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La purificación de lipopolisacáridos (LPS) o endotoxinas y su caracterización es un aspecto esencial para estudios que buscan aclarar el papel de estas biomoléculas de bacterias Gram negativas presentes en la cavidad oral y su relación con enfermedades locales periodontales y sistémicas. Este estudio implementa una metodología para la extracción, purificación y caracterización de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83, utilizando técnicas previamente descritas. La extracción cruda de LPS se realizó con fenol-agua caliente; la purificación se realizó con tratamiento enzimático con nucleasas y proteasa, seguido de cromatografía de exclusión por tamaño (Sephacryl S-200 HR) con deoxicolato de sodio como fase móvil. La caracterización de los extractos purificados se realizó por barrido espectrofotométrico, pruebas bioquímicas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromogénica de LAL. Como control para la identificación y caracterización de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodología implementada permitió la obtención de LPS de elevada pureza con la identificación de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endotóxica a bajas concentraciones. La metodología implementada en este estudio para la purificación y caracterización de LPS a partir de bacteria completa fue eficiente al compararla con los LPS comerciales.
Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarify the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed by using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low concentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when it was compared with commercial LPS.
Asunto(s)
Cromatografía , Eikenella , Endotoxinas , Lipopolisacáridos , Periodontitis , PorphyromonasRESUMEN
OBJECTIVE:To establish a method for detecting bacterial endotoxins in methylthioninium chloride injection (MCI) quantitatively. METHODS: Kinetic turbidimetric limulus test was applied to detect bacterial endotoxins in MCI quantitatively, and compared with gel-clot method. RESULTS: MCI was un-interfered with the test for bacterial endotoxins at the concentration of 0.125 mg?mL-1; the content of bacterial endotoxins in all samples tested (10 mg?mL-1) were not more than 0.25 EU?mg -1, which were in accordance with the result of gel-clot method. CONCLUSIONS: Kinetic turbidimetric limulus test provides a new way to detect bacterial endotoxins in MCI quantitatively.
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OBJECTIVE: To establish a method for endotoxin assay in aminoglycoside antibiotics for injection.METHODS: Kinetic turbidimetric limulus test was used for endotoxin assay in aminoglycoside antibiotics for injection.The recovery rate of bacterial endotoxin was determined though pre-interference experiments in which quantitative endotoxin was added to different concentrations of aminoglycosides with the concentration optimized,then an interference test on 4 different samples was conducted.RESULTS: At a concentration of 0.5 mg?mL-1,aminoglycosides didn't interfere with the limulus test if the amount of standard endotoxin was at 5.000,0.500,and 0.050 EU?mL-1,respectively.CONCLUSIONS: The kinetic turbidimetric limulus test could be used for endotoxin assay in aminoglycoside antibiotics for injection.
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AIM: The kinetic turbidimetric Limulus test was used in the endotoxin assay of Ceftazidime injection. METHODS: The validation test was fulfiled, when the amount of endotoxin spiked in Ceftazidime injection sample (Lot. No. 010202) in which concentration of its dilution solution were 10, 5,2,1,0.5 g·L-1. They were recovered in 60.08%, 82.90%, 85.22%, 94.00%, 92.67%; with the amount of endotoxin spiked in Ceftazidime sample (Lot. No. 010103, 010104, 010202), their 1 g·L-1 dilution were recovered in 109.9%, 101.7% and 86.56%. RESULTS: With standard endotoxin working curve is 5.00, 0.500, 0.0500 ZU·L-1. The 1 g·L-1 solution to Ceftazidime was effective to eliminate the interference in Limulus test. CONCLUSION: The kinetic turbidimetric Limulus test provide a new way to the detection of endotoxin in Ceftazidime injection.
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100MIC.The endotoxin could be released more and more with the time.CONCLUSION:It is reasonable to choose antibiotics to refer to the characteristics of the endotoxin release by germs.
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OBJECTIVE:To apply the kinetic turbidimetric Limulus test to the endotoxin assay of Levocarnitine sodium chloride injection.METHODS:The16-fold dilution of Levocarnitine sodium chloride injection was prepared.The content of bacterial endotoxin in Levocarnitine sodium chloride injection was determined with kinetic turbidimetric Limulus test after screen test and validation test.RESULTS:The16-fold dilution of Levocarnitine sodium chloride injection was effective to e?liminate the interference in Limulus test.The average recovery was in the range of50%～200%.CONCLUSION:The kinetic turbidimetric Limulus test provides a new and quick method for the quantitative determination of bacterial endotoxin in Levo?carnitine sodium chloride injection.
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Objective: The dynamic turbidimetry Limulus test was used to assay endotoxin content in Chuan Hu Ning injection. Methods: When the standardized endotoxin added in Chuan Hu Ning injection sample (Lot.No.000409) and in its 40-fold dilution, 80-fold dilution, 120-fold dilution and 200-fold dilution , the recovery was 0 %, 21.18 %, 76.69 %, 101.1 %and 117.8 %and as the standardized endotoxin added in the three batches of 200-fold dilution of Chuan Hu Ning injection (Lot.No.000408?000409?001023), the recovery was 110.5 %, 124.4 %and 87.98 %. Results: As standardized endotoxin concentration being 5.00, 0.500, 0.0500Eu/mL , the 200-fold dilution of Chuan Hu Ning injection is effective to eliminate the interference in the test. Conclusion: The dynamic turbidimetry Limulus test provides a new way for the determination of endotoxin in Chuan Hu Ning injection.
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Object To determine the bacterial endotoxin in BREVISCAPINI INJECTION (BI) by kinetic turbidimetric limulus test Methods By spiking the amount of standard endotoxin into BI sample, the interference test was fulfiled to detect the recovery in the range of 50% 200% Results A 12 fold dilution to BI sample is effective to eliminate the interference in limulus test Conclusion The kinetic turbidimetric limulus test provides an efficient way to detect the bacterial endotoxin in BI sample
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Objective To study the interference of Huangyangning Injection in bacterial endotoxin test and to establish bacterial endotoxin test method for this specimen.Methods According to China Pharmacopoeia 2000,inhibition or en-hancement test for bacterial endoto xins test was carried out for Huangyangning Injection.Results l ∶150solution of Huangyangning Injection had no inte rference on the test.Conclusion Reliable data and result are obtained and bacterial endotoxin test method for Huangyangning Injection has been established.
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Objective: The kinetic turbidimetric limulus test was used in the endotoxin assay of Sodium Ferulate for injection. Methods: The preliminary validation test was fulfilled, when the standard endotoxin spiked in Sodium Ferulate for injection sample (Lot. No. 20000704) and its 10 fold dilution、 20 fold dilution、 40 fold dilution、 60 dilution、 80 fold dilution. The recoveries were 0.20%, 74.70%, 87.93%, 87.09%, 98.55%, 119.2% respectively. The optimum detection concentration found was 60 fold dilution. During the formal interference tests of 3 batches of Sodium Ferulate for injection (Lot. No. 20000308 20000704 20000315), the recoveries were 120.4%,98.55% and 85.40% respectively. Results: 5.00,0.500,0.0500 Eu/mL of standard endotoxin were used. The standard endotoxin was quantitatively added into the 60 fold dilution of Sodium Ferulate for injection. The recoveries were all 50%～200% and no interference action was found.Conclusion: The kinetict turbidimetric limulus test provided a new way to the detection of endotoxin in Sodium Ferulate for injection.
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OBJECTIVE:To establish the method for assaying bacterial endotoxins in levofloxacin hydrochloride injec?tion.METHODS:The interference test of3batches of levofloxacin hydrochloride injection with2kinds of limulus test agent was studied.RESULTS:The interference between the samples and limulus test agents was eliminable(below1.0mg/ml).The detection results were up to standard.CONCLUSION:The bacterial endotoxin in the sample can be examined by limulus test instead of pyrogen test in rabbits.
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OBJECTIVE:To approach the feasibility of replacing pyrogen test by bacterial endotoxin test(BET)in assaying the injectio puerarin.METHODS:Kinetic turbimetric assay(KTA)was used to determine the bacterial endotoxin,and an inhi?bition and strengthen test by using the EDS-99bacterial endotoxin systems was adopted for assaying injectio puerarin.RES_ ULTS:The regression equation of standard curve was logT=2.7957—0.2422logC,r=—0.9973;Injectio puerarin did not interfere with limulus test agent in1∶60dilution.CONCLUSION:This method is slight in interference and accurate in de?tection result,and can be used in routine assay.
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OBJECTIVE:To study the bacterial endotoxin test for lomefloxacin hydrochloride and glucose injection.METHODS:According to the assigned method and guiding principle for bacterial endotoxin test in CHINESE PHARMACOPOEIA 2000,the interference test was performed.RESULTS:Lomefloxacin and glucose injection could interfere with limulus test,however,the interference could be eliminated after adjusting the pH of injection to 6.8～7.0.The proper concentration of limulus test agent of bacterial endotoxin test was 0.25EU/ml.CONCLUSION:Bacterial endotoxin test can be used for quality control of lomefloxacin and glucose injection.
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OBJECTIVE:To establish a bacterial endotoxin test for ofloxacin injection METHODS:Interference tests in ofloxacin injection were performed with limulus test agents provided by different factories RESULTS:Ofloxacin injection did not interfer with bacterial endotoxin CONCLUSION:Bacterial endotoxin test can take the place of pyrogen test for ofloxacin injection
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AIM The kinetic turbidimetric Limulus test was used in the endotoxin assay of Ceftazidime injection. METHODS The validation test was fulfiled ,when the amount of endotoxin spiked in Ceftazidime injection sample(Lot.No.010202) in which concentration of its dilution solution were 10,5,2,1,0 5 g?L -1 . They were recovered in 60 08%, 82 90%, 85 22%, 94 00%, 92 67%; with the amount of endotoxin spiked in Ceftazidime sample (Lot.No. 010103, 010104, 010202),their 1 g?L -1 dilution were recovered in 109 9%, 101 7% and 86 56%. RESULTS With standard endotoxin working curve is 5 00, 0 500, 0 0500 ZU?L -1 . The 1 g?L -1 solution to Ceftazidime was effective to eliminate the interference in Limulus test. CONCLUSION The kinetic turbidimetric Limulus test provide a new way to the detection of endotoxin in Ceftazidime injection.
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The antagonistic effects of ruthenium red (RuR) on the biological activi-ties of endotoxin (ET) which included the limulus amebocytes lysate test, the local Shw-artzman reaction, decreasing the white blood cell counts, and increase of zinc in periphe-ral blood were observed. The destroyed meshes of endotoxin have been seen under electronmicroscope. The amino-groups of endotoxin increased markedly, which it was treated withRuR. These results indicated that the polycationic residues of RuR may be the effectiveunits of antiendotoxin activity.
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The effects of E. coli endotoxin on acute hepatic insufficiency induced by galactosamine were studied. Results indicated that endotoxin exhibited significant effects of promoting the nerosis of hepatocyte, prolonging prothrombin time, elevating the serum levels of billrubin and urea nitrogen on acute hepatic insufficiency in rats induced by galactosamtne. Lethargy and coma in the latter rats treated with endotoxin occured more quickly and more seriously than those in the controls, but no significant difference of amino acids in plasma and brain was found except for taurine which showed a significant increase in brain. These results suggest that endotoxin is an important factor in the development and progress of acute hepatic insufficiency.
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The effect of E. coli endotoxin (LPS) on early and advanced experimental cirrhosis have been studied. Results indicate that LPS has exhibited a significant promoting effect on liver damage and fibrosis, exerted inhibitory effect on colcollagen resorption. The rats with early and advanced cirrhosis were administered with LPS. Their parenchymal cells of the liver had more prominent lesion than those of the controls. The experiments demgnstrated that the hepatic renal dysfunction, hepatic encephalopathy, hilirubin metabolic disorder of the cirrhotic rats were induced and aggravated by LPS. Thus it proved that LPS is an importent pathogenic factor in the development and progress of hepatie insufficiency.