A Simple Automated Colorimetric Method for Determination of N-Acetyl- β-d-Glucosaminidase

Abstract

SUMMARYAn automated method for the determination of N-acetyl-~-D-glucosaminidasein serum or plasma using p-nitrophenol (pNP) N-acetyl-~-D-glucosamineas substrate and a Pye Unicam AURA system is described. Normal samples had activities of 853 ± 146 (SO) nmol pNP liberated! ml/h, with intra-assay coefficient of variation I' 2 % and inter-assay coefficient of variation I· 6 %.Inhibition of enzyme activity by heparin in plasma samples can be reversed by the addition of calcium chloride to the buffer. liberated/ml plasma/hour. The standard was stable for one week at 4°C in a dark bottle, and the substrate for 48 hours at 4°C in a dark bottle.The effect of heparin upon enzyme activity was investigated by incubating serum samples at 37°C with heparin of different concentrations for times from 10 minutes to 4 hours and then determining enzyme activity using 2·9 mmol/l substrate. In addition, enzyme activities were measured in four blood samples collected into heparinised tubes which had been separated immediately and after 24 hours at 4°C. Material and methodsThe activity of N-acetyl-~-D-glucosaminidase in plasma and serum has been shown to be elevated in diabetics.I" It has been suggested that an increased activity of the enzyme might indicate development of microangiopathic changes," although this has not been confirmed.v" Considerable interest in monitoring the enzyme has encouraged requests for enzyme determinations. We report a simple colorimetric method which lends itself readily to automation and is suitable for performance on a routine basis. In addition, inhibition of enzyme activity by heparin is shown, and this inhibition is reversed by addition of calcium chloride to the reaction mixture.Optimum conditions for the automated assay were determined manually using freshly collected specimens. AUTOMATED METHODThe automated method used a Pye Unicam AURA system, which consisted of an ACI Chemistry Unit and an SP6-300 spectrophotometer coupled to a Hewlett Packard 9815A calculator.The instrument was set at 37"C with a 30-minute delay between dispensing of substrate and glycine/ NaOH. The first rack of sample cups and reaction MANUAL METHOD tubes contained alternate aliquots of water and All determinations were carried out in triplicate standard, 750 fJ.mol/1 p-nitrophenol, with which to at 37°C. To 0·1 ml plasma was added 0·9 ml calibrate the instrument, equivalent to 1500 nmol substrate, o·1-5·0 rnrnol/l p-nitrophenol N-acetyl-liberated/rnl plasma/hour. Subsequent racks con--D-glucosamine (Sigma) in 0·05 M Na citrate tained double cups with sample in both sides, one for buffer, pH 4·5. After 30 minutes incubation the test and one for sample blank. Plasma, 50 fJ.l, was reaction was stopped by the addition of I ·0 mil· 0 M washed into the system with 300 fJ.l of o·05M citrate NaOH containing 0·5 M glycine, and the optical buffer,0·25M CaCI 2 , pH 4·5. Reactionofthetestwas density was measured at 400 nm. Blanks, in which started by addition of 200 fJ.1 8·1 mmol/l substrate in the glycine/NaOH was added before the subs...

Renal Function in Adult Beta-Thalassemia/Hb E Disease

“…Microscopic examination and urine cultures were carried out to exclude cases with infection. Assay for β 2 -microglobulin was carried out by ELISA [13], creatinine by modified alkaline picrate method [14], urinary N-acetyl glucosaminidase by an enzymatic method [15], urinary citrate by citrate lyase method [16], and malondialdehyde (MDA) by the thiobarbituric method [17]. Detection of aminoaciduria was carried out by a chromatographic method [18].…”

Ameliorative Effect of Taurine and Niacin on Paraquat-Induced Early Lung Injury in Rats.

“…The trachea was cannulated and the lung was lavaged with ice-cold sterile physiological saline 5 times at a volume of 3 ml per wash. The BALF from each rat was centrifuged at 300 x g for 10 min to remove cells prior to assay for total protein [27], albumin [28], lactate [29], lactate dehydrogenase [30], angiotensin-converting enzyme [31], N-acetyl-fl-D-glucosaminidase [32], acid phosphatase, and alkaline phosphatase [33] in a cold room at 4°C.…”

Study of Renal enzymuria and albuminuria activity in the Petals of Pink Nelumbo nucifera Gaertn.

“…The activity of N-acetyl-β-D-glusosaminidase (Moore and Morris, 1982), Gama glutamyl transpeptidase (Rosalki and Rau, 1972), alkaline phosphatase (King, 1965b) and Lactate dehydrogenase (King, 1965c) were determined using the abovecited references. Albumin levels in urine were assayed using commercially available kits from Accurex biomedical Pvt.…”