Oxylipin Profiles in Plasma of Patients with Wilson's Disease

Abstract

Wilson's disease (WD) is a rare autosomal recessive metabolic disorder resulting from mutations in the copper-transporting, P-type ATPase gene ATP7B gene, but influences of epigenetics, environment, age, and sex-related factors on the WD phenotype complicate diagnosis and clinical manifestations. Oxylipins, derivatives of omega-3, and omega-6 polyunsaturated fatty acids (PUFAs) are signaling mediators that are deeply involved in innate immunity responses; the regulation of inflammatory responses, including acute and chronic inflammation; and other disturbances related to any system diseases. Therefore, oxylipin profile tests are attractive for the diagnosis of WD. With UPLC-MS/MS lipidomics analysis, we detected 43 oxylipins in the plasma profiles of 39 patients with various clinical manifestations of WD compared with 16 healthy controls (HCs). Analyzing the similarity matrix of oxylipin profiles allowed us to cluster patients into three groups. Analysis of the data by VolcanoPlot and partial least square discriminant analysis (PLS-DA) showed that eight oxylipins and lipids stand for the variance between WD and HCs: eicosapentaenoic acid EPA, oleoylethanolamide OEA, octadecadienoic acids 9-HODE, 9-KODE, 12-hydroxyheptadecatrenoic acid 12-HHT, prostaglandins PGD2, PGE2, and 14,15-dihydroxyeicosatrienoic acids 14,15-DHET. The compounds indicate the involvement of oxidative stress damage, inflammatory processes, and peroxisome proliferator-activated receptor (PPAR) signaling pathways in this disease. The data reveal novel possible therapeutic targets and intervention strategies for treating WD.

Keywords: COX; CYP450; LOX; PUFAs; UPLC-MS/MS; Wilson’s disease; copper; lipidomics; oxylipins.

Figure 1

(A) Volcano plot indicating significantly changed compounds. The X-axis indicates a log2 fold change of wilson disease WD to HC (healthy control) patients. Y-axis indicates −log10 p-values (adjusted). The cut-off for p-values is indicated based on Bonferroni correction. Compounds that changed insignificantly are indicated in gray, compounds whose means changed in WD (relative to HCs) more than twofold or less than twofold but insignificantly are indicated in green. Red dots stand for compounds, which changed more than twofold and had a p-value (adjusted < 0.05). (B) Relative concentrations of separate metabolites that changed significantly in WD patients in comparison with HCs. Pairwise comparison of adjusted means was conducted taking into account the age and sex of patients. * p < 0.05 (adjusted for multiple testing).

Figure 2

(A) The principal component analysis (PCA) performed to verify outliers. The 95% Hotelling T2 confidence interval is indicated as an ellipse. (B) The partial least square discriminant analysis (PLS-DA) model discriminating healthy control (HC) and Wilson disease patients (WD). The explained variance of each component is indicated in brackets on the corresponding axis. (C) PLS2-DA model represented in 3-D showing separation among the HC and WD patients.

Figure 3

A clustered image map was generated using the Euclidean distance and the complete linkage clustering algorithm. On the figure, each entry of the matrix is colored according to its value; rows represent metabolites, columns represent subjects. Dendrograms are shown on the left side (for patients) and on top (for metabolites). Color bars on the bottom of the picture indicate: (A) whether the subjects belongs to the Wilson disease (WD) group or healthy control (HC) group; (B) sex distribution: male (M) or female (F); (C) nephropathy status: no, yes, HC or data not available (N.A.); (D) psychosomatic status: no, yes, HC or N.A.; (E) form of the disease: trembling, akinetic-rigid, extrapyramidal, beforeneurology, or HC.

Figure 4

A clustered image map was performed using the Euclidean distance and complete linkage clustering algorithm. All polyunsaturated fatty acids (PUFA) derivatives were summed up according to their (A) initial substrate of biochemical pathways or (B) pathways’ enzyme origin. In the figure, each entry of the matrix is colored according to its value, rows represent subjects, columns represent metabolites. Dendrograms are shown on the top (for metabolites). The color bar on the left side of the picture indicates whether a subject is WD (black) or a HC (red). Abbreviations: AA: arachidonic acid; DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid; EA: AEA and OEA; LA: linoleic acid; DGLA: dihomo-γ-linolenic acid; ALA: α-linolenic acid; CYP: cytochrome P450 monooxygenase; LOX: lipoxygenase; ROS: reactive oxygen species; COX: cyclooxygenase.