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The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

The kinetic V/K isotope effect upon peroxidation of (1R)-[1-2H1]ethanol by liver catalase was studied by using a radiochemical assay with both 3H ad 14C as tracers. The value of 1.9 found is in agreement with the value of 2.52 for peroxidation of (1R)-[1-3H1]ethanol according to the 'Swain equation' [Swain, Stivers, Ruver & Schaad (1958) J. Am. Chem. Soc. 80, 5885-5893]. There was no isotope effect on V, the catalase haem up to 500 min-1. Even at rates 20-fold higher the isotope effect on V may not be different from 1. A detailed description of the synthesis of the 2H- and 3H-substituted ethanol compounds and the analysis of these has been deposited as Supplementary Publication SUP 50110 (5 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169,5.

1. Rats were treated for 4 weeks with liquid diets that contained, on the basis of energy content, 35% fat, 18% protein and 47% carbohydrate (high-fat diet) or 35% fat, 18% protein, 11% carbohydrate and 36% ethanol (high-fat/ethanol diet). 2. The livers were perfused with 1mm-[1-(14)C]palmitate and with 0, 10mm- or 80mm-ethanol. The oxidation and esterification of palmitate was measured. Two subcellular pools of triacylglycerol were separated; one contained triacylglycerol from cytoplasmic lipid droplets and the other contained triacylglycerol from the endoplasmic reticulum and Golgi apparatus. 3. In the presence of ethanol, liver from rats fed on the high-fat diet esterified about 70% of the [1-(14)C]palmitate taken up compared with 90% in liver from rats fed chow (containing 11% fat on the basis of energy content). Compared with chow diet the high-fat diet did not potentiate the effect of ethanol on storage of [1-(14)C]palmitate in hepatic triacylglycerol. The relation between the fat content of the diet and the degree of fatty liver induced by by ethanol [Lieber & DeCarli (1970) Am. J. Clin. Nutr.23, 474-478] is discussed. 4. The ethanol-containing diet increased the hepatic content of triacylglycerol 4-fold and the increase was exclusively found in the fraction suggested to contain lipid from cytoplasmic lipid droplets. The ethanol-induced fatty liver, perfused with ethanol, esterified and oxidized palmitate at rates that were quite similar to the rates found in high-fat control livers perfused without ethanol. This suggests that the fatty liver had adapted to the presence of ethanol with respect to palmitate metabolism. 5. O(2) and ethanol uptake by the livers were not affected by the ethanol-containing diet.

Determination of acetaldehyde in human blood by the semicarbazide method has been studied. An artefactual production of acetaldehyde from ethanol in blood and plasma of about 20 mumol/l was observed at concentrations of ethanol of about 60 mmol/l. This artefact was reduced to less than 1 mumol/l after addition of thiourea. The presumably non-enzymatic production of acetaldehyde from ethanol was demonstrated by the release of 3H from (1R)-[3H]ethanol added to the blood immediately after sampling. The results demonstrate that oxidation of ethanol is the major cause of the artefactual acetaldehyde formation. In human volunteers, metabolising ethanol, very low concentrations of acetaldehyde were found by the modified method, which includes thiourea.

1. In the preceding paper [Kondrup (1979) Biochem. J.184, 63-71] the separation of two major fractions of hepatic triacylglycerol was described. One fraction contained triacylglycerol from the endoplasmic reticulum and from the Golgi apparatus. The other fraction contained triacylglycerol from the cytoplasmic lipid droplets. In the present paper possible precursor-product relationships between the two fractions were investigated by means of computer models. 2. The fatty acids present in di- and tri-acylglycerol in the fractions isolated in the time studies were analysed by gas chromatography. From this analysis the relative specific radioactivities, and contents, of palmitate in acylglycerols in the two fractions at the various time points were calculated. 3. A computer was used to predict relative specific radioactivities of pools in defined models of hepatic triacylglycerol metabolism. The acceptability of the models was evaluated by comparing predicted with measured relative specific radioactivities. 4. It is suggested that triacylglycerol in cytoplasmic lipid droplets does not originate (a) directly from triacylglycerol in the endoplasmic reticulum, (b) from a sub-pool of it or (c) directly from non-esterified fatty acids entering the cell. Rather, it is formed from diacylglycerol (and acyl-CoA) in the endoplasmic reticulum. Diacylglycerol, on the other hand, is furnished in part by hydrolysis of triacylglycerol in the endoplasmic reticulum. 5. This suggestion is discussed in relation to previous models of hepatic fatty acid metabolism.

Some alcoholics have a regular daily alcohol consumption of more than 100 g. In preliminary observations we had the impression that the claimed alcohol intake in such 'heavy drinkers' was higher than could be accounted for by the ethanol elimination rate as measured routinely at 10 mmol/l (0.5 g/l). We therefore measured the ethanol elimination rate at very high blood ethanol concentrations of 40-80 mmol/l (2-4 g/l) found in eight alcoholics following heavy alcohol intake by measuring the falling blood ethanol concentrations until being less than 1 mmol/l. The elimination rate, on average 83 mumol/min per 1 blood, was about 49% higher than the elimination rate measured at 10 mmol/l in the same subject, being on average 58 mumol/min per 1/blood (paired t-test, P less than 0.05). The elimination rate following the high initial ethanol concentrations remained high until the concentration was below 5 mmol/l. Calculations of elimination rates are based on a number of assumptions concerning the physiologic and metabolic conditions. We examined specifically if the concentration-time curves could be adequately described by assuming metabolism according to a Michaelis-Menten pathway with a low Km value (simulating alcoholdehydrogenase with Km 0.2 mmol/l) or by assuming metabolism by two pathways with an alternative high-Km pathway with Km about 10 mmol/l. It was not necessary, in the statistical analysis, to include an alternative high-Km pathway. On the other hand, the data does give room for up to 50% elimination via such alternative pathways. The elimination rate at the high concentrations corresponded roughly to the claimed daily alcohol intake; furthermore the measured elimination rate at the lower concentrations were similar to values in non-alcoholics.

1. Simultaneous determination of the rate of appearance of 3H in water from [(1R)-1-3H1] ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37 degrees C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25 degrees C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. 2. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the two rates measured in the assay. 3. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supports the view that both values are close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. 4. The constancy of the isotope effect under various conditions makes it possible to use it for interpretations in vivo. 5. It was established that beta-D-galactose dehydrogenase exhibits B-specificity towards the nicotinamide ring in NAD.

1. The effect of ethanol on the metabolism of [1-(14)C]palmitate in rat liver was investigated in a single-pass perfusion system at concentrations of 10mm- or 80mm-ethanol and 0.2mm- or 1mm-palmitate. 2. After the perfusion the hepatic lipid was isolated in subcellular fractions. The two major fractions contained triacylglycerol from cytoplasmic lipid droplets and from endoplasmic reticulum plus Golgi apparatus respectively. 3. In experiments with 0.2mm-palmitate perfusion with 10mm- or 80mm-ethanol did not measurably increase the esterification, and the oxidation was markedly decreased and the fatty acid uptake was not affected. 4. Perfusion with ethanol, at 1mm-palmitate, increased the fatty acid uptake, increased esterification and decreased oxidation. The effects of 10mm- and 80mm-ethanol were similar. The incorporation of [1-(14)C]palmitate into triacylglycerol in cytoplasmic lipid droplets was not affected statistically significantly by ethanol. Ethanol increased the incorporation of [1-(14)C]palmitate into di- and tri-acylglycerol in the membranous fraction. Estimated chemically, the contents of di- and tri-acylglycerol were only slightly affected by ethanol. These results suggest that the effect of ethanol was to increase the turnover of fatty acids in triacylglycerol rather than to increase its accumulation. 5. The results indicate that an increased concentration of fatty acids is more important for the formation of acute fatty liver in fed rats than are the direct effects of ethanol on hepatic fatty acid metabolism.