Studies on the determination of total 5-oxoproline in blood serum

Abstract

This study deals with the development of a solution technique for the quantitative determination of 5-oxoproline and is partly based upon existing qualitative methods. An attempt was made to render this procedure applicable to routine use in the clinical laboratory.

The assay involves a series of steps leading to the ultimate formation and measurement of triiodide ion in aqueous solutions. First, N-chlorination of 5-oxoproline is achieved by the use of sodium hypochlorite. After the excess sodiuim hypochlorite is reduced by ethanol, acetate buffer is added for pH control. Potassium iodide is added and is immediately oxidized to iodine. The iodine complexes with the excess iodide ion to form triiodide ion. The characteristic yellow solution can then be measured spectrophotometrically at 390-393 nm.

In order to obtain workable standard curves, it was necessary to optimize the conditions of the reactions--namely the concentrations of reagents and the reaction times. Experimentation revealed that a 10 minute chlorination reaction followed by a 45 minute period for the reduction of excess sodium hypochlorite produced the best results.

After obtaining a standard curve, an attempt was made to assay for 5-oxoproline in extracts of serum obtained from normal individuals and patients suffering from renal failure. The assay values obtained were higher than the reported literature values for 5-oxoproline and no significant differences was evident between the assay values for the normal individuals and the patients.

The sensitivity of the assay procedure and the reliability of the extraction process were tested by assaying solutions of caffeine, urea, and glutamic acid, their "extracts," and their acid hydrolysates. The acid hydrolyzed serum of one normal individual and five anephric patients was also assayed.

Thin layer chromatography experiments supported the results and conclusions of the solution experiments. The presence of 5-oxoproline in serum was indicated by this procedure. In addition, the N-chloro derivative of 5-oxoproline was also detected.

This work demonstrated that the assay procedure could be used as a method for the determination of 5-oxoproline. However, in order to be of practical use, some other extraction technique must be used for the separation of 5-oxoproline from other interfering compounds in the serum.