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

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/isoLGhydroxylactams. 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 timeconsuming 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-PEhydroxylactam (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 agerelated macular degeneration (5.2 ± 0.4 ng/ml, n = 15) were elevated ~53% (P < 0.0001) compared to 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.

Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

“…Within this general background, the pro-fibrogenic features of IsoLGs are largely unknown although the formation of IsoLGs has been demonstrated by the detection of their adducts in several animal models of liver disease [16,28,29,50]. This paucity of information is largely consequent to the difficulty of precisely assessing the in vivo levels of IsoLG in physiological and pathological conditions, and to the fact that they do not exist in free form due to their extreme rapidity of adduction [16].…”

“…On the other hand, the concentration of IsoLG-phosphatidylethanolamine (IsoLG-PE) adducts in whole livers of ethanol-fed mice was shown to be approximately 30 nM [28]. In addition, previous experimental evidence obtained in in vitro studies has suggested that IsoLG-PE may have a limited incorporation into the cells leading to the need of employing higher doses in order to elicit biological effects [20].…”

“…With respect to their potential role in liver disease, IsoLGs adducts have been described in livers of rodents exposed acutely to carbon tetrachloride [16], and chronically to ethanol [28,29], or high fat diet [30]. However, IsoLGs's effects on specific hepatic cell types and their potential contribution to disease pathogenesis have not been investigated.…”

Isolevuglandin-modified phosphatidylethanolamine is metabolized by NAPE-hydrolyzing phospholipase D

“…While the formation of a family of al-PEs including IsoLG-PE has been recently established ( 11,18 ), the mechanism by which these compounds are metabolized has been unknown. Our studies demonstrate that cells utilize NAPE-PLD to rapidly degrade IsoLG-PE to IsoLG-Etn, which then undergoes further metabolism ( Fig.…”

Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1

“…IsoLGs have been shown to posttranslationally modify free primary amino groups present in biomolecules with an avidity greatly exceeding other reactive oxidized lipids ( 26,27 ). Targets of isoLG modifi cation include proteins and phospholipids (PLs) in the serum, the vasculature, and the eye, forming stable covalent adducts with isoLGs (26)(27)(28)(29). IsoLG adduct levels have been shown to be a biomarker of oxidative stress, increased in patients with atherosclerosis, renal disease, and AMD ( 28,30,31 ).…”