Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, alpha-oxidation, Hantzsch reaction, peroxisomes, phytanic acid, sphingosine-1-phosphate, SPHINGOSINE-1-PHOSPHATE LYASE ACTIVITY, LIQUID-CHROMATOGRAPHIC DETERMINATION, SJOGREN-LARSSON-SYNDROME, MASS-SPECTROMETRY, 2-HYDROXYPHYTANOYL-COA LYASE, THIAMINE PYROPHOSPHATE, ALPHA-OXIDATION, FATTY-ACIDS, RAT-LIVER, CLONING, α-oxidation, Acyl Coenzyme A, Aldehyde-Lyases, Aldehydes, Animals, Brain, Carbon-Carbon Lyases, Cells, Cultured, Chromatography, High Pressure Liquid, Chromatography, Reverse-Phase, Enoyl-CoA Hydratase, Enzyme Assays, Female, Fibroblasts, Fluorescence, Humans, Liver, Male, Mice, Mice, Inbred C57BL, Models, Chemical, Molecular Structure, Recombinant Proteins, Sphingosine, Substrate Specificity, 0601 Biochemistry and Cell Biology, 1101 Medical Biochemistry and Metabolomics, 3101 Biochemistry and cell biology, 3205 Medical biochemistry and metabolomics

Abstract:

Long chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, non radioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were employed as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by RP-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in pmol range and different long chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates.