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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 )

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ENDOTOXINS

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  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

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