Biomedical subjects

Seon Hwa Lee

Publications and source records attributed to Seon Hwa Lee.

At least 19 recordsLinked to original sources

PubMed2006

Targeted quantitative analysis of fatty acids in atherosclerotic plaques by high sensitivity liquid chromatography/tandem mass spectrometry.

The quantitative analysis of fatty acid composition in atherosclerotic plaques provides a way to monitor the underlying etiology of atherosclerosis. Previously, the method of choice for analyzing fatty acids in biological samples was gas chromatography/mass spectrometry (GC/MS); however, recent developments in electrospray ionization (ESI)/liquid chromatography (LC)/tandem mass spectrometry have made it a superior alternative. Previous research has largely focused on global analyses of intact lipids rather than more targeted analysis of the fatty acids themselves. We have now developed a targeted, stable isotope dilution LC-electrospray ionization/multiple reaction monitoring/MS method for the quantitative analysis of 10 fatty acids (myristic, palmitic, stearic, oleic, linoleic, alpha-linolenic, gamma-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids) using their trimethylaminoethyl ester (TMAE) derivatives to improve sensitivity. The method was validated, had a detection limit in the fmol range, and was used in the analysis of fatty acids in atherosclerotic plaques from carotid arteries.

Atherosclerosis↗

PubMed2006

Endogenous lipid hydroperoxide-mediated DNA-adduct formation in min mice.

Despite intensive research over the last two decades, there are still no specific markers of endogenous lipid hydroperoxide-mediated DNA damage. We recently demonstrated that heptanone-etheno-2'-deoxyguanosine adducts are formed in the DNA of rat intestinal epithelial cells that stably express cyclooxygenase-2. Heptanone-etheno adducts can only arise from the reaction of lipid hydroperoxide-derived 4-oxo-2(E)-nonenal with DNA. This raised the possibility that similar adducts would be formed in vivo in settings where cyclooxygenase-2 expression is increased. Therefore, DNA-adduct formation was studied in C57BL/6JAPC(min) mice, a colorectal cancer mouse model in which cyclooxygenase-2 is up-regulated. 15(S)-Hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid is the major lipid hydroperoxide produced endogenously by cyclooxygenase-2. It undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, which forms heptanone-etheno adducts with DNA. A quantitative comparison was made of the heptanone-etheno-DNA adducts present in C57BL/6J and C57BL/6JAPC(min) mice. Using highly specific and sensitive methodology based on stable isotope dilution liquid chromatography/tandem mass spectrometry, we have detected the endogenous formation of heptanone-etheno adducts in mammalian tissue DNA for the first time. In addition, we found that there were statistically significant increased levels of the heptanone-etheno-2'-deoxyguanosine and heptanone-etheno-2'-deoxycytidine adducts in the C57BL/6JAPC(min) mice when compared with the control C57BL/6J mice.

Animals↗

PubMed2006

Characterization of a lipid hydroperoxide-derived RNA adduct in rat intestinal epithelial cells.

Five major products (adducts A(1a), A(1b), A(2), A(3,) and B) from the reaction of guanosine (Guo) with 4-oxo-2(E)-nonenal (ONE) were detected by liquid chromatography-mass spectrometry (LC-MS). Tandem MS (MS/MS) analysis of these compounds suggested that modifications to the nucleoside had been introduced. Adducts A(1a), A(1b), A(2), and A(3) were heptanone-ethano-2'-Guo adducts that all decomposed to adduct B. Adducts A(1a) and A(1b) were isomeric hemi-ketal forms. Adducts A(2) and A(3) were diastereomers of the open chain ketone form. The structure of adduct B was shown by LC-MS/MS and NMR spectroscopy to be the heptanone-etheno-Guo (HepsilonGuo) adduct, 3-(D-erythropentafuranosyl)imidazo-7-(heptane-2' '-one)-9-hydroxyl[1,2-alpha]purine. The overall reaction of Guo with ONE was very similar to its reaction with 2'-deoxyguanosine. Reaction of ONE with yeast transfer RNA also resulted in the formation of HepsilonGuo. Finally, HepsilonGuo was detected and quantified in the RNA from rat intestinal epithelial cells that stably express cyclooxygenase-2. These data show that RNA is modified by the same bifunctional reactive electrophiles derived from lipid peroxidation that covalently modify DNA.

Animals↗

PubMed2006

LC/ESI/MS analysis of saturated and unsaturated fatty acids in rat intestinal epithelial cells.

Reactive oxygen species (ROS) can mediate damage to cellular macromolecules and lipids. Lipid peroxidation is considered to be a major pathway by which ROS can cause tissue damage and alterations in cell membranes. Other factors affecting oxidative damage include the target molecules such as fatty acids, which are readily oxidized by ROS. Thus, lipid peroxidation may depend upon the cellular fatty acid composition. Analysis of saturated fatty acids that are present by liquid chromatography/mass spectrometry (LC/MS) is difficult because they are poorly ionized under electrospray ionization (ESI) conditions. The separation of short to very long chain saturated and unsaturated fatty acids is also very challenging when LC is employed instead of gas chromatography. The use of trimethylaminoethyl (TMAE) ester iodide derivatization has been shown previously to improve the sensitivities of saturated fatty acids in the ESI mode. A reversed-phase LC method using a diphenyl column was employed to separate 14 fatty acids as their TMAE derivatives. Stable isotope dilution LC/ESI/multiple reaction monitoring/MS methodology was then developed for the quantitative analysis of seven saturated and seven unsaturated forms of short (C14) to very long (C26) chain fatty acids as their TMAE ester iodide derivatives. This methodology has allowed the analysis of fatty acid composition from parental rat intestinal epithelial cell and rat intestinal epithelial cells transfected with cyclooxygenase-2, a model system of oxidative stress.

Animals↗

PubMed2005

Cyclooxygenase-2-mediated DNA damage.

Rat intestinal epithelial cells that express the cyclooxygenase-2 (COX-2) gene permanently (RIES cells) were used as a model of in vivo oxidative stress. A targeted lipidomics approach showed that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) was the major hydroxylated non-esterified lipid formed in unstimulated intact cells. The corresponding hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE) undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, a precursor of heptanone-etheno-2'-deoxyguanosine. This etheno adduct was identified in the DNA of RIES cells. A dose-dependent increase in adduct levels was observed in the presence of vitamin C. This suggested that vitamin C increased lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation in the cells. The selective COX-2 inhibitor NS-398 was protective against cellular DNA damage but was less effective if vitamin C was present. Prostaglandin E(2) and 15(S)-HETE biosynthesis were completely inhibited by 110 mum NS-398 in the intact RIES cells. No inhibition of COX-1 was detected in the wild-type RIE cells at this concentration of NS-398. Arachidonic acid treatment of RIES cell lysates and ionophore stimulation of intact RIES cells produced significantly more 15(R)-HETE than the untreated intact cells. These preparations also both produced 11(R)-HETE, which was not detected in the intact cells. Aspirin treatment of the intact unstimulated RIES cells resulted in the exclusive formation of 15(R)-HETE in amounts that were slightly higher than the original 15(S)-HETE observed in the absence of aspirin, implying that significant amounts of 15(R)-HPETE had also been formed. 15(R)-HPETE should give exactly the same amount of heptanone-etheno-2'-deoxyguanosine as its 15(S)-enantiomer. However, no increase in heptanone-etheno adduct formation occurred in the aspirin-treated cells. The present study suggests a potential mechanism of tumorigenesis that involves DNA adduct formation from COX-2-mediated lipid peroxidation rather than prostaglandin formation. Therefore, inhibition of COX-2-mediated lipid hydroperoxide formation offers a potential therapeutic alternative to COX-2 inhibitors in chemoprevention strategies.

Animals↗

PubMed2005

Analysis of FeII-mediated decomposition of a linoleic acid-derived lipid hydroperoxide by liquid chromatography/mass spectrometry.

Intracellular Fe(II), which is up-regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic omega-6 polyunsaturated fatty acid, linoleic acid, to 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)-HPODE during oxidative stress. Subsequent Fe(II)-mediated decomposition to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans-4,5-epoxy-2(E)-decenal (EDE), cis-EDE, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH(+) ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13-HPODE was treated with 50 microM Fe(II). At this concentration of Fe(II), the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of Fe(II) (500 microM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by Fe(II). The HNE is thought to arise from a one-electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an alpha-cleavage of the HPNE-derived alkoxy radical or by direct dehydration of HPNE.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

Liquid chromatography/mass spectrometry analysis of bifunctional electrophiles and DNA adducts from vitamin C mediated decomposition of 15-hydroperoxyeicosatetraenoic acid.

Reactive oxygen species convert the omega-6 polyunsaturated fatty acid arachidonic acid into 15-hydroperoxy-5,8,11,13-(Z,Z,ZE)-eicosatetraenoic acid (15-HPETE). Cyclooxygenases and lipoxygenases can also convert arachidonic acid into 15-HPETE. Vitamin C mediated decomposition of 15(S)-HPETE to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI-MS). The individual bifunctional electrophiles, trans-4,5-epoxy-2(E)-decenal (t-EDE), cis-4,5-epoxy-2(E)-decenal (c-EDE), 4-oxo-2(E)-nonenal (ONE), and 4-hydroxy-2(E)-nonenal (HNE), exhibited protonated molecules at m/z 169, 169, 155, and 157, respectively. The MH+ ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. All five bifunctional electrophiles were formed when 15-HPETE was treated with vitamin C. The LC/MS-based methodology showed that t-EDE was the major bifunctional electrophile formed during vitamin C mediated 15(S)-HPETE decomposition. Stable isotope dilution LC/MS studies revealed that this did not result in the formation of increased levels of unsubstituted etheno-dGuo adducts in calf thymus DNA when compared with 13(S)-hydroperoxy-9,10-(Z,E)-octadecadienoic acid [13(S)-HPODE], a lipid hydroperoxide derived from linoleic acid. However, the formation of heptanone-etheno-dGuo adducts in calf thymus DNA was reduced when compared with the 13(S)-HPODE. This was attributed to the reduced formation of ONE from 15-HPETE when compared with its formation from 13-HPODE. In contrast to reactions with dGuo or DNA conducted using 13(S)-HPODE, no carboxy-containing adducts were observed with 15(S)-HPETE.

Ascorbic Acid↗

PubMed2005

Targeted chiral lipidomics analysis.

Genomics, transcriptomics, and proteomics are proving to be very useful techniques, which have impacted significantly on our understanding mechanisms of human disease. However, this systems biology approach has several drawbacks than can be overcome by the integration of metabonomics and lipidomics. We have developed a targeted lipidomics approach that makes it possible to directly analyze chiral lipids generated in cellular systems. Bioactive lipids are usually present in trace amounts as enanatiomers and regioisomers that require separation before they can be analyzed by mass spectrometry. Normal phase chiral chromatography is generally used to resolve bioactive lipid enanatiomers. However, conventional electrospray and atmospheric pressure chemical ionization/tandem mass spectrometry have limited sensitivity when normal phase solvents are used, which makes it difficult to conduct studies when only trace amounts of the bioactive lipids are present. The use of electron capture atmospheric pressure chemical ionization/tandem mass spectrometry overcomes this problem. Enantiomers and regioisomers of targeted bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal phase chiral chromatography and electron capture atmospheric chemical ionization/tandem mass spectrometry. A targeted lipidomics profile from rat epithelial cells transfected with cyclooxygenase-2 and maintained in culture was obtained. Inhibition with the non-selective cyclooxygenase inhibitor aspirin increased the formation of 15(R)-hydroxyeicosatetraenoic acid in the cells although it completely inhibited formation of the 15(S)-enantiomer and prostaglandin E2. New mass spectrometry instrumentation with an improved atmospheric pressure chemical ionization source was found to be an order of magnitude more sensitive than existing instruments for analysis of bioactive lipids using electron capture methodology. This type of mass spectrometer will permit a more detailed analysis of cellular bioactive lipid production than has been possible previously. It will also permit in vivo targeted lipidomics studies to be conducted using biological fluids derived from animal models and human subjects.

Animals↗

PubMed2005

Viniferin formation by COX-1: evidence for radical intermediates during co-oxidation of resveratrol.

Resveratrol (1) is a polyphenolic natural product, which functions as both a mechanism-based inactivator and a co-reductant of the COX-1 peroxidase. These functions are mediated through different moieties on the molecule, namely, the m-hydroquinone moiety (mechanism-based inactivator) and the phenol moiety (co-reductant). Implicit in this bifunctionality is the notion that resveratrol is oxidized at the peroxidase active site of COX-1, resulting in the formation of two hypothetical radical species. Oxidation of the m-hydroquinone moiety can generate a hypothetical m-semiquinone radical, which is unstabilized and leads to irreversible enzyme inactivation. Oxidation of the phenol moiety can generate a hypothetical phenoxy radical, which is stabilized and leads to co-reduction during peroxidase catalysis. These two radicals have been trapped as the resveratrol dimers, cis-epsilon-viniferin (4, trapped m-semiquinone radical) and trans-delta-viniferin (5, trapped phenoxy radical), and identified by liquid chromatography (LC), absorbance spectroscopy, and LC/tandem mass spectrometry (MS(n)) methods. Methoxy-resveratrol analogues, in which either the m-hydroquinone or the phenol moiety were protected as methyl ethers, were used to confirm the proposed mechanism of viniferin production by COX-1.

Benzofurans↗

PubMed2005

Unexpected formation of etheno-2'-deoxyguanosine adducts from 5(S)-hydroperoxyeicosatetraenoic acid: evidence for a bis-hydroperoxide intermediate.

Analysis of products from the reaction between 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid and 2'-deoxyguanosine in the presence of FeII, FeIII, or vitamin C by liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry revealed the presence of four DNA adducts. Surprisingly, adducts I and II had mass spectral characteristics identical to those for 1,N2-etheno-2'-deoxyguanosine and heptanone-1,N2-etheno-2'-deoxyguanosine. These adducts have previously been shown to arise from the homolytic decomposition of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid. It appears that under the reaction conditions, 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid was subjected to a previously unknown peroxidation reaction to give a bis-hydroperoxide intermediate that underwent a Hock rearrangement to produce 3(Z)-nonenal from the omega-terminus. The 3(Z)-nonenal was then converted to 4-hydroperoxy-2-nonenal, a precursor to the formation of 4-oxo-2-nonenal. 4-Oxo-2-nonenal forms heptanone-1,N2-etheno-adducts with 2'-deoxyguanosine, whereas 4-hydroperoxy-2-nonenal forms 1,N2-etheno-2'-deoxyguanosine. Two novel carboxylate adducts were also identified. The structure of the more abundant adduct (III) was characterized as its methyl ester derivative by NMR spectroscopy as 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(5' '-carboxypenta-2' '-one)-9-oxo[1,2-alpha]purine (5-carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine). This etheno adduct was formed by the reaction of 2'-deoxyguanosine with 5,8-dioxo-6(E)-octenoic acid. The bifunctional electrophile is proposed to arise from the alpha-terminus during the Hock rearrangement of bis-hydroperoxide derived from 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid. 5-Carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine may serve as a biomarker of 5-lipoxygenase-mediated oxidative stress. The less abundant carboxylate adduct IV arose from a quite different pathway and was tentatively characterized as 6-carboxy-3-hydroxy-1-hexene-1,N2-etheno-2'-deoxyguanosine.

DNA Adducts↗

PubMed2005

4-Hydroperoxy-2-nonenal-induced formation of 1,N2-etheno-2'-deoxyguanosine adducts.

Analysis of the reaction between 4-hydroperoxy-2-nonenal (HPNE) and 2'-deoxyguanosine (dGuo) by liquid chromatography/mass spectrometry (LC/MS) revealed the formation of 1,N2-etheno-dGuo as well as heptanone-etheno-dGuo and trace amounts of dihydroxyheptane-etheno-dGuo. Identities of the dGuo adducts were confirmed by comparison with authentic standards. The minor dihydroxyheptane-etheno-dGuo adducts could be generated from 2,3-epoxy-4-hydroxynonanal (EHN), the epoxidation product of 4-hydroxy-2-nonenal (HNE). An LC/MS method was developed for the analysis of EHN. No EHN was detected by LC/MS during the decomposition of HPNE. Therefore, the dihydroxyheptane-etheno-dGuo adducts are either generated from a direct reaction between HPNE and dGuo or from another intermediate that cannot be detected by LC/MS. In addition, no HNE-derived hydroxypropano-dGuo adducts were observed. On the basis of these findings, we conclude that HPNE, a primary product of lipid peroxidation, is a major precursor to the formation of 1,N2-etheno-dGuo. We propose that it arises from the reaction of dGuo and HPNE through the intermediate formation of a cyclic hydroxy-ethano-epoxide derivative. The minor amounts of heptanone-ethano-dGuo adducts that were formed from HPNE in the absence of vitamin C suggest that heptanone-etheno-dGuo can be generated directly from HPNE without the intermediate formation of ONE. Therefore, HPNE can be considered as another lipid hydroperoxide-derived bifunctional electrophile with the potential for biological activities that are similar to HNE and ONE.

Aldehydes↗

PubMed2005

Dioxododecenoic acid: a lipid hydroperoxide-derived bifunctional electrophile responsible for etheno DNA adduct formation.

It has been proposed that 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE]-mediated formation of 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal arises from a Hock rearrangement. This suggested that a 4-oxo-2(E)-nonenal-related molecule, 9,12-dioxo-10(E)-dodecenoic acid (DODE), could also result from the intermediate formation of 9-hydroperoxy-12-oxo-10(E)-dodecenoic acid. A recent report has described the formation of DODE-derived etheno adducts when 13(S)-HPODE was allowed to decompose in the presence of 2'-deoxynucleosides or DNA. However, the regioselectivity of lipid hydroperoxide-derived DODE addition to 2'-deoxyguanosine (dGuo) or other 2'-deoxynucleosides was not determined. The structure of carboxynonanone-etheno-dGuo formed from vitamin C-mediated 13(S)-HPODE decomposition has now been established by a combination of 1H and 13C NMR spectroscopy studies of its bis-methylated derivative. The site of dGuo methylation was first established as being at N-5 rather than at O-9 from NMR analysis of a methyl derivative of the model compound, heptanone-etheno-dGuo. (1)H,(13)C 2D heteronuclear multiple bond correlations were then used to establish unequivocally that the bis-methyl derivative of carboxynonanone-etheno-dGuo was 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxymethylnona-2' '-one)-9-oxo-5-N-methyl[1,2-a]purine rather than its 6-(9' '-carboxymethylnona-2"-one)-9-oxo-5-N-methyl[1,2-a]purine regioisomer. Therefore, etheno adduct formation occurred by initial nucleophilic attack of the exocyclic N(2) amino group of dGuo at the C-12 aldehyde of DODE to form an unstable carbinolamine intermediate. This was followed by intramolecular Michael addition of the pyrimidine N1 of dGuo to C-11 of the resulting alpha,beta-unsaturated ketone. Subsequent dehydration gave 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxynona-2' '-one)-9-oxo-[1,2-a]purine (carboxynonanone-etheno-dGuo). An efficient synthesis of DODE was developed starting from readily available 1,8-octanediol using a furan homologation procedure. This synthetic method allowed multigram quantities of DODE to be readily prepared. Synthetic DODE when reacted with dGuo gave carboxynonanone-etheno-dGuo that was identical with that derived from vitamin C-mediated 13(S)-HPODE decomposition in the presence of dGuo.

DNA Adducts↗

PubMed2003

Altered urinary profiles of polyamines and endogenous steroids in patients with benign cervical disease and cervical cancer.

The risk of cancer of the cervix is linked with sexual behavior. Although infectious agents, such as human papillomaviruses (HPVs) are implicated, these alone may be insufficient to induce the disease. We investigated the potential role of estrogen, androgen, and polyamine metabolism as co-factors in the development of cervical cancer. We obtained urine samples from patients with benign cervical disease (n=18) and cervical cancer (n=18) and from age-matched normal female subjects (n=25). For 11 polyamine determination, an improved and sensitive gas-chromatographic with nitrogen/phosphorus-detection (GC/NPD) procedure was used. The urinary levels of 25 androgens and corticoids and 16 estrogens were quantitatively determined by gas chromatography-mass spectrometry-selected ion-monitoring (GC/MS/SIM). In the patients with cervical cancer, the ratio of 16alpha-hydroxy estrone (16alpha-OH E1)/2-hydroxy estrone (2-OH E1), putrescine (Put)/N(1)-acetylspermidine (N(1)-acSpd) and 5beta-tetrahydrocortisol (THF)/5alpha-tetrahydrocortisol (5alpha-THF) were significantly increased in comparison to the values of the normal controls. These data suggest: (1) an increase of 16alpha-hydroxylation in estrogen metabolism; (2) the high activity of polyamine oxidase (PAO) in polyamine metabolism; and (3) the low activity of 5alpha-reductase in androgen metabolism may play a significant role in the development of cervical cancer. Although additional research is necessary, the combination of 16alpha-OH E1/2-OH E1 and THF/5alpha-THF may provide a dual marker for the discrimination of benign cervical disease and cervical cancer.

Adrenal Cortex Hormones↗

PubMed2003

A novel lipid hydroperoxide-derived cyclic covalent modification to histone H4.

Previous studies have established that 4-hydroxy-2-nonenal is a lipid hydroperoxide-derived aldehydic bifunctional electrophile that reacts with DNA and proteins. However, it has now been recognized that 4-oxo-2-nonenal is also a major product of lipid hydroperoxide decomposition. Furthermore, 4-oxo-2-nonenal is more reactive than 4-hydroxy-2-nonenal toward the DNA-bases 2'-deoxyguanosine, 2'-deoxyadenosine, and 2'-deoxycytidine and proteins. The formation of 4-oxo-2-nonenal can be induced through vitamin C-mediated or transition metal ion-mediated homolytic decomposition of polyunsaturated omega-3 lipid hydroperoxides such as 13(S)-hydroperoxyoctadecadienoic acid. We have discovered that synthetic 4-oxo-nonenal or 4-oxo-2-nonenal-generated from 13(S)-hydroperoxyoctadecadienoic acid recognizes the specific amino acid motifs of His75, Ala76, and Lys77 in bovine histone H4. Reaction of the histidine and lysine residues with 4-oxo-2-nonenal results in the formation of a novel cyclic structure within the protein. The cyclic structure incorporates the histidine imidazole ring and a newly formed pyrrole derived from the lysine. The cyclic imidazole-pyrrole derivative that is formed from the small Nalpha-acetyl-His-Ala-Lys peptide exists as a mixture of two atropisomers that inter-convert upon heating. Such lipid hydroperoxide-derived modifications could potentially modulate transcriptional activation in vivo. Furthermore, the ability to synthesize cyclic peptides using 4-oxo-2-nonenal will facilitate the preparation of novel structural analogs with potential biological activity.

Aldehydes↗

PubMed2003

Estrogens in female thyroid cancer: alteration of urinary profiles in pre- and post-operative cases.

To evaluate the potential effect of estrogens in premenopausal female thyroid cancer, the concentrations of 14 estrogens were quantitatively determined in the urine of pre- and post-operative patients with thyroid papillary cancer (18 patients case, 26 approximately 54 years) and normal female subjects (20 cases, 31 approximately 52 years). The highly sensitive gas chromatography-mass spectrometry-selected ion-monitoring method was used for estrogens analysis. And an estrogen-oxidative metabolism and 16alpha-hydroxyestrone to 2-hydroxyestrone (16alpha-OH E1/2-OH E1) which is the two primary and competing site of estrogen-oxidation, were determined. Catechol estrogens, including 2-OH E1, were also increased without significant changes of the other estrogen metabolites in pre-operative patients with thyroid papillary cancer compared with normal subjects. The lowest mean value of 16alpha-OH E1/2-OH E1 was remarked in pre-operative patients, and it was significantly different from the ratio of post-operative cases. As a result, it is suggested that the increase of 2-hydroxylation in estrogen metabolism may have a significant association with female thyroid cancer.

Adult↗

PubMed2003

Targeted lipidomics using electron capture atmospheric pressure chemical ionization mass spectrometry.

There is an increasing need to be able to conduct quantitative lipidomics analyses as a complement to proteomics studies. The highest specificity for proteomics analysis can be obtained using methodology based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS). For lipidomics analysis it is often necessary to be able to separate enantiomers and regioisomers. This can be very challenging when using methodology based on conventional reversed-phase chromatography. Normal-phase chromatography using chiral columns can provide dramatic improvements in the resolution of enantiomers and regioisomers. However, conventional ESI- and APCI-MS/MS has limited sensitivity, which makes it difficult to conduct studies in cell culture systems where only trace amounts of non-esterified bioactive lipids are present. The use of electron capture APCI-MS/MS overcomes this problem. Enantiomers and regioisomers of diverse bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal-phase chiral chromatography and electron capture APCI-MS/MS. This methodology has allowed a lipidomics profile from rat epithelial cells maintained in culture to be delineated and allowed the effect of a non-selective lipoxygenase inhibitor to be assessed.

Animals↗

PubMed2003

Characterization of 2'-deoxycytidine adducts derived from 4-oxo-2-nonenal, a novel lipid peroxidation product.

Analysis of the reaction between 2'-deoxycytidine and 4-oxo-2-nonenal by LC/MS revealed the presence of three major products (adducts A(1), A(2), and B; [M + H](+) = 364). Adducts A(1) and A(2) were isomeric, and each dehydrated to form adduct B. The structure of adduct B was shown by LC/MS and NMR spectroscopy to be an etheno-2'-deoxycytidine adduct 1' '-[1-(2'-deoxy-beta-d-erythro-pentofuranosyl)-1H-imidazo[2,1-c]pyrimidin-2-oxo-4-yl]heptane-2' '-one. A time course experiment performed at 65 degrees C (pH 5-8) showed that the transformation of both A(1) and A(2) was pH-dependent. In acidic conditions, adducts A(1) and A(2) dehydrated primarily to adduct B. In contrast, in basic conditions, adducts A(1) and A(2) hydrolyzed primarily to dCyd. The data are consistent with adducts A(1) and A(2) being substituted ethano adducts that dehydrate to adduct B, a substituted 3,N(4)-etheno-2'-deoxycytidine adduct.

Aldehydes↗

PubMed2003

A novel lipid hydroperoxide-derived modification to arginine.

The guanidine group present in the amino acid arginine was found to react with the lipid hydroperoxide-derived bifunctional electrophile, 4-oxo-2-nonenal. The reaction between N(alpha)-tert-butoxycarbony-l-arginine and 4-oxo-2-nonenal resulted in the formation of an adduct (adduct A) that subsequently dehydrated on heating to adduct B. Liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy were used to assign the structure of adduct B as (N(delta),N(omega)(')-etheno-2'-heptanon-2' '-one)-N(alpha)-t-Boc-arginine. The reaction proceeded from initial reaction of the primary N(omega)-amino group at the C-1 aldehyde of 4-oxo-2-nonenal. Subsequently, an intramolecular Michael addition of a secondary N(delta)-amino group occurring at C-3 resulted in formation of the cyclic carbinolamine adduct A. Dehydration and rearrangement of the exocyclic imine resulted in the formation of adduct B, which contained a stable imidazole ring. The tetra peptide LRDE reacted with 4-oxo-2-nonenal primarily at arginine rather than at the amino terminus. This suggests that arginine-containing proteins can react with lipid hydroperoxide-derived 4-oxo-2-nonenal to form a novel imidazole modification.

Aldehydes↗