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Lack of behavioral effects of monoamine depletion in healthy subjects
Lack of Behavioral Effects of Monoamine Depletion in Healthy Subjects Ronald M. Salomon, Helen L. Miller, John H. Krystal, George R. Heninger, and Dennis S. Charney
This study was designed to determine the behavioral effects of a reduction in catecholamine and indoleamine function in healthy subjects. Eight healthy subjects received the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (AMPT) in combination with a full-strength tryptophan-depleting amino acid drink during one 4-day test session, and AMPT and tryptophan-supplemented amino acid drink ( n = 2), or a 25% strength tryptophan-depleting amino acid drink ( n = 6 ) during a second 4-day test session. The combined administration of AMPT and the tryptophan-free amino acid drink did not produce statistically signzjicant or even clinically noticeable changes in mood among the healthy subjects. The implications of these observations for the monoamine hypotheses of depression are discussed. 0 1997 Society of Biological Psychiatry Key Words: Norepinephrine, dopamine, AMPT, behavior, catecholamine, indoleamine
Introduction The predominant hypotheses of the pathophysiology of depression involve alterations in brain monoamine systems. These hypotheses have primarily taken the form of proposing abnormal regulation in norepinephrine (Bunney and Davis 1965; Schatzberg and Schildkraut 1995), dopamine (Kapur and Mann 1992), and serotonin (Charney and Delgado 1992; Coppen 1967) function. The most consistent evidence supporting these hypotheses is found in reports of altered neuroendocrine function by monoamines and the efficacy of specific inhibitors of monoamine reuptake in the treatment of depression From the Department of Psychiatry, Yale University School of Medicine, and the Department of Veterans Affairs Medical Center, West Haven, Connecticut. Address reprint requests to Dennis S. Charney, MD, Department of Veterans Affairs Medical Center, (116A) Yale University School of Medicine, Chief Psychiatry Service, 950 Campbell Avenue, West Haven, CT 06516. Received April 13, 1995; revised December 20, 1995.
O 1997 Society of Biological Psychiatly
(Heninger and Charney 1987). Simple monoamine deficiency hypotheses have, in general, not been supported. Acute administration of monoamine precursors or agonists, such as bromocriptine, L-dopa, tryptophan, and fenfluramine do not improve mood in depressed patients. Conversely, acute depletion of monoamine levels using alpha-methyl-para-tyrosine (AMPT) or a tryptophan-depleting amino acid drink do not markedly worsen depressed mood in depressed patients (Delgado et al 1991, 1993). There have been suggestions that functional interactions among monoamine systems are important in the regulation of mood. For example, profound changes in mood may only occur when concentrations of two or more of the monoamines are disrupted. In this context, the purpose of the present investigation was to determine if substantial reductions in norepinephrine, doparnine, and serotonin levels produced by combined administration of AMPT and 0006-3223/97/$17.00 SSDI 0006-3223(95)00670-2
Monoamine Depletion
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a tryptophan-depleting amino acid drink would produce dysphoric mood and associated symptoms in healthy subjects.
Methods Subjects Eight healthy subjects (4 men, 4 women) who were psychiatrically and medically healthy, and drug and alcohol free for at least 3 weeks, completed the study. The ages ranged from 20 to 50 years (35.8 2 12.5); this and all future values expressed as mean t standard deviation. The Structured Clinical Interview for DSM-111-R (Spitzer et a1 1990), non patient edition for all subjects was used to validate the lack of mental illness. Each healthy subject was found to be medically healthy based upon a physical examination and routine laboratory testing, including blood tests for hematopoetic, liver, and kidney function, and an electrocardiogram. Written informed consent was obtained for each healthy subject.
Procedure Each healthy subject participated in two 4-day test sessions. Test sessions were conducted approximately 1 week apart. During both sessions active open-label AMPT was administered as described below. In addition, the first 2 healthy subjects who participated in the study during one session ingested a 1-day low-tryptophan diet and a tryptophan-depleting amino acid drink (depletion session). During the other test session, they ingested a 1-day tryptophan-supplemented diet followed by an amino acid drink containing tryptophan (control session). The remaining 6 healthy subjects ingested a tryptophan-depleting amino acid drink during the depletion session. During the control session, they received an amino acid drink only 25% by weight of each amino acid contained in the drink ingested during the depletion session. These 6 healthy subjects did not receive either a 1-day low-tryptophan diet (depletion session) or a 1-day tryptophan-supplemented diet (control session). The change in paradigm for these 6 healthy subjects was necessitated by reports of an association between the ingestion of tryptophan and the eosinophilia myalgia syndrome, reports that resulted in discontinuation of the use of tryptophan in this study. None of the patients in the current study experienced this syndrome. Baseline behavioral ratings and biochemical measurements were taken on day 1 at 8:00 AM. On day 2 behavioral ratings and biochemical measurements were repeated at 9:00 AM, prior to the initiation of AMPT administration. AMPT (1 g) was administered at 9:00 AM, 1:00 PM, and 4:00 PM during day 2 and 9:00 AM and 1:00 PM during day 3. Behavioral ratings were obtained at each
of the five time points. On day 2, 2 of the 8 healthy subjects were given a structured three-meal diet that was low in tryptophan (160 mg for the day) (Delgado et al 1994). In addition, these 2 healthy subjects received one capsule with each meal that contained either 500 mg of tryptophan (control session) or placebo (depletion session). The other 6 healthy subjects ingested a normal diet on day 2. On day 3 at 9:00 AM each healthy subject ingested the amino acid drink. At the end of day 3, 8 hours after the drink, a meal containing normal amounts of tryptophan was given and followed by a freely chosen diet. Day 4 was a follow-up day on which behavioral ratings and biochemical measurements were repeated at 12 noon. The amino acid drink ingested during the depletion session was composed of 15 amino acids. These included L-alanine (5.5 g), L-methionine (3.0 g), L-valine (8.9 g), L-arginine (4.9 g), L-phenylalanine (5.7 g) L-leucine (13.5 g) L-cysteine (2.7 g) L-proline (12.2 g), L-tyrosine (6.9 g), glycine (3.2 g), L-threonine (6.5 g), L-serine (6.9 g), L-histidine (3.2 g), L-isoleucine (8.0 g), and L-lysine (1 1.0 g). Because of the unpleasant taste of the sulfur-containing amino acids (methionine, cysteine, and arginine) these were placed into capsules and taken 15 min before consuming the remaining amino acids in drink form. The drink was prepared by adding the remaining amino acid powder to water at room temperature to a final volume of 300 mL and shaken vigorously. The amino acid solution was flavored with approximately 5 mL of chocolateflavored syrup. Patients drank the solution quickly through a straw. As noted above, the amino acid drink ingested during the control session by 2 healthy subjects consisted of these 15 amino acids plus 2.3 g of tryptophan. The other 6 ingested a tryptophan-free drink containing identical proportions of the amino acids as in the original tryptophan depletion drink, but containing only 25% (by weight) of each amino acid. The double blind was maintained by having a research pharmacist who did not have direct contact with the raters or patients mix the amino acid mixtures with water and chocolate syrup on the morning of each test. Patients and raters were unable to distinguish the quarter-strength from the full-strength drinks.
Behavioral Ratings The healthy subjects were rated using the Hamilton Depression Rating Scale [HDRS, using a structured research interview protocol, the Yale Depression Inventory (Mazure et a1 1986)], Beck Depression Inventory, Profile of Mood States scores (McNair et a1 1988), and a 23-item side effect symptom checklist at day 1 8:00 AM, day 2 8:00 AM, day 2 4:00 PM, day 3 8:00 AM, day 3 4:00 PM, and day
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amino add drink
I 1 control amino add drink I
L
1
AMPT Administration Amino Acid D
a
Figure 1. The effect of AMPT administration and the full-strength tryptophan (TRP)-free or control amino acid drink on Hamilton Depression Rating Scale (HDRS) scores. The amino acid drinks were given at 9:00 AM on day 3. The control drink consisted of a tryptophan-supplemented amino acid drink (n = 2) or a 25% by weight tryptophan-free amino acid drink (n = 6) (see Methods). There were no significant effects on the HDRS compared to day 1 baseline.
4 noon. During times when the patient was not needed for behavioral ratings or biochemical measurements, the healthy subjects were permitted to go about their usual daily activities.
Biochemical Measurements Plasma samples for levels of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were obtained at day 1 9:00 AM,day 2 9:00 AM, day 3 9:00 AM, day 3 4:00 PM, and day 4 noon. Plasma samples for total and free tryptophan concentrations were obtained during day 3 at 9:00 AM and 4:00 PM. Total plasma tryptophan was assayed by high-performance liquid chromatography with flourometric detection (HPLC-F). Free plasma tryptophan was assayed by obtaining the ultrafiltrate of plasma from Arnicon filters centrifuged (100 X g) at room temperature and subjecting the ~ l t r ~ l t r ato t ethe HPLC-F method (Delgado et al 1994). Plasma HVA and MHPG were measured by gas chromatography and mass spectrometry with the use of internal standards (Bacopou10s et a1 1979; Maas et a1 1976).
Data Analysis The effects of AMPT and tryptophan depletion on behavior, and on free and total tryptophan, MHPG, and HVA plasma levels, were initially analyzed using analysis of variance (ANOVA) for repeated measures. This allowed an assessment of the main effects of drug (AMPT and tryptophan depletion vs. AMPT without tryptophan depletion), time (changes over time points sampled), and the
drug and time interaction. Post hoc analyses to examine changes at specific time points were calculated using the Dunnett's multiple t tests. Results are reported as significant whenp < .05 with a two-tailed test. Data analysis and graphic representations utilized the Statistical Program for the Social Sciences@ (SPSS) and DeltaGraph Pro@ software programs.
Results Behavioral Effects of Combined AMPT and Tryptophan Depletion The combined administration of AMPT and the tryptophan-free amino acid drink did not produce statistically significant or even clinically noticeable changes in mood among the healthy subjects. The ANOVA of HDRS scores revealed a nonsignificant main effect of drug (F = .34, df = 1, p = .58), a significant main effect of time (F = 5.82, df = 5, p < .001), and a nonsignificant interaction of drug and time (F = .33, df = 5,35, p = .89). Post hoc analysis indicated no significant changes in HDRS score during AMPT administration and amino acid drink ingestion compared to baseline measurements (day 1 AM,day 2 AM) (Figure 1). The ANOVA of the Profile of Mood States scale subscales (tension-anxiety, depression-dejection, angerhostility, vigor, fatigue, confusion-bewilderment) revealed nonsignificant drug and time interactions. In addition, post hoc analysis indicated there was no significant change from baseline measurements on any of the subscales (Table 1).
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Table 1. Effects of AMPT Administration and Control or Full-Strength Tryptophan-Free Amino Acid Drink on the POMS in Healthy Subjects AMPT Administration
POMS scale ( 2 SD)
Amino + acid drink"
Amino Acid Drink Day 3
Day 2 Day 1 AM
AM
PM
AM
PM
Day 4
AM
Tension-anxiety D C Depression-dejection D C Anger-hostility D C Vigor D C Fatigue D C Confusion- bewilderment D C POMS, Profile of Mood States rating scale; D, tryptophan depletion; C, control drink (consisted of tryptophan-supplemented amino acid drink (n tryptophan-free amino acid drink ( n = 6), see Methods). "Amino acid drinks were given at 9 AM on day 3.
Effect of AMPT on Plasma MHPG and HVA Levels As expected, AMPT produced robust and decreases in plasma MHPG during both test sessions. significant decreases from baseline were found on day 3 AM and pM measurements. AMPT produced significant decreases in H V during ~ the control test session, but only a toward significance during the depletion test session (see Figure 2).
Effect of Amino Acid Drinks on Tryptophan Levels Both free and total tryptophan levels decreased dramatically during tryptophan depletion testing but were unchanged by the control amino acid drink (see Figure 3). There was a significant decrease in total (1 1.0 1.9 p,g/mL to 3.1 + 3.9 p,g/mL) and free (2.0 f- 0.2 to 0.6 t 0.9 p,g/mL) tryptophan levels 420 min after ingestion of the full-strength tryptophan-free amino acid drink on day 3 during the depletion session. It is noteworthy, however, that the 25% tryptophan-free amino acid drink decreased both total (1 1.2 + 1.8 to 6.6 -t 4.1 pg/mL) and free (2.3 + 0.2 to 1.2 0.9 p,g/mL) tryptophan levels in the 6 healthy subjects who received it.
+
+
Discussion Interpretations of the current investigation depend upon the ability of AMPT to lower brain levels of norepineph-
=
2) or 25%
rine and dopamine, and the tryptophan-free amino acid drink to decrease brain serotonir Reclinical studies have demonstrated that AMPT produces a reduction in brain biosynthesis and concentrations of both catecholamines (Rech et a1 1966; Nagatsu et al 1964; Spector et 1965). Consistent with these observations and previous clinical investigations demonstrating an AMPT-induced reduction in urinary and cerebrospinal fluid (CSF) catecholamine metabolites (Brodie et al 1971; Bunney et a1 1971; Miller et a1 in press a, in press b), AMPT in the present study significantly decreased plasma MHPG; however, AMPT significantly decreased plasma HVA only during the control session, raising the possibility that dramatic reductions in plasma tryptophan and consequently serotonin may interact with the AMPT effect on dopamine turnover. It is also possible that, given the short period of AMPT administration, robust reductions in brain monoamines were not achieved. In the present study, the full-strength tryptophan-free amino acid drink led to robust and significant decreases in plasma free tryptophan and total tryptophan levels within several hours after the tryptophan-free amino acid drink. In laboratory animals, this degree of plasma tryptophan depletion leads to a 60-70% decrease in brain serotonin level in a similar time period, based upon in vivo microdialysis and tissue sampling of the frontal cortex of the rat, and the decrease in brain serotonin level correlated best with the pool of free plasma tryptophan available (Del-
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MHPG
L.
Free Tryptophan
Day 1 Day 2 Day3 Day3 Day 4 A M ~ A M AM P M PM ~ AMPT Administration
-
Amino Add Drink
-
AMPT Administration
1
$
15
$
10
5
5
amino add drink control amino add drink
1
HVA
IN
-g --
(
Total Tryptophan
;1.5 0.
z
5
amino add drink contml amino add drink
10
I-
r
5
,E
0
c
0
AMPT Adminismation Amino Acid Drink
Figure 2. The effect of AMPT administration and the fullstrength tryptophan (TRP)-free or control amino acid drink on plasma MHPG and HVA levels. The amino acid drinks were given at 9:00 AM on day 3. The control drink consisted of a tryptophan-supplemented amino acid drink (n = 2 ) or a 25% by weight tryptophan-free amino acid drink (n = 6) (see Methods). *p < .05, **p < .O1 (Dunetts c test compared to day 1 baseline).
gad0 et al 1994; Heslop et a1 1991). Decreased levels of CSF tryptophan and 5-hydroxyindoleaceticacid have been measured in verret monkeys after tryptophan was depleted by an amino acid mixture (Young et al 1989). The 25% tryptophan-free amino acid drink also lowered total and free tryptophan levels, indicating that new methods to control for the tryptophan-depleting effects of the fullstrength tryptophan-free amino acid drink are needed. Therefore, if effects identified in laboratory animals also occur in humans, the combination of AMPT and the tryptophan-free amino acid drinks should have produced substantial decreases in brain norepinephrine and serotonin. In this context, it is of considerable theoretical interest that this combination did not produce depressive symptoms in healthy subjects with no personal or family history of depression. It is not known if such decreases would exacerbate depressed mood in depressed patients, or provoke a relapse in remitted depressives. Consistent with the results of this investigation, McCann and colleagues (1993, 1995) have observed that AMPT did not robustly affect mood in healthy subjects.
-
AMPT Adminismation Amino Acid Drink
Figure 3. The effect of AMPT administration and the fullstrength tryptophan (TRP)-free or control amino acid drink on total and free tryptophan levels. Of note, the 25% by weight tryptophan-free amino acid drink also significantly reduced plasma total and free tryptophan levels, but less so than the full-strength tryptophan-free amino acid drink (see Results). *p < .05, **p < .O1 (Dunetts t test compared to day 1 baseline).
The results of this study provide an opportunity to critically reexamine the role of brain monoamine function in the pathophysiology of depression. Previous investigations have found that administration of the tryptophan-free amino acid dnnk or AMPT separately do not produce marked alterations of mood in drug-free depressed patients (Delgado et a1 1994; Miller et al in press a; but see Bunney et al 1971). In contradistinction to the above conclusion, Benkelfat and colleagues found that tryptophan depletion lowered mood in 30% of healthy men with no personal history, but a positive family history of affective illness, but not in healthy men with neither a personal nor family history of depression (Benkelfat et a1 1994). These results suggest that responses to lowered tryptophan and serotonin might reveal a genetic vulnerability to depression in some individuals. The effects of tryptophan depletion and AMPT need to be evaluated in patients with a history of depression who are not receiving medication and are in remission.
Monoamine Depletion
Recent investigations have found depletion of norepinephrine and doparnine, as a consequence of AMPT administration, reverses the remission induced by norepinephrine (desipramine) and a norepinephrine and dopamine (mazindol) reuptake inhibitor, but not selective and specific serotonin reuptake inhibitors (SSRIs) (Miller et a1 in press b). In contrast, tryptophan depletion reverses the therapeutic effects of specific SSRIs, but not drugs that potently and selectively inhibit norepinephrine reuptake (Delgado et a1 1991, 1993). These data suggest that the efficacy of antidepressant drugs may not be due to a common mechanism involving a single monoamine system. SSRIs and norepinephrine reuptake inhibitors may work via primary actions on serotonin and noradrenaline function, respectively. These studies, considered with the
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current report, also raise the possibility that both the mechanism of action of antidepressant drugs and the pathophysiology of depression may be related to neuronal systems, as yet unidentified, that are regulated by both monoarnines and indoleamines.
This study was funded by grants from the Department of Veterans Affairs (HLM, DSC). Plasma HVA and MHPG assays were performed by Harold Landis and Robert Reynolds at the Yale Mental Health Clinical Research Center, Gas Chromotography-Mass Spectroscopy Laboratory (MH-30929). The assistance of Kathleen Colonese, Lisa Roach, and Melissa Giunti in conducting this study is gratefully acknowledged. Claire O'Connor and Evelyn Testa provided excellent statistical and technical assistance.
References Bacopoulous NG, Redmond DE, Roth RH (1979): Serotonin and dopamine metabolites in brain regions and cerebrospinal fluid of a primate species: Effects of ketamine and fluphenazine. J Neurochem 32: 1215-1218. Benkelfat C, Ellenbogen MA, Dean P, Palmour RM, Young SN (1994): Mood lowering effect of tryptophan depletion. Arch Gen Psychiatry 5 1:687-697. Brodie HKH, Murphy DL, Goodwin FK, Bunney WE (1971): Catecholamines and mania: The effect of alpha-methyl-paratyrosine on manic behavior and catecholamine metabolism. Clin Pharmacol Exp Ther 12:218-224. Bunney WE, Davis JM (1965): Norepinephrine in depressive reactions. Arch Gen Psychiatry 13:483-494. Bunney WE, Brodie HK, Murphy DL, Goodwin FK (1971): Studies of alpha-methyl-para-tyrosine,L-dopa, and L-tryptophan in depression and mania. Am J Psychiatry 1272372- 88 1. Charney DS, Delgado PL (1992): Current concepts of serotonin neuronal function and the pathophysiology of depression. In Elliott M, Heal DJ, Marsden CA (eds), Experimental Approaches to Anxiety and Depression. London: Wiley, pp 197-218. Coppen A (1967): The biochemistry of affective disorders. Br J Psychiatry 113:1237-1264. Delgado PL, Price LH, Miller HL, et a1 (1991): Rapid serotonin depletion as a provocative challenge test for patients with major depression: Relevance to antidepressant action and the neurobiology of depression. Psychophamacol Bull 27:321330. Delgado PL, Miller HL, Salomon RM, et al(1993): Monoamines and the mechanism of antidepressant action: Effects of catecholamine depletion on mood of patients treated with antidepressants. Psychopharmacol Bull 29:389-396. Delgado PL, Price LJ, Miller JL, Salomon RM, Heninger GH, Charney DS (1994): Serotonin and the neurobiology of depression: Effects of tryptophan depletion in drug-free depressed patients. Arch Gen Psychiatry 5 12365-874. Heninger GR, Charney DS (1987): Mechanism of action of antidepressant treatments: Implications for the etiology and
treatment of depressive disorders. In Meltzer HY (ed), Psychophannacology, A Generation of Progress. New York: Raven Press, pp 535-554. Heslop K, Portas CM, Curzon G (1991): Effect of altered tryptophan availability on tissue and extracellular serotonin in rat cortex. In Rollema J, Westerink B, Drijshout WJ (eds), Monitoring Molecules in Neuroscience. Mepple, The Netherlands: Krits Repro, pp 259-261. Kapur S, Mann JJ (1992): Role of the dopaminergic system in depression. Biol Psychiatry 32:l-17. Maas JW, Hattox SE, Landis DH, Roth RH (1976): The determination of a brain arteriovenous difference for 3-methoxy-4-hydroxyphenelethylene glycol (MHPG). Brain Res 118:167-173. Mazure CM, Nelson JC, Price LH (1986): Reliability and validity of the symptoms of major depressive illness. Arch Gen Psychiatry 43:45 1-456. McCann UD, Penetar DM, Shaham Y, et a1 (1993): Effects of catecholamine depletion on alertness and mood in rested and normal volunteers. Neuropsychopharmacology 8:345-356. McCann UD, Thorne D, Hall M, et a1 (1995): The Effects of L-dihydroxyphenylalanine on alertness and mood in a-methyl-para-tyrosine-treatedhealthy humans: Further evidence for the role of catecholamines in arousal and anxiety. Neuropsychophamacology 13:41-52. McNair DM, Lorr M, Droppleman LF (1988): Manual for the Profile of Mood States. San Diego, CA: Educational and Industrial Testing Service. Miller HL, Delgado PL, Salomon RM, Heninger GR, Charney DS (1996): Effects of alpha-meta-para-tyrosine (AMPT) in drug-free depressed patients. Neuropsychophamacology 14: 151-157. Miller HL, Delgado PL, Salomon RM, et a1 (in press b): The behavioral effects of catecholamine depletion on antidepressant induced remission of depression: Implications for monom i n e hypothesis of antidepressant action. Arch Gen Psychiatry 53:117-128. Nagatsu T, Levitt M, Udenfriend S (1964): Tyrosine hydroxy-
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lase, the initial step in norepinephrine biosynthesis. J Biol Chem 239:2910-2917. Rech RH, Borys HK, Moore ICE (1966): Alterations in behavior and brain catecholamine levels in rats treated with alphamethyl-para-tyrosine. J P h a m c o l Exp Ther 153:412-419. Schatzberg AF, Schildkraut JJ (1995): Recent studies on norepinephrine systems in mood disorders. In Bloom FE, Kupfer DJ (eds), Psychophamcology: The Fourth Generation of Progress. New York: Raven Press, pp 91 1-920. Spector S, Sjoerdsma A, Udenfriend S (1965): Blockade of
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endogenous norepinephrine synthesis by alpha-methyl-paratyrosine, an inhibitor of tyrosine hydroxylase. J P h a m c o l Exp Ther 147:86-95. Spitzer RL, Williams JBW, Gibbon M, First MB (1990): Structured Clinical Interview for DSM-111-R, nonpatient edition (SCID-NP, version 1.0). Washington, DC: American Psychiatric Press. Young SN, Emin FR, Pihl RO (1989): Biochemical aspects of tryptophan depletion in primates. Psychophannacology 98: 508-511.
This study was designed to determine the behavioral effects of a reduction in catecholamine and indoleamine function in healthy subjects. Eight healthy subjects received the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (AMPT) in combination with a full-strength tryptophan-depleting amino acid drink during one 4-day test session, and AMPT and tryptophan-supplemented amino acid drink ( n = 2), or a 25% strength tryptophan-depleting amino acid drink ( n = 6 ) during a second 4-day test session. The combined administration of AMPT and the tryptophan-free amino acid drink did not produce statistically signzjicant or even clinically noticeable changes in mood among the healthy subjects. The implications of these observations for the monoamine hypotheses of depression are discussed. 0 1997 Society of Biological Psychiatry Key Words: Norepinephrine, dopamine, AMPT, behavior, catecholamine, indoleamine
Introduction The predominant hypotheses of the pathophysiology of depression involve alterations in brain monoamine systems. These hypotheses have primarily taken the form of proposing abnormal regulation in norepinephrine (Bunney and Davis 1965; Schatzberg and Schildkraut 1995), dopamine (Kapur and Mann 1992), and serotonin (Charney and Delgado 1992; Coppen 1967) function. The most consistent evidence supporting these hypotheses is found in reports of altered neuroendocrine function by monoamines and the efficacy of specific inhibitors of monoamine reuptake in the treatment of depression From the Department of Psychiatry, Yale University School of Medicine, and the Department of Veterans Affairs Medical Center, West Haven, Connecticut. Address reprint requests to Dennis S. Charney, MD, Department of Veterans Affairs Medical Center, (116A) Yale University School of Medicine, Chief Psychiatry Service, 950 Campbell Avenue, West Haven, CT 06516. Received April 13, 1995; revised December 20, 1995.
O 1997 Society of Biological Psychiatly
(Heninger and Charney 1987). Simple monoamine deficiency hypotheses have, in general, not been supported. Acute administration of monoamine precursors or agonists, such as bromocriptine, L-dopa, tryptophan, and fenfluramine do not improve mood in depressed patients. Conversely, acute depletion of monoamine levels using alpha-methyl-para-tyrosine (AMPT) or a tryptophan-depleting amino acid drink do not markedly worsen depressed mood in depressed patients (Delgado et al 1991, 1993). There have been suggestions that functional interactions among monoamine systems are important in the regulation of mood. For example, profound changes in mood may only occur when concentrations of two or more of the monoamines are disrupted. In this context, the purpose of the present investigation was to determine if substantial reductions in norepinephrine, doparnine, and serotonin levels produced by combined administration of AMPT and 0006-3223/97/$17.00 SSDI 0006-3223(95)00670-2
Monoamine Depletion
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1997;41:58-64
a tryptophan-depleting amino acid drink would produce dysphoric mood and associated symptoms in healthy subjects.
Methods Subjects Eight healthy subjects (4 men, 4 women) who were psychiatrically and medically healthy, and drug and alcohol free for at least 3 weeks, completed the study. The ages ranged from 20 to 50 years (35.8 2 12.5); this and all future values expressed as mean t standard deviation. The Structured Clinical Interview for DSM-111-R (Spitzer et a1 1990), non patient edition for all subjects was used to validate the lack of mental illness. Each healthy subject was found to be medically healthy based upon a physical examination and routine laboratory testing, including blood tests for hematopoetic, liver, and kidney function, and an electrocardiogram. Written informed consent was obtained for each healthy subject.
Procedure Each healthy subject participated in two 4-day test sessions. Test sessions were conducted approximately 1 week apart. During both sessions active open-label AMPT was administered as described below. In addition, the first 2 healthy subjects who participated in the study during one session ingested a 1-day low-tryptophan diet and a tryptophan-depleting amino acid drink (depletion session). During the other test session, they ingested a 1-day tryptophan-supplemented diet followed by an amino acid drink containing tryptophan (control session). The remaining 6 healthy subjects ingested a tryptophan-depleting amino acid drink during the depletion session. During the control session, they received an amino acid drink only 25% by weight of each amino acid contained in the drink ingested during the depletion session. These 6 healthy subjects did not receive either a 1-day low-tryptophan diet (depletion session) or a 1-day tryptophan-supplemented diet (control session). The change in paradigm for these 6 healthy subjects was necessitated by reports of an association between the ingestion of tryptophan and the eosinophilia myalgia syndrome, reports that resulted in discontinuation of the use of tryptophan in this study. None of the patients in the current study experienced this syndrome. Baseline behavioral ratings and biochemical measurements were taken on day 1 at 8:00 AM. On day 2 behavioral ratings and biochemical measurements were repeated at 9:00 AM, prior to the initiation of AMPT administration. AMPT (1 g) was administered at 9:00 AM, 1:00 PM, and 4:00 PM during day 2 and 9:00 AM and 1:00 PM during day 3. Behavioral ratings were obtained at each
of the five time points. On day 2, 2 of the 8 healthy subjects were given a structured three-meal diet that was low in tryptophan (160 mg for the day) (Delgado et al 1994). In addition, these 2 healthy subjects received one capsule with each meal that contained either 500 mg of tryptophan (control session) or placebo (depletion session). The other 6 healthy subjects ingested a normal diet on day 2. On day 3 at 9:00 AM each healthy subject ingested the amino acid drink. At the end of day 3, 8 hours after the drink, a meal containing normal amounts of tryptophan was given and followed by a freely chosen diet. Day 4 was a follow-up day on which behavioral ratings and biochemical measurements were repeated at 12 noon. The amino acid drink ingested during the depletion session was composed of 15 amino acids. These included L-alanine (5.5 g), L-methionine (3.0 g), L-valine (8.9 g), L-arginine (4.9 g), L-phenylalanine (5.7 g) L-leucine (13.5 g) L-cysteine (2.7 g) L-proline (12.2 g), L-tyrosine (6.9 g), glycine (3.2 g), L-threonine (6.5 g), L-serine (6.9 g), L-histidine (3.2 g), L-isoleucine (8.0 g), and L-lysine (1 1.0 g). Because of the unpleasant taste of the sulfur-containing amino acids (methionine, cysteine, and arginine) these were placed into capsules and taken 15 min before consuming the remaining amino acids in drink form. The drink was prepared by adding the remaining amino acid powder to water at room temperature to a final volume of 300 mL and shaken vigorously. The amino acid solution was flavored with approximately 5 mL of chocolateflavored syrup. Patients drank the solution quickly through a straw. As noted above, the amino acid drink ingested during the control session by 2 healthy subjects consisted of these 15 amino acids plus 2.3 g of tryptophan. The other 6 ingested a tryptophan-free drink containing identical proportions of the amino acids as in the original tryptophan depletion drink, but containing only 25% (by weight) of each amino acid. The double blind was maintained by having a research pharmacist who did not have direct contact with the raters or patients mix the amino acid mixtures with water and chocolate syrup on the morning of each test. Patients and raters were unable to distinguish the quarter-strength from the full-strength drinks.
Behavioral Ratings The healthy subjects were rated using the Hamilton Depression Rating Scale [HDRS, using a structured research interview protocol, the Yale Depression Inventory (Mazure et a1 1986)], Beck Depression Inventory, Profile of Mood States scores (McNair et a1 1988), and a 23-item side effect symptom checklist at day 1 8:00 AM, day 2 8:00 AM, day 2 4:00 PM, day 3 8:00 AM, day 3 4:00 PM, and day
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amino add drink
I 1 control amino add drink I
L
1
AMPT Administration Amino Acid D
a
Figure 1. The effect of AMPT administration and the full-strength tryptophan (TRP)-free or control amino acid drink on Hamilton Depression Rating Scale (HDRS) scores. The amino acid drinks were given at 9:00 AM on day 3. The control drink consisted of a tryptophan-supplemented amino acid drink (n = 2) or a 25% by weight tryptophan-free amino acid drink (n = 6) (see Methods). There were no significant effects on the HDRS compared to day 1 baseline.
4 noon. During times when the patient was not needed for behavioral ratings or biochemical measurements, the healthy subjects were permitted to go about their usual daily activities.
Biochemical Measurements Plasma samples for levels of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were obtained at day 1 9:00 AM,day 2 9:00 AM, day 3 9:00 AM, day 3 4:00 PM, and day 4 noon. Plasma samples for total and free tryptophan concentrations were obtained during day 3 at 9:00 AM and 4:00 PM. Total plasma tryptophan was assayed by high-performance liquid chromatography with flourometric detection (HPLC-F). Free plasma tryptophan was assayed by obtaining the ultrafiltrate of plasma from Arnicon filters centrifuged (100 X g) at room temperature and subjecting the ~ l t r ~ l t r ato t ethe HPLC-F method (Delgado et al 1994). Plasma HVA and MHPG were measured by gas chromatography and mass spectrometry with the use of internal standards (Bacopou10s et a1 1979; Maas et a1 1976).
Data Analysis The effects of AMPT and tryptophan depletion on behavior, and on free and total tryptophan, MHPG, and HVA plasma levels, were initially analyzed using analysis of variance (ANOVA) for repeated measures. This allowed an assessment of the main effects of drug (AMPT and tryptophan depletion vs. AMPT without tryptophan depletion), time (changes over time points sampled), and the
drug and time interaction. Post hoc analyses to examine changes at specific time points were calculated using the Dunnett's multiple t tests. Results are reported as significant whenp < .05 with a two-tailed test. Data analysis and graphic representations utilized the Statistical Program for the Social Sciences@ (SPSS) and DeltaGraph Pro@ software programs.
Results Behavioral Effects of Combined AMPT and Tryptophan Depletion The combined administration of AMPT and the tryptophan-free amino acid drink did not produce statistically significant or even clinically noticeable changes in mood among the healthy subjects. The ANOVA of HDRS scores revealed a nonsignificant main effect of drug (F = .34, df = 1, p = .58), a significant main effect of time (F = 5.82, df = 5, p < .001), and a nonsignificant interaction of drug and time (F = .33, df = 5,35, p = .89). Post hoc analysis indicated no significant changes in HDRS score during AMPT administration and amino acid drink ingestion compared to baseline measurements (day 1 AM,day 2 AM) (Figure 1). The ANOVA of the Profile of Mood States scale subscales (tension-anxiety, depression-dejection, angerhostility, vigor, fatigue, confusion-bewilderment) revealed nonsignificant drug and time interactions. In addition, post hoc analysis indicated there was no significant change from baseline measurements on any of the subscales (Table 1).
Monoamine Depletion
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BIOL PSYCHIATRY 1997;41:58-64
Table 1. Effects of AMPT Administration and Control or Full-Strength Tryptophan-Free Amino Acid Drink on the POMS in Healthy Subjects AMPT Administration
POMS scale ( 2 SD)
Amino + acid drink"
Amino Acid Drink Day 3
Day 2 Day 1 AM
AM
PM
AM
PM
Day 4
AM
Tension-anxiety D C Depression-dejection D C Anger-hostility D C Vigor D C Fatigue D C Confusion- bewilderment D C POMS, Profile of Mood States rating scale; D, tryptophan depletion; C, control drink (consisted of tryptophan-supplemented amino acid drink (n tryptophan-free amino acid drink ( n = 6), see Methods). "Amino acid drinks were given at 9 AM on day 3.
Effect of AMPT on Plasma MHPG and HVA Levels As expected, AMPT produced robust and decreases in plasma MHPG during both test sessions. significant decreases from baseline were found on day 3 AM and pM measurements. AMPT produced significant decreases in H V during ~ the control test session, but only a toward significance during the depletion test session (see Figure 2).
Effect of Amino Acid Drinks on Tryptophan Levels Both free and total tryptophan levels decreased dramatically during tryptophan depletion testing but were unchanged by the control amino acid drink (see Figure 3). There was a significant decrease in total (1 1.0 1.9 p,g/mL to 3.1 + 3.9 p,g/mL) and free (2.0 f- 0.2 to 0.6 t 0.9 p,g/mL) tryptophan levels 420 min after ingestion of the full-strength tryptophan-free amino acid drink on day 3 during the depletion session. It is noteworthy, however, that the 25% tryptophan-free amino acid drink decreased both total (1 1.2 + 1.8 to 6.6 -t 4.1 pg/mL) and free (2.3 + 0.2 to 1.2 0.9 p,g/mL) tryptophan levels in the 6 healthy subjects who received it.
+
+
Discussion Interpretations of the current investigation depend upon the ability of AMPT to lower brain levels of norepineph-
=
2) or 25%
rine and dopamine, and the tryptophan-free amino acid drink to decrease brain serotonir Reclinical studies have demonstrated that AMPT produces a reduction in brain biosynthesis and concentrations of both catecholamines (Rech et a1 1966; Nagatsu et al 1964; Spector et 1965). Consistent with these observations and previous clinical investigations demonstrating an AMPT-induced reduction in urinary and cerebrospinal fluid (CSF) catecholamine metabolites (Brodie et al 1971; Bunney et a1 1971; Miller et a1 in press a, in press b), AMPT in the present study significantly decreased plasma MHPG; however, AMPT significantly decreased plasma HVA only during the control session, raising the possibility that dramatic reductions in plasma tryptophan and consequently serotonin may interact with the AMPT effect on dopamine turnover. It is also possible that, given the short period of AMPT administration, robust reductions in brain monoamines were not achieved. In the present study, the full-strength tryptophan-free amino acid drink led to robust and significant decreases in plasma free tryptophan and total tryptophan levels within several hours after the tryptophan-free amino acid drink. In laboratory animals, this degree of plasma tryptophan depletion leads to a 60-70% decrease in brain serotonin level in a similar time period, based upon in vivo microdialysis and tissue sampling of the frontal cortex of the rat, and the decrease in brain serotonin level correlated best with the pool of free plasma tryptophan available (Del-
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R.M. Salomon et a1
BIOL PSYCHIATRY 1997;41:58-64
MHPG
L.
Free Tryptophan
Day 1 Day 2 Day3 Day3 Day 4 A M ~ A M AM P M PM ~ AMPT Administration
-
Amino Add Drink
-
AMPT Administration
1
$
15
$
10
5
5
amino add drink control amino add drink
1
HVA
IN
-g --
(
Total Tryptophan
;1.5 0.
z
5
amino add drink contml amino add drink
10
I-
r
5
,E
0
c
0
AMPT Adminismation Amino Acid Drink
Figure 2. The effect of AMPT administration and the fullstrength tryptophan (TRP)-free or control amino acid drink on plasma MHPG and HVA levels. The amino acid drinks were given at 9:00 AM on day 3. The control drink consisted of a tryptophan-supplemented amino acid drink (n = 2 ) or a 25% by weight tryptophan-free amino acid drink (n = 6) (see Methods). *p < .05, **p < .O1 (Dunetts c test compared to day 1 baseline).
gad0 et al 1994; Heslop et a1 1991). Decreased levels of CSF tryptophan and 5-hydroxyindoleaceticacid have been measured in verret monkeys after tryptophan was depleted by an amino acid mixture (Young et al 1989). The 25% tryptophan-free amino acid drink also lowered total and free tryptophan levels, indicating that new methods to control for the tryptophan-depleting effects of the fullstrength tryptophan-free amino acid drink are needed. Therefore, if effects identified in laboratory animals also occur in humans, the combination of AMPT and the tryptophan-free amino acid drinks should have produced substantial decreases in brain norepinephrine and serotonin. In this context, it is of considerable theoretical interest that this combination did not produce depressive symptoms in healthy subjects with no personal or family history of depression. It is not known if such decreases would exacerbate depressed mood in depressed patients, or provoke a relapse in remitted depressives. Consistent with the results of this investigation, McCann and colleagues (1993, 1995) have observed that AMPT did not robustly affect mood in healthy subjects.
-
AMPT Adminismation Amino Acid Drink
Figure 3. The effect of AMPT administration and the fullstrength tryptophan (TRP)-free or control amino acid drink on total and free tryptophan levels. Of note, the 25% by weight tryptophan-free amino acid drink also significantly reduced plasma total and free tryptophan levels, but less so than the full-strength tryptophan-free amino acid drink (see Results). *p < .05, **p < .O1 (Dunetts t test compared to day 1 baseline).
The results of this study provide an opportunity to critically reexamine the role of brain monoamine function in the pathophysiology of depression. Previous investigations have found that administration of the tryptophan-free amino acid dnnk or AMPT separately do not produce marked alterations of mood in drug-free depressed patients (Delgado et a1 1994; Miller et al in press a; but see Bunney et al 1971). In contradistinction to the above conclusion, Benkelfat and colleagues found that tryptophan depletion lowered mood in 30% of healthy men with no personal history, but a positive family history of affective illness, but not in healthy men with neither a personal nor family history of depression (Benkelfat et a1 1994). These results suggest that responses to lowered tryptophan and serotonin might reveal a genetic vulnerability to depression in some individuals. The effects of tryptophan depletion and AMPT need to be evaluated in patients with a history of depression who are not receiving medication and are in remission.
Monoamine Depletion
Recent investigations have found depletion of norepinephrine and doparnine, as a consequence of AMPT administration, reverses the remission induced by norepinephrine (desipramine) and a norepinephrine and dopamine (mazindol) reuptake inhibitor, but not selective and specific serotonin reuptake inhibitors (SSRIs) (Miller et a1 in press b). In contrast, tryptophan depletion reverses the therapeutic effects of specific SSRIs, but not drugs that potently and selectively inhibit norepinephrine reuptake (Delgado et a1 1991, 1993). These data suggest that the efficacy of antidepressant drugs may not be due to a common mechanism involving a single monoamine system. SSRIs and norepinephrine reuptake inhibitors may work via primary actions on serotonin and noradrenaline function, respectively. These studies, considered with the
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current report, also raise the possibility that both the mechanism of action of antidepressant drugs and the pathophysiology of depression may be related to neuronal systems, as yet unidentified, that are regulated by both monoarnines and indoleamines.
This study was funded by grants from the Department of Veterans Affairs (HLM, DSC). Plasma HVA and MHPG assays were performed by Harold Landis and Robert Reynolds at the Yale Mental Health Clinical Research Center, Gas Chromotography-Mass Spectroscopy Laboratory (MH-30929). The assistance of Kathleen Colonese, Lisa Roach, and Melissa Giunti in conducting this study is gratefully acknowledged. Claire O'Connor and Evelyn Testa provided excellent statistical and technical assistance.
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