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The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap. We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species. Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision, detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed lipid analysis of PC and SPM species in a variety of sample materials.

Prevention of contrast agent-induced nephropathy is of crucial importance for a number of diagnostic studies. N-Acetylcysteine (NAC) was recently reported to decrease serum creatinine levels in this setting, and its administration before radiocontrast medium administration has been widely recommended. The objective of this prospective study was to investigate whether there are effects of NAC on serum creatinine levels that are independent of alterations in GFR. Volunteers with normal renal function who did not receive radiocontrast medium were studied. Fifty healthy volunteers completed the study protocol. NAC was administered orally at a dose of 600 mg every 12 h, for a total of four doses. Surrogate markers of renal function, such as serum creatinine, urea, albumin, and cystatin C levels, were measured and estimated GFR (eGFR) was assessed immediately before the administration of NAC and 4 and 48 h after the last dose. There was a significant decrease in the mean serum creatinine concentration (P < 0.05) and a significant increase in the eGFR (P < 0.02) 4 h after the last dose of NAC. The cystatin C concentrations did not change significantly. In several studies, a protective effect of NAC on renal function after radiocontrast medium administration has been postulated. This is the first study to demonstrate an effect of NAC on creatinine levels and eGFR, surrogate markers of renal injury, without any effect on cystatin C levels. Before renoprotective effects of NAC against contrast agent-induced nephropathy are considered, the direct effects of NAC on creatinine levels, urea levels, and eGFR should be assessed.

Plasminogen activator inhibitor-1 (PAI-1) levels were found to be associated with obesity, indicating that adipocytes might influence PAI-1 plasma levels. In addition, the 4G/5G promoter polymorphism of the PAI-1 gene possibly modulates PAI-1 gene transcription and, as a consequence, PAI-1 plasma levels. Metabolic parameters, diabetes complications, PAI-1 plasma levels, and PAI-1 promoter genotypes were determined and were tested for correlation in 547 Caucasian patients with type 2 diabetes. Genotyping was performed by using allele-specific PCR, and PAI-1 plasma levels were measured in 547 well-characterized subjects with type 2 diabetes. The allelic frequencies of the polymorphism (0.56 for the 4G-genotype, 0.44 for the 5G-genotype) were not different from those observed in nondiabetic controls. The PAI-1 concentration was positively associated with MI, but not with the 4G/5G polymorphism. Statistical analysis of metabolic parameters, diabetic complications, and the 4G/5G polymorphism revealed that serum fibrinogen levels were significantly higher in the 4G/4G subgroup compared with the 4G/5G and 5G/5G subgroups. The correlation between serum fibrinogen and 4G allele remained significant, even when additional variables, such as gender, age, BMI, duration of diabetes, and HbA1c, were controlled. In patients with type 2 diabetes mellitus, the PAI-1 4G/5G promoter polymorphism does not predict PAI-1 plasma levels and is not associated with common metabolic parameters besides fibrinogen levels.

Recent data indicate that ceramide (Cer) and lysophosphatidylcholine (LPC) regulate immune cell functions. Since these bioactive lipids are generated in blood plasma by inflammatory lipases, we hypothesized that they may be involved in the process of acute systemic sepsis. In order to provide support for this hypothesis, we analyzed the plasma levels of Cer and LPC by quantitative tandem mass spectrometry in 102 sepsis patients starting with the day at which the sepsis criteria were fulfilled for the first time, as well as on day 4 and day 11. The values were compared with 56 healthy controls and correlated with sepsis-related mortality within 30 days of study entry. Most Cer species were increased in sepsis patients, while all LPC species were markedly decreased. In addition, we determined the molar ratios with their precursor molecules sphingomyelin (SPM) and phosphatidylcholine (PC), which reflect the enzymatic reactions responsible for their formation. Species-specific as well as total Cer-SPM ratios were increased, whereas LPC-PC ratios were decreased in sepsis patients. The increased Cer-SPM ratios as well as the decreased LPC-PC ratios showed a strong predictive power for sepsis-related mortality. Together with existing data from in vitro experiments and animal models, the results provide the first ex vivo indication for the role of Cer and lysophospholipids in systemic inflammation in humans.

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) ‘Lubrol rafts’, defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

Background & Aims: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism. Methods: ABCA1-deficient mice (ABCA1 −/− ) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays. Results: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl–coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1 −/− mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1 −/− mice occurs in peripheral tissues rather than the liver. Conclusions: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function. GASTROENTEROLOGY 2001;120:1203-1211

The zinc finger gene 202 (ZNF202) located within a hypoalphalipoproteinemia susceptibility locus on chromosome 11q23 is a transcriptional repressor of various genes involved in lipid metabolism. To provide further evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia, we investigated the effect of ZNF202 expression on ATP binding cassette transporter A1 (ABCA1) and ABCG1. ABCA1 is a key regulator of the plasma high density lipoprotein pool size, whereas ABCG1 is another mediator of cellular cholesterol and phospholipid efflux in human macrophage. We demonstrate here that the full-length ZNF202m1 isoform binds to GnT repeats within the promotors of ABCA1 (-229/-210) and ABCG1 (-572/-552). ZNF202m1 expression in HepG2 cells dose-dependently repressed the promotor activities of ABCA1 and ABCG1. This transcriptional effect required the presence of the SCAN domain in ZNF202 and the functional integrity of a TATA box at position -24 of ABCA1, whereas the presence of GnT binding motifs was nonessential. The state of ZNF202 SCAN domain oligomerization affected the ability of the adjacent ZNF202 Krüppel-associated box domain to recruit the transcriptional corepressor KAP1. Overexpression of ZNF202m1 in RAW264.7 macrophages prevented the induction of ABCA1 gene expression by 20(S)OH-cholesterol and 9-cis-retinoic acid, further substantiating the interference of ZNF202 in critical elements of transcriptional activation. Finally, HDL and apoAImediated lipid efflux was significantly reduced in RAW264.7 cells stably expressing ZNF202m1. In conclusion, we have identified ABCA1 and ABCG1 as target genes for ZNF202-mediated repression and thus, provide evidence for a functional linkage between ZNF202 and hypoalphalipoproteinemia.

Background The aim of this study was to analyse the human adipocyte-specific apM-1 gene for sequence variations. Methods Sequence analysis was performed in 344 randomly chosen blood samples using a capillary sequencer. Results Whereas no mutations were detected in intronic regions and in 2.7 kb of the promoter, two sequence variations were found within the coding sequence of apM-1. For both mutations, a polymerase chain reaction-(PCR) based restriction fragment length polymorphism (RFLP) analysis was developed, which provided a rapid screening method. A conservative T G transition at nucleotide + 45 within exon-2 [Gly15Gly] was detected with an allelic frequency of 0.9 for the wild-type allele and 0.1 for the mutated allele. In addition, a missense point mutation at nucleotide + 331 within exon-3 [Tyr111His] was detected with an allelic frequency of 0.97 for the wild-type allele and 0.03 for the mutated allele. This mutation replaces a tyrosine by an histidine within the carboxyterminal globular domain of apM-1. Concerning the Gly15Gly polymorphism, the TT genotype was found in 275 subjects (79.9%), the TG genotype in 67 subjects (19.5%) and the GG genotype in 2 subjects (0.6%): one with maturity onset diabetes of young age (MODY-diabetes) and one with Lipoatrophic Diabetes Syndrome (LPDS). Concerning the Tyr111His polymorphism, the TT genotype was found in 328 subjects (95.4%), the TC genotype in 15 subjects (4.3%) and the CC genotype in 1 subject (0.3%). Conclusion The existence of two yet unknown mutations within the apM-1 gene was demonstrated and RFLP analysis was established for rapid screening. Well defined cohorts of patients are necessary to determine the putative role of apM-1 gene mutations in the pathogenesis of metabolic disorders.

Background The recently described A→G (−3826) point mutation within the distal region of the UCP-1 promoter is possibly involved in the development of obesity, diabetes and related metabolic disorders. It was the aim of this study to examine the allelic frequency and the prevalence of the three UCP-1 genotypes in a broad caucasian cohort and to investigate the significance of this polymorphism for obesity and diabetes. Methods 1020 subjects were randomly chosen from 6450 participants in the Diabetomobile Study. The UCP-1 genotype was determined by genomic PCR and Bcl-I-RFLP analysis in 1020 subjects and tested for association with a variety of metabolic parameters. In addition, the influence of this mutation on adipocyte nuclear factor binding was investigated by electrophoretic mobility shift assays (EMSA). Results The genotype frequencies in 1020 subjects were: AA genotype, 57.0%; AG genotype, 35.4%; GG genotype, 7.6%; with allelic frequencies of 0.75 for allele A and 0.25 for allele G. No significant differences between the genotypes and age, gender, BMI, leptin, glucose, fasting insulin, C-peptide, HbA1c, diabetes manifestation, total cholesterol, and HDL cholesterol were found. Analysis of the Trp64Arg polymorphism of the β3-adrenergic receptor in a subgroup of 343 subjects revealed no additive effect to the UCP-1 polymorphism. An yet unknown adipocyte-specific factor of nuclear extracts from 3T3-L1 adipocytes during differentiation is able to bind specifically to the distal UCP-1 promoter region and this binding ability can not be abolished by the mutation. Conclusions We determined the genotype and allelic frequency of the UCP-1 promoter polymorphism in the largest known population-based study. The results from genotyping demonstrate clearly that this polymorphism does not play a major role in the pathogenesis obesity and diabetes. A yet unknown adipocyte derived and differentiation-dependent regulated transcription factor is able to bind to the distal UCP-1 promoter surrounding −3826 bp. This binding is not affected by presence of the mutation.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 μg of cell protein corresponding to about 20,000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.—Liebisch, G., W. Drobnik, M. Reil, B. Trümbach, R. Arnecke, B. Olgemöller, A. Roscher, and G. Schmitz. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J. Lipid Res. 1999. 40: 1539–1546.

Activated lipid-laden macrophages in the vascular wall are key modulators of the inflammatory processes underlying atherosclerosis. We demonstrate here that the ATP-binding cassette (ABC) transporter ABCA1 is induced during differentiation of human monocytes into macrophages. ABCA1 has been implicated in macrophage interleukin-1β secretion and apoptosis. Moreover, ABCA1 mRNA and protein levels are strongly upregulated by uptake of modified LDL and downregulated by HDL3-mediated lipid efflux in macrophages. Mutation analysis in patients with the classical Tangier disease (TD), a monogenetic disorder characterized by hypersplenism, macrophage accumulation and deposition of cholesteryl esters in the reticuloendothelial system, low plasma HDL and premature atherosclerosis, revealed deleterious mutations in their ABCA1 gene. The localization pattern of the mutations within the ABCA1 protein appears to determine the tropism for either the reticuloendothelial system, as seen in the classical TD phenotype, or the artery wall, as in the case of HDL deficiency in the absence of splenomegaly. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified additional cholesterol-responsive genes that are induced during monocyte differentiation into macrophages. Our results indicate a dual regulatory function for ABCA1 in macrophage lipid metabolism and inflammation.

We isolated a full-length cDNA encoding human caveolin 3, which is 72% and 59% homologous to human caveolin 1 and caveolin 2, respectively. Human caveolin 3 protein contains the `caveolin signature sequence' and the 33 amino acids spanning intramembrane domain common to all caveolins. Northern blot analysis indicates that the caveolin 3 transcript is 1.6 kb in size and exclusively detectable in muscle tissue.

Microgliosis is a common phenomenon in neurodegenerative disorders including retinal dystrophies. We performed a detailed characterization of activated microglia in the retinoschisin (Rs1h)-deficient (Rs1h(-/Y)) mouse model of inherited retinal degeneration. To visualize and isolate microglia, we crossed Rs1h(-/Y) animals with transgenic MacGreen mice, which express green fluorescent protein under the control of the macrophage-specific csf1r promoter. Activated microglia were detected in retinal sections and whole-mounts of early postnatal MacGreen/Rs1h(-/Y) mice before the onset of overt neuronal cell death. These activated microglia contained prominent lipid droplets and analysis of the retinal lipid composition showed decreased docosahexaenoic acid (DHA) levels in Rs1h(-/Y) retinas. To establish a link between microglia activation, reduced DHA levels, and neurodegeneration, a dietary intervention study was performed. Female Rs1h(-/-) mice and their Rs1h(-/Y) litter were either subjected to a diet enriched with DHA, or a control chow lacking DHA. Supplementation with DHA enhanced photoreceptor survival and converted activated microglia to a quiescent phenotype. Furthermore, DHA, but not docosapentaenoic acid or adrenic acid reduced pro-inflammatory gene expression, migration, and lipid accumulation of cultured BV-2 microglia. We conclude that retinal DHA levels control the activity of microglia and thereby may affect the progression and extent of retinal degeneration.

Aim of the study: Investigation, whether water-filtered infrared-A (wIRA) irradiation during moderate bicycle ergometer endurance exercise has effects especially on local fat reduction and on weight reduction beyond the effects of ergometer exercise alone. Methods: Randomised controlled study with 40 obese females (BMI 30-40 (median: 34.5), body weight 76-125 (median: 94.9) kg, age 20-40 (median: 35.5) years, isocaloric nutrition), 20 in the wIRA group and 20 in the control group. In both groups each participant performed 3 times per week over 4 weeks for 45 minutes bicycle ergometer endurance exercise with a constant load according to a lactate level of 2 mmol/l (aerobic endurance load, as determined before the intervention period). In the wIRA group in addition large parts of the body (including waist, hip, and thighs) were irradiated during all ergometries of the intervention period with visible light and a predominant part of water-filtered infrared-A (wIRA), using the irradiation unit “Hydrosun® 6000” with 10 wIRA radiators (Hydrosun® Medizintechnik, Müllheim, Germany, radiator type 500, 4 mm water cuvette, yellow filter, water-filtered spectrum 500-1400 nm) around a speed independent bicycle ergometer. Main variable of interest: change of “the sum of circumferences of waist, hip, and both thighs of each patient” over the intervention period (4 weeks). Additional variables of interest: body weight, body mass index BMI, body fat percentage, fat mass, fat-free mass, water mass (analysis of body composition by tetrapolar bioimpedance analysis), assessment of an arteriosclerotic risk profile by blood investigation of variables of lipid metabolism (cholesterol, triglycerides, high density lipoproteins HDL, low density lipoproteins LDL, apolipoprotein A1, apolipoprotein B), clinical chemistry (fasting glucose, alanin-aminotransferase ALT (= glutamyl pyruvic transaminase GPT), gamma-glutamyl-transferase GGT, creatinine, albumin), endocrinology (leptin, adiponectin (= adipo Q), homocysteine, insulin). All variables were at least measured before and after the intervention period. Ergometry (ECG, blood pressure behaviour, lactate curve with power at 2, 3 and 4 mmol/l) before the intervention period. In addition: nutrition training ahead of and during the intervention period with a nutrition protocol over one week for assessment of the daily energy intake; calculation of basic metabolic rate and total energy requirement. Assessment of undesired effects. Only methods of non-parametric statistics were used, both descriptive (median, percentiles of 25 and 75 (= interquartile range), minimum, maximum) and confirmatory (two-sided Mann-Whitney U test for unpaired samples for the only one main variable of interest). Total error probability: .05 (5%). An intention to treat analysis ITT with last observed carry forward method was used preferably (presented results) and in addition an on treatment analysis OT. Only 2 (treatment group) and 4 (control group) drop-outs occurred (mostly due to lack of time). Results: The “sum of circumferences of waist, hip, and both thighs of each patient” decreased during the 4 weeks significantly more (p

Plasminogen activator inhibitor-1 (PAI-1) levels were found to be associated with obesity indicating that adipocytes influence PAI-1 plasma levels. In addition, the 4 G/5 G promoter polymorphism of the PAI-1 gene may modulate PAI-1 transcription. We investigated the transcriptional regulation of the human PAI-1 gene in adipocytes and analyzed the genetic contribution of the 4 G/5 G polymorphism. The PAI-1 promoter was analyzed using electrophoretic mobility shift assays (EMSAs) and luciferase reporter gene assays. A putative binding site for the upstream stimulatory factor-1/2 (USF-1/2) at the polymorphic region of the PAI-1 promoter was identified. The binding of USF-1/2 was studied using nuclear extracts prepared from adipocytes and was similar in all the promoter variants as analyzed by EMSA. A 257 bp PAI-1 promoter fragment including the 4 G/5 G site was transcriptionally active in adipocytes and was not influenced by the polymorphism. The present data indicate for the first time that USF-1/2 is transcriptionally active in differentiated adipocytes. However, USF-1/2 binding activity and PAI-1 transcription are not influenced by the 4 G/5 G-allele. These data possibly explain the observation that PAI-1 secretion from adipose tissue is not influenced by the PAI-1 promoter polymorphism.

Central aspects of the cellular lipid trafficking mechanisms that occur during keratinocyte differentiation are still not well understood. In the past years, evidence has accumulated to suggest that members of the superfamily of adenosine triphosphate binding cassette (ABC) transporters are critically involved in the transmembrane transport of cellular lipids. To test the hypothesis that ABC molecules are potentially involved in the epidermal transport of sphingolipids, glycerophospholipids, cholesterol, and fatty acids, we performed mRNA expression profiling of all currently known ABC molecules during in vitro differentiation of human keratinocytes and HaCaT cells. We identified six ABC molecules that displayed significant regulation during differentiation of these cells. The recently cloned transporter ABCA7 was highly expressed in keratinocytes and HaCaT cells and upregulated during differentiation. Overexpression of ABCA7 in HeLa cells resulted in increased expression of intracellular and cell surface ceramide and elevated intracellular phosphatidylserine levels. Given the observation that during terminal keratinocyte differentiation intracellular and surface ceramide levels are increased, our results render ABCA7 a candidate regulator of ceramide transport in this process. In addition to ABCA7, the cholesterol transporters ABCB1 and ABCG1 and the glutathione/glucuronide sulfate transporters ABCC1, ABCC3, and ABCC4, were strongly upregulated during keratinocyte and HaCaT cell differentiation. These findings support the notion that ABCB1 and ABCG1 are potentially implicated in cholesterol transport, whereas ABCC1, ABCC3, and ABCC4 are candidate regulators of the translocation of sulfated lipids during stratum corneum keratinization. Our results suggest specific biologic functions for members of the ABC transporter family in epidermal lipid reorganization during terminal keratinocyte differentiation.

Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.

The fatty acid transport protein family is a group of evolutionarily conserved proteins that are involved in the cellular uptake and metabolism of long and very long chain fatty acids. However, little is known about their respective physiological roles. To analyze the functional significance of fatty acid transport protein 4 (Fatp4, Slc27a4), we generated mice with a targeted disruption of the Fatp4 gene. Fatp4-null mice displayed features of a neonatally lethal restrictive dermopathy. Their skin was characterized by hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. The rigid skin consistency resulted in an altered body shape with facial dysmorphia, generalized joint flexion contractures, and impaired movement including suckling and breathing deficiencies. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides, in particular a decrease in the C26:0 and C26:0-OH fatty acid substitutes. These findings reveal a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier.

Cholesterol-lowering therapy is the central approach in the primary and secondary prevention of cardiovascular disease, the leading cause of death in industrialized countries. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are currently the most potent and widely used cholesterol-lowering drugs. Large-scale clinical trials unequivocally demonstrated the efficacy of statin treatment in reducing the risk of cardiovascular events. In general, HMG-CoA reductase inhibitors are well tolerated, although in a minority of patients severe adverse effects like myopathy or rhabdomyolysis may develop. The incidence of this potentially life-threatening side effects increases with co-adminstration of drugs that are metabolized via the same pharmacokinetic pathways or at high-dose statin therapy. The recent focus on the pleiotropic effects of statins that are more frequently observed at higher doses and the conclusion drawn from the large statin trials that low-density lipoprotein (LDL)-cholesterol is "the lower the better", may need careful consideration in individuals at risk of adverse drug reactions. On the other hand, not all patients respond to statin therapy with a reduction in coronary heart disease (CHD) risk. It is therefore of interest to develop diagnostic test systems, which would allow to identify patients at increased risk of adverse drug reactions or patients with a lack of therapeutic effect. Beside exogenous factors, genetic variability determines the response of an individual to drug therapy and the analysis of genetic variants affecting pharmacokinetic or pharmacodynamic aspects of drug therapy is the subject of pharmacogenomics. This review summarizes current knowledge of the pharmacology and the pharmacogenomics of statin therapy.

The adipocyte-derived hormone, leptin, has been implicated in the regulation of appetite, weight gain and glucose homeostasis as well as in liver fibrogenesis, hematopoiesis and immune function. No previous reports have clearly defined pathologically elevated or decreased serum leptin levels for Caucasian adults. The aim of this study was to define and characterize subjects with relative hyper- and hypoleptinemia in a large population-based German cohort. Percentiles of leptin levels by body mass index (BMI) were calculated from 4971 adult Germans, and the participants with leptin levels above the 95th and below the 5th percentile were defined as relatively hyperleptinemic and relatively hypoleptinemic, respectively, for their BMI. These participants were compared with the intermediate group with respect to anthropometric and clinical data and parameters of glucose and iron metabolism, lipid status, renal, adrenal and reproductive function. Relatively hyperleptinemic participants (HL) showed higher insulin, c-peptide, and total cholesterol levels than the hypoleptinemic subjects; in males, ferritin levels were higher and testosterone levels lower in the HL group. In conclusion, we report the first percentile curves for serum leptin by BMI in a large Caucasian population. Relatively low leptin values may be associated with a lower metabolic risk than relatively high serum leptin values.

ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol and phospholipid homeostasis. Its function has not been fully characterized and may depend on the association with additional proteins. To identify ABCA1-interacting proteins a human liver yeast two-hybrid library was screened with the 144 C-terminal amino acids of ABCA1. Fas-associated death domain protein (FADD) was identified to bind to ABCA1, and this interaction was confirmed by pull-down assays and co-immunoprecipitations. Recombinant expression of a dominant negative form of FADD or the C terminus of ABCA1 in the human hepatoma cell line HepG2 markedly reduced the transfer of phospholipids to apoA-I. This indicates that the binding of additional proteins, one of them being full-length FADD, is required for ABCA1 function. The association of FADD with ABCA1 provides an unexpected link between high density lipoprotein metabolism and an adaptor molecule mainly described in death receptor signal transduction.

Numerous ATP-binding cassette (ABC) transporters are expressed in monocyte-derived macrophages and are subject to sterol-dependent regulation. ABCA1 has been identified as a key regulator of macrophage cholesterol efflux and HDL-mediated reverse cholesterol transport. Although the precise mechanisms of ABCA1 function are not completely understood, recent data suggest that the ABCA1 pathway regulates vesicular traffic, filipodia formation and lipid microdomains, thereby controlling susceptibility to atherosclerosis. Nuclear hormone receptors including LXR/RXR and PPAR/RXR heterodimers are recognized as direct or indirect regulators of ABCA1 expression and are discussed as potential targets for pharmacological intervention in cardiovascular disease. Future studies clarifying the processes involved in the ABCA1 pathway at the cellular level are expected to identify new and possibly more specific pharmaceutical targets.

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca 2+ dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region.

In an open, uncontrolled pilot study, 5 men and 1 woman with suspected gram-negative sepsis were treated with a new whole-blood endotoxin adsorption system. Lipopolysaccharide (LPS) adsorption was carried out by hemoperfusion over high-affinity polymethacrylate-bound albumin (Fresenius Endotoxin Adsorber EN 500). All patients suffered from endotoxemia (>20 pg/ml LAL) and met at least two systemic inflammatory response syndrome (SIRS) criteria. Four patients suffered from pneumonia due to mechanical ventilation, one from peritonitis, and one from pneumonia and peritonitis. Endotoxin adsorption was very well tolerated, and efficient LPS removal was shown in all patients. Apache II score immediately before immunoadsorption was 23.5 and was 22.3 after the last treatment. All 6 critically ill patients improved substantially and were discharged from the intensive care unit. LPS whole blood immunoadsorption is a promising new method. No side effects have been observed thus far. A large controlled study to prove clinical efficacy in patients with severe sepsis is under way.

The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.

The objective of the current study was to characterize the influence of high density lipoproteins (HDL) on processes related to the vascular recruitment of human monocytes, which may contribute to the anti-atherogenic properties of these lipoproteins. We show that HDL(3) and apo AI inhibit the following processes in primary human monocytes: (1) M-CSF induced cell spreading; (2) M-CSF stimulated expression of surface molecules involved in adhesion, migration, and scavenging; (3) fMLP induced chemotaxis. These processes are obviously modulated by the regulation of cellular cholesterol pools as indicated by the following findings. In Tangier monocytes with defective apo AI induced cholesterol efflux, apo AI had no influence on the spreading response. In control cells, stimulation of cholesterol efflux by p-cyclodextrin mimicked the effect of apo AI and HDL(3) on spreading and chemotaxis, whereas cholesterol loading with enzymatically modified LDL (E-LDL) showed the opposite effect. Finally, a similar inverse regulation by E-LDL and apo AI/HDL(3) was also observed in regard to the surface expression of beta(1)- and beta(2)-integrins as well as the hemoglobin/haptoglobin scavenger receptor CD163 and the Fcgamma-IIIaR CD16. CDC42 was identified as a potential downstream target linking changes in cellular cholesterol content to monocyte spreading and chemotaxis. Thus, CDC42 antisense markedly reduced spreading and, in parallel with their influence on monocyte spreading, HDL(3), apo AI and p-cyclodextrin down-regulated CDC42 expression while E-LDL had the inverse effect. The apo AI induced decrease of CDC42 protein expression was paralleled by the reduction of active GTP-bound CDC42. In summary, we provide evidence that HDL(3) and apo AI are able to inhibit processes in primary human monocytes, which are related to the recruitment of monocytes into the vessel wall and probably involve regulation of cellular cholesterol pools and CDC42 function.

We report the identification of the full-length cDNA for a novel ATP-binding cassette (ABC) transporter from human macrophages. The mRNA is of 6.8 kb size and contains an open reading frame encoding a polypeptide of 2146 amino acids with a calculated molecular weight of 220 kDa. The predicted protein product is composed of two transmembrane domains and two nucleotide binding folds indicating that it pertains to the group of full-size ABC transporters. The novel transporter shows highest protein sequence homology with the recently cloned human cholesterol and phospholipid exporter ABCA1 (54%) and the human retinal transporter ABCR (49%), both members of the ABC transporter subfamily A. In accordance with the currently proposed classification, the novel transporter was designated ABCA7. ABCA7 mRNA was detected predominantly in myelo-lymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Expression of ABCA7 is induced during in vitro differentiation of human monocytes into macrophages. In macrophages, both the ABCA7 mRNA and protein expression are upregulated in the presence of modified low density lipoprotein and downregulated by HDL3. Our results suggest a role for ABCA7 in macrophage transmembrane lipid transport.

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL 3 , suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL 3 -dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL 3 -mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.

The human adipocyte-specific apM-1 gene encodes a secretory protein of the adipose tissue that has been suggested to play a role in the pathogenesis of obesity. The regulation of apM-1 was studied along adipocyte differentiation. While apM-1-mRNA and apM-1 protein were absent in preadipocytes and in 48 h differentiated adipocytes, they were found upregulated from day 4 to day 9 of adipocyte differentiation as shown by RNase protection assay and Western blot analysis. These data indicate that apM-1 may be a late marker of adipocyte differentiation. In human sera apM-1 protein is also detectable by Western blots using a polyclonal antibody raised against a synthetic peptide sequence of the human apM-1. The genomic structure of the human apM-1 gene together with a total of 2.7 kb of the 5'-flanking region with putative transcription factor binding sites is presented. Interestingly, sequence comparisons link the apM-1 gene to the family of TNF's and to genes expressed in activated T-cells. The chromosomal localization of apM-1 was investigated by FISH and mapped to human chromosome 1q21.3-1q23, a region that was identified as a susceptibility locus for Familial Combined Hyperlipidaemia (FCH) and polygenic NIDDM. These data and the chromosomal localization on chromosome 1q21.3-q23 raises the possibility that apM-1 as an adipocyte-specific secretory protein may play a role in the pathogenesis of FCH and associated insulin resistance. Exon- and intron-specific primer sequences are presented as a basis for mutation screening of patients affected with FCH.

Abstract —We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL 3 -mediated lipid efflux and G i -protein–mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4±0.3% S-phase and 19.1±0.5% G 2 /M-phase cells compared with 9.7±0.6% and 7.8±0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G 2 /M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide and threo -1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither G i protein function by pertussis toxin nor PLD by butanol resulted in a G 2 /M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G 2 /M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits α i 1, α i 2, and α i 3; phosphatidylinositol transfer proteins-α and -β; and ADP ribosylation factors 1 and 3 were found to be normal. Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction. Because PDMP was also found to decrease HDL 3 -mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.

Abstract We compared HDL 3 - and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL 3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the α and ε isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

The signalling mechanisms whereby high-density lipoproteins (HDL) and low-density lipoproteins (LDL) affect a number of cellular functions in fibroblasts are unclear. This study has analyzed the influence of HDL3 and LDL on the phosphatidylinositol specific phospholipase C pathway in human skin fibroblasts. Exposure of myo-[2-3H]-inositol prelabelled fibroblasts to HDL3 or LDL elicited major increases in IP1 and minor increases in IP2 and IP3 within 30 s. In fura-2 loaded suspended fibroblasts, HDL3 and LDL increased intracellular Ca2+ concentrations ([Ca2+]i) with comparable rapid, transient kinetics. The dose-profiles for HDL3- and LDL-induced increases in [Ca2+]i were also comparable, with half-maximally and maximally effective concentrations being approximately 15 micrograms/mL and approximately 50 micrograms/mL, respectively. HDL3- and LDL-induced increases in [Ca2+]i were diminished by approximately 60% (vs. control fibroblasts) in thapsigargin-pretreated fibroblasts, indicating that release of Ca2+ from intracellular pools is the major contributor toward lipoprotein-induced increases in [Ca2+]i. Pertussis toxin-pretreatment of cells completely abolished lipoprotein induced Ca(2+)-transient, indicating the involvement of a guanine nucleotide-binding protein in the signalling process. In [3H]-palmitic acid-prelabelled fibroblasts, both HDL3 and LDL were observed to stimulate production of DAG. Activation of protein kinase C (PKC) was analysed by determining the cytosol-to-membrane translocation of both enzymatic activity and immunoreactivity of specific PKC isoforms (alpha, delta, epsilon, and zeta). Stimulation with HDL3 and LDL evoked a rapid (within 2.5 min) translocation of PKC activity, with PKC alpha and PKC epsilon being the isoforms translocated. It is concluded that HDL3 and LDL acutely stimulate a phosphoinositide-specific phospholipase C pathway in human skin fibroblasts. However, the specific cell membrane events mediating this signal transduction remain to be further elucidated.

The removal of excess cholesterol from peripheral cells is the first step in reverse cholesterol transport and many studies demonstrated a net removal of cellular cholesterol in response to HDL3 1,2.This function has been suggested to account for the inverse correlation between coronary heart disease risk and plasma levels of HDL-cholesterol3. In addition to unspecific cholesterol desorption from the plasma membrane4, depletion of cholesterol may be achieved by binding of HDL apolipoproteins to specific surface receptors and subsequent induction of cholesterol transport from internal stores to the plasma membrane 5,6

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.

Cholesterol efflux to apolipoprotein A-1 (apoA-1) from cholesterol-loaded macrophages is an important anti-atherosclerotic mechanism in reverse cholesterol transport. We recently provided kinetic evidence for two distinct pathways for cholesterol efflux to apoA-1 [Gaus et al. (2001) Biochemistry 40, 9363]. Cholesterol efflux from two membrane pools occurs sequentially with different kinetics; a small pool rapidly effluxed over the first hour, followed by progressive release from a major, slow efflux pool over several hours. In the present study, we propose that the rapid and slow cholesterol efflux pools represent cholesterol derived from lipid raft and nonraft domains of the plasma membrane, respectively. We provide direct evidence that apoA-1 binds to both lipid raft and nonraft domains of the macrophage plasma membrane. Conditions that selectively deplete plasma membrane lipid raft cholesterol, such as incorporation of 7-ketocholesterol or rapid exposure to cyclodextrins, block apoA-1 binding to these domains but also inhibit cholesterol efflux from the major, slow pool. We propose that cholesterol exported to apoA-1 from this major slow efflux pool derives from nonraft regions of the plasma membrane but that the interaction of apoA-1 with lipid rafts is necessary to stimulate this efflux.

Background: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification.Methods: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros.Results: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was ∼12% for the major and 25% for the minor species. The detection limit was Conclusion: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.