4-oxo-2-hexenal, a mutagen formed by omega-3 fat peroxidation: occurrence, detection and adduct formation

The purpose of this review is to summarize our recent studies of a novel mutagen, 4-oxo-2-hexenal. To identify the mutagens formed in a model reaction of lipid peroxidation, linolenic acid methyl ester and hemin were reacted with dG. A 4-oxo-2-hexenal-dG adduct (dG*) was identified in the model reaction mixture. The 4-oxo-2-hexenal (4-OHE) showed mutagenic activity in the Salmonella typhimurium strains TA100 and TA104. 4-OHE reacts with DNA to form dG, dC, and 5-methyl-dC(5-Me-dC)-adducts (dG*, dC*, 5-Me-dC*) in vitro. After 4-OHE was orally administered to mice, these adducts were detected in esophageal, stomach and intestinal DNA by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). We also confirmed the formation of 4-OHE during the heat processing of edible vegetable oil, and during cooking. It was present at an especially high concentration in broiled saury. 4-OHE is probably generated by the oxidation of omega-3 fats. These results provide a warning to humans, who may be exposed to this mutagen. Since 4-OHE induces DNA adduct formation in experimental animal organs, further studies on the carcinogenicity of 4-OHE and the detection of 4-OHE-DNA adducts in human tissue will be required.     

Identification of hydroxyalkenals formed from omega-3 fatty acids

The highly toxic lipid peroxidation product, 4-hydroxynonenal, is formed from the decomposition of hydroperoxides of omega-6 fatty acids. In this study the analogous hydroxyalkenals formed from the decomposition of hydroperoxides of omega-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) were isolated and identified using TLC densitometry, HPLC and GC/Mass Spectrometry. The major hydroxyalkenal formed from both fatty acids was a diene analog of 4-hydroxynonenal, 4-hydroxynona(2,6)dienal, while 4-hydroxyhexanal was a minor product. Measurement of specific omega-3 lipid peroxidation products may be important in studies using dietary fish oil.     

Current development in non-enzymatic lipid peroxidation products,isoprostanoids and isofuranoids,in novel biological samples

Abstract

Isoprostanoids and isofuranoids are lipid mediators that can be formed from omega-3 and omega-6 polyunsaturated fatty acids (PUFAs). F₂-isoprostanes formed from arachidonic acid, especially 15-F_2t-isoprostane, are commonly measured in biological tissues for decades as the biomarker for oxidative stress and diseases. Recently, other forms of isoprostanoids derived from adrenic, eicosapentaenoic, and docosahexaenoic acids namely F₂-dihomo-isoprostanes, F₃-isoprostanes, and F₄-neuroprostanes respectively, and isofuranoids including isofurans, dihomo-isofurans, and neurofurans are reported as oxidative damage markers for different metabolisms. The most widely used samples in measuring lipid peroxidation products include but not limited to the blood and urine; other biological fluids, specialized tissues, and cells can also be determined. In this review, measurement of isoprostanoids and isofuranoids in novel biological samples by gas chromatography (GC)–mass spectrometry (MS), GC–MS/MS, liquid chromatography (LC)–MS, and LC–MS/MS will be discussed.     

Identification of spin trapped carbon-centered radicals in soybean lipoxygenase-dependent peroxidations of omega-3 polyunsaturated fatty acids by LC/ESR,LC/MS,and tandem MS

With the combined techniques of on-line liquid chromatography/electron spin resonance (LC/ESR) and on-line liquid chromatography/mass spectrometry (LC/MS), we have previously characterized all classes of lipid-derived carbon-centered radicals (*Ld) formed from omega-6 polyunsaturated fatty acids (PUFAs: linoleic acid and arachidonic acid). In the present study, the carbon-centered radicals formed from two omega-3 PUFAs (linolenic acid and docosahexaenoic acid) resulting from their reactions with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butylnitrone (POBN) were investigated using the combination of LC/ESR and LC/MS techniques. A total of 16 POBN trapped carbon-centered radicals formed from the peroxidation of linolenic acid and 11 formed from the peroxidation of docosahexaenoic acid were detected by LC/ESR, identified by LC/MS, and structurally confirmed by tandem mass analysis (MS/MS). The on-line ESR chromatograms and MS chromatograms obtained from two omega-3 PUFAs closely resembled each other not only because the four major beta-scission products, including an ethyl radical and three isomeric pentenyl radicals, were formed from each PUFA, but also because isomeric POBN adducts of lipid dihydroxyallylic radicals from both PUFAs had almost identical chromatographic retention times.     

Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of omega-6 polyunsaturated fatty acids via LC/ESR,LC/MS,and tandem MS

Using the combined techniques of on-line high performance liquid chromatography/electron spin resonance (LC/ESR) and mass spectrometry (MS), we previously identified spin-trapped adducts of all expected carbon-centered lipid-derived radicals ((*)L(d)) formed in linoleic acid peroxidation. In the present study, spin trapped lipid-derived carbon-centered radicals formed from the reactions of two omega-6 polyunsaturated fatty acids (PUFAs: linoleic and arachidonic acids) with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) were identified using a combination of LC/ESR and LC/MS. All expected lipid-derived carbon-centered radicals in lipoxygenase-dependent peroxidations of linoleic acid and arachidonic acid were detected and identified by the combination of LC/ESR and LC/MS with confirmation by tandem mass spectrometry (MS/MS). The five classes of (*)L(d) formed from both omega-6 PUFAs including lipid alkyl radicals (L(*)), epoxyallyic radicals (OL(*)), dihydroxyallyic radicals ((*)L(OH)(2)), and a variety of R(*) and (*)RCOOH from beta-scission of lipid alkoxyl radicals, gave distinct retention times: POBN/(*)L(OH)(2) approximately 4-6 min, POBN/R(*) and POBN/(*)RCOOH approximately 8-22 min, POBN/L(*) and PBON/OL(*) approximately 25-36 min. The major beta-scission products in peroxidations of omega-6 PUFAs were the pentyl radicals. The ratio of beta-scission products, however, varied significantly depending on pH, [PUFA], as well as [O(2)].     

Formation of highly reactive cyclopentenone isoprostane compounds (A3/J3-isoprostanes) in vivo from eicosapentaenoic acid

Omega-3 (omega-3) polyunsaturated fatty acids (PUFAs) found in marine fish oils are known to suppress inflammation associated with a wide variety of diseases. Eicosapentaenoic acid (EPA) is one of the most abundant omega-3 fatty acids in fish oil, but the mechanism(s) by which EPA exerts its beneficial effects is unknown. Recent studies, however, have demonstrated that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and anti-proliferative effects. Very few studies to date have investigated which EPA oxidation products are responsible for this bioactivity. Our research group has previously reported that anti-inflammatory prostaglandin A(2)-like and prostaglandin J(2)-like compounds, termed A(2)/J(2)-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether cyclopentenone-IsoP compounds are formed from the oxidation of EPA in vivo. Herein, we report the formation of cyclopentenone-IsoP molecules, termed A(3)/J(3)-IsoPs, formed in abundance in vitro and in vivo from EPA peroxidation. Chemical approaches coupled with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to structurally characterize these compounds as A(3)/J(3)-IsoPs. We found that levels of these molecules increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 +/- 0.4 ng/mg EPA to 196 +/- 23 ng/mg EPA after 36 h. We also detected these compounds in significant amounts in fresh liver tissue from EPA-fed rats at basal levels of 19 +/- 2 ng/g tissue. Amounts increased to 102 +/- 15 ng/g tissue in vivo in settings of oxidative stress. These studies have, for the first time, definitively characterized novel, highly reactive A/J-ring IsoP compounds that form in abundance from the oxidation of EPA in vivo.     

Analysis of bone marrow fatty acid composition using high-resolution proton NMR spectroscopy

High-resolution 1H NMR spectroscopy as a complementary method in the analysis of human bone marrow fatty acid (FA) composition was examined. Marrow FA composition in 10 bone samples measured by NMR and gas chromatography (GC) were compared. NMR T1 relaxation time of FA was determined and reproducibility tests were performed to assess the variability. Good correlations were obtained between the NMR and GC results for omega-6 polyunsaturated fatty acid (PUFA) (Spearman r, 0.878), omega-3 PUFA (0.895), monounsaturated FA (0.964) and saturated FA (0.939). The NMR method tended to overestimate saturated FA and underestimate omega-3/omega-6 ratio compared to GC results. T1 relaxation time of marrow FA was 0.56-3.65s. Coefficient of variation of the NMR method was 0.6-8.2% in intra-experimental and 0.2-8.4% in inter-experimental measurements. This study demonstrates a complementary role for 1H NMR spectroscopy as an additional analytical tool in human lipid research.     

Lipid peroxidation and cellular damage caused by the pyrrolizidine alkaloid senecionine,the alkenal trans-4-hydroxy-2-hexenal,and related alkenals

Lipid peroxidation was examined as a possible mechanism for cell injury by trans-4OH-2-hexenal, the macrocyclic pyrrolizidine alkaloid senecionine and related alkenals in isolated rat hepatocytes. Each compound elicited a positive dose response for peroxidation of cellular lipids as measured by the formation of thiobarbituric acid-reactive products. The addition of the anti-oxidant N, N'-diphenyl-p-phenylenediamine to the hepatocyte suspensions inhibited the production of thiobarbituric acid-reactants. However, the presence of the anti-oxidant had no protective effects on the cell membrane integrity as evidence by the leakage of lactate dehydrogenase from the cells into the sorrounding media. These results suggest that lipid peroxidationwhich occurs in the presence of senicone, trans-4-OH-2-hexenal or related alkenals is not entirely responsible for the cellular damage is isolated rat hepatocytes.abbreviations DPPD, N,N diphenyl-p-phenylenediamine - LDH lactate dehydrogenase - PF, I, PF II perfusates I and II - SKF-525A 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride - TBARs thiobarbituric acid reactants - t-2H trans-2-hexenal - t-2N trans-2-nonenal - t-4HH trans-4-OH-2-hexenal - t-4HN trans-4-OH-2-nonenal     

Suppression of fatty acid synthase by dietary polyunsaturated fatty acids is mediated by fat itself,not by peroxidative mechanism

This study examined the effect of dietary polyunsaturated fatty acids (PUFA) that were supplemented with vitamin E on lipid peroxidation, glutathione-dependent detoxifying enzyme system activity, and lipogenic fatty acid synthase (FAS) expression in rat liver. Male Sprague-Dawley rats were fed semipurified diets containing either 1% (w/w) corn oil or 10% each of beef tallow, corn oil, perilla oil, and fish oil for 4 wk. Alpha-tocopherol was supplemented in perilla oil (0.015%) and fish oil (0.019%). Hepatic thiobarbituric acid reactive substances, an estimate of lipid peroxidation, were not significantly different among the dietary groups. The glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities were all elevated by the polyunsaturated fats, especially fish oil. The activity of FAS was reduced in the polyunsaturated fat-fed groups in the order of fish oil, perilla oil, and corn oil. The mRNA contents decreased in rats that were fed the 10% fat diets, particularly polyunsaturated fats, compared with the rats that were fed the 1% corn oil diet. Similarly, the inhibitory effect was the greatest in fish oil. These results suggest that lipid peroxidation can be minimized by vitamin E; PUFA in itself has a suppressive effect on lipogenic enzyme.     

Engineering oilseeds to produce nutritional fatty acids

There is a growing body of evidence suggesting that regular consumption of foods rich in omega-3 long chain polyunsaturated fatty acids has multiple positive health benefits. The fats and oils from marine fish contain high contents of these beneficial fatty acids but increased consumer demand has also increased strain on the ability of the world's fisheries to meet demand from wild capture. Many consumers are choosing fish oil supplements or are eating foods that have been complemented with fish oils instead of consuming fish directly. However, removing undesirable odors, flavors and contaminants is expensive. In contrast, oils derived from land plants such as soybean are inexpensive and contaminant free. Recent strides in plant molecular biology now allow the engineering of oilseeds for the production of novel fats and oils, including those synthesized by complex, multigene biosynthetic pathways such as the omega-3 long chain polyunsaturated fatty acids. Given the potential benefits to the environment with regards to overfishing and the health prospects of increased consumption of these healthy fatty acids, producing these fatty acids in oilseeds is a desirable and worthy goal. In this review, we will describe the recent advances in this field along with some of the technical hurdles encountered thus far.     

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.     

Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment.

In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)- plus (2E)-hexenal were identified as 2,4-dinitro-phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol.g-1 fresh weight to 45 nmol.g-1 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol.g-1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol.g-1 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment.     

Antarctic microorganisms as source of the omega-3 polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are long-chain polyunsaturated fatty acids (PUFAs) that belong to the omega-3 group. They are essential fatty acids found in phospholipid of cell membranes. There is strong evidence that these nutrients may also favorably modulate many diseases. Primary sources of omega-3 PUFAs in the human diet are fish and fish-derived products. The fishing industry worldwide, however, is becoming unable to satisfy the growing demand for these PUFAs. A promising cost-effective alternative source of PUFAs is bacterial production. We identified 40 Antarctic marine bacterial isolates by 16S rRNA gene sequence analysis. Fifteen genera in three phyla were represented in the collection. Isolates were tested for ability to produce EPA using a method in which their ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC) is determined and by gas chromatography coupled to mass spectrometry (GC–MS). All isolates could reduce TTC, and GC–MS analysis showed that four produced EPA and that six produced DHA. We show for the first time that isolates identified as Cellulophaga, Pibocella and Polaribacter can produce EPA and DHA, only DHA or only EPA, respectively. One isolate, Shewanella sp. (strain 8-5), is indicated to be a good candidate for further study to optimize growth and EPA production. In conclusion, a rapid method was tested for identification of new EPA producing strains from marine environments. New EPA and DHA producing strains were found as well as a potentially useful PUFA production strain.     

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.     

Effect of fish oil supplementation on plasma oxidant/antioxidant status in rats

The aim of this study was to investigate effect of dietary omega-3 fatty acid supplementation on the indices of in vivo lipid peroxidation and oxidant/antioxidant status of plasma in rats. The plasma thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, and activities of xanthine oxidase (XO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were studied in male Wistar Albino rats after ingestion of 0.4 g/kg fish oil (rich in omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid) for 30 days and compared to untreated control rats. The rats in the treated group had significantly higher SOD activity (P < 0.001), NO levels (P < 0.01) and decreased TBARS levels (P < 0.05) with respect to controls whereas GSH-Px and XO activities were not significantly different between the groups. None of the measured parameters had significant correlation with each other in both groups. We conclude that dietary supplementation of omega-3 fatty acids may enhance resistance to free radical attack and reduce lipid peroxidation. These results support the notion that omega-3 fatty acids may be effective dietary supplements in the management of various diseases in which oxidant/antioxidant defence mechanisms are decelerated.     

Identification of novel autoxidation products of the omega-3 fatty acid eicosapentaenoic acid in vitro and in vivo

Increased intake of fish oil rich in the omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5 omega-3) and docosahexaenoic acid (DHA, C22:6 omega-3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-of-the-art mass spectrometric techniques including Ag(+) coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides. Systematic study of the peroxidation of EPA provides the basis to examine the role of specific oxidation products as mediators of the biological effects of fish oil.     

Lipids and fatty acids in muscle, liver and skin of three edible fish from the Senegalese coast: Sardinella maderensis, Sardinella aurita and Cephalopholis taeniops

Lipid content and fatty acid composition were determined in three species of edible fish caught in Senegalese waters during the upwelling season (January, 1993). Sardinella maderensis and Sardinella aurita are fat fish containing more than 5% (fresh wt.) of lipids, whereas Cephalopholis taeniops is a lean fish with approximately 1% of lipids. Skin, liver and muscle were studied for each fish species. About 40 fatty acids were identified by GC and GC/MS as methyl esters and N-acyl pyrrolidides. Palmitic acid was the main acid in the muscle and skin of all samples studied (20-33% of total fatty acids). Oleic acid was the main fatty acid in the liver of S. maderensis (27.2%+/-0.1) and S. aurita (44.7%+/-0.1). Arachidonic acid was a minor component in all samples. The flesh (muscle) of the three fish species contained high concentrations of omega3 polyunsaturated fatty acids (PUFA), ranging from 16.0 to 29.1% and including 20:5 omega3 (eicosapentaenoic acid, EPA) and 22:6 omega3 (docosahexaenoic acid, DHA) acids as major components. These two acids together accounted for 24.7%+/-0.1 and 12.9%+/-0.1 of total acids in the skin of S. maderensis and S. aurita, respectively. The percentages of PUFA found in the fish studied were very similar to those in fish used commercially as sources of PUFA. Muscle sterols, which accounted for 9-11% of total lipids, consisted mainly of cholesterol (up to 97% of total sterols).     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Binding of chloroform to the cysteine of hemoglobin

The products of the covalent binding of chloroform to rat hemoglobin during microsomal metabolism were isolated and identified by gas chromatography (GC) and mass spectroscopy (MS). After isolation by Proteinase K hydrolysis, amino acid analysis and cellulose thin-layer chromatography (TLC), the major product was identified by GC/MS as N-hydroxymethyl cysteine and a minor product as 2-hydroxythiazolidine-4-carboxylic acid. N-Hydroxymethyl cysteine is proposed to be formed during isolation from the 2-oxothiazolidine-4-carboxylic acid present in the intact hemoglobin.     

Effect of randomized supplementation with high dose olive, flax or fish oil on serum phospholipid fatty acid levels in adults with attention deficit hyperactivity disorder

Dietary intake of omega-3 fatty acids has been positively correlated with cardiovascular and neuropsychiatric health in several studies. The high seafood intake by the Japanese and Greenland Inuit has resulted in low ratios of the omega-6 fatty acid arachidonic acid (AA, 20:4n-6) to eicosapentaenoic acid (EPA, 20:5n-3), with the Japanese showing AA:EPA ratios of approximately 1.7 and the Greenland Eskimos showing ratios of approximately 0.14. It was the objective of this study to determine the effect of supplementation with high doses (60 g) of flax and fish oils on the blood phospholipid (PL) fatty acid status, and AA/EPA ratio of individuals with Attention Deficit Hyperactivity Disorder (ADHD), commonly associated with decreased blood omega-3 fatty acid levels. Thirty adults with ADHD were randomized to 12 weeks of supplementation with olive oil (< 1% omega-3 fatty acids), flax oil (source of alpha-linolenic acid; 18:3n-3; alpha-LNA) or fish oil (source of EPA and docosahexaenoic acid; 22:6n-3; DHA). Serum PL fatty acid levels were determined at baseline and at 12 weeks. Flax oil supplementation resulted in an increase in alpha-LNA and a slight decrease in the ratio of AA/EPA, while fish oil supplementation resulted in increases in EPA, DHA and total omega-3 fatty acids and a decrease in the AA/EPA ratio to values seen in the Japanese population. These data suggest that in order to increase levels of EPA and DHA in adults with ADHD, and decrease the AA/EPA ratio to levels seen in high fish consuming populations, high dose fish oil may be preferable to high dose flax oil. Future study is warranted to determine whether correction of low levels of long-chain omega-3 fatty acids is of therapeutic benefit in this population.