Parathyroid Hormone Fragments Research Articles

Intraoperative parathyroid hormone measurement pitfalls: parathyroid hormone spikes with carboxyl-terminal parathyroid hormone fragments in primary hyperparathyroidism—a case report

BackgroundIntraoperative parathyroid hormone (IOPTH) monitoring is a critical surgical adjunct for determining the extent of surgery for primary hyperparathyroidism (PHPT), with reported false-positive and false-negative rates of up to 10%. Surgeons must understand the parathyroid hormone (PTH) dynamics and select the appropriate IOPTH protocol and interpretation criteria for curative surgery.Case presentationWe present the case of a 64-year-old woman with a large cystic parathyroid tumor and PHPT who experienced a significant delay in IOPTH decrease but was cured without additional surgery. The patient’s basal intact PTH was 96.2 pg/mL, which decreased to 93.3 pg/mL at 25 min and 72.4 pg/mL at 55 min after removal of the parathyroid tumor. In an attempt to elucidate its pathophysiology, 1–84 PTH levels were measured in stored serum. These results can also be attributed to the relatively low basal PTH levels, intact PTH spike, and high ratio of large carboxyl-terminal PTH fragments present. The patient had normal intact PTH and calcium levels at the 9-month postoperative visit.ConclusionsAs detailed reports on these phenomena are scarce, we discuss the causes of false-negative IOPTH results in terms of PTH production, secretion, metabolism, and differences in measurement methods to avoid unnecessary surgery.

Interaction between p21-activated kinase 4 and β-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total β-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with β-catenin, and disruption of β-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with β-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Diagnostic pitfalls and therapeutic challenges of hypercalcaemia in chronic kidney disease: a case report and a narrative review

BackgroundEvaluation of hypercalcaemia in a patient with chronic kidney disease (CKD) is challenging, especially in low-resource settings. Hormone assays should be interpreted with caution as CKD affects both parathyroid hormone (PTH) and vitamin D. Therapies such as bisphosphonates are contraindicated in CKD, while fluid resuscitation can lead to volume overload. We report the diagnostic workup of a patient with stage V CKD who presented with symptomatic hypercalcaemia and discuss the diagnostic pitfalls and therapeutic challenges.Case presentationA 72-year-old Sri Lankan woman with stage V, non-oliguric CKD presented with a 2-week history of worsening lassitude, increased thirst and constipation. She was clinically euvolemic and did not have signs of uraemia. Bilateral lung fields had occasional coarse crepitations. The rest of the physical examination was normal. Her serum creatinine level was similar to her baseline (4.7 mg/dl, eGFR 9 ml/min). She was found to have a high serum calcium (14.3 mg/dl) and phosphate (5.0 mg/dl) levels. Her PTH level was 24.1 pg/ml (15–68), and she was deficient in 25-hydroxycholecalciferol (9 mg/ml (30–100)). She was not on calcium or vitamin D supplementation. Disseminated tuberculosis was diagnosed after detecting granulomata in the lungs and abdomen in the contrast-enhanced computed tomography (CECT) and mycobacterial DNA in sputum. She was hydrated with 0.9% NaCl with meticulous use of frusemide. The effect of frusemide waned off by the 10th day, requiring haemodialysis to control the hypercalcaemia. Vitamin D was replaced intramuscularly with 200,000 IU, after which the calcium levels increased. She was treated with IV pamidronate 30 mg, and the calcium levels started reducing drastically. Antituberculous therapy (ATT) was initiated 7 days after pamidronate treatment. The calcium levels normalised 2 days after ATT and sustained beyond 2 months.ConclusionInterpretation of PTH and phosphate levels should be done with caution when evaluating hypercalcaemia in patients with advanced chronic kidney disease. First- and second-generation assays detect PTH fragments which accumulate in CKD, leading to false positives. Hypophosphataemic effects of PTH/PTHrP can be masked by accumulation of phosphate in CKD. Bisphosphonates might have a role in treating calcitriol-induced hypercalcaemia, although this needs further evaluation.

A-042 Comparison of the Second-Generation Parathyroid Hormone Assays: ELSA-PTH and Alinity i Intact PTH

Abstract Background Measurement of parathyroid hormone (PTH) level is essential in diagnosing and treating hyper- and hypoparathyroidism. Because of the various presenting forms of PTH (biologically active full-length PTH and also PTH fragments) in the blood and the lack of standardization, there can be a difference in results between the assays even when the same assay generation is used. We compared the two second-generation PTH assays, ELSA-PTH and Alinity i Intact PTH, and evaluated the analytical performance of Alinity i Intact PTH, newly adopted in the laboratory. Methods For method comparison, 56 remnant samples of routinely ordered sera for the PTH test were collected. ESLA-PTH assay tested on Gamma-10 contains the anti-[39-84, amino acids] PTH antibody coated on the tube, which captures PTH in the sample and is measured by the radioactivity through the bound I125-labelled anti-[1-34] PTH antibody. Alinity i Intact PTH assay performed on Alinity i system uses the anti-[39-84] PTH antibody coated on a paramagnetic microparticle and is analyzed by the emitted light through the captured acridinium-labeled anti-[1-34] PTH antibody. The correlation between the two tests was evaluated. Additionally, precision, linearity, and reference range verification were analyzed to investigate the performance of the Alinity i Intact PTH assay. Results Of the 56 samples, 35 (62.5%) were requested by the Nephrology department for evaluating chronic kidney disease, 16 (28.6%) by the General surgery department during and after parathyroid and thyroid surgery, and 5 (8.9%) by other departments. 52.3% of the patients were female, and the median age was 61.5 years (interquartile range: 45.3–71). The median values of ELSA-PTH and Alinity i Intact PTH were 69.0 pg/mL and 101.9 pg/mL, respectively, and the PTH values of Alinity i Intact PTH were significantly higher (P < 0.0001) by the Wilcoxon test. The Pearson correlation coefficient (r) was 0.959, and the average difference between the two tests was 78.57 pg/mL. In Alinity i Intact PTH, the coefficient of variation obtained at each low (10.5 pg/mL), medium (66.6 pg/mL), high (254.6 pg/mL) levels were 1.9%, 2.4%, and 1.8% for repeatability, and 2.03%, 2.71%, and 2.65% for within-laboratory precision, respectively. Linearity was observed within the analytical measuring range of 3–3000 pg/mL. The reference range (15–68.3 pg/mL) recommended by the manufacturer was validated with 20 normal sera. Conclusion The PTH values of Alinity i Intact PTH were significantly higher than those of the ELSA-PTH, and both assays showed a strong correlation. Additionally, the Alinity i Intact PTH assay demonstrated acceptable analytical performance.

Combined and sequential therapies with anabolic and antiresorptive drugs in the management of patients with postmenopausal osteoporosis

The aim of osteoporosis therapy is to reduce the increased fracture risk associated with osteoporosis-related bone fragility. Prevention of fragility fracture relies on balanced nutrition, weight-bearing and balance-improving physical exercises, and pharmacological therapies. Among the latter, the antiresorptive drugs are the most widely used. Bone formation stimulators (anabolics) are second line-therapy with reversible effects once discontinued. For patients at very high risk or imminent risk of fracture, the question arises of whether combining drugs with different modes of action, or using sequential regimens with these agents, could achieve early, higher-magnitude antifracture efficacy than is obtained with usual antiresorptives, and sustained antifracture efficacy. As regards combination therapies, until we have clear evidence that using drugs together provides greater fracture risk reduction than monotherapy, these therapies are not recommended. Anabolic treatments like teriparatide, the amino-terminal fragment of parathyroid hormone, abaloparatide, an analog of parathyroid hormone related protein, and romosozumab, the monoclonal antibody against sclerostin, decrease vertebral and non-vertebral fracture risk and are more efficacious in fracture risk reduction than antiresorptives, as shown in head-to-head trials. However, an anabolic agent should be followed by an antiresorptive drug to maintain and even further increase its antifracture efficacy, which is otherwise rapidly reversible. Because of their early, high-magnitude and sustained antifracture efficacy, such sequential regimens should become the standard of care for patients at very high or imminent risk of fracture.

Whole-Body Vibration Therapy as a Modality for Treatment of Senile and Postmenopausal Osteoporosis: A Review Article.

Whole-body vibration therapy is an intentional biomechanical stimulation of the body using various frequencies of vibrations with the motive of health improvement. Ever since its discovery, this therapy has been extensively used in physiotherapeutic measures and the sports industry. For its property of increasing bone mass and density, space agencies use this therapy on astronauts who return to Earth after long-term space missions to regain lost bone and muscle mass. The potential of this therapy to restore bone mass encouraged researchers to look for its scope in the treatment of age-related bone degenerative diseases such as osteoporosis and sarcopenia, as well as in the correction of posture control and gait in geriatrics and post-menopausal women. Osteoporosis and osteopenia account for roughly half of all fractures worldwide. These degenerative diseases also cause gait and posture changes. Bisphosphonates, monoclonal antibodies, parathyroid hormone fragments, hormone replacement therapies, and calcium and vitamin D supplementation are among the medical treatments available. Lifestyle changes and physical exercise are advised. However, vibration therapy's scope as a treatment option is yet to be explored. The safe range of frequency, amplitude, duration, and intensity of the therapy is still to be determined. This article is a review of the results of various clinical trials done in the last 10 years that target the effect of vibration therapy in both osteoporotic women and the elderly for the treatment of such ailments and deformities. We collected data from PubMed using advanced search and applied the exclusion criteria. In total, we analyzed nine clinical trials.

Calcium-Regulating Hormonal System and HMGB1 in Cardiomyopathies.

Calcium ions play a key role in the heart's functional activity. The steadystate levels of calcium are contingent on the calcium regulating hormonal system, impairment of which might result in the development of cardiac pathology. An important role in these processes is also attributed to the specific inflammatory mediator, HMGB1, one of the damage-associated molecular patterns (DAMPs) released by immune cells or cell damage. This study investigated the cardioprotective potential of the calcium-regulating hormonal system in cardiomyopathies with an emphasis on the possible role of HMGB1. Ca2+ and inorganic phosphate levels were determined in the serum using an electrolyte analyzer and spectrophotometric analyzer correspondingly. The 1-34 fragment of parathyroid hormone (PTH), calcitonin, vitamin D, and HMGB1 were detected using ELISA kits. The levels of PTH, calcitonin, phosphate, and HMGB1 were found elevated in females suffering from cardiomyopathy. The same tendency was observed in men; however, statistically significant changes were registered only for PTH and phosphate. It can be suggested that among other reasons, the decrease of the left ventricular function in cardiomyopathy patients can be linked to the high HMGB1, whereas the activation of the calciumregulating system as manifested by the elevated PTH aims at restoration of calcium homeostasis and thus have positive, i.e. cardioprotective consequences.

P8.033: Teriparatide in Kidney Transplant Patients With Bisphosphonate-Resistent Osteoporosis

Introduction: Bisphosphonates increase Bone mineral density (BMD) by suppressing bone turnover inhibiting osteoclastic function with risk of inducing adynamic bone disease. Teriparatide is a fragment of the natural human parathyroid hormone (PTH) consisting of the first 34 amino acids counting from the N-terminus end of the natural PTH. Is anabolic drugs stimulate bone formation by increasing osteoblast activity and regulates both bone formation and resorption reducing the risk of vertebral and nonvertebral fractures. Arm: Teriparatide (Parathormone 1-34) treatment in renal trasplant patients with bisphosphonate-resistent osteoporosis.Metods: observational describe study of cases series of 10 kidney transplant patients with bone fragility without response to prolonged treatment with bisphosphonates (greater than 3 years). They were treated with daily subcutaneous injections of 20 microg teriparatide (PTH 1-34) by 18 months. All the patients had 6 months of bisphosphonate wash out. Measurements: we investigated bone turnover markers and effect of teriparatide over BMD of the femoral neck and lumbar spine as well also fractures, graft rejection, cardiovascular events, death and drug related adverse event. Results: 10 patients 9 women, mean age 64.4±14.15 years, BMI 24.3 ±2.64, etiology 3GMN, 3 PKD, 3 unknown, 1 NDBT, HD 9, preempive 1, transplant Time 10.12±5.21 years, 1 preemptive, 8 DBD and 1 LRD, Induction: 8 thymoglobulin, 2 basiliximab, Maintenance 5 belatacept/mycophenolate/MTP, 2 rapamune/mycophenolate/MTP, 3 tacrolimus/mycophenolate/MTP, 6 patients with fractures prior to treatment. Mean of treatment with bisphosphonates 4.1±1.79 years. It is observed at 18 months of treatment with 1-34 PTH increased levels of bone alkaline phosphatase (13.26 UI/L ± 5.75 vs 17.90 ± 7.28, p = 0.045), with NS changes in the rest of the analytes (Table 1) and increase of lumbar bone density of 11.27% (0.8816 gr/cm2 (TS-2.6) vs 0.9941 gr/cm2 (TS-1.38)) and femoral neck of 7.07% (0.6752 gr/cm2(TS-2.5) vs 0.7230 gr/cm2(TS-2.19)). 2 patients presented wrist fractures during treatment with low response(lumbar BMD) to treatment. No cardiovascular events, rejections, deaths were reported. Adverse Events: 1 patient presented hypotension during the first month of treatment. Conclusion: In this group of patients, and adequate response to teriparatide treatment was observed however our limitation were small number of patients and bone biopsy prior teriparatide treatment.

Quantification of Anti-Osteoporotic Anabolic Peptide in Stealth Lipid Nanovesicles Through Validated RP-HPLC Method.

Teriparatide is a recombinant fragment of human parathyroid hormone, a potent osteoanabolic agent used for osteoporosis. The present study endeavored to develop a simple, rapid, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of teriparatide in pegylated lipid nanovesicles for rapid formulation development/optimization. A rapid RP-HPLC-based analytical method was developed for the quantification of teriparatide in pegylated lipid nanovesicles. The method was optimized on a Waters XBridge C18 (4.6 × 150 mm, 10 μm) column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile both in a linear gradient program. In the method, a short run time of 9 min was achieved at a flow rate of 1.0 mL/min with an injection volume of 50 µL at a detection wavelength of 210 nm. The developed method was validated according to the ICH Q2 (R2) guideline. The method was applied for the quantification of teriparatide in prepared pegylated lipid nanovesicles. Teriparatide encapsulated pegylated lipid nanovesicles were prepared by the ethanol injection method. Further, these vesicles were characterized for % entrapment efficiency (%EE), particle size, zeta potential, and morphology by Cryo-SEM. The teriparatide was eluting at 4.8 min in the run. Further, for the method validation, the linear relationship between concentration and response was established over the concentration range of 50-250 µg/mL with the R2 > 0.999. The method sensitivity was shown with LOD and LOQ with the value of 100 ng/mL and 500 ng/mL, respectively. The method was found to be accurate and precise with the recovery ranging in 100 ± 2% and RSD <2%, respectively. Minor deliberate changes proved the robustness of the developed method. These results indicated that the developed and validated method is accurate, precise, rapid, reliable, and fit for the quantification of teriparatide in different formulations. The RP-HPLC method was developed and validated for the quantification of teriparatide from novel pegylated lipid nanovesicles.

Intelligent and thermo-responsive Au-pluronic® F127 nanocapsules for Raman-enhancing detection of biomolecules

Thermo-responsive Raman-enhanced nanocapsules were successfully fabricated by Pluronic® F127 (F127) decorated with gold nanoparticles (AuNPs) for surface-enhanced Raman scattering (SERS) detection of biomolecules. F127 nanocapsules changes from hydrophilicity (swelling) to hydrophobicity (de-swelling) when the temperature increases from 15 °C to 37 °C, owing to the lower critical solution temperature (LCST) of F127 is about 26.5 °C. The size of nanocapsules would be enormous shrinking from 160 nm to 20 nm, resulting in a significant decrease in the distance between AuNPs to enhance hot spot effect, which increases the sensitivity of SERS detection. Based on the thermo-sensitive behavior, the ratio of AuNPs and F127 would be manipulated to find the optimal SERS enhancement effect. SERS nanocapsules can rapidly detect biomolecules (adenine and R6G) with limit of detection (LOD) lower than 10−6 M. In addition, the relatively difficult to detect clinical samples, carboxyl-terminal parathyroid hormone fragments (C-PTH), can also be measured by the thermo-responsive SERS nanocapsules developed in this work. It is expected the biomolecules can be adsorbed at low temperature (15 °C), as well as collected and concentrated at high temperature (37 °C) for SERS detection, to increase the sensitivity and stability of SERS detection.

Relationship between Body Anthropometric Measurement and Parathyroid Hormone in Female Subjects

The distribution of fat tissue is related to the risk of metabolic diseases. Parathyroid Hormone (PTH) is an essential hormone for calcium homeostasis. According to several types of research, body fat affects PTH levels. Currently, Body Mass Index (BMI) is not the only parameter needed to identify the body fat distribution in accordance with chronic disease risks such as Waist Circumference (WC), Waist to Hip Ratio (WHR), and Waist to Height Ratio (WHtR). The study aimed to determine the relationship between body anthropometric measurement and PTH. A cross-sectional study was performed on a healthy population of 75 healthy female volunteers with a BMI ≥ 23 kg/m2. Waist circumference, WHR, WHtR, and BMI measurements were carried out and followed by the PTH fragment 1-84 (PTH1-84) test. Data were analyzed using the Spearman test with a significance of p&lt;005. There was no significant correlation between PTH and WHR (r=0.057; p=0.628). There was weak correlation between PTH and BMI (r=0.268; p=0.020), WC (r=0.287; p=0.012) and WHtR (r=0.238; p=0.04). Body mass index, WC, and WHtR can be used as anthropometric parameters to determine PTH disorders

Quantification of Parathyroid Hormone and its Fragments in Serum by Liquid Chromatography-High-Resolution Mass Spectrometry.

Parathyroid hormone (PTH), an 84-amino acid peptide hormone, is a major regulator of calcium homeostasis. Quantification of PTH in serum is used clinically to investigate calcium imbalances and for monitoring osteodystrophy in patients with renal failure. In addition to intact PTH, several PTH fragments are found in circulation. Recent studies have shown that accurate quantification of PTH fragments may provide valuable clinical information in certain scenarios. In this chapter, a high-resolution mass spectrometry-based method for quantification of PTH (1-84) and its fragments is described. This method involves immunoaffinity capture of intact PTH and PTH-fragments followed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

Bioanalytical method development and validation of highly selective and sensitive LC-MS/MS method for determination of teriparatide (parathyroid hormone fragment 1–34) in human serum through direct detection of intact teriparatide molecule

Teriparatide is a novel recombinant peptide fragment of the first 1–34 amino acids of human parathyroid recommended for treatment of osteoporosis. Therapeutic proteins and peptides are routinely estimated using ligand binding assay formats however LC-MS/MS technique which is routinely used in bioanalysis of small molecules has now gained importance in large molecule bioanalysis for the advantages it can offer over LBAs in terms of improved accuracy, selectivity and anti-body free method development. This paper presents a sensitive bioanalytical method for determination of teriparatide in human serum using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. Teriparatide was isolated from human serum using solid phase extraction. The intact peptide was separated on a chromatograph and the multiply charged ion (+7) was detected using a mass spectrometer. The total run time was 4.0 min. The internal standard used was rat PTH 1–34 fragment. The mass transitions of m/z 589.3 > 656.3 for teriparatide and m/z 677.4 > 778.6 for internal standard were used for MS/MS detection. The sample extraction involved a solid phase extraction method followed by concentration of the eluent by evaporation and subsequent reconstitution. The non-specific binding effect caused by the adherence of the peptides/proteins to the vials/tube walls was significantly reduced by using BSA solution as blocking agent. The method has been validated over a linear range of 15.07–913.3 pg/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 6.36 to 10.85 and accuracy was within 96.71% to 100.88%. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between test and reference formulation (20 mcg/80 mL – solution for injection) and the method was successfully applied to quantify teriparatide in serum samples of this clinical study and about 1220 human serum samples were analyzed to determine teriparatide. This method is a promising anti-body free LC-MS/MS based methodology for estimation of teriparatide in human serum and may be applied as starting method for other such peptide molecules.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Improving the Accuracy and Reliability of Parathyroid Hormone Levels Through the Mass-Spectrometric Measurement of Full-length PTH and C-Terminal PTH Fragments

Parathyroid hormone (PTH) is a key biomarker for hypo/hyperparathyrioidism as well as chronic kidney disease, one of the leading health conditions in the USA. As a result, the most recent update of the Kidney Disease Improving Global Outcomes (KDIGO) guideline for CKD-MBD management emphasized the role of PTH as one of the key biomarkers of this disorder. The earlier stages of CKD generate few symptoms and only until the kidney is significantly impaired do patients begin experiencing signs of renal failure. Therefore, the measurement of parathyroid hormone (PTH) in serum and/or plasma is critical not only for the correct detection, diagnosis, and prevention of renal failure, but also calcium, phosphate, and vitamin D disorders. Current laboratory methods for PTH show high variability and inaccuracy, thus creating the need for a reference measurement procedure that can help laboratories and assay manufacturers improve their measurement accuracy and reliability to avoid the misclassification of patients. Due to the short half-life of PTH, this 84 amino-acid polypeptide hormone is produced at low circulating levels in normal conditions ranging from 10 - 65 pg/mL. In addition, N-terminal and C-terminal peptides, which have shown to interfere with clinical analyzer platforms, account for over 80% of all circulating PTH levels. Therefore, a highly specific and sensitive method is needed for the accurate detection of full-length PTH and these PTH fragments. There is clinical relevancy in the ability to measure C-terminal PTH fragments as the ratio of these PTH fragments to full-length PTH has been diagnostic for severe or end-stage renal disease, non-dynamic bone disease, and hyperparathyroid-associated bone loss. Bottom-up proteomics approaches that incorporate enzymatic digestion steps during sample preparation will result in the loss of information for the fragments. Therefore, a new, innovative, top-down proteomics method was developed to measure full-length PTH and its breakdown products (fragments) by mass spectrometry (UHPLC-HRMS). This method enabled, for the first time, the detection of full-length PTH at very low concentrations typically observed in patients with hypoparathyroidism as well as C-terminal fragments that may interfere with regular immunoassays typically used in patient care. The highly specific and sensitive method for PTH and related peptides in CKD patient sera demonstrated no interference from the internal standards and other PTH fragments. Therefore, the optimized method was applied for the screening of normal and CKD serum at various stages of disease progression. Preliminary results demonstrated that certain PTH fragments are correlated with eGFR and different stages of chronic kidney diseases (CKD). This method along with the respective findings from this study will help to improve the diagnosis, treatment, and prevention of CKD-MBD.

Surfactant‐modified parathyroid hormone fragments with high potency and prolonged action: Structure‐informed design using glycolipid surfactant conjugation

AbstractThe pharmaceutical properties of peptides (high potency and specificity with low toxicity) make them an attractive therapeutic class but typically short duration of action and low bioavailability can limit their usefulness. Here, we begin evaluation of a new class of peptide modification, glycolipid surfactant conjugation, designed to extend the half‐life of peptide therapeutics. This work illustrates ease of synthesis and conjugation, range of modulation of pharmacokinetic/pharmacodynamic behavior and acceptability in vivo of this approach. Proof of concept used parathyroid hormone and showed that an N‐terminal fragment can be modified, informed by the parathyroid hormone receptor x‐ray structure, to produce high potency, enhanced intrinsic efficacy and prolonged action in vivo. This suggests daily (hypoparathyroidism) or weekly (osteoporosis) administration with biological sequelae (stem cell maturation to osteoblasts) throughout the week. Compound 7 was chosen for advanced study. Exploration of the physical properties and development potential of glycolipid surfactant‐modified peptides are underway with additional peptide therapeutics.

Parathyroid Hormone Concentrations in Maintenance Hemodialysis: Longitudinal Evaluation of Intact and Biointact Assays

Rationale & ObjectiveManagement of chronic kidney disease mineral and bone disorder requires parathyroid hormone (PTH) concentrations. “Biointact” PTH immunoassays detect “whole” PTH (wPTH), whereas “intact” immunoassays measure PTH plus PTH fragments (iPTH). We aimed to determine whether longitudinal changes in PTH concentrations can be evaluated using biointact and intact immunoassays alike.Study DesignOpen noninterventional longitudinal cohort study.Setting & ParticipantsPTH concentrations were measured quarterly up to 5 times in 102 hemodialysis patients.Predictors & Tests ComparedAge, sex, phosphate levels, and others as clinical predictors for PTH trend. Tests compared were iPTH immunoassays from Siemens and Roche and wPTH immunoassays from Roche and DiaSorin.OutcomesPTH concentration trend; regression equations; test bias.Analytical ApproachPredictive regression-to-the-mean model for PTH slope; Bland-Altman plots, Passing-Bablok regression, and reference change values for test comparisons.ResultswPTH concentrations were similar with both immunoassays (wPTH-Roche = 11.7 + 0.97 × wPTH-DiaSorin, r = 0.99; mean ± 1.96 SD bias, 8.2 ± 43.3 pg/mL [17.5% ± 40.9%], by Bland-Altman plots). iPTH-Siemens concentrations were higher than iPTH-Roche concentrations (iPTH-Siemens = −5.4 + 1.33 × iPTH-Roche, r = 0.99; mean ± 1.96 SD bias, 84.0 ± 180.2 pg/mL [21.1% ± 29.8%], by Bland-Altman plots). iPTH-Roche and iPTH-Siemens concentrations were 2- and 2.5-fold higher than wPTH concentrations, respectively. Full agreement among all 4 immunoassays in detecting both significant and insignificant changes in PTH concentrations, upward or downward from one quarter to the next, was reached in 87% of consecutive measurements. In a predictive model, baseline PTH concentrations > 199 pg/mL (wPTH-Roche), 204 pg/mL (wPTH-DiaSorin), 386 pg/mL (iPTH-Roche), and 417 pg/mL (iPTH-Siemens) correctly predicted declining PTH concentration trend in 62% to 68% of patients, but age, sex, hemodialysis vintage, and calcium and phosphate levels were no significant predictors.LimitationsLimited number of immunoassays, only 59 patients attended all quarterly samplings.ConclusionswPTH-Roche and wPTH-DiaSorin concentrations were similar, while iPTH was higher than wPTH concentrations. The iPTH-Siemens immunoassay is either higher calibrated or detects more fragments than iPTH-Roche. However, longitudinal PTH concentration changes largely coincided with all tested immunoassays.

Chemical Characterization and Quantitation of Circulating Intact Parathyroid Hormone and Parathyroid Hormone Fragments by High-Resolution Mass Spectrometry in Chronic Renal Failure

Chemical Characterization and Quantitation of Circulating Intact Parathyroid Hormone and Parathyroid Hormone Fragments by High-Resolution Mass Spectrometry in Chronic Renal Failure

SAT-LB64 Teriparatide and Its Bone Healing Power

Introduction:Teriparatide, a parathyroid hormone analog, is an important anabolic agent approved by the U.S. Food and Drug Administration to increase bone mineral density in osteoporotic patients. Parathyroid hormone (PTH) regulates calcium, phosphate, and active vitamin-D metabolites.The amino terminal peptide fragments of PTH has been known to increase bone mass and are being used in clinical practice for osteoporosis management (1).Current literature shows the efficacy of teriparatide in increasing bone density of lumbar spine and femoral neck, and decreasing the risk of vertebral and non-vertebral fractures both in postmenopausal women and men. It is also known to prevent fractures in patients with osteoporosis and promote healing of fractures (2).Case Description:A 79-year-old Hispanic female with history of osteopenia and major lumbar spine wedge compression fractures presented to our clinic for consultation. She was on ibandronate for the past four months and was having symptoms of pill esophagitis. Her last bone mineral density done on August 2017 revealed T-score of -2.5 at the lumbar spine, -1.5 at the left femoral neck, and 3.3% bone loss on the left femoral head. Rather than being started on teriparatide, zoledronic acid, or denosumab, she continued ibandronate along with calcium and vitamin D. Two months after the initial consultation, she sustained a traumatic fracture of the posterior arch and body of C2 bilaterally following a motor vehicle accident. There were discussions about starting anabolic treatment, as serial imaging did not show any significant improvement in the healing process despite the use of a collar. Two months after sustaining C2 fracture, she was started on teriparatide. Repeat cervical spine x-ray three months later showed complete healing of the C2 fracture.Discussion:There are a limited number of cases reported in regards to teriparatide induced healing of non-osteoporotic fractures (3). Our case is one of the very few reported to have shown complete radiographic and clinical healing of a traumatic, non-osteoporotic fracture after use of teriparatide for 12 weeks.

Parathyroid Hormone Measurement in Chronic Kidney Disease: From Basics to Clinical Implications.

Accurate measurement of parathyroid hormone (PTH) is crucial for therapeutic decision-making in patients with chronic kidney disease-mineral and bone disorder (CKD-MBD). The second-generation PTH assays, often referred to as “intact PTH” assays, are the current standard and most available assays in clinical practice. However, intact PTH assays measure both full-length biologically active PTH and heterogeneous PTH fragments in the circulation, providing the equivocal value of PTH measurement in patients with CKD-MBD. Due to the variability of PTH assays, preanalytical sample errors, and the phenomenon of end-organ PTH hyporesponsiveness, current CKD-MBD guidelines recommend a wide range for serum PTH targets (2–9 the upper normal limit of the intact PTH assay) in dialysis patients to diminish the risk of developing adynamic bone disease. Nevertheless, a sizeable proportion of CKD patients still experience renal osteodystrophy despite having serum PTH levels within the recommended range. The primary cause of this inconsistency is the analytical interference of various PTH fragments and oxidized PTH forms that considerably accumulate in CKD patients. Therefore, a new mass spectrometry-based assay, which is capable of specifically measuring the whole spectra of PTH fragments, can potentially improve diagnostic accuracy for renal osteodystrophy. However, the effects of different PTH fragments on bone metabolism, vascular calcification, and mortality in CKD patients warrant further research.