New photometric assay for chymotrypsin in stool

Area of Science:

• Biochemistry
• Clinical Chemistry
• Enzymology

Background:

• Accurate measurement of fecal enzyme activity is crucial for diagnosing various gastrointestinal conditions.
• Traditional methods for enzyme determination can be time-consuming and may not fully solubilize particle-bound enzymes.
• Particle-bound enzymes in fecal samples present challenges for accurate and efficient activity assays.

Purpose of the Study:

• To develop and validate a novel photometric assay for determining fecal enzyme activity.
• To improve the solubilization and enhance the activity of particle-bound fecal enzymes.
• To establish a reliable and automatable method for fecal enzyme analysis.

Main Methods:

• Fecal samples undergo pretreatment with detergent and high salt concentrations.
• Kinetic enzyme determination utilizes the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-4-nitroanilide.
• The assay allows enzyme activity measurement in suspension or supernatant after centrifugation.

Main Results:

• Pretreatment ensures near-complete solubilization of particle-bound enzymes.
• The assay demonstrates enhanced enzyme activity and a decreased Km value.
• Results show strong correlation with classical titrimetry methods.

Conclusions:

• The new photometric assay provides efficient and accurate fecal enzyme activity measurement.
• The pretreatment significantly improves enzyme accessibility and assay sensitivity.
• The method's adaptability to automated systems facilitates high-throughput clinical applications.