COUPLED ENZYMATIC ASSAY FOR THE DETERMINATION OF SUCROSE

An enzymatic spectrophotometric assay for the determination of sucrose in unextracted samples of serum and urine was developed, The method e ntailed the coupling of invertase-catalyzed sucrose hydrolysis with a fructose dehydrogenase-catalyzed oxidation of the liberated fructose. The latter reaction generated reducing equivalents that were transferr ed to a tetrazolium salt with a concomitant increase in absorbance at 570 nm, The assay, which was carried out in microtiter plates, had a m inimum detectable sucrose concentration of 0.03 mmol/liter and run-to- run and within-run coefficients of variation of 7.5 and 6.7%, respecti vely, and showed a good correlation with urine sucrose determination b y GLC (r = 0.92). The assay range of 0.03-2.10 mmol/liter is suitable for the quantitation of serum sucrose following iv administration and for the quantitation of urine sucrose at basal levels and following th e consumption of an oral test dose of sucrose. This method was used to analyze urine samples from a group of human subjects who consumed 20 g of sucrose for the assessment of gastroduodenal permeability. This c onvenient assay provides for the rapid and specific estimation of sucr ose and has the potential to be used in a variety of manual, semiautom ated, or automated formats. (C) 1997 Academic Press, Inc.