Age-Related Changes in the Chorioretinal Junction: An Immunohistochemical Study.

The chorioretinal junction comprises the retinal pigment epithelium, Bruch's membrane (BM), and adjacent choroidal capillaries. Its significance lies in its ability to support the retina mechanically and metabolically. The aim of this cross-sectional study was to record the senescent changes affecting all the constituents of the chorioretinal junction in 40 histological specimens across the whole spectrum of the adult age range. This study included light microscopy, with hematoxylin and eosin and PAS stains, and fluorescent microscopy. Immunohistochemistry was done using antibodies against neurofilament, synaptophysin, S-100, and collagen IV. The descriptive microanatomy was corroborated by morphometry. The amount of melanin and lipofuscin granule and drusens were noted. The ratio of thickness of BM to capillary diameter reduced from 1:6 or less in the 2nd decade to 1:3 in the 10th decade. Complete hyalinization of intercapillary pillars was seen in the 10th decade. The accumulation of lipofuscin with age was documented with the diminution in the size of epithelial cells. The subepithelial accumulation of drusen was first noted in the specimen from the late 60s. We have described all senescent changes in the chorioretinal junction chronologically. Similar changes are found in a more pronounced form in age-related macular degeneration. These data might serve as a reference baseline for clinicians and pathologists.

Morphology and age-related changes in calcospherites of human teeth: an ultrastructural study.

BACKGROUND: Dentine has been examined extensively for age-related physiological changes, but there are limited data on age-related changes at ultrastructural level of dentine. AIM: The present study aimed to examine age-related ultrastructural changes in calcospherites of human dentine under the scanning electron microscope. MATERIALS AND METHODS: Thirty single rooted teeth of North Western adult Indians (18-75 years) were collected from the Department of Oral Health Sciences, PGIMER, Chandigarh. Labiolingual sections were prepared and morphology of calcospherites was studied at different locations (coronal, cervical, midroot and apical) of the tooth. RESULTS: Morphologically, four types (I [small, unfused and discrete], II [partially fused and globular], III [large, completely fused] and IV [structureless]) of calcospherites were seen in the sample. With advancing age, type I calcospherites approached neighbouring crystals and changed their form to type II, which ultimately coalesced and transformed to type III. Results revealed that among different age groups (young, middle and old), calcospherites of only type I, II and III showed statistically significant differences in their shapes using Pearson's Chi-square test. Statistically non-significant differences were obtained in the shapes at different locations of the tooth. CONCLUSIONS: Results showed that calcospherites get fused as age advances. There is a change in the shape of calcospherites based on the location of the tooth.

Human retinal senescence measured histologically: as an aid to clinical diagnosis by ocular coherence tomography

Spectral domain optical coherence tomography (SD-OCT) has become an established diagnostic tool for the clinical assessment of retinal pathology and its progression, in OPD setting. The aim of our study was to do in vitro quantification of relevant retinal layers to collect baseline data at different age groups, against which OCT findings can be interpreted. Thirty eyeballs (20-99 years) were used to study the retinal nerve fibre layer (RNFL), ganglion cells and inner plexiform layer (GC+IPL) and outer nuclear layer (ONL) thickness using V-Test software, 198035 on the H&E stained histological specimens. Mean thickness of these retinal layers was studied. To estimate the decrease from the optimal stage, absolute percentage decline (APD) was calculated for each decade. The mean thickness of RNFL at was 77.8 µm, 77.1 µm, 73.6 µm, 70.6 µm, 69.2µm, 54.1µm, 36.5µm 26.8µm from 3rd to 10th decade respectively. Significant APD of 30% was evident between 7th and 8th decades. APD graphs for RNFL and GC+IPL were almost parallel to each other. The absolute percentage decline (APD) for the thickness of ONL was 0.2%, 4.6%, 13%, 35%, 60%, 62% and 64% for 4th, 5th, 6th, 7th, 8th, 9th and 10th decade respectively. This study has provided normative base line histological data for ready reference. Decade wise changes in thickness of different layers can be used by ophthalmologist to differentiate senescent from pathological changes and to monitor progression of disease

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Methotrexate modulates folate phenotype and inflammatory profile in EA.hy 926 cells.

EA.hy 926 cells grown under low folate conditions adopt a "pro-atherosclerotic" morphology and biochemical phenotype. Pharmacologically relevant doses of the antifolate drug methotrexate (MTX) were applied to EA.hy 926 cells maintained in normal (Hi) and low (Lo) folate culture media. Under both folate conditions, MTX caused inhibition of cell proliferation without significantly compromising metabolic activity. MTX treated Hi cells were depleted of folate derivatives, which were present in altered proportions relative to untreated cells. Transcript profiling using microarrays indicated that MTX treatment modified the transciptome in similar ways for both Hi and Lo cells. Many inflammation-related genes, most prominently those encoding C3 and IL-8, were up-regulated, whereas many genes involved in cell division were down-regulated. The results for C3 and IL-8 were confirmed by quantitative RT-PCR and ELISA. MTX appears to modify the inflammatory potential of EA.hy 926 cells such that its therapeutic properties may, at least under some conditions, be accompanied by the induction of a subset of gene products that promote and/or maintain comorbid pathologies.

Pemetrexed alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions.

Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low ("Lo") and high ("Hi") folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency.

8-Oxo-2'-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[(15)N(5)]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [(13)C(10)(15)N(5)]dGuo and monitoring of its conversion to 8-oxo-[(13)C(10)(15)N(5)]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72µg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements.

11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid.

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 µM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 µM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ(2).

Synthesis of deuterium-labeled analogs of the lipid hydroperoxide-derived bifunctional electrophile 4-oxo-2(E)-nonenal.

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4-hydroxy-2(E)-nonenal and 4-oxo-2(E)-nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide-derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α,ß-unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope-labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)-based assays. An efficient onestep cost-effective method has been developed for the preparation of C-9 deuterium-labeled ONE. In addition, a method for specific deuterium labeling of ONE at C-2, C-3 or both C-2 and C-3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins.

Optimization-based prediction of asymmetric human gait.

An optimization-based formulation and solution method are presented to predict asymmetric human gait for a large-scale skeletal model. Predictive dynamics approach is used in which both the joint angles and joint torques are treated as unknowns in the equations of motion. For the optimization formulation, the joint angle profiles are treated as the primary unknowns, and velocities and accelerations are calculated using them. In numerical implementation, the joint angle profiles are discretized using the B-spline interpolation. An algorithm is presented to inversely calculate the joint torques and the ground reaction forces. The sum of the joint-torques squared, called the dynamic effort, is minimized as the human performance measure. Constraints are imposed on the joint strengths (torques) and joint ranges of motion along with other physical constraints. The formulation is validated by simulating a symmetric gait and comparing the results with the experimental data. Then asymmetric gait motion is simulated, where the left and right step lengths are different. The kinematics and kinetics results from the simulation are presented and discussed. Predicted ground reaction forces are explained by using the inverted pendulum model. Predicted kinematics and kinetics have trends that are similar to those reported in the literature. Potential practical applications of the formulation and the solution approach are discussed.

Chemoselective N-acylation via condensations of N-(benzoyloxy)amines and alpha-ketophosphonic acids under aqueous conditions.

A new amide-forming reaction with N-benzoyloxyamines and alpha-ketophosphonic acids was investigated. A mixed solvent of t-BuOH/water (1:1) at 40 degrees C provided the desired amide in high yield (71-96%). Both phosphonic acids ( 9, 12, or 13) and their disodium salts (e.g., 10) were shown to react with the respective N-benzoyloxyamines ( 1b and 4) in excellent yields. The phosphonic acid methyl ester monosodium salt 11 did not react under these conditions. However, compound 11 did provide the desired amide in 22% yield upon addition of 2 equiv of TFA. The N-acylation reaction is highly chemoselective for N-benzoyloxyamines as both aliphatic amines and N-hydroxylamines were shown not to react productively with the alpha-ketophosphonic acids under the conditions tested. Moreover, the alpha-ketophosphonic acids are more selective than the related alpha-ketocarboxylic acid systems, which react with both the N-hydroxylamines and N-benzoyloxyamines. In this regard, this novel phosphonic acid methodology provides a new high-yielding, chemoselective acylating reagent for further study.

Dynamic motion planning of 3D human locomotion using gradient-based optimization.

Since humans can walk with an infinite variety of postures and limb movements, there is no unique solution to the modeling problem to predict human gait motions. Accordingly, we test herein the hypothesis that the redundancy of human walking mechanisms makes solving for human joint profiles and force time histories an indeterminate problem best solved by inverse dynamics and optimization methods. A new optimization-based human-modeling framework is thus described for predicting three-dimensional human gait motions on level and inclined planes. The basic unknowns in the framework are the joint motion time histories of a 25-degree-of-freedom human model and its six global degrees of freedom. The joint motion histories are calculated by minimizing an objective function such as deviation of the trunk from upright posture that relates to the human model's performance. A variety of important constraints are imposed on the optimization problem, including (1) satisfaction of dynamic equilibrium equations by requiring the model's zero moment point (ZMP) to lie within the instantaneous geometrical base of support, (2) foot collision avoidance, (3) limits on ground-foot friction, and (4) vanishing yawing moment. Analytical forms of objective and constraint functions are presented and discussed for the proposed human-modeling framework in which the resulting optimization problems are solved using gradient-based mathematical programming techniques. When the framework is applied to the modeling of bipedal locomotion on level and inclined planes, acyclic human walking motions that are smooth and realistic as opposed to less natural robotic motions are obtained. The aspects of the modeling framework requiring further investigation and refinement, as well as potential applications of the framework in biomechanics, are discussed.

Induction of endothelial cell apoptosis by lipid hydroperoxide-derived bifunctional electrophiles.

Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2(E)-nonenal (ONE; 5-30 microm) and 4,5-epoxy-2(E)-decenal (EDE; 10-20 microM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10(E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2-10 microM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d,l-buthionine-(R,S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.

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.

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.

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.

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.

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.