An Enzymatic Assay for Chloramphenicol with Partially Purified Chloramphenicol Acetyltransferase

Abstract

In an enzymatic assay for chloramphenicol, chloramphenicol acetyltransferase partially purified by affinity chromatography was used; [3H]acetyl coenzyme A served as a substrate. The purified enzyme was sensitive to p-hydroxymercuribenzoate but insensitive to 5,5'-dithio-bis-2-nitrobenzoic acid. The Michaelis-Menten constant was 10.5 muM. The operating range of the enzymatic assay was 0-30 mug/ml. The coefficient of variation was 1.9% at a concentration of 10 mug/ml. The method correlated well with a microbiological agar-paper disk method for mock unknown sera (r = 0.997) and serum or urine specimens (r = 0.993). The enzymatic assay was unaffected by the presence of any of 12 other antibiotics in tested serum.

Comparison of methods available for assay of chloramphenicol in clinical specimens.

“…'9 High bilirubin values and lipaemic sera are known to interfere with the reaction. 33 Our investigation suggests that, of nine methods tested, only the diffusion assays using S. lutea/yeast extract agar and B. subtilis are suitable for routine use, although chloramphenicol cannot be measured at less than 6 mg/l with the latter method. The assay using S. lutea/yeast extract agar is sensitive, easy to perform, and reproducible and uses acceptable volumes of sample.…”

“…The toxic side effects are enumerated by Wade and Reynolds.4 While irreversible and sometimes fatal aplastic anaemia (incidence 1: 20 000-1:80 000) appears to be idiosyncratic and is, perhaps, genetically determined, depression of the bone marrow and the gray syndrome in premature and other newborn infants are dose related.5 6 Reversible depression of the marrow occurs usually when plasma chloramphenicol concentrations reach [25][26][27][28][29][30][31][32][33][34][35] mg/I, and the gray syndrome has been reported most frequently in infants given more than 50 mg/kg body weight daily. Reduced doses are advised for those with impaired renal or liver function, but there is little uniformity of view on appropriate dosage regimens for premature and other neonates.…”

High-Performance Liquid Chromatographic Assay of Chloramphenicol in Serum

“…This method appears to be specific for CAP in the presence of many other antibiotics, CAP esters, and glucuronide metabolite. The procedure is time consuming (2 to 4 h), requires a large sample volume (1.0 ml of serum), and has a sensitivity limit of approximately 5 ,tg/ml. Gas-liquid chromatographic methods have been reported (15,18).…”

“…The destructive nature of the method of detection prevents further study of separated compounds. Finally, enzymatic assays for CAP have recently been presented (5,11). Aside from the technical problems of producing the acetyltransferase enzyme and the requirement for a liquid scintillation counter, these assays appear to offer excellent precision, accuracy, sensitivity, and specificity.…”

High-Pressure Liquid Chromatographic Assay of Chloramphenicol in Biological Fluids

“…The colorimetric method requires large sample volumes and several extractions to distinguish between the biologically active and inactive forms of chloramphenicol (3). The radioenzymatic assay (2,8,10) offers improved sensitivity, precision, and accuracy; however, more technician time and skill are required. The gas-liquid chromatographic method (7) uses 0.5 ml of serum, requires chemical derivatization, and also requires more technician time and skill.…”