Rapid determn. of 1,5-anhydro:glucitol for diabetes diagnosis - by reaction with pyranose oxidase and measuring amt. of hydrogen peroxide formed

摘要

Quantitative determn. of 1,5-anhydroglucitol (I) comprises reacting it with a pyranose oxidase (PO) at pH 7.2-8.5 (pref. 7.5-8). Pref. sugars other than (I) present in the sample are selectively remove by phosphorylation with hexokinase and excess ATP (esp. at pH 7.5-8), leaving (I) unchanged in the sample. Pref. PO is derived from Polyporus obtusus (ATCC 26733). For sugar removal, ATP is present at 10-1000 times (molar basis) the amt. of glucose. Partic. ATP is 5-500 mM, used with 5-100 units/ml.hexokinase and 5-50 mM Mg. Reaction of (I) and PO is quantified by measuring the amt. of H2O2 generated, e.g. H2O2 is reacted with a colour-forming system in presence of peroxidase. Most pref. is 4-aminoantipyrine (APP) and N-ethyl-N-(2-hydroxysulphopropyl) -m-toluidine (II), both at 0.1-10 mM, used with 5-500 units/ml. PO and 2-20 U/ml. horse radish peroxidase. Reaction is at 25-40 deg.C for 2-30 mins. USE/ADVANTAGE - Low levels of (I) in the blood are used to diagnose diabetes. This method is rapid, simple and suitable for use in automated analysers, and (unlike the known chromatographic or enzymatic assays) does not require any physical sepn. steps. At the specified pH values, ATP has practically no inhibitory effect on PO