title: RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1.
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, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (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 picomolar 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.
authors: Department Cellular and Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
authors: Serena
authors: S
authors: Mezzar
authors: Evelyn
authors: E
authors: de Schryver
authors: Paul P
authors: PP
authors: Van Veldhoven
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D019755:Carbon-Carbon Lyases; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004746:Enoyl-CoA Hydratase / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008956:Models, Chemical; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000378:metabolism; D013110:Sphingosine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D000447:Aldehydes; D011994:Recombinant Proteins; C120366:sphingenine-1-phosphate; C025987:2-hydroxyoctadecanoyl-coenzyme A; D019755:Carbon-Carbon Lyases; C561547:HACL1 protein, human; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D004746:Enoyl-CoA Hydratase; D013110:Sphingosine
keywords: Hantzsch reaction; peroxisomes; phytanic acid; sphingosine-1-phosphate; α-oxidation