Determination of N-acetyl-β-hexosaminidase in serum from hemolyzed blood.

Background: Determination of lysosomal N-acetyl-β-hexosaminidase (HEX) in serum from hemolyzed blood, creates serious analytical problems, because hemoglobin absorbs light at a similar wavelength like 4-nitrophenol, which is released from artificial substrate.

Objective: The objective of the work was to adapt a manual method to allow analysis of HEX in hemolyzed samples.

Methods: Serums without and with hemolysis were incubated with 4-nitrophenol-N-acetylglucosamine as a substrate. Released 4-nitrophenol was determined colorimetrically. After the incubation of the serum from hemolyzed blood with substrate, hemoglobin was precipitated with trichloroacetic acid (TCA) before 4-nitrophenol determination.

Results: The mean concentration of HEX activity in non-hemolyzed and hemolyzed blood of the same patients, determined with non-modified and modified methods had no significant differences, and they are: 243.12±119.76 and 233.99±108.76pkat/mL, respectively. A coefficient of correlation between non-modified and modified methods equals the 0.98. For HEX determination with the modified method in serum from hemolyzed blood, optimal reaction time was 60min, pH of reaction mixture was 4.7, and Km was 0.11mMm.

Conclusion: HEX determinations in the same serums from non-hemolyzed blood by the non-modified method and hemolyzed blood with the modified method, gave similar results with a 0.98 coefficient of correlation. The modified method is appropriate for HEX determination in serum from hemolyzed blood.