Increased Nitric Oxide Radicals in Postmortem

Brain From Patients With Schizophrenia

by Jeffrey K. Yao, Sherry Leonard, and Ravinder D. Reddy

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Abstract consumption, lipid content, and transition metals are at
particular risk (Smith 1992). Neuronal membranes are
Alterations in antioxidant status in schizophrenia sug- therefore vulnerable to radical-mediated damage.
gest free radical-mediated neurotoxicity; this finding Complex antioxidant defense systems exist to protect
can be a consequence of increased free radical produc- against oxidative stress. In schizophrenia, evidence for the
tion. There are multiple pathways to excess free radi- dysregulation of free radical metabolism includes abnor-
cal generation and subsequent oxidative stress. One mal activities of critical antioxidant enzymes (Abdalla et
such pathway is the formation of peroxynitrite by a al. 1986; Reddy et al. 1991; Yao et al. 1998a, 1999);
reaction of nitric oxide (NO) and superoxide radical. reduced levels of antioxidants (Suboticanec et al. 1990;
NO is formed from L-arginine by nitric oxide synthase McCreadie et al. 1995; Brown et al. 1998; Yao et al.
(NOS). A constitutive cytosolic isoform, neuronal NOS 19986, 1998c; Yao et al. 2000); and other indices of lipid
(nNOS), appears to be fairly stable in the postmortem peroxidation in plasma, red blood cells, and cerebrospinal
brain tissues. Utilizing a sensitive fluorometrie assay, fluid (Prilipko 1992; Peet et al. 1993; McCreadie et al.
NO levels were measured by its stable metabolites, 1995; Mahadik et al. 1998). Such abnormalities have been
nitrate and nitrite, in the caudate region of post- associated with tardive dyskinesia, negative symptoms,
mortem brain tissues from patients and control sub- neurological signs, poor premorbid function, and com-
jects. In the human brain, NO is metabolized primar- puted tomography scan abnormalities (Reddy and Yao
ily in the form of nitrate. A significantly increased level 1999). Studies to date have generally been exploratory.
of NO was found in schizophrenia patients (241 ± 146 Further elucidation of the role of free radicals and antioxi-
pmol/mg dry weight, n = 18) than was found in those dants in schizophrenia and its treatment will require sys-
of normal (142 ± 65 pmol/mg dry weight, n = 20) and tematic investigation.
psychiatric controls without schizophrenia (125 ± 83 Biological systems have evolved complex protective
pmol/mg dry weight, n = 16) (analysis of covariance strategies against free radical toxicity. Under physiologi-
[ANCOVA], F = 6.446, df= 2,51,p = 0.003). These find- cal conditions, the potential for free radical-mediated
ings were independent of age, brain weight, post- damage is kept in check by the antioxidant defense sys-
mortem interval (PMI), sample storage time, or ciga- tem, which comprises several enzymatic and nonenzy-
rette smoking. Elevated NO levels in the brains of matic components. The critical antioxidant enzymes
schizophrenia patients lend further support for the include superoxide dismutase, catalase, and glutathione
free radical pathology in schizophrenia. peroxidase (GSH-Px). These enzymes act cooperatively at
Keywords: Nitric oxide, caudate, postmortem different sites in the metabolic pathway of free radicals.
changes, schizophrenia. Superoxide dismutase catalyzes the conversion of super-
Schizophrenia Bulletin, 30(4): 923-934, 2004. oxide radicals to hydrogen peroxide. Catalase and glu-
tathione peroxidase convert hydrogen peroxide to water.
Evidence suggests that free radicals are involved in mem- GSH-Px uses glutathione (GSH) to yield the oxidized
brane pathology and may play a role in schizophrenia form of glutathione, which is converted back to GSH by
(Cadet and Lohr 1987; Mahadik and Mukherjee 1996; glutathione reductase. Hydrogen peroxide is susceptible
Smythies 1997; Reddy and Yao 1999; Yao et al. 2001Z?).
Free radicals are reactive chemical species generated dur-
Send reprint requests to Dr. J.K. Yao, VA Pittsburgh Healthcare
ing normal metabolic processes and, in excess, can dam- System (Bldg. #13), 7180 Highland Drive, Pittsburgh, PA 15206; e-mail:
age lipids, proteins, and DNA. Regions of high oxygen jkyao@pitt.edu.

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Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

to autoxidation to form hydroxyl radicals, particularly in disorders including Alzheimer's disease (Norris et al.
the presence of metal catalysts such as iron. 1996; Thorns et al. 1998), multiple sclerosis (Heales et
In addition to superoxide and hydroxyl radicals, a al. 1999), and Parkinson's disease (Bockelmann et al.
pathway to excess free radical generation and subse- 1994; Hunot et al. 1996; Gerlach et al. 1999). We under-
quent oxidative stress is the formation of peroxynitrite took this investigation to determine whether increased
by a reaction of nitric oxide radical and superoxide radi- NO production is also present in schizophrenia. We mea-
cal (figure 1). NO can also produce hydroxyl radicals as sured NO in the caudate region of postmortem brain tis-
well as nitrogen dioxide radicals. NO is a free radical sues by its stable metabolites, nitrate and nitrite, using a

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because of its unpaired electron. Because NO radicals fluorometric assay.
cannot produce initiation or propagation reactions, they
do not generate free radical chain reactions. However,
NO is a unique second-messenger molecule that medi-
Methods
ates a number of cellular functions including neurotrans- Postmortem Brain Tissues. Tissues were collected at
mission, neurotoxicity and plasticity, vasodilation, regu- autopsy from several sources in the Denver area:
lation of blood flow, and inhibition of platelet University Hospital, Denver Veterans Administration
aggregation (Moncada et al. 1991; Bredt and Snyder Hospital, the coroner's offices of Arapahoe and Denver
1992; Ignarro et al. 1999; Krukoff 1999). Elevated NO counties, and the Mile High Tissue Bank. Complete med-
production has been linked to various neurodegenerative ical histories of the subjects, including their age, sex, race,

Figure 1. Production of nitric oxide radicals and decomposition of peroxynitrite pathways

NOS Arginase
L-Citrulline <-^ L-Arginine • Ornithine + Urea

NO SOD
H2O2

ONOO" ONOOH

Radical scavengers
•NO,
NO, Lipid Peroxidation
Note.— GSH = glutathione; GSSG = oxidized glutathione; H2O2 = hydrogen peroxide; NOS = nitric oxide synthase; NO = nitric oxide;
NO2~ = nitrite; O2«~ = reactive oxygen species; »OH = hydroxy radical; ONOO" = peroxynitrite anion; ONOOH = peroxynitrous acid;
SOD = superoxide dismutase.

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Increased Nitric Oxide Radicals in Postmortem Brain Schizophrenia Bulletin, Vol. 30, No. 4, 2004

cause of death, drug regimen, PMI, and detailed smoking Measurement of Nitrite and Nitrate Levels. Nitrite lev-
and drinking histories, were taken from hospital charts, els were determined using the same procedure described
physicians, and family members. All patients with schizo- above, except that nitrate reductase and enzyme cofactor
phrenia and other mental disorders (e.g., bipolar disorder, were omitted in the incubation mixture. Nitrite contents
depression, alcoholism, and drug addiction) were on were determined using a standard curve generated by a
antipsychotic medications at the time of death. At the time nitrite standard, whereas levels of nitrate were calculated
of autopsy, the brain was removed and hemisectioned in by subtraction of nitrite contents from the total NO pro-
the sagittal plane. One-half of the cerebrum, cerebellum, duced.

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and brainstem was immersed in 10 percent formalin and
fixed for 1 to 2 weeks for neuropathology evaluation. The Statistical Analyses. We conducted multiple regression
other hemisphere was sliced coronally and transported on analyses to determine whether the brain collection and
aluminum plates, previously chilled on ice, to the labora- storage variables (age, PMI, brain weight, and storage
tory for dissection. To avoid hemispheric bias, alternate time) were significantly associated with the biochemical
(left/right) hemispheres were collected. Immediately after measures of interest. Separate regression analyses were
sample collection, tissue was dissected from approxi- conducted for each of the biochemical measures. The
mately 50 areas in 1-gram pieces and frozen on dry ice brain collection and storage measures were entered into
snow. The remainder of the hemisphere was also frozen each of the regression analyses as predictor variables.
on slabs of dry ice and stored with the dissected parts at We examined the distributions for all variables with nor-
-80 °C. In the present study, caudate regions from schizo- mality plots and the Kolmogorov-Smirnov test (Siegel
phrenia and control groups were used for NO analyses. 1956), which quantifies the discrepancy between data
Tissues were shipped on dry ice by Federal Express to Dr. distribution and an ideal Gaussian distribution. We used
Yao's laboratory at the Highland Drive VA Pittsburgh the Dallal and Wilkinson approximation (Dallal and
Healthcare System, Pittsburgh, PA. Wilkinson 1986) to Lilliefors' test to compute the p val-
ues. The data pass the normality test if p > 0.10. For nor-
Tissue Preparation. Approximately 0.2 g of brain sam- mally distributed data, we compared means and vari-
ple was first lyophilized before the dry weight was taken. ances with / tests and F tests. Fisher's exact test was
Samples were then homogenized in 1.2 mL of 20 mM used to compare the frequency distributions of smoking
Tris-HCl (pH = 7.4). Following centrifugation for 10 min- and alcohol use between the schizophrenia and control
utes at 3,000 rpm, the cell-free homogenate was further groups.
deproteinized by addition of 100 JJLL of 5 percent We conducted one-way analysis of variance
metaphosphoric acid. The protein precipitates were (ANOVA) to evaluate group differences for brain collec-
removed by centrifugation for 10 minutes at 3,000 rpm. tion and storage parameters among the schizophrenia
The resulting protein-free supernatant was used to per- group and the two control groups: those with and with-
form the NO assays. out other mental disorders. We conducted analysis of
covariance (ANCOVA) to control statistically for the
potential effects of the brain collection and storage vari-
Determination of Nitric Oxide Levels. Because nitric
ables on the group differences for the biochemical mea-
oxide is rapidly converted to nitrate and nitrite in vivo, we
sures of interest. Group means for the NO measures
measured the sum of nitrate and nitrite (using
were compared using ANOVAs, with pairwise compar-
Fluorometric Nitric Oxide kits by Calbiochem of La Jolla,
isons (2-tailed unpaired t tests) following significant
CA) as an index of total NO produced. In brief, 20 (JLL of
findings.
each protein-free homogenate was added to a 96-well flu-
orometric detection plate. To each sample well we added
80 (xL of assay buffer, 10 (JLL of enzyme cofactors, and 10 Results
|xL of nitrate reductase. The plates were covered and incu-
bated at 24 °C for 2 hours. After incubation, 20 nL of 2,3- Brain Collection and Storage Parameters. We con-
diaminonaphthalene (DAN) reagent and 10 (xL of sodium ducted ANOVAs to evaluate group differences in brain
hydroxide were added to each well. Detection was per- collection and storage parameters (table 1). Significant
formed with a 1420 Victor2 multilabel counter using an differences between the schizophrenia and control sam-
excitation wavelength of 375 nm and an emission wave- ples were found in brain weights, PMI, and storage time.
length of 415 nm. We calculated NO levels using a stan- The distribution of PMI among three groups is shown
dard curve generated by a nitrate standard. The assay is in figure 2. Only three samples from the schizophrenia
linear to 500 pmol. The sensitivity of this assay is less group have a PMI exceeding 40 hours; the PMI of the
than 5 pmol. remaining samples are all within 30 hours.

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Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

Table 1. Brain collection and storage parameters (mean ± SD)

Schizophrenia Controls

Parameters patients Without MD With other MD

n 18 20 16

Age, yrs 55.4 ±15.3 55.7 ±14.5 47.3 ±13.3

Sex (male/female) 16/2 13/7 16/0

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Smoker (yes/no) 14/4 14/6 13/3
1
Postmortem interval (hrs) 24.2±12.6 14.4 ±7.5 16.4 ±6.6
2
Brain weight (g) 1410 ±169 1258 ±143 1410 ± 9 8
3
Storage time (mos) 39.7 ± 21.9 25.2 ±15.3 24.1 ± 1 2 . 2
Note.—SD = standard deviation; ANOVA = analysis of variance; MD = mental disorders.
1
Significantly higher than both control groups (ANOVA, F = 3.18, df= 2,51, p = 0.0058).
2
Significantly higher than control group without MD (ANOVA, F = 3.18, df = 2,51; p = 0.0016).
3
Significantly higher than both control groups (ANOVA, F = 3.18, d/=2,51, p = 0.0138).

Figure 2. Distribution of PMI between schizophrenia and control groups

••
•

40

30
• •"• • • T TT
• •• A
A*
20 •: ^
T

• A AA
• _
•
10 • A
T
T
T
•
• AA
A A
A
0
Schizophrenia Patients Controls Controls - MD

Groups
Note.—MD = mental disorders; PMI = postmortem interval.

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Increased Nitric Oxide Radicals in Postmortem Brain Schizophrenia Bulletin, Vol. 30, No. 4, 2004

The distributions for all variables were examined Quantitative Determination of NO Levels. To mea-
using normality plots and the Kolmogorov-Smirnov test. sure the distribution of NO metabolites, we measured
The data pass the normality test if p > 0.10. Results did both nitrate and nitrite levels. More than 90 percent of
not indicate that normality transformations of the data NO metabolites in the human brain are in the form of
were necessary. In addition, the frequency of smoking nitrates (figure 3).
was not significantly different between the schizophrenia The mean levels of NO in the caudate region of
and the control groups (Fisher's exact test). postmortem brain tissue were significantly higher
(ANCOVA, F = 6.446; df= 2,51; p = 0.003) in the schiz-

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Edema Measurement in Caudate Samples. Cerebral ophrenia group than in the control groups (table 3). To
edema is commonly seen in various pathological avoid the confounding effect of PMI, three samples with
processes in brain. To determine the water content in PMI greater than 30 hours in the schizophrenia group
caudate samples, we measured both the fresh and dry (figure 2) were omitted from the statistical analyses. The
weight of each sample and used the formula of Elliott mean levels of NO in the caudate region of postmortem
and Jasper (1949) to calculate the percentage of brain tissue were still significantly higher (ANCOVA,
swelling (edema) or shrinkage. Water levels in the post- F = 5.481; df= 2,48; p = 0.007) in schizophrenia group
mortem caudate were significantly higher in the schizo- than in the control groups (table 3 and figure 4).
phrenia group than in the control groups (table 2). Individual regression analyses were conducted to
Samples from the schizophrenia group showed an aver- determine whether age, PMI, brain weight, or storage
age of 15 percent swelling compared with the control time were significantly associated with NO measures.
samples. Therefore, we lyophilized our samples before None of these collection and storage parameters were sig-
measuring NO. nificantly associated with NO measures (table 4).

Table 2. Measurement of edema in brain caudate region (mean ± SD)

Groups Fresh weight(mg) Dry weight (mg) Dry weight (%) Edema1 (%
Controls
Without MD 159.7 ±32.2 35.4 ±9.9 22.3 ± 3.8
With MD 141.9 ±22.8 33.2 ±10.2 22.6 ±4.0
Schizophrenia patients 137.0 ±24.8 27.1 ±6.4 18.9±2.42 15.3
Note.—SD = standard deviation; MD = mental disorder.
1
The percentage of swelling (edema) or shrinkage was calculated using the formula of Elliott and Jasper (1949).
2
The dry weight of samples from schizophrenia subjects is significantly lower than that of control groups either with (p = 0.0033) or with-
out (p = 0.0024) other mental disorders.

Table 3. Comparison of nitric oxide levels in the caudate region of postmortem brain tissue between
schizophrenia patients and control groups
Controls
(pmol/mg dry wt) ANCOVA
Schizophrenia
patients Without MD With other MD
Methods dry wt) (mean ± SD) (mean ± SD) df
All samples 241 ±146 142 ±65 125 ±83 6.446 2,51 0.003
1
Samples with
PMI < 30 hrs 209 ± 80 142 ±65 125 ±83 5.481 2,48 0.007
Note.— ANCOVA = one-way analysis of variance with covariates including age, postmortem interval (PMI), brain weight, and storage
time; MD = mental disorders; SD = standard deviation.
1
Only three samples with PMI > 30 hours from the schizophrenia patients group were omitted from the comparison.

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Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

Figure 3. Distribution of nitric oxide metabolites in brain caudate region

100

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80

60-

40-

20

Nitric oxide Nitrite Nitrate Nitrate

(pmol) (pmol) (pmol)

Table 4. Correlations between nitric oxide levels and brain collection and storage parameters
r (Correlation Coefficient)
Groups n Age Brain weight PMI Storage time
Schizophrenia patients 18 -0.0041 0.1219 0.4486 -0.2744
Controls without MD 20 -0.0709 -0.1266 0.3011 -0.1320
Controls with other MD 16 -0.1413 0.2508 0.2107 -0.2374
Note.— MD = mental disorder; PMI = postmortem interval.

Effect of Cigarette Smoking on NO Levels. Cigarette Discussion

smoke contains many pro-oxidants. To test whether NO
levels were affected by cigarette smoking, NO measures Increased NO Production in Schizophrenia. The pre-
were correlated with the number of cigarettes smoked by sent study provides the first evidence of increased NO
each subject daily. Cigarette smoking data were available in production in the postmortem brain tissue of patients with
81.5 percent of total samples examined. No significant cor- schizophrenia (figure 4). NO is the product of a five-elec-
relations existed between NO levels and cigarette smoking tron oxidation of the amino acid L-arginine by NOS
in either total samples (r = 0.028, n = 44, p = 0.855) or the (Palmer et al. 1988). The three isoforms of the NOS fam-
schizophrenia group (r = 0.078, n - \5,p- 0.782). ily are neuronal, inducible, and endothelial (Forstermann

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Increased Nitric Oxide Radicals in Postmortem Brain Schizophrenia Bulletin, Vol. 30, No. 4, 2004

et al. 1991). In the human brain, nNOS is responsible for (Das et al. 1996). Such a reduction may be associated
the synthesis of NO (Blum-Degen et al. 1999). Major with an increased level of plasma asymmetrical dimethy-
metabolites of NO appear to be in the form of nitrate (fig- larginine, which is an inhibitor of NOS. These investiga-
ure 3). Therefore, nitrate reductase is necessary to assay tors suggest that this endogenous inhibitor of NOS may
NO levels in the brain tissues. provide a novel regulatory mechanism of NO synthesis in
Previously, Karson et al. (1996) demonstrated an brain. Taken together, the present finding further supports
increased concentration of NOS in the cerebellum region the theory that NO is involved in the free radical pathol-
of postmortem brain tissue from patients with schizophre- ogy of schizophrenia (see Yao et al. 200\b for review).

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nia. NOS activity was also found to be significantly
higher in platelets of drug-naive schizophrenia patients NO Measures Were Independent of Brain Collection
compared with normal controls, drug-treated schizophre- and Storage Parameters. Although significant differ-
nia patients, and panic disorder subjects (Das et al. 1995). ences in brain weight, PMI, and storage time were found
Recently, Herken et al. (2001) showed a significant among the schizophrenia group and two control groups
increase of erythrocyte NO levels in neuroleptic-treated (table 1), no significant correlations between NO mea-
patients with schizophrenia. In contrast, plasma levels of sures and brain collection and storage parameters were
nitrate were significantly lower in drug-naive first-episode present (table 4). Our findings are thus in accordance with
schizophrenia patients than in normal control subjects the recent report of Blum-Degen et al. (1999), who

Figure 4. Nitric oxide contents in the caudate region of postmortem brain tissue of schizophrenia and
control groups

NO, pmol/mg dwt

400

300

200

100

Controls Controls Schizophrenia

without MD with MD Patients

Note.—NO = nitric oxide; dwt = dry weight; MD = mental disroder.

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Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

demonstrated that NOS activity remains unchanged dur- production (Yao et al. 2001a). Thus, the reaction of NO
ing aging and is independent of PMI, gender, or sample with free thiols may compete with a substrate such as
storage time. GSH for decomposition of hydrogen peroxide by GSH
peroxidase (figure 1).
Regional Distribution of NO Levels in the Brain.
Nitric oxide is a highly diffusible cell-signaling mole- NO-Induced Brain Edema. Previously, Brown et al.
cule that is widely distributed in various regions of the (1986) showed that the brains of patients with schizo-
brain. Recently, Blum-Degen et al. (1999) found NOS phrenia were 6 percent lighter and had lateral ventricles

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activity in 28 regions of the human brain, with the high- that were 19 percent larger in the anterior. They sug-
est levels of activity in the cerebellar cortex, nucleus gested that such enlargement is associated with tissue
accumbens, substantia innominata, and subthalamic loss in the temporal lobe. The present study also demon-
nucleus. They also demonstrated the presence of argi- strated a decreased level of dry weight or increased water
nine and citrulline, precursor and product, respectively, content in caudate region in patients with schizophrenia
of the NOS reaction, in these same regions of the brain. compared with the control groups (table 2), suggesting
Although present data only show the increased NO pro- the presence of brain edema in schizophrenia.
duction in the caudate region of the postmortem brain Breakdown of the blood-brain barrier (BBB) is known to
from patients with schizophrenia, elevated NO may also lead to vasogenic edema and secondary brain damage.
exist in other regions of the brain. We are currently Free radicals have long been considered contributors to
exploring this possibility. the alteration of BBB permeability that underlies brain
edema (Demopoulous et al. 1972). Recent studies also
Possible Mechanisms Involving NO Production. In demonstrated that NO can increase the permeability of
addition to the nNOS in the brain, various immunomodu- the BBB that allows plasma constituents to enter the
lators such as lipopolysaccharide and interferon can brain (Schilling and Wahl 1997; Thiel and Audus 2001).
induce NO production through the inducible NOS Moreover, the association of brain edema with increased
(Lorsbach et al. 1993; Lowenstein et al. 1993). A recent NO production has been demonstrated in various experi-
study on effects of bilateral infusion of interleukin-6 (IL- mental models for brain injury including hyperthermic
6) in the rat hippocampus indicated that IL-6 could induce brain injury (Aim et al. 1998; Sharma et al. 2000), pneu-
NOS (Ma and Zhu 2000). Elevated levels of plasma IL-6 mococcal meningitis (Koedel et al. 1995), hyperammone-
have previously been reported in patients with schizo- mia (Larsen et al. 2001), and cold injury (Gotoh et al.
phrenia (Shintani et al. 1991; Ganguli et al. 1994; Maes et 1998; Nag et al. 2000, 2001). On the other hand, inhibit-
al. 1995; Lin et al. 1998; van Kammen et al. 1999). Thus, ing or deleting NOSs (Hara et al. 1996; Calapai et al.
increased NO production in the brains of schizophrenia 2000; Sharma et al. 2000; Takemori et al. 2000), free rad-
patients may be mediated through an IL-6 induction. ical scavengers (Petty et al. 1996), and antioxidants
Alternatively, NO levels can be regulated by the (Kawamata et al. 1997) could attenuate edema formation.
enzyme arginase, which is responsible for the hydrolysis Taken together, our present data further support the
of arginine to ornithine and urea. Reciprocal regulatory notion that NO plays a pivotal role in the pathogenesis of
mechanisms exist between NOS and arginase pathways. brain damage.
Increased arginase activity leads to substrate depletion
and thereby reduction of the NOS reaction (Corraliza et Effect of Cigarette Smoking on NO Production.
al. 1995), whereas inhibition of arginase by L-hydrox- Cigarette smoking has long been associated with an
yarginine (metabolite of NO pathway) may increase NO increased risk of cardiovascular and pulmonary diseases,
production (Daghigh et al. 1994). which may be modulated by endogenous NO (Kharitonov
et al. 1995a). Specifically, cigarette smoking reduces the
Interaction Between NO Radicals and Thiol levels of exhaled (Kanazawa et al. 1996), nasal (Olin et
Scavengers. Under physiological conditions, NO and its al. 1998), and serum (Node et al. 1997) NO but increases
metabolites react with various thiols (e.g., GSH) to form NO levels in breath condensate in healthy subjects (Balint
stable S-nitrosothiols (Kharitonov et al. 1995fc). These et al. 2001). In addition, the platelet-derived NO release is
thiol compounds play an important role in the suppression significantly impaired in long-term smokers, which leads
of NO functions in cellular membranes. Recently, Gegg et to an increase in platelet aggregability (Ichiki et al. 1996).
al. (2003) demonstrated an increase of GSH in astrocytes On the other hand, smoking cessation is associated with
exposed to NO. The present findings suggest that, to sup- an increased level of exhaled NO (Robbins et al. 1997).
press the NO radical function, the level of cytosolic thiol Thus, it appears that cigarette smoking may inhibit the
compounds may be elevated in response to increased NO enzyme NOS. In the present study, most samples were

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Increased Nitric Oxide Radicals in Postmortem Brain Schizophrenia Bulletin, Vol. 30, No. 4, 2004

obtained from subjects who smoked (table 1). No signifi- mitochondria after haloperidol treatment. Nitric Oxide,
cant correlation between NO measures and cigarette 3:235-243, 1999.
smoking was present. Therefore, the increased levels of Balint, B.; Donnelly, L.E.; Hanazawa, T ; Kharitonov,
caudate NO in the schizophrenia group appear to be inde- S.A.; and Barnes, P.J. Increased nitric oxide metabolites
pendent of cigarette smoking. in exhaled breath condensate after exposure to tobacco
smoke. Thorax, 56:456-461, 2001.
Effect of Antipsychotic Drugs on NO Production.
Blum-Degen, D.; Heinemann, T.; Lan, J.; Pedersen, V.;
Arnaiz et al. (1999) recently demonstrated that haloperidol
Leblhuber, F.; Paulus, W.; Riederer, P.; and Gerlach, M.

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inhibits NO production in submitochondrial particles from
Characterization and regional distribution of nitric oxide
mice, suggesting inhibited mitochondria] electron transfer
synthase in the human brain during normal ageing. Brain
with enhanced superoxide radical and hydrogen peroxide
Research, 834:128-135, 1999.
production. This finding is in accordance with that of
Rengasamy and Johns (1993), which showed an inhibition Bockelmann, R.; Wolf, G.; Ransmayr, G.; and Riederer, P.
of NO synthase by a superoxide generating system. NADPH-diaphorase/nitric oxide synthase containing neu-
Moreover, Prince et al. (1997) reported that cytochrome rons in normal and Parkinson's disease putamen. Journal
oxidase enzyme activity increased in specific regions of the of Neural Transmission, Parkinson's Disease and
rat brain following chronic neuroleptic treatment. In Dementia Section, 7:115-121, 1994.
humans, platelet NOS was found to be significantly higher Bredt, D.S., and Snyder, S.H. Nitric oxide, a novel neu-
in drug-naive schizophrenia patients than in normal con- ronal messenger. Neuron, 8:3-11, 1992.
trols. Treatment with chlorpromazine, haloperidol, or cloza- Brown, K.; Reid, A.; White, T; Henderson, T; Hukin, S.;
pine appeared to normalize platelet NOS levels in patients Johnstone, C ; and Glen, A. Vitamin E, lipids, lipid perox-
with schizophrenia (Das et al. 1995). In the present study, idation products and tardive dyskinesia. Biological
subjects with schizophrenia and those with other mental Psychiatry, 43:863-867, 1998.
disorders were taking antipsychotic medications at the time
of death. However, no significant differences were detected Brown, R.; Colter, N.; Corsellis, J.A.; Crow, T.J.; Frith,
in caudate NO levels between control subjects with and C D . ; Jagoe, R.; Johnstone, E.C.; and Marsh, L.
without other mental disorders. Taken together, antipsy- Postmortem evidence of structural brain changes in schiz-
chotic medications are unlikely to contribute significantly ophrenia. Differences in brain weight, temporal horn area,
to increased NO production in the caudate region of the and parahippocampal gyms compared with affective dis-
brains of schizophrenia patients. order. Archives of General Psychiatry, 43:36—42, 1986.
Cadet, J.L., and Lohr, J.B. Free radicals and the develop-
Therapeutic Treatment. If NO does play a role in schiz- mental pathology of schizophrenic burnout. Integrative
ophrenia, pursuing interventions against NO toxicity may Psychiatry, 5:40-48, 1987.
have merit. Deutsch et al. (1997) showed a modest but Calapai, G.; Marciano, M.C.; Corica, F.; Allegra, A.;
statistically significant decrease in the severity of psy- Parisi, A.; Frisina, N.; Caputi, A.P.; and Buemi, M.
chopathology after patients were treated with methylene Erythropoietin protects against brain ischemic injury by
blue (MB). Further studies are needed to illustrate thera- inhibition of nitric oxide formation. European Journal of
peutic efficacy of MB or other NOS inhibitors. Pharmacology, 401:349-356, 2000.
Corraliza, I.M.; Soler, G.; Eichmann, K.; and Modolell,
References M. Arginase induction by suppressors of nitric oxide syn-
thesis (IL-4, IL-10 and PGE2) in murine bone-marrow-
Abdalla, D.S.P.; Monteiro, H.P.; Oliviera, J.A.C.; and derived macrophages. Biochemical and Biophysical
Bechara, E.J. Activities of superoxide dismutase and glu- Research Communications, 206:667-673, 1995.
tathione peroxidase in schizophrenic and manic-depres- Daghigh, E; Fukuto, J.M.; and Ash, D.E. Inhibition of rat
sive patients. Clinical Chemistry, 32:805-807, 1986. liver arginase by an intermediate in NO biosynthesis, NG-
Aim, P.; Sharma, H.S.; Hedlund, S.; Sjoquist, P.O.; and hydroxy-L-arginine: Implications for the regulation of
Westman, J. Nitric oxide in pathophysiology of hyperther- nitric oxide biosynthesis by arginase. Biochemical and
mic brain injury: Influence of a new anti-oxidant com- Biophysical Research Communications, 202:174—180,
pound H-290/51; a pharmacological study using immuno- 1994.
histochemistry in the rat. Amino Acids, 14:95-103, 1998. Dallal, G.E., and Wilkinson, L. An analytic approximation
Arnaiz, S.L.; Coronel, M.F.; Boveris, A. Nitric oxide, to the distribution of Lilliefors's test statistic for normal-
superoxide, and hydrogen peroxide production in brain ity. The American Statistician, 40:294-296, 1986.

931
Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

Das, I.; Khan, N.S.; Puri, B.K.; and Hirsch, S.R. Elevated Heales, S.J.R.; Bolanos, J.P; Stewart, V.C.; Brookes, P.S.;
endogenous nitric oxide synthase inhibitor in schizo- Land, J.M.; and Clark, J.B. Nitric oxide, mitochondria
phrenic plasma may reflect abnormalities in brain nitric and neurological disease. Biochimica et Biophysica Ada,
oxide production. Neuroscience Letters, 215:209-211, 1410:215-228, 1999.
1996. Herken, H.; Uz, E.; Ozyurt, H.; and Akyol, O. Red blood
Das, I.; Khan, N.S.; Puri, B.K.; Sooranna, S.R.; de cell nitric oxide levels in patients with schizophrenia.
Belleroche, J.; and Hirsch, S.R. Elevated platelet calcium [Letter]. Schizophrenia Research, 52:289-290, 2001.
mobilization and nitric oxide synthase activity may reflect Hunot, S.; Boissiere, F.; Faucheux, B.; Brugg, B.; Mouatt-

Downloaded from https://academic.oup.com/schizophreniabulletin/article-abstract/30/4/923/1931278 by guest on 01 May 2019

abnormalities in schizophrenic brain. Biochemical and Prigent, A.; Agid, Y.; and Hirsch, E.C. Nitric oxide syn-
Biophysical Research Communications, 122:375-380, thase and neuronal vulnerability in Parkinson's disease.
1995. Neuroscience, 72:355-363, 1996.
Demopoulous, H.B. Molecular aspects of membrane Ichiki, K.; Ikeda, H.; Haramaki, N.; Ueno, T.; and
structure in cerebral edema. In: Reulen, H.J., ed. Steroids Imaizumi, T. Long-term smoking impairs platelet-derived
and Brain Edema. Berlin, Germany: Springer-Verlag, nitric oxide release. Circulation, 94:3109-3114, 1996.
1972. pp. 29-39. Ignarro, L.J.; Cirino, G.; Casini, A.; and Napoli, C. Nitric
Deutsch, S.I.; Rosse, R.B.; Schwartz, B.L.; Fay- oxide as a signaling molecule in the vascular system: An
McCarthy, M.; Rosenberg, P.B.; and Fearing, K. overview. Journal of Cardiovascular Pharmacology,
Methylene blue adjuvant therapy of schizophrenia. 34:879-886, 1999.
Clinical Neuropharmacology, 20:357-363, 1997. Kanazawa, H.; Shoji, S.; Hirata, K.; Kurthara, N.; and
Elliott, K.A.C., and Jasper, H. Measurement of experi- Yoshikawa, J. Role of endogenous nitric oxide in airflow
mentally induced brain swelling and shrinkage. American obstruction in smokers. Chest, 110:927-929, 1996.
Journal of Physiology, 157:122-129, 1949. Karson, C.N.; Griffin, W.S.; Mrak, R.E.; Husain, M.;
Forstermann, U.; Schmidt, H.H.; Pollock, J.S.; Sheng, H.; Dawson, T.M.; Snyder, S.H.; Moore, N.C.; and Sturner,
Mitchell, J.A.; Warner, T.D.; Nakane, M.; and Murad, F. W.Q. Nitric oxide synthase (NOS) in schizophrenia:
Isoforms of nitric oxide synthase: Characterization and Increases in cerebellar vermis. Molecular and Chemical
purification from different cell types. Biochemical Neuropathology, 27:275-284, 1996.
Pharmacology, 42:1849-1857, 1991. Kawamata, T.; Katayama, Y.; Maeda, T.; Mori, T.;
Ganguli, R.; Yang, Z.; Shurin, G.; Chengappa, K.N.; Brar, Aoyama, N.; Kikuchi, T.; and Uwahodo, Y. Antioxidant,
J.S.; Gubbi, A.V.; and Rabin, B.S. Serum interleukin-6 OPC-14117, attenuates edema formation and behavior
concentration in schizophrenia: Elevation associated with deficits following cortical contusion in rats. Ada
duration of illness. Psychiatry Research, 51:1-10, 1994. Neurochirurgica. Supplement, 70:191-193, 1997.
Gegg, M.E.; Beltran, B.; Salas-Pino, S.; Bolanos, J.P.; Kharitonov, S.A.; Robbins, R.A.; Yates, D.; Keatings, V.;
Clark, J.B.; Moncada, S.; and Heales, S.J.R. Differential and Barnes, P.J. Acute and chronic effects of cigarette
effect of nitric oxide on glutathione metabolism and mito- smoking on exhaled nitric oxide. American Journal of
chondrial function in astrocytes and neurons: Implications Respiratory and Critical Care Medicine, 152:609-612,
for neuroprotection/neurodegeneration? Journal of 1995a.
Neurochemistry, 86:228-237, 2003. Kharitonov, V.G.; Sundquist, A.R.; and Sharma,V.S.
Gerlach, M.; Blum-Degen, D.; Lan, J.; and Riederer, P. Kinetics of nitrosation of thiols by nitric oxide in the pres-
Nitric oxide in the pathogenesis of Parkinson's disease. ence of oxygen. Journal of Biological Chemistry,
Advanced Neurology, 80:239-245, 1999. 270:28158-28164, \995b.
Gotoh, K.; Kikuchi, H.; Kataoka, H.; Nagata, I.; Nozaki, Koedel, U.; Bernatowicz, A.; Paul, R.; Frei, K.; Fontana,
K.; Takahashi, J.C.; and Hazama, F. Nitric oxide synthase A.; and Pfister, H.W. Experimental pneumococcal menin-
immunoreactivity related to cold-induced brain edema. gitis: Cerebrovascular alterations, brain edema, and
Neurological Research, 20:637-642, 1998. meningeal inflammation are linked to the production of
nitric oxide. Annals of Neurology, 37:313-323, 1995.
Hara, H.; Huang, PL.; Panahian, N.; Fishman, M.C.; and
Moskowitz, M.A. Reduced brain edema and infarction Krukoff, T.L. Central actions of nitric oxide in regulation
volume in mice lacking the neuronal isoform of nitric of autonomic functions. Brain Research: Brain Research
oxide synthase after transient MCA occlusion. Journal of Reviews, 30:52-65, 1999.
Cerebral Blood Flow and Metabolism, 14:605-611, Larsen, F.S.; Gottstein, J.; and Blei, A.T. Cerebral hyper-
1996. emia and nitric oxide synthase in rats with ammonia-

932
Increased Nitric Oxide Radicals in Postmortem Brain Schizophrenia Bulletin, Vol. 30, No. 4, 2004

induced brain edema. Journal of Hepatology, 34:548-554, breakdown and repair after cold injury. Laboratory
2001. Investigation, 81:41-49,2001.
Lin, A.; Kenis, G.; Bignotti, S.; Tura, G.J.; De Jong, R.; Node, K.; Kitakaze, M.; Yoshikawa, H.; Kosaka, H.; and
Bosnians, E.; Pioli, R.; Altamura, C ; Scharpe, S.; and Hori, M. Reversible reduction in plasma concentration of
Maes, M. The inflammatory response system in treatment- nitric oxide induced by cigarette smoking in young adults.
resistant schizophrenia: Increased serum interleukin-6. American Journal of Cardiology, 79:1538-1541, 1997.
Schizophrenia Research, 32:9-15, 1998. Norris, P.J.; Faull, R.L.; and Emson, PC. Neuronal nitric
Lorsbach, R.B.; Murphy, W.J.; Lowenstein, C.J.; Snyder, oxide synthase (nNOS) mRNA expression and NADPH-

Downloaded from https://academic.oup.com/schizophreniabulletin/article-abstract/30/4/923/1931278 by guest on 01 May 2019

S.H.; and Russell, S.W. Expression of the nitric oxide diaphorase staining in the frontal cortex, visual cortex and
synthase gene in mouse macrophages activated for tumor hippocampus of control and Alzheimer's disease brains.
cell killing. Journal of Biological Chemistry, Molecular Brain Research, 41:36-49, 1996.
268:1908-1913, 1993. Olin, A.C.; Hellgren, J.; Karlsson, G.; Ljungkvist, G.;
Lowenstein, C.J.; Alley, E.W.; Raval, P.; Snowman, Nolkrantz, K.; and Toren, K. Nasal nitric oxide and its
A.M.; Snyder, S.H.; Russell, S.W.; and Murphy, W.J. relationship to nasal symptoms, smoking and nasal
Macrophage nitric oxide synthase gene: Two upstream nitrate. Rhinology, 36:117-121, 1998.
regions mediate induction by interferon and lipopolysac- Palmer, R.M.J.; Ashton, D.S.; and Moncada, S. Vascular
charide. Proceedings of the National Academy of endothelial cells synthesize nitric oxide from L-arginine.
Sciences of the United States of America, 90:9730-9734, Nature, 333:664-666, 1988.
1993.
Peet, M.; Laugharne, J.; Rangarajan, N.; and Reynolds,
Ma, T.C., and Zhu, X.Z. Effects of intrahippocampal infu- G.P. Tardive dyskinesia, lipid peroxidation, and sustained
sion of interleukin-6 on passive avoidance and nitrite and amelioration with vitamin E treatment. International
prostaglandin levels in the hippocampus in rats. Clinical Psychopharmacology, 8:151-153, 1993.
Arzneimittel-Forschung, 50:227-231, 2000.
Petty, M.A.; Poulet, P.; Haas, A.; Namer, I.J.; and Wagner,
Maes, M.; Bosmans, E.; Calabrese, J.; Smith, R.; and J. Reduction of traumatic brain injury-induced cerebral
Meltzer, H.Y. Interleukin-2 and interleukin-6 in schizo- oedema by a free radical scavenger. European Journal of
phrenia and mania: Effects of neuroleptics and mood sta- Pharmacology, 307:149-155, 1996.
bilizers. Journal of Psychiatric Research, 29:141-152,
Prilipko, L.L. The possible role of lipid peroxidation in
1995.
the pathophysiology of mental disorders. In: Packer, L.;
Mahadik, S.P., and Mukherjee, S. Free radical pathology Prilipko, L.; and Christen, Y, eds. Free Radicals in the
and antioxidant defense in schizophrenia: A review. Brain. Berlin, Germany: Springer-Verlag, 1992. pp.
Schizophrenia Research, 19:1-17, 1996. 146-152.
Mahadik, S.P.; Mukherjee, S.; Scheffer, R.E.; Correnti, Prince, J.A.; Yassin, M.S.; and Oreland, L. Neuroleptic-
E.E.; and Mahadik, J.S. Elevated plasma lipid peroxides induced mitochondrial enzyme alterations in the rat brain.
at the onset of nonaffective psychosis. Biological Journal of Pharmacology and Experimental Therapeutics,
Psychiatry, 43:674-679, 1998. 280:261-267, 1997.
McCreadie, R.G.; MacDonald, E.; Wiles, D.; Campbell, Reddy, R.; Sahebarao, M.P.; Mukherjee, S.; and Murthy,
G.; and Paterson, J.R. The Nithsdale Schizophrenia J.N. Enzymes of the antioxidant system in chronic schizo-
Surveys: XIV. Plasma lipid peroxide and serum vitamin E phrenic patients. Biological Psychiatry, 30:409-412,
levels in patients with and without tardive dyskinesia, and 1991.
normal subjects. British Journal of Psychiatry, Reddy, R.D., and Yao, J.K. Membrane protective strate-
167:610-617, 1995. gies in schizophrenia: Conceptual and treatment issues.
Moncada, S.; Palmer, R.M.J.; and Higgs, E.A. Nitric In: Peet, M.; Glen, I.; and Horrobin, D.F., eds.
oxide: Physiology, pathophysiology, and pharmacology. Phospholipid Spectrum Disorder in Psychiatry.
Pharmacology Review, 43:109-142, 1991. Lancashire, U.K.: Marius Press, 1999. pp. 75-88.
Nag, S.; Picard, P.; and Stewart, D.J. Increased Rengasamy, A., and Johns, R.A. Inhibition of nitric oxide
immunolocalization of nitric oxide synthases during synthase by a superoxide generating system. Journal of
blood-brain barrier breakdown and cerebral edema. Acta Pharmacological and Experimental Therapeutics,
Neurochirurgica. Supplement, 76:65-68, 2000. 267:1024-1027, 1993.
Nag, S.; Picard, P.; and Stewart, D.J. Expression of oxide Robbins, R.A.; Millatmal, T; Lassi, K.; Rennard, S.; and
synthases and nitrotyrosine during blood-brain barrier Daughton, D. Smoking cessation is associated with an

933
Schizophrenia Bulletin, Vol. 30, No. 4, 2004 J.K. Yao et al.

increase in exhaled nitric oxide. Chest, 112:313-318, dant capacity in schizophrenia. Schizophrenia Research,
1997. 32:1-8, 1998a.
Schilling, L., and Wahl, M. Brain edema: Pathogenesis Yao, J.K.; Reddy, R.; McElhinny, L.G.; and van Kammen,
and therapy. Review. Kidney International Supplement, D.P. Effects of haloperidol on antioxidant defense system
59:S69-S75,1997. enzymes in schizophrenia. Journal of Psychiatric
Sharma, H.S.; Drieu, K.; Aim, P.; and Westman, J. Role of Research, 32:385-391, 1998ft.
nitric oxide in blood-brain barrier permeability, brain Yao, J.K.; Reddy, R.; and van Kammen, D.P. Reduced
edema and cell damage following hyperthermic brain level of plasma antioxidant uric acid in schizophrenia.

Downloaded from https://academic.oup.com/schizophreniabulletin/article-abstract/30/4/923/1931278 by guest on 01 May 2019

injury: An experimental study using EGB-761 and Psychiatric Research, 80:29-39, 1998c.
Gingkolide B pretreatment in the rat. Ada Yao, J.K.; Reddy, R.; and van Kammen, D.P. Human
Neurochirurgica Supplement, 76:81-86, 2000. plasma glutathione peroxidase and symptom severity in
Shintani, F.; Kanba, S.; Maruo, N.; Nakaki, T.; Nibuya, schizophrenia. Biological Psychiatry, 45:1512-1515,
M.; Suzuki, E.; Kinoshita, N.; and Yagi, G. Serum inter- 1999.
leukin-6 in schizophrenic patients. Life Sciences, Yao, J.K.; Reddy, R.D.; and van Kammen, D.P. Abnormal
49:661-664, 1991. age-related changes of plasma antioxidant proteins in
Siegel, S. Nonparametric Statistics. New York, NY: schizophrenia. Psychiatry Research, 97:137-151, 2000.
McGraw-Hill, 1956. Yao, J.K.; Reddy, R.D.; and van Kammen, D.P. Oxidative
Smith, C.V. Free radical mechanisms of tissue injury. In: damage and schizophrenia: An overview of the evidence
Moslen, M.T., and Smith, C.V., eds. Free Radical and its therapeutic implications. CNS Drugs, 15:287-310,
Mechanisms of Tissue Injury. Boca Raton, FL: CRC 200 \b.
Press, 1992. pp. 1-22.
Smythies, J. Oxidative reactions and schizophrenia: A
review-discussion. Schizophrenia Research, 24:357-364,
Acknowledgments
1997. This study was supported in part by Merit Review grants
Suboticanec, K.; Folnegovic-Smalc, V.; Korbar, M.; (J.K.Y., S.L.) and Research Career Scientist Awards
Mestrovic, B.; and Buzina, R. Vitamin C status in chronic (J.K.Y., S.L.) from the Office of Research and
schizophrenia. Biological Psychiatry, 28:959-966, 1990. Development, Department of Veterans Affairs, and the
Takemori, K.; Ito, H.; Suzuki, T. Effects of inducible Highland Drive VA Pittsburgh Healthcare System. The
nitric oxide synthase inhibition on cerebral edema in authors are grateful to B. Bland and B. Sullivan for their
severe hypertension. Acta Neurochirurgica Supplement, technical assistance.
76:335-338, 2000.
Thiel, V.E., and Audus, K.L. Nitric oxide and blood-brain The Authors
barrier integrity: Review. Antioxidants and Redox
Signalling, 3:273-278, 2001. Jeffrey K. Yao, Ph.D., is Research Career Scientist, VA
Thorns, V.; Hansen, L.; and Masliah, E. nNOS expressing Pittsburgh Healthcare System; Research Professor of
neurons in the entorhinal cortex and hippocampus are Psychiatry, Western Psychiatric Institute and Clinic,
affected in patients with Alzheimer's disease. University of Pittsburgh Medical Center; and Research
Experimental Neurology, 150:14-20, 1998. Professor of Pharmaceutical Sciences, University of
Pittsburgh School of Pharmacy, Pittsburgh, PA. Sherry
van Kammen, D.P.; McAllister-Sistilli, C.G.; Kelley,
Leonard, Ph.D., is Research Career Scientist, Denver
M.E.; Gurklis, J.A.; and Yao, J.K. Elevated interleukin-6
VA Medical Center, and Associate Professor,
in schizophrenia. Psychiatry Research, 87:129-136, 1999.
Departments of Psychiatry and Pharmacology,
Yao, J.K.; Leonard, S.; and Reddy, R. Increased level of University of Colorado Health Sciences Center, Denver,
mercaptans in postmortem brains from schizophrenic CO. Ravinder D. Reddy, M.D., is Associate Professor of
patients. Biological Psychiatry, 49:172S, 2001a. Psychiatry, Western Psychiatric Institute and Clinic,
Yao, J.K.; Reddy, R.; McElhinny, L.G.; and van University of Pittsburgh Medical Center, Pittsburgh,
Kammen, D.P. Reduced status of plasma total antioxi- PA.

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