Albumin Preparations Research Articles

Ammonium Sulfate Precipitation but Not Delipidation is a Valuable Method for Human Albumin Preparation for Biological Studies

Background: Glycated hemoglobin level is considered as one of the main clinical parameters used for diabetes diagnosis. With a shorter half-life than hemoglobin, glycated human serum albumin appears to be an alternative and relevant marker for glycemic control and for the diagnosis of numerous associateddiabetes complications. The characterization of albumin as an alternative marker in clinical studies required its delipidation after the protein purification from plasma. The aim of this study was to find a reliable and convenient method for albumin delipidation and purification which does not have significant impact on the redox state, intrinsic structural and functional integrities of human serum albumin. Methods: Affinity chromatography and ammonium sulfate precipitation were the two different techniques used for albumin isolation from plasma and delipidation was performed with ethanol-ether mixture. The biochemical (redox state), structural and functional (antioxidant, affinity and esterase properties) integrities of purified HSA were determined by using biochemical assays and fluorescence spectroscopy measurements. Results: The two-step precipitation of HSA with ammonium sulfate enabled to purify albumin exhibiting preserved structural and functional properties in terms of antioxidant and enzymatic activity and through a rapid and efficient protocol. By contrast, batch- or chromatography affinity-mediated purification presented more backwards than ammonium sulfate precipitation method. In addition, our results showed the delipidation step following the purification should not to be considered as it impacted both structure and function of albumin. Conclusion: Such novel insights may in turn unlock novel protocol developments that should help reach a better understanding of modified albumin involvement in insulin resistance progression.

Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture

Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na+/K+ ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior.

Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a light-activated local treatment modality that is under intensive preclinical and clinical investigations for cancer. To enhance the treatment efficiency of phototherapy and reduce the light-associated side effects, it is highly desirable to improve drug accumulation and precision guided phototherapy for efficient conversion of the absorbed light energy to reactive oxygen species (ROS) and local hyperthermia. In the present study, a programmed assembly strategy was developed for the preparation of human serum albumin (HSA)-indocyanine green (ICG) nanoparticles (HSA-ICG NPs) by intermolecular disulfide conjugations. This study indicated that HSA-ICG NPs had a high accumulation with tumor-to-normal tissue ratio of 36.12±5.12 at 24 h and a long-term retention with more than 7 days in 4T1 tumor-bearing mice, where the tumor and its margin, normal tissue were clearly identified via ICG-based in vivo near-infrared (NIR) fluorescence and photoacoustic dual-modal imaging and spectrum-resolved technology. Meanwhile, HSA-ICG NPs efficiently induced ROS and local hyperthermia simultaneously for synergetic PDT/PTT treatments under a single NIR laser irradiation. After an intravenous injection of HSA-ICG NPs followed by imaging-guided precision phototherapy (808 nm, 0.8 W/cm2 for 5 min), the tumor was completely suppressed, no tumor recurrence and treatments-induced toxicity were observed. The results suggest that HSA-ICG NPs generated by programmed assembly as smart theranostic nanoplatforms are highly potential for imaging-guided cancer phototherapy with PDT/PTT synergistic effects.

CT and histological findings and treatment outcomes after carotid endarterectomy for symptomatic cervical carotid artery stenosis in octogenarians

Symptomatic cervical carotid artery stenosis is one of the common causes of ischemic stroke in octogenarians. The records for 90 consecutive patients with symptomatic cervical carotid artery stenosis treated by carotid endarterectomy(CEA)were analyzed retrospectively. The patients were divided into two groups:21 patients aged 80 years or over and 69 patients aged less than 80 years. CT angiography revealed that ulcer formation was significantly more common and the length of carotid artery stenosis was significantly greater in the octogenarians. Histological examination of the plaque revealed no significant difference between the two groups in terms of ulcer formation, lipid content, and intraplaque hemorrhage. However, all three of these pathological findings were present in a significantly higher number of octogenarians. No significant difference was observed for new ischemic lesions on diffusion-weighted MR imaging, neurological complications, or cardiac and pulmonary complications. However, treatment with an albumin preparation and loop diuretics was significantly more common in octogenarians after CEA. The present study suggests that with careful postoperative management CEA can be safely performed in symptomatic patients aged 80 years or over with almost the same risk as that for patients aged less than 80 years.

Preparation and evaluation of bovine serum albumin immobilized chiral monolithic column for affinity capillary electrochromatography

A system of capillary silica monolith with bovine serum albumin (BSA) functionalized through two approaches for affinity monolithic capillary electrochromatography (AMCEC) was developed. Covalent immobilization conditions for two different Schiff base methods, which employed 3-glycidopropyl trimethoxysilane (GPTS) and 3-aminopropyl trimethoxysilane (APTS) as starting materials, respectively, were investigated to obtain good and stable chiral separation. The BSA immobilized silica monoliths were evaluated in terms of morphology, electroosmotic flow, retention time, column efficiency and resolution of model compound (±)-tryptophan. The columns exhibited satisfactory run-to-run, column-to-column repeatability and maintained their enantioselectivity for more than 3months. Both developed methods can baseline separate tryptophan enantiomers, whereas shorter retention time, better column efficiency, and enantiomeric recognition between two pairs of drug enantiomers (pantoprazole and atenolol) were obtained by the GPTS method.

Quality evaluation of human serum albumin prepared by heat denaturation in Iran: An experience for developing countries

Blood and plasma are unique resources and access to these products save life. In this study, albumin demand and surplus plasma makes it possible to use local experiences in plasma industry for preparation of albumin so plasma was heated after stabilization; afterward denatured proteins were precipitated and separated by continuous centrifuge system. The supernatant contained albumin was filtrated, diafiltrated, ultrafiltrated, formulated and pasteurized. Albumin preparation in pilot scale with heat denaturation was performed for the first time in Iran. This method using surplus plasma is recommended for all countries that have no access to plasma fractionation industry. Therefore with more attention it has potential for use in the production of safe plasma derived products and thereby it can be used as a safe product in clinic.

Endoscopic Submucosal Dissection for Early Gastric Cancer Complicated by Gastric Gland Heterotopia: A Case Report

A 72-years-old male visited our institution because of severe abdominal distention. Abdominal computed tomography revealed liver cirrhosis with massive retention of ascites. Ascites was treated using diuretic drugs and albumin preparation. Gastroendoscopy revealed esophageal varices, which was successfully treated with endoscopic variceal ligation. A follow-up gastroendoscopy performed 4 months later revealed the disappearance of esophageal varices and the presence of a depressed lesion on the anterior wall of the lesser curvature of the midgastric body with a small orifice near the anal side of the depressed lesion, suggesting gastric gland heterotopia. A biopsy from the depressed lesion revealed group 5. Endoscopic ultrasonography revealed anechoic lesions in the third layer and type 0-IIc lesion with invasion to the third layer, suggesting that the IIc lesion invaded thesubmucosal layers. On the basis of endoscopic findings, the IIc lesion was considered to be within the submucosal layer; therefore, Endoscopic Submucosal Dissection (ESD) was performed, and pathological findings of the resected specimen revealed moderately differentiated tubular adenocarcinoma within the mucosallayer and multiple cystic dilated lesions in the submucosal layer. The post-ESD course wasuneventful and recurrence or de novo lesion has not been detected by regular gastroendoscopy

Severe Intraoperative Albumin Transfusion Reaction and Review of the Literature

Reactions to human albumin are rare, but happen at a frequency to warrant caution since it is routinely produced from batched human plasma. Albumin preparations have advanced substantially in purity since their inception, but despite this achievement severe reactions may still occur. These reactions may include a rash, urticarial, hypotension, dyspnea, or even anaphylaxis. The patient is a 74 year old male with history of recurrent squamous cell carcinoma of gingiva undergoing a neck dissection for lymph nodes. The patient was given 500 mL of albumin and suddenly developed intractable hypotension. In addition, once the surgical drapes were removed, the patient exhibited a rash and flushing on both the chest and abdomen. The patient was treated with pressor agents and a severe reaction to albumin was logged in the allergy alert section of the patient chart. Although human albumin has an excellent safety profile, it is important to recognize allergic transfusion reaction symptoms quickly and initiate an appropriate investigation. * IgA : immunoglobulin A IgG : immunoglobulin G PPF : plasma protein fraction FDA : United States Food and Drug Administration BP : blood pressure BPM : beats per minute RR : respiratory rate

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.

Preparation of Bovine Serum Albumin (BSA) nanoparticles by desolvation using a membrane contactor: A new tool for large scale production

Albumin nanoparticles are attractive drug delivery systems as they can be prepared under soft conditions and incorporate several kinds of molecules. The aim of this study was to upscale the desolvation process for preparing Bovine Serum Albumin (BSA) nanoparticles using a membrane contactor. At a first step, the BSA nanoparticles were prepared at small scale using a syringe pump. BSA nanoparticles of 139nm in size, with a polydispersity index of 0.046, were obtained at the optimal conditions: pH 8.2, 100mgmL−1 BSA albumin solution (2mL), and 1mLmin−1 flow rate of ethanol addition (8mL). The upscaling with a membrane contactor was achieved by permeating ethanol through the pores of a Shirasu Porous Glass (SPG Technology Co., Japan) membrane and circulating the aqueous phase tangentially to the membrane surface. By increasing the pressure of the ethanol from 1 to 2.7 bars, a progressive decrease in nanoparticle size was obtained with a high nanoparticles yield (around 94–96%). In addition, the flow rate of the circulating phase did not affect the BSA nanoparticle characteristics. At the optimal conditions (pH 8.2, 100mgmL−1 BSA albumin solution, pressure of ethanol 2.7 bars, flow rate of the circulating phase 30.7mLs−1), the BSA nanoparticles showed similar characteristics to those obtained with the syringe pump. Large batches of BSA nanoparticles were prepared up to 10g BSA. The BSA nanoparticles were stable at least during 2months at 4°C, and their characteristics were reproducible. It was then concluded that the membrane contactor technique could be a suitable method for the preparation of albumin nanoparticles at large scale with properties similar to that obtained at small scale.

Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children

Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children

Concentrated Ascites Reinfusion Therapy for Sinusoidal Obstructive Syndrome After Hematopoietic Stem Cell Transplantation

Sinusoidal obstruction syndrome (SOS) is one of the severe complications of hematopoietic stem cell transplantation (HSCT). Systemic management including respiratory and circulatory support is necessary. In addition, abdominal paracentesis is often needed for pain relief and to reduce the pressure of tense ascites. Concentrated ascites reinfusion therapy (CART) involves the filtration, concentration, and reinfusion of drained ascites, which contributes to reuse of autologous proteins. CART has been reported as supportive therapy for patients with liver cirrhosis and cancer. We retrospectively reviewed the efficacy and safety of CART in three patients (two with acute myelogenous leukemia and one with chronic myeloid leukemia) who developed SOS after allo-HSCT. They all had symptomatic, tense, and diuretic-refractory ascites with right costal pain and marked weight gain. Two patients showed immediate improvement after CART. However, one patient experienced four CARTs with slow recovery. All patients are now alive and are being monitored as outpatients over 2 years with remission. No severe adverse event was observed related to CART, and 25.2-98.0 (median 30.2) grams of albumin was collected and reinfused. CART after paracentesis reduces protein loss in ascites by reinfusion of autologous protein instead of exogenous albumin preparations. Although transient fever is reported as a frequent adverse event, no events like severe bleeding or infection were observed. While its safety has not been fully established in patients with hematological disease after HSCT, CART may be a considerable supportive therapy for SOS with tense ascites.

Robust size control of bovine serum albumin (BSA) nanoparticles by intermittent addition of a desolvating agent and the particle formation mechanism

In polymeric nanoparticle preparation, despite similar conditions, large fluctuations in particle size distributions are usually observed. Herein, we demonstrate that the intermittent addition of a desolvating agent can improve reproducibility in the preparation of polymeric bovine serum albumin (BSA) nanoparticles. Using this modification, BSA nanoparticles of controlled size can be manufactured with narrow particle size distributions. In our study, ethanol as a desolvating agent was added intermittently to 1% BSA solutions at different pHs with stirring at 700rpm. The effect of the preparation parameters on size and optical density of the fabricated nanoparticles were studied. The average particles sizes of BSA nanoparticles prepared at pH values of 6, 7 and 9 were approximately 100, 200 and 300nm, respectively. As ethanol addition increased, desolvation of BSA molecules resulted in formation of loose-structured particles with pH-dependent size. Beyond that, only particle density increased, but size remained unchanged with further addition of ethanol. Consistently uniform particle size distribution was achieved by adding ethanol intermittently.

Variability in protein quality used for embryo culture: embryotoxicity of the stabilizer octanoic acid

To screen human serum albumin (HSA) preparations for toxicity and investigate causes of variation. Experimental laboratory study. University-based laboratory. FVB and CF1 mice crossed to create embryos used in experiments. Mouse embryo assay performed with 5% or 15% HSA (100 mg/mL albumin) from three samples from three separate manufacturers (A, B, C). Blastocyst rates calculated at 96 hours of culture (experiments repeated in triplicate). The HSA preparations were desalted to remove stabilizers added during HSA processing, then mass spectrometry was used to determine the relative variation in stabilizer concentrations; the effect of the stabilizer octanoic acid on embryo development was tested. At 5% HSA, all samples had blastocyst rates ≥ 70%; at 15% HSA, the blastocyst rates for samples B and C were <50%. Desalting did not affect sample B but did improve the blastocyst rates of sample C. Mass spectrometry revealed high levels of octanoic acid in sample C compared with sample A. The addition of octanoic acid to sample A produced toxicity similar to sample C. The stabilizer octanoic acid varies by lot and inhibits embryo development. Because octanoic acid is known to cause disruptions in mitochondrial bioenergetics, reduce intracellular pH, and induce oxidative damage in peripheral tissues, its use in embryo culture should be monitored and limited.

PREPARATION OF BOVINE SERUM ALBUMIN MONOMER FOR CONJUGATION EXPERIMENTS BY USING DIFFERENT TYPES OF CHROMATOGRAPHY MEDIA

The presence of some sugar contaminants and high molecular mass (HMW) derivatives detected in commercially available bovine serum albumin (BSA) preparations precluded the use of such reagents for basic molecular biology experiments. In this study the effectiveness of three separation methods of monomeric BSA from contaminants were examined: gel filtration on Sephadex G-200, HW-55S resin, and ion-exchange chromatography on DEAE-Sephadex A-50. Only chromatography on HW-55S allowed obtaining protein lacking HMW derivates and freeing of sugar contaminants, which was confirmed in SDS/PAGE and phenol/sulfuric acid analysis, respectively. Moreover, the determination of protein particle size by dynamic light scattering (DLS) at a level of 7 nm was in accordance with data for BSA monomeric molecules previously reported. Finally, the monomer BSA was subjected to modification with natural core oligosaccharide derived from Shigella lipopolysaccharide. The SDS/PAGE analysis of reaction products demonstrated different amounts of glycoconjugates compared to values obtained for commercial BSA. Only the pure protein modification permitted correct appraisal of obtained products. We propose gel filtration chromatography on HW-55S resin as simple and effective method of monomer BSA separation from commercial reagent before basic and applied research.

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

BackgroundAlbumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties. MethodsMost studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin. ResultsBoth in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells. Conclusions and general significanceOur results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.

Preparation of albumin from human plasma by heat denaturation method in plasma bag

In many countries, there is surplus plasma which is thrown out. Due to the high cost of albumin as a pharmaceutical product, it is not widely available for all patients. Therefore, we considered direct preparation of albumin in the plasma bag. Human plasma has many unique features compared with prepared pharmaceutical products because of its human origin. In countries which do not have a plasma fractionation industry, shortages for plasma products are commonplace. Albumin stabiliser was added to the plasma in the bag to stabilised albumin molecule and make it resistant to heat. When the plasma bag was heated, other plasma proteins are irreversibly precipitated and can be separated by centrifugation. Finally, albumin is filtered and pasteurized in plasma bag. The purity of prepared albumin is >99% and the yield is 21 g per liter of plasma. Polymer content of pasteurized albumin measuring with High Performance Liquid Chromatography (HPLC) was less than 1%. Since albumin preparation in plasma bag is a simple and cheap technology, it has the potential for use in the production of a safe plasma derived product, although suitability for human use would require appropriate clinical assessment for safety. Using this method, albumin could be prepared from all types of plasma. This method is suggested as a possible method of albumin production for all countries which do not have a plasma fractionation industry and even for the countries with low plasma supplies.

Use of patatin, a protein extracted from potato, as alternative to animal proteins in fining of red wine

The use of plant-derived proteins as wine fining agent has gained increased interest owing to the potential allergenicity of animal proteins in susceptible subjects. Patatin P is the name of a family of glycoproteins that can be recovered from potato aqueous by-product. In this study, a comparative fining trial simulating industrial procedures with 10, 20 and 30 g/hL of commercial preparations of patatin, potassium caseinate, gelatin and egg albumin on an Aglianico (Vitis vinifera L.) red wine was performed. Color indexes and phenolics were analyzed by spectrophotometric methods and HPLC. The potential astringency has been evaluated by an index based on the ability of wine to precipitate salivary proteins (SPI, Saliva Precipitation Index). Patatin is a suitable alternative to animal proteins used as fining agent because: (i) a decrease in total phenolics and tannins after the treatments with 10, 20 and 30 g/hL of commercial preparation containing P was detected; (ii) Patatin, as well as all the fining agents used in this experiment, is able to diminish astringency and the content of red wine phenolics able to react with salivary proteins. Considering all concentrations tested, the effectiveness in reducing proteins reactive towards wine polyphenols was patatin = gelatine > egg albumin > casein (p < 0.05); (iii) at each concentration considered, the treatment with patatin causes no depletion of chromatic characteristics of red wine although a significant slight loss of individual anthocyanins was observed.

Preparation, Characterization and Optimization of Egg Albumin Nanoparticles as Low Molecular‐Weight Drug Delivery Vehicle

AbstractProtein nanoparticles have been recognized as carriers to deliver low molecular‐weight drugs, anticancer drug, DNA, vaccines, oligonucleotides, peptides and etc. The purpose of this research was preparation of Egg Albumin (EA) nanoparticle with suitable size/size distribution and good surface properties for drug delivery application based on simple coacervation method along with optimization of the nanoparticles by employing Taguchi method. Several synthesis parameters were examined to characterize their impacts on nanoparticle size and topography. These variables were including temperature, EA concentration, desolvating agent volume, pH value and agitation speed. In addition, size and morphology of prepared nanoparticles were analyzed by photon correlation spectroscopy (PCS) as well as atomic force microscopy (AFM). As result of Taguchi analysis in this research, desolvating agent volume and pH were most influencing factors on particle size. The minimum size of nanoparticles (∼51 nm) were obtained at Temperature 55 °C, 30 mg/ml EA concentration, desolvating agent volume 50 ml, agitation speed of 500 rpm and pH 4. The mechanistic of optimum conditions for preparing protein nanoparticles from Egg Albumin for the first time and their characterization as delivering nano system are discussed.

Albumin Coated Microbubble Optimization: Custom Fabrication and Comprehensive Characterization

Gas microbubbles are used routinely to improve contrast in medical diagnostic imaging. The emerging fields of microbubble-enhanced quantitative imaging and microbubble-enhanced drug delivery have further enhanced the drive toward microbubble characterization and design techniques. The quest to improve efficiency, particularly in the field of drug delivery, presents a requirement to develop methods to manipulate microbubble properties to improve utility. This article presents an investigation in to the feasibility of influencing albumin shelled microbubble properties through the variation of albumin availability during fabrication. Microbubbles were fabricated from albumin suspensions of varying concentration before thorough physical and acoustic characterization. Microbubbles with shells fabricated from a 2% albumin suspension had a greater scattering to attenuation ratio (STAR) than 10% albumin preparations (4.4% and 2.2%, respectively) and approximately double the nonlinear STAR (from 0.7% to 1.5%). The 2% microbubbles also exhibited greater (up to 40%), more violent radial oscillations during high speed imaging than 5% and 10% preparations. The results show that microbubble characteristics can be simply manipulated in the lab and indicate that for a given application this may provide the opportunity to further enhance favorable characteristics.