Biomedical subjects

Xianlin Han

Publications and source records attributed to Xianlin Han.

At least 19 recordsLinked to original sources

PubMed2026

Multi-omic analyses of the same sample using metabolomics, lipidomics, proteomics, phosphoproteomics, and glycoproteomics.

Mass spectrometry (MS)-based multi-omics offers powerful tools to comprehensively characterize proteins, post-translational modifications, metabolites, and lipids. However, these measurements are typically performed using separate sample preparation workflows and modality-specific liquid chromatography mass spectrometry (LC-MS) platforms, limiting integration and constraining applications to small amounts of sample materials, especially scarce clinical specimens. Here, we describe a unified nano-LC-MS framework that enables metabolomic, lipidomic, proteomic, phosphoproteomic, and glycoproteomic analyses from the same starting material using a single nano-LC-MS platform, with only the chromatographic conditions, acquisition methods, and enrichment procedures tailored to each omics. This integrated strategy reduces workflow complexity and sample consumption while improves analytical continuity across molecular layers. By enabling deep multi-omics characterization from the same sample, this platform provides a practical foundation for comprehensive analysis of precious clinical samples.

Proteomics↗

PubMed2007

Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma.

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Animals↗

PubMed2007

Neurolipidomics: challenges and developments.

The field of lipidomics is one of the most rapidly expanding areas of systems biology research. Considering the uniqueness and complexity of the lipidome in the nervous system (i.e., neurolipidome), neurolipidomics remains quite challenging but exciting. With the recent development of mass spectrometry (MS)-based lipidomics, particularly the rapid improvement of multi-dimensional MS-based shotgun lipidomics, much progress has been made in neurolipidomics. As the accelerated development of future technologies enables lipidomics penetrance into lower and lower abundance regions of mass contents of individual lipid molecular species, it can be anticipated that many biochemical mechanisms underlying lipid metabolism critical to neuronal disease states will be increasingly uncovered. Through exploiting the information content inherent in the complexity of neuronal lipid composition and kinetic turnover which can be revealed by neurolipidomics, substantial insights into neuronal plasticity and gene function can be gathered. Through neurolipidomics, the markers for these neuronal diseases which identify pathological alterations and are diagnostic of disease onset, progression or severity may potentially be discovered. Accordingly, with neurolipidomics, our understanding of the complexities of the nervous system will be undoubtedly accelerated, as many mysteries are resolved.

Animals↗

PubMed2007

Characterization of oxysterols by electrospray ionization tandem mass spectrometry after one-step derivatization with dimethylglycine.

We report a novel approach to derivatize the primary, secondary, and tertiary hydroxy group(s) of oxysterols with N,N-dimethylglycine (DMG) in the presence of both 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-(N,N-dimethylamino)pyridine to yield their corresponding mono- or di-DMG esters. Eight oxysterols including 7-oxocholesterol, 5alpha,6alpha- and 5beta,6beta-epoxycholesterols, as well as 7alpha-, 7beta-, 24(S)-, 25-, and 27-hydroxycholesterols, were studied. Electrospray ionization tandem mass spectrometric characterization of these singly or doubly protonated derivatives demonstrates the presence of an informative fragmentation pattern for each oxysterol derivative. Potential dissociation pathways for the production of these unique fragmentation patterns are proposed and discussed. Collectively, these informative and unique fragmentation patterns allow rapid and direct discrimination of the identities of 7alpha-, 7beta-, 24(S)-, 25-, and 27-hydroxycholesterol isomers, as well as 5alpha,6alpha- and 5beta,6beta-epoxycholesterol isomers, thereby potentially providing a foundation for quantitative analysis of oxysterols in biological samples in combination with a chromatographic separation.

Ketocholesterols↗

PubMed2006

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 muM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing beta-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.

Animals↗

PubMed2006

Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach.

Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO(3)](-)) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/mul concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.

Aged↗

PubMed2006

Abundance of triacylglycerols in ganglia and their depletion in diabetic mice: implications for the role of altered triacylglycerols in diabetic neuropathy.

Herein, we report the first study on the mass distribution and molecular species composition of abundant triacylglycerols (TAG) in ganglia. This study demonstrates five novel findings. First, unanticipated high levels of TAG were present in all examined ganglia from multiple species (e.g. mouse, rat and rabbit). Second, ganglial TAG mass content is location-dependent. Third, the TAG mass levels in ganglia are species-specific. Fourth, dorsal root ganglial TAG mass levels in streptozotocin-induced diabetic mice are dramatically depleted relative to those found in untreated control mice. Fifth, mouse ganglial TAG mass levels decrease with age although molecular species composition is not changed. Collectively, these results indicate that TAG is an important component of ganglia and may potentially contribute to pathological alterations in peripheral neuronal function in diabetic neuropathy.

Age Factors↗

PubMed2006

A regulatory role for 1-acylglycerol-3-phosphate-O-acyltransferase 2 in adipocyte differentiation.

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBPbeta and PPARgamma, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed2006

Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples.

Cardiolipin is a prominent component of the mitochondrial inner membranes contributing to the regulation of multiple discrete mitochondrial functions. Here, we extend shotgun lipidomics to identify and quantitate cardiolipin molecular species directly from lipid extracts of biological samples. Three shotgun lipidomics approaches for analyses of cardiolipin molecular species were developed using either a continuous ion-transmission instrument (i.e., triple-quadrupole type) with either low or high mass resolution settings or a high mass resolution hybrid pulsed instrument [i.e., quadrupole time-of-flight (QqTOF) type]. Three chemical principles were used for the development of these approaches. These include the marked enrichment of linoleate in cardiolipin to maximize the signal-to-noise ratio, the specific neutral loss of ketenes from doubly charged cardiolipin molecular ions to yield doubly charged triacyl monolysocardiolipins, and the doubly charged character of two phosphates in each cardiolipin molecular species. Through these techniques, we identified and quantified the specific molecular species profiles of cardiolipin directly from lipid extracts of mouse heart, liver, and skeletal muscle. The accuracy ( approximately 5%) and the low end of the linear dynamic range (10 fmol/microl) for quantitation make these approaches useful for studying alterations in cardiolipin metabolism in multiple disease states using either type of mass spectrometer.

Animals↗

PubMed2006

Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids.

The external electric field induces a separation of cations from negative electrolyte ions in the infusate while differential ionization of molecular species that possess differential electrical propensities can be induced in either the positive- or negative-ion mode during the electrospray ionization process. These physical and electrical processes that occur in the electrospray ion source have been used to selectively ionize lipid classes possessing different electrical propensities that are now known as "intrasource separation and selective ionization". However, the chemical principles underlying charge-dependent alterations in ionization efficiencies responsible for the selective ionization of lipid classes are not known with certainty. Herein, we examined the multiple factors that contribute to intrasource separation and selective ionization of lipid classes under optimal instrumental conditions. We demonstrated that many different lipid classes could be selectively ionized in the ion source and that intrasource resolution of distinct molecular constituents was independent of lipid concentration, flow rate, and residual ions under most experimental conditions. Moreover, the presence of alkaline conditions facilitates the selective ionization of many lipid classes through a mechanism independent of the design of the ESI ion source. Collectively, this study provides an empirical foundation for understanding the chemical mechanisms underlying intrasource separation and selective ionization of lipid classes that can potentially be used for global analysis of cellular lipidomes without the need for chromatographic separation.

Algorithms↗

PubMed2006

Lysosomal phospholipase A2 and phospholipidosis.

A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2-/-) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2-/- mice was normal. Lpla2-/- mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2-/- mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2-/- mice. By 1 year of age, Lpla2-/- mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2-/- mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice.

Alleles↗

PubMed2006

Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy.

Dysregulated sphingolipid metabolism causes neuronal cell death and is associated with insulin resistance and diseases. Thus, we hypothesized that diabetes-induced changes in retinal sphingolipid metabolism may contribute to neuronal pathologies in diabetic retinopathy. ESI-MS/MS was used to measure ceramide content and ceramide metabolites in whole retinas after 2, 4, and 8 weeks of streptozotocin-induced diabetes. After 4 and 8 weeks of diabetes, a approximately 30% decrease in total ceramide content was observed, concomitant with a significant approximately 30% increase in glucosylceramide levels in fed diabetic rats compared with their age-matched controls. Acute insulin therapy as well as a short-term lowering of glucose via fasting did not affect the increase in glucosylceramide composition. To assess the putative biological consequences of the increase in glucosylceramide composition, R28 retinal neurons were treated with glucosylceramide synthase inhibitors. Inhibiting glycosphingolipid metabolism increased insulin sensitivity in retinal neurons. Glycosphingolipid inhibitors augmented insulin-stimulated p70 S6kinase activity in the presence of inhibitory concentrations of high glucose or glucosamine. Inhibition of glycosphingolipid synthesis also suppressed glucosamine- and interleukin-1beta-induced death. Consistent with these inhibitor studies, pharmacological accumulation of glycosphingolipids increased activation of the endoplasmic reticulum stress response, a putative modulator of insulin resistance and neuronal apoptosis. It is speculated that an increase in glucosylceramide, and possibly higher-order glycosphingolipids, could contribute to the pathogenesis of diabetic retinopathy by contributing to local insulin resistance, resulting in neuronal cell death. Thus, dysfunctional glycosphingolipid metabolism may contribute to metabolic stress in diabetes, and therapeutic strategies to restore normal sphingolipid metabolism may be a viable approach for treatment of diabetic retinopathy.

Animals↗

PubMed2005

Shotgun lipidomics identifies cardiolipin depletion in diabetic myocardium linking altered substrate utilization with mitochondrial dysfunction.

Diabetic cardiomyopathy is characterized by excessive utilization of fatty acid substrate, diminished glucose transport, and mitochondrial dysfunction. However, the chemical mechanisms linking altered substrate utilization to mitochondrial dysfunction are unknown. Herein, we use shotgun lipidomics and multidimensional mass spectrometry to identify dramatic decreases in the critical mitochondrial inner membrane lipid, cardiolipin, in diabetic murine myocardium (from 7.2 +/- 0.3 nmol/mg of protein in control hearts to 3.1 +/- 0.1 nmol/mg of protein in diabetic myocardium; p < 0.001, n = 7). Moreover, the direct metabolic precursor of cardiolipin, phosphatidylglycerol, was also substantially depleted (2.5 +/- 0.2 nmol/mg of protein in control hearts vs 1.3 +/- 0.1 nmol/mg of protein in diabetic myocardium; p < 0.001, n = 7). Similarly, glycerol 3-phosphate, necessary for the penultimate step in phosphatidylglycerol production, decreased by 58% in diabetic myocardium (from 4.9 +/- 0.9 to 2.2 +/- 0.3 nmol/mg of protein; n = 4). Since Barth's syndrome (a disorder of cardiolipin metabolism) induces mitochondrial dysfunction and cardiomyopathy, and since decreases in cardiolipin content precipitate mitochondrial dysfunction, these results provide a unifying hypothesis linking altered substrate utilization and metabolic flux in diabetic myocardium with altered lipid metabolism, cardiolipin depletion, mitochondrial dysfunction, and resultant hemodynamic compromise.

Animals↗

PubMed2005

Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity: a novel mechanism potentially underlying fatty acid induced insulin resistance.

The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 microM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [(14)C]palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at approximately 35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide (232)VPTPNVSVVDLTRC*R(245) was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.

Amino Acid Sequence↗

PubMed2005

Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study.

The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.

Animals↗

PubMed2005

The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2gamma: identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling.

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2gamma (iPLA2gamma) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA2gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2gamma effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2gamma with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA2gamma. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA2gamma-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2gamma and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA2gamma expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA2gamma-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.

Animals↗

PubMed2005

Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization.

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.

Animals↗

PubMed2005

Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics.

Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.

Animals↗