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Originally published In Press as doi:10.1074/jbc.M103768200 on June 18, 2001

J. Biol. Chem., Vol. 276, Issue 33, 30964-30970, August 17, 2001
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Formation of Highly Reactive gamma -Ketoaldehydes (Neuroketals) as Products of the Neuroprostane Pathway*

Nathalie Bernoud-Hubac, Sean S. Davies, Olivier Boutaud, Thomas J. Montine, and L. Jackson Roberts IIDagger

From the Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee 37232-6602

Received for publication, April 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuroprostanes are prostaglandin-like compounds produced by free radical-induced peroxidation of docosahexaenoic acid, which is highly enriched in the brain. We previously described the formation of highly reactive gamma -ketoaldehydes (isoketals) as products of the isoprostane pathway of free radical-induced peroxidation of arachidonic acid. We therefore explored whether isoketal-like compounds (neuroketals) are also formed via the neuroprostane pathway. Utilizing mass spectrometric analyses, neuroketals were found to be formed in abundance in vitro during oxidation of docosahexaenoic acid and were formed in greater abundance than isoketals during co-oxidation of docosahexaenoic and arachidonic acid. Neuroketals were shown to rapidly adduct to lysine, forming lactam and Schiff base adducts. Neuroketal lysyl-lactam protein adducts were detected in nonoxidized rat brain synaptosomes at a level of 0.09 ng/mg of protein, which increased 19-fold following oxidation in vitro. Neuroketal lysyl-lactam protein adducts were also detected in vivo in normal human brain at a level of 9.9 ± 3.7 ng/g of brain tissue. These studies identify a new class of highly reactive molecules that may participate in the formation of protein adducts and protein-protein cross-links in neurodegenerative diseases and contribute to the injurious effects of other oxidative pathologies in the brain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isoprostanes (IsoPs)1 are prostaglandin (PG)-like compounds that are generated in vivo nonenzymatically as products of free radical-induced peroxidation of arachidonoyl lipids (1, 2). Their formation proceeds via PGH2-like bicyclic endoperoxide intermediates, which are reduced to form F-ring IsoPs (F2-IsoPs) (1) or undergo rearrangement to form D-ring and E-ring IsoPs (2) and isothromboxanes (3). Recently, we reported that IsoP endoperoxide intermediates also undergo rearrangement to form highly reactive gamma -ketoaldehyde levuglandin-like compounds (4).2 We propose to term these nonenzymatically generated gamma -ketoaldehydes isoketals (IsoKs) to distinguish them from levuglandins formed by rearrangement of the cyclooxygenase endoperoxide intermediate, PGH2 (5). These extremely reactive molecules form covalent adducts with lysine residues on proteins at a rate that exceeds that of 4-hydroxy-2-nonenal (4-HNE) by orders of magnitude, which is considered to be one of the most reactive aldehydes generated as a product of lipid peroxidation (6). Moreover, they exhibit a remarkable proclivity to cross-link proteins. We have previously shown that IsoKs initially form a reversible Schiff base adduct, which then proceeds through a pyrrole to stable lactam and hydroxylactam adducts (7).2

Docosahexaenoic acid (DHA) (22:6omega 3) is a polyunsaturated fatty acid uniquely enriched in the brain and retina, especially in synaptic membranes and in photoreceptor cells (8-10). Astrocytes play an important role in the delivery of DHA to the blood-brain barrier endothelial cells and to neurons (11, 12). Although the physiologic basis for why DHA is enriched in the brain and retina remains unclear, reduced levels of DHA are associated with disturbances in visual acuity, behavior, and learning in young animals and humans (13-15). We had previously demonstrated the formation of IsoP-like compounds in vivo from free radical-catalyzed peroxidation of DHA (16, 17). Because DHA is highly concentrated in nervous system tissue, we have termed these compounds neuroprostanes (NPs) (17). Analogous to the formation of IsoPs, the formation of NPs also proceeds through bicyclic endoperoxide intermediates that not only are reduced to F-ring compounds but also undergo rearrangement in vivo to form D- and E-ring NPs (17, 18). Therefore, we explored the hypothesis that IsoK-like compounds could also be generated as rearrangement products of the NP pathway, for which we propose the term neuroketals (NKs).

Our interest in the possibility that NKs could be formed derives from the fact that free radicals have been implicated in the pathogenesis of a wide variety of neurodegenerative disorders, including Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (19-23). Furthermore, reactive aldehydes are thought to be key mediators of oxidant injury because of their capacity to covalently modify proteins and DNA (4, 24-28).2 Thus, generation of NKs may induce neuronal injury due to their reactivity and could potentially be involved in the formation of protein cross-links, a common feature in neurodegenerative diseases. The notion that NKs, if formed, may at least participate in the pathogenesis of Alzheimer's disease is strengthened by our previous finding that F4-NP levels are significantly increased in cerebrospinal fluid from patients with this disease (17).

The pathway by which NKs can be generated is shown in Fig. 1, A-C. Five docosahexaenoyl radicals are initially formed that are then converted to eight peroxyl radicals following the addition of oxygen. These undergo endocyclization followed by further addition of molecular oxygen to form eight bicyclic endoperoxide intermediate regioisomers, which can then rearrange to form eight D4-NK and eight E4-NK regioisomers. The designation "D" and "E" is a carryover from the established prostaglandin nomenclature for PGD and PGE and levuglandins E and D to indicate the location of the keto group. Each regioisomer is theoretically comprised of eight racemic diastereoisomers for a total of 128 D4-type and 128 E4-type NKs. In accordance with the nomenclature system for IsoPs that has been approved by the Eicosanoid Nomenclature Committee, the eight regioisomers are designated by the carbon number on the side chain of the precursor endoperoxide intermediates where the hydroxyl group was located, with the carboxyl carbon as C1 (29).


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  		Fig. 1.   A-C, pathway for the formation of NKs by nonenzymatic free radical-induced peroxidation of DHA. Five DHA radicals are initially generated to form eight D4-NK and eight E4-NK regioisomers. Each regioisomer is theoretically composed of eight racemic diastereoisomers for a total of 256 compounds.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Docosahexaenoic acid was purchased from Nu-Chek-Prep, Inc. (Elysian, MN). Undecane, N-N-dimethylformamide, ammonium acetate, Trolox, and triphenylphosphine were from Aldrich; pentafluorobenzyl bromide, methoxyamine HCl, sodium borohydride, and butylated hydroxytoluene were from Sigma; Pronase and porcine aminopeptidase M (60 units/ml) were from Calbiochem; C18 Sep Pak cartridges and Oasis cartridges were from Waters Associates (Milford, MA); and [13C6]L-lysine and [2H3]methoxyamine HCl were from Cambridge Isotope Laboratories, Inc. (Andover, MA). L-[4,5-3H]Lysine was from PerkinElmer Life Sciences; N,O-bis(trimethylsilyl)trifluoroacetamine was from Regis Chemical (Morton Grove, IL); N,O-[2H9]bis(trimethylsilyl)trifluoroacetamine was from CDP isotopes (Pointe-Claire, Quebec, Canada). 4.6 × 250-mm Macrosphere 300 C18 column and 2.1 × 15-mm XD8-C8 column were from MacMod Analytical (Chadds Ford, PA). Male Harlan Sprague-Dawley rats were from Harlan Sprague-Dawley, Inc. (Indianapolis, IN).

Oxidation of DHA-- 5 mg of DHA were oxidized in vitro in 1× phosphate-buffered saline using an iron/ADP/ascorbate mixture (1 mM/200 mM/100 mM) for 2 h as described (30).

Purification and Analysis of NKs by Gas Chromatography (GC)/Negative Ion Chemical Ionization (NICI)/Mass Spectrometry (MS)-- The purification and analysis of NKs followed similar procedures used for purification and analysis of IsoKs (4). Following oxidation of DHA, compounds were converted to O-methyloxime derivatives by treatment with a 3% aqueous solution of methoxyamine HCl. The pH of the reaction mix was then adjusted to 3, and the samples were extracted using a C18 Sep Pak cartridge. The compounds were then converted to a pentafluorobenzyl ester derivative, purified by TLC using the solvent heptane/ethyl acetate (60:40, v/v), converted to a trimethylsilyl ether derivative, and quantified by GC/NICI/MS using [2H4]PGE2 as an internal standard and a modification of the method used to purify and analyze IsoKs (4). The region extending from 1.5 cm above to 2.5 cm above an O-methyloxime pentafluorobenzyl ester derivative of [2H4]PGE2 standard was scraped. This area was determined to contain NKs by analyzing sequential small cuts of the TLC plates. NKs were detected by GC/NICI/MS employing selected ion monitoring for the M-·CH2C6F5 ions (m/z 505 for NKs and m/z 528 for the [2H4]PGE2 internal standard. Catalytic hydrogenation was performed as described previously (31).

Purification and Analysis of F4-NPs and IsoKs by GC/NICI/MS-- Purification and analysis of F4-NPs and IsoKs by GC/NICI/MS was performed as described (4, 17).

Formation and Analysis of NK-lysyl Adducts-- 10 mg of DHA was oxidized as described above in the presence of 10 mg of lysine. To reduce and stabilize Schiff base adducts, <FR><NU>1</NU><DE>10</DE></FR> volume of 1 M sodium borohydride in DMF was added and allowed to incubate for 30 min at 4 °C. The sample was extracted with an Oasis cartridge and analyzed by liquid chromatography (LC)/electrospray ionization (ESI)/MS/MS in the positive ion mode as described (4). The auxiliary gas pressure was 10 p.s.i., and the sheath gas pressure was 70 p.s.i. The voltage on the capillary was 20 V, and the tube lens voltage was 80 V. The capillary temperature was 200 °C. Collision-induced dissociation (CID) of molecular ions of putative NK-lysyl adducts was performed from -20 eV to -40 eV with 2.6-millitorr collision gas, scanning daughter ions between 50 and 550.

Preparation and Oxidation of Rat Brain Synaptosomes-- Synaptosomes were prepared from brain of Harlan Sprague-Dawley rats according to the method of Janowsky et al. (32). Lipid peroxidation was initiated by the addition of an iron/ADP/ascorbate mixture as described above. Incubations were carried out at 37 °C for 4 h, and the samples were then placed at -80 °C to terminate the reactions.

Analysis of NK-lysyl Adducts in Rat Brain Synaptosomes-- Following oxidation, 1 volume of 0.4 N KOH (containing 3 mM Trolox) was added for base hydrolysis, and the mixture was incubated under argon for 2 h at 37 °C. After neutralization of the sample with 5 N HCl, 10 volumes of cold ethanol (containing 5 mg of butylated hydroxytoluene (BHT) and 50 mg of triphenylphosphine (TPP)/100 ml) were added, and the proteins were precipitated by centrifugation at 2000 rpm at 4 °C for 10 min. Proteins were then reprecipitated in 10 volumes of cold Folch solution and washed with 10 volumes of MeOH (each containing BHT and TPP). Proteins were resuspended in 1× phosphate-buffered saline and heated to 98 °C for 5 min. After cooling, Pronase (3 mg/mg of starting protein weight) was added, and the mixture was incubated overnight at 37 °C. Samples were then heated at 98 °C for 5 min to inactivate the Pronase, and after cooling, aminopeptidase M (1 µl/mg starting protein weight) was added, and the digest was incubated at 37 °C for 18 h. The digest was extracted with an Oasis cartridge as described above and purified by HPLC using a 4.6 × 250 mm Macrosphere 300 C18 column. The solvent system employed was a gradient consisting of 20 mM ammonium acetate with 0.1% acetic acid (solvent A) to 5 mM ammonium acetate/MeOH/acetic acid (10:90:0.1, v/v/v) (solvent B). The flow rate was 1 ml/min beginning at 100% A, followed by an increase to 40% B over 5 min and then to 100% B over 14 min. The column was then washed with 100% B for 10 more min. HPLC fractions containing radioactivity from NK-lysyl adduct internal standards were combined, reextracted with Oasis cartridges, and analyzed by LC/ESI/MS/MS as described above. Internal standards were formed by oxidation of 25 mg of DHA in the presence of [13C6]lysine (2 mg) and [3H]lysine (50 × 106 cpm). Adducts were extracted with Oasis cartridge and HPLC as described above. Fractions were collected every min, and aliquots containing radioactivity were analyzed by LC/ESI/MS/MS. HPLC fractions containing NK-lysyl adducts were combined, and the concentration was calculated from the specific activity of the [3H]lysine.

Analysis of NK-lysyl Adducts in Human Brain-- Human cerebral cortices were ground in cold Folch solution (containing BHT and TPP). Proteins were precipitated and resuspended in 3 ml of cold MeOH (containing BHT and TPP) and 3 ml of 0.4 N KOH (containing Trolox) for the base hydrolysis. Proteins were then precipitated, washed, and subjected to complete enzymatic digestion to individual amino acids. Adducts were then extracted by Oasis cartridge and HPLC (using the same solvents as above and a flow rate of 1 ml/min beginning at 100% A followed by an increase to 40% B over 14 min and then to 100% B over 16 min) and analyzed by LC/ESI/MS/MS as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Evidence of the Formation of NKs during Oxidation of DHA in Vitro-- Previously, we have shown that oxidation of arachidonic acid (AA) in vitro results in the formation of IsoKs (4). Thus, we initially explored whether NKs are also formed in vitro during oxidation of DHA with iron/ADP/ascorbate. A representative selected current chromatogram obtained from this analysis is shown in Fig. 2. The two peaks in the lower m/z 528 chromatogram represent the syn- and anti-O-methyloxime isomers of the internal standard [2H4]PGE2. The predicted M-·CH2C6F5 ion for the pentafluorobenzyl ester, O-methyloxime, trimethylsilyl ether derivative NKs is m/z 505. In the upper ion current chromatogram are a series of m/z 505 peaks eluting at a longer retention time compared with PGE2. These peaks would be consistent with NKs, which would be expected to have a longer GC retention time than PGE2 because of their two additional carbon atoms.


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  		Fig. 2.   Selected ion current chromatograms obtained from GC/NICI/MS analysis for NKs generated during Fe/ADP/ascorbate-induced oxidation of DHA in vitro. The series of peaks in the m/z 505 chromatogram represent putative NKs analyzed as a pentafluorobenzyl ester, O-methyloxime, trimethylsilyl ether derivative, and the m/z 528 chromatogram represents the syn- and anti-O-methyloxime isomers of the [2H4]PGE2 internal standard.

Additional analyses further supported the identity of these compounds as NKs. No peaks were seen in the m/z 504 ion current chromatogram, indicating that the peaks in the m/z 505 chromatogram are not natural isotope peaks of compounds generating an ion of less than m/z 505. Analysis of the putative NKs as a [3H9] trimethylsilyl ether derivative resulted in a shift in the 505 peaks eluting after the arrow in Fig. 2 upwards to m/z 514, indicating the presence of one hydroxyl group. When analyzed as a [2H3]-O-methyloxime derivative, the m/z 505 peaks eluting after the arrow shifted upwards 6 Da to m/z 511, indicating the presence of two carbonyl groups (data not shown). Analysis following catalytic hydrogenation is shown in Fig. 3. Prior to catalytic hydrogenation, there were no peaks present 8 Da above m/z 505 at m/z 513. However, following hydrogenation, intense new peaks appear at m/z 513 with a concomitant loss of the peaks in the m/z 505 ion current chromatogram. This indicated that the compounds contained four double bonds. Collectively, these data indicate that the compounds represented by the chromatographic peaks eluting after, but not before, the arrow in the m/z 505 ion current chromatogram have the type and number of functional groups and double bonds predicted for NKs.


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  		Fig. 3.   Analysis of putative NKs before and after catalytic hydrogenation. In the absence of hydrogenation, intense peaks are present in the m/z 505 ion current chromatogram representing NKs as shown in Fig. 2. No peaks are present 8 Da above m/z 505 at m/z 513 prior to hydrogenation. Following catalytic hydrogenation, intense peaks appear in the m/z 513 current chromatogram, indicating that the m/z 505 compounds contain four double bonds.

We then compared the relative amounts of NKs and F4-NPs formed during oxidation of DHA. The amounts of NKs formed are less than the amounts of F4-NPs (Fig. 4A). Interestingly, however, the amount of NKs generated exceeded the amount of IsoKs formed during co-oxidation of equal molar amounts of DHA and AA with iron/ADP/ascorbate by 3.1-fold (Fig. 4B).


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  		Fig. 4.   Comparison of the relative amounts of F4-NPs and NKs (A) and NKs and IsoKs (B) formed during co-oxidation of equal amounts of DHA and AA in vitro.

Evidence for the Formation of NK-lysyl Adducts-- We previously demonstrated that IsoKs covalently adduct to lysine residues with remarkable rapidity (within seconds) (4). IsoKs initially form an unstable reversible Schiff base adduct, which then proceeds through a pyrrole to stable lactam and hydroxylactam adducts2 (4, 7) (Fig. 5). To determine whether NKs form covalent adducts with lysine in vitro, DHA was oxidized with iron/ADP/ascorbate in the presence of lysine. Adducts were then analyzed, after reduction by sodium borohydride, by LC/ESI/MS. Selected ion current chromatograms monitoring m/z 491 and 503 from these analyses are shown in Fig. 6. The predicted [MH]+ ion for the dehydrated reduced Schiff base NK lysine adduct is m/z 491. This is consistent with our previous observation that IsoKs not only undergo reduction during treatment with the sodium borohydride but also dehydration (7). The predicted [MH]+ ions for the NK lysine lactam adducts is m/z 503. The presence of multiple m/z 491 and 503 peaks is consistent with the formation of multiple NK-lysyl adduct isomers, as would be predicted (see Fig. 1).


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  		Fig. 5.   Proposed mechanism of formation of NK-lysyl adducts (adapted from Ref. 2).


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  		Fig. 6.   LC/ESI/MS analysis of NK-lysyl adducts formed during oxidation of DHA in the presence of lysine. Shown are the selected ion current chromatograms of the [MH]+ ions m/z 491 for the dehydrated reduced Schiff base adducts (A) and m/z 503 for the lactam adducts (B).

To further substantiate the structural identity of these compounds as Schiff base and lactam adducts, the compounds were analyzed by LC/ESI/MS/MS. CID of the putative dehydrated reduced Schiff base adducts produced daughter ions at m/z 473 and 346 (Fig. 7A). CID of the putative NK-lysyl lactam adducts produced relevant daughter ions at m/z 485, m/z 467, m/z 356, m/z 338, and m/z 84 (Fig. 7B). The ions at m/z 473 in the Schiff base CID spectrum and the ions at m/z 485 and m/z 467 in the lactam CID spectrum represent the loss of one molecule of H2O (m/z 473, 485) and two molecules of H2O (m/z 467). Other daughter ions present in these CID spectra can be assigned the structures shown in Fig. 8 based on analogous ions present in the CID spectra of IsoK-lysyl adducts (4, 7).


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  		Fig. 7.   LC/ESI/MS/MS analysis of NK-lysyl adducts formed during oxidation of DHA in the presence of lysine. The [MH]+ ions m/z 491 of the dehydrated reduced Schiff base adducts (A) and m/z 503 of the lactam adducts (B) were subjected to CID at -25 eV, and daughter ions were scanned from m/z 50 to m/z 550. The proposed structures of individual ions are shown in Fig. 8.


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  		Fig. 8.   Interpretation of the structures of individual daughter ions of the dehydrated NK-lysyl reduced Schiff base adducts (A) and NK-lysyl lactam adducts (B).

Formation of NK-lactam Protein Adducts in Rat Brain Synaptosomes-- We then examined whether NK-lactam protein adducts could be detected in synaptosomes isolated from adult rat brain. Synaptosomes (composed of sealed off neuronal and glial processes) are a widely used model for the study of central nervous system gray matter metabolism (33). We compared the formation of NK-lactam adducts in nonoxidized synaptosomes and in synaptosomes following oxidation for 4 h with iron/ADP/ascorbate. NK-lysyl lactam adducts were isolated and after complete enzymatic digestion of proteins to individual amino acids and quantified following base hydrolysis. Adducts were analyzed by LC/ESI/MS/MS utilizing selected reaction monitoring of the transition of the [MH]+ ions for the synaptosomal lactam adducts (m/z 503) and NK [13C6]lysine lactam internal standards (m/z 509) to the specific respective CID ions m/z 84 and 89. The internal standards were obtained by oxidation of DHA in the presence of [13C6]lysine and [3H]lysine. The lactam adducts were detected in nonoxidized synaptosomes at a level of 0.09 ng/mg of protein (Fig. 9A), and levels increased 19-fold to 1.71 ng/mg of protein following oxidation (Fig. 9B). The pattern of peaks representing synaptosomal lactam adducts differs somewhat from the pattern obtained for the internal standard. This can be explained by our previous observation that there appears to be a steric influence of phospholipids on the formation of different isomers from esterified substrate. The pattern of peaks representing lactam adducts in nonoxidized synaptosomes also differs somewhat from the patterns detected in oxidized synaptosomes. This is due to variation in absolute recovery of the NK adduct isomers in the pooled HPLC fractions collected due to the large number of isomers that elute over a broad range, as was also seen for peaks representing the internal standards.


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  		Fig. 9.   LC/ESI/MS/MS analysis of NK-lysyl lactam adducts in non-oxidized synaptosomes (A) and oxidized synaptosomes (B). Lipid peroxidation was initiated with Fe/ADP/ascorbate. Synaptosomes were delipidated, and proteins were then subjected to complete enzymatic digestion to individual amino acids. Internal standards of [13C6]lactam, [3H]lactam adducts were formed by oxidation of DHA in the presence of [13C6]lysine and [3H]lysine. Selected reaction monitoring of the transition m/z 503 to m/z 84 and m/z 509 to m/z 89 for synaptosomal lactam adducts and the internal standards, respectively, was performed.

Detection of NK-lysyl Adducts in Vivo in Human Brain-- We then sought to determine whether NK-lactam protein adducts are present in detectable quantities in vivo in human brains obtained from individuals who had no known neurological disease at the time of death. Proteins from frozen human cerebral cortex were precipitated, delipidated, and treated with or without base hydrolysis before complete proteolysis. Levels of NK-lysyl lactam adducts in the cerebral cortex were 9.9 ± 3.7 ng/g of brain tissue (n = 4) (Fig. 10). The amount of adducts detected was not different from the levels measured when proteins had not been subjected to base hydrolysis, indicating that NK-lactam adducts were not esterified to phospholipids This is consistent with our observation that IsoK-lactam protein adducts are not associated with phospholipids.2


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  		Fig. 10.   LC/ESI/MS/MS analysis of NK-lysyl lactam adducts from human cerebral cortex. Selected reaction monitoring is shown of the transitions from m/z 503 to m/z 84 and m/z 509 to m/z 89 for NK-lysyl lactam protein adducts in brain and the [13C6]NK-lysyl lactam internal standard, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These studies have identified a novel class of IsoK-like compounds that are formed by free radical-induced peroxidation of DHA both in vitro and in vivo. Our motivation for exploring whether IsoK-like compounds are formed via the NP pathway stems from the fact that DHA is uniquely enriched in neural and retinal tissues; DHA comprises about one-third and 30-65% of total fatty acids in aminophospholipids of gray matter and rod outer segments, respectively (8-10, 34).

Oxidative damage has been strongly implicated in the pathogenesis of a number of neurological disorders (19-21, 35, 36). The brain is especially sensitive to oxidative injury because of its high content of polyunsaturated fatty acids, its high oxygen consumption rate, and its relative paucity of antioxidant defenses compared with other tissues (37). In this regard, NPs and IsoPs are readily detectable in normal brain tissue, suggesting a level of ongoing oxidant stress in the brain (17, 18). It was of interest to find that NK-lysyl protein adducts are also readily detectable in normal brain tissue, suggesting that proteins are being covalently modified by NKs even in the normal state. At present, it is not known if NPs exert biological activity. However, because of their capacity to covalently modify proteins, adduction of key proteins by NKs may be highly injurious to neurons. This may take on particular relevance in pathologic disorders involving oxidant injury. This notion is supported by our previous findings that levels of NPs and/or IsoPs are significantly increased, indicative of enhanced oxidant injury, in both Huntington's disease and Alzheimer's disease (38-40). Reactive aldehydes derived from lipid peroxidation have been suggested to play a key role in the pathogenesis of neurodegenerative processes. The reactive aldehydes most intensively studied have been 4-HNE and 4-hydroxy-2-hexenal, formed from oxidation of AA and DHA, respectively, and malondialdehyde (6, 41). Protein-bound 4-HNE levels are increased in Alzheimer's disease ventricular fluid (42), pyramidal neuron cytoplasm and neurofibrillary tangles in Alzheimer's disease brain (43-45) and in Parkinson's disease nigral neurons (46). Modification of proteins by 4-HNE impairs the function of neuronal glucose transporter GLUT-3 (47) and the astrocytic glutamate transporter GLT-1 (48) and causes disruption of neuronal microtubules (49). Although 4-HNE is considered to be one of the most cytotoxic reactive aldehydes formed from lipid peroxidation (6), it is of interest and particular relevance that we previously showed that IsoKs adduct to lysine residues at a rate that exceeds that of 4-HNE by several orders of magnitude (4). Relevant to the hypothesis that NKs could be important effector molecules in the pathobiology of oxidative neuronal injury are the data obtained from the 2,5-hexanedione, the reactive metabolite that is responsible for the neurotoxicity of n-hexane. 2,5-Hexanedione is a gamma -diketone that reacts with the epsilon -amine group of lysine with reaction chemistry similar to that of NKs. gamma -Diketone neuropathy is characterized by cross-linking of neurofilaments, via the formation of pyrrole adducts, leading to axonal atrophy and swelling (50, 51). Thus, the neurotoxic effects of NKs would be expected to be similar to that of 2,5-hexanedione.

It is interesting to note that the amounts of NKs generated during co-oxidation of equivalent amounts of DHA and AA in vitro were greater than the amounts of IsoKs formed. This is consistent with the fact that DHA is more susceptible than AA to oxidation (52). This is also in accord with the findings that (a) NP levels are higher than levels of IsoPs in normal human brain, (b) levels of NPs are increased in brain from patients with Alzheimer's disease, whereas IsoPs are not, and (c) levels of NPs are higher than levels of IsoPs in cerebrospinal fluid from both control subjects and patients with Alzheimer's disease (17, 40). Collectively, this suggests that NKs formed by the NP pathway may play a more prominent role as neurotoxins in settings of oxidant injury to the brain than IsoKs formed by the IsoP.

In summary, these studies have elucidated the formation of highly reactive gamma -ketoaldehydes NKs as products of the NP pathway of free radical-induced peroxidation of DHA, both in vitro and in vivo. This identifies a new class of molecules that may be involved in the formation of protein adducts and protein cross-links in neurodegenerative diseases, a common feature of these disorders, and contribute to the injurious effects of other oxidative pathologies in the brain.

    ACKNOWLEDGEMENT

We thank M. Lisa Manier for valuable technical support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM42056, GM15431, CA68485, and DK26657.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602. Tel.: 615-343-1816; Fax: 615-343-9446; E-mail: jack.roberts@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, June 18, 2001, DOI 10.1074/jbc.M103768200

2 C. J. Brame, O. Boutaud, S. S. Davies, T. Yang, D. Roden, J. A. Oates, J. D. Morrow, and L. J. Roberts II, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: IsoP, isoprostane; AA, arachidonic acid; BHT, butylated hydroxytoluene; DHA, docosahexaenoic acid; CID, collision-induced dissociation; ESI, electrospray ionization; GC, gas chromatography; 4-HNE, 4-hydroxy-2-nonenal; LC, liquid chromatography; HPLC, high pressure liquid chromatography; IsoK, isoketal; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NICI, negative ion chemical ionization; NP, neuroprostane; NK, neuroketal; PG, prostaglandin; TPP, triphenylphosphine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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