Nicot G, Lachâtre G, Gonnet C, Dupuy J L, Valette J P
Département de Pharmacologie Clinique, Centre Hospitalier Régional Universitaire, Hôpital Dupuytren, Limoges, France.
Clin Chem. 1987 Oct;33(10):1796-800.
We describe a new method for separating and measuring urinary N-acetyl-beta-D-glucosaminidase isoenzymes by "high-performance" liquid chromatography. Isoenzymes are eluted from the anion-exchange column with a one-step linear gradient of NaCl solution. For continuous post-column quantification of the activities of these isoenzymes, we use an on-line post-column reactor and 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide as substrate; the methylumbelliferone formed in this reaction is quantified fluorimetrically. We discuss the effects of varying different components of the assay: NaCl concentration, the pH of the mobile phase and of the reaction reagent, substrate concentration, incubation temperature, and the geometry of the post-column reactor.
