Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis.

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

Structural Lesions of Proteins Connected to Lipid Membrane Damages Caused by Radical Stress: Assessment by Biomimetic Systems and Raman Spectroscopy.

Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insight into the effects of the propagation of an initial radical damage on protein to the lipid compartment. The latter is based on liposome technology and allows measuring the trans unsaturated fatty acid content as a result of free radical stress on proteins. Two kinds of sulfur-containing proteins were chosen to connect their chemical reactivity with membrane lipid transformation, serum albumins and metallothioneins. Biomimetic systems based on radiation chemistry were used to mimic the protein exposure to different kinds of free radical stress and Raman spectroscopy to shed light on protein structural changes caused by the free radical attack. Among the amino acid residues, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting that metal-Cys clusters are good interceptors of reactive species in metallothioneins, together with disulfides moieties in serum albumins. Met is another important site of the attack, in particular under reductive conditions. Tyr and Phe are sensitive to radical stress too, leading to electron transfer reactions or radical-induced modifications of their structures. Finally, modifications in protein folding take place depending on reactive species attacking the protein.

Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

Assessment of the targeting specificity of a fluorescent albumin conceived as a preclinical agent of the liver function.

In the context of increasing liver diseases, no contrast agent is currently available in Europe and the United States to directly assess the liver function. Only neolactosylated human serum albumin is being clinically used in Asia. In order to perform preclinical studies in the context of liver diseases, we conceived a fluorescent lactosylated albumin for the quantification of liver functional cells (l-Cyal). Precise characterization was achieved in order to determine the amounts of lactose and Cyanine 5 (Cy5) coupled to the albumin. In addition, potential aggregation was characterized by asymmetrical flow field-flow fractionation hyphenated to multi-angle light scattering (AF4-MALS). The optimal functionalized albumin exhibited a mass greater than 87 kDa which corresponds to the addition of 34 lactose moieties per protein and 1-2 Cy5 labels. Also, no significant formation of aggregates could be identified due to the modification of the native albumin. In healthy mice, the accumulation of l-Cyal in the liver and its selectivity for hepatocyte cells were shown by optical imaging and flow cytometry. Administration of l-Cyal to mice bearing liver metastases showed a reduced signal in the liver related to a decrease in the number of hepatocytes. The l-Cyal bioimaging contrast agent could be particularly useful for assessing the state of liver related diseases.

Welcome to volume 9 of Therapeutic Delivery.

Happy New Year to all of our readers! Welcome to volume 9 of Therapeutic Delivery. We would like to take the opportunity to look back over 2017, which was another excellent year for us. We thank all our readers, reviewers, authors and Editorial Board members for their continued support and very much look forward to working with all our contributors again in 2018.

Effectiveness and Safety Assessment of Citrate Anticoagulation During Albumin Dialysis in Comparison to Other Methods of Anticoagulation.

Liver failure is a serious and often deadly disease often requiring MARS (Molecular Adsorbent Recirculating System) therapy. Choosing the safe and effective method of anticoagulation during artificial liver support systems seems to be very difficult and extremely important. The aim of this study was to assess effectiveness and safety of regional anticoagulation with citrate in liver failure patients during MARS. We used a single center observational study. We analyzed 158 MARS sessions performed in 65 patients: 105 (66.5%) sessions in 41 patients with heparin anticoagulation, 40 (25.3%) sessions in 19 patients with citrate, and 13 (8%) sessions in only five patients without anticoagulation, that were excluded from part of the analysis. To determine the effectiveness of regional anticoagulation with citrate, probability of filter survival and changes in laboratory parameters were analyzed according to the applied method of anticoagulation. The safety of citrate was determined by Ca/Ca2+ ratio, acid-base balance, bleeding complications, and the need for blood product transfusions. The probability of filter survival in the citrate group was 94% and in the heparin group 82% (P = 0.204). There was no relationship between the method of anticoagulation and effectiveness of MARS therapy in lowering the levels of the analyzed parameters. Only one patient had a Ca/Ca2+ ratio higher than he safety margin. There were no statistically significant changes in pH and lactate level irrespective of anticoagulation; bicarbonate dropped significantly only in the heparin group (P = 0.03). The frequency of bleeding complications and the need for transfusions did not differ significantly between groups. Regional anticoagulation with citrate can be an effective and safe method of anticoagulation during MARS therapy, but requires attentive monitoring and further studies in liver failure patients.

Multiparametric Assessment of Gold Nanoparticle Cytotoxicity in Cancerous and Healthy Cells: The Role of Size, Shape, and Surface Chemistry.

In recent years, we and others have become interested in evaluating the use of surface-enhanced Raman scattering (SERS) tags for early cancer detection and in designing new approaches to demonstrate the applicability of this spectroscopic technique in the clinic. SERS-based imaging in particular offers ultra sensitivity up to the single molecule, multiplexing capability, and increased photostability and has been shown to outperform fluorescence. However, to employ SERS tags for early cancer detection, it is important to understand their interaction with cells and determine their cytotoxicity. We have been particularly interested for quite some time in determining if and how gold nanostars, which have been demonstrated as outstanding SERS enhancing substrates, can be safely employed in living systems and translated to the clinic. In this study, we carried out a multiparametric in vitro study to look at the cytotoxicity and cellular uptake of gold nanoparticles on human glioblastoma and human dermal fibroblast cell lines. Cytotoxicity was evaluated by incubating cells with three different morphologies of AuNPs, namely nanospheres, nanorods, and nanostars, each having three different surface chemistries (cetyltrimethylammonium bromide (CTAB), poly(ethylene glycol) (PEG), and human serum albumin (HSA)). Our results showed that the surface chemistry of the nanoparticles had predominant effects on cytotoxicity, and the morphology and size of the nanoparticles only slightly affected cell viability. CTAB-coated particles were found to be the most toxic to cells, and PEGylated nanostars were determined to be the least toxic. Caspase-3 assay and LDH assay revealed that cell death occurs via apoptosis for cancerous cells and via necrosis for healthy ones. Cellular uptake studies carried out via TEM showed that the particles retain their shape even at long incubation times, which may be beneficial for in vivo SERS-based disease detection. Overall, this study provides valuable information on gold-nanoparticle-induced cytotoxicity that can be leveraged for the development of safe and effective nanoparticle-based therapeutic and diagnostic systems.

Optimization of amino acid-stabilized erythropoietin parenteral formulation: In vitro and in vivo assessment.

The aim of this study was to optimize the formulation of erythropoietin (EPO) using amino acids instead of human serum albumin (HSA) and to evaluate its in vivo stability in order to avoid the risk of viral contamination and antigenicity. Different EPO formulations were developed in such a way as to allow studying the effects of amino acids and surfactants on the EPO stability profile. The main techniques applied for EPO analysis were ELISA, Bradford method, and SDS gel electrophoresis. The in vivo stability was evaluated in a Balb-c mouse animal model. The results showed that the presence of surfactant was very useful in preventing the initial adsorption of EPO on the walls of vials and in minimizing protein aggregation. Amino acid combinations, glycine with glutamic acid, provided maximum stability. Formulation F4 (containing glycine, glutamic acid and Tween 20) showed minimum aggregation and degradation and in vivo activity equivalent to commercially available HSA-stabilized EPO (Eprex®).

Synthesis, cytotoxicity assessment, and interaction and docking of novel palladium(II) complexes of imidazole derivatives with human serum albumin.

Imidazole analogs are the agents that attract both bioinorganic chemist and drug designer. Numerous methods have been proposed for synthesis of imidazole derivatives. In this study, a series of heterocyclic system with p-conjugated system such as 2-aryl-imidazo[4,5-f][1,10]phenanthroline analogs were synthesized. Then, three new palladium(II) complexes containing 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1,10]Phenanthroline (FIP) and 2-(thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (TIP) ligands were synthesized. The structures of the compounds, [Pd(Phen)(TIP)](NO3)2, [Pd(Phen)(FIP)](NO3)2, and [Pd(FIP)2]Cl were determined by spectroscopic methods and elemental analysis. Biological activity of the complexes synthesized was assessed against chronic myelogenous leukemia cell line, K562. Also, the interactions of human serum albumin with complexes were investigated using isothermal titration in the Tris buffer, pH 7.4. According to the results obtained, it was found that there is a set of six binding sites for these complexes on HSA with positive cooperativity in the binding process. Docking technique was also applied to confirm the experimental results. The results showed that smaller complexes have higher interaction affinity.

Assessment of the Dissociation Energetics of Some Selected Ligand Drugs Bound on Human Serum Albumin by Differential Scanning Calorimetry.

Drug-protein binding may play a role in the thermal energetics of protein denaturation and could lead to the determination of its equilibrium dissociation parameter. The aim of this study was to assess the energetics of a drug that was bound to human serum albumin (HSA) during thermal denaturation. Drugs that were bound at a single high-affinity primary binding site on HSA, including diazepam and ibuprofen, were employed. Commercial HSA was treated with charcoal to remove stabilizers and adjusted to 20% w/v in a pH 7.4 buffered solution. Serial concentrations of individual drugs up to 0.16 mmole/g-protein were added to the cleaned HSA solutions whereas diazepam was added to a commercial HSA solution. Samples were subjected to differential scanning calorimetry (DSC) set to run from 37 to 90°C at 3.0°C/min. Binding of the drug slightly increased the denaturing temperature of the cleaned HSA due to a shift in the equilibrium toward the native protein bound with the drug. Diazepam depressed the denaturing temperature of the commercial HSA by competing with the stabilizers already bound to the primary site of the HSA. This yielded not only the HSA-stabilizer but also the HSA-diazepam type complexes that exhibited a different denaturation process. A rational approximation of the Lumry-Eyring protein denaturation model was used to treat the DSC endotherms. The approximated scheme: [Formula: see text] was successfully fitted to the data. It was used to determine the dissociation parameters for diazepam and ibuprofen bound to the HSA. These results were comparable to those obtained from other methods.

[Probabilistic calculations of biomolecule charge states that generate mass spectra of multiply charged ions].

In this work, information relating to charge states of biomolecule ions in solution obtained using the electrospray ionization mass spectrometry of different biopolymers is analyzed. The data analyses have mainly been carried out by solving an inverse problem of calculating the probabilities of retention of protons and other charge carriers by ionogenic groups of biomolecules with known primary structures. The approach is a new one and has no known to us analogues. A program titled "Decomposition" was developed and used to analyze the charge distribution of ions of native and denatured cytochrome c mass spectra. The possibility of splitting of the charge-state distribution of albumin into normal components, which likely corresponds to various conformational states of the biomolecule, has been demonstrated. The applicability criterion for using previously described method of decomposition of multidimensional charge-state distributions with two charge carriers, e.g., a proton and a sodium ion, to characterize the spatial structure of biopolymers in solution has been formulated. In contrast to known mass-spectrometric approaches, this method does not require the use of enzymatic hydrolysis or collision-induced dissociation of the biopolymers.

Distributions of experimental protein structures on coarse-grained free energy landscapes.

Predicting conformational changes of proteins is needed in order to fully comprehend functional mechanisms. With the large number of available structures in sets of related proteins, it is now possible to directly visualize the clusters of conformations and their conformational transitions through the use of principal component analysis. The most striking observation about the distributions of the structures along the principal components is their highly non-uniform distributions. In this work, we use principal component analysis of experimental structures of 50 diverse proteins to extract the most important directions of their motions, sample structures along these directions, and estimate their free energy landscapes by combining knowledge-based potentials and entropy computed from elastic network models. When these resulting motions are visualized upon their coarse-grained free energy landscapes, the basis for conformational pathways becomes readily apparent. Using three well-studied proteins, T4 lysozyme, serum albumin, and sarco-endoplasmic reticular Ca(2+) adenosine triphosphatase (SERCA), as examples, we show that such free energy landscapes of conformational changes provide meaningful insights into the functional dynamics and suggest transition pathways between different conformational states. As a further example, we also show that Monte Carlo simulations on the coarse-grained landscape of HIV-1 protease can directly yield pathways for force-driven conformational changes.

A TEM protocol for quality assurance of in vitro cellular barrier models and its application to the assessment of nanoparticle transport mechanisms across barriers.

We report here a protocol to characterise and monitor the quality of in vitro human cellular barrier models using Transmission Electron Microscopy (TEM), which can be applied for transport assays, mechanistic studies and screening of drug/compound (including nanoparticle) penetration across such biological barriers. Data from two examples of biological barriers are given, namely the hCMEC/D3 endothelial blood-brain barrier model, and the Caco-2 intestinal epithelial barrier model, to show the general applicability of the method. Several aspects of this method are applicable to the quality assurance of in vitro barrier models, e.g., assessment of the multi or mono-layer structure of the endothelial cells; identification of any potential "holes" in the barrier that could confound transport assay results; validation of tight junction expression; and determination of the types and amounts of key cellular organelles present in the barrier to account for any significant changes in phenotype that may occur compared to the in vivo situation. The method described here provides a key advantage in that it prevents loss of the filter membrane during monolayer sectioning, thereby preserving critical details associated with the basal cell membrane. Applicability of the protocol for other in vitro biological barriers, such as the blood-foetus, blood-testes, blood-cerebrospinal fluid (CSF) and lung alveolar-capillary barriers is also discussed. Additionally, we demonstrate the use of the method for assessment of nanoparticle transport across cellular barriers and elucidation of transcytosis mechanisms. Sequential events of cellular endocytosis, localisation and transcytosis can be described in detail by TEM imaging, revealing useful sub-cellular details that provide evidence for the mechanism of nanoparticle transport in the hCMEC/D3 blood-brain barrier model and the Caco-2 intestinal epithelial cell model. Potential artefacts resulting from the nanoparticles interacting with the Transwell membranes can also be assessed.

A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin. Isopropanol with 1.0% trichloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60-80%. The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. This assay was successfully applied to a toxicological study. When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method.

Replica exchange Monte Carlo simulation of human serum albumin-catechin complexes.

Replica exchange Monte Carlo simulation equipped with an orientation-enhanced hydrophobic interaction was utilized to study the impacts of molar ratio and ionic strength on the complex formation of human serum albumin (HSA) and catechin. Only a small amount of catechins was found to act as bridges in the formation of HSA-catechin complexes. Selective binding behavior was observed at low catechin to HSA molar ratio (R). Increase of catechin amount can suppress HSA self-aggregation and diminish the selectivity of protein binding sites. Strong saturation binding with short-range interactions was found to level off at around 4.6 catechins per HSA on average, while this number slowly increased with R when long-range interactions were taken into account. Meanwhile, among the three rings of catechin, the 3,4-dihydroxyphenyl (B-ring) shows the strongest preference to bind HSA. Neither the aggregation nor the binding sites of the HSA-catechin complex was sensitive to ionic strength, suggesting that the electrostatic interaction is not a dominant force in such complexes. These results provide a further molecular level understanding of protein-polyphenol binding, and the strategy employed in this work shows a way to bridge phase behaviors at macroscale and the distribution of binding sites at residue level.

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods.

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800 nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.

Qualitative and quantitative assessment of marketed erythropoiesis-stimulating agents by capillary electrophoresis.

Formulated erythropoiesis stimulating agents (ESAs) containing erythropoietin (EPO)-alpha, EPO-beta or darbepoetin-alpha were analyzed by capillary electrophoresis with a previously published method requiring no sample pre-treatment [1]. In this study, the method proved to be applicable to all formulations encountered, that is, in the presence of polysorbate 80, polysorbate 20 or human serum albumin as major excipients, thus extending the range of products that can be analyzed without pre-treatment. Method performance was evaluated and showed good linearity, range, precision and sensitivity. No significant matrix effects were observed for the various formulations. The ability of the method to resolve isoforms of each of the three active ingredients enabled comparison of the isoform distribution of finished products with that of the respective drug substance. In general, finished products and their corresponding drug substances showed similar isoform distribution and all were within manufacturer specifications. In addition, the content in active ingredient in the various dosage strengths was found to be in close agreement with the label claims with the exception of 2 out of 131 containers analyzed. Overall, this study demonstrated that the capillary zone electrophoresis method could be successfully applied to the analysis of most of the ESA products currently on the market in North America and Europe and that all products were found to have good batch-to-batch consistency.

Experimental-like affinity constants and enantioselectivity estimates from flexible docking.

Experimental-like affinity constants and enantioselectivity estimates, not predicted so far computationally, were obtained using a novel flexible modeling/docking combined strategy. The S- and R-warfarin-human serum albumin (HSA, site I) complexes were used as an interaction model. The process for a verified estimation includes the following: (i) ionized open chain forming at physiological pH (a recent focus); (ii) conformational search (molecular mechanics and Monte Carlo methods); (iii) rigid protein-flexible ligand docking (GlideXP) generating low energy paired S- and R-poses; (iv) graphical comparison against the X-ray crystal structure (unsatisfactory verification step); (v) quantum polarized ligand docking (insufficient verification step); (vi) induced fit docking (one pose satisfying the verification criterion; selection step); (vii) converting docking scores to affinity and enantioselectivity estimates (log K(S) = 5.43, log K(R) = 5.34, ES = K(S)/K(R) = 1.23) and numerical comparison against equivalent literature data from bioanalytical techniques (validation step); (viii) intermolecular forces explaining ES (hydrogen bonding and π-π interactions).

Insignificant role of the N-terminal cobalt-binding site of albumin in the assessment of acute coronary syndrome: discrepancy between the albumin cobalt-binding assay and N-terminal-targeted immunoassay.

The aim of this study was to evaluate whether the N-terminus of human serum albumin (HSA) has a role in the cobalt binding detected using albumin cobalt-binding (ACB) assay. We compared the results obtained using an enzyme-linked immunosorbent assay (ELISA) for N-terminal-modified HSA with those of a conventional ACB assay in two groups: acute coronary syndrome (n = 43) and non-ischemic chest pain (n = 39). ACB and cardiac troponin-I levels were higher in the acute coronary syndrome group. No significant correlation between ACB assay and ELISA results was observed in either of the two patient groups. In acute chest pain patients, the N-terminal site of HSA has a negligible or limited role in cobalt binding in the ACB assay.