The role of circulating N-terminal parathyroid hormone fragments in the early postparathyroid adenomectomy period.

Calcium metabolism and hormonal control after parathyroid adenomectomy are poorly understood. During the first postoperative hours, biologically active intact parathyroid hormone (PTH) (hPTH 1-84) levels are subnormal and, in spite of down-regulation of PTH peripheral receptors (caused by hypercalcemia before surgery), total and ionized calcium concentrations are maintained in the normal range. Serum samples from 20 patients with primary hyperparathyroidism were collected in the immediate preoperative period and 4 and 48 hours after excision of one parathyroid adenoma. Total and ionized calcium, intact (iPTH), midregion (mrPTH) specific PTH (hPTH 53-68), and N-terminal PTH (N-PTH) serum concentrations were determined. Levels of N-PTH were obtained with a radioimmunoassay by a modified reverse immunoextraction procedure that measures N-PTH fragments after exclusion of the interfering iPTH. No significant correlation was found between ionized and total calcium, mrPTH, and iPTH. However, total and ionized calcium levels correlated well with N-PTH (r = 0.9999, p = 0.0054, and r = 0.9993, and p = 0.0226, respectively). The data suggest that the relatively moderate decrease in calcium levels, in spite of marked decrease in circulating iPTH during the first postoperative hours, may be attributable to the minimal decrease of the bioactive N-PTH epitope concentrations. We would hypothesize that hPTH (1-34) fragments may play a significant role in regulating serum calcium levels in the early postoperative period.     

Radioimmunoassays for the 28–48 region of parathyroid hormone detect intact hormone but not hormone fragments

Summary Metabolism of parathyroid hormone (PTH) is known to involve cleavage in the 28-48 region. With the goal of establishing a radioimmunoassay specific for the intact PTH molecule, we have tested the hypothesis that assays specific for the 28-48 region might fail to recognize the hormone fragments arising in vivo. Antisera against native bovine PTH (bPTH) contain antibody species directed at the 28-48 region of bPTH, since (a) 5 of 6 such antisera showed inhibition of binding of¹²⁵I-labeled bPTH-(1-84) by unlabeled synthetic bPTH-(28-48), and (b) 9 of 9 such antisera bound¹²⁵I-labeled bPTH-(28-48). The latter radioligand yields assays specific for the 28-48 region; 5 of 5 such assays recognized intact bPTH-(1-84), but none recognized bPTH-(1-34), bPTH-(37-84), or both fragments together. Binding of¹²⁵I-bPTH-(28-48) to anti-bPTH sera was not inhibited by intact human PTH (hPTH). Likewise, only 1 of 6 antisera against native hPTH would bind¹²⁵I-bPTH-(28-48) or show inhibition of¹²⁵I-bPTH-(1-84) binding by unlabeled bPTH-(28-48). This poor immunological cross-reactivity between the 28-48 regions of bPTH and hPTH implies a major conformational transition between the two hormones in the 40–47 region of the molecule where the primary structure differs at 5 of 8 positions. A 28-48 specific assay employing an antiserum raised against both bPTH and hPTH did recognize both bPTH-(1-84) and hPTH-(1-84), but not bPTH-(1-34), hPTH-(1-34), bPTH-(37-84), or hPTH-(44-68). Thus, 6 different 28-48 region-specific assays have been shown to recognize intact PTH but not hormone fragments resembling those thought to be formed in vivo.     

Formation and serum disappearance of fragments of parathyroid hormone in the infused dog

Summary Radioiodinated parathyroid hormone [¹²⁵I-PTH(1-84)] was infused into intact dogs. At various times venous samples were chromatographed to determine the levels of intact hormone (1-84) and large fragments in the circulation. Both bioactive (electrolytically iodinated) and inactive (chloramine-T-labeled) preparations were used. In both instances, plateau concentrations of intact hormone and of large metabolite(s) were quickly reached. After cessation of infusion the levels of intact hormone and metabolite(s) quickly declined. Clearly, in the dog, the peripheral formation and disappearance of large fragments of exogenous PTH occur at rates comparable to the clearance of the intact hormone itself.     

Uremic cardiomyopathy: potential role of vitamin D and parathyroid hormone

44 patients receiving regular hemodialysis therapy were investigated using M-mode echocardiography and systolic time intervals to examine the effects of parathyroid hormone (PTH) and vitamin D on left ventricular function. 12 patients were treated with 1 microgram daily of 1, alpha-hydroxycholecalciferol for 6 weeks, which produced a decrease in plasma PTH concentration from 1,883 +/- 226 to 1,123 +/- 289 ng/l. Fractional fibre shortening (FS) increased from 34.6 to 37.6% (p less than 0.025) and mean velocity of fibre shortening (Vcf) increased from 1.21 to 1.32 circ/s (p less than 0.01). A second group of 20 patients was studied before and after the plasma magnesium concentration was increased from 1.25 to 1.70 mmol/l, resulting in a fall in plasma PTH concentration from 546 to 418 ng/l (p less than 0.001). This was associated with an increase in both FS from 32.4 to 34.3%, and Vcf from 1.19 to 1.21 circ/s. A third group of 6 patients with severe hyperparathyroidism underwent total parathyroidectomy, FS increased from 34.9 to 36.3% and Vcf increased from 1.22 to 1.38 circ/s. In conclusion, our results indicate that vitamin D and PTH do influence left ventricular function in uremic patients on chronic hemodialysis, and that a reduction in plasma PTH levels is beneficial to the uremic heart.     

Regulation of intestinal calcium-binding protein in rats: role of parathyroid hormone.

Intestinal calcium-binding protein (CaBP) levels of rats fed a high (1.5%) Ca diet were the same whether the animals were parathyroidectomized (PTX), sham-operated controls pair-fed with the PTX animals, or sham-operated controls fed ad libitum. Consequently, a given base level of CaBP seems to be parathyroid hormone independent and not closely related to feed intake. On the other hand, whereas the ad libitum fed controls more than doubled their intestinal CaBP in response to a 2-day low-calcium (0.02%) regimen, neither the parathyroidectomized animals nor the pair-fed sham-operated controls were able to do so. Since the latter two groups consumed less feed and therefore less vitamin D than the ad libitum fed animals, the inability to increase CaBP in response to a low-calcium diet may have been caused by a restricted vitamin D intake rather than by the absence of parathyroid hormone.     

Inhibition of erythropoiesis in chronic renal failure: the role of parathyroid hormone

Sera from 20 anemic patients with chronic renal failure (CFR) were studied for their effect on bone marrow in vitro erythroid colony formation (CFUE) and the observations correlated with parathyroid hormone (PTH) and ionized calcium levels in the patients' sera. Results demonstrated that 17 out of 20 patients' sera significantly inhibited in vitro erythropoiesis by 47% to 97%. No significant elevation in ionized calcium was found in 16 of the patients tested. Furthermore, assay of PTH levels in these patients revealed that 9 out of 20 had elevated levels of PTH. No correlation was found between PTH serum levels and the degree of in vitro inhibition of erythropoiesis (CFUE) by the patients' sera. Addition of up to 2,000 pg/mL (far above the patients' levels) of exogenous N-terminal or C-terminal PTH with in vitro bone marrow cultures resulted in no inhibitory effect on CFUE. It is concluded that the circulating inhibitor of erythropoiesis which has been shown to exist in the sera of this particular group of patients with CRF, is not PTH.     

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1-34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1-34), such as human PTH(1-31) [hPTH-(1-31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1-31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3-34) [bPTH(3-34)], hPTH(3-34)NH2, and hPTH(13-34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3-34)NH2 and hPTH(13-34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1-34)NH2 and hPTH(1-31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1-34)NH2 and hPTH(1-31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1-34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.     

Activation and inhibition of protein kinase C in cultured bovine parathyroid cells: effect on the release of C-terminal fragments of parathyroid hormone.

The response of the parathyroid gland to low Ca2+ may be mediated in part by protein kinase C (PKC). We assessed the effect of two PKC activators, SC-9 and SC-10, and one PKC inhibitor, H-7, on Ca(2+)-regulated PTH release and degradation in primary cultures of bovine parathyroid cells. Both SC-9 and SC-10 stimulated PTH release, compared to high Ca2+ alone, in parathyroid cells incubated in high Ca2+, with maximal PTH release of at least twofold occurring at a concentration of either activator of 10 nM (p less than 0.05). We have previously shown that another PKC activator, PMA, not only enhances PTH release in the presence of high Ca2+ but suppresses low Ca(2+)-stimulated PTH secretion. In the present study, neither SC-9 nor SC-10 caused a comparable suppression of PTH release at low Ca2+. However, the PKC inhibitor, H-7 (1 microM), blocked low Ca(2+)-stimulated (compared to the low Ca2+ control) PTH secretion by approximately 50% (p less than 0.01) and did not affect high Ca2+ suppression of PTH secretion. H-7 (1 microM) was able to oppose the stimulation of PTH release by the PKC activators SC-9, SC-10, and PMA at high Ca2+ and negated the PTH release-inhibiting effect of PMA at low Ca2+. Culture medium from these experiments was subjected to reversed-phase HPLC and the eluted fractions analyzed by RIA for the presence of intact and C-terminal fragments of PTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effect of carboxyl-terminal parathyroid hormone fragments on osteoclast-like cell formation and osteoclastic activity

The controversy exists about the biological activity of carboxyl (C)-terminal parathyroid hormone (PTH) fragments. The present study was performed to examine the effect of C-terminal PTH fragments on osteoclast-like cell formation and bone-resorbing activity. Human (h) PTH-(1-84) caused a stimulation of osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures more effectively than hPTH-(1-34). All of the C-terminal fragments examined [hPTH-(35-84), hPTH-(53-84) and hPTH-(69-84)] significantly stimulated osteoclast-like cell formation. The conditioned medium from osteoblastic UMR-106 cells pretreated with C-terminal PTH as well as amino-terminal PTH significantly stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony stimulating factor. Moreover, all of the C-terminal fragments also caused a stimulation of osteoclast-like cell formation from hemopoietic blast cells even in the absence of osteoblasts. As for bone-resorbing activity of mature osteoclasts, all of the C-terminal fragments stimulated bone-resorbing activity in osteoblast-containing mouse bone cell cultures, while these fragments did not affect bone-resorbing activity of isolated rabbit osteoclasts. The present study indicates that C-terminal PTH fragments stimulate osteoclast-like cell formation as well as bone-resorbing activity of mature osteoclasts in the presence of osteoblast and also accelerate osteoclast-like cell formation from hemopoietic blast cells in the absence of osteoblasts.     

Analysis of parathyroid hormone in bovine parathyroid cysts

Parathyroid cysts were found in 2-5% of beef steers and heifers while collecting parathyroids at a local abattoir. The cyst fluid contained parathyroid hormone (PTH), averaging 15 ng/ml of fluid. Reversed-phase high-performance liquid chromatographic (HPLC) analysis of the fluid using an acetonitrile-trifluoroacetic acid solvent system, followed by carboxyl-terminal specific PTH radioimmunoassay of the eluant fractions, revealed two major peaks of PTH immunoreactivity. The first eluted at approximately 25% acetonitrile, a position similar to the commercially available human carboxyl terminal fragment of PTH-(39-84). The second peak, at approximately 37% acetonitrile, corresponded to the elution position for bovine PTH-(1-84). The cyst fluid was tested for enzyme activity by incubation of 125I-labeled bovine PTH with cyst fluid for 30 minutes at 37 degrees C, which resulted in no fragmentation of the labeled peptide. Immunohistochemistry of the cells lining the parathyroid cyst indicated that they contained PTH.     

Beyond minerals and parathyroid hormone: role of active vitamin D in end-stage renal disease

Secondary hyperparathyroidism is a common complication of end-stage renal disease (ESRD) that is often treated with activated forms of intravenous vitamin D. The natural course and treatment of secondary hyperparathyroidism in hemodialysis patients is punctuated by episodes of hypercalcemia, hyperphosphatemia, and increased calcium-phosphate product, which in previous studies were linked to increased mortality. Historically these episodes have been attributed to vitamin D, leading some authorities to favor decreased vitamin D use. However, the studies that examined the impact of mineral levels and parathyroid hormone (PTH) on survival did not consistently account for vitamin D therapy itself on hemodialysis patient survival. The current review examines in detail two recent large-scale studies of hemodialysis patients: one that demonstrated a survival advantage of paricalcitol over calcitriol and a second that demonstrated a significant survival advantage of any intravenous vitamin D formulation versus none. In both studies, the effects were independent of mineral and PTH levels, suggesting "nontraditional" actions of vitamin D contributed to the observed survival advantage. Several of these nontraditional actions are reviewed with an emphasis on those that might impact hemodialysis outcomes.     

7-84 parathyroid hormone fragments are proportionally increased with the severity of uremic hyperparathyroidism

AIMS: Recent progress in PTH assay has revealed that the intact PTH assay kit in current use does not differentiate between the truncated 7-84 PTH molecule and the 1-84 PTH molecule. In our series, we examined the effectiveness of a new PTH assay as a noninvasive method of evaluating severity of uremic hyperparathyroidism. METHODS AND MATERIALS: Two hundred and seventy hemodialysis (HD) patients recruited from three HD centers were included and divided into subgroups according to the conventional iPTH assay results. Pre-dialysis blood samples were collected and subjected to two different PTH assays: "intact" PTH assay (iPTH) and "whole" PTH (wPTH) assay. Two biochemical markers of bone remodeling were also examined. RESULTS: In all cases, PTH levels determined by the wPTH assay were in the average 32.3% lower than those determined by the iPTH assay. The difference of the results of the two PTH assay methods, which indicated the portion of 7-84 PTH fragments of the total PTH molecules measured with the iPTH assay, was gradually increased while the severity of uremic hyperparathyroidism increased. Biochemical markers of bone formation/resorption showed a similar change. CONCLUSION: The portion of the 7-84 PTH fragments and markers of increased bone turnover increased in proportion to the severity of uremic hyperparathyroidism. This finding disproves the hypothetical role of 7-84 PTH fragments alone as the noninvasive marker of low-turnover bone disease in HD patients.     

The role of aluminium and parathyroid hormone in erythropoietin resistance in haemodialysis patients

Recombinant human erythropoietin (rHuEpo) is an effective therapy for anaemia in most patients with end-stage renal disease (ESRD). However, there remain a minority of patients with ESRD who are resistant to the effects of rHuEpo. The present study examined the role of aluminium overload and hyperparathyroidism of the biological effects of rHuEpo. Twenty-two patients aged 26-74 (mean 53 +/- SD 15.5) received rHuEpo 50-200 U/kg per week for 16.5 +/- 8.0 months (range 3-27). Haemoglobin was maintained at 11.5-13.0 g/dl by appropriate dose adjustment. Iron supplements were provided to maintain serum ferritin greater than 200 ng/ml. The mean time to rHuEpo response (Hb greater than 2 g/dl over baseline) was 6.1 +/- 2.6 weeks. Mean pretreatment serum aluminium correlated with time to Hb response (r = 0.48; P less than 0.05) and pretreatment mean corpuscular volume (r = 0.43; P less than 0.05) but not with eventual rHuEpo maintenance dose. PTH did not correlate with either Hb response or eventual maintenance rHuEpo dose. In summary, elevated serum aluminium concentrations were associated with an initial resistance to the biological effects of rHuEpo but had no effect on long-term dose requirements. In contrast, no impact of PTH on either immediate or long-term rHuEpo dose was evident.     

The role of parathyroid hormone measurements after surgery for primary hyperparathyroidism

BACKGROUND: During parathyroidectomy for primary hyperparathyroidism (pHPT), intraoperative parathyroid hormone (IOPTH) levels are used to confirm removal of all hyperfunctioning parathyroid tissue. The phenomenon of elevated parathyroid hormone (PTH) levels with normocalcemia after curative parathyroidectomy, seen in up to 40% of patients, continues to be an unexpected and unexplained finding. We therefore investigated whether postoperative PTH levels are as reliable as IOPTH levels in predicting cure after surgery for pHPT. METHODS: We reviewed our prospective database of consecutive patients undergoing surgery for pHPT between December 1999 and November 2004. Curative parathyroidectomy was defined as normocalcemia 6 months or longer postoperatively. RESULTS: A total of 328 patients who underwent 330 operations for pHPT had IOPTH measurements and serum follow-up calcium levels at 6 months or longer. Surgery was curative in 315 (95.5%) operations. IOPTH levels correctly predicted operative success in 98.2% (positive predictive value [PPV]. Postoperatively, the PPV of a normal PTH level at 1 week, 3 months, and 6 months was 97.1%, 97.3%, and 96.5%, respectively. Of all patients with an elevated postoperative PTH level at 1 week, 3 months, or 6 months, only 13.7%, 14.3%, and 14%, respectively, were not cured. CONCLUSIONS: Normal postoperative PTH levels reliably predict operative success. However, they do not improve upon results predicted by IOPTH levels. Elevated postoperative PTH levels do not predict operative failure in most patients. We propose that PTH measurements after surgery for pHPT may be misleading, costly, and not indicated in normocalcemic patients.     

Intraoperative parathyroid hormone assay and parathyroid reoperations

BACKGROUND: The purpose of this study was to determine whether intraoperative parathyroid hormone (IOPTH) assay improved results of reoperations. METHODS: One hundred two patients with persistent/recurrent sporadic primary hyperparathyroidism underwent 108 reoperations (1996-2002). IOPTH was not used (n=58) from 1996-1998 (group 1). IOPTH was used (n=50) from 1999-2002 (group 2). Sensitivity and positive predictive value of IOPTH and its influence on surgical strategy were analyzed. A 50% decrease occurring 10 minutes after removal of parathyroid tumor was used to determine if all abnormal tissue had been removed. RESULTS: Groups 1 (58 patients) and 2 (50 patients) were comparable except for duration of follow-up. The cure rate was 84% (group 1, 87%; group 2, 82%, P=0.7). Hypocalcemia developed in 20 patients (permanent in 2 patients). There was 1 permanent vocal cord paralysis and 1 patient died of toxic shock syndrome. IOPTH successfully predicted cure in 44 of 49 patients (sensitivity, 90%); the positive predictive value was 90%. Values for parathyroid hormone level and the ratio parathyroid hormone/calcium at day 1 were at least as accurate as IOPTH in predicting cure. IOPTH was helpful in 1 patient but misleading in 4 patients. It failed to modify intraoperative strategy in most other patients. CONCLUSIONS: IOPTH testing was relatively reliable in patients with persistent or recurrent sporadic primary hyperparathyroidism, but the test unfortunately failed to improve the overall success rate at reoperation.