A novel and sensitive assay for heme oxygenase activity

Area of Science:

• Biochemistry
• Enzymology
• Mass Spectrometry

Background:

• Heme oxygenase (HO) is a crucial renoprotective enzyme.
• HO activity is vital for cellular protection but difficult to measure accurately.
• Existing spectrophotometric assays for HO activity are limited by sample requirements and bilirubin instability.

Purpose of the Study:

• To develop a novel and sensitive assay for quantifying heme oxygenase (HO) activity.
• To establish a reliable method for measuring biliverdin production as an indicator of HO activity.
• To overcome the limitations of current spectrophotometric methods for HO activity determination.

Main Methods:

• Developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin.
• Utilized biliverdin-d4 as an internal standard for accurate mass spectrometry-based detection.
• Incubated mouse kidney lysates with hemin, NADPH, and bilirubin oxidase, measuring biliverdin at different temperatures to determine HO activity.

Main Results:

• The LC-MS/MS method demonstrated high sensitivity and specificity for biliverdin quantification.
• HO activity was successfully measured in mouse kidney samples.
• Treatment with cobalt protoporphyrin significantly increased HO activity, as evidenced by elevated biliverdin production.

Conclusions:

• The developed LC-MS/MS assay provides a sensitive and specific method for measuring HO activity.
• This novel assay overcomes the instability of bilirubin and the limitations of spectrophotometric methods.
• The method allows for the detection of subtle changes in HO activity in various tissues.