The Journal of Lipid Research current issue

[Thematic Reviews] Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism

Reue, K., Brindley, D. N. — 2008-11-14

Phosphatidate phosphatase-1 (PAP1) enzymes have a key role in glycerolipid synthesis through the conversion of phosphatidate to diacylglycerol, the immediate precursor of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. PAP1 activity in mammals is determined by the lipin family of proteins, lipin-1, lipin-2, and lipin-3, which each have distinct tissue expression patterns and appear to have unique physiological functions. In addition to its role in glycerolipid synthesis, lipin-1 also operates as a transcriptional coactivator, working in collaboration with known nuclear receptors and coactivators to modulate lipid metabolism gene expression. The requirement for different lipin activities in vivo is highlighted by the occurrence of lipodystrophy, insulin resistance, and neuropathy in a lipin-1-deficient mutant mouse strain. In humans, variations in lipin-1 expression levels and gene polymorphisms are associated with insulin sensitivity, metabolic rate, hypertension, and risk for the metabolic syndrome. Furthermore, critical mutations in lipin-2 result in the development of an inflammatory disorder in human patients. A key goal of future studies will be to further elucidate the specific roles and modes of regulation of each of the three lipin proteins in key metabolic processes, including triglyceride and phospholipid synthesis, fatty acid metabolism, and insulin sensitivity.

[Research Articles] Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study

2008-11-14

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.

[Research Articles] Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

2008-11-14

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPAR) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

[Research Articles] Cholesterol accumulation and diabetes in pancreatic {beta}-cell-specific SREBP-2 transgenic mice: a new model for lipotoxicity

2008-11-14

To determine the role of cholesterol synthesis in pancreatic β-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in β-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in β-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in β-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of β-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in β-cells caused severe diabetes by loss of β-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of β-cell function.

[Research Articles] HRASLS3 is a PPAR{gamma}-selective target gene that promotes adipocyte differentiation

Hummasti, S., Hong, C., Bensinger, S. J., Tontonoz, P. — 2008-11-14

The prevalence of obesity and its associated metabolic diseases worldwide has focused attention on understanding the mechanisms underlying adipogenesis. The nuclear receptor PPAR has emerged as a central regulator of adipose tissue function and formation. Despite the identification of numerous PPAR targets involved in a range of processes, from lipid droplet formation to adipokine secretion, information is still lacking on targets downstream of PPAR that directly affect fat cell differentiation. Here we identify HRASLS3 as a novel PPAR regulated gene with a role in adipogenesis. HRASLS3 expression increases during the differentiation of preadipocyte cell lines and is highly expressed in white and brown adipose tissue in mice. HRASLS3 expression is induced by PPAR ligands in preadipocyte cell lines as well in adipose tissue in vivo. We demonstrate that the HRASLS3 promoter contains a functional PPAR response element and is a direct target for regulation by PPAR/RXR heterodimers. Finally, we show that overexpression of HRASLS3 augments PPAR-driven lipid accumulation and adipogenesis, whereas siRNA-mediated knockdown of HRASLS3 expression decreases differentiation. Together, these results identify HRASLS3 as one of the downstream effectors of PPAR action in adipogenesis.

[Research Articles] Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer

Kiebish, M. A., Han, X., Cheng, H., Chuang, J. H., Seyfried, T. N. — 2008-11-14

Otto Warburg first proposed that cancer originated from irreversible injury to mitochondrial respiration, but the structural basis for this injury has remained elusive. Cardiolipin (CL) is a complex phospholipid found almost exclusively in the inner mitochondrial membrane and is intimately involved in maintaining mitochondrial functionality and membrane integrity. Abnormalities in CL can impair mitochondrial function and bioenergetics. We used shotgun lipidomics to analyze CL content and composition in highly purified brain mitochondria from the C57BL/6J (B6) and VM/Dk (VM) inbred strains and from subcutaneously grown brain tumors derived from these strains to include an astrocytoma and ependymoblastoma (B6 tumors), a stem cell tumor, and two microgliomas (VM tumors). Major abnormalities in CL content or composition were found in all tumors. The compositional abnormalities involved an abundance of immature molecular species and deficiencies of mature molecular species, suggesting major defects in CL synthesis and remodeling. The tumor CL abnormalities were also associated with significant reductions in both individual and linked electron transport chain activities. A mathematical model was developed to facilitate data interpretation. The implications of our findings to the Warburg cancer theory are discussed.

[Research Articles] Strong activation of cyclooxygenase I and II catalytic activity by dietary bioflavonoids

Bai, H.-W., Zhu, B. T. — 2008-11-14

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins (PGs), thromboxanes, and hydroxyeicosatetraenoic acids. In the present study, we investigated several dietary bioflavonoids for their ability to modulate the catalytic activity of COX I and II in vitro and also in cultured cells. We found that some of them are the most powerful direct stimulators of the catalytic activity of COX I and II known to date, increasing the formation of prostaglandin products in vitro by up to 11-fold over the controls. This stimulatory effect of bioflavonoids is enzyme specific because none of them stimulates the catalytic activity of a number of lipooxygenases tested. Compared with phenol, a prototypical COX stimulator commonly used in vitro, the naturally occurring bioflavonoids are up to 29 times more efficacious in stimulating the COX activity. Additional studies using intact cells in culture showed that some of the dietary compounds that were active in the biochemical assays also activated the formation of PGE₂ (a representative PG) when they were present at 0.01 to 1 µM concentrations. The stimulatory effect of dietary compounds on COX-mediated PG formation is far more potent in intact cells than in the in vitro assays. Mechanistically, bioflavonoids mainly acted to slow down the suicidal inactivation of the COX enzymes, but they did not appear to reactivate the inactivated enzymes. The finding of this study suggests that some of the bioflavonoids likely will serve as the naturally occurring cofactors for the COX enzymes in humans.

[Research Articles] Ceramide accelerates ultraviolet-induced MMP-1 expression through JAK1/STAT-1 pathway in cultured human dermal fibroblasts

Kim, S., Kim, Y., Lee, Y., Chung, J. H. — 2008-11-14

Ultraviolet (UV) irradiation accelerates formation of ceramide through hydrolysis of sphingomyelin and de novo synthesis. Here, we investigated the effects of ceramide on UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Our results showed that acidic-sphingomyelinase (aSMase) and MMP-1 mRNA expression were increased by UV irradiation. Treatment of D609 (aSMase inhibitor) decreased the level of basal and UV-induced MMP-1 expression. On the other hand, basal and UV-induced MMP-1 expression was increased through induction of intracellular ceramide by D-MAPP, a ceramidase inhibitor. Our results also showed that MMP-1 protein expression was dose-dependently increased by C₂-ceramide or SMase treatment. The activation of ceramide pathway by C₂-ceramide enhanced phosphorylation of signal transducer and activators of transcription-1 (STAT-1), whereas ceramide-induced MMP-1 expression was potently prevented by piceatannol; Janus kinase (JAK1) inhibtor; and WHI-P131, a specific inhibitor of JAK3; but not by AG490, JAK 2 inhbitor, in human dermal fibroblasts. We also found that UV induced the phosphorylation of STAT-1, and UV-induced MMP-1 expression was significantly decreased by JAK1 inhibitor, piceatannol. Overall, we demonstrate that induction of intracellular ceramide by UV may activate MMP-1 gene expression via JAK1/STAT-1 pathway. Therefore, we suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting MMP-1 expression, which is a causing gene of skin aging.

[Research Articles] Multiple genetic variants along candidate pathways influence plasma high-density lipoprotein cholesterol concentrations

2008-11-14

The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR_q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR_q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR_q = 0.221 for rs3135506; P = 0.018, FDR_q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further.

[Research Articles] Regulation of the human thromboxane A2 receptor gene by Sp1, Egr1, NF-E2, GATA-1, and Ets-1 in megakaryocytes

Gannon, A. M., Kinsella, B. T. — 2008-11-14

The and β isoforms of the human thromboxane A₂ (TXA₂) receptor (TP) are encoded by a single gene but are transcriptionally regulated by distinct promoters, termed promoter 1 (Prm1) and Prm3, respectively. Herein, it was sought to identify factors regulating Prm1 within the megakaryocytic human erythroleukemia 92.1.7 cell line. Through gene deletion and reporter assays, the core Prm1 was localized to between nucleotides –6,320 and –5,895, proximal to the transcription initiation site. Furthermore, two upstream repressor and two upstream activator regions were identified. Site-directed mutagenesis of four overlapping Sp1/Egr1 elements and an NF-E2/AP1 element within the proximal region substantially reduced Prm1 activity. Deletion/mutation of GATA and Ets elements disrupted the upstream activator sequence located between –7,962 and –7,717, significantly impairing Prm1 activity. Electrophoretic mobility shift assays and chromatin immunoprecipitations confirmed that Sp1, Egr1, and NF-E2 bind to elements within the core promoter, whereas GATA-1 and Ets-1 factors bind to the upstream activator sequence (between –7,962 and –7,717). Collectively, these data establish that Sp1, Egr1, and NF-E2 regulate core Prm1 activity in the megakaryocytic-platelet progenitor cells, whereas GATA-1 and Ets-1 act as critical upstream activators, hence providing the first genetic basis for the expression of the human TXA₂ receptor (TP) within the vasculature.

[Research Articles] Origins of intestinal ABCA1-mediated HDL-cholesterol

Field, F. J., Watt, K., Mathur, S. N. — 2008-11-14

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.

[Research Articles] Stable carbon isotope discrimination by human 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Ludke, S. M., Flenker, U., Schanzer, W., Schomburg, D. — 2008-11-14

The aim of this study was to investigate the possible existence and magnitude of stable carbon isotope discrimination by human 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). The catalytic portion of HMGR was expressed and purified. The reaction product mevalonate was lactonized and extracted from the reaction mixture by a solid-phase extraction protocol. Stable carbon isotope ratios of mevalonolactone (MVL) were analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. An average fractionation factor ¹²k/¹³k of 1.0031 ± 0.0004 for all carbon atoms contained in MVL was estimated by the method of internal competition. The value was calculated by nonlinear curve fitting, where the ratio ¹³C/¹²C of MVL was plotted versus the fraction of reaction.

[Research Articles] Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

2008-11-14

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 µmol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.

[Patient-Oriented and Epidemiological Research] VAP II analysis of lipoprotein subclasses in mixed hyperlipidemic patients on treatment with ezetimibe/simvastatin and fenofibrate

Farnier, M., Perevozskaya, I., Taggart, W. V., Kush, D., Mitchel, Y. B. — 2008-11-14

This analysis evaluates the effects on lipoprotein subfractions and LDL particle size of ezetimibe/simvastatin with or without coadministration of fenofibrate in patients with mixed hyperlipidemia. This multicenter, double-blind, placebo-controlled, parallel-group study included 611 patients aged 18–79 years randomized in 1:3:3:3 ratios to one of four 12 week treatment groups: placebo; ezetimibe/simvastatin 10/20 mg/day; fenofibrate 160 mg/day; or ezetimibe/simvastatin 10/20 mg/day + fenofibrate 160 mg/day. At baseline and study endpoint, cholesterol associated with VLDL, intermediate density lipoprotein (IDL), LDL, and HDL subfractions was quantified using the Vertical Auto Profile II method. LDL particle size was determined using segmented gradient gel electrophoresis. Whereas fenofibrate reduced cholesterol mass within VLDL and IDL, and shifted cholesterol from dense LDL subfractions into the more buoyant subfractions and HDL, ezetimibe/simvastatin reduced cholesterol mass within all apolipoprotein B-containing particles without significantly shifting the LDL particle distribution profile. When administered in combination, the effects of the drugs were complementary, with more-pronounced reductions in VLDL, IDL, and LDL, preferential loss of more-dense LDL subfractions, and increased HDL, although the effects on most lipoprotein subfractions were not additive. Thus, ezetimibe/simvastatin + fenofibrate produced favorable effects on atherogenic lipoprotein subclasses in patients with mixed hyperlipidemia.

[Patient-Oriented and Epidemiological Research] Comprehensive evaluation of apolipoprotein H gene (APOH) variation identifies novel associations with measures of lipid metabolism in GENOA

2008-11-14

Apolipoprotein H (apoH, also named β-2 glycoprotein I) is found on several classes of lipoproteins, and is involved in the activation of lipoprotein lipase in lipid metabolism. We have comprehensively investigated the association of variation in the apoH gene (APOH) with lipid traits in hepatic cholesterol transport, dietary cholesterol transport (DCT), and reverse cholesterol transport (RCT). Our study population consisted of families from the Genetic Epidemiology Network of Arteriopathy multicenter study that include African Americans, Mexican Americans, and European Americans. We individually tested 36 single-nucleotide polymorphisms (SNPs) that span the APOH locus, including nonsynonymous variants that result in known apoH charge isoforms. In addition, we constructed haplotypes from SNPs in the 5' promoter region that comprise cis-acting regulatory elements, as well as haplotypes for multiple amino acid substitutions. We found point-wise significant associations of APOH variants with various lipid measures in the three racial groups. The strongest associations were found for DCT traits (triglyceride and apoE levels) in Mexican Americans with a nonsynonymous variant (SNP 14917, Cys306Gly) that may alter apoH protein folding in a region involved in phospholipid binding. In conclusion, family-based analyses of APOH variants have identified associations with measures of lipid metabolism in three American racial groups.

[Patient-Oriented and Epidemiological Research] A pilot study of the effects of pioglitazone and rosiglitazone on de novo lipogenesis in type 2 diabetes

2008-11-14

Treatment of type 2 diabetes mellitus (T2DM) patients with pioglitazone results in a more favorable lipid profile, and perhaps more favorable cardiac outcomes, than treatment with rosiglitazone. Pioglitazone treatment increases VLDL-triacylglycerol clearance, but the role of de novo lipogenesis (DNL) has not been explored, and no direct comparison has been made between the thiazolidinediones (TZDs). Twelve subjects with T2DM and hypertriacylglyceridemia were randomized to either rosiglitazone or pioglitazone treatment. Stable isotope infusion studies were performed at baseline and after 20 weeks of treatment. Both treatments reduced glucose and HbA_1c concentrations equally. Pioglitazone treatment resulted in a 40% reduction in hepatic DNL (P < 0.01) and in a 25% reduction in hepatic glucose production (P < 0.05), while rosiglitazone did not significantly change either parameter, although comparisons of changes between treatments were not significantly different. These pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not. Larger follow-up studies are required to confirm differential effects of these agents definitively. The reduction in DNL may underlie altered assembly or atherogenicity of lipoprotein particles and may reflect PPAR or other non-PPAR actions on the liver by pioglitazone. These differences might help explain previously reported differences in lipid profiles and cardiovascular disease outcomes for rosiglitazone and pioglitazone.

[Patient-Oriented and Epidemiological Research] CYP7A1 promoter polymorphism -203A>C affects bile salt synthesis rate in patients after ileal resection

2008-11-14

Cholesterol 7-hydroxylase (CYP7A1) plays a crucial role in cholesterol metabolism and has been implicated in genetic susceptibility to atherosclerosis. Thus, an understanding of its transcriptional regulation is of considerable importance. We evaluated the effect of a common –203A>C polymorphism in the CYP7A1 promoter region on the activity of CYP7A1, estimated as the ratios of serum 7-hydroxycholest-4-en-3-one (C4) to either total or non-HDL-cholesterol. The study was performed on patients after resection of the distal ileum, leading to upregulation of CYP7A1 activity (n = 65). Healthy volunteers served as the control group (n = 66). Whereas higher CYP7A1 activity was associated with the –203A allele in the patient group (C4/cholesterol ratio, 29.0 vs. 14.8 µg/mmol, P = 0.032; C4/non-HDL-cholesterol ratio, 53.3 vs. 21.3 µg/mmol in –203AA and –203CC, P = 0.017, respectively), no differences were observed in the healthy controls. We conclude that under physiological conditions, the –203A>C polymorphism in the CYP7A1 gene promoter region does not seem to have any clinically relevant effect. However, in patients with severe bile salt malabsorption, this polymorphism markedly affects CYP7A1 activity.

[Methods] Preparation of pure lipid hydroperoxides

2008-11-14

Increasing evidence of lipid peroxidation in food deterioration and pathophysiology of diseases have revealed the need for a pure lipid hydroperoxide (LOOH) reference as an authentic standard for quantification and as a compound for biological studies in this field. Generally, LOOH is prepared from photo- or enzymatically oxidized lipids; however, separating LOOH from other oxidation products and preparing pure LOOH is difficult. Early studies showed the usability of reaction between hydroperoxide and vinyl ether for preparation of pure LOOH. Because the reactivity of vinyl ether with LOOHs other than fatty acid hydroperoxides has never been reported, here, we employed the reaction for preparation of a wide variety of pure LOOHs. Phospholipid, cholesteryl ester, triacylglycerol, or fatty acid was photo- or enzymatically oxidized; the resultant crude sample containing hydroperoxide was allowed to react with a vinyl ether [2-methoxypropene (MxP)]. Liquid chromatography (LC) and mass spectrometry confirmed that MxP selectively reacts with LOOH, yielding a stable MxP adduct (perketal). The lipophilic perketal was eluted at a position away from that of intact LOOH and identified and isolated by LC. Upon treatment with acid, perketal released the original LOOH, which was finally purified by LC. Using our optimized purification procedures, for instance, we produced 75 mg of pure phosphatidylcholine hydroperoxide (>99%) from 100 mg of phosphatidylcholine. Our developed method expands the concept of the perketal method, which provides pure LOOH references. The LOOHs prepared by the perketal method would be used as "gold standards" in LOOH methodology.

[Methods] Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Ikeda, K., Shimizu, T., Taguchi, R. — 2008-11-14

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.

[Methods] Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

Vertzoni, M., Archontaki, H., Reppas, C. — 2008-11-14

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50–100 µl) and minimal sample pretreatment. A Hypersil BDS RP-C₁₈ column (250 x 4.6 mm, 5 µm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5–500 µM. Limits of detection and quantification were in the range of 0.07–0.60 µM and 0.20–1.80 µM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.

[Calendar] Calendar

2008-11-14

[Errata] ERRATA

2008-11-14