Improved fluorometric enzymatic sorbitol assay in human blood

Abstract

Samples for use in the fluorometric enzymatic assay of sorbitol in erythrocytes are normally prepared using HClO 4 and K 2CO 3. We have replaced these reagents with NaOH and ZnSO 4. Human whole blood, erythrocyte and plasma samples prepared with NaOH and ZnSO 4 are colorless and clear, while erythrocyte samples prepared with HClO 4 and K 2CO 3 are a pale yellow–brown color. The sorbitol dehydrogenase reaction in the supernatant of the mixture of NaOH and ZnSO 4 is inhibited, but ethylenediaminetetraacetate completely eliminates this effect. The sorbitol assay in erythrocytes prepared with NaOH and ZnSO 4 shows higher sensitivity and reproducibility than did that with HClO 4 and K 2CO 3. Recovery of sorbitol added to erythrocytes is similar in both assay methods. Concentrations of whole blood and erythrocyte sorbitol assayed by the present method are significantly higher in diabetics than in normals. Poorly controlled diabetics had higher whole blood and erythrocyte sorbitol than well-controlled diabetics. Whole blood sorbitol concentrations differed more between diabetic and normal subjects than did erythrocyte sorbitol concentrations.