112
BIOL PSYCHIATRY 1992;31:112-118
CSF Amine Metabolites in Depression P. Lakshmi Reddy, Sumant Khanna, M. N. Subhash, S. M. Channabasavanna, and B. S. Sridhara Rama Rao
The amine metabolites, namely homovanillic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA ) were measured in cerebrospinal fluid (CSF) of depressives (n = 30) and controls (n = 30). Depressed patients had significantly lower HVA levels than controls. No significant diffe;ences were noted between the two groups in 5-HIAA levels. However, the differences between the groups for the CSF HVA/5-HIAA ratio were larger than those Jbr the CSF HVA alone (p < 0.01 versus p < 0.025, respectively). HVA levels correlated positively with monoamine oxidase activity and adenosine deaminase activity.
The concentration of amine metabolites, namely hcmovan~llic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA) in lumbar cerebrospina.~ fluid (CSF) has been extensively investigated with a view toward evaluating the turaover of central nervous system (CNS) neurotransmitters in affective disorders. Low concentration of the serotonin metabolite, 5..HIAA, has been reported in the CSF of patien:s with depression without probenecid (Coppen et al 1972; Ashcroft and Glen 1974; Banki and Molnar 1981; Asberg et al 1984) and with probenecid treatment (Ross and Sjostrand 1969; vanPraag et al 1970). Recently the probenecid procedure has been criticized because the postprobenecid values might be less valid than the baseline values (Chaudry et al 1983; Thiemann et al 1984). Some of the well-controlled studies have not given much support to the finding of low CSF 5HIAA in depression (Koslow et al 1983; Gemer and Bunney 1986). However, a more consistent finding has been the association of low 5-HIAA levels with suicidal behavior in depressed patients (Agren 1980; Oreland et al 1981; vanPraag 1982; Banki et al 1984). In manic patients CSF 5-HIAA levels have been reported to be decreased or normal (Mendels et al 1972). The dopamine metabolite HVA has been investigated in the CSF of patients with depression. In some studies, a lower level of HVA has been found in depressives as compared to controls (Ashchroft et al 1976; Kasa et al 1982; Asberg et al 1984). However, others did not find any significant difference (Post et al 1980). On the other hand, it has been observed that a positive correlation exists between CSF HVA levels and psychomotor activity (Banki 1977). CSF HVA levels have been reported to be increased (Mendels et al 1972) or normal (Bowers et al 1969) in mania. Recently, Peabody et al (1987) observed a negative correlation between CSF HVA and 5-HIAA levels and scores on the Hamilton Rating Scale for depressien in depressive subjects.
From the Department of Neurochemistry (PLR, MNS, BSSRR), and the Department of Psychiatry (SK, SMC), National Institute of Mental Health and Neuroscience, Bangalore-560029, India. Address reprint requests to Dr. P.L. Reddy, Post Doctoral Fellow, Department of Pharmacodynamics, College of Pharmacy, University of lllinois at Chicago, Box 6998, Chicago, illinois 60680. Received August 15, 1990, revised May 31, 1991. © 1992 Society of Biological Psychiatry
0006-3223/92/$05.00
CSF Amine Metabolites in Depression
BIOL PSYCHIATRY |992;31:I12-I18
I 13
The results relating to CSF amine metabolites in affective disorders are hence equivocal. We are presently performing various neurochemical profiles in patients with affective disorders of different types in which we elucidate the relationships among these parameters. As a part of this study we here summarize the correlation between the CSF amine metabolites, homovanillic acid and 5-hydroxy indoleacetic acid and various laboratory results in patients with major depressive disorder. Material and Methods
Subjects The clinical material consisted of 30 drag-free, unipolar (UP) depressed inpatients (14 men and 16 women); their mean age was 36.4 years. All patients met the Research Diagnostic Criteria (RDC) of Spitzer et al (1978). Thirty age- and sex-matched subjects (15 men and 15 women) with no major physical or mental illness, who came for consultation to the department of anesthesia of a general hospital were used as controls. Major medical illness was ruled out after thorough physical examination and routine clinical laboratory investigations. The possibility of mental illness was ruled out by taking a cutoff score ->20 on the Brief Psychiatric Rating Scale The mean age of control subjects was 35 years. All subjects were admitted to the hospital for the study.
Collection of Samples In both patients and controls, 5 ml of CSF was collected between 8 AM and 9 AM into a polypropylene tube and immediately placed in ice. The CSF was obtained from the 4 and 5 intervertebral space of the lumbar region. Most of the samples were analyzed on the same day, but in a few (3 control samples and 2 patient samples) the analysis was done 48 hr after collection and preservation at -20°C. In patients, the blood was also collected for the analysis of platelet monoamine oxidase (MAO), plasma dopamine beta hydroxylase (DBH), erythrocyte Na +,K +-ATPase, cortisol, growth hormone (GH), thyroid stimulating hormone (TSH), prolactin (PRL), luteinizing hormone (LH), erythrocyte adenosine deaminase activity (ADA), and serum immunoglobulins (IgG, IgA, and IgM).
Methods The high pressure liquid chromatography with electrochemical detector (HPLC-ED) method of Subhash et al (1988) was used for the analysis of HVA and 5-HIAA in CSF samples. For separation of amine metabolites, the reverse phase Bondapac ~ C-18 (25 cm × 4 mm) obtained from M/s. Dupont Inc., USA was used. The mobile phase consisted of sodium acetate buffer (0.1 mol/L, pH - 4.0) and 5% methanol. Both the mobile phase and the samples were filtered through 0.4 ttm membrane (Sartorious Inc.). The mobile phase was pumped at a rate of 1.5 ml/min. Using hydroxyindole (HI) as in internal standard the calibration was made and peat:s integrated using the Hewlett-Packard integrator. A calibration mixture of 100 ttL containing 2 ng of HI, 5 ng of 5-HIAA, and 10 ng of HVA was injected. The run time for separation of HVA and 5-HIAA was 20 rain. After calibration with standards, 100 ttl. of CSF was injected and the amounts of HVA and 5-HIAA were directly obtained from the integrator as ng/ml and expressed
P.L. Reddy et al
BIOL PSYCHIATRY 1992;31:112-118
114
Table 1. CSF Amine Metabolites in Depression Parameter
Controls (n = 30)
Depressives (n = 30)
HVA (nmol/L) 5-HIAA (nmol/L) HVA/5-HIAA ratio
171.53 4- 53.31 79.28 4- 29.17 2.55 ± 1.41
141.48 ± 45.62 a 88.62 _ 37.07 1.82 ± 0.85 b
ap < 0.025. bp < 0.01.
as nmol/L. To check the efficacy of the HPLC-ED method for analysis of HVA and 5HIAA in CSF, experiments were made to determine th,. recovery of known amounts of the standard added to the CSF sample. The method followed for erythrocyte Na+,K+-ATPase activity determination was described elsewhere (Reddy et al 1989). Platelet MAO was assayed fluorimetfically, but DBH (plasma and CSF) erythrocyte adenosine deaminase was assayed spectrophotometrically (Reddy 1989). Plasma hormones (cortisol, growth hormone, thyroid stimulating hormone, prolactin, and luteinizing hormone) were analyzed by the radioimmunoassay (RIA) method and the serum immunoglobulins analyzed by radioimmuno diffusion technique (Reddy 1989). Students t-test and Pearson's correlation coefficient methods were used for the statistical evaluation of the data.
Results The HVA and 5-HIAA levels in CSF of controls and depressives are iP~dicated in Table !. Compared to controls, significantly lowered HVA levels (t ffi 2.34, df ffi 58, p < 0.025) were noted in depressives. Levels of 5-HIAA showed no difference (t - 1.09, df - 58, NS) between controls and depressives. However, a significant difference was noted in the HVA/5-HIAA ratio (r - 2.43 df -- 58, p < 0.01) between the two groups of subjects. The correlation analysis between amine metabolites and other parameters in depression are indicated in Table 2. It could be noted that the CSF HVA level correlated with platelet MAO activity (r = 0.39, n = 30 p < 0.025), erythrocyte adenosine deaminase activity (r = 0.48, n = 23, p < 0.01), and 5-HIAA levels (r = 0.82, n ffi 30, p < 0.001).
Discussion During the present study, significantly lower CSF-HVA levels were noted in depressed subjects compared with controls. The finding of a lowered CSF-HVA level is not in agreement with some of the findings of earlier investigators who reported no change in CSF HVA levels (Ashcroft et al 1976; Kasa et al 1982; Asberg et al 1984; Post et al 1980). This discrepancy could be due to the method of selection of subjects or the collection of samples on an outpatient basis and/or the analytical technique used. It might be relevant to point out that in the present study the, Stand~d Research Diagnostic Criteria of Spitzer et al (1978) was employed for the selection of subjects. All the subjects were inpatients. A sensitive method of HPLC with electrochemical detector was used for the determination of monoamine metabolite levels. However, the Brief Psychiatric Rating
CSF Amine Metabolites in Depression
BiOL PSYCHIATRY 1992;31:112-118
115
Table 2. Correlationa of CSF Monoamine Metabolites with Various Other Parameters in Depression Parameters
CSF HVA (r)
CSF 5-HIAA (r)
Age (n = 30) Plasma DBH (n - 30) CSF DBH (n - 30) MAO (n - 30) Na +, K + -ATPase (n = 30) Cortisol (n = 30) GH (n = 30) TSH ( = 30) Prolactin (n = 30) LH (n ffi 21) IgG (n ffi 23) IgA (n = 23) IgM (n = 23) ADA (n - 23) CSF HVA (n -- 30)
-0.18 0.26 -0.32 0.39 b 0.12 0.09 0.20 -0.14 0.08 0.31 -0.24 - 0.16 0.06 0.48 b u
-0.10 0.09 -0.15 0.06 0.20 0.19 0.19 --0.20 0.02 --0.21 .....0.17 0.04 0.24 0.29 0.82 b
°Pearson's Correlation Coefficient method was used. bStatistically significant (p < 0.05).
Scale used in selection of controls could only rule out the possibility of acute symptomatology. The concentration of HVA in CSF reflects the metabolism of dopamine in the brain, as there is no contribution from the spinal cord (Ashcroft 1982). The low HVA levels in the CSF, found in some depressed patients, may thus reflect low levels of dopamine in the brain. Low CSF metabolite levels may result from a reduced rate of dopamine production or, alternatively, a reduction in functional release of the amine. A reduction in the synthesis of amine might result from reduced availability of the precursor amino acid, tyrosine, or a change in the enzymatic mechanisms involved in the synthesis. The finding of unaffected 5-HIAA levels in UP depression does not directly support the hypothesis of a 5-HT deficiency in depression. The hypothesis is based ,n the finding of low 5-HIAA levels in the CSF of depressed patients. As mentioned earlier in the introductory section, consensus on whether CSF 5-HIAA is low in depression has not yet been reached; even increased concentration of CSF 5-HIAA in depressed patients was noted in a study by Koslow et al (1983). Charney et al (1981), referring to the down regulation of amine receptor function induced by chronic treatment with antidepressant drugs, put forward the hypothesis that amine function may be increased in depression. The early reports of depressives who showed low 5-HT in certain brain areas (Shaw et al 1967; Pare et al 1969; Lloyd et al 1974) have to be taken with certain reservations due to inherent methodological errors/artifacts (Gottfries 1980). Low CSF-5-HIAA concentrations noted in most studies on depressed patients have been strongly associated with certain individual symptoms, such as suicidal behavior and not with diagnosis of depression (Banki et al 1984). The negative results in the 5-HIAA part of the study, even when matched pairs were investigated, are not surprising as there is some inconsistency in reports concerning CSF 5-HIAA. This inconsistency may be due to variables such as age, body height, sex, vertebral interspace for lumbar puncture and seasonal variation. In many studies these factors have not been controlled. The present report is based on population samples with
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P.L. Reddy et al
no differences between controls and depressed patients in age and sex distribution. Recently, Gibbons et al (1990) pointed ol, t that there is a considerable variation in normal control values for 5-HIAA among different centers. Hence the studies that found differences between controls and depressives in 5-HIAA levels should be treated with caution. The positive correlation between CSF 5-HIAA and HVA has been documented in depression. Evidence exists to show that there is an association between serotonrergic and dopaminergic activity in patients with depression. For instance, bromocriptine, a relatively selective dopamine agonist affects the serotonergic system (Nordin et al 1981). In addition, zimeldine, a specific serotonin uptake blocker alters the HVA levels (Bertilsson et al 1980). The correlation between serotonin and dopamine metabolites has also been reported in schizophrenic patients and in animals (Faull et al 1984). The clinical significance of this association remains to be seen. The CSF HVA levels correlated positively with both monoamine oxidase and adenosine deaminase activities. This could be explained as follows: adenosine deaminase is a catabolic enzyme in the adenosine metabolism. High adenosine deaminase activity results in decreased adenosine content, which in turn facilitates the release of neurotransmitter since adenosine inhibits the neurotransmiCer release (Fredholm and Dunwiddie 1988). The released neurotransmitter, especially dopamine, could have been easily accessible to MAO, resulted in higher metabolite formation. Conversely, low adenosine deaminase activity results in lower metabolite formation. We need further exploration of the role of adenosine as neuromodulator in depressive disorder. The CSF metabolite levels reported in the present study may be a measure of the central turnover of dopamine and serotonin. Calculating a ratio of the CSF levels of HVA and 5-HIAA might indicate a relationship between the turnover of these two transmitters (Agren et al 1986). Thus, it is noteworthy that the differences for the CSF HVA/5-HIAA ratio were relatively larger between the groups than those for CSF HVA alone (p < 0.01 versus p < 0.025, respectively). Although this suggests that depressed patients have an imbalance between the turnover of dopamine and serotonin, a strong positive correlation (p < 0.001) between CSF HVA and 5HIAA and lowered CSF HVA levels together definitely indicates an impaired dopamine turnover in depression.
References Agren H (1980): Symptom patterns in UP and BP depression correlating with monoamine metabolites in CSF. Psychiatry Res 2:225-232. Agren H, MeffordJ, Rudoffer M, LinnoilaM, Potter W (1986): Interactingneurotransmittersystem. An nonexperimental approach to the 5-HIAA HVA correlation on human CSF. J Psychiatry Res 20:175-193. Asberg M, Bertilsson L, Mortensson B (1984): CSF monoamine metabolites in depression and suicide. In Usdin E, Asberg M, Bertilsson L, Sioquist F (eds), Frontiers in Biocl~emical and Pharmacological Research in Depression. New York: Raven. Ashcroft G (1982): Biochemistry and pathology of the affective psychoses. In Wing JK (ed) Handbook of Psychiatry. Cambridge: Cambridge University Press, pp 160-165. Ashcroft GW, Glen AIM (1974): Mood and neuronal function: A modified amine hypothesis. Adv Biochem Psychopharmacol I 1:335-342. Ashcroft GW, Dow RC, Yaten CM, Pullar IA (1976): Significance of lumbar C5~ meiaboli~e measures in affective illness. In Tuomisto J, Paasonen MK (eds), CNS and Behavioral Pharmacology (3.) Helsinki, Finland: University of Helsinki.
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Banki CM (1977): Correlation between CSF amine metabolites and psychomotor activity in affective disorders. J Neurochem 28:255-257. Banki CM, Molnar G (1981): CSF 5HIAA as an index of central serotonergic processes. Psychiatry Res 5:23-28. Banki CM, Arato M, Pap Z, Kuvez M (1984): Biochemical markers in suicidal patients: Investigations with CSF amine metabolites and neuroendocrine tests. J Affective Disord 6:341-346. Bertilsson L, Tuck Jr, Siwers B (1980): Biochemical effects of zimeldine in man. Fur J Clin Pharmacol 18:483-485. Bowers MB, Heninger GR, Gerbode F (1969): CSF 5HIAA and HVA in psychiatric patients, lnt J Neuropharmacol 8:255-262. Charney DS, Menkes DB, Heninger GR (1981): Receptor seesitivity and the mechanism of action of antidepressant treatment. Arch Gen Psychiatry 38:1160-1180. ' Chaudry RW, Ebert MH, Van Kammen DP, Post RM, Goodwin FK (1983): CSF probenecid studies: A reinterpretation. Biol Psychiatry 18:1287-1292. Coppen A, Brookshank BWL, Peet M (1972): Tryptophan concentration in CSF of depressive patients. Lancet 1:1393-1399. Faull KF, King RJ, Berger PA, Barchas JD (1984): Systems theory as a tool for integrating functional interactions among biogenic amines. In Usdin E, Carlsson A, Dahlstrom A, Engle J (eds), Catecholamines, Part C: Neuropharmacology and Central Nervous System--Therapeutic Aspects. New York: Alan R. Liss. Fredholm BB, Dunwiddie TV (1988): How does adenosine inhibit transmitter release? TIPS 9:130134. Gerner RH Bunney WE Jr (1986): Biological hypothesis of affective disorders. In Berger PA, Brodie HKH (eds), The American Handbook of Psychiatry: Biological P~ychi;~,try. Vol VIII. New York: Basic Books, pp 265-270. Gibbons RD, Davis JM, Hedeker DR (1990): A comment on the selection of healthy cor~trols for psychiatric experiments. Arch Gen Psychiatry 47:785-786. Gottfries GC (1980): Human brain levels of monoamines and their metabolites. Postmortem investigation. Acta Psychiat Scand (Suppl. 280) 61:49--61. Kasa J, Otsuki S, Yamamoto M, Sato M, Karuoda H, Ogawa N. (1982): CSF GABA and HVA in depressive disorders. Biol Psychiatry 17:877-887. Koslow SH, Mass JW, Bowden CL, Davis M, Hanin I, Javaid J (1983): CSF and urinary biogenic amines and metabolites in depression and mania. Arch Gen Psychiatry 40:999.-1003. Lloyd KJ, Farley IJ, Deek JHN, Homykiewicz O (1974): 5HT and 5HIAA in discrete areas of the brainstem of suicide victims and control patients. Adv Bwchem Psychopharmacol 11:387-395. Mendels J, Frazer A, Fitzgerald RG, Ramsey TA, Stokes JN (1972): Biogenic amine metabolites in CSF of depressed and manic patients. Science 175:1380-1381. Nordin L, Siwers B, Bertilsson L (1981): Bromocriptine treatment of depressive disorders. Clinical and biochemical effects. Acta Psychiatr Scand 64:25-28. Oreland L, Wiberg A, Asbert M (1981): Piatelet MAO activity and monoamine metabolites in CSF of depressed and suicidal patients and the healthy controls. Psychiatry Res 4:2 ~-29. Pare GMB, Young DPH, Price KS, Stacy RS (1969): 5HT, noradrenaline and dopamine in brainstem, hypothalamus and caudate nucleus of controls and of patients committing suicide by coalgas poisoning. Lancet ii: 133-135. Peabody CA, Faull KF, Kiny RJ, White HA, Bauches JD, Berger PA (1987): CSF amine metabolites and depression. Psychiatry Res 21:1-7. Post RM, Ballenger JC, Goodwin FK (1980): Cerebrospinal fluid studies of neurotransmitter function in manic and depressive illness. In Wood JH (ed), Neurobiolc_y of Cerebrospinal Fluid. New York: Plenum, pp 685-717.
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Reddy PL (1989): Doctoral dissertation submitted to the Bangalore University, Bangalore. Reddy PL, Khanna S, Subhash MN, Channabasavanna SM, Rao BSS (1989): Ery:hrocyte membrane Na-K ATPase activity in affective disorder. Biol Psychiatry 26:533-537. Ross BE, Sjostrand R (1969): 5HIAA, HVA levels in CSF after probenecid application in patients with MDP. Pharmacol Clin 1:153-160. Shaw DM, Camps FE, Eccleston EG (1967): 5HT in the hindbrain of depressive suicides. Br J Psychiatry 113:1407-1412. Spitzer RL, Endicott J, Robins E (1978): Research diagnostic criteria: Rationale and reliability. Arch Gen Psychiatry 35:773-782. Subhash MN, Padmashree TS, Swamy HS (1988): CSF monoamine metabolites---HPLC/EDMethod. lnd J Clin Biochem 3:53-57. Thiemann S, Faull KF, Kraemer HC, Bacehar JD, Berger PA (1984): CSF probenecid and biogenic amines. Biol Psych~ ~try 19:1357-1360. vanPraag HM (1982): Depression, suicide and the metabolism of 5HT in the brain. J Affective Disord 4:275-285. vanPraag HM, Paite J (1970): 5HIAA levels in CSF of depressive patients treated with probenecid. Nature 225:1259-1261.
BIOL PSYCHIATRY 1992;31:112-118
CSF Amine Metabolites in Depression P. Lakshmi Reddy, Sumant Khanna, M. N. Subhash, S. M. Channabasavanna, and B. S. Sridhara Rama Rao
The amine metabolites, namely homovanillic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA ) were measured in cerebrospinal fluid (CSF) of depressives (n = 30) and controls (n = 30). Depressed patients had significantly lower HVA levels than controls. No significant diffe;ences were noted between the two groups in 5-HIAA levels. However, the differences between the groups for the CSF HVA/5-HIAA ratio were larger than those Jbr the CSF HVA alone (p < 0.01 versus p < 0.025, respectively). HVA levels correlated positively with monoamine oxidase activity and adenosine deaminase activity.
The concentration of amine metabolites, namely hcmovan~llic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA) in lumbar cerebrospina.~ fluid (CSF) has been extensively investigated with a view toward evaluating the turaover of central nervous system (CNS) neurotransmitters in affective disorders. Low concentration of the serotonin metabolite, 5..HIAA, has been reported in the CSF of patien:s with depression without probenecid (Coppen et al 1972; Ashcroft and Glen 1974; Banki and Molnar 1981; Asberg et al 1984) and with probenecid treatment (Ross and Sjostrand 1969; vanPraag et al 1970). Recently the probenecid procedure has been criticized because the postprobenecid values might be less valid than the baseline values (Chaudry et al 1983; Thiemann et al 1984). Some of the well-controlled studies have not given much support to the finding of low CSF 5HIAA in depression (Koslow et al 1983; Gemer and Bunney 1986). However, a more consistent finding has been the association of low 5-HIAA levels with suicidal behavior in depressed patients (Agren 1980; Oreland et al 1981; vanPraag 1982; Banki et al 1984). In manic patients CSF 5-HIAA levels have been reported to be decreased or normal (Mendels et al 1972). The dopamine metabolite HVA has been investigated in the CSF of patients with depression. In some studies, a lower level of HVA has been found in depressives as compared to controls (Ashchroft et al 1976; Kasa et al 1982; Asberg et al 1984). However, others did not find any significant difference (Post et al 1980). On the other hand, it has been observed that a positive correlation exists between CSF HVA levels and psychomotor activity (Banki 1977). CSF HVA levels have been reported to be increased (Mendels et al 1972) or normal (Bowers et al 1969) in mania. Recently, Peabody et al (1987) observed a negative correlation between CSF HVA and 5-HIAA levels and scores on the Hamilton Rating Scale for depressien in depressive subjects.
From the Department of Neurochemistry (PLR, MNS, BSSRR), and the Department of Psychiatry (SK, SMC), National Institute of Mental Health and Neuroscience, Bangalore-560029, India. Address reprint requests to Dr. P.L. Reddy, Post Doctoral Fellow, Department of Pharmacodynamics, College of Pharmacy, University of lllinois at Chicago, Box 6998, Chicago, illinois 60680. Received August 15, 1990, revised May 31, 1991. © 1992 Society of Biological Psychiatry
0006-3223/92/$05.00
CSF Amine Metabolites in Depression
BIOL PSYCHIATRY |992;31:I12-I18
I 13
The results relating to CSF amine metabolites in affective disorders are hence equivocal. We are presently performing various neurochemical profiles in patients with affective disorders of different types in which we elucidate the relationships among these parameters. As a part of this study we here summarize the correlation between the CSF amine metabolites, homovanillic acid and 5-hydroxy indoleacetic acid and various laboratory results in patients with major depressive disorder. Material and Methods
Subjects The clinical material consisted of 30 drag-free, unipolar (UP) depressed inpatients (14 men and 16 women); their mean age was 36.4 years. All patients met the Research Diagnostic Criteria (RDC) of Spitzer et al (1978). Thirty age- and sex-matched subjects (15 men and 15 women) with no major physical or mental illness, who came for consultation to the department of anesthesia of a general hospital were used as controls. Major medical illness was ruled out after thorough physical examination and routine clinical laboratory investigations. The possibility of mental illness was ruled out by taking a cutoff score ->20 on the Brief Psychiatric Rating Scale The mean age of control subjects was 35 years. All subjects were admitted to the hospital for the study.
Collection of Samples In both patients and controls, 5 ml of CSF was collected between 8 AM and 9 AM into a polypropylene tube and immediately placed in ice. The CSF was obtained from the 4 and 5 intervertebral space of the lumbar region. Most of the samples were analyzed on the same day, but in a few (3 control samples and 2 patient samples) the analysis was done 48 hr after collection and preservation at -20°C. In patients, the blood was also collected for the analysis of platelet monoamine oxidase (MAO), plasma dopamine beta hydroxylase (DBH), erythrocyte Na +,K +-ATPase, cortisol, growth hormone (GH), thyroid stimulating hormone (TSH), prolactin (PRL), luteinizing hormone (LH), erythrocyte adenosine deaminase activity (ADA), and serum immunoglobulins (IgG, IgA, and IgM).
Methods The high pressure liquid chromatography with electrochemical detector (HPLC-ED) method of Subhash et al (1988) was used for the analysis of HVA and 5-HIAA in CSF samples. For separation of amine metabolites, the reverse phase Bondapac ~ C-18 (25 cm × 4 mm) obtained from M/s. Dupont Inc., USA was used. The mobile phase consisted of sodium acetate buffer (0.1 mol/L, pH - 4.0) and 5% methanol. Both the mobile phase and the samples were filtered through 0.4 ttm membrane (Sartorious Inc.). The mobile phase was pumped at a rate of 1.5 ml/min. Using hydroxyindole (HI) as in internal standard the calibration was made and peat:s integrated using the Hewlett-Packard integrator. A calibration mixture of 100 ttL containing 2 ng of HI, 5 ng of 5-HIAA, and 10 ng of HVA was injected. The run time for separation of HVA and 5-HIAA was 20 rain. After calibration with standards, 100 ttl. of CSF was injected and the amounts of HVA and 5-HIAA were directly obtained from the integrator as ng/ml and expressed
P.L. Reddy et al
BIOL PSYCHIATRY 1992;31:112-118
114
Table 1. CSF Amine Metabolites in Depression Parameter
Controls (n = 30)
Depressives (n = 30)
HVA (nmol/L) 5-HIAA (nmol/L) HVA/5-HIAA ratio
171.53 4- 53.31 79.28 4- 29.17 2.55 ± 1.41
141.48 ± 45.62 a 88.62 _ 37.07 1.82 ± 0.85 b
ap < 0.025. bp < 0.01.
as nmol/L. To check the efficacy of the HPLC-ED method for analysis of HVA and 5HIAA in CSF, experiments were made to determine th,. recovery of known amounts of the standard added to the CSF sample. The method followed for erythrocyte Na+,K+-ATPase activity determination was described elsewhere (Reddy et al 1989). Platelet MAO was assayed fluorimetfically, but DBH (plasma and CSF) erythrocyte adenosine deaminase was assayed spectrophotometrically (Reddy 1989). Plasma hormones (cortisol, growth hormone, thyroid stimulating hormone, prolactin, and luteinizing hormone) were analyzed by the radioimmunoassay (RIA) method and the serum immunoglobulins analyzed by radioimmuno diffusion technique (Reddy 1989). Students t-test and Pearson's correlation coefficient methods were used for the statistical evaluation of the data.
Results The HVA and 5-HIAA levels in CSF of controls and depressives are iP~dicated in Table !. Compared to controls, significantly lowered HVA levels (t ffi 2.34, df ffi 58, p < 0.025) were noted in depressives. Levels of 5-HIAA showed no difference (t - 1.09, df - 58, NS) between controls and depressives. However, a significant difference was noted in the HVA/5-HIAA ratio (r - 2.43 df -- 58, p < 0.01) between the two groups of subjects. The correlation analysis between amine metabolites and other parameters in depression are indicated in Table 2. It could be noted that the CSF HVA level correlated with platelet MAO activity (r = 0.39, n = 30 p < 0.025), erythrocyte adenosine deaminase activity (r = 0.48, n = 23, p < 0.01), and 5-HIAA levels (r = 0.82, n ffi 30, p < 0.001).
Discussion During the present study, significantly lower CSF-HVA levels were noted in depressed subjects compared with controls. The finding of a lowered CSF-HVA level is not in agreement with some of the findings of earlier investigators who reported no change in CSF HVA levels (Ashcroft et al 1976; Kasa et al 1982; Asberg et al 1984; Post et al 1980). This discrepancy could be due to the method of selection of subjects or the collection of samples on an outpatient basis and/or the analytical technique used. It might be relevant to point out that in the present study the, Stand~d Research Diagnostic Criteria of Spitzer et al (1978) was employed for the selection of subjects. All the subjects were inpatients. A sensitive method of HPLC with electrochemical detector was used for the determination of monoamine metabolite levels. However, the Brief Psychiatric Rating
CSF Amine Metabolites in Depression
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115
Table 2. Correlationa of CSF Monoamine Metabolites with Various Other Parameters in Depression Parameters
CSF HVA (r)
CSF 5-HIAA (r)
Age (n = 30) Plasma DBH (n - 30) CSF DBH (n - 30) MAO (n - 30) Na +, K + -ATPase (n = 30) Cortisol (n = 30) GH (n = 30) TSH ( = 30) Prolactin (n = 30) LH (n ffi 21) IgG (n ffi 23) IgA (n = 23) IgM (n = 23) ADA (n - 23) CSF HVA (n -- 30)
-0.18 0.26 -0.32 0.39 b 0.12 0.09 0.20 -0.14 0.08 0.31 -0.24 - 0.16 0.06 0.48 b u
-0.10 0.09 -0.15 0.06 0.20 0.19 0.19 --0.20 0.02 --0.21 .....0.17 0.04 0.24 0.29 0.82 b
°Pearson's Correlation Coefficient method was used. bStatistically significant (p < 0.05).
Scale used in selection of controls could only rule out the possibility of acute symptomatology. The concentration of HVA in CSF reflects the metabolism of dopamine in the brain, as there is no contribution from the spinal cord (Ashcroft 1982). The low HVA levels in the CSF, found in some depressed patients, may thus reflect low levels of dopamine in the brain. Low CSF metabolite levels may result from a reduced rate of dopamine production or, alternatively, a reduction in functional release of the amine. A reduction in the synthesis of amine might result from reduced availability of the precursor amino acid, tyrosine, or a change in the enzymatic mechanisms involved in the synthesis. The finding of unaffected 5-HIAA levels in UP depression does not directly support the hypothesis of a 5-HT deficiency in depression. The hypothesis is based ,n the finding of low 5-HIAA levels in the CSF of depressed patients. As mentioned earlier in the introductory section, consensus on whether CSF 5-HIAA is low in depression has not yet been reached; even increased concentration of CSF 5-HIAA in depressed patients was noted in a study by Koslow et al (1983). Charney et al (1981), referring to the down regulation of amine receptor function induced by chronic treatment with antidepressant drugs, put forward the hypothesis that amine function may be increased in depression. The early reports of depressives who showed low 5-HT in certain brain areas (Shaw et al 1967; Pare et al 1969; Lloyd et al 1974) have to be taken with certain reservations due to inherent methodological errors/artifacts (Gottfries 1980). Low CSF-5-HIAA concentrations noted in most studies on depressed patients have been strongly associated with certain individual symptoms, such as suicidal behavior and not with diagnosis of depression (Banki et al 1984). The negative results in the 5-HIAA part of the study, even when matched pairs were investigated, are not surprising as there is some inconsistency in reports concerning CSF 5-HIAA. This inconsistency may be due to variables such as age, body height, sex, vertebral interspace for lumbar puncture and seasonal variation. In many studies these factors have not been controlled. The present report is based on population samples with
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no differences between controls and depressed patients in age and sex distribution. Recently, Gibbons et al (1990) pointed ol, t that there is a considerable variation in normal control values for 5-HIAA among different centers. Hence the studies that found differences between controls and depressives in 5-HIAA levels should be treated with caution. The positive correlation between CSF 5-HIAA and HVA has been documented in depression. Evidence exists to show that there is an association between serotonrergic and dopaminergic activity in patients with depression. For instance, bromocriptine, a relatively selective dopamine agonist affects the serotonergic system (Nordin et al 1981). In addition, zimeldine, a specific serotonin uptake blocker alters the HVA levels (Bertilsson et al 1980). The correlation between serotonin and dopamine metabolites has also been reported in schizophrenic patients and in animals (Faull et al 1984). The clinical significance of this association remains to be seen. The CSF HVA levels correlated positively with both monoamine oxidase and adenosine deaminase activities. This could be explained as follows: adenosine deaminase is a catabolic enzyme in the adenosine metabolism. High adenosine deaminase activity results in decreased adenosine content, which in turn facilitates the release of neurotransmitter since adenosine inhibits the neurotransmiCer release (Fredholm and Dunwiddie 1988). The released neurotransmitter, especially dopamine, could have been easily accessible to MAO, resulted in higher metabolite formation. Conversely, low adenosine deaminase activity results in lower metabolite formation. We need further exploration of the role of adenosine as neuromodulator in depressive disorder. The CSF metabolite levels reported in the present study may be a measure of the central turnover of dopamine and serotonin. Calculating a ratio of the CSF levels of HVA and 5-HIAA might indicate a relationship between the turnover of these two transmitters (Agren et al 1986). Thus, it is noteworthy that the differences for the CSF HVA/5-HIAA ratio were relatively larger between the groups than those for CSF HVA alone (p < 0.01 versus p < 0.025, respectively). Although this suggests that depressed patients have an imbalance between the turnover of dopamine and serotonin, a strong positive correlation (p < 0.001) between CSF HVA and 5HIAA and lowered CSF HVA levels together definitely indicates an impaired dopamine turnover in depression.
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