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To estimate ethanol consumption by university students attending a student health facility, a biochemical marker of alcohol intake [whole blood associated acetaldehyde (WBAA)] was quantified by fluorimetric HPLC. Over a two year period we studied blood samples, coded by date and sex, from 645 females and 332 males, and compared the results to previously established reference ranges for teetotalers by sex. Men had higher absolute values for WBAA than women (9.9 versus 9.5 microM in the present study). However, significantly greater numbers of women (74%) than men (44%) had WBAA levels above the 99th percentile for teetotalers. Variations occurred during the academic year, with significant elevations occurring in the late fall and winter months. Testing of WBAA levels in a student health service may be important especially for women to facilitate counseling on the dangers of alcohol abuse.

Background: About half of certain Asians have a deficiency of the low-Km aldehyde dehydrogenase (ALDH2) isoenzyme. This deficiency results from inheritance of a mutant ALDH2*2 allele., Objective: To determine whether Asian Americans with ALDH2*2 alleles differ from Asian Americans without this mutation in terms of blood levels of alcohol and acetaldehyde after ingestion of a moderate amount of alcohol., Design: Double-blind, crossover study., Setting: Private research institute., Participants: 35 healthy Asian-American men. Three men who became ill after alcohol ingestion and one who had outlying data were excluded., Intervention: Alcoholic beverage, containing 0.56 g of alcohol per kg of body weight, or placebo beverage, containing 3 mL of alcohol, given orally on separate occasions., Measurements: Blood levels of alcohol and acetaldehyde measured before and several times after ingestion of the alcoholic or placebo beverage., Results: Participants with ALDH2*2 alleles had significantly higher blood acetaldehyde levels after ingesting alcoholic and placebo beverages than did participants with ALDH2*1 alleles, despite similar blood alcohol concentrations., Conclusions: Blood acetaldehyde levels rather than blood alcohol concentration may mediate enhanced alcohol sensitivity among Asians with ALDH2*2 alleles.

Acetaldehyde is the first oxidation product of ethanol in vivo. Our earlier work showed that with sufficient acetaldehyde, five of the six possible sites of the peptide pentalysine were modified as a Schiff base (Braun KP, et al: J Biol Chem 270:11263-11266, 1995). However, we were unable to deduce unequivocally which site was unmodified. Lysine residues, as well as the amine terminal valine residues, in hemoglobin have been implicated as target structures for acetaldehyde adducts resulting from ethanol consumption. Hemoglobin adducts of acetaldehyde have been used clinically as a marker of ethanol consumption, but the chemical nature of these adducts remains undefined. As part of our continuing structural characterization of acetaldehyde-protein adduct formation, we studied the peptides Val-His-Leu-Thr-Pro and Val-His-Leu-Thr-Pro-Val-Glu-Lys, from the amine terminus of the beta-globin chain of hemoglobin, in vitro. Both peptides have at least one potential site for adduct formation. In the octapeptide, the N-terminal amine group of Val as well as the epsilon-amine group of the lysine sidechain can potentially be modified by acetaldehyde. We used mass spectrometry, carbon-13 nuclear magnetic resonance, and Raman spectroscopy and characterized stable Schiff base acetaldehyde adducts of these two peptides at both reactive sites. The identification of stable Schiff base adducts with the N-terminal peptides of the beta-chain of hemoglobin as well as with epsilon-amino groups of lysine provides another possible means of monitoring ethanol consumption. The functional implications of these stable Schiff bases remains undefined.

Stable hemoglobin-acetaldehyde adducts present in hemoglobin fractions separated by polyaspartic acid cation exchange chromatography were quantified by fluorimetric HPLC. The fluorescent species eluted from the HPLC was confirmed by mass spectrometry to be consistent with the expected product from reaction of acetaldehyde, 1,3-cyclohexanedione (CHD), and ammonium ion. Hemolysate (2.2 mM hemoglobin) was incubated in equivalent volumes of either phosphate-buffered saline or 5 mM acetaldehyde at 37 degrees C for 30 min and washed three times with H2O to remove free acetaldehyde and labile adducts before the injection of 14.7 mg hemoglobin onto the cation exchange column. Fluorimetric HPLC analysis of hemolysate samples either with or without in vitro reaction with acetaldehyde revealed that most acetaldehyde resides in the hemoglobin A0 fraction. The reaction with acetaldehyde in vitro resulted in a significant increase in fast-eluting minor hemoglobin species on cation exchange chromatography concomitant with increased acetaldehyde in the HbA1a+b, HbA1c, and HbA1-AcH fractions. We report three new cation exchange chromatographic peaks after reaction with acetaldehyde: HbA1-AcH-3, HbA1c-1, and HbA0-1. Each new peak was found to associate with a significant quantity of CHD-reactive acetaldehyde. These experiments provide additional evidence that stable adducts form between acetaldehyde and hemoglobin and that these adducts occur in multiple hemoglobin species separated by cation exchange chromatography. Further characterization and structural assignment of these species are warranted in view of their potential utility as markers for ethanol intake.

Acetaldehyde is the first oxidation product of ethanol in vivo. Lysine residues in proteins such as hemoglobin have been implicated as target structures for acetaldehyde adducts resulting from ethanol consumption. Although the presence of both stable and unstable acetaldehyde-hemoglobin adducts has been established, the structural characterization of the adducts has received relatively little attention. As a model for such adduct formation, we studied the peptide pentalysine in vitro. Pentalysine has several potential sites for adduct formation. The amino-terminal amine group as well as the epsilon-amine groups of each lysine side chain can serve as potential sites for modification by acetaldehyde. Mass spectrometry, nuclear magnetic resonance, and Raman spectroscopy were employed to demonstrate that acetaldehyde forms a stable linkage to lysine amine groups via a Schiff base.

Variable immunobiological changes occur with alcohol consumption. Previous studies have shown that acetaldehyde forms stable adducts with serum proteins, including albumin. These adducts are elevated in persons and animals consuming ethanol. We examined the effect of serum protein-acetaldehyde adducts formed with fetal bovine serum (FBS) on concanavalin A-stimulated murine splenocytes. Interleukin-2 (IL-2) secretion and IL-2 receptor (IL-2R) expression were determined as a function of the effect of the acetaldehyde-protein adduct(s). FBS was incubated with acetaldehyde (500, 100, 50, 25, 10, and 0 microM) for 1 hr at 37 degrees C. Excess acetaldehyde was removed by ultrafiltration using a 500 molecular weight cut-off membrane in 3 volumes. Free as well as bound acetaldehyde was quantified using fluorigenic HPLC before and after incubation. Recovered acetaldehyde correlated with the amount added (r2 = 0.996). Splenocytes were cultured for 48 hr in complete medium containing 5% acetaldehyde-treated and 5% untreated FBS with 4 micrograms/ml concanavalin A. Although cell viability was unchanged, acetaldehyde-treated FBS mixed with native FBS decreased IL-2 secretion in a dose-dependent manner. The percentage of cells expressing IL-2R was reduced only at the highest acetaldehyde-FBS dose. Therefore, immunological effects ascribed to ethanol may result in part from the toxic properties of acetaldehyde-protein adducts on IL-2 secretion.

We noted a rise in acetaldehyde levels in clinical samples of venous whole blood containing ethanol that did not occur in samples from teetotalers. Experiments were performed to define the mechanism involved in acetaldehyde production. The addition of 0.10% ethanol to whole blood produced an immediate increase in acetaldehyde due to acetaldehyde in the stock solution followed by a subsequent increase that became statistically significant by 48 hr. Separation of blood into components documented that the increase in acetaldehyde was associated with the red cell but not plasma fraction. Incubation of isolated hemoglobin with ethanol produced a rise in acetaldehyde levels. Incubation of oxygenated whole blood with ethanol produced a linear increase in acetaldehyde, whereas nitrogen-exposed blood produced no increase. The rise of acetaldehyde in the presence of ethanol was dependent on the concentration of oxygenated hemoglobin A0. Addition of inhibitors of catalase, alcohol dehydrogenase, and glycolytic enzymes (aminotriazole, azide, pyrazole, sodium fluoride, sodium citrate, and iodoacetate) did not inhibit the rise of acetaldehyde, but addition of the hemoglobin ligand cyanide abolished the rise in acetaldehyde. Kinetic analysis with oxygenated whole blood plus inhibitors revealed a Km of 2.5 mM and Vmax of 1.42 microM/min. We conclude that oxyhemoglobin contributes to the metabolism of ethanol to acetaldehyde. These findings may explain in part the high levels of acetaldehyde found in red cells compared with plasma. The results also have implications for the optimum storage of blood samples for acetaldehyde analysis.

We hypothesized that while moderate drinking is associated with increasing levels of high density lipoprotein (HDL) levels, excessive drinking of ethanol might, in fact, be associated with lower HDL levels and by implication increased cardiovascular risk. We therefore performed analyses of whole blood-associated acetaldehyde (WBAA) as a measure of drinking behavior and HDL on blood samples from 2780 individuals applying for life insurance. Whole blood-associated acetaldehyde correlated positively with HDL in the entire sample set throughout the range of values (R = 0.101, p = 0.0001). The relationship held for females (N = 477, p = 0.036) but was stronger for males (N = 2277, p = 0.0001). We conclude that ethanol consumption correlates positively with HDL for both males and females and that the relationship persists through higher ranges of ethanol consumption.

We measured whole blood-associated acetaldehyde (WBAA) levels in 225 teetotalers (123 females, 102 males) between the ages of 18 and 86 years. Values were normally distributed, but mean values for females were significantly lower than for males (7.6 +/- 0.6 vs. 7.9 +/- 0.7 microM, p < 0.0001). There was a significant positive correlation with age for the entire group (r2 = 0.149, p = 0.001) and for both sexes. The correlation with WBAA and age was stronger for females. Significant but lesser positive correlations were found between WBAA and other variables that increase with age, including glucose, fructosamine, cholesterol, alkaline phosphatase, serum glutamate pyruvate transaminase (SGPT), gamma glutamyl transpeptidase (GGT), and creatinine in the entire data set. Partial r analyses indicated that the correlations were mediated through the primary association of WBAA and age. We conclude that in individuals who do not consume ethanol there are significant sex differences in whole blood acetaldehyde and that the values increase throughout life.

We evaluated three markers of ethanol intake [whole blood associated acetaldehyde (WBAA), serum beta-hexosaminidase, and gamma-glutamyl transpeptidase (GGT)] in four groups of subjects: teetotalers (n = 104), random insurance applicants or "normals" (n = 1,010), subjects enrolling in an alcohol treatment program or "alcoholics" (n = 31), and subjects attending outpatient drug/alcohol treatment follow-up clinics (n = 128). Significant differences (p < 0.004 for each assay and each comparison) were found in the mean values between teetotalers and normals and normals and alcoholics. Male teetotalers and normals had significantly (p < 0.002) higher levels of WBAA than females of the same group. Male normals had significantly higher levels of GGT than females (p < 0.001). GGT increased with age in the normal population into the fifth decade and decreased thereafter. WBAA was the most sensitive assay with 97% of alcoholics having values above the 99th percentile for the teetotaler population (vs. 66% for serum beta-hexosaminidase and 70% for GGT). None of the alcoholic subjects had values for all three assays below the 99th percentile for teetotalers compared with 21% of those in follow-up and 72% of normals. We conclude that WBAA appears to be the best of the three markers studied and that measurement of multiple markers for ethanol use appears clinically useful and incremental.

Five pairs of volunteers were studied to determine the effect of drinking ethanol on breath acetaldehyde levels. On a given study day, samples of breath were obtained for measurement of acetaldehyde and ethanol from both participants at t = -1 h, t = -0.5 h, and at t = 0 to obtain baseline values. The drinkers were then given ethanol (0.3 g/kg body weight), and the controls given an equal volume of tap water. Breath samples were then taken at 0.5, 1, 1.5, 2 h, and hourly until t = 6 h. The last sample taken was at t = 23.5 h. Acetaldehyde levels in breath were quantified with a fluorigenic high-performance liquid chromatographic assay. Blood ethanol was approximated using a breath analyzer. Acetaldehyde in breath rose 50-fold at the 0.5-h, time point and returned to levels not significantly different from baseline values by 3-4 h. The mean peak blood ethanol values reached 0.055%. The t 1/2 elimination for ethanol was 1.6 h, and that for acetaldehyde was 2.25 h. Elimination of both acetaldehyde and ethanol in breath were initially 0 order. A significant correlation (r = 0.74) was found between baseline breath acetaldehyde levels and peak acetaldehyde levels. We conclude that acetaldehyde resulting from ethanol intake rapidly partitions into breath. The correlation of baseline breath acetaldehyde values with peak values found after an ethanol challenge indicate that measurement of breath acetaldehyde may be useful in the identification of individual differences in ethanol metabolism.

A study was undertaken in 16 male C57BL mice to evaluate the effect of ethanol intake via the drinking water (10% v/v) on urinary-associated acetaldehyde. Eight received ethanol and 8 served as controls. Urinary-associated acetaldehyde (UAA) was measured using a fluorigenic high performance chromatographic assay. Ethanol consumption did not impair growth over the two weeks of the experiment. Following administration of ethanol, UAA increased and remained significantly elevated over levels seen in controls until ethanol administration ceased (11.3 +/- 3.6 SEM microM for ethanol-consuming mice vs. 0.69 +/- 0.33 microM for controls). Ethanol in the urine was found to interfere with the assay for acetaldehyde. However, following cessation of ethanol, acetaldehyde in urine was found to be significantly elevated in urine at 24 hours, after ethanol levels were no longer detectable. In conclusion, measurement of urinary-associated acetaldehyde discriminates ethanol-consuming from nonconsuming mice during ethanol ingestion as well as 24 hours following cessation of ethanol when ethanol levels are no longer detectable in urine. Thus measurement of urinary acetaldehyde may be a useful marker for monitoring ethanol intake.

Twenty seven subjects with Type II diabetes mellitus underwent analysis of the Self-Administered Alcoholism Screening Test (SAAST) and hemoglobin associated acetaldehyde levels (HbAA). The SAAST scores and HbAA levels correlated with one another (r = .48, p = 0.009). No correlation between glycated hemoglobin levels (GHb) and HbAA levels or SAAST was found. Glucose incubation of whole blood led to an increase in GHb but no change in HbAA. We conclude that HbAA and SAAST correlate with each other when measured in patients with diabetes. Therefore each test appears clinically useful in quantifying alcohol consumption in individuals with Type II diabetes mellitus.

A study was undertaken in C57BL mice to evaluate the effect of gender on whole blood associated acetaldehyde following exposure to ethanol in the drinking water (10% v/v). Whole blood associated acetaldehyde (WBAA) was measured from capillary blood samples using a fluorigenic high performance chromatographic assay on days 0, 7, 15 and 27. Ethanol consumption did not impair growth of either male or female mice when compared to controls. Following administration of ethanol, WBAA increased in both male and female mice but marked gender differences were apparent. Female mice consumed more fluid relative to body weight than males (155 +/- 27 S.D. vs. 124 +/- 19 ml/kg/day, p less than 0.001), but had lower mean WBAA levels during the four weeks of ethanol administration (137 +/- 37 vs. 318 +/- 66 nmol/g hemoglobin, p less than 0.001). WBAA levels in male mice were stable over the course of the experiment. Female mice were found to have peak WBAA levels on day seven after which time levels decreased significantly. These experiments emphasize gender differences in ethanol metabolism as well as the need to establish norms based on gender for assays of ethanol consumption which use acetaldehyde adducts with blood proteins.

Blood samples were obtained from miniature swine maintained on 0, 2, or 6 g/kg/24 hr ethanol for 8 months (N = 6 in each group). Samples from drinking pigs were taken after 8 hr of ethanol abstinence and all were coded and sent for "blinded" analysis. A fluorigenic high performance liquid chromatographic assay was used to quantify whole blood-associated acetaldehyde, hemoglobin-associated acetaldehyde, plasma-associated acetaldehyde, platelet-associated acetaldehyde, and lymphocyte-associated acetaldehyde. Detectable levels of acetaldehyde were found in each sample in both drinking and nondrinking pigs. Analysis of whole blood-associated acetaldehyde was most discriminatory in distinguishing nondrinking from drinking pigs (mean 21.4 +/- 1.0 microM for nondrinkers vs. 24.6 +/- 1.5 SD for the group consuming 2 g/kg ethanol, p = 0.001). Measurements of hemoglobin-associated acetaldehyde normalized to protein concentration (250 +/- 47 nmoles/g vs. 203 +/- 33 SD, p less than 0.05 drinking vs. nondrinking pigs) and platelet-associated acetaldehyde (0.46 0.34 vs. 0.15 +/- 0.16 nmoles/3 x 10(8) platelets, p = 0.05 drinking vs. nondrinking pigs) were also useful in discriminating drinking from nondrinking animals. Analysis of plasma-associated acetaldehyde and lymphocyte-associated acetaldehyde were not useful as markers of ethanol consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty subjects underwent analysis of the Self-Administered Alcoholism Screening Test (SAAST) and hemoglobin-associated acetaldehyde levels (HbAA). Eleven alcoholic individuals reporting for treatment, 10 self-defined "social drinkers" and 10 self-defined "teetotallers" who had consumed less than 6 drinks of ethanol per year ever in their lifetime and had not knowingly had ethanol for 6 months were included in the study. The SAAST scores and HbAA levels correlated with one another (r = .55, p = 0.002). For alcoholic individuals, the mean HbAA level was 180 +/- 64 nm/g Hb and the mean SAAST score was 17.3 +/- 6.3. Both analyses could distinguish the alcoholic from the teetotaller group (p = 0.03 for HbAA and p less than 0.001 for the SAAST) and from the group of social drinkers (p = 0.04 for HbAA and p less than 0.001 for the SAAST). The difference in HbAA levels between the social drinkers and teetotallers was not significant (132 +/- 25 vs. 129 +/- 15 nm/g Hb). After modification of the SAAST analysis for teetotallers, SAAST scores were significantly different between social drinkers and teetotallers (3.5 +/- 2.2 vs. 1.1 +/- 1.2, p = 0.009). We conclude that HbAA and SAAST correlate with each other may be clinically useful in distinguishing alcoholic from nonalcoholic individuals. While the SAAST appears to be the more sensitive test, it requires modification in the case of teetotallers.

Six pairs (1 habitual smoker and 1 nonsmoking control) of volunteers were studied to determine the effect of smoking tobacco on breath and whole blood acetaldehyde levels. On a given study day, samples of blood and breath were obtained from both participants at -0.25, 0, 0.25, 0.50, 0.75, 1.5, 2.5, and 3.5 hour time points. The smoking volunteer was told to smoke 1-3 cigarettes between the 0 and 0.25 hour time points. Acetaldehyde levels in breath and whole blood were quantified with a fluorigenic high performance liquid chromatographic assay. Acetaldehyde in breath rose six-fold in smokers at the 0.25 hour time point and returned to levels not significantly different from baseline values found in smokers or nonsmokers by 0.50 hr. Whole blood-associated acetaldehyde measurements remained unchanged in smokers throughout the experiment and were not different from nonsmokers. In conclusion, while smoking produces appreciable levels of acetaldehyde in expired air, the partitioning of acetaldehyde associated with smoking to blood or blood proteins appears to be below the level of detection of the assay employed (picomolar). Smoking of tobacco products may not interfere with assays designed to quantify ethanol intake by measuring acetaldehyde adducts with blood proteins.

The effect of various concentrations of acetaldehyde (0, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 mM) on the relative rates of formation of hemoglobin acetaldehyde adducts detected in fractions eluted from cation exchange high-pressure liquid chromatography (HPLC) was investigated. When the hemoglobin and acetaldehyde mixtures were incubated at 37 degrees C for various time intervals up to 24 hr, increased amounts of HbA1c could be observed after 2 hr incubation with 1 mM or greater concentrations of acetaldehyde, or after 4 hr incubation with at least 0.5 mM acetaldehyde. An increase in the HbA1a + b fraction was not observed with 4 hr incubation time until the acetaldehyde level reached 1 mM. The HPLC method detected no difference in minor hemoglobins from alcoholic and normal subjects. Incubation of red blood cells at 37 degrees C for 1 hr with six consecutive pulses of 0.05 mM [14C]acetaldehyde showed no differences in the amounts of minor hemoglobins determined chromatographically at various pulse intervals. However, the measure of the 14C-label incorporation into hemoglobin showed that adducts eluting in the HbA1a+b fraction were formed at a faster rate than those eluting in the HbA1c or HbA0 fraction, respectively. The specific activities of the HbA1a+b fractions at 2, 4, and 6 pulses were 34, 128, and 949 cpm/mg hemoglobin; those of the HbA1c fraction were 15, 58, and 174 cpm/mg hemoglobin. This evidence of modification of hemoglobin by physiological levels of acetaldehyde from 14C-label incorporation suggests that an assay more sensitive than chromatographic separation of adducts might be clinically useful in detecting alcoholism or monitoring alcohol detoxification programs.

Clinical studies on the minor hemoglobins (hemoglobin A1a-c) have suggested that a novel adduct may form in people abusing alcohol. Such patients were found to have an elevated concentration of minor hemoglobins, but normal or subnormal amounts of glycosylated hemoglobin (hemoglobin A1c) as determined by radioimmunoassay, Acetaldehyde, a reactive metabolite of ethanol, was postulated to form adducts with hemoglobin A that change its chromatographic properties. At physiological concentrations, acetaldehyde was found to form adducts with hemoglobin that were stable to extensive dialysis for several days. The amount of hemoglobin adducts formed were a function of the concentration and number of exposures to acetaldehyde. The reaction of acetaldehyde with hemoglobin A produced chromatographic variants, some of which migrated in the hemoglobin A1a-c region. Analysis of stable acetaldehyde-hemoglobin adducts demonstrated that valine, lysine, and tyrosine residues of globin were sites of reaction. The acetaldehyde-modified amino acid residues appear to exist in interconvertible conformations, only one of which is reducible by sodium borohydride. The amount of these adducts was found to be significantly elevated in hemoglobin from alcoholics as compared with normal volunteers.

Acetaldehyde has been found to form adducts with human hemoglobin, a portion of which (15-25%) are stable to dialysis. The reaction is nonenzymatic and occurs with purified hemoglobin A. As determined by incorporation of radioactivity, the amount of stable hemoglobin adducts formed is proportional to the amount of acetaldehyde to which hemoglobin is exposed, or to the number of intermittent pulses. Reaction of hemoglobin A with 3 to 30 mM acetaldehyde significantly increases the amount of minor hemoglobins recovered following chromatography on cation exchange resin. Acetaldehyde adducts with hemoglobin involve primarily the beta chain and at least three different amino acid residues (valine, lysine and tyrosine), and two modified residues (glucosyl-valine and glucosyl-lysine). The acetaldehyde appears to be reacting with the epsilon-amino group of lysine and alpha-amino group of valine probably through an initial Schiff's base reaction. The secondary amines of glycosylated valine or glycosylated lysine residues are also proposed to be at the sites of reaction with acetaldehyde. Disubstitution of amino groups is known to occur with hexose sugar (Schwartz, Gray 1977) and by analogy, acetaldehyde might also react with the secondary amine of glycosylated residues. Acetaldehyde adduct formation with tyrosine residues may involve either a nucleophilic attack by the third or fifth carbon of the phenolic ring, analogous to formaldehyde modification of proteins (Blass, Bizzini, Raynaud 1965) or alternatively by reaction with the hydroxyl group of tyrosine. Only a portion of the stable hemoglobin-acetaldehyde adducts which were stable to 24h of dialysis could be irreversibly fixed by sodium borohydride or cyanoborohydride reduction. A greater portion however appeared to be in a non-reducible (non-carbonyl, non-amino) form. Up to 45% of the dialysis stable adducts could be reduced by sodium cyanoborohydride and be hydrolyzed to amino acid adducts if given either sufficient reduction time (2-3 weeks at 22 degrees C) or increased temperature (1-2 days at 50 degrees C). An increase in reducible adduct recovery occurred in all 5 residues detected by amino acid analysis. This suggests that the adducts that are stable to acid hydrolysis form and reverse through a reducible (e.g. Schiff base) form but that most of the time the adducts occur in a non-reducible state. At present, assay systems are not available which can detect acetaldehyde adducts in the blood of humans consuming alcohol.(ABSTRACT TRUNCATED AT 400 WORDS)

A fluorigenic high performance liquid chromatographic (HPLC) assay for the determination of acetaldehyde in plasma and hemoglobin-associated acetaldehyde is described. The assay is based on the reaction of acetaldehyde with two molecules of 1,3-cyclohexanedione in the presence of ammonium ion to form a fluorescent species followed by reverse phase HPLC separation of specific aldehyde derived compounds. The assay is specific; and has sensitivity in the picomole range, with intraassay precision of less that 3.5% and interassay precision of less than 15%. The total run time is less than 6 minutes on HPLC. Hemoglobin-associated acetaldehyde levels were higher than the levels found in plasma. Endogenous levels of acetaldehyde in samples obtained from teetotalers were found to be 0.43 +/- 0.04 (S.D.) microM in plasma and 74.2 +/- 16.1 nmol/g protein as hemoglobin-associated acetaldehyde. The levels of both plasma acetaldehyde and hemoglobin-associated acetaldehyde were significantly higher in patients reporting to a center for alcohol treatment than the levels encountered in teetotalers.

Acetaldehyde forms stable adducts with proteins, and rapidly eluting hemoglobins on cation exchange chromatography have been found to be elevated in persons consuming excess alcohol. Incubation of hemoglobin hemolysate with 5 mM acetaldehyde at 37 degrees C for various time intervals resulted in linear increases in the amounts of hemoglobin (Hb)A1a+b and HbA1c fractions determined by cation exchange high pressure liquid chromatography. The rate of formation of the HbA1c fraction was significantly higher (p less than 0.001) than that of the HbA1a+b fraction. No increases in the amounts of minor hemoglobins were observed when hemoglobin was incubated with 0.05 mM acetaldehyde. Incubation of hemoglobin with 5 mM acetaldehyde followed by reduction with sodium borohydride (NaBH4) resulted in a significant increase in both HbA1a+b and HbA1c fractions. The rate of formation of the HbA1c fraction was again significantly faster than that of HbA1a+b. Dialysis of nonreduced acetaldehyde-modified hemoglobin had no effect on the amounts of the two minor hemoglobin fractions. Dialysis of NaBH4-reduced acetaldehyde-modified hemoglobin resulted in decreased amounts of the HbA1a+b fractions but no changes in the HbA1c fractions. Incubation with sodium cyanoborohydride led to minimal changes in chromatographic properties of hemoglobin. The clinical utility of acetaldehyde-modified hemoglobin eluting in the HbA1c fraction in the detection of excess alcohol consumption appears to be limited by the high concentration of acetaldehyde required. Furthermore, attempts to stabilize acetaldehyde-Schiff base adducts of hemoglobin with reducing agents must include appropriate controls, since the reductive step alone may lead to changes in the chromatographic properties of hemoglobin.

Thirty C57BI mice were randomized into two groups. Group 1 served as controls while Group 2 was given 10% V/V ethanol with the drinking water. Whole blood- associated acetaldehyde (WBAA) was measured on capillary blood samples using a fluorigenic high performance chromatographic assay. WBAA peaked at Day 2. A stable mean plateau of 263 +/- 71 SD with a range of 160-400 nmoles/g hemoglobin WBAA was found in the group consuming ethanol compared with 122 +/- 17 SD and a range of 88-150 nmoles/g hemoglobin for controls (p less than 0.001). When ethanol was discontinued, levels of WBAA declined and became similar to those of controls by 9 days following cessation of ethanol. The quantitative difference between ethanol-consuming and control animals and also the rapid rise of whole blood-associated acetaldehyde and the relatively slow decline following cessation of ethanol intake indicate that such a test might be a useful monitor of drinking behavior.

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.

A fluorigenic high performance liquid chromatographic (HPLC) method was used to determine plasma (PA) and hemoglobin-associated (HbAA) acetaldehyde levels following a pulse of 0.3 g/kg ethanol to volunteers from whom bloods were drawn serially for 8 hours on the clinical research unit. On discharge from the research unit, the volunteers were instructed to avoid ethanol for 28 days. The results were compared to previously published results in teetotalers and alcoholic individuals reporting for treatment at an inpatient detoxification facility. Following ethanol ingestion, the peak levels of ethanol and both plasma and hemoglobin-associated acetaldehyde were detected at the 30 min time point and plasma levels were less than those associated with hemoglobin (31 +/- 16 S.D. and 159 +/- 48 S.D. nmol/g respectively, p less than 0.001). PA and HbAA returned to baseline values following ethanol ingestion within 3.5 hours. PA returned to within 1 standard deviation of levels found in teetotalers by 5 days, whereas HbAA remained elevated for the 28 days of the study. These data provide evidence that measurement of PA and HbAA may provide a useful marker for relatively acute and chronic ethanol ingestion respectively.

The stabilities of 14C-acetaldehyde with various hemoglobin fractions (HbA1a + b, HbA1a, and HbA0) were examined by determining amounts of adducts remaining after dialysis at 4 degrees C for various time intervals. Significant differences were found in the stabilities of adducts formed with various hemoglobin fractions. Acetaldehyde adducts formed with HbA1a + b were more stable to dialysis than adducts formed with HbA0 or HbA1c (7-8% of total adducts formed with HbA1a + b were stable to dialysis, compared with 4-5% stable adducts formed with either HbA0 or HbA1c). While only 37-57% of the trichloroacetic acid (TCA) precipitable adducts from HbA0 or HbA1c samples were stable to dialysis, 72-75% of TCA precipitable adducts from HbA1a + b were retained after dialysis. Posttranslational modification of hemoglobin by phosphorylated glycolytic intermediates appears to alter the physical properties of hemoglobin following further modification with acetaldehyde. In view of the increased amounts of glycosylated proteins found in patients with diabetes, these observations may be relevant to the pathogenesis of the sequelae of diabetes and/or alcoholism and the influence of one chronic illness on the other.