OBJECTIVETo establish an ELISA approach to study the interaction of polysaccharides with cytokine in vitro.

METHODSThe heparin BSA complexes (HBC) were synthesized with a chemistry method and separated using a 1 X 90 cm column of Separose 4B. After identification of the complex via SDS-PAGE,the wells of ELISA plates were coated with HBC and the interaction of HBC with interferon-gamma (IFN-gamma) was detected. The effects of heparin, low molecular weight heparin (LMW heparin), chondroitin sulfate (CS), hyaluronic acid (HA) and carrageenans on the binding of HBC to IFN-gamma were tested in this system.

RESULTHuman recombinant IFN-gamma bound to heparin in a concentration dependent manner, the binding of IFN-gamma to HBC was detected at the concentration of 0.25 ng, and saturated at around 2 ng. Free heparin, LMW heparin, CS,HA and carrageenans competed for the binding of IFN-gamma to HBC with significant different ability. The IC(50)concentrations of heparin and LMW heparin were 2.40 microg/ml and 18.60 microg/ml respectively.

CONCLUSIONIFN-gamma is a cytokine with high binding affinity to heparin and carrageenans family but poor to CS-A and CS-C. ELISA is a simple, sensitive approach to detect the interaction of polysaccharides with cytokine in vitro.

Enzyme-Linked Immunosorbent Assay ; methods ; Heparin ; metabolism ; Interferon-gamma ; metabolism ; Polysaccharides ; metabolism ; Serum Albumin, Bovine ; metabolism

OBJECTIVETo explore the mechanism of hypoalbuminemia in patients with severe sepsis.

METHODSI(125)-labeled albumin was administered intravenously to 10 health volunteers and 10 patients with severe sepsis. Blood samples were taken at 0, 1, 2, 4, 8, 12, 24 hours and 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 18, 22, 25 days for the measurement of the dose of gamma-radiation and the curve of concentration and time. Then the half-life time (t(1/2)), apparent volume of distribution (V(d)) and transportation rate (K(12)) from center compartment to side compartment of albumin were calculated.

RESULTSThe half-life time in septic group was obviously shorter than that in control group (8.2 +/- 1.4 vs. 12.5 +/- 1.7, P < 0.01). The transportation rate in the septic group was higher than that in the control group [(4.4 +/- 1.9) x 10(-2)/h vs. (2.4 +/- 0.6) x 10(-2)/h, P < 0.05]. There was no significant difference in apparent volume of distribution between the two groups.

CONCLUSIONSIn patients with severe sepsis, the distribution rate of albumin from vessel to tissue was obviously increased and the decomposition rate of albumin was markedly improved.

Adult ; Aged ; Female ; Half-Life ; Humans ; Kinetics ; Male ; Middle Aged ; Sepsis ; metabolism ; Serum Albumin ; metabolism

OBJECTIVETo establish a direct and fast method separating calcium levofolinate and calcium dextrofolinate in a bovine serum albumin stationary phase chiral column.

METHODSUsing EC150/4 RESOLVOSIL BSA-7(150 mm x 4 mm) chiral separation column, with 0.20 mol/L, pH=5.0 phosphate buffer as mobile phase HPLC method was performed to separate calcium folinate enantiomers.

RESULTThe capacity factor and resolution of the two calcium folinate enantiomers were greatly affected by mobile phase buffer concentration,pH and the column temperature. And the retention time of calcium levofolinate and calcium dextrofolinate were 18.5 min and 22.6 min, respectively. The resolution, R(s)=1.49.

CONCLUSIONCalcium folinate enantiomers are separated successfully using this method.

Chromatography, High Pressure Liquid ; Hydrogen-Ion Concentration ; Leucovorin ; chemistry ; metabolism ; Serum Albumin, Bovine ; metabolism ; Stereoisomerism

With the availability of the method of analysis of serum protein using minute amounts of material, it was felt desirable to understand the protein metabolism and physiologic function in the body. The present study was undertaken to clarify the serum albumin, globulin and total protein at term to demonstrate the normal concentration and correlation between the 30 mother and newborn infant pairs. Serum albumin, globulin and total protein were determined by the Biuret method with pooled human serum. The A/G ration was calculated by formula of A/G. The following result were obstained. 1) In comparing the newborn infants of nonanemic mothers a albumin and total protein concentrations were higher and globlin concentrations decreased in the anemic mothers. 2) In comparing the nonanemic mothers and anemic mothers the mean albumin concentrations were nearly equal but globulin and total protein were slightly increased in the nonanemic mothers. 3) The mean serum albumin(of maternal and umbilical cord blood) was 3. 8+/-0. 35 gm/100 ml and 3. 8+/-0. 49 gm/100 ml respectively. 4) The mean serum globulin of mate. nal and umbilical cord blood was 2. 7+/-0. 41 gm/100 ml and 2. 32+/-0. 47 gm/100 ml respectively. The correlation of the globulin status between mot-hers and their newborn infants was not significant(r=0. 32). 5) The m-an serum total protein of maternal and umbilical cord blood was 6. 59+/-0. 59 gm/100 ml and 6. 02+/-0.57gm/100ml respectively.
Biuret ; Fetal Blood ; Humans ; Infant, Newborn* ; Metabolism ; Mothers* ; Serum Albumin* ; Umbilical Cord

The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.
Fermentation ; physiology ; Humans ; Models, Biological ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

Critical Illness ; Humans ; Hypoalbuminemia ; drug therapy ; Male ; Middle Aged ; Serum Albumin ; metabolism

Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

AIMTo study the protein binding of glimepiride.

METHODSAn HPLC-FA method is performed by using Pinkerton GFF II-S5-80 internal-surface reversed-phase silica support (150 mm x 4.6 mm ID, 5 microm) at pH 7.4 in a 67 mmol x L(-1) isotonic sodium phosphate buffer at 37 degree C. Other conditions included flow rate of 0.2 mL x min(-1), UV detection at wavelength 230 nm and injection volume 900 microL.

RESULTSNonlinear regression parameter estimation was used for the association constant measurement of glimepiride to both primary and secondary sites, which were 5.1 (micromol x L(-1)-1 and 1 for K1 and n1, and 0.017 (micromol x L(-1))-1 and 7 for K2 and n2, respectively.

CONCLUSIONThe method is shown to be suitable for investigation of protein binding of glimepiride.

Chromatography, High Pressure Liquid ; methods ; Humans ; Hypoglycemic Agents ; metabolism ; Protein Binding ; Serum Albumin ; metabolism ; Sulfonylurea Compounds ; metabolism

AIMTo study the binding of sibutramine hydrochloride (SH) and bovine serum albumin (BSA) in physiological condition by spectroscopic method.

METHODSThe quenching mechanism of the fluorescence of bovine serum albumin by sibutramine hydrochloride was studied with the fluorescence and the absorption spectroscopy. The binding constants K and the number of binding sites were determined at different temperatures according to Scatchard equation and the main binding force was discussed by thermodynamic equations. The effect of the drug on bovine serum albumin conformation was also studied by using synchronous fluorescence spectroscopy.

RESULTSThe quenching mechanism of sibutramine hydrochloride to bovine serum albumin was static quenching. The binding constants K at 8 degrees C, 25 degrees C, 37 degrees C were 1.21 x 10(5), 8.31 x 10(4), 6.97 x 10(4) L x mol(-1) with one binding site, respectively. The thermodynamic parameters of the reaction were deltaH = -9.70 kJ x mol(-1), deltaS = 56.41 J x mol(-1) x K(-1).

CONCLUSIONThe binding force is electrostatic interaction. Sibutramine hydrochloride can be deposited and transported by serum protein in vivo. Sibutramine hydrochloride has nearly no effect on the serum protein conformation.

Animals ; Binding Sites ; Cattle ; Cyclobutanes ; metabolism ; Protein Binding ; Serum Albumin, Bovine ; metabolism ; Spectrometry, Fluorescence ; methods ; Spectrophotometry, Ultraviolet ; methods

Albumin is the most abundant protein in human serum. A dye-binding method is commonly used in clinical laboratories for its estimation using different types of dyes. However, all these dye methods were interfered by a variety of compounds. Here we present a method for the detection of albumin in human serum and other biological fluids. The principle is based on the fact that lactate dehydrogenase isoenzyme-5 (LDH-5) binds specifically to Dextran-Blue (DB). Albumin inhibits the binding of LDH-5 with DB. Absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa.
Chemistry, Clinical/*methods ; Chromatography, Gel ; Human ; Isoenzymes/metabolism ; Lactate Dehydrogenase/metabolism ; Protein Binding ; Sepharose/chemistry ; Serum Albumin/*analysis