The L-arginine Pathway in Acute Ischemic Stroke and Severe Carotid Stenosis: Temporal Profiles and Association with Biomarkers and Outcome Tihamer Molnar, MD, PhD,* Gabriella Pusch, MD,† Viktoria Papp, MD,† Gergely Feher, MD, PhD,† Laszlo Szapary, MD, PhD,† Bernadett Biri, PhD,‡ Lajos Nagy, PhD,‡ Sandor Keki, PhD,‡ and Zsolt Illes, MD, PhDxjj
Background: Endothelial dysfunction is associated with increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) resulting in a decreased production of nitric oxide, which regulates the vascular tone. Methods: Patients with acute ischemic stroke (AIS, n 5 55) and asymptomatic significant carotid stenosis (AsCS, n 5 44) were prospectively investigated. L-arginine, ADMA, SDMA, S100 B, and high-sensitivity C-reactive protein (hsCRP) were serially measured within 6 hours after the onset of stroke, at 24 and 72 poststroke hours. All markers were compared with healthy subjects (n 5 45). The severity of AIS was daily assessed by National Institute of Health Stroke Scale scoring. Results: Even within 6 hours after the onset of stroke, L-arginine, ADMA, and SDMA were significantly higher in patients with AIS compared with both AsCS and healthy subjects. S100 B reflecting infarct size, positively correlated with the level of SDMA at 72 poststroke hours; changes in concentration of S100 B positively correlated with changes in the concentration of ADMA by 72 hours. Change in concentration of both ADMA and SDMA correlated with the change in concentration of hsCRP. Concentrations of Larginine and hsCRP at 72 poststroke hours, respectively, were independent predictors of poststroke infection. S100 B level measured within 6 hours after the onset of AIS and hsCRP at 72 poststroke hours were independent predictors of death. Conclusions: Metabolites of the L-arginine pathway were elevated in the very acute phase of ischemic stroke indicating a more pronounced endothelial dysfunction compared with AsCS. An increased basal L-arginine level in patients with AIS might be an adaptive mechanism; such transient elevation of the L-arginine/ADMA ratio at 24 poststroke hours may suggest that a temporary increase of L-arginine along with decrease of ADMA might be related to the protective role of L-arginine. Changes in the L-arginine pathway are predictive of poststroke infections. Key Words: L-arginine— asymmetric and symmetric dimethylarginine—stroke—carotid stenosis. Ó 2014 by National Stroke Association
From the *Department of Anesthesiology and Intensive Care, University of Pecs, Pecs, Hungary; †Department of Neurology, University of Pecs, Pecs, Hungary; ‡Department of Applied Chemistry, University of Debrecen, Debrecen, Hungary; xDepartment of Neurology, Odense University Hospital, Odense, Denmark; and jjInstitute of Clinical Research, University of Southern Denmark, Odense, Denmark. Received January 10, 2014; revision received April 29, 2014; accepted May 5, 2014.
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This study was supported by grants from the Hungarian Research Fund OTKA K 77892 and ETT 50053-2006 to Z.I. and a grant from University of Pecs AOK-KA- 34039/10-17 to T.M. Address correspondence to Tihamer Molnar, MD, PhD, Department of Anesthesiology and Intensive Care, University of Pecs, Hungary, Pecs, Ifjusag u. 13., Pecs 7623, Hungary. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.002
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 8 (September), 2014: pp 2206-2214
METHYLARGININE DERIVATIVES IN AIS AND ASYMPTOMATIC CAROTID STENOSIS
Stroke is the primary cause of disability and the second leading cause of death worldwide.1 Ischemic stroke is the most frequent type of stroke.2 Despite substantial efforts, strategies to reduce the incidence of stroke and to restrict acute brain injury are limited. Identifying basic factors in stroke pathogenesis and detrimental cascade of brain injury, such as endothelial dysfunction and oxidative stress, may help to define novel strategies of treatment.3,4 Nitric oxide (NO) can play a role in maintaining vascular integrity.5 NO is synthesized by a stereospecific oxidation of the terminal guanidino nitrogen of the amino acid L-arginine by the action of a family of nitric oxide synthases (NOSs). The synthesis of NO can be blocked with guanidino-substituted analogs of L-arginine, such as asymmetric dimethylarginine (ADMA) by inhibition of the NOS active site.6 ADMA and symmetric dimethylarginine (SDMA) are protein breakdown products of Larginine. ADMA directly inhibits endothelial nitric oxide synthase (eNOS) thus reducing NO production, whereas SDMA competes with the NO precursor arginine for uptake into endothelial cells.6,7 Numerous data support the beneficial effect of eNOS and vascular NO in the early stages of cerebral ischemia playing a prominent role in maintaining cerebral blood flow and preventing neuronal injury.8 Indeed, eNOS knockout mice show decreased blood flow in the ischemic border zone and develop larger cerebral infarctions.9 By contrast, enhancing NO production by administration of the eNOS substrate L-arginine increases regional cerebral blood flow in the ischemic territory and confers protection from stroke.10 Nevertheless, some NOS isoforms play a harmful role during cerebral ischemia; inducible nitric oxide synthase (iNOS) is induced by inflammation associated with ischemia, and contributes to secondary late-phase damage.8 In clinical and experimental studies, elevated level of ADMA is associated with endothelial dysfunction and atherosclerotic burden.5,6,11-13 Because high ADMA levels positively correlated with cardiovascular risk factors, the predictive value of ADMA for cardiovascular events and recently also for cerebrovascular events has been investigated.7,14 After the acute event of stroke, ADMA levels increase depending on stroke severity,15 which plays a role in brain injury by reducing the cerebral blood flow and promoting oxidative stress and inflammation.16,17 Although prognostic value of serum concentrations of methylated arginine derivatives has not been fully elucidated in stroke, an increase in both ADMA and SDMA plasma levels within the first 72 hours after the onset of ischemic stroke may predict poor outcome.14,15 Besides stroke, studies have revealed excessive ADMA accumulation in the presence of atherosclerotic risk factors and/or endothelial dysfunction even in patients without clinical evidence of atherosclerosis.18 ADMA has been considered to play a significant role in athero-
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sclerosis by inhibiting both eNOS and iNOS; iNOS triggers atherosclerosis via peroxynitrite formation.19 Higher plasma ADMA levels were found in cardiac syndrome X patients compared with normal controls, which positively correlated with common carotid artery (CCA) intima-media thickness (IMT).19 Higher plasma ADMA concentrations were also associated with greater internal carotid artery (ICA) or bulb IMT but not with CCA IMT, suggesting that ADMA may promote subclinical atherosclerosis in a site-specific manner.20 The aim of this study was to investigate the following: (1) the L-arginine pathway in patients with asymptomatic significant carotid stenosis (AsCS) and acute ischemic stroke (AIS) compared with age-matched healthy controls; (2) the longitudinal change of these metabolites during the acute phase of AIS; and (3) correlation of the L-arginine pathway with biomarkers reflecting infarct size and infection, such as S100 B and C-reactive protein.21-25 S100 B is an astroglial protein, which is elevated in the peripheral blood of patients with ischemic stroke, and its peak concentration around day 3 after stroke correlates with the infarct volume and clinical outcome.21-24 (4) We also examined the predictive value of these biomarkers on death, poststroke infection, and outcome defined by change in National Institute of Health Stroke Scale (NIHSS).
Material and Methods The study protocol was approved by the regional ethics committee and informed consent was obtained from each patient.
Patients and Samples A total of 55 patients suffering from AIS, 44 patients with AsCS were prospectively selected for this study. Forty-five age-matched (mean age, 64 6 16; male, 13) healthy blood donors served as normal controls. Medical history concerning previous coronary artery disease, chronic obstructive pulmonary disease, diabetes, hypertension, current cigarette smoking, serum creatinine levels, and body mass index was obtained from each patient (Table 1). All AIS patients were admitted to the Department of Neurology, University of Pecs, Hungary. Patients with acute neurologic deficits were examined by a certified neurologist; general medical history was collected, standard laboratory tests, 12-lead ECG, and cerebral computed tomography were performed in all cases on admission. Definitive stroke was defined according to international guidelines.26 Severity of stroke was measured by NIHSS and by the concentration of S100 B in the sera.27,28 To define worsening or improvement, NIHSS scale was daily assessed from day 1 to day 7 after admission. All AsCS patients were enrolled following carotis duplex scan and neurologic examination at the outpatient clinic of Department of Neurology, University of Pecs,
T. MOLNAR ET AL.
2208
Table 1. Demographic and clinical data of patients with acute ischemic stroke and asymptomatic significant carotid stenosis
Variables
AIS, N 5 55
AsCS, N 5 44
P value
Age, y 70 6 10 66 6 10 NS Male 28 (51) 29 (66) NS CAD 8 (14) 7 (16) NS COPD 7 (13) 5 (11) NS Smoking 12 (22) 13 (29) NS Hypertension 40 (72) 39 (88) NS Diabetes 7 (13) 8 (18) NS Creatinine (mmol/L) 75.5 6 14.5 74.5 6 13.8 NS Statin therapy 25 (45) 38 (86) ,.01 Clopidogrel 5 (9) 29 (66) ,.01 Aspirin 32 (58) 24 (55) NS NIHSS on admission 11.5 6 5.4 NA NA NIHSS on emission 8.4 6 6.4 NA NA Poststroke infection 16 (29) NA NA Death 13 (24) NA NA Abbreviations: AIS, acute ischemic stroke; AsCS, asymptomatic significant carotid stenosis; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; NIHSS, National Institute of Health Stroke Scale; NA, not applicable. Values are presented as mean 6 standard deviation or as numbers (relative percentages), respectively (chi-square test and independent samples t test).
Hungary. Duplex scanning was performed bilaterally with a Philips HD7XE by using a 5-10 MHz linear probe. Duplex ultrasound examination included grayscale and color Doppler imaging in the transverse and sagittal planes. Duplex scan measurements of ICA peak systolic velocity (PSV), ICA end-diastolic velocity, and the ratio of ICA PSV to CCA PSV were analyzed to identify a 70%-99% ICA stenosis. We used the Carotid Consensus Panel criteria to diagnose significant carotid artery stenosis (PSV .230 cm/second, end-diastolic velocity .100 cm/second, ICA/CCA ratio .4).29 In agreement with international guidelines, significant carotid atherosclerosis was defined by 70%-100% stenosis of ICA determined by duplex scan sonography.30 Scans were performed to detect carotid stenosis as a common comorbidity of peripheral arterial disease or coronary disease or because of nonspecific symptoms (ie, dizziness). Exclusion criteria were infectious disease, fever ,4 weeks before stroke, an elevated white blood cell (WBC), erythrocyte sedimentation rate, high-sensitivity C-reactive protein (hsCRP, cutoff value ,10 mg/L), hemorrhagic stroke defined by an acute cranial computed tomography scan, chronic renal failure (creatinine .120 mmol/L), and decline to participate in the study.
Infectious Complications An evidence-based guideline was followed to detect infectious complications (in short, physical and laboratory
measures including WBC, erythrocyte sedimentation rate, hsCRP, procalcitonin (PCT), fever, abnormal urine, chest X-ray, or positive cultures).31
Blood Sampling Venous blood samples from AIS patients were taken serially for measurement of ADMA, SDMA, L-arginine, hsCRP, and S100 B within 6 hours after onset of first symptoms, at 24 and 72 hours. Blood samples were centrifuged at 3000 3 g for 10 minutes. Supernatants were frozen and stored at 280 C until analysis. Serum levels of S100 B were examined by automated electrochemiluminescent immunoassay (Liaison Sangtec 100 system; DiaSorin, Bromma, Sweden) and serum levels of hsCRP were examined by automated fluorescence immunoassay (BRAHMS Kryptor, Berlin, Germany).32
Determination of L-arginine and Arginine Derivatives The amino acid content of the blood serum samples was retrieved by solid-phase extraction method and was quantified by High Performance Liquid Chromatography (HPLC) after derivatization. Solid-phase extraction of the analytes was performed as previously described.33,34 Arginine and homoarginine were detected at lex 5 337 nm, lem 5 520 nm, and lem 5 454 nm was used for ADMA and SDMA.
Statistical Analysis Statistical calculation was performed using the SPSS 11.0 (Chicago, IL, USA) package. One-way ANOVA, Chisquare test, Spearman correlation and Mann-Whitney U test were used. Multiple logistic regression was applied to confirm independent predictors. Data were presented as mean, 95% confidence intervals (CIs) and median, interquartile ranges. A P value ,.05 was regarded as significant.
Results Demographics, risk factors, outcome measures, and medication are summarized in Table 1.
Baseline Levels of Metabolites within 6 Hours after Onset of Stroke Concentrations of all 3 metabolites (L-arginine, ADMA, and SDMA) within 6 hours after the onset of stroke were significantly higher in the sera of patients with AIS compared with both healthy controls and patients with AsCS (P ,.001, respectively). SDMA was also lower in patients with AsCS compared with healthy controls (Fig 1).
Longitudinal Changes of Metabolites of the L-arginine Pathway in the Acute Phase of AIS We investigated changes in the serum concentrations of L-arginine, ADMA, and SDMA within 6 hours after onset
METHYLARGININE DERIVATIVES IN AIS AND ASYMPTOMATIC CAROTID STENOSIS L-arginine (umol/l) 160
Figure 1. Comparison of L-arginine and methylarginine derivatives in acute ischemic stroke (AIS), asymptomatic significant carotid stenosis (AsCS), and healthy subjects (HS). Serum concentration of L-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) in patients with AIS within 6 hours after onset of symptoms, with AsCS, and in HS. Data are presented as mean and 95% confidence interval.
p
Background: Endothelial dysfunction is associated with increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) resulting in a decreased production of nitric oxide, which regulates the vascular tone. Methods: Patients with acute ischemic stroke (AIS, n 5 55) and asymptomatic significant carotid stenosis (AsCS, n 5 44) were prospectively investigated. L-arginine, ADMA, SDMA, S100 B, and high-sensitivity C-reactive protein (hsCRP) were serially measured within 6 hours after the onset of stroke, at 24 and 72 poststroke hours. All markers were compared with healthy subjects (n 5 45). The severity of AIS was daily assessed by National Institute of Health Stroke Scale scoring. Results: Even within 6 hours after the onset of stroke, L-arginine, ADMA, and SDMA were significantly higher in patients with AIS compared with both AsCS and healthy subjects. S100 B reflecting infarct size, positively correlated with the level of SDMA at 72 poststroke hours; changes in concentration of S100 B positively correlated with changes in the concentration of ADMA by 72 hours. Change in concentration of both ADMA and SDMA correlated with the change in concentration of hsCRP. Concentrations of Larginine and hsCRP at 72 poststroke hours, respectively, were independent predictors of poststroke infection. S100 B level measured within 6 hours after the onset of AIS and hsCRP at 72 poststroke hours were independent predictors of death. Conclusions: Metabolites of the L-arginine pathway were elevated in the very acute phase of ischemic stroke indicating a more pronounced endothelial dysfunction compared with AsCS. An increased basal L-arginine level in patients with AIS might be an adaptive mechanism; such transient elevation of the L-arginine/ADMA ratio at 24 poststroke hours may suggest that a temporary increase of L-arginine along with decrease of ADMA might be related to the protective role of L-arginine. Changes in the L-arginine pathway are predictive of poststroke infections. Key Words: L-arginine— asymmetric and symmetric dimethylarginine—stroke—carotid stenosis. Ó 2014 by National Stroke Association
From the *Department of Anesthesiology and Intensive Care, University of Pecs, Pecs, Hungary; †Department of Neurology, University of Pecs, Pecs, Hungary; ‡Department of Applied Chemistry, University of Debrecen, Debrecen, Hungary; xDepartment of Neurology, Odense University Hospital, Odense, Denmark; and jjInstitute of Clinical Research, University of Southern Denmark, Odense, Denmark. Received January 10, 2014; revision received April 29, 2014; accepted May 5, 2014.
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This study was supported by grants from the Hungarian Research Fund OTKA K 77892 and ETT 50053-2006 to Z.I. and a grant from University of Pecs AOK-KA- 34039/10-17 to T.M. Address correspondence to Tihamer Molnar, MD, PhD, Department of Anesthesiology and Intensive Care, University of Pecs, Hungary, Pecs, Ifjusag u. 13., Pecs 7623, Hungary. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.002
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 8 (September), 2014: pp 2206-2214
METHYLARGININE DERIVATIVES IN AIS AND ASYMPTOMATIC CAROTID STENOSIS
Stroke is the primary cause of disability and the second leading cause of death worldwide.1 Ischemic stroke is the most frequent type of stroke.2 Despite substantial efforts, strategies to reduce the incidence of stroke and to restrict acute brain injury are limited. Identifying basic factors in stroke pathogenesis and detrimental cascade of brain injury, such as endothelial dysfunction and oxidative stress, may help to define novel strategies of treatment.3,4 Nitric oxide (NO) can play a role in maintaining vascular integrity.5 NO is synthesized by a stereospecific oxidation of the terminal guanidino nitrogen of the amino acid L-arginine by the action of a family of nitric oxide synthases (NOSs). The synthesis of NO can be blocked with guanidino-substituted analogs of L-arginine, such as asymmetric dimethylarginine (ADMA) by inhibition of the NOS active site.6 ADMA and symmetric dimethylarginine (SDMA) are protein breakdown products of Larginine. ADMA directly inhibits endothelial nitric oxide synthase (eNOS) thus reducing NO production, whereas SDMA competes with the NO precursor arginine for uptake into endothelial cells.6,7 Numerous data support the beneficial effect of eNOS and vascular NO in the early stages of cerebral ischemia playing a prominent role in maintaining cerebral blood flow and preventing neuronal injury.8 Indeed, eNOS knockout mice show decreased blood flow in the ischemic border zone and develop larger cerebral infarctions.9 By contrast, enhancing NO production by administration of the eNOS substrate L-arginine increases regional cerebral blood flow in the ischemic territory and confers protection from stroke.10 Nevertheless, some NOS isoforms play a harmful role during cerebral ischemia; inducible nitric oxide synthase (iNOS) is induced by inflammation associated with ischemia, and contributes to secondary late-phase damage.8 In clinical and experimental studies, elevated level of ADMA is associated with endothelial dysfunction and atherosclerotic burden.5,6,11-13 Because high ADMA levels positively correlated with cardiovascular risk factors, the predictive value of ADMA for cardiovascular events and recently also for cerebrovascular events has been investigated.7,14 After the acute event of stroke, ADMA levels increase depending on stroke severity,15 which plays a role in brain injury by reducing the cerebral blood flow and promoting oxidative stress and inflammation.16,17 Although prognostic value of serum concentrations of methylated arginine derivatives has not been fully elucidated in stroke, an increase in both ADMA and SDMA plasma levels within the first 72 hours after the onset of ischemic stroke may predict poor outcome.14,15 Besides stroke, studies have revealed excessive ADMA accumulation in the presence of atherosclerotic risk factors and/or endothelial dysfunction even in patients without clinical evidence of atherosclerosis.18 ADMA has been considered to play a significant role in athero-
2207
sclerosis by inhibiting both eNOS and iNOS; iNOS triggers atherosclerosis via peroxynitrite formation.19 Higher plasma ADMA levels were found in cardiac syndrome X patients compared with normal controls, which positively correlated with common carotid artery (CCA) intima-media thickness (IMT).19 Higher plasma ADMA concentrations were also associated with greater internal carotid artery (ICA) or bulb IMT but not with CCA IMT, suggesting that ADMA may promote subclinical atherosclerosis in a site-specific manner.20 The aim of this study was to investigate the following: (1) the L-arginine pathway in patients with asymptomatic significant carotid stenosis (AsCS) and acute ischemic stroke (AIS) compared with age-matched healthy controls; (2) the longitudinal change of these metabolites during the acute phase of AIS; and (3) correlation of the L-arginine pathway with biomarkers reflecting infarct size and infection, such as S100 B and C-reactive protein.21-25 S100 B is an astroglial protein, which is elevated in the peripheral blood of patients with ischemic stroke, and its peak concentration around day 3 after stroke correlates with the infarct volume and clinical outcome.21-24 (4) We also examined the predictive value of these biomarkers on death, poststroke infection, and outcome defined by change in National Institute of Health Stroke Scale (NIHSS).
Material and Methods The study protocol was approved by the regional ethics committee and informed consent was obtained from each patient.
Patients and Samples A total of 55 patients suffering from AIS, 44 patients with AsCS were prospectively selected for this study. Forty-five age-matched (mean age, 64 6 16; male, 13) healthy blood donors served as normal controls. Medical history concerning previous coronary artery disease, chronic obstructive pulmonary disease, diabetes, hypertension, current cigarette smoking, serum creatinine levels, and body mass index was obtained from each patient (Table 1). All AIS patients were admitted to the Department of Neurology, University of Pecs, Hungary. Patients with acute neurologic deficits were examined by a certified neurologist; general medical history was collected, standard laboratory tests, 12-lead ECG, and cerebral computed tomography were performed in all cases on admission. Definitive stroke was defined according to international guidelines.26 Severity of stroke was measured by NIHSS and by the concentration of S100 B in the sera.27,28 To define worsening or improvement, NIHSS scale was daily assessed from day 1 to day 7 after admission. All AsCS patients were enrolled following carotis duplex scan and neurologic examination at the outpatient clinic of Department of Neurology, University of Pecs,
T. MOLNAR ET AL.
2208
Table 1. Demographic and clinical data of patients with acute ischemic stroke and asymptomatic significant carotid stenosis
Variables
AIS, N 5 55
AsCS, N 5 44
P value
Age, y 70 6 10 66 6 10 NS Male 28 (51) 29 (66) NS CAD 8 (14) 7 (16) NS COPD 7 (13) 5 (11) NS Smoking 12 (22) 13 (29) NS Hypertension 40 (72) 39 (88) NS Diabetes 7 (13) 8 (18) NS Creatinine (mmol/L) 75.5 6 14.5 74.5 6 13.8 NS Statin therapy 25 (45) 38 (86) ,.01 Clopidogrel 5 (9) 29 (66) ,.01 Aspirin 32 (58) 24 (55) NS NIHSS on admission 11.5 6 5.4 NA NA NIHSS on emission 8.4 6 6.4 NA NA Poststroke infection 16 (29) NA NA Death 13 (24) NA NA Abbreviations: AIS, acute ischemic stroke; AsCS, asymptomatic significant carotid stenosis; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; NIHSS, National Institute of Health Stroke Scale; NA, not applicable. Values are presented as mean 6 standard deviation or as numbers (relative percentages), respectively (chi-square test and independent samples t test).
Hungary. Duplex scanning was performed bilaterally with a Philips HD7XE by using a 5-10 MHz linear probe. Duplex ultrasound examination included grayscale and color Doppler imaging in the transverse and sagittal planes. Duplex scan measurements of ICA peak systolic velocity (PSV), ICA end-diastolic velocity, and the ratio of ICA PSV to CCA PSV were analyzed to identify a 70%-99% ICA stenosis. We used the Carotid Consensus Panel criteria to diagnose significant carotid artery stenosis (PSV .230 cm/second, end-diastolic velocity .100 cm/second, ICA/CCA ratio .4).29 In agreement with international guidelines, significant carotid atherosclerosis was defined by 70%-100% stenosis of ICA determined by duplex scan sonography.30 Scans were performed to detect carotid stenosis as a common comorbidity of peripheral arterial disease or coronary disease or because of nonspecific symptoms (ie, dizziness). Exclusion criteria were infectious disease, fever ,4 weeks before stroke, an elevated white blood cell (WBC), erythrocyte sedimentation rate, high-sensitivity C-reactive protein (hsCRP, cutoff value ,10 mg/L), hemorrhagic stroke defined by an acute cranial computed tomography scan, chronic renal failure (creatinine .120 mmol/L), and decline to participate in the study.
Infectious Complications An evidence-based guideline was followed to detect infectious complications (in short, physical and laboratory
measures including WBC, erythrocyte sedimentation rate, hsCRP, procalcitonin (PCT), fever, abnormal urine, chest X-ray, or positive cultures).31
Blood Sampling Venous blood samples from AIS patients were taken serially for measurement of ADMA, SDMA, L-arginine, hsCRP, and S100 B within 6 hours after onset of first symptoms, at 24 and 72 hours. Blood samples were centrifuged at 3000 3 g for 10 minutes. Supernatants were frozen and stored at 280 C until analysis. Serum levels of S100 B were examined by automated electrochemiluminescent immunoassay (Liaison Sangtec 100 system; DiaSorin, Bromma, Sweden) and serum levels of hsCRP were examined by automated fluorescence immunoassay (BRAHMS Kryptor, Berlin, Germany).32
Determination of L-arginine and Arginine Derivatives The amino acid content of the blood serum samples was retrieved by solid-phase extraction method and was quantified by High Performance Liquid Chromatography (HPLC) after derivatization. Solid-phase extraction of the analytes was performed as previously described.33,34 Arginine and homoarginine were detected at lex 5 337 nm, lem 5 520 nm, and lem 5 454 nm was used for ADMA and SDMA.
Statistical Analysis Statistical calculation was performed using the SPSS 11.0 (Chicago, IL, USA) package. One-way ANOVA, Chisquare test, Spearman correlation and Mann-Whitney U test were used. Multiple logistic regression was applied to confirm independent predictors. Data were presented as mean, 95% confidence intervals (CIs) and median, interquartile ranges. A P value ,.05 was regarded as significant.
Results Demographics, risk factors, outcome measures, and medication are summarized in Table 1.
Baseline Levels of Metabolites within 6 Hours after Onset of Stroke Concentrations of all 3 metabolites (L-arginine, ADMA, and SDMA) within 6 hours after the onset of stroke were significantly higher in the sera of patients with AIS compared with both healthy controls and patients with AsCS (P ,.001, respectively). SDMA was also lower in patients with AsCS compared with healthy controls (Fig 1).
Longitudinal Changes of Metabolites of the L-arginine Pathway in the Acute Phase of AIS We investigated changes in the serum concentrations of L-arginine, ADMA, and SDMA within 6 hours after onset
METHYLARGININE DERIVATIVES IN AIS AND ASYMPTOMATIC CAROTID STENOSIS L-arginine (umol/l) 160
Figure 1. Comparison of L-arginine and methylarginine derivatives in acute ischemic stroke (AIS), asymptomatic significant carotid stenosis (AsCS), and healthy subjects (HS). Serum concentration of L-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) in patients with AIS within 6 hours after onset of symptoms, with AsCS, and in HS. Data are presented as mean and 95% confidence interval.
p
