The measurement and analysis of sedimentation equilibrium provides one of the most powerful and widely applicable methods for the characterization of reversible associations of macromolecules in solution. Recent developments in instrumentation, experimental design, and data analysis have substantially broadened the range of systems to which this technique may be applied, simplified its application, and reduced the cost of acquiring analytical capability.
Animal ; Human ; Kinetics ; Macromolecular Systems* ; Molecular Weight ; Proteins/chemistry ; Proteins/analysis ; Solutions ; Ultracentrifugation/methods ; Ultracentrifugation/instrumentation*

OBJECTIVEApplying for the activity of enzyme in vitro,the research optimized the best preparation procedure for the anticoagulated blood region from Lumbricus.

METHODAll through our experiment, the content of protein and theactivity of enzyme were examined. The extraction process, the refining technology, concentration processes of Lumbricus were optimized with single factor checking and orthogonal design method.

RESULTAt 37 degrees C, the coarse powder of Lumbricus soaking with 15 fold of 0.9% sodium chloride and ultrasonic extracting 40 minites for three times was the best ultrasonic extraction. Utrafiltration membrane with molecular weights of 30 x 10(3) for refining and 10 x 10(3) for concentrating were selected.

CONCLUSIONUltrasonic extraction and membrane separation technology, to well improve the effect of purification for the anticoagulant site of Lumbricus, is conducive to further study.

Animals ; Anticoagulants ; chemistry ; isolation & purification ; Drug Compounding ; methods ; Oligochaeta ; chemistry ; enzymology ; Temperature ; Ultracentrifugation ; Ultrasonics

OBJECTIVETo present an improved method to obtain pure, viable, freshly isolated hepatic stellate cells.

METHODSAdult male SD rats were used. All procedures were performed with the animals under sodium pentobarbital anesthesia. Three days after the single intravenous administration of 1 ml liposome-encapsulated CL2MDP, which has selective cytotoxicity of Kupffer cells, livers were perfused with D-Hank's solution containing 100 U/ml heparin for 10 to 15 minutes, and then with 0.05% collagenase dissolved in D-Hank's solution for 25 to 30 minutes. The liver was then gently homogenized and further incubated in 0.025% collagenase, and 0.005% DNAase I for 30 minutes at 37 degrees C under constant stirring. This suspension was filtered through stainless steel gauze and centrifuged for 2 minutes at 50 x g to remove parenchymal cells. Sinusoidal cells in the supernatant were recovered by centrifugation for 10 minutes at 300 x g. The cells were resuspended in the presence of 28.7% Nycodenz stock solution. The final concentration of Nycodenz at this stage was 11.5%. Following centrifugation for 17 minutes at 1400 x g, The cells at the top of this Nycodenz solution were collected. Cells were resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, The cells were seeded in 50 ml culture flask at a density of 500,000 cells/ml, The cell viability was determined by trypan blue exclusion staining, the purity of hepatic stellate cells was identified by the expression of Desmin using immunocytochemistry method. Endogenous peroxidase staining was used to detect Kupffer cells.

RESULTSThe yield rate of hepatic stellate cells was 3 x 10(7) per rat, the cell viability was more than 95%, the desmin positive cell rate was 90%, no endogenous peroxidase positive cells were detected.

CONCLUSIONThe method for the isolation of hepatic stellate cells was developed without Kupffer cells confusion. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.

Animals ; Cell Culture Techniques ; Cell Separation ; methods ; Kupffer Cells ; cytology ; Liver ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Ultracentrifugation ; methods

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods—LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.
Alanine Transaminase ; Alkaline Phosphatase ; Aspartate Aminotransferases ; Bilirubin ; Calcium ; Centrifugation ; Creatinine ; Glucose ; Hyperlipidemias* ; Laboratories, Hospital ; Liquid-Liquid Extraction ; Methods ; Potassium ; Sodium ; Spain ; Transferases ; Ultracentrifugation ; Urea