Schi-_ophrenia Research, 6 (1992) 3 l-39 r> 1992 Elsevier Science Publishers B.V. All rights reserved

SCHIZO

31 0920.9964/92/$05.00

00189

CSF chromogranin Assessment

A-like immunoreactivity of clinical and biochemical

in schizophrenia relationships

Daniel P. van Kammen’, Jeffrey Peters’, Jeffrey Yao’, Thomas Neylan’, Edward Pontius’ and Daniel T. O’Connor2 1Highland Drive DVAMC,

Pittsburgh,

(Received

PA, and Western Psychiatric Institutc~ and Clinic. Pitt.&qh, Universit?; cf Cal@nia, San Diego, CA, U.S.A.

13 November

1990, revised

received 4 March

1991, accepted

Mark

PA, U.S.A.,

IO March

Beuger’, and 2D VAMC and

1991)

Chromogranin A (CgA) is co-released with catecholamines and peptides and has a wide distribution in the brain. Chromogranin A provides a measure of tonic arousal. CSF CgA-like immunoreactivity (CgA-LI) was studied in 42 drug-free male schizophrenic patients. 33 of these patients were first studied during chronic haloperidol maintenance treatment. Withdrawal from haloperidol maintenance treatment was associated with a significant increase in CSF CgA-LI, particularly in the patients who did not relapse. Contrary to expectation CSF CgA-LI was higher in drug-free patients who slept longer the night before the lumbar puncture. Significant relationships were observed between CSF CgA-LI and CSF homovanillic acid, acetylcholinesterase, neuropeptide Y-L1 and 5-hydroxs -indole acetic acid, but not with CSF norepinephrine or 3-methoxy-4-hydroxyphenylglycol. Ventricular brain ratios correlated negatively with CSF CgA-LI levels. Key words: Chromogranin

A-LI;

Haloperidol

withdrawal;

Relapse;

INTRODUCTION

Chromogranin A (CgA) (Smith and Kirshner, 1967; Smith and Winkler, 1967) is the major soluble protein co-stored and co-released with catecholamines from storage vesicles, particularly in the adrenal medulla. Human CgA is a 49,918 Da, acidic (~1: 4.5774.68; 31.5% (w/w) glutamic acid), monomeric protein (O’Connor and Frigon, 1984; Helman et al., 1988). This peptide has recently been identified in a variety of human endocrine (O’Connor and Deftos, 1986) and immune tissues (Angeletti and Hickey, 1985) and in the cerebrospinal fluid (CSF) (O’Connor et al., unpublished data). CgA may be co-released with

Correspondence to: D.P. van Kammen, Highland Drive DVAMC, Pittsburgh, PA 15206, U.S.A. (Tel: 412-3654730; Fax: 412-3654209).

Cerebral

spinal fluid; Catecholamine:

(Schizophrenia)

catecholamines and several peptides in the brain. Chromagranin A-like immunoreactivity (CgA-LI) can be used as a marker of a subset of monoaminergic neurons and may reflect exocytotic release of neurotransmitters or neurohormones. CgA has been associated with regulation of sympathetic arousal (O’Connor and Bernstein, 1984). This is of interest to the study of the biology of schizophrenia in which disturbed arousal (Zahn et al., 1987) and immune regulation (McAllister et al., 1989) have been reported. These reports suggested to us that CSF CgA-LI would be related to several clinical and biochemical measures purportedly disturbed in schizophrenia. Several groups have reported that CSF homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5-HIAA) levels were decreased in schizophrenic patients with so called brain atrophy, i.e., increased ventricular brain ratios (VBRs) and global cortical atrophy (Nyback et al., 1983; Potkin et al., 1983;

32

Van Kammen et al., 1983, 1986; Losonszky et al., 1987). This decrease in CSF monoamine metabolites may reflect presumably decreased release of dopamine and serotonin. We hypothesized that CgA-LI would be negatively correlated with brain atrophy indexes in our drug-free schizophrenic patients. In other words, an increase in atrophy would result in less CgA-LI released. Chronic haloperidol treatment is associated with a decreased firing rate of locus coeruleus cells (Dinan and Aston-Jones, 1985) and DA neurons (Grace and Bunney, 19X0), while haloperidol withdrawal is associated with increases in HVA; we therefore also hypothesized that withdrawal from chronic haloperidol treatment should be associated with increases in CSF CgA-LT.

METHODS Subjects 42 physically healthy, male schizophrenic patients, diagnosed according to DSM-IIIR guidelines were admitted consecutively to the Schizophrenia Research Unit at the Highland Drive VA Medical Center, Pittsburgh, PA. They participated in this study after having signed informed consent forms. Mean ( f SD) age was 34 k 7.6 years (range 20-50 years). We obtained ratings of premorbid functioning with the Premorbid Adjustment Scale (PAS) (Cannon-Spoor et al., 1982). Table 1 displays the clinical and demographic variables on these patients including age, age of onset, duration of illness, height and weight. All patients were on a low-monoamine, alcohol-free, and caffeine restricted diet. 37 of the drug-free patients had been placed on placebo for 2-6 weeks after having been withdrawn from haloperidol maintenance treatment (van Kammen et al., 1990). 12 patients met relapse criteria which consisted of an increase of at least 3 points on the Bunney Hamburg global psychosis score (Bunney and Hamburg, 1963) for 3 days compared with the mean of the daily psychosis ratings of the last week on haloperidol treatment (van Kammen et al., 1989, 1990). 23 had not relapsed at the time of the LP; relapse status could not be determined in two others. The other drug-free patients were drug-naive (n = 4) or had been drug-free for a month before admission (n= 1).

Behavioral ratings Symptom severity was measured daily by the nursing staff with the global psychosis item of the Bunney-Hamburg rating scale (Bunney and Hamburg, 1963). The Brief Psychiatric Rating Scale (BPRS) (Overall et al., 1967) the Scale for Negative Symptoms (SANS) ratings (Andreasen, 1982) and weight were obtained weekly. We used the ratings closest to the time of the LP, at the most 3 or 4 days before or after the LP, and on the same medication status as the LP. The daily psychosis ratings included in the analysis were taken on the three consecutive days before the LP. Similarly, hours of sleep during the night were collected by the well trained nursing staff for the three consecutive nights before the LP. Spinal,fluid Spinal fluid was obtained according to previously described standard methodology (van Kammen and Sternberg, 1980). All patients were at least 2 weeks drug-free (36k 14.1 days, range 14-67 days. excluding the four drug-naive patients). After overnight bed rest, lumbar punctures (LPs) were performed with the patient lying in bed in the lateral decubitus position between 8:00 and 8:30 a.m. Subjects had been without food or drinks, except water, since IO:30 p.m. the night before the LP. All LPs were nontraumatic, and the CSF was free of blood, as determined by cell counts. CSF was collected on ice in two 12 ml aliquots (pool A and B) which were then divided into aliquots of 0.5 and 1 ml and stored at ~ 80°C until assay. CSF CgA-LI was determined in the B pool using a double (polyclonal) antibody radioimmunoassay assay with polyethylene glycol (PEG) separation of bound from free (O’Connor et al., 1989). Because there was a variable storage time until assay, we correlated CSF CgA-LI values with storage time (r = - 0.06, n = 75. p = NS), which assured us that storage time did not affect the levels of CgALI. Duplicate determinations of CgA-LI were done on 0.1 ml aliquots of CSF. The intra-assay coefficient of variation was 449% (O’Connor et al., 1989). The antibody does not cross-react with related peptides or any other known peptide. We included in our analysis other CSF monoamine and peptide data available on these patients such as CSF NE, MHPG, HVA, 5-HIAA obtained with high pressure liquid chromatography (HPLC) (van

33 TABLE

1

Descriprives

Mean

Vuriable

Age Onset Duration Cspoortavg Psychosis Psychosis Sleep 1 Sleep 3 Daysdflp LP weight LP height

of demographics: 011 drug:free

1 3

SD

caws correlations with CgA-LI and demographics Min

Max

Haloperidol CgA-LI Correlations

Drug .free CgA-LI Correlations n

r

P

n

r

P

34.12 22.74 Il.36 2.19

7.63 4.78 7.34 1.10

20 13 0 0.28

50 35 30 5.00

42 42 42 41

- 0.20 - 0.09 -0.17 0.07

NS NS NS NS

* * * *

* + * *

* * * *

6.86 6.92 6.21 6.07 36.45

3.02 2.94 1.63 I .41 14. I I

1 1.70 0 0.33 14

12 13.30 8.00 8.00 67

42 42 41 41 38

-0.0001 - 0.08 0.30 0.34 0.09

NS NS 0.062 0.029 NS

32 33 32 32 *

-0.04 0.03 0.21 0.06 *

NS NS NS NS *

81.76 175.46

16.34 6.11

58.70 165

128.50 188

42 41

- 0.03 - 0.02

NS NS

33 32

-0.19 0.04

NS NS

Pearson correlation coefficient, two-tailed significance Key: Age = age at the time of the lumbar puncture; Onset = age of patient when he experienced his first psychotic episode; Duration = years from onset until date of study participation; Cspoortavg = Premorbid Assessment Scale total average score; Psychosis 1= patients’ psychosis rating from the day prior to CSF collection; Psychosis 3 = mean of patient’s psychosis rating across the 3 days prior to CSF collection; Sleep I = hours of sleep the night before CSF collection; Sleep 3 = means hours slept per night across the 3 nights prior to CSF collection; Daysdflp= the number of days drug-free prior to CSF collection; LP weight = patient’s weight (kg) at the time of the lumbar puncture; LP height = patient’s height (cm) at the time of the lumbar puncture.

Kammen et al., 1989) neuropeptide Y-like immunoreactivity (NPY-LI) (Peters et al., 1990), acetylcholinesterase (AChE) and butylcholinesterase (BChE) (Huff et al., 1988). The CT scans without contrast were performed on a GE CT/T 8800 scanner. Brain slices 10 mm thick with additional 5 mm slices through the lateral ventricles, at 15% to the canthomeatal line, were obtained. After inspection for gross abnormalities, the cortical or prefrontal atrophy, ventricular brain ratio (VBR) and maximum width of the third ventricle were measured. The description of the methodology has been published elsewhere (Rieder et al., 1983; Houston et al., 1984; van Kammen et al.. 1986). Two independent raters achieved a high interrater reliability for global sulcal widening (GSW) (ice = 0.96) VBR (ice= 0.96) and third ventricle width (ice =0.79) (van Kammen et al., 1988) and cerebellar atrophy scored on a 2 point scale (K= 1.O). The scans were obtained during the patients hospitalization on the

unit. Table 2 shows the means k SD of the CT scan measures. VBRs of 8.3% or more, cortical atrophy scores of 0.75 or more, and the presence of cerebellar atrophy were considered abnormal (van Kammen et al., 1983). In the 42 drug-free patients 25 patients had GSW scores 20.5 (two could not be measured), ten had VBR> 8.2%, and five showed cerebellar atrophy. 12 patients had no evidence of brain atrophy. Huloperidol treatment 33 of the 42 participating

patients received the two lumbar punctures before and after haloperidol withdrawal. The mean dose of haloperidol for these 33 patients was 13 + 8.9 mg/day (range 2-40 mg/day). There were no significant differences between the relapsers (n = 12) and nonrelapsers (n= 19) during haloperidol treatment in the psychosis subscales of the Brief Psychiatric Rating Scale (BPRS) except in the paranoid disturbance subscale. Hours of sleep for one night or across

CgA-LI HVA SHIAA MHPG NE NPY ACHE BCHE Pearson

30.9 1 I104.82

56.30 174.83 118.24 44.49 0.59 154.65 23.65 6.76 correlation

41.53 16.91 0.44 24.9 1 7.32 2.35 coefficients,

20.2 62.0 51.0 12.0 0.0200 110.40 12.740 4.00

two-tailed

155.1 630.0 216.0 73.0 2.057 201.60 40.8 IO 15.260

42 38 38 34 38 32 27 29

0.67 0.31 -0.25 0.05 0.54 0.58 0.43

0.000 1 0.056 NS NS 0.002 0.002 0.02 I

29 30 27 31 26 25 25

0.25 -0.09 0.23 0.24 ~ 0.62 - 0.55 - 0.42

NS NS NS NS 0.00 I 0.004 0.036

sgnificance.

Key:

CgA-LI = CSF chromogranin A-like immunoreactivity: HVA = CSF homovanillic acid; SHIAA = CSF 5-hydroxyindole acetic acid; MHPG = CSF 3-methoxy-4-hydroxyphenylglycol: NE = CSF norepinephrine; NPY = CSF neuropeptide-y-like immunoreactivity; ACHE = CSF acetylcholine esterase.

three nights before the LP, dose of haloperidol, or CT scan measures (van Kammen et al., 1989) were also not significantly different between groups. Pearson correlation coefficients were obtained to determine whether drug-free CSF CgA-LI levels (n=44) were related to clinical descriptors (positive), CT scan measures (negative), and symptom severity (positive). Group means k SD were compared by Student’s t tests. Haloperidol treated versus drug-free values were assessed with paired t tests in the relapsers (n= 12). nonrelapsers (tl= 19) and in all patients (n = 33); relapse and nonrelapse were defined according to relapse criteria met within 6 weeks of placebo replacement. All tests were two-tailed.

RESULTS Drug:free

condition

Demographics.

CSF CgA-LI did not correlate significantly with age (Y= - 0.23, p = NS), duration of illness (Y= - 0.17, p = NS), premorbid functioning (PAS: total score, r = 0.07, p= NS), psychosis (r = - 0.08, p= NS), number of days drug-free (r=O.O9, p=NS), weight (r= -0.03, p= NS) or

height (r = - 0.02, p = NS). CSF CgA-LI correlated with the duration of sleep of the three nights before the LP (r= 0.34, p=O.O29) and the sleep the night before the LP (r = 0.30, p = 0.06). Behavioral ratings. CSF CgA-LI did not correlate significantly with the total BPRS score (r = 0.16, p = NS), the psychosis subscale (r = 0.17, p = NS), the paranoia subscale (r = - 0.01, p = NS). or Bunney-Hamburg global psychosis ratings (r = 0.04, p= NS). No significant correlations were observed with the SANS (total score or subscales) and CgA-LI. CT scan measures. CSF CgA-LI correlated significantly with VBR (r= -0.39, n=42, p=O.Ol) but not with the other CT measures. CSF CgALI correlated significantly with VBR in the patients with normal (r= -0.61, n= 12, ~~0.04) and abnormal CT scans (r= -0.41, n= 30, p