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Anion exchange HPLC isolation of high-density lipoprotein (HDL) and on-line estimation of proinflammatory HDL. PLoS One 2014;9(3):e91089

Date

03/13/2014

Pubmed ID

24609013

Pubmed Central ID

PMC3946658

DOI

10.1371/journal.pone.0091089

Scopus ID

2-s2.0-84897390408   8 Citations

Abstract

Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.

Author List

Ji X, Xu H, Zhang H, Hillery CA, Gao HQ, Pritchard KA Jr

Author

Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Anemia, Sickle Cell
Animals
Anion Exchange Resins
Case-Control Studies
Cell-Free System
Cholesterol
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Hemoglobins
Humans
Inflammation Mediators
Lipoproteins, HDL
Mice, Inbred C57BL
Online Systems
Oxidation-Reduction
Reference Standards
Triglycerides
Xanthine
Xanthine Oxidase
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a