Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.

Effects of enzymes on elastic modulus of low-density lipoproteins were investigated using atomic force microscopy.

Oxidation of low-density lipoproteins (LDLs) induces development of cardiovascular disease. Recently, reports of studies using atomic force microscopy (AFM) have described that the elastic modulus of metal-induced oxidized LDLs is lower than the modulus before oxidation. However, the mechanisms of change of the elastic modulus have not been well investigated. We postulated that disorder of the LDL structure might decrease the elastic modulus. This study measured the elastic modulus of LDLs before and after enzyme treatment with V8 protease, α-chymotrypsin, and phospholipase A2. After LDLs were obtained from serum by ultracentrifugation, LDLs or enzyme-treated LDLs were physically absorbed. They were crowded on a mica surface. Although V8 protease and α-chymotrypsin did not induce the elastic modulus change, treatment with PLA2 decreased the elastic modulus. The LDL particle size did not change during the enzyme treatment. Results suggest that disordering of the lipid structure of the LDL might contribute to the elastic modulus change. Results show that AFM might be a useful tool to evaluate disorders of complex nanoscale particle structures from lipids and proteins such as lipoproteins.

Identification of molecular species of oxidized triglyceride in plasma and its distribution in lipoproteins.

BACKGROUND: The role of triglycerides carried in the triglyceride-rich lipoproteins (TRL) in the progression of atherosclerosis is uncertain. Identification of oxidized triglycerides and its possible association with atherosclerosis were largely ignored. Here we applied mass spectrometric approach to detect and identify triglyceride hydroperoxides (TGOOH) in human plasma and lipoproteins. METHODS: EDTA plasma was collected from healthy human volunteers (n=9) after 14-16 h of fasting. Very low-density lipoprotein (VLDL) (d<1.006) and intermediate-density lipoprotein (IDL) (d=1.006-1.019) were isolated from the plasma (n=6) by sequential ultracentrifugation in KBr, followed by the isolation of LDL and high-density lipoprotein (HDL) using size-exclusion high-performance liquid chromatography (HPLC). Total lipids from the plasma and isolated lipoproteins were extracted, and analyzed for the detection and identification of TGOOH using liquid chromatography/LTQ ion trap Orbitrap mass spectrometry. All the processes, from specimen collection to the mass spectrometric analysis, were carried out at 4 °C in the presence of antioxidant to prevent oxidation of lipoproteins. RESULTS: We identified 11 molecular species of TGOOH in either plasma or VLDL and IDL, of which TGOOH-18:1/18:2/16:0, TGOOH-18:1/18:1/16:0, TGOOH-16:0/18:2/16:0, TGOOH-18:1/18:1/18:1, and TGOOH-16:0/20:4/16:0 were most dominant. These TGOOH molecules are carried by TRL but not by LDL and HDL. Mean concentration of TGOOH in plasma, VLDL and IDL were, respectively, 56.1 ± 25.6, 349.8 ± 253.6 and 512.5 ± 173.2 µmol/mol of triglycerides. CONCLUSIONS: This is the first report to identify several molecular species of oxidized triglycerides in TRL. Presence of oxidized triglyceride may contribute to the atherogenicity of TRL. Further work is needed to elucidate the association of the oxidized triglyceride in atherosclerosis.

Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer.

Herein, we represent a simple method for the detection and characterization of molecular species of triacylglycerol monohydroperoxides (TGOOH) in biological samples by use of reversed-phase liquid chromatography with a LTQ Orbitrap XL mass spectrometer (LC/LTQ Orbitrap) via an electrospray ionization source. Data were acquired using high-resolution, high-mass accuracy in Fourier-transform mode. Platform performance, related to the identification of TGOOH in human lipoproteins and plasma, was estimated using extracted ion chromatograms with mass tolerance windows of 5 ppm. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO4 to generate oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No TGOOH molecular species were detected in the nLDL and nHDL, whereas 11 species of TGOOH molecules were detected in the oxLDL and oxHDL. In positive-ion mode, TGOOH was found as [M + NH4](+). In negative-ion mode, TGOOH was observed as [M + CH3COO](-). TGOOH was more easily ionized in positive-ion mode than in negative-ion mode. The LC/LTQ Orbitrap method was applied to human plasma and three molecular species of TGOOH were detected. The limit of detection is 0.1 pmol (S/N = 10:1) for each synthesized TGOOH.

Cholesterol sulfate induces expression of the skin barrier protein filaggrin in normal human epidermal keratinocytes through induction of RORα.

Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased RORα expression. Further studies are required to fully elucidate the mechanisms involved.

Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by use of an Orbitrap mass spectrometer.

Oxidation of cholesteryl esters in lipoproteins by reactive oxygen species yields cholesteryl ester hydroperoxides (CEOOH). In this study, we developed a novel method for identification and characterization of CEOOH molecules in human lipoproteins by use of reversed-phase liquid chromatography with an hybrid linear ion trap-Orbitrap mass spectrometer (LC-LTQ Orbitrap). Electrospray ionization tandem mass spectrometric analysis was performed in both positive-ion and negative-ion modes. Identification of CEOOH molecules was completed by use of high-mass-accuracy (MA) mass spectrometric data obtained by using the spectrometer in Fourier-transform (FT) mode. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO(4), furnishing oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No CEOOH molecules were detected in the nLDL and the nHDL, whereas six CEOOH molecules were detected in the oxLDL and the oxHDL. In positive-ion mode, CEOOH was detected as [M + NH(4)](+) and [M + Na](+) ions. In negative-ion mode, CEOOH was detected as [M + CH(3)COO](-) ions. CEOOH were more easily ionized in positive-ion mode than in negative-ion mode. The LC-LTQ Orbitrap method was applied to human plasma and six species of CEOOH were detected. The limit of detection was 0.1 pmol (S/N = 5:1) for synthesized CEOOH.

Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry.

1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8-12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n = 10) with CuSO(4) for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.

Oxidative Stress Linked Organ Lipid Hydroperoxidation and Dysregulation in Mouse Model of Nonalcoholic Steatohepatitis: Revealed by Lipidomic Profiling of Liver and Kidney.

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.

The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) Prevents Enterocyte Cell Death under Oxygen-Dissolving Cold Conditions through Polyphyletic Antioxidant Actions.

Cold preservation in University of Wisconsin (UW) solution is not enough to maintain the viability of the small intestine, due to the oxidative stress. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual properties to reduce oxidative stress, radical scavenging, and antioxidant protein induction, in other cells. This study was designed to determine whether DHMBA reduces cold preservation injury of enterocytes, and to identify the effector site. Enterocytes were subjected to 48-h cold preservation under atmosphere in UW solution (±DHMBA), and then returned to normal culture to replicate reperfusion of the small intestine after cold preservation. At the end of cold preservation (ECP) and at 1, 3, 6, and 72 h after rewarming (R1h, R3h, R6h, and R72h), we evaluated cell function and the injury mechanism. The results showed that DHMBA protected mitochondrial function mainly during cold preservation, and suppressed cell death after rewarming, as shown by the MTT, ATP, mitochondrial membrane potential, LDH, and lipid peroxidation assays, together with enhanced survival signals (PI3K, Akt, p70S6K) and induction of antioxidant proteins (HO-1, NQO-1, TRX-1). We found that DHMBA mitigates the cold-induced injury of enterocytes by protecting the mitochondria through direct and indirect antioxidative activities.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs' plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

A Simple and Efficient Method for Synthesis of sn-Glycero-Phosphoethanolamine.

An efficient three-step strategy for the convenient synthesis of Sn-glycero-3-phosphoethanolamine (GroPEtn) from a commercially available 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) is reported. Direct hydrolysis of DPPE produces a complex inseparable mixture, hence a protection and deprotection strategy is employed to prepare GroPEtn. The primary amine of DPPE is protected with a highly stable acid-labile trityl group, followed by strong base hydrolysis of N-trityl-DPPE gives N-trityl-GroPEtn. Further a mild, rapid, and efficient deprotection method is established using trifluoroacetic acid to remove N-trityl moiety, affords GroPEtn as a single product. This is the first semisynthetic approach and efficient method to produce GroPEtn with a total yield of 66% in three steps. GroPEtn did not show any cytotoxicity against human kidney (HK-2) cells and reporter gene assay for activation of Keap1-Nrf2-mediated antioxidant defense mechanism showed no significant effects.

Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes.

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 µm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0-87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics.

Flazin as a Promising Nrf2 Pathway Activator.

Flazin is a ß-carboline-derived alkaloid found in Japanese fermented foods. Here, the potential of flazin as an antioxidant food was studied with particular reference to its effect on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system in human hepatocytes (C3A). Flazin and flazin analogues including the decarboxylated derivative perlolyrine were chemically synthesized and compared with each other and with chlorogenic acid and curcumin. Among these compounds, flazin showed the lowest cytotoxicity (IC50 < 500 µM) and the highest capacity to activate the Keap1-Nrf2 system. It provided the largest (>3-fold of the control) cytoprotection ability against a pro-oxidant, although its radical absorbance capacity was relatively low. Flazin increased the expressions of Nrf2-dependent phase II enzyme genes and their products (NQO1, GSTP, and GSH proteins). The strong cytoprotection ability of flazin associated with low log P (0-3) is shared by sulforaphane and 3,5-dihydroxy-4-methoxybenzyl alcohol, suggesting the potential value of flazin and flazin-rich foods for the prevention of oxidation-related health disorders.

Regulation of SULT2B1a (pregnenolone sulfotransferase) expression in rat C6 glioma cells: relevance of AMPA receptor-mediated NO signaling.

The neurosteroid pregnenolone sulfate (PREGS), which is synthesized in glial cells, plays a significant role in learning and memory performance. The aim of this study was to investigate the regulation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes the conversion of pregnenolone to PREGS, using the rat C6 glioma cell line. Rat C6 glioma cells expressed the SULT2B1a isoform, which sulfonates pregnenolone, but, neither the SULT2B1b isoform, which catalyzes cholesterol, nor the prototypical steroid sulfotransferase SULT2A1 were expressed in these cells. Increasing concentrations of l-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression; however, neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide also inhibited SULT2B1a expression. Attenuation of SULT2B1a expression by L-glutamic acid was reversed by the selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and partially reversed by the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI). Induction of inducible NOS by TNF-alpha in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression; this was partially reversed by the specific inducible NOS inhibitor N(6)-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Furthermore, exposure to exogenous NO donors inhibited SULT2B1a mRNA expression, and exposure to sodium nitroprusside, LPS/TNF-alpha and L-glutamic acid in combination with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.

Serotonin (5-HT) receptor subtypes mediate specific modes of 5-HT-induced signaling and regulation of neurosecretion in gonadotropin-releasing hormone neurons.

Serotonin (5-HT), the endogenous nonselective 5-HT receptor agonist, activates the inositol 1,4,5-triphosphate/calcium (InsP3/Ca2+) signaling pathway and exerts both stimulatory and inhibitory actions on cAMP production and GnRH release in immortalized GnRH neurons. The high degree of similarity between the signaling and secretory responses elicited by GnRH and 5-HT prompted us to target specific 5-HT receptor subtypes to deconvolute the complex actions of these agonists on signal transduction and GnRH release. Specific mRNA transcripts for 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7 were identified in immortalized GnRH neurons (GT1-7). The rate of firing of spontaneous action potentials (APs) by hypothalamic GnRH neurons and cAMP production and pulsatile GnRH release in GT17 cells were profoundly inhibited during activation of the Gi-coupled 5-HT1A receptor. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor increased the rate of firing of spontaneous APs, stimulated InsP3 production and caused a delayed increase in GnRH release. Selective activation of the Gs-coupled 5-HT4 receptor also increased the rate of firing of APs, stimulated cAMP production, and caused a sustained and robust increase in GnRH release. The ability of 5-HT receptor subtypes expressed in GnRH neurons to activate single or multiple G proteins in a time- and dose-dependent manner differentially regulates the phospholipase C/InsP3/Ca2+, and adenylyl cyclase/cAMP signaling pathways, and thereby regulates the frequency and amplitude of pulsatile GnRH release. This process, in conjunction with the modulation of spontaneous electrical activity of the GnRH neuron, contributes to the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro.

Cloning, characterization and tissue expression of rat SULT2B1a and SULT2B1b steroid/sterol sulfotransferase isoforms: divergence of the rat SULT2B1 gene structure from orthologous human and mouse genes.

cDNAs for rat SULT2B1 steroid/sterol sulfotransferase isoforms were cloned, and the encoded proteins overexpressed, purified and characterized. The rat SULT2B1a isoform avidly sulfonates pregnenolone but poorly utilizes cholesterol as a substrate, whereas cholesterol is more efficiently sulfonated than pregnenolone by the SULT2B1b isoform; on the other hand, neither isoform sulfonates dehydroepiandrosterone to any significant degree. Real-time PCR revealed that SULT2B1a was only expressed in brain and testis, whereas SULT2B1b was mainly expressed in skin, intestine and kidney. The SULT2B1 gene is unique among steroid/sterol sulfotransferase genes in that it encodes for two isoforms as a result of an alternative exon I. Interestingly, whereas the orthologous human and mouse SULT2B1 gene structures are identical, the rat SULT2B1 gene structure diverges. Similar to human and mouse SULT2B1 genes the rat SULT2B1 gene consists of an alternative exon I; however, as a result of exonic rearrangement, the genic locations of exons IA and IB are reversed in the rat gene. Where exon IA is located downstream of exon IB in the human and mouse SULT2B1 genes, in the rat SULT2B1 gene exon IA is located upstream of exon IB. Furthermore, unlike the case with human and mouse SULT2B1 genes where differential splicing is necessitated since a portion of exon IA is fused with exon IB to complete the SULT2B1b mRNA, this step is not required with the rat gene.

Imaging Mass Spectrometry Reveals a Decrease of Cardiolipin in the Kidney of NASH Model Mice.

Non-alcoholic steatohepatitis (NASH) can be complicated with chronic kidney disease (CKD). In this study, changes in the distribution of biomolecules in the kidney were studied in NASH model mice with the use of imaging mass spectrometry (IMS). The mass spectra and ion images of IMS showed that the signals of cardiolipin (CL) species were decreased in the kidney cortex of the NASH mice. The decrease of CL might therefore suggest the kidney involvement of NASH.

Rapid tin-mediated access to a lysophosphatidylethanolamine (LPE) library: Application to positional LC/MS analysis for hepatic LPEs in non-alcoholic steatohepatitis model mice.

Even though lysophospholipids have attracted much interest in recent years on account of their unique bioactivity, research related to lysophospholipids is usually hampered by problems associated with standard sample preparation and discrimination of regioisomers. Herein, we demonstrate a quick tin-chemistry-based synthetic route to lysophosphatidylethanolamines (LPEs) and its application in the positional analysis of hepatic LPEs in non-alcoholic steatohepatitis (NASH) model mice. We found that the preference of hepatic LPE regioisomer largely depends on the unsaturation of acyl chain in both control and NASH model mice. In addition, hepatic C18:2-LPE and C20:5-LPE levels were significantly lower in the NASH model mice than those in the control. The LC/MS technique based on the library of LPE regioisomers allows an accurate observation of hepatic LPE metabolism and might provide useful information to elucidate yet ambiguous pathogenesis of NASH.

Synthesis of (2ß,3α,6-²H3cholesteryl linoleate and cholesteryl oleate as internal standards for mass spectrometry.

The accurate analysis of trace component in complex biological matrices requires the use of reliable standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled derivatives of the analyte molecules are the most appropriate internal standards. We report here the synthesis of (2ß,3α,6-(2)H3)cholesteryl linoleate and oleate containing three non-exchangeable deuterium in the steroid ring. The principal reactions used were: (1) trans diaxial opening of 2α,3α-epoxy-6-oxo-5α-cholestane with LiAlD4 and subsequent oxidation of the resulting (2ß,6α-(2)H2)-3α,6ß-diol with Jones' reagent, followed by reduction of the resulting (2ß-(2)H)-3,6-dione with NaBD4 leading to the (2ß,3α,6α-(2)H3)-3ß,6ß-dihydroxy-5α-cholestane, (2) selective protection of the 3ß-hydroxy group as the tert-butyldimethylsilyl ether, (3) dehydration of the 6ß-hydroxy group with POCl3 and removal of tert-butyldimethylsilyloxy groups with 5M HCl in acetone, and (4) esterification of the resultant (2ß,3α,6-(2)H3)cholesterol with linoleic and oleic acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The isotopic purity was found to be satisfactory by mass spectrometry, and nuclear magnetic resonance properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.

Expression of cholesterol sulfotransferase (SULT2B1b) in human platelets.

BACKGROUND: Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. METHODS AND RESULTS: We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4 degrees C but substantially diminished over a period of 4 hours at 37 degrees C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. CONCLUSIONS: Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37 degrees C is subject to regulation by the apoA-I component of HDL.