Rapid determination of erythrocyte pyruvate kinase activity

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.