Isolevuglandins covalently modify phosphatidylethanolamines in vivo: detection and quantitative analysis of hydroxylactam adducts.
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.
Li
Wei
W
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
Laird
James M
JM
Lu
Liang
L
Roychowdhury
Sanjoy
S
Nagy
Laura E
LE
Zhou
Rong
R
Crabb
John W
JW
Salomon
Robert G
RG
D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism*; D056487:Chemical and Drug Induced Liver Injury, Chronic / Q000097:blood / Q000201:enzymology* / Q000503:physiopathology; D000431:Ethanol / Q000008:administration & dosage / Q000633:toxicity; D006801:Humans; D007769:Lactams / Q000097:blood* / Q000737:chemistry; D008268:Macular Degeneration / Q000097:blood / Q000201:enzymology* / Q000503:physiopathology; D013058:Mass Spectrometry; D051379:Mice; D018384:Oxidative Stress; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D054467:Phospholipases A2 / Q000378:metabolism; D011458:Prostaglandins E / Q000737:chemistry / Q000378:metabolism*
D001095:Arachidonic Acids; D015415:Biomarkers; D007769:Lactams; D010714:Phosphatidylethanolamines; D011458:Prostaglandins E; D000431:Ethanol; D054467:Phospholipases A2