ENDOTOXINS Richard Marchand MD Associate professor University of Montreal

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

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

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

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

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

DOSAGE : Gel clot method n Principle : the smallest dilution ( λ ) capable of inducing a clot form a reactant lot of LAL is determined n 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 n The concentration of endotoxin in he sample is claculated by multiplying the sensibility factor ( λ ) by the titre of the dilution – Ex. : If – Ex. : If λ = EU/mL and titre = 1/16 then the sample contains 16 X = 2.0 EU/mL

DOSAGE : DOSAGE : chromogenic methods n Principle : instead of turbidity, the colour produced by the utilization of a chromogenous synthetic substrate is measured. n Static method : the sample is incubated with LAL reactant for a specific duration and the final colour intensity is measured with a spectrophotometre. n 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 n The required duration is plottted on a curve of standard duration time in order to measure the quantity in the sample.

PHARMACOPEIA : depyrogenization n Dry heat: 4 hours at 160 o C (3 log minimum ) n 2-45 minutes at 250 o C (average: 30 min) Walsh 1945 (penicillin) 30 min at 250 o C Bacillus subtilis spores have a D value 160 o C = 1.46 min Sterilization-SAL (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 ?????

ENDOTOXINS Kinetics to dry heat n Tsuji and Harisson (Upjohn) n Second order R2 – D 170 = 251 – D 190 = 99.4 – D 210 = 33.3 – D 250 = 4.99 n Log Y = A + B x 10 C x (x = duration in minutes) n Z value = 46.4 minutes approx.

Destruction in terms of 2 curves of first order

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

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

n Steam (n=3) 121 o C Normal cycle – Recovery : 1.4 % (56 EU) – Max. destruction : 98.6 % – WCS : 91 % 9 % on the instrument n Plasma (n 1 =3, n 2 =3) H 2 O Sterrad 100S – Recovery : n 1 = 0.4 % (18 EU) n 2 = 0.1 % (4 EU) – Max. destruction 99.6 and 99.9 % – WCS : 94 and 96 % 6 and 4 % on the instrument RESULTS

n Steam (n=3) 121 o C Normal cycle – Recovery : 1.4 % (56 EU) – Max. destruction : 98.6 % – WCS : 91 % 9 % on the instrument n Ozone – Recovery : n 1 = 0.x % ( EU) n 2 = 0.x % ( EU) – Max. destruction 99.x and 99.x % – WCS : 9x and 9x % a and b % on the instrument RESULTS