1 / 15

ENDOTOXINS

ENDOTOXINS. Richard Marchand MD Associate professor University of Montreal. ENDOTOXINS : definition. Large molecules coming from bacterias The major part comes from the cell wall Gram neg >>> Gram pos Composed of lipids and sugars (polysaccharids) : lipo-polysaccharids ( LPS )

shanon
Download Presentation

ENDOTOXINS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. ENDOTOXINS Richard Marchand MD Associate professor University of Montreal

  2. ENDOTOXINS : definition • Large molecules coming from bacterias • The major part comes from the cell wall • Gram neg >>> Gram pos • Composed of lipids and sugars (polysaccharids) : lipo-polysaccharids (LPS) • Resistant to heat (> 100 oC) • Syn.: pyrogens

  3. ENDOTOXINS LPS structure Lipid A Core Polysaccharids O Lipid A (lipidA) : diglucosamine containing fatty acids chains of 10 to 20 carbon atoms Core : core made of sugar (carbohydrates) acid residus 2 kéto -3 déoxy -D- mannoctulosinic (KDO) Polysaccharid O : repetitive branch of linear sugars

  4. ENDOTOXINS • The toxic strength varies according to the type • fentograms per bacteria (4.0 X 10-15 gr) • Strong molecular variations and antigenicity between species (antibodies against one type ineffective against the other) • Forms a major component of the bacteria cell wall • Free endotoxins concept (those are not in the cell wall) • Buffer effect concept (according to internal proteic state)

  5. ENDOTOXINS • They cause :- fever and tachycardia - increase in cardiac output -decrease in peripheral resistances • Mode of action : via the immune system and the liberation of cytokins • Sensitiveness = genetically determined • (*Man is extremely sensitive : few micrograms per Kg are sufficient to produce a state of shock) (*Women also !)

  6. DOSAGE OF ENDOTOXINS • Before 1987 : poor rabbits • Since: 3 methods derived from Levine’s method (1979) • Gel-clot reaction • Turbimetric methods: static and dynamic • Chromogenic methods : static et dynamic • Uses Limulus polyphemus lysate (LAL)

  7. DOSAGE : Gel clot method • Principle : the smallest dilution (λ) capable of inducing a clot form a reactant lot of LAL is determined • Method : -a standard reference (RSE) by double dilutions of a known endotoxin is determined -the largest dilution capable to induce the clot in the sample to be measured is determined • The concentration of endotoxin in he sample is claculated by multiplying the sensibility factor (λ) by the titre of the dilution • Ex. : If λ = 0.125 EU/mL and titre = 1/16 then the sample contains 16 X 0.125 = 2.0 EU/mL

  8. DOSAGE : chromogenic methods • Principle : instead of turbidity, the colour produced by the utilization of a chromogenous synthetic substrate is measured. • Static method : the sample is incubated with LAL reactant for a specific duration and the final colour intensity is measured with a spectrophotometre. • Dynamic method : consists in taking multiple readings during the course of the incubation in order to determine the required time to obtain a threshold of a given intensity • The required duration is plottted on a curve of standard duration time in order to measure the quantity in the sample.

  9. PHARMACOPEIA : depyrogenization • Dry heat: 4 hours at 160 oC (3 log minimum) • 2-45 minutes at 250 oC (average: 30 min) Walsh 1945 (penicillin) 30 min at 250oC Bacillus subtilis spores have a D value160oC = 1.46 min Sterilization-SAL 10-6 (6 X 1.46) = 8.76 minutes (27 times less) Would radiant heat be more effective than heat by convection What is it about alternatives to dry heat ?????

  10. ENDOTOXINSKinetics to dry heat • Tsuji and Harisson (Upjohn) • Second order R2 • D170 = 251 • D190 = 99.4 • D210 = 33.3 • D250 = 4.99 • Log Y = A + B x 10C x (x = duration in minutes) • Z value = 46.4 minutes approx.

  11. Destruction in terms of 2 curves of first order

  12. DEPYROGENIZATION ASSAYS Test bottles (ACC) : 0,5 microgr. 5,000 EU /vial Recuparation : Int. cont. 4,540 +/- 1107 4 contrôles : 4,088 +/- 1280 82 % By projection of the extraction curves : • extraction is approx. 50 % for high [ Q ] • extraction is less then 10 % for low [ Q ], • possibly less then 1 % for very low[ Q ]

  13. RESULTS • ETO 100% (n=3) normal cycle 55 oC • Recovery 65 % (2650 +/- 1101) • Max. destruction : 35 % • WCS : 17 % 83 % on the instrument • Dry heat (n=3) 1 hour at 170 oC ( 1/3 cycle) • Recovery 54 % (2204 +/- 220) • Max. destruction : 46 % • WCS : 23 % 77 % on the instrument

  14. RESULTS • Steam (n=3) 121 oC Normal cycle • Recovery : 1.4 % (56 EU) • Max. destruction : 98.6 % • WCS : 91 % 9 % on the instrument • Plasma (n1=3, n2=3) H2O Sterrad 100S • Recovery : n1= 0.4 % (18 EU) n2 = 0.1 % (4 EU) • Max. destruction 99.6 and 99.9 % • WCS : 94 and 96 % 6 and 4 % on the instrument

  15. RESULTS • Steam (n=3) 121 oC Normal cycle • Recovery : 1.4 % (56 EU) • Max. destruction : 98.6 % • WCS : 91 % 9 % on the instrument • Ozone • Recovery : n1= 0.x % ( EU) n2 = 0.x % ( EU) • Max. destruction 99.x and 99.x % • WCS : 9x and 9x % a and b % on the instrument

More Related