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. 1986 Jan;23(1):11–16. doi: 10.1128/jcm.23.1.11-16.1986

Determination of endotoxin in injectable antibiotic preparations by the chromogenic assay method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate.

S Yano, Y Hotta, S Takahashi
PMCID: PMC268564  PMID: 3700595

Abstract

The effects of 50 antibiotics on the detection and determination of bacterial endotoxins by the chromogenic method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate of p-nitroaniline derivatives were tested, and the antibiotic concentration for 50% inhibition of the chromogenic reaction in the presence of 0.5 ng of endotoxin (Escherichia coli 0111:B4) per ml was estimated. All the antibiotic preparations were depyrogenized by ultrafiltration treatment before they were subjected to the test. The reaction was conducted in the presence of a high concentration (0.5 M) of Tris buffer to constantly maintain the pH of the reaction mixture, and liberated p-nitroaniline was determined by high-pressure liquid chromatography. Several aminoglycosides (amikacin, bekanamycin, kanamycin, and streptomycin sulfate), bleomycin hydrochloride, and fosfomycin disodium showed no inhibition of the reaction up to 20 mg/ml. However, other antibiotics, including penicillins, cephalosporins, macrolides, and tetracyclines, inhibited the reaction concentration dependently. Polymyxin B sulfate was the most potent inhibitor, with less than 8 micrograms/ml for 50% inhibition. It was concluded that the chromogenic method can be applied to the detection and determination of endotoxin in most of the antibiotic preparations. An application of this method to carbenicillin disodium preparations was exemplified.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Case M. J., Ryther S. S., Novitsky T. J. Detection of endotoxin in antibiotic solutions with Limulus amoebocyte lysate. Antimicrob Agents Chemother. 1983 May;23(5):649–652. doi: 10.1128/aac.23.5.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cooperstock M. S. Inactivation of endotoxin by polymyxin B. Antimicrob Agents Chemother. 1974 Oct;6(4):422–425. doi: 10.1128/aac.6.4.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fujita Y., Nakahara C. Preparation and application of a new endotoxin determination kit, PYRODICK, using a chromogenic substrate. Prog Clin Biol Res. 1982;93:173–182. [PubMed] [Google Scholar]
  4. Greisman S. E., Hornick R. B. Comparative pyrogenic reactivity of rabbit and man to bacterial endotoxin. Proc Soc Exp Biol Med. 1969 Sep;131(4):1154–1158. doi: 10.3181/00379727-131-34059. [DOI] [PubMed] [Google Scholar]
  5. Gutteridge J. M., Shute D. J., Lightbown J. W. Chromatographic separation of endotoxin and bleomycin for pyrogenicity testing. J Biol Stand. 1980;8(1):15–21. doi: 10.1016/s0092-1157(80)80043-6. [DOI] [PubMed] [Google Scholar]
  6. Harada-Suzuki T., Morita T., Iwanaga S., Nakamura S., Niwa M. Further studies on the chromogenic substrate assay method for bacterial endotoxins using horseshoe crab (Tachypleus tridentatus) hemocyte lysate. J Biochem. 1982 Sep;92(3):793–800. doi: 10.1093/oxfordjournals.jbchem.a133991. [DOI] [PubMed] [Google Scholar]
  7. Kakinuma A., Asano T., Torii H., Sugino Y. Gelation of Limulus amoebocyte lysate by an antitumor (1 leads to 3)-beta-D-glucan. Biochem Biophys Res Commun. 1981 Jul 30;101(2):434–439. doi: 10.1016/0006-291x(81)91278-x. [DOI] [PubMed] [Google Scholar]
  8. Kobayashi M., Yamamoto M. [Studies on the gelation reaction of Limulus lysate (pre-gel). IV. Effect of alkaline earth metals and chelating agents on the gelation reaction of Limulus lysate (author's transl)]. Yakugaku Zasshi. 1975 Aug;95(8):959–965. doi: 10.1248/yakushi1947.95.8_959. [DOI] [PubMed] [Google Scholar]
  9. Lüderitz O. Recent results on the biochemistry of the cell wall lipopolysaccharides of Salmonella bacteria. Angew Chem Int Ed Engl. 1970 Sep;9(9):649–663. doi: 10.1002/anie.197006491. [DOI] [PubMed] [Google Scholar]
  10. McCullough K. Z., Scolnick S. A. Effect of semisynthetic penicillins on the Limulus lysate test. Antimicrob Agents Chemother. 1976 May;9(5):856–858. doi: 10.1128/aac.9.5.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Morrison D. C., Jacobs D. M. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry. 1976 Oct;13(10):813–818. doi: 10.1016/0019-2791(76)90181-6. [DOI] [PubMed] [Google Scholar]
  12. Nakamura S., Morita T., Harada-Suzuki T., Iwanaga S., Takahashi K., Niwa M. A clotting enzyme associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus): its purification and characterization. J Biochem. 1982 Sep;92(3):781–792. doi: 10.1093/oxfordjournals.jbchem.a133990. [DOI] [PubMed] [Google Scholar]
  13. Nakamura S., Takagi T., Iwanaga S., Niwa M., Takahashi K. A clottable protein (coagulogen) of horseshoe crab hemocytes. Structural change of its polypeptide chain during gel formation. J Biochem. 1976 Sep;80(3):649–652. doi: 10.1093/oxfordjournals.jbchem.a131323. [DOI] [PubMed] [Google Scholar]
  14. Newsome P. M. Penicillins and the limulus amoebocyte lysate test for endotoxin. J Pharm Pharmacol. 1977 Nov;29(11):704–706. doi: 10.1111/j.2042-7158.1977.tb11442.x. [DOI] [PubMed] [Google Scholar]
  15. Schleef R. R., Kenney D. M., Shepro D. Effect of sodium chloride on limulus amebocyte lysate. Inhibition of endotoxin activation of procoagulase. Thromb Haemost. 1979 Apr 23;41(2):329–336. [PubMed] [Google Scholar]
  16. Sweadner K. J., Forte M., Nelsen L. L. Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation. Appl Environ Microbiol. 1977 Oct;34(4):382–385. doi: 10.1128/aem.34.4.382-385.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takahashi S., Yano S., Nagaoka Y., Kawamura K., Minami S. A highly sensitive pyrogen test for antibiotics I: detection of trace amounts of endotoxin in injectable sodium ampicillin preparations. J Pharm Sci. 1983 Jul;72(7):739–742. doi: 10.1002/jps.2600720706. [DOI] [PubMed] [Google Scholar]
  18. Tarmina D. F., Milner K. C., Ribi E., Rudbach J. A. Modification of selected host-reactive properties of endotoxin by treatment with sodium deoxycholate. J Bacteriol. 1968 Nov;96(5):1611–1616. doi: 10.1128/jb.96.5.1611-1616.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tsuji K., Steindler K. A. Use of magnesium to increase sensitivity of Limulus amoebocyte lysate for detection of endotoxin. Appl Environ Microbiol. 1983 Apr;45(4):1342–1350. doi: 10.1128/aem.45.4.1342-1350.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. WEINBERG E. D. The mutual effects of antimicrobial compounds and metallic cations. Bacteriol Rev. 1957 Mar;21(1):46–68. doi: 10.1128/br.21.1.46-68.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]

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