Improved spectrophotometric assay for beta-lactam residues in kidney tissue.
This paper describes a detection system for beta-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, N alpha-N epsilon-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD+ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red-mauve colour that is proportional to CPase activity. The presence of beta-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g-1 compared with 50 ng g-1 by the same assay but using a R-d-ala-d-ala substrate from a commercial kit.
Everest
D J
DJ
Analytical Chemistry Unit, Central Veterinary Laboratory, Addlestone, Surrey, UK.
Jackman
R
R
Thorne
L
L
Everest
S J
SJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D008058:Dihydrolipoamide Dehydrogenase; D004350:Drug Residues / Q000032:analysis*; D007202:Indicators and Reagents; D007668:Kidney / Q000737:chemistry*; D007672:Kidney Cortex / Q000737:chemistry; D007770:L-Lactate Dehydrogenase; D010400:Penicillin G / Q000032:analysis*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D011933:Reagent Kits, Diagnostic; D007770:L-Lactate Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase; D010400:Penicillin G