Lipidomic Analysis of Ether-Linked and Oxidized Lipids in Obesity and Endothelial Dysfunction

Title: Lipidomic Analysis of Human Plasma Reveals Ether-Linked Lipids That Are Elevated in Morbidly Obese Humans Compared to Lean

Journal: Diabetology & metabolic syndrome

Published: 2013

Background

Obesity is often associated with dyslipidemia and oxidative stress, contributing to the development of metabolic syndrome, cardiovascular disease, and other health issues. Elevated lipid levels, particularly oxidized low-density lipoproteins (LDL), can lead to vascular inflammation and endothelial dysfunction. While previous studies have focused on oxidized phospholipids, recent research highlights the role of ether-linked lipids and plasmalogens. These lipids, characterized by unique ether or vinyl ether linkages, may act as antioxidants and reservoirs for arachidonic acid. However, their specific role in obesity-related metabolic disturbances remains poorly understood. This study used a lipidomics approach to compare plasma lipid profiles between lean and morbidly obese subjects, aiming to identify lipids associated with obesity and investigate their effects on endothelial cell function.

Materials & Methods

Ethics Statement

Approved by Colorado State University and Poudre Valley Hospital. Informed consent was obtained, following the Declaration of Helsinki.

Participants

15 morbidly obese gastric bypass patients (BMI > 40 kg/m²)
13 non-obese controls (BMI < 30 kg/m²)
Recruited through the Northern Colorado Surgical Associates.

Sample Collection and Analysis

Fasting blood samples were collected, with plasma stored at −80°C.
HbA1C levels were measured using a DCA Vantage analyzer.

Lipid Extraction

Lipids were extracted using the MTBE method under argon gas to prevent oxidation.

LC/MS Analysis

Lipid profiling was performed using UPLC coupled with a Q-ToF Micromass spectrometer.
MarkerLynx software was used for peak detection and integration.

Data Analysis

PCA and OPLS-DA models were used for group discrimination.
Significant lipid ions were identified using MS/MS and confirmed with lipid standards.

Cell Culture and Lipid Treatments

HCAECs were treated with lipid standards, and protein expression was assessed using Western blot.
MCP-1 levels were measured using ELISA.

Statistical Analysis

Unpaired t-tests were conducted, with significance set at p ≤ 0.05.

Results

Subject Characteristics:

No significant difference in average age between groups.
Morbidly obese group had significantly higher BMI (49.87 ± 11.27 kg/m²) compared to the control group (25.76 ± 4.39 kg/m², p < 0.01).
HbA1C levels were significantly higher in the obese group (6.41 ± 0.35%) than in the control (5.15 ± 0.10%, p < 0.01).

Global Lipidomic Analysis:

Lipid extracts were analyzed using LC/MS via shotgun and targeted approaches.
PCA showed poor separation between groups, indicating heterogeneity.
OPLS-DA modeling improved group differentiation, identifying 43 significantly altered lipid ions, with 26 ions elevated in obese subjects.

Lipid Ion Identification:

MS/MS analysis identified 22 lipid ions, predominantly ether-linked phospholipids, with higher abundance in obese subjects.
Ion identifications were confirmed by comparing MS/MS spectra to published spectra and standards.

Endothelial Cell Response to Lipids:

Treatment with ether-linked PE (P-18:0/20:4) on HCAEC cells increased ICAM (158% of control, p = 0.028) and VCAM (144% of control, p = 0.038) expression, without affecting MCP-1 levels.
Similar effects on ICAM and VCAM were observed with oxidized PC, but not with un-oxidized PC.

Targeted Oxidized Phosphatidylcholine Analysis:

Targeted LC/MS detected a significant increase in a potential PC ion in obese subjects (p = 0.040).
Oxidized PC species (PGPC) were nearly significantly elevated (p = 0.052, p = 0.055) in obese subjects, whereas PEIPC and POVPC were not significantly different.

Principle components analysis models for all ions for all subjects detected in positive and negative mode.

OPLS-DA models and representative scores plots from positive mode analysis (A) and negative mode analysis (B).

Representative spectrogram of the ion m/z 750.5399 from MS/MS analysis that was identified as the plasmalogen phosphatidylethanolamine PE(P-18:0/20:4(5Z,8Z,11Z,14Z)), along with a previously published MS/MS spectrogram of the same species. Peak patterns match with the exception of a fragment of 331 in the previously published spectra resulting from presence of a minor additional isomer.

Chromatography and MS/MS spectra of ion m/z 885.5499 (PI(18:0/20:4(5Z,8Z,11Z,14Z)) from the purchased standard (top chromatogram and spectra) and a sample (bottom chromatogram and spectra). Fragmentation patterns match at major peaks 885- parent ion, 581- parent ion with loss of sn-2 arachidonic acid side chain m/z 303, 303- liberated arachidonic acid, 283- liberated stearic acid, and 241- inositol head group.

Reference

Martín-Ortiz, Andrea, et al. "Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry." Journal of chromatography A 1428 (2016): 143-153. http://dx.doi.org/10.1016/j.chroma.2015.09.060

* For Research Use Only. Not for use in diagnostic procedures.

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