Pyrogens

Pyrogens are Endotoxin, (or)lipopolysaccharide(LPS),is highly toxic to mammalian cells and is one of the most potent  modulators of the immune system.they areproduced from  gram –ve microorganisms.

COMPOSITION: pyrogens are  is composed of hydrophobic fatty acid and hydrophilic carbohydrate domains

The chemical nature of LPS makes pyrogen removal problematic . LPS is unusually thermostable and fairly insensitive to pH changes.

Both the United States Pharmacopeia and the European Pharmacopoeia specify the rabbit pyrogen test and the Limulus amebocyte lysate

PHYSIOLOGICAL EFFECTS:

1.First,pyrogens elevate the circulating levels of inflammatory cytokines (e.g.,IL-1,IL-6, TNF-_ and IL-8) followed by the clinically relevant events of fever,hypotension, lymphopenia, neutrophilia, and elevated levels of plasma cortisol and acute-phase proteins (e.g., C-reactive protein)

2.Low doses of pyrogens induce inflammatory reactions without any clinicallysignificant symptoms.

3. Moderatedoses of pyrogens induce fever and significant changes in plasma composition.

4. Highdoses of pyrogens can lead to septic shock characterized by cardiovascular dysfunction,including myocardial depression and dilatation, vasodilation, vasoconstriction,endothelium

dysfunction,and organ dysfunction (e.g., kidney, liver, lung, or brain) followed by multiple organ failures and death.

IDENTIFICATION:

Thereare 2 tests for the identification of endotoxins in parenterals

1 LALtest

2.Rabbit fever test

LAL TEST:

                                                        

 LALvials

The rabbit fever test was the standard FDA approved test for endotoxins, until the approval of the LAL test by the FDA in the 1980’s.

THE HORSE SHOE CRAB: LAL reagent is prepared from circulating blood cells of the horse shoe crab(limuluspolyphemus).

Ø     Substituted ffor USP pyrogen test (Rabbitffever test)..

Ø     Single test vial of LAL contains 0.2ml Lyophilized LAL

TEST PROCEDURE:

LAL is an aqueous extract of the blood cells of horseshoecrabs which forms a clot or change in color, depending on the technique, in thepresence of bacterial endotoxin. 

PROCEDURE:

Addition of  Lal reagent to our sample  causes clot formation, if  endotoxins are present.in our sample.

 The main MECHANISM involved in this is

Gram-negative bacterial endotoxin catalyzes the activationof a proenzyme in the Limulus Amebocyte Lysate. The initial rate of activationis determined by the concentration of endotoxin present. The activated enzyme(coagulase) hydrolyzes specific bonds within a clotting protein (coagulogen)also present in Limulus Amebocyte Lysate. Once hydrolyzed, the resultan tcoagulin self-associates and forms a gelatinous clot.

The test sample is compared to a standard series of Control Standard Endotoxin (CSE) dilutions. 

 The endpoint orreaction times of these dilutions are used to calculate the amount of endotoxinpresent in the sample. All tests are performed in at least duplicate. 

A positive control of the sample and negative controlusing non-pyrogenic water are also performed. .

RABBIT FEVER TEST:

The test involvesmeasuring the rise in temperature of rabbits following intravenous injection ofa test solution.

Dose : not to exceed10 ml per kg injected iv with in a period of not more than 10 mins.

Temperature ismeasured by using thermometers or temperature probes, here we measure rectaltemp.of rabbit.

Procedure:

Take 3 healthyrabbits , inject the sample into the ear vein of 3 healthy rabbits of 10 ml perkg of body wt.

 And record the rectal temp. of the rabbit  at 30 min intervals between 1 and 3 hrssubsequent to the inlection.

INTERPRETATION:

 If no rabbit shows an individual rise in temp.of  0.5 0  c. or more and sum of the temp . of therabbits  shows not more than 1.4  0 c. indicates absence of pyrogens in the sample

If any rabbit showsan individual temp. rise of more than 0.5 0  c., continue the test using five otherrabbits, now total 8 rabbits

if not more than 3 of8 rabbits show individual rise in temp. of 0.5 0  c. or more and sum of the 8 individual max temp rises does nt exceed 3.3 0  c.

Indicates absence ofpyrogens and vice versa.