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Increased ratio of quinolinic acid to kynurenic acid in cerebrospinal fluid of D retrovirusЧinfected rhesus macaques Relationship to clinical and viral status.

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Increased Ratio of Quinolinic Acid to
Kynurenic Acid in Cerebrospinal Fluid of
D Retrovirus-infected Rhesus Macaques:
Relationshp to Clinical and Viral Status
Melvyn P. Heyes, PhD,” Ivan N. Mefford, PhD,t Bonnie J. Quearry, BS,” Manish Dedhia,”
and Andrew Lackner, DVMS.
Increased concentrations of excitotoxin quinolinic acid in cerebrospinal fluid (CSF) are associated with infection with
the human immunodeficiency virus (HIV-1) and have been implicated in the pathogenesis of the acquired immune
deficiency syndrome (AIDS) dementia complex. In the present study, inoculation of macaques with D/ l/California, an
immunosuppressive serotype 1 type D retrovirus, was associated with acute and chronic increases in CSF and serum
quinolinic acid concentrations in macaques that had developed SAIDS, a simian disease analogous to AIDS in humans-particularly macaques with demonstrable opportunistic infections. Kynurenic acid, an antagonist of excitatory
amino acid receptors as well as the excitotoxic effects of quinolinic acid, was also increased in the CSF of SAIDS
macaques, but to a significantly lesser degree than was quinolinic acid (kynurenic acid, 1.8-fold; quinolinic acid, 15.6fold). CSF quinolinic acid, but not kynurenic acid, was also increased in viremic macaques with SAIDS-related
complex (2.4-fold) and asymptomatic virus positive carriers (3.4-fold). Macaques that had recovered from
D/ Kalifornia infection and were antibody positive and virus negative had normal CSF quinolinic acid and kynurenic
acid concentrations. Increased activity of indoleamine-2,3-dioxygenase,the first enzyme of the kynurenine pathway,
was indicated in the macaques with SAIDS by reduced serum Gtryptophan and elevated serum Gkynurenine concentrations. Macaques infected with D/ l/California may provide a primate model for investigation of the mechanisms
involved in increases in CSF quinolinic acid in retrovirus and other infectious diseases, including HIV-1. It remains to
be determined whether the increased CSF quinolinic acid concentrations and the increased ratio of quinolinic acid to
kynurenic acid have neurological significance or are a useful “marker” of infection.
Heyes MP, Mefford IN, Quearry BJ, Dedhia M, Lackner A. Increased ratio of quinolinic
acid to kynurenic acid in cerebrospinal fluid of D retrovirus-infected rhesus macaques:
relationship to clinical and viral status. Ann Neurol 1990;27:666-675
Quinolinic acid (QUIN) is a neuroactive metabolite of
L-trytophan (L-TRP) synthesized in systemic tissues via
the kynurenine pathway (Fig 1).O n local application of
QUIN to neurons of the central nervous system and
spinal cord, QUIN acts as an agonist of N-methyl-Daspartate (NMDA)-type excitatory amino acid receptors [I] and in large quantities may cause seizures {2]
and nerve cell death {3}. In endotoxin-treated mice,
the activity of indoleamine-2,3-dioxygenase(IDO), the
first enzyme of the kynurenine pathway that synthesizes QUIN, is increased in several extrahepatic tissues, plasma L-kynurenine (L-KYN) is elevated [4],
and QUIN, 3-hydroxykynurenine L-TRP and
5-hydroxyindoleacetic acid (5-HIAA) concentrations
are increased in cerebral cortex [5, 61. In infectious
diseases and other conditions with immune system activation, concentrations of QUIN in brain may be increased and the neuropathological effects of QUIN
become manifest 161.
A strikmg example of an infection with concurrent
neurological deficits is the acquired immune deficiency
syndrome (AIDS) in humans. This disease is characterized by chronic infection with the human immunodeficiency virus (HIV-l), superimposed on which are opportunistic infections. Some patients also develop the
AIDS dementia complex [7-91. We have discovered
that the concentration of QUIN is increased in the
cerebrospinal fluid (CSF) of patients with AIDS and
have implicated QUIN in the pathogenesis of the
AIDS dementia complex [10-12). Some of our cur-
From the *Section on Analytical Biochemistry and the ?Section on
Clinical Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD, and the $California Primate
Research Center, University of California, Davis, CA.
Received Sep 15, 1989, and in revised form Dec 4 and Dec 29.
Accepted for publication Jan 5, 1990.
Address correspondence
Dr Heyes, Section on Analytical
Biochemistry, Laboratory of Clinical Science, Building 10, Room
3D40, National Institute of Mental Health, Bethesda, MD 20892.
666 Copyright 0 1790 by the American Neurological Association
Material and Methods
Thirty-seven rhesus monkeys (Macaca mulatta), including 6
uninfected control animals, were studied retrospectively.
Macaques were defined as (1) SAIDS, (2) SAIDS-related
complex (SRC), (3) healthy carrier, or ( 4 ) recovered, by previously established criteria 114, 16, 18, 21). Briefly, a rhesus
monkey diagnosed with SAIDS due to type D retroviruses
had persistent generalized lymphadenopathy and four or
more of the following clinical signs: splenomegaly; weight
loss greater than 10%; anemia (<30% packed erythrocyte
volume); neutropenia; persistent lymphopenia; lymphoid depletion; marrow hyperplasia; persistent diarrhea unresponsive to appropriate therapy; and opportunistic infections,
such as generalized cytomegalovirus infection, intestinal
cryptosporidiosis, oral and esophageal candidiasis, or multiple bacterial infections unresponsive to appropriate therapy.
SAIDS-related complex (SRC) was defined by the presence
of generalized lymphadenopathy and fewer than four of the
criteria used to define SAIDS. Animals with SAIDS or SRC
were viremic and may or may not have antibodies to the type
D retrovirus. Healthy carriers were defined as persistently
viremic animals that lack clinical signs of SAIDS. Recovered
animals were viremic at one time but at the time of sample collection were virus negative, antibody positive, and
healthy, without any sign of SAIDS. The 6 control animals
were from the SAIDS-free colony at the California Primate
Research Center and were negative for D retrovirus and
simian immunodeficiency virus by serology and virus isolation. The macaques studied are presented in Table 1 and
classified by experimental group, with viral status in peripheral blood mononuclear cells (PBMC) and CSF, antibody
status in peripheral blood, and other relevant data at the time
of sample collection listed.
Animals were inoculated intravenously with defined
stocks of plasma from animals infected with D/l/California
(n = 8) 1227, tissue culture grown virus (n = 12) {17, 231,
or molecularly cloned virus (n = 7) 1141, with the exception
of the 4 healthy carriers that were spontaneously occurring
cases from an outdoor field cage with unknown dates of
initial infection 116). The disease course and the clinical outcome have both been shown to be independent of inoculum
Serum, PBMC, and CSF were collected while the animals
were immobilized by an intramuscular injection of ketamine
hydrochloride (10 mg/kg). Serial samples were obtained
from 16 of the animals including 2 of the controls (see Table
1). None of the macaques had either pleocytosis or red
blood cells in the CSF. Virus was isolated by cocultivation of
CSF and PBMC with Raji cells as described previously 122,
247. Serum and CSF not needed for viral isolation were
stored at -70°C. Serum was tested for antibodies to
D/ l/California by an enzyme-linked immunosorbent assay
using whole virus and peroxidase conjugated anti-macaque
immunoglobulin as previously described 1221.
All of the macaques with SAIDS, 1 of the 9 animals with
SRC, and 2 of the carrier macaques were humanely killed. A
complete set of tissues was collected for routine histological
examination. Samples of intestinal content and selected tissues were submitted for parasitology, microbiology, and mycology in selected cases [25-27).
Fig 1 . Metabolism of L-tqptophan (L-TRP) through the kynurenine pathway. L-TRP is converted t o formylkynurenine by
the action of indoleamine-2,3-dioxygenase (IDO) in extrahepatic
tissues and tvyptophan-2,3-dioxygenasein liver. ID0 is activated in several cell types by interferon-gamma 132, 33, 56,
5 7). In mice, systemic endotoxin administration acutely decreases
blood L-TRP concentrations 15, 61, increased blood L-kynurenine
concentrations {4), and increases brain quinolinic acid concentrations (5, 6). L-kynurenine may enter brain (38, 39) and be
converted to kynurenic acid in brain tissue 133).
rent investigations involve a search for animal models
for brain Q U I N metabolism in infectious diseases, particularly in retroviral infections. Macaques in captivity
may also develop acquired immune deficiency syndromes, analogous to AIDS in humans, following
inoculation with retroviruses, including the type D
retrovirus D/ 1/California (formerly simian acquired
immune deficiency syndrome [SAIDS)) retrovirus
serotype-1, SRV-1 [13-18). The symptoms of SAIDS
due to D / l/California increase in severity with disease
progression and the macaques die with opportunistic
bacterial, viral, protozoan, or fungal infections, including generalized cytomegalovirus infection, cryptosporidiosis, noma, and oral candidiasis El8).
In the present study, the concentrations of QUIN
and kynurenic acid (KYNA), a related metabolite that
can attenuate the excitotoxic effects of QUIN 119,
201, were measured in samples of CSF collected from
rhesus macaques infected with DI l/California and
compared to uninfected control macaques. The concentrations of L-TRP, 5-HIAA, the dopamine metabolite homovanillic acid (HVA), and the norepinephrine metabolite 3-methoxy,4-hydroxy-phenylglycol
(MHPG) were measured in the CSF, and QUIN,
L-TRP, L-KYN, and MHPG were quantified in the
Heyes et al: CSF Quinolinic Acid in SAIDS 667
Table 1. Data on Macaques Infected with D / l /California"
Virus Isolation
Group Macaque
8016 124, 261
16416 124, 261
16868 1261
16989 1261
19411 [26]
C S P Statusd
lo3 160
19494 124,271
19496 124, 27)
21903 124, 271
12.5 X lo3 160
6.4 x lo3 160
12.5 x lo3 40
23067 [26}
20132 {24, 261
20249 124,261
lo3 160
lo3 80
25 x 103 20
3.2 x 103 20
3.2 X lo3 80
3.2 x 103 -
x 103 x lo3 40
21083 ~ 2 3 ~ 2 4 , 2 7 1 1.6
21092 123, 24, 271
21319 [24, 251
21915 114, 241
Asymptomatic Carriers'
17500 [16, 241
17569 1241
17636 ~ 1 6 , 2 7 1
19729 116, 24, 271
x 103 x lo3 40
668 Annals of Neurology Vol 27
Time PI CSF QUIN Pathology, Postmortem Findings,
and Microbiology
N o 6 June 1990
Cachexia, anemia, microbiology negative
Pancytopenia, thrombocytopenic purpura, microbiology negative
Pancytopenia, multiple anaerobes cultured from necrotizing gingivitis
Pancytopenia,endometritis, E . coli from
Multiple anaerobes cultured from severe
noma lesion
Oral candidiasis
Coagulase positive staphylococci from
lung abscess
Disseminated intravascular coagulation
and disseminated cytomegalovirus infection, Morganelkz, from esophageal
Classified as SRC at this time
Anemia, thrombocytopenia, focal skin
ulcer, coagulase-positive staphylococci
from skin only
Recurrent unresponsive diarrhea, enterocolitis with atrophy, microbiology
Multifocal fibrosarcoma, generalized
amyloidosis, coagulase + staphylococci
from lung
Table I . (Continued)a
Virus Isolation
Group Macaque
20325 I241
21315 E241
21713 {14, 241
21765 {24]
21968 [25}
22838 [26]
C S P Statusd
Pathology, Postmortem Findings,
and Microbiology
Classified as SRC at this time
“In macaques for which more than one sample was available, the average value is used in the calculation of group mean concentrations.
Pathological data and microbiology results are included for only those macaques that were killed at the time of sample collection. The
immunological and pathological features of some of these macaques have been presented previously 114, 16, 23-27).
bNumber of D/I/California infected cells/lO‘ peripheral blood mononuclear cells (PBMC). Virus negative ( - ) is < 1 syncytia-inducing unit per
8.0 x 104 PBMC.
‘Number of syncytial induction units/ml of cerebrospinal fluid (CSF).
dDetermined by enzyme-linked immunoabsorbent assay.
‘All are spontaneous cases. Weeks post inoculation (PI) is time from first positive viral culture.
not determined, SAIDS = simian acquired immune deficiency syndrome, SRC = SAIDS-related complex.
Biochemical Analyses
hexafluoroisopropanol, zinc acetate, sodium octylsulfate,
sodium chloride, Tris buffer, and ethoxy hydroxyphenylglycol were obtained from Sigma Chemical (St Louis, MO), and
trifluoroacetylimidazole was obtained from Pierce Chemical
(Rockford, IL). Sodium acetate, ammonium acetate, sodium
EDTA, methanol, ethyl acetate, and acetonitrile were obtained from Fisher (Fair Lawn, NJ).
QUIN was quantified by electron capture negative chemical ionization gas chromatography/mass spectrometry C28,
271. CSF (30 pl), serum (10 pl), and QUIN standards (3150 pmol diluted in deionized water) were mixed with 100 pl
of deionized water containing 30 pmol of [‘80]-QUIN as
internal standard. Samples were freeze dried overnight and
QUIN and { ‘80)-QUIN esterified to their dihexafluoroisopropanol esters. After extraction into heptane, samples were
analyzed using a Kratos MS-80 magnetic sector mass spectrometer (Urmston, England) and QUIN and [‘80}-QUIN
monitored as the molecular ions.
KYNA concentrations in CSF were quantified as peak
heights by high performance liquid chromatography (HPLC)
and fluorescence detection following post-column mixing
with zinc acetate {30; Heyes and Quearry, unpublished data,
19701. Undiluted CSF (50 pl) was injected directly onto a
4.6 x 75 mm Altec (Beckman Instruments, Inc., San Ramon, CA) ultrasphere 3-pm octadecylsilane column by a
Gilson model 23 1 (Gilson, Middleton, WI) autosampler.
The mobile phase consisted of 10 mmol/L sodium acetate
and 6 ml/100 ml acetonitrile at a flow rate of 0.7 ml/min. An
LKB 2249 pump (Pharmacia LKB,Piscataway, NJ) was used
to mix 1 mol/L zinc acetate post-column at a flow rate of 0.7
ml/min. KYNA was quantified using a Hewlett Packard
(Palo Alto, CA) 1046A fluorescence detector (excitation,
254 nm; emission, 404 nm).
For the quantification of serum L-KYN by ultraviolet light
absorbance spectrophotometry {31}, 100 pI of serum was
mixed with 100 p1 of 2.5 mol/L perchloric acid, centrifuged,
and a 7O-pl aliquot collected into 25O-pl polypropylene
tubes. The HPLC system consisted of an LKB 2150 pump,
an Altec ultrasphere 5 pm 4.6 x 75 mm column, a Kratos
spectroflow 773 spectrophotometer, and a mobile phase consisting of 0.1 mol/L ammonium acetate and 60 ml/L acetonitrile at p H 3.9. A Gilson model 23 1 autosampler was used to
inject 50 p1of sample. Sample peak heights were compared
to freshly prepared standards.
L-TRP and 5-HIAA were quantified in CSF and L-TRP
was quantified in serum by HPLC. Ten microliters of serum
was mixed with 500 p1 of 1 mol/L hydrochloric acid, centrifuged, and the supernatant transferred to 250-pl polypropylene tubes. The HPLC system consisted of a 4.6 X 75
mm Altec ultrasphere 3-pm octadecylsilane column, an
LC4B amperometric detector (BAS, Lafayette, IN), an electrochemical detector cell, and an LKB 2150 pump. The
mobile phase 150 mmol/L sodium acetate, 258 pmollL
sodium octylsulfate, 134 pmol/L sodium EDTA, 40 ml (for
serum) or 5 ml (for CSF) acetonitrile per liter of buffer at p H
3.9 and a flow rate of 1 ml/min. Oxidation voltage across the
glassy carbon electrode was set at 0.7 V versus an AgIAgC1
reference electrode. A Wisp 7 10B autoinjector (Waters,
Milford, MA) or a Gilson model 231 autosampler was used
to inject 20 pl of undiluted CSF, 10 +I of serum extract, or
freshly prepared standards.
Undiluted CSF was analyzed for HVA and MHPG by
HPLC. The system consisted of a 15 cm X 4.1 mm internal
diameter column packed in house with 3 pm ODS Hypersil
Heyes et al: CSF Quinolinic Acid in SAIDS 669
Table 2. Serum QUIN, L-TRP, L-KYN, and MHPG of D / l /California Inoculated Macaques
QUIN (nmol/L)
L-TRP (pmol/L)
L-KYN (pmol/L)
MHPG (nmol/L)
1735 t 673
43.0 2 5.4
1.71 ? 0.20
16.1 ? 1.4
1118 ? 100
44.3 5 3.5
1.76 5 0.14
20.5 t 1.5
2951 t 1047
41.0 ? 10.8
2.65 ? 0.33
29.8 ? 7.4
2430 t 515
41.9 ? 2.4
2.57 ? 0.27
15.7 ? 1.3
9104 t 2431"""
26.8 2 3.5"
6.52 2 1.52"""
27.2 +- 3.2""
QUIN = quinolinic acid, L-TRP = L-tryptophan, L-KYN = L-kynurenine, MHPG = 3-methoxy, 4-hydroxyphenylglycol, SAIDS = simian
acquired immune deficiency syndrome, SRC = SAIDS-related complex.
" p < 0.05, compared with controls.
"*p < 0.01, compared with controls.
**"p < 0.0001, cornpared with controls.
(Shandon, Southern Products Ltd, Runcorn, England). A
mobile phase of 50 mmol/L sodium acetate, 4 ml/100 ml
methanol, and 100 mg/L sodium EDTA adjusted to p H 5.06
was used. Detection was accomplished using an applied potential of + 0.75 volts vs Ag/AgCl reference at a glassy
carbon electrode using a model LC4B amperometric detector (BAS). Thirty pl of undiluted CSF was applied to the
column using a Gilson model 231 autosampler. Concentrations were determined by comparison of sample peak
heights with authentic standards.
To quantify MHPG in serum, 200 to 300 pl of serum was
mixed with 100 p1of 1 x 10.' mol/L ethoxy hydroxyphenylglycol as internal standard. Proteins and lipds were removed
by ultraliltration with Amicon (WR Grace and Co, Danvers,
MA) centrifree filters by centrifugation at 5,OOOg for 45 minutes. To this filtrate was added 1.0 ml of 1.5 mol/L Tris
buffer, p H 8.6,400 mg sodium chloride, and 4.0 ml of ethyl
acetate. Each sample was then mixed for 30 seconds and the
phases separated by centrifugation for 10 minutes at 600g.
The ethyl acetate phase was recovered and evaporated to
dryness under nitrogen. The residue was then suspended in
100 p1 of 1% acetic acid and 30 pl was applied to the chromatographic system described for separation of MHPG in
CSF. Peak heights of MHPG and ethoxyhydroxyphenylglycol were measured and compared to peak heights of
standards for correction for recovery.
Statistical Analyses
For macaques in which more than one sample of serum or
CSF was available, the average value of each macaque was
used in group data analyses, provided the macaque remained
in the same clinical group. Macaque 22838 was classified as
SRC at one week post inoculation but was classified as recovered by week 5. Macaque 23067 was classihed as SRC at
one week post inoculation and had progressed to SAIDS by
week 5. Values from both classifications in these macaques
are used in the data analyses. One of the SAIDS macaques
(19411) had severe noma (a rapidly progressive necrotizing
gingivitis, stomatitis, and osteomyelitis) with a serum QUIN
concentration of 579377.0 nmol/L and was not included in
the statistical analyses of serum QUIN.
Values presented are mean plus or minus one standard
error of the mean or percent of control macaques. The
ranges of values are for the full data set. Results were analyzed by one-way analysis of variance with Dunnett's t-test
after logarithmic transformation. Correlation coefficients
670 Annals of Neurology Vol 27 No 6 June 1990
were calculated by the method of least squares. A value of
p < 0.05 was considered statistically significant.
Serum measures are presented in Table 2. L-TRP concentrations were reduced in serum of SAIDS macaques (to 62% of control, p < 0.05) and were associated with increased serum L-KYN concentrations
(381%, p < 0.0001) and elevated serum QUIN concentrations (50396, p < 0.0001). The small increases
in serum L-KYN and Q U IN in carrier and SRC macaques were not statistically significant. MHPG concentrations in serum were increased in SAIDS macaques
only (26096,p < 0.02).
CSF measures are presented in Table 3. The concentration of QU IN in the CSF was significantly
higher in macaques with clinical SAIDS (1559%, p <
O.OOOl), SRC (24296, p < 0.005), and carrier macaques (345%, p < 0.05) compared to controls. Although it would appear that carrier macaques had
higher CSF Q U IN concentrations than did SRC macaques, this larger mean value is attributable to macaque
17636, an animal that had high virus isolation from
PBMC, a lung positive for stuphylocci, and a multifocal
fibrosarcoma (see Table 1). CSF QUIN was not significantly different from control in the recovered macaques (115%). The concentration of KYNA in the
CSF was increased in the SAIDS macaques (181%,
p < 0.01) but in no other group. In the control macaques, the ratio of the concentrations of QUIN to
KYNA was 4.22 f 0.82 (Fig 2). This ratio was increased to 821% of control in the SAIDS macaques
(34.64 ? 6.12; p < O.OOOl), SRC (8.56 ? 0.93,p <
O.Ol), carrier (16.6 f 3.8, p < 0.01), but not recovered macaques (4.21 ? 0.48).
CSF L-TRP and 5-HIAA concentrations were reduced in the SAIDS macaques (6496, p < 0.05 and
69%, p < 0.05, respectively), whereas CSF HVA concentrations were not significantly different from control macaques in any group. MHPG concentrations in
the CSF were increased in SAIDS macaques only
(145%,p < 0.05).
Table 3. CSF QUIN, KYNA, L-TRP, 5-HIAA, HVA, and MHPG of D/1/California Inoculated Macaques
QUIN (nmol/L)
KYNA (nmol/L)
L-TRP (pmol/L)
HVA (nmol/L)
MHPG (nmol/L)
21.9 t 1.6
5.73 t 0.82
2.02 t 0.13
304.8 ? 32.5
1639 ? 132
94.6 ? 5.7
t 3.6
t 0.32
* 0.26
t 41.4
t 146
* 14.5
t 0.28
t 46.9
t 111
t 21.6
t 5.5"*
t 0.50
t 0.11
t 15.0
t 91
t 1.3
t 81.9"""
t 0.90""
t 43.8"
CSF = cerebrospinal fluid, QUIN = quinolinic acid, KYNA = kynurenic acid, L-TRF' = L-tryptophan, 5-HIAA = 5-hydroxyindoleacetic
SAIDS = simian acquired immune deficiency syndrome, SRC
acid, HVA = homovanillic acid, MHPG = 3-methoxy,4-hydroxyphenylglycol,
= SAIDS-related complex.
" p < 0.05, compared with controls.
**p < 0.01, compared with controls.
*"*p < 0.0001, compared with controls.
Acute Changes in CSF and Serum QUIN and CSF
KYNA Following Inoculation w i t h Dl1 /California
Fig 2. Bar graph of the ratio of quinolinic acid (QUIN) to
kynurenic acid (KYNA) in cerebrospinaljuid (CSF) of control
and DIl ICalifornia inoculated macaques. Increases in CSF
QUIN were observed in carrier, simian acquired immune
deficiency (SAIDS) and SAIDS-related complex (SRC) macaques
but not virus negative antibody positive recovered macaques.
Whereas CSF KYNA concentrations were also increased in
SAIDS macaques, the magnitude of this increase was less than
the increase in QUIN, such that the ratio of QUIN:KYNA
was increased in the SAIDS macaques. "p < 0.01, ""p <
Early consequences of inoculation with D1 11California
were studied in 6 macaques (Fig 3). The changes in
QUIN concentrations in CSF (see Fig 3A) and serum
(see Fig 3B) and the ratio of QUIN to KYNA (see Fig
3D) are plotted on a logarithmic scale, whereas the
changes in CSF KYNA concentrations are plotted on
a linear scale (see Fig 3C).
CSF QUIN concentrations increased one week after
inoculation in 3 macaques, which were classified as
SRC at this time, compared to control macaques (see
Figure 3A: 22838,22906, and 23067;p < 0.001). In 5
viremic macaques (SRC: 22906 and 7575; SAIDS:
8016, 16989, and 23067), CSF QUIN concentrations
were elevated. In the macaque that was classified as
SRC at week 1 (22838), CSF QUIN was elevated but
had returned to control values at week 5 when the
macaque became virus negative and antibody positive
(recovered). In serum, QUIN concentrations (see Fig
3B) were elevated in 3 SAIDS macaques (23067,
16989, and 8016). One of the macaques in the SRC
group (22906) also had increased serum QUIN,
whereas the other SRC macaque was within the normal range (7575). The macaque that was classified as
SRC at week 1 and recovered by week 5 (22838) had
serum QUIN concentrations within the normal range.
CSF KYNA concentrations were increased in 1
macaque with SAIDS (23067) and SRC (22906)
whereas the remaining macaques were within the normal range (see Fig 3C). Consequently, the ratio of the
concentrations of QUIN to KYNA in the CSF (see
Fig 3D) followed the same pattern as that found for
CSF QUIN concentrations presented in Figure 3A.
The decreases in serum L-TRP concentrations and associated increases in serum L-KYN and QUIN levels
in SAIDS macaques are consistent with increased
Heyes et al: CSF Quinolinic Acid in SAIDS 671
Time (Weeks)
Time (Weeks)
10 -
l o
Time (Weeks)
Time (Weeks)
Fig 3. Scatter plot of quinolinic acid (QUIN) and kynurenic
acid (KYNA)concentrations in control, simian acquired immune deficiency (SAIDS) and SAIDS-related complex (SRC)
macaques infected with DIl ICaliforniafor 1 to 13 weeks
plotted on a logarithmic scale. Note that macaque 22838 was
classified as SRC at week 1 but reclassified as recovered at
672 Annals of Neurology Vol 27 No 6 June 1990
week S. (A)The results show acute increases in cerebrospinal
j u i d (CSF) QUIN concentrations in uiremic macaques. (B)
Serum QUIN showed a similar pattern. (C) CSF KYNA was
slightly increased in one SRC macaque (22906) and one SAIDS
macaque (23067). (0)Tendency for increased ratio of QUIN:
KYNA in DIl ICalifrnia uiremic macaques.
L-TRP catabolism through the kynurenine pathway
and may in part reflect activation of I D 0 in systemic
tissues (see Fig 1). Interferon-gamma increases I D 0
activity in lung and macrophages in vitro 132, 331, and
it is possible that interferon-gamma activates I D 0 in
the D/ l/California infected macaques. In this context,
QUIN may be viewed as a nonspecific “marker” of
activation of the immune system.
The source of the increased QUIN and KYNA in
the CSF of viremic macaques may reflect several responses. Movement of QUIN across the blood-brain
barrier in rodents is reported to be low 1341, and in
macaques with SAIDS there is no evidence of increased blood-brain barrier permeability, based on the
ratio of albumin in CSF and serum 1241. However, the
permeability of the blood-brain barrier to small molecules such as QUIN and KYNA in macaques is not
known. Macaque 19411, which had a markedly increased serum QUIN (334-fold higher than control),
had a CSF Q U IN concentration of 592.4 nmol/L,
which ranked only second highest in the SAIDS group
(see Table 1). Although these observations suggest an
intracerebral source for QUIN, it is possible that some
of the increased CSF QUIN originated from blood,
particularly as the increases in serum QUIN were sustained over a prolonged period.
Reductions in CSF L-TRP concentrations (see Table
3) in the SAIDS macaques could be secondary to the
decreases in serum L-TRP (see Table 2) or may reflect
activated intracerebral IDO. Macrophages in vitro, activated by endotoxin or interferon-gamma, have increased I D 0 activity and synthesize increased amounts
of L-KYN 1331, a precursor of KYNA in brain {351,
and also increased amounts of 3-hydroxyanthranilic
acid, an established precursor of QUIN in brain in
vivo 1361. The increased CSF QUIN {lo] and KYNA
{Heyes et al. Unpublished data, 19907 in HIV-1 infected patients may reflect activation by interferongamma of I D 0 within macrophages in brain. Although
D/ l/California also infects macrophages 1371, infiltrates of macrophages in brain or CSF pleocytosis are
not a reported characteristic of D/ UCalifornia-infected
macaques, including macaques with SAIDS 118, 241.
This may indicate that if activation of I D 0 in brain is
involved in the decrease in CSF L-TRP and the increase
in QUIN and KYNA concentrations in the SAIDS macaques, I D 0 is localized in cells other than macrophage
infiltrates. The choroid plexus, which has high I D 0
activity 138, 391, is also a site for D/l/California
localization 1241 and is a possible source of QUIN.
Increased CSF KYNA in the SAIDS macaques may
reflect increased synthesis from L-KYN produced
either in brain or following entry of L-KYN from
blood 138, 391.
The formation enzyme for QUIN in brain and systemic tissues is 3-hydroxyanthranilate-3,4-dioxyge-
nase, which is localized in astrocytes 140). Gliosis
and glial nodules are a feature of the HIV-1-infected
brain 18, 411, and increased CSF QUIN concentrations in HIV- 1-infected patients could reflect increased
3-hydroxyanthranilate-3,4-dioxygenaseactivity. However, increased brain 3-hydroxyanthranilate-3,4-dioxygenase activity is found in patients with Huntington’s
disease 1421, where the concentrations of QUIN are
not increased in either brain tissue 1431 or CSF 1441.
Furthermore, in D/ 1/California infected macaques,
gliosis is not a feature of the brain 118, 241. In a preliminary study, Shasken 1451 reported that the activity
of 3-hydroxyanthranilate-3,4-dioxygenasein glioblastoma C6 cells in vitro was increased following acute
infection with herpes simplex virus, although the effects of retroviral infections are not known. Whereas
HIV-1 may be localized within glial cells 1411, D/1/
California has not been reported to infect these cells
It is not known to what degree CSF QUIN and
KYNA concentrations are an accurate reflection of
their concentrations in the extracellular fluid space of
brain, where QUIN and KYNA would have access to
excitatory amino acid receptors. If CSF QUIN concentrations do reflect their extracellular fluid levels, then
the concentration of QUIN achieved in the SAIDS
macaques exceeds concentrations reported to be neurotoxic to an organotypic corticostriatal neuronal system in vitro (100 nmol/L) after 49 days of exposure
1461, but is lower than QU IN concentrations of 100
pmol/L that are neurotoxic in other neuronal culture
systems over shorter periods of time 1471. Infection
with D-type retroviruses, including D/ 1/California, are
not associated with neurodegeneration, brain atrophy,
or convulsions, and the brain from D-type retrovirusinfected macaques does not resemble the brains of patients with Huntington’s disease. Conceivably the small
increase in CSF KYNA concentrations in the SAIDS
macaques may have “protected” neurons against the
excitotoxic effects of QUIN.
Although much interest is focused on the neurotoxic and convulsant effects of Q U IN and the potential “neuroprotective” effects of KYNA, increases in
extracellular fluid QUIN and KYNA could have neurological significance other than excitotoxicity {48].
QUIN is an agonist of NMDA receptors [I, 191,
which are involved in synaptic plasticity and nerve differentiation 1491 as well as long-term potentiation,
learning, and memory 1501. Increased QUIN and
KYNA in the SAIDS macaques, HIV-1-infected patients, and other infectious disease states could disrupt
the transfer of information via excitatory amino acid
receptors with consequential neurological dysfunction
independent of cytotoxic lesions. Such disruptions may
be reversible.
Finally, divergent responses of 5-HIAA, HVA, and
Heyes et al: CSF Quinolinic Acid in SAIDS 673
MHPG occurred in the SAIDS macaques (see Table
3). The reductions in 5-HIAA concentrations in CSF
may be secondary to reduced concentrations of L-TRP
in brain [Sl], although small reductions in CSF
5-HIAA also occurred in SRC and carrier macaques.
The decrease in CSF L-TRP concentrations in the
SAIDS macaques contrasts with the acute increase in
brain L-TRP and 5-HIAA concentrations that follow
acute systemic administration of either endotoxin, pokeweed mitogen, concanavalin-A, or interleukin-1 [5, 6,
52-55}, which emphasizes the importance of primate
models of infection. Increased concentrations of MHPG
in CSF and serum in the SAIDS macaques are consistent with the enhanced norepinephrine metabolism
and turnover previously observed in rodent brain
in response to acute systemic endotoxin, pokeweed
mitogen, concanavalin-A, or interleukin-1 administration [54, 551 and could reflect the opportunistic infections in the SAIDS macaques. The increased MHPG
concentrations in the CSF (+ 145%) may also reflect
the increase in MHPG levels in serum ( + 260%) because the blood-brain barrier is relatively permeable to
The present results demonstrate sustained increases
in both CSF and serum Q U I N concentrations in
D/ I /California virus positive macaques, particularly
those with opportunistic infections, coupled with
smaller increases in CSF KYNA concentrations. A
role for increased I D 0 activity is suggested, and so it is
likely that CSF QUIN and KYNA will be increased in
other types of infection. The usefulness of CSF Q U I N
measures as a marker of infection remains to be determined. Macaques infected with D/ l/California may offer a model for investigating the mechanisms involved
in the increases of Q U I N in the CSF in HIV-1infected patients. However, the pathological and functional significance of the increases in CSF Q U I N and
KYNA remain to be determined.
This research was supported in part by Public Health Service grants
RR00169 from the Division of Research Resources, National Institutes of Health, and A120573 from the National Institute of Allergy
and Infectious Diseases. A. Lackner was supported by a Public
Health Service Special Emphasis Research Career Award in Laboratory Animal Science from the Division of Research Resources,
We appreciate the useful discussions with Dr S. P. Markey and the
assistance of J. A. Yergey, R. Sherman, D. Mackenzie, and M. Der.
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