PTH (1-84) N15 Human

What is PTH (1-84) N15 Human recombinant protein and how is it produced?

Parathyroid Hormone (1-84) N15 Human recombinant protein is a stable isotope-labeled version of the full-length 84-amino acid parathyroid hormone. It is produced in Escherichia coli expression systems as a single, non-glycosylated polypeptide chain . The recombinant protein undergoes multiple purification steps, including RP-HPLC and SDS-PAGE analysis, to achieve a purity greater than 97.0% . The N15 labeling refers to the incorporation of the stable nitrogen-15 isotope throughout the protein structure, which makes it particularly valuable for tracking studies and mass spectrometry applications in research settings. The protein is typically formulated as a sterile filtered white lyophilized powder and is reconstituted before use .

What is the amino acid sequence of PTH (1-84) N15 and how does it relate to its biological function?

The full amino acid sequence of PTH (1-84) N15 Human recombinant protein is: SVSEIQLMHN LGKHLNSMER VEWLRKKLQD VHNFVALGAP LAPRDAGSQR PRKKEDNVLV ESHEKSLGEA DKADVNVLTK AKSQ . This sequence corresponds to the complete 84-amino acid native human parathyroid hormone, which is critical for its full biological activity. The biological activity of the recombinant protein is typically measured using the UMR106 cell/cAMP method, corresponding to a specific activity of approximately 9,000 Units/mg . The intact PTH (1-84) molecule contains regions that interact with PTH receptors, particularly in bone and kidney tissues, to regulate calcium homeostasis. While the truncated PTH (1-34) fragment contains the primary receptor-binding region, the full-length PTH (1-84) exhibits different pharmacokinetics and potentially different biological effects, making it valuable for comprehensive research applications .

How should PTH (1-84) N15 be stored and handled to maintain its stability?

The proper storage and handling of PTH (1-84) N15 are critical for maintaining its biological activity. The lyophilized protein should be stored desiccated below -18°C, although it remains stable at room temperature for up to 3 weeks . Upon reconstitution, PTH (1-84) N15 should be stored at 4°C if used within 2-7 days . For longer-term storage post-reconstitution, the solution should be kept below -18°C . To enhance stability during storage, it is recommended to add a carrier protein such as 0.1% HSA (Human Serum Albumin) or BSA (Bovine Serum Albumin) . It is critical to prevent freeze-thaw cycles as these can significantly degrade the protein structure and function . For reconstitution, it is recommended to use sterile water or appropriate buffer solutions to achieve a concentration not less than 100 μg/ml, which can then be further diluted to other aqueous solutions as needed for experimental protocols .

How can PTH (1-84) N15 be used in mass spectrometry-based research applications?

PTH (1-84) N15 Human recombinant protein serves as an excellent internal standard for mass spectrometry-based quantitative proteomics due to its stable isotope labeling. Researchers can utilize the N15-labeled protein alongside unlabeled samples to achieve accurate quantification of endogenous PTH levels. The experimental protocol typically involves:

• Adding a known quantity of PTH (1-84) N15 to biological samples before processing

• Processing both the N15-labeled internal standard and the unlabeled endogenous protein identically

• Analyzing the samples using liquid chromatography-mass spectrometry (LC-MS/MS)

• Identifying peptides by their mass-to-charge ratios

• Calculating the ratio between labeled and unlabeled peptide signals to determine absolute quantities

The mass shift created by the N15 labeling (approximately 1 Da per nitrogen atom) allows for clear discrimination between the internal standard and endogenous protein while maintaining identical chemical properties and chromatographic behavior . This approach is particularly valuable in clinical research studying PTH metabolism, clearance rates, and pharmacokinetics in various disease states.

What experimental models are appropriate for studying PTH (1-84) N15 biological activity?

Several experimental models have been validated for studying the biological activity of PTH (1-84) N15:

• Cell-based assays: The UMR106 rat osteosarcoma cell line is the gold standard for measuring PTH activity through cAMP production. This method confirms the specific activity of approximately 9,000 Units/mg for recombinant PTH (1-84) N15 .

• Animal models: Hypoparathyroid animal models allow for the investigation of PTH replacement effects on calcium homeostasis, bone metabolism, and organ systems.

• Clinical studies: Human trials have demonstrated that PTH (1-84) administration can maintain serum calcium levels while reducing or eliminating requirements for calcium and active vitamin D supplementation in patients with hypoparathyroidism .

When designing experiments, researchers should consider the administration route, dosing frequency, and concentration. Clinical trials have established that subcutaneous injection into the thigh is an effective administration route, with pharmacokinetic data showing that PTH (1-84) has a longer half-life (approximately 2.5-3 hours) compared to the truncated PTH (1-34) fragment (75 minutes) . This allows for less frequent dosing in experimental protocols.

How can PTH (1-84) N15 be used as a reference standard in PTH assay development?

PTH (1-84) N15 Human recombinant protein offers significant advantages as a reference standard in assay development due to its high purity and stable isotope labeling. The methodological approach for utilizing PTH (1-84) N15 in assay development includes:

• Calibration curve generation: Creating a series of dilutions of PTH (1-84) N15 to establish a standard curve with known concentrations

• Assay validation: Determining assay parameters including limit of detection, linear range, precision, and accuracy using the N15-labeled standard

• Specificity testing: Evaluating cross-reactivity with PTH fragments and other related molecules

• Matrix effect assessment: Analyzing how biological matrices affect assay performance by comparing recovery of the N15-labeled standard

The high purity (>97%) of the recombinant protein ensures reliable standardization . The N15 labeling enables the development of isotope dilution mass spectrometry methods, which are considered reference methods for accurate quantification. This is particularly valuable for clinical research laboratories developing harmonized assays for measuring PTH levels in conditions such as hypoparathyroidism, hyperparathyroidism, and chronic kidney disease.

How does the pharmacokinetic profile of PTH (1-84) differ from PTH (1-34), and what are the research implications?

Research has revealed significant differences in the pharmacokinetic profiles between PTH (1-84) and the truncated PTH (1-34) fragment, with important implications for experimental design:

PTH (1-34) exhibits a shorter half-life with circulating peak concentration reached by 30 minutes after injection and an exponential clearance with a half-life of approximately 75 minutes . In contrast, PTH (1-84) demonstrates a slower pharmacokinetic profile with an approximate half-life of 2.5-3 hours . These differences result in distinct administration requirements in research protocols: PTH (1-34) typically requires multiple daily doses, while PTH (1-84) can be administered daily or even every other day in some experimental models .

The pharmacokinetic studies by Clarke and colleagues demonstrated that plasma PTH levels increased rapidly after administration of PTH (1-84) 50 or 100 μg, with levels declining to predose measurements after approximately 12-24 hours . The extended half-life of PTH (1-84) may provide more sustained effects on target tissues compared to PTH (1-34), potentially influencing outcomes in bone metabolism studies and calcium homeostasis research. When designing longitudinal studies, researchers should consider these pharmacokinetic differences to optimize dosing regimens for the specific research questions being addressed.

What are the methodological considerations for studying PTH (1-84) effects on bone metabolism?

Investigating the effects of PTH (1-84) on bone metabolism requires careful methodological planning with multiple complementary techniques:

• Bone density measurements:

  • Dual-energy X-ray absorptiometry (DXA) to measure areal bone mineral density

  • Quantitative computed tomography (QCT) to assess volumetric bone density

  • Research indicates that PTH (1-84) therapy results in different effects depending on skeletal site, with potential increases at the lumbar spine and decreases at peripheral sites

• Bone turnover markers:

  • Measure serum markers of bone formation (e.g., P1NP, bone-specific alkaline phosphatase)

  • Assess markers of bone resorption (e.g., CTX, NTX)

  • Studies show PTH (1-84) can increase bone turnover markers up to 13-fold above baseline values

• Bone histomorphometry:

  • Bone biopsy specimens for structural and dynamic analysis

  • Tetracycline labeling to assess bone formation rates

  • Evaluation of trabecular and cortical bone parameters

• Molecular signaling studies:

  • Analysis of Wnt signaling pathway activation, which is involved in the osteoanabolic effects of PTH administered intermittently in lower doses

  • Investigation of RANKL/OPG ratio modulation by PTH treatment

Research protocols must consider the paradoxical effects of PTH: continuous administration can be catabolic to bone, while intermittent administration at lower doses can be osteoanabolic . The balance between these effects depends on dosing regimen, frequency, and underlying bone metabolism status of the experimental model.

What are the research findings regarding PTH (1-84) as a treatment for hypoparathyroidism, and what methodological approaches were used?

Several key clinical studies have investigated PTH (1-84) as a treatment for hypoparathyroidism using rigorous methodological approaches:

• Sikjaer et al. (2011):

  • Study design: Randomized, double-blind, placebo-controlled trial with 62 subjects aged 25-80 years

  • PTH dosing: 100 μg daily for 24 weeks

  • Key findings: PTH (1-84) reduced calcium and active vitamin D requirements by 75% and 73%, respectively

  • Bone effects: Significantly decreased BMD at the lumbar spine by 1.8% and at the hip by 1.6%; volumetric bone density at the lumbar spine significantly increased by 12.2%

  • Bone turnover: Markers increased up to 13-fold above baseline values

• Cusano et al. (2013):

  • Study design: Open-label study with 27 subjects aged 25-68 years

  • PTH dosing: 100 μg every other day for 4 years

  • Key findings: PTH (1-84) reduced calcium and active vitamin D requirements by 37% and 45%, respectively

  • Bone effects: Significantly increased BMD at the lumbar spine by 5.5%; forearm showed a significant decline of 2.0% at 2 years; hip unchanged

  • Biomarkers: Bone turnover markers significantly increased up to threefold above baseline at 6-12 months

• Mannstadt et al. (2013) - REPLACE trial:

  • Study design: Randomized, double-blind, multicenter trial with 134 subjects aged 18-85 years

  • PTH dosing: Starting at 50 μg daily, titrated to biochemistries, for 24 weeks

  • Key findings: PTH (1-84) reduced calcium and active vitamin D requirements by 52% and 78%, respectively

  • Primary outcome: 53% of PTH-treated subjects versus 2% of placebo subjects achieved the primary outcome (≥50% reduction in oral calcium and active vitamin D with normalized serum calcium)

These studies employed rigorous methodological approaches including randomization, blinding, placebo controls, and long-term follow-up. They utilized comprehensive assessment techniques including biochemical markers, bone density measurements, and quality of life assessments. The findings consistently demonstrate that PTH (1-84) replacement therapy can maintain serum calcium while reducing supplemental calcium and active vitamin D requirements in hypoparathyroidism .

What are the common challenges in PTH (1-84) N15 reconstitution and how can they be addressed?

Researchers working with PTH (1-84) N15 may encounter several challenges during reconstitution that can affect experimental outcomes:

• Protein aggregation: The hydrophobic regions of PTH can promote aggregation during reconstitution.

  • Solution: Reconstitute slowly with gentle swirling rather than vigorous shaking

  • Add carrier proteins (0.1% HSA or BSA) to prevent protein-surface interactions

  • Maintain concentration above 100 μg/ml during initial reconstitution

• Activity loss during storage: Repeated freeze-thaw cycles can substantially reduce biological activity.

  • Solution: Prepare single-use aliquots immediately after reconstitution

  • Store aliquots at -20°C or below for long-term storage

  • Use temperature-controlled thawing procedures

• Buffer incompatibility: Some buffer components may affect protein stability or downstream applications.

  • Solution: Initially reconstitute in sterile water or 1xPBS (pH 7.4) as recommended

  • If alternative buffers are required for specific experiments, perform gradual buffer exchange using dialysis or desalting columns

• Adsorption to containers: PTH can adsorb to plastic surfaces, leading to concentration inconsistencies.

  • Solution: Use low-binding microcentrifuge tubes and pipette tips

  • Pre-coat containers with carrier proteins

  • Account for potential losses when calculating working concentrations

Following these methodological recommendations will help ensure consistent protein quality and experimental reproducibility when working with PTH (1-84) N15 recombinant protein.

How can researchers troubleshoot unexpected results in PTH (1-84) N15 biological activity assays?

When researchers encounter unexpected results in PTH (1-84) N15 biological activity assays, a systematic troubleshooting approach is essential:

• Protein integrity verification:

  • Run SDS-PAGE to confirm protein size and integrity

  • Verify concentration using spectrophotometry or protein assay methods

  • Consider mass spectrometry analysis to confirm intact N15 labeling

• Cell-based assay optimization:

  • Verify UMR106 cell responsiveness using a positive control

  • Confirm proper cell passage number and culture conditions

  • Check cAMP detection reagent quality and shelf-life

  • Include proper negative controls and vehicle-only treatments

  • Establish a standard curve with each experiment

• Receptor sensitivity analysis:

  • Test multiple concentrations to determine EC50 values

  • Investigate potential receptor desensitization from previous PTH exposure

  • Evaluate expression levels of PTH receptors in the cell model

• Interfering factors investigation:

  • Examine buffer components for potential interference

  • Test for presence of proteases that might degrade PTH

  • Evaluate interactions with other experimental compounds

The expected activity of PTH (1-84) N15 should correspond to approximately 9,000 Units/mg as determined by the UMR106 cell/cAMP method . Significant deviations from this value warrant careful methodology review using the systematic approach outlined above.

What methodological approaches can address variability in PTH (1-84) N15 isotope incorporation?

Variability in N15 isotope incorporation can impact the accuracy of quantitative studies using PTH (1-84) N15 as an internal standard. Researchers can implement several methodological approaches to address this challenge:

These methodological approaches ensure that variability in isotope incorporation does not compromise experimental results when using PTH (1-84) N15 as a quantitative standard in proteomics or pharmacokinetic studies.

What are the emerging research applications for PTH (1-84) N15 in metabolic bone disease studies?

Several innovative research applications for PTH (1-84) N15 are emerging in the field of metabolic bone disease:

• Tracer kinetic studies: Using PTH (1-84) N15 as a metabolic tracer to track PTH metabolism, clearance, and tissue distribution in various disease states such as chronic kidney disease, where PTH metabolism is altered. The stable isotope labeling allows for non-radioactive in vivo tracking.

• PTH fragment analysis: Investigating the generation of bioactive PTH fragments in vivo by administering full-length PTH (1-84) N15 and subsequently analyzing blood and tissue samples for N15-labeled fragments. This approach helps elucidate the physiological processing of PTH and the biological significance of various fragments.

• Comparative effectiveness research: Directly comparing the molecular and cellular effects of PTH (1-84) versus PTH (1-34) using differential isotope labeling to distinguish between the two molecules in the same experimental system.

• Bone-kidney-parathyroid axis modeling: Developing comprehensive mathematical models of calcium homeostasis using PTH (1-84) N15 kinetic data to better understand the complex interactions between these organ systems.

• Proteomics of PTH signaling: Identifying novel protein interaction partners and signaling pathways activated by PTH using N15-labeled protein coupled with cross-linking and mass spectrometry approaches.

These emerging applications leverage the unique properties of the N15-labeled full-length hormone to address fundamental questions about PTH biology and pathophysiology that could not be adequately studied with conventional techniques.

How might PTH (1-84) N15 be utilized in developing new therapeutic approaches for hypoparathyroidism?

PTH (1-84) N15 offers unique capabilities for advancing therapeutic approaches for hypoparathyroidism:

• Personalized medicine development:

  • Using PTH (1-84) N15 to identify patient-specific metabolic clearance rates

  • Correlating clearance rates with optimal dosing regimens

  • Identifying biomarkers that predict response variability

• Combination therapy optimization:

  • Tracking the pharmacokinetics of PTH (1-84) N15 when administered alongside other agents (e.g., calcimimetics)

  • Determining optimal timing between multiple therapeutic agents

  • Evaluating potential drug-drug interactions at the molecular level

• Controlled release formulation development:

  • Using PTH (1-84) N15 to evaluate the in vivo release profile from novel delivery systems

  • Comparing tissue distribution patterns between different formulations

  • Correlating release kinetics with biological effects

• Long-term safety assessment:

  • Tracking potential accumulation in tissues during extended treatment

  • Identifying metabolic products that may arise from long-term administration

  • Correlating bone effects with serum levels and tissue distribution

Clinical trials have already demonstrated that PTH (1-84) replacement therapy for hypoparathyroidism can maintain serum calcium while reducing supplemental calcium and active vitamin D requirements . The REPLACE trial showed that 53% of PTH-treated subjects achieved the primary outcome compared to only 2% of placebo subjects . Further research using N15-labeled PTH could help refine these therapeutic approaches and address remaining challenges in long-term management of hypoparathyroidism.