTH and (7-84) PTH levels, while improved SDANN was related with decreased (1-84) PTH levels. No antagonistic effects of (1-84) PTH and (7-84) PTH on HRV were found in the CKD5 patients.     

Specific measurement of PTH (1-84) in various forms of renal osteodystrophy (ROD) as assessed by bone histomorphometry

BACKGROUND: Parathyroid hormone (PTH) measurements serve as a noninvasive, diagnostic tool for the assessment of renal osteodystrophy (ROD). Their value has been questioned following reports indicating that all commercially available intact PTH (I-PTH) assays cross-react with amino terminally truncated PTH fragments. Because these fragments can account for 50% of total PTH, their detection will overestimate the true PTH concentration and may lead to diagnostic inaccuracies. The aim of this study was to evaluate the specific Bio-Intact PTH (1-84) Assay (BI-PTH) in patients with various types of ROD confirmed by bone biopsy. METHODS: Bone biopsies were taken from 132 patients with chronic kidney disease (CKD) stages 3 to 5, and quantitative bone histomorphometry was done. Plasma PTH levels were measured using both the BI-PTH and I-PTH assays on an automated analyzer. RESULTS: Patients with CKD stages 3/4 and low turnover skeletal lesions had BI-PTH values (pg/mL, mean +/- SD) of 35 (+/-34) and I-PTH values of 59 (+/- 63). Corresponding values for BI-PTH and I-PTH in those with high turnover lesions were 141 (+/-60) and 221 (+/-106). Patients with CKD stage 5 and low turnover skeletal lesions had BI-PTH and I-PTH levels of 51 (+/-38) and 90 (+/-60), respectively, whereas the corresponding results for BI-PTH and I-PTH in those with high turnover lesions were 237 (+/-214) and 461 (+/-437). The areas under the receiver operating characteristic (ROC) curves for distinguishing low turnover from high turnover lesions were 0.94 for BI-PTH and 0.91 for I-PTH in CKD stages 3/4 and 0.86 for BI-PTH and 0.85 for I-PTH in CKD stage 5. Among all patients, BI-PTH levels are approximately 50% lower than I-PTH levels, but the results of the two assays are correlated highly (R2 = 0.92). CONCLUSION: Plasma PTH measurements using either the BI-PTH or I-PTH assay effectively identify patients with reduced bone turnover and serve to distinguish this subgroup from those with high turnover lesions of renal bone disease. Both assays provide better diagnostic discrimination for this purpose than calculated values for the ratio of PTH (1-84)/amino terminally truncated PTH fragments.     

Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34)

BACKGROUND: Recombinant human parathyroid hormone (PTH [1-34]; teriparatide) is a new treatment for postmenopausal osteoporosis that can be systemically administered for the primary purpose of increasing bone formation. Because several studies have described the enhancement of fracture-healing and osteointegration in animals after use of PTH, we sought to critically analyze this skeletal effect. METHODS: Two hundred and seventy male Sprague-Dawley rats underwent standard, closed femoral fractures and were divided into three groups that were administered daily subcutaneous injections of 5 or 30 mug/kg of PTH (1-34) or vehicle (control). The dosing was administered for up to thirty-five days. Groups were further divided into three subgroups and were killed on day 21, 35, or 84 after the fracture. The bones were subjected to mechanical torsion testing, histomorphometric analysis, or microquantitative computed tomography. RESULTS: By day 21, calluses from the group treated with 30 mug of PTH showed significant increases over the controls with respect to torsional strength, stiffness, bone mineral content, bone mineral density, and cartilage volume. By day 35, both groups treated with PTH showed significant increases in bone mineral content and density and total osseous tissue volume, and they demonstrated significant decreases in void space and cartilage volume (p < 0.05). Torsional strength was significantly increased at this time-point in the group treated with 30 mug of PTH (p < 0.05). While dosing was discontinued on day 35, analyses performed after eighty-four days in the group treated with 30 mug of PTH showed sustained increases over the controls with respect to torsional strength and bone mineral density. No change was noted in osteoclast density at the time-points measured, suggesting that treatment with PTH enhanced bone formation but did not induce bone resorption. CONCLUSIONS: These data show that daily systemic administration of PTH (1-34) enhances fracture-healing by increasing bone mineral content and density and strength, and it produces a sustained anabolic effect throughout the remodeling phase of fracture-healing.     

Efficacy of whole PTH assay and 1-84/non-(1-84) PTH ratio in patients on hemodialysis or undergoing parathyroidectomy

For measurement of parathyroid hormone (PTH), the intact PTH (I-PTH) assay is generally used. However, I-PTH assay apparently detects PTH 7-84 fragments, in addition to PTH 1-84. The whole PTH (W-PTH) assay exhibits no cross-reactivity with PTH 7-84. Ratio of PTH 1-84 to non-(1-84) PTH has been proposed as a non-invasive indicator of bone turnover in patients with end-stage renal disease (ESRD). We evaluated the efficacy of W-PTH assay and 1-84/non-(1-84) PTH ratio in hemodialysis patients and patients who had undergone parathyroidectomy. PTH levels were measured using W-PTH and I-PTH assays in 138 hemodialysis patients, 27 healthy controls and 10 patients who were scheduled to undergo parathyroidectomy for secondary hyperparathyroidism. Alkaline phosphatase, bone alkaline phosphatase and intact osteocalcin were also measured for comparison with 1-84/non-(1-84) PTH ratio. W-PTH was strongly correlated with I-PTH in both groups, and with all three bone metabolic markers in hemodialysis patients. In hemodialysis patients, both PTH and bone metabolic markers were significantly lower in the subgroup with a PTH ratio < 1.0 than in the subgroup with a PTH ratio > or = 1.0. However, PTH ratio exhibited no significant correlation with bone metabolism markers. PTH ratio was higher after parathyroidectomy than before. W-PTH and I-PTH assays offer identical indicators of bone metabolism in ESRD patients. However, 1-84/non-(1-84) PTH ratio may be of limited diagnostic use regarding bone turnover if a cut-off value of 1.0 is used. PTH 1-84 may be secreted relatively more than non-(1-84) PTH after parathyroidectomy, due to decreases in serum calcium. As the W-PTH assay allows easy calculation of non-(1-84) PTH by subtracting the I-PTH value, this assay contributes to identification of the function of non-(1-84) PTH fragments in various conditions.     

Parathyroid hormone and large related C-terminal fragments increase at different rates with worsening of renal function in chronic kidney disease patients. A possible indicator of bone turnover status?

BACKGROUND: The intact parathyroid hormone (PTH) serum value has been the non-invasive biomarker of choice for the early diagnosis of renal bone disease in the chronic kidney disease (CKD) patient population. It has now been known that the intact PTH assay value is the sum of 1-84 PTH (true hypercalcemic PTH) and large C-terminal PTH fragments, mainly 7-84 PTH, a fragment with hypocalcemic hormone actions. AIM: The aim of this study was to investigate the differences among the different functional stages of CKD in the following PTH parameters: intact PTH, 1-84 PTH, 7-84 PTH, and the ratio 1-84 PTH/7-84 PTH. GFR (clearance of 99mTc-DTPA) was measured in 164 (85 males and 79 females) adult CKD patients with different degrees of renal function impairment (serum creatinine 0.50 12.1 mg/dl, mean 2.00). PATIENTS AND METHODS: Plasma concentrations of calcium, phosphate, 1-84 PTH and intact PTH were also measured. The value of 7-84 PTH was calculated as the difference between intact PTH and 1-84 PTH. The reduction of, GFR was accompanied by an increase of intact PTH, with a prevalent increase of 7-84 PTH over 1-84 PTH, resulting in a decrease of the ratio 1-84 PTH/7-84 PTH. RESULTS: The values of 7-84 PTH showed a discrimination between Stages 1 and 2 (GFR > 60 ml/min ) and Stage 3 (GFR 30 60 ml/ min) CKD patient populations. In fact, 7-84 PTH was already significantly increased in patients at CKD Stage 3. The analysis of individual patients indicated that a low value (< 1.4) of the ratio 1-84 PTH/7-84 PTH, suggestive for low bone turnover, was already found in more than 20% of CKD Stage 3 patients. CONCLUSION: The results of the present study demonstrate that the reduction in GFR is accompanied by a higher increase in 7-84 PTH with respect to 1-84 PTH, which suggests the possibility that bone metabolism and calcemic status are already reduced in patients with moderate renal failure (CKD Stage 3).     

Comparison of Hypotensive Response following Intravenous Injection of Parathyroid Hormone 1-84 and 1-34 in Conscious Rats

Activation of parathyroid hormone 1 (PTH-1) receptors on vascular smooth muscle cells causes relaxation and decreases blood pressure in rats and humans. However, when PTH(1-84) and PTH(1-34) were injected in anesthetized rats, PTH(1-34) produced a greater decrease in blood pressure. This study quantified the dose-response relationship of the hypotensive response to intravenously injected PTH(1-84) and PTH(1-34) in conscious rats and assessed the role that the C-terminal region of PTH(1-84) played in the differences. Mean arterial pressure (MAP) decreased rapidly following injection of both peptides (0–100 nmol/kg) and reached a nadir at 1–2 minutes before increasing at a rate that was dose- and time-dependent. PTH(1-34) produced a greater hypotensive effect than PTH(1-84) at most doses tested and was significantly different from PTH(1-84) at 1–10 nmol/kg. The greatest difference in MAP decrease between PTH(1-84) and PTH(1-34) (24 and 35 mm Hg, respectively) occurred at 10 nmol/kg. Median effective dose (ED₅₀) values for PTH(1–84) and PTH(1-34) were significantly different (5.9 and 1.3 nmol/kg, respectively). The C-terminal PTH fragments PTH(7–84), PTH(39–84), and PTH(53–84) did not affect MAP when injected alone (10 nmol/kg), nor did they influence the hypotensive response when given at a 10–fold molar excess in combination with PTH(1-84) or PTH(1-34) (1.4 nmol/kg). In conclusion, PTH(1-84) is a less potent but, because it induced the same maximum response, not a less efficacious hypotensive agent than PTH(1-34) when administered by bolus intravenous injection in conscious rats. We found no evidence to support the concept that the C-terminal region of PTH is responsible for this difference in potency.     

Effect of combined humanPTH(1-34) and calcitonin treatment in ovariectomized rats

We examined the combined effects of human parathyroid hormone 1-34 (hPTH) and elcatonin (ECT: a synthetic derivative of eel calcitonin) to prevent loss of bone mass, architecture and strength in ovariectomized (OVX) rats. Fifty-four female rats (aged 13 weeks) were assigned to one of nine groups: Sham (fake surgery performed), OVX, ECT (15 U/kg administered), PTH5, PTH10 and PTH20 (5, 10 or 20 microg/kg administered), and E+PTH5, E+PTH10 and E+PTH20 (15 U/kg of ECT and 5, 10 or 20 microg/kg of hPTH administered). The drug or vehicle was subcutaneously administered three times a week for 12 weeks. The femurs were removed at the completion of the experiment. The right distal femoral metaphysis was used for measuring bone mineral density (BMD), analyzing trabecular bone structure by micro-computed tomography (microCT), and conducting the bone strength test, and the left femur was used for histomorphometric analysis. Trabecular bone volume (BV/TV) and other bone mass parameters were greater in the ECT and PTH groups than in the OVX group. The number of nodes (N.Nd/TV) and trabecular number (Tb.N) were significantly greater in the ECT group, and trabecular thickness (Tb.Th) and trabecular bone pattern factor (TBPf) were significantly greater in the PTH group. These results indicate that these drugs preserve the bone architecture by different means. Analysis by means of microCT revealed that BV/TV, Tb.N, fractal D and N.Nd/TV were significantly greater in the E+PTH groups than in the PTH groups at each concentration. Trabecular separation (Tb.Sp) was significantly lower in the E+PTH5 and E+PTH10 groups than in the respective PTH5 and PTH10 groups. When the maximum load was applied in a compression test on the distal femur, the E+PTH groups had higher values than the PTH groups, however, the three point bending strength of the diaphysis of femur in the E+PTH10 and E+PTH20 groups tended to be low compared to those in the PTH10 and PTH20 groups. These results indicate that combination therapy using PTH and ECT preserves the trabecular microarchitecture better than single-drug therapy using ECT or PTH in OVX rats, however, it is necessary to optimize the calcitonin (CT) dosage and administration in order to achieve the optimal combined effect of PTH and CT.     

Effect of endothelin receptor antagonist on parathyroid gland growth, PTH values and cell proliferation in azotemic rats.

BACKGROUND: A variety of stimuli are involved in the pathogenesis of parathyroid gland hyperplasia in renal failure. Recently, it was shown that blocking the signal from the endothelin-1 (ET-1) receptor (ET(A)R/ET(B)R) by a non-selective receptor antagonist, bosentan, reduced parathyroid cell proliferation, parathyroid gland hyperplasia and parathyroid hormone (PTH) levels in normal rats on a calcium deficient diet. Our goal was to determine whether in 5/6 nephrectomized (NPX) rats with developing or established hyperparathyroidism, the endothelin receptor blocker, bosentan, reduced the increase in parathyroid cell proliferation, parathyroid gland hyperplasia and PTH values. METHODS: High (HPD, 1.2%) or normal phosphorus diets (PD) (NPD, 0.6%) were given to 5/6 NPX rats for 15 days (NPX(15)). In each dietary group, one-half the rats were given bosentan (B) i.p. 100 mg/kg/day. The four groups of rats were: (1) NPX(15)-1.2% P; (2) NPX(15)-1.2% P+B; (3) NPX(15)-0.6% P; and (4) NPX(15)-0.6% P+B. In a second study in which hyperparathyroidism was already established in 5/6 NPX rats fed a HPD for 15 days, rats were divided into two groups in which one group was maintained on a HPD and the other group was changed to very low PD (VLPD, <0.05%) for an additional 15 days. In each dietary group, one-half the rats were given bosentan i.p. 100 mg/kg-day. The four groups of rats were: (1) NPX(30)-1.2% P; (2) NPX(30)-1.2% P+B; (3) NPX(30)-0.05% P and (4) NPX(30)-0.05% P+B. Parathyroid cell proliferation was measured by proliferating cell nuclear antigen (PCNA) staining and ET-1 expression by immunohistochemical techniques. RESULTS: In the study of developing hyperparathyroidism, bosentan reduced ET-1 expression in the parathyroid glands of rats on the NPD and HPD (P<0.05). But only in rats on the NPD did bosentan result in a reduced increase in parathyroid gland weight (P<0.05). In the study of established hyperparathyroidism, in which 5/6 NPX rats were given a HPD for 15 days, bosentan started on day 15 reduced (P<0.05) ET-1 expression in rats maintained for 15 additional days on the HPD or the VLPD. On the VLPD, parathyroid gland weight was less (P<0.05) than that in rats on the HPD sacrificed at 15 or 30 days. Bosentan did not reduce parathyroid cell proliferation or parathyroid gland weight in rats maintained on the HPD or further reduce these parameters beyond that obtained with dietary phosphorus restriction. PTH values were lowest in the VLPD group, intermediate in the NPD group, and highest in the HPD group, but in none of the three groups did bosentan decrease PTH values. CONCLUSIONS: In azotemic rats with developing hyperparathyroidism, bosentan resulted in a reduced increase in parathyroid gland weight when dietary phosphorus content was normal. Despite a reduction in ET-1 expression in rats on a HPD with developing or established hyperparathyroidism, bosentan did not reduce the increase in parathyroid cell proliferation, parathyroid gland growth or PTH values. Thus, ET-1 blockade with bosentan did not prevent parathyroid gland growth in the azotemic rat.     

Separate binding sites for intact PTH 1-84 and synthetic PTH 1-34 in canine kidney

Summary The present studies examine the characteristics of parathyroid hormone (PTH) receptor binding in canine basolateral renal cortical membranes using iodinated preparations of intact bovine PTH 1-84 and [Nle⁸, Nle¹⁸, Tyr³⁴] bPTH 1-34 amide. A solid phase lactoperoxidase technique was used to iodinate the bPTH 1-84. The PTH 1-34 analog was iodinated using chloramine T. Both radioligands were purified by reverse phase high pressure liquid chromatography (HPLC). Specific binding of¹²⁵I PTH 1-84 reached equilibrium at 3 hours whereas binding of the¹²⁵I PTH 1-84 analog reached equilibrium at 45 minutes. Excess bPTH 1-84 resulted in complete inhibition of binding of¹²⁵I bPTH 1-84, whereas 22±1.6% of the bound radioligand remained bound in the presence of excess synthetic bPTH 1-34. These data suggested the possibility of a binding site for the carboxy-terminal region of intact PTH, or binding sites selective for intact hormone. Therefore, additional studies were performed with PTH fragments, PTH 28-53, PTH 35-84, and PTH 53-84. In contrast to previous studies in other systems, these fragments did not result in significant displacement of¹²⁵I PTH 1-84. Analysis of binding of¹²⁵I PTH 1-84 and¹²⁵I [Nle⁸, Nle¹⁸, Tyr³⁴] PTH 1-34 amide, using LIGAND, both indicated a single site model with similar affinities. Thus, the data are consistent either with multiple receptors with similar affinities or a second binding site for bPTH 1-84 on the same receptor. In the canine kidney membranes, bPTH 1-84 and synthetic bPTH 1-34 were equipotent in activating adenylate, although the dose-response curve for bPTH 1-84 was shifted slightly to the right (Kact 2 nm for synthetic bPTH 1-34 versus 5 nm for bPTH 1-84). The present studies suggest that there are binding sites in canine kidney which are selective for the intact hormone and support the existence of more than one class of PTH receptors or a second binding site for intact PTH on a single PTH receptor.     

Maintenance of normocalcemia by continuous infusion of the synthetic bovine parathyroid hormone (1–34) in parathyroidectomized rats

Summary This work was conducted to estimate the replacement dose of the synthetic bovine parathyroid hormone [PTH(1–34)] that is required for maintenance of serum calcium (Ca) in parathyroidectomized (PTX) rats. Male rats were PTX and used in this study only if serum Ca was reduced to at least 7 mg/dl. We found that a solution of 2% cysteine, 150 mM NaCl, and 1 mM HCl was superior to 20 mM acetic acid for maintenance of biological activity of PTH (1–34) in situ during the period of hormone infusion studied. The PTH dose—calcemic response relationship was investigated using PTH in doses of 0.6, 1, and 3 U/h. The infusion of 1 U PTH per hour raised Ca to the normal level, whereas rats infused with 0.6 U/h were hypocalcemic and 3 U/h resulted in marked hypercalcemia. To extend this observation we carried out an infusion of 1 U PTH per hour for 14 days. We found that this infusion rate of bovine PTH (1–34) provided a relatively stable level of serum calcium with modest fluctuation from normocalcemic to somewhat hypercalcemic levels for the entire 14-day period of PTH infusion. Serum calcitonin was also elevated during the infusion period and then returned to the initial level when PTH treatment was stopped. After the minipumps containing PTH were removed, the serum Ca dropped rapidly to 5 mg/dl, which was significantly lower than the control (vehicle-infused) or initial values of serum Ca (7 mg/dl). Infusion of PTH at 3 U/h for 4 days did not produce this rebound hypocalcemia after the pumps were removed. Serum Ca in those experiments returned to the initial level after hormone treatment was discontinued.     

Changes in total parathyroid hormone (PTH), PTH-(1-84) and large C-PTH fragments in different stages of chronic kidney disease

INTRODUCTION: Loss of renal function is accompanied by progressive increase in serum levels of intact parathyroid hormone (iPTH) in patients with end-stage renal disease (ESRD). There is a paucity of data regarding levels of PTH-(1-84) and its large carboxyl-terminal fragments (large C-PTH fragments) and progressive loss of kidney function in patients with chronic kidney disease (CKD). The current study was undertaken to describe the glomerular filtration rate (GFR)-dependent plasma concentrations of PTH-(1-84) and related large C-PTH fragments in adult patients with CKD by using different commercially available PTH assays. METHODS: We studied 80 Caucasian patients with CKD stages 1-5 without renal replacement therapy. Creatinine clearance was calculated by the Modification of Diet in Renal Disease (MDRD) formula. Levels of iPTH were determined by second-generation assays (iPTH Elecsys system, Roche Diagnostics; DUO total iPTH, Scantibodies Laboratory, Inc.; iPTH, Nichols Institute Diagnostics). Third-generation assays were used to measure PTH-(1-84) (CAP (cyclase activating PTH), Scantibodies; Bio-Intact PTH, Nichols). Levels of large C-PTH fragments and ratios of PTH-(1-84)/large C-PTH fragments were calculated and statistical analyses performed. RESULTS: Levels of iPTH and PTH-(1-84) showed CKD stage-dependent increases. Variations among the assays increased with progressive loss of kidney function. The assay from Scantibodies showed a GFR-dependent decrease of the ratio 1-84 PTH / large C-PTH fragment that was not observed with the Nichols assay. CONCLUSION: Increasing variations among the assays with progression of CKD emphasize the fact that the interpretation of measurements must take into consideration the specific assay. We found evidence for a possible preferential increase of the level of large C-PTH fragments over 1-84 PTH in a CKD stage-dependent manner (Scantibodies). The clinical implications of this finding have to be further evaluated by bone biopsy studies.     

Combined treatment with PTH (1-34) and OPG increases bone volume and uniformity of mineralization in aged ovariectomized rats

The combination of PTH with OPG has been proposed as a potential therapy in patients with severe osteoporosis. In the present study, we examined the bone material of aged ovariectomized (OVX) rats treated either with PTH (1-34) or OPG alone or in combination of both. The micro- and nanostructural characteristics of the mineralized bone were evaluated using quantitative backscattered electron imaging (qBEI) and small-angle X-ray scattering (SAXS). Rats (n=68) were either sham-operated or ovariectomized (OVX) at the age of 3 months, and 15 months later, OVX animals were treated either with vehicle, OPG (10 mg/kg), PTH (80 microg/kg) or a combination of both during 5.5 months. All treatments were by subcutaneous injection, 3 days per week. Secondary metaphyseal spongiosa from distal femora was assessed for mineralized bone volume (BV/TV), for the mean Ca-concentration (Camean), the width of the bone mineralization density distribution (Cawidth), as well as the average mineral particle thickness parameter (T) and the degree of alignment of the mineral particles (rho). A remarkable increase of BV/TV up to 139% (P<0.001) was observed in the PTH-treated groups independently of OPG. Camean was slightly increased (+1.7%, P<0.05) in the OPG-treated group. Cawidth was reduced (-6.4%, P<0.01, and -8.9%, P<0.001) in animals treated with OPG and PTH+OPG, respectively. In contrast, Cawidth in sham-operated rats was 16.0% (P<0.001) higher than in OVX. The T parameter was not altered in the trabecular bone within the group of treated and untreated OVX rats. However, the non-ovariectomized animals exhibited a significantly lower T value (-7.1%, P<0.01) with respect to OVX. In conclusion, qBEI and SAXS data of OVX rats suggest that PTH alone was responsible for increase of bone volume, whereas OPG positively influenced the homogeneity and density of mineralization without affecting the nanostructure of the bone material.     

Fracture repair: modulation of fracture-callus and mechanical properties by sequential application of IL-6 following PTH 1-34 or PTH 28-48

Fracture healing presents a sequence of three major stages: inflammation and granulation tissue formation, callus formation and remodeling. Our working hypothesis was that fracture-repair might be enhanced by stimulating proliferation of chondrocytes and osteoblasts in the early stages of fracture healing followed by sequential acceleration of the remodeling process. In the present study we employed a novel device developed by us implementing a standardized fracture in rat tibiae. We investigated the effect of PTH 28-48 or PTH 1-34 alone or in sequence combination with IL-6 together with its soluble receptor (IL-6sR) on fracture repair. PTH 28-48 or PTH 1-34 was applied locally into the hematoma of fractures on days 4, 5 and 6 and IL-6+ its soluble receptor on days 7, 9, and 11. Post-fracture callus volume as measured 14 days post-fracture was increased significantly only by PTH 1-34 (20%; P<0.01). When one of the PTH fragments and IL-6+IL-6sR were applied sequentially callus volume was increased significantly (33%; P<0.01). X-rays radiography at 5 weeks post-fracture showed enlarged callus volume following treatment by either PTH fragments alone, and complete union following the sequential injection of both PTH fragments and IL-6+IL-6sR, only. Only the combination of one of the PTH fragments with IL-6+IL-6sR, as measured 6 weeks post-fracture by three point bending, changed dramatically the quality of the regenerating bone as presented by a 300% increase in mechanical resistance when PTH 1-34 was combined and 200% when PTH 28-48 was combined relative to vehicle-treated fractured bones. We conclude that the sequential application of IL-6+IL-6sR with both PTH fragments has the potential of enhancing fracture healing in long bones and should be further explored in preclinical and in clinical studies.     

Mechanical Vibration Associated With Intermittent PTH Improves Bone Microarchitecture in Ovariectomized Rats

Introduction: Intermittent 1-34 parathyroid hormone (iPTH) administration, a bone-forming treatment, is widely used as a therapy for severe osteoporosis. It can only be used for a maximum of 24 mo and must be followed by an antiresorptive drug to retain the new formed tissue. Mechanical load, in the form of low-intensity and high-frequency vibration, has received considerable attention due to its ability to prevent bone loss. Aim: To investigate the ability of whole body mechanical vibration (MV) to potentiate the anabolic effects of iPTH and to inhibit bone resorption following discontinuation of iPTH treatment in estrogen-deficient rats. Methodology: Fifty-four 6-month-old female Wistar rats were ovariectomized (OVX) or sham-operated. After 5 mo, they were divided into 7 groups: Sham – non-OVX; Control – OVX, vehicle for 60 d; MV – OVX, submitted to MV for 60 d; PTH60d – OVX, injected with iPTH for 60 d; PTH+MV – OVX, injected with iPTH combined with MV for 60 d; PTH30d – OVX, injected with iPTH for 30 d, and untreated for 30 d; PTH30d/MV30d – OVX, injected with iPTH for 30 d, followed by MV for 30 d. Bone mineral density (BMD) and body composition (lean mass and fat) were evaluated at OVX (T0), the beginning (T1), and at the end (T2) of treatments by dual X-ray absorptiometry (DXA). Femurs were processed for histomorphometry (bone volume - BV/TV and cortical thickness - Ct.Th) and tibias for biomechanical test. Results: Body composition and BMD were similar among the groups at T0. In T2, MV presented higher fat than other groups (except PTH60d) and PTH30d/MV30d showed greater lean mass than Control. At T1, Sham presented the highest BMD, but between T1 vs T2 there was an increase in all iPTH-treated groups. At T2, BMD was higher in PTH60d and PTH+MV than in the Control and MV groups. The highest BV/TV was observed in the PTH+MV group, followed by PTH60d. Cortical thickness was increased in PTH60d and PTH+MV compared to Sham. Vibration applied post-iPTH (PTH30d/MV30d) improved the force at failure in tibias when compared to Sham and Control groups. Conclusion: MV potentiated iPTH anabolic effects in cancellous bone; however, MV was unable to maintain bone mass after stopping iPTH in ovariectomized rats.     

Comparison of bone formation responses to parathyroid hormone(1-34), (1-31), and (2-34) in mice

In this study we used a mouse model system to compare the in vivo effects of parathyroid hormone(1-34) [PTH(1-34)] with that of PTH(1-31) or PTH(2-34) analogs. Daily subcutaneous administration of PTH(1-34) for 15 days caused a dose-dependent increase in the serum osteocalcin level and bone extract alkaline phosphatase activity, markers of bone formation. PTH(2-34) was much less potent, whereas PTH(1-31) was equipotent in stimulating bone formation parameters in mice. PTH(1-34) caused significant increases in serum calcium (after 4 h) and tartrate-resistant acid phosphatase activity in bone extract (after 4 h), whereas PTH(2-34) and PTH(1-31) were less potent. Because PTH(1-31) caused a smaller increase in bone resorption parameters compared to PTH(1-34), despite similar effects on bone formation parameters, we evaluated the long-term anabolic effects of PTH(1-31) and PTH(1-34) in mice. Weekly evaluations of serum osteocalcin levels demonstrated that daily injections of PTH(1-34) and PTH(1-31) at 80 microg/kg body weight increased serum osteocalcin levels within 1 week of the start of treatment, which were maintained during the entire 22 week treatment. Assessment of bone density at the end of the treatment period with peripheral quantitated computed tomography (pQCT) revealed that PTH(1-34) caused a significantly greater increase in femoral bone density compared to PTH(1-31) at the middiaphysis (18% vs. 9% over vehicle control; p < 0.001). Both PTH(1-34) and PTH(1-31) increased periosteal circumference compared to vehicle (p < 0.01) without a significant difference between the two treatments. In contrast, PTH(1-34) caused a significantly greater reduction in endosteal circumference than PTH(1-31) (p < 0.001). Both analogs significantly increased maximum load and area of moment of inertia over the vehicle group. In conclusion, our findings suggest that PTH(1-34) and PTH(1-31) may exhibit different anabolic effects at the periosteum vs. endosteum in the long bones of mice.     

重组人甲状旁腺素（1-34)与雌激素对去卵巢大鼠骨代谢的影响

目的观察重组人甲状旁腺素(1-34)与雌激素单用和联用对去卵巢大鼠骨代谢的影响。方法选用雌性4月龄SD大鼠45只,随机分为5组:①假手术(Sham)8只;②去卵巢(OVX)9只;③雌激素治疗组(OVX E)10只:OVX2个月后给予苯甲酸雌二醇治疗6w;④PTH治疗组(OVX PTH)9只:OVX2个月后给予rhPTH(1-34)治疗6w;⑤雌激素与PTH联合治疗组(OVX E PTH)9只:OVX2个月后给予rhPTH(1-34)和E2联合治疗6w。观察各组大鼠胫骨近端松质骨骨小梁形态计量学参数,椎体生物力学指标及部分血清骨生化代谢指标。结果雌激素治疗组和PTH治疗组的骨静态参数均表现为骨量增加;雌激素治疗组的骨形成参数和骨吸收参数降低;PTH治疗组的骨形成指标明显升高,而骨吸收指标虽较Sham高,但较OVX组有所降低;E2与PTH联合治疗组与单用E2组和单用PTH组比较,骨量明显提高,骨转换率参数介于单用E2和单用PTH组之间。3个治疗组的生物力学指标较OVX组均有明显提高,其中E2与PTH联合治疗组改善最明显。结论雌激素和PTH可使去卵巢大鼠松质骨骨量增加和改善生物力学性能,两者联合治疗有协同作用。     

Recent Advances Toward the Clinical Application of PTH (1-34) in Fracture Healing

PTH 1-34, an active form of parathyroid hormone, has been shown to enhance osteoblastic bone formation when administered as a daily subcutaneous injection. The effect of the intermittent administration of PTH (1-34) is an uncoupling of bone turnover with an increase in bone mass and density and decrease in risk of vertebral and nonvertebral fractures. While PTH (1-34) has been used clinically to increase bone mass and reduce fracture risk in postmenopausal women with osteoporosis, there is increasing evidence that PTH (1-34) may promote fracture healing. Animal studies have demonstrated accelerated callus formation with enhanced remodeling and biomechanical properties of the healing fracture. Given these effects, PTH (1-34) will likely be used clinically to enhance fracture union in poor healing situations such as osteoporosis and recalcitrant nonunions.     

Assessment of maintenance therapy with reduced doses of PTH(1-34) in combination with a raloxifene analogue (LY117018) following anabolic therapy in the ovariectomized rat.

This experiment was designed to evaluate the ability of a raloxifene analogue (RA), LY117018, with or without reduced dosing of human parathyroid hormone (hPTH)(1-34) to maintain gains in bone mass after a fully anabolic treatment regimen given to aging osteopenic rats. Six-month-old rats were ovariectomized (ovx) or sham-operated (sham). After 1 month, ovx rats were treated with an anabolic regimen consisting of subcutaneous hPTH(1-34) 80 microg/kg/day and oral raloxifene 3 mg/kg/day, each given 5 days/week for 3 months. Thereafter, the treated ovx rats went on to an 8 week maintenance phase of treatment with either RA alone at the same dose, hPTH(1-34) at a reduced dosing interval (twice a week), or a combination of the two. Bone mineral density (BMD) was measured ex vivo at four skeletal sites, lumbar spine (L2-4), proximal hemipelvis, whole femur, and tibia, by dual-energy X-ray densitometry. All four sites showed a similar pattern of response. After the 3 month anabolic phase, the sham group had significantly higher BMD values than ovx rats at all skeletal sites (p < or = 0.002). The ovx rats treated with PTH + RA during the anabolic phase of the protocol had significantly higher BMD than the sham group in the femur, tibia, and spine (p < or = 0.02) and higher but not significantly different values in the pelvis. Following the 2 month maintenance phase, comparisons were made with the PTH-RA group at the end of the anabolic phase. Decrements in BMD were seen in all three maintenance therapy groups, but they were not statistically significant in the RA plus reduced PTH dose group. However, reduced hPTH(1-34) dosing and RA alone resulted in significant reductions of bone mass measurements at several skeletal sites during the maintenance phase. We conclude that the raloxifene analogue LY117018 may be useful in maintaining bone mass in aging ovx rats following anabolic therapy with hPTH(1-34) and raloxifene analogue, but that this strategy only allows for dose reduction of hPTH(1-34) rather than its discontinuation.