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Improved fluorometric enzymatic sorbitol assay in human blood
R Shinohara1, Y Ohta, M Yamauchi
1Department of Biochemistry, School of Health Sciences, Fujita Health University, Toyoake, Aichi, Japan.
Summary
This study introduces a new method for measuring sorbitol in red blood cells using sodium hydroxide and zinc sulfate, improving assay sensitivity and reproducibility. The new method reveals higher sorbitol levels in diabetic patients compared to healthy individuals.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Analytical Chemistry
Background:
- Sorbitol measurement in erythrocytes is crucial for understanding diabetic complications.
- Traditional methods using perchloric acid (HClO4) and potassium carbonate (K2CO3) yield colored samples and potential assay interference.
Purpose of the Study:
- To develop and validate a novel, improved method for fluorometric enzymatic assay of sorbitol in erythrocytes.
- To compare the new method with the conventional HClO4/K2CO3 method in terms of sample clarity, assay performance, and clinical utility.
Main Methods:
- Replaced HClO4 and K2CO3 with sodium hydroxide (NaOH) and zinc sulfate (ZnSO4) for sample preparation.
- Evaluated sample clarity, sorbitol dehydrogenase reaction inhibition, and the effect of ethylenediaminetetraacetate (EDTA).
- Assessed assay sensitivity, reproducibility, and sorbitol recovery in human blood, erythrocyte, and plasma samples.
Main Results:
- NaOH/ZnSO4 method produced clear, colorless samples, unlike the pale yellow-brown color from HClO4/K2CO3.
- NaOH/ZnSO4 method showed higher sensitivity and reproducibility for erythrocyte sorbitol assay.
- Ethylenediaminetetraacetate (EDTA) completely reversed the observed inhibition of the sorbitol dehydrogenase reaction.
Conclusions:
- The NaOH/ZnSO4 method offers a superior alternative for erythrocyte sorbitol determination due to improved sample quality and assay performance.
- Elevated whole blood and erythrocyte sorbitol concentrations were observed in diabetic patients, correlating with glycemic control.
- Whole blood sorbitol levels showed a greater difference between diabetic and normal individuals than erythrocyte sorbitol levels.