Autoxidation Products Research Articles

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Nano-liquid chromatography–tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation

Oxysterols are present in mammalian brain at ng/g–μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MS n ). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC–MS n provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3 ± 3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [ 2H 7]-labelled cholesterol and cholesterol autoxidation products identified.

Cytotoxic molecular mechanisms and cytoprotection by enzymic metabolism or autoxidation for glyceraldehyde, hydroxypyruvate and glycolaldehyde

Previously, we showed that dietary fructose or its carbonyl metabolites, glyceraldehyde and glycolaldehyde, could be oxidized by inflammatory reactive oxygen species (ROS), products of immune cells, to form highly toxic and genotoxic products, such as glyoxal. Glycolaldehyde-caused hepatocyte protein carbonylation likely resulted from glyoxal, an autoxidation product formed by ROS. Although hepatocyte protein carbonylation by glyoxal or d-glycolaldehyde was rapid, the product was unstable. Glyceraldehyde-induced protein carbonylation was slower and was also less cytotoxic. Non-toxic concentrations of H 2O 2 were then used to mimic inflammation and oxidative stress associated with fructose-induced non-alcoholic steatohepatitis (NASH). A slow infusion of H 2O 2 markedly increased glyoxal, glyceraldehyde, and glycolaldehyde-induced cytotoxicity and protein carbonylation. However, it had a smaller effect on glyceraldehyde-induced protein carbonylation. The cytotoxicities of both aldehydes were increased if glutathione (GSH)-depleted hepatocytes were used, presumably because of the increased ROS formation and subsequent glyoxal-induced protein carbonylation. Catalytic amounts of Cu or Fe increased the glycolaldehyde and glyceraldehyde-induced cytotoxicity and protein carbonylation resulting from autoxidation to glyoxal. Glyceraldehyde and glycolaldehyde were also detoxified by mitochondrial aldehyde dehydrogenase (ALDH2) as ALDH2 inhibitors increased their cytotoxicity. Hydroxypyruvate has not been previously tested for toxicity and was found to be the most toxic fructose metabolite. Catalytic amounts of Cu or Fe caused hydroxypruvate autoxidation, which formed extensive ROS, glycolaldehyde and glyoxal. Iron chelators EGTA or deferoxamine inhibited cytotoxicity as well as the extensive ROS formation. The Girard assay confirmed that glyoxal was a common autoxidation product from glyceraldehyde, glycolaldehyde and hydroxypyruvate.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape(TM) from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

Isotope-reinforced polyunsaturated fatty acids protect yeast cells from oxidative stress

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4 h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D 2-linoleic or 11,11,14,14-D 4-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.

ChemInform Abstract: Characterization of Autoxidation Products of the Antifungal Compounds Econazole Nitrate and Miconazole Nitrate.

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Abstract 2918: Molecular identification of the cholesterol-5,6-epoxide hydrolase: A new target for selective estrogen receptor modulators and unsaturated fatty acids

Abstract The cholesterol-5,6-epoxide hydrolase (ChEH) is the last epoxyde hydrolase which coding gene remains to be identified. The ChEH catalyzes the hydration of 5,6α-epoxy-cholestanol (α-CE) and 5,6β-epoxy-cholestanol (β-CE), two autoxydation products of cholesterol, into cholestane-3β,5α,6β-triol (CT). We earlier reported that the antiestrogen binding site (AEBS) was a hetero-oligomeric complex made of two subunits: the 3β-hydroxysterol-Δ7-reductase (DHCR7) and 3β-hydroxysterol- Δ8- Δ7-isomerase (D8D7I), two enzymes involved in post-lanosterol cholesterol biosynthesis (Kedjouar et al, J Biol Chem, 2004). We have recently reported that AEBS ligands induced differentiation and apoptosis of human breast cancer cell lines through the intracellular accumulation of autoxydation products of sterols such as α-CE and β-CE (Payre et al, Mol Cancer Ther, 2008; de Medina et al, Cell Death & Differ, 2009). Because the ChEH and AEBS displayed some biochemical similitudes, we have investigated, in the present study, the potential relationship that may exist between the ChEH and AEBS. We established that α-CE and β-CE (the substrates of the ChEH) and CT (the product of the catalytic activity of the ChEH), were high-affinity and competitive ligands of AEBS. AEBS ligands include selective estrogen receptors modulators (SERMs) such as tamoxifen and raloxifene, poly unsaturated fatty acids such as docosahexaenoic acid (DHA), oxysterols such as 7-hydroxycholesterol and 7-ketocholesterol, and diphenylmethane compounds such as N-Pyrrolidino-4-(phenylmethyphenoxyl)-ethanamine (PBPE) and tesmilifene. We established that all AEBS ligands were inhibitors of the ChEH at pharmacological concentrations. We demonstrated a positive correlation between the binding to AEBS and the inhibition of the ChEH catalytic activity by AEBS ligands (r2 = 0.95, n = 39, p < 0.0001) and showed that AEBS and the ChEH had the same pharmacological profile. Finally, the structural similarity between the ChEH and AEBS was established by co-expression or genetic invalidation in mammalian cells of the D8D7I and the DHCR7, which respectively allowed the reconstitution or the impairment of both AEBS and the ChEH activity. Collectively, these data demonstrate that AEBS and the ChEH are molecularly and pharmacologically similar. These results provide the first evidence of the molecular identification of the ChEH, which coding gene had never been reported. In conclusion, we have identified a mechanism by which AEBS ligands induce the accumulation of sterol autoxidation products that were previously shown to induce breast cancer cell differentiation and apoptosis. The ChEH constitutes a new target for the chemoprevention and the treatment of breast cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2918.

Thermal and oxidative disproportionation in amine degradation—O 2 stoichiometry and mechanistic implications

Three modes of amine degradation have been previously identified—direct oxidation by metal ions, thermal disproportionation and autoxidation. Results are presented in which products of disproportionation reactions of quaternary compounds are compared to those seen in the thermal degradation of methyldiethanolamine, MDEA. This strongly suggests a role of a quaternary or a protonated amine intermediate in this reaction. Products of MDEA autoxidation at 140 °C also show characteristics of disproportionation reactions. Potential mechanisms are presented to account for the products seen.

Isotope‐Reinforced Polyunsaturated Fatty Acids Protect Yeast Cells from Oxidative Stress

Polyunsaturated fatty acids (PUFAs) are exquisitely sensitive to autoxidation damage. The enhanced vulnerability of PUFAs to such autoxidation stems from the labile nature of the bis‐allylic hydrogen atoms. PUFAs synthesized to contain Deuterium atoms uniquely at the bis‐allylic sites (termed isotope‐reinforced PUFAs) would be expected to be more resistant to autoxidation reactions due to the isotope effect. This hypothesis was tested by making use of yeast coq mutants with defects in biosynthesis of coenzyme Q (CoQ, or ubiquinone). CoQ plays a well‐known role in respiratory energy metabolism and also functions as a lipid soluble chain terminating antioxidant. Although yeast cannot synthesize PUFAs, they are able to incorporate exogenously supplied PUFAs. Yeast coq mutants incubated in the presence of linolenic acid (C18:3) exhibit profound loss of viability as ascertained by greater than 99% loss of colony formation at 4 hours. In contrast, the coq mutants treated with the isotope‐reinforced linolenic acid (bis‐allylic D4‐C18:3) retain 80–90% viability, a value similar to wild‐type or CoQ‐replete yeast. These results indicate that isotope reinforced PUFAs are stabilized as compared to standard PUFAs, and the coq mutant yeast cells containing the D4‐linolenic acid are protected against the toxic effects of lipid autoxidation products. This research was supported by NIH GM45952.

Analysis of Carbonaceous Solid Deposits from Thermal Oxidative Stressing of Jet-A Fuel on Iron- and Nickel-Based Alloy Surfaces

Thermal stressing of Jet-A was conducted in a flow reactor on iron- and nickel-based metal surfaces at a fuel flow rate of 1 mL/min for 5 h at a wall temperature of 350 °C and 3.5 MPa (500 psig) so that both liquid-phase autoxidation and thermal decomposition of autoxidation products contribute to the formation of carbonaceous deposits. The deposits produced were characterized by field emission scanning electron microscopy (FESEM) and temperature programmed oxidation (TPO). The effect of metal surface on deposit formation increases in the following order: AISI316 < AISI 321 ≈ AISI 304 < Inconel 600 < AISI 347 < Inconel 718 < FecrAlloy < Inconel-750X. The variation in the activity of the metal substrates is attributed to their reaction with reactive sulfur compounds in the fuel and interaction of oxygenated intermediates formed by autoxidation during thermal stressing.

Dioxygenase catalysis by d 0 metal–catecholate complexes containing vanadium and molybdenum with H 2(3,5-DTBC) and H 2(3,6-DTBC) substrates

Facile synthetic dioxygenases, catalysts that can split O 2 and place both oxygen atoms selectively into 2 olefins to yield 2 epoxides or into 2 C–H bonds to yield 2 alcohols, remain a “Holy Grail” of oxidation catalysis. Recently, it was shown that [VO(3,5-DTBC)(3,5-DBSQ)] 2 (where 3,5-DTBC and 3,5-DBSQ are 3,5-di- tert-butylcatecholate and 3,5-di- tert-butylsemiquinone, respectively) is the catalytic-cycle resting state of a record catalytic lifetime catechol dioxygenase catalyst (100,000 total catalytic turnovers) for the substrate 3,5-di- tert-butylcatechol, H 2(3,5-DTBC) [C.-X. Yin, R.G. Finke, J. Am. Chem. Soc. 127 (2005) 9003–9013]. Herein we show that the precatalyst V(3,6-DTBC) 2(3,6-DBSQ) also gives dioxygenase products for the substrate H 2(3,5-DTBC), notably the same dioxygenase products in similar yields as seen for [VO(3,5-DTBC)(3,5-DBSQ)] 2. EPR studies show that the same g = 2.003–2.004 species are present in solution throughout the oxidation reaction for both [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ). The similar products, product yields, and EPR spectra observed suggest that both [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ) are feeding into the same catalytic cycle. In addition we have expanded the substrates to include H 2(3,6-DTBC), a substrate of interest for its higher symmetry, structurally simplified organic products and steric hindrance expected to favor the formation of monomeric metal–catecholate complexes. The precatalysts [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ) were examined with H 2(3,6-DTBC) and found to give the same intradiol and extradiol dioxygenase products in the same yields. EPR studies of [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ) with H 2(3,6-DTBC) show that the same spectra are observed throughout the oxidation, but at different reaction times. These EPR observations, along with the product studies, suggest that [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ) have a common mechanistic cycle en route to the H 2(3,6-DTBC) dioxygenase products, albeit a mechanism different than that observed for H 2(3,5-DTBC) based on observed EPR spectra and differing product distributions. We also show that oxidation catalysis with [MoO(3,5-DTBC) 2] 2 follows primarily an oxidase path leading to the undesired benzoquinone autoxidation products for both H 2(3,5-DTBC) and H 2(3,6-DTBC) and with reaction times one to two orders of magnitude longer than seen for the vanadium precatalysts. The dramatic difference in product distribution and slower rate for [MoO(3,5-DTBC) 2] 2, along with its lack of a semiquinone ligand in at least this precatalyst, suggests the hypothesis that the d 0 vanadium(V) bonded to a semiquinone ligand in [VO(3,5-DTBC)(3,5-DBSQ)] 2 and V(3,6-DTBC) 2(3,6-DBSQ) is a necessary component of these dioxygenase precatalysts able to produce primarily dioxygenase products.

La oxidación lipídica en la cadena de producción acuícola

&lt;p align="left"&gt;En el campo alimenticio, la industria acuícola ha sustentado su desarrollo en la utilización de materias primas provenientes de la cadena de procesamiento especies de peces de origen marino. Los principales productos industriales de esta cadena son la harina y el aceite de pescado, los cuales tienen un alto valor nutricional y económico. En el caso particular del aceite de pescado, se ha encontrado que es una materia prima escasa, que presenta un alto contenido de ácidos grasos poliinsaturados (AGPI omega 3) y que ha sido catalogada como alimento funcional por los beneficios para la salud del consumidor. Paradójicamente el alto contenido de AGPI n-3 hace del aceite de pescado una materia prima altamente susceptible a los procesos de auto oxidación que promueven la transformación de los ácidos grasos en Peróxidos, aldehídos, cetonas y polímeros, responsables del daño celular oxidativo. En el presente documento describimos los procesos de generación de productos primarios y secundarios de la oxidación, los mecanismos de protección en contra del daño oxidativo y el efecto negativo de los productos primarios y secundarios de la auto oxidación lipídica sobre calidad nutricional de los alimentos balanceados, la salud de los peces y la calidad del producto cárnico para consumo humano.&lt;/p&gt;

Synthesis and Characterization of VV(3,6-DBSQ)(3,6-DBCat)2, a d0 Metal Complex with Dioxygenase Catalytic Activity

Transition-metal complexes containing redox-active quinoid ligands are of interest because of their catalytic capabilities in multielectron, substrate-activation reactions such as dioxygenase catalysis using O(2). The new catecholate complex V(V)(3,6-DBSQ)(3,6-DBCat)(2) (where 3,6-DBSQ = 3,6-di-tert-butylsemiquinone and 3,6-DBCat = 3,6-di-tert-butylcatecholate) was synthesized by combining VO(acac)(2) with 1 equiv of 3,6-DBBQ (where 3,6-DBBQ = 3,6-di-tert-butylbenzoquinone) and 2 equiv of H(2)(3,6-DBCat) in dry methanol under an inert atmosphere. The resultant complex was characterized by single-crystal X-ray diffraction, elemental analysis, near-IR, UV/vis, and electron paramagnetic resonance (EPR) spectroscopy. The crystallography as well as the near-IR and EPR studies suggest that the radical spin is localized on the 3,6-DBSQ ligand at room temperature, making V(V)(3,6-DBSQ)(3,6-DBCat)(2) a type 1 mixed-valence complex. Initial dioxygenase catalysis studies reveal that V(V)(3,6-DBSQ)(3,6-DBCat)(2) is a good dioxygenase precatalyst for the substrate H(2)(3,6-DBCat) with O(2) in ca. 600 total turnovers to >93% intra- and extradiol products with only 1-2% of the undesired benzoquinone autoxidation product.

Autoxidation of lipids in parchment

Historic parchment is a macromolecular material, which is complex due to its natural origin, inhomogeneity of the skin structure, unknown environmental history and potential localised degradation. Most research into its stability has so far focussed on thermal and structural methods of analyses. Using gas chromatographic analysis of the atmosphere surrounding parchment during oxidation, we provide the experimental evidence on the production of volatile aldehydes, which can be the products of lipid autoxidation. Oxidation of parchment with different aldehyde emissions was additionally followed in situ using chemiluminometry and the same techniques were used to evaluate the oxidation of differently delipidised parchment. It was shown that the production of peroxides and the emission of aldehydes from the material decrease with lower lipid content. Building on this evidence, we can conclude that the presence of lipids (either initially present in the skin or resulting from conservation intervention) leads to oxidative degradation of collagen and that the non-destructive analysis of the emission of volatiles could be used as a quick tool for evaluation of parchment stability.

Monitoring changes in the volatile profile of cheese crackers during storage using GC–MS and PTR–MS

AbstractThe primary objective of this work was to determine the feasibility of using PTR–MS ion profiles to monitor changes in the volatile profile of cheese crackers during storage. This was done by obtaining PTR–MS ion profiles of cheese crackers during the early shelf‐life of the product (first 4 months of storage) and comparing these data to those obtained using a more traditional approach, i.e. purge and trap–GC–MS (PT–GC–MS). The PTR–MS profile of the samples was monitored for 47 ion masses, mainly corresponding to low molecular weight compounds or fragments of low mass. From the application of multivariate statistical analysis (cluster analysis, PCA, ANOVA) to the PTR–MS data of the samples of different storage times, it was shown that the age of the sample was an important factor for their classification. It was possible to differentiate two main groups of samples, the first group included samples of age &lt;3 months (1, 1.5 and 2 months). This group had low ion intensities relative to the second group. The second group comprised samples of age ≥3 months (3 and 4 months) that showed higher ion intensities. PT–GC–MS data on the same samples found 38 volatile compounds, primarily aldehydes, that increased during storage. A good correlation was found between changes in some PTR–MS masses and major volatile compounds of crackers. The compounds that increased were generally products of autoxidation. It appears that a PTR–MS could be a useful tool for monitoring volatile changes during storage in cheese crackers. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Effect of temperature on lipid‐derived volatile compounds in boiled potato during storage

AbstractBACKGROUND: The aroma of boiled potatoes is greatly appreciated by consumers. Although it is formed by 150 volatile compounds, only a few seem to actually contribute to the characteristic aroma. In addition, potatoes have to be stored at a low temperature after harvest to guarantee a year‐round supply. However, even at low temperatures, tuber volatile composition is modified during storage. The aim of the present study was to investigate the effect of storage temperature on boiled potato volatile constituents, mainly focused on lipid‐derived components, by considering different varieties stored at 4 °C and 8 °C.RESULTS: It was found that fewer lipid‐derived components were obtained when the potato tubers were stored at 4 °C rather than 8 °C. Specifically, the amounts of 2,4‐decadienal isomers and hexanal increased with the storage time, particularly at 8 °C. Similarly, pentanal and nonanal, which are autoxidation products, occurred to a greater extent at higher temperatures. In contrast, the levels of sugar‐derived compounds, in general, increased during storage at 4 °C rather than 8 °C as a result of higher content of sugars at low temperature.CONCLUSION: The effect of storage temperature on lipid‐derived volatile components in boiled potato appears to depend on the variety studied. Generally speaking, lower temperatures during storage promote sugar content and, as a consequence, sugar‐derived products. At the same time, lower temperatures result in less lipid oxidation and, therefore, a fewer lipid‐derived compounds. Copyright © 2009 Society of Chemical Industry

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.

Identification of autoxidation oligomers of flavan‐3‐ols in model solutions by HPLC‐MS/MS

Autoxidation of flavan-3-ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The results showed that (+)-catechins and (-)-epicatechins generated autoxidation reaction with each other to form a series of oligomers that had the same [M - H](-) molecular ions (MS(1)) as those of natural procyanidins, but had completely different fragment ions (MS(2)). According to MS/MS analysis, the major fragments of these oligomers were derived not only from the retro-Diels-Alder (RDA) dissociations on the C-rings of the flavan-3-ol units, but also from the quinone-methide (QM) cleavage of the interflavan linkages (IFL), and thus they were identified as B-type dehydrodicatechins, B-type dehydrotricatechins and A-type dehydrotricatechins, respectively. The potential structures of their [M - H](-) molecular ions and partial fragment ions were deduced on the basis of the MS/MS characterization and the oxidation of flavan-3-ols in previous reports. Some specific fragment ions were found to be very useful for identifying the autoxidation oligomers (the B-type dehydrodicatechins at m/z 393, the B-type dehydrotricatechins at m/z 681 and the A-type dehydrotricatechins at m/z 725).

Use of lipids and their degradation products as biomarkers for carbon cycling in the northwestern Mediterranean Sea

Changes in phytoplankton composition and degradation of particulate organic matter (POM) in the northwestern Mediterranean Sea were studied using time-series sediment trap samples collected during the spring of 2003 at the DYFAMED station. Lipid biomarkers (pigments, fatty acids, sterols, acyclic isoprenoids, alkenones and n-alkanols) were used to identify the main contributors to the POM produced during two phytoplankton blooms, while the effects of photooxidation, autoxidation and biodegradation were differentiated using characteristic lipid degradation products. Traps collected material corresponding to pre-bloom, bloom and post-bloom periods. Pigment analyses in the integrated (0-200 m) water column samples indicated that diatoms dominated the initial stages of the bloom event, with smaller amounts of haptophytes and pelagophytes. During the second part of bloom event there was a switch to haptophyte dominance with significant contributions from diatoms and pelagophytes, and an increased contribution from cryptophytes. Fatty acid distributions in the trap samples reflected contributions from marine bacteria, phytoplankton and zooplankton. Photooxidation and autoxidation products of monounsaturated oleic, cis-vaccenic and palmitoleic acids were detected along with photooxidation products from the chlorophyll side-chain. The relatively good correlation between the variation of U 37 K ′ index and specific phytol autoxidation product percentage allowed us to attribute the alterations of U 37 K ′ observed during the pre-bloom period and in the deeper traps to the involvement of selective autoxidative degradation processes. A variety of sterol oxidation products formed by biohydrogenation, autoxidation and photooxidation were detected. Sterol degradation products appeared to be less suited than oxidation products of monounsaturated fatty acids for the precise monitoring of the degradation state of POM, but their stable functionalized cyclic structure constitutes a useful tool to estimate the part played by biotic and abiotic processes. In these waters, biotic degradation generally predominates, but abiotic degradation is not negligible and, as expected, the extent of biotic degradation increases with depth. To obtain a more complete picture of POM degradation, the use of a pool of lipid degradation products (i.e. from unsaturated fatty acids, the phytyl side-chain and sterols) should be employed.

The fate of marine lipids: Biotic vs. abiotic degradation of particulate sterols and alkenones in the Northwestern Mediterranean Sea

Sterol and alkenone compositions in suspended particle and surface sediment samples collected in the Northwestern Mediterranean Sea during the MEDFLUX program were used to evaluate the relative importance of biotic and abiotic degradation processes on marine organic matter. Alkenone concentrations decreased much more rapidly (~ 500 fold) between 5 and 800 m than Δ 5-sterols (~ 100-fold) or POC (~ 100-fold). The diverse functional groups attached to the stable tetracyclic carbon skeleton of Δ 5-sterols appeared to be useful for estimating the relative effects of biotic vs. abiotic (photooxidation and autoxidation) degradation. Products of abiotic degradation predominated over products of biotic degradation in suspended particles in the NW Mediterranean. For alkenones, the U 37 K′ index increased from 0.43 to 0.55 with increasing water depth, and a good correlation between variations of U 37 K′ and concentrations of specific Δ 5-sterol autoxidation products points to selective autoxidation of alkenones in suspended particles. Stereomutated alkenones (with cis double bonds) were detected in the surface sediment, allowing us to estimate that stereomutation resulted in a + 0.05 increase in U 37 K′ . Therefore, abiotic degradation may be another factor effect on alkenone-derived paleothermometry.