ENDOTOXINSENDOTOXINS

Richard Marchand MDRichard Marchand MD

Associate professor Associate professor

University of MontrealUniversity of Montreal

ENDOTOXINS : definition

Large molecules coming from bacteriasLarge molecules coming from bacterias The major part comes from the cell wallThe major part comes from the cell wall Gram neg >>> Gram posGram neg >>> Gram pos Composed of lipids and sugars Composed of lipids and sugars (polysaccharids) :(polysaccharids) :

lipo-polysaccharids (lipo-polysaccharids (LPSLPS)) Resistant to heat (> 100 Resistant to heat (> 100 ooC)C) Syn.: Syn.: pyrogenspyrogens

ENDOTOXINSENDOTOXINS

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

ENDOTOXINSENDOTOXINS

The toxic strength varies according to the typeThe toxic strength varies according to the type fentograms per bacteria (4.0 X 10-15 gr)

Strong molecular variations and antigenicity between Strong molecular variations and antigenicity between species species (antibodies against one type ineffective against the other)(antibodies against one type ineffective against the other)

Forms a major component of the bacteria cell wallForms a major component of the bacteria cell wall Free endotoxins concept (Free endotoxins concept (those are not in the cell wall)those are not in the cell wall)

Buffer effect concept Buffer effect concept (according to internal proteic state)(according to internal proteic state)

ENDOTOXINSENDOTOXINS

They cause :They cause : - - fever and tachycardiafever and tachycardia

- increase in cardiac output- increase in cardiac output

-decrease in peripheral resistances-decrease in peripheral resistances

Mode of action : Mode of action : via the immune system and the via the immune system and the

liberation of cytokinsliberation of cytokins

Sensitiveness = genetically determinedSensitiveness = genetically determined (*Man is extremely sensitive : few micrograms per Kg are sufficient to (*Man is extremely sensitive : few micrograms per Kg are sufficient to

produce a state of shock)produce a state of shock)

(*Women also !)(*Women also !)

DOSAGE OF ENDOTOXINS

Before 1987 : poor rabbitsBefore 1987 : poor rabbits

Since: 3 methods derived from Levine’s Since: 3 methods derived from Levine’s

method (1979)method (1979)

– Gel-clot reactionGel-clot reaction

– Turbimetric methods: static and dynamic Turbimetric methods: static and dynamic

– Chromogenic methods : static et dynamicChromogenic methods : static et dynamic Uses Uses Limulus polyphemusLimulus polyphemus lysate (LAL) lysate (LAL)

DOSAGE : Gel clot methodDOSAGE : Gel clot method

PrinciplePrinciple : the smallest dilution ( : the smallest dilution (λλ) capable of inducing a clot ) capable of inducing a clot form a reactant lot of LAL is determinedform a reactant lot of LAL is determined

MethodMethod : : -a standard reference (RSE) by double dilutions -a standard reference (RSE) by double dilutions of of a known endotoxin is determined a known endotoxin is determined

-the largest dilution capable to induce the -the largest dilution capable to induce the clot in clot in the sample to be measured is determined the sample to be measured is determined

The concentration of endotoxin in he sample is claculated by The concentration of endotoxin in he sample is claculated by

multiplying the sensibility factor (multiplying the sensibility factor (λλ) by the titre of the dilution) by the titre of the dilution

– Ex. : If Ex. : If λ = 0.125 EU/mL and titre = 1/16then the sample contains 16 X 0.125 = 2.0 EU/mL

DOSAGE : DOSAGE : chromogenic methods

PrinciplePrinciple : instead of turbidity, the colour produced by the : instead of turbidity, the colour produced by the utilization of a chromogenous synthetic substrate is measured.utilization of a chromogenous synthetic substrate is measured.

Static method Static method : the sample is incubated with LAL reactant for a : the sample is incubated with LAL reactant for a specific duration and the final colour intensity is measured with a specific duration and the final colour intensity is measured with a

spectrophotometre.spectrophotometre. Dynamic method Dynamic method : consists in taking multiple readings during the : consists in taking multiple readings during the

course of the incubation in order to determine the required time course of the incubation in order to determine the required time to obtain a threshold of a given intensityto obtain a threshold of a given intensity

The required duration is plottted on a curve of standard duration The required duration is plottted on a curve of standard duration time in order to measure the quantity in the sample.time in order to measure the quantity in the sample.

PHARMACOPEIA : PHARMACOPEIA : depyrogenization depyrogenization

Dry heatDry heat: 4 hours at 160 : 4 hours at 160 ooC C (3 log (3 log minimumminimum))

2-45 minutes at 250 2-45 minutes at 250 ooC C (average: 30 min) (average: 30 min)

Walsh 1945 (penicillin) 30 min at 250Walsh 1945 (penicillin) 30 min at 250ooCC

Bacillus subtilisBacillus subtilis spores have a D value spores have a D value160160ooCC = 1.46 min = 1.46 min Sterilization-SAL 10Sterilization-SAL 10-6 -6 (6 X 1.46) = 8.76 minutes (6 X 1.46) = 8.76 minutes (27 times less)(27 times less)

Would radiant heat be more effective than heat by convection Would radiant heat be more effective than heat by convection

What is it about alternatives to dry heat ?????What is it about alternatives to dry heat ?????

ENDOTOXINSENDOTOXINSKinetics to dry heatKinetics to dry heat

Tsuji and HarissonTsuji and Harisson (Upjohn) (Upjohn)

Second order R2Second order R2– DD170170 = 251 = 251– DD190190 = 99.4 = 99.4– DD210210 = 33.3 = 33.3– DD250250 = 4.99 = 4.99

Log Y = A + B x 10Log Y = A + B x 10C x (x = duration in minutes)C x (x = duration in minutes)

Z value = 46.4 minutes approx.Z value = 46.4 minutes approx.

Destruction in terms of 2 curves of Destruction in terms of 2 curves of first orderfirst order

0,001

0,01

0,1

1

10

0min

10min

20min

30min

40min

100 C

160 C

250 C

DEPYROGENIZATION ASSAYSDEPYROGENIZATION ASSAYS

Test bottles (ACC) : 0,5 microgr. Test bottles (ACC) : 0,5 microgr.

5,000 EU /vial5,000 EU /vial

Recuparation : Recuparation : Int. cont.Int. cont. 4,540 +/- 1107 4,540 +/- 1107

4 contrôles : 4,088 +/- 1280 82 %4 contrôles : 4,088 +/- 1280 82 %By projection of the extraction curves :By projection of the extraction curves :

extraction is approx. 50 % for high [ Q ]extraction is approx. 50 % for high [ Q ] extraction is less then 10 % for low [ Q ],extraction is less then 10 % for low [ Q ], possibly less then 1 % for very low[ Q ]possibly less then 1 % for very low[ Q ]

RESULTSRESULTS

ETO 100% (n=3) normal cycle 55 ETO 100% (n=3) normal cycle 55 ooC C – Recovery 65 % Recovery 65 % (2650 +/- 1101)(2650 +/- 1101)

– Max. destruction : 35 %Max. destruction : 35 %– WCS : 17 %WCS : 17 % 83 % on the instrument 83 % on the instrument

Dry heat (n=3) 1 hour at 170 Dry heat (n=3) 1 hour at 170 ooC C ( 1/3 cycle)( 1/3 cycle)

– Recovery 54 % Recovery 54 % (2204 +/- 220)(2204 +/- 220)

– Max. destruction : 46 %Max. destruction : 46 %– WCS : 23 %WCS : 23 % 77 % on the instrument 77 % on the instrument

SteamSteam (n=3) 121 (n=3) 121 ooC Normal cycleC Normal cycle– Recovery : 1.4 % Recovery : 1.4 % (56 EU)(56 EU)

– Max. destruction : 98.6 %Max. destruction : 98.6 %– WCS : 91 %WCS : 91 % 9 % on the instrument 9 % on the instrument

PlasmaPlasma (n (n11=3, n=3, n22=3) H=3) H22O Sterrad 100SO Sterrad 100S

– Recovery : nRecovery : n11= 0.4 % = 0.4 % (18 EU)(18 EU) n n22 = 0.1 % = 0.1 % (4 EU)(4 EU)

– Max. destruction 99.6 and 99.9 %Max. destruction 99.6 and 99.9 %– WCS : 94 and 96 %WCS : 94 and 96 % 6 and 4 % on the instrument 6 and 4 % on the instrument

RESULTSRESULTS

SteamSteam (n=3) 121 (n=3) 121 ooC Normal cycleC Normal cycle– Recovery : 1.4 % Recovery : 1.4 % (56 EU)(56 EU)

– Max. destruction : 98.6 %Max. destruction : 98.6 %– WCS : 91 %WCS : 91 % 9 % on the instrument 9 % on the instrument

Ozone Ozone – Recovery : nRecovery : n11= 0.x % = 0.x % ( EU)( EU) n n22 = 0.x % = 0.x % ( EU)( EU)

– Max. destruction 99.x and 99.x %Max. destruction 99.x and 99.x %– WCS : 9x and 9x %WCS : 9x and 9x % a and b % on the instrument a and b % on the instrument

RESULTSRESULTS