Serum kynurenic acid positively correlates with cardiovascular

Serum kynurenic acid positively correlates with cardiovascular

Pharmacological Reports
2006, 58, 507–511
ISSN 1734-1140
Copyright © 2006
by Institute of Pharmacology
Polish Academy of Sciences
Serum kynurenic acid positively correlates with
cardiovascular disease risk factor, homocysteine:
a study in stroke patients
Ewa M. Urbañska1,2, Piotr Luchowski1,3, El¿bieta Luchowska1, Jaros³aw
Pniewski4, Rados³aw WoŸniak4, Ma³gorzata Chodakowska-¯ebrowska4,
Jerzy Lazarewicz5
!
Department of Pharmacology and Toxicology, Department of Cardiosurgery, Skubiszewski Medical University
in Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland
Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland
"
Department of Neurology, Central Clinical Hospital MSWiA, Warszawa, Poland
#
Department of Neurochemistry, Medical Research Centre, Polish Academy of Science, Warszawa, Poland
Correspondence: Ewa M. Urbañska, e-mail: [email protected]
Abstract:
KYNA, an antagonist of ionotropic glutamate receptors and a7 nicotinic receptors, has been found as well in the brain as in the periphery.
The altered metabolism of KYNA, especially its deficiency, can lead to the enhanced glutamate-mediated excitotoxicity, and was
suggested to be a factor contributing to the development of neurodegeneration and seizures. Elevated serum concentration of
homocysteine is considered to be an independent risk factor of atherosclerosis and is an emerging risk factor of cognitive dysfunction and
stroke. In the present study, serum level of KYNA, homocysteine and other biochemical parameters were assessed in patients at early (up
to 24 h after infarct) stage of stroke. Serum KYNA and homocysteine levels were similar in control (N = 26) and stroke (N = 24) groups.
KYNA level correlated positively with the level of homocysteine in control and in stroke group, with p = 0.018; r = 0.462 and p = 0.027;
r = 0.451, respectively. In control group, KYNA correlated positively also with age (p = 0.007; r = 0.514) and with creatinine level
(p = 0.002; r = 0.581). In stroke group, serum KYNA correlated positively with creatinine (p = 0.001; r = 0.644) and with urea level
(p < 0.001; r = 0.716). Homocysteine level correlated inversely with folate level in control (p = 0.01; r = –0.499) but not in stroke group
(p = 0.13; r = –0.317). Serum homocysteine in stroke group correlated positively also with age (p = 0.001; r = 0.6401), and with urea level
(p = 0.017; r = 0.4813). Clinical significance of the association between serum KYNAand homocysteine levels requires further investigation.
Key words:
kynurenic acid, stroke, homocysteine, serum
Abbreviations: CSF – cerebrospinal fluid, HPLC – high performance liquid chromatography, KAT – kynurenine aminotransferase, KYNA – kynurenic acid
Introduction
A putative role of kynurenic acid (KYNA), the endogenous metabolite of tryptophan, in the physiology
and pathology of the central nervous system has been
studied for over two decades [31]. KYNA is an antagonist of ionotropic glutamate receptors and a7
nicotinic receptors [8, 31]. The altered metabolism of
KYNA, especially its deficiency, can lead to the enhanced glutamate-mediated excitotoxicity, and was
suggested to be a factor contributing to the development of neurodegeneration and seizures [24]. Experimentally applied KYNA displays neuroprotective and
anticonvulsant properties [31]. In the models of focal
and transient global ischemia in rodents, KYNA was
Pharmacological Reports, 2006, 58, 507–511
507
capable of decreasing the size of stroke-induced brain
damage and of improving neurological outcome [6,
23]. KYNA levels seem to decline in neurodegenerative disorders and to increase in some psychiatric illnesses. The content of KYNA in the striatum is reduced, and the activity of KYNA biosynthetic enzymes is low in the putamen of Huntington’s disease
victims [10]. KYNA level in the frontal cortex and
substantia nigra is diminished in the course of Parkinson’s disease and in the cerebrospinal fluid (CSF) during relapsing-onset multiple sclerosis [19, 22]. The
increased level of KYNA was demonstrated in CSF
and cortex of schizophrenic patients [5, 25].
Much less is known about the function of KYNA in
the periphery. Peripheral synthesis of KYNA occurs in
the heart, kidneys or liver [1, 2, 18]. A prominent proportion of serum KYNA seems to be derived also from vascular endothelium which produces and liberates substantial amounts of KYNA in vitro and in vivo [28, 29]. The
endothelial formation of KYNA is regulated by various
factors including hypoglycemic/anoxic conditions [28].
Elevated serum concentration of homocysteine,
a methionine-derived sulfur-containing amino acid, is
considered an independent risk factor of atherosclerosis
and is an emerging risk factor of cognitive dysfunction
[15, 17]. The elevated serum homocysteine was suggested to be a risk factor for atherothrombotic stroke. The
strong association of stroke with hyperhomocysteinemia
was revealed in numerous studies [12, 30], but the lack of
such correlation has also been demonstrated [11, 16].
We have recently shown that homocysteine biphasically modulates brain synthesis of KYNA [13]. Homocysteine at low concentrations stimulates KYNA production, whereas its high levels inhibit KYNA formation in vitro and in vivo [13]. Moreover, homocysteine
affects in a similar manner the endothelial KYNA production in rat aortic rings [29]. The aim of the present
study was to evaluate the serum level of KYNA, homocysteine and other biochemical parameters in patients at early (up to 24 h after infarct) stage of stroke.
Materials and Methods
Patients
Twenty four patients who where diagnosed with the
first ever ischemic stroke within the last 24 h (14
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Pharmacological Reports, 2006, 58, 507–511
males, 10 females) and twenty six volunteers without
stroke in past medical history (4 males, 22 females)
participated in the study. The diagnosis of ischemic
stroke was based on anamnesis, neurological and radiological examination (computer tomography brain
scan). Cases were recruited from a series of 418 patients entering the clinic from April 2002 to June
2003. Exclusion criteria included intracranial hemorrhage, brain tumor, and the admission to the hospital
24 h after the onset of stroke. The detailed medical
history and demographic data of each patient were
gathered based on a standard questionnaire (Tab. 1).
Patient group more often had hypertension and
ischemic heart disease compared with control subjects. Prior to the examination, an informed consent
was obtained from each patient. The studies were performed according to the Declaration of Helsinki and
with the permission of local Ethics Committee.
Experimental procedures
The samples of venous blood were collected within
24 h from the stroke onset and after 8 h fasting. The
blood was immediately centrifuged, plasma was separated, aliquoted and stored (–72°C) until further
analyses. The homocysteine level was measured using
fluorescence polarization immunoassay (Imx, Abbott,
USA). The level of folate and vitamin B12 was assessed using electrochemiluminescence method
(ElecSys 2010 analyzer, Roche, Germany).
KYNA level was measured as previously described
[14]. Briefly, on the day of analysis plasma samples
were acidified with 50% trichloroacetic acid and 1M
HCl. Denatured protein was removed by centrifugation and the supernatant was applied to a Dowex
50W+ column that was prewashed with 0.1 M HCl.
Columns were subsequently washed with 1 ml of
0.1 M HCl and 1 ml of water. KYNA was eluted with
2 ml of water. Eluate was subjected to high performance liquid chromatograph (HPLC) and KYNA was
detected fluorimetrically (Varian HPLC system; ESA
catecholamine HR-80, 3 µm, C18 reverse-phase column) as described before [14].
Mean differences in normally distributed data between cases and controls were analyzed using Student’s t-test. When the data were not distributed normally, the statistical comparisons were performed using Mann-Whitney test. Correlations were assessed
by the method of Spearman. Statistical analyses were
performed using Statistica 4.0 software package.
Serum kynurenic acid correlates with homocysteine level
Ewa M. Urbañska et al.
Tab. 1. General characteristic of patients within stroke and control
group
Stroke
N = 24
Age (years)
Control
N = 26
p
Creatinine (µmol/l)
71.16 ± 10.39 68.38 ± 8.33
Males, N (%)
0.229
14 (58.3%)
4 (15.4%)
0.057
Acetylsalicylic acid intake, N (%)
6 (25%)
7 (26.2%)
0.677
Infarction in the past, N (%)
7 (29.2%)
2 (7.7%)
0.199
Ischemic heart disease, N (%) 17 (70.8%)
8 (30.8%)
0.163
Hypertension, N (%)
11 (42.3%)
0.330
18 (75%)
Tab. 2. Biochemical parameters in control and stroke group
Data are presented as the mean values ± SD or proportion of cases
representing a given feature in the group. Statistical comparisons
were performed using Mann-Whitney test
Results
Stroke and control patients did not differ as regards
the average age, and serum levels of creatinine, urea,
vitamin B12, folate and total homocysteine (Tab. 1, 2).
Serum KYNA level in stroke group was similar to
control (4.41 ± 2.41 vs. 3.91 ± 1.84 nmol/l) (Tab. 2).
Homocysteine serum level correlated inversely
with folate level in control (p = 0.01; r = –0.499) but
not in stroke group (p = 0.13; r = –0.317) (Tab. 3). Se-
Urea (mmol/l)
Stroke
N = 24
Control
N = 26
p
92.54 ± 39.9
77.44 ± 13.5
0.143
6.65 ± 2.24
6.33 ± 1.3
0.992
Vit. B12 (pmol/l)
270.2 ± 120.4
295.6 ± 121.0
0.087
Folate (nmol/l)
18.02 ± 4.94
22.38 ± 9.40
0.116
Homocysteine (µmol/l)
12.37 ± 4.27
13.36 ± 4.07
0.225
KYNA (nmol/l)
4.41 ± 2.41
3.91 ± 1.84
0.580
Data are presented as the mean values ± SD. Statistical comparisons were performed using Mann-Whitney test
rum homocysteine in stroke group correlated positively also with age (p = 0.001; r = 0.6401), and with
urea level (p = 0.017; r = 0.4813) (Tab. 3).
KYNA serum level correlated positively with the
level of homocysteine in both control and stroke group,
with p = 0.018; r = 0.462 and p = 0.027; r = 0.451, respectively (Tab. 3). Serum KYNA in control group correlated positively also with age (p = 0.007; r = 0.514),
and with creatinine (p = 0.002; r = 0.581) (Tab. 3). In
stroke group, serum KYNA correlated with creatinine
(p = 0.001; r = 0.644) and with urea level (p < 0.001;
r = 0.716) (Tab. 3).
Tab. 3. Correlation matrix (Spearman’s correlation coefficients) in control and stroke group.
STROKE
Age
Creatinine
Urea
Vit. B
Folate
Homocysteine
KYNA
p = 0.287
r = 0.2266
p = 0.05*
r = 0.4050
p = 0.848
r = –0.0414
p = 0.996
r = 0.0010
p = 0.001***
r = 0.6401
p = 0.066
r = 0.3814
p < 0.001***
r = 0.6681
p = 0.743
r = 0.0707
p = 0.618
r = –0.1073
p = 0.081
r = 0.3634
p = 0.001***
r = 0.6437
p = 0.940
r = –0.0162
p = 0.327
r = –0.2090
p = 0.017**
r = 0.4813
p < 0.001***
r = 0.7150
p = 0.321
r = –0.2114
p = 0.354
r = –0.1978
p = 0.980
r = –0.0054
p = 0.131
r = –0.3172
p = 0.614
r = –0.1084
CONTROL
Age
Creatinine
p = 0.642
r = 0.0955
Urea
p = 0.554
r = 0.1215
p = 0.321
r = 0.2025
Vit. B
p = 0.038**
r = –0.4100
p = 0.331
r = –0.1985
p = 0.281
r = –0.2197
Folate
p = 0.625
r = 0.1005
p = 0.209
r = –0.2551
p = 0.311
r = 0.2066
p = 0.781
r = –0.0573
Homocysteine
p = 0.251
r = 0.2336
p = 0.071
r = 0.3600
p = 0.722
r = 0.0733
p = 0.313
r = –0.2057
p = 0.01**
r = –0.4986
KYNA
p = 0.007**
r = 0.5143
p = 0.002**
r = 0.5809
p = 0.103
r = 0.3271
p = 0.114
r = –0.3171
p = 0.283
r = –0.2186
p = 0.035*
r = 0.4314
p = 0.018**
r = 0.4619
Correlations for controls are shown in the lower left corner and for cases in the upper right corner of the table; r-correlation coefficient; *p £ 0.05,
**p £ 0.01, ***p £ 0.001
Pharmacological Reports, 2006, 58, 507–511
509
Discussion
Hyperhomocysteinemia is considered to be an independent risk factor of cardiovascular disease, including myocardial infarction and atherosclerosis, as well as arterial
and venous thrombosis [4]. Accumulating data demonstrate that homocysteine may promote endothelial injury,
thus contributing to the development of vascular pathology. In vitro homocysteine induces oxidative stress,
evokes vascular inflammation, stimulates platelet aggregation or enhances binding of lipoproteins to fibrin [27].
A majority of clinical studies seem to support the view
that the increased serum homocysteine correlates with an
increased risk of stroke [3, 12, 30]. Other reports, however, do not confirm such an association [11, 16].
It was recently suggested that homocysteine may be
released from the damaged tissues, such as heart or
brain. Therefore, early after stroke or myocardial infarction, homocysteine level may be unchanged, whereas
later on it may increase [9, 16, 26]. It is also possible
that a lack of any association between serum homocysteine concentration and the risk of coronary heart disease or stroke is seen in the population of patients with
no evidence of pre-existing cardiovascular disease [20].
Indeed, a strong correlation between homocysteine and
the risk of ischemic stroke occurs among patients with
already existing atherosclerotic vascular disease [32].
Our results indicate that serum homocysteine concentration is not changed within 24 h after the stroke,
as compared to healthy subjects. These data are,
therefore, in agreement with studies showing no correlation between plasma homocysteine concentrations
and the occurrence of an acute vascular event [11, 16].
KYNA, detected in the mammalian brain and in
periphery, is known mainly as an endogenous antagonist of central glutamate and a7 nicotinic receptors.
Under experimental conditions, it displays strong
neuroprotective and anticonvulsant properties [31]. In
contrast, the role played by KYNA in the periphery is
not well recognized, but it has been established that
endothelium is a rich source of KYNA which is released to circulating blood [28, 29].
As demonstrated here, KYNA level correlated with
age of patients and with serum creatinine. These correlations were not shown before, but might be relatively easily explained. KYNA is excreted mainly
with urine, thus a renal dysfunction (frequently occurring among elderly, and manifested with increased serum creatinine) may cause the elevation of its serum
510
Pharmacological Reports, 2006, 58, 507–511
level [21]. Brain KYNA concentration correlates with
age, and parallels the increasing activity of KATs in
rodents [7]. Therefore, similar age-dependent increase
in serum KYNA in humans might be associated with
an enhanced activity of KYNA biosynthetic enzymes
and/or with the impaired renal function.
KYNA concentration was not increased among
stroke patients early after the insult. Similarly, global
ischemic episodes in gerbils did not change the hippocampal content of KYNA 24 h post-ischemia [14].
However, KYNA level correlated positively with the
serum level of homocysteine in both control and
stroke groups. Experimental studies in vitro and in
vivo demonstrated that homocysteine may modulate
KYNA levels in the brain and in the periphery [13,
29]. Homocysteine at pathophysiologically relevant
concentrations, i.e. 50–100 µM, stimulates KYNA formation in rat aortic rings, while at higher levels (> 0.4 mM)
it diminishes KYNA production [29]. In vivo, application of homocysteine increases serum KYNA level as
well as the content of KYNA within endothelium [29].
In the light of available data, it may be suggested
that the increased serum homocysteine level can be
followed by the enhanced production of endogenous
glutamate receptor antagonist, KYNA. This hypothesis, as well as the clinical significance of such correlation requires further studies on a larger cohort of patients, currently being in progress.
Acknowledgments:
This study was supported by a grant from the State Committee for
Scientific Research KBN PBZ – K018/P05/2001 and grant from
Medical University in Lublin D.S. 318/05.
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Received:
December 7, 2005; in revised form: June 26, 2006.
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