Post-traumatic stress disorder and serum cytokine and chemokine concentrations in patients with rheumatoid arthritis✰.

OBJECTIVE: Although post-traumatic stress disorder (PTSD) is identified as a risk factor in the development of rheumatoid arthritis (RA), associations of PTSD with disease progression are less clear. To explore whether PTSD might influence disease-related measures of systemic inflammation in RA, we compared serum cytokine/chemokine (cytokine) concentrations in RA patients with and without PTSD. METHODS: Participants were U.S. Veterans with RA and were categorized as having PTSD, other forms of depression/anxiety, or neither based on administrative diagnostic codes. Multiplex cytokines were measured using banked serum. Associations of PTSD with cytokine parameters (including a weighted cytokine score) were assessed using multivariable regression, stratified by anti-CCP status and adjusted for age, sex, race, and smoking status. RESULTS: Among 1,460 RA subjects with mean (SD) age of 64 (11) years and disease duration of 11 (11) years, 91% were male, 77% anti-CCP positive, and 80% ever smokers. Of these, 11.6% had PTSD, 23.7% other depression/anxiety, and 64.7% had neither. PTSD, but not depression/anxiety, was associated with a higher cytokine score and number of high-concentration analytes in adjusted models, though this was limited to anti-CCP positive subjects. PTSD was associated with heightened expression of several individual cytokines including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-17, IFN-γ, GM-CSF, MCP-1, and TNF-α. CONCLUSION: Anti-CCP positive RA patients with PTSD have higher serum cytokine concentrations than those without PTSD, demonstrating that systemic inflammation characteristic of RA is heightened in the context of this relatively common psychiatric comorbidity.

Gingival tissue, an extrasynovial source of malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins.

BACKGROUND AND OBJECTIVE: Postranslational modification of proteins can lead to the production of autoantibodies and loss of immune tolerance. This process has been hypothesised to be a critical factor in the pathogenesis of rheumatoid arthritis. The objective of this study was to demonstrate that inflamed human gingival tissue provides an extrasynovial source of malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins all of which are considered to be linked to the development of rheumatoid arthritis. Identification of such modified proteins in inflamed gingiva may explain, in part, how inflammation of the periodontal tissues may influence the development of rheumatoid arthritis. MATERIAL AND METHODS: Gingival biopsies of healthy, mild and moderate periodontitis were triple stained with antibodies against malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins. RESULTS: Assessment of healthy gingival tissue revealed negligible staining for carbamylated, malondialdehyde-acetaldehyde (MAA), or citrullinated proteins. Mild periodontitis was positive for all three modifications. Furthermore, there was an increase in staining intensity for carbamylated, citrullinated and MAA-modified proteins in moderate periodontitis. Negative staining results were observed for the isotype controls. CONCLUSION: This study provides evidence for the presence of citrullinated, carbamylated and MAA adduct modified proteins in inflamed periodontal tissues. The potential for these proteins to play a role in autoimmunity in a multi-system inflammatory syndromic disease model now needs to be determined.

Celastrol inhibits inflammatory stimuli-induced neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are web-like structures released by activated neutrophils. Recent studies suggest that NETs play an active role in driving autoimmunity and tissue injury in diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The purpose of this study was to investigate if celastrol, a triterpenoid compound, can inhibit NET formation induced by inflammatory stimuli associated with RA and SLE. We found that celastrol can completely inhibit neutrophil oxidative burst and NET formation induced by tumor necrosis factor alpha (TNFα) with an IC50 of 0.34 µM and by ovalbumin:anti-ovalbumin immune complexes (Ova IC) with an IC50 of 1.53 µM. Celastrol also completely inhibited neutrophil oxidative burst and NET formation induced by immunoglobulin G (IgG) purified from RA and SLE patient sera. Further investigating into the mechanisms, we found that celastrol treatment downregulated the activation of spleen tyrosine kinase (SYK) and the concomitant phosphorylation of mitogen-activated protein kinase kinase (MAPKK/MEK), extracellular-signal-regulated kinase (ERK), and NFκB inhibitor alpha (IκBα), as well as citrullination of histones. Our data reveals that celastrol potently inhibits neutrophil oxidative burst and NET formation induced by different inflammatory stimuli, possibly through downregulating the SYK-MEK-ERK-NFκB signaling cascade. These results suggest that celastrol may have therapeutic potentials for the treatment of inflammatory and autoimmune diseases involving neutrophils and NETs.

N-(methylamino)isobutyric acid inhibits proliferation of CFSC-2C hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) involves the induction of ECM protein synthesis and rapid cell proliferation. Thus, agents that interfere with either process could potentially mitigate the development of liver disease by reducing the synthesis of proteins associated with fibrosis or by reducing the number of activated HSC. Previously, we described that the non-metabolizable amino acid analog N-(methylamino)isobutyric acid (MeAIB) reduced hepatic collagen content of rats in a model of CCl(4)-induced liver injury, and in vitro studies using CFSC-2G cells indicated that MeAIB directly reduced collagen synthesis. However, the MeAIB-mediated reduction of hepatic collagen, in vivo, following liver injury was associated with a decrease in hepatic alpha-smooth muscle actin (alpha-SMA) which suggested that MeAIB also inhibited the activation of HSCs. Because HSC activation is inseparable from proliferation, the purpose of this study was to examine the effect of MeAIB treatment on the proliferation of HSCs in an in vitro model utilizing CFSC-2G cell cultures. In these studies, MeAIB effectively inhibited the proliferation of CFSC-2G cells by interfering with the progression of the cells through the G(1)-phase of the cell cycle which delayed entry into S-phase. MeAIB prevented the phosphorylation of p70S6 kinase (p70S6K) at Thr389 and reduced the phosphorylation at Thr421/Ser424. Because p70S6K is required for G(1)-cell cycle progression and is known to be regulated by nutrient availability, this correlates well with MeAIB interfering with the proliferation of CFSC-2G HSCs. In addition, the rate of protein synthesis was reduced by MeAIB treatment following mitogenic stimulation, which agrees with a p70S6K-mediated reduction in translation. These data are consistent with MeAIB inhibiting the proliferation of CFSC-2G cells by altering the mitogen activated pathway(s) leading to phosphorylation of p70S6K by a yet to be described mechanism.

Elucidation of reaction scheme describing malondialdehyde-acetaldehyde-protein adduct formation.

Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MDHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.

The chemistry and biological effects of malondialdehyde-acetaldehyde adducts.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Geoffrey M. Thiele and Simon Worrall. The presentations were (1) The chemistry of malondialdehyde-acetaldehyde (MAA) adducts, by Dean J. Tuma; (2) The formation and clearance of MAA adducts in ethanol-fed rats, by Simon Worrall; (3) Immune responses to MAA adducts may play a role in the development of alcoholic liver disease, by Lynell W. Klassen; (4) Unique biological responses to MAA-modified proteins that may play a role in the development and/or progression of alcoholic liver disease, by Geoffrey M. Thiele; (5) MAA-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells, by Todd A. Wyatt; and (6) An enzyme immune assay for serum antiacetaldehyde adduct antibody using low-density lipoprotein-adduct and its significance in alcoholic liver injury and ALDH2 heterozygotes, by Naruhiko Nagata.

Detection of circulating antibodies against malondialdehyde-acetaldehyde adducts in patients with alcohol-induced liver disease.

Acetaldehyde and malonildialdehyde can form hybrid protein adducts, named MAA adducts that have strong immunogenic properties. The formation of MAA adducts in the liver of chronic alcohol-fed rats is associated with the development of circulating antibodies that specifically recognized these adducts. The aim of this study was to examine whether MAA adducts might participate in the immune response associated with human alcohol-induced liver disease. Circulating antibodies against MAA adducts were evaluated in 50 patients with alcohol-induced hepatitis or cirrhosis, in 40 patients with non-alcohol-induced liver disease, in 15 heavy drinkers without liver damage and in 40 healthy controls by enzyme-linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) reacting with MAA-modified proteins were significantly increased in the patients with alcohol-induced cirrhosis or hepatitis. The individual levels of anti-MAA IgG in those patients were associated with the severity of liver damage. Anti-MAA antibodies were also positively correlated with the levels of IgG recognizing epitopes generated by acetaldehyde and malonildialdehyde. However, competitive inhibition experiments indicated that the anti-MAA antibodies were unrelated to those against acetaldehyde- or malonildialdehyde-derived antigens and mainly recognized a specific, cyclic MAA epitope. Some degree of immune reactivity towards MAA adducts was also observed in patients with non-alcohol-induced liver injury. However, competitive ELISA showed that the antigens recognized by these sera were not the cyclic MAA adducts. Altogether, these results showed the formation of MAA antigens during alcohol-induced liver disease and suggest their possible contribution to the development of immunologic reactions associated with alcohol-related liver damage.

Conversion of acetaldehyde-protein adduct epitopes from a nonreduced to a reduced phenotype by antigen processing cells.

Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.

Chronic ethanol consumption impairs receptor-mediated endocytosis of formaldehyde-treated albumin by isolated rat liver endothelial cells.

Receptor-mediated endocytosis (RME) by a scavenger receptor on sinusoidal liver endothelial cells (LECs) for formaldehyde-treated bovine serum albumin (f-Alb) has previously been shown to be impaired following chronic ethanol consumption. These studies were initially performed by in situ perfusion, making it difficult to determine the point in the process at which RME is affected. Therefore, it was the purpose of this study to use isolated LECs to begin elucidating at what point in the process chronic ethanol consumption affects RME. Initial studies showed that degradation at the single-cell level were similarly decreased at levels that had been observed for in situ studies, suggesting that the ethanol effects can be repeated using isolated LECs, making them useful for in vitro studies. Binding studies with 125I-formaldehyde-treated bovine serum albumin (125I-f-Alb) demonstrated there was a slight, but significantly different, decrease in binding by LECs from ethanol-fed rats when compared with pair-fed or chow-fed rats. However, the affinity of these receptors was not different between these groups. In contrast, a defect in the initial stages of receptor-ligand internalization was indicated as less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared with controls. Additionally, once the data were adjusted for the amount of ligand internalized, the degradation of the internalized ligand was only slightly impaired. These results indicate that chronic ethanol feeding impairs the process of RME by the liver; the major cause of this impairment appears to be caused by a decreased ability of these cells to internalize all of the surface-bound ligand, with a minimal defect in postinternalization events.

Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant.

Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.

Association of malondialdehyde-acetaldehyde (MAA) adducted proteins with atherosclerotic-induced vascular inflammatory injury.

Atherosclerosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor-alpha (TNF-alpha), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF-alpha, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF-alpha concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.

Monoclonal and polyclonal antibodies recognizing acetaldehyde-protein adducts.

Studies have investigated the hypothesis that metabolically derived acetaldehyde (AA) is capable of complexing with liver cell proteins to form AA-protein adducts that are capable of acting as antigens and inducing an immune response, as detected by the formation of unique antibodies. In an effort to better characterize and describe these adducts, mouse monoclonal and rabbit polyclonal antibodies specific for antigens prepared with AA under non-reducing (physiologic) and reducing (presence of sodium cyanoborohydride) conditions have been prepared. Two monoclonal antibodies were developed. The first antibody was RT1.1, which is specific to N-ethyl lysine (NEL); it is of the IgG2b isotype and recognizes all proteins modified with AA under reducing conditions. The other monoclonal antibody, NR-1, was of the IgG3 isotype; it recognizes proteins modified with AA under non-reducing conditions and cannot be inhibited by NEL. Affinity-purified and/or absorbed polyclonal antibodies were also produced to these epitopes. Using this panel of monoclonal and affinity-purified polyclonal antibodies, unique antigen-antibody binding occurred that: (1) detected only NEL; (2) reacted with the alpha-amino group on proteins prepared under reducing conditions; and (3) detected adducts on proteins prepared under non-reducing conditions. However, the only antibodies that recognized antigen(s) from alcohol-fed rat livers were those that were not specific to NEL or the alpha-amino group modified under reducing conditions. These data indicate that the relevant adduct in alcohol-fed rat livers is not NEL, and that it presumably is related to proteins modified with AA under non-reducing conditions.

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats.

BACKGROUND & AIMS: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.

Epitope characterization of malondialdehyde-acetaldehyde adducts using an enzyme-linked immunosorbent assay.

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.

Long-term ethanol administration alters the degradation of acetaldehyde adducts by liver endothelial cells.

Previous reports have shown that long-term ethanol administration alters receptor-mediated endocytosis (RME) of a variety of macromolecules by liver endothelial cells (LEC). Acetaldehyde is the major metabolic product of ethanol metabolism and has been shown to bind to proteins to form adducts. In this study, the level of protein modification by acetaldehyde necessary for the uptake and degradation of acetaldehyde-modified proteins by LEC was investigated. Bovine serum albumin (BSA) acetaldehyde adducts were prepared by incubation of albumin with acetaldehyde at 100 mmol/L for 1 hour at 37 degrees C, and 1 mmol/L or 0.2 mmol/L for 5 days at 37 degrees C. In situ liver perfusion in the presence of these adducts resulted in the degradation of 107 +/- 10.02 microg, 69.82 +/- 5 microg, and 2.5 +/- 0.42 microg of acetaldehyde-adducted albumin, respectively, during a 4-hour period. These values were decreased by 53%, 67%, and nearly 100%, respectively, in livers from ethanol-fed rats. Additionally, modification of protein with 1 mmol/L of acetaldehyde for different periods of time and/or pH altered the amount of 14C-acetaldehyde binding, but no significant changes in degradation were observed. Finally, an excess of formaldehyde-modified albumin totally inhibited the degradation of acetaldehyde adducts, suggesting that they use the same receptor. These data show that acetaldehyde-modified proteins may be taken up and degraded by the scavenger receptor on LEC. This uptake and degradation are dependent on the extent modification of the protein by acetaldehyde, and long-term ethanol consumption decreases the degradation of acetaldehyde-protein adducts.

Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration.

Acetaldehyde and the lipid peroxidation-derived aldehyde malondialdehyde (MDA), are reactive compounds that are generated during ethanol metabolism in the liver, and both aldehydes have been shown to be capable of binding to proteins and forming stable adducts. Because similar concentrations of MDA and acetaldehyde can coexist in the liver during ethanol oxidation, protein adduct formation in the presence of both of these aldehydes was studied under both in vitro and in vivo conditions. When proteins were incubated in the presence of both MDA and acetaldehyde, MDA caused a marked and concentration-dependent increase in the stable binding of acetaldehyde to proteins. Maximum stimulation of binding occurred at approximately a fourfold molar excess of MDA relative to acetaldehyde when concentrations of 1.0 mmol/L and 0.1 mmol/L were tested. The formation of highly fluorescent product or products was associated with the MDA stimulation of acetaldehyde binding, indicating that new and distinct products were being generated. These hybrid adducts of MDA and acetaldehyde have been designated as MAA adducts. An affinity-purified polyclonal antibody was produced that specifically recognized MAA epitopes on proteins and did not cross-react with carrier proteins or proteins modified with either acetaldehyde or MDA alone. A quantitative competitive enzyme-linked immunosorbent assay (ELISA) was developed and detected the presence of MAA-modified proteins in liver cytosol from ethanol-fed rats but not in pair-fed controls. Quantification of the data from the competitive ELISA indicated the presence of approximately 75 pmoles protein-bound MAA per milligram liver cytosol proteins of the ethanol-fed animals. These results indicate that acetaldehyde and MDA can react together in a synergistic manner and generate hybrid adducts (MAA-adducts) and further suggest that MAA adducts may represent a major species of adducts formed in the liver during ethanol metabolism in vivo.

Specificity of N-ethyl lysine of a monoclonal antibody to acetaldehyde-modified proteins prepared under reducing conditions.

A monoclonal antibody has been developed that recognizes only protein-acetaldehyde (AA) adducts prepared under reducing conditions: 5 mM AA with 30 mM sodium cyanoborohydride overnight at 37 degrees. This monoclonal antibody is a mouse IgG2b that has been designated RT1.1. The primary adduct formed when proteins are exposed to acetaldehyde under reducing conditions is N-ethyl lysine (NEL). To examine the epitope specificity of RT1.1, inhibition ELISAs were developed using NEL and other possible inhibitors, such as arginine, ethylamine, lysine and proteins modified with AA under non-reducing conditions. RT1.1 (at half-maximum optical density, 50 ng/mL) was inhibited only by NEL and was independent of the carrier or the pH of the buffer used in the ELISA. Further evidence indicating that NEL is the epitope recognized by RT1.1 was obtained using mouse and human epidermal growth factor (EGF). Both proteins contain one alpha amino group but only the human-EGF contains lysine residues with epsilon amino groups. In experiments where these two proteins were modified with AA under reducing conditions, RT1.1 reacted only with human-EGF. These studies demonstrate that RT1.1 is specific for NEL that is formed by the ethylation of proteins with acetaldehyde under reducing conditions. Additionally, these studies demonstrate that the procedures and methods used herein may be useful for characterizing other antibodies prepared to AA-modified proteins under a variety of defined in vitro chemical conditions.