Endotoxins measurement

Mielniczuk, Z., Mielniczuk, E., and Larsson, L. (1993), Gas chromatography-mass spectrometry methods for analysis of 2- and 3-hydroxylated fatty acids Application for endotoxin measurement, I. Microbiol. Meth., 17, 91-102. 

The observation of gel formation in a test tube as an endpoint for an endotoxin assay provides the means for a very simple test. The need for more objective quantitative methods has led scientists to develop a variety of automated methods for endotoxin measurement. This discussion is limited to the three methods that were accepted by the Food and Drug Administration (FDA). 

The LAL assay is at present the most widely accepted assay for endotoxin measurements, having been adopted as the standard assay for endotoxin detection by the United States Food and Dmg Administration (FDA) in 1980. The more recent kinetic chromogenic versions of the LAL assay are very sensitive and have a broad measurement range (0.01 -100 Endotoxin Units (EU)/ml w 1 pg/ml-10 ng/ml). The detection limit for airborne endotoxin measurements is approximately 0.05 EU/m (5 pg/m ). Since different test batches may give different results an internal standard must be used. The U.S. FDA... 

Milton D.K., Feldman H.A., Neuberg D.S., Bruckner R.J. and Greaves I.A.(I992) Environmental endotoxin measurement the kinetic limulus assay with resistant-parallel-line estimation. Envir. Res., 57, 212-230. 

Milton D.K., Johnson D.K., Park J.-H. (1997) Environmental endotoxin measurement interference and sources of variation in the Limulus assay of house dust. Am. Ind. Hyg. Assoc. J. 5B. 861-867. 

Pyrogenic A fever-producing substance. The presence of these substances is determined by the Limulus Amebocyte Lysate (LAL) test and measured in EU/ml (endotoxin units per milliliter). 

R. Hernanz, M.J. Alonso, H. Zibrandtsen, Y. Alvarez, M. Salaices, and U. Simonsen, Measurements of nitric oxide concentration and hyporeactivity in rat superior mesenteric artery exposed to endotoxin. Cardiovasc. Res. 62, 202-211 (2004). 

Cliff RO, Kwasiborski V, Rudolph A8. A comparative study of the accurate measurement of endotoxin in liposome-encapsulated hemoglobin. Artif Cells Blood 8ubstit Immobil Biotechnol 1995 23 331. 

Roth RI, Levin J. Measurement of endotoxin levels in hemoglobin preparations. Meth Enzymol 1994 231 75. 

Endotoxin. Endotoxin is the lipopolysaccharide that comprises a major portion of the cell wall of the gram-negative bacteria. The endotoxins from each species of bacteria are different but the lipid A moiety is similar for the Enterobacterlaceae, and has a similar series of biological actions regardless of its source. The lipid A material is different in some of the gram-negative rods present in cotton (i.e. in the Pseudomonas species). This variety of compounds makes quantitation of endotoxin difficult. Hence, it is usually measured by its biological activity as compared to a standard endotoxin (usually that of E. coll). (See Table VIII.)... 

Cinkotal et al. (11) measured the endotoxin content In the cardroom of seven cotton mills, and two cotton waste mills. None was found in a tea packing factory or a pipe tobacco factory and the level was low in the willowing mills. Bysslnotic symptoms correlated with the level. 

On completion of intermediate purification, most contaminants are removed. For example, endotoxin concentrations are reduced by more than two orders of magnitude, while the specific activity of recombinant protein, a measure of purity, is increased several-fold. The purified material emerging from this stage of downstream processing is composed of recombinant... 

The acceptance criteria may also specify limits on microbial counts, endotoxin, particulate matter, and other parameters appropriate or significant for the particular product. The author remembers one protocol that specified the absence of a characteristic odor that was indicative of a certain residue and was a sensitive yet simple measure of whether or not the equipment was adequately cleaned. 

Melgert et al. studied the delivery of the corticosteroid dexamethasone to fibrotic livers [240], Dexamethasone has more potent and broader anti-inflammatory effects compared with naproxen. It inhibits the release of inflammatory mediators like TNF-a, IFN-y, and IL-6 and acts as an NFkB inhibitor [241, 242], Dexamethasone coupled to albumin (Dexa-HSA) was specifically taken up by sinusoidal cells in fibrotic rat livers, whereas dexamethasone itself was mainly taken up by hepatocytes. In vivo, Dexa-HSA promoted survival in endotoxin-induced liver inflammation in rats [240], In vitro, anti-inflammatory effects of the conjugate were measured in endotoxin-challenged liver slices. Dexa-HSA inhibited the release of nitric oxide and TNF-a in a dose-dependent manner (Melgert et al. unpublished data). To further enhance the delivery to KC at present dexamethasone is coupled to manHSA, and this conjugate is studied with respect to the pharmacokinetic profile and pharmacotherapeutic effects in fibrotic rats. 

As a measure of the functional selectivity of Cox inhibitors in an in vitro or ex vivo setting, human whole blood assays were used. The PGE2 produced in human whole blood challenged with bacterial endotoxin arises from monocyte-derived Cox-2, and thromboxane B2 produced in clotting blood arises from platelet Cox-1 (Patrignani et al., 1994 Brideau et al., 1996). These assays are widely used and are the generally accepted standards for selectivity of Cox inhibitors relevant to an in vivo setting... 

Acknowledgement The authors are indebted to the German ministry BMBF (project 01GU0824) and the DFG (project SCHR621/2-3), for financial support, and to Dr. Walter Richter (Universitatsklinikum Jena) for performing the freeze-fracture electron microscopy measure-ments.The authors would like to thank Prof. Ulrich Seydel, former head of the Division of Biophysics, for continous support and development of new concepts in the biophysics of endotoxins. 

Based on rFC, a modern and yet simple, rapid, specific and sensitive diagnostic test for endotoxin has been developed (Ding and Ho, 2001). rFC is a proenzyme until it encounters trace levels of endotoxin where it unequivocally exhibits full enzymatic activity, hence, acting as a very sensitive and specific biosensor for endotoxin. The resulting activated rFC acts as a catalyst to hydrolyse a synthetic substrate to form a quantifiable product, which measures the level of endotoxin. A fhiorimetric assay... 

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