Metabolomics using GC–TOF–MS followed by subsequent GC–FID and HILIC–MS/MS analysis revealed significantly altered fatty acid and phospholipid species profiles in plasma of smokers

Abstract

Mass spectrometry is an ideal tool for investigations of the metabolome in human plasma. To investigate the impact of smoking on the human metabolome, we performed an untargeted metabolic fingerprinting using GC-TOF-MS with EDTA-plasma samples from 25 smokers and 25 non-smokers. The observed elevated levels in the monounsaturated fatty acids (MUFAs) in smokers were verified by a targeted analysis using GC-FID, which revealed also significantly alterations in saturated and polyunsaturated fatty acids in smokers (p<0.05, Mann-Whitney U test). Since the main fraction of fatty acids in plasma is esterified to phospholipids, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species composition in the plasma samples of the same subjects. The profiles of 39 PC and 40 PE species were analyzed with a newly developed and validated HILIC-ESI-MS/MS method. We were able to baseline separate the two lipid classes (PC from PE) by maintaining co-elution of individual lipid species of each class. The method shows a linear range from 0.5μM to 2000μM and an inter- and intraday coefficient of variation (CV)<20% across all analytes. Application of the validated method to the plasma samples of smokers and non-smokers, derived from a diet-controlled smoking study, revealed significantly elevated levels of PC and PE species containing MUFAs in smokers. In summary, we could demonstrate that there is a significantly altered total fatty acid profile, with increased MUFAs, in the plasma of smokers compared to non-smokers. Results obtained with the new HILIC-MS/MS method indicate that the altered fatty acid profile is also reflected in the PC and PE profile of smokers.