View article

In 1955, Roe [1] described a modification of a method to measure dextran in blood and urine [2] for estimation of sugar in blood and spinal fluid. In these methods, total carbohydrates are measured using the anthronesulfuric acid reaction. Here, heat and a strong acidic environment produce both hydrolysis of glycosidic bonds and dehydration of monomers to produce furfuraldehyde derivatives. These compounds react with anthrone and produce colored products. However, routine measurement of a large number of samples requires the use of a significant amount of concentrated sulfuric acid and needs strict safety precautions as well as many test tubes. Previous attempts of automation have been hampered by the exothermic nature of this reaction (G. Palacios, personal communication). The use of microtitration plate technology offers a way to overcome these problems and reduce cost, time, and hazard. Therefore, our aim was to adapt the well-known anthrone-sulfuric acid assay for reliable quantification of glucose-based carbohydrates using 96-well microtitration plates.

This assay requires a total of 12mL of anthronesulfuric acid reagent (2g/L anthrone solution in concentrated sulfuric acid) and 0.5 mL standard solution (0.4 g/L glucose solution in water) per plate. The anthrone-sulfuric acid reagent must be prepared just before use. Aliquots of 24mg of anthrone were made in bulk into 30-mL universal plastic containers with leakproof caps and kept protected from light until used. Just before addition of the reagent to the plate, 12mL of concentrated sulfuric acid, stored at 4 C, was added to