A new continuous automated assay for the determination of intestinal lactase.
BACKGROUND
A reliable, sensitive and practicable method for the measurement of intestinal lactase activity is needed.


METHODS
The assay is based on the continuous measurement of released glucose by a coupled hexokinase/glucose-6-phosphate dehydrogenase (HK/G6PDH) reagent.


RESULTS
The procedure was first optimized for lactase from rat intestinal mucosa. The optimum pH is 6.5 and apparent Km was 17 mmol/l for lactose. The procedure was also adapted on a Cobas Mira automated analyzer; progress curves of the reaction rate can be continuously monitored. The automated assay correlated strongly with the conventional method of Dahlqvist (r(2) = 0.996). The described method has also been applied to human intestinal mucosa biopsies after determination of the catalytic properties of human enzyme (optimum pH 6.0 and apparent Km 34 mmol/l).


CONCLUSIONS
The HK/G6PDH method is reliable, rapid, sensitive and easy to perform both manually as well as in the automated version. It is optimized for human and rat intestinal lactase.
Laboratoire de Biochimie Appliquée (Faculté de Pharmacie), Université Louis-Pasteur de Strasbourg, Illkirch, France. isall@evc.net
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D000328:Adult; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019411:Clinical Laboratory Techniques / Q000295:instrumentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology*; D043322:Lactase; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods; D013393:Sucrase / Q000032:analysis / Q000378:metabolism; D000520:alpha-Glucosidases / Q000032:analysis / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D043322:Lactase; D000520:alpha-Glucosidases; D001616:beta-Galactosidase; D013393:Sucrase