Elevated levels of hydroxylated phosphocholine lipids in the blood serum of breast cancer patients Loubna A. Hammad 1 y , Guangxiang Wu 1 y , Marwa M. Saleh 1 , Iveta Klouckova 1 , Lacey E. Dobrolecki 2 , Robert J. Hickey 2,3 , Lauren Schnaper 4 , Milos V. Novotny 1 and Yehia Mechref 1 * 1 METACyt Biochemical Analysis Center, Department of Chemistry, Indiana University, Bloomington, Indiana 47405 2 Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202 3 Indiana University Cancer Center, Indianapolis, Indiana 46202 4 Breast Cancer Center, Greater Baltimore Medical Center, Baltimore, MD 21204 Received 11 September 2008; Revised 9 January 2009; Accepted 10 January 2009 The difference in serum phospholipid content between stage-IV breast cancer patients and disease- free individuals was studied by employing a combination of chemometric statistical analysis tools and mass spectrometry. Chloroform-extracted serum samples were profiled for their lipid class composition and structure using precursor ion, neutral loss, and product ion tandem mass spectro- metric (MS/MS) scanning experiments. Changes in the relative abundance of phospholipids in serum as a consequence of cancer progression, measured through electrospray ionization (ESI) mass spectrometry of flow-injected serum samples collected from 25 disease-free individuals and 50 patients diagnosed with stage-IV breast cancer, were statistically evaluated using principal component analysis (PCA), analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis. Lipids whose abundance changed significantly as a consequence of cancer progression were structurally characterized using product ion spectra, and independently quantified using precursor ion scan experiments against an internal standard of known concentration. Phosphocholine lipids that displayed a statistically significant change as a consequence of cancer progression were found to contain an oxidized fatty acid moiety as determined by MS 3 experiments. Copyright # 2009 John Wiley & Sons, Ltd. Lipids are an important class of biomolecules that exhibit a wide variety of functions within cells and play a major role in cell signaling and subcellular structure. 1–3 Human mammary tissues metabolize lipids from plasma, which serves as the transport medium for lipids circulating in the body. This process is affected by female gonadal hormones. Lipids circulating in the body do not appear in their free form, but are embedded in a matrix named lipoprotein. 4 Lipoproteins are globular micelle-like particles that consist of a non-polar core of mobile triacylglcerols and cholesteryl esters sur- rounded by a single layer of phospholipid, into which a mixture of cholesterol and proteins is inserted. At various sites in the body, lipoproteins interact with specific receptors and enzymes that transfer or modify their lipid cargoes. Unesterified fatty acids are merely bound to serum albumin and other proteins in blood plasma. Phospholipids account for approximately 60 mol% of lipid mass in eukaryotic cells. They consist of a polar head group with a phosphate moiety and two fatty acids that are attached to the glycerol backbone. The physiological importance of phospholipids is illustrated by the numerous diseases to which lipid abnormalities contribute such as atherosclerosis, Alzhei- mer’s disease, and cancer. 5–11 As a branch of metabolomics, lipidomics has advanced to be a systems-based study of all lipids, the molecules with which they interact, and their functions within the cell. The first step in lipidomics is the determination of a total lipid profile or lipidome. This can be obtained from the mass spectrum of a crude lipid-extracted sample and can be used to monitor temporal changes in cellular concentration of diverse lipids over time and in response to particular stimuli. ‘Lipid Arrays’ (or more precisely virtual arrays) can be generated based on the mass spectra acquired through flow injection electrospray ionization mass spectrometry (ESI-MS) and constructed with statistical algorithms. These arrays represent a qualitative map of molecular species changes as a consequence of biological perturbations. 12,13 Recent studies have demonstrated that both benign and malignant proliferation of breast tissue in women is associated with changes in plasma lipids and lipoprotein levels, and that alterations in serum total triacylglycerols, cholesterol, high-density lipoprotein-cholesterol, and low- density lipoprotein-cholesterol are closely related to the progression of breast cancer. 10,14–18 However, details about RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2009; 23: 863–876 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3947 *Correspondence to: Y. Mechref, Department of Chemistry, Indi- ana University, 800 E Kirkwood Ave., Bloomington, IN 47405, USA. E-mail: ymechref@indiana.edu y These authors contributed equally to this work. Contract/grant sponsor: The Indiana Metabolomics and Cytomics Initiative (METACyt) for the METACyt Biochemical Analysis Center. Copyright # 2009 John Wiley & Sons, Ltd.