Celle-fri biokemiske fluorometriske enzymatisk Assay for høj overførselshastighed måling af Lipid fedtstoffer i High Density Lipoprotein
Summary
Vi beskriver her en fluorometriske celle-fri biokemiske assay for bestemmelse af HDL-lipid fedtstoffer. Denne hurtige og reproducerbare assay kan bruges til at bestemme HDL funktion i stor skala undersøgelser og kan bidrage til vores forståelse af HDL funktion i menneskelige sygdom.
Abstract
Lav high-density lipoprotein kolesterol (HDL-C) niveauer er en af de mest magtfulde uafhængige negative prædiktorer af aterosklerotisk hjerte-kar-sygdom (CVD). Struktur og funktion af HDL snarere end HDL-C kan mere præcist at forudsige åreforkalkning. Flere HDL proteiner og lipid kompositoriske ændringer at forringer HDL funktion forekommer i inflammatoriske stater som åreforkalkning. HDL funktion bestemmes normalt af celle-baserede assays såsom cholesterol efflux assay, men disse analyser har talrige ulemper mangel på standardisering. Celle-fri assays kan give mere robust foranstaltninger af HDL funktion i forhold til celle-baserede assays. HDL oxidation forringer HDL funktion. HDL har en vigtig rolle i lipid peroxid transport og høj mængde af lipid peroxider er relateret til unormal HDL funktion. Lipid-sonde interaktioner bør overvejes når fortolkningen af ikke-enzymatiske fluorescens undersøgelser til måling af lipid oxidativt tilstand. Dette motiverede os til at udvikle en celle-fri biokemiske enzymatisk metode til at vurdere HDL lipid peroxid indhold (HDLox), bidrager til HDL dysfunktion. Denne metode er baseret på enzymet peberrodsperoxidase (HRP) og fluorokrom Amplex Red, der kan kvantificere (uden kolesterol oxidase) lipid peroxid indhold pr. mg af HDL-C. Her er en protokol describedfor bestemmelse af HDL-lipid fedtstoffer ved hjælp af fluorokrom reagenset. Assay variation kan reduceres ved strenge standardisering af forsøgsbetingelser. Højere HDLox værdier er forbundet med nedsat HDL antioxidant funktion. Udlæsning af denne analyse er forbundet med udlæsninger af validerede celle-baserede assays, erstatningsmålinger af hjerte-kar-sygdom, systemisk inflammation, immune dysfunktion og tilknyttede kardiovaskulære og metaboliske risiko fænotyper. Denne tekniske tilgang er en robust metode til at vurdere HDL funktion i menneskelige sygdom hvor systemisk inflammation, oxidativ stress og oxiderede lipider har en central rolle (som åreforkalkning).
Introduction
Aterosklerotisk hjerte-kar-sygdom (CVD) er den hyppigste årsag til dødsfald på verdensplan1,2. Epidemiologiske undersøgelser har vist, at lave niveauer af high-density lipoprotein (HDL) kolesterol er generelt omvendt forbundet med risiko for udvikling af åreforkalkning1,2. Selv om flere undersøgelser støtter en atheroprotective rolle for HDL1,2, er den mekanisme, hvormed HDL dæmper indledningen og progression af aterosklerose komplekse 3,4. Således, det er blevet foreslået, at den komplekse struktur og funktion af HDL i stedet for absolutte niveau kan mere præcist at forudsige åreforkalkning 5,6,7,8. Flere HDL proteiner og lipid kompositoriske ændringer at forringer HDL funktion forekommer i inflammatoriske stater som åreforkalkning. Disse i) reducere dens cholesterol efflux potentielle 9, ii) mindske anti-inflammatorisk og øge HDL-associerede pro-inflammatoriske proteiner 6,7, iii) faldet antioxidant faktor niveauer og aktivitet og HDLs evne til at hæmme oxidation af Low Density Lipoprotein (LDLox)10 og iv) øge lipid hydroperoxyd indhold og redox aktivitet (HDLox)9,11. Robust assays, der evaluerer pleotropic funktionerne af HDL (såsom cholesterol efflux, antioxidant funktion) kan supplere bestemmelse af HDL-HDL-C i klinikken.
HDL funktion vurderes normalt af celle-baseret metoder såsom cholesterol efflux assay8,12,13,14. Disse metoder har store begrænsninger herunder betydelige uensartethed med hensyn til typer af celler, der bruges, type af udlæsning rapporteret, mangel på standardisering og forstyrrende virkninger af triglycerider 7,15. Disse ulemper frembyde vanskeligheder for store kliniske studier16. Celle-fri assays kan give mere robust foranstaltninger af HDL funktion i forhold til celle-baserede assays. Cholesterol efflux er en af de vigtigste funktioner af HDL, men det kan kun afgøres ved celle-baserede assays. Andre metoder til at bestemme HDL funktion såsom proteomics17,18,19,20,21,22,23, 24 og cellebaserede monocyt chemotaxis assays af HDL funktion 17,22,25 er ikke blevet standardiseret og kan ikke bruges i stor skala humane undersøgelser.
HDL har betydelig antioxidant atheroprotective virkning5,6,7,8. Funktionen antioxidant af HDL er fastslået i overværelse af LDL i forrige celle gratis fluorometriske assays 26. Disse biokemiske fluorometriske metoder af HDL antioxidant funktion blev oprindeligt udviklet af Mohamad Navab og Alan Fogelman og deres kolleger26. Selv om mange humane undersøgelser har brugt disse metoder til at bestemme HDL funktion 17,18,19,20,21,22,23 ,24, lipid (HDL)-lipid (LDL) og lipid-fluorokrom interaktioner kan begrænse reproducerbarhed af disse celle gratis ikke-enzymatiske biokemiske assays af HDL funktion27,28.
Nylige interesse har fokuseret på de funktionelle konsekvenser af HDL oxidation, der er resultatet af oxidation af lipider og proteiner i HDL 27,29,30. Tidligere undersøgelser har vist, at oxidation af HDL forringer HDL funktion 27,29,30. HDL har en vigtig rolle i lipid peroxid transport og høj mængde af lipid peroxider er relateret til unormal HDL funktion. Dermed kan HDL lipid peroxid indhold bruges til at bestemme HDL funktion 9,17,20,31 og givet de kendte begrænsninger af forudgående assays af HDL funktion7, 15,27,32, vi udviklet alternative fluorometriske metoder der kvantificerer HDL lipid peroxid indhold (HDLox) 32. Denne metode er baseret på enzymet peberrodsperoxidase (HRP) og fluorokrom Amplex Red, der kan kvantificere (uden kolesterol oxidase) lipid peroxid indhold pr. mg af HDL-C 32. Det biokemiske princip i analysen er vist i figur 1. Vi har vist, at denne fluorescens-baserede tilgang ikke har begrænsninger af forudgående HDL funktion assays27,28. Denne analyse har været yderligere raffineret og standardiseret i vores laboratorium, således at det kan pålideligt anvendes i stor skala humane undersøgelser selv med befrugtede plasma 32,33,34, 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42. udlæsning af denne analyse er forbundet med udlæsninger af validerede celle-baserede assays, erstatningsmålinger af hjerte-kar-sygdom, systemisk inflammation, immune dysfunktion og tilknyttede kardiovaskulære og metaboliske risiko fænotyper 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39. her, vi beskriver denne enkle, men alligevel robust metode til at måle HDL lipid peroxid indhold (HDLox). Denne analyse kan bruges som et redskab til at besvare vigtige forskningsspørgsmål vedrørende rollen af HDL funktion i menneskelige sygdom hvor systemisk inflammation, oxidativ stress og oxiderede lipider har en central rolle (som åreforkalkning)32.
Protocol
Representative Results
Discussion
Protokollen beskrevet her tilbyder en robust værktøj for at besvare vigtige forskningsspørgsmål vedrørende rollen af HDL funktion i åreforkalkning og sygdom hos mennesker. Analysen kvantificerer HDL lipid peroxid indhold pr. mg af HDL-C ved hjælp af enzymatiske forstærkning (HRP). Denne metode undgår kendte begrænsninger af forudgående HDL funktion assays (fx cholesterol efflux assayet) herunder betydelige uensartethed med hensyn til typer af celler, der bruges, type af udlæsning rapporteret, mangel på sta…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Forfatterne parlamentsarbejdet Dr Mohamad Navab, Alan Fogelman og Srinivasa Reddy arbejde for deres centrale rolle i udviklingen af tidligere gentagelser af denne model. T.A.A. understøttes af en RMIT University Vice-kansler postdoc stipendium. AJ og AH understøttes af NHMRC project grant 1108792. TK er støttet af NIH tilskud NIH K08AI08272, NIH/NCATS Grant # µL1TR000124.
Materials
| Experimental Reagents | |||
| HDL PEG (Polyethylene Glycol) Precipitating Reagent | Pointe Scientific | H7511 | |
| Amplex Red reagent. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| DMSO. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| Horse Radish Peroxidase (HRP) | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| Cholesterol Esterase. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| Cholesterol Reference standard | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| Resorufin fluorescense Reference standard | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| 5x Reaction Buffer. | Life Technologies, Grand Island, NY | A12216 | Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light |
| HDL Cholesterol Automated Reagent | ThermoFisher Scientific Co., San Jose, CA, USA. | TR39601 | |
| Name | Company | Catalog Number | Comments |
| Plasticware | |||
| 96-well plates (polypropylene, flat bottom, clear). | Sigma Aldrich | M0687 | |
| 96-well plates (polypropylene, flat bottom, black). | Sigma Aldrich | M9936 | |
| 1.5 mL Eppendorf tubes | Eppendorf | 0030 125.150 | |
| ClipTip 200, sterile | ThermoFisher Scientific Co., San Jose, CA, USA. | 14-488-058 | |
| Thermo Scientific Multichannel Pipettes, 8-channel, 125 | ThermoFisher Scientific Co., San Jose, CA, USA. | 14-387–955 | |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Gen5 2.01 software | Biotek, Vermont, USA | NA | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Gen5 Plate reader | Biotek, Vermont, USA | NA |
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