Alcohol Research Unit, Department of Biochemistry, University of Queensland, Queensland 4072, Australia
Received 13 September 1999; in revised form 21 October 1999; accepted 5 November 1999
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of modified proteins
This study required the use of two modified proteins. Human plasma protein was modified using each procedure described below and used to generate antisera reactive with each type of modification. Similarly, bovine haemoglobin was also modified using each procedure and used as the coating protein in the ELISAs used to detect antibodies generated in vivo against each type of adduct.
Modification by AcH. AcH-modified proteins were produced by incubating protein (10 mg/ml) with 1 mM AcH in phosphate-buffered saline (PBS), pH 7.4. Unreduced adducts were generated by a 5 h incubation at 37°C, whereas reduced adducts were produced by a 1 h incubation at 37°C followed by the addition of 1 ml of 40% (w/v) sodium cyanoborohydride/10 ml of reaction mixture. A further 30 min incubation at 37°C was then carried out to allow the reduction of Schiff bases.
Modification by HER HER-modified proteins were prepared by incubating 10 mg/ml protein with 50 mM ethanol, 100 µM ferric ammonium sulphate, 200 µM EDTA, and 100 µM hydrogen peroxide, in 50 mM sodium phosphate buffer (pH 7.4) at 37°C for 30 min as described by Moncada et al. (1994).
Modification by MDA MDA-modified protein was produced by incubating 10 mg/ml protein with 1 mM MDA for 12 h at 37°C in phosphate-buffered saline, pH 7.4. MDA was produced immediately before use by the hydrolysis of the dimethylacetal with 1 M HCl for 30 min at 37°C. A portion of the acidic solution was then diluted with water and adjusted to pH 7.4 with 1 M NaOH to prepare neutral MDA solution for use in the modification reactions.
Modification by AcH and MDA Tuma et al. (1996) have shown that a mixture of MDA and AcH reacts with proteins to produce MDAAcH adducts which they termed MAA. Protein, 10 mg/ml in 100 mM sodium phosphate (pH 7.4), was incubated with 1 mM AcH and 1 mM MDA for 72 h or 3 days at 37°C.
At the end of each incubation, the reaction mixtures were dialysed extensively against buffer for 12 h at 4°C before being used as immunogens or in ELISAs. Modified proteins were stored in small aliquots at 80°C for less than 1 month before use.
Generation of anti-adduct antisera
New Zealand white rabbits (8 months old) were immunized with modified human plasma protein in three sets of multi-site injections as described at Worrall et al. (1989). After the final booster injection, the rabbits were exsanguinated by cardiac puncture under nembutal anaesthesia (60 mg/kg, i.p.). Blood was collected using heparinized needles and tubes and the resulting plasma was stored at 80°C.
Purification of anti-adduct antisera
To decrease binding to unmodified proteins, antisera were purified as described by Worrall et al. (1989) and using the modification of Nicholls et al. (1994). Briefly, 1 ml of rabbit antiserum and 1 ml of buffer A (20 mM TrisHCl, pH 8.0, containing 28 mM NaCl) were mixed and loaded onto a Biogel P-6 DG desalting column (Biorad). The eluate was collected as a single fraction and applied to a DEAEAffigel blue column (Biorad) pre-equilibrated with Buffer A. The column was eluted with Buffer A, and 2-ml fractions collected. Those containing the highest protein concentrations were pooled and applied to an immunoadsorption column for 4 h at 4°C. The eluate from the immuno-adsorption column was collected as a single fraction and stored at 4°C for less than a week prior to use in the ELISAs described below. The immuno-adsorption column was made by reacting 2 ml of rat plasma (from a control) with 10 ml of Affigel-10 activated ester gel (Biorad) suspended in coupling buffer (40 mM HEPES, pH 7.5, containing 160 mM CaCl2) for 4 h at room temperature. The gel was washed with 20 bed-volumes of buffer to remove unbound protein.
Biotinylation of antibodies
Commercially obtained rabbit anti-rat albumin or rat IgG polyclonal antibodies (Accurate Chemical & Scientific Corporation, Westbury, NY, USA) were biotinylated using a commercially available kit (Amersham plc, Amersham, UK). Briefly, the antibody concentration was adjusted to 1 mg/ml and dialysed overnight against PBS at 4°C. Biotinylation reagent was added to the solution (40 µl/mg of protein) and incubated for 2 h at 20°C. Unreacted biotinylation reagent was removed from the biotinylated antibody by chromatography on a Sephadex G-25 column. Biotinylated antibody was stored at 4°C and used within a week.
Detection of modified proteins and rat plasma, haemolysate and liver homogenate
The presence of modified proteins in liver homogenate, plasma or haemolysate was detected by indirect ELISA. Briefly, samples were diluted to 100 µg protein/ml in PBS, and 100 µl were added to each well. After 2 h at 4°C, the plates were washed with Tris-buffered saline (TBS), pH 7.4. Non-specific binding sites were blocked by incubation with 200 µl of saturated casein solution [2.0% (w/v), pH 7.58.0] for 1 h at 4°C. Purified rabbit anti-adduct antiserum (100 µl) was then added to each well and incubated for 4 h at 4°C. The plate was washed with TBScasein (TBS containing 0.5% casein) and 100 µl of biotinylated anti-rabbit immunoglobulin antibody was added. After 2 h at 4°C, the plates were washed again with TBScasein and 100 µl of streptavidinalkaline phosphatase complex added. After 1 h at 37°C, the plates were again washed with TBS. The final step was to add 100 µl of p-nitrophenyl phosphate (1 mg/ml) in diethanolamine buffer (10 mM diethanolamine, 0.5 mM MgCl2, pH 9.5) and incubate at 37°C. The absorbance of each well at 405 nm was measured using a Titertek multiscan plate reader after a 30 min3 h incubation, depending on the type of adduct and whether liver, plasma or haemolysate was assayed.
Modification of plasma albumin and IgG was detected by using a sandwich ELISA. Microtitre plates were coated with 100 µl of purified rabbit anti-modified human plasma protein diluted to 20 µg/ml in TBS, pH 7.4. After 4 h at 4°C, the plates were washed with TBS and the samples added (50 µl plasma or liver homogenate mixed with 150 µl TBS) and incubated for 2 h at 4°C. The plate was then washed with TBS and 100 µl of biotinylated anti-rat albumin or IgG diluted 1:100 with TBS containing 0.1% (w/v) casein was added to each well. After 2 h at 4°C, the plate was washed with TBS, and streptavidin alkaline phosphatase complex added. The rest of the assay was as described above. The absorbance of each well at 405 nm was measured using a Titertek multiscan plate reader after a 13 h incubation, depending on the protein to be measured.
Detection of anti-adduct antibodies in rat plasma
The method used to determine plasma immunoreactivity with modified protein was essentially the same ELISA as that described in Worrall et al. (1991a). However, for these assays, the microtitre plates were coated with 100 µl of a solution containing 50 µl/ml unmodified or modified bovine haemoglobin in PBS, pH 7.4. After blocking with casein solution, 100 µl of a 1:10 dilution of plasma in TBS containing 0.5% (w/v) bovine casein (TBScasein) were added to each well and incubated for 1 h at 37°C. The wells were then washed with TBScasein and incubated with biotinylated antibodies for total rat Ig, or class-specific ones for IgA, IgG or IgM (1:1000 dilution in TBScasein buffer). From this point, the assay was carried out as described previously.
Definitions and statistical evaluation of results
Antibody binding to modified epitopes was defined as the difference in absorbance between wells coated with unmodified and AcH-modified bovine haemoglobin and was termed adduct-specific reactivity (ASR). Statistical analyses were performed on an IBM-compatible PC using SigmaStatTM for Windows 95 Version 2.03 (Jandel Scientific Software, San Rafael, CA, USA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Modified proteins in liver
Figure 1 shows that multiple types of modification occur in the livers of rats fed ethanol for up to 10 weeks. In each case, the amount of modification increased gradually over the first 6 weeks of feeding, but was relatively steady after that time. In the control animals, the levels of modification were stable throughout the experiment and were always lower than those of the ethanol-fed group.
|
|
Modified proteins in haemolysate
Unlike the liver, where multiple types of modification were detected, only a single type of modification was found in haemolysate. Figure 3 shows the development of unreduced AcH adducts in haemolysate from these animals. In the early stages of ethanol feeding, there was no elevation in modification of the haemolysate when compared to controls. However, by 6 weeks, there was a significantly higher level of modification in the ethanol-fed animals which was further increased by 10 weeks.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The liver is the main site of ethanol oxidation and it would be expected that this tissue would have the highest levels of modification and the widest variety of adduct types. The liver oxidizes >90% of imbibed ethanol to produce AcH by the action of alcohol dehydrogenase and the microsomal ethanol-oxidizing system (MEOS), an important component of which is cytochrome P-450 IIE1 (Lieber, 1999). During chronic ethanol oxidation, appreciable levels of AcH can accumulate due to the inability of aldehyde dehydrogenase to oxidize all AcH to acetate. Studies using freshly isolated rat hepatocytes suggest that a free AcH concentration of about 1 mM can be obtained at an ambient ethanol concentration of 25 mM (Irving et al., 1985
). The MEOS system can also produce highly reactive HER as a secondary reaction to the production of AcH (Lieber, 1999
). An indirect effect of ethanol metabolism is to decrease the hepatic concentration of glutathione, making cellular membranes more prone to peroxidation and leading to the increased production of lipid peroxide breakdown products, such as MDA and 4-HNE. Thus, ethanol oxidation leads to the production of two well-characterized groups of reactive species, namely aldehydes (AcH and MDA) and free radicals (HER). Each of these species can react individually with macromolecules, such as proteins, to produce covalent modifications. In addition, it has recently been shown that AcH and MDA can act in concert to produce an adduct which contains two molecules of MDA and one molecule of AcH, and another which contains one molecule of each. These adducts have been designated MAA (malondialdehydeacetaldehyde adducts) by Tuma et al. (1996).
The tissue samples analysed in the study were from male Wistar rats maintained on the LieberDeCarli liquid diet. Those animals fed the ethanol-containing form of the diet consumed 814 g ethanol/kg/day, whereas the control animals were pair-fed the control form of the diet in which the ethanol was isocalorically replaced by maltosedextrin. After 6 weeks on the liquid diet, blood-ethanol concentrations ranged from 85138 mg/dl 1 h after consumption in the ethanol-fed animals, whereas ethanol was not detectable in the blood of control animals. Thus, comparison between tissues from the ethanol-fed and control animals will show whether the quantity or type of adduct is related to ethanol consumption.
Previous studies have shown that proteins modified by AcH (Barry et al., 1987; Behrens et al., 1988
; Lin et al., 1988
; Worrall et al., 1991b
), MDA (Niemela et al., 1994
) and HER Albano et al., 1993; Clot et al., 1996) are formed in the liver of ethanol-fed animals. However, no simultaneous measurement of the generation of multiple adduct types has been previously carried out. In this study, ethanol-fed animals exhibited higher levels of each type of adduct after 10 weeks. Furthermore, as expected, the amount of each type of adduct increased after the initiation of ethanol feeding until a plateau was reached, in most cases at about 6 weeks. However, the time taken to reach significantly elevated levels of each adduct varied. Reduced AcH adducts (thought to be largely N-ethylated amino groups) and MAA adducts were elevated after 2 weeks, whereas unreduced AcH adducts (chemical nature unknown, but probably includes imidazolidinone and thiazolidine derivatives), HER-derived modifications, and N-ethylated lysine residues (recognized by the monoclonal antibody RT1.1) were only elevated after 4 weeks. The slow increase in HER-modified proteins is probably related to the induction of MEOS activity, which takes about 2 weeks of chronic ethanol feeding to become maximal (Lieber, 1999
). However, MDA adducts took 10 weeks to become significantly elevated in the ethanol-fed animals. The discrepancy between the generation of reduced AcH adducts and the N-ethylated amino groups detected by RT1.1 is interesting given that N-ethylated amino groups were thought to be the main type of reduced adduct formed. The reduced AcH adducts are significantly elevated in the ethanol-fed animals 2 weeks prior to the N-ethylated residues, suggesting that other types of adduct are initially formed in vivo that are recognized by the antiserum raised against proteins incubated with AcH and then reduced with sodium cyanoborohydride, but are not recognized by RT1.1. Further characterization of the adducts formed by this modification protocol will help to identify the nature of the other adducts. A comparison of the generation of MDA-modified protein and MAA adducts is also of interest, as both types of modification require MDA. It appears that, at least in the initial stages, MDA produced reacts together with AcH to produce MAA adducts, such that they are significantly elevated after 2 weeks, whereas MDA adducts are not elevated until the concentration of MAA adducts becomes maximal.
Plasma proteins from ethanol-fed animals were also found to carry elevated amounts of each adduct type after 10 weeks of feeding, when compared to control animals. Again in the same way as hepatic modification, the degree of modification of plasma protein was found to increase over time, and to then plateau. However, with the exception of the N-ethylated residues, each type of adduct took longer to be significantly elevated in plasma than it did within the liver. A potential explanation for these data is that ethanol metabolites initially react with internal components of hepatocytes and only react with secretory proteins such as albumin during the secretory process once the initial sites have been modified. However, this raises the question of whether plasma proteins are modified prior to secretion by the hepatocyte or are modified in the circulation by reactive metabolites which have leaked out of the liver. In an attempt to answer this question, we compared the types of modification carried by albumin, the major plasma protein which is secreted by hepatocytes, and IgG, a protein secreted by circulating plasma cells (i.e. non-hepatic origin). Albumin was found to carry all of the types of modification studied. However, IgG carried only unreduced and reduced AcH adducts. These data suggest that at least some types of modification carried by albumin probably occur prior to its release into the circulation. For example, we have shown that plasma albumin carries HER-derived modifications. However, due to the highly reactive nature of these free radicals, it is unlikely that they are able to diffuse out of the liver cells to react with extra-hepatic albumin. Furthermore, the production of HER outside the liver is likely to be low. This is further supported by the fact that circulating IgG does not carry this type of modification, presumably because it is secreted by cells which do not contain the MEOS activity required to make HER and do not encounter the radical in the circulation. Similar arguments can be made for MDA-derived modification and for MAA adducts, both of which do not appear to form on circulating IgG.
Several previous studies have shown that AcH reacts with haemoglobin in vivo to produce covalent adducts (Niemela et al., 1990; Sillanaukee et al., 1991
; Lin et al., 1993
). In this study, we also found significantly elevated immunoreactivity with AcH-derived adducts in haemolysates from ethanol-fed rats, compared to control animals. However, the adducts were only of the unreduced type, with no reduced adducts being detected. This is again an interesting finding, since it demonstrates that adducts formed in erythrocytes are different from those formed in the plasma. This is not surprising, given that mature erythrocytes do not contain endoplasmic reticulum and therefore do not have MEOS activity. Thus, they are unlikely to generate large amounts of HER and would not therefore contain HER-modified proteins. The AcH adducts formed may be derived from AcH produced inside the erythrocyte or from AcH that has leaked out of the liver. Under most conditions, erythrocytes are well protected against free radical damage, being amply supplied with reduced glutathione and a glutathione regenerating system (al-Turk et al., 1987
). This probably explains the absence of MDA and MAA adducts.
Many researchers have shown that adduct formation leads to the generation of neoantigens and to the production of antibodies reactive with the modification (hapten) and the carrier protein. In this study, we were able to detect antibodies reactive with each type of modification. While previous studies have shown that antibodies reactive with a single type of modification are generated in vivo (Israel et al., 1986; Hoerner et al., 1988
; Worrall et al., 1989
; Clot et al., 1995
; van de Vijver et al., 1996
), this is the first study to compare the generation of antibodies against multiple types of adduct in the same animal. Initially, we analysed the total immunoglobulin reactivity (IgG, IgA, and IgM together) against each type of modification. After 610 weeks on the diet, the ethanol-fed animals had elevated reactivity with each type of modification. Indeed, the immune responses were similar for most adducts with a maximal response being obtained after about 6 weeks of ethanol feeding. However, the immunoreactivity with reduced AcH and MAA adducts was still increasing after 10 weeks of feeding. This was especially true of the reactivity against MAA adducts, where a similar response was seen in each ethanol-fed animal tested (data not shown). We then dissected the antibody responses by looking at the classes of immunoglobulin involved in the response to each type of adduct. An elevated IgM response against all of the adduct types was observed after 10 weeks on the diet. An elevated IgG response of each adduct type other than MDA adducts was also observed in the ethanol-fed animals. However, only ethanol-fed animals exhibited a significantly elevated IgA response against unreduced and reduced AcH adducts. We have previously demonstrated a relationship between elevated IgA reactivity with AcH-modified proteins and ethanol abuse in humans (Worrall et al., 1996
). The causal relationship for the elevated IgA reactivity has not yet been delineated, but must lie in the close relationship between IgA and the liver (Brown and Kloppel, 1989
).
The LieberDeCarli liquid diet is the most commonly used paradigm to study the pathological effect of ethanol. However, it does not cause significant liver damage, such as hepatitis or cirrhosis unless an additional stimulus such as lipopolysaccharide (Pennington et al., 1997) is used. However, we have previously shown that rats fed the ethanol-containing form of the diet and injected with AcH-modified proteins, leading to the generation of a high titre of anti-AcH adduct antibodies, exhibited panlobular piecemeal necrosis, whereas control-fed animals did not (Worrall et al., 1992
). These data suggest that the LieberDe Carli liquid diet is a good starting point for the generation of advanced liver disease. Comparison between the adducts formed by feeding the LieberDeCarli liquid diet and the TsukamotoFrench paradigm, in which ethanol feeding does not require additional factors to produce hepatitis and fibrosis (Nanji et al., 1989
), may help to identify which adducts are important in the aetiology of significant liver damage. In this study, we have demonstrated that multiple types of protein modification are generated as a result of chronic ethanol feeding of rats. We have also shown that different types of modification occur in the liver when compared to plasma and erythrocytes. These data show that it is likely that modification of plasma proteins occurs at two sites. Plasma proteins may be modified during their synthesis and secretion by hepatocytes, but the data generated by analysis of IgG (not synthesized and secreted by the liver) suggest that modification can also occur in the plasma. Whether this is as a result of ethanol metabolism in the circulation or due to ethanol metabolites leaking out of the liver remains to be determined. We are now completing a similar study using human tissue.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albano, E., Clot, P., Morimoto, M., Tomasi, A., Ingelman Sundberg, M. and French, S. W. (1996) Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol. Hepatology 23, 155163.[ISI][Medline]
al-Turk, W. A., Stohs, S. J., el-Rashidy, F. H., Othman, S. and Shaheen, O. (1987) Glutathione, glutathione S-transferase and glutathione reductase in himan erythrocytes and lymphocytes as a function of sex. Drug Design and Delivery 1, 237243.[Medline]
Barry, R. E., Williams, A. J. and McGivan, J. D. (1987) The detection of acetaldehyde/liver plasma membrane protein adduct formed in vivo by alcohol feeding. Liver 7, 364368.[ISI][Medline]
Behrens, U. J., Hoerner, M., Lasker, J. M. and Lieber, C. S. (1988) Formation of acetaldehyde adducts with ethanol-inducible P450IIE1 in vivo. Biochemical and Biophysical Research Communications 154, 584590.[ISI][Medline]
Brown, W. R. and Kloppel, T. M. (1989) The liver and IgA: immunological, cell biological and clinical implications. Hepatology 9, 763784.[ISI][Medline]
Clot, P., Bellomo, G., Tabone, M., Arico, S. and Albano, E. (1995) Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis. Gastroenterology 108, 201207.[ISI][Medline]
Clot, P., Albano, E., Eliasson, E., Tabone, M., Arico, S., Israel, Y., Moncada, C. and Ingelman-Sundberg, M. (1996) Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics. Gastroenterology 111, 206216.[ISI][Medline]
Fleisher, J. H., Lung, C. C., Meinke, G. C. and Pinnas, J. L. (1988) Acetaldehydealbumin adduct formation: possible relevance to an immunologic mechanism in alcoholism. Alcohol and Alcoholism 23, 133141.[ISI][Medline]
Hoerner, M., Behrens, U. J., Worner, T. M., Blacksberg, I., Braly, L. F., Schaffner, F. and Lieber, C. S. (1988) The role of alcoholism and liver disease in the appearance of serum antibodies against acetaldehyde adducts. Hepatology 8, 569574.[ISI][Medline]
Irving, M. G., Simpson, S. J., Brooks, W. M., Holmes, R. S. and Dodrell, D. M. (1985) Application of the reverse DEPT polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy. International Journal of Biochemistry 17, 471478.[ISI][Medline]
Israel, Y., Hurwitz, E., Niemela, O. and Arnon, R. (1986) Monoclonal and polyclonal antibodies against acetaldehyde-containing epitopes in acetaldehydeprotein adducts. Proceedings of the National Academy of Sciences of the USA 83, 79237927.[Abstract]
Koskinas, J., Kenna, J. G., Bird, G. L., Alexander, G. J. and Williams, R. (1992) Immunoglobulin A antibody to a 200-kilodalton cytosolic acetaldehyde adduct in alcoholic hepatitis. Gastroenterology 103, 18601867.[ISI][Medline]
Lieber, C. S. (1999) Microsomal ethanol-oxidizing system (MEOS): the first 30 years (19681998) a review. Alcoholism: Clinical and Experimental Research 23, 9911007.[ISI][Medline]
Lieber, C. S. and DeCarli, L. M. (1980) Alcoholic fatty liver. Model 30, Supplemental update. In Handbook: Animal Models of Disease, Capen, C. C., Hackel, D. B., Jones, J. C. and Migaki, G. eds. Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, DC.
Lin, R. C., Smith, R. S. and Lumeng, L. (1988) Detection of a protein acetaldehyde adduct in the liver of rats fed alcohol chronically. Journal of Clinical Investigation 81, 615619.[ISI][Medline]
Lin, R. C., Shahidi, S., Kelly, T. J., Lumeng, C. and Lumeng, L. (1993) Measurement of hemoglobinacetaldehyde adduct in alcoholic patients. Alcoholism: Clinical and Experimental Research 17, 669674.[ISI][Medline]
Moncada, C., Torres, V., Varghese, G., Albano, E. and Israel, Y. (1994) Ethanol-derived immunoreactive species formed by free radical mechanisms. Molecular Pharmacology 46, 786791.[Abstract]
Nanji, A. A., Tsukamoto, H. and French, S. W. (1989) Relationship between fatty liver and subsequent development of necrosis, inflammation and fibrosis in experimental alcoholic liver disease. Experimental and Molecular Pathology 51, 141148.[ISI][Medline]
Nicholls, R. M., Fowles, L. F., Worrall, S., de Jersey, J. and Wilce, P. A. (1994) Distribution and turnover of acetaldehyde-modified proteins in liver and blood of ethanol-fed rats. Alcohol and Alcoholism 29, 149157.[Abstract]
Niemela, O., Klajner, F., Orrego, H., Vidins, E., Blendis, L. and Israel, Y. (1987) Antibodies against acetaldehyde-modified protein epitopes in human alcoholics. Hepatology 7, 12101214.[ISI][Medline]
Niemela, O., Israel, Y., Mizoi, Y., Fukunaga, T. and Eriksson, C. J. P. (1990) Hemoglobin-acetaldehyde adducts in human volunteers following acute ethanol ingestion. Alcoholism: Clinical and Experimental Research 14, 838841.[ISI][Medline]
Niemela, O., Parkkila, S., Yla Herttuala, S., Halsted, C., Witztum, J. L., Lanca, A. and Israel, Y. (1994) Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation. Laboratory Investigation 70, 537546.[ISI][Medline]
Niemela, O., Parkkila, S., Yla Herttuala, S., Villanueva, J., Ruebner, B. and Halsted, C. H. (1995) Sequential acetaldehyde production, lipid peroxidation, and fibrogenesis in micropig model of alcohol-induced liver disease. Hepatology 22, 12081214.[ISI][Medline]
Pennington, H. L., Hall, P. M., Wilce, P. A. and Worrall, S. (1997) Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis. Journal of Gastroenterology and Hepatology 12, 305313.[ISI][Medline]
Sillanaukee, P., Seppa, K. and Koivula, T. (1991) Association of a haemoglobinacetaldehyde adduct with questionnaire results on heavy drinkers. Alcohol and Alcoholism 26, 519525.[ISI][Medline]
Tuma, D. J., Thiele, G. M., Xu, D., Klassen, L. W. and Sorrell, M. F. (1996) Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration. Hepatology 23, 872880.[ISI][Medline]
van de Vijver, L. P., Steyger, R., van Poppel, G., Boer, J. M., Kruijssen, D. A., Seidell, J. C. and Princen, H. M. (1996) Autoantibodies against MDA-LDL in subjects with severe and minor atherosclerosis and healthy population controls. Atherosclerosis 122, 245253.[ISI][Medline]
Worrall, S., de Jersey, J., Shanley, B. C. and Wilce, P. A. (1989) Ethanol induces the production of antibodies to acetaldehyde-modified epitopes in rats. Alcohol and Alcoholism 24, 217223.[ISI][Medline]
Worrall, S., de Jersey, J., Shanley, B. C. and Wilce, P. A. (1991a) Antibodies against acetaldehyde-modified epitopes: an elevated IgA response in alcoholics. European Journal of Clinical Investigation 21, 9095.[ISI][Medline]
Worrall, S., de Jersey, J., Shanley, B. C. and Wilce, P. A. (1991b) Detection of stable acetaldehyde-modified proteins in the livers of ethanol-fed rats. Alcohol and Alcoholism 26, 437444.[ISI][Medline]
Worrall, S., de Jersey, J. and Wilce, P. A. (1992) Liver damage in ethanol-fed rats injected with acetaldehyde-modified proteins. Alcoholism: Clinical and Experimental Research 16, 623A.
Worrall, S., de Jersey, J., Wilce, P. A., Seppa, K., Hurme, L. and Sillanaukee, P. (1996) Relationship between alcohol intake and immunoglobulin A immunoreactivity and acetaldehyde-modified bovine serum albumin. Alcoholism: Clinical and Experimental Research 30, 836840.