Clinica Chimica Acta, 202 (1991) 211-218

211

0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50

CCA 05084

Effect of alcohol on &aminolevulinic acid dehydratase and porphyrin metabolism in man Ina Sieg i, Manfred

0. Doss i, Hubert

Kandels

* and Jiirgen Schneider

*

’ Abteilung fiir Klinische Biochemie im Interdiszipliniiren Medizinlschen Zentrum and 2 Abteilung Endokrinologie und Stoffwechsel im Medizinischen Zentrum fdr Innere Medizin, Klinikum der Philipps-Uniuersitiit, Marburg / Lahn (FRG) (Received 15 August 1990; revision received 23 July 1991; accepted 26 July 1991)

Key words: S-aminolevulinic acid dehydratase; Alcohol; Enzyme inhibitor; Enzyme reactivator;

b-Aminolevulinic acid; Coproporphyrin

Summary The influence of alcohol on porphyrin metabolism was investigated in 6 healthy non-alcoholics and 19 patients with chronic alcohol abuse. In the healthy subjects, blood and urine samples were obtained before and after acute alcohol exposure, whereas in the chronic alcoholics only one examination was performed. In both groups a significant inhibition of &aminolevulinic acid dehydratase was demonstrated. The activity was partially restored in vitro by addition of zinc ions or dithiothreitol. A combination of both activators produced reactivation to normal levels. Coproporphyrinuria was more prominent in chronic alcoholics (373 nmo1/24 h on average, upper norm 119 nmo1/24 h) compared to non-alcoholics (140 nmo1/24 h). Urinary porphobilinogen and &amino-levulinic acid were normal except for a moderately increased S-aminolevulinic acid in four healthy individuals. In conclusion, alcohol causes reversible inhibition of &aminolevulinic acid dehydratase. The metabolic changes reflect both an inhibition of S-aminolevulinic acid dehydratase and coproporphyrinogen oxidase; a simultaneous, moderate induction of hepatic S-aminolevulinic acid synthase is suggested. Erythrocyte b-aminolevulinic acid dehydratase activity could serve as a sensitive indicator for both acute and chronic alcohol consumption even better than coproporphyrinuria.

Correspondence Deutschhausstr.

to: Prof. Dr. M. Doss, Abteilung fur Khnische Biochemie, 17 l/2, D-3550 Marburg/Lahn, Federal Republic of Germany.

Philipps-Universitat,

212

Introduction Alcohol is known to be a significant and frequent factor in the induction and maintenance of subclinical and clinical disturbances of porphyrin metabolism, including hepatic porphyrias [ll. In healthy adults the excessive consumption of alcohol often leads to a secondary hepatic coproporphyrinuria, which may persist in the case of alcoholic liver defects and can be accompanied by a secondary protoporphyrinemia [l-3]. In the genetically determined acute and chronic hepatic porphyrias alcohol often initiates the biochemical and clinical manifestation of the diseases [4]. Among those enzymes of porphyrin metabolism which are affected by alcohol, &aminolevulinic acid dehydratase (ALA-D,.porphobilinogen synthase, EC 4.2.1.24) is second in the heme biosynthetic pathway and catalyses the condensation of two ALA molecules to porphobilinogen, the immediate precursor of porphyrinogens. Biochemically, this enzyme consists of 8 identical subunits and contains SH-groups and zinc, which are essential for full activity [5-71. In this study, erythrocyte ALA-D activity was investigated in healthy non-alcoholics before and after alcohol uptake and in patients with chronic alcohol abuse. As a point of special interest we examined the reversibility of alcohol-evoked ALA-D inhibition in vitro. Finally, the effects of alcohol-mediated enzyme disturbances on heme biosynthesis were analysed by determining the urinary excretion of heme precursors. Patients and methods Investigations were carried out on two groups of individuals: 1. Chronic alcoholics (n = 19, age ranging from 28 to 62 yrs with a mean value of 45 yrs, 18 male and one female) with a daily alcohol consumption of > 150 g. Alcohol uptake was assessed by anamnestic information given. A single blood and urine sample per patient were obtained 3 to 5 days after admission to hospital. Although blood alcohol had not been determined, it was assumed to be normal at the time the bood sample was taken. 2. Healthy male non-alcoholics (n = 6) between 18 and 28 yrs of age, who voluntarily took part in the experiment. None of them had been consuming alcohol during the 4 days prior to the experiment. One hour before the alcohol uptake started, they had a light meal with low fat content. Over a period of 4 h they drank 160-230 g of alcohol in the form of beer, wine and liqueur (alcohol. content 4, 10 or 20% of volume, respectively). AI1 of the individuals had normal blood lead levels of between 0.49 and 0.72 pmol/l. Porphyrin metabolism was studied by analyses of blood and urine. Blood samples were drawn immediately before and 7 h after stopping alcohol uptake. Calculated concentrations of blood alcohol, assessed according to the method by Widmark, ranged between 1.06 and 3.59 g %o for the 7-h blood sample (middle body weight 70 kg, reduced body mass 49 kg, alcohol elimination rate 0.1-0.2 g %o per h). Determination of ALA-D activity was performed according to the European standard method with the slight modification that 100 ~1 of packed erythrocytes

213

were analysed IS]. Enzyme activity was reactivated by addition of zinc ions (zinc chloride) at a final concentration of 10e4 M, dithiothreitol (lo-* M) and the combination of both activators 191. Reactivation factors were determined as the quotient from [ALA-D activity in the presence of the respective reactivator] and [AL&D activity without reactivator]. Urinary excretions of ALA and porphobilinogen were assayed spectrophotometrically after isolation by ion exchange chromatography [lo], porphyrins by high performance thin-layer chromatography [ 111. Urine samples were analysed immediately after alcohol uptake and 24 h Iater. Statistical evaluation of the data was performed by using Student’s t-test. Results The non-alcoholics showed a significant ALA-D inhibition of 44% after acute alcohol intake (Table I). In the presence of zinc ions there was still a significantly reduced enzyme activity, although the inhibitory effect was markedly diminished to an average of 30%, corresponding with a reactivation factor of 1.4. The addition of dithiothreitol resulted in a reversal of ALA-D inhibition, showing an average enzyme activity that reached, in spite of alcohol, 75% of the initial value, with a reactivation factor of 1.7. The most potent effect on restoration of ALA-D activity occurred, with 78%, reactivation factor 1.9, under combined addition of zinc and dithiothreitol (Table I). In the group of alcoholics, ALA-D activity was diminished to a level of 43% on average (Table II) and thus underwent a stronger inhibition in comparison to the healthy control group with 56% residual activity after acute alcohol uptake. Reactivation factors, too, were with 1.7, 2.8 and 3.4 on a higher level than those found in non-alcoholics (1.4, 1.7 and 1.9). The determination of urinary ALA, porphobilinogen and coproporphyrin revealed for all non-alcoholics increased levels after alcohol consumption, except for

TABLE I Inhibition and reactivation of erythrocyte ALA-D activity in healthy non-alcoholics Healthv adults ;

ALA-D activitv (FmoI/l.h) _ a

Mean f SD Range P

b

Reactivation factors (%)

zinc ions (1O-4 M)

dithiothreitol (lo-’ M)

zinc ions + dithiothreitol

a

b

a

b

a

b

1019 f 149

584 * 19.5

(56 f 14)

1.2 & 0.05

1.4 *0.2

1.2 f 0.08

1.7 * 0.2

1.4 t 0.05

1.9 +0.3

866 -1250

333 -776

(36 -70)

1.1 -1.2

1.1 -1.8

1.1 -1.3

1.4 -2.0

1.3 -1.4

1.6 -2.5

< 0.005

(a) Before, (b) after alcohol uptake. * Mafe, 18-28 yrs of age.

< 0.02

< 0.005

< 0.005

214 TABLE II Inhibition and reactivation of ALA-D activity and excretion of urinary heme precursors in chronic alcoholics Patients

Erythrocyte Reactivation factors ALA-D activity dithioboth (pmol/l. h) (%I a zinc threitol

Urinary S-ALA b &mo1/24 h)

Urinary copro ’ (nmo1/24 h)

Mean f SD

534 5189

47 +16

1.7 f 0.6

2.8 +1.3

3.4 f 1.4

29 f 14

373 + 175

Range

149 -8.58

13 -75

1.0 -2.9

1.3 -4.9

1.5 -5.7

9 -53

139 -759

Normal values (mean + SD, or range)

1136 +116

80 -120

< 1.2

< 1.3

< 1.4

2-49

21-119

(n = 19)

a Of healthy controls; b d-Aminolevulinic the same as in Table I.

acid; ’ coproporphyrin.

Concentrations

of reactivators are

a single ALA value. While porphobilinogen excretion did not exceed the normal range, in three individuals a pathological increase of ALA and coproporphyrin was observed, another one showed an isolated elevation of ALA (Table III). In contrast, among the group of chronic alcoholics, ALA excretion remained, with one exception, within the normal range. However, values for coproporphyrin were found to be distinctly or even strongly increased (Table II>. The determination of porphobilinogen revealed, in comparison to the findings in non-alcoholics, no abnormal results (data not shown). Discussion This study revealed an ALA-D inhibition of about 50% in healthy adults after alcohol consumption and in chronic alcoholics. The finding is in agreement with in vitro [12] and in vivo experiments [1,3,13-151 of other authors. As one cause for the ALA-D inhibition an oxidation of SH-groups, which are essential for the full

TABLE III Urinary excretion of porphyrin precursors and coproporphyrin

Mean f SD Range Normal range

in healthy adults

&Aminolevulinic acid &mol/24 h)

Porphobilinogen (pmol/24 h)

Coproporphyrin (nmol/24 h)

a

b

a

a

21+18 2-43

74+31 36-106

1.1+0.3 5.4+2.1 0.7-1.3 2.4-7.2 0.5-7.5

2-49

b

25+11 10-44

(a) Before, (b) after alcohol uptake. Individuals are the same as in Table I.

b 140+69 58-221 21-119

215

TABLE IV Inhibitors of ALA-D activity in man or laboratory animals Alcohol Benzene Bromobenzene Gasoline Kerosine

Lead Styrene Succinylacetone Trichloroethylene

enzyme activity, is being discussed [1,12], as well as a diminution of SH-donors like cystein and/or glutathion by binding to the alcohol metabolite acetafdehyde [16]. Several inhibitors of ALA-D are known in man and animals (Table IV) [17-243. Further investigations indicated that lead induces a reversible ALA-D inhibition 1253, while styrene leads to an irreversible decrease of enzyme activity [26]. Therefore, it was part of this study to find out to which type of inhibitor alcohol would have to be assigned. In all cases investigated a reactivation of ALA-D activity was found in vitro. Comparable to lead poisoning [27], the combination of zinc and dithiothreitol proved to be the most effective enzyme reactivator, followed by dithiothreitol; in both conditions the difference between initial ALA-D activity and restored activity was no longer si~ificant, meaning that enzyme activity could be reactivated into the so-called normal range (Table I, II). Similar observations have been made in lead intoxication, where ALA-D inhibition was accompanied by a loss of bound zinc and active sulfhydryl groups 271, a process which is probably also of importance in alcohol-mediated ALA-D inhibition. The precise mechanism of ALA-D reactivation, resulting in a reconstitution of reduced SH-groups [7,28], has not yet been completely elucidated. The significance of zinc is probably to be seen in its close structural position next to the sulfhydryl groups of the active site, thus offering stabilization and protection against oxidizing agents [7,22,29]. In contrast to the findings in lead exposure, the extent of ALA-D inhibition due to alcohol, as well as enzyme reactivation, were markedly reduced. While in cases of acute lead intoxication the loss of ALA-D activity usually lies over 90%, with corresponding activation factors of well over 6 [9], alcohol-indu~d enzyme inhibition mainly ranged from 40 to 70%, accompanied by maximum reactivation factors of 3.0 on average (normal: < 1.4, Table I, II). The mechanism of ALA-D inhibition seems to be different with irreversible inhibitors like styrene, where a direct enzyme inhibition, caused by covalent binding of styrene metabolites to the sulfhydryl groups, is amplified by an additional suppression of ALA-D synthesis f241. Before assessing the effects of ALA-D inhibition on heme biosynthesis by determination of urinary ALA, porphobilinogen and coproporphyrin, two preconsiderations were taken into account: (1) ALA-D is not the only enzyme of porphyrin biosynthesis that is affected by alcohol. Moreover, alcohol also suppresses the activity of uroporphyrinogen decarboxylase, copropo~hyrinogen oxidase and ferrochelatase [1,3,14], while it has an inductive effect on ALA synthase

216

Fig. 1. Alcohol effects on the enzymatic chain of heme biosynthesis: [T] induction; [&I inhibition; [ALA-S] b-aminolevulinic acid synthase; [ALA-D] &aminolevulinic acid dehydratase; [PBG-D] porphobilinogen deaminase; [URO-III-S] uroporphyrinogen III synthase; [LJD] uroporphyrinogen decarboxylase; [CO] coproporphyrinogen oxidase; [PO] protoporphyrinogen oxidase; [FC] ferrochelatase.

[1,30,31] and porphobilinogen deaminase [3,14] (Fig. 1). (2) Although present in all body tissues, hepatic enzymes of porphyrin metabolism are the ones which primarily determine the excretion of heme precursors in urine and feces [4]. As there was no clinical indication for a liver biopsy in any of the individuals, ALA-D analyses in this study were restricted to red blood cells. However, there is indirect evidence for a similar reaction of liver and erythrocyte ALA-D after alcohol uptake: (a) as was demonstrated before, close parallels exist in ALA-D inhibition by alcohol and by lead; in lead intoxication, a correlation of hepatic and erythrocyte ALA-D activity in humans has been proven by Secci et al. [32]. (b) The increase of urinary ALA after alcohol consumption in most of the non-alcoholics was far too prominent to be explained by a mere reduction of erythrocyte ALA-D activity, and most likely is the result of a simultaneous ALA-D inhibition in the liver [33]. (c) From animal experiments it is known that in rats alcohol significantly suppresses erythrocyte, hepatic and renal ALA-D [12]. Determination of urinary heme precursors in the groups of non-alcoholics and chronic alcoholics revealed no pathological changes of porphobilinogen excretion, although the six non-alcoholics showed a distinct elevation of porphobilinogen after alcohol uptake. Thus, alcohol-induced ALA-D inhibition seems to be completely compensated by the physiological reserve capacity of the enzyme, probably in combination with an enhanced availability of substrate resulting from a slight induction of ALA synthase. Another indication for the apparently high ALA-D reserve capacity can be deduced from the clinical observation that even patients with a residual ALA-D activity of 2% of normal, due to an inherited enzyme deficiency in the homozygous state [34], do not develop anemia and hence still maintain a sufficient heme production in the bone marrow. The increased excretion of ALA in 5 of the healthy non-alcoholics can be interpreted as resulting from ALA-D inhibition. Another synergistic effect could be seen in the induction of ALA synthase, which is a physiological process of counter-regulation to compensate

217

for the suppressive effects of alcohol and its metabolites on the enzymes of heme synthesis (Fig. 1). In the group of alcoholics only 2 patients revealed a moderate enhancement of urinary ALA.Therefore, chronic alcohol uptake seems to lead to a reduction of surplus ALA synthase activity, thus adapting ALA synthase induction to the appropriate level for sufficient heme production, while sudden, acute alcohol uptake results in an excessive induction of ALA synthase. Urinary coproporphyrin excretion turned out to be the parameter affected most by alcohol uptake: half of the healthy volunteers and most of the alcoholics revealed a pathological coproporphyrinuria, probably resulting from an alcohol-induced inhibition of coproporphyrinogen oxidase in the liver. Comparable with the metabolic findings both in acute hepatic porphyrias and acute lead poisoning [4], alcohol-induced urinary excretion profiles of heme metabolites are of hepatic origin and can be explained by a synergism of inhibitory and inducing mechanisms (Fig. 1). In conclusion, ALA-D activity may well be regarded as a sensitive biochemical indicator for chronic alcohol consumption, as has been proposed by Krasner et al. [13], and also for acute alcohol uptake. The easily performed in vitro reactivation of this enzyme is an additional help in recognizing and excluding irreversible inhibitory effects such as those caused by styrene [26] or hereditary enzyme deficiency [9,34]. Acknowledgements

We are indebted to Mrs. Sabine Preis for skilful laboratory assistance. This study was supported by the Deutsche Forschungsgemeinschaft, Grant Do. 134. References Doss M. Alcohol and porphyrin metabolism. In: Seitz HK, Kommerell B, eds. Alcohol related diseases in gastroenterology. Berlin, Heidelberg, New York: Springer, 1985;232-252. Orten JM, Doehr SA, Bond C, Johnson H, Pappas A. Urinary excretion of porphyrins and porphyrin intermediates in human alcoholics. Q J Stud Alcohol 1963;24:598-609. McCall KEL, Moore MR, Thompson GG, Goldberg A. Abnormal haem biosynthesis in chronic alcoholics. Em J Clin Invest 1981;11:461-468. Doss MO. Hepatic porphyrias: pathobiochemical, diagnostic, and therapeutic implications. In: Popper H, Schaffner F, eds. Progress in liver diseases, vol. VII. New York: Grune and Stratton, 1982;573-597. Shemin D. 5Aminolaevulinic acid dehydratase: structure, function, and mechanism. Phil Tram R Sot Land B 1976;273:109-115. Anderson PM, Desnick RJ. Purification and properties of &aminolevulinate dehydratase from human erythrocytes. J Biol Chem 1979;254:6924-6930. Tsukamoto I, Yoshinaga T, Sano S. The role of zinc with special reference to the essential thiol groups in &aminolevulinic acid dehydratase of bovine liver. Biochim Biophys Acta 1979;570:167-178. Geisse S, Briiller H-J, Doss M. Porphobilinogen synthase (d-aminolevulinic acid dehydratase) activity in human erythrocytes: reactivation by zinc and dithiothreitol depending on influence of storage. Clin Chim Acta 1983;135:239-245. Doss M, Laubenthal F, Stoeppler M. Lead poisoning in inherited &aminolevulinic acid dehydratase deficiency. Int Arch Environ Health 1984;54:55-63.

218 10 Doss M, Schmidt A. Quantitative Bestimmung von &Aminolevulinsaure und Porphobilinogen im Urin mit Ionenaustauschchromatographie-Fertigslulen. Z Klin Chem Klin Biochem 1971;9:99-102. 11 Doss M. Trennung, Isolierung und Bestimmung von Proto-, Kopro-, Pentacarboxy-, Hexacarboxy-, Heptacarboxy- und Uroporphyrin. Hoppe-Seyler’s Z Physiol Chem 1969;350:499-502. 12 Moore MR, Beattie AD, Thompson GG, Goldberg A. Depression of &aminolevulinic acid dehydratase activity by ethanol in man and rat. Clin Sci 1971;40:81-88. 13 Krasner N, Moore MR, Thompson GG, McIntosh W, Goldberg A. Depression of erythrocyte d-aminolaevulinate acid dehydratase activity in alcoholics. Clin Sci Mol Med 1974;46:415-418. 14 McCall KEL, Thompson GG, Moore MR, Goldberg A. Acute ethanol ingestion and haem biosynthesis in healthy subjects. Eur J Clin Invest 1980;10:107-112. 15 Kondo M, Urata G, Shimizu Y. Decreased liver d-aminolevulinate dehydratase activity in porphyria cutanea tarda and in alcoholism. Clin Sci Mol Med 1983;46:415-418. 16 Lieber CS. Metabolism and metabolic effects of alcohol. Semin Hematol 1980;17:85-99. 17 Bonsignore D. &4rninolaevulinate dehydratase activity of erythrocytes as a diagnostic test in occupational lead poisoning. Med Lavoro 1966;57:647-652. 18 Beattie AD, Briggs JD, Canavan JSF, Doyle D, Mullin PJ, Watson AA. Acute lead poisoning. Five cases resulting from self-injection of lead and opium. Quart J Med 1975;(XLIV)174:275-284. 19 Lindblad B, Lindstedt S, Steen G. On the enzymic defects in hereditary tyrosinemia. Proc Nat1 Acad Sci USA 1977;74:4641-4645. 20 Rao GS, Pandya KP. Hepatic metabolism of heme in rats after exposure to benzene, gasoline and kerosine. Arch Toxicol 1980;46:313-317. 21 Tschudy DP, Hess RA, Frykholm BC. Inhibition of S-aminolevulinic acid dehydratase by 4,6-dioxohepatonic acid. J Biol Chem 1981;256:9915-9923. 22 Fujita H, Koizumi A, Yamamoto M, Kumai M, Sadamoto T, Ikeda M. Inhibition of d-aminolevulinate dehydratase in trichloroethylene-exposed rats, and the effects on heme regulation. Biochim Biophys Acta 1984;800:1- IO. 23 Koizumi A, Fujita H, Sadamoto T, Ohmachi T, Watanabe M. Ikeda M. Cytochrome P-450 system dependent depression of d-aminolevulinic dehydratase activity by bromobenzene in rats. Toxicol 1984;32:1-10. 24 Fujita H, Koizumi A, Hayashi N, Ikeda M. Reduced synthesis of 5-aminolevulinate dehydratase in styrene-treated rats. Biochim Biophys Acta 1986;867:89-96. 25 Sakai T, Yanagihara S, Ushio K. Restoration of lead-inhibited &aminolevulinate-dehydratase activity in whole blood by heat, zinc ion and (or) dithiothreitol. Clin Chem 1980;26:625-628. 26 Fujita H, Koizumi A, Furusawa T, Ikeda M. Decreased erythrocyte S-aminolevulinate dehydratase activity after styrene exposure. Biochem Pharmacol 1987;36:711-716. 27 Fujita H, Orii Y, Sano S. Evidence of increased synthesis of b-aminolevulinic acid dehydratase in experimental lead-poisoned rats. Biochim Biophys Acta 1981;678:39-50. 28 Tsukamoto I, Yoshinaga T, Sano S. Zinc and cystein residues in the active site of bovine liver &aminolevulinic acid dehydratase. Int J Biochem 1980;12:751-756. 29 Bevan RB, Bodlaender P, Shemin D. Mechanism of porphobilinogen synthase. J Biol Chem 1980;255(5):2030-2035. 30 Shanley BC, Zail SS, Joubert SM. Effect of ethanol on liver &aminolevulinate synthase activity and urinary porphyrin excretion in symptomatic porphyria. Br J Haematol 1969;17:389-396. 31 Lane SE, Stewart ME. Alcohol-mediated effects on the level of cytochrome P-450 and heme biosynthesis in cultured rat hepatocytes. Biochim Biophys Acta 1983;755:313-317. 32 Secchi GC, Erba L, Cambiaghi G. Delta-aminolevulinic acid dehydratase activity of erythrocytes and liver tissue in man. Arch Environ Health 1974;28:130-132. 33 Doss M, Miiller WA. Acute lead poisoning in inherited porphobilinogen synthase t&aminolevulinic acid dehydrase) deficiency. Blut 1982;45:131-139. 34 Doss M, v Tiepermann R, Schneider J, Schmid H. New type of hepatic porphyria with porphobilinogen synthase defect and intermittent acute clinical manifestation. Klin Wochenschr 1979;57:11231127.

Effect of alcohol on delta-aminolevulinic acid dehydratase and porphyrin metabolism in man.

The influence of alcohol on porphyrin metabolism was investigated in 6 healthy non-alcoholics and 19 patients with chronic alcohol abuse. In the healt...
604KB Sizes 0 Downloads 0 Views