Rapid microplate method for PAH estimation

Area of Science:

• Nephrology
• Biochemistry
• Analytical Chemistry

Background:

• Effective renal plasma flow (ERPF) is crucial for evaluating renal hemodynamics.
• Traditional methods for measuring p-aminohippuric acid (PAH) clearance are often complex, costly, and time-consuming.
• There is a need for a more efficient and accessible assay for PAH determination.

Purpose of the Study:

• To develop and validate a rapid, precise, and accurate microplate-based assay for p-aminohippuric acid (PAH).
• To compare the performance of the new microplate assay with a standard High-Performance Liquid Chromatography (HPLC) method for PAH measurement.
• To assess the reliability and cost-effectiveness of the microplate assay for clinical and research applications.

Main Methods:

• A novel microplate assay utilizing p-dimethylaminocinnamaldehyde to detect PAH via a colorimetric reaction was developed.
• The microplate assay was validated against a reference High-Performance Liquid Chromatography (HPLC) method.
• Renal PAH clearances were measured in 10 healthy volunteers using both the microplate and HPLC methods.

Main Results:

• The microplate assay demonstrated excellent agreement with the reference HPLC method, with an average ratio close to 1.0.
• Limits of agreement were within acceptable ranges for plasma (17.2%), urine (19.3%), and clearances (25.5%).
• Both intraday (<7%) and interday (<6%) coefficients of variation were low for urine and plasma PAH measurements, indicating high precision.

Conclusions:

• The microplate-based PAH assay is a reliable, rapid, and accurate alternative to existing HPLC methods.
• This assay significantly reduces costs, time, and reagent requirements for measuring effective renal plasma flow.
• The developed method offers a practical solution for routine renal hemodynamic assessment in clinical and research settings.