Automated enzymatic assay for plasma ammonia

Area of Science:

• Clinical Chemistry
• Enzymology
• Analytical Biochemistry

Background:

• Accurate plasma ammonia determination is crucial for diagnosing hepatic encephalopathy and other metabolic disorders.
• Existing methods may suffer from interferences or require lengthy pre-incubation steps.
• The Du Pont Automatic Clinical Analyzer (ACA) offers a platform for automated clinical assays.

Purpose of the Study:

• To develop and validate a sensitive enzymatic method for plasma ammonia measurement using the ACA.
• To optimize reaction conditions for enhanced assay performance.
• To assess potential interferences in the developed assay.

Main Methods:

• An enzymatic assay utilizing glutamate dehydrogenase and monitoring NADPH depletion at 340 nm.
• Kinetic measurement performed with a sample volume of 500 muL on the ACA.
• Optimization of reaction conditions using one-factor-at-a-time and response surface methodology.
• Evaluation of interferences from anticoagulants and common endogenous blood components.

Main Results:

• The assay demonstrated linearity up to 1000 mumol/L of plasma ammonia.
• No significant interferences were observed from anticoagulants, pyruvate, or oxalacetate.
• Use of NADPH eliminated the need for pre-incubation, reducing assay time.
• Optimized conditions yielded maximum assay sensitivity.

Conclusions:

• The described enzymatic ACA method provides a sensitive, linear, and interference-free assay for plasma ammonia.
• This method offers a reliable and efficient tool for clinical laboratories.
• The assay's robustness makes it suitable for routine diagnostics.