Albúmina en cirrosis: perspectiva inflamatoria / Albumin in cirrhosis: inflammatory perspective

La albúmina sérica humana es la proteína más abundante en el plasma, su estructura molecular le confiere estabilidad, pero también flexibilidad para ligar y transportar un amplio rango de moléculas. Su función oncótica es la propiedad más reconocida que la lleva a introducirse en la terapéutica médica como un expansor de volumen. Sin embargo, en los últimos años se le han adicionado funciones con carácter antioxidante, inmunomodulador y de estabilización endotelial, que hacen presumir que su impacto terapéutico está más allá de sus funciones volumétricas. En los últimos años, específicamente en la cirrosis y la falla hepática aguda sobre crónica, se ha tenido un cambio en el paradigma fisiológico, desde una perspectiva netamente hemodinámica hacia una perspectiva inflamatoria, en donde las funciones oncóticas y no oncóticas de la albúmina están alteradas y tienen un carácter pronóstico en estas entidades. Este conocimiento creciente, desde una perspectiva inflamatoria, hace que se fortalezca el uso terapéutico de la albúmina sérica humana desde las indicaciones tradicionales como prevención de la disfunción circulatoria posparacentesis, prevención y tratamiento de lesión renal aguda, hasta las discusiones para administración a largo plazo en pacientes cirróticos con ascitis.

Human serum albumin is the most abundant protein in plasma, with a molecular structure that provides stability while also allowing flexibility to bind and transport a wide range of molecules. Its oncotic function is the most recognized property, leading to its introduction in medical therapy as a volume expander. However, in recent years, additional functions with antioxidant, immunomodulatory, and endothelial stabilization properties have been identified, suggesting that its therapeutic impact extends beyond its volumetric functions. Specifically, in cirrhosis and acute-on-chronic liver failure, there has been a shift in the pathophysiological paradigm from a purely hemodynamic perspective to an inflammatory perspective, where both oncotic and non-oncotic functions of albumin are altered and have prognostic significance in these conditions. This growing understanding from an inflammatory perspective strengthens the therapeutic use of human serum albumin, not only for traditional indications such as the prevention of post-paracentesis circulatory disfunction, prevention and treatment of acute kidney injury, but also for discussions regarding long-term administration in cirrhotic patients with ascites.

Preparation and efficacy evaluation of polyclonal antibodies against serum albumin of five species / 中国生物制品学杂志

@#Objective To prepare polyclonal antibodies against the serum albumin of human,cattle,sheep,pig and horse,and evaluate their efficacy in the identification of human serum albumin(HSA). Methods The specific polypeptides of human,cattle,sheep,pig and horse serum albumin were prepared by bioinformatics and polypeptide synthesis method,which were coupled with keyhole limpet hemocyanin(KLH)to prepare the peptide antigen after the purity was identified by high performance liquid chromatography(HPLC). Male New Zealand white rabbits were immunized with polypeptide antigens of five species subcutaneously,with 2 for each kind of antigen. The antiserum was then obtained and purified by Protein A affinity chromatography to prepare the polyclonal antibody. The titers and specificity of the polyclonal antibodies were determined by ELISA and Western blot respectively,and the prepared five species of serum albumin were used to identify HSA products. Results The synthetic peptides of human,cattle,sheep,pig and horse serum albumin had a purity of over 91%,and the corresponding polyclonal antibodies all had the titer of 1∶160 000,which showed specific binding with the corresponding antigens and effectively identified the HSA products. Conclusion The polyclonal antibodies of human cattle,sheep,pig and horse serum albumin prepared in this study have good specificity and the preparation process is simple and rapid,suitable for the mass production,which lays a foundation of the development of HSA rapid identification kit.

Drug-free targeted thrombolytic strategy based on gold nanoparticles-loaded human serum albumin fusion protein delivery system / 药学学报

Thrombus is a major factor leading to cardiovascular diseases such as myocardial infarction and stroke. Although fibrinolytic anti-thrombotic drugs have been widely used in clinical practice, they are still limited by narrow therapeutic windows, short half-lives, susceptibility to inactivation, and abnormal bleeding caused by non-targeting. Therefore, it is crucial to effectively deliver thrombolytic agents to the site of thrombus with minimal adverse effects. Based on the long blood circulation and excellent drug-loading properties of human serum albumin (HSA), we employed genetic engineering techniques to insert a functional peptide (P-selectin binding peptide, PBP) which can target the thrombus site to the N-terminus of HSA. The fusion protein was expressed using Pichia pastoris and purified by Ni-chelating affinity chromatography. After being loaded with gold nanoparticles (Au NPs), the fusion protein formed homogeneous and stable nanoparticles (named as PBP-HSA@Au) with a diameter of 17.7 ± 1.0 nm and a zeta potential of -11.3 ± 0.2 mV. Cytotoxicity and hemolysis tests demonstrated the superb biocompatibility of PBP-HSA@Au. Platelet-targeting experiments confirmed the thrombus-targeting ability conferred by the introduction of PBP into PBP-HSA@Au. Upon near-infrared ray (NIR) irradiation, PBP-HSA@Au rapidly converted light energy into heat, thereby disrupting fibrinogen and exhibiting outstanding thrombolytic efficacy. The designed HSA fusion protein delivery system provides a precise, rapid, and drug-free treatment strategy for thrombus therapy. This system is characterized by its simple design, high biocompatibility, and strong clinical applicability. All animal experiments involved in this study were carried out under the protocols approved by the Animal Experiment Ethics Committee of Jiangnan University [JN. No20230915S0301015(423)].

Análise de custos de pacientes cirróticos descompensados recebendo albumina a longo prazo versus tratamento médico padrão nos sistemas de saúde público e privado do Brasil / Albumin versus standard medical treatment in brazilian public and private healthcare systems

ABSTRACT Background: Cirrhosis is one of the final stages of chronic liver disease. Common causes of cirrhosis include alcoholism and viral hepatitis infections. Cirrhosis can progress from an asymptomatic, compensated phase to decompensation and the appearance of overt symptoms. There is no specific treatment for decompensated cirrhosis. The ANSWER trial positioned long-term albumin infusions as a potential treatment for patients with cirrhosis and uncomplicated ascites. Objective: This study assesses the economic impact of albumin infusions following the ANSWER trial regimen in Brazilian patients with decompensated cirrhosis from the public and private healthcare systems perspectives. Methods: The incremental cost per patient per year was calculated for standard medical treatment (SMT) plus long-term albumin infusions versus SMT alone. Costs of diuretics and albumin were obtained from Banco de Preços em Saúde and the Drug Market Regulation Chamber. Costs for complication and procedures were gathered from the published literature. Costs were transformed to 2021 Brazilian reals (BRL). Incidences of clinical complications and treatments were gathered from the ANSWER trial. Univariate sensitivity analysis was performed by increasing and decreasing all inputs by 20%. Results: The cost per patient per year was 118,759 BRL and 189,675 BRL lower for patients treated with SMT and albumin (compared to SMT only) for the public and private healthcare systems, respectively. The additional cost of albumin was offset by reduced complications and treatments (149,526 BRL and 249,572 BRL, respectively). The univariate sensitivity analysis showed cost savings for both healthcare systems in all the scenarios assessed. Conclusion: This economic analysis suggests that, if the ANSWER trial clinical outcomes translate into real-world effectiveness, addition of albumin infusions to SMT in patients with decompensated cirrhosis may lead to cost savings for the public and private healthcare systems in Brazil.

RESUMO Contexto: A cirrose representa o estágio final da doença hepática crônica. Causas comuns de cirrose incluem alcoolismo e infecções por hepatite viral. A cirrose pode progredir de uma fase compensada assintomática para descompensação e aparecimento de sintomas evidentes. Não há tratamento específico para cirrose descompensada. O estudo ANSWER demonstrou que a administração de albumina a longo prazo pode representar um potencial tratamento para pacientes com cirrose e ascite não complicada. Objetivo: Nosso estudo avalia o impacto econômico da administração de albumina a longo prazo seguindo o protocolo do estudo ANSWER em pacientes brasileiros com cirrose descompensada, sob a perspectiva dos sistemas de saúde público e privado. Métodos: O custo incremental por paciente por ano foi calculado para o tratamento médico padrão (SMT) associado a administração de albumina a longo prazo comparado a SMT apenas. Os custos de diuréticos e albumina foram obtidos no Banco de Preços em Saúde e na Câmara de Regulação do Mercado de Medicamentos. Os custos de complicações e procedimentos foram coletados da literatura publicada. Os custos foram transformados em Reais de 2021 (BRL). As incidências de complicações clínicas e tratamentos foram coletadas do estudo ANSWER. Uma análise de sensibilidade univariada foi realizada aumentando e diminuindo todas as variáveis em 20%. Resultados: O custo por paciente por ano foi de R$ 118.759 e R$ 189.675 menor para pacientes tratados com SMT e albumina (comparado apenas com SMT) para os sistemas de saúde público e privado, respectivamente. O custo adicional da albumina foi compensado pela redução de complicações e tratamentos (149.526 BRL e 249.572 BRL, respectivamente). A análise de sensibilidade univariada mostrou redução de custos para ambos os sistemas de saúde em todos os cenários avaliados. Conclusão: Esta análise econômica sugere que, se os resultados clínicos do estudo ANSWER se confirmarem no mundo real, a administração de albumina associada ao SMT em pacientes com cirrose descompensada pode levar a redução de custos para os sistemas de saúde público e privado no Brasil.

High level expression of recombinant human serum albumin in CHO cells and optimization of its culture technology / 中国生物制品学杂志

@#ObjectiveTo achieve efficient expression of human serum albumin(HSA)in Chinese hamster ovary(CHO)cells and optimize its culture technology,so as to lay a foundation of the large-scale production of HSA.MethodsThe eukaryotic expression vector of HSA was constructed by gene recombination technology,and then electrotransfected into fully suspended CHO cells. The monoclonal cell lines with stable and high expression of HSA were screened by G418 and limited dilution method. By adding glucose,sodium butyrate and supplementalmedium to the basal medium,the cell culture process was optimized to improve the expression of HSA. Finally,the scale-up culture verification was carried out in a 5 L bioreactor.ResultsThe recombinant expression vector pcDNA3.1-HSA was successfully constructed and expressed in fully suspended CHO cells. After two monoclonal screening,the secondary monoclonal cell lines CHO-rHSA-7H2A9 and CHOrHSA-7H2D12 were obtained with high HSA expression of 29. 37 mg/L and 25. 26 mg/L respectively. The HSA expression level reached about 100. 00 mg/L by optimizing the culture process and wasfinally increased to 166. 16 mg/L in the 5 L bioreactor,which was about 30 times higher than that in the supernatant of the first monoclonal cells.Conclusion The high level expression of HSA in CHO cells was achieved,which laid a foundation of the further large-scale production of HSA in the field of biological products and solving the market supply problems.

Preparation of quercetin-human serum albumin-nanoparticles and its inhibitory effects against NASH-induced liver fibrosis in vivo and in vitro / 中国药房

OBJECTIVE To prepare quercetin-human serum albumin-nanoparticles （Que-HSA-NPs），and to evaluate the in vivo and in vitro inhibitory effects of Que-HSA-NPs on hepatic fibrosis of non-alcoholic steatohepatitis （NASH）. METHODS Que-HSA-NPs were prepared by desolvation-chemical cross-linking method ，their appearance characteristics were observed ，and their particle size ，polydispersity index （PDI），Zeta potential and drug loading were detected. Quercetin （Que）and Que-HSA-NPs were applied to murine HSC-T 6 cells. The effects of them on survival rate of HSC-T 6，mRNA expression of transforming growth factor β（TGF-β），Type Ⅰ collagen α1（COL1A1）and α-smooth muscle actin （α-SMA）were compared. Que and Que-HSA-NPs were applied to mice fed with low methionine and choline deficient high-fat diet. The serum levels of liver injury indexes ，liver pathological characteristics ，mRNA expressions of TGF-β，COL1A1 and α-SMA，protein expression of α-SMA in liver tissue were determined to evaluate the improvement effects of them on hepatic fibrosis of NASH in mice. RESULTS The prepared Que-HSA-NPs was spherical ，the particle size was （172.9±2.2）nm，the PDI was 0.233，the Zeta potential was －29.2 mV，and the drug loading was 2.99％. Que and Que-HSA-NPs were nontoxic to HSC-T 6 at concentrations of 0-250 μg/mL. Both of them could significantly decrease mRNA expressions of TGF-β，COL1A1 and α-SMA，especially Que-HSA-NPs （P＜0.05）. They also could significantly decrease the serum levels of liver inju ry index ，relieve liver injury and down-regulate mRNA expressions of TGF-β，COL1A1 and α-SMA and protein expression of α-SMA， especially Que-HSA-NPs （P＜0.05）. CONCLUSIONS Que- HSA-NPs is successfully prepared ，and confirm that its anti- NASH hepatic fibrosis effect is better than that of Que .

Effect of Stains on the Purity Determination of Human Serum Albumin by Agarose Gel Electrophoresis / 中国药学杂志

OBJECTIVE: To study the effect of stains on the purity detection results of human serum albumin by agarose gel electrophoresis, thus to select appropriate stain so that the detection result of agarose gel electrophoresis method can be basically consistent with the method specified in the 2015 edition of Chinese Pharmacopoeia, cellulose acetate membrane electrophoresis method. METHODS: Acid blue, amino black and ponceau were used in agarose gel electrophoresis to detect the purity of 30 batches of human serum albumin samples, and the results were compared with those obtained by cellulose acetate membrane electrophoresis. RESULTS: Impurity bands could not be detected by ponceau stain; the results of amino black staining was significantly lower than that of cellulose acetate membrane electrophoresis. There was no statistical difference between the results of amino black staining and cellulose acetate membrane electrophoresis. The results of 25 batches of samples detected by cellulose acetate membrane electrophoresis were within the 95% confidence interval of the results detected by amino black staining. CONCLUSION: The category of stain has great influence on the detected purity of human serum albumin when using agarose gel electrophoresis. The results of amino black staining is consistent with those of the method recorded in the Chinese Pharmacopoeia in use.

Evaluation of I-JS001 for hPD1 immuno-PET imaging using sarcoma cell homografts in humanized mice

JS001 (toripalimab) is a humanized IgG monoclonal antibody which strongly inhibits programmed cell death protein 1 (PD1). In this study, we used a different iodine isotype (I) to label JS001 probes to target the human PD1 (hPD1) antigen. , the half maximal effective concentration (EC) value of I-JS001 did not significantly differ from that of JS001. The uptake of I-JS001 by activated T cells was 5.63 times higher than that by nonactivated T cells after 2 h of incubation. The binding affinity of I-JS001 to T cells of different lineages after phytohemagglutinin (PHA) stimulation reached 4.26 nmol/L. Humanized C57BL/6 mice bearing mouse sarcoma S180 cell tumors were validated for immuno-positron emission tomography (immuno-PET) imaging. Pathological staining was used to assess the expression of PD1 in tumor tissues. The homologous Ihuman IgG (IhIgG) group or blocking group was used as a control group. Immuno-PET imaging showed that the uptake in the tumor area of the I-JS001 group at different time points was significantly higher than that of the blocking group or the I-hIgG group in the humanized mouse model. Taken together, these results suggest that this radiotracer has potential for noninvasive monitoring and directing tumor-specific personalized immunotherapy in PD1-positive tumors.

Two Cases of Hypersensitivity Reactions Caused by Human Serum Albumin During Therapeutic Plasma Exchange

Iso-oncotic human serum albumin (HSA) is the primary replacement fluid of choice during therapeutic plasma exchange (TPE). Hypersensitivity reactions to HSA are rare, but require proper evaluation and management. In this article, we report two cases of hypersensitivity reactions to 5% HSA during TPE and discuss strategies to address this problem. The first case was a 60-year-old female patient, who was scheduled for TPE for treatment of recurrent focal segmental glomerulosclerosis after ABO-incompatible kidney transplantation. She developed a pruritic rash on her entire body during the first two sessions of TPE using 5% HSA. The third session was conducted using 500 mL normal saline, 1,000 mL 10% pentastarch, and 750 mL 5% HSA, where she eventually developed a pruritic rash when HSA was infused. There were no adverse events during the fourth and fifth session when fresh frozen plasma was used in place of HSA. The second case was a 50-year-old male patient diagnosed with optic neuritis, who was admitted for five sessions of TPE. The patient developed a pruritic rash on his entire body during the first session of TPE using 5% HSA. The patient experienced no adverse events during the following four sessions using fresh frozen plasma. Certain elements contained in HSA, such as albumin aggregates, prekallikrein activator, and caprylate-modified albumin, might be the reason for these hypersensitivity reactions. Careful selection of alternative replacement fluids is important to avoid premature termination of TPE procedures and secure optimal treatment options for patients.

Comparison and theoretical modeling of capillary electrophoresis analysis for the interaction between caffeine and serum proteins / 药学学报

The method for analyzing the interaction between caffeine and human serum albumin (HSA) was established by capillary electrophoresis. Under physiological conditions, the interaction between ligand (caffeine)-receptor (HSA) was studied with frontier-analysis (FA) method, Hummel-Dreyer (HD) method and plug-plug kinetic (PPK) method. The interaction parameters of caffeine-HSA system were obtained using non-linear equation, Scatchard equation and Klotz equation. The results showed that FA, HD and PPK methods were suitable for caffeine-HSA system, among them, HD method was the best, and the Non-linear equation was the best theoretical model to caffeine-HSA system. Interaction parameter tests showed that caffeine-HSA interaction was a single site interaction and the binding stability was moderate. The mechanism of caffeine-HSA interaction has been elucidated, which can provide valuable information for further research of alkaloids.

Metabolism and pharmacokinetics of covalent tyrosine kinase inhibitors / 药学学报

Covalent tyrosine kinase inhibitors (TKIs) can inhibit the signaling pathway of tumor cells by covalent binding with cysteine residues of target proteins, which has the advantages of high potency, extended duration of action and overcoming drug resistance. In this article, we will review the metabolism and pharmacokinetics of some covalent TKIs. Currently, the covalent TKIs approved by US food and drug administration (FDA) are afatinib, neratinib, dacomitinib, osimertinib, ibrutinib and acalabrutinib. Pyrotinib have been approved by National Medical Products Administration (NMPA) to reach the market recently. Covalent TKIs can covalently bind with plasma proteins, especially human serum albumin, thus effected the pharmacokinetics of these drugs.

Design and expression of FGF23 inhibitor in Pichia pastoris / 药学学报

Hypophosphatemia is a common metabolism disease in humans. Fibroblast growth factor 23 (FGF23) inhibits phosphate reabsorption by targeting on the renal tubules. FGF23C-tail contains 73 amino acids from C-terminus of FGF23, serves as an inhibitor of FGF23, and can increase phosphate reabsorption. Therefore, FGF23C-tail is an important drug for hypophosphatemia. In this paper, we constructed a fusion protein of FGF23C-tail with HSA, and investigated the expression of the fusion protein in the Pichia pastoris system. The recombinant gene was constructed by fusion PCR. A high-yield strain was selected by G418 resistance and fermentation yield, and the expression yield was 43.7 mg·L-1 in flask. In 5 L fermenters, the highest expression yield could reach 265.6 mg·L-1. FGF23C-tail-HSA could be used as an inhibitor for FGF23, and could significantly increase blood phosphorus levels in rats. The procedures for care and use of animals were approved by the Ethics Committee of YiChun University. This paper provided a basis research for further studying physiological activity of FGF23C-tail-HSA.

Anti-liver fibrosis activities of the extracellular domain of transforming growth factor beta type II receptor fusion protein in vivo / 中国药科大学学报

@#This study is performed to analyze the anti-liver fibrosis effect of the fusion protein of human serum albumin and extracellular domain of transforming growth factor beta type II receptor(eTGFBR2)in vivo to looking for the more stable anti-liver fibrosis drug. The mice model of liver fibrosis was constructed by CCl4 induction and the following groups are included in the study: the control group, CCl4 model group, the positive control group, eTGFBR2 treatment group, HSA-eTGFBR2 treatment group, and HSA group. Hematoxylin eosin staining, serum liver function index detection, and western blot are used to identify the anti-liver fibrosis activities. The results showed that: (1)CCl4 caused liver structure disorder, hepatocellular necrosis, collagen fibers proliferation, and induced liver fibrosis at last; (2)HSA-eTGFBR2 and its monomer drug improved the symptoms of liver fibrosis significantly, as well as reduced the damage of liver cells and collagen deposition, and recovered the liver basic structure to normal. Both of HSA-eTGFBR2 and its monomer drug improved liver function and reduced the expression level of liver fibrosis marker α-SMA and COL I. Moreover, the anti-liver fibrosis effect of the fusion protein is comparable to the monomer drug. In contrast, the albumin had no effect on therapeutic effect; (3)Reducing the injection frequency of HSA-eTGFBR2 achieved the comparable effects to the monomer drug with the normal injection frequency. In summary, the fusion protein HSA-eTGFBR2 has good anti-liver fibrosis effect. In addition, reducing the injection frequency of the fusion protein could also achieve the comparable treatment with the monomer drug, indicating that the fusion protein is stable and has longer half-lives and then a relatively positive application prospect in future.

Interaction between human serum albumin and cholesterol-grafted polyglutamate as the potential carriers of protein drugs

Currently there is no successful platform technology for the sustained release of protein drugs. It seems inevitable to specifically develop new materials for such purpose, and hence the understanding of protein-material interactions is highly desirable. In this study, we synthesized cholesterol-grafted polyglutamate (PGA--Chol) as a hydrophobically-modified polypeptide, and thoroughly characterized its interaction with a model protein (human serum albumin) in the aqueous solution by using circular dichroism, fluorescence methods, and light scattering. With the protein concentration fixed at 5 μmol/L, adding PGA--Chol polymers into the solution resulted in continuous blue shift of the protein fluorescence (from 339 to 332 nm), until the polymer molar concentration reached the same value as the protein. In contrast, the un-modified polyglutamate polymers apparently neither affected the protein microenvironment nor formed aggregates. Based on the experimental data, we proposed a physical picture for such protein-polymer systems, where the polymer first bind with the protein in a 1:1 molar ratio a fraction of their hydrophobic pendant cholesterol resides along the polymer chain. In this protein/polymer complex, there are excess unbound cholesterol residues. As the polymer concentration increases, the polymers form multi-polymer aggregates around 200 nm in diameter the same hydrophobic cholesterol residues. The protein/polymer complex also participate in the aggregation their excess cholesterol residues, and consequently the proteins are encapsulated into the nanoparticles. The encapsulation was also found to increase the thermal stability of the model protein.

Inter-laboratory Comparison of UPLC Determination of Polymers in Human Albumin and Evaluation of the Method Applicability / 中国药学杂志

OBJECTIVE: To conduct an inter-laboratory comparison of UPLC method for polymer determination in human serum albumin and verify the method applicability. METHODS: National reference of human serum albumin and 20 samples of human serum albumin from domestic and foreign manufactures were distributed to six laboratories in order to carry out inter-laboratory comparison and demonstration of applicability. UPLC was used to determine the content of polymer and HPLC method was used for parallel comparison. RESULTS: There was no significant difference in the determination results between the UPLC method and current HPLC method in four laboratories (P>0.05) equipped with both HPLC and UPLC. The mean values of 21 samples measured with UPLC method by six laboratories were matched with those measured with HPLC method by four laboratories (t test P>0.05). The mean values of standard deviation (SD) and relative standard deviation (RSD) for 21 samples by UPLC method were only 0.06 and 1.02% respectively. The mean values of standard deviation (SD) and relative standard deviation (RSD) were 0.14 and 2.33% for parallel 21 samples determination by HPLC method, suggesting that the difference of UPLC test results between laboratories was smaller. CONCLUSION: The results of UPLC method are in good agreement with those of HPLC method. UPLC method is more effective and efficient, with smaller inter-laboratory difference, thus is significantly better than the HPLC method.

Recombinant human serum albumin promotes differentiation of human pluripotent stem cells into cardiomyocytes in vitro / 中国胸心血管外科临床杂志

@#Objective    To investigate whether recombinant human serum albumin (rHSA) can replace traditional B27 as a basic medium for differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes. Methods     hPSCs were seeded at a cell density of 1.2×104/cm2; until up to 75% confluency hPSCs were induced by differentiation medium containing various concentration of rHSA (0, 50, 100, 200 g/L). Light microscope and fluorescence microscope recorded the whole process of stem cells differentiating into myocardium. Flow cytometry was used to detect the cardiac differentiation efficiency at different concentrations of rHSA. Immunofluorescence staining was used to detect the cardiac specific protein α-actinin and troponin T (cTnT) and electron microscope to observe the ultrastructure of human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) and beating rates of hPSC-CMs response to drugs. Results     A large number of spontaneous beating cardiomyocytes were observed 9 days after induction and differentition. The percentage of colonies showing beating cardiomyocytes was 60.4% at the concentration of 200 g/L of rice derived-rHSA. Beating cardiomyocytes were α-actinin and cTnT positive. Ultrastructural analysis showed scattered sarcomeres and mitochondrial. hPSC-CMs were dose-dependent on isopropyl adrenaline and verapamil. Conclusion     Using such simple media to differentiate hPSCs into functional cardiomyocytes is cost-effective and highly efficient, and can be used in the clinical research.

Oxidized human serum albumin as a possible correlation factor for atherosclerosis in a rural Japanese population: the results of the Yakumo Study

BACKGROUND@#The effect of the redox state of human serum albumin (HSA) on the antioxidant properties of the entire body has been a focus of recent research. The usefulness of HSA redox state as a biomarker for reducing oxidative stress has been investigated in clinical settings; however, evidence for its significance as a health index in non-clinical settings is yet to be established. This study aimed to examine the associations between HSA redox state and the atherosclerotic indices of carotid intima-media thickness (IMT) and plaque formation in a rural Japanese population.@*METHODS@#We conducted a cross-sectional study as part of a health check-up program in the rural area of Hokkaido, Japan, at the end of August 2013. A total of 281 residents (124 men and 157 women) were included in the final analysis. Lifestyle-related data were obtained through a self-reported questionnaire, and ultrasound examinations were performed to measure IMT and determine plaque formation. The high-performance liquid chromatography postcolumn bromocresol green method was used to separate HSA into human nonmercaptalbumin and human mercaptalbumin (HMA).@*RESULTS@#We found a significant negative relationship between the fraction of HMA [f(HMA)] and IMT (standardized β = - 0.132, p = 0.03). Moreover, f(HMA) was significantly associated with plaque formation (p < 0.01) with an odds ratio of 0.89 (95% confidence interval, 0.81-0.97) for every 10% increment in f(HMA).@*CONCLUSIONS@#We found that the HSA redox state, as determined by f(HMA), was associated with atherosclerotic indices in Japanese subjects. These results suggest that the HSA redox state indicates the risk of developing atherosclerosis.

Application of UPLC/VION Mass Spectrometry in Quality Control of Recombinant Human Albumin / 中国药学杂志

OBJECTIVE: To evaluate the applicability of UPLC/MS method for the identification test of human serum albumin (HSA) products including plasma derived and recombinant HSA samples. METHODS: ACQUITY UPLC with Vion IMS QT of LC/MS system was used combined with on-line HSA sample desalting with ACQUITY UPLC BEH C18 column. The acquired multiplycharged mass spectrum was processed with MaxEnt1 automatic protein deconvolution software in UNIFI, which can transfer the raw mass spectrometry data to zero charge molecular mass or mass distribution of the intact protein. RESULTS: Intact protein mass analysis not only provided the accurate mass of HSA, but also provided an overall view of the heterogeneity of HSA and the relative amounts of various forms. From this study, a very specific mass signal [(66 437 ± 1), which is the theoretical average MW of human serum albumin ]was obtained from all the six HSA samples. And the characteristic spectra of different samples were also got. CONCLUSION: UPLC/MS method has very good specificity and high sensitivity and can distinguish HSA products made by different manufacturers and processes. The total analytical time is 10 min, which is ideal for the QC identification test of HSA products.

Establishment of a Method for Determination of Molecular-Size Distribution of Human Serum Albumin by Ultra Performance Liquid Chromatography / 中国药学杂志

OBJECTIVE: To establish a determination method of molecular-size distribution of human serum albumin (HSA) by ultra performance liquid chromatography (UPLC). METHODS: An UPLC method was developed to specifically determine the polymers and other components in HSA on UPLC PROTEIN BEH SEC analysis column with Waters Alliance UPLC system and Waters UPLC TUV detector. The separation was performed using a mobile phase consisting of PBS at a flow rate of 0.6 mL•min-1 and the UV detection wavelength was set at 280 nm. HSA samples were diluted to different concentrations (0.5-20 mg•mL-1) to confirm the optimal concentration range of the injection. The change of component percentage and the linear relationship between HSA concentration and chromatographic peak height were confirmed and the molecular-size distribution was calculated by area normalization method. Within the optimum injection concentration range, the national control sample for HSA was diluted to 12 mg•mL-1 and tested by UPLC method and the methodology was confirmed. Twenty batches of HSA samples were determined by both UPLC and existing HPLC methods, and the samples were determined in parallel. The consistency of the methods was compared and the method was reconfirmed. RESULTS: The UPLC retention time of HSA polymer was 1.469 min, of dimer was 1.972 min, and of monomer was 2.267 min, respectively. The resolution of dimer and monomer was 2.20 and the USP tailing of monomer peak was 1.18 respectively. In the range of the injection concentrations, 0.5-20 mg•mL-1, there was linear relationship between the concentrations of the components of 11 HAS samples, including polymer, dimer and monomer peak, and the peak area%, peak height, peak area, and the squares of linear correlation coefficient were all greater than 0.997 0. The components peak area percentage of HSA samples remained relatively stable within the concentration range of 10-16 mg•mL-1 (total injection amount of 100-160 μg). The RSDs of the percentage of polymers were 0.00% (n=3, 10 mg•mL-1), 0.10% (n=3, 12 mg•mL-1), and 0.10% (n=3, 16 mg•mL-1), respectively. The UPLC method was used to determine the national control sample for human albumin of 12 mg•mL-1, and the mean value of peak area% was 5.62% (n=10). The results were consistent with those of the parallel determination by HPLC (5.58%), both of which were in accordance with the quality control range of the national standard for human albumin. The RSD of the percentage of the peak area of the polymers in national standard for human albumin by UPLC was 0.40% (n=10). The HPLC and UPLC methods were used to determine the polymer peak area percentage of 20 batches of HSA samples from 7 domestic and foreign enterprises at the concentration of 12 mg•mL-1. The correlation coefficient of the two methods was 0.996 0 (P> 0.05) and there was no significant difference between the two methods (P>0.05). CONCLUSION: Compared with the traditional HPLC method, the detection time of HSA SEC by the proposed UPLC method is shortened by at least 10 times, and the accuracy and repeatability of the determination are satisfactory. UPLC method can save much more analysis time, is simple and much faster, and can be used for high-throughput determination of molecular-size distribution of human serum albumin.