Cyclohexanone extraction: An improvement in the thiobarbituric acid method for the determination of nonenzymatic glycosylation of hair and epidermal keratin

Abstract

Colorimetric techniques such as the thiobarbituric acid assay are widely used for the determination of nonenzymatic glycosylation of proteins. One of the major problems associated with this technique is the high nonspecific background absorbance which, due to its variability, results in loss of sensitivity. This report describes a method of removing the nonspecific absorbance by extracting the final chromogen into cyclohexanone. Using this method for the determination of nonenzymatic glycosylation of hair and epidermal keratin, at least 90% of the background absorbance is removed, increasing the sensitivity of the technique. This allows more effective discrimination of the level of glycolsylation of proteins from control and diabetic patients, by reducing the degree of overlap. The requirement for individual borohydride reduced samples is also avoided, thus simplifying the technique. Cyclohexanone extraction provides a simple addition to the standard thiobarbituric acid technique with significant improvement in results.

Key Words:

• hair
• keratin
• nonenzymatic glycosylation
• thiobarbituric acid