Recently, we have identified the dramatic depletion of cardiolipin (CL) in diabetic myocardium 6 weeks after streptozotocin (STZ) injection that was accompanied by increases in triacylglycerol content and multiple changes in polar lipid molecular species. However, after 6 weeks in the diabetic state, the predominant lipid hallmarks of diabetic cardiomyopathy were each present concomitantly, and thus, it was impossible to identify the temporal course of lipid alterations in diabetic myocardium. Using the newly developed enhanced shotgun lipidomics approach, we demonstrated the dramatic loss of abundant CL molecular species in STZ-treated hearts at the very earliest stages of diabetes accompanied by a profound remodeling of the remaining CL molecular species including a 16-fold increase in the content of 18:2-22:6-22:6-22:6 CL. These alterations in CL metabolism occur within days after the induction of the diabetic state and precede the triacylglycerol accumulation manifest in diabetic myocardium. Similarly, in ob/ob mice, a dramatic and progressive redistribution from 18:2 FA-containing CL molecular species to 22:6 FA-containing CL molecular species was also identified. Collectively, these results demonstrate alterations in CL hydrolysis and remodeling at the earliest stages of diabetes and are consistent with a role for alterations in CL content in precipitating mitochondrial dysfunction in diabetic cardiomyopathy.Diabetic cardiomyopathy is characterized by the presence of marked alterations in the lipid composition of myocardium, inefficient substrate utilization, and diastolic dysfunction (1-9). Many studies have implicated mitochondrial dysfunction (1,2,4,5,(7)(8)(9)(10)(11) as the underlying mechanism that precipitates hemodynamic dysfunction and contributes to the untoward sequelae of events in diabetic patients following myocardial ischemia. Persistent changes in substrate utilization occur in diabetic myocardium with an increased utilization of fatty acid substrate and a decreased dependence on glucose. Increased fatty acid utilization promotes the generation of reactive oxygen species which can oxidize highly unsaturated lipids in the mitochondrial compartment such as cardiolipin (CL) 1 and impair mitochondrial function. Collectively, these features each contribute to the accumulation of toxic lipids in diabetic myocardium [e.g., acylcarnitines, acyl-CoAs, and triacylglycerols (TG)] that compromise the functional integrity of many membrane systems (2,4,5,7,9). In early studies, we and others identified profound alterations in the myocardial lipid composition in obese and diabetic rats, which were accompanied by physiologic dysfunction (1, 2). The abnormalities in lipid metabolism present in diabetic myocardium include the accumulation of acylcarnitines, thereby directly implicating mitochondrial dysfunction as a likely contributing mechanism to the compromised metabolic and hemodynamic efficiency of diabetic myocardium. The recent appreciation of these processes has led to the widespread agreem...

A matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as matrix. The linearity of response (r 2 =0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analytematrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.

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Identification and quantification of unsaturated fatty acid (FA) isomers in a biological system are significant in the study of lipid metabolism and catabolism, membrane biophysics, and pathogenesis of diseases, but are challenging in lipidomics. We developed a novel approach for identification and quantitation of unsaturated FA isomers by exploiting two facts: (1) unsaturated FA anions yield fragment ion(s) from loss of CO2 or H2O from the anions upon collision-induced dissociation; and (2) the fragment ions yielded from discrete FA isomers have distinct profiles of the fragment ion intensity vs. collision conditions. These distinct profiles likely result from the differential interactions of the negative charge of the fragment ion with the electron clouds of the double bonds due to their different distances in discrete FA isomers. The novel approach was also extended to analyze the double bond isomers of FA chains present in phospholipids by multi-stage tandem mass spectrometry. Collectively, we developed a new approach for identification and quantification of the double bond isomers of endogenous FA species or FA chains present in intact phospholipid species. We believe that this approach should further advance the lipidomic power for identification of the biochemical mechanisms underlying metabolic diseases.

Herein, a systematic study on the identification and quantitation of choline-containing phospholipid molecular species, including choline glycerophospholipid (PC), lysoPC, and sphingomyelin (SM), is described using multi-dimensional mass spectrometry-based shotgun lipidomics after intrasource separation (MDMS-SL). Current methods for analysis of choline-containing lipids were improved through multiple modifications leading to: (1) identification of constituents present in the PC and SM classes, subclasses of PC, and individual molecular species using MDMS-SL analysis in the positive-ion mode; (2) identification of the fatty acyl constituents and their regiospecificity in diacyl PC molecular species through the neutral loss of trimethylamine plus fatty acids; (3) direct identification of the alkenyl chains of plasmenylcholine species using precursor ion scans of the fragment ions carrying the alkenyl chains; (4) elimination of the effects of polyunsaturation on the quantitation of PC species by multiple ratiometric comparisons; (5) accurate identification and quantitation of lysoPC molecular species including regioisomers by diagnostic fragment ions; and (6) accurate identification and quantitation of SM molecular species by neutral loss scans of phosphocholine plus methyl aldehyde which is specific to SM molecular species. With these enhancements, the application of MDMS-SL for the analyses of choline-containing phospholipid molecular species in biomedical research has been extended to a much larger number of molecular species with greater quantitative accuracy and an increased depth of structural information.