Ethanol Clearance Rates Research Articles

Circadian rhythm of blood ethanol clearance rates in rats: response to reversal of the L/D regimen and to continuous darkness and continuous illumination.

Phase relationships of the circadian rhythms of blood ethanol clearance (metabolic) rates and body temperature were studied in rats successively exposed to 4 illumination regimens: LD (light from 0800-2000 hr), DL (light from 2000-0800 hr), constant darkness (DD) and, lastly, constant light (LL). After a 4-wk standardization to each regimen, body temperatures were taken at 9 X 4-hr intervals to establish baseline circadian profiles. One week later, groups (N = 8) received 1.5 g/kg ethanol (i.p.) at 6 equally spaced timepoints during a 24-hr span, when temperatures were again measured. Ethanol clearance rates were estimated from decreasing blood ethanol levels sampled every 20 min from 60-200 min after dosing, and the resultant elimination curves were subjected to cosinor analysis. These studies show for the first time that the high amplitude circadian rhythm in ethanol metabolism persists under constant conditions of illumination (DD and LL), demonstrating that it may well be a truly internal circadian rhythm and not a response to exogenous cues of the light/dark cycle. During both LD and DL, maximal and minimal ethanol clearance rates fell near the end of the dark and light phases, respectively, and followed circadian peak and trough control temperatures by approximately 6 hr. A fixed internal phase relationship between the core body temperature and the circadian rhythm in ethanol metabolism is demonstrated, thus establishing the rhythm in body temperature as a suitable and convenient internal marker rhythm for studies of the metabolism of low-to-moderate ethanol doses. These studies demonstrate that the phase relationships of blood ethanol clearance rate and body temperature can be manipulated by the illumination regimen selected, an observation of both basic and practical importance.

Regulation of ethanol metabolism in the rat.

The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 mumol/(min X g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 mumol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.

Effect of Hypothermia on Breath-Alcohol Analysis

Abstract Mild hypothermia, induced by experimental immersion of ten subjects in cold water, distorted the decay curve of breath ethanol of intoxicated subjects by as much as 22% while not altering overall ethanol clearance rate. The results provide in vivo verification of the in vitro temperature correction factor of 6.8% · °C−1, and support previous recommendations that temperature monitoring be included in procedures for breath-ethanol testing. We recommend that mouth temperature be obtained before breath sampling to screen for abnormal body temperature and to allow for potential use of a temperature correction factor. This modification to existing analytical procedures would help to optimize the reliability of breath-ethanol analysis in predicting blood-ethanol concentration.

Assessment of the role of non-ADH ethanol oxidation In Vivo and in hepatocytes from deermice

Deermice genetically lacking alcohol dehydrogenase (ADH −) were used to quantitate the effect of 4-methylpyrazole (4-MP) on non-ADH pathways in hepatocytes and in vivo. Although primarily an inhibitor of ADH, 4-methylpyrazole was also found to inhibit competitively the activity of the microsomal ethanol-oxidizing system (MEOS) in deermouse liver microsomes. The degree of 4-MP inhibition in ADH − deermice then served to correct for the effect of 4-MP on non-ADH pathways in deermice having ADH (ADH +). In ADH + hepatocytes, the percent contributions of non-ADH pathways were calculated to be 28% at 10 mM and 52% at 50 mM ethanol. When a similar correction was applied to in vivo ethanol clearance rates in ADH + deermice, non-ADH pathways were found to contribute 42% below 10 mM and 63% at 40–70 mM blood ethanol. The catalase inhibitor 3-amino-1,2,4-triazole, while reducing catalase-mediated peroxidation of ethanol by 83–94%, had only a slight effect on blood ethanol clearance at ethanol concentrations below 10 mM, and no effect at all at 40–70 mM ethanol. These results indicate that non-ADH pathways (primarily MEOS) play a significant role in ethanol oxidation in vivo and in hepatocytes in vitro.

An evaluation of the role of ethanol clearance rate in the differential response of long-sleep and short-sleep mice to ethanol

Ethanol (ETOH) clearance rates were determined in Long-Sleep (LS) and Short-Sleep (SS) mice, which were selectively bred for differential soporific response to ETOH. Determination of blood ethanol levels at 45 min intervals following administration of 3.8 g/kg IP indicated that SS mice clear ETOH at a faster rate. Repeatedly sampled mice of each line cleared ETOH more slowly than those where samples were taken only once. A second experiment utilized pairs of LS and SS mice matched on body weight. These were treated with one of several doses of ETOH, and duration of loss of the righting reflex measured for one member of the pair. Blood ETOH levels were determined in samples taken from both members of the pair when the tested member regained the righting reflex. LS mice had small but consistently higher blood ETOH levels in these pairs. The magnitude of the differences between the LS and SS mice in these experiments indicate that clearance rate differences can account for only a small portion of the differences in behavioral responses of these mice to ETOH.

Effects of different liquid diets and sustained ethanol release on alcohol metabolism

The effects of two liquid diets, Sustacal and Shorey-AIN, on liver alcohol dehydrogenase (ADH) activity and ethanol clearance were tested in rats under conditions of high ethanol exposure for nine days. High blood ethanol levels (BEL) were produced through a combination of an initial intubated dose of ethanol, sustained ethanol release tube (SERT), and ethanol as 37% of total energy in the liquid diet. Under free-feeding conditions, rats consumed slightly more ethanol per unit body weight in the Shorey-AIN diet, a diet formulated for rodent nutrition, than in the Sustacal diet, a diet originally intended for human consumption. However, BEL were significantly higher in the Sustacal group than in the Shorey-AIN group. No differences in ethanol clearance rates were observed between the groups. On the other hand, total liver ADH activity was significantly reduced in both the Shorey AIN/ethanol and the Sustacal/ethanol groups, compared to lab chow controls. When the Sustacal diet was fortified with casein and methionine so that the protein content matched that of the Shorey AIN diet, the BEL were no longer significantly higher than those produced by the Shorey AIN/ethanol diet. The results demonstrate the effect of nutritional factors on BEL under conditions of high ethanol load. However, these factors do not appear to alter major characteristics of ethanol metabolism and clearance in our short-term experiments.

Circadian variation in blood ethanol clearance rates in female rats

Abstract Pregnant female rats, challenged with a single ethanol dose near the mid‐gestation period, displayed a significant circadian variation in the rates of both blood ethanol clearance and ethanol metabolism. Maximal rates occurred near the dark‐to‐light change of the LD 12:12 regimen. These rhythms may have been synchronized by pregnancy‐induced hormonal cycling since these same animals, challenged as virgins with asynchronous estrus cycling, showed no difference in ethanol metabolic rate over the 24‐h span and circadian variation in blood ethanol clearance rates of questionable significance.

Effects of ethanol: I. Acute metabolic tolerance and ethnic differences.

Forty-five young men were given an initial dose of ethanol calculated to achieve a blood alcohol concentration (BAC) of 100 mg of ethanol/100 ml of blood. Using breath analysis, the rates of ethanol clearance were calculated over a period of a few hours. When each individual reached a BAC level exactly one-half (about 50 mg%) that seen at his peak BAC, he was given a second dose of ethanol amounting to 45% of his initial dose, and the clearance rate was again determined. Estimates of absorption time, peak BAC, volume of distribution, clearance rate, and other metabolic parameters were made separately following each dose of ethanol. As intended, there was no significant difference between peak 1 and peak 2; however, there was a significant increase in clearance rate (beta 60) after dose 2. We term this phenomenon acute metabolic tolerance to ethanol ( AMTE ). When the sample was stratified by ethnicity, there was a trend for men of Oriental ancestry to clear the drug at a faster rate than was the case for men of Caucasian or Polynesian ancestry. The data also indicated that prior drinking history may have had an effect on clearance rate, though this differed by ethnicity. Two different methods for estimating volume of distribution (V) were used. The correlation between the separate estimates obtained was poor, and is probably an indication of the need for development of more accurate methods for estimating V.

Circadian rhythms of alcohol dehydrogenase and MEOS in the rat.

The circadian peak in alcohol dehydrogenase (ADH) activity fell near the time of maximal blood ethanol clearance rates both in groups of rats injected with a single ethanol dose (acute group) and in rats continuously exposed to ethanol for 22 weeks (chronic group). However, at all timepoints investigated ADH activity levels were lower and fluctuated less in the chronic group than in either the acute or control (ethanol naive) groups. In contrast, activity levels of the microsomal ethanol oxidizing system (MEOS) revealed a prominent rhythm that was 180 degrees out of phase with the ADH rhythm in the chronic group, while MEOS activity showed very low levels in the acute and control groups and did not vary over the circadian span.

Factors influencing rates of ethanol oxidation in isolated rat hepatocytes

The stimulation of ethanol oxidation by fructose which has frequently been observed in isolated hepatocytes was found to occur only in unsupplemented cells. In the presence of other substrates (lactate, pyruvate) which accelerate ethanol oxidation, fructose had no additional effect. Acceleration of ethanol oxidation by fructose was not directly related to the ATP demand created by fructose. The effects of fructose on ethanol oxidation rates were not due to changes in acetaldehyde concentration. In cells from fed animals, acetaldehyde concentrations rose as high as 200 μM in some incubations, and therefore became a significant factor limiting ethanol oxidation rates. In hepatocytes isolated from starved rats incubated with pyruvate, where acetaldehyde concentrations were very low (1–2 μM) it was possible to assess the effect of changes in [lactate]/[pyruvate] (and hence free cytosolic NADH) on rates of ethanol oxidation. The results showed that the increase in free cytosolic [NADH] usually found during ethanol oxidation in vivo would inhibit rates of ethanol clearance by a maximum of 20%.

Effect of pulse pressure on fluid exchange between blood and tissues in trout gills

Clearance of ethanol from gill tissues was measured during both pulsatile and nonpulsatile perfusion in an isolated saline-perfused trout head preparation. Pulsatile perfusion increased the rate of ethanol clearance from the respiratory tissues. Compartmental analysis of ethanol washout curves indicated that ethanol clearance during pulsatile perfusion was increased principally by more rapid exchange of ethanol between slow (tissue) and fast (vascular) compartments. Analysis of fluid movements between blood and tissues showed that 25–45% of the total flux could pass from blood to tissues and back within each beat of the heart. This dynamic component represents a previously overlooked paracellular pathway for exchange of water and solutes which is dependent upon pulsatile vascular pressures.

A simple cannulation technique for determination of accurate blood ethanol clearance rates in rats.

A simple cannulation technique for determination of accurate blood ethanol clearance rates in rats.

Meal timing dominates the lighting regimen as a synchronizer of the circadian blood ethanol clearance rate rhythm in rats

Meal timing dominates the lighting regimen as a synchronizer of the circadian blood ethanol clearance rate rhythm in rats

Meal Timing Dominates the Lighting Regimen as a Synchronizer of the Circadian Blood Ethanol Clearance Rate Rhythm in Rats

The effects of timing of a single daily meal on the circadian rhythm of blood ethanol clearance rates were investigated in two groups of rats maintained on opposite 12 hours light:12 hours dark (LD) schedules. Initially, the rats were fed ad libitum. Then feeding was restricted to 4 mid-light (ML) hours for one group and 4 mid-dark (MD) hours for the other. Finally, the meal timing was reversed; the ML-fed group became the MD-fed group and vice versa. Following acclimatization to each of the 3 feeding regimens, blood ethanol clearance rates were determined for subgroups (n = 8) of both groups injected (i.p.) with ethanol at 5 times during 24-hour spans. The LD schedule synchronized the clearance rate rhythm during ad libitum feeding. Although the rhythm persisted without significant amplitude changes during restricted feeding regimens, minimal clearance rates during ML and MD feeding approximated the time of food presentation, with maximal rates 8–12 hours later. This relationship remained constant when the feeding phases were reversed. Thus, when food availability is limited, the single daily meal dominates the lighting regimen as synchronizer of the circadian blood ethanol clearance rate rhythm.circadian rhythm ethanol meal timing

Rates of ethanol disappearance from blood and hypothermia following acute and prolonged ethanol inhalation

An inhalation procedure was employed for the acute and prolonged administration of ethanol. The rate of ethanol clearance from blood of rats following 6 hr of inhalation was significantly faster than the rates of clearance associated with the ip or po routes of administration. Also, the blood clearances from ip and po ethanol differed significantly. However, the rates of ethanol disappearance were constant for a given route, such as inhalation or ip, regardless of the dose administered. Immediately after 10 days of ethanol inhalation, the rate of ethanol elimination was not accelerated when compared to the results obtained for 6 and 24 hr of inhalation. When the animals were again treated for 6 hr with ethanol vapor 48 hr post-10-day exposure, a significant enhancement of the rate of ethanol disappearance from the blood was observed. In another experiment, utilizing a 24-hr period of ethanol exposure, it was possible 48 hr later to separatethe accelerated rate of ethanol clearance from blood and the concomitant loss of hypothermia, which previously had been observed to occur 48 hr after prolonged ethanol inhalation. In addition, the data suggested that an increased rate of ethanol clearance is not a contributory factor to the loss of hypothermia, when these two events occur concurrently. Therefore, it appears that functional tolerance rather than metabolic tolerance was observed. Finally, when various ip doses of ethanol were administered to naive rats, it was found that a minimal peak blood ethanol concentration of 1.48 mg/ml had to be attained to produce hypothermia.

A study on diazepam and postwithdrawal drinking of ethanol solution in rats

A 10-day ethanol inhalation procedure without the use of pyrazole was used to produce physical dependence on ethanol in Sprague-Dawley rats. During this 10-day period, weight changes in the control animals reflected the weight changes in the ethanoltreated animals. At the end of the inhalation procedure the rate of ethanol clearance from the blood was 0.44 ± 0.2 mg/ml/hr in the dependent animals; also, signs of withdrawal were manifested in all ethanol vapor-treated animals, but not in control animals. Twenty-four hours after removal from the chamber, a 5-day drinking study was started. Control animals and physically dependent animals were each divided into two groups, one for diazepam (5 mg/ kg, orally) administration and one for vehicle administration. The results from the free-choice drinking study (water and ethanol solution, 7%) indicated that diazepam did not enhance ethanol consumption in animals which were physically dependent on ethanol but significantly suppressed ethanol drinking in nondependent animals. Also when examining the effect of physical dependence alone on postwithdrawal drinking, it appeared that no increase in ethanol preference was observed with animals under our experimental design.

Physical dependence on ethanol: Rate of ethanol clearance from the blood and effect of ethanol on body temperature in rats

An inhalation procedure for the administration of ethanol without the use of pyrazole was employed to produce physical dependence on ethanol in rats. A reduction in body temperature was noted at the latter part of the induction phase and in the early hours of withdrawal. In the withdrawal phase the animals experienced convulsions on handling and sponataneous signs of withdrawal. To investigate the rates of ethanol clearance from the blood po and ip routes of ethanol administration were used. A significant enhancement of the rate of ethanol disappearance from the blood was measured in the animals at 48 hr following 10 days of ethanol exposure. Also at this 48-hr time period the usual hypothermic response to ethanol administration was lost in these animals as compared to a control group and to animals receiving ethanol ip, at 7 days after exposure to ethanol vapor. The data indicated that the loss of hypothermic effect from ethanol might be a factor in the increased rate of ethanol clearance. However, in studying the rate of ethanol disappearance from the blood in nondependent animals at 34°C which prevented hypothermia, there was no difference in the rates of ethanol clearance as compared to controls at 23.5°C where the hypothermic effect from ethanol was present. It appears that body temperature does not play a role in the observed acceleration of ethanol clearance from the blood in animals which have undergone withdrawal.

Effects of Phenobarbital Administration on Rates of Ethanol Clearance and on Ethanol-Oxidizing Enzymes in Man

Abstract The effects of phenobarbital administration on rates of ethanol disappearance from the blood and on the hepatic activities of the ethanol-oxidizing enzymes were determined in 4 male chronic alcoholic patients without significant clinical evidence of liver disease. Phenobarbital administration resulted in significant increases, both in the rates of ethanol disappearance from the blood and in the concentration of protein per gram of liver wet weight; however, no changes were observed in the activities of either alcohol dehydrogenase or the nicotinamide adenine dinucleotide phosphate-dependent ethanol-oxidizing system determined in liver homogenates. On the other hand, the urinary excretion of D-glucaric acid, an index of hepatic enzyme induction, was increased. While this study demonstrates an enhancement of ethanol clearance from the blood after phenobarbital administration, the mechanism for this enhancement remains uncertain.

Factors involved in ethanol narcosis: Analysis in mice of three inbred strains

We have studied the effects of genotype and dose on the time of onset of ethanol-induced sleep, as measured by fall time, and on the length of sleep. We have also investigated the relationships among genotype, dose and the blood ethanol levels at time of fall and time of awakening in three inbred mouse strains (C57BL/6J, DBA/2J and BALB/cJ). The sleep-time dose response curves are linear for the dose range tested in all three strains. There was no significant linear correlation between dose and blood ethanol level at awakening in any of the three strains. Between-strain comparisons showed significant differences in rate of ethanol clearance from the blood, and differences in tissue sensitivity to ethanol among the strains were demonstrated. Our data suggest that the major factor influencing sleep time is blood alcohol clearance, reflecting differences in alcohol metabolic rates. Between-strain comparisons of fall time showed significant differences, over the dosage range tested, in nervous system tissue sensitivity (as inferred from blood alcohol level at time of fall) between the BALB/cJ strain and the C57BL/6J and DBA/2J strains. Differences between C57BL/6J and DBA/2J strains were significant only at the lowest dose tested. The rank-ordering of the strains with respect to tissue sensitivity to ethanol is identical for all three tissue sensitivity measures obtained in these experiments.

Effects of Ethanol and Fat on the Transport of Reducing Equivalents into Rat Liver Mitochondria

Abstract Chronic ethanol intoxication in rats accelerated the rate of ethanol metabolism, but decreased the of alcohol dehydrogenase. Chronic consumption of ethanol decreased the activities of cytochrome oxidase and succinic dehydrogenase in mitochondria and in cell-free homogenates, whereas total hepatic protein was not altered by ethanol feeding. Thus, total mitochondrial activity was decreased and cannot account for the increased rate of blood ethanol elearance. We therefore investigated the activities of enzymes and those of systems for the transport of reducing equivalents into mitochondria. Reconstituted malate-aspartate, fatty acid, and α-glycerophosphate shuttles were equally effective in transporting reducing equivalents into the mitochondria from ethanol-fed and control rats. The activities of enzymes involved in the shuttles, such as cytoplasmic and α-glycerophosphate dehydrogenase and glutamic oxalacetic transaminase, were either decreased or unchanged by chronic ethanol consumption. The permeability of the mitochondria to NADH and to substrate anions which participate in the shuttles was also not altered by ethanol feeding. It is therefore unlikely that the increased rates of ethanol oxidation produced by chronic ethanol consumption are caused by enhanced transport of reducing equivalents into the mitochondria. A high fat diet alone (35% of total calories) increased both the endogenous and total rates of shuttle activity, compared with the rates obtained with mitochondria from rats which had consumed a low fat diet. However, the rates of blood ethanol clearance were comparable in rats consuming high fat and low fat diets. Therefore, the transport of reducing equivalents into the mitochondria under these conditions does not appear to be rate-limiting for ethanol metabolism.