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DEFINITIONS PYROGEN : Any substance or agent causes a rise in body temperature or induces a feveris consider as pyrogenic. fever reduces the endotoxin levels. Water producing this reaction is said to be a pyrogenic and water free from this effect is apyrogenic. Pyrogen as produced by gram –ve bacteria and may from a part of endotoxin(o somatic antigen). Body reduce the endotoxin level by inducing fever

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Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. The relationship of endotoxin (lipopolysaccharide) to the bacterial cell surface is illustrated in the Figure1

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Figure 1. Structure of the cell envelope of a Gram-negative bacterium (e.g. E. coli ). periplasm - The space between the inner (plasma) and outer membranes. The peptidoglycan sheet is actually in the pariplasm outer membrane The inner face of the outer membrane is composed of phospholipid , the same as in the plasma membrane. The outer face of the outer membrane contains primarily composed of lipopolysaccharide which has the amphipathic qualities of a phospholipid . Lipopolysaccharide is endotoxin .

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The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity ( pyrogenicity )is associated with the lipid component ( Lipid A ) immunogenicity is associated with the polysaccharide components. The cell wall antigens ( O antigens ) of Gram-negative bacteria are components of LPS (lipid A). which activates complement by the alternative ( properdin ) pathway, results elicits a variety of inflammatory responses (fever) in an animal . In vivo , Gram-negative bacteria probably release minute amounts of endotoxin while growing. This may be important in the stimulation of natural immunity

Difference between endotoxin and classical exotoxin:

Difference between endotoxin and classical exotoxin PROPERTY ENDOTOXIN EXOTOXIN CHEMICAL NATURE Lipopolysaccharide (mw = 10kDa) Protein (mw = 50-1000kDa) RELATIONSHIP TO CELL part of outer membrane Extracellular, diffusible DENATURED BY BOILING No Usually ANTIGENIC Yes Yes FORM TOXOID No Yes POTENCY Relatively low (>100ug) Relatively high (1 ug) SPECIFICITY Low degree High degree ENZYMATIC ACTIVITY No Often PYROGENICITY Yes Occasionally

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CHEMISTRY OF LYPOPOLYSACCHARIDE Lipopolysaccharides are complex amphiphilic molecules with a mw of about 10kDa, It varies widely in chemical composition for both between and among bacterial species. LIPID A Structure

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LPS consists of three components or regions: Lipid A, R polysaccharide O polysaccharide . Region I. Lipid A (lipid component of LPS ) It contains the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached. Usually 6 saturated FA Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present..

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Region II. Core (R) antigen or R polysaccharide – it is attached to the 6 position of one NAG. The R antigen consists of a short chain of sugars. For example: KDO - Hep - Hep - Glu - Gal - Glu - GluNAc – it is structurally distinct in other genera of Gram-negative bacteria. 2-keto-3-deoxyoctonoic acid KDO is unique and invariably present in LPS and so it has been used as an indicator in assays for LPS (endotoxin). With minor variations, the core polysaccharide is common to all members of a bacterial genus

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Region III. Somatic (O) antigen or O polysaccharide It is attached to the core polysaccharide. The O polysaccharide is much longer than the core polysaccharide, IT is a major antigenic determinant (antibody-combining site) of the Gram-negative cell wall Virulence , and the property of " smoothness ",  is associated with an intact O polysaccharide

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LPS and virulence of Gram-negative Bacteria : Lipid A (the toxic component of LPS) polysaccharide side chains (the nontoxic but immunogenic portion of LPS) act as determinants of virulence in Gram- ve bacteria. The O polysaccharide and virulence : The involvement of the  polysaccharide chain in virulence is shown by the fact that small changes in the sugar sequences in the side chains of LPS result in major changes in virulence.

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1. O-specific antigens allows organisms to adhere specifically to certain tissues, especially epithelial tissues. 2. Smoothness of antigens makes it resistance to phagocytes , since rough mutants are more readily engulfed and destroyed by phagocytes. 3. The hydrophilic O polysaccharides could act as water- solubilizing carriers for toxic Lipid A . It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface. 4. in such a way O antigens could provide protection from damaging reactions with antibody and complement .

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The mechanism of endotoxin In humans, LPS binds to a lipid binding protein (LBP) in the serum. Which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2 , which associates with Toll-like receptor-4 (TLR4) . This triggers macrophage/endothelial cells to secrete pro-inflammatory midiator

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three types of events are triggered due to TLR4 receptor activation Production of cytokines: including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. These, in turn, stimulate production of prostaglandins and leukotrienes. These are powerful mediators of inflammation Activation of complement sytem -C 3a , C 5a pathway which release histamine. 3 . Activation of coagulation cascade : Initial activation of Hageman factor (blood-clotting Factor XII)  can activate several humoral systems resulting in

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A) coagulation B) activation of the complement alternative pathway . C) plasmin activation which leads to fibrinolysis and hemorrhaging. D) kinin activation releases bradykinins and other vasoactive peptides which causes hypotension. The net effect is to induce inflammation, intravascular coagulation, hemorrhage and shock.

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Production: cultivation of bacteria harvesting isolation & purification of L.p.s Identification chemical analysis biological activity

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Eg: 1.preparation of national reference endotoxins 2. osomatic antigen of shigella dysenterae.

Pyrogen Testing:

Pyrogen Testing The presence of pyrogenic substance in perenteral preparation is determined by qualitative biological test based on the fever response of rabbit is the basic of Pyrogen test. Rabbit used because they show physiological response to pyrogen similar to human being. The specification limits and procedurals details are given in official pharmacopeias.

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Test Details - Selection of animal - Rabbit - use rabbit of either of sex preferably of same weight not less than 1.5 kg. Diet -Feed complete balance diet so that there is no loss of weight during the week proceeding the test. Use specification - at least 2 week must be allowed to elapse before the animal is used again. Withhold food for over night before injection and withhold water through out the test.

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2. Materials - Thermometer -(clinical thermometer or thermostat )- Should indicate the tempreature with precision of + 1 ̊ c and maximum reading reached within 4 min. Insert the thermometer to the ractum of test rabbits to a depth of about 5 cm/as per monograph. Electrical device - If it is used it should be inserted to rabbit 90 min before the injection and left in position through out the test.

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Retaining boxes - it is the box for rabbit in which temperature is measured . It should be made in such a way that animal can retain by loosely fitting neek stocks and rest of the body remains free. Temperature should be + 3 ̊ c of of rabbit leaving quarter where rabbit kept for last 18 h. Condition should be maintained 1 hour before test. Other equipment - The glassware ,syringe, needle is washed with water for injection. And heated in hot air oven at 250̊ c for 30 min or 200 ̊ c for 1 hour.

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3. priIliminary test (sham test)- 1 to 3 days before testing the substance inject intravenously into the test rabbits of10 ml/kg body wt of Pyrogen free isotonic warmed to about 38.5̊ c normal saline/ NaCl solution. Record the temperature of animal at least 90 min before the injection of test sample continue for 3 hours after injection Any animal showing temprature of 0.6 ̊ c or more is not used fot main test

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4 . Main test- carry the test using a group of three rabbits. Preparation of sample :- sample is diluted by Pyrogen free saline solution/ NaCl solution. Warm the liquid up to 38.5 ̊ c before injection. Inject the sample :-Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period of maximum 4 min. The volume of injection a) Should not more than 10 ml/kg b) should not less than 0.5ml/kg

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Recording of temperature:- Record the temperature at an interval of 30 min. Begins at least 90 min before the injection and last up to 3 hours after the injection of test sample. Initial temperature – it is the mean of two temperature recorded for each rabbit at an interval of 30 min in first 40 min period. Maximum temperature- it is the highest temperature recorded for each rabbit in the whole testing period. Interference :- If difference between initial and maximum temperature is negative then it is consider as zero response. Rabbit showing temperature variation more than 2 ̊ c in two successive reading is not used Any Rabbit of a group used as test animal whose initial temperature don’t vary more than 1 ̊ c than other rabbit of same group

5. Interpretation of results:- The interpretation is specified in different pharmacopeias shown in following table. :

5 . Interpretation of results :- The interpretation is specified in different pharmacopeias shown in following table .

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Step I.P B.P Selection of animal Rabbit Rabbit Use Specification Not specified Don’t Use rabbit during the preceding 3 days Unless the material being examined passed the test Materials Same same Preliminary test Same Same Solution used for dilution Pyrogen free saline solution Pyrogen free NaCl solution Main test procedure same Same Temperature recording Same Same Quantity specified 0.5ml/kg – 10ml /kg 0.5ml/kg – 10ml /kg Injection site Same Same Interpretation No of rabbits Material passed if summed response does not exceed No of rabbits Material passed if summed response does not exceed Material passed if summed response exceed 1 0.6 0.6 1 0.6̊ c 3 1.15 2.65 3 1.4̊ c 6 2.80 4.30 9 4.45 5.95 8 3.7̊ c 12 6.60 06.6

Limulus amebocytes lysate test:

Limulus amebocytes lysate test Recently in vitro test has been developed for pyrogen test utilizing the gelling property of the lysate of amebocytes horse hoe crab. In the presence of pyrogenic Endotoxins from gram –ve bacteria, a firm gel is formed within 60 min when incubated at 37 ̊ c. The LAL test has been found to be 5 to 10 times more sensitive than rabbit test.

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