f15d990ef700f38d229a766b608d9cca pmc_9820682.pdf a6b996ab1cb7e69b970fab19cd3f402b1c43155e pmc_9820682.pdf 665b5aab050e7e2d69f26fb4eed09a002c2f8f9cd61f9d96d70c6e32f4d580d1 pmc_9820682.pdf Title: Presence of Ceramidase Activity in Electronegative LDL Subject: Electronegative low-density lipoprotein (LDL(-)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(-). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(-) incubated alone at 37 C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(-). In addition, LDL(-) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(-). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(-). Keywords: electronegative LDL; ceramide; sphingosine; ceramidase; sphingomyelinase Author: Núria Puig, Jose Rives, Montserrat Estruch, Ana Aguilera-Simon, Noemi Rotllan, Mercedes Camacho, Núria Colomé, Francesc Canals, José Luis Sánchez-Quesada and Sonia Benitez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Dec 22 06:33:13 2022 CET ModDate: Thu Dec 22 06:37:55 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1088867 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CIQLLF+VnURWPalladioL Type 1 Custom yes yes no 10 0 CLWXUP+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 NBLFCV+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 KTGUUB+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 28 0 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no 309 0 CNALGD+PalatinoLinotype CID TrueType Identity-H yes yes yes 312 0 CNALGE+PalatinoLinotype TrueType WinAnsi yes yes no 318 0 CNALGF+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 321 0 CNALGG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 327 0 CNALGB+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 418 0 CNALGC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 424 0 CNALGD+PalatinoLinotype CID TrueType Identity-H yes yes yes 427 0 CNALGE+PalatinoLinotype TrueType WinAnsi yes yes no 433 0 CNALGF+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 436 0 CNALGG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 442 0 CNAMMP+PalatinoLinotype TrueType MacRoman yes yes no 445 0 CNBBIE+Arial CID TrueType Identity-H yes yes yes 448 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 493 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-07-22 02:39:54 CEST RepresentationInformation: pmc_9820682.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-07-20 02:26:35 CEST Size: 1088867 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 714 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Sph and Cer Content in LDL(-) Increase at 37 C Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: NEFA Species Increase at 37 C in LDL(-) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Effect of CDase Inhibitor on Sph, Cer, and NEFA Content in LDL(-) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: LDL(-) Is Able to Degrade Cer from an External Source Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Proteomic Analysis of LDL(-) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Western Blot of Neutral CDase Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: LDL Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: LDL Incubation at 37 C Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: TLC Lipid Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Lipid Determination via Liquid Chromatography-Mass Spectrometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: Sphingolipid Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: NEFA Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Hydrolysis of External Cer by LDL(-) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Proteomic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Western Blot of Neutral CDase Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Statistical Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Presence of Ceramidase Activity in Electronegative LDL Author: Núria Puig, Jose Rives, Montserrat Estruch, Ana Aguilera-Simon, Noemi Rotllan, Mercedes Camacho, Núria Colomé, Francesc Canals, José Luis Sánchez-Quesada and Sonia Benitez Subject: Electronegative low-density lipoprotein (LDL(-)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(-). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(-) incubated alone at 37 C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(-). In addition, LDL(-) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(-). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(-). 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