Human liver aldehyde dehydrogenase: Subcellular distribution in alcoholics and nonalcoholics

Alcohol, Vol. 5, pp. 73-80. ©Pergamon Journals Ltd., 1988. Printed in the U.S.A.

0741-8329/88 $3.00 + ,00

Human Liver Aldehyde Dehydrogenase: Subcellular Distribution in Alcoholics and Nonalcoholics D. M E I E R - T A C K M A N N , G . C. K O R E N K E , 1 D. P. A G A R W A L 2 A N D H. W E R N E R G O E D D E 3

Institute o f Human Genetics, University o f Hamburg, D 2000 Hamburg 54, F R G R e c e i v e d 3 A p r i l 1987; A c c e p t e d 4 A u g u s t 1987 MEIER-TACKMANN, D., G. C. KORENKE, D. P. AGARWAL AND H. W. GOEDDE. Human liver aldehyde dehydrogenase: Subcellular distribution in alcoholics and nonalcoholics. ALCOHOL 5(1) 73--80, 1988.--Activity assay and isoelectric focusing analysis of human biopsy and autopsy liver specimens showed the existence of two major aldehyde dehydrogenases (ALDH I, ALDH II). Subceilular distribution of these isozymes was determined in autopsy livers from alcoholics and nonalcoholics. Nearly 70% of the total ALDH activity was recovered in the cytosol which contained both the major isozymes. Densitometric evaluation of isozyme bands showed that about 65% of the cytosolic enzyme activity was due to ALDH II and the rest due to ALDH I isozyme. Only about 5% of the total ALDH activity was found in the mitochondrial fraction (70% ALDH I and 30% ALDH II). Significantly reduced total and specific ALDH activities were noted in all the suhoellular fractions from cirrhotic liver specimens. Apparently, ALDH" I isozyme from cytosol and mitochondria is primarily responsible for the oxidation of small amounts of acetaldehyde normally found in the blood of nonalcoholics after drinking moderate amounts of alcohol. However, in alcoholics who exhibit higher blood acetaldehyde concentrations after drinking alcohol, ALDH II isozyme may be of greater physiological significance. Human Biopsy and autopsy liver Alcoholics Nonalcoholics

Aldehyde dehydrogenase

Isozymes

Subcellular distribution

respectively. However, the subcellular localization of different A L D H isozymes in human liver is still not finally established and the so-called cytosolic and mitochondrial isozymes may, in fact, be present in more than one subcellular fraction [5, 16, 36, 37]. In recent years, a considerable controversy has developed on the possible role of A L D H isozymes in alcoholrelated organ and tissue damage. Impaired acetaldehyde metabolism by way of reduced A L D H activity may be the primary cause of tissue injury. While a selective reduction of cytosolic A L D H has been observed in alcoholics with fatty liver [13,30], decreased activity o f mitochondrial A L D H was observed in the livers o f chronic alcoholics [29, 31, 33]. Moreover, decreased liver cytosolic A L D H activity has been claimed to be a primary abnormality and could represent genetic vulnerability to alcoholism [39]. However, these results are equivocal since in another study [15], hepatic A L D H activity was found to rise again when alcohol intake was reduced. Erythrocyte ALDH activity, which resembles the cytosolic A L D H II isozyme, has been also found to be

A L D E H Y D E dehydrogenase ( A L D H , aldehyde: N A D oxidoreductase, EC 1.2.1.3) catalyses the oxidation o f acetaldehyde in human liver and other organs. At least two major isozymes o f hepatic A L D H have been characterized in humans which differ in their structural and functional properties like primary structure, molecular weight, electrophoretic migration, isoelectric point, catalytic constants, inhibition with disulfn'am and subcellular distribution [7, 8, 11, 12, 16, 32]. The isozyme with the most cathodic migration in electrophoresis and lowest isoelectric point is designated as A L D H I or E2 and the next fast migrating isozyme is designated as A L D H II or El. Although both the isozymes show a K m in the micromolar range, A L D H I is usually referred to as "low K i n " isozyme and A L D H II as "high K m " isozyme. Animal and human tissue fractionation studies reported so far have shown that whereas the low Km isozyme is predominantly localized in the mitochondria, the high K m isozyme is of cytoplasmic origin [6, 14, 16, 17, 38, 40]. Therefore, the low K m and high Km isozymes are frequently called simply mitochondrial or cytosolic isozymes,

IA substantial part of this work is included in the MD dissertation submitted by G. C. Korenke to the Medical Faculty, University of Hamburg. 2Preliminary data of this work were presented at the Workshop on Aldehyde Dehydrogenase/Aldo-Keto-Reductaseand Alcohol Dehydrogenase held at Kingston, Ontario, July 1--4, 1984, 3Requests for reprints should be addressed to Dr. H. Werner Goedde, Institut f'tir Humangenetik, Universit~t Hamburg, Butenfeld 32, D 2000 Hamburg .54, FRG.

73

MEIER-TACKMANN ET AL.

74 reduced in chronic alcoholics and returns to normal values on reducing the alcohol intake [l, 2, 21]. The purpose of the present investigation was to understand the pathogenetic role of ALDH isozymes in alcoholic liver damage by studying their localization and quantitative distribution in biopsy and autopsy liver specimens from alcoholics and nonalcoholics. In order to discuss the possible implications of reduced liver ALDH isozyme activities, the acetaldehyde oxidation rate in peripheral blood of nonalcoholics was theoretically calculated from the in vitro ALDH isozyme activities found in the cytosolic fraction.

CRUDE LIVER HOMOGENATE

1

Step

SUPERNATANT

SEDrHE~T

Step 4a

Step 2

f- . . . . . .

1

SUPERNATANT

SEDI NENT S t e p 4b

I

r

7

HW

SEDINENT

..

I

,

]

SUPERNIt TEN T

SED{MENT

METHOD

Subjects and Liver Samples Seven needle biopsy liver specimens were obtained at the Kreiskrankenhaus Eckernfrrde during abdominal surgery from patients with normal liver status. The samples were transported in moist petri dishes kept over crushed ice. Enzyme assays were performed within 3-6 hours after biopsy. Homogenates of biopsy samples were prepared by grinding 1 part of tissue with 19 parts of aqua dest. using a Potter-Elvehjem glass homogenizer with a Teflon pestle Braun, Melsungen, Germany). For activity assay and isozyme separation by isoelectric focusing (IEF), the clear supernatants were obtained by subjecting the liver homogenates and the various subcellular fractions to 3 cycles of freezing at -80°C and thawing before centrifugation at 27,750×g for 15 min. For the subcellular fractionation studies the crude liver homogenates were not subjected to this procedure. Autopsy livers from 3 alcoholics and 4 nonalcoholics (healthy controis) were obtained from the Departments of Legal Medicine and Pathology, University Hospital Eppendorf, Hamburg. The autopsy specimens were obtained within 2-3 days after death, the bodies being maintained at 4°C until post-mortem examination. The cause of death of alcoholics (mean age=62.7 years) was given as heart failure and bleeding of oesophagus varicose veins; for nonalcoholics (mean age=69.3 years) as cardiac infarction of air embolism. The classification of livers into healthy controls and cirrhotic degeneration was done at these institutes according to the standard practice. Since the sample number in each group was small (n<5), no statistical analysis was done.

Aldehyde Dehydrogenase Activity Assay The biopsy samples were analysed for total and specific enzyme activity as well as isozyme profile by IEF using crude homogenates. The autopsy specimens were subjected to subcellular fractionation and the homogenates and various fractions were analysed for ALDH activity and isozyme profile. The ALDH activity was measured by following the production of NADH at 25°C using a double beam spectrophotometer attached with a recorder (Hitachi, Model 100-60 or 220 A). The standard reaction mixture consisted of 0.1 M sodium pyrophosphate buffer, pH 9.0, 5 mM acetaldehyde, 2 mM NAD ÷, 10 mM pyrazole, and sample aliquot in a final volume of 500/xl. After 5 min preincubation, the reaction was started by adding acetaldehyde in the sample cuvette and aqua dest. in the reference cuvette. The NADH production was measured at 340 nm for about 15 rain. Optical density units were converted to international units by using an extinction coefficient for NADH of 6.22. The total ALDH activity is expressed as mU per g wet weight of liver

[

1

SUPERNATANT

SEDI BENT

,,

[ MW

SEDIHENT

Step

Step 7 -

CYTOSOLI C FRACTION

-

t

f__ HI CROSOHAL PRt*CTION

MW

I

....

•

SUPERNATANT

i I HI TOCHONDR] AL PEACTION

I NUCLE/~R PRItCTION

FIG. 1. Scheme of subcellular fractionation of human autopsy liver. Steps 1.3: c e ~ e x l for 15 min at 1,~Xg; stel~ 4-5: centrifuged for 10 rain at 29,000xg; step 6: c e n t r i ~ for 15 rain at 20,2~xg; step 7: centrifuged for 20 min at 8,400xg; step 8-: ceatrifuged for 90 min at 147,000 x g. *, **pooled; MW=mit~ho~trial washings (pooled). tissue and the specific activity as mU per mg total soluble protein.

lsoelectric Focusing (IEF) ALDH isozymes were separated from 20/~1 of homogenate or subcellular fraction of different protein concentration by IEF in polyacrylamide gels. The analytical details of IEF have been published before [9].

Staining of lsozyme Bands For visualization of the isozyme bands, the potyacrylamide gel after IEF was overlaYed with an agarose gel containing a specific enzyme staining solution [9] and incubated first in the dark for 1 hr at 37°C and subsequently for 14 hr at 4°C. Dark forrnazan bands showing isozyme activity zones were visible.

Densitometric Evaluation of lsozyme Bands For densitometric evaluation of the isozyme band intensities, the agarose layer was removed and the gel was scanned using a laser densitometer (LKB, Ultrosean 2202) attached with a recorder and an integrator (Hewtett Packard, Model 3390A). The area under the curve values were convened to/zmoles NADH produced per min per g wet tissue. For this purpose, purified ALDH I and II preparations with known enzyme activities determined y were subjected to IEF followed by the densitometry. With the help of standard curves, a conversion factor for ALDH activity from the area under the curve values was obtained.

Protein Determination The protein content in crude homol~nat¢s and various subcellular fractions was determined by the method OfBrad-

LIVER ALDEHYDE DEHYDROGENASE ISOZYMES TABLE 1 COMPARATIVEALDH ACTIVITYIN CRUDE LIVER HOMOGENATES FROM AUTOPSY AND BIOPSY SPECIMENS

75 TABLE 2 DISTRIBUTION OF MARKER ENZYME ACTIVITIESIN DIFFERENT SUBCELLULARFRACTIONSOF NONALCOHOLICAND CIRRHOTIC HUMAN LIVER

ALDH Activity Autopsy Liver

Specimen 1 2 3 4 5 6 7 Median Mean

Total (mU/g wet wt.)

Specific (mU/mg protein)

Percent Enzyme Activity*

Biopsy Liver Total (mU/g wet wt.)

Specific (mU/mg protein)

Subcellular Fraction

Marker Enzyme

Nonalcoholic Liver

Cirrhotic Liver

Nuclear

NMNA SDH G-6-P LDH

24 17 11 5

23 6 5 2

832.0 912.4 1321.0 1445.2 ----

10.4 15.8 15.7 38.5 ----

749.3 767.7 964.8 1322.4 1706.0 3754.0 5336.9

29.0 5.7 17.7 14.2 13.3 22.0 28.7

Mitochondrial

NMNA SDH G-6-P LDH

18 31 33 3

16 26 13 2

1116.7 1127.7

15.75 20.1

1322.4 2085.9

17.5 18.6

Mitochondrial washings

NMNA SDH G-6-P LDH

0 30 22 6

0 43 48 4

Microsomal

NMNA SDH G-6-P LDH

15 19 24 5

8 17 17 4

Cytosolic

NMNA SDH G-6-P LDH

43 2 10 81

53 8 17 88

ford [3] as modified by Macart and Gerbaut [25] using a bovine serum albumin as a standard (Behring-Werke, Marburg, Germany).

Subcellular Fractionation The fractionation procedure was a modifcation of the method described before for human placental tissue [27]. F o r preliminary work to establish the validity of the method for fractionation of autopsy livers, rat livers were used for subcellular distribution of A L D H activity under different storage conditions. The human liver crude homogenates were prepared by grinding 1 part o f tissue with 4 parts of fractionation buffer (0.25 M sucrose in 10 mM Tris/HCl, pH 7.4). A schematic outline o f the fractionation procedure is shown in Fig. 1. The unwashed mitochondrial pellet obtained after step 4a was resuspended in buffer and centrifuged once more for 10 min at 29,000xg (step 4b). The sediments obtained from steps 4b and 5 were pooled and suspended in the fractionation buffer before re-centrifugation to get the mitochondrial pellet (step 6).

Marker Enzymes The following marker enzymes specific for each subcellular fraction were assayed as described earlier [27]: Nuclei: Nicotinamide mononucleotide-adenylyl-transferase (NMNA, EC 2.7.7.1); Mitochondria: Succinate dehydrogenase (SDH, EC 1.3.99.1); Microsomes: Glucose-6-phosphatase (G-6-P, EC 3.1.3.9); Cytosol: Lactate dehydrogenase (LDH, EC 1.1.1.27). RESULTS

Nonalcoholic Livers In both biopsy and autopsy samples from nonalcoholics, a large variation in individual and mean values for total A L D H activity expressed per g fresh tissue was observed (Table 1). However, the mean specific activity (per nag soluble protein) values were very similar in both kinds of liver samples.

*Mean values from 4 nonalcoholic and 3 cirrhotic livers.

Subcellular Distribution of A L D H Activity Since the needle biopsy samples were too small in size, subcellular distribution of A L D H activity was studied by fractionation of autopsy livers only. The fractionation procedure found optimal for fresh human placental tissue in an earlier study [27] could not be directly applied to the autopsy livers. On subjecting human livers to this fractionation method, the mitochondrial fraction was found to be heavily contaminated with microsomes and vice versa. An additional washing (Fig. I, step 4b) reduced this contamination considerably. However, there was a significant loss in subcellular proteins as judged by the presence of marker enzymes in the mitochondrial washings. Similar observations were made with fresh rat livers subjected to subcellular fractionation using this procedure. In rat livers stored for 72 hr at 4°C, the mitochondrial and microsomal fractions were still more contaminated with each other as judged by the presence of respective marker enzymes (results not shown here). Table 2 shows the distribution of marker enzymes in various subcellular fractions from control and cirrhotic autopsy livers. The combined recovery of individual marker enzymes in various fractions as percentage of the total activity in the nonalcoholic liver homogenate was: N M N A = 7 6 ; S D H = 9 3 ; G-6-P=88 and L D H = 9 3 . The corresponding recovery figures in cirrhotic livers were very similar. The distribution of A L D H activity in homogenates and subcellular fractions from control and cirrhotic livers is

M E I E R - T A C K M A N N ET A L.

76

ALDH I ALDH I ALOH~.

1

2

4"

AL.DH II

3

I

2

3

I.

5

FIG. 2. Isoelectric focusing profile of ALDH isozymes in crude liver homogenates from biopsy samples (2,3) and autopsy specimens (1) of nonalcoholics.

FIG. 3. lsoetectric focusing profile of ALDH isozymes from different subcellular fractions of nonalcoholic human liver. l=homogenate; 2=nuclear fractions; 3=mitochondrial fraction; 4=microsomal fraction; 5=cytosolic fraction.

shown in Table 3. About 90% of the total enzyme activity present in the homogenates was recovered in various fractions. Although A L D H was detectable in all the fractions, the highest activity was found in the cytosol. The mitochondrial fractions from nonalcoholic livers contained only about one tenth of the total enzyme activity measured in the cytosol.

TABLE 3 DISTRIBUTION OF ALDH ACTIVITYIN NONALCOHOLIC AND CIRRHOTIC HUMAN LIVERS

Isoelectric Focusing Profile of A L D H Isozymes On I E F of homogenates, two prominent isozyme bands ( A L D H I and A L D H II) were invariably visible. While the A L D H III isozym¢ band was not always detectable, a weak A L D H IV band was present in each o f the biopsy and autopsy sample. The isozyme profile and activity band intensities from biopsy and autopsy specimens were identical (Fig. 2). In all the subcellular fractions, too, both A L D H I and A L D H II were detected (Fig. 3). Whereas the homogenares and the cytosolic fractions both showed strong A L D H I and A L D H II isozyme bands, other fractions exhibited a prominent A L D H I and a weak A L D H II isozyme band after staining. The mitochondrial fraction was composed o f a very strong A L D H I and a very weak A L D H II band. A L D H i isozyme was usually the first isozyme visible on the gels but the intensity of A L D H II increased gradually as the gels were left in the dark at 4°C. After about 15 hr there was no change in the band intensities.

Cirrhotic Livers The total and specific A L D H activity was considerably reduced in the cirrhotic liver homogenates as compared to nonalcoholic control liver extracts (Table 3), However, the distribution profile of the enzyme activity in different subcellulax fractions from cirrhotic livers was not considerably different from that of nonalcoholics. There was no remarkable difference between nonalcoholic and cirrhotic livers regarding the distribution of marker enzyme activities except for a very low S D H activity in cirrhotic liver (Table 4). In ,comparison to nonalcoholic livers, cirrhotic livers also showed poor band intensities for both the A L D H isozymes in crude homogenates as well as in the cytosolic and mitochondrial fractions (Fig. 4). The relative diauibution of A L D H I and A L D H II isozyme activities as d e t e ~ from the isozyme band intensities by densitometric evaluation is

Fraction Crude homogenate Nonalcoholic Cirrhotic

Total (mU/g wet wt.)

ALDH Activity* Specific (% (mU/mg loss) protein)

(% loss)

1128 454

60

20. l 8.1

60

Nuclear Nonalcoholic Cirrhotic

150 11

90

15.6 3.0

80

Mitochondrial Nonalcoholic Cirrhotic

53 11

80

6.2 1.4

75

Microsomal Nonalcoholic Cirrhotic

52 24

50

7.4 2. I

70

Cytosolic Nonalcoholic Cirrhotic

711 328

60

30.0 14.4

50

Mitochondrial washings Nonalcoholic Cirrhotic

71 30

60

9.2 5.7

45

*Mean values from 4 nonalcoholic and 3 cirrhotic livers.

shown in Table 5. The cirrhotic livers had considerable poor A L D H I isozyme activity both in the mitochondrial as well as in the cytosolic fractions. The loss in relative activities in the cytosolic fraction show that A L D H I isozyme was much more reduced than the A L D H II isozyme. DISCUSSION Although it is generally agreed that excessive alcohol

LIVER ALDEHYDE DEHYDROGENASE ISOZYMES

77

TABLE 4 ~,.I:IH I

MARKER ENZYME ACTIVITIES IN NONALCOHOLIC AND CIRRHOTIC LIVER I-IOMOGENATES

A i . ~ 11

Enzyme Activity* (U/g wet liver) Homogenate

NMNA

SDH

G-6-P

LDH

Nonalcoholic Cirrhotic

0.78 0.99

8.01 1.97

0.33 0.34

25.36 27.37

*Mean activity from 4 nonalcoholic, 3 cirrhotic livers. I

2

3

~

5

6

FIG. 4. Isoelectric focusing profile of ALDH isozymes in crude homogenates, cytosolic and mitochondrial fractions from nonalcoholics livers (1-3) and cirrhotic livers (4-6). 1,4=homogenate; 2,5=cytosolic fraction; 3,6=mitochondrial fraction.

TABLE 5 SUBCELLULAR DISTRIBUTION OF LIVER ALDH ACTIVITY DETERMINED BY DENSITOMETRIC EVALUATION OF THE ISOZYME BANDS AFTER IEF

ALDH Activity* (%) Mitochondria

Cytosol

Liver Sample

ALDH I

ALDH II

ALDH I

ALDH II

Nonalcoholic Cirrhotic

70 60

30 40

35 15

65 85

*The combined intensity of ALDH I and ALDH II bands was taken as 100%.

consumption is a major factor in liver injury, the mechanisms involved in the pathogenesis of alcoholic liver disease remains largely unresolved. Acetaldehyde, the active metabolite of ethanol oxidation, has been implicated to be mainly responsible for the diverse hepatotoxic effects of alcohol drinking [19, 20, 26, 35]. Significantly higher blood acetaldehyde concentrations have been recorded in alcoholics than in nonalcoholic control subjects [18, 22, 23, 28, 34]. This may be the consequence of an impaired hepatic oxidation of acetaldehyde via liver ALDH. A number of studies have indeed demonstrated a significantly reduced in vitro ALDH activity in surgical biopsy samples from alcoholics as compared to nonalcoholic controls [4, 13, 15, 29, 30, 31, 33, 34, 39, 41]. Our results in the present study clearly demonstrate that chronic ethanol consumption diminshes ALDH isozyme activities in human liver. Considerably low SDH activity found in the cirrhotic liver extracts hints to a mitochondrial damage. This observation is further supported by a very weak ALDH I isozyme band found in cirrhotic liver extracts on IEF. Such a change in isozyme composition, however, might have resulted from the 3 freeze/thaw cycles used for the preparation of homogenate supernatants. The present study also confirms earlier observations of Pietruszko et al. [32] showing a significant loss in the mitochondrial ALDH activity on storage of livers. Apparently autopsy livers are not suitable for fractionation studies as during post-mortem conditions, lysis of cell membranes occur leading to a possible aggregation of microsomes with

mitochondria, which indicates a possible formation of artefactual submitochondrial particles. This was evident from the difficulties in getting these fractions free from each other. Rat livers on storage under conditions identical with human autopsy livers showed similar findings. Data reported in the literature for the subcellular distribution of human liver ALDH isozymes are equivocal. It is generally assumed that the low Km isozyme (ALDH I) is predominantly localized in the mitochondria and the high Km isozyme (ALDH II) in the cytosol [14,40]. Whereas Koivula [16] reported the presence of two isozymes in the mitochondrial and cytosolic fractions, Takada et al. [36] detected both ALDH I and ALDH II isozymes in the cytosol only. The present fractionation data using autopsy livers of nonalcoholics and alcoholics clearly showed that both the major ALDH isozymes were distributed in each subcellular fraction. In the present study, although the cytosolic fraction was contaminated with nuclei as evident from the presence of the marker enzyme NMNA, there was a negligible contamination from microsomes and mitochondria. Likewise, the mitochondrial and microsomal fractions were almost free from the cytosolie marker enzyme. Moreover, since the volume of cytoplasm is considerably greater than the volume of other fractions, the leakage from cytoplasm to mitochondria is not significant, as shown in Table 2. Hence, the bulk of ALDH I isozyme present in the cytosolic fraction cannot be accounted for alone due to a mitochondrial contamination. Thus, it is misleading to designate only ALDH I as the mitochondrial isozyme and only ALDH II as the cytosolic

78

MEIER-TACKMANN E T A I a

rl

13

e

q

ti

©

T me ol reac~lc~r m J r l

FIG. 5. Oxidation rates of acetaldehyde in liver cytosol (a-c) and by cytosolic ALDH I and ALDH II isozymes from nonalcoholic livers (d-f) and cirrhotic livers (g-i) at different blood acetaldehyde concentrations. A=nonalcoholic liver cytosol; &--cirrhotic liver cytosol: O=cytosolic ALDH I isozyme; O=cytosolic ALDH II isozyme.

isozyme. As seen by the marker enzyme distribution, there was some leakage from the particles. However. since the distribution proffie of the marker enzymes does not considerably vary in both nonalcoholic and cirrhotic liver fractions. a comparison between nonalcoholic and alcoholic liver ALDH may be done. The complexities of in vitro and in vivo conditions make it difficult to compare the involvement of mitochondrial and cytosolic ALDH I and ALDH II isozymes in hepatic and peripheral blood acetaldehyde oxidation [24,42]. However. the relative contribution of the two isozymes in acetaldehyde oxidation in nonacloholic and cirrhotic livers may be calculated by using Michaelis-Menten equation. The Km values for ALDH I and ALDH II with acetaldehyde are known to be 3/~M and 32 ~M, respectively [8,10]. Since the maximal ALDH activity was found in the cytosol, the relative contribution of ALDH isozymes in acetaldehyde oxidation in this fraction was calculated. Based on the mean total activity found in the nonalcoholic livers (711 mU/g wet wt.) the Vmax value (p,mol/min/g wet wt.) for ALDH I isozyme, which makes 35% of the cytosolic fraction, would be 0.249 tzM and for ALDH II with 65% share, the Vmax would be 0.465 izM (see Table 5). Likewise, in cirrhotic livers with only 328 mU/g wet wt. total activity and composed of 15% ALDH I and 85% ALDH II, the corresponding Vmax values would be still lower (49.2 and 279). In alcoholics, the blood acetaldehyde concentration may range between 1 and 100 izmol 1-1 depending upon the dose of alcohol and the oxidation capacity of the liver [29], whereby the intrahepatic acetaldehyde concentration may be much higher than in the blood during ethanol metabofism. Thus, taking into account the observed isozyme composition of the cytosofic fraction, disappearance rates of blood acetaldehyde were calculated in the cytosol from nonalcoholic and cirrhotic livers. As demonstrated in Fig. 5a--c, a considerable decrease in the oxidation rate o f acetaldehyde in cirrhotic rivers as compared to nonalcoholic livers would be expected at 1, 10 and

100 /xmol 1-~ blood acetaldehyde concentrations. For example, at 1 ~mol 1-1 acetaldehyde concentration, the total amount of substrate could be oxidized in 16 min by nonalcoholic liver cytosol but would require at least 60 min for the corresponding cirrhotic liver fraction. The rate of acetaldehyde oxidation by ALDH I and ALDH II isozymes independent of each other, both in nonalcoholic and cirrhotic liver cytosolic fractions, is also apparently different (Fig. 5d-h). At blood acetaldehyde concentrations below 10/zmol 1-1. the low Km A L D H t isozyme shows much faster oxidation capacity than the high Km ALDH II isozyme. Namely, at 1 /~mol 1-1 blood acetaldehyde concentration, 50% of the substrate would be oxidized by ALDH I isozyme in 8 min but would require 50 rain to be oxidized by ALDH II isozyme alone (Fig+ 5d). However, at higher blood acetaldehyde concentration, the ALDH II isozyme may be almost equally active as ALDH I in the oxidation of acetaldehyde (Fig. 5f, h, i). These inferences are in agreement with the general rules of Michaelis-Menten kinetics and suggest that the ALDH I isozyme in both the cytosol and mitochondria may be primarily responsible for the oxid~afion for small amounts of acetaldehyde normally found in the blood of nonalcoholics after moderate drinking. However, in alcoholics with higher blood acetaldehyde concentrations, ALDH II isozyme predominantly found in the cytosol may be of greater physiological significance.

ACKNOWLEDGEMENTS The authors are indebted to Prof. Dr. W. Janssen, Institute of Legal Medicine, University of Hamburg, and to Prof.Dr. G. Seifen and Prof. Dr. G. t~lltng, P~,_lmlollicatInstitute, Ultivenfityof Hamburg, for kindly providing autopsy s ~ , ~ tolPD Dr. Lahrtz, Kreiskrankentmus Ec!~mt~rde, for kifid~yp r o v ~ b ~ y tissues. They also thank Mrs. E. Losenhauscn for ~ ! ~/j~ieJl:assistante. Our thanks are also due to the Paul-M~rti¢li-S"ti~a~g, Malnz. for financial support.

LIVER ALDEHYDE DEHYDROGENASE

ISOZYMES

79

REFERENCES

I. Agarwal, D. P., L. Tobar-Rojas, S. Harada and H. W. Goedde. Comparative study of erythrocyte aldehyde dehydrogenase in alcoholics and control subjects, Pharmacol Biochem 18" Suppl 1, 89-95, 1983. 2. Agarwal, D. P., C. Miiller, G. C. Korenke, U. Mika, S. Harada and H. W. Goedde. Changes in erythrocyte and liver aldehyde dehydrogenase isozymes in alcoholics. In: Enzymology of Carbonyl Metabolism 2. Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, edited by T. G. Flynn and H. Weiner. New York: Alan R. Liss, Inc., 1985, pp. 113-127. 3. Bradford, M. B. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248--254, 1976. 4. Doffoel, M., M. Ledig, M. Ziessei, D. Vetter, D. Paris-Bockel, H. Bernard, P. Mandel and R. Bockel. La diminution de l'activit~ de l'acetaldehyde dehydrogenase hepatique est-elle cause de l'alcoolisme? Extrait Rev Alcoolisme 31: 1-6, 1986. 5. Duley, J. A., O. Harris and R. S. Holmes. Analysis of human alcohol- and aldehyde-metabolizing isozymes by electrophoresis and isoelectric focusing. Alcohol: Clin Exp Res 9: 263-271, 1985. 6. Eckfeldt, J. H. and T. Yonetani. Subcellular localization of the F1 and F2 isozymes of horse liver aldehyde dehydrogenase. Arch Biochem Biophys 175: 717-722, 1976. 7. Forte-McRobbie, C. R. and R. Pietruszko. Aldehyde dehydrogenase content and composition of human liver. Alcohol 2: 375-381, 1985, 8. Goedde, H. W., D. Meier-Tackmann, D. P. Agarwal and S. Harada. Physiological role of ALDH izozymes. In: Progress in Clinical and Biological Research, Vol 114, Enzymology of Carbonyl Metabolism, Aldehyde Dehydrogenase and Aldo-Keto Reductase, edited by H. Weiner and B. Werrnuth. New York: Alan R. Liss, Inc. 1982, pp. 329-346. 9. Goedde, H. W., D. P. Agarwal, S. Harada, D. MeierTackmann, R. Du, U. Bienzle, A. Kroeger and L. Hussein. Population genetic studies on aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity. Am J Hum Genet 35: 769772, 1983. 10. Goedde, H. W., D. P. Agarwal and S. Harada. The role of alcohol dehydrogenase isozymes in alcohol metabolism, alcohol sensitivity and alcoholism. In: lsozymes: Curr Top Biol Med Res Vol 8: Cellular Localization, Metabolism and Physiology, edited by M. C. Rattazzi, J. G. Scandalios and G. S. Whitt. New York: Alan R. Liss, Inc., 1983, pp. 175-193. II. Greenfield, N. J. and R. Pietruszko. Two aldehyde dehydrogenases from human liver: Isolation via affinity chromatography and characterization of the isozymes. Biochem Biophys Acta 483: 35-45, 1977. 12. Harada, S., D. P. Agarwal and H. W. Goedde. Eiectrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues. Life Sci 26: 1773-1780, 1980. 13. Jenkins, W. J. and T. J. Peters. Selectively reduced hepatic acetaldehyde dehydrogenase in alcoholics. Lancet i: 628--629, 1980. 14. Jenkins, W. J. and T. J. Peters. Subcellular localizatoin of acetaldehyde dehydrogenase in human liver. Cell Biochem Funet 1: 37-40, 1983. 15. Jenkins, W. J., K. Cakebread and K. R. Palmer. Effect of alcohol consumption on hepatic aldehyde dehydrogenase activity in alcohol patients. Lancet i: 1048-1049, 1984. 16. Koivula, T. Subcellular distribution and characterization of human liver aldehyde dehydrogenase fractions. Life Sci 16: 1563-1570, 1975. 17. Koivula, T. and M. Koivusalo. Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution. Bioehim Biophys Acta 397: 9-23, 1975. 18. Korsten, M. A., S. Matsuzaki, L. Feinman and C. S. Lieber. High blood acetaldehyde levels after ethanol administration. N Engl J Med 292: 386--389, 1975.

19. Lebsack, M. E., E. R. Gordon and C. S. Lieber. Effect of chronic ethanol consumption on aldehyde dehydrogenase activity in the baboon. Biochem Pharmacol 30: 2273-2277, 1981. 20. Lieber, C. S., E. Barona, Y. Matsuda, M. Salaspuro, Y. Hasumura and S. Matsuzaki. Hepatotoxicity of acetaldehyde. Adv Exp Med Biol 126: 397--411, 1980. 21. Lin, C., J. J. Potter and E. Mezey. Erythrocyte aldehyde dehydrogenase activity in alcoholism. Alcohol: Clin Exp Res 8: 539541, 1984. 22. Lindros, K. O., A. Stowell, P. H. Pikkarainen and M. Salaspuro. Elevated blood acetaldehyde on alcoholics and accelerated ethanol elimination. Pharmacol Biochem Behav 13:119124, 1980. 23. Lindros, K. O. Human blood acetaldehyde levels: With improved methods, a clearer picture emerges. Alcohol: Clin Exp Res 7: 70--75, 1983. 24. Lundquist, F. Acetaldehyde and aldehyde dehydrogenasecentral problems in the study of alcoholism. Eur J Clin Invest 13: 183--184, 1983. 25. Macart, M. and L. Gerbaut. An improvement of the Coomassie blue dye binding method allowing an equal sensitivity to various proteins. Application to cerebrospinal fluid. Clin Chem Acta 122: 93-101, 1982. 26. Matsuzaki, S. and C. S. Lieber. Increased susceptibility of hepatic mitochondria to the toxicity of acetaldehyde after chronic ethanol consumption. Biochim Biophys Res Commun 75: 1059-1065, 1977. 27. Meier-Tackmann, D., G. C. Korenke, D. P. Agarwal and H. W. Goedde. Human placental aldehyde dehydrogenase: Subcellular distribution and properties. Enzyme 33: 153-161, 1985. 28. Nuutinen, N., M. Valle, M. Salaspuro and K. O. Lindros. Blood acetaldehyde concentration gradient between hepatic and antecubital venous blood in ethanol-intoxicated alcoholics and controls. Eur J Clin Invest 14" 306-311, 1984. 29. Nuutinen, H., K. O. Lindros and M. Salaspuro. Determinants of blood acetaldehyde level during ethanol oxidation in chronic alcoholics. Alcohol: Clin Exp Res 7: 163-168, 1983. 30. Palmer, K. R. and W. J. Jenkins. Impaired acetaldehyde oxidation in alcoholics. Gut 23: 729-733, 1982. 31. Pares, A., X. Soler, J. Panes, X. Pares, J. Caballeria, J. Farres, R. Raz and J. Rodes. Hepatic alcohol and aldehyde dehydrogenases in liver disease. 3rd ISBRA Cong. Helsinki. Alcohol Alcohol 42: A52, 1986. 32. Pietruszko, R., N. J. Greenfield and C. R. Edson. Human liver aldehyde dehydrogenase. In: Alcohol and Aldehyde Metabolizing Systems, vol H. New York: Academic Press, 1977, pp. 195-202. 33. Ricciardi, B. R,, J. B. Saunders, R. Williams and D. A. Hopkinson. Hepatic AHD and ALDH isoenzymes in different racial groups and chronic alcoholism. Pharmacol Biochem Behav 18: Suppl 1, 61-65, 1983. 34. Salaspuro, M., H. Nuutinen, P. H. Pikkarainen and K. L. Lindros. Elevated blood acetaldehyde in alcoholics with accelerated ethanol elimination and decreased hepatic aldehyde dehydrogenase. Liver 1: 146, 1981. 35. Sorell, M. F. and D. J. Tuma. Hypothesis: Alcoholic liver injury and the covalent binding of acetaldehyde. Alcohol: Clin Exp Res 9: 306-309, 1985. 36. Takada, A., S. Takase, J. Nei and Y. Matsuda. Subceilular distribution of ALDH isozvmes in the human liver. 2nd ISBRA Cong. Alcohol: Clin Exp Res 8: 123, 1984. 37. Takada, A., M. Tsutsumi and K. Sugata. Species differences of hepatic acetaldehyde dehydrogenase isozymes. 3rd ISBRA Cong. Helsinki. Alcohol Alcohol A3, 1986. 38. Tank, A. W., H. Weiner and J, A. Thurman. Enzymology and subceilular localization of aldehyde oxidation in rat liver. Biochem Pharmacol 30: 3265-3275, 1981.

80 39. Thomas, M., S. Halsall and T. J. Peters. Role of hepatic aldehyde dehydrogenase in alcoholism: Demonstration of persistent reduction of cytosolic activity in abstaining patients. Lancet ii: 1057-1059, 1982. 40. Tipton, K. F. and G. T. M, Henehan. Distribution of aldehyde dehydrogenase activities in human liver. 2nd ISBRA Cong., Alcohol: Clin Exp Res 8: 131, 1984.

MEIER-TACKMANN

El+ ,41.

41. Tipton, K. F., J. M. McCrodden, D. G. WeJv aml K. Ward. lsozymes of human liver alcohol dehydrogenase and aldehyde dehydrogenase in alcohofic and non-alcoholic subjects+ Alcohol Alcohol 18: 21%225, 1983. 42. Von Wartburg, J. P. and R. Bi~hler. Biology of disease. Alcoholism and Aldehydism: New biomedical concepts. Lab Invest 50: 5-15, 1984.