Quantitative fluorometric screening test for fecal porphyrins

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Porphyrins are crucial biomarkers in various physiological and pathological conditions.
• Accurate quantification of porphyrins in biological matrices like stool is essential for diagnosis.
• Existing methods for stool porphyrin analysis can be complex and lack sensitivity.

Purpose of the Study:

• To develop and validate a novel fluorometric method for screening and quantifying porphyrins in stool.
• To establish a sensitive, rapid, and reliable assay for stool porphyrin analysis.
• To compare the new method with existing spectrophotometric techniques.

Main Methods:

• Stool samples were extracted with concentrated hydrochloric acid (HCI) and diluted.
• Fluorometric analysis involved excitation scanning (350–450 nm) and emission monitoring at 603 nm.
• Total porphyrin quantification utilized the isosbestic point (402.5 nm); HPLC compatibility was assessed.

Main Results:

• The fluorometric method demonstrated high sensitivity, detecting as low as 1 nmol/g dry weight of stool.
• Linearity was observed for stool porphyrin concentrations up to 4000 nmol/g dry weight.
• The method showed good correlation with a spectrophotometric assay, offering higher sensitivity and easier sample processing.
• Within-run imprecision was determined to be 3%.

Conclusions:

• The developed fluorometric method provides a sensitive and efficient means for stool porphyrin analysis.
• This assay simplifies sample preparation and enhances detection capabilities compared to previous methods.
• The reference interval for healthy individuals was established at <300 nmol/g dry weight.