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. 2010 Oct;51(10):3046-54.
doi: 10.1194/jlr.M007096. Epub 2010 Jul 14.

Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Ariel E Feldstein  1 , Rocio Lopez, Tarek Abu-Rajab Tamimi, Lisa Yerian, Yoon-Mi Chung, Michael Berk, Renliang Zhang, Thomas M McIntyre, Stanley L Hazen
Affiliations

Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Ariel E Feldstein et al. J Lipid Res. 2010 Oct.

Abstract

Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic fatty liver disease (NAFLD). However, the pathways that contribute to oxidative damage in vivo are poorly understood. Our aims were to define the circulating profile of lipid oxidation products in NAFLD patients, the source of these products, and assess whether their circulating levels reflect histological changes in the liver. The levels of multiple structurally specific oxidized fatty acids, including individual hydroxy-eicosatetraenoic acids (HETE), hydroxy-octadecadenoic acids (HODE), and oxo-octadecadenoic acids (oxoODE), were measured by mass spectrometry in plasma at time of liver biopsy in an initial cohort of 73 and a validation cohort of 49 consecutive patients. Of the markers monitored, 9- and 13-HODEs and 9- and 13-oxoODEs, products of free radical-mediated oxidation of linoleic acid (LA), were significantly elevated in patients with nonalcoholic steatohepatitis (NASH), compared with patients with steatosis. A strong correlation was revealed between these oxidation products and liver histopathology (inflammation, fibrosis, and steatosis). Further analyses of HODEs showed equivalent R and S chiral distribution. A risk score for NASH (oxNASH) was developed in the initial clinical cohort and shown to have high diagnostic accuracy for NASH versus steatosis in the independent validation cohort. Subjects with elevated oxNASH levels (top tertile) were 9.7-fold (P < 0.0001) more likely to have NASH than those with low levels (bottom tertile). Collectively, these findings support a key role for free radical-mediated linoleic acid oxidation in human NASH and define a risk score, oxNASH, for noninvasive detection of the presence of NASH.

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Figures

Fig. 1.
Fig. 1.
Detection and quantification of oxFA profile by ESI/LC/MS/MS. Individual isomers of HETEs, EETs, HODEs, and oxoODEs formed by oxidation of arachidonic acid and linoleic acid are quantified with one single injection. Lipid extracts are resolved by HPLC and monitored online by ESI/LC/MS/MS as detailed under “Methods.” Abbreviations: EET, epoxy eicosatetraenoic acid; HETE: hydroxy eicosatetraenoic acid; HODE: hydroxy octadecadenoic acid; oxFA, oxidized fatty acid; oxoODE, oxo-octadecadenoic acid; 15-HETE-d8, internal standard.
Fig. 2.
Fig. 2.
OxFA levels are markedly increased in the blood of patients with NASH. The box-whisker plot is represented with the lower boundary of the box indicating the 25th percentile, the line within the box indicating the median value, and the upper boundary of the box indicating the 75th percentile. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. P values represent differences among groups. Abbreviations: HETE: hydroxy-eicosatetraenoic acid; HODE: hydroxy-octadecadienoic acid; NASH, nonalcoholic steatohepatitis; oxFA, oxidized fatty acid; oxoODE: oxo-octadecadienoic acid.
Fig. 3.
Fig. 3.
Circulating oxFA levels as predictors of NASH in patients with suspected NAFLD. A scoring system (oxNASH) that included 13-HODE/LA ratio, age, BMI, and AST showed best prediction of NASH diagnosis. (A) The AUC curve on the initial training cohort (n = 73) for oxNASH was estimated to be 0.83 (95% CI: 0.73, 0.93) and was found to be significantly higher than the AUCs of both that of serum AST 0.69 (95% CI: 0.56, 0.81) and serum ALT 0.56 (95% CI: 0.43, 0.70) (P < 0.01). (B) The box-whisker plot for oxNASH in the three groups of patients is represented with the lower boundary of the box indicating the 25th percentile, the line within the box indicating the median value, and the upper boundary of the box indicating the 75th percentile. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. (C) The AUC in the independent validation group (n = 49) was estimated to be 0.74 (95% CI: 0.6, 0.88). Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; AUC, area under the curve; BMI, body mass index; HODE, hydroxy-octadecadenoic acid; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; oxFA, oxidized fatty acid.
Fig. 4.
Fig. 4.
Risk of having NASH on liver biopsy based on oxNASH levels. Forest plot illustrating the odds ratio and 95 confidence intervals for risk of histopathologic diagnosis of NASH based upon oxNASH tertiles. Numbers in parentheses represent oxNASH tertile ranges. For each comparison, the first tertile served as the reference group. Abbreviation: NASH, nonalcoholic steatohepatitis.
Fig. 5.
Fig. 5.
Free radical-mediated processes are key mediators of lipid oxidation in NAFLD. Specific oxidized lipid species were separated by liquid chromatography on a chiral phase to identify and quantify the structural isomers and their chiral distribution. A significant increase in peak area of both 13-S HODE and 13-R HODE were observed in patients with NASH compared with patients with hepatic steatosis and patients with normal liver biopsy. The peak area of 13-S HODE was similar to that of 13-R HODE in the three groups of patients. Abbreviations: HODE, hydroxy-octadecadenoic acid; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.
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