Dynamics of arachidonic acid mobilization by inflammatory cells

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.     

Lipidomic approaches to the study of phospholipase A2-regulated phospholipid fatty acid incorporation and remodeling

The distribution of fatty acids among cellular glycerophospholipids is finely regulated by the CoA-dependent acylation of lysophospholipids followed by transacylation reactions. Arachidonic acid is the fatty acid precursor of a wide family of bioactive compounds called the eicosanoids, with key roles in innate immunity and inflammation. Because availability of free AA constitutes a rate-limiting step in the generation of eicosanoids by mammalian cells, many studies have been devoted to characterize the processes of arachidonate liberation from phospholipids by phospholipase A₂s and its re-incorporation and further remodeling back into phospholipids by acyltransferases and transacylases. These studies have traditionally been conducted by using radioactive precursors which do not allow the identification of the phospholipid molecular species involved in these processes. Nowadays, lipidomic approaches utilizing mass spectrometry provide a new frame for the analysis of unique phospholipid species involved in fatty acid release and phospholipid incorporation and remodeling. This review focuses on the mass spectrometry techniques applied to the study of phospholipid fatty acid trafficking and the recent advances that have been achieved by the use of this technique.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Regulation of rat brain polyunsaturated fatty acid (PUFA) metabolism during graded dietary n-3 PUFA deprivation

Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation may elucidate dietary regulation of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (α-LNA). Compared with control diet (4.6% α-LNA), plasma DHA fell significantly at 1.7% dietary α-LNA while brain DHA remained unchanged down to 0.8% α-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA₂ and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA₂, sPLA₂ and COX-2 activities were increased at or below 0.8% dietary α-LNA, possibly in response to elevated brain DPAn-6. In summary, homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary DHA despite reduced plasma DHA, when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA₂ and COX-1 activities may reduce brain DHA loss.     

Purification and characterization of a cytosolic Ca<Superscript>2+</Superscript>-independent phospholipase A<Subscript>2</Subscript> from bovine brain

The Ca²⁺-independent phospholipase A₂ (iPLA₂) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA₂ from bovine brain. iPLA₂ was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA₂ activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on twodimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃), Triton X-100, iron, and Ca²⁺. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA₂, and adenosine triphosphate (ATP). The spot with the iPLA₂ activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA₂.     

β-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E2 synthesis in rat hepatocytes–Kupffer cells

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E₂ (PGE₂) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE₂ synthesis in a rat hepatocyte–Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) β-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE₂ synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased β-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE₂ synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.     

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca²⁺-independent phospholipase A₂ (iPLA₂), Ca²⁺-independent plasmalogen PLA₂ or secretory PLA_{2 (sPLA2)}, but not by Ca²⁺-dependent cytosolic PLA₂ (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca²⁺ into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca²⁺-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca²⁺ and of cPLA₂. DHA signaling may be mediated by iPLA₂, plasmalogen PLA₂, or other enzymes insensitive to low concentrations of Ca²⁺. Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.     

Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species

Mitochondrial cardiolipin undergoes extensive remodeling of its acyl groups to generate uniformly substituted species, such as tetralinoleoyl-cardiolipin, but the mechanism of this remodeling has not been elucidated, except for the fact that it requires tafazzin. Here we show that purified recombinant Drosophila tafazzin exchanges acyl groups between cardiolipin and phosphatidylcholine by a combination of forward and reverse transacylations. The acyl exchange is possible in the absence of phospholipase A₂ because it requires only trace amounts of lysophospholipids. We show that purified tafazzin reacts with various phospholipid classes and with various acyl groups both in sn-1 and sn-2 position. Expression studies in Sf9 insect cells suggest that the effect of tafazzin on cardiolipin species is dependent on the cellular environment and not on enzymatic substrate specificity. Our data demonstrate that tafazzin catalyzes general acyl exchange between phospholipids, which raises the question whether pattern formation in cardiolipin is the result of the equilibrium distribution of acyl groups between multiple phospholipid species.     

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca^2 + concentration ([Ca^2 +]_i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca^2 +]_i. Chelating Ca^2 + ions in the extracellular medium suppressed the intracellular Ca^2 + signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca^2 +- and P2X7-independent transport mechanism in macrophages.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.     

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.     

Regulation of lung surfactant phospholipid synthesis and metabolism

The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.     

A mathematical model for the determination of steady-state cardiolipin remodeling mechanisms using lipidomic data

Technical advances in lipidomic analysis have generated tremendous amounts of quantitative lipid molecular species data, whose value has not been fully explored. We describe a novel computational method to infer mechanisms of de novo lipid synthesis and remodeling from lipidomic data. We focus on the mitochondrial-specific lipid cardiolipin (CL), a polyglycerol phospholipid with four acyl chains. The lengths and degree of unsaturation of these acyl chains vary across CL molecules, and regulation of these differences is important for mitochondrial energy metabolism. We developed a novel mathematical approach to determine mechanisms controlling the steady-state distribution of acyl chain combinations in CL . We analyzed mitochondrial lipids from 18 types of steady-state samples, each with at least 3 replicates, from mouse brain, heart, lung, liver, tumor cells, and tumors grown in vitro. Using a mathematical model for the CL remodeling mechanisms and a maximum likelihood approach to infer parameters, we found that for most samples the four chain positions have an independent and identical distribution, indicating they are remodeled by the same processes. Furthermore, for most brain samples and liver, the distribution of acyl chains is well-fit by a simple linear combination of the pools of acyl chains in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). This suggests that headgroup chemistry is the key determinant of acyl donation into CL, with chain length/saturation less important. This canonical remodeling behavior appears damaged in some tumor samples, which display a consistent excess of CL molecules having particular masses. For heart and lung, the "proportional incorporation" assumption is not adequate to explain the CL distribution, suggesting additional acyl CoA-dependent remodeling that is chain-type specific. Our findings indicate that CL remodeling processes can be described by a small set of quantitative relationships, and that bioinformatic approaches can help determine these processes from high-throughput lipidomic data.     

Dynamic simulation of cardiolipin remodeling: greasing the wheels for an interpretative approach to lipidomics

Cardiolipin is a class of mitochondrial specific phospholipid, which is intricately involved in mitochondrial functionality. Differences in cardiolipin species exist in a variety of tissues and diseases. It has been demonstrated that the cardiolipin profile is a key modulator of the functions of many mitochondrial proteins. However, the chemical mechanism(s) leading to normal and/or pathological distribution of cardiolipin species remain elusive. Herein, we describe a novel approach for investigating the molecular mechanism of cardiolipin remodeling through a dynamic simulation. This approach applied data from shotgun lipidomic analyses of the heart, liver, brain, and lung mitochondrial lipidomes to model cardiolipin remodeling, including relative content, regiospecificity, and isomeric composition of cardiolipin species. Generated cardiolipin profiles were nearly identical to those determined by shotgun lipidomics. Importantly, the simulated isomeric compositions of cardiolipin species were further substantiated through product ion analysis. Finally, unique enzymatic activities involved in cardiolipin remodeling were assessed from the parameters used in the dynamic simulation of cardiolipin profiles. Collectively, we described, verified, and demonstrated a novel approach by integrating both lipidomic analysis and dynamic simulation to study cardiolipin biology. We believe this study provides a foundation to investigate cardiolipin metabolism and bioenergetic homeostasis in normal and disease states.     

Statistical analysis of the processes controlling choline and ethanolamine glycerophospholipid molecular species composition

The regulation and maintenance of the cellular lipidome through biosynthetic, remodeling, and catabolic mechanisms are critical for biological homeostasis during development, health and disease. These complex mechanisms control the architectures of lipid molecular species, which have diverse yet highly regulated fatty acid chains at both the sn1 and sn2 positions. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serve as the predominant biophysical scaffolds in membranes, acting as reservoirs for potent lipid signals and regulating numerous enzymatic processes. Here we report the first rigorous computational dissection of the mechanisms influencing PC and PE molecular architectures from high-throughput shotgun lipidomic data. Using novel statistical approaches, we have analyzed multidimensional mass spectrometry-based shotgun lipidomic data from developmental mouse heart and mature mouse heart, lung, brain, and liver tissues. We show that in PC and PE, sn1 and sn2 positions are largely independent, though for low abundance species regulatory processes may interact with both the sn1 and sn2 chain simultaneously, leading to cooperative effects. Chains with similar biochemical properties appear to be remodeled similarly. We also see that sn2 positions are more regulated than sn1, and that PC exhibits stronger cooperative effects than PE. A key aspect of our work is a novel statistically rigorous approach to determine cooperativity based on a modified Fisher's exact test using Markov Chain Monte Carlo sampling. This computational approach provides a novel tool for developing mechanistic insight into lipidomic regulation.     

Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease.