Plasma Human Serum Albumin Research Articles

Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry (ITC) and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. Whereas AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

Albumin Is a Component of the Esterase Status of Human Blood Plasma.

The esterase status of blood plasma can claim to be one of the universal markers of various diseases; therefore, it deserves attention when searching for markers of the severity of COVID-19 and other infectious and non-infectious pathologies. When analyzing the esterase status of blood plasma, the esterase activity of serum albumin, which is the major protein in the blood of mammals, should not be ignored. The purpose of this study is to expand understanding of the esterase status of blood plasma and to evaluate the relationship of the esterase status, which includes information on the amount and enzymatic activity of human serum albumin (HSA), with other biochemical parameters of human blood, using the example of surviving and deceased patients with confirmed COVID-19. In experiments in vitro and in silico, the activity of human plasma and pure HSA towards various substrates was studied, and the effect of various inhibitors on this activity was tested. Then, a comparative analysis of the esterase status and a number of basic biochemical parameters of the blood plasma of healthy subjects and patients with confirmed COVID-19 was performed. Statistically significant differences have been found in esterase status and biochemical indices (including albumin levels) between healthy subjects and patients with COVID-19, as well as between surviving and deceased patients. Additional evidence has been obtained for the importance of albumin as a diagnostic marker. Of particular interest is a new index, [Urea] × [MDA] × 1000/(BChEb × [ALB]), which in the group of deceased patients was 10 times higher than in the group of survivors and 26 times higher than the value in the group of apparently healthy elderly subjects.

Isoaspartate as a biomarker for early diagnostics of neurodegenerative diseases

AbstractBackgroundThe formation of isoAsp via spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization has been regarded as a molecular clock of aging that significantly correlates with neurodegenerative diseases (NDDs). Our earlier findings established a strong link between isoAsp and Alzheimer’s disease (AD). In this study, we aimed at validating the diagnostic value of blood isoAsp‐related biomarkers for NDD prediction.MethodsThe levels of isoAsp in plasma human serum albumin (HSA) and the immunoglobulin G (IgG) specific against artificially aged HSA were measured in two cohorts by indirect enzyme‐linked immunosorbent assay (ELISA). Cohort 1 contained patients with AD and controls, while Cohort 2 recruited subjects with mild cognitive impairment (MCI), vascular dementia (VD), frontotemporal dementia (FTD), Parkinson disease (PD) and healthy controls. Apart from the memory tests, other plasma biomarkers reported in literature were quantified as well, including amyloid beta (Aβ) peptides Aβ40 and Aβ42, neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP) and phosphorylated Tau 181 (pTau181) protein.ResultsCohort 1 confirmed as expected a significant increase of isoAsp in HSA and reduction of IgG against aged HSA in AD group compared to controls (P < 0.0001). Cohort 2 revealed similar trends in comparisons between controls and subjects of different NDDs (P < 0.05). The most exciting finding was the best performance of isoAsp‐related biomarkers in MCI prediction among all blood biomarkers. In addition, the levels of our biomarkers were strongly associated with the memory test scores (P < 0.01).ConclusionWe demonstrated the diagnostic capacity of isoAsp‐related biomarkers in predicting NDDs at the early stage of disease. The biomarker levels correlated with cognitive decline, supporting their roles in NDD development. However, the specificity of these novel biomarkers seemed to be not limited to AD. Additional longitudinal studies with follow‐up data shall be conducted for validation of these findings.

A Computational Approach for Molecular Characterization of Covaxin (BBV152) and Its Ingredients for Assessing Its Efficacy against COVID-19

SARS-CoV-2 vaccination is a life-saving strategy for the entire population living in this pandemic. Several vaccines were developed using different platforms such as nucleic acids, viral vectors recombinant proteins, live attenuated, and inactivated virus modalities, etc. Although immunogenicity and efficacy of these COVID vaccines were investigated, Covaxin (a vaccine code-named BBV152), an inactivated COVID-19 vaccine, has not been well studied yet. This study aimed to explore the interactions between biomolecules with vaccine adjuvants by analyzing molecular and protein–protein interactions of S protein, angiotensin-converting enzyme 2 (ACE2), and human serum albumin (HSA) with the ingredients of Covaxin (2-phenoxyethanol and imidazoquinolinone) by computational methods using Autodock Vina, Cluspro, and Swiss ADME. In addition, its drug-likeness property was investigated. The binding energies using Autodock Vina showed stronger interactions of 2-phenoxyethanol and imidazoquinolinone with viral surface protein, S protein, human cell membrane receptor ACE2, and drug carrier plasma HSA (−5.2, −5.3 and −5.3 kcal/mol; −8.5, −8.5 and −9.1 kcal/mol, respectively). The interaction between S protein with ACE2 in the presence of 2-phenoxyethanol and imidazoquinolinone hindered the S protein function by reducing the binding energy between these proteins. In addition, imidazoquinolinone may have the drug-likeness property based on pharmacokinetic and physicochemical parameters. These results suggest that the Covaxin vaccine, owing to these ingredients, may impart greater efficacy in averting the virus and thus it may be more effective in producing herd immunity. In conclusion, for the first time, this computational study predicts the possible useful effects of these two adjuvants of Covaxin in therapeutic and drug-likeness strategies against SARS-CoV-2.

Is the Protein-Mediated Uptake of Drugs by Organic Anion Transporting Polypeptides a Real Phenomenon or an Artifact?

Plasma proteins or human serum albumin (HSA) have been reported to increase the in vitro intrinsic uptake clearance (CLint,uptake) of drugs by hepatocytes or organic anion transporting polypeptide (OATP)-transfected cell lines. This so-called protein-mediated uptake effect (PMUE) is thought to be due to an interaction between the drug-protein complex and the cell membrane causing an increase in the unbound drug concentration at the cell surface, resulting in an increase in the apparent CLint,uptake of the drug. To determine if the PMUE on OATP-mediated drug uptake is an artifact or a real phenomenon, we determined the effect of 1%, 2%, and 5% HSA on OATP1B1-mediated [human embryonic kidney (HEK)293 transfected cells] and passive CLint,uptake (mock HEK293 cells) on a cocktail of five statins. In addition, we determined the non-specific binding (NSB) of the statin-HSA complex to the cells/labware. The increase in uptake of atorvastatin, fluvastatin, and rosuvastatin in the presence of HSA was completely explained by the extent of NSB of the statin-HSA complex, indicating that the PMUE for these statins is an artifact. In contrast, this was not the case for OATP1B1-mediated uptake of pitavastatin and passive uptake of cerivastatin, suggesting that the PMUE is a real phenomenon for these drugs. Additionally, the PMUE on OATP1B1-mediated uptake of pitavastatin was confirmed by a decrease in its unbound IC50 in the presence of 5% HSA versus Hank's balanced salt solution buffer (HBSS). These data question the utility of routinely including plasma proteins or HSA in uptake experiments and the previous findings on PMUE on OATP-mediated drug uptake. SIGNIFICANCE STATEMENT: Here we report, for the first time, that the protein-mediated uptake effect (PMUE) on organic anion transporting polypeptide (OATP)-transported drugs could be an artifact of the non-specific binding (NSB) of the drug-albumin complex to cells/labware. Future experiments on PMUE must take into consideration such NSB. In addition, mechanisms other than PMUE need to be explored to explain the underprediction of in vivo OATP-mediated hepatic drug clearance from in vitro uptake studies.

Human serum albumin presents isoform variants with altered neonatal Fc receptor interactions.

Human serum albumin (HSA) is the most abundant protein in plasma and presents the particularity, with IgG, to have an extraordinary long serum half-life conferred by its interaction with the neonatal Fc receptor (FcRn). If the impact of IgG post-translational modifications (PTMs) on FcRn binding is well documented, it is far less reported for HSA despite numerous PTMs occurring on the protein in plasma. HSA is susceptible to numerous degradation reactions in plasma, because of aging, oxidative stress or liver and pancreas related pathologies. In the present study, we combined FcRn affinity chromatography and mass spectrometry to investigate the impact of HSA PTMs upon FcRn binding. This methodology presents the advantage to distinguish the effect of a single modification from a plasma HSA preparation made of a mixture of different isoforms. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation, which are modifications related to several pathological conditions, were demonstrated to act negatively on HSA-FcRn interaction. The HSA-FcRn binding alteration generated by these modifications is consistent with their vicinity with the interaction interface of the two proteins. Results were discussed regarding altered half-life of HSA observed in several disease states and pave the way toward new understandings of the hypoalbuminemia pathogenesis. SIGNIFICANCE STATEMENT: In this study, we investigated the impact of several post-translational modifications of HSA toward its ability to bind to the neonatal Fc receptor using in vitro affinity chromatography, mass spectrometry, and surface plasmon resonance. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation were demonstrated to decrease HSA-FcRn binding. These modifications occurring in circulating HSA were discussed in relation to several pathologies as well as for the use of HSA as a therapeutic protein.

MOLECULAR DOCKING OF AMITRIPTYLINE TO CERULOPLASMIN, RETINOL-BINDING PROTEIN, AND SERUM ALBUMIN

Objective: A drug’s efficiency depends on the binding capacity of the drug with the particular plasma protein. The less bound drug can be easily diffused through cell membranes. The present study deals with in silico studies of amitriptyline binding to three plasma proteins human ceruloplasmin (HCP), cellular retinol-binding protein (CRBP), and human serum albumin (HSA) and tries to establish the binding capacity behavior with the frontier molecular orbital approach.Methods: Amitriptyline is selected as legend and docked with three plasma proteins HCP, HCP PDB ID 1KCW, CRBP PDB ID 5LJC, and HSA. Docking calculations were carried out using docking server. frontier molecular orbital calculations are performed through web-based computational chemistry interface WEBMO version 17.0.012e using server Buchhner.chem.hope.edu. on computational engine MOPAC.Results: HCP and HSA predominantly show polar and hydrophobic interactions, whereas CRBP forms hydrogen bond apart from polar and hydrophobic interactions. Favorable values of inhibition constant, Ki, is obtained which is equal to 1.13 μM for CRBP, 6.00 μM for HCP, and 2.00 μM for has.Conclusion: A studies prove that amitriptyline can bind to all three plasma proteins, namely, HCP, CRBP, and HSA. Amitriptyline binds to an HSA and HCP through polar and hydrophobic interactions while weak electrostatic interactions felicitate diffusion of amitriptyline through the plasma membrane. Comparatively, strong hydrogen bond in CRBP may make the bound drug to be released at a slow rate. Strong binding of amitriptyline to CRBP is also evident from the least value of inhibition constant, Ki, which is equal to 1.13 μM for CRBP, 6.00 μM for HCP, and 2.00 μM for has.

Atomic-resolution map of the interactions between an amyloid inhibitor protein and amyloid β (Aβ) peptides in the monomer and protofibril states

Self-association of amyloid β (Aβ) peptides is a hallmark of Alzheimer's disease and serves as a general prototype for amyloid formation. A key endogenous inhibitor of Aβ self-association is human serum albumin (HSA), which binds ∼90% of plasma Aβ. However, the exact molecular mechanism by which HSA binds Aβ monomers and protofibrils is not fully understood. Here, using dark-state exchange saturation transfer NMR and relaxation experiments complemented by morphological characterization, we mapped the HSA-Aβ interactions at atomic resolution by examining the effects of HSA on Aβ monomers and soluble high-molecular weight oligomeric protofibrils. We found that HSA binds both monomeric and protofibrillar Aβ, but the affinity of HSA for Aβ monomers is lower than for Aβ protofibrils (Kd values are submillimolar rather than micromolar) yet physiologically relevant because of the ∼0.6-0.7 mm plasma HSA concentration. In both Aβ protofibrils and monomers, HSA targets key Aβ self-recognition sites spanning the β strands found in cross-β protofibril structures, leading to a net switch from direct to tethered contacts between the monomeric Aβ and the protofibril surface. These HSA-Aβ interactions are isoform-specific, because the HSA affinity of Aβ monomers is lower for Aβ(1-42) than for Aβ(1-40). In addition, the HSA-induced perturbations of the monomer/protofibrils pseudo-equilibrium extend to the C-terminal residues in the Aβ(1-42) isoform but not in Aβ(1-40). These results provide an unprecedented view of how albumin interacts with Aβ and illustrate the potential of dark-state exchange saturation transfer NMR in mapping the interactions between amyloid-inhibitory proteins and amyloidogenic peptides.

Can Chiroptical Spectroscopy be Used for the Analysis of Blood Plasma?

As an improvement on currently used methods of molecular spectroscopy, we used chiroptical techniques (electronic circular dichroism, fluorescence detected circular dichroism, and Raman optical activity [ROA]) to investigate the human blood plasma. To avoid the degradation of plasma samples, we measured them directly without any further preparation. We also tested cutoff weight filters (Amicon Ultra 100, 30, 10, and 3 kDa by Merck Millipore) to reduce undesirable fluorescence in the ROA and Raman spectra and also to remove the most abundant protein in the plasma-human serum albumin. The obtained spectra show that the ultrafiltration has a positive effect on undesirable fluorescence in ROA and Raman and also could reduce the amount of albumin in the plasma. Our results suggest that blood plasma can be successfully measured by the aforementioned methods. Therefore, these methods can potentially be useful for following research in the development of new, noninvasive, and reliable screening methods of clinical diagnostics.

Effect of plasma proteins on buprenorphine transfer across dually perfused placental lobule

Objective. The aim of this investigation is to determine the effect of human serum albumin (HSA) and α-acid glycoprotein (AAG) on buprenorphine (BUP) transplacental transfer and distribution.Methods. The technique of dual perfusion of placental lobule (DPPL) was utilised. BUP was co-perfused with the marker compound antipyrine (AP). In each experiment, the radiolabelled isotopes [3H]-BUP and [14C]-AP were added to enhance their detection limits. Human plasma proteins, HSA and AAG, were added to both the maternal and fetal circuits separately and in combination at their physiological concentrations in maternal and fetal circulations close to term.Results. Transplacental transfer of BUP, in absence of plasma proteins, is a two-step process: the first is its uptake by the syncytiotrophoblast from the maternal circuit, and the second is its transfer/release from the tissue to the fetal circuit. The addition of HSA to the perfusion medium affected only the second step of BUP transfer, but AAG affected both steps. The combined effect of HSA and AAG was not different from that observed in presence of the latter alone.Conclusions. Binding of BUP to circulating AAG could have an important role in the transfer of the drug from the maternal to fetal circulation.

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.

Complete determination of disulfide forms of purified recombinant human serum albumin, secreted by the yeast Pichia pastoris.

In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.

Binding of hydralazine and a major metabolite, pyruvate hydrazone, to rat plasma protein and human serum albumin.

The interaction of hydralazine (HP) and a major metabolite, pyruvate hydrazone (HPH), with rat plasma protein or human serum albumin (HSA) was studied both in vivo (by the ultrafiltration method) and in vitro (at 20 and 30°C, by the equilibrium dialysis method). The binding of HP and HPH to plasma proteins was fairly high (96.0 and 81.7%. respectively) after i.v. administration to rats. HPH, which had an association constant higher than HP, was found to interact with a single class of site on HSA, while HP was bound to at least two heterogeneous binding sites. The interaction between HP and rat plasma protein or HSA was temperaturedependent at the secondary binding site, suggesting that a nonionic mechanism is involved in the binding, and the interaction at the primary site was ionic strength-dependent. In contrast, the interaction of HPH with HSA showed less temperature-dependence. The thermodynamic analyses of the HP binding process at the secondary site showed a negative value for ΔG°, a large contribution of ΔH° to ΔG° and a positive ΔS°, while at the primary site a large contribution to ΔG° by ΔS° was seen. Thus, these findings suggest that the main binding energies at the primary and secondary sites are derived from electrostatic and nonionic sources, respectively. For HPH binding, however, the predominantly bound species was ionic. HP did not induce competitive displacement of fluorescent probes except for a slight displacement of dansylamide in the presence of a high concentration of HP. This suggests that HP is bound weakly to sites other than sites I and II, two specific sites for acidic drugs, on HSA. The present results lead us to postulate that HP interacts with the receptor sites mainly by hydrophobic and hydrogen bonding forces and partly by electrostatic forces.