Isolevuglandins covalently modify phosphatidylethanolamines in vivo: detection and quantitative analysis of hydroxylactam adducts

Free Radic Biol Med. 2009 Dec 1;47(11):1539-52. doi: 10.1016/j.freeradbiomed.2009.09.003. Epub 2009 Sep 12.

Abstract

Levuglandins (LGs) and isolevuglandins (isoLGs, also called "isoketals" or "isoKs") are extraordinarily reactive products of cyclooxygenase- and free radical-induced oxidation of arachidonates. We now report the detection in vivo and quantitative analysis of LG/isoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) into LG/isoLG-hydroxylactams. Notably, LC-MS/MS detection of these hydroxylactams is achieved with samples that are an order of magnitude smaller and sample processing is much simpler and less time consuming than required for measuring protein-derived LG/isoLG-lysyl lactams. A key feature of our protocol is treatment of biological phospholipid extracts with phospholipase A(2) to generate mainly 1-palmitoyl-2-lysoPE-hydroxylactams from heterogeneous mixtures of phospholipids with a variety of acyl groups on the 2 position. Over 160% higher mean levels of LG/isoLG-PE-hydroxylactam (P<0.001) were detected in liver from chronic ethanol-fed mice (32.4+/-6.3 ng/g, n=6) compared to controls (12.1+/-1.5 ng/g, n=4), and mean levels in plasma from patients with age-related macular degeneration (5.2+/-0.4 ng/ml, n=15) were elevated approximately 53% (P<0.0001) compared to those of healthy volunteers (3.4+/-0.1 ng/ml, n=15). Just as LG/isoLG-protein adducts provide a dosimeter of oxidative injury, this study suggests that LG/isoLG-PE-hydroxylactams are potential biomarkers for assessing risk for oxidative stress-stimulated diseases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arachidonic Acids / chemistry
  • Arachidonic Acids / metabolism
  • Biomarkers / chemistry
  • Biomarkers / metabolism*
  • Chemical and Drug Induced Liver Injury, Chronic / blood
  • Chemical and Drug Induced Liver Injury, Chronic / enzymology*
  • Chemical and Drug Induced Liver Injury, Chronic / physiopathology
  • Ethanol / administration & dosage
  • Ethanol / toxicity
  • Humans
  • Lactams / blood*
  • Lactams / chemistry
  • Macular Degeneration / blood
  • Macular Degeneration / enzymology*
  • Macular Degeneration / physiopathology
  • Mass Spectrometry
  • Mice
  • Oxidative Stress
  • Phosphatidylethanolamines / chemistry
  • Phosphatidylethanolamines / metabolism
  • Phospholipases A2 / metabolism
  • Prostaglandins E / chemistry
  • Prostaglandins E / metabolism*

Substances

  • Arachidonic Acids
  • Biomarkers
  • Lactams
  • Phosphatidylethanolamines
  • Prostaglandins E
  • Ethanol
  • Phospholipases A2