Detection of Reduced Acetaldehyde Protein Adducts Using a Unique Monoclonal Antibody

Acetaldehyde (AA), the major product of alcohol metabolism, has been shown to bind to proteins in vivo and form chemical adducts. These AA-protein adducts have been shown to alter protein structure and function and may result in tissue damage. Recent reports have shown that polyclonal antibodies can be produced that recognize proteins modified in vitro with AA in the presence of sodium cyanoborohydride (NaCNBH₃), a strong reducing (R) agent. Antibodies prepared in this way have been shown to recognize proteins in the livers of rats fed alcohol chronically. Because multiple AA-protein adducts can be recognized by polyclonal antisera, and a variety of adducts may be formed in vitro or in vivo, this study was designed to develop monoclonal antibodies specific for proteins modified by AA. In addition, adducts formed under R conditions are probably chemically different than those formed under nonreducing (NR) conditions, and monoclonal antibodies may provide the specificity required to distinguish these chemical differences. Balb/c mice were immunized with bovine brain tubulin that was modified by treatment with 5 mM AA for 7 days under NR conditions. Sera from immunized animals were tested for antibody activity to the immunogen (protein-NR) and for cross-reactivity to protein-R and unmodified protein. Although the highest serum antibody titers were seen toward the NR adduct, antibodies to the R adduct were also detected. This activity difference was independent of the carrier protein, because NR and R bovine serum albumin, keyhole limpet hemocyanin, and actin also gave similar results when used as the adducted protein. Surprisingly, all the monoclonal antibodies (RT1.1, RT1.2, RT1.3, and RT1.4) produced by hybridomas generated from spleen cells from NR-tubulin immunized mice recognized the R and not the NR adduct. One of these hybridomas (RT1.1) produces an lgG2b antibody that reacts with all tested proteins that have been modified with AA under chemical R conditions. Because of its monoclonal specificity, this antibody may be useful in probing for the presence of R AA-protein adducts made both in vitro and in vivo.     

Urinary Markers of Chronic Excessive Ethanol Consumption

A gas chromatographic mass spectrometric procedure is described for the measurement of free ethanol and conjugates of ethanol and acetaldehyde. It was demonstrated that conjugates of ethanol and acetaldehyde were present in urine of alcoholics. Urinary levels of free ethanol and the conjugates in abstaining alcoholics were higher than those of social drinkers. On the average, alcoholics excreted equal amounts of free ethanol and ethanol conjugates while controls eliminated three parts of ethanol conjugates to one part of free ethanol. After 14 days of abstinence, 16/23 (70%) of alcoholics had levels of free ethanol and acetaldehyde conjugates higher than the mean +2 SD of social drinkers for these substances. When the molar ratio of ethanol conjugates/free ethanol was considered, five more subjects who were alcoholics could be classified as being alcoholics. There was one false positive among 46 control subjects.     

The ethanol stimulus in rats with differing ethanol preferences

Alcohol-accepting (AA) and alcohol-nonaccepting (ANA) rats (Alko, Finland) were tested for their ability to master a shock-motivated (0.6 mA scrambled AC current) T-maze discrimination in which IP injections of ethanol (1.0 g/kg, 10% w/v in saline, 20-min latency) and, on alternate days, equivolume saline were employed as a discriminative stimuli signalling the safety of right or left goal compartments. ANA rats reached the criterion level of performance (eight of ten correct first-trial responses) more quickly than did AA rats (12.3±2.3 versus 31.4±7.7 sessions, P<0.01) and also maintained a superior level of performance throughout the course of the experiment, suggesting that ethanol may have been a more salient cue for ANA rats than for AA rats. Injections of acetaldehyde (0.1–0.25 g/kg, 1.0–2.5% w/v in saline) produced ethanol-appropriate responding to a greater extent in ANA rats than in AA rats, indicating that the actions of acetaldehyde may contribute importantly to the stimulus condition produced by the injection of ethanol in ANA rats. Sodium pentobarbital (10.0 g/kg) was equally effective in mimicking the action of ethanol in both AA and ANA rats. AA and ANA rats did not differ significantly in the impairment of motor performance produced by a range of ethanol doses, suggesting that differences in stimuli related to motor impairment do not contribute to the differences observed in the cue value of ethanol for AA and ANA rats.     

Ethanol Patch Test: A Simple Method for Identifying the Effectiveness of Cyanamide in Alcoholics

Background: To identify the pharmacological effectiveness of cyanamide, 144 alcoholics treated with cyanamide were subjected to a test that used an acetaldehyde dehydrogenase (ALDH) inhibitor, the ethanol patch test, which is considered to be a good indicator of ALDH2 phenotype. Methods: We placed 100 μl of 70% ethanol on a lint pad and, as a control, placed the same volume of distilled water on a second pad. The ethanol patch test was performed on 144 alcoholics more than 2 weeks after abstinence from alcohol before and after treatment with cyanamide for 1 week. The dose of cyanamide was increased up to 150 mg until the patch test yielded a positive result. Results: In the ethanol patch test, 36 alcoholics (25.0%) gave a positive result before treatment with cyanamide and might have been ALDH2¹/2² heterozygotes. Among 108 alcoholics who were not positive, the distribution of the cyanamide dose that yielded a positive ethanol patch test result was 30 mg in 42 cases (38.9%), 50 mg in 33 cases (30.6%), 70 mg in 5 cases (4.6%), 100 mg in 6 cases (5.6%), and 150 mg in 2 cases (1.9%). Prevalence of liver cirrhosis was significantly higher in alcoholics who showed a positive ethanol patch test result at doses of less than 50 mg cyanamide than those at doses more than 70 mg (p = 0.029). The prevalence of adverse effects was significantly higher in alcoholics who showed a positive ethanol patch test result at doses of more than 70 mg than at doses of less than 50 mg cyanamide (p = 0.002). Conclusions: The ethanol patch test is a useful method for identifying pharmacological effectiveness of cyanamide and may reduce the prevalence of side effects in cyanamide-treated alcoholics.     

Fine organic particle, formaldehyde, acetaldehyde concentrations under and after the influence of fire activity in the atmosphere of Riverside, California

Concentrations of gas-phase organic carbons (formaldehyde (HCHO) and acetaldehyde (CH₃CHO)) and fine particle-phase carbons (organic carbon (OC) and elemental carbon (EC)) were measured under and after the influence of fire activity in southern California. The measurement was conducted after the start of the wildfire activities from October 27 through November 6, 2003 at a site in Riverside, southern California. Under the influence of the fire activities, HCHO, CH₃CHO and EC concentrations were found to be over two times as high as those after the fire activities ended. The total lifetime cancer risk estimated by HCHO and CH₃CHO concentrations measured was significantly higher under the influence of the wildfire activities than that after the activities ended. OC showed a larger difference in concentrations between the two event periods as compared with gas-phase organic carbons and EC. OC/EC ratios ranged from 3.7 to 12.5 during the study period with the highest OC/EC ratio observed when the study area was under the influence of the fire activities. Correlation analysis and multiple linear regressions between OC/EC concentrations and visibility were performed. It was found that the visibility was even worse under the influence of fire activity as compared to the period after fire activity ended. EC was a stronger contributor to the visibility reduction compared to OC. The influence of air mass pathways on HCHO, CH₃CHO, OC, and EC concentrations during the wildfire activities was addressed using a backward trajectory model developed by NOAA.     

顶空气相色谱法测定注射用氨苄西林钠舒巴坦钠中残留溶剂

目的：建立一种气相色谱法同时测定注射用氨苄西林钠舒巴坦钠中有机溶剂乙醛、乙醇、异丙醇、异丁醛含量的方法。方法：采用外标法顶空自动进样,在非极性弹性毛细管柱上进行分离,效果良好。结果：方法重现性好,定量准确,便于操作。结论：平均回收率乙醛99.3%-102.5%;乙醇100.0%-102.5%;异丙醇99.2%-102.3%;异丁醛100.3%-102.0%。变异系数乙醛2.2%;乙醇2.2%;异丙醇1.5%;异丁醛1.9%。     

Ethanol-induced CTA mediated by acetaldehyde through central catecholamine activity

The possible involvement of catecholamines (CA) in the mediation of acetaldehyde's conditioned taste aversion (CTA) was examined by testing the effects of alpha-methyl-para-tyrosine (AMPT, a tyrosine hydroxylase inhibitor) on the CTAs produced by acetaldehyde. AMPT blocked the acquisition of the CTA normally produced by a low dose of acetaldehyde (0.2 g/kg), but had no significant effect on CTA produced by a high dose of acetaldehyde (0.3 g/kg). In a second study, acetaldehyde's role in the CTA produced by ethanol was investigated using the pre-exposure conditioned taste aversion paradigm. Pre-exposure to acetaldehyde (both doses) blocked the ethanol CTAs but when pre-exposure with acetaldehyde was coupled with AMPT, only the larger dose of acetaldehyde blocked the ethanol aversion. These results suggest that while the CTA to the low dose of acetaldehyde may be primarily central and catecholamine-mediated, the mechanism underlying the high dose CTA is probably peripheral and emetic in nature. These findings support the conclusion that acetaldehyde may be mediating many of the actions of ethanol.     

Effects of endogenous acetaldehyde production by disulfiram and ethanol feeding on rat pancreas

Exogenous acetaldehyde infusion can induce pancreatitis-like injury of the pancreas in some isolated pancreas models, whereas in vivo such treatment has failed to induce pancreatitis. In vivo exogenous acetaldehyde may not be effective because it is rapidly metabolized. The aim of this study was to investigate whether endogenous acetaldehyde accumulates in the pancreas after ethanol feeding when acetaldehyde metabolism is blocked by disulfiram, and whether this treatment can induce pancreatitis-like injury in the rat. The liver was studied for comparison. In part I of the experiment, adult male Wistar rats were given water (n = 24), ethanol (n = 24), disulfiram (n = 24), and ethanol plus disulfiram for 1 week (n = 24) or 3 weeks (n = 24) and for 3 weeks with (n = 6) and without (n = 6) hypovolemia. In part II of the experiment, rats were given water (n = 6), ethanol (n = 6), and high-dose disulfiram (n = 6) and ethanol plus high-dose disulfiram (n = 6). Ethanol and acetaldehyde concentrations in blood, liver, and pancreas were measured. Animal behavior was monitored, and weight changes, plasma amylase activity, water content, and histomorphology of the pancreas and liver were studied without knowing the group. No increases in plasma amylase activity and no histomorphologic changes in the pancreas were observed under light or electron microscopy in part I of the experiment. In part II, treatment with ethanol induced acetaldehyde accumulation in the liver (33.6 ±2.6 (μmol/L), but to a lesser degree in the blood (9.6 ±1.6 μmol/L) and pancreas (5.0 ±1.2 μ,mol/L). Ethanol plus disulfiram induced marked accumulation of acetaldehyde in the liver (83.2 ±15.9 μmol/L), blood (280.0 ±47.4 μnol/L), and pancreas (43.6 ±4.7 μ,mol/L). When tissue acetaldehyde levels reached 30 to 40 μ,mol/L, we found a decrease in zymogen granules along with formation of small intracytoplasmic vacuolizations in the acinar cells and accumulation of lipid droplets in the hepatocytes, whereas physiologic signs of pancreatitis (hyperatnylasemia, edema) or increases in liver enzymes did not develop. High levels of acetaldehyde accumulate in the liver and pancreas w ith the treatment described. Although this was accompanied by lipid degeneration of the hepatocytes and some subcellular changes in the acinar cells, physiologic signs of pancreatitis did not develop. Thus acetaldehyde accumulation alone, or in combination with hypovolemia, is not responsible for the induction of acute pancreatitis. Supported by grants from the Paulo Foundation and the Medical Research Fund of Tampere University Hospital.     

A correlation between voluntary ethanol consumption and brain catalase activity in the rat

The relationship between voluntary ethanol consumption and brain catalase activity was investigated in male Long Evans rats. In the first study, rats which were voluntarily consuming alcohol or water for 25 days were sacrificed by decapitation immediately (group A) or 15 days (group B) following withdrawal of alcohol and their brains analysed for catalase activity. Mean brain catalase activity did not differ among the two groups of rats exposed to ethanol and the ones exposed to water only. Furthermore, there were significant positive correlations between individual voluntary ethanol intake and catalase activity in both groups, (group A:r = .69, p less than or equal to 0.05; group B:r = .54, p less than or equal to 0.05). In the second study, rats were forced to drink high levels of ethanol presented as the only source of fluid for 25 days. Rats were sacrificed and brain, liver, muscle and heart tissue were extracted and analysed for catalase activity. There were no differences in mean brain catalase activity between water and forced ethanol drinking rats indicating that the enzyme was not inducible by high volume ethanol consumption. The results suggest that inherent differences in brain catalase activity may be one of the factors in determining an animal's propensity to voluntarily consume ethanol.