ENDOTOXINS

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ENDOTOXINS

• Richard Marchand MD
• Associate professor
• University of Montreal

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ENDOTOXINS definition

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

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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

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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)

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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 !)

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DOSAGE OF ENDOTOXINS

• Before 1987 poor rabbits
• Since 3 methods derived from Levines
  method (1979)
• Gel-clot reaction
• Turbimetric methods static and dynamic
• Chromogenic methods static et dynamic
• Uses Limulus polyphemus lysate (LAL)

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DOSAGE Gel clot method

• Principle the smallest dilution (?) 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
  (?) by the titre of the dilution
• Ex. If ? 0.125 EU/mL and titre
  1/16 then the sample contains 16 X 0.125 2.0
  EU/mL

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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.

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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 ?????

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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.

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Destruction in terms of 2 curves of first order
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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

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RESULTS

• ETO 100 (n3) normal cycle 55 oC
• Recovery 65 (2650 /- 1101)
• Max. destruction 35
• WCS 17 83 on the instrument
• Dry heat (n3) 1 hour at 170 oC ( 1/3 cycle)
• Recovery 54 (2204 /- 220)
• Max. destruction 46
• WCS 23 77 on the instrument

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RESULTS

• Steam (n3) 121 oC Normal cycle
• Recovery 1.4 (56 EU)
• Max. destruction 98.6
• WCS 91 9 on the instrument
• Plasma (n13, n23) 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

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RESULTS

• Steam (n3) 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