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ACUTE INTERACTION BETWEEN ETHANOL AND SEROTONIN METABOLISM IN THE RAT
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ELSEVIER
Scienq Vol.61,No.5, pp.S?7.5S3,1997 %@@t e 1~ Ekvier ScienceInc. Printedin theUSA. All rightsresawd cm4-33a5/97 $17.W+ al
PII S0024-3205(97)00417-7
ACUTE INTERACTIONBETWEENETHANOLAND SEROTONIN METABOLISMIN THERAT Margareta Some, Olof Beck’ and Anders Helander* Karolinska Institute, Department of Clinical Neuroscience, Psychiatry Section at St. Gorans Hospital, and ‘Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden (Receivedin finalformMay6, 1997)
The effect of acute ethanol on peripheral serotonin (5HT) metabolism was studied in Sprague-Dawley rats. Four hours after a single dose of ethanol (1.0 g/kg) administered into the stomach, a significant increase in the 5HT level in stomach tissue and a decrease in ileum was observed. The level of 5-hydroxyindole-3-acetic acid (5HL4A) was increased in urine, while increased concentrations of 5-hydroxytryptophol (5HTOL) occurred in jejunum, ileum, spleen and urine. After 7-9 h when the blood ethanol concentration had returned to zero, 5HTOL levels were still higher than control values in jejunurn, ileum and urine. At 4 h, an elevated ratio of 5HTOL to 5HIAA was observed in urine and ileum (by -2-fold), liver (-3-fold), and spleen (-5-fold), whereas the ratio was reduced in stomach. In urine and spleen, this metabolic shifi persisted after 7-9 h. The 5HTOL level in bile was increased by -3.5-fold after 8 h. 5HIAA was not detectable in bile. The present results indicate that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. The rat does not appear to be an ideal model for studying the interaction between ethanol and 5HT metabolism in man. Key WordT:acuteethano~5-hydroxytrypt*
serotinirr turnover,rat
When ethanol is present in the body, several endogenous NAD-dependent reactions are disturbed including the metabolism of biogenic amines such as serotonin (5-hydroxytryptamine, or 5HT). In man, 5-hydroxytryptophol (5HTOL) is a minor metabolize of 5HT but after acute alcohol ingestion 5HTOL levels increase dramatically in a dose-dependent manner (l-3). At the same time, the level of the predominant metabolic product 5-hydroxyindole-3-acetic acid (5HIAA) is concomitantly decreased. Under normal conditions, 5HTOL constitutes less than 1°/0of 5HIAA in the urine, whereas the 5HTOL concentration may exceed that of 5HIAA after drinking has occurred. This metabolic shift towards reduction of 5HT is also observable by anrdysis of plasm% and it does not normalize until several hours after ethanol has been eliminated (3). On the basis of this time lag, an increased urinary 5HTOL/5HIAA ratio has been proposed as a biochemical
*Corresponding author: Dr. A. Helander, Alcohol & Drug Dependence Unit, St. Gorans Hospital, S-112 81 Stockholm, Sweden. Fax: (+46)8 6721994.
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marker of recent alcohol use, for example to reveal relapse in drinking during rehabilitation of alcohol dependent subjects (4,5). The metabolizes of 5HT excreted in urine originate largely from 5HT in the enterochromaffii cells of the gastrointestinal tract, which is the main source of 5HT in the body, whereas only a small amount comes from the central nervous system (6,7). Biogenic amines and ethanol share some catabolic enzymes, and experiments with liver homogenates suggest that the shift in 5HT metabolism after acute ethanol occurs because of competitive inhibition of aldehyde dehydrogenase by acetaldehyde derived from ethanol (8,9). In addition, the excess of NADH resulting from oxidation of ethanol and acetaldehyde favors reduction of the intermediate 5-hydroxyindole-3-acetaldehyde by alcohol dehydrogenase (ADH) to form 5HTOL (10). The location in the body of this metabolic interaction has not been elucidated in detail. The liver is the major organ responsible for ethanol detoxification and, in experiments with isolated perfused rat liver, biotransformation of administered 5HT to yield 5HTOL was observed (11). However, 5HTOL is widely distributed in the rat although the highest concentrations occur in liver, lung and small intestine (12). The present study was conducted in order to determine the location of the metabolic interaction between ethanol and endogenous 5HT in the periphery, and to examine whether the rat is a useful model for studying this interaction in man.
~.
5HT, 5HIAA, 5HTOL, 5-hydroxyindoleand sulfatase (type Hl, containing &glucuro-
nidase activity) were obtainedfrom Sigma Chemical Co. (St. Louis, MO), diethyl ether and ethyl
acetate were from Merck (Darmstadt, Germany), yeast ADH from Boehringer (Mannheim, Germany), pentafluoropropionic anhydride from Supelco (Bellefonte, PA), and trifluoroethanol from Aldrich (Steinheim, Germany). The deuterated internal standards (5HT-2H,,5HIAA-2H2and 5HTOL-2H.J were synthesized as previously described (13,14). All other chemicals were of analytical grade and all solutions were prepared in deionized HPLC-grade water. mocedut%l. Female Sprague-Dawley rats (250-350 g) received a standard laboratory diet (withdrawn 12 h before start of experiment) and tap water ad libitum. The animals were anesthetized with pentobarbital (60 mgkg i.p.; Mebumal Vet, Pharmacia, Uppsala, Sweden) and the body temperature was maintained at -37°C by heat lamps. After cannulation of the bile duct and urine bladder, a bile and a urine specimen was collected and blood was sampled from the tip of the tail in heparinized micropipets. Thereafter, the rats were administered ethanol (20°/0,v/v, in srdine) at a single dose of 1.0 gikg of body weight into the stomach. Bile and urine was collected continuously at l-h intervals, and blood was sampled every 0.5 h. After 4 h or 7-9 h, the rats were decapitated and liver, lung, spleen, kidney, stomach, jejunum, ileum, and colon tissue isolated and placed on ice. Control samples were collected from rats not given any ethanol. All samples were stored at -80°C until analysis. The study was approved by the local Ethics Committee. ~ay of 5HT. 5~. 5HT and metabolizeswere assayed using highly sensitive and specific gas chromatographic-mass spectrometric (GC-MS) technique. The concentrations of 5HIAA and total (free+ conjugated) 5HTOL were determined after enzymatic hydrolysis of bile, urine, and tissue samples, whereas 5HT was determined in the tissue samples only and without hydrolysis. Sample preparation was carried out essentially as described in detail elsewhere (15). The tissue samples (0.5 g) were homogenized in 5.0 mL of 0.8 mol/L HCIOq.After neutralization, 1.0 mL of the extract was used for quantification of 5HT as previously described (16) except that the solid-phase extraction step was omitted. Bile and urine specimens (100 pL; in a few cases two
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subsequent samples were pooled to obtain a sufficient volume for the analysis) and the tissue extracts (2.5 mL) were incubated at 37°C for 16 h after addition of 0.5 mL KHzPOdbuffer (0.1 mol/L, pH 6.0) and 5 mg sulfatase. The fi-actionused for 5HIAA determination was acidified with 100 pL of 4 mol/L HCOOH per mL of water phase. Both 5HTOL and 5HIAA were extracted with diethyl ether (proportion to water phase 3:1, v/v). Separation of the perfluoroacylated derivatives of 5HT, 5HTOL, and 5HIAA was achieved on a capillary column (DB5MS, 30 m x 0.25 mm id.; J&W Scientific, Folsom, CA) using splitless injections. A GC-MS system (HP 5972, Hewlett Packard, Palo Alto, CA) was used for the selected ion monitoring at ndz 346 (5HT), m/z 350 (5HT-2H,), In/Z 451 (5HT0L), 211/Z 454 (5HT0L-2H,), In/Z438 (5HL4A), and in/Z 440 (5H1AA‘H,). For quantitation, calibration curves were constructed by plotting the peak area ratio of the standard samples and their internal standard versus the concentration. Standard samples were water solutions of 5HT, 5HTOL and 5HIAA prepared as tissue samples for analysis. Levels of 5HT, 5HTOL and 5HIAA in tissues and body fluids were determined from the response ratio by reference to the calibration curves. . . The ethanol concentration in blood was determined with Determu@m of e~ an enzymatic method using yeast ADH ~17). . . ~. If significance was reached by analysis of variance (one-way ANOVA), comparison of group means for tissues were made by the Wilcoxon test. For urine, a t-test for paired observations was used as post-hoc test. Ap value of <0.05 was required for significance. Restdls Figure 1 shows the levels of 5HT, 5HIAA and 5HTOL in rat tissue and urine samples. In control animals, the highest concentrations of 5HT were demonstrated within the gastrointestinal tract and in spleen (Fig. la). The highest concentrations of 5HIAA (Fig. lb) were found in the gut, in lung tissue, and urine, whereas colon and urine showed the highest levels of 5HTOL (Fig. Ic). The highest baseline ratios of 5HTOL/5HIAA were observed in colon and liver (-1.9), and the lowest ratios were found in ileum (0.16) and lung tissue (0.14) (Fig. 2). After an acute dose of ethanol (1.0 g/kg) was administered into the stomach, blood ethanol concentrations reached a maximum of 11.1+ 4.9 mmol/L (mean+ SD) at 1 h and returned to zero within 7-8 h (data not shown). After 4 h, the 5HT concentration in stomach tissue was higher than control values whereas the level in ileum was reduced (Fig. la). 5HIAA levels were elevated in urine after 4 h (Fig. lb). At 4 h after ethanol administration, the 5HTOL concentrations in jejunum, ileum, spleen and urine were higher than baseline values, and in jejunum, ileum and urine this effect persisted after 7-9 h (Fig. Ic). At 4 h, a rise in the 5HTOL/5HIAA ratio was observed in urine and ileum (by -2-fold), liver (-3-fold), and spleen (-5-fold), whereas the ratio was reduced in stomach (Fig. 2). In urine and spleen, the ratio remained elevated after 7-9 h. In the bile samples collected before administration of ethanol, the concentration of total (free + conjugated) 5HTOL was 0.7 + 0.3 pmol/L (n = 3). When the ethanol had been administered, a continuous rise over time was observed and the concentration reached after 8 h was 2.4 + 0.2 ~mol/L (data not shown). 5HIAA was not detectable in bile. In the control rats, ratios of 5HIAW5HT and (5HIAA+5HTOL)/5HT were calculated as indirect estimates of 5HT turnover (Fig. 3). With either method, the highest ratios occurred in lung tissue followed by kidney, whereas they were lowest (<1’%.of lung) in jejunum and spleen.
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Ethanolon SerotoninMetaboliasra
5s0
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ileum
colon
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Fig. 1. Levels of a) 5HT, b) 5HIAA, and c) total (free+ conjugated) 5HTOL in rat tissue (nmol/g) and urine (nmolhnL) samples in control animals (O h), and at 4 h and 7-9 h after ethanol administration (1.0 ~g). Results are mean values + SD (n = 4). * p <0.05 compared to controls; +p <0.05 compared to 4-h values.
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Ratios of 5HTOL/5HIAA in rat tissue and urine samples in control animals (Oh), and at 4 h and 7-9 h after ethanol administration (1.0 @g). Results are mean values+ SD (n = 4). *p <0.05 compared to control (Oh) values; +p <0.05 compared to 4-h values.
The highest baseline concentrations of 5HT were found within the gastrointestinal tract and in spleen. In the intestinal mucosa, 5HT is synthesized and stored in enterochromaffii cells (18) and this pool accounts for -90% of all 5HT in the mammalian body (7). A high 5HT content in spleen is in accordance with previous observations (19) and may be related to the fact that platelets, which also contain high concentrations of 5HT, are stored within this organ. It has been reported that the kidney has the capacity to synthesize 5HT from circulating tryptophan (20-22). In the present study, the 5HT concentration in kidney was relatively low, which might be due to a rapid elimination, and/or metabolism, of renal 5HT. The baseline concentrations of 5HT, 5HTOL, and 5HIAA in liver and lung tissue concur with earlier findings (12,15,23). In the liver, the 5HTOL concentration actually exceeded that of 5HIAA which also agrees with previous reports (12,15). The 5HT concentration in lung was rather low. However, the high concentration of 5HIAA relative to 5HT may reflect a high turnover, and the lungs have previously been demonstrated to play an important role in the removal of 5HT via uptake from the circulation and subsequent inactivation by the action of monoamine oxidase (24). The baseline levels of 5HTOL and 5HIAA in the small intestine are in good agreement with earlier reports (12). In that study, it was also indicated that biotransformation of 5HT to 5HTOL occurs mainly in liver, intestine and lung. Acute ethanol induces a shift in 5HT metabolism leading to increased levels of 5HTOL and a concomitant drop in 5HIAA. Human studies have demonstrated the 5HTOL excretion curve to parallel that of ethanol, however, with a considerable time lag (3). As a consequence, 5HTOL levels remain increased for several hours after ethanol is no longer detectable. The precise mechanism underlying this delay in normalization is not known, but it has been proposed to depend on the retention of accumulated 5HTOL owing to enterohepatic circulation (25,26). However, in the present study, the basal concentration of 5HTOL in bile was -0’7. of that in urine, and the slow increase over time observed throughout the experiment was not correlated to
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14
10 6 2
I
1.0 0.8 0.6 I
stomach
jejunum
ileum
colon
spleen
lung
liver
kidney
Fig. 3. Estimation of 5HT turnover in tissues from control rats by calculating ratios of 5HIAA/5HT and (5HIAA+5HTOL)/5HT. The results are mean values of four rats.
the urinary output. This would suggest that enterohepaticcirculation is not a major cause of the delay in 5HTOL normalization after acute ethanol. Another possible mechanism for a delayed
urinary excretion is a kinetic effect related to the extra metabolic step necessary to form 5HTOL conjugates. While 5HL4A is eliminated in the free form, the more lipophilic 5HTOL is conjugated with glucuronic acid or sulfate to facilitate urinary excretion (27). It should be noted that a delay in 5HTOL excretion compared with 5HIAA may occur also under normal conditions but becomes evident only when 5HTOL levels are dramatically increased. In humans, a distinct rise (>IOO-fold)in the 5HTOL/5HIAA ratio in urine is observed after acute alcohol ingestion with the maximum reached in about six hours (3). In the present study on rats, however, only a 2-fold rise in this ratio occurred in the urine after a corresponding dose of ethanol. Because the basal level of the 5HTOL/5HIA4 ratio was much higher in the rat (-0.25) than that found in man (<0.01) (3), the relative magnitude of the metabolic shifi after receiving ethanol will be much lower in the rat. Significant increases in the 5HTOL/5HL4A ratio after ethanol administration were also seen in liver, ileum and spleen. However, the low 5HT turnover in spleen, as estimated from a low 5HLQV5HT ratio, may imply that this change in 5HTOL/5HIAA ratio merely reflects the situation in the circulating blood, rather than a metabolic interaction within this organ. Taken together, this suggests that an interaction between ethanol and endogenous 5HT metabolism carI take place both in the gut and, after release of 5HT into portal circulation, in the liver. The turnover of 5HT is often estimated from the ratio of the major metabolize 5HIAA to parent amine. However, the 5HIAA/5HT ratio is influenced in part by factors other than the 5HT turnover rate (28). Furthermore, the present work demonstrated that in the rat, where 5HTOL is a quantitatively much more abundant metabolize than in man, measurement of 5HL4A alone will result in an underestimation of 5HT catabolism. This is especially important in tissues with a high concentration of 5HTOL relative to 5HIAA, such as liver and colon where the 5HT turnover index
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was more than twofold higher when instead using the ratio of (5HIAA+5HTOL)/5HT, but also when the proportion of 5HT metabolizes is altered, such as after alcohol ingestion and during treatment with inhibitors of aldehyde dehydrogenase (29). To conclude, the present results show that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. A metabolic interaction between acute ethanol and endogenous 5HT resulting in elevated 5HTOL/5HIAA ratios was demonstrated in liver, spleen and ileum tissue as well as in urine. The change in urinary ratio was, however, much less dramatic than that occurring in man. Further studies will be performed to find an animal species in which the magnitude of this metabolic interaction more resembles that in humans.
1. V.E. DAVIS, H. BROWN, J.A. HUFF and J.L. CASHAW, J Lab Clin Med 6$! 132-140
(1967). 2.0. BECK, S. BORG, L. ERIKSSON and A. LUNDMAN, Naunyn-Schmiedeberg’s Arch Pharmacol W 293-297 (1982). 3. A. HELANDER, O. BECK, G. JACOBSSON, C. LOWENMO and T. WIKSTROM, Life Sci U 847-855 (1993). 4. A. VOLTAIRE, O. BECK and S. BORG, Alcohol Clin Exp Res .lfi 281-285 (1992). 5. A. HELANDER, O. BECK and S. BORG, Alcohol Alcohol -497-502 (1994). 6. M.L. AIZENSTEIN and J. KORF, J Neurochem 321227-1233 (1979). 7. G.W. LAMBERT, D.M. KAYE, H.S. COX, M. VAZ, A.G. TURNER, G.L. JENNINGS and M.D. ESLER, LifeSci$1255-267 (1995). 8. R.A. LAHTI and E. MAJCHROWICZ, Quart J Stud Alc ~ 1-14 (1974). 9. A. HELANDER and O. TOTTMAR, Biochem Pharmacol 363981-3985 (1987). 10. A. FELDSTEIN and O. WILLIAMSON, LifeSci2777-783 (1968). 11. G.M. TYCE, E.V. FLOCK and C.A. OWEN JR, Am J Physiol U 611-619 (1968). 12. S. CHEIFETZ and J.J. WARSH, J Neurochem 341093-1099 (1980). 13.0. BECK and G. SEDVALL, J Lab Comp Radiopharm J-L57-61 (1975). 14. T. HESSELGREN and O. BECK, J Lab CompRadiopharmU411-419 (1980). 15.0. BECK, S. BORG, G. JONSSON, A. LUNDMAN and P. VALVERIUS, J Neural Transm W 57-67 (1984). 16.0. BECK, N.H. WALLEN, A. BROIJERSEN, P.T. LARSSON and P. HJEMDAHL, Biochem Biophys Res Commun l%i 260-266 (1993). 17. A. HELANDER and O. TOTTMAR, Alcohol Clin Exp Res 12643-646 (1988). 18. G.A. BUBENIK, R.O. BALL and S.-F. PANG, J Pineal Res 127-16 (1992). 19. S. UDENFRIEND and H. WEISSBACH, Proc Soc Exp Biol Med n 748-751 (1958). 20. Y.-L. CHAN and K.C. HUANG, Am J Physiol 224140-143 (1973). 21. C.T. STIER JR and H.D. ITSKOVITZ, Proc Soc Exp Biol Med -1-?N 550-557 (1985). 22. M.J. SOLE, A. MADAPALLIMATTAM and A.D. BAINES, Kidney Int 29689-694 (1986). 23. P. CELADA, F. MARTIN and F. ARTIGAS, Life Sci 5.51237-1243 (1994). 24. C.N. GILLIS and J.A. ROTH, Biochem Pharmacol 252547-2553 (1976). 25. M. KEKKI, P. PENTI~EN and O. MUSTALA, Gastroenterology a 700-708 (1974). 26. M. KEKKI, P. PENTI~EN and O. MUSTALA, Quart J Stud Alc ~ 1195-1204 (1974). 27. A. HELANDER, O. BECK and L. BOYSEN, Life Sci 561529-1534 (1995). 28. D.M. KUHN, W.A. WOLF and M.B.H. YOUDIM, Neurochem Int 8141-154 (1986). 29.0. BECK, A. HELANDE~ S. CARLSSON and S. BORG, Pharmacol Toxicol n 323-326 (1995).
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Scienq Vol.61,No.5, pp.S?7.5S3,1997 %@@t e 1~ Ekvier ScienceInc. Printedin theUSA. All rightsresawd cm4-33a5/97 $17.W+ al
PII S0024-3205(97)00417-7
ACUTE INTERACTIONBETWEENETHANOLAND SEROTONIN METABOLISMIN THERAT Margareta Some, Olof Beck’ and Anders Helander* Karolinska Institute, Department of Clinical Neuroscience, Psychiatry Section at St. Gorans Hospital, and ‘Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden (Receivedin finalformMay6, 1997)
The effect of acute ethanol on peripheral serotonin (5HT) metabolism was studied in Sprague-Dawley rats. Four hours after a single dose of ethanol (1.0 g/kg) administered into the stomach, a significant increase in the 5HT level in stomach tissue and a decrease in ileum was observed. The level of 5-hydroxyindole-3-acetic acid (5HL4A) was increased in urine, while increased concentrations of 5-hydroxytryptophol (5HTOL) occurred in jejunum, ileum, spleen and urine. After 7-9 h when the blood ethanol concentration had returned to zero, 5HTOL levels were still higher than control values in jejunurn, ileum and urine. At 4 h, an elevated ratio of 5HTOL to 5HIAA was observed in urine and ileum (by -2-fold), liver (-3-fold), and spleen (-5-fold), whereas the ratio was reduced in stomach. In urine and spleen, this metabolic shifi persisted after 7-9 h. The 5HTOL level in bile was increased by -3.5-fold after 8 h. 5HIAA was not detectable in bile. The present results indicate that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. The rat does not appear to be an ideal model for studying the interaction between ethanol and 5HT metabolism in man. Key WordT:acuteethano~5-hydroxytrypt*
serotinirr turnover,rat
When ethanol is present in the body, several endogenous NAD-dependent reactions are disturbed including the metabolism of biogenic amines such as serotonin (5-hydroxytryptamine, or 5HT). In man, 5-hydroxytryptophol (5HTOL) is a minor metabolize of 5HT but after acute alcohol ingestion 5HTOL levels increase dramatically in a dose-dependent manner (l-3). At the same time, the level of the predominant metabolic product 5-hydroxyindole-3-acetic acid (5HIAA) is concomitantly decreased. Under normal conditions, 5HTOL constitutes less than 1°/0of 5HIAA in the urine, whereas the 5HTOL concentration may exceed that of 5HIAA after drinking has occurred. This metabolic shift towards reduction of 5HT is also observable by anrdysis of plasm% and it does not normalize until several hours after ethanol has been eliminated (3). On the basis of this time lag, an increased urinary 5HTOL/5HIAA ratio has been proposed as a biochemical
*Corresponding author: Dr. A. Helander, Alcohol & Drug Dependence Unit, St. Gorans Hospital, S-112 81 Stockholm, Sweden. Fax: (+46)8 6721994.
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marker of recent alcohol use, for example to reveal relapse in drinking during rehabilitation of alcohol dependent subjects (4,5). The metabolizes of 5HT excreted in urine originate largely from 5HT in the enterochromaffii cells of the gastrointestinal tract, which is the main source of 5HT in the body, whereas only a small amount comes from the central nervous system (6,7). Biogenic amines and ethanol share some catabolic enzymes, and experiments with liver homogenates suggest that the shift in 5HT metabolism after acute ethanol occurs because of competitive inhibition of aldehyde dehydrogenase by acetaldehyde derived from ethanol (8,9). In addition, the excess of NADH resulting from oxidation of ethanol and acetaldehyde favors reduction of the intermediate 5-hydroxyindole-3-acetaldehyde by alcohol dehydrogenase (ADH) to form 5HTOL (10). The location in the body of this metabolic interaction has not been elucidated in detail. The liver is the major organ responsible for ethanol detoxification and, in experiments with isolated perfused rat liver, biotransformation of administered 5HT to yield 5HTOL was observed (11). However, 5HTOL is widely distributed in the rat although the highest concentrations occur in liver, lung and small intestine (12). The present study was conducted in order to determine the location of the metabolic interaction between ethanol and endogenous 5HT in the periphery, and to examine whether the rat is a useful model for studying this interaction in man.
~.
5HT, 5HIAA, 5HTOL, 5-hydroxyindoleand sulfatase (type Hl, containing &glucuro-
nidase activity) were obtainedfrom Sigma Chemical Co. (St. Louis, MO), diethyl ether and ethyl
acetate were from Merck (Darmstadt, Germany), yeast ADH from Boehringer (Mannheim, Germany), pentafluoropropionic anhydride from Supelco (Bellefonte, PA), and trifluoroethanol from Aldrich (Steinheim, Germany). The deuterated internal standards (5HT-2H,,5HIAA-2H2and 5HTOL-2H.J were synthesized as previously described (13,14). All other chemicals were of analytical grade and all solutions were prepared in deionized HPLC-grade water. mocedut%l. Female Sprague-Dawley rats (250-350 g) received a standard laboratory diet (withdrawn 12 h before start of experiment) and tap water ad libitum. The animals were anesthetized with pentobarbital (60 mgkg i.p.; Mebumal Vet, Pharmacia, Uppsala, Sweden) and the body temperature was maintained at -37°C by heat lamps. After cannulation of the bile duct and urine bladder, a bile and a urine specimen was collected and blood was sampled from the tip of the tail in heparinized micropipets. Thereafter, the rats were administered ethanol (20°/0,v/v, in srdine) at a single dose of 1.0 gikg of body weight into the stomach. Bile and urine was collected continuously at l-h intervals, and blood was sampled every 0.5 h. After 4 h or 7-9 h, the rats were decapitated and liver, lung, spleen, kidney, stomach, jejunum, ileum, and colon tissue isolated and placed on ice. Control samples were collected from rats not given any ethanol. All samples were stored at -80°C until analysis. The study was approved by the local Ethics Committee. ~ay of 5HT. 5~. 5HT and metabolizeswere assayed using highly sensitive and specific gas chromatographic-mass spectrometric (GC-MS) technique. The concentrations of 5HIAA and total (free+ conjugated) 5HTOL were determined after enzymatic hydrolysis of bile, urine, and tissue samples, whereas 5HT was determined in the tissue samples only and without hydrolysis. Sample preparation was carried out essentially as described in detail elsewhere (15). The tissue samples (0.5 g) were homogenized in 5.0 mL of 0.8 mol/L HCIOq.After neutralization, 1.0 mL of the extract was used for quantification of 5HT as previously described (16) except that the solid-phase extraction step was omitted. Bile and urine specimens (100 pL; in a few cases two
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subsequent samples were pooled to obtain a sufficient volume for the analysis) and the tissue extracts (2.5 mL) were incubated at 37°C for 16 h after addition of 0.5 mL KHzPOdbuffer (0.1 mol/L, pH 6.0) and 5 mg sulfatase. The fi-actionused for 5HIAA determination was acidified with 100 pL of 4 mol/L HCOOH per mL of water phase. Both 5HTOL and 5HIAA were extracted with diethyl ether (proportion to water phase 3:1, v/v). Separation of the perfluoroacylated derivatives of 5HT, 5HTOL, and 5HIAA was achieved on a capillary column (DB5MS, 30 m x 0.25 mm id.; J&W Scientific, Folsom, CA) using splitless injections. A GC-MS system (HP 5972, Hewlett Packard, Palo Alto, CA) was used for the selected ion monitoring at ndz 346 (5HT), m/z 350 (5HT-2H,), In/Z 451 (5HT0L), 211/Z 454 (5HT0L-2H,), In/Z438 (5HL4A), and in/Z 440 (5H1AA‘H,). For quantitation, calibration curves were constructed by plotting the peak area ratio of the standard samples and their internal standard versus the concentration. Standard samples were water solutions of 5HT, 5HTOL and 5HIAA prepared as tissue samples for analysis. Levels of 5HT, 5HTOL and 5HIAA in tissues and body fluids were determined from the response ratio by reference to the calibration curves. . . The ethanol concentration in blood was determined with Determu@m of e~ an enzymatic method using yeast ADH ~17). . . ~. If significance was reached by analysis of variance (one-way ANOVA), comparison of group means for tissues were made by the Wilcoxon test. For urine, a t-test for paired observations was used as post-hoc test. Ap value of <0.05 was required for significance. Restdls Figure 1 shows the levels of 5HT, 5HIAA and 5HTOL in rat tissue and urine samples. In control animals, the highest concentrations of 5HT were demonstrated within the gastrointestinal tract and in spleen (Fig. la). The highest concentrations of 5HIAA (Fig. lb) were found in the gut, in lung tissue, and urine, whereas colon and urine showed the highest levels of 5HTOL (Fig. Ic). The highest baseline ratios of 5HTOL/5HIAA were observed in colon and liver (-1.9), and the lowest ratios were found in ileum (0.16) and lung tissue (0.14) (Fig. 2). After an acute dose of ethanol (1.0 g/kg) was administered into the stomach, blood ethanol concentrations reached a maximum of 11.1+ 4.9 mmol/L (mean+ SD) at 1 h and returned to zero within 7-8 h (data not shown). After 4 h, the 5HT concentration in stomach tissue was higher than control values whereas the level in ileum was reduced (Fig. la). 5HIAA levels were elevated in urine after 4 h (Fig. lb). At 4 h after ethanol administration, the 5HTOL concentrations in jejunum, ileum, spleen and urine were higher than baseline values, and in jejunum, ileum and urine this effect persisted after 7-9 h (Fig. Ic). At 4 h, a rise in the 5HTOL/5HIAA ratio was observed in urine and ileum (by -2-fold), liver (-3-fold), and spleen (-5-fold), whereas the ratio was reduced in stomach (Fig. 2). In urine and spleen, the ratio remained elevated after 7-9 h. In the bile samples collected before administration of ethanol, the concentration of total (free + conjugated) 5HTOL was 0.7 + 0.3 pmol/L (n = 3). When the ethanol had been administered, a continuous rise over time was observed and the concentration reached after 8 h was 2.4 + 0.2 ~mol/L (data not shown). 5HIAA was not detectable in bile. In the control rats, ratios of 5HIAW5HT and (5HIAA+5HTOL)/5HT were calculated as indirect estimates of 5HT turnover (Fig. 3). With either method, the highest ratios occurred in lung tissue followed by kidney, whereas they were lowest (<1’%.of lung) in jejunum and spleen.
vol. 61, No. 5, 1997
Ethanolon SerotoninMetaboliasra
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Fig. 1. Levels of a) 5HT, b) 5HIAA, and c) total (free+ conjugated) 5HTOL in rat tissue (nmol/g) and urine (nmolhnL) samples in control animals (O h), and at 4 h and 7-9 h after ethanol administration (1.0 ~g). Results are mean values + SD (n = 4). * p <0.05 compared to controls; +p <0.05 compared to 4-h values.
vol. 61, No.5, 1997
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Ratios of 5HTOL/5HIAA in rat tissue and urine samples in control animals (Oh), and at 4 h and 7-9 h after ethanol administration (1.0 @g). Results are mean values+ SD (n = 4). *p <0.05 compared to control (Oh) values; +p <0.05 compared to 4-h values.
The highest baseline concentrations of 5HT were found within the gastrointestinal tract and in spleen. In the intestinal mucosa, 5HT is synthesized and stored in enterochromaffii cells (18) and this pool accounts for -90% of all 5HT in the mammalian body (7). A high 5HT content in spleen is in accordance with previous observations (19) and may be related to the fact that platelets, which also contain high concentrations of 5HT, are stored within this organ. It has been reported that the kidney has the capacity to synthesize 5HT from circulating tryptophan (20-22). In the present study, the 5HT concentration in kidney was relatively low, which might be due to a rapid elimination, and/or metabolism, of renal 5HT. The baseline concentrations of 5HT, 5HTOL, and 5HIAA in liver and lung tissue concur with earlier findings (12,15,23). In the liver, the 5HTOL concentration actually exceeded that of 5HIAA which also agrees with previous reports (12,15). The 5HT concentration in lung was rather low. However, the high concentration of 5HIAA relative to 5HT may reflect a high turnover, and the lungs have previously been demonstrated to play an important role in the removal of 5HT via uptake from the circulation and subsequent inactivation by the action of monoamine oxidase (24). The baseline levels of 5HTOL and 5HIAA in the small intestine are in good agreement with earlier reports (12). In that study, it was also indicated that biotransformation of 5HT to 5HTOL occurs mainly in liver, intestine and lung. Acute ethanol induces a shift in 5HT metabolism leading to increased levels of 5HTOL and a concomitant drop in 5HIAA. Human studies have demonstrated the 5HTOL excretion curve to parallel that of ethanol, however, with a considerable time lag (3). As a consequence, 5HTOL levels remain increased for several hours after ethanol is no longer detectable. The precise mechanism underlying this delay in normalization is not known, but it has been proposed to depend on the retention of accumulated 5HTOL owing to enterohepatic circulation (25,26). However, in the present study, the basal concentration of 5HTOL in bile was -0’7. of that in urine, and the slow increase over time observed throughout the experiment was not correlated to
EthanolonSerotoninMetabolism
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14
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stomach
jejunum
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Fig. 3. Estimation of 5HT turnover in tissues from control rats by calculating ratios of 5HIAA/5HT and (5HIAA+5HTOL)/5HT. The results are mean values of four rats.
the urinary output. This would suggest that enterohepaticcirculation is not a major cause of the delay in 5HTOL normalization after acute ethanol. Another possible mechanism for a delayed
urinary excretion is a kinetic effect related to the extra metabolic step necessary to form 5HTOL conjugates. While 5HL4A is eliminated in the free form, the more lipophilic 5HTOL is conjugated with glucuronic acid or sulfate to facilitate urinary excretion (27). It should be noted that a delay in 5HTOL excretion compared with 5HIAA may occur also under normal conditions but becomes evident only when 5HTOL levels are dramatically increased. In humans, a distinct rise (>IOO-fold)in the 5HTOL/5HIAA ratio in urine is observed after acute alcohol ingestion with the maximum reached in about six hours (3). In the present study on rats, however, only a 2-fold rise in this ratio occurred in the urine after a corresponding dose of ethanol. Because the basal level of the 5HTOL/5HIA4 ratio was much higher in the rat (-0.25) than that found in man (<0.01) (3), the relative magnitude of the metabolic shifi after receiving ethanol will be much lower in the rat. Significant increases in the 5HTOL/5HL4A ratio after ethanol administration were also seen in liver, ileum and spleen. However, the low 5HT turnover in spleen, as estimated from a low 5HLQV5HT ratio, may imply that this change in 5HTOL/5HIAA ratio merely reflects the situation in the circulating blood, rather than a metabolic interaction within this organ. Taken together, this suggests that an interaction between ethanol and endogenous 5HT metabolism carI take place both in the gut and, after release of 5HT into portal circulation, in the liver. The turnover of 5HT is often estimated from the ratio of the major metabolize 5HIAA to parent amine. However, the 5HIAA/5HT ratio is influenced in part by factors other than the 5HT turnover rate (28). Furthermore, the present work demonstrated that in the rat, where 5HTOL is a quantitatively much more abundant metabolize than in man, measurement of 5HL4A alone will result in an underestimation of 5HT catabolism. This is especially important in tissues with a high concentration of 5HTOL relative to 5HIAA, such as liver and colon where the 5HT turnover index
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was more than twofold higher when instead using the ratio of (5HIAA+5HTOL)/5HT, but also when the proportion of 5HT metabolizes is altered, such as after alcohol ingestion and during treatment with inhibitors of aldehyde dehydrogenase (29). To conclude, the present results show that the rat has a much higher proportion of 5HTOL formation than man under normal conditions. A metabolic interaction between acute ethanol and endogenous 5HT resulting in elevated 5HTOL/5HIAA ratios was demonstrated in liver, spleen and ileum tissue as well as in urine. The change in urinary ratio was, however, much less dramatic than that occurring in man. Further studies will be performed to find an animal species in which the magnitude of this metabolic interaction more resembles that in humans.
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