Human Serum Albumin Levels Research Articles

Optical microchips based on high-affinity recombinant protein binders—Human serum albumin detection in urine

Recent developments in molecular evolution technologies have led to novel types of high-affinity recombinant protein binders (PB) able to substitute antibodies in many diagnostic and therapeutic applications. Despite almost a decade of research, they have so far only been sporadically used for biosensor construction. Here, we present a proof-of-principle comparative study focused on the application of three types of PB recognizing human serum albumin (HSA) in the fabrication of diagnostic optical microchips detecting clinically relevant HSA levels in urine. The PB tested were: (i) biotinylated anti-HSA Affibody (AF) (IgG binding domain of protein A, Staphylococcus aureus); (ii) biotinylated protein construct based on albumin-binding domain (ABD) of protein G (Streptococcus G148) fused with long TolA spacer (6xHis-WT-ABD-TolA-AviTag) and (iii) WT-ABD-Trp leader-streptavidin tetrameric fusion protein (SA-ABD-WT). Open glass microchips with 24 independent microwells (volume 8 μL) and micropatterned detection zones were prepared and used for oriented binding of proteins through the biotin/streptavidin chemistry. The analytical performance of the optical microchips was tested by performing direct specific detection of fluorescently labelled HSA in various environments. Results show that the length of peptide spacer present between the binding protein domain and sensor surface is a key factor influencing biosensor performance. The biosensor based on SA-ABD-WT reached the limit of detection (LOD) for HSA in urine (LOD = 0.65 μg/ml) sufficient to identify the chronic kidney disease caused by high blood pressure or diabetes. Furthermore, it offers the highest signal intensity, low noise and significant simplification of microchip preparation due to a simple one-step immobilization procedure. Our results may be further exploited in development of diagnostic microchips dedicated to the detection of a wide range of molecular targets recognized by specific ABD protein binders.

An ultrasensitive rapid-response fluorescent probe for highly selective detection of HSA

A fluorescent probe HCAB based on twisted intramolecular charge transfer (TICT) mechanism has been designed and synthesized by introducing benzoyl moiety to 4-dimethylamino-2′-hydroxychalcone. HCAB showed excellent selectivity towards human serum albumin (HSA) among different proteins with a remarkable 160-fold fluorescence enhancement and a wider linear range 0–100 mg/L of HSA. Job’s Plot analysis suggested that the formation of HCAB-HSA complex followed a 1:1 stoichiometry. Molecular docking and the displacement assay demonstrated the binding site of HCAB was subdomain IIA and IB of HSA. We also tested HSA levels in human plasma for practical application, the results obtained from HCAB method were similar to those obtained from the standard clinical method.

Two-step separation-free quantitative detection of HSA and FIB in human blood plasma by a pentaphenylpyyrrole derivative with aggregation-enhanced emission properties

A novel fluorescence based strategy, employing the aggregation-enhanced emission (AEE) property of sodium 4,4′,4”-(3,4-diphenyl-1H-pyrrole-1,2,5-triyl)tribenzoate (DP-TPPNa), was proposed to determine the concentrations of human serum albumin (HSA) and fibrinogen (FIB) in human plasma without separation required. HSA level was determined at concentrations lower than 110μg/mL by the selective PL response of DP-TPPNa to HSA and FIB was quantified at high HSA concentrations in the range of 140–200μg/mL by the synergistic effects of FIB and HSA on the PL intensity of DP-TPPNa. Compared with authoritative instruments, the method developed in the present work is rapid, simple, extremely sensitive, highly selective and precise for the quantitative detection of proteins with one single system, which is an ideal detection system candidate for the early diagnosis of diseases. The results also indicate that the interactions between proteins can affect their configuration and conformation blood plasma. This can be used to study the correlations among the serum components.

Monitoring human serum albumin cell cultures using surface plasmon resonance (SPR) spectroscopy

Abstract. Continuously monitoring cell cultures is essential for both controlling critical parameters and improving understanding of key processes. An ideal technique in this context is surface plasmon resonance (SPR) spectroscopy, which essentially exploits changes in the angle of incident light that occur when molecules bind to a surface. It provides the ability to monitor real-time changes in small concentrations of various molecules, with no need for additional labels or sample preparation. Here we present an SPR-based immunoassay for monitoring concentrations of human serum albumin (HSA), and compare its sensitivity when used in conjunction with a Biacore platform and the cheaper, smaller liSPR system. In conjunction with either system, the immunoassay can detect HSA (a hepatocyte viability marker) at concentrations typically present in three-dimensional hepatocyte cultures mimicking the liver used to evaluate effects of drug candidates before exposure to humans or animals. Furthermore, in conjunction with the liSPR system, it is sufficiently sensitive to measure the much lower HSA levels present in skin–hepatocyte co-cultures.

Influences of Artificial Heart-Lung Machine Operation on the Binding Sites of Albumin: Possibility of an Effective Administration Plan

The purpose of this study is to investigate the influence of changes in the human serum albumin (HSA) and free fatty acid (FFA) on alteration of the binding abilities of sites I and II after the operation of an artificial heart-lung machine. The binding abilities of phenytoin (site I) and diazepam (site II) to patients' sera collected before and after the operation of an artificial heart-lung machine and pseudopatient serum samples were examined by ultra-filtration. The binding ability of site I markedly decreased after the operation of an artificial heart-lung machine in all patients, and the binding ability of site II unexpectedly increased in some patients. The variation in pseudopatient serum was similar to that in the patient sera. The difference in the binding ability between sites I and II was due to that the fact that the binding ability of site I is more strongly influenced by HSA level reduction than by [FFA]/[HSA] reduction, whereas the binding ability of site II is more strongly influenced by [FFA]/[HSA] reduction than by HSA level reduction in some patients. Therefore, it may be possible to predict the binding ability of site I by monitoring the HSA level without directly monitoring the free phenytoin fraction (%).

Interference-free determination of ischemia-modified albumin using quantum dot coupled X-ray fluorescence spectroscopy

Ischemia-modified protein (IMA) is the most sensitive diagnostic biomarker of ischemic heart disease, but differentiation of IMA from human serum albumin (HSA), a ubiquitous serum protein, is still challenging owing to the shared antigenicity. In this investigation, we developed a rapid and interference-free approach for IMA determination using quantum dots-coupled X-ray Fluorescence Spectroscopy (Q-XRF). In a typical Q-XRF assay, serum total HSA is quantified using quantum dot-coupled sandwich immunoassay, and intact HSA (iHSA) is determined using a XRF spectroscopy, by measuring XRF intensity of Co (II) bonded to iHSA. IMA concentration is automatically determined within 30min by calculating the difference between total HSA and iHSA. This strategy can effectively eliminate the interference from native HSA level. Results show that no significant influences have been observed from hemolysis or high levels of cholesterol (7mg/L), triglyceride (5.2mg/L), IgG (10g/L), and fibrinogen (4g/L). A linearity of 1–100mg/mL is obtained in iHSA determination using XRF (r2=0.979). The proposed Q-XRF assay demonstrates a lowest detection limit of 0.05U/mL. Receiver-operating characteristic (ROC) curves reveal that Q-XRF assay provide an improved sensitivity than ACB assay (95.9% vs. 82.9%) in differentiating ischemic patients from health individuals, at an optimal cutoff point of 79.2U/mL. The proposed approach provides a new strategy for interference-free, simple and rapid evaluation of IMA concentration by combining sandwich immunoassay and XRF spectroscopy.

A sandwich ELISA for measuring benzo[a]pyrene–albumin adducts in human plasma

Exposure to polycyclic aromatic hydrocarbons has often been quantified via DNA or human serum albumin (HSA) adducts of the carcinogenic metabolite benzo[a]pyrene diol epoxide (BPDE). We previously reported a sandwich ELISA, using 8E11 as capture antibody and anti-HSA as detection antibody, that detected intact BPDE adducts in HSA isolated from plasma. After confirming that BPDE binds to HSA at His146 and Lys195, we modified the ELISA to measure intact BPDE–HSA directly in human plasma. To adjust for interference due to nonspecifically bound HSA on well surfaces and to cross-reactivity of the antibodies, the ELISA employs paired wells with and without addition of BPDE tetrols to deactivate 8E11. By performing assays in quadruplicate, a series of sample-specific adjustments and screening steps are used to reduce measurement errors that are a consequence of detecting low BPDE–HSA concentrations in the general population. ELISA measurements of BPDE–HSA in plasma from smoking and nonsmoking subjects (range 0.280–2.88 ng BPDE–HSA/mg HSA) and from highway workers with and without exposure to asphalt emissions (range 0.346–13.9 ng BPDE–HSA/mg HSA) detected differences in BPDE–HSA levels in the a priori expected directions.

Human Hepatoma HepaRG Cell Line Engraftment in Severe Combined Immunodeficient × Beige Mice Using Mouse-Specific Anti-Fas Antibody

Chimeric mice with repopulated human hepatocytes are widely used for drug development research. HepaRG cell line is a naturally immortalized human liver cell line with progenitor properties and inducible bipotent differentiation capability. It would be useful if HepaRG cells could repopulate damaged livers severe, combined immunodeficient × beige (SCID/bg) mice and exhibit special human hepatic features. After inducing mouse hepatocyte apoptosis with an antimouse agonistic Fas monoclonal antibody (Jo2 mAb), HepaRG cells with bipotent differentiation capability were repopulated in SCID/bg mouse livers. HepaRG cells were transplanted intrasplenically into SCID/bg mice treated with 0.2 mg/kg Jo2 mAb once a week for 10 weeks. Human hepatocyte repopulation was characterized by immunohistochemistry for human serum albumin (HSA), Hep Par1, and CK18 and by immunofluorescence staining for HSA. Human albumin mRNA was detected by reverse-transcription polymerase chain reactions. HSA levels were quantified by Western blotting and enzyme-linked immunosorbent assays. Our results showed that HepaRG cell engraftment protected mice from the effects of Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. At 2 weeks after transplantation, increase concentrations of HSA were detected in recipient blood and liver. At 12 weeks after transplantation, approximately 15%–20% of mice showed livers repopulated with human hepatocytes. In conclusion, normal SCID/bg mice were suitable recipients for HepaRG cell transplantation when induced with Jo2 mAb. This chimeric mouse model with HepaRG cells could serve as a useful model for in vivo studies of drug metabolism and hepatitis virus infections.

The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform.

An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved. This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.

Erythrocyte uptake and protein binding of cyclosporin A (CyA) in human blood: factors affecting CyA concentration in erythrocytes.

To further the understanding of the complexity of cyclosporin A (CyA) pharmacokinetics, we conducted an erythrocyte uptake and efflux study, and a protein binding study in human blood. The uptake study showed that the transport of CyA from the extracellular fraction to erythrocytes was retarded by increased human serum albumin (HSA) and lipid levels in this fraction. In addition, the concentration of CyA in erythrocytes increased with increases in CyA concentration in blood and reductions in hematocrit. The efflux study showed that the transport of CyA from erythrocytes to the extracellular fraction was essentially enhanced by increases of HSA and lipid levels in that fraction, but that these effects were relatively small. There were two affinity binding sites for CyA in ghost-free erythrocyte hemolysate, but not in the plasma fraction. The affinity binding constants for these binding sites were reduced by elevations in temperature, and under physiological conditions, 37 degrees C, almost all the CyA in erythrocytes was bound to a CyA binding protein, namely, cyclophillin. These findings suggest that CyA distribution in blood is of two different types which are present in the erythrocyte and plasma fractions, respectively. Monitoring of blood biochemistry variables showed that the concentration of CyA in erythrocytes had an interlocking relationship with these physiological factors, which were related to patient disease state, i.e., hematocrit, lipids, albumin, and total protein; the concentration of CyA in erythrocytes could be predicted from these physiological factors.

Antiinflammatory effects of a corticosteroid and protease inhibitor agents on antigen-induced otitis media in chinchillas

Using an antigen-induced otitis media (OM) model in chinchillas sensitized with human serum albumin (HSA), we studied the antiinflammatory effect of a corticosteroid (triamcinolone) and a protease (kallikrein) inhibitor (aprotinin) by conducting both biochemical and cytologic analyses of middle ear fluid (MEF). The levels of HSA, alpha 1-antitrypsin, alpha 2-macroglobulin, and total protein concentration were measured in the MEF to evaluate the degree of OM. Both vascular leakage and leukocyte infiltration were significantly reduced by corticosteroid and inhibitor treatments. HSA levels in the MEF were markedly reduced after HSA challenge, and both treatments reduced the drop in HSA levels. In conclusion, the kallikrein-kinin system is related to the early stage of antigen-induced OM. Both corticosteroid and inhibitor treatments effectively reduce the degree of antigen-induced OM in chinchillas, suggesting that both may be useful local therapeutic agents in the treatment of human OM.

The relationship between antigen levels and middle ear inflammation in antigen-induced otitis media in the chinchilla.

The relationship between antigen levels in middle ear effusions (MEE) and the degree of middle ear inflammation was studied in an antigen-induced otitis media model, using chinchillas sensitized with human serum albumin (HSA). The degree of middle ear inflammation was evaluated by both tympanometric analysis, and cytological and biochemical analyses of the MEE. Middle ear inflammation develops after HSA challenge with a remarkable decrease in HSA levels in the MEE. This inflammation persists even when HSA levels are no longer detectable in the MEE. These findings show that local challenges with an antigen induce a certain degree of middle ear inflammation, which continues even after complete elimination of the antigen from the middle ear through an immunological defense mechanism.

The relation of antigen levels in the middle ear and round window membrane permeability in antigen-induced otitis media

By using an antigen induced (Arthus) otitis media (OM) model in chinchillas sensitized with human serum albumin (HSA), we investigated the passage of HSA through the round window membrane (RWM) by measuring HSA levels in both the middle ear fluid (MEF) and the perilymph (PL). The effect of corticosteroid treatment on HSA passage was also studied. HSA (0.5, 5, and 50mg/ml; 1ml) was instilled into the left middle ear bulla (MEB) of both control and sensitized animals (control and Arthus group), and HSA containing 12.5mg/ml of Triamcinolone diacetate was instilled into the right MEB of both animals (control+steroid and Arthus+steroid groups). Mean values of HSA levels in both the MEF and the PL were proportional to the original three different HSA levels (0.5, 5 and 50mg/ml) instilled into the MEB. HSA levels in the MEF differed significantly among the three groups in the following order (Arthus<Arthus+steroid<control). In the control, HSA levels in the PL were significantly reduced by a steroid treatment (p<0.025, 50mg/ml and p<0.05, 5mg/ml). The percentage of HSA positive PL samples was significantly lower in the Arthus group than the control group (p<0.05, 5mg/ml). HSA levels in the PL appear to depend on HSA levels in the MEF which were reduced in the sensitized animals. These results suggest that an immunological reaction may be involved in the reduction of HSA passage through the RWM by reducing HSA levels in the MEF. Corticosteroid treatment may reduce the passage of HSA into the PL in case of non-sensitized animals.

Relationship between Antigen Levels in Middle Ear and Antigen Passage through Round Window Membrane in Antigen-induced Otitis Media

By using an antigen-induced (Arthus) otitis media (OM) model in chinchillas sensitized with human serum albumin (HSA), we investigated the passage of HSA through the round window membrane (RWM) by measuring HSA levels in both the middle ear fluid (MEF) and the perilymph (PL). The effect of corticosteroid treatment on HSA passage was also studied. Mean HSA levels in both the MEF and the PL were proportional to the HSA dose administered (0.5, 5, 50 mg/ml). The proportion of HSA-positive PL samples was significantly lower in the sensitized group than that of the control (p less than 0.01). HSA levels in the PL appear to depend on HSA levels in the MEF, which were significantly depressed in the sensitized animals. These results suggest that an immunological reaction may be involved in the reduction of antigen (HSA) passage through the RWM by reducing antigen concentrations in the MEF. Corticosteroid treatment reduces the passage of HSA into the PL in non-sensitized animals.

Permeability of round window membrane to human serum albumin in chinchillas

The round window membrane (RWM) permeability to human serum albumin (HSA) was investigated in sensitized chinchillas with HSA (Arthus otitis media). The effect of a corticosteroid agent (Triamcinolone) on the RWM permeability was also studied. HSA could not be detected in either the perilymph (PL) and CSF of normal chinchilla within 1hr after instillation into the middle ear bulla. However, significant level of HSA could be quantified in the PL of which peak was observed at 24 hrs after instillation. In sensitized chinchillas, HSA level in the PL at 12 hrs after instillation was significantly higher than that of normal chinchilla (p<0.01), and had large individual variations after 24 hrs. HSA levels in the PL significantly decreased by a steroid treatment (p<0.025 at 24 hrs and p<0.05 at 48 hrs). It is concluded that only a small amount of HSA could pass through the normal RWM for a short time, and certain period of time is necessary for HSA to pass, and that the RWM permeability to HSA increases in the early stage of Arthus otitis media (about 12 hrs) and has large variations with the development of Arthus otitis media. A corticosteroid agent reduces the passage of HSA by suppressing the degree of Arthus otitis media.

Round window membrane permeability to human serum albumin in antigen-induced otitis media

The round window membrane (RWM) permeability to human serum albumin (HSA) was investigated in both normal chinchillas and chinchillas sensitized with HSA. The effect of a corticosteroid agent (triamcinolone) on the RWM permeability was also analyzed. It was found that HSA could not be detected in either the perilymph or the cerebrospinal fluid of normal chinchillas within 1 hour after instillation into the middle ear bulla. Perilymph levels of HSA peaked 24 hours after instillation. In antigen-induced otitis media, the HSA level in the perilymph at 12 hours after challenge was significantly higher than that in normal chinchillas (P less than .01), but it did not increase with the development of otitis media. A significant difference of HSA level in the perilymph of animals with and without steroid treatment was noted (P less than .025 at 24 hours and P less than .05 at 48 hours). It is concluded that little HSA passes through the normal RWM in a short time and that the RWM permeability to HSA increases in the early stage (about 12 hours) of antigen-induced otitis media. Corticosteroids can affect the RWM permeability to HSA by reducing the level of antigen-induced otitis media.

Metabolism of Radio-iodinated Human Serum Albumin in Patients With Monoclonal Gammopathy

Abstract (mean HSA level 3.3 g/100 ml, normal with 125I-labeled human serum albumin (HSA) in five patients with monoclonal gammopathy; three with Waldenström’s macroglobulinemia (one also had a γG monoclonal protein) and one patient each with γG multiple myeloma and γG benign gammopathy. Several abnormalities in HSA metabolism occurred in different combinations in different patients and no single consistent pattern was seen. The abnormalities included hypoalbuminemia (means HSA level 3.3 g/100, normal controls 4.7 g/100 ml); hypervolemia due to an expanded plasma volume (mean plasma volume 54.4 ml/kg, normal controls 41.0 ml/kg); decreased plasma and total body albumin pools; increased plasma localization of albumin; shortened plasma T ½; reduced fractional turnover rate; and reduced albumin synthesis rate. The most marked changes were noted in two patients with Waldenström’s macroglobulinemia who required plasma-pheresis for the hyperviscosity syndrome.