C-PTH Fragments Research Articles

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

Introduction Circulating 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. Methods In 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. Results The 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. Conclusions The 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.

Acute and chronic regulation of circulating PTH: Significance in health and in disease

Circulating human parathyroid hormone (PTH) is immunoheterogenous. It is composed of 80% carboxyl-terminal (C) fragments and of 20% PTH(1-84). This composition contrasts with the biological activity of the hormone, which is only related to PTH(1-84), creating a paradox between circulating PTH composition and PTH bioactivity. PTH molecular forms are either secreted by the parathyroid glands or generated by the peripheral metabolism of PTH(1-84) in the liver. The kidney has a major role in the disposal of C-PTH fragments. Secretion of PTH molecular forms by the parathyroid glands is highly regulated under a variety of clinical conditions, suggesting that C-PTH fragments could exert some biological effects of their own. Recent data suggest that C-PTH fragments can exert biological actions opposite to those of PTH(1-84) by acting on a C-PTH receptor not yet cloned. They can decrease calcium concentration, phosphate excretion, bone resorption and 1,25(OH)₂ synthesis. The clinical implications of this new concept are reviewed.

Parathyroid hormone, kidney disease, evidence and guidelines

Parathyroid hormone (PTH) is released from the parathyroid glands predominantly as an 84 amino-acid peptide (PTH (1--84)). Classical biological activity, mediated via interaction with the PTH receptor PTH1R, resides in the N-terminus of the molecule. C-terminal (CPTH) fragments are also directly secreted by the parathyroid gland. Following release, PTH (1--84) undergoes proteolytic degradation in Kupier cells in the liver: resulting CPTH, but not the N-terminal fragments, may re-enter the circulation. The variously derived PTH fragments and PTH (1--84) are then excreted renally. In the setting of chronic kidney disease (CKD), assessment of plasma PTH concentration is used as a surrogate measure of underlying renal bone disease. Measurement of PTH in the presence of renal impairment has always been problematic. In the 1970s, it became apparent that PTH radioimmunoassays crossreacted with CPTH fragments that accumulated in the presence of renal failure. The development of a two-site immunoassay byNichols Institute Diagnostics (Nichols Allegro immunoradiometric assay [IRMA]) in the 1980s, described as an ‘intact’ PTH assay, seemed to resolve this issue. This assay became the bench mark against which other assays were developed. Further, the somewhat limited evidence correlating renal bone disease and plasma PTH concentration, and upon which guidelines have been based, relates largely to experience with this prototype second-generation assay. Currently a range of manufacturers oier intact PTH assays on a variety of automated platforms. Notably, Nichols Institute Diagnostics can no longer be included in that list following the demise of the company in 2006. In the mid-1990s, a Canadian research group discovered that all was not as it might seem. In fact, intact PTH assays seemed to cross-react to a large degree with a CPTH fragment thought to be PTH (7--84) and other less prevalent fragments of PTH that accumulated in renal failure. It would appear that cross-reactivity with this fragment had been accounting all along for approximately 50% of the immunoreactive PTH we had been measuring in such patients, although the exact degree of cross-reactivity varies between methods. Putting aside the perhaps more trivial matter of whether you should continue to market your assay as an intact PTH assay when it is clear that it is not (many manufacturers still do so), the question of how well diierent PTH assays agree with each other and whether measuring PTH (7--84) has clinical relevance in kidney disease needs addressing. It is evident that second-generation PTH assays do not agree among kidney disease patients, either with each other or with the original Nichols intact PTH assay. Among dialysis patients, a recent study has shown that commonly used commercial assays may overor underestimate PTH by up to 50% compared with the Nichols Allegro IRMA. Such betweenmethod variability is also con¢rmed on a regular basis by external quality assessment data (Figure 1). This is due to a variety of factors including lack of common standardization, variation in recovery of PTH (1--84) (Figure 2) and diierences in the extent of crossreactivity with PTH (7--84). For example, data from a United Kingdom National External QualityAssessment Service (UK NEQAS) study in July 2004 estimated approximately 69% cross-reactivity with PTH (7--84) for the Diagnostic Products Corporation (DPC) method, 73% with the Roche ElecSys method, 102% with the Bayer Advia method and 95% with the former Nichols Advantage method. Indeed all 14 ‘intact PTH’assays tested appeared to cross-react with PTH (7--84). PTH results may be interpreted in the setting of a range of clinical recommendations by nephrologists. The UK Renal Association standard states: ‘PTH concentration should be less than four times the upper limit of normal of the assay used in patients beingmanaged for chronic renal failure or after transplantation and in patients who have been on haemodialysis or peritoneal dialysis for longer than three months’. Using a cut-oi based on the upper limit of normal requires that the upper limit of normal is accurate and appropriate for the assay used.There is evidence to suggest that this may not always be the case. A survey conducted by UK NEQAS in July 2005 indicated that the majority of participants have an upper limit of normal close to 65 ng/L, irrespective of the assay used (Figure 3). Is there evidence that variations in

Variant of Adynamic Bone Disease in Hemodialysis Patients: Fact or Fiction?

Adynamic bone disease is a type of renal osteodystrophy characterized by low bone turnover and paucity of bone cells. It was proposed that a new type of this disease featuring high osteoclastic resorption without parathyroid hormone stimulus and designated adynamic bone disease variant occurs in hemodialysis patients. The present study is designed to evaluate the frequency and characteristics of both diseases in a large series of bone biopsy specimens. We reviewed 1,160 bone biopsy specimens from hemodialysis patients. Specimens in which adynamic bone disease was diagnosed were selected and categorized as classic or variant based on osteoclastic surface. In 218 bone biopsy specimens (18.8%), adynamic bone disease was identified, whereas the variant form was identified in 35 specimens (38.8%). Biopsy specimens categorized as the variant form were from patients who were younger and had greater phosphorus and parathyroid hormone levels. Histologically, the variant form presented greater osteoid volume, fibrosis volume, osteoid surface, osteoblast surface, and eroded surface. Similarly, values for all dynamic parameters were greater in the variant group. Osteoclastic surface correlated with phosphorus level, parathyroid hormone level, and osteoblast surface. Age and osteoblast surface were identified as independent determinants of the variant form. Adynamic bone disease variant seems to occur in younger hemodialysis patients with greater levels of parathyroid hormone, which acts on cell-covered bone surfaces. It probably is a transitional phase from low- to high-turnover status, rather than a true entity within the spectrum of renal osteodystrophy.

Influence of the parathyroid glands on bone metabolism

Bone is a classic target tissue for parathyroid hormone (PTH), whose calciotropic effect is mediated largely via catabolic actions on this tissue. Paradoxically, PTH also exerts anabolic actions, with intermittent injections of PTH or its amino-terminal fragments causing an increase in bone formation and bone mass, actions that form the basis for the use of PTH in the treatment of osteoporosis. Besides vitamin D, PTH is the only other known bone anabolic agent. High-affinity PTH receptors (PTH-1R) have been detected on osteoblasts and osteoclasts (albeit in lower numbers). Bone turnover, which includes activation of osteoclasts and osteoblasts, appears to be best reflected not by absolute concentrations of PTH (which can vary based on the assay and antibody used) but by a balance of circulating full-length PTH-(1-84) and amino-terminally truncated C-PTH fragments. When PTH-(1-84) is predominant, bone turnover is promoted. Among PTH fragments, PTH-(7-84) appears to be the most potent antagonist of PTH-(1-84). The mechanisms involved in these effects are unclear although mediation via unique C-terminal receptors has been suggested. We propose that, within the range of total PTH (100-1000 pg mL(-1)), the ratio of PTH-(1-84)/C-PTH fragment is a valuable tool for diagnosis of bone turnover. Data indicate that at PTH levels < 100-150 pg mL(-1) and > 1000 pg mL(-1), the ratio looses its predictive power. Assay type, patient characteristics (race, underlying renal disease) and treatment attributes (vitamin D, corticosteroids, phosphate binders) have an impact on the PTH ratio, and care should be used in interpreting assay results and making subsequent treatment decisions.

Circulating PTH molecular forms: What we know and what we don't

Circulating parathyroid hormone (PTH) molecular forms have been identified by three generations of PTH assays after gel chromatography or high-performance liquid chromatography fractionation of serum. Carboxyl-terminal (C) fragments missing the amino-terminal (N) structure of PTH(1-84) were identified first. They represent 80% of circulating PTH in normal individuals and up to 95% in renal failure patients. They are regulated by calcium (Ca) slightly differently than PTH(1-84), occurring in a relatively smaller proportion relative to the latter in hypocalcemia but in a much larger proportion in hypercalcemia. Synthetic C-PTH fragments do not bind to the PTH/PTHrP type I receptor and are not implicated in the classical biological effect of PTH(1-84). They bind to a different C-PTH receptor and exert biological actions on bone that are opposite to those of PTH(1-84). The integrity of the distal C-structure appears to be important for these biological effects, and it is uncertain if all C-PTH fragments are intact up to position 84. A second category of C-PTH fragment has a partially preserved N-structure. They are called non-(1-84) PTH or N-truncated fragments. They react in Intact (I)-PTH assays but not in PTH assays with a 1-4 epitope. They are acutely regulated by Ca(2+) concentration. They also exert similar hypocalcemic and antiresorptive effects but have 10-fold greater affinity for the C-PTH receptor compared to other C-PTH fragments. Even if they represent only 10% of all C-PTH fragments, they could be as relevant biologically. An N form of PTH other than PTH(1-84) has been identified in the circulation. It reacts very well in PTH assays with a 1-4 epitope but poorly in I-PTH assay with a 12-18 epitope. It is oversecreted in severe primary and secondary hyperparathyroidism and in parathyroid cancers. Its biological activity is still unknown. Overall, these studies suggest that PTH(1-84) and C-PTH fragments are regulated differently to exert opposite biological effects on bone via two different receptors. This may serve to control bone turnover and Ca concentration more efficiently.

Inter-method variability in PTH measurement: Implication for the care of CKD patients

The National Kidney Foundation/Kidney-Dialysis Outcome Quality Initiative guidelines recommend to maintain the serum intact parathyroid hormone (PTH) concentration between 150 and 300 ng/l in chronic kidney disease (CKD) stage 5 patients. As these limits were derived from studies that used the Allegro intact PTH assay, we aimed to evaluate whether they were applicable to other PTH assays. We compared the PTH concentrations measured with 15 commercial immunoassays in 47 serum pools from dialysis patients, using the Allegro intact PTH assay as the reference. We also evaluated the recovery of graded amounts of synthetic 1-84 and 7-84 PTH added separately to a serum pool. Although the assays were highly correlated, the concentrations differed from one assay to another. The median bias between the tested assays and the Allegro intact PTH assay ranged from -44.9 to 123.0%. When the PTH concentrations were 150 or 300 ng/l with the Allegro intact PTH assay, they ranged with other assays from 83 to 323 ng/l and from 160 to 638 ng/l, respectively. The tested assays recognized 7-84 PTH with various cross-reactivities, whereas a given amount of 1-84 PTH was recovered differently by these assays. We found important inter-method variability in PTH results owing to both antibody specificity and standardization reasons. The unacceptable consequence is that opposite therapeutic attitudes may be reached in a single patient depending on the PTH assay used. We propose to use assay-specific decision limits for CKD patients, or to apply a correcting factor to the PTH results obtained with a given assay.

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

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. 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. 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. 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.

Acute Regulation of Circulating Parathyroid Hormone (PTH) Molecular Forms by Calcium: Utility of PTH Fragments/PTH(1–84) Ratios Derived from Three Generations of PTH Assays

The quantitative evaluation of circulating PTH peaks revealed by PTH assays after HPLC separation constitutes the best way to study the behavior of PTH molecular forms, but it is also impractical. The objective of the study was to investigate the regulation of circulating PTH molecular forms by calcium through the use of PTH fragments/PTH (1-84) ratios derived from PTH assays with different specificities before and after HPLC separation of circulating PTH. CaCl2 and Na citrate were infused in eight volunteers. PTH was measured in serum and HPLC fractions at different calcium concentrations in PTH assays reacting with regions 1-2 (CA), 12-18 (T), and 65-69 (C) of the PTH structure. From hypo- to hypercalcemia, the C/CA ratio had the highest range (1.92 to 9.75; P < 0.001), and the C/T ratio had a higher range (1.69 to 6.11; P < 0.01) than the T/CA ratio (1.15 to 1.86). Human (h) PTH (1-84) represented 32.7 and 4.3% of circulating PTH in hypo- and hypercalcemic HPLC profiles, respectively. These numbers were 5 and 0.9% for amino-terminal (N)-PTH, an amino-terminal form of PTH distinct from hPTH (1-84), 7.3 and 6.8% for non-(1-84) PTH or large C-PTH fragments with a partially preserved N structure, and 54.9 and 88.1% for C-PTH fragments missing a N structure. The HPLC C-PTH fragments to hPTH (1-84) ratio had the most extensive range (1.67 to 20.58). Despite their quantitative differences, all ratios identified identical behavior of PTH fragments relative to PTH (1-84). PTH assay ratios are an adequate tool to investigate the modulation of PTH molecular forms, even if all PTH assays show some undesirable cross-reactivity with certain circulating forms of PTH.

Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism

Non-(1-84) parathyroid hormone (PTH) fragments are large circulating carboxyl-terminal (C) fragments with a partially preserved amino-terminal (N) structure. hPTH (7-84), a synthetic surrogate, has been demonstrated to exert biologic effects in vivo and in vitro which are opposite to those of hPTH (1-34) on the PTH/PTHrP type I receptor through a C-PTH receptor. We wanted to determine the N structure of non-(1-84) PTH fragments. Parathyroid cells isolated from glands obtained at surgery from three patients with primary hyperparathyroidism and three patients with secondary hyperparathyroidism were incubated with 35S-methionine to internally label their secretion products. Incubations were performed for 8 hours at the patient-ionized calcium concentration and in the presence of various protease inhibitors. The supernatant was fractionated by high-performance liquid chromatography (HPLC) and fractions were analyzed with PTH assays having (1 to 4) and (12 to 23) epitopes, respectively. The serum of each patient was similarly analyzed. Peaks of immunoreactivity identified were submitted to sequence analysis to recover the 35S-methionine residues in positions 8 and 18. Three regions of interest were identified with PTH assays. They corresponded to non-(1-84) PTH fragments (further divided in regions 3 and 4), a peak of N-PTH migrating in front of hPTH (1-84) (region 2) and a peak of immunoreactivity corresponding to the elution position of hPTH (1-84) (region 1). The last corresponded to a single sequence starting at position 1. Region 2 gave similar results in all cases (a major signal starting at position 1) but also sometimes minor sequences starting at position 4 or 7. Regions 3 and 4 always identified a major sequence starting at positions 7 and minor sequences starting at positions 8, 10, and 15. Surprisingly, a major signal starting at position 1 was also present in region 3. The HPLC profile obtained from a given patient's parathyroid cells was qualitatively similar to the one obtained with his/her serum in each case. These results indicate that non-(1-84) PTH fragments are composed of a family of fragments which may be generated by specific or progressive cleavage at the N region. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1-84) PTH fragments. The generation process is similar to the one described for smaller C-PTH fragments a number of years ago, suggesting a similar production mechanism and source for all C-PTH fragments.

Overproduction of an amino‐terminal form of PTH distinct from human PTH(1–84) in a case of severe primary hyperparathyroidism: influence of medical treatment and surgery

Rare patients with severe primary hyperparathyroidism present with large parathyroid tumours, severe hypercalcaemia, very high PTH levels and osteitis fibrosa cystica. Some of these patients display a large amount of C-PTH fragments in circulation and present with a higher C-PTH/I-PTH ratio than seen in less severe cases of primary hyperparathyroidism. We wanted to determine how PTH levels and circulating PTH high-performance liquid chromatography (HPLC) profiles analysed with PTH assays having different epitopes could be affected by medical and surgical treatment in such patients. A 55-year-old man with severe hypercalcaemia (Ca(2+): 2.01 mmol/l), very high PTH levels (CA-PTH 82.1 and T-PTH 72 pmol/l) caused by a large parathyroid tumour (7.35 g) and accompanied by significant bone involvement (alkaline phosphatase of 185 UI/l and subperiostal bone resorption of hands) was referred to us. Blood was obtained at various time points during his medical treatment, before and after surgery, to measure parameters of calcium and phosphorus metabolism, and of bone turnover. HPLC separations of circulating PTH molecular forms were performed and analysed with PTH assays having 1-4 (CA), 12-18 (T), 26-32 (E) and 65-84 (C) epitopes. Before surgery, serum Ca2+ was nearly normalized with hydratation, intravenous (IV) pamidronate and oral vitamin D administration. Despite a decrease in Ca2+ to 1.31 mmol/l, CA-PTH and T-PTH levels decreased by half in relation to a threefold increase in basal 1,25-dihydroxyvitamin D [1,25(OH)2D] level (94 to 337 pmol/l). After this initial positive response, hypercalcaemia and elevated CA- and T-PTH levels recurred even if 1,25(OH)2D levels remained elevated. The tumour was removed surgically and proved to be poorly differentiated with nuclear atypia and mitosis. After surgery, the Ca2+ level and PTH secretion normalized. The higher CA-PTH level relative to the T-PTH level observed before surgery in this patient was related to the oversecretion of an amino-terminal (N) form of PTH recognized by PTH assays with (1-4) or (26-32) epitopes but not by the T-PTH assay with a (12-18) epitope. This molecular form represented 50% of CA-PTH measured in this patient, but only 7% in less severe cases of primary hyperparathyroidism. It was unaffected by medical therapy and disappeared after surgery. The relationship between the overexpression of this N-PTH molecular form and severe primary hyperparathyroidism remains unclear. Further studies will be required in these rare patients to see whether N-PTH is a marker of less well differentiated parathyroid tumours and/or relates to the overproduction of C-PTH fragments in the presence of severe hypercalcaemia.

Receptors Specific for the Carboxyl-Terminal Region of Parathyroid Hormone on Bone-Derived Cells: Determinants of Ligand Binding and Bioactivity

PTH comprises 84 amino acids of which the first 34 are sufficient for full activation of the classical PTH/PTHrP receptor, the type 1 PTH receptor. It is known that multiple carboxyl (C)-terminal fragments of PTH are present in the blood and that they comprise the majority of circulating PTH. C-PTH fragments, previously regarded as by-products of PTH metabolism, are directly secreted by the parathyroid glands or arise from the peripheral cleavage of the intact hormone. Compelling evidence now strongly suggests that these C-PTH fragments mediate biological effects via activation of a receptor that specifically recognizes the C-terminal portion of intact PTH, and this receptor is therefore named the carboxyl-terminal PTH receptor (CPTHR). We have previously reported that osteocytes abundantly express this novel receptor and that its activation is involved in cell survival and communication. Here we report the characterization of determinants of PTH that are required for high-affinity binding to the CPTHR. Using synthetic PTH peptides harboring alanine substitution or truncations, we showed the existence of discrete binding domains and critical residues within the intact hormone. We have furthermore identified eight amino acids within the PTH sequence that play key roles in optimizing the binding affinity of C-PTH fragments to CPTHRs. These include the tripeptide sequence Arg(25)-Lys(26)-Lys(27), the dibasic sequence Lys(53)-Lys(54), and three additional residues within the PTH (55-84) sequence, Asn(57), Lys(65), and Lys(72). Functional analysis of these residues demonstrated a strong correlation between binding affinity and biological effect and points to a potential role of CPTHR activation in regulating bone cell survival.

Intravenous Vitamin D Therapy Reduces PTH-(1–84)/Large C Fragments Ratio in Chronic Hemodialysis Patients

Background: Renal osteodystrophy is one of the major complications in patients with chronic renal failure. Large C-PTH fragments are secreted from the parathyroid glands and exert antagonistic actions against PTH-(1–84). The PTH-(1–84)/large C-PTH fragments ratio reflects both biosynthesis and processing of PTH; however the alteration of the ratio under vitamin D therapy has not been investigated. Methods: Seventeen hemodialysis patients with intact PTH levels of >300 pg/ml were enrolled. Calcitriol or maxacalcitol were administered intravenously for 78 weeks. Intact PTH, PTH-(1–84), and the PTH-(1–84)/large C-PTH fragments ratio were measured at 0, 13, 26, 52 and 78 weeks. Results: Intact PTH and PTH-(1–84) levels, which were 492.0 ± 115.7 and 303.4 ± 105.4 pg/ml, respectively, at baseline, significantly decreased at the end of the study to 268.9 ± 121.9 (p < 0.0001) and 190.7 ± 106.9 pg/ml (p = 0.0008), respectively. In contrast, large C-PTH fragments, which were 152.7 ± 53.5 pg/ml at baseline, did not significantly change at 78 weeks (144.5 ± 72.2 pg/ml, p = 0.7612). Consequently, the PTH-(1–84)/large C-PTH fragments ratio was significantly reduced from 2.25 ± 1.31 to 1.47 ± 0.89 (p = 0.0004). Conclusion: The PTH-(1–84)/large C-PTH fragments ratio reflects the change of PTH biosynthesis, processing and secretion from the parathyroid glands, and it may be a beneficial marker to evaluate the overall biological PTH action and predict bone turnover status in hemodialysis patients under intravenous vitamin D therapy.

Diagnosis, assessment, and treatment of bone turnover abnormalities in renal osteodystrophy

HE ABNORMALITIES of the skeleton in chronic kidney disease (CKD), collectively known as renal osteodystrophy, are an important cause of morbidity and decreased quality of life. In the management of patients with kidney disease, it is necessary to have a rational approach to the diagnosis and assessment of renal osteodystrophy in order to devise a treatment plan that hopefully will lead to an improved outcome. In the past, the term renal osteodystrophy was mainly equated only with abnormalities of bone turnover, but as described in the article by Cunningham and Sprague 1 in this same issue, renal osteodystrophy is a complex disorder of compromised bone strength in CKD patients. While osteoporosis is a term used to describe fragile bones prone to fracture in the general population and is most often assessed by dual x-ray absorptiometry (DEXA), renal osteodystrophy should be the principal term to describe fragile bones prone to fracture and other morbidities in CKD. Renal osteodystrophy is a function of bone turnover (assessed by bone biopsy), bone density (assessed by DEXA or quantitative CT [qCT]), and bone architecture, but the principal determinant of bone fragility in CKD is abnormal bone turnover. However, diagnosing and treating bone turnover abnormalities remains challenging. Our discussion group met to assess the current state of knowledge, understand the basis of our current therapy, and identify the information that needs to be gathered to improve the therapy of bone turnover and thereby improve the disorder of renal osteodystrophy. A number of important questions, listed in Table 1, were considered.

Circulating Forms of Parathyroid Hormone: Peeling Back the Onion

Circulating parathyroid hormone (PTH) regulates normal bone and mineral ion homeostasis and is central in the pathogenesis of bone disease in primary and secondary hyperparathyroidism, especially in advanced renal insufficiency (1). The major biological effects of PTH, a linear polypeptide 84 amino acids in length, reflect activation of the G-protein-coupled PTH/PTHrP receptor (PTHR) expressed on target cells in the renal tubules and the osteoblastic lineage of bone (2). Activation of PTHRs requires only the most N-terminal portion of PTH [PTH(1–34) is a full agonist] and is mostly eliminated if the N-terminal serine residue of the human hormone is removed. PTH, secreted by the parathyroid glands primarily in response to changes in blood ionized calcium, undergoes rapid endopeptidic cleavage, mainly by Kupffer cells in the liver (3). This event generates a series of N-truncated “CPTH” fragments (the corresponding N-terminal segments are destroyed in situ) that reenter the circulation and are cleared mainly by glomerular filtration, an important route for PTH clearance as well (4). Chemical analysis of PTH fragments isolated from rat blood after administration of radiolabeled bovine PTH established that hepatic metabolism yields a family of CPTH fragments with NH2 termini ranging between residues 34 and 43, although the methods used likely would not have detected substantially larger CPTH fragments (5). Interestingly, chemically similar CPTH fragments are secreted directly from the parathyroid glands as well, and the ratio of secreted CPTH fragments to intact PTH increases as blood calcium increases (6)(7). CPTH fragments typically circulate at a three- to fivefold molar excess over intact PTH, but this ratio may increase dramatically in end-stage renal disease (ESRD), depending on the severity and chronicity of renal insufficiency and the blood concentrations of calcium, phosphate, and 1,25-dihydroxyvitamin D, all of which regulate PTH secretion (1)(8). Measurement of …

Use and indication of vitamin D and vitamin D analogues in patients with renal bone disease.

Vitamin D plays a pivotal role in the pathogenesis and treatment of renal bone disease. Vitamin D levels decline in the early phase of renal failure, however, through a compensatory mechanism parathyroid hormone (PTH) stimulates the production of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol) to return it to normal circulating concentrations. Nevertheless, resistance to calcitriol is observed and may be related to the decreased presence of the heterodimeric, DNA-binding partner for the vitamin D receptor protein. In end-stage kidney disease (ESKD) the circulating levels of calcitriol are invariably low. The indications of vitamin D therapy are the replacement of the missing hormone vs suppression of hyperparathyroidism (HPT) requiring daily low-dose oral vs intermittent 'pulse' or oral administration. However, this therapy must be accompanied by careful patient monitoring to avoid hypercalcaemia and low bone turnover. Low bone turnover is not merely a histologic entity, but a clinical condition associated with a high risk of extraosseous calcifications, in particular in the cardiovascular system, leading to increased morbidity. Thus, determination of bone turnover in patients with ESKD is essential. Bone biopsy is the gold standard to assess bone turnover, however, it is not always available and nephrologists rely on PTH levels. The intact PTH assay measures PTH(1-84) and large C-PTH fragments, which may antagonize the PTH(1-84) effects on bone. An assay that measures exclusively PTH(1-84) has recently become available and a calculated PTH(1-84)/C-PTH fragment ratio has been shown to be the best predictor of bone turnover in patients with ESKD not treated with vitamin D or with other medications known to affect bone metabolism. 1,25-dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT) is a vitamin D analogue that could control serum PTH concentrations without deleterious effects on bone.

Origin of parathyroid hormone (PTH) fragments detected by intact-PTH assays.

Intact parathyroid hormone (I-PTH) assays react with non-(1-84)PTH, large carboxyl-terminal (C) fragments with a partially preserved amino-terminal (N) structure. They account for up to 50% of I-PTH in renal failure and may be implicated in PTH resistance. We wanted to know if they were secreted by the parathyroid glands and generated by peripheral metabolism of PTH(1-84). Anesthetized normal and nephrectomized (NPX) rats were injected i.v. with 1.5 microg human (h) PTH(1-84). Blood was obtained from 8 rats at 2, 4, 6, 8, 12, 24, 48 and 96 min. I-PTH (Allegro I-PTH) was measured in all samples. Pools of serum were fractionated by HPLC at each time point and the fractions assayed to quantitate hPTH(1-84) and non-(1-84)PTH. Secretion studies were performed with dispersed cells from 5 parathyroid adenomas. The serum of 10 patients with primary hyperparathyroidism and cell supernatants were fractionated by HPLC and were analyzed as described. hPTH(1-84) disappeared from serum biexponentially. The half-life of the first exponential was similar in normal (2.08 min) and NPX (1.94 min) rats, while that of the second was longer in NPX rats (32.4 vs 20.9 min). The residual quantity of hPTH(1-84) under the curve was greater in NPX (6964+/-2392 pmol) than in normal rats (3229+/-561 pmol; P<0.001). Non-(1-84)PTH concentration was maximal at 8 min in both groups and was higher in NPX (92.8+/-13.8 pmol/l) than in normal rats (38.8+/-7.2 pmol/l; P<0.01). The area under the curve of non-(1-84)PTH was also greater in NPX (1904+/-405 pmol) than in normal rats (664+/-168 pmol; P<0.001). All parathyroid adenomas secreted non-(1-84)PTH. It represented 21.1+/-3.9% of secreted and 32.5+/-1.3% of circulating I-PTH in primary hyperparathyroidism. Non-(1-84)PTH, like other C-PTH fragments, originates from both the peripheral metabolism of hPTH(1-84) and from parathyroid gland secretion. Renal failure influences its concentration by increasing the amount of substrate available and by reducing non-(1-84)PTH clearance. Its higher proportion in serum relative to cell supernatants in primary hyperparathyroidism reflects the added role of peripheral metabolism and the longer half-life of fragments.

Use and indication of vitamin D and vitamin D analogues in patients with renal bone disease

Vitamin D plays a pivotal role in the pathogenesis and treatment of renal bone disease. Vitamin D levels decline in the early phase of renal failure; however, through a compensatory mechanism parathyroid hormone (PTH) stimulates the production of calcitriol to return it to normal circulating concentrations. Nevertheless, resistance to calcitriol is observed and may be related to the decreased presence of the heterodimeric, DNA-binding partner for the vitamin D receptor protein. In end-stage kidney disease (ESKD) the circulating levels of calcitriol are invariably low. The indications of vitamin D therapy are the replacement of the missing hormone versus suppression of hyperparathyroidism requiring daily low-dose oral versus intermittent pulse or oral administration. However, this therapy must be accompanied by careful patient monitoring to avoid hypercalcemia and low bone turnover. Low bone turnover is not merely a histologic entity, but a clinical condition associated with high risk of extraosseous calcifications, in particular in the cardiovascular system, leading to increased morbidity. Thus, determination of bone turnover in patients with ESKD is essential. Bone biopsy is the gold standard to assess bone turnover, however, it is not always available and nephrologists rely on PTH levels. The intact PTH assay measures PTH (1-84) and large C-PTH fragments, which may antagonize the PTH (1-84) effects on bone. An assay that measures exclusively PTH (1-84) has recently become available and a calculated PTH (1-84) /C-PTH fragment ratio has been shown to be the best predictor of bone turnover in patients with ESKD not treated with vitamin D or with other medications known to affect bone metabolism. 22-Oxacalcitriol is a vitamin D analogue that could control serum PTH concentrations without deleterious effects on bone.

Improved assessment of bone turnover by the PTH-(1-84)/large C-PTH fragments ratio in ESRD patients

The "intact" parathyroid hormone (PTH) assay recognizes PTH-(1-84) as well as amino terminally truncated PTH fragments, that is, large carboxyterminal PTH fragments (C-PTH fragments). The present study investigated whether the use of the plasma PTH-(1-84)/C-PTH fragment ratio enhances the noninvasive assessment of bone turnover in patients on dialysis. Bone biopsies and blood samples for determinations of routine indices of bone turnover and PTH peptides were obtained in 51 adult patients on dialysis not treated with drugs affecting bone such as vitamin D or corticosteroids. Blood levels of large C-PTH fragments were calculated by subtracting PTH-(1-84) from "intact" PTH. Patients were classified according to their levels of bone turnover based on histomorphometrically obtained results of activation frequency. Prediction of bone turnover by the various blood indices was done by using proper statistical methods. In addition, hypercalcemia was induced by calcium gluconate infusion in a subset of patients, and levels of PTH-(1-84), "intact" PTH, and PTH-(1-84)/C-PTH fragment ratio were determined. The PTH-(1-84)/C-PTH fragment ratio was the best predictor of bone turnover. A ratio> 1 predicted high or normal bone turnover (sensitivity 100%), whereas a ratio <1 indicated a high probability (sensitivity 87.5%) of low bone turnover. Calcium infusion resulted in decrease in PTH-(1-84)/C-PTH fragment ratio. The PTH-(1-84)/C-PTH fragment ratio predicts bone turnover with acceptable precision for biological measurements. Moreover, a change in serum calcium levels is one of the regulators of the relative amount of circulating PTH-(1-84) and its large C-PTH fragments.

Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTH-related peptide receptor.

Even if the carboxyl-terminal (C-) fragments/intact (I-) PTH ratio is tightly regulated by the ionized calcium (Ca(2+)) concentration in humans and animals, in health and in disease, the physiological roles of C-PTH fragments and of the C-PTH receptor remain elusive. To explore these issues, we studied the influence of synthetic C-PTH peptides of various lengths on Ca(2+) concentration and on the calcemic response to human (h) PTH-(1-34) and hPTH-(1-84) in anesthetized thyroparathyroidectomized (TPTX) rats. We also looked at the capacity of these PTH preparations to react with the PTH/PTHrP receptor and with a receptor for the carboxyl (C)-terminal portion of PTH (C-PTH receptor) in rat osteosarcoma cells, ROS 17/2.8. The Ca(2+) concentration was reduced by 0.19 +/- 0.03 mmol/liter over 2 h in all TPTX groups. Infusion of solvent over 2 more h had no further effect on the Ca(2+) concentration (-0.01 +/- 0.01 mmol/liter), whereas infusion of hPTH-(7-84) or a fragment mixture [10% hPTH-(7-84) and 45% each of hPTH-(39-84) and hPTH-(53-84)] 10 nmol/h further decreased the Ca(2+) concentration by 0.18 +/- 0.02 (P<0.001) and 0.07+/-0.04 mmol/liter (P< 0.001), respectively. Infusion of hPTH-(1-84) or hPTH-(1-34) (1 nmol/h) increased the Ca(2+) concentration by 0.16 +/- 0.03 (P < 0.001) and 0.19 +/- 0.03 mmol/liter (P < 0.001), respectively. Adding hPTH-(7-84) (10 nmol/h) to these preparations prevented the calcemic response and maintained Ca(2+) concentrations equal to or below levels observed in TPTX animals infused with solvent alone. Adding the fragment mixture (10 nmol/h) to hPTH-(1-84) did not prevent a normal calcemic response, but partially blocked the response to hPTH-(1-34), and more than 3 nmol/h hPTH-(7-84) prevented it. Both hPTH-(1-84) and hPTH-(1-34) stimulated cAMP production in ROS 17/2.8 clonal cells, whereas hPTH-(7-84) was ineffective in this respect. Both hPTH-(1-84) and hPTH-(1-34) displaced (125)I-[Nle(8,18),Tyr(34)]hPTH-(1-34) amide from the PTH/PTHrP receptor, whereas hPTH-(7-84) had no such influence. Both hPTH-(1-84) and hPTH-(7-84) displaced (125)I-[Tyr(34)]hPTH-(19-84) from the C-PTH receptor, the former preparation being more potent on a molar basis, whereas hPTH-(1-34) had no effect. These results suggest that C-PTH fragments, particularly hPTH-(7-84), can influence the Ca(2+) concentration negatively in vivo and limit in such a way the calcemic responses to hPTH-(1-84) and hPTH-(1-34) by interacting with a receptor different from the PTH/PTHrP receptor, possibly a C-PTH receptor.