Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal

Markus A Keller,Katrin Watschinger,Georg Golderer,Manuel Maglione,Bettina Sarg,Herbert H Lindner,Gabriele Werner-Felmayer,Alessandro Terrinoni,Ronald J.A Wanders,Ernst R Werner

doi:10.1194/jlr.d002220

Abstract

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.

Highlights

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids
In order to establish an assay for fatty aldehyde dehydrogenase activity that follows the formation of products and eventual by-products, we modified an existing HPLC assay for quantification of alkylglycerol monooxygenase [12]
Pyrenedecanal, a fluorescently labeled aldehyde harboring a pyrene moiety at the distal end of the hydrocarbon chain, was used as substrate and formation of pyrenedecanoic acid served as readout

Summary

Introduction

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients’ skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.—Keller, M.

Methods

Results

Conclusion