De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

Endogenous ligand recognition and structural transition of a human PTH receptor.

Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

Mutation of Methionine to Asparagine but Not Leucine in Parathyroid Hormone Mimics the Loss of Biological Function upon Oxidation.

Human parathyroid hormone (PTH) is an 84-amino acid peptide that contains two methionine (Met) residues located at positions 8 and 18. It has long been recognized that Met residues in PTH are subject to oxidation to become Met sulfoxide, resulting in a decreased biological function of the peptide. However, the mechanism of the lost biological function of PTH oxidation remains elusive. To characterize whether the shift from the hydrophobic nature of the native Met residue to the hydrophilic nature of Met sulfoxide plays a role in the reduction of biological activity upon PTH oxidation, we conducted in silico and in vitro site-directed mutagenesis of Met-8 and Met-18 to the hydrophilic residue asparagine (Asn) or to the hydrophobic residue leucine (Leu) and compared the behavior of these mutated peptides with that of PTH oxidized at Met-8 and/or Met-18. Our results showed that the biological activity of the Asn-8 and Asn-8/Asn-18 mutants was significantly reduced, similar to Met-8 sulfoxide and Met-8/Met-18 sulfoxide analogues, while the functions of Asn-18, Leu-8, Leu-8/Leu-18 mutants, or Met-18 sulfoxide analogues were similar to wild-type PTH. This is rationalized from molecular modeling and immunoprecipitation assay, demonstrating disruption of hydrophobic interactions between Met-8 and Met-18 of PTH and type-1 PTH receptor (PTHR1) upon mutation or oxidation. Thus, these novel findings support the notion that the loss of biological function of PTH upon oxidation of Met-8 is due, at least in part, to the conversion from a hydrophobic to a hydrophilic residue that disrupts direct hydrophobic interaction between PTH and PTHR1.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.

Molecular Mechanism of Ca2+ in the Allosteric Regulation of Human Parathyroid Hormone Receptor-1.

Parathyroid hormone (PTH) is an endogenous ligand that activates the PTH type 1 receptor (PTH1R) signaling. Ca2+, a common second messenger, acts as an allosteric regulator for prolonging the activation of PTH1R. However, a clear picture of the underlying allosteric mechanism is still missing. Herein, extensive molecular dynamics (MD) simulations are performed for PTH1R-PTH complexes with and without Ca2+ ions, allowing us to delineate the molecular details of calcium-induced allostery. Our results indicate that acidic residues in the extracellular loop 1 (ECL1) (D251, E252, E254, and E258-E260) and PTH (E19 and E22) serve as key determinants for local Ca2+-coupling structures and rigidity of ECL1. Moreover, the binding of Ca2+ induces conformational changes of transmembrane domain 6/7 (TM6/7) that are related to PTH1R activation and strengthens the residue-residue communication within PTH and TMD allosterically. Moreover, our results demonstrate that the presence of Ca2+ ions potentiates the interaction between PTH and PTH1R via steered molecular dynamics (SMD) simulations, while the point mutation in the PTH (PTHR25C) weakens the binding of PTH and PTH1R. These results support that Ca2+ ions might further prolong the residence time of PTH on PTH1R and facilitate the positive allostery of PTH1R. Together, the present work provides new insights into the allosteric regulation mechanism of GPCRs induced by ions and related drug design targeting the PTH1R allosteric pathway.

Short carboxyl terminal parathyroid hormone peptides modulate human parathyroid hormone signaling in mouse osteoblasts.

BACKGROUND: Novel human parathyroid hormone (hPTH) peptides of unknown biological activity have recently been identified in the serum of subjects with normal renal function, chronic renal failure, and end-stage renal disease through the application of liquid chromatography-high resolution mass spectrometry. PURPOSE: of experiments: To determine the bioactivity of these peptides, we synthesized hPTH28-84, hPTH38-84, and hPTH45-84 peptides by solid phase peptide synthesis and tested their bioactivity in MC3T3-E1 mouse osteoblasts, either individually or together with the native hormone, hPTH1-84, by assessing the accumulation of 3´,5´-cyclic adenosine monophosphate (cAMP) and the induction of alkaline phosphatase activity. RESULTS: Increasing doses of hPTH1-84 (1-100 nM) increased the accumulation of cAMP and alkaline phosphatase activity in osteoblasts. hPTH28-84, hPTH38-84, and hPTH45-84 in concentrations of 1-100 nM were biologically inert. Surprisingly, 100 nM hPTH38-84 and hPTH45-84 increased the accumulation of cAMP in osteoblasts treated with increasing amounts of hPTH1-84. Human PTH28-84 had no effects on cAMP activity alone or in combination with hPTH1-84. Conversely, 100 nM hPTH38-84, hPTH45-84, and hPTH28-84 blocked the activation of alkaline phosphatase activity by hPTH1-84. CONCLUSIONS: The data show that the short carboxyl-terminal hPTH peptides, hPTH38-84 and hPTH45-84, increase the amount of cellular cAMP generated in cultured osteoblasts in response to treatment with full-length hPTH1-84 when compared to full-length hPTH1-84 alone. Human PTH28-84 had no effect on cAMP activity alone or in combination with hPTH1-84. Human PTH28-84, hPTH38-84 and hPTH45-84 reduced the effects of hPTH1-84 in osteoblasts with respect to the induction of alkaline phosphatase activity compared to hPTH1-84 alone. Short carboxyl peptides of human PTH are biologically inert but when administered together with full-length hPTH1-84 modulate the bioactivity of hPTH1-84 in osteoblasts.

Relationship between GFR, intact PTH, oxidized PTH, non-oxidized PTH as well as FGF23 in patients with CKD.

Fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) are regulators of renal phosphate excretion and vitamin D metabolism. In chronic kidney disease (CKD), circulating FGF23 and PTH concentrations progressively increase as renal function declines. Oxidation of PTH at two methionine residues (positions 8 and 18) causes a loss of function. The impact of n-oxPTH and oxPTH on FGF23 synthesis, however, and how n-oxPTH and oxPTH concentrations are affected by CKD, is yet unknown. The effects of oxidized and non-oxidized PTH 1-34 on Fgf23 gene expression were analyzed in UMR106 osteoblast-like cells. Furthermore, we investigated the relationship between n-oxPTH and oxPTH, respectively, with FGF23 in two independent patients' cohorts (620 children with CKD and 600 kidney transplant recipients). While n-oxPTH stimulated Fgf23 mRNA synthesis in vitro, oxidation of PTH in particular at Met8 led to a markedly weaker stimulation of Fgf23. The effect was even stronger when both Met8 and Met18 were oxidized. In both clinical cohorts, n-oxPTH-but not oxPTH-was significantly associated with FGF23 concentrations, independent of known confounding factors. Moreover, with progressive deterioration of kidney function, intact PTH (iPTH) and oxPTH increased substantially, whereas n-oxPTH increased only moderately. In conclusion, n-oxPTH, but not oxPTH, stimulates Fgf23 gene expression. The increase in PTH with decreasing GFR is mainly due to an increase in oxPTH in more advanced stages of CKD.

Receptor selectivity from minimal backbone modification of a polypeptide agonist.

Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share ∼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single ß-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The ß-scan series allowed us to identify two homologs, each containing two αâ†’ß replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both ß2- and ß3-residues, which differ in terms of side-chain location.

Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB.

Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 105-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical-"hotspot"-residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.

Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located "outside the box" explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

Parathyroid Hormone Derivative with Reduced Osteoclastic Activity Promoted Bone Regeneration via Synergistic Bone Remodeling and Angiogenesis.

Osteogenesis, osteoclastogenesis, and angiogenesis are the most important processes in bone repair. Parathyroid hormone (PTH) has pro-osteogenic, pro-osteoclastogenic, and proangiogenic effects and may be a candidate for use in bone defect repair. However, the local application of PTH to bone defects is counterproductive due to its excessive osteoclastic and bone resorptive effects. In this study, a PTH derivative, PTHrP-2, is developed that can be applied to local bone defects. First, a modified peptide with a calcium-binding repeat glutamine tail undergoes controlled local release from a ceramic material and is shown to be a better fit for the repair process than the unmodified peptide. Second, the modified peptide is shown to have strong pro-osteogenic activity due to mineralization and its facilitation of serine (Ser) phosphorylation. Third, the modified peptide is shown to maintain the pro-osteoclastogenic and proangiogenic properties of the unmodified peptide, but its pro-osteoclastogenic activity is reduced compared to that of the unmodified peptide. The reduced pro-osteoclastogenic and increased pro-osteogenic properties of the modified peptide reverse the imbalance between osteoblasts and osteoclasts with local PTH application and shift bone resorption to bone regeneration.

A Visual FRET Immunofluorescent Biosensor for Ratiometric Parathyroid Hormone (1-84) Antigen Point-of-Care Detection.

Excessive secretion of PTH leads to disturbance of calcium and phosphorus metabolism in the body, which promotes bone, kidney, digestive system and nervous system diseases. Due to the short half-life of PTH, it becomes a difficult issue for PTH detection in the clinical diagnosis field. We explored a competitive immunofluorescent sensing mode based on FRET of two-color CdTe QDs for ratiometric PTH 1-84 antigen detection. The FRET effect and ratiometric fluorescence between the two-color CdTe QDs motivated accurate quantification of PTH 1-84 antigen concentration from 0.01 ng mL-1 to 0.08 ng mL-1 with a limit of detection of 3 pg mL-1. More importantly, under UV irradiation, samples with different concentrations of PTH 1-84 antigen achieved fluorescence visualization, which provides huge possibility for the practical application of PTH 1-84 antigen point-of-care detection.

The contribution of cross-talk between the cell-surface proteins CD36 and CD47-TSP-1 in osteoclast formation and function.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

O-GlcNAc Engineering of GPCR Peptide-Agonists Improves Their Stability and in Vivo Activity.

Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.

Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates.

Covalent conjugates between a synthetic polymer and a peptide hormone were used to probe the molecular extension of these macromolecules and how the polymer modifies the fibril formation of the hormone. NMR spectroscopy of 15 N labeled parathyroid hormone (PTH) was employed to visualize the conformation of the conjugated synthetic polymer, triggered by small temperature changes via its lower critical solution temperature. A shroud-like polymer conformation dominated the molecular architecture of the conjugated chimeras. PTH readily forms amyloid fibrils, which is probably the physiological storage form of the hormone. The polyacrylate based polymers stimulated the nucleation processes of the peptide.

[The association between vitamin D/calcium deficiency and cardiovascular events.]

According to some case reports, extreme hypocalcemia induced by vitamin D and calcium deficiency leads to heart failure. This rare clinical entity "Hypocalcemic cardiomyopathy" is also reported in elderly patients as well as infants. In patients with chronic kidney disease and heart failure, hypocalcemia is reported to predict worse outcome. Prescription of active vitamin D or its analogues is associated with lower rates of cardiovascular events in predialysis and dialysis patients, however indication biases often seen in observational studies cannot preclude the possibility that the benefit of these agents is limited to patients with high parathyroid hormone(PTH)levels. In fact, J-DAVID study, a randomized controlled trial from Japan, clearly showed that oral administration of alfacalcidol of 0.5 µg/day did not reduce cardiovascular events in hemodialysis patients with intact PTH<180 pg/mL.

[CKD-MBD and cardiovascular disease.]

Cardiovascular disease(CVD)is a crucial cause of death in patients with chronic kidney disease and various factors play a role in the progression of CVD. Among the various factors, mineral bone disorder has been focused on in recent year. Phosphate is an important factor because it affects cardiovascular system not only directly but also indirectly. Phosphate can influence the serum and cellar levels of parathyroid hormone, fibroblast growth factor 23, and active vitamin D and thereby leading to the progression of CVD. Thus, it is essential to understand the mechanisms of CVD progression and think about a control of mineral bone disorder.

Cellular Effects and Delivery Propensity of Penetratin Is Influenced by Conjugation to Parathyroid Hormone Fragment 1-34 in Synergy with pH.

The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH(1-34)-penetratin conjugates and coadministered penetratin with PTH(1-34) regarding transepithelial permeation of PTH(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH(1-34)-penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH(1-34)-penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH(1-34)-penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4.

Oxidation of PTH: in vivo feature or effect of preanalytical conditions?

BACKGROUND: Posttranslational oxidation of parathyroid hormone (PTH) modifies its biological activity. Measurement of non-oxidized PTH (n-oxPTH) could be an improvement in assessing PTH status, as intact PTH may rather reflect oxidative stress. However, it is debated whether oxidation of PTH occurs in vivo, or whether it is mainly an in vitro artifact. The aim of this study was to investigate the influence of different preanalytical conditions on the oxidation of PTH within a wide range of plasma PTH concentrations and oxidation propensity. METHODS: n-oxPTH was separated from its oxidized form using an affinity column capturing the oxidized PTH. n-oxPTH was measured in eluate using commercially available PTH assays. The study included ethylenediaminetetraacetic acid plasma samples from 17 patients undergoing hemodialysis and 32 healthy subjects. We determined effects of storage temperature, time until centrifugation and freeze-thaw cycles. PTH and n-oxPTH concentrations were measured in each sample using six different immunoassays. RESULTS: n-oxPTH concentrations remained unchanged up to 180 min until centrifugation, two freeze-thaw cycles or after storage at -20°C or -80°C up to 79 days. Various methods for n-oxPTH and PTH measurements yielded highly comparable results, apart from standardization differences between various PTH and n-oxPTH assays. CONCLUSIONS: n-oxPTH concentrations were stable under our study conditions, indicating negligible ex vivo oxidation of PTH. In addition, PTH immunoassays have a different sensitivity for n-oxPTH than for total PTH. For this reason, the n-oxPTH/total PTH ratio cannot be used in absence of a n-oxPTH standard. Clinical implications of determining n-oxPTH require additional study.