Protein binding study of isoflavone, perillyl alcohol and S-ibuprofen by high-performance frontal analysis

High-performance frontal analysis (HPFA) was used for a protein binding study of isoflavones (daidzein, genistin, and genistein), enantiomers of perillyl alcohol and S-ibuprofen to human serum albumin (HSA). The analyses were performed on a Develosil and Inertsil 100-Diol-5 column (10 cm×4.6mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 ml/min. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes were each fixed up the zonal profile with an evident plateau appears. The unbound drug concentration was determined from a plateau height of the plateau region after that experimental data were fitted by Scatchard equation. The binding constants (K) and total binding affinities (nK) of drugs to HSA were calculated, respectively.     

Unbound DHA causes a high blank value in β‐oxidation assay: a concern for in vitro studies

The information on binding capacity of different fatty acids (FAs) to albumin is incomplete, however, in the majority of in vitro experiments, FAs and albumin were simply mixed and their affinity believed to be complete. In this study, seven [1‐¹⁴C] FAs were mixed with albumin and assayed for β‐oxidation in rat liver homogenates. In the process of identifying the radioactive background of control assay by LCMS/MS, the results indicated different binding capacity of FAs to albumin. The percentage of unbound FAs recovered in clarified acidic solution was lower than 2% with 16:0 and 18:1n‐9, nearly 5% with EPA, 7% with 18:2n‐6, 18:3n‐3 and 20:4n‐6, and surprisingly high to 41% with DHA. Various FA/albumin molar ratios as well as different types of albumin only marginally affected these data. Thus, the big mass of unbound free DHA led to a high blank value, which is 60 times higher than the real value in the procedure of β‐oxidation measurement in vitro. In the design of future FA research in vitro, the binding capacity of FA to albumin or other proteins must be considered, especially for DHA research.     

Tribological process induced conformational transformation of protein may change the friction of cartilage

In-vitro testing procedures have been successfully developed to investigate the effects of tribological process induced transformation of protein-based lubricant on the friction change of articular cartilages. Serum and albumin solutions were the biological lubricants used in this study. The results indicated that the lubricating ability for cartilages deteriorates after the biological lubricants were articulated between polyethylene and stainless steel materials. In addition, the secondary structure change of the albumin molecule has been characterized after the molecules were articulated by the artificial joint materials. We have provided evidence that the conformational change of protein lubricants leads to the friction increase of articular cartilage.     

Functional properties of protein preparations from amaranth seeds in model system

The gross chemical composition and functional properties (solubility, emulsifying and foaming properties) of different amaranth protein preparations were studied in model systems and were compared to those of casein and soy protein isolates. Preparations of alkaline-soluble total protein, albumin, globulin, and glutelin-like alkaline-soluble residual protein were produced from two different types of defatted amaranth meals by extraction and fractionation. Although similarity can be shown between protein patterns of legumes (including soy) and amaranth, the emulsifying and foaming properties of amaranth protein preparations are relatively poor in comparison to the reference proteins, except foaming properties of albumin preparations. Nevertheless, taking in mind that these properties depend on interactions with other food components and textural requirements of individual food products, the amaranth protein preparations may be treated as potential protein sources and food ingredients.     

The influence of proteins on the fretting-corrosion behaviour of a Ti6Al4V alloy

The effects of collagen and albumin on the fretting-corrosion behaviour of a Ti6Al4V alloy contacting an Al₂O₃ counter-piece was investigated in pH buffered saline solutions at 37 °C using a tribo-electrochemical apparatus. Phosphate ion and hydroxyethyl-piperazinyl-ethanesulfonic acid (HEPES) were used as the pH buffer agents. Tests were conducted under two applied electrochemical potentials and two loads. Potentiodynamic polarisation curves were measured to assess the effect of proteins and pH buffer agents on the corrosion behaviour. Surfaces were characterised by XPS analysis, secondary electron spectroscopy and laser profilometry.Fretting wear of the Ti6Al4V alloy increased with increasing applied potential and load but was not significantly affected by the presence of collagen or albumin. Only a small lubricant effect of collagen could be observed at cathodic potentials. In phosphate buffer saline (PBS) solutions, those proteins were found to act as cathodic inhibitor by shifting the corrosion potential and the cathodic current towards more cathodic values. Phosphate ions were found to be incorporated on the Ti6Al4V alloy and to cause sedimentation of wear particles around the wear trace. In HEPES solutions wear particles were dispersed away from the wear trace.     

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 μg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 μg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 μg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.     

Use of proteins to minimize the physical aging of EUDRAGIT sustained release films

The objective of this study was to investigate the influence of two proteins, albumin and type B gelatin, on the physical aging of EUDRAGIT® RS 30 D and RL 30 D coated theophylline pellets. The physicomechanical properties of sprayed films, thermal properties of cast films, influence of proteins on the zeta potential and particle size of the dispersion, and the release of proteins from cast films under simulated dissolution conditions were investigated. The release rate of theophylline decreased significantly over time from pellets coated with an acrylic dispersion containing 10% albumin when there was no acidification of the acrylic dispersion; however, when pellets were coated with an acidified EUDRAGIT®/albumin dispersion, the theophylline release rate was stable for dosage forms stored in the absence of humidity. The drug release rate was faster for pellets coated with acrylic dispersions containing 10% gelatin compared to the albumin-containing formulations. When sprayed films were stored at 40°C/75% RH, the water vapor permeability decreased significantly for both EUDRAGIT® films and those containing EUDRAGIT® and albumin; however, there was no significant change in this parameter when 10% gelatin was present. Albumin was released from the acrylic films when the pH of the dissolution media was below the isoelectric point of the protein while no quantitative release of gelatin was observed in pH 1.2 or 7.4 media. The effect of gelatin to prevent the decrease in drug release rate was due to stabilization in water vapor permeability of the film. Acidification of the polymeric dispersion resulted in electrostatic repulsive forces between albumin and the acrylic polymer, which stabilized the drug release rate when the dosage forms were stored in aluminum induction sealed containers at both 40°C/75% RH and 25°C/60% RH.     

Albumin-based biomaterial for lung tissue engineering applications

The role of albumin-based biomaterials in tissue engineering (TE) cannot be overemphasized. The authors review the role of albumin in lungs scaffold grafting, which promotes cell seeding. Albumin grafted on decellularized lungs scaffold is presented as a great support material for cell-tissue interaction as well as for ease in attachment, growth, and differentiation when seeded with different types of cells. Albumin scaffold fabrication from different sources is a promising approach that may facilitate medical treatments from bench-to-bed, although the role of this scaffold in lungs surfactant proteins regeneration and binding needs to be fully elucidated.     

Purification of pregastric lipases of caprine origin

Pregastric lipases from kid (KPGL) and goat (GPGL) were purified from the commercial extracts by different chromatographic procedures. The total recovery of activity for both purification methods was ca. 10%, and the specific activities of KPGL and GPGL were 533 and 546 U/mg, respectively, at pH 6.5, 35°C for tributyrylglycerol (TBG) as substrate in a casein/lecithin emulsion. The purification factors were 130- and 76-fold for the goat and kid lipases, respectively. The purified lipases from kid and goat showed the same 50 kDa protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an identical sequence for the first 11 amino acids. The optimal pH for the lipases was within the pH range 6–7, with maximal activity at pH 6.5. The stability of the purified lipases was decreased dramatically at pH>6.5, but was enhanced by the addition of albumin. Presented at the AOCS 88th Annual Meeting & Expo, May 11–14, 1997, Seattle, Washington.     

Aqueous Preparation and Evaluation of Albumin‐Chitosan Microspheres Containing Indomethacin

Controlled‐release egg albumin‐chitosan microspheres containing indomethacin as a model drug were successfully prepared by coacervation method. The proposed method can offer a simple method for microsphere preparation in an aqueous system with the elimination of the use of organic solvents that are usually needed in conventional techniques of microencapsulation. The interaction between negatively charged egg albumin molecules in phosphate buffer, pH 7.2, or sodium hydroxide solution and positively charged chitosan molecules dissolved in diluted acetic acid to form an insoluble precipitate was the principle for the formation of the microspheres. The effects of many process variables, such as amount of formaldehyde as a cross‐linking agent, stirring time, final pH of encapsulation medium, initial drug loading, and albumin concentration or albumin‐to‐chitosan weight ratio, on the properties of the prepared microspheres were investigated. Incorporation efficiencies of the microspheres to the drug were high in most cases and ranged between 63.3 ± 3.6% and 92.39 ± 3.2%, while particle sizes were 435.2 ± 12.6 up to 693.9 ± 34.6 µm for the different tested batches. On the other hand, the values of angles of repose and compressibility indices were in the range of 23.5 ± 0.4 to 32.0 ± 0.7 degrees and 11.1 ± 0.7% to 23.6 ± 0.7% respectively, which indicate overall good free flowing nature of the microspheres of all batches. The maximum required amount of the cross‐linking agent was determined to avoid excessive unnecessary chemicals. It was also noticed that excessive time of stirring and excessive initial drug loading are not recommended as it may lead to microspheres of low properties. The pH of the encapsulation media (pH 3.77 up to pH 4.91) significantly affected the properties of the microspheres. As the pH of the encapsulation media was increased, the incorporation efficiency, particle size, and flowability decreased, along with increase of drug release rate, which could be related to incomplete cross linking of the microspheres matrix. It was also observed that high concentration of albumin solution and accordingly the increase of albumin‐to‐chitosan weight ratio were accompanied with increases in incorporation efficiency and particle size with improved microsphere flowability and slow indomethacin release. Thus, the proposed microspheres showed the ability to control the release of indomethacin, and their properties were highly affected by many process variables that could be controlled to obtain an optimized system.