Coupled enzymatic assay for the determination of sucrose.

E W Holmes

Index: Anal. Biochem. 244(1) , 103-9, (1997)

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Abstract

An enzymatic spectrophotometric assay for the determination of sucrose in unextracted samples of serum and urine was developed. The method entailed 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 transferred to a tetrazolium salt with a concomitant increase in absorbance at 570 nm. The assay, which was carried out in microtiter plates, had a minimum detectable sucrose concentration of 0.03 mmol/liter and run-to-run and within-run coefficients of variation of 7.5 and 6.7%, respectively, and showed a good correlation with urine sucrose determination by 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 the 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 convenient assay provides for the rapid and specific estimation of sucrose and has the potential to be used in a variety of manual, semiautomated, or automated formats.