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The High Drinking in the Dark mouse lines (HDID-1 and HDID-2) were selectively bred to achieve high blood ethanol concentrations (BECs) in the Drinking in the Dark (DID) task, a widely used model of binge-like intake of 20% ethanol. There are several components that differentiate DID from other animal models of ethanol intake: time of day of testing, length of ethanol access, single-bottle access, and individual housing. Here, we sought to determine how some of these individual factors contribute to the high ethanol intake observed in HDID mice. HDID-1, HDID-2, and non-selected HS/NPT mice were tested in a series of DID experiments where one of the following factors was manipulated: length of ethanol access, fluid choice, number of ethanol bottles, and housing condition. We observed that 1) HDID mice achieve intoxicating BECs in DID, even when they are group-housed; 2) HDID mice continue to show elevated ethanol intake relative to HS/NPT mice during an extended access session, but this is most apparent during the first 4 h of access; and 3) offering a water choice during DID prevents elevated intake in the HDID-1 mice, but not necessarily in HDID-2 mice. Together, these results suggest that the lack of choice in the DID paradigm, together with the length of ethanol access, are important factors contributing to elevated ethanol intake in the HDID mice. These results further suggest important differences between the HDID lines in response to procedural manipulations of housing condition and ethanol bottle number in the DID paradigm, highlighting the distinct characteristics that each of these lines possess, despite being selectively bred for the same phenotype.

Hemolysis, a common occurrence in blood collected for chemical analysis, has been reported to affect analytical test results for some analytes depending upon the material tested and the analytical technique employed. The potential for hemolysis to impact blood ethanol determinations using headspace gas chromatography of samples diluted with an internal standard was investigated. A sample of non-hemolyzed blood and a matched sample of hemolyzed blood were both analyzed thirty times for ethanol concentration using headspace gas chromatography. The mean ethanol concentration measured for the non-hemolyzed samples was 0.0639 g/dl. The mean ethanol concentration measured for the hemolyzed samples was 0.0642 g/dl. The calculated t value, 1.897, was less than the critical t value, 2.002, at a 0.05 level of significance. There was no measured statistical difference detected between the mean blood ethanol concentration determined for a hemolyzed whole blood sample and a non-hemolyzed whole blood sample.

Ethanol can have both an aversive and rewarding effect, which may have a significant relationship to its individual preference. So far, the reasons for the high and low ethanol preference in the WHP (Warsaw High Preferring) and WLP (Warsaw Low Preferring) lines have not been found. WHP rats spontaneously drink over 5 g/kg/day of ethanol, while WLP rats drink under 2 g/kg/day. The purpose of the work was to study the sensitivity of WHP and WLP rats to the aversive effects of ethanol at doses of 1.5 g/kg and 2.0 g/kg in the conditioned taste aversion (CTA) procedure. Lower doses (0.5 and 1.0 g/kg, i.p. [intraperitoneally]) were tested earlier and only 1.0 g/kg produced a slight aversion in WLP rats. The secondary aim was to check the additional potential factors (blood ethanol concentration, pain sensitivity, anxiety-related behavior, learning, and memory) that may constitute an important differentiating feature of the WHP and WLP lines. For this purpose, the following tests were conducted: blood ethanol concentration, novel object recognition (NOR), flinch-jump, hot-plate, and elevated plus maze (EPM). The 1.5 g/kg i.p. dose of ethanol caused the development of an aversion only in WLP rats and the aversion extinguished in the post-conditioning phase. The 2.0 g/kg i.p. dose of ethanol resulted in the development of an aversion in both the tested groups, with the aversion being maintained throughout the whole post-conditioning period only in the WLP rats. There were no differences between the lines in terms of the blood ethanol concentration and the EPM tests. WHP rats had a higher pain sensitivity compared to WLP rats in flinch-jump and hot-plate tests. WLP rats showed a shorter exploration time for both objects compared to WHP in the NOR test. In conclusion, WHP and WLP rats differ in sensitivity to the aversive effects of ethanol. This difference may partially explain their opposite ethanol preference.

Variability in the blood-breath ratio (BBR) of alcohol is important, because it relates a measurement of the blood-alcohol concentration (BAC) with the co-existing breath-alcohol concentration (BrAC). The BBR is also used to establish the statutory BrAC limit for driving from the existing statutory BAC limits in different countries. Thein-vivoBBR depends on a host of analytical, sampling and physiological factors, including subject demographics, time after end of drinking (rising or falling BAC), the nature of the blood draw (whether venous or arterial) and the subjects breathing pattern prior to exhalation into the breath analyzer. The results from a controlled drinking study involving healthy volunteers (85 men and 15 women) from three ethnic groups (Caucasians, Hispanics and African Americans) were used to evaluate various factors influencing the BBR. Ethanol in breath was determined with a quantitative infrared analyzer (Intoxilyzer 8000) and BAC was determined by headspace gas chromatography (HS-GC). The BAC and BrAC were highly correlated (r = 0.948) and the BBR in the post-absorptive state was 2 382 +/- 119 (mean +/- SD). The BBR did not depend on gender (female: 2 396 +/- 101 and male: 2 380 +/- 123,P > 0.05) nor on racial group (Caucasians 2 398 +/- 124, African Americans 2 344 +/- 119 and Hispanics 2 364 +/- 104,P > 0.05). The BBR was lower in subjects with higher breath- and body-temperatures (P < 0.05) and it also decreased with longer exhalation times into the breath-analyzer (P < 0.001). In the post-absorptive state, none of the 100 subjects had a BBR of less than 2 100:1.

Background Beyond yielding high blood ethanol (EtOH) concentrations (BECs), binge-drinking models allow examination of drinking patterns which may be associated with EtOH's rewarding effects, including front-loading and consummatory successive negative contrast (cSNC), a decrease in intake when only water is available to subjects expecting EtOH. The goals of the current study were to broaden our understanding of these reward-related behaviors during binge EtOH access in high alcohol-preferring (HAP) replicate lines (HAP2 and HAP3) of mice selectively bred to prefer alcohol. We hypothesized that both lines would show evidence of front-loading during binge EtOH access and that we would find a cSNC effect in groups where EtOH was replaced with water, as these results have been shown previously in HAP1 mice. Methods HAP replicate 2 and replicate 3 female and male mice were given 2 hours of EtOH or water access in the home cage for 15 consecutive days using "drinking in the dark" (DID) procedures. Mice received the same fluid (either 20% unsweetened EtOH or water) for the first 14 days. However, on the 15th day, half of the mice from these 2 groups were provided with the opposite assigned fluid (EtOH groups received water and vice versa). Intake was measured in 1-minute bins using specialized sipper tubes, which allowed within-session analyses of binge-drinking patterns. Results EtOH front-loading was observed in both replicates. HAP3 mice displayed front-loading on the first day of EtOH access, whereas front-loading developed following alcohol experience in HAP2 mice, which may suggest differences in initial sensitivity to EtOH reward. Consummatory SNC, which manifests as lower water intake in mice expecting EtOH as compared to mice expecting water, was observed in both replicates. Conclusions These findings increase confidence that defined changes in home cage consummatory behavior are driven by the incentive value of EtOH. The presence of cSNC across HAP replicates indicates that this reaction to loss of reward is genetically mediated, which suggests that there is a biological mechanism that might be targeted.

The stability of ethanol in antemortem blood stored under various conditions has been widely studied. Antemortem blood samples stored at refrigerated temperature, at room temperature, and at elevated temperatures tend to decrease in ethanol concentration with storage. It appears that the stability of ethanol in blood exposed to temperatures greater than 38°C has not been evaluated. The case presented here involves comparison of breath test results with subsequent analysis of blood drawn at the time of breath testing. However, the blood tubes were in a refrigerator fire followed by refrigerated storage for 5 months prior to analysis by headspace gas chromatography. The subject's breath was tested twice using an Intoxilyzer 8000. The subject's blood was tested in duplicate using an Agilent headspace gas chromatograph. The measured breath ethanol concentration was 0.103 g/210 L and 0.092 g/210 L. The measured blood ethanol concentration was 0.0932 g/dL for both samples analyzed. Although the mean blood test result was slightly lower than the mean breath test result, the mean breath test result was within the estimated uncertainty of the mean blood test result. Even under the extreme conditions of the blood kit being in a refrigerator fire, the measured blood ethanol content agreed well with the paired breath ethanol test.

The correlation between the rising consumption of new psychoactive drugs (NPS), including that of cathinones, and the occurrence of death has not been sufficiently backed up with published analytic data. In fact, the identification of cathinones in human biological samples remains difficult mainly due to the diversity of these substances and their high turnover. In this context, this manuscript aims at documenting a fatal case of a 39-year-old man: autopsy findings consisted in unspecific asphyxic syndrome. Blood ethanol concentration determination and toxicological screenings were performed using gas chromatography with flame ionization detection, liquid chromatography with diode array detection and gas chromatography with mass spectrometry detection, respectively. Liquid chromatography with high-resolution mass spectrometry detection allowed the confirmation of the presence of NPS and the subsequent metabolic study. The analyses have shown the presence of ethanol, tetrahydrocannabinol and two cathinones, 4′-methyl-α-pyrrolidinohexanophenone (MPHP) and N-ethyl-4′-methylnorpentedrone (4-MEAP). MPHP/4-MEAP concentrations were 47/1.6, 97/3.5 and 2380/49,700 µg/L in femoral blood, cardiac blood and urine, respectively. The in vitro metabolic study has highlighted the presence of five metabolites derived from MPHP and three from 4-MEAP but only two metabolites of these products have been detected in biological samples. The 4′-carboxy-PHP, one of the metabolites of MPHP, was detected in every biological sample with higher chromatographic signals than MPHP itself. The number of fatalities related to cathinones use is expected to increase in the coming years. This manuscript reports useful analytical data about MPHP, one of its metabolites (4′-carboxy-PHP) and 4-MEAP.

In clinical laboratories, a common practice used to verify tests prior to reporting is repeat testing. Our objective was to evaluate the differences between the results of blood ethanol concentration (BEC) test repetitions and report on the role of repeat testing to prevent reporting of incorrect results.We conducted a retrospective study of data retrieved from the Bursa Yuksek Ihtisas Training and Research Hospital's document management system by calculating the percentage change between repeated BEC test runs. To assess for clinical relevance, the bias between two results from the same sample was compared using the 1988 Clinical Laboratory Improvement Amendments' (CLIA) proficiency testing allowable total error (TEa) limits.From a total of 1,627 BEC tests performed between January 2017 and January 2018, 70% (1,133) were repeat tested. Of these, 830 resulted in BECs between 0-5 mmol/L, of which 237 (28.5%) were above the 25% acceptable TEa. Two hundred seventy-six BEC test results were greater than14 mmol/L, and there was a good consensus between the initial and repeat test results (99%). In this group, the mean bias was 0.0% (95%, CI = -9.8-9.8%). However, three of the repeat test results were considered significantly different. There were two discordant results in the 5-14 mmol/L ethanol level, and the mean bias was 2.1% (95%, CI = -15.0-19.1%).The majority of the repeated BEC test values were the same as the baseline value; therefore, there may be limited benefit in continuing such frequent repeated analyses.U kliničkim laboratorijama uobičajena je praksa da se izvrši evaluacija testa koji će biti korišćen. Nama je bio cilj da se izvrši evaluacija testa radi utvrđivanja razlika između rezultata pri određivanju koncentracije etanola u krvi (BESC) ponovljenim određivanjem i da iste objavimo kako bi se izbeglo izdavanje netačnih rezultata.Izučavanja su rađena u Bursa Yuksek Training i Research Hospital primenom menadžment sistema radi izračunavanja procenta odstupanja u ponovljenim određivanjima primenom BEC testa. Odgovarajuća klinička procena između rezultata dobijenih u istim uzorcima upore|ivana je primenom 1988 Clinical Laboratory Improvement Amendments (CLIA) testa koji omogućava izračunavanje granica ukupne greške (TEa).Od ukupno 1 627 BEC testova izvedenih između januara 2017 i januara 2018, 70% (1 133) su bili ponovljeni testovi. Od ovih, 830 BEC rezultata bili su između 0–5 mmol/L, a samo je 237 (28,5%) bilo iznad 25% prihvatljive TEa. Dvesta sedamdeset šest rezultata BEC testova bilo je veće od14 mmol/L, i dobijen je dobar konsenzus izmeđuinicijalnih i ponovljenih rezultata testova (99%). U ovoj grupi, srednje odstupanje je bilo 0,0% (95%, CI = -9,8–9,8%). Međutim, tri od ponovljenih rezultata su se značajno razlikovala. Postojala su dva neslaganja između rezultata kod nivoa etanola 5–14 mmol/L, pa je srednje odstupanje bilo 2,1% (95%, CI = -15,0–19,1%).Većina vrednosti ponovljenih BEC testova bila je ista kao i osnovna vrednost, što znači da nema opravdanja da se često vrednosti testova ponavljaju.

This study assessed the ethanol and methanol contents of homemade spirit (Kachasu) sold in Blantyre, Malawi. The likelihood of ethanol and methanol toxicity, respectively, was determined through Monte Carlo simulations using reported Kachasu intake volumes of 21 consumers and the determined methanol and ethanol contents. Ethanol concentration, in samples from 20 different distillers, ranged from 11 to 55% v/v. Methanol was detected in 10 of the 20 samples (0.01–0.28% v/v). The likely mean ethanol intake of drinkers in Blantyre was found to be 214 ± 93 mL per day (90% CI, 68.9–373.4 mL), and mean methanol intake was 0.44 ± 0.37 mL (90% CI, 0.03–1.17 mL). The intake values translated to mean blood ethanol and methanol concentrations of 38 ± 16 mg/mL and 0.05 ± 0.04 mg/mL, respectively. Therefore, the risk of methanol toxicity was considered as negligible. However, there was a high risk of ethanol toxicity. Since production and selling of Kachasu are already illegal in Malawi, enforcement of regulations should be strengthened to reverse the current situation where Kachasu is being distilled and sold openly even within cities. Consumers should also be sensitized about the likely risks associated with consumption of Kachasu in Malawi so that they can make informed choices.

Since the accuracy of headspace gas chromatographic analysis of blood for ethanol concentration has been so well established over the past several decades, it has become commonplace in court proceedings to attack preanalytical handling of the blood samples including the lack of measuring sample temperature prior to sample preparation. The impact on measured ethanol concentration of allowing refrigerated (~4℃) samples varying amounts of time to equilibrate with room temperature, 24, 4, 3, 2, and 1 h, prior to sample preparation was evaluated. Samples were diluted 1:10 with an internal standard using a diluter/dispenser and analyzed using headspace gas chromatography. The mean ethanol concentration measured for the sixteen samples at each of the five equilibration times was 0.153 g/dl. The F-critical from the one-way ANOVA was 2.4937. The calculated F value was 0.4209. Additionally, the effect on measured ethanol concentration of having calibrators at different temperatures than case samples was investigated. Three groups were analyzed: all calibrators, controls, and samples given 24 h to equilibrate with room temperature, all calibrators, controls, and samples prepared immediately after removal from refrigeration, and calibrators sampled immediately after removal from refrigerator with samples and controls allowed 24 h to equilibrate with room temperature. The mean ethanol concentration measured for the thirty blood samples in each of the three groups was 0.197 g/dl. The F-critical from the one-way ANOVA was 3.1013. The calculated F value was 0.0188. Measured ethanol concentrations were insensitive to the variations in preanalytical conditions evaluated in this study.

Animal models of alcohol drinking and dependence are a critical resource for understanding the neurobiological mechanisms and development of more effective treatments for alcohol use disorder (AUD). Because most rat strains do not voluntarily consume large enough quantities of alcohol to adequately model heavy drinking, dependence, and withdrawal-related symptoms, researchers frequently turn to experimenter administered methods to investigate how prolonged and repeated exposure to large quantities of alcohol impacts brain and behavior. Vaporized ethanol is a common method used for chronically subjecting rodents to alcohol and has been widely used to model both binge and dependence-inducing heavy drinking patterns observed in humans. Rodent strain, sex, and age during exposure are all well-known to influence outcomes in experiments utilizing intraperitoneal or intragastric methods of repeated ethanol exposure. Yet, despite its frequent use, the impact of these variables on outcomes associated with ethanol vapor exposure has not been widely investigated. The present study analyzed data generated from over 700 rats across an eight-year period to provide a population-level assessment of variables influencing level of intoxication using vapor exposure. Our findings reveal important differences with respect to strain, sex, and age during ethanol exposure in the relationship between blood ethanol concentration and behavioral signs of intoxication. These data provide valuable scientific and practical insight for laboratories utilizing ethanol vapor exposure paradigms to model AUD in rats.

A 72-year-old male was arrested for impaired driving. He had a history of chronic alcohol1 consumption and limited food intake on the day of his arrest. The Intoxilyzer® 8000C provided the following communication messages during testing: “Interferent Detect”, “Invalid Sample” and then an additional “Interferent Detect.” This information from the Intoxilyzer® 8000C prompted a medical investigation. The individual was taken to the hospital where ketoacidosis was part of the medical diagnosis. Blood samples were collected and analyzed at two hospital laboratories and the Centre of Forensic Sciences. The results from the hospital analyses were serum ethanol concentrations of 172 and 161 mg/100 mL and an acetone concentration of 18 mg/100 mL; the results from the forensic analyses were blood ethanol, acetone, isopropanol and beta-hydroxybutyrate concentrations of 139 mg/100 mL, 21 mg/100 mL

Alcohol use disorder affects millions of people each year. Currently approved pharmacotherapies have limited success in treating this disorder. Evidence suggests that this lack of success is partly due to how these pharmacotherapies are tested in preclinical settings. The vast majority of preclinical studies assessing the effects of pharmacotherapies on alcohol or drug self-administration are done in individually housed animals. However, it is known that alcohol and drug intake are heavily influenced by social settings. Here, we adapted radio frequency tracking technology to determine the effects of oxytocin, a potential therapy for alcohol use disorder, on alcohol consumption in socially housed male and female prairie voles. Voluntary alcohol consumption in these animals resulted in high daily alcohol intakes, blood ethanol concentrations that are considered intoxicating, and central changes in FosB immunoreactivity, indicative of changes in neural activity. Prairie voles that received oxytocin temporarily reduced alcohol consumption but not alcohol preference, compared with control prairie voles regardless whether their cagemates received a similar treatment or not. Our results demonstrate that oxytocin can decrease consummatory behaviors in the presence of peers that are not receiving this treatment, and therefore, its potential use in clinical trials is warranted. Moreover, effectiveness of other pharmacotherapies in preclinical studies can be tested in mixed-treatment socially housed animals similarly to clinical studies in humans.

Anxiety and aggression are associated with ethanol self-administration, but these behaviors can serve as either risk factors for or consequences of heavy drinking in rodents and humans. Baseline levels of aggressive-like and anxious-like behavior in non-human primates have not yet been characterized in relation to future or prior ethanol intake. The objective of the study was to test the association between temperament at baseline with future ethanol self-administration in late adolescent male (n = 21) and female (n = 11) rhesus monkeys. Shortly after entering the laboratory and before exposure to ethanol, the Human Intruder Test (HIT) and the Novel Object Test (NOT) were used to determine baseline anxious-like and aggressive-like behavior in age-matched male and female rhesus monkeys (Macaca mulatta). The monkeys were induced to drink ethanol 4 % (w/v) using a schedule-induced polydipsia procedure, followed by “open-access” ethanol self-administration in which the monkeys were allowed a choice of water or 4 % ethanol (w/v) for 22 h/day for 52 weeks. Aggressive monkeys self-administered more ethanol and attained higher blood ethanol concentrations (BECs). No significant differences in ethanol intakes or BECs were found between anxious and non-anxious monkeys or between behaviorally inhibited and non-inhibited monkeys. Baseline aggressive behavior positively correlated with ethanol intake and intoxication. Baseline reactive aggression was associated with higher future ethanol intake and intoxication. While significant sex differences in HIT reactivity were observed, the relationship between aggression and ethanol drinking was observed across sex and is not sex-specific.

Background: The stability of ethanol was investigated in blood specimens in glass or plastic evacuated tubes after storage in a refrigerator at 4 °C for up to 12 months. Methods: Sterile blood, from a local hospital, was divided into 50 mL portions and spiked with aqueous ethanol (10% w/v) to give target concentrations of 0.20, 1.00, 2.00 and 3.00 g/L. Ethanol was determined in blood by headspace gas chromatography (HS-GC) with an analytical imprecision of

The aversive action of the pharmacological properties of ethanol was studied in selectively bred Warsaw Alcohol High-Preferring (WHP) and Warsaw Alcohol Low-Preferring (WLP) rats. For this study, a conditioned-taste aversion test was used. Male WHP and WLP rats were submitted to daily 20-min sessions for 5 days, in which a saccharin solution (1.0 g/L) was available (pre-conditioning phase). Next, this drinking was paired with the injection of ethanol (0, 0.5, 1.0 g/kg), intraperitoneally [i.p.] immediately after removal of the saccharin bottle (conditioning phase). Afterward, the choice between the saccharin solution and water was extended for 18 subsequent days for 20-min daily sessions (post-conditioning phase). Both doses of ethanol did not produce an aversion to saccharin in WLP and WHP rats in the conditioning phase. However, injection of the 1.0 g/kg dose of ethanol produced an aversion in WLP rats that was detected by a decrease in saccharin intake at days 1, 3, 7, and 10 of the post-conditioning phase, with a decrease in saccharin preference for 16 days of the post-conditioning phase. Conditioned taste aversion, measured as a decrease in saccharin intake and saccharin preference, was only visible in WHP rats at day 1 and day 3 of the post-conditioning phase. This difference between WLP and WHP rats was apparent despite similar blood ethanol levels in both rat lines following injection of 0.5 and 1.0 g/kg of ethanol. These results may suggest differing levels of aversion to the post-ingestional effects of ethanol between WLP and WHP rats. These differing levels of aversion may contribute to the selected line difference in ethanol preference in WHP and WLP rats.

The role of ethanol metabolism system in adaptation of laboratory animals to cold temperatures was shown. Cold stress (1-2°C) modeled in male Wistar rats over 7 weeks significantly modulated endogenous ethanol metabolism and led to reorganization of many physiological systems, which resulted in activation of metabolic processes. Under these conditions, endogenous ethanol was utilized as the most easily and fast metabolized energy substrate, due to which its blood concentration decreased and was replenished at the expense of exogenous ethanol. Normalization of blood ethanol concentration led to better adaptation to cold.

Background: Several studies have analyzed the functions of foods and dietary constituents in the dynamics of alcohol metabolism. However, few studies have reported the function of dietary fibers in the dynamics of alcohol metabolism. Objective: We assessed the effects of botanical foods that contain dietary fibers on alcohol metabolism. Methods: The ability of the water-insoluble fraction (WIF) of 18 kinds of botanical foods to maintain 15% (v/v) ethanol solution was examined using easily handled filtration. A simple linear regression analysis was performed to examine the correlation between the filtered volumes and blood ethanol concentration (BEC) in F344 rats 4 h after the ingestion of 4.0 g/kg of ethanol following dosage of 2.5% (w/v) WIF of the experimental botanical foods. Furthermore, the supernatant (6.3 Brix; water-soluble fraction) and precipitate (WIF of tomato), with a strong ethanol-maintaining ability, were obtained and BEC and the residual gastric ethanol in rats were determined 2 h after the administration of 4.0 g/kg of ethanol and the individuals fractions. Results: The filtered volumes of dropped ethanol solutions containing all the botanical foods tested except green peas were decreased compared with the ethanol solution without WIF (control). There was a significant correlation between the filtered volumes and blood ethanol concentration (BEC). There was no significant difference in the residual gastric ethanol between controls and the supernatant group; however, it was increased significantly in the WIF group than in controls or the supernatant group. Consistent with this, BEC reached a similar level in controls and the supernatant group but significantly decreased in the WIF group compared with controls or the supernatant group. Conclusions: These findings suggest that WIFs of botanical foods, which are mostly water-insoluble dietary fibers, possess the ability to absorb ethanol-containing solutions, and this ability correlates strongly with the inhibition of the blood ethanol response, likely by delaying gastric emptying. Key words: Botanical food, tomato, water-insoluble fraction, dietary fiber, blood ethanol concentration

Introduction: Measurements of blood ethanol concentrations must be accurate and reliable. The most important factors affecting blood ethanol stability are temperature and storage time. In this study, we aimed to compare ethanol stability in plasma samples at-20 °C for the different storage periods. Materials and methods: Blood samples were collected from intoxicated drivers (N = 80) and initial plasma ethanol concentrations were measured immediately. Plasma samples were then stored at-20 °C and re-assessed after 2, 3, 4, or 5 months of storage. Differences between the initial and stored ethanol concentrations in each group (N = 20) were analyzed using Wilcoxon matched-pairs test. The deviation from the initial concentration was calculated and compared with Clinical Laboratory Improvement Amendments (CLIA’88) Proficiency Testing Limits. Relationships between the initial concentrations and deviations from initial concentrations were analyzed by Spearman’s correlation analysis. For all statistical tests, differences with P values of less than 0.05 were considered statistically significant. Results: Statistically significant differences were observed between the initial and poststorage ethanol concentrations in the overall sample group (P < 0.001). However, for the individual storage duration groups, analytically significant decreases were observed only for samples stored for 5 months, deviations from the initial concentrations exceeded the allowable total error (TEa). Ethanol decreases in the other groups did not exceed the TEa. Conclusion: According to our results, plasma ethanol samples can be kept at-20 °C for up to 3-4 months until re-analysis. However, each laboratory should also establish its own work-flow rules and criterion for reliable ethanol measurement in forensic cases. © by Croatian Society of Medical Biochemistry and Laboratory Medicine.

Effects of Opuntia ficus indica (OFI) extracts on ethanol-induced psychomotor alterations were studied using Sprague-Dawley rats orally administered 4 g/kg of ethanol (EtOH group) or distilled water (control group). An OFI-group received OFI extracts (50, 100, and 200 mg/kg) 30 min prior to EtOH administration. Behavioral and hematological tests were evaluated 1, 2, 4, and 8 h after EtOH administration. Electroencephalogram (EEG) assessment was also performed. EtOH significantly (p

Schedules of intermittent food delivery induce excessive fluid intake, termed schedule-induced polydipsia (SIP), and hypothalamic-pituitary-adrenal (HPA) axis activation is important for the expression and maintenance of this adjunctive behavior. Previous work has focused on examining the relationship between water intake and plasma corticosterone (CORT) in rats at a single or a limited range of fixed time (FT) intervals. However, little remains known regarding SIP and the corresponding stress response (1) across the bitonic function that epitomizes adjunctive behavior, (2) when ethanol is the available fluid, and (3) when a species other than rat or multiple strains are studied. Here we report the findings from ethanol-preferring C57BL/6J (B6) and non-preferring DBA/2J (D2) mice serially exposed to progressively larger FT intervals (0 → 60 min) and given access to either water or a 5% (v/v) ethanol solution. Following 2 weeks of experience with each schedule, blood samples were collected at the conclusion of the last 60-min session to evaluate CORT and the blood ethanol concentration (BEC) achieved. While both strains exhibited a bitonic function of ethanol intake and BEC that peaked at or near a 5-min interval, only D2 mice showed a similar response with water. In contrast, CORT levels rose monotonically with incremental increases in the FT interval regardless of the strain examined or fluid type offered, indicating that glucocorticoid release likely reflects the aversive aspects of increasing intervals between reinforcement rather than engagement in adjunctive behavior. These findings also caution against the use of a single intensity stressor to evaluate the relationship between stress and ethanol intake, as the magnitude of stress appears to affect ethanol consumption in a non-linear fashion.

Background Concurrent alcohol use among cocaine abusers is common but the behavioral variables that promote co-abuse are not well understood. The present study examined the effects of intragastric (i.g.) ethanol (EtOH) administration in monkeys responding under a schedule of cocaine reinforcement in which extensive drug seeking was maintained by conditioned stimuli. Methods Four adult male cynomolgus monkeys ( Macaca fascicularis ) were trained to respond under a second-order fixed-interval (FI) 600 s (fixed-ratio (FR) 30:S) schedule of cocaine (0.003–0.56 mg/kg/injection) presentation. Sessions ended after 5 injections or 90 min had elapsed. Different EtOH doses (0.5–2.0 g/kg, i.g.) were administered 30 min before the session, typically on Tuesdays and Fridays. Blood ethanol concentrations (BECs) were also assessed. Pattern of FI responding was assessed by determining quarter-life (QL) values. Results Cocaine self-administration was characterized as an inverted U-shaped function of dose; QL values increased monotonically with dose. EtOH pretreatments dose-dependently decreased self-administration at several cocaine doses in 3 of 4 monkeys. In one animal, EtOH increased low-dose cocaine-maintained responding. For all monkeys, QL values were increased by EtOH when low- and high-cocaine doses were self-administered, suggesting additive effects of EtOH and cocaine. Furthermore, BECs were not altered following cocaine self-administration. Conclusions The reductions in cocaine self-administration and the increases in QL values following EtOH, suggest that EtOH was enhancing cocaine-related conditioned reinforcement. A better understanding of the behavioral mechanisms that mediate the co-abuse of alcohol and cocaine will lead to improved treatments for both drugs.

In the last century, several mathematical models have been developed to calculate blood ethanol concentrations (BAC) from the amount of ingested ethanol and vice versa. The most common one in the field of forensic sciences is Widmark's equation. A drinking experiment with 10 voluntary test persons was performed with a target BAC of 1.2 g/kg estimated using Widmark's equation as well as Watson's factor. The ethanol concentrations in the blood were measured using headspace gas chromatography/flame ionization and additionally with an alcohol dehydrogenase (ADH)-based method. In a healthy 75-year-old man a distinct discrepancy between the intended and the determined blood ethanol concentration was observed. A blood ethanol concentration of 1.83 g/kg was measured and the man showed signs of intoxication. A possible explanation for the discrepancy is a reduction of the total body water content in older people. The incident showed that caution is advised when using the different mathematical models in aged people. When estimating ethanol concentrations, caution is recommended with calculated results due to potential discrepancies between mathematical models and biological systems.

The adverse reaction to 1.25 g/kg ethanol was monitored in male Fischer rats given durian or cabbage (2.4 g FW/100 g BW/day), administered intragastrically. During the first ethanol challenge, a reduced rate of blood acetaldehyde clearance and hypothermia, which is associated with the disulfiram-ethanol reaction, was observed in rats given durian or cabbage. Blood ethanol levels and rate of acetaldehyde elimination were lowest 30 min after the first ethanol challenge in rats given cabbage, while a similar but more exacerbated trend was observed at 60 min in rats given durian. When subjected to conditioned taste aversion using saccharine solution (0.2% v/v) paired with ethanol administration, the rats given durian or cabbage exhibited aversion, with the former showing the earliest and most pronounced response, persisting through to the last ethanol challenge. Rats given cabbage exhibited delayed aversion, which progressively increased to the same level as that observed in rats given durian.

Mouse lines are being selectively bred in replicate for high blood ethanol concentrations (BECs) achieved after limited access of ethanol (EtOH) drinking early in the circadian dark phase. High Drinking in the Dark-1 (HDID-1) mice are in selected generation S21, and the replicate HDID-2 line in generation S14. Tolerance and withdrawal symptoms are 2 of the 7 diagnostic criteria for alcohol dependence. Withdrawal severity has been found in mouse studies to be negatively genetically correlated with EtOH preference drinking.To determine other traits genetically correlated with high DID, we compared naïve animals from both lines with the unselected, segregating progenitor stock, HS/Npt. Differences between HDID-1 and HS would imply commonality of genetic influences on DID and these traits.Female HDID-1 and HDID-2 mice tended to develop less tolerance than HS to EtOH hypothermia after their third daily injection. A trend toward greater tolerance was seen in the HDID males. HDID-1, HDID-2, and control HS lines did not differ in the severity of acute or chronic withdrawal from EtOH as indexed by the handling-induced convulsion (HIC). Both HDID-1 and HDID-2 mice tended to have greater HIC scores than HS regardless of drug treatment.These results show that tolerance to EtOH's hypothermic effects may share some common genetic control with reaching high BECs after DID, a finding consistent with other data regarding genetic contributions to EtOH responses. Withdrawal severity was not negatively genetically correlated with DID, unlike its correlation with preference drinking, underscoring the genetic differences between preference drinking and DID. HDID lines showed greater basal HIC scores than HS, suggestive of greater central nervous system excitability.

Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes.

We designed and built a novel, miniature gas chromatograph (mGC) to use exhaled breath to estimate blood ethanol concentrations that may offer GC quality sensitivity and specificity, but with portability, reduced size, and decreased cost. We hypothesized that the mGC would accurately estimate the serum ethanol concentration using exhaled breath. Human subjects (n = 8) were dosed with ethanol employing the Widmark criteria, targeting a blood concentration of 0.08 g/dL. Serum and breath samples were collected concurrently over an hour. Ethanol concentrations in serum were measured using a CLIA-approved laboratory. Ethanol concentrations in conventional breath were assayed using a calibrated mGC or Intoxilyzer 400PA. Data were analyzed using Bland-Altman analysis using serum concentrations as a "gold standard". For the mGC, the regression line (correlation coefficient), bias, and 95% limits of agreement were y = 1.013x - 0.009 (r = 0.91), -0.008 g/dL, and -0.031 to 0.016 g/dL, respectively, for 30 specimens. For the Intoxilyzer 400PA, the regression line (correlation coefficient), bias, and 95% limits of agreement were y = 0.599x + 0.008 (r = 0.86), -0.024 g/dL, and -0.049 to 0.002 g/dL, respectively, for 71 specimens with a large magnitude effect. We concluded that the mGC, using exhaled breath, performed well to estimate the serum ethanol concentrations.

Recent studies indicate that the infant rat has high affinity for ethanol ingestion and marked sensitivity to the drug's reinforcing effects [Spear, N.E., Molina, J.C. Fetal or infantile exposure to ethanol promotes ethanol ingestion in adolescence and adulthood: a theoretical review. Alcohol Clin Exp Res 2005; 29: 909-29.]. A novel operant technique was developed to analyze reinforcing effects of ethanol delivery during the third postnatal week. The impact of this ethanol-reinforcement experience upon subsequent ethanol consumption during adolescence (postnatal weeks 5-6) was also examined. In Experiment 1, pups (postnatal days 14-17) were given an explicit contingency between nose-poking behavior and intraoral delivery of either water or 3.75% v/v ethanol (paired groups). Yoked controls (pups receiving either reinforcer independently of their behavior) were also included. Paired subjects reinforced with ethanol exhibited rapid and robust operant conditioning leading to blood ethanol concentrations in the 25-48 mg% range. In Experiment 2, a higher ethanol concentration (7.5% v/v) provided significant reinforcement. During adolescence, animals originally reinforced with 3.75% v/v ethanol exhibited greater ingestion of ethanol than control animals without prior ethanol reinforcement. These results indicate that, without extensive initiation to ethanol, infant rats rapidly learn to gain access to ethanol and that this experience has a significant impact upon later ethanol intake patterns.

The Warsaw High Preferring (WHP) and Warsaw Low Preferring (WLP) lines were bred from Wistar foundation stock to obtain lines of rats that differ in their preference for ethanol solutions. The WHP line has met several major criteria for an animal model of alcoholism. The WHP rats voluntarily drink excessive amounts of ethanol while the WLP rats consume negligible amounts of ethanol. The WHP rats attain physiologically active blood ethanol concentrations with chronic free-choice drinking. They also develop subtle but visible signs of physical dependence (the withdrawal signs). The patterns of ethanol consumption in WHP and WLP lines are stable in time and independent of the manner of access to ethanol solutions. Notably, when exposed to the increasing ethanol concentrations WHP rats gradually increased total ethanol intake whereas the WLP rats consumed invariably very low amounts of ethanol. Furthermore, the WHP rats show an increased responsiveness to the stimulatory effects of low dose of ethanol.

The word “endogenous” means produced or originating from within the body, so endogenous ethanol therefore implies a spontaneous auto regulation of ethanol through various human metabolic processes. In the current research, endogenous ethanol concentrations in blood were determined by sensitive headspace gas chromatography/mass Spectrophotometry in 1400 residents of Saudi Arabia. The subjects were from 14 nationalities, of both sexes and of different age groups. There was no significance difference in blood ethanol concentration between nationalities or between sexes within and between nationalities. The data was extracted and the overall mean ± SD, minimum, maximum, 5% percentile and 95% percentile were 0.14, ± 0.35, 0.00, 1.53 , 0.00, 1.20 mg/dl respectively. The values of blood ethanol concentration as reported in this study indicate they are far too low to have any forensic significance.

Non-human primates are widely used in research, yet relatively few studies have addressed potential pharmacokinetic differences between males and females. The present study examined the relationship between total body water, sex, age, and weight in the rhesus macaque (Macaca mulatta). Ethanol-naive, adolescent rhesus macaques (n = 119) were administered ethanol (males, 2.1 g/kg; females, 2.0 g/kg) intravenously, and blood samples for blood ethanol concentration obtained at 5, 10, and 60 minutes following the end of the infusion. Non-linear regression was used to compare and contrast a series of pharmacokinetic models examining the relationship between weight, sex, age, V(d) and zero-order elimination rate. V(d) (mean +/- SEM) for male rhesus was 0.771 +/- 0.008 l/kg and for females was 0.730 +/- 0.008 l/kg, different at P < 0.00001. There were no sex differences in the rate of zero-order ethanol elimination, estimated to be 0.0032 +/- 0.0004 g/kg/minute. The data reported here may be useful in designing and interpreting pharmacokinetic studies using rhesus monkeys.

The elimination rate of blood ethanol usually depends on the activity of liver alcohol dehydrogenase (ADH). During acute alcohol intoxication, however, it is unclear how liver ADH activity changes with dose and time and what the involvement is of the two major isozymes of liver ADH: the classically known class I ADH and the very high Km class III ADH. We investigated dose- and time-wise changes in liver ADH activity and the contents of both ADHs by administering ethanol to mice, and analyzed the relationship among these ADH parameters to assess the contributions of these ADHs to liver ADH activity and ethanol metabolism in vivo.Mice were given ethanol doses of 0, 1, 3 or 5 g/kg body weight and killed 0.5, 1, 2, 4, 8 or 12 h after administration. The elimination rate of blood ethanol was calculated from the regression line fitted to the blood ethanol curve. The liver ADH activity of crude extract was conventionally measured with 15 mM ethanol as a substrate. The liver class I and class III ADH contents were determined by enzyme immunoassay. These three ADH parameters were statistically analyzed.The change in liver ADH activity depended on both dose and time (P0.001 by two-way ANOVA, n=74), but the change in the class I content depended on dose alone (P0.0001). The class III content depended on both dose and time (P0.001) with a time course similar to that of liver ADH activity for each dose. The sum of the class I and class III contents exhibited a higher correlation with liver ADH activity (r=0.882, P0.0001) than the class I content alone did (r=0.825). The mean liver ADH activity during ethanol metabolism for each dose correlated significantly with the elimination rate of blood ethanol (r=0.970, P0.0001).Liver ADH activity changes dose and time dependently during acute alcohol intoxication and governs the elimination rate of blood ethanol through the involvement not only of class I but also of class III ADH.

It is important to select an appropriate model system for studies examining the mechanisms of ethanol-induced injury. The most common model systems use either mice or rats with ethanol administered by means of intragastric gavage or intraperitoneal injection, yet few studies have compared directly the blood ethanol concentration (BEC) profiles that result from each of these model systems. In the current study, Sprague-Dawley rats and C57BL/6J mice were given ethanol by means of intragastric gavage or intraperitoneal injection at 40 days of age. Blood samples were collected at consistent time intervals to determine BECs. Blood ethanol concentrations in mice were sharper, with a more rapid rise to a sharp peak BEC, followed by a relatively rapid decline. In contrast, rat BEC profiles showed an initial rapid rise, followed by a more gradual rise to peak concentrations, and, then, a relatively gradual decline. This difference was particularly evident in rats receiving ethanol intragastrically. The differences found in BEC profiles between rats and mice and between ethanol administration paradigms may yield differences in the extent or mechanism of damage induced by ethanol, an important consideration when selecting an appropriate model for the investigation of ethanol-induced tissue damage.

In experimental studies, liquid ethanol diets are usually given as the sole source of nutrition and fluid. Two series of experiments were conducted to examine the effect of freely accessible water on the consumption of ethanol liquid diets in male Long–Evans rats. The consumption of diets and subsequent learning ability of rats were first examined in animals given twice-daily saline injections. One group received diet with no access to water for 12 weeks and was subsequently given free access to water with diets for an additional 12 weeks. A second group was given diet and water ad libitum for 24 weeks. Control animals received an isocaloric sucrose-containing diet (with or without ad libitum access to water). Subsequently, rats were tested for active avoidance learning. In the first 12 weeks, animals with ad libitum access to water drank more diet than did water-restricted animals, and previously water-restricted animals increased their diet consumption when access to water was freely available. All water-restricted animals, in both ethanol- and sucrose-treated groups, showed deficits in active avoidance learning, whereas only ethanol-treated animals in groups with ad libitum access to water showed learning deficits. In the second series of experiments, the effect of saline injections on diet consumption, both in the presence and absence of water, was examined. Although saline injections were associated with decreased diet consumption, there was no effect of free access to water. No differences in blood ethanol concentration were seen among groups. Findings obtained from both series of studies demonstrate that consumption of a Sustacal-based liquid ethanol diet does not decrease if access to water is freely available.

Background “Work” for ethanol, that is, the ability of a laboratory animal to press a lever to gain access to ethanol, has been proposed as (a) a requirement for definition of an animal model of alcoholism and (b) a measure of ethanol-reinforcing properties. The present study evaluated oral self-administration of ethanol under an operant (lever pressing) procedure in selectively bred Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. Methods Rats from both lines were initiated to self-administer 10% ethanol, on a fixed ratio 1 schedule and in daily 30 min sessions, by using the Samson sucrose fading procedure. Subsequently, rats were exposed to increasing concentrations of ethanol up to 30% on a fixed ratio 4 schedule. Finally, the extinction responding for ethanol, defined as the maximal number of lever responses reached by each rat in the absence of ethanol reinforcement, was determined. Results The results indicated that sP rats acquired and maintained lever pressing for ethanol, self-administering mean amounts of ethanol in the range of 0.6 to 1.1 g/kg/session, which gave rise to mean blood ethanol levels in the 30 to 45 mg% range. Extinction responding for ethanol in sP rats averaged 73. In contrast, once sucrose was faded out, sNP rats displayed minimal levels of responding for ethanol, and extinction responding averaged 6. Conclusions The results of the present study extend to the sP/sNP rat lines the finding that ethanol can be established as a reinforcer in selectively bred alcohol-preferring rats, whereas it has modest, if any, reinforcing properties in alcohol-nonpreferring rats.

Remoxipride, a dopamine D 2 antagonist, decreases responding that results in the presentation of small amounts (~0.1 ml) of ethanol in limited-access paradigms. This type of operant response is a combined appetitive/consummatory response that is differentially affected by changing stimulus properties of consumed ethanol (i.e., taste, pharmacology) over the course of the session. In the present experimental design, ethanol-directed appetitive and consummatory responses were procedurally separated to investigate the specific effects of remoxipride on these distinct behaviors. Male Long–Evans rats were trained to make a series of lever-press responses once each day that resulted in access to a sipper tube spout containing 10% ethanol for 20 min. Three doses of remoxipride were tested: 5.0, 10.0, and 15.0 mg/kg (–30 min, i.p.). In Experiment 1, a response requirement of 20 was used, and both reinforced and nonreinforced sessions were examined. In nonreinforced sessions, subjects were permitted to lever press for 20 min, after which the session ended without sipper tube presentation. These sessions were conducted to remove the possibility that limiting responding might obscure a drug effect on the seeking response. In Experiment 2, a low response requirement (4) was used to investigate the effects of remoxipride on ethanol intake. Average baseline ethanol intake (Experiment 1) was 0.69 g/kg, with blood ethanol concentrations at the end of the session at 64 mg%. At all doses tested, remoxipride had no effect on the measures of ethanol consumption (e.g., total intake, lick latency, lick rate) in either experiment. However, remoxipride dose dependently decreased the number of appetitive responses made, while having no effect on response latency or rate, during both reinforced and nonreinforced sessions in Experiment 1. In these experiments, the systemic antagonism of the dopamine D 2 receptor decreased ethanol seeking without causing a general impairment of motor function. The procedural separation of seeking and intake responses revealed that appetitive responding was more sensitive than consummatory responding to remoxipride treatment.

Acute alcohol exposure has been shown to produce differential motor impairments between aged and adult rats and between adolescent and adult rats. However, the effects of acute alcohol exposure among adolescent, adult, and aged rats have yet to be systematically investigated within the same project using a dose-dependent analysis. We sought to determine the age- and dose-dependent effects of acute alcohol exposure on gross and coordinated motor performance across the rodent lifespan. Adolescent (PD 30), adult (PD 70), and aged (approximately 18 months) male Sprague–Dawley rats were tested on 3 separate motor tasks: aerial righting reflex (ARR), accelerating rotarod (RR), and loss of righting reflex (LORR). In a separate group of animals, blood ethanol concentrations (BEC) were determined at multiple time points following a 3.0 g/kg ethanol injection. Behavioral tests were conducted with a Latin square repeated-measures design in which all animals received the following doses: 1.0 g/kg or 2.0 g/kg alcohol or saline over 3 separate sessions via intraperitoneal (i.p.) injection. During testing, motor impairments were assessed on the RR 10 min post-injection and on ARR 20 min post-injection. Aged animals spent significantly less time on the RR when administered 1.0 g/kg alcohol compared to adult rats. In addition, motor performance impairments significantly increased with age after 2.0 g/kg alcohol administration. On the ARR test, aged rats were more sensitive to the effects of 1.0 g/kg and 2.0 g/kg alcohol compared to adolescents and adults. Seven days after the last testing session, animals were given 3.0 g/kg alcohol and LORR was examined. During LORR, aged animals slept longer compared to adult and adolescent rats. This effect cannot be explained solely by BEC levels in aged rats. The present study suggests that acute alcohol exposure produces greater motor impairments in older rats when compared to adolescent and adult rats and begins to establish a procedure to determine motor effects by alcohol across the lifespan.

One of the greatest challenges that scientists face when studying the neurobiology and/or genetics of alcohol (ethanol) consumption is that most pre-clinical animal models do not voluntarily consume enough ethanol to achieve pharmacologically meaningful blood ethanol concentrations (BECs). Recent rodent models have been developed that promote binge-like levels of ethanol consumption associated with high BECs (i.e., 100 mg/dl or higher). This paper describes procedures for an animal model of binge-like ethanol drinking which has come to be called “drinking in the dark” (DID). The “basic” variation of DID involves replacing the water bottle with a bottle containing 20% ethanol for 2 to 4 hours, beginning 3 hours into the dark cycle, on cages of singly-housed C57BL/6J mice. Using this procedure, mice typically consume enough ethanol to achieve BECs greater than 100 mg/dl and to exhibit behavioral evidence of intoxication. An alternative 2-bottle (ethanol and water) procedure is also described.

Nonhuman primates (NHPs), in particular old-world monkeys, are excellent human surrogates for understanding putative risk factors, including genetic and epigenetic factors, behavioral traits, and stress and hormonal interactions. NHPs have been very informative in alcohol research because they will repeatedly drink intoxicating doses of ethanol, including drinking until signs of physical dependence upon alcohol appear. Perhaps even more informative is that NHPs show a wide distribution in the daily intake of alcohol (with resultant blood ethanol concentrations) when they have nearly constant access to alcohol (22 hrs/day). Given the individual differences in drinking alcohol repeatedly to intoxication, this chapter will address several main risk factors for developing alcohol dependence that have been documented in humans and can be studied in monkeys. These main factors include genetic mechanisms, stress and the HPA axis, age of onset of drinking alcohol, and temperament.

The purpose of this study was to produce data on the ethanol concentrations in ambient air that result from hand rubbings with hydroalcoholic solutions (HAS) or the use of ethanol-based varnishes, and then to predict the blood ethanol levels (BELs) that result from these procedures. The concentration of ethanol in air at the volunteer’s nose after the application of HAS on hands was measured with five volunteers who performed five tests in two different environments: 1) in an inhalation chamber (air change rate ~18 h-1), and 2) in a closed office (poorly ventilated) with two different amounts (1.5 and 3 g) of HAS. In the case of varnish, 125 ml were applied on a 1-m 2 wood surface placed in the middle of an inhalation chamber (n=4). The ethanol concentration was measured 20 cm and 40 cm from the center of the board for the next 60 minutes. As for HAS we noted a large intra- and inter-individual variability in ethanol levels in inhaled air. As expected the highest concentration in the inhalation chamber (~1250 ppm) was lower than in the office (~2352 ppm). For the application of the varnish, the ethanol concentrations greatly exceeded 1000 ppm for a short duration (< 4 min). Physiologically-based pharmacokinetic (PBPK) modeling of ethanol concentrations based on ethanol levels measured in inhaled air predicted the following maximum BELs in women (men): 0.39 and 0.37 mg/L (0.37 and 0.35 mg/L) in the office, and 0.26 and 0.42 mg/L (0.25 and 0.40 mg/L ) in the inhalation chamber for 1.5 g and 3 g, respectively. The total dose of ethanol absorbed estimated for a working day involving 42 hand rubbings with 1.5 or 3 g of HAS averaged 0.20 g. For the varnish, the predicted highest BELs for men and women were 0.77 and 0.79 mg/L, respectively. In all cases, the BELs remained below 1 mg/L. The results of this study should make it easier to assess the risk related to chronic inhalation of low levels of ethanol in the general population and among workers associated with these practices.

This review provides an overview of an animal model of binge-like ethanol drinking that has come to be called “drinking in the dark” (DID), a procedure that promotes high levels of ethanol drinking and pharmacologically relevant blood ethanol concentrations (BECs) in ethanol-preferring strains of mice. Originally described by Rhodes, Best, Belknap, Finn, and Crabbe (2005), the most common variation of the DID procedure, using singly housed mice, involves replacing the water bottle with a bottle containing 20% ethanol for 2e4 h, beginning 3 h into the dark cycle. Using this procedure, high ethanol drinking strains of mice (e.g., C57BL/6J) typically consume enough ethanol to achieve BECs greater than 100 mg/dL and to exhibit behavioral evidence of intoxication. This limited access procedure takes advantage of the time in the animal’s dark cycle in which the levels of ingestive behaviors are high, yet high ethanol intake does not appear to stem from caloric need. Mice have the choice of drinking or avoiding the ethanol solution, eliminating the stressful conditions that are inherent in other models of binge-like ethanol exposure in which ethanol is administered by the experimenter, and in some cases, potentially painful. The DID procedure is a high throughput approach that does not require extensive training or the inclusion of sweet compounds to motivate high levels of ethanol intake. The high throughput nature of the DID procedure makes it useful for rapid screening of pharmacological targets that are protective against binge-like drinking and for identifying strains of mice that exhibit binge-like drinking behavior. Additionally, the simplicity of DID procedures allows for easy integration into other paradigms, such as prenatal ethanol exposure and adolescent ethanol drinking. It is suggested that the DID model is a useful tool for studying the neurobiology and genetics underlying binge-like ethanol drinking, and may be useful for studying the transition to ethanol dependence.

Aims—To determine the concentrations of ethanol in femoral venous blood (FVB) and vitreous humour (VH) obtained during forensic necropsies. The ratios of ethanol concentrations in VH and FVB, the reference interval, and the associated confidence limits were calculated to provide information about the uncertainty in estimating FVB ethanol concentrations indirectly from that measured in VH. Methods—Ethanol concentrations were determined in specimens of FVB and VH obtained from 706 forensic necropsies.The specimens were analysed in duplicate by headspace gas chromatography (HS-GC), with a precision (coeYcient of variation) of 1.5% at a mean ethanol concentration of 500 mg/litre. The limit of detection of ethanol in body fluids by HS-GC in routine casework was 100 mg/litre. Results—In 34 instances, ethanol was present in VH at a mean concentration of 154 mg/litre, whereas the FVB ethanol concentration was reported as negative (< 100 mg/litre). These cases were excluded from the statistical analysis. The concentration of ethanol in FVB was higher than in VH in 93 instances, with a mean diVerence of 160 mg/litre (range 0 to 900). The mean concentration of ethanol in FVB (n = 672) was 1340 mg/litre (SD, 990) compared with 1580 mg/litre (SD, 1190) in VH. The arithmetic mean VH/ FVB ratio of ethanol was 1.19 (SD, 0.285) and the 95% range was 0.63 to 1.75. The mean and SD of the diVerences (log VH ˛ log FVB) was 0.063 (SD, 0.109), which gives 95% limits of agreement (LOA) from ˛0.149 to 0.276. Transforming back to the original scale of measurement gives a geometric mean VH/FVB ratio of 1.16 and 95% LOA from 0.71 to 1.89. These parametric estimates are in good agreement, with a median VH/FVB ratio of 1.18 and 2.5th and 97.5th centiles of 0.63 and 1.92. Conclusions—The ethanol distribution ratios (VH/FVB) show wide variation and this calls for caution when results of analysing VH at necropsy are used to estimate the concentration in FVB. Dividing the ethanol concentration in VH by 2.0 would provide a very conservative estimate of the ethanol content in FVB, being less than the true value, with a high degree of confidence. (J Clin Pathol 2001;54:699‐702)

Background: Investigations of oral ethanol self-administration in nonhuman primates have revealed important parallels with human alcohol use and abuse, yet many fundamental questions concerning the individual risk to, and the biological basis of, excessive ethanol consumption remain unanswered. Moreover, many conditions of access to ethanol in nonhuman primate research are largely unexplored. This set of experiments extends within- and across-session exposure to ethanol to more fully characterize individual differences in oral ethanol self-administration. Methods: Eight male and eight female adult cynomolgus monkeys (Macaca fascicularis) were exposed to daily oral ethanol self-administration sessions for approximately 9 months. During the first 3 months, a fixed-time (FT) schedule of food delivery was used to induce the consumption of an allotted dose of ethanol in 16-hr sessions. Subsequently, the FT schedule was suspended, and ethanol was available ad libitum for 6 months in 16- or 22-hr sessions. Results: Cynomolgus monkeys varied greatly in their propensity to self-administer ethanol, with sex and individual differences apparent within 10 days of ethanol exposure. Over the last 3 months of ethanol access, individual average ethanol intakes ranged from 0.6 to 4.0 g/kg/day, resulting in blood ethanol concentrations from 5 to 235 mg/dl. Males drank approximately 1.5-fold more than females. In addition, heavy-, moderate-, and light-drinking phenotypes were identified by using daily ethanol intake and the percentage of daily calories obtained from ethanol as criteria. Conclusions: Cynomolgus monkeys displayed a wide intersubject range of oral ethanol self-administration with a procedure that used a uniform and prolonged induction that restricted early exposure to ethanol and subsequently allowed unlimited access to ethanol. There were sex and stable individual differences in the propensity of monkeys to consume ethanol, indicating that this species will be important in characterizing risk factors associated with heavy-drinking phenotypes.

Background: The alcohol deprivation effect (ADE) is a temporary increase in the voluntary intake of ethanol solutions following a period of alcohol deprivation. Multiple deprivations can prolong the expression of an ADE. This study examined the effects of initial deprivation length, concurrent exposure to multiple ethanol concentrations, and number of deprivation exposures on the magnitude and duration of the ADE in alcohol-preferring (P) rats. Methods: Adult female P rats received 24-hr free-choice access to 10, 20, and 30% ethanol and water for 6 weeks. Rats were then randomly assigned to three groups; one group served as a nondeprived control, whereas the other two groups were initially deprived of ethanol for 2 or 8 weeks. The ethanol solutions were restored to both deprived groups for 2 weeks before the groups were deprived of ethanol for another 2 weeks. This cycle was repeated three times for a total of four deprivations. Results: After the initial ethanol deprivation period, both deprived groups displayed a similar 2-fold increased ethanol intake (g/Kg/day) during the initial 24-hr period when ethanol was restored. Both deprived groups showed greater than 2-fold increases in intake of the 20 and 30% ethanol solutions after re-exposure. Ethanol consumption returned to baseline levels within 2 weeks, before the subsequent deprivation period. Multiple deprivations increased the magnitude of the ADE over that observed in the first deprivation during the initial 24-hr period of re-exposure and prolonged the duration of the ADE. In addition, repeated deprivations increased ethanol intake in the first 2-hr period of re-exposure and produced blood ethanol levels in excess of 150 mg/100 ml. Conclusions: Alterations in the reinforcing and/or aversive effects of alcohol occurred after a single prolonged deprivation and were enhanced with repeated deprivations.

Background: It has been proposed that development of tolerance to the behavioral effects of ethanol depends on the degree of impairment produced by the drug; that is, more sensitive individuals should develop greater tolerance. Tests of this hypothesis with respect to acute functional tolerance have produced contradictory results. We tested the hypothesis by examining the genetic relationship between initial sensitivity and acute functional tolerance in the LSXSS recombinant inbred mice. Methods: We tested mice for initial sensitivity to the ataxic effects of 1.75 g/kg of ethanol in a stationary dowel balance test by determining blood and brain ethanol concentrations at fall. Acute tolerance to the ataxic effects of ethanol was determined by measuring blood ethanol concentration (BEC) at regain of dowel balance ability after the first injection (BEC1RB) and after a second ethanol injection of 2.0 g/kg (BEC2RB). Acute tolerance was quantified by the difference in ethanol concentration at the two regains of balance (BEC2RB − BEC1RB) or by the difference between the second regain and one of the initial sensitivity measures (BEC2RB − initial sensitivity). Results: Four different measures of initial sensitivity were taken: two that used BEC values and two that used forebrain or hindbrain ethanol concentrations. We calculated acute tolerance values by using each of these initial sensitivity measures plus BEC2RB. No evidence of a genetic relationship between initial sensitivity and acute tolerance was found, which suggests that these are two independent phenomena with respect to stationary dowel balance. Conclusions: Three conclusions can be drawn from this work: (1) Orbital sinus BEC at early time points (

Oxidative stress contributes to the pathogenesis of alcoholic liver disease. The purpose of this study is to estimate the amount of oxidative stress that is present when healthy humans consume moderate amounts of ethanol. Blood was collected from healthy volunteers before, 1 h, and 3 h after drinking 400 ml of Japanese rice wine at the rate of 100 ml per 5 min. The aldehyde dehydrogenase 2 genotype and the concentrations of blood ethanol, total lipid hydroperoxides (LOOH), and cholesterol hydroperoxides were determined. The plasma LOOH was found to have significantly increased 1 h after drinking. Cholesterol hydroperoxides were not detected in plasma, either before or after drinking. There was no relationship between the LOOH and the ethanol concentration. We showed that one-shot of moderate ethanol consumption temporarily increases the plasma LOOH in healthy volunteers but excessive plasma LOOH compounds were eliminated within a short time.

To determine the effects of body temperature, ethanol use, electrolyte status, and acid-base status on the electrocardiograms (ECGs) of hypothermic patients.Prospective, two-year, observational study of patients presenting to an urban ED with temperatureor =95 degrees F (or =35 degrees C). All patients had at least one ECG obtained. Electrocardiograms were interpreted by a cardiologist blinded to the patient's temperature. J-point elevations known as Osborn waves were defined as present if they were at least 1 mm in height in two consecutive complexes.100 ECGs were obtained in 43 patients. Presenting temperatures ranged between 74 degrees F and 95 degrees F (23.3 degrees C-35 degrees C). Initial rhythms included normal sinus (n = 34), atrial fibrillation (n = 8), and junctional (n = 1). Osborn waves were present in 37 of 43 initial ECGs. Of the six initial ECGs that did not have Osborn waves present, all were obtained in patients whose temperatures wereor =90 degrees For =32.2 degrees C). For the entire group, the Osborn wave was significantly larger as temperature decreased (p = 0.0001, r = -0.441). The correlation between temperature and size of the Osborn wave was strongest in six patients with four or more ECGs (range r = -0.644 to r = -0.956, p = 0.001). No correlation could be demonstrated between the height of the Osborn waves and the serum electrolytes, including sodium, chloride, potassium, bicarbonate, BUN, creatinine, glucose, anion gap, and blood ethanol levels.The presence and size of the Osborn waves in hypothermic patients appear to be a function of temperature. The magnitude of the Osborn waves is inversely correlated with the temperature.

Background: The addition of sucrose to ethanol solutions results in a substantial increase in ethanol self-administration by rats that are deprived of neither food nor water. However, if sucrose alters ethanol absorption or metabolism, resulting in blood ethanol concentrations (BECs) not different from those resulting from lower intakes of ethanol/water solutions, then the usefulness of sucrose/ethanol mixtures in increasing ethanol consumption is questionable. The present study was conducted to determine whether the addition of sucrose to ethanol solutions altered BECs in an operant self-administration paradigm. Methods: Tail blood (from male Long-Evans rats) was collected 30 min after the intake of four different solutions, i.e., 5% sucrose/20% ethanol, 5% sucrose/10% ethanol, 2% sucrose/10% ethanol, and 10% ethanol. Results: Ethanol intakes (mean, 1.57 ± 0.21 g/kg) and BECs (mean, 78.4 ± 9.3 mg/100 ml) were highest when 5% sucrose was added to the ethanol solution. Moreover, the ratios between ethanol intakes and resulting BECs were approximately the same for all solutions. Conclusions: These findings indicate that, under the conditions of this procedure, the BEC reached is dependent on the amount of ethanol consumed and is not influenced by the addition of sucrose to the solution.

The effect of ethanol on rats was investigated at increasing rates of acceleration for bar rotation speed. Ethanol was given to rats by a liquid diet starting with 2.4% ethanol (v/v) for 3 days. Then the ethanol concentration was increased to 4.8% (v/v) for 3 days and finally to 7.2% (v/v) for 15 days. Accelerod performance was recorded before and throughout 20 days of ethanol intake. Mean blood ethanol levels were 266.34+/-13.11 and 285.20+/-9.77 mg/dl on the 7th and 15th days of ethanol (7.2% v/v) consumption, respectively, as measured in a parallel group of animals. Ethanol produced significant concentration-dependent impairments in the accelerod performance of rats. The motor impairment effect of ethanol was most prominent in the test using the greatest rate of acceleration (from 0 to 79 rpm within 2 min). The impairment effect of ethanol on accelerod performance occurred throughout the period of ethanol exposure. Our results indicate that motor impairment on the accelerod performance test produced by an ethanol liquid diet depends on the concentration of ethanol and the rate of acceleration. In addition, under free-access conditions accelerod performance may not be a suitable behavioral test for detecting tolerance development to ethanol in rats.