Pentafluorobenzyloxime Derivatives Research Articles

Strategies for the analysis of chlorinated lipids in biological systems

Myeloperoxidase-derived HOCl reacts with the vinyl ether bond of plasmalogens yielding α-chlorofatty aldehydes. These chlorinated aldehydes can be purified using thin-layer chromatography, which is essential for subsequent analysis of extracts from some tissues such as myocardium. The α-chlorofatty aldehyde 2-chlorohexadecanal (2-ClHDA) is quantified after conversion to its pentafluorobenzyl oxime derivative using gas chromatography–mass spectrometry and negative-ion chemical ionization detection. 2-ClHDA accumulates in activated human neutrophils and monocytes, as well as in atherosclerotic lesions and infarcted myocardium. Metabolites of 2-ClHDA have also been identified, including the oxidation product, 2-chlorohexadecanoic acid (2-ClHA), and the reduction product, 2-chlorohexadecanol. 2-ClHA can be quantified using LC–MS with selected reaction monitoring (SRM) detection. 2-ClHA can be ω-oxidized by hepatocytes and subsequently β-oxidized from the ω-end, leading to the production of the dicarboxylic acid, 2-chloroadipic acid. This dicarboxylic acid is excreted in the urine and can also be quantified using LC–MS methods with SRM detection. Quantitative analyses of these novel chlorinated lipids are essential to identify the role of these lipids in leukocyte-mediated injury and disease.

Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry

Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20μg and 40min, respectively. The Km value obtained (6μM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.

Formation of a vitamin C conjugate of acrolein and its paraoxonase-mediated conversion into 5,6,7,8-tetrahydroxy-4-oxooctanal.

Vitamin C (ascorbic acid) has been reported to participate in Michael addition reactions in vitro to form vitamin C conjugates with alpha,beta-unsaturated aldehydes, such as acrolein. This study shows evidence for the formation and metabolism of the vitamin C conjugate of acrolein (AscACR) in cultured human monocytic THP-1 cells exposed to acrolein diacetate. By using (18)O and (13)C labeling in combination with liquid chromatography-tandem mass spectrometry, AscACR was shown to undergo hydrolytic conversion of the ascorbyl lactone into an intermediate carboxylic acid. Subsequent decarboxylation of the carboxylic acid yielded 5,6,7,8-tetrahydroxy-4-oxooctanal (THO). When THP-1 cells were pretreated with ascorbic acid (1 mM, 18 h) and then exposed to acrolein diacetate, THO was detected as its pentafluorobenzyl oxime derivative in the cell lysates and medium. Treatment of THP-1 cells with both ascorbic acid and acrolein diacetate was required for THO formation. The formation of THO from AscACR was facilitated by the lactonase enzymes, human recombinant paraoxonases 1 and 2. THP-1 cells exhibited PON activity, which explains the catalytic conversion of AscACR into THO in these cells. THO was formed in addition to metabolites of the glutathione conjugate of acrolein, indicating that THO formation contributes to the elimination of acrolein in a cellular environment.

Quantification of Pentafluorobenzyl Oxime Derivatives of Long Chain Aldehydes by GC–MS Analysis

Negative ion mass spectrometric techniques, for compounds having good ionization properties, such as pentafluorobenzyl derivatives, are believed to be more sensitive than positive ion methods. Preparation of PFB oximes of fatty aldehydes from crude lipid extracts is problematic due to the release of free aldehydes from plasmalogens during derivatization. Accordingly, in these studies plasmalogens were removed by silicic acid column chromatography prior to pentafluorobenzyl derivatization. This simple purification step to remove plasmalogens is shown to facilitate the quantification of long-chain aldehydes by analysis of their pentafluorobenzyl oxime derivatives utilizing gas chromatography-mass spectrometry in the negative ion chemical ionization mode. The limit of detection for long chain fatty aldehydes using this method is 0.5 pmol and it is linear over two orders of magnitude. Silicic acid column chromatography followed by electrospray ionization mass spectrometry demonstrated that plasmalogens were removed (the detection limit for this analyses was <or=0.3 pmol). Furthermore, we have exploited the utility and sensitivity of this method to identify increases in hexadecanal and octadecanal in 3-amino-1,2,4-triazole treated human neutrophils.

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.

Halogenated Monoterpene Aldehydes from the South African Marine Alga Plocamium corallorhiza

Four new halogenated monoterpene aldehydes (1-4) have been isolated from the South African marine red alga Plocamium corallorhiza, along with the known compounds 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene (5) and 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene (10). The structures of the new compounds were determined by interpretation of their spectroscopic data and synthesis and mass spectrometric analysis of their pentafluorobenzyloxime (PFBO) derivatives.

Selective Determination of Dihydroxyacetone in Self-Tanning Creams by HPLC as Pentafluorobenzyloxime Derivative

A new rapid and specific method for the determination of dihydroxyacetone (DHA) in self-tanning creams by reversed phase HPLC with UV detection was developed. DHA, together with hydroxyacetone (HA), used as internal standard, was extracted from the cream samples in a NaCl saturated aqueous solution and a small aliquot derivatized with pentafluorobenzylhydroxylamine (PFBHA) solution (10 mg/mL). The data on the time-course of the reaction showed that derivatization was completed after 5 min at room temperature in a citrate buffer at pH 4.0. The quickly formed oximes were isolated from the reaction mixture in acetonitrile after the addition of NaCl saturated solution. The rapid chromatographic separation on a reversed phase C-18 column using a simple acetonitrile/water mixture followed by detection at 262 nm allowed a selective estimation of DHA in self-tanning creams currently on the national market. Quantitative yields of the oxime formation was verified by comparing responses of DHA subjected to the described procedure and of synthetic derivative previously prepared. Linearity (r 2 ≥ 0.997), recovery (97–105%), LOD (20 nmol) and precision (RSD ≤ 3.52%) of the method were tested.

Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal

Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing alpha-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid (alpha-ClFA) and chlorinated fatty alcohol (alpha-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the alpha-ClFOH and alpha-ClFA pools. After 1 h, the radiolabel was predominantly found in the alpha-ClFOH pool. Cell-derived alpha-ClFOH and alpha-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to alpha-ClFA and alpha-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to alpha-ClFA and reduced to alpha-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from alpha-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease.

GC‐MS analysis of aldehydes reveals diverse pathways of lipid hydroperoxide metabolism with myoglobin

A comprehensive profile of aldehyde production is being developed as a tool to probe the factors that influence lipid hydroperoxide reactions by hemeproteins. Cryofocusing coupled with GC-TOFMS provided a real-time method for monitoring the volatile metabolite hexanal, and formation of pentafluoro benzyl oxime or pentafluro phenyl hydrazine derivatives and GC analysis were used to monitor malondialdehyde (MDA) and 4hydroxynonenal (4HNE) production. Studies with equine metmyoglobin (metMb) indicate that hydroperoxides in arachidonic acid (AA) are required for MDA and 4HNE production, that the molar ratio of MDA to 4HNE formed is greater for AA than for AA in phospholipid, and that trace metals in the metMb preparations contributed to some extent to the formation of MDA and 4HNE. The results suggest that the form of AA presented to metMb influences disposition of lipid hydroperoxides. Treatment of metMb with H2O2 forms a protein-bound heme adduct (MbH). MbH is more effective than Mb at generating TBARS products with phospholipids containing AA, although much less capable of forming MDA, 4HNE, and hexanal. MbH has optimal activity at low ionic strength (0.2 mM NaOAc, pH 6 or 7), whereas Mb has optimal activity at 2 mM NaOAc, pH 5. MbH is very active at supporting formation of lipid hydroperoxides, but does not influence as extensively as does metMb their conversion to aldehydes. Supported by NSF grant 9987063.

A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm.

A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).

Mass spectrometric analysis of pentafluorobenzyl oxime derivatives of reactive biological aldehydes

We recently demonstrated that a battery of reactive aldehydes can be generated by human neutrophils through the action of myeloperoxidase on α-amino acids. To determine the aldehydes, we formed the pentafluorobenzyloxime (PFBO) derivatives by reacting them with pentafluorobenzylhydroxylamine (PFBHA) and submitting the derivatives to gas chromatography electron-capture mass spectrometry (GC/EC/MS). Two geometric isomers are formed for each of the aldehydes, and they are separable by gas chromatography (GC) and exhibit distinguishable electron-capture (EC) mass spectra. Major fragment ions include [M − HF] · −, which probably has a six-membered ring formed via HF loss from the molecular radical anion. Subsequent decomposition of this intermediate yields other characteristic ions (e.g. those formed by elimination of NO and the anion of m/ z 178). We proposed fragmentation pathways and mechanisms that are consistent with the data derived from collisionally-activated dissociations (CAD) coupled with tandem mass spectrometry, exact-mass measurements, studies of deuterium-labeled analogs, and calculations by PM3 semiemperical and ab initio methods. The generality of the structurally informative fragmentation pattern together with GC separation is the basis of a powerful means for the identification of reactive aldehydes in biological processes and the pathogenesis of disease.

Sensitive determination of volatile aliphatic aldehydes in autoxidized fats and oils by gas chromatographic/electron-capture detection of their O -pentafluorobenzyloxime derivatives

This paper describes a novel method for the sensitive determination of the volatile aliphatic aldehydes formed by autoxidation in fats and oils. In a solution of fats, the aldehydes are derivatized to the O-pentafluorobenzyloximes (PFBOs) without any isolation procedure. From the mixture, the lipids are removed by silica gel column chromatography using pentane/dichloromethane (4+1) as the eluant. The eluate is evaporated to dryness and the PFBOs are extracted from the residue with a small volume of acetonitrile. They are separated by capillary column gas chromatography and are determined with high selectivity and sensitivity by using an electron-capture detector. The determination limit was as low as 0.01 mg/kg for hexanal. Analysis of a series of corn oil samples yielded high recoveries for 14 aldehydes added at the 1 mg/kg level, with low relative standard deviations of between 3 and 17%.

GC/MS characterization of effluents front the ozone bleaching of eucalypt kraft pulps

Concerns regarding the possible environmental effects of organochlorine by‐products from bleaching of pulp with chlorine‐based compounds have led to the pulp and paper industry developing new bleaching sequences. Ozone, oxygen and hydrogen peroxide are the main reagents in these Totally Chlorine Free (TCF) bleaching processes. In this study, eucalypt kraft pulps from a variety of Australian wood sources were subjected to bleaching sequences comprising oxygen, ozone and hydrogen peroxide/alkali extraction stages. The aqueous liquid effluents from each stage were analyzed by GC/MS for aldehydes, ketones, alcohols, carboxylic acids and other by‐products. Pentafluorobenzyl oxime derivatives of the aldehydes and ketones were analyzed by electron impact GC/MS. The major carbonyl compounds detected were formaldehyde, glyoxal, dimethylglyoxal and acetone. An homologous series of n‐aldehydes corresponding to cleavage of ω‐3, 6, 9 and 12 unsaturated fatty acids also was detected. Aromatic aldehydes were identified in the oxygen stage and high consistency ozone stages, but not in any medium consistency ozone or post‐ozone bleach stages. In all stages a series of saturated alkyl carboxylic acids from formic to octacosanoic acid was detected. Formic and acetic acids were present in the highest yield. Only trace quantities of unsaturated fatty acids were detected. Details of these and other compounds detected are discussed.

Increase of aldehydic compounds derived from plasmalogens in the brain of aged cattle

The content of plasmalogens in bovine brain was investigated with respect to age. No difference between the plasmalogen content in brain of young and old individuals was detected. In old individuals, plasmalogens suffer much easier hydrolysis to corresponding aldehydes than in young ones. In addition, the brain of old animals contain an approx. 30-fold higher amount of free aldehydes and plasmalogen epoxides compared to young ones. Aldehydes, as well as derived α-hydroxyaldehydes, were trapped by addition of pentafluorobenzylaminehydrochloride. The resulting pentafluorobenzyloxime derivatives were enriched by thin-layer chromatography, transferred to trimethylsilyl derivatives and further investigated by GC/MS. Quantification was achieved by GC/FID as well as by GC/ECD.

Gas chromatographic-electron impact mass spectrometric screening procedure for unknown hydroxyaldehydic lipid peroxidation products after pentafluorobenzyloxime derivatization

Aldehydic lipid peroxidation products can be detected after transformation to pentafluorobenzyloxime derivatives by GC-MS screening using characteristic ion traces. Thus the rather unstable unsaturated hydroxyaldehyde, 6-hydroxy-2,4-undecadienal, was identified as autoxidation product of linoleic acid. Its structure was unambiguously confirmed by comparison with an authentic sample. After Fe 2+ -ascorbate induced lipid peroxidation of oleic acid several 4-hydroxy-2-alkenals and 4-hydroxyalkanals were detected. These represent previously unknown secondary oxidation products of lipid peroxidation of oleic acid. Nevertheless oleic acid proved about 1000 times more stable against peroxidation than linoleic or higher unsaturated acids.

Direct Characterization of Protein Adducts of the Lipid Peroxidation Product 4-Hydroxy-2-nonenal Using Electrospray Mass Spectrometry

Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.

Quantitative Measurement of 4-Hydroxyalkenals in Oxidized Low-Density Lipoprotein by Gas Chromatography-Mass Spectrometry

Previously, we reported on the determination of 4-hydroxyalkenals as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry. In this study, an improved method is presented which allows quantitative detection of 4-hydroxyalkenals in tissues by using two stable isotope-labeled internal standards, 9 D 3-4-hydroxynonenal and 9 D 3-4-hydroxyhexenal. This assay was used to quantitate 4-hydroxyalkenals in copper-oxidized human low-density lipoprotein, and we found more 4-hydroxynonenal than 4-hydroxyhexenal. This is consistent with the fact that there are more ω-6 than ω-3 fatty acids in human low-density lipoprotein samples, which are the sources of 4-hydroxynonenal and 4-hydroxyhexenal, respectively.

誘導体化ガスクロマトグラフィー及び高速液体クロマトグラフィーによる大気中アルデヒドの測定

Derivatization techniques used for the analysis of volatile aldehydes in air samples are reviewed. Pentafluorobenzyloxime derivatives of aldehydes are useful for gas-chromatographic analysis because the reactions proceed in mild conditions and the derivatives show good volatility. Thiazolidine derivatives have some characteristics; i, e., the reaction is rapid and mild, produces only one derivative from one aldehyde, and the products are selectively detected by NPD. DNPH/HPLC method combined with solid sorbent sampling system is suitable for routine analysis.

A thermospray mass spectrometric assay for Fe-induced 4-hydroxynonenal in tissues.

A method is presented for the determination of 4-hydroxynonenal (HNE) in tissue homogenates following in vitro lipid peroxidation induced by iron (Fe++). HNE is measured as the pentafluorobenzyl oxime derivative using liquid chromatography thermospray mass spectrometry. In vitro metabolism of HNE via the glutathione/glutathione-S-transferase pathway was inhibited using iodoacetic and iodobenzoic acids. The assay has been used as an indicator of the peroxidizability of tissue samples from animals both adequate in and depleted of alpha-tocopherol. The concentrations of HNE produced in tissues taken from animals depleted of alpha-tocopherol were found to be up to 8 times higher than those taken from animals supplemented with alpha-tocopherol.

Gas chromatographic and gas chromatographic/tandem mass spectrometric characterization of precursors on the sesterterpene pathway for steroid biosynthesis.

An increasing amount of evidence is accumulating to support the proposal that steroidogenesis can occur by a sesterterpene pathway as well as the cholesterol pathway. Key intermediates on the sesterterpene pathway are 23,24-dinor-5-cholen-3 beta-ol (guneribol) and some of its metabolites, e.g. 23,24-dinor-4-cholen-3-one (guneribone). It has been reported that these intermediates are biosynthesized and converted to steroid hormones by a range of endocrine tissues in vitro. Monitoring the pentafluorobenzyloxime derivatives by negative ion chemical ionization mass spectrometry in the electron capture mode provided evidence for the presence of guneribone in extracts of bovine testicular and human adrenal tumour tissue. Complementary evidence was obtained from gas chromatographic/tandem mass spectrometric data generated on a triple-quadrupole instrument by monitoring daughter ions (in the multiple ion detection, MID mode) of the molecular anion of derivatized guneribone in both standards and tissue extracts. The present findings that sesterterpene pathway intermediates are present as endogenous compounds in tissue extracts, together with the previously reported radiochemical data, give further support to the sesterterpene pathway hypothesis.