GASTROENTEROLOGY

1991;101:1716-1723

Intraacinar Profiles of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Activities in Human Liver IRENEUSZ Anatomisches

P. MALY and DIETER SASSE Institut, Basel, Switzerland

The intraacinar activity profiles of alcohol dehydrogenase and the aldehyde dehydrogenases (I, I plus II, and total) were determined, using liver biopsy samples from eight male and eight female patients. Microchemical assays were performed in microdissected tissue samples from the whole length of the sinusoid. Alcohol dehydrogenase activity in men < 53 years of age showed a maximum in the intermediate zone, whereas in women < 50 years of age an increase in the gradient toward the perivenous zone was observed. Furthermore, alcohol dehydrogenase activity in the livers of women was significantly higher than in men. After the age of 53 in men and 50 in women, the sex specificity of the distribution profiles was no longer apparent. The intraacinar profiles of aldehyde dehydrogenase isoenzymes showed only minor variations in the different groups; they were not statistically significant. This is also true for low-Michaelis constant (KJ aldehyde dehydrogenase, which is most important for acetaldehyde oxidation in vivo. Thus, of the variations in zonal heterogeneity of ethanol-degrading enzymes, it is mainly the activity of alcohol dehydrogenase that may contribute to the sex- and age-related susceptibility of liver parenchyma.

here are many reports in the literature confirming that hepatocytes are functionally heterogeneous, depending on their position within the microvasculatory unit, i.e., the liver acinus (1,~). Quantitative measurements of enzyme activity in the different acinar zones have not only allowed reasonable interpretation of antagonistic metabolic processes such as gluconeogenesis and glycolysis, but they have also contributed to the understanding of zonal differences in the susceptibility to various injurious agents. The characteristic finding that alcoholic liver injury is at its onset not ubiquitous but favors the perivenous

T

zone (3) also seems to suggest a heterotopic distribution pattern of alcohol metabolizing enzymes. Ethanol is degraded predominantly in the liver, where the first step is catalyzed by alcohol dehydrogenase (ADH). Alternative pathways are related to the microsomal ethanol oxidizing system (MEOS) and to peroxisomal catalase, although their relative contribution to ethanol degradation in humans is not fully resolved (4). The product of these enzyme activities is acetaldehyde, which serves as a substrate for the different isoenzymes of aldehyde dehydrogenase The major role in the oxidation of (ALDH) (5,6). ethanol-derived acetaldehyde is played by ALDH I, with its low Michaelis constant (K,,,) in the micromolar range. Various techniques, mostly in rats, have been used to determine differences in the metabolic activity of ADH and ALDH within the liver parenchyma. Qualitative histochemistry (7-g), immunohistochemistry (lO,ll), separation of periportal and perivenous cells by different perfusion techniques (12-l 7), and centrifugation techniques (18,19) have all been used but yield contradictory results. Thus, periportal and perivenous maxima, as well as an even intraacinar distribution of ADH have been described. However, because every method providing quantitative data allows only indirect conclusions of the former intraacinar position of the respective samples, many of the conflicting results may be related to the uncertainty of localization. At present, only the technique of microdissecting intraacinar strips of tissue is capable, under visual control, of relating quantitative data on

Abbreviations used in this paper: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; BSA, bovine serum albumin; DTT, ditbiothreitol; NADH, nicotinamide adenine; dinucleotide phosphate; pp, periportal; pv, perivenous. o 1991by the American Gastroenterological Association 0016-5085/91/$3.00

December 1991

ALCOHOL AND ALDEHYDEDEHYDROGENASEIN HUMAN LIVER 1717

ADH and ALDH activity to defined parts of the human liver parenchyma (20-22). Using these microquantitative methods, exact intraacinar activity profiles of ADH and of three different isoenzymes of ALDH could for the first time be determined and sex- and agedependent variations be shown.

Materials and Methods Human liver biopsy specimens were obtained from the Department of Surgery after approval by the Ethical Committee of the Medical Faculty of the University of Basel. Liver tissue was obtained from eight male and eight female patients, none of whom had any metabolic disease or were malnutritioned. None of the women used pharmaceutical contraceptives. The livers of the men (16, 24, 28, 52, and 54 years of age] and of the women (42, 46, and 64 years of age) were removed from patients who had suffered traumatic head injury and whose kidneys were designated for transplantation. The other patients (men: 36, 54, and 56 years of age; women: 31,40, 51,and 58 years of age] underwent surgery for cholelithiasis, with the exception of one woman, 54 years old, whose liver was examined after splenectomy for the staging of Hodgkin’s disease. Only liver tissue without pathohistological findings was included in this study. The tissue was immediately frozen in liquid N, and stored in air-tight tubes at -80°C until required. Preparation

of Tissue Samples

Fifteen-micrometer sections were cut at a cryostat temperature of -20°C. Zonal heterogeneity was delineated by the histochemical demonstration of glucose-6-phosphatase activity (23). Sequential sections were lyophilized under vacuum (5 x lo-"torr) at -40°C for about 24 hours (24). Comparison between stained sections and the corresponding lyophilized sections allowed the recognition and precise microdissection of periportal-to-perivenous (pp/pv) strips of tissue. To avoid possible errors caused by differences in the width of the strips, it is necessary to attain exactly parallel edges. Because the beginning of each strip is defined by the terminal afferent vessels and the end by the terminal efferent venule, care was taken to ensure that the strips contained neither vessel wall nor surrounding connective tissue. Seven to eight pp/pv strips were microdissected from each patient. Each strip was then subdivided into five subsequent samples of 50-150 ng dry wt, as determined on a quartz fiber balance. The recorded weight was reduced by 2% to allow for the uptake of N, and 0, and by a further 1% for each 10% of the recorded relative humidity to allow for the uptake of H,O.

ing 0.5PL reagent 1 (in the ADH assay] or reagent 3 (in the ALDH assay] was inserted into each oil droplet. The tissue samples were subsequently introduced through the oil into the medium.

Enzymatic Assay for Alcohol Dehydrogenase In preliminary studies it was observed that the molarity of the Tris HCl buffer (100 mmofi) used for the ADH assay in rat liver (20) was insufficient to inhibit human low-K, ALDH. The concentration of this buffer was therefore increased to 300 mmol/L, which is sufficient to bind acetaldehyde (25). The tissue samples were “dissolved” under oil in reagent 1 [300 mmol/L Tris HCl buffer, pH 7.4; 0.02% bovine serum (DTT); 1.0mmol/L albumin (BSA); 1 mmol/L dithiothreitol amytal; and 2.8 mmol/L nicotinamide adenine dinucleotide (NAD)]. After a preincubation of 15 minutes at 4”C, the holders were warmed to 37”C, and the specific reaction was started by the addition under oil of 0.5 pL of reagent 2 (300 mmol/L Tris HCl buffer, pH 7.4; 0.02% BSA; 1 mmol/L DTT; 1.0 mmol/L amytal; 2.8 mmol/L NAD; and 20.0 mmol/L ethanol). Thereafter, the brass holders were kept at 37°C for a further 20 minutes, and the reaction was finally stopped by the addition of 4 PL 2-mmol/L 4-methylpyrazole in 0.2N NaOH and by heating to 70°C fi>r 15 minutes. For the determination of tissue blanks, reagent 2 was used without ethanol.

Enzymatic Assay for Aldehyde

Dehydrogenase

Preliminary studies had shown that treating the tissue samples under oil with 0.3% sodium deoxycholate leads to the release of maximum total ALDH activity without inhibition of the low-K, ALDH. In this assay, the tissue samples were introduced through the oil into 0.5 FL reagent 3 (70mmol/L sodium pyrophosphate buffer, pH 8.0; 1.5mmol/L NAD; 5.0 mmol/L 4-methylpyrazole; 1.0 mmol/L amytal; 0.02% BSA; and 5.0 mmol/L DTT) at 4°C.After 10 minutes, 0.5 PL of reagent 4 were added (reagent 4 = reagent 3 + 0.696 sodium deoxycholate). Preincubation was then continued for additional 20 minutes at 4°C. Thereafter, the specific reaction was started by adding 4 FL reagent 5 [70 mmol/L sodium pyrophosphate buffer, pH 8.0;1.5mmol/L NAD; 5.0 mmol/L methylpyrazole; 1.0 mmol/L amytal; 0.04% BSA; 5.0mmol/L DTT; and either 37.5Fmol/L acetaldehyde (ALDH I) or 1.875 mmol/L acetaldehyde (ALDH I + ALDH II) or 18.75 mmol/L acetaldehyde (total ALDH)]. For the determrnation of tissue blanks, reagent 4 was used without substrate. Incubation time was 20 minutes at 37°C. The reaction was stopped by the addition of 4 PL 0.2 NaOH and heating tl3 70°C for 15 minutes.

Procedure All reactions took place in the hollow caps of propylene microtubes arranged serially in small brass holders. Each of these cavities was filled with 10 FL silicon oil (specific gravity, 1.12;Silopren K 1000;Fluka AG, Buchs, Switzerland). Under a stereomicroscope, a droplet compris-

Luminometric

Measurements

The amount of specifically produced nicotinamide adenine dinucleotide phosphate (NADH) was measured by recording the stable level of light emitted as a result of two coupled enzyme reactions. The first is catalyzed by NADH-

1718

MALY AND SASSE

GASTROENTEROLOGY

specific flavin mononucleotide oxidoreductase and the second, the light-producing reaction, by bacterial luciferase (NADH monitoring kit, LKB 1243-103; LKB, Lucerne, Switzerland). Initially, 65 I_LLlOO-mmol/L potassium phosphate buffer, pH 6.8 (for ADH), or 61 p,L lOO-mmol/L potassium phosphate buffer, pH 6.8 (for ALDH), was added to the medium under oil and mixed. Thereafter, an aliquot of 50 t.r,Lwas taken with a Hamilton diluter and transferred to the assay cuvettes and diluted with 150 ~.LLof the same buffer. Immediately before luminometric determination, the cuvettes were warmed to 25°C for 10 minutes in the luminometer; 50-PL NADH monitoring kit was then added and mixed in the instrument, and the instantaneous light output was continuously recorded with a LKB-Wallac 1250 luminometer coupled to a recorder and a printer. When emission had reached a constant value, 10 ~.LL1 kmol/L NADH (standard) was added by means of the 1291 LKB dispenser, and the further increase in light emission was recorded until the next constant level was reached. For the calculation of liver ADH- and ALDH-specific NADH production, reagent blanks were determined, using the whole incubation medium, but without tissue samples. Tissue blanks were determined by measuring the light emission of the incubation medium plus the tissue sample, but without substrate. The values of the tissue blanks are a function of the mass and incubation time only, independent of the acinar localization. The rate of NADH production in the tissue blank, V, (pm01 . min-’ . g-‘) is given by

I, - I*

vt= NmHX 1

m

nNADH

X-Xl4 t

.t

are light emissions from the tissue where I, I, and INADH blank, reagent blank and NADH standard, respectively (mV); m is mass of tissue (g, as dry weight); n is amount of NADH standard (pmol); and t is time (minutes). The rate of NADH production in the sample, V, (Km01 . min-’ . g-‘), is given by

v, =

4- 4 x

1

NADH

’

m

%ADH t

x

1.4 - v,,

where 1, is the light emission (mV) from the sample.

Graphics To show the microquantitative distribution profiles of ADH and ALDH activity in the liver acinus, the total mass of the subunits of each microdissected strip was related to 100% of the whole sinusoidal pplpv distance. According to its mass, each subunit represented a certain part of this total length. In this way it was possible to plot the enzyme activity of each sample against its relative size and position within the acinus, thus creating the profile for each microdissected strip. Strips from individual patients or a group of patients could then be summated. The pplpv length on the abscissa was subdivided into 200 points, for each of which the mean and standard deviation of the enzyme activity was calculated and plotted by computer. Results were statisti-

Vol. 101, No. 6

cally analyzed by analysis of variance (ANOVA) to assess the influence of age and sex on the mean activity. Only ADH activity showed a significant result (P < 0.01); additional unpaired t tests were performed.

Results Alcohol Dehydrogenase The determination of total ADH activity in the livers of 16 patients showed a marked increase of activity in men after the age of 53 and in women after the age of 50. This increase in enzyme activity is statistically significant (P < 0.001). Furthermore, ADH activity shows a significant sex difference; in the livers of younger women, enzyme activity is higher by 22% than in younger men. This difference is no longer detectable in older persons (Table 1). Further age- and sex-related differences become evident in the intraacinar distribution patterns of ADH activity. Men < 53 years. The intraacinar distribution pattern shows ADH activity at the periportal beginning to have a value of 7. I 3 + 0.29 pmol . min-’ . g-’ (Figure lA). Activity then increases to an intermediary maximum of 12.62 + 2.02 kmol . min-’ . g-‘. Towards the perivenous end, activity decreases to a value of 11.05 + 1.43 kmol * min-l . g-‘. The intermediary maximum surpasses the periportal values by a factor of 1.77 and the perivenous values by a factor of 1.14 (minimum/maximum difference, P < 0.001). Men > 53 years. The distribution profile of ADH activity shows a gradual increase along the sinusoid (Fig 1B). The periportal values of 10.66 & 2.11 pmol . min-’ . g-’ climb to the perivenous maximum of 19.73 + 2.22 kmol . min-’ . g-‘, which is thus higher by a factor of 1.85 (minimum/maximum difference, P < 0.001). Women < 50 years. The intraacinar distribution of enzyme activity shows a distinct perivenous maximum (Figure 1C). In the periportal zone, ADH activity was determined at 7.87 -+ 1.51 pmol . min-’ * g-‘; from there activity increases to a perivenous value of 16.65 + 2.41 p,mol *min-’ . g-‘, which amounts to a factor of 2.11 (minimum/maximum difference, P < 0.001). Women > 50 years. The distribution profile of ADH activity shows highest values in the perivenous zone of the liver acinus (Figure 1D). Periportally ADH was determined at 12.05 ? 2.6 kmol . min-’ . g-‘; activity then increases to values of 20.76 + 1.81 pmol . min-’ . g-’ at the perivenous end of the sinusoid. This maximum surpasses the periportal values by a factor of 1.72 (minimum/maximum difference, P < 0.001).

December

1991

ALCOHOL

AND ALDEHYDE

DEHYDROGENASE

IN HUMAN

LIVER

1719

Table I. Activities ofAlcohol Dehydrogenase, Aldehyde Dehydrogenase I, Aldehyde Dehydrogenase I Plus II, and Total Aldehyde Dehydrogenase in Human Livers Calculated From All Microdissected Samples of Individual Liver Biopsy Specimens ADH activity Age

(pm01 mine’

Groups

(yr)

g-‘)

1. Mean 53yr 3. Women 50yr

ALDH I activity”

ALDHI+II activityb

(pm01. min-’ Mean

f SD

10.85 k 1.34

16.35 k 2.05

13.21 k 2.01

16.66 2 2.68

g-7 11.86 f 1.00 12.58 + 1.43 14.86 k 1.41 12.87 Il.24 12.70 ?I0.95 16.03 + 1.31 12.91 2 1.37 9.86 2 1.65 7.43 + 0.58 12.42 2 1.70 10.73 k 1.01 9.15 k 0.69 13.36 k 1.05 10.35 2 0.72

Total ALDH activity’

(pm01 mine’ . Mean

2 SD

12.97 k 1.12

12.93 + 3.09

9.93 2 2.14

11.91 ? 1.23

11.96k 0.48 11.95k 0.98

(pm01 min.’ Mean

g-‘1 22.77 k 1.63 19.53 " 1.31 27.46 2 1.46 22.79 IT 1.93 29.20 " 2.13 22.24 -+ 2.16 21.77 -t 1.74 19.85 + 2.01 15.52 ? 1.06 22.69 -c 2.22 16.00 t 1.45 21.75 k 2.18 22.98 f 2.29 19.79 -c 1.54

k SD

24.35 rt 3.92

21.29 2 1.27

18.99 ? 3.75

21.16 zip 2.83

17.86t 0.90 23.99+ 2.77

g-7 26.46 f 2.13 30.65 k 4.03 32.43 2 0.73 29.69 2 3.31 29.18 ? 1.66 35.77 f 2.78 29.85 -fr 2.18 29.21 _' 2.28 21.73 k 1.42 28.06 rk 2.62 29.53 zi 3.26 28.03 I!I 2.97 29.72 t 1.74 27.15 + 1.20

Mean

t SD

29.68 rt 2.19

31.61 2 3.62

26.84 ? 3.48

28.28 2 1.27

27.272 1.64 28.97t 2.14

NOTE. Significance of groups: 1:2, P < 0.001; 3:4, P < 0.001; 1:3, P < 0.001; 2:4, NS; 1:4, P < 0.001; and 2:3, I’ < 0.001. “Amount of acetaldehyde, 30 pmol; ?.5 mmol/L; "15mmol/L.

Aldehyde

Dehydrogenase

I

The activity of low-K,,, ALDH I with 30 p,mol/L acetaldehyde as substrate shows no statistically significant differences between the groups (Table 1). Men < 53 years. Enzyme activity shows minimum values of 12.04 +- 1.70 p.mol . min’ . g-’ at the periportal beginning of the sinusoid (Figure 2A). The maximum is in the intermediary part of the acinus (13.69 ? 0.88 p_mol . mini’ . g-l); from there the values remain at this level until the perivenous end (13.07 ‘-c 1.70 pmol . min-’ . g-‘). The maximum surpasses the minimum by a factor of only 1.14 (minimum/maximum difference, not significant). Men > 53 years. There is a rather flat distribution profile (Figure 2B). Minimum values are found in the periportal zone (12.17 + 3.35 prnol . min-’ . g-‘); themaximumvalues (13.81 + 3.88 Fmol . mini’ . g-‘) are only higher by a factor of 1.13 (minimum/ maximum difference, not significant). Women < 50 years. Enzyme activity shows a gradual increase from lowest values (7.91 + 1.03 Km01 . min’ . g-‘) in the periportal zone to highest values (11.24 k 2.50 pmol . min-’ . g-‘) at the beginning of the perivenous zone (Figure 2C). From there enzyme activity again decreases toward the sinusoidal end. The maximum differs from the minimum by a factor of 1.42 (P < 0.001). Women > 50 years. The intraacinar distribution profile shows lowest values of 10.77 * 2.53 pmol . min-’ . g-’ in the periportal zone (Figure 2D). At the end of the sinusoidal length, maximum values of

12.86 ? 2.09 kmol . min-’ . g-’ are reached. This perivenous maximum is higher than the periportal minimum by a factor of only l:t9 (P < 0.05).

Aldehyde

Dehydrogenass

I Plus II

The activity of ALDH I plus II with 1.5 mmol/L acetaldehyde as substrate shows no age- or sexspecific differences (Table 1). Men < 53years. Enzyme activity shows lowest values in the periportal zone (22.92 + 4.39 km01 . min-’ . g-l). Maximum values are attained in the intermediary part with values of 25.28 +- 3.85 km01 . min’ . g-‘. Activity r:hen remains on this plateau until the perivenous end of the sinusoid. The maximum differs from the minimum by a factor of 1.1 (P, not significant). Men > 53 years. There is a flat peak at the perivenous end. Periportal values amount to 19.33 -+ 1.44 pm01 . min-’ . g-'. In the perivenous zone they are higher by a factor of only 3.18 (P < O.OOl), with values of 22.86 ? 1.54 p,mol . min’ . g-‘. Women 50 years. The intraacinar distribution profile is almost flat; the values in the periportal

1720 h4ALYAND

24

ADH,

SASSE

men

GASTROENTEROLOGY

years

c.53

A

24

ADH,

men>53

Vol. 101,No. 6

6

years

Figure 1 Intraacinar distribution profiles of liver ADH activity, mean (black) it SD.

A. Men 53 years old (three patients). Profile is calculated from 100 microdissected samples from 20 pp/pv strips of tissue.

D

24

C. Women 50 years old (four patients). Profile is calculated from 140 microdissected strips from 28 pp/pv strips of tissue.

PV

ALDH

I,

men53

ALDH

I,

women>50

years

6

201816-

Figure z Intraacinar distrilmtion profiles of liver ALDH I, 30 pmol/L acetaldehyde. Mean (black) 2 SD.

A. Men

53 years old (three patients). Profile is calculated from 105 microdissected samples from 21 pp/pv strips of tissue.

ALDH

I,

womenc:SO

years

C

years

D

C. Women 50 years old (four patients). Profile is calculated from 150 microdissected strips from 30 pp/pv strips of tissue.

PP

PV

December

ALCOHOL AND ALDEHYDE DEHYDROGENASE IN HUMAN LIVER

1991

zone (20.11 rfr 2.99 prnol s min-’ . g-‘) are lower by a factor of only 1.15 than the maximum in the perivenous zone (23.132 4.62Fmol . min-’ 1g-l). Total Aldehyde

Dehydrogenase

Total ALDH activity, determined with 15 mmol/L acetaldehyde, differs only slightly between the male groups of different ages. The higher activity in older women than in younger women is also not significant. In neither sex do the older age groups differ from the younger ones (Table 1). Men < 53years. The distribution pattern shows a periportal activity of 28.04+ 2.76pmol 1 min-’ . g-‘. A relative maximum is attained in the intermediary part of the acinus with values of 31.52-t2.81pmol * min-’ . g-‘, and activity then decreases again toward the perivenous end (29.48 ? 3.12pmol * min-’ . g-l). Total ALDH activity in the intermediary part is therefore higher by a factor of 1.12 compared with the periportal zone and by a factor of 1.07 compared with the perivenous zone (P, not significant). Men > 53years. The distribution profile shows a flat maximum at the perivenous end. The periportal values are 30.29 + 3.56 pmol . min-’ . g-‘; the perivenous values are 33.28 + 3.83 pmol *min-’ *g-‘. This makes up a factor of only 1.1, and the minimum maximum difference is not significant. Women < 50years. A gradual increase of activity is evident. From lowest values in the periportal area (24.43 + 3.43 krnol . min-’ . g-l), activity increases toward the perivenous end, where values of 29.22 ? 5.18 krnol . min-’ . g-’ are attained. The minimum/maximum difference is 1.2 (P < 0.05). Women > 50 years. The distribution profile is nearly flat. Periportal values of 27.03 ? 1.18 kmol . min-’ . g-’ are only slightly lower than the perivenous maximum (29.73 + 2.13 prnol. min-’ *g-l). This makes up a factor of only 1.1, and the difference is not significant. Discussion

Most of the data on ADH activity in human liver were obtained under divergent assay conditions. The values presented here are in agreement with measurements of crude tissue homogenates made by von Wartburg and Papenberg (26) and by Zorzano et al. (27). Using microquantitative techniques, the first study on the intraacinar localization of ADH activity in the human liver was performed by Morrison and Brock (28). However, the values of periportal and perivenous samples from a 47-year-old man and a K&year-old woman were combined, and a general perivenous ADH maximum was described. Later, using immuno-

1721

histochemical techniques, the perivenous maximum of ADH was confirmed. However, in this case the age and sex of the patient were not quoted (10). Once different intraacinar profiles of ADH activity in rats due to sex had been described (20,21), it became reasonable to look for similar patterns in the human liver. In this study it has been shown that in men < 53 years of age ADH activity was maximum in the intermediate zone, whereas women < 50 years of age show an increasing gradient of ADH activity from the periportal to the perivenous zone. There are, however, not only differences in distribution profiles; the total ADH activity is also significantly higher in women than in men. These results seem to be at variance with the findings of Arthur et al. (29) who found similar elimination rai.es of ethanol in both sexes. But it has to be remembered that, depending on the stage of the menstrual cycle, the elimination of ethanol in women can be either faster or slower than in men (30). The main sex difference becomes evident when the intraacinar profiles of ADH activity are compared. In women, the perivenous activity shows a 1.5 times higher capacity for ethanol degradation than that of the same zone in younger men. Consequently, the hepatocytes of this parenchymal area should, after ingestion of ethanol, be exposed to higher concentrations of acetaldehyde and/or a more pronounced shifting of the redox status. These factors are considered to contribute to the higher susceptibility of the female liver to ethanol damage (31,32). Age was also found to influence ADH activity to a significant degree. In this respect both sexes showed an increase in ADH activity. Moreover, in men after the age of 53, this increase in enzyme activity was accompanied by a change in the distribution profile, which then became similar to the intraacinar pattern in women. On the basis of this alteration of the ADH profile, it was possible to discriminate the two age groups in men; in a similar manner a subdivision was made in the female group before and after the age of 50. When comparing ADH activity in the groups of younger and older persons, one finds an increase of 50% in men (P < 0.001) and an increase of 15% in women (P < 0.001). Moreover, in older men the ADH activity of the perivenous zone is increased by 78% with all the consequences mentioned above. In spite of the higher ADH activity in older persons, it has been reported that rates of ethanol elimination are not affected by age (33). This observation is not necessarily contradictory to our results, because it is not only ADH activity that is responsible for the rate of ethanol elimination but also the capacity for NADH reoxidation (34). This might be different in the periportal and perivenous zones; such an assumption is supported by results of investigations in baboons,

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Received June 21,199O. Accepted July 17,1991. Address requests for reprints to: Prof. Dr. Dieter Sasse, Anatomisches Institut, Pestalozzistrasse 20, CH-4056 Basel, Switzerland. This work was supported by the Schweizerische Stiftung fiir Alkoholforschung. We are greatly indebted to Prof. Dr. F. Harder, Head of the Department of Surgery, Basel, for his decisive cooperation; we also thank M. Arnold for her skillful technical assistance, P. Krause for typing the manuscript, and Dr. F. Steel for his help with the English text.