How to measure NAG

Until now the laboratory measurement of NAG activities has been complex. The early fluorimetric assays posed standardisation problems whilst the early chromogenic substrates based on p-nitrophenol, due to their yellow colour, had very high blanks and thus a low sensitivity. More recently two new chromogenic substrates have been developed for the urinary NAG assay, namely methylcresol phenolphthalein glucosaminide and omega-nitrostyryl glucosaminide. The former has a high extinction coefficient when the chromophor is released but the substrate itself has a high absorption in the visible region so the colour change is less apparent.

Figure 1 - MNP Glc NAc

Substrate of choice

The latter, namely 2-methoxy-4-(2-nitrovinyl) phenyl- glucosaminide is pale yellow in acid solution and the released chromophoric phenol is red coloured and has a high extinction coefficient at 505nm in alkaline solution. This substrate has proved to be highly sensitive and is the basis of a new NAG test pack researched and developed by scientists at King's College, London.

Figure 2

Effect of pH on the absorption maximum and the molar extinction coefficient of MNP

Absorption of MNP at concentrations (a) 0.1mM in buffer of pH 5.0, (b) 0.05mM in buffer of pH 9.5. The insert shows the increase of molar extinction coefficient, E(M-1cm-1) with increasing pH at 505nm.

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