A micromethod for the determination of fructose in human erythrocytes and plasma

Abstract

Fructose makes up approximately one-sixth to one-third of the total carbohydrate intake and is cleared from the portal blood after absorption more rapidly and completely than glucose. In tissues, fructose can also be formed from glucose by the polyol pathway in which glucose is first reduced to sorbitol by aldose reductase with the mediation of NADPH, and subsequently sorbitol is oxidized to fructose by sorbitol dehydrogenase using NAD as the cofactor. Usually the level of fructose and sorbitol in the body fluid is low. However, in hyperglycemia, increased levels of sorbitol and fructose in several tissues have been observed, possibly due to increased flux of glucose through the polyol pathway [l-3]. The methods for the determination of free fructose in biological samples such as gas chromatography (GC), high performance liquid chromatography (HPLC) and enzymatic [4-61 are of little use with samples containing high concentrations of glucose which is the case for tissues from hyperglycemic subjects. The presence of much higher concentrations of glucose as compared to fructose markedly interferes with the resolution of fructose peak in GC and HPLC analysis of sugars because of the close resolution time for glucose and fructose. In addition, analysis of sugars by GC and HPLC is cumbersome and time consuming as compared to enzymatic methods [4,5]. The most commonly used enzymatic method for fructose determination which utilizes hexokinase, phosphoglucoisomerase and glucose-6-P-dehydrogenase is less accurate when the fructose/glucose ratio in the sample is approximately 0.05 or less [6]. D-Fructose dehydrogenase [7] or fructokinase [8] has also been used to determine fructose in biological samples, but these enzymes are not commercially available. In the present communication we have described a specific and sensitive fluorometric method based upon the quantification of NAD formed as a result of reduction of fructose by NADH in the presence of sorbitol dehydrogenase [9].