Direct MALDI-MS analysis of cardiolipin from rat organs sections

Hay-Yan J. Wang 0 1 Shelley N. Jackson 0 1 Amina S. Woods 0 1 0 Published online December 6, 2006 Address reprint requests to Dr. A. S. Woods, National Institute on Drug Abuse Abuse-Intramural Research Program, National Institute of Health , 333 Cassell Drive, Baltimore, MD 21224, USA 1 National Institute on Drug Abuse-Intramural Research Program, National Institute of Health , Baltimore, Maryland, USA Cardiolipins (CL) are mitochondria specific lipids. They play a critical role in ATP synthesis mediated by oxidative phosphorylation. Abnormal CL distribution is associated with several disease states. MALDI-MS and MALDI-MS/MS were used to demonstrate in situ analysis and characterization of CL from tissue sections of organs containing high concentrations of mitochondria. Once the experimental parameters were established, a survey of CL distribution in heart, liver, kidney, leg muscle, and testis was undertaken. The major CL specie in the heart muscle, leg muscle, liver, and kidney is the (18:2)4 CL, while liver and kidney also contain a minor specie, (18:2)3/(18:1) CL. The major CL specie in testis is the (16:0)4 CL. The CL species distribution in various organs appeared to be in agreement with prior reports. Overall, proper matrix selection, tissue section handling, instrument tuning, and the inclusion of cesium ion in matrix ensured successful in situ MALDI-MS and MALDI-MS/MS analysis of CL. Upon modification and standardization, this method could be streamlined for rapid pathological diagnosis with short turnaround time in clinical settings. (J Am Soc Mass Spectrom 2007, 18, 567-577) 2007 American Society for Mass Spectrometry - Ddyl)-sn-glycerol; cardiolipin (CL)] belongs to a iphosphatidylglycerol [1,3-bis(sn-3=-phosphatiunique category of lipid. It consists of two phosphatidic acids linked by a central glycerol. The CL molecule contains four fatty acid chains, three glycerols, and two phosphates [1] (Structure 1). The name cardiolipin was given to a lipid from beef heart first purified by Pangborn in 1942 [2]. CL substituents were later quantitatively determined by Macfarlane and colleagues, who subsequently proposed the dimeric structure of this molecule based on their quantitative analysis [35]. The two phosphatidic acids in CL connect to the central glycerol at the two terminal positions, leaving the central 2=- hydroxyl group of glycerol unmodified. Each phosphatidyl moiety of CL contains a distinct chiral center. The diphosphatidyl arrangement in CL could give rise to diastereomers and create a unique chemical environment for each phosphate group. The central 2=- hydroxyl group is thought to result in intramolecular hydrogen bonding that alters the pK2 of CL and to generate the acid-anion in the head group domain of the molecule. This proposal was supported by a titration experiment where the pK2 of regular CL was above 8, but became equivalent to its pK1 (less than 4.0) when the 2=-hydroxyl group was replaced by a hydrogen [1]. Thus, in spite of the presence of two phosphate groups, CL carries only one negative charge under normal physiological conditions. In mammalian cells, CL is located in the inner membrane of mitochondria, and accounts for 10 to 20% of total mitochondrial lipids [6]. Hence, a high concentration of CL in organ tissue usually indicates a high amount of mitochondria in such organs whose high metabolic activities are fueled by large amounts of ATP generated from mitochondrial oxidative phosphorylation. In mammals, high amounts of mitochondria can be found in myocardium, liver, kidney, and skeletal muscle. In prokaryote circle, CL is also associated with organisms in the eubacteria subkingdom that utilize oxidative phosphorylation as their energy source. Much of mitochondrial cytochrome c (Cyt C) is tightly bound to the CL in its membrane. The Cyt C-CL complex functions as a peroxidase to scavenge excessive H2O2 generated by oxidative phosphorylation. CL is also an integral part of Complexes III and IV, which are an integral part of the electron transport chain generating electrochemical proton gradients across the inner mitochondrial membrane. In addition, CL is also the critical component for the assembly of mitochondria supercomplex consisting of Complexes III and IV [7]. Several earlier studies of oxidative stress-mediated apoptosis demonstrated that mitochondria directly or indirectly subjected to oxidative stress disrupted the homeostasis between CL and Cyt C [79] and caused the dissociation of Cyt C-CL complexes. Upon dissociation, Cyt C is released from the inner mitochondrial membrane and eventually escapes to the cytoplasm, where it triggers a cascade of events leading to programmed cell death [7]. Many pathological studies have associated the presentation and progression of pathological conditions with changes in the fatty acyl moieties of CLs, as seen in Barth syndrome, an X-linked disease, where a change in the fatty acid composition of CL is detected [10 14]. The onset of heart failure in spontaneous hypertensive rats was also linked to the shifting of fatty acids in myocardial CL from saturated C18 fatty acids to highly unsaturated C20 and C22 species [14]. These examples highlight the biological and pathological importance of CL. Conventional mass spectrometric analysis of CL in tissue uses the extraction methods developed by Folch et al. [15] or Bligh and Dyer [16] as the starting point. The tissue is homogenized in a chloroform-methanol mixture, and the homogenate is subjected to various extraction steps followed by laborious chromatographic separation and purification before mass spectrometric analysis [14]. Such sample handling could provide detailed information on tissue lipid composition. However, the procedures consume substantial amounts of time and sample. Further, multistep sample extraction and processing could potentially cause significant amounts of sample loss and renders such approach unsuitable for the analysis of trace amounts of the molecules of interest. In this study, we report an alternative approach for direct detection of CLs from tissue sections without extensive sample processing. Upon establishing suitable experimental parameters, we surveyed the major CL species from rat organs tissue sections containing high concentrations of mitochondria, such as heart, liver, kidney, muscle, and testis. Materials and Methods Animals All animal use and handling were approved by the Animal Care and Use Committee (ACUC) in NIDAIRP, NIH. Male Sprague-Dawley rats between 280 and 400 g were used in this study. Rats were euthanized by overdose isoflurane inhalation. Upon cessation of respiration, the animals were rapidly decapitated and dissected to harvest the liver, heart, kidney, testes, and a small piece of muscle (m. rectus femoris). The harvested organs were briefly washed in ice-cold normal saline three times and trimmed into tissue cubes. The tissue cubes were blot-dried and rapidly frozen in isopent (...truncated)