Proteolytic activity and free amino acid concentrations in polymorphonuclear leucocytes

Area of Science:

• Biochemistry
• Cell Biology
• Hematology

Background:

• Polymorphonuclear granulocytes (PMN) are studied for amino acid (AA) metabolism.
• Variations in reported AA concentrations can arise from cell separation and lysis methods.
• Protease activation during sample preparation may lead to artifactual AA levels.

Purpose of the Study:

• To investigate the impact of protease activation on free AA concentrations in PMN.
• To determine if antiproteolytic agents are necessary for accurate AA quantification in PMN.

Main Methods:

• Measurement of granulocyte protease activity.
• Quantification of AA concentrations in PMN cell suspensions.
• Comparison of samples processed with and without antiproteolytic agents.

Main Results:

• Granulocyte AA concentrations were 8-15 times lower in samples treated with antiproteolytics.
• Protease activity was significantly reduced by the addition of antiproteolytic agents.
• This indicates that proteolysis significantly contributes to measured free AA levels.

Conclusions:

• Protease activation is a significant source of analytical variation in PMN AA measurements.
• The use of protease inhibitors throughout sample preparation is essential for reliable estimation of free AA in granulocytes.
• Accurate assessment of AA metabolism in PMN necessitates controlling for protease activity.