Amount Of Human Serum Albumin Research Articles

Updated review on neuromodulators

As botulinum toxin is increasingly used cosmetically and medically, it is important to understand the differences between each formulation of this product. While the active molecule in each is a 150 kDa protein that prevents neurotransmitter release, there are currently five products FDA-approved for clinical use, with a sixth currently in its Phase 3 trial and a seventh applied under FDA review, each with different nontoxic accessory proteins and varying amounts of human serum albumin. These properties affect diffusion rates and storage methods, which are outlined in this review. Common complications and recommendations to avoid them are discussed.

Fluorescence Enhancement Based Quantification of Human Serum Albumin from Biological Sample Using Indole Based Nanosuspension: Molecular Interactions and Molecular Docking Studies.

Fluorescent 3-[(E)-(2-phenylhydrazinylidene) methyl]-1H-indole (PHI) was synthesized by condensation of indole-3-carboxaldehyde and phenyl hydrazine in presence of acetic acid and ethanol and after spectral characterization used further to prepare its aqueous nano suspension by reprecipitation method using polyvinylpyrrolidone (PVP) as stabilizer. The average particle size of nano suspension measured by Dynamic Light Scattering (DLS) was found 77.5nm while FESEM microphotograph showed spherical morphology. The blue shift in the absorption spectrum and stokes shifted fluorescence of nanosuspension of PHI compared to its monomer spectrum in dilute solution indicate formation of H-type aggregate by face to face overlapping of the molecules.The aggregation induced enhanced emission (AIEE) of PVP capped nanosuspension of PHI is increased appreciably by presence of aqueous solution of human serum albumin (HSA). A suitable mechanism of molecular binding interactions based on complex formation between PHI nanoaggregate and HSA through PVP is proposed. Fluorescence life time, zeta potential and particle size data of PHI nanoparticles (PHINPs) obtained in presence of different amounts of HSA are in support of molecular interactions leading to complex formation. The molecular docking studies showed that HSA and PVP capped PHINPs exhibit strong hydrogen bonding interaction. The fluorescence enhancement effect induced in PHI nanosuspension is used further to develop analytical method for quantitative estimation of HSA in aqueous biological sample solution.

Design of Ultra-Thin PEO/PDMAEMA Polymer Coatings for Tunable Protein Adsorption.

Protein adsorption on solid surfaces provides either beneficial or adverse outcomes, depending on the application. Therefore, the desire to predict, control, and regulate protein adsorption on different surfaces is a major concern in the field of biomaterials. The most widely used surface modification approach to prevent or limit protein adsorption is based on the use of poly (ethylene oxide) (PEO). On the other hand, the amount of protein adsorbed on poly(2-(dimethylamine)ethyl methacrylate) (PDMAEMA) coatings can be regulated by the pH and ionic strength of the medium. In this work, ultra-thin PEO/PDMAEMA coatings were designed from solutions with different ratios of PEO to PDMAEMA, and different molar masses of PEO, to reversibly adsorb and desorb human serum albumin (HSA), human fibrinogen (Fb), lysozyme (Lys), and avidine (Av), four very different proteins in terms of size, shape, and isoelectric points. X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM), and atomic force microscopy (AFM) were used to characterize the mixed polymer coatings, revealing the presence of both polymers in the layers, in variable proportions according to the chosen parameters. Protein adsorption at pH 7.4 and salt concentrations of 10−3 M was monitored by QCM. Lys and Av did not adsorb on the homo-coatings and the mixed coatings. The amount of HSA and Fb adsorbed decreased with increasing the PEO ratio or its molar mass in a grafting solution. It was demonstrated that HSA and Fb, which were adsorbed at pH 7.4 and at an ionic strength of 10−3 M, can be fully desorbed by rinsing with a sodium chloride solution at pH 9.0 and ionic strength 0.15 M from the mixed PEO5/PDMAEMA coatings with PEO/PDMAEMA mass ratios of 70/30, and 50/50, respectively. The results demonstrate that mixed PEO/PDMAEMA coatings allow protein adsorption to be finely tuned on solid surfaces.

A Proteomic Study of Atherosclerotic Plaques in Men with Coronary Atherosclerosis.

Background: To study the changes in protein composition of atherosclerotic plaques at different stages of their development in coronary atherosclerosis using proteomics. Methods: The object of research consisted of homogenates of atherosclerotic plaques from coronary arteries at different stages of development, obtained from 15 patients. Plaque proteins were separated by two-dimensional electrophoresis. The resultant protein spots were identified by the matrix-assisted laser desorption ionization method with peptide mass mapping. Results: Groups of differentially expressed proteins, in which the amounts of proteins differed more than twofold (p < 0.05), were identified in pools of homogenates of atherosclerotic plaques at three stages of development. The amounts of the following proteins were increased in stable atherosclerotic plaques at the stage of lipidosis and fibrosis: vimentin, tropomyosin β-chain, actin, keratin, tubulin β-chain, microfibril-associated glycoprotein 4, serum amyloid P-component, and annexin 5. In plaques at the stage of fibrosis and calcification, the amounts of mimecan and fibrinogen were increased. In unstable atherosclerotic plaque of the necrotic–dystrophic type, the amounts of human serum albumin, mimecan, fibrinogen, serum amyloid P-component and annexin were increased. Conclusion: This proteomic study identifies the proteins present in atherosclerotic plaques of coronary arteries by comparing their proteomes at three different stages of plaque development during coronary atherosclerosis.

Impedimetric Immunosensor Utilizing Polyaniline/Gold Nanocomposite-Modified Screen-Printed Electrodes for Early Detection of Chronic Kidney Disease

The presence of small amounts of human serum albumin (HSA) in urine or microalbuminuria (30–300 µg/mL) is a valuable clinical biomarker for the early detection of chronic kidney disease (CKD). Herein, we report on the development of an inexpensive and disposable immunosensor for the sensitive, specific, and label-free detection of HSA using electrochemical impedance spectroscopy (EIS). We have utilized a simple one-step screen-printing protocol to fabricate the carbon-based three-electrode system on flexible plastic substrates. To enable efficient antibody immobilization and improved sensitivity, the carbon working electrode was sequentially modified with electropolymerized polyaniline (PANI) and electrodeposited gold nanocrystals (AuNCs). The PANI matrix serves as an interconnected nanostructured scaffold for homogeneous distribution of AuNCs and the resulting PANI/AuNCs nanocomposite synergically improved the immunosensor response. The PANI/AuNCs-modified working electrode surface was characterized using scanning electron microscopy (SEM) and the electrochemical response at each step was analyzed using EIS in a ferri/ferrocyanide redox probe solution. The normalized impedance variation during immunosensing increased linearly with HSA concentration in the range of 3–300 µg/mL and a highly repeatable response was observed for each concentration. Furthermore, the immunosensor displayed high specificity when tested using spiked sample solutions containing different concentrations of actin protein and J82 cell lysate (a complex fluid containing a multitude of interfering proteins). Consequently, these experimental results confirm the feasibility of the proposed immunosensor for early diagnosis and prognosis of CKD at the point of care.

Bence-Jones protein as the form of nano-scaled β-stacked supramolecular aggregates

Abstract. The formation in β-structured protein aggregates in tissues and fluids of the body is one of the most dangerouse complications of various diseases. The most famous of them are amyloidoses, but they such deposits are observed at other, much more widespread, diseases. The generally accepted approach to amyloids’detectionis based on high-specific coloring by Congo Red dye. However, the Abbe's diffraction limit excludes the seeing of the objects smaller than 0.61 wavelengths (about 240 nm). Such nanoscale formations are capable to disrup the functioning of surrounding tissues, to causethe complications and recurrences of the disease, and to pass through biological barriers with the following accumulation in body’s fluids. It’s likely that these conditions are the cause of the urinary congophilia, that is associated with preeclampsia at pregnancy and chronic kidney disease. Nor the less suspicious object is the Bens-Jones protein that appears in the urine at multiple myeloma and some other diseases, which are in more or less extent,are related to the disturbance of protein metabolism.&#x0D; The purpose of this study was to clarify the aggregate state of the Bens-Jones protein as a possible β-structured supramolecular associate.&#x0D; Methods.The subject of the study was the freshly received urine from a patient with multiple myeloma. The presence of the Bens-Jones protein was checked by thermopacification of the acidified sample. For control, the urine was used by a healthy person with the addition of certain amounts of human serum albumin ("Reanal", Hungary) with a concentration of 0, 0.01, 0.1 and 1%.&#x0D; Result. The obtained data testify to the appropriateness of such a point of view and create preresquites for the expanding of diagnostic possibilities.&#x0D; Conclusions.The results obtained during the study testify to the peculiarity of the structure of the Bens-Jones protein, which is nano-sized beta-structured supramolecular

Human Alpha-1-Antitrypsin (hAAT) therapy reduces renal dysfunction and acute tubular necrosis in a murine model of bilateral kidney ischemia-reperfusion injury.

Several lines of evidence have demonstrated the anti-inflammatory and cytoprotective effects of alpha-1-antitrypsin (AAT), the major serum serine protease inhibitor. The aim of the present study was to investigate the effects of human AAT (hAAT) monotherapy during the early and recovery phase of ischemia-induced acute kidney injury. Mild renal ischemia-reperfusion (I/R) injury was induced in male C57Bl/6 mice by bilateral clamping of the renal artery and vein for 20 min. hAAT (80 mg/kg, Prolastin®) was administered daily intraperitoneally (i.p.) from day -1 until day 7 after surgery. Control animals received the same amount of human serum albumin (hAlb). Plasma, urine and kidneys were collected at 2h, 1, 2, 3, 8 and 15 days after reperfusion for histological and biochemical analysis. hAAT partially preserved renal function and tubular integrity after induction of bilateral kidney I/R injury, which was accompanied with reduced renal influx of macrophages and a significant decrease of neutrophil gelatinase-associated lipocalin (NGAL) protein levels in urine and plasma. During the recovery phase, hAAT significantly decreased kidney injury molecule-1 (KIM-1) protein levels in urine but showed no significant effect on renal fibrosis. Although the observed effect size of hAAT administration was limited and therefore the clinical relevance of our findings should be evaluated carefully, these data support the potential of this natural protein to ameliorate ischemic and inflammatory conditions.

Clinical efficacy of tranexamic acid administrationviadifferent routes during total hip arthroplasty: study protocol for a prospective randomized controlled trial

Background: Intravenous injection or topical application of tranexamic acid during total hip arthroplasty has been shown to effectively reduce blood loss. There is no consensus on differences in clinical efficacy and safety between these two administration methods. To determine the optimal clinical efficacy of tranexamic acid during hip replacement, we will compare differences between these two administration methods during total hip arthroplasty. Methods/Design: The study protocol is a prospective, paired (1:1:1), double-blind, randomized controlled clinical trial. A total of 174 patients who will receive hip replacement at Ruijin Hospital, Shanghai Jiao Tong University, China will be randomly divided into three groups, with 58 patients per group. Before the end of surgery, in the placebo group, 60 mL of physiological saline will be used to soak the articular cavity for at least 3 minutes and then be sucked away; in the topical tranexamic acid group, 60 mL of physiological saline containing 3 g tranexamic acid will be used and then sucked away; in the intravenous tranexamic acid group, 100 mL of physiological saline containing 10 mg/kg tranexamic acid will be intravenously administered 15 minutes before surgery and then again 3 hours later. The primary outcome is total blood loss on postoperative day 3, which is calculated using Nadler's and Gross's formula. Secondary outcomes include blood transfusion rates, drainage output within 2 days postoperation, amount of human serum albumin used during hospitalization, postoperative blood coagulation indices, and incidences of deep venous thrombosis and other thromboembolic events during postoperative day 1-week 6. Discussion: This protocol is powered to provide reference information for the rational use of tranexamic acid by comparing clinical efficacy of the drug between intravenous and topical administration routes. Trial registration: ClinicalTrials.gov identifier: NCT02312440; registered on 28 November 2014. Ethical approval: This study protocol was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University, China (permission No. (2014)-47-2) and will be performed in accordance with the Declaration of Helsinki.

Abstract B14: A novel strategy for the treatment of melanoma

Abstract Introduction: Approximately 76,000 individuals are diagnosed with melanoma, and about 9,000 die as a result of the disease each year in the United States. Even though there are many options for standard treatment of melanoma such as surgical resection, radiotherapy, intraregional therapy with sclerosing agents, isolated limb perfusion, and immunotherapy, the mortality rate is still high. The high mortality reflects a lack of effective therapeutic methods for the treatment of melanoma. The purpose of this study is to develop an effective therapeutic approach for the treatment of melanoma, using mild/moderate (less-than 41oC) hyperthermia (HT) to actively modulate the delivery of the FDA approved anticancer drug nab-paclitaxel (human serum albumin (HSA) based paclitaxel-nanoparticle) to tumors. Physiologically, HSA serves as a natural carrier to deliver nutrients, including itself as an amino acid source to cells with metabolic and/or reproductive needs by binding to its receptor albondin on the cell surfaces. We and other investigators have reported that the rate of cellular metabolism increases as temperature rises to the mild/moderate temperature range of 38oC to 41oC, implying that heat increase cellular uptake of HSA to meet the demands of the higher metabolic rate. In the clinic, nab-paclitaxel has demonstrated preferential uptake in tumors with good solubility, stability and longer circulation time with limited toxicity. In this study, we used HT to deliver nab-paclitaxel to melanoma xenograft in athymic mice. Materials and Methods: Human melanoma A375 and G361 cells were purchased from ATCC (American Type Culture Collection) and maintained in a growth medium of Dulbecco's Modified Eagle's Medium (A375) and McCoy's 5A medium (G361) supplemented with 10% human serum, 100 units/ml penicillin and 100 μg/ml streptomycin. Both melanoma cells were adapted to human serum containing medium by maintaining them in the medium for at least 6 months. Human fibroblast GM05399 cells were obtained from Coriell Cell Repositories (Coriell Institute, Camden, NJ). Near infrared (NIR) dye (IRDye 800CW) Protein Labeling Kits were purchased from Li-Cor Inc. Nude mice, 4 or 5-weeks-old, were bought from the National Cancer Institute at Frederick, MD. Immunofluorescence staining and flow cytometry methods were applied to measure HSA in tumor cells in vitro after heat treatment. To observe the biodistribution of nab-paclitaxel in vivo, we labeled nab-paclitaxel with NIR dye using an IRDye 800CW Protein Labeling Kit following the manufacturer's instructions. The integrity of the nab-paclitaxel after a single freezing and thawing cycle, and nab-paclitaxel-NIR dye conjugates were measured using Dynamic Light Scattering (DLS). Biodistribution and kinetic changes of the nab-paclitaxel-NIR dye conjugates were examined and monitored after injection of the conjugates and heat treatment with the Pearl Imager in melanoma xenograft carrying animals. Results: (1). HSA accumulated in melanoma cells after heating at 41oC for as little as 0.5 hour; there was no increase in the amount of HSA in human normal fibroblasts after the same heat treatment. (2). Integrity of the nab-paclitaxel was not interrupted after a single freezing and thawing cycle, and by the labeling procedures with fluorescent dye. (3). Most interestingly nab-paclitaxel and its NIR dye conjugates are stable after heating at 41oC for 1 hr. (4). Mild/moderate HT actively modulated the delivery of nab-paclitaxel-NIR dye conjugates to tumors, accumulated and retained nab-paclitaxel-NIR conjugates in the tumor for at least 72 hrs. Conclusion and Future Studies: HT has been previously used to improve blood circulation and increase cell membrane permeability for antitumor chemical delivery to tumors in the clinic. In this study, the accumulation of nab-paclitaxel in heated tumor cells appears to be receptor-mediated. Because of size constraints, nab-paclitaxel could not passively diffuse into tumor cells, even cells permeabilized by heat treatment. Therefore, our preliminary results have demonstrated that mild/moderate hyperthermia can actively facilitate delivery of nab-paclitaxel to tumors in vitro and in vivo. HT promoted retention of nab-paclitaxel-NIR conjugates in tumor xenograft for a prolonged period of time as compared to controls (without heat treatment). A study is currently underway to evaluate the therapeutic efficacy of HT combined with nab-paclitaxel in the treatment of melanoma in vivo. A study to assess whether whole body hyperthermia can modulate delivery of nab-paclitaxel to metastatic tumors is being considered. Magnetic Resonance-guided High Intensity Focused Ultrasound mediated HT in combination with nab-paclitaxel may be an effective approach for the treatment of melanoma. Citation Format: Mai Xu, Albert C. Lockhart, Robert J. Myerson, Imran Zoberi, Gerald P. Linette, Andrea Wang-Gillam. A novel strategy for the treatment of melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B14.

Biochemical characterization of the interactions between doxorubicin and lipidic GM1 micelles with or without paclitaxel loading.

Doxorubicin (Dox) is an anthracycline anticancer drug with high water solubility, whose use is limited primarily due to significant side effects. In this study it is shown that Dox interacts with monosialoglycosphingolipid (GM1) ganglioside micelles primarily through hydrophobic interactions independent of pH and ionic strength. In addition, Dox can be incorporated even into GM1 micelles already containing highly hydrophobic paclitaxel (Ptx). However, it was not possible to incorporate Ptx into Dox-containing GM1 micelles, suggesting that Dox could be occupying a more external position in the micelles. This result is in agreement with a higher hydrolysis of Dox than of Ptx when micelles were incubated at alkaline pH. The loading of Dox into GM1 micelles was observed over a broad range of temperature (4°C–55°C). Furthermore, Dox-loaded micelles were stable in aqueous solutions exhibiting no aggregation or precipitation for up to 2 months when kept at 4°C–25°C and even after freeze–thawing cycles. Upon exposure to blood components, Dox-containing micelles were observed to interact with human serum albumin. However, the amount of human serum albumin that ended up being associated to the micelles was inversely related to the amount of Dox, suggesting that both could share their binding sites. In vitro studies on Hep2 cells showed that the cellular uptake and cytotoxic activity of Dox and Ptx from the micellar complexes were similar to those of the free form of these drugs, even when the micelle was covered with albumin. These results support the idea of the existence of different nano-domains in a single micelle and the fact that this micellar model could be used as a platform for loading and delivering hydrophobic and hydrophilic active pharmaceutical ingredients.

The use of laser-induced fluorescence or ultraviolet detectors for sensitive and selective analysis of tobramycin or erythropoietin in complex samples

Complex samples analysis is a challenge in pharmaceutical and biopharmaceutical analysis. In this work, tobramycin (TOB) analysis in human urine samples and recombinant human erythropoietin (rhEPO) analysis in the presence of similar protein were selected as representative examples of such samples analysis. Assays of TOB in urine samples are difficult because of poor detectability. Therefore laser induced fluorescence detector (LIF) was combined with a separation technique, micellar electrokinetic chromatography (MEKC), to determine TOB through derivatization with fluorescein isothiocyanate (FITC). Borate was used as background electrolyte (BGE) with negative-charged mixed micelles as additive. The method was successively applied to urine samples. The LOD and LOQ for Tobramycin in urine were 90 and 200ng/ml respectively and recovery was >98% (n=5). All urine samples were analyzed by direct injection without sample pre-treatment. Another use of hyphenated analytical technique, capillary zone electrophoresis (CZE) connected to ultraviolet (UV) detector was also used for sensitive analysis of rhEPO at low levels (2000IU) in the presence of large amount of human serum albumin (HSA). Analysis of rhEPO was achieved by the use of the electrokinetic injection (EI) with discontinuous buffers. Phosphate buffer was used as BGE with metal ions as additive. The proposed method can be used for the estimation of large number of quality control rhEPO samples in a short period.

Magnetic nanoparticles–serum proteins bioconjugates for binding of irinotecan

The binding of irinotecan to serum proteins (hemoglobin, globulin and human serum albumin) was studied on the surface of epoxide modified superparamagnetic iron oxide nanoparticles (GPTS-SPIONs), which were synthesized by the coprecipitation of ferrous and ferric salts with NH4OH and then modified with [3-(2,3-epoxypropoxy)propyl] trimethoxy silane (GPTS) to obtain functional epoxide groups on the SPIONs’ surface. Results were compared to find an alternative as drug carries system. Data showed that binding amount of human serum albumin (HSA), globulin (Glb) and hemoglobin (Hb) found to be as 44, 21.2 and 32.6μg per 20mg of GPTS modified SPIONs, respectively. The thermal behavior of the serum protein–Ir interaction on GPTS-SPIONs was also studied by using thermo gravimetric analysis (TGA) technique and then the kinetic parameters for the thermal decomposition were determined using Horowitz–Metzger method.

Role of the blood‐brain barrier in limiting CNS uptake of pyrethroid insecticides: findings with hCMEC cells as a proxy (1064.14)

Pyrethroids, the most widely‐used insecticides in the U.S., generally exhibit low neurotoxicity in humans and other mammals. Kinetic studies of the pyrethroid deltamethrin (DLM) have revealed that levels of the hydrophobic, neurotoxic parent compound are &lt;20% of blood levels in rats. Experiments with hCMEC cells, a human brain endothelial cell line, were undertaken as part of an effort to understand the BBB’s role in limited CNS deposition of DLM. hCMEC cells were grown in EGM‐2 medium, seeded in T‐75 flasks, and allowed to grow to confluency within 3‐4 days. DLM uptake by the cells was concentration‐dependent, indicating a saturable process. Cyclosporin inhibited uptake, suggesting an active influx transport process. Increasing the amount of human serum albumin reduced the free/unbound fraction of DLM, which correlated with decreased cellular uptake. In conclusion: DLM’s pronounced plasma protein/lipoprotein binding (~90+%) appears to limit its availability for BBB passage; and a BBB influx transporter of low capacity and low affinity contributes to hCMEC uptake.Grant Funding Source: Council for the Advancement of Pyrethroid human Risk Assessment (CAPHRA)

Optimizing the Properties of the Protein Corona Surrounding Nanoparticles for Tuning Payload Release

We manipulate the passive release rates of DNA payloads on protein coronas formed around nanoparticles (NPs) by varying the corona composition. The coronas are prepared using a mixture of hard and soft corona proteins. We form coronas around gold nanorods (NRs), nanobones (NBs), and carbon nanotubes (CNTs) from human serum (HS) and find that tuning the amount of human serum albumin (HSA) in the NR-coronas (NR-HS-DNA) changes the payload release profile. The effect of buffer strength, HS concentration, and concentration of the cetyltrimethylammonium bromide (CTAB) passivating the NP surfaces on passive release is explored. We find that corona properties play an important role in passive release, and concentrations of CTAB, HS, and phosphate buffer used in corona formation can tune payload release profiles. These advances in understanding protein corona properties bring us closer toward developing a set of basic design rules that enable their manipulation and optimization for particular biological applications.

Effect of pullulan nanoparticle surface charges on HSA complexation and drug release behavior of HSA-bound nanoparticles.

Nanoparticle (NP) compositions such as hydrophobicity and surface charge are vital to determine the presence and amount of human serum albumin (HSA) binding. The HSA binding influences drug release, biocompatibility, biodistribution, and intercellular trafficking of nanoparticles (NPs). Here, we prepared 2 kinds of nanomaterials to investigate HSA binding and evaluated drug release of HSA-bound NPs. Polysaccharides (pullulan) carboxyethylated to provide ionic derivatives were then conjugated to cholesterol groups to obtain cholesterol-modified carboxyethyl pullulan (CHCP). Cholesterol-modified pullulan (CHP) conjugate was synthesized with a similar degree of substitution of cholesterol moiety to CHCP. CHCP formed self-aggregated NPs in aqueous solution with a spherical structure and zeta potential of −19.9±0.23 mV, in contrast to −1.21±0.12 mV of CHP NPs. NPs could quench albumin fluorescence intensity with maximum emission intensity gradually decreasing up to a plateau at 9 to 12 h. Binding constants were 1.12×105 M−1 and 0.70×105 M−1 to CHP and CHCP, respectively, as determined by Stern-Volmer analysis. The complexation between HSA and NPs was a gradual process driven by hydrophobic force and inhibited by NP surface charge and shell-core structure. HSA conformation was altered by NPs with reduction of α-helical content, depending on interaction time and particle surface charges. These NPs could represent a sustained release carrier for mitoxantrone in vitro, and the bound HSA assisted in enhancing sustained drug release.

A combined spectroscopic, docking and molecular dynamics simulation approach to probing binding of a Schiff base complex to human serum albumin

The molecular mechanism of a Schiff base complex ((E)-((E)-2-(3-((E)-((E)-3(mercapto (methylthio) methylene)cyclopentylidene) amino) propylimino) cyclopentylidene) (methylthio) methanethiol) binding to Human Serum Albumin (HSA) was investigated by fluorescence quenching, absorption spectroscopy, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of HSA was quenched by this Schiff base complex that has been analyzed for estimation of binding parameters. The titration of Schiff base solution by various amount of HSA was also followed by UV–Vis absorption spectroscopy and the corresponding data were analyzed by suitable models. The results revealed that this Schiff base has an ability to bind strongly to HSA and formed 1:1 complex. Energy transfer mechanism of quenching was discussed and the value of 5.45±0.06nm was calculated as the mean distance between the bound complex and the Trp residue. This is implying the high possibility of energy transfer from HSA to this Schiff base complex.Molecular docking results indicated that the main active binding site for this Schiff base complex is site III in subdomain IB. Moreover, MD simulation results suggested that this Schiff base complex can interact with HSA, without affecting the secondary structure of HSA but probably with a slight modification of its tertiary structure. MD simulations, molecular docking and experimental data reciprocally supported each other.

A pilot study on potency determination of human follicle-stimulating hormone: A comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay

Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman–Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BAIU=0.9925 RP-HPLCIU−1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was −1.4%, with a 95% confidence interval of −9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical–chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.

Sorption of Proteins onto Porous Single-Component Poly(vinyl amine) Multilayer Thin Films

Porous multilayer thin films consisting solely of cross-linked poly(vinyl amine) ((PVAm)(n)) were generated by a selective cross-linking of the PVAm layers in the [PVAm/poly(acrylic acid) (PAA)](n) thin films, followed by the removal of PAA. A regular increase in the (PVAm/PAA)(n) multilayer onto silica particles was observed by potentiometric titration when PVAm with a low molar mass (M(w) = 15 000 g mol(-1)) was used in the construction of LbL thin films. The amount of human serum albumin (HSA) loaded on the single-component (PVAm)(5) thin film was approximately 32 mg HSA/g of hybrid when PVAm with a molar mass of 15 000 g mol(-1) was used compared with the single-component film prepared with PVAm with a molar mass of 340 000 g mol(-1) when the amount of HSA loaded was 15.5 mg HSA/g of hybrid. The sorption modalities of HSA and bovine serum albumin (BSA) onto the single-component thin films deposited on silicon wafers as a function of the number of PVAm layers was investigated by atomic force microscopy and contact angle measurements. The decrease in the film roughness and the increase in the film wettability after the loading of both proteins showed the protein was mainly absorbed into the porous (PVAm)(n) thin film.

Albumin Dialysis Molecular Adsorbents Recirculating System: Impact of Dialysate Albumin Concentration on Detoxification Efficacy

Albumin dialysis with the molecular adsorbent recirculating system (MARS) or single pass albumin dialysis (SPAD) uses human serum albumin (HSA) as an addendum of the dialysate fluid. The purpose of this in vitro study was to evaluate the impact of the dialysate albumin concentration on removal efficacy. Heparinized human plasma (3 L/test) was spiked with creatinine (1000 mg/L), unconjugated bilirubin (100 mg/L), chenodeoxycholic acid (CDCA) (100 mg/L), and diazepam (3 mg/L). The MARS albumin circuit was primed with different amounts of HSA (150, 100, 60, and 40 g). The plasma, albumin, and dialysate flow rates were 200, 200, and 40 mL/min, respectively. Clearances were calculated based on repeated sampling during the experiments, which lasted 480 min. The effective HSA concentrations in the dialysate were 175, 115, 77, and 46 g/L, respectively. They decreased over treatment time to 147, 99, 63, and 41 g/L, respectively, due to surface adsorption. The plasma-HSA concentration remained unchanged over time in all experiments (average 39 g/L). The creatinine clearance was not impacted by dialysate HSA concentration. For the albumin-bound markers a clear correlation between HSA-concentration and clearance was demonstrated with the highest clearances for the 100 and 150 g HSA experiments. The 100 g HSA setup appeared to be the one with best cost-benefit ratio, resulting in clearances (after 1 h of treatment) of 31 mL/min creatinine, 0.3 mL/min unconjugated bilirubin, 11 mL/min CDCA, and 35 mL/min diazepam. Low albumin concentrations, such as in SPAD, result in low clearance rates for albumin-bound substances. The optimal clearances in these experiments were reached with a priming dose of 100 g HSA.

Influences of protein films on antibacterial or bacteria-repellent surface coatings in a model system using silicon wafers

Immobilization of defined chemical functionalities to biomaterial surfaces is employed to optimize them not only for tissue compatibility but also for prevention of bacterial infection. Grafting surfaces with chains of poly(ethylene glycol) (PEG) results in bacterial repellence whereas modification with cationic groups conveys them with bactericidal properties. Since biomaterials in situ will become exposed to a protein-rich environment, it is necessary to investigate the influence of prior protein adsorption on the antibacterial activity of this type of chemical surface modification. In the present study, we immobilized short-chain PEG and two pyridinium group-containing methacrylate monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and 6-methacryloyloxyhexylpyridinium chloride (MHPC), to silicon wafer model surfaces to investigate the influence of prior protein adsorption on the bactericidal activity of the surface coating towards subsequently attached bacteria. Adsorbed amounts of human serum albumin and salivary proteins were found to be two times higher on cationic compared to PEG-modified surfaces. An analogous tendency was found for attachment of Streptococcus gordonii and Streptococcus mutans to the same surfaces without prior protein exposure. However, most bacteria attached to cationic surfaces were found to be dead. Prior exposure of cationic surfaces to protein solutions drastically altered bacterial attachment dependent on the type of protein solution and bacterial species employed. Significantly, the original bactericidal activity of pyridinium-coated surfaces was found greatly reduced upon adsorption of a protein film. As a conclusion we propose that future approaches should combine the protein- and bacteria-repellent properties of PEG-coatings with the bactericidal function of charged cationic groups.