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ApothéCure - Featured

Pyrogens & Sterility

What are Endotoxins or Pyrogens?

Endotoxins are the integral part of gram-negative bacteria.  The outer cell wall of gram-negative bacteria consists of a thick layer of peptidoglycan and it is surrounded by a layer of outer membrane.  Endotoxins also known as Lipopolysacharides (PLS) are a structural component of the gram-negative bacterial outer membrane. The LPS composed of three regions.  Region 1 consists of O- specific polysaccharide, known as Somatic O- antigen.  It is responsible for serological activity.

Region 11- is the core polysaccharide of the gram negative cell wall. Region 111- is known as Lipid A and is responsible for toxicity and pyrogenic activity.  The biologic and toxic activities of endotoxin are broad.  Nanogram quantity of endotoxin cause fever in humans and to the release of endogenous pyrogen.

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"Glutathione-L" Certificate of Analysis - DYNA Labs

"Procaine HCL" Certificate of Analysis - DYNA Labs

"EDTA Disodium" Certificate of Analysis - DYNA Labs

The major properties of endotoxins:

• It is a heat stable glycolipid also known as Lipid A.
• Its other properties include pyrogenicity, induce shock in hosts, lethality, tissue necrosis activity, complement activation, B cell mitogenicity in mice, immunoadjuvant activity and anti tumor activity.

Detection of Endotoxin activity:

The presence and quantitation of endotoxin can be determined by several ways.  The test procedures include clot formation as well as chromogenic tests (Cape Code Inc.,) using Limulus Amebocyte Lysate (LAL) from the horse shoe crab Limulus polyphemus.  LAL is an aqueous extract of blood cells (amebocytes) from Limulus polyphemus.  The endotoxin test results are expressed in endotoxin units (EU) per ml and the test systems will detect as low as 0.005 EU/ml.

The biological principle of the Chromogenic test:

LAL contains enzymes that are activated in a series of reactions in the presence of endotoxin.  All steps of the reactions are not fully understood.  The last enzyme activated in the cascade, react with a chromogenic substrate producing an end product with an yellow color and its intensity is measured photo metrically.  The end product released (color intensity) is proportional to the amount of the endotoxin present in the test system.

Important considerations in endotoxin testing:

Use aseptic techniques at all times during the test procedures

All materials used in the test procedures must be free of detectable endotoxin including the test reagents except the known endotoxin positive control.

Glassware and other heat stable materials used in the test procedures must be free of any detectable endotoxin.  This can be achieved by exposure to dry heat for three hours at 180 C..

Sample for endotoxin should be collected aseptically in non-pyrogenic containers and ship frozen to the laboratory for testing.  Always use depyrogenated glassware or disposable polystyrene plastic tubes are recommended to minimize adsorption of endotoxin to container surfaces.  Both in Clot formation and Chromogenic testing for endotoin presence in clinical samples; it is critical to include appropriate positive and negative controls.

References:

• Guideline on validation of the Limulus Amebocyte Lysate test as an End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices. U S Department of Health and Human Services, Public Health Service, Food and Drug Administration, December 1987
• Bacterial Endotoxin Tests, USP 26 NF21
• Bang, F.B.1953. The toxic effect of a marine bacterium on Lumulus and the formation of blood clots. Biol.Bull.(Wood Hole, MA) 105:361-362
• Levin,J., and F.B.Bang 1964. The role of endotoxin in the extra cellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. 115: 265-274
• Progress in clinical and biological research vol.231. Detection of Bacterial Endotoxins with Limulus Amebocyte Lysate Test. 1987.  Watson, S.W., J. Levin and T.J. Novitsky (Eds), Alan R. Liss, Inc., NY.
• Tsuji, K and S.J. Harrison 1978. Dry-heat destruction of lipopolysaccharide: Dry- heat destruction kinetics, Appl. Env. Microbiol. 36: 710-714
• Sweet, B.H. and J.F. Huxsoll. Depyrogenation by dry heat, Ch.12, p.101-108. in: Depyrogenation, Technical Report No.7, 1985. Parenteral Drug Association, Inc., Philadelphia, PA.

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Lobo Tejas Laboratories
Lobo Tejas Laboratories, owned by Gary Osborn, RPh, CCN, is operated by his medical staff at the Texas Institute of Functional Medicines.
 
Gary believes in providing meaningful and cost effective lab testing to his patients at the Texas Institute of Functional Medicines, his customers at ApothéCure and his fellow sterile compounders. He has contact with many top-notch labs and suppliers and is able to introduce a one-stop shopping experience to the thousands of physicians and pharmacists he deals with nationally and internationally.
 
Lobo Tejas Laboratories was started in response to the federal government's action against a pharmacist in California. He was accused of compounding a non-sterile injection with the resulting death of patients. He had no way of proving the sterility of his compounds in this case, as he only did in-house incubator controls. The rest is history in California!