Study on alanine aminotransferase kinetics by microchip electrophoresis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Alanine aminotransferase (ALT) is crucial for diagnosing liver diseases.
• Accurate measurement of ALT enzyme kinetics is essential for clinical assessment.
• Existing methods may require improvement in sensitivity and efficiency.

Purpose of the Study:

• To develop a microanalytical method for evaluating ALT enzyme kinetics.
• To quantify substrates L-glutamic acid (L-Glu) and L-alanine (L-Ala) using microchip electrophoresis laser-induced fluorescence.
• To determine kinetic parameters (Km and Vmax) for both forward and reverse ALT reactions.

Main Methods:

• Utilized a microchip electrophoresis laser-induced fluorescence system for separation and detection.
• Optimized conditions for the quantitative analysis of L-Glu and L-Ala.
• Monitored substrate concentration decrease to determine enzyme kinetic constants.

Main Results:

• Achieved effective separation of amino acid mixtures, including ALT substrates.
• Established limits of detection for L-Glu (4.0 × 10⁻⁷ M) and L-Ala (2.0 × 10⁻⁷ M).
• Determined kinetic constants: forward reaction (Km=10.12 mM, Vmax=0.48 mM/min) and reverse reaction (Km=3.22 mM, Vmax=0.22 mM/min).

Conclusions:

• The developed microchip electrophoresis method is suitable for ALT enzyme kinetic studies.
• The assay demonstrates high sensitivity and accuracy for quantifying amino acids.
• The method shows significant potential for practical applications in clinical diagnostics.