Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study

<p>Abstract</p> <p>Background</p> <p>Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. <it>In vitro </it>and <it>ex vivo </it>cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer.</p> <p>Results</p> <p>Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition.</p> <p>Conclusion</p> <p>The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.</p

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection

<p>Abstract</p> <p>Background</p> <p>There are currently no accurate serum markers for detecting early risk of colorectal cancer (CRC). We therefore developed a non-targeted metabolomics technology to analyse the serum of pre-treatment CRC patients in order to discover putative metabolic markers associated with CRC. Using tandem-mass spectrometry (MS/MS) high throughput MS technology we evaluated the utility of selected markers and this technology for discriminating between CRC and healthy subjects.</p> <p>Methods</p> <p>Biomarker discovery was performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Comprehensive metabolic profiles of CRC patients and controls from three independent populations from different continents (USA and Japan; total <it>n </it>= 222) were obtained and the best inter-study biomarkers determined. The structural characterization of these and related markers was performed using liquid chromatography (LC) MS/MS and nuclear magnetic resonance technologies. Clinical utility evaluations were performed using a targeted high-throughput triple-quadrupole multiple reaction monitoring (TQ-MRM) method for three biomarkers in two further independent populations from the USA and Japan (total <it>n </it>= 220).</p> <p>Results</p> <p>Comprehensive metabolomic analyses revealed significantly reduced levels of 28-36 carbon-containing hydroxylated polyunsaturated ultra long-chain fatty-acids in all three independent cohorts of CRC patient samples relative to controls. Structure elucidation studies on the C28 molecules revealed two families harbouring specifically two or three hydroxyl substitutions and varying degrees of unsaturation. The TQ-MRM method successfully validated the FTICR-MS results in two further independent studies. In total, biomarkers in five independent populations across two continental regions were evaluated (three populations by FTICR-MS and two by TQ-MRM). The resultant receiver-operator characteristic curve AUCs ranged from 0.85 to 0.98 (average = 0.91 ± 0.04).</p> <p>Conclusions</p> <p>A novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC.</p

Human serum-derived hydroxy long-chain fatty acids exhibit anti-inflammatory and anti-proliferative activity

<p>Abstract</p> <p>Background</p> <p>Circulating levels of novel long-chain hydroxy fatty acids (called GTAs) were recently discovered in the serum of healthy subjects which were shown to be reduced in subjects with colorectal cancer (CRC), independent of tumor burden or disease stage. The levels of GTAs were subsequently observed to exhibit an inverse association with age in the general population. The current work investigates the biological activity of these fatty acids by evaluating the effects of enriched human serum extracts on cell growth and inflammation.</p> <p>Methods</p> <p>GTAs were extracted from commercially available bulk human serum and then chromatographically separated into enriched (GTA-positive) and depleted (GTA-negative) fractions. SW620, MCF7 and LPS stimulated RAW264.7 cells were treated with various concentrations of the GTA-positive and GTA-negative extracts, and the effects on cell growth and inflammation determined.</p> <p>Results</p> <p>Enriched fractions resulted in poly-ADP ribose polymerase (PARP) cleavage, suppression of NFκB, induction of IκBα, and reduction in NOS2 mRNA transcript levels. In RAW264.7 mouse macrophage cells, incubation with enriched fractions prior to treatment with LPS blocked the induction of several pro-inflammatory markers including nitric oxide, TNFα, IL-1β, NOS2 and COX2.</p> <p>Conclusions</p> <p>Our results show that human serum extracts enriched with endogenous long-chain hydroxy fatty acids possess anti-inflammatory and anti-proliferative activity. These findings support a hypothesis that the reduction of these metabolites with age may result in a compromised ability to defend against uncontrolled cell growth and inflammation, and could therefore represent a significant risk for the development of CRC.</p

Sclerotinia sclerotiorum : phytotoxins and metabolism of phytoalexins

Sclerotinia sclerotiorum (Lib.) de Bary is a plant pathogenic fungus causing serious yield losses in a broad range of cultivated plants, excluding cereals. Most of the economically important brassicas such as canola, rapeseed, mustards, cabbages and others such as sunflower, peanut, bean, soybean, lettuce, and carrot are susceptible to this pathogen. No host specificity has been demonstrated in S. sclerotiorum and there is no specific resistance known in the host species. The main trust of this research project was to establish biotransformation pathways used by S. sclerotiorum to detoxify phytoalexins produced by host plants. As well, the potential production of phytotoxins and cytotoxic compounds by S. sclerotiorum was analyzed. The metabolite sclerin was isolated from cultures of S. sclerotiorum and its phytotoxicity to crucifers established for the first time. Fatty acids isolated from sclerotia of S. sclerotiorum of which oleic acid was a major component were found to be cytotoxic to the brine shrimp (Artemia salina). Chemical defences, i.e. phytoalexins, were elicited and isolated from a resistant plant (Erucastrum gallicum, dog mustard): indole-3-acetonitrile, arvelexin, 1-methoxyspirobrassinin and erucalexin (new phytoalexin, a structural isomer of 1-methoxyspirobrassinin). As well, the biotransformations of the phytoalexins brassinin, produced by rapeseed, canola and brown mustard plants, camalexin, and 6-methoxycamalexin, produced by wild crucifers like Arabidopsis thaliana, Capsella bursapastoris and Camelina sativa, were investigated. It was established that S. sclerotiorum could efficiently metabolize these phytoalexins using a remarkable glucosylation reaction of their indole ring. Overall, results of these biotransformation studies followed by antifungal bioassays indicated that metabolism of brassinin, camalexin, and 6-methoxycamalexin were detoxification processes. Analogues of these phytoalexins were designed based on structures of the detoxification products to probe the specificity or otherwise of the enzyme(s) involved in the metabolism of the phytoalexins. All the analogues tested were metabolized by the fungus though at slower rates. 6-Fluorocamalexin, one of the analogues, significantly slowed down the metabolism of brassinin both in cell cultures and in enzymatic assays with cell homogenates. Partial purification (five fold) of the brassinin detoxifying enzyme (brassinin glucosyltransferase) of S.sclerotiorum was achieved