Intercorrelations among monoamine metabolite concentrations in human lumbar CSF are not due to a shared acid transport system

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Intercorrelations Among Monoamine Metabolite Concentrations in Human Lumbar CSF Are Not Due to a Shared Acid Transpo System Michael Jibson, Kym F. Faull, and John G. Csernansky

lntercorrelations among homovanillic acid (HVA ), 5-hydroxyindoleacetic acid (5-HIAA ), and 3-methoxy-hydroxy-phenylglycol (MHPG) concentrations in lumbar cerebrospinal fluid (CSF) were examined before and after blockade of the acid transport system by probenecid in 59 psychiatric inpatients. The three compeunds remained intercorrelated despite acid transport blockade, suggesting that the common transport system does not account for their covariance. Other possibilities to explain the interrelationship among these compounds are discussed. Introduction _The monoam,_'nes--dopamine (DA), serotonin (5-HT), and norepinephrine (NE)--are important neurotransmitters in the central nervous system (CNS) and have been implicated in a wide variety of illnesses, of which schizophrenia and mood disorders are of special interest to psychiatrists. DA is most often linked to schizophrenia (Losonczy et al. 1987) and NE and 5-HT are frequently related to mood d~sorders (van Praag 1978; Siever 1987; Meltzer and Lowy 1987). Much of the evidence for these hyl~theses, however, is indirect, based on associations between neuroleptic efficacy and DA receptor blockade (Fotter et al. 1987), antidepressant efficacy and NE, or 5-HT reuptak¢; and receptors (Potter et al. 1987; Traskman et al. 1979). Attempts to more directly relate psychiatric disorders to nfmrotransmitter systems have proved to be difficult. Given the technical and ethical problems of studying human brain function, alternate means of detecting changes in monoamine systems have been sought. Prominent among these has been measurement of the major metabolites of DA, 5-HT, and NE, namely, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxy-phenylglycol (MHPG) in cerebrospinal fluid (CSF). Numerous studies have attempted to relate CSF monoamine metabolite concentrations to psychiatric diagnosis (Berger et al. 1980; Gerner et al. 1984; Roy et al. 1988; Asberg et al. 1973; Sedvall

From the Department of Psychiatry and Behavioral Sciences, Stanford Uni,,ersity School of Medicine, Stanford, CA (M.J., K.F.F., J.G.C.); and the Palo Alto VA Medical Center (M.J., J.G.C.). ,.~aloAlto, CA. Supported in part by grants from the National Institute of Mental Health, MH- 30854 to the VA-Stanford Mental Health Clinical Research Center at S'~anfordUniversity, and a special grant of the Medical Research Service of the Veterans Administration (now Department of Veterans Affairs) to the Schizophrenia Biologic Research Center (SBRC) at the Palo Alto VA Medical Center. Address reprint requests to Dr. Csernansky, Department of Psychiatry, Washington Uawersity Medical School, 4940 Audubon Avenue, St. Louis, MO 63110. Received September 25, 1989: revised March 5, 1990. Published 1990 by Elsevier Science Publishing Company, inc.

0006-3223/90/$00.00

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and Wode-Helgodt 1980), symptom type (Peabody et al. 1987), severity of symptoms (Peabody et al. 1987; Wode-Helgodt et al. 1977; Davis et al. 1985), or ventricular size (Goetz and van Kammen, 1986; Losonczy et al. 1986; Houston et ai. 1986; Potkin et al. 1983; van Kammen et al. 1983). An implicit assumption underlying the linking of specific disorders to particular neurotransmitter systems is that these systems play separate and distinct roles in brain function. However, this functional independence might be compromised by interactions among various CNS neurotransmitter systems, and has not been supported by studies of the CSF monoamine metabolites. To the contrary, a direct correlation between HVA and 5-HIAA has generally been noted (Faull et al. 1984; Risby et al. 1987), and less pronounced correlations between these metabolites and MHPG have also been reported (Gerner et al. 1984; Nyback et al. 1983; Koslow et al. 1983; Faull et al. 1984). A probenecid-sensitive organic acid transport system in the CSF (Forn 1972) actively moves HVA and 5-HIAA from CSF to the venous circulation (Wood 1980). MHPG, in contrast, is only slightly increased by probenecid blockade of this active transport (Gordon et al. 1973; Berger et al. 1980; Faull et al. 1981), and diffuses freely across the blood/ brain and blood/CSF barriers (Wolfson and Escriva 1976; Fanll et al., unpublished data). As a result, concentration gradients from ventricles to lumbar spine have been found for HVA and 5-HIAA, but not for MHPG (Wood 1980; Scheinin 1985; Faull and Csemansky, unpublished data). The presence of this acid transport system raises the possibility that at least CSF HVA and 5-HIAA intercorrelations are a result of the effects of this system. We tested this hypothesis directly by looking at intercorrelations among the levels of HVA, 5-HIAA, and MHPG in lumbar CSF of medication-free schizophrenic, schizoaffective, and depressed patients before and after blockade of the acid trangport system by probenecid. We reasoned that if metabolite intercorrelations persisted following probenecid administration, particularly with high CSF probenecid levels and essentially complete blockade, acid transport could be eliminzted as a major cause of the phenomena.

Methods The data were retrospectively drawn from a database at the Clinical Research Center of the Palo Alto VA Medical Center compiled over many years. Fifty-nine male subjects were selected from the database who met the RDC criteria for schizophrenia, schizoaffective disorder, or major depression. Subjects had been free from psychotropic drugs for at least 2 weeks prior to the collection of CSF, except for chloral hydrate. Each participant had given written informed consent. Lumbar punctures were performed in the lateral decubitus position around 8:00 AM on 2 consecutive days. Patients fasted and remained in bed overnight prior to the procedures. Subjects were given a total of 100 mg/kg of probenecid in six oral doses of 12.5 mg/kg at 18, 16, 14, 12, 10, and 8 hr, and a final oral dose of 25 mg/kg, 3 hr before the second lumbar puncture. Nausea and, less often, vomiting were encountered in many patients. Twenty-four milliliters of CSF were collected in 6-ml aliquots on each day, and the fifth to tenth aliquot was used for analysis. Immediately after the collection of CSF, 1 ~tmol/ml ascorbic acid was added, and the samples were frozen and stored at - 7 0 ° until analyzed. The concentrav'.ons of HVA, MHPG, 5-HIAA, and probenecid were determined by selected ion monitoring with combined gas d~omatography/mass spectrometry as described previously (FauU et al. 1978, 1979). Spearman corzelation coefficients, Chi-square tests with Yate's correction for conti-

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Table 1. Mean (4- sD) CSF Concentrations (ng/ml) of Monoamine Metabofites Before and After Probenecid Administration Pre-probenecid

HVA 5-HIAA MHPG

Post-pm~necid

Low group (n -- 30)

High group (n --- 29)

Low group (n -- 30)

High group (n = 29)

32.7 - 12.8 23.6 - 11.6 7.6 - 2.1

32.0 _+ 16.0 20.5 - 9.6 8.1 .4- 3.9

180.1 - 86.2 96.8 - 42.8 10.1 -+ 3.2

195.9 _ 77,5 124.3 _ 72,3 10.5 -+ 4.7

nuity, and Fisher's Z-transformations were calculated to test our hypotheses. Spearman rather than Pearson correlation coefficients were estimated to minimize the effects of any outlying points, and because this approach is more appropriate for distributions created by a median split of the data. Two-tailed p-values are reported for the homogeneity of correlations test. In all cases, p-values greater than 0.05 were considered nonsignificant.

Results The patients were divided into two groups, based on a median CSF probenecid level of 15 ttg/ml. This was done to avoid spurious correlations among the metabolites due to differing concentrations of probenecid and differing degrees of probenecid-induced metabolite elevation. At levels above 15 ttg/ml, we reasoned that acid transport blockade would be essentially complete (see Discussion). The high probenecid group included 29 patients, with a mean probenecid concentration of 21.9 I~g/ml -+ 7.7 ttg/ml (SD). The low probenecid group consisted of 30 patients, with a mean probenecid coticenwation of 11.1 I~g/ml + 3.4 ttg/ml (SD). The mean concentrations of HVA, 5-HIAA, and MHPG found for each of the groups are given in Table 1. The categorization of patients by diagnosis in the high and low probenecid groups is summarized in Table 2. There was no diagnostic group difference between the high and low probenecid groups. Further, age did not discriminate the high [44 years _+ 12 years (SD)] and low [39 years 4- 13 years (SD)] probenecid groups. The relationships among HVA, 5-HIAA, and MHPG concentrations in the two groups of patients before and after the drug was administered are shown in Figure 1. In the high probenecid group, positive correlations among HVA, 5-HIAA, and MHPG were found before and after drug administration. The low probenecid group showed a ~ignificant positive correlation between HVA and MHPG prior to administration of probenecid,

Table 2. Distribution of Subjects by CSF Probenecid Group and Diagnosis

High CSF Probenecid Low CSF Probenecid Total X2 = 2.058, v -- l , p = 0.15.

Schizophrenia and schizoaffective disorder

Major depression

Total

11 18 29

18 12 30

29 30 59

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PRE-PROBENECID

All Subjects

Low Probenecid Group (n-30)

(n-59)

5-HIAA

MIIPG

5-tlIAA

+.38""

,

MHPG

High Probenecid Gr6up (n=29)

MHPG

5-1tlA A

+.16

+.55""

POST-PROBENECID

HVA

5-HI A A

HVA

MitPG

+.47""

5-ltl AA

*.50""

HVA

MHPG

5-HI AA

÷.43"

MHPG

Figure 1. Intercorrelations (Spearman) among monoamine metabolite concentrations before and after probenecid administration. Key: *p < 0.05; **p < 0.01; ***p < 0.001; ****p = 0.0001.

whereas HVA-5-HIAA and MHPG-5-HIAA correlations were positive but not significant. Following probenecid treatment, significant positive HVA-5-HIAA and MHPG-5-HL~A correlations were found, :rod the HVA-MHPG correlation, although positive, was no longer statistically significant. The post-probenecid interrelationships among HVA, 5HIAA, and MHPG in the high probenecid group are shown in Figure 2. t41-1PG L3, 23.40

Ti

16.57

•

-.

i

),

9.73 358

2.90

4O

339

HVA

64

Figure 2. Interrelationships of CSF HVA, 5-HIAA, and MHPG concentrations (ng/ml) following probenecid administration in the high pmbenecid group (n = 29).

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Table 3. Correlation Coefficients (Spearman) of Post-Probenecid Monoamine Metabolite Concentrations with Age and CSF Probenecid Concentrations HVA

5 -HIAA

MHPG

High CSF Probenecid group (n = 29)

Age Probenecid level

- 0.008 - 0.19

- O. 11 - 0.09

+ 0.007 - O. 16

Low CSF Probenecid group

Age Probenecid level

-0.19 +0.28

+0.23 +0.63*

-0.21 + 0 . 4 8 ~'

(n = 30) °p = 0.0002; bp = 0.007.

Monoamine metabolite intercorrelations in the high probenecid group were compared with those found for the low group using a test for homogeneity procedure. Prior to probenecid administration, the HVA-5-HIAA correlation was significantly greater (z = 2.54; p = 0.011) in the high probenecid group, whereas the HVA-MHPG and MHPG5-HIAA correlations did not differ between the two groups. After probenecid administration there were no significant differences between high and low groups. Table 3 shows the relationships between post-probenecid metabolite concentrations and age or CSF probenecid concentration in both probenecid groups. In the high probenecid group, no metabolite was significantly correlated with either of these variables. In the low probenecid group, MHPG and 5-HIAA showed significant positive correlations with probenecid level, whereas HVA did not. No metabolite was colrelated with age.

Discussion We predicted that, if the positive correlation between CSF HVA and 5-HIAA concentrations were largely a function of the acid transport mechanism, it would be abolished by transport blockade. Examination of the high probenecid group suggests that fltis is not the case. Direct correlations between HVb. and 5-HIAA, between HVA end MHPG, and between 5-HIAA and MHPG remained significant even in the presence of CSF probenecid concentrations sufficient to increase HVA and 5-HIAA levels four- to sixfold. Several years ago, Bowers (1972) found a similar correlation between CSF 5-HIAA and HVA, assayed fluorometrically, following probenecid administration. Our findings support this important early work, and strongly suggest that the acid transport system does not account for correlations among any of the major monoamine metabolites in CSF. In this study, we assumed that the degree of inhibition of the acid transport system was greater in the high probenecid group that in the low probenecid group. However, CSF probenecid concentrations at which the acid transport system is completely blocked in humans remain unknown. CSF sampled from individual canine subjects at the cisterna magna following various probenecid doses showed a plateau in accumulation of acid metabolites at around 15 pLg,/ml(Faull et al. 1982). In humans, the situation is less clear due to the fact that no data are available from individuals following a variety of probenecid doses. Acress-subject data is co,.ffounded by variance in pre-pro~necid membolite concentrations. Cowdry et al. (1983), after reviewing the literature and reporting data of &eir ewn, suggested that patients with primary affective disorders show a maximal probenecid response at a lumbar CSF level of 15-20 Ixg/ml, whereas schizophrenic and schizoaffective patients do not.

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Another approach to determining the threshold for complete acid transport blockade in humans is to test for the presence or absence of CSF probenecid/metabolite correlations. Following complete blockade, no correlation should exist between CSF probenecid concentrations and the concentrations of a probenecid-sensitive metabolite. Fatql et al. (1981) and Ebert et al. (1980) have both reported significant direct correlations of probenecid with HVA and 5-l-l~,A in lumbar CSF of primary affective disorder patients, even after including patients whose probenecid concentrations exceeded 20 ttg/ml. However, these correlations might have been produced by the majority of patients who had lower CSF probenecid levels. In our low probenecid subjects, probenecid concentrations did correlate significantly with 5-HIAA, but not HVA concentrations (see Table 3). In our high probenecid subjects, there were no probenecid/metabolite correlations at all, a result consistent with complete acid transport blockade. The data also indicated that pre-probenecid metabolite intercorrelations were stronger in those patients who later had higher CSF probenecid concentrations (i.e., the high probenecid group). There is no immediate explanation for this finding. Diagnosis, age, or age/metabolite correlations (see Table 3) did not distinguish the high and low probenecid groups. Further, there were no correlations between CSF probenecid concentrations and pre-probenecid HVA (rs = -0.19, p NS), 5-HIAA (rs = -0.09, p NS), or MHPG (r~ = -0.16, p NS) concentrations. Therefore, it seems likely that this unexpected finding was either spurious, or based on some unknown neurochemical factor that would distinguish the two groups. The origin of monoamine metabolite intercorrelations in human lumbar CSF remains the subject of ongoing discussion (Scheinin 1985; Hsaio et al. 1987; Risby et al. 1987; Agren et al. 1986). Functional interactions among the CNS monoamine systems are highly likely, based on a variety of arguments. The monoamines share both synthetic and degradative enzymes (Kopin 1985), and as Agren et al. (1986) point out, metabolite concentrations reflect turnover rather than function. Anatomical interconnections among the systems in animals provide a basis for the modulation of one monoamine by another (Hsaio et al. 1987; Risby et al. 1987; Agren et al. 1986). Several studies have attempted to deduce the nature of functional interactions among the monoamine neurotransmitters in humans from CSF correlation data, such as we have presented here (Risby et al. 1987; Agren et al. 1986). However, the statistical arguments presented are based on path analysis, the theoretical limits of which are easily and frequently violated (Freedman 1987). Systems analysis has also been used to investigat~ lumbar CSF metabolite intercorrelations in canines (Faull et al. 1984). Interestingly, the pattern of intercorrelations among these metabolites may differ in lumbar and cervical CSF. Greene and Faull (unpublished results) have found that HVA and 5-HIAA, but not HVA and MHPG or 5HIAA and MHPG, are correlated in cervical CSF drawn from drug-naive squirrel monkeys. The present study should direct further attention to the study of functional interactions among the monoamine neurotransmitters. In our opinion, these results suggest that active transport cannot account for intercorrelationsobserved among monoamine metabolites in human lumbar CSF.

The authors thank Pamela J. Elliott for manuscript preparation and editorial advice.

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