Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Inhibition of macrophage fatty acid ß-oxidation exacerbates palmitate-induced inflammatory and endoplasmic reticulum stress responses.

AIMS/HYPOTHESIS: Saturated fatty acids (SFAs) such as palmitate activate inflammatory pathways and elicit an endoplasmic reticulum (ER) stress response in macrophages, thereby contributing to the development of insulin resistance linked to the metabolic syndrome. This study addressed the question of whether or not mitochondrial fatty acid ß-oxidation (FAO) affects macrophage responses to SFA. METHODS: We modulated the activity of carnitine palmitoyl transferase 1A (CPT1A) in macrophage-differentiated THP-1 monocytic cells using genetic or pharmacological approaches, treated the cells with palmitate and analysed the proinflammatory and ER stress signatures. RESULTS: To inhibit FAO, we created THP-1 cells with a stable knockdown (KD) of CPT1A and differentiated them to macrophages. Consequently, in CPT1A-silenced cells FAO was reduced. CPT1A KD in THP-1 macrophages increased proinflammatory signalling, cytokine expression and ER stress responses after palmitate treatment. In addition, in human primary macrophages CPT1A KD elevated palmitate-induced inflammatory gene expression. Pharmacological inhibition of FAO with etomoxir recapitulated the CPT1A KD phenotype. Conversely, overexpression of a malonyl-CoA-insensitive CPT1A M593S mutant reduced inflammatory and ER stress responses to palmitate in THP-1 macrophages. Macrophages with a CPT1A KD accumulated diacylglycerols and triacylglycerols after palmitate treatment, while ceramide accumulation remained unaltered. Moreover, lipidomic analysis of ER phospholipids revealed increased palmitate incorporation into phosphatidylethanolamine and phosphatidylserine classes associated with the CPT1A KD. CONCLUSIONS/INTERPRETATION: Our data indicate that FAO attenuates inflammatory and ER stress responses in SFA-exposed macrophages, suggesting an anti-inflammatory impact of drugs that activate FAO.

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

Analysis of Diacylglycerol Molecular Species in Cellular Lipid Extracts by Normal-Phase LC-Electrospray Mass Spectrometry.

The quantitative determination of 48 molecular species of regioisomeric diacylglycerols has been made in a single analysis of an extract of bone marrow derived macrophages. The analytical procedure involves solvent extraction of neutral lipids, including diacylglycerols, derivatization of free hydroxyl moieties as 2,4-difluorophenyl urethane, and analysis by normal phase liquid chromatography-tandem mass spectrometry. The derivatization step not only prevents fatty acyl group migration, thus allowing determination of both 1,2- and 1,3-diacylglycerols, but also yields species that are sensitively and uniquely determined by constant neutral loss mass spectrometry. The method also detected monoacylglycerols, which were characterized by unique retention time and collisional spectra, and were present in mouse bone marrow derived macrophage extracts.

Glycerolipid and cholesterol ester analyses in biological samples by mass spectrometry.

Neutral lipids are a diverse family of hydrophobic biomolecules that have important roles in cellular biochemistry of all living species but have in common the property of charge neutrality. A large component of neutral lipids is the glycerolipids composed of triacylglycerols, diacylglycerols, and monoacylglycerols that can serve as cellular energy stores as well as signaling molecules. Another abundant lipid class in many cells is the cholesterol esters that are on one hand sterols and the other fatty acyl lipids, but in either case are neutral lipids involved in cholesterol homeostasis and transport in the blood. The analysis of these molecules in the context of lipidomics remains challenging because of their charge neutrality and the complex mixtures of molecular species present in cells. Various techniques have been used to ionize these neutral lipids prior to mass spectrometric analysis including electron ionization, atmospheric chemical ionization, electrospray ionization and matrix assisted laser desorption/ionization. Various approaches to deal with the complex mixture of molecular species have been developed including shotgun lipidomics and chromatographic-based separations such as gas chromatography, reversed phase liquid chromatography, and normal phase liquid chromatography. Several applications of these approaches are discussed. .

Sources and distribution of organic compounds using passive samplers in Lake Mead national recreation area, Nevada and Arizona, and their implications for potential effects on aquatic biota.

The delineation of lateral and vertical gradients of organic contaminants in lakes is hampered by low concentrationsand nondetection of many organic compounds in water. Passive samplers (semipermeable membrane devices [SPMDs] and polar organic chemical integrative samplers [POCIS]) are well suited for assessing gradients because they can detect synthetic organic compounds (SOCs) at pg L(-1) concentrations. Semi-permeable membrane devices and POCIS were deployed in Lake Mead, at two sites in Las Vegas Wash, at four sites across Lake Mead, and in the Colorado River downstream from Hoover Dam. Concentrations of hydrophobic SOCs were highest in Las Vegas Wash downstream from waste water and urban inputs and at 8 m depth in Las Vegas Bay (LVB) where Las Vegas Wash enters Lake Mead. The distribution of hydrophobic SOCs showed a lateral distribution across 10 km of Lake Mead from LVB to Boulder Basin. To assess possible vertical gradient SOCs, SPMDs were deployed at 4-m intervals in 18 m of water in LVB. Fragrances and legacy SOCs were found at the greatest concentrations at the deepest depth. The vertical gradient of SOCs indicated that contaminants were generally confined to within 6 m of the lake bottom during the deployment interval. The high SOC concentrations, warmer water temperatures, and higher total dissolved solids concentrations at depth are indicative of a plume of Las Vegas Wash water moving along the lake bottom. The lateral and vertical distribution of SOCs is discussed in the context of other studies that have shown impaired health of fish exposed to SOCs.

Lipidomics reveals a remarkable diversity of lipids in human plasma.

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Identification of methyl triclosan and halogenated analogues in male common carp (Cyprinus carpio) from Las Vegas Bay and semipermeable membrane devices from Las Vegas Wash, Nevada.

Methyl triclosan and four halogenated analogues have been identified in extracts of individual whole-body male carp (Cyprinus carpio) tissue that were collected from Las Vegas Bay, Nevada, and Semipermeable Membrane Devices (SPMD) that were deployed in Las Vegas Wash, Nevada. Methyl triclosan is believed to be the microbially methylated product of the antibacterial agent triclosan (2, 4, 4'-trichloro-4-hydroxydiphenyl ether, Chemical Abstract Service Registry Number 3380-34-5, Irgasan DP300). The presence of methyl triclosan and four halogenated analogues was confirmed in SPMD extracts by comparing low- and high-resolution mass spectral data and Kovats retention indices of methyl triclosan with commercially obtained triclosan that was derivatized to the methyl ether with ethereal diazomethane. The four halogenated analogues of methyl triclosan detected in both whole-body tissue and SPMD extracts were tentatively identified by high resolution mass spectrometry. Methyl triclosan was detected in all 29 male common carp from Las Vegas Bay with a mean concentration of 596 microg kg(-1) wet weight (ww) which is more than an order of magnitude higher than previously reported concentrations in the literature. The halogenated analogs were detected less frequently (21%-76%) and at much lower concentrations (<51 microg kg(-1) ww). None of these compounds were detected in common carp from a Lake Mead reference site in Overton Arm, Nevada.