Department of Biochemistry and Biophysics, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois.
* The technical assistance and cooperation of Mostafa Bigdeli, Frank Letizia, Cynthia Weiner, and Diane Konrad are gratefully acknowledged. Supported in part by Loyola GRS and USPHS NIAAA 00266.
Several aromatic amines of neural significance are known to cyclize or condense with aldehydes. The principal products of these reactions are compounds structurally akin to certain phenolic plant alkaloids--1,2,3,4-tetrahydroisoquinolines when catecholamines are the reactants, and 1,2,3,4-tetrahydro-Bcarbolines when indoleamines are involved. The aldehyde of importance has been acetaldehyde, a metabolic product of ethanol (1,2). Another physiological aldehyde that has received attention is 3,4-dihydroxyphenylacetaldehyde, a normal metabolic intermediate which can cyclize with dopamine to form a 1-benzyl alkaloid, tetrahydropapaveroline (3,4). Also, formaldehyde has received limited consideration in terms of these condensation reactions (5) because it is a methanol metabolite, and is possibly circulating in the alcoholic during chronic ethanol ingestion (6).
Several other chapters in this book and articles elsewhere (1,7) adequately present the background work on catecholamine-derived tetrahydroisoquinolines, and the rationale for suggesting that the alkaloids may be aberrant intraneuronal substances implicated in alcohol dependence or even tolerance. However, in contrast to the rapid expansion of provocative pharmacological findings on tetrahydroisoquinolines which are discussed in the accompanying chapters, research on alkaloid formation appears quite dismal. Indeed, researchers and clinicians must appreciate that it is still not known whether tetrahydroisoquinolines are found in significant amounts in alcoholic drinkers. There are probably a number of reasons why this is unanswered. For example, the methods for tetrahydroisoquinoline detection are still expensive, highly specialized and fairly difficult to carry out (with the exception of high pressure liquid chromatography, vide infra), and well-controlled human intoxication studies apparently have not been done, or at least have not been reported. At any rate, urinary excretion profiles from such studies conceivably could be unenlightening, even in the face of significant tissue alkaloid synthesis.
In this chapter, recent and past data on the in vivo formation of catechol tetrahydroisoquinolines in intoxicated rats will be presented, and its relevance to human alcoholism considered. The data has been obtained mainly from experiments in our laboratory using gas chromatography with electron capture detection (ECD). Several new developments in the methodology of tetrahydroisoquinoline detection, as well as new directions in alkaloid formation, will be stressed.
The experiments discussed here used non-fasted SpragueDawley male rats 180-350 g. Drugs (ethanol, pyrogallol, disulfiram) were used as obtained from commercial sources; pargyline was a gift from Dr. S. Speciale. Injection solutions and ethanol gavage solutions were made up in isotonic saline. Disulfiram (Ayerst) was mulled in 1N acetic acid, shaken ten minutes, and titrated to pH 6.75-7.1 with 1N NaOH.
CATECHOL EXTRACTIONS (8)
Immediately following decapitation, brains were removed and rinsed in cold isotonic saline. Either the entire catecholamine-rich areas (pyrogallol experiments) or the caudates and hypothalami (disulfiram experiments) were dissected out, weighed and homogenized in 1N perchloric acid. Following centrifugation, the supernatants were extracted for catechols with aluminum oxide (A1203). The hydrochloric acid extracts from A1203 were lyophilized and stored at -20°C prior to derivatization.
For acetaldehyde and ethanol, 200 ul blood samples were quantitated on Porapak QS gas chromatographic columns by the head space technique, as described elsewhere (9). Tetrahydroisoquinolines were determined according to the procedure of Bigdeli and Collins (8). Lyophilized acid extracts from tissues were derivatized with heptafluorobutyryl anhydride in acetonitrile, separated on relatively polar (5% OV-17 on 80/100 gas chrom Q) and non-polar (5% SE-54 on 80/100 GCQ) 6 ft. glass columns, and analyzed by 3H- or 63 Ni-ECD (Varian). Derivatized catechols from adrenal glands were separated on 5% GEXF1105 columns. In ethanol experiments using pyrogallol, 14c_ catecholamines were carried through each sample in order to determine recoveries (10). In the disulfiram-ethanol experiments, recoveries were obtained by adding known quantities of salsolinol to selected perchloric acid extracts.
As discussed in other reports (10,11), treatment of rats with acute or chronic ethanol by liquid diet or gavage failed to produce detectable concentrations of brain salsolinol (SAL), the tetrahydroisoquinoline product of acetaldehyde with dopamine, or of N-methyl-4-hydroxy-salsolinol (MOSAL), adrenal tetrahydroisoquinoline derived from acetaldehyde and epinephrine (Figure 1).
The combination of pyrogallol (250 mg/kg i.p. sixty minutes before ethanol) and acute ethanol (three i.p. injections, 9 g/kg total, over five hours, with sacrifice two hours later) elevated acetaldehyde blood levels 10-20 fold. Evidence was obtained on two gas chromatographic columns for the formation of SAL in the combined caudate, midbrain, and brain stem areas of pyrogallol/ethanol-treated rats (10). As shown in Table 1, DOPA administration (100 mg/kg i.p.) thirty and 150 minutes after pyrogallol did not significantly change SAL concentrations. However, pargyline pretreatment (100 mg/kg i.p. twenty-four hours and one hour before pyrogallol) caused a dramatic increase in the concentrations of the dopamine-derived tetrahydroisoquinoline (Table 1). Although not consistently assayed, acetaldehyde blood levels were generally similar at sacrifice between these three groups. Gas chromatograms of catechols from controls (saline rather than ethanol) showed no evidence for the presence of SAL.
Rats were treated as described in the text. Number of animals in parentheses. *p < 0.02 when compared to pyrogallol/ ethanol or pyrogallol/ethanol + DOPA.
Adrenals from pyrogallol/ethanol rats contained a new catechol which chromatographed identically with MOSAL, the suspect epinephrine derivative (Figure 2). DOPA treatmeq as described above had little effect on adrenal epinephrine and MOSAL concentrations (Table 2). Furthermore, pargyline, contrary to the brain situation, did not significantly elevate either the catecholamine or the adrenal tetrahydroisoquinoline. Ethanol intoxication did reduce adrenal epinephrine levels significantly in pyrogallol-treated rats, and as shown in Table 2, pargyline appeared to block this reduction while DOPA did not.
In similar studies underway with disulfiram and ethanol, disulfiram was intubated daily (25 mg/kg) for one week, and a single dose of 25% ethanol or saline was intubated on the last day. In these studies (Letizia and Collins, in preparation), acetaldehyde blood levels ninety minutes after 3 g/kg and 5 g/kg ethanol were 379 nmoles/ml and 551 nmoles/ml, respectively (Table 3). "Endogenous" acetaldehyde blood levels in disulfiram/saline controls were low but measurable (30-60 nmoles/ml).
Using the dual column technique with 3H-ECD, SAL was identified and quantitated in the caudatal and hypothalamic areas of disulfiram/ethanol-treated rats. Concomitant with a 1.5-fold increase in acetaldehyde blood levels, the concentrations of SAL increased by 60-85% (Table 3). There was no indication of salsolinol in the respective brain areas from disulfiram-saline rats. Adrenal MOSAL analyses are in progress.
Fig. 2. Three gas chromatograms of the heptafluorobutyryl (HFB)-derivatized catechols in rat adrenals. Chromatogram on the left is the control (pyrogallol-saline) profile, with E (epinephrine), DA (dopamine) and NE (norepinephrine). The center (experimental) chromatogram from rats treated acutely with ethanol following pyrogallol shows the presence of a new catechol (arrow) with a retention time identical to the E-derived tetrahydroisoquinoline, N-methyl-4-hydroxy-SAL (MOSAL). Addition of the HFB-derivatized N -methy1-4 -hydroxySAL to the experimental sample potentiates this new peak, as shown on the chromatogram on the right.
Conditions: Rats were treated and adrenal catechols were prepared as described in the text. Individual samples derived from single pairs of adrenals were diluted with 10-20 ml. ethyl acetate, and 1-2 11/ portions were analyzed with 63Ni-ECD on 5% GEXF1105 gas chromatography columns at 175°C. N2=35 ml/min, att=2 x 10-10 AFS.
When its oxidation in rats is inhibited by pyrogallol or disulfiram, acetaldehyde derived from administered ethanol cyclizes with brain dopamine (and perhaps other brain catecholamines) to form salsolinol in quantities of 15-50 ng/g tissue; combined pretreatment with pargyline and pyrogallol raises this value to nearly 120 ng/g. As indicated in the preliminary disulfiram/ethanol results (Table 3), salsolinol formation is approximately proportional to the high levels of blood acetaldehyde.
Rats were treated with disulfiram for one week and ethanol on day 7 as described in the text. Acetaldehyde levels and salsolinol concentrations were assayed according to the procedures in METHODS. Number of rats in parentheses.
As discussed in those chapters by Cohen, Hirst et al. and Blum et al., salsolinol, although not a highly active drug, has definite pharmacological and behavioral effects. In our own studies we have found that peripheral administration of the alkaloid in a dose range of 50-250 mg/kg significantly reduces brain and heart catecholamines (12). This may be the general result of tetrahydroisoquinoline release and inhibition of uptake of catecholamines (1). An alternative is that it reflects, in part, inhibition of catecholamine biosynthesis at the tyrosine hydroxylase locus. Salsolinol and several other catechol tetrahydroisoquinolines, with the notable exception of tetrahydropapaveroline, are reasonably good in vitro inhibitors of this regulatory enzyme (12).
Another acetaldehyde-derived alkaloid which appears to be present in our in vivo experiments, MOSAL (Figure 1, the derivative of adrenal epinephrine), is of particular physiological interest. This tetrahydroisoquinoline causes long-lasting and nearly complete depletion of guinea pig hypothalamic norepinephrine when given peripherally (in a crude form) at doses of 1-3 mg/kg (13). Such a striking "6-hydroxy-dopamine" effect certainly warrants further study, but facile, practical syntheses of pure 4-hydroxy-tetrahydroisoquinolines are needed (14).
Now considering other analytical approaches, gas chromatography with mass spectrometric (multiple or single ion) detection (GC/MS) has been used to detect low concentrations of dopamine-derived tetrahydroisoquinolines. The first "physiological" demonstration of salsolinol and tetrahydropapaveroline, in fact, was in GC/MS (and ECD) studies of urines from a limited number of Parkinsonian patients undergoing DOPA therapy (15). The following year, researchers in Milan reported GC/MS evidence for tetrahydropapaveroline in whole brain from DOPA-treated rats (16). GC/MS remains the definitive and perhaps the most sensitive technique for the identification and assay of endogenous neuroamines and their metabolites (17).
An important new development in catechol tetrahydroisoquinoline analysis is high performance liquid chromatography with thin-layer electrochemical detection. Riggin and Kissinger have applied this highly sensitive, inexpensive technique [previously developed for analysis of catecholamines (18)] to the detection and quantitation of dopamine-derived tetrahydroisoquinolines (19). They have found SAL, norSAL (6,7-dihydroxy-tetrahydroisoquinoline, from dopamine and formaldehyde) and tetrahydropapaveroline excretion by rats treated only with 100 mg/kg DOPA (19). High performance liquid chromatography provides a relatively simple alternative to gas chromatography with ECD, and if used in conjunction with the latter technique, would substantiate the identities and amounts of amine-derived alkaloids.
If radioactive precursors are employed, gas chromatography with radioactivity detection (gas radiochromatography) can be a valuable identification tool. Frequently used in 14C-biosynthetic studies, gas radiochromatography has been used recently to identify the dopamine-formaldehyde product, norSAL (6,7-dihydroxy-tetrahydroisoquinoline), during incubations of the catecholamine and methyl-tetrahydro[ uj5_14-1 folic acid with a "formaldehyde-forming" enzyme preparation from rat brain (20). The principal enzyme is believed to be 5,10-methylenetetrahydrofolate reductase (21,22) rather than a biogenic amine N-methylating enzyme. Tetrahydroisoquinolines (23) or tetrahydro-0-carbolines (24) were found to be the apparent in vitro products, and gas radiochromatography helps to confirm this. The general technique could be applied to in vivo studies with ethanol and 14C-precursors, providing the necessary cold carrier alkaloid products are available to aid in the collection of the gas chromatographic peaks.
The results of our gas chromatography/ECD experiments on tetrahydroisoquinoline formation in high acetaldehyde situations provoke questions about the actual feasibility of extensive aldehyde cyclization in viva. Since the quantities of SAL and MOSAL are admittedly low in the face of high blood acetaldehyde, are there tissue mechanisms which preclude the cyclization pathway? It appears to be so. Possibilities include low acetaldehyde levels within tissues, rapid acetaldehyde oxidation, unavailability of the major proportions of catecholamines, and thiol group interactions with the aldehyde. The first possibility, perhaps a factor for the brain--Sipple finds brain acetaldehyde to be ca. 40-60 nmoles/g at 300 nmoles/ml blood levels (25), and Tabakoff et al. report that acetaldehyde levels in mouse brain to be 10% of the blood levels (26)--is regarded as unlikely because of the limited tetrahydroisoquinoline formation in the adrenals, which should have no great acetaldehyde barrier. Acetaldehyde oxidation may be an important factor, although aldehyde dehydrogenase inhibitors were administered in these experiments. More likely is protection of stored or vesicular catecholamines from the cyclization reactions [a factor which may be overcome by pargyline treatment in our studies, or by DOPA ingestion (16)]. We are also considering the fourth (but not final) possibility that in vivo acetaldehyde is highly and reversibly bound to tissue thiol groups and thus is relatively unavailable for interaction with biogenic amines.
Our failure to detect SAL or MOSAL in rat tissues following ethanol intoxication in the absence of enzyme inhibitors has been confirmed in part by Riggin and Kissinger (19). Furthermore, Rahwan and O'Neill, using gas chromatography with ECD, were unable to detect SAL in whole brains of mice following six days exposure to ethanol vapor (27). These authors conclude that SAL has no role in ethanol effects in mice.
Presently, although others may differ (28,29), we do not interpret these data to mean that acetaldehyde interactions with nucleophilic functions in general and with catecholamines, specifically, are necessarily insignificant molecular processes in human alcoholism. Because of possible differences between rodents and humans in the physiology of alcohol dependence, and perhaps in such biochemical aspects as acetaldehyde tissue uptake, levels and turnover, biogenic amine availability and tetrahydroisoquinoline disposition, results from intoxicated rodents can not be extrapolated readily. Biochemical studies to date in rodents may even be overlooking more important acetaldehyde/nucleophile interactions, e.g., with thiol-containing (endogenous) substrates (30) or membrane proteins. Clearly, further animal work and rigorous, direct human exploratory studies using several of the sensitive techniques reviewed here are needed before alkaloid formation can be discounted.
With dual column gas chromatography and sensitive electron capture detection (ECD), catechol tetrahydroisoquinoline derivatives of endogenous catecholamines can be identified and assayed in neural tissues of rats treated with ethanol and inhibitors of acetaldehyde oxidation. Thus, salsolinol (SAL) and N-methyl-4-hydroxy-salsolinol (MOSAL), alkaloidal products of acetaldehyde condensation with dopamine and epinephrine, respectively, are present in low levels in rat brain (SAL) and adrenals (MOSAL) following pyrogallol and ethanol. Disulfiram pretreatment followed by acute intoxication also results in amounts of caudatal SAL which are proportional to the elevations in blood acetaldehyde as ethanol dosage is increased. Attempts to demonstrate tetrahydroisoquinoline biosynthesis in intoxicated rats with low acetaldehyde blood levels (no inhibitors) have been unsuccessful to date with gas chromatography and ECD. Other methods--gas chromatography with mass spectrometric or radiochemical detection, or the promising new high performance liquid chromatography with electrochemical detection--may have the sensitivity required. Other species would offer more promise than rodents, and, of course, rigorous examination of body fluids from human alcoholics during conditions of controlled diet and subsequent intoxication may provide answers to the controversy over the importance of acetaldehyde and tetrahydroisoquinolines in alcoholism.
1. Cohen, G.: Alkaloid products in the metabolism of alcohol and biogenic amines. Biochem. Pharmacol. 25:1123-1128 (1976).
2. Cohen, G. and Collins, M.: Alkaloids from catecholamines in adrenal tissue: Possible role in alcoholism. Science /67:1749-1751 (1970).
3. Holtz, P., Stock, K. and Westermann, E.: Formation of tetrahydropapaveroline from dopamine in vitro. Nature 203:656-658 (1964).
4. Davis, V. and Walsh, M.: Alcohol, amines and alkaloids: A possible biochemical basis for alcohol addiction. Science 167:1005-1007 (1970).
5. Collins, M. and Cohen, G.: Isoquinoline alkaloid biosynthesis from adrenal catecholamines during 14C-methyl alcohol metabolism in rats. Fed. Proc. 29:608 (1970).
6. Majchrowicz, E.: Metabolic correlates of ethanol, acetaldehyde, acetate and methanol in humans and animals, Biochemical Pharmacology of Ethanol (Adv. Exp. Med. Biol.) 56:111-140 (1975).
7. Rahwan, R.: Toxic effects of ethanol: Possible role of acetaldehyde, tetrahydroisoquinolines and tetrahydro-8- carbolines. Toxicol. Appl. Pharmacol. 34:3-27 (1975).
8. Bigdeli, M. and Collins, M.: Tissue catecholamines and potential tetrahydroisoquinoline alkaloid metabolites: A gas chromatographic assay method with electron capture detection. Biochem. Medicine 12:55-65 (1975).
9. Collins, M., Custod, J., Rubenstein, J. and Tabakoff, B.: Studies on the effects of pyrogallol and the structurally related DOPA decarboxylase inhibitor, R044602, on acetaldehyde metabolism. Ann. N.Y. Acad. Sci. 273:227232 (1976).
10. Collins, M. and Bigdeli, M.: Tetrahydroisoquinolines in vivo. I. Rat brain formation of salsolinol, a condensation product of dopamine and acetaldehyde, under certain conditions during ethanol intoxication. Life Sciences 16: 585-602 (1975).
11. Collins, M. and Bigdeli, M.: Biosynthesis of tetrahydroisoquinoline alkaloids in brain and other tissues of ethanol-intoxicated rats, Alcohol Intoxication and Withdrawal II. Edited by Gross, M.M., Plenum Press, New York, p. 79-91 (1975).
12. Collins, M., Weiner, C. and Letizia, F.: Effects of catechol tetrahydroisoquinolines on catecholamine biosynthesis, Alcohol Intoxication and Withdrawal III. Edited by Gross, M.M., Plenum Press, New York, in press (1976).
13. Osswald, W., Polonia, J. and Polonia, M.: Preparation and pharmacological activity of the condensation product of adrenaline with acetaldehyde. Naunyn-Schmiedeberg's Arch. Pharmacol. 289:275-290 (1975).
14. Collins, M.: Tetrahydroisoquinoline alkaloids from condensation of alcohol metabolites with norepinephrine: Preparative synthesis and potential analysis in nervous tissue by gas chromatography. Ann. N.Y. Acad. Sci. 215: 92-97 (1973).
15. Sandler, M., Bonham-Carter, C., Hunter, K. and Stern, G.: Tetrahydroisoquinoline alkaloids: In vivo metabolites of L-DOPA in man. Nature 241:439-443 (1973).
16. Turner, A., Baker, K., Algeri, S., Frigerio, A. and Garattini, S.: Tetrahydropapaveroline: Formation in vivo and in vitro in rat brain. Life Sciences 14:22472257 (1974).
17. Weisel, F.A.: Mass fragmentographic determination of dopamine and its metabolites in striatum from one mouse brain, Antipsychotic Drugs, Pharmacodynamics and Pharmacokinetics. Edited by Sedvall, G., Urnas, B. and Zotterman, Y., Pergamon Press, Oxford (1975).
18. Refshauge, C., Kissinger, P., Dreilling, R., Blank, L., Freeman, R. and Adams, R.: New high performance liquid chromatographic analysis of brain catecholamines. Life Sciences 14:311-322 (1974).
19. Riggin, R. and Kissinger, P.: Assay of tetrahydroisoquinolines in biological fluids using high performance liquid chromatography. Anal. Biochem. (submitted, 1976).
20. Vandenhewel, W., Gruber, V., Mandel, L. and Walker, R.: Identification of 6,7-dihydroxytetrahydroisoquinoline as an in vitro reaction product by gas-liquid chromatography. J. Chrom. 114:476-479 (1975).
21. Pearson, A. and Turner, A.: Folate-dependent 1-carbon transfer to biogenic amines mediated by methylenetetrahydrofolate reductase. Nature 258:173-174 (1975).
22. Taylor, R. and Hanna, M.: 5-methyltetrahydrofolate aromatic alkylamine N-methyltransferase: An artifact of 5,10-methylenetetrahydrofolate reductase activity. Life Sciences 17:111-120 (1975).
23. Meller, E., Rosengarten, H., Friedhoff, A., Stebbins, R. and Silber, R.: 5-Methyltetrahydrofolic acid is not a methyl donor for biogenic amines: Enzymatic formation of formaldehyde. Science 187:171-173 (1975).
24. Hsu, L. and Mandell, A.: Enzymatic formation of tetrahydro-8-carboline from tryptamine and 5-methyltetrahydrofolic acid in rat brain fractions: regional and subcellular distribution. J. Neurochem. 24:631-636 (1975).
25. Sippel, H.: The acetaldehyde content in rat brain during ethanol metabolism. J. Neurochem. 23:451-452 (1974).
26. Tabakoff, B., Anderson, R. and Ritzmann, R.: Brain acetaldehyde after ethanol administration. Biochem. Pharmacol. 25:1305-1309 (1976).
27. Rahwan, R. and O'Neill, P.: Absence of formation of brain salsolinol during chronic ethanol administration to mice. Pharmacologist 18:190 (1976).
28. Thurman, R. and Pathman, D.: Withdrawal symptoms from ethanol: Evidence against involvement of acetaldehyde, The Role of Acetaldehyde in the Actions of Ethanol. Edited by Lindros, K. and Eriksson, C., Finn. Fdn. for Alcohol Studies, Helsinki, p. 217-231 (1976).
29. Goldstein, D.: Pharmacological aspects of physical dependence on ethanol. Life Sciences 18:553-562 (1975).
30. Nagasawa, H., Goon, D., Constantino, N. and Alexander, C.: Diversion of ethanol metabolism by sulfhydryl amino acids. D-penicillamine-directed excretion of 2,5,5-trimethyl-d-thiazolidine-4-carboxylic acid in the urine of rats after ethanol administration. Life Sciences 17:707714 (1975).