Determination of acid sphingomyelinase activity in biological samples with ultra-performance liquid chromatographic assay

Abstract

A simple, precise and accurate ultra-performance liquid chromatographic (UPLC) method was developed for the quantitative determination of the acid sphingomyelinase (ASM) activity in L-02 hepatocytes. The biological samples of cell homogenate were collected from L-02 hepatocytes and incubated with the fluorescent substrate BODIPY C₁₂-Spm for 2 hr. The samples were determined using ACQUITY UPLC^® BEH Amide chromatographic column with mobile phase containing acetonitrile and 13 mM sodium acetate (97:3, v/v) at a flow rate of 0.8 mL/min. The injection volume was 5 μL. The eluent was monitored using a fluorescence detector set to excitation and emission wavelengths of 435 nm and 525 nm, respectively. Fluorescent product B₁₂-Cer in chromatography analytical can be effectively separated in 2 min, showing good linearity in the range of 6.25–300 ng/mL with the correlation coefficient of 0.9926. The mean ASM activity in L-02 hepatocytes with different sample pretreatment methods, lysis buffer, ultrasonic disruption, and freeze-thawing with liquid nitrogen was 3225.76 ± 323.63, 3330.98 ± 277.99 and 3355.05 ± 267.77 ng/mgprot/h, respectively. Thus we think the current method can be used to detect ASM activity in traces of biological samples.

Graphical Abstract

Keywords:

• ASM activity
• biological sample
• L-02 hepatocytes
• UPLC

Acknowledgments

The authors thank all of the individuals in this laboratory for their valuable ideas and suggestions.

Conflict of Interest

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was financially supported by National Natural Science Foundation of China [No. 81773478].