alcohol dehydrogenases: gene multiplicity and differential functions of five classes of isozymes

Drug and Alcohol Review (i993) I2, 99-iio

Alcohol dehydrogenases: gene multiplicity and differential functions of five classes of isozymes

ROGER S. HOLMES

Faculty of Science and Technolo , 7:th vnive,-sit:, S,Csbane, Queensland Abstract

Mammalian alcohol dehydrogenases (ADHs) constitute an enzyme family of multiple forms (isozymes) which are differentially distributed throughout the body. Subunit types C~, fl and y in dimeric combinations constitute the isozymes of human liver class I ADH, and are > 9 4 % homologous in structure. Human rc and X subunits form homodimeric Class II and III A D H isozymes, rC-ADH is liver specific whereas X-ADH is widely distributed throughout the body. A sixth human ADH subunit (des igna ted / /o r or), forming a new dimeric human stomach ADH, has been recently reported as Class IV ADH. Evidence for a seventh human A D H subunit has also been described, designated as Class V, the transcripts having been reported in the stomach and liver. All five classes of A D H represent isozymes which are homologous but exhibit at least 30% sequence differences in primary srtructure. Kinetic analyses of four of these classes of ADH indicated differential functions, serving either in the oxidative or reduetive mode. Studies from various laboratories indicate the following respective functions: oxidation of aliphatic and aromatic alcohols--liver Class I and Class II, and stomach Class IV ADHs; reduction of peroxidic aldehydes--Classes I, II and IV; 'biogenic' alcohol oxidation--Classes I and II; and glutathione- dependent formaldehyde dehydrogenase-Class III. [Holmes RS. Alcohol dehydrogenases: gene multiplicity and differential functions of five classes of isozymes. Drug Alcohol Rev I993; I2: 99-Iio.]

Key words: alcohol dehydrogenase; classes; functions; isozymes.

Introduction

Alcohol dehydrogenases (ADHs; E.C.I.I.I.I) are widely distributed in tissues of humans and experimental mammals [-1-41, and function in the reversible interconversion of a wide range of alcohols into the corresponding aldehydes and ketones. The mammalian enzyme is a zinc metalloenzyme, which has a dimeric subunit structure, and which has been extensively investi-

gated in terms of the biochemical and genetic basis of multiplicity for this enzyme. Until recently, mammalian ADHs have been divided into three classes of isozymes, designated Classes I - I I I [5-71" Evidence for additional ADH Classes has recently been described [8-r2], and it is the purpose of this paper to review current reports for the existence of at least five distinct Classes of

Roger Holmes, PhD DSc, Professor of Biochemistry and Molecular Biology, Deputy Vice-Chancellor (Research), Griffith University, Brisbane, Queensland, 4rib Australia. Correspondence and requests for reprints to Professor Holmes.

99

Ioo Roger S. Holmes

ADH, and to review published work regarding the differential function of these enzymes in alcohol and aldehyde metabolism.

Liver alcohol dehydrogenases--molecular biology

Liver ADH is predominantly responsible for catalyzing the first step in the metabolism of alcohol in the body, by way of the following reaction:

CH3CHzOH + NAD + ~__ CH3CHO + NADH + H +

Human Liver ADHs

The human liver Class I ADHs comprise a number of distinct isozymes, formed by the dimeric association of three polypeptides, O~, fl and y, which are encoded by closely linked genes located in the chromosomal region, 4q2x-4q24, and designated as ADHI, ADH2 and ADH3, respectively [x,x3]. The resultant isozymes exist as six separate forms in homozygous individuals, generated following homodimeric and heterodi- meric associations of these subunits:

O/0/ a,8 a7

B,8 ,6y yy

Biochemical [i4-x6] and molecular genetic [x7-i9] analyses of these ADH subunits have shown extensive sequence homology, with the respective coding sequences showing identity of 95.6% between fl and y; 95.I% between ~ and fl; and 94% between 0t and y. These data, as well as comparative molecular genetic studies of the fl subunit of baboon liver ADH [2o], support an hypothesis of successive ADH Class I gene duplications occurring during primate evolution, in the following sequence [i8,2o]: ancestral Class I6°-+sMr~> ~ + D ~ y ] 44---SM)'a > fl-'~-y ADH gene

Population genetic studies of human Class I ADHs have revealed variants in primary structure and cDNA nucleotide sequence for the fl and y subunits, resulting in three polymorphic forms for fl and two forms for y. The fll variant is predominant in Caucasians, whereas the f12 variant is the major form in Oriental populations

[2I]. This enzyme has a histidine in place of an arginine at position 47, which causes a lower pH optimum and about an 8o-fold increase in specific activity at neutral pH, as compared to the f l l f l l isozyme [see 22]. This allelic form of ADH may contribute to the 'flushing' response commonly observed in Orientals, as a result of a more rapid production of acetaldehyde [2x], particularly in association with a 'low activity' variant of human liver mitochondrial aldehyde dehydrogenase (ALDH) [23,24]. The f13 variant has been reported only from Negroid populations, and results from an arginine/cysteine substitution at position 369 [22]. This form is characterized kinetically by a higher Michaelis constant (Km) for ethanol (64 mM), as compared with 49/AM and 9.4 ° ]./M respectively, for the f l , f l l and f12f12 isozymes. A variant form of the y-ADH subunit has also been described, for which two amino acid replacements have been reported (arginine for glutamine at position 271 i and isoleucine for valine at position 349), for the y l and y2 subunits, respectively [26].

Human liver Class II ADH (also designated-- rCADH) occurs as a single form exhibiting a 'high- Km' for ethanol as substrate [26], and has a primary structure and cDNA nucleotide sequence with more than 6o% homology with Class I ADH isozymes [27] , establishing ~-ADH as a distinct structural and genetic class of enzyme. Human liver Glass Ill ADH (or ZADH) exists as a major and minor form of activity, and exhibits negligible activity with ethanol at concentrations of < o . 5 M [28]. This enzyme also shares more than 60% sequence homology with the other two classes of human liver ADH [291 , and the responsible gene (designated ADH5) has been mapped to the same q2I-q24 region of chromosome 4, described for the Class I ADHs [i3].

Baboon liver ADHs

The baboon (Papio hamadryas) has been used as an animal model to study alcoholic liver injury [29] and the effects of alcohol consumption upon ethanol elimination rates [3 o] and liver ADH patterns of activity [31,32]. Baboon liver ADHs have been examined using agarose-isoelectric focusing and histochemical staining methods, to assess the multiplicity and substrate specificity of these enzymes (Fig. i). The major liver isozymes, using ethanol as substrate, are designated as

A D H classes--differential functions xoI

5raM 25raM IOOmM 500raM

..... ~ . . . . . . . . . . . . . ~ . . . . . . .~%.~ . :~ ~.

p H 7 - ~' ~ ' '

- A D H " I

L K S k g S L K S k K S

- A D H - 5 - A D H - 4 ( ~ ' }

Figure x. Isoelectric focusing patterns of baboon liver (L), kidney (K), and stomach (S) alcohol dehydrogenase (ADH) isozymes, p H range: 3-zo. Zymograms were stained with 5 mm ethanol, 25 mM ethanol, zoo mM ethanol, and 500 mM ethanol, to show differential substrate specificities of Glass I (ADH-x and ADH-z), Glass II (ADH-4) and Glass 1[" (ADH-3) isozymes. The Glass 111 isozyme (ADH-5) showed no detectable activity under these staining conditions.

(Reprinted from [3], with permission.)

ADH-2 (the major activity) and ADH-4 [3], which resemble Glass I and Class II enzymes, respectively, in their properties. In contrast with human liver Class I ADHs, which possess three genetically distinct subunits (a, fl and y), only a single major form is present in baboon liver. A cDNA clone for baboon liver ADH-2 has recently been isolated and examined, with the deduced amino acid sequence closely resembling that of human liver fl-ADH, with 363 of 374 residues being identical [2o]. Hence, baboon liver ADH-z has been designated as ADH f12. A second Class I ADH isozyme is observed in baboon kidney (ADH-x), which is immunologically cross-reactive with the baboon liver f12 enzyme [32], and is more homologous with the human y-ADH subunit in cDNA nucleotide sequence [33]. Southern blot analyses of baboon genomic DNA, using a baboon ADH-fl cDNA probe, as well as DNA sequencing studies of exon 5 of baboon Glass I ADH genes, demonstrate that there are three such genes in the baboon genome, even though only two are apparently expressed (forming the ADH-J~ subunit in liver and the ADH-y in kidney) [33]" Agarose- IEF analyses of baboon liver ADHs indicate the presence of isozymes with similar properties to those of human liver ~-ADH and X-ADH [3], although confirmation of the Class designation for these enzymes awaits further studies.

Mouse liver A D H s

The mouse, Mus musculus, has also been widely

used as an animal model in biochemical, behavioural and genetic research associated with alcohol, and as a result, alcohol metabolizing enzymes such as ADH have been extensively investigated in this organism [see 34]. Biochemical and genetic studies on mouse liver ADHs have reported a single Class I isozyme (designated ADH-A2), encoded by a gene (Adh-z) on chromosome 3 [35], which is the only form of ADH capable of using alcohol at physiological concentrations in the mouse ( < 5 ° mM) [6]. Genetic variants for this enzyme, among inbred strains of mice showing distinct behavioural responses in the voluntary consumption of alcohol, alcohol-induced modifi- cation of activity, or alcohol withdrawal severity, have been used to show no obvious correlation with ADH phenotype and such alcohol-related behaviours [36].

Molecular genetic studies of mouse liver ADH- A 2 have demonstrated extensive homology with human, horse and rat Class I ADHs, with 84.5% amino acid sequence identity with horse liver ADH (E subunit) [37]. The gene encoding the mouse ADH-A subunit (Adh-z) contains nine exons, and is differentially expressed among some strains, yielding high (YBR/Ki) and low (BALB/c) liver ADH activity [38]. Comparisons of the 5'- nontranslated region, and of the first 225 bps 5' to the transcription point, revealed identity in nucleotide sequence in each case. In contrast, sequences of the first intron, which contained 25 copies of a hexanucleotide repeat in the high activity strain,

Io2 Roger S. Holmes

also showed a deletion of ioi bp of this region in the low activity strain. Hence, these differences in gene expression for mouse liver ADH may be due to differences in intronic sequences, rather than in the 5" region of the gene. More recent studies of Carr et al. [39] have examined protein-DNA interactions in the 5' region of the mouse Adh-1 gene, and have identified a number of sequence elements that resemble consensus regulatory sequences, similar to those observed in human [4o] and baboon [33] 5' regions of Class I ADH genes. Bond and Singh [41] have investigated the effects of ethanol consumption upon liver ADH transcription rates among inbred strains of mice, and reported some interesting differences. C57BL/6 J mice (an alcohol-preferring strain) exhibited a 200% increase in ADH-A mRNA levels following ethanol feeding, whereas alcohol-avoiding strains (BALB/c and i29/ReJ) showed either a marginal increase or decrease in mRNA levels, as compared with isocaloric-fed control animals.

A second form of mouse liver ADH, designated ADH-B2 by Algar et al. [6], has been purified and characterized, showing kinetic [6] and immunolo- gical [42] properties similar to those for human Class III liver ADH. It is relevant to note, however, that the corresponding liver Class II isozyme, observed in human [26] and baboon [3] liver, was absent from mouse liver extracts, and from other mouse tissues examined [43].

Rat l iver A D H s

Investigations of biological phenomena associated with mammalian alcohol consumption have predominantly used the rat (Rattus rattus) as animal models, particularly in the study of the neurobiolo- gical mechanisms associated with alcohol-seeking behaviour [44], and in examining hormonal effects upon alcohol metabolism [see 45]. Rat liver ADHs exist as two major forms, and show patterns of activity and properties that are directly comparable to mouse liver ADHs, for which single forms of Class I (designated ADH 3 in the rat) and Class III (ADH2) activities have been described [7]. Pro- tein sequencing studies [46] and nucleotide sequencing of the rat liver ADH 3 cDNA [47] have shown that this enzyme contains a single amino acid insertion of asparagine at position ii6, as compared to the human, horse and mouse Class I ADHs, which has resulted from a shift in the 3'

splice junction of the fourth intron, to include an extra three base pairs to the fifth exon [45]. The gene encoding rat ADH 3 spans 13 kb and comprises nine exons and eight introns, and shares many of the consensus regulatory 5" sequences [45] observed in the corresponding human [4o], mouse [39] and baboon [33] genes.

Rat liver ADH 3 activity is modulated by castration, thyroidectomy, diabetes and hypophy- sectomy, which has been shown to be correlated with corresponding changes in ethanol elimination rates [see 48]. Castration, for example, increased liver ADH activity in male rats, apparently caused by a reduction in the degradation rate of the enzyme, in vivo [49]. Corticosteroids have been shown to have a direct effect upon rat liver ADH 3 gene expression, resulting in an increase in mRNA levels in hepatoma cells, following dexa- methasone treatment [50].

Rat liver ADH2 has been purified and characterized in its physical and enzymatic properties [7], and in primary structure [5i]. The enzyme closely resembles the sequence and properties of other mammalian Class III ADHs [52], which share the characteristic kinetic feature for this enzyme in being inactive with ethanol at concentrations of ~ o . 5 M. Putative three-dimensional structures for this ADH Class, based upon the known configuration for the horse liver Class I isozyme [53], indicate a significant increase in the hydrophilicity of the substrate barrel typical for this enzyme, which may explain why ethanol is such a poor substrate, and the reason for the functioning of Class III ADH as a glutathione- dependent formaldehyde dehydrogenase [54].

Stomach alcohol dehydrogenases--evidence for a Class IV enzyme

A number of recent studies have reported that a proportion of orally ingested alcohol is metabolized in the upper gastrointestinal tract, in both rats [55,56 ] and humans [57]. This gastric oxidation of ethanol, designated as 'first-pass metabolism', may account for up to 20% of the total rate of ethanol clearance from the body, presumably by way of stomach ADH and ALDH activities [58].

The earliest biochemical and genetic analysis of ADHs from mammalian stomach was from Smith et al. [59], who reported variants for the Y2 isozyme, which was later classified as a Class I

ADH [I]. Cederbaum and co-workers [-60] subsequently reported a 'high-Km' form of ADH, which exhibited high activity in rat stomach and hepatocellular carcinoma cells, but was absent from liver extracts. Mouse stomach ADH was also investigated [2,6,6i], the results of which showed that the stomach isozyme (designated as ADH- C2) is encoded by a distinct, but closely linked locus (.4dh-3) to that encoding liver Class I ADH (Adh-I), and exhibits a Km value of 232 mM for ethanol as substrate. In contrast, baboon stomach exhibits at least two forms of ADH active with ethanol as substrate, although showing distinct staining properties at different concentrations of ethanol (Fig. i). ADHI exhibited Class I ADH properties, being active with ethanol over the 5-500 mM concentration range studied, whereas ADH 3 showed activity only at relatively high concentrations ( > i o o mM), and required 5oo mM ethanol for high activity, thus confirming the respective 'low-Kin' and 'high-Km' properties for these enzymes. Agarose-isoelectric focusing studies for human stomach ADH have shown similar results, with a single Class I enzyme (Y2) and a 'high-Kin' form of ADH being observed [62]. Yin et al. [io] have confirmed this observation for the latter ADH isozyme (designated //-ADH) in human stomach extracts, and kinetically characterized a partially purified ADH preparation./A- ADH showed a moderately high Km (as compared with Class I ADH isozymes) of 18 mM for ethanol as substrate, which was, however, much lower than that reported for mouse [67] , rat [7] and baboon [63] stomach ADHs, with values of 232 mM, 34 ° mM and 539 mM being reported. Moreno and Pares [9] have recently purified the 'high-Km' form of human stomach ADH (named 0"-ADH) to homogeneity, and reported a Km value of 41 mM with ethanol as substrate. Both groups have suggested that t h i s / /o r o" human stomach ADH may significantly contribute towards the 'first-pass metabolism' of ethanol.

As a result of the 'high' Km status for gastric ADH, with ethanol as substrate, and the lower sensitivity of the mouse, rat, baboon and human enzymes to 4-methyl pyrazole inhibition, this isozyme was tentatively assigned Class II ADH status [2,3,7,%1o ]. Recent studies by Pares and co- workers [8,i2], however, have established the rat and human stomach enzyme as a separate form of ADH (designated Class IV). Partial amino acid

.4DH classes--differential functions Io 3

sequence data, covering six separate regions of the enzyme, showed 32-4o% residue differences from rat and human ADH Classes I-III , which suggested that the stomach ADHs are a product of a separate ADH gene Class, which has been present during evolutionary time, well prior to the appear- ance of eutherian mammals.

Evidence for a Class V ADH gene

Recent studies by Yasunami and co-workers [ii] have reported evidence for an additional class of ADH gene (ADH6-designated Class V) by isolat- ing and analyzing genomic clones from a human genomic DNA library, using a Class I ADH cDNA probe. The deduced amino acid sequence for the coding region showed 60% homology with Class I - I I I human ADHs, and a similar degree of homology was reported with gastric Class IV ADH It2], based upon partial sequence data. Slot- blot hybridization of human tissue RNAs revealed the presence of ADH6 mRNA in liver and stomach. These results are consistent with the presence of an additional ADH gene Class within the human genome, however, further studies will be required to demonstrate the presence of this ADH isozyme in vivo.

A D H classes--differential functions in metabolism

More than a decade has passed since Pietruszko [641 published her review of nonethanol substrates for ADH, which clearly identified a broad detox- ifying role for this enzyme against toxic alcohols, aldehydes and ketones ingested in the diet of mammals. Since then, our knowledge of the catalytic and molecular biological properties of mammalian ADHs has been considerably ad- vanced, and in particular, at least four separate Classes of ADHs have been identified and bioche- mically characterized. Moreover, many 'natural' in vivo substrates for ADH have been recognized, and distinct metabolic roles for the major Classes of ADH have been proposed, on the basis of their differential tissue distributions and kinetic properties. It is the purpose of this review to identify these potential separate roles for the different ADH Classes, and to compare their catalytic activities with 'natural' alcohol and aldehyde substrates, occurring both in the diet and as a metabolic by-products within the body.

lO4 Roger S. Holmes

First, it should be emphasized that ADH catalyses the reversible interconversion of alcohols and their corresponding aldehydes and ketones; and secondly, that the overall equilibrium constant of the reaction varies between io -12 and xo - s M, depending on the nature of the substrate and product [65]. Thus, ADH catalyses a reaction which is thermodynamically geared towards aldehyde reduction, rather than alcohol oxidation, although N A D + / N A D H ratios must play a key role in determining metabolic flow. In addition, many tissues exhibit very high levels of aldehyde dehydrogenase activity, which is compartmental- ized within cytoplasm, mitochondria and the endoplasmic reticulum [661, and this enzyme also plays a major role in facilitating the metabolism of alcohols to aldehydes, and subsequently to the corresponding acids, by way of the sequential activities of ADH and ALDH.

Ethanol metabolism

The predominant tissue distribution of the Class I ADHs in mammalian liver, as well as their 'low- Km' properties with ethanol as substrate, confirm their major role in alcohol metabolism in the body [n,221. Approximately, 75-800/0 of consumed alcohol is metabolized by the liver in humans [74], and of the three human liver ADH Classes, the major liver Class I isozyme (f12) exhibits the highest kcat/Km value, which gives an excellent measure of substrate efficacy, and confirms the major role for Class I ADH in ethanol metabolism (Table i). In addition, this enzyme is the only human A D H Crass with a Km value, which is

lower than circulating ethanol concentrations in the body, observed following moderate alcohol consumption (i.e. < i o mM ethanol). In contrast, the liver Class II ADH (/r 2 isozyme) exhibits a Km value more than ten times higher than such levels of circulating ethanol, and is therefore unlikely to contribute significantly to ethanol metabolism in the body (Table i) [691. Class III ADH utilizes ethanol poorly as a substrate, and is essentially inactive with this substance at physiological concentrations (Table i) [28,70 ].

Recent studies on rats have suggested that the balance of alcohol clearance of orally ingested ethanol is metabolized in the upper gastrointestinal tract, and does not enter the systemic circulation [55,75,76]. These investigations reported differences in the rate of ethanol elimination according to whether oral or intravenous routes of administration were used, and the proposed gastric oxidation of ethanol described as 'first-pass metabolism' of alcohol [761. Subse- quently, Caballeria and co-workers [56] reported similar results in humans, and proposed that gastric ethanol oxidation accounts for approximately 20°/o of the total rate of ethanol clearance in man. Most recently, Frezza and co-workers [57] have published evidence that 'first-pass metabolism' is lower among women and alcoholic individuals, which correlates with lower levels of gastric ADH activity observed in these individuals.

Human stomach exhibits two major forms of ADH activity active with ethanol as substrate, designated as Class I (Y2 isozyme) and Class IV (g or aADH) ADHs [9,io 1. The former exhibits typical Class I kinetic properties with ethanol as

Table i. Kinetic parameters for human .4DH isozyme classes with various alcohols

Ethanol 5-HTOL Benzyl alcohol Isozyme

Glass kcat/Km kcat Km kcat/Km kcat Km kcat/Km kcat Km kcat/Km

I fl 'fl ' 9.1 0.55 I6 a 7.8 0.80 I O a II O.I2 92b II ~ 47 ° i2o 3.9 ¢ 6. 9 0.28 25 ' 55 ° 0.007 7857 oc III XZ no saturation ~ no activity ~ no saturation ~ IV o'0" 280 4x 6.9 f not analyzed 620 0. 7 89 of

Kin, mM; kcat, min-1; kcat/Km, mM -I min -1. Assay conditions as described in designated references: a [67] i b [68] i ¢ [69]; a [28; e [7o] ~ f[9]- 5-HTOL, 5-hydroxytryptophol (alcohol derived from serotonin metabolism).

A D H classes--differential functions lO 5

substrate, with a Km value of 0.38 mM, but with only a modest kcat of 23 rain - l [77], whereas human stomach Class IV ADH exhibits a loo-fold higher Km value, but with a i2-fold higher kcat value (Table 1) [9]. This latter isozyme may account for up to 5o% of ADH activity in human stomach following ethanol consumption [lO], for which ethanol concentrations would be expected to be much higher than those observed in the circulation following absorption. Thus, both Class I and Class IV human stomach ADHs may play an important role in 'first-pass metabolism' of ethanol.

Formaldehyde metabolism

Formaldehyde is a toxic by-product of oxidative demethylation and some other metabolic reactions in the body, which serves as substrate for formaldehyde dehydrogenase, according to the following reaction [78]:

formaldehyde -F GSH -F NAD + S-formylglutathione + NADH -F H +

A second enzyme, S-formyl-glutathione hydrolase, irreversibly hydrolyses the reaction product, S- formylglutathione, to GSH and formate, thereby completing the oxidative conversion of formaldehyde to formate [79]. Recently, Koivusalo and co-workers [54] have shown that formaldehyde dehydrogenase and the Class III ADH isozyme share amino acid sequence and kinetic property identity. In addition, the gene for the human formaldehyde dehydrogenase and the Class III ADH have been mapped to the same region of the genome (4q 21-24) [I2], and both of these enzymes are widely distributed throughout the body [70,79]. Thus, formaldehyde dehydrogenase and Class III ADH are apparently identical enzymes, which play a major role in formaldehyde metabolism.

Biogenic alcohol metabolism

Biogenic amines such as serotonin, dopamine and norepinephrine are nornmally metabolized in the body by the sequential activities of monoamine oxidase (MAO; EC 1.4.3-4) and aldehyde dehydrogenase (ALDH; EC 1.u.1.3) , resulting in the formation of the corresponding carboxylic acid [8o]. Aldehyde reductase (AHR; EC 1.1.1.2), an NADPH-dependent enzyme, has also been impli-

cated in biogenic amine metabolism, forming the biogenic alcohol [81], which may be transported to the liver for further metabolism.

Recent studies from Vallee's laboratory have examined the possible role of liver ADHs in biogenic alcohol and aldehyde interconversions [67,72,77] , for which separate roles for the major ADH Classes have been proposed. Investigations of the kinetic properties of the human liver ADH Classes with intermediary alcohol products of norepinephrine metabolism, for example, have shown that these serve as excellent substrates for the Class I isozymes, in vitro, whereas Class II and Class III ADHs exhibited no detectable activity [77]. Similar results were observed for the alcohol by-products ofdopamine metabolism [72]. In contrast, the intermediary alcohol of serotonin metabolism, 5-hydroxytryptophol (5-HTOL), is efficiently oxidized in vitro by both liver Class I and Class II ADH Classes (Table 1) [67] , thereby indicating that both of these Classes may be involved in 5 -HTOL metabolism. Again, Class III ADH showed no detectable activity with this biogenic alcohol [67]. Thus, liver Class I ADHs are capable of oxidizing biogenic alcohols derived from norepinephrine, dopamine and serotonin, in vitro, and may function in the metabolism of these compounds in the body. Class II ADH is selectively active with 5-HTOL, derived from serotonin metabolism, and may also function as such in vivo.

Peroxidic aldehyde reduction

A range of cytotoxic aldehydes have been reported in mammalian cells following lipid peroxidation processes, including alkanals, u-alkenals and 4- hydroxyalkenals [82], for which liver ADH has been proposed to function as a 'peroxidic aldehyde' reductase [83]. Recently, Sellin et al. [73] have examined the kinetic properties of the three Classes for human liver ADHs with a range of 4-hydroxyalkenals. The Class I and Class II ADHs efficiently reduced these aliphatic 4-hydroxyalkenals with chain lengths from 5 to 15 carbons, exhibiting comparable kcat and Km values to those of simple aliphatic aldehydes of comparable chain lengths. The liver Class II ADH was particularly efficient in the reduction of 4-hydroxyalkenals, showing kcat values which were 5-8 times higher than for the liver Class I

io6 Roger S. Holmes

isozymes. Moreno and Pares [9] have examined the kinetic properties of human stomach Class IV ADH with the peroxidic aldehyde, trans-2-hexe- hal, and shown high values for the specificity constant (kcat/Km) for this substrate. Algar et al. [63] have reported similar results for the corresponding baboon stomach ADH. Thus, two of the major classes of liver ADH (I and II) and the major Class of stomach ADH (IV) may serve to protect these tissues against cytotoxic peroxidic aldehydes.

Aliphatic and aromatic alcohol metabolism

A wide range of aliphatic and aromatic alcohols, aldehydes and ketones have been reported in foods and beverages, and serve as substrates for ADHs in the body [reviewed in 64]. A number of alcohols, for example, have been reported in beer and wine, including methanol, ethanol, propanol, isobuta- nol, isoamyl alcohol, 2-methyl i-butanol, 3-methyl x-butanol, 2,3-butanediol , t-hexanol, i-octanol, benzyl alcohol and 2-phenethanol [84]. In particular, isoamyl alcohol and 2,3-butanediol have been reported at relatively high concentrations in wine, at 2 mM and 7-5 mM, respectively [85].

Studies on mammalian liver Class I ADHs have shown that aliphatic alcohols of a wide range of chain length serve as excellent substrates for these enzymes [64] , showing a decrease in Km value with the increase of alcohol chain length, but with kcat values remaining relatively constant. This is consistent with the proposed Theorell-Chance mechanism for Class I ADHs, with enzyme- coenzyme dissociation serving as the rate determining step [see 64]. The human liver Class II [26,29] and Class III [5,28] ADHs, and the human stomach Class IV ADH [9], all show a similar trend towards lower Km values with increase in alcohol chain length, but exhibit distinct properties in each case. The latter enzyme, in particular, showed high catalytic efficiency with medium to long chain alcohol substrates, which may assist in the 'first-pass' clearance of these compounds ingested in the diet. Aromatic alcohols, such as benzyl alcohol, also serve as excellent substrates for stomach Class IV ADH [9], although the liver Class II form exhibited the lowest Km for this substrate, and maintained a higher level of efficiency, as compared to the liver Class I isozymes (Table i).

We can conclude from these investigations that the liver and stomach are particularly capable of oxidizing a wide range of aliphatic and aromatic alcohols, ingested in the diet. Human [9], baboon [3], mouse [6] and rat [7] stomach ADHs, for example, exhibit a high level of catalytic efficiency towards such substrates, providing the capacity for first-pass clearance of ingested compounds. More- over, liver Class I and Class II ADHs also show substrate preference towards alcohols of longer chain length, and would be expected to be predominantly responsible for 'clearing' such compounds from the circulation, by way of liver oxidative metabolism.

Conclusions

Substantial progress has been made since x979, following the last major review of the functions of ADH in alcohol and aldehyde metabolism [64]. At that time, only the major Class I liver ADHs had been subjected to detailed kinetic study, and an initial study performed on the liver Class II enzyme [26]. Following the recognition of human Z-ADH as a distinct Class III form [28], all three Classes were subsequently subjected to intensive biochemical, molecular genetic and kinetic analysis during the i98os. The three human Class I ADH polypeptides have been fully characterized at the protein and cDNA level, and one of the genes (ADH2) subjected to detailed molecular analysis. Chromosomal mapping studies [see x2] have shown that all five genes encoding the liver ADHs of Classes I - I I I are closely located in the 4q u*-25 region, and with the following gene order for the Class I ADH genes:

5 ' -ADH3-ADH2-ADHI- 3' [86].

The major variants for ADH2 and ADH 3 in human populations have been characterized, and associated functional changes in biochemical properties explained at the molecular level. The liver Class II and Class III ADHs have also been fully characterized in terms of their amino acid and cDNA sequences, with the results confirming that these are distinct structural classes. More recent studies on rat stomach ADH have provided evidence for a Class IV enzyme, based upon partial amino acid sequences [8], and biochemical studies of human stomach ADHs have reported a new form (designated 3/or or) of ADH [9,to].

A D H classes--differential functiom lO 7

Kinetic analyses of these four Classes of A D H s have indicated a variety of functions, for which some apparently overlap between Classes. The following respective functions are indicated from the results of those studies: oxidation of e tha- n o l - l i v e r Class I and stomach Class IV ADHs; other aliphatic and aromatic a lcohols-- l iver Classes I and II and stomach Class IV ADHs; 'biogenic ' alcohol metabol i sm-- l iver Class I and Class II (alcohol metabolite of dopamine only) ADHs ; reduction of peroxidic a ldehydes-- l iver Classes I and II, and stomach Class IV ADHs; and glutathione-dependent formaldehyde dehydrogenase--Class III A D H .

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[3] Holmes RS, Courmey YR, VandeBerg JL. Alco- hol dehydrogenase isozymes in baboons: tissue distribution, catalytic properties and variant phe- notypes in liver, kidney, stomach and testis. Alcoholism: Clin Exp Res I986;1o:623-3o.

[4] Boleda MD, Julia P, Moreno A, Pares X. Role of extrahepatic alcohol dehydrogenase in rat ethanol metabolism. Arch Biochem Biophys 1989; 274:74-81.

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