Plasma levels of lipophilic antioxidant vitamins in acute ischemic stroke patients: correlation to inflammation markers and neurological deficits

Nutrition 21 (2005) 987–993 www.elsevier.com/locate/nut

Applied nutritional investigation

Plasma levels of lipophilic antioxidant vitamins in acute ischemic stroke patients: correlation to inflammation markers and neurological deficits Chia-Yu Chang, M.S., M.D.a, Jen-Yin Chen, M.D.b, Dershin Ke, M.D., Ph.D.a, and Miao-Lin Hu, Ph.D.c,ⴱ a Department of Neurology, Chi-Mei Foundation Hospital, Tainan, Taiwan, ROC Department of Anesthesiology, Chi-Mei Foundation Hospital, Tainan, Taiwan, ROC c Department of Food Science, National Chung-Hsing University, Taichung, Taiwan, ROC b

Manuscript received November 28, 2004; accepted February 2, 2005.

Abstract

Objectives: Acute ischemic stroke is a clinical condition accompanied by inflammation and oxidative stress. In this study, we compared levels of plasma lipophilic antioxidants and inflammation markers between patients with stroke and healthy controls and assessed the associations of antioxidants, inflammation markers, and neurologic deficits among patients with stroke. Methods: We measured plasma levels of lipophilic antioxidant vitamins (retinol, lycopene, ␣-carotene, ␤-carotene, ␣-tocopherol, and ␥-tocopherol), inflammation markers (high-sensitivity C-reactive protein [hs-CRP], fibrinogen, erythrocyte sedimentation rate, and white blood cell count), and neurologic deficits (indicated by the score of the National Institute of Health Stroke Scale) in 68 patients with acute ischemic stroke within 48 h after stroke onset in comparison with 41 normal controls. Results: Plasma ␣- and ␤-carotene concentrations were lower and levels of inflammation markers were higher among patients with acute ischemic stroke compared with normal controls. Levels of ␣- and ␤-carotene in patients with stroke were negatively associated with hs-CRP level (R ⫽ ⫺0.29 and ⫺0.41, respectively, P ⬍ 0.01) and with neurologic deficits (R ⫽ ⫺0.28 and ⫺0.27, respectively, P ⬍ 0.05). The negative association between neurologic deficits and combined plasma levels of ␣- and ␤-carotene remained after adjustment for age and sex (P ⫽ 0.04). However, the magnitude of association decreased after adjustment of hs-CRP (P ⫽ 0.08). Conclusion: Plasma concentrations of ␣- and ␤-carotene are lower in patients with acute ischemic stroke than in healthy controls and are negatively correlated with hs-CRP level and neurologic deficits. The negative association between neurologic deficits and combined plasma ␣- and ␤-carotene levels is confounded by hs-CRP. © 2005 Elsevier Inc. All rights reserved.

Keywords:

Acute ischemic stroke patients; Antioxidant vitamins; Inflammation markers

Introduction Acute ischemic stroke is a sudden loss of brain function resulting from interference with the blood supply to the

This research was supported by grant CMFH 9021 from the Chi-Mei Foundation Hospital (Tainan, Taiwan) and grant nsc-89-2320-b005-004 from the National Science Council. * Corresponding author. Tel.: ⫹886-4-281-2363; fax: ⫹886-4-2876211. E-mail address: [email protected] (M.-L. Hu). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.02.010

central nervous system. It is a leading cause of mortality and disability in many countries [1]. Brain injury after acute ischemic stroke develops from a complex series of pathophysiologic events including oxidative stress and inflammation [2]. An increased free radical generation during cerebral ischemia/reperfusion injury has been shown in experimental studies using several techniques such as microdialysis, salicylate spin trapping, and electron paramagnetic resonance [3–5]. In human studies, most plasma carotenoids have been shown to be decreased immediately after an ischemic stroke, possibly a consequence of increased

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oxidative stress, as indicated by a concomitant increase in malondialdehyde concentration [6]. In a previous study, we also found that patients with acute ischemic stroke had lower levels of cholesterol-adjusted carotenoids and ␣-tocopherol but higher levels of thiobarbituric acid reactive substances in plasma than did controls [7]. Further, Cherubini et al. [8] found that most antioxidants are decreased immediately after an acute ischemic stroke, and that plasma levels of vitamin C are directly correlated with functional status and neurologic impairment, whereas plasma vitamin A concentrations are inversely correlated with both parameters. All these findings suggest that measurements of antioxidant concentrations in patients with ischemic stroke may have some clinical implications because the status of antioxidants is related to our defense capacity against oxidative stress and may be related to clinical outcome. Recently, accumulating evidence has suggested that ischemia-induced inflammation plays a critical role in the acute phase after stroke [9]. A marked inflammatory reaction initiated by ischemia subsequently induces expression of cytokines, adhesion molecules, and other inflammatory mediators that could contribute to further ischemic damage [10]. After acute ischemic stroke, a sustained inflammatory response has been reported in about 75% of patients, as suggested by increased level of C-reactive protein (CRP) detected after stroke [11]. A strong and persistent inflammatory response is associated with a worsened outcome [12]. Moreover, CRP at discharge has been shown to be related to a 1-year outcome [13]. However, other inflammatory markers such as erythrocyte sedimentation rate (ESR) and fibrinogen concentrations were not found to be correlated with an early stroke outcome, although ESR was subsequently noted to be correlated with stroke outcome measured 8 to 12 mo after the acute insult [14]. Inflammation may cause changes in some nutritional biomarkers such as serum retinol and carotenoids as a consequence of the acute-phase response of patients [15]. For instance, recent re-evaluations of epidemiologic studies of the Third National Health and Nutrition Examination Survey have shown that serum retinol concentrations are lower in subjects with high serum CRP concentrations (ⱖ10 mg/ dL) than in other subjects of the same age and sex [16]. A nationally representative survey also showed that serum ␤-carotene concentration is strongly and inversely associated with systemic markers of inflammation (CRP level and white blood cell [WBC] count), suggesting that the relation between serum ␤-carotene concentration and disease risk may be confounded by inflammation [17]. In addition, the Third National Health and Nutritional Survey of 1988 to 1994, in which 4557 non-smoking participants 25 to 55 y old were analyzed, showed that adjusted concentrations of all five carotenoids (␣-carotene, ␤-carotene, ␣-cryptoxanthin, lycopene, and lutein/zeaxanthin) are significantly lower in those with CRP levels above 0.88 mg/dL (P ⫽ 0.001) [18]. Moreover, the Nun study in older women

showed that significant decreases in plasma concentrations of lycopene, ␣-carotene, ␤-carotene, and total carotenoids are associated with an increased serum concentration of CRP [19]. Although plasma antioxidant levels and inflammation status in patients with acute ischemic stroke could be correlated with stroke outcome, more studies are required to clarify their relations. In this study we assessed whether plasma lipophilic antioxidants are associated with inflammation status in patients with acute ischemic stroke and whether the two parameters are predictors of early stroke outcome. We hypothesized that the plasma levels of lipophilic antioxidant vitamins are lower in patients with acute ischemic stroke than in healthy controls and that the antioxidant levels are correlated with measurements of inflammation and of neurologic deficits after acute ischemic stroke.

Materials and methods Subjects Sixty-eight patients who had their first-ever ischemic stroke and were admitted to the Neurological Ward of the Chi-Mei Medical Center (Tainan, Taiwan) within 48 h after stroke onset between April 2002 and August 2003 were consecutively enrolled. Ischemic stroke was defined as a sudden focal neurologic deficit persisting for longer than 24 h with no evidence of hemorrhage in the initial cranial image study. Forty-one control subjects were healthy volunteers from among relatives of outpatients or hospital employees. Those who had taken vitamin or herbal supplements and had an infection during the 3 mo previously to their recruitment were excluded from the study. The study was approved by the institutional review committee of the Chi-Mei Medical Center and informed consent was obtained from each participant. All patients underwent brain computed tomography or magnetic resonance imaging and detailed neurologic and physical examinations. Basic information including demographics, body mass index, and vascular risk factors was collected. Ischemic stroke subtypes were classified into five categories: 1) large-artery atherosclerosis, 2) cardiac embolism, 3) small-artery occlusion, 4) stroke of other determined etiology, and 5) stroke of undetermined etiology [20]. However, we included only those patients who were classified as having large- or small-artery atherosclerosis. Patients in the three other categories were not included because of uncertainty of their classification. Severity of neurologic deficit was measured with the National Institute of Health (NIH) Stroke Scale [21], which was administered at times of admission and discharge. The NIH Stroke Scale is a 24-point scale (11 items), with 0 being a normal score; the higher the score, the worse the neurologic deficit. Functional status of patients was evaluated by the Barthel Index [22] and the Modified Rankin Scale [23] at time of discharge.

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Blood collection and plasma preparation In the patient group, blood was collected in tubes containing ethylene-diaminetetra-acetic acid or Vacutainer tubes containing no anticoagulant (Becton Dickinson gel separator tubes, Becton Dickinson, New Jersey, USA) the morning after admission after an overnight fast for measurement of biochemical and lipophilic antioxidant vitamins. In the control group, blood was obtained in the morning after an overnight fast for the same measurements. After centrifugation at 3000g at 4°C for 10 min, plasma was frozen at ⫺80°C and shipped by express mail to the Redox Functional Medicine Laboratory at Taipei for antioxidant measurements. Measurements of lipophilic antioxidants [24,25] An aliquot of plasma (120 ␮L) was deproteinized with 300 ␮L of ethanol and then extracted with 4.0 mL of hexane. The supernatant was centrifuged (2000g for 5 min), the hexane layer was evaporated under nitrogen at 45°C, and the residue was reconstituted by mobile phase. A 20-␮L portion of this solution was injected into the highperformance liquid chromatographic system for measurements of lipophilic antioxidants. Stock solutions (stored at ⫺20°C) of pure retinol and tocopherols (Sigma Chemical Co., St. Louis, MO, USA) were dissolved in hexane; lycopene and the two carotenes (Sigma Chemical Co.) were first dissolved in small quantities of dichloromethane and then diluted with n-hexane for daily calibration. High-performance liquid chromatographic grade acetonitrile, tetrahydrofuran, methanol, and a Lichrospher 100 RP-18 column (5 ␮m, 4 ⫻ 125 mm) were purchased from Merck (Darmstadt, Germany). The highperformance liquid chromatograph was equipped with the following modules: Waters 2478 dural ␭-absorbance detectors, Waters 486 scanning fluorescence detectors, and Waters 2690 separation modules (Waters and Associates, Milford, MA, USA). The mobile phase (acetonitrile:tetrahydrofuran: methanol:water, 70:10:15:5 v/v/v/v) was filtered by a 0.45-␮m GHP acrodisc syringe (Gelman Sciences, Ann Arbor, MI, USA) and pumped at 1.0 mL/min. The ultraviolet-visible detector was programmed to monitor at 325 nm for retinol from 0 to 3 minutes, at 470 nm for lycopene from 3 to 6.5 minutes, and at 445 nm for ␣- and ␤-carotene from 6.5 to 10 minutes. The fluorescence detector was programmed to monitor at 298/ 328 nm (excitation/emission) for ␣- and ␤-tocopherol from 3 to 5 minutes. The reproducibility of the method was checked by multiple determinations of the pooled human plasma on the same day (intrabatch precision). The following coefficients of variance were obtained: 2.5% for retinol (n ⫽ 8), 5.2% for lycopene (n ⫽ 8), 2.9% for ␣-carotene (n ⫽ 8), 3% for ␤-carotene (n ⫽ 8), 4% for ␣-tocopherol (n ⫽ 8), and 3.6% for r-tocopherol (n ⫽ 8). The same pooled human plasma was used for each assay on 8 consecutive days to test day-to-day reproducibility of the method. The interbatch coefficients of variance were 4.5% for retinol (n ⫽ 8), 3.5%

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for lycopene (n ⫽ 8), 5.3% for ␣-carotene (n ⫽ 8), 6.2% for ␤-carotene (n ⫽ 8), 4.2% for ␣-tocopherol (n ⫽ 8), and 3.7% for r-tocopherol (n ⫽ 8). Biochemical measurements Plasma total cholesterol, high-density lipoprotein cholesterol, and triacylglycerols were determined by using commercially available enzymatic kits. Low-density lipoprotein (LDL) cholesterol concentration was calculated by Friedewald’s formula. Blood glucose was determined colorimetrically by the D-glucose oxidase method [26]. Serum high-sensitivity CRP (hs-CRP) concentrations were measured by high-sensitive latex-enhanced immunonephelometry (BN II nephelometer, Dade Behring, Deerfield, Illinois, USA). Plasma fibrinogen was assayed by using Dade fibrinogen determination reagents on a CA-1500 analyzer (Sysmex, Mundelein, Illinois, USA). ESR was measured by Westergreen’s method [27]. Statistical analysis Data are expressed as mean ⫾ standard deviation, percentage, or median with interquartile ranges, as appropriate. Statistics differences were evaluated with the KruskalWallis test for comparison across three groups, followed by Wilcoxon’s rank-sum test for comparison between two groups with Bonferroni’s correction. Chi-square test was used for analysis of categorical variables such as sex and smoking among groups. Bivariate correlations were performed using Spearman’s rank correlation test. Multivariate linear regression analysis was performed by using the score of the NIH Stroke Scale as a dependent variable and stroke type (large or small artery), ␣- plus ␤-carotene (␣/␤-carotene), and logarithmically transformed hs-CRP as independent variables. Statistical analysis was carried out with SPSS 10.0.7 (SPSS, Inc., Chicago, IL, USA). Probability values lower than 0.05 were considered statistically significant.

Results Sixty-eight patients with acute ischemic stroke and 41 control subjects formed the study population (Table 1). There were no significant differences in age, sex, body mass index, smoking status, plasma total cholesterol, and LDL cholesterol between patients and control subjects. Patients who had large- or small-artery ischemic stroke had significantly lower plasma levels of high-density lipoprotein cholesterol and albumin but a higher level of blood glucose than did control subjects. Hypertension and diabetes mellitus were more prevalent in patients with ischemic stroke than in control subjects. However, no significant differences were found in any of the above measurements between patients who had small-artery ischemic stroke and those who had large-artery ischemic stroke.

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Table 1 Clinical characteristics of control subjects and patients with acute ischemic stroke*

Age (y) Men (%) Body mass index (kg/m2) Smoking (%) Hypertension (%) Diabetes mellitus (%) Albumin (mg/dL) Total plasma cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Plasma triacyglycerols (mg/dL) Blood glucose (mg/dL)

Control subjects (n ⫽ 41)

Patients with ischemic stroke Small artery (n ⫽ 35)

Large artery (n ⫽ 33)

60 ⫾ 8 (60) 56 24.5 ⫾ 3.2 (25.0) 27 27a 5a 4.5 ⫾ 0.3 (4.5)a 209 ⫾ 30 (208) 61 ⫾ 20 (59)a 134 ⫾ 34 (136) 145 ⫾ 125 (114) 102 ⫾ 21 (95)a

65 ⫾ 10 (66) 57 24.4 ⫾ 3.5 (25.0) 29 60b 40b 3.9 ⫾ 0.4 (3.9)b 208 ⫾ 43 (215) 50 ⫾ 15 (48)b 137 ⫾ 36 (140) 141 ⫾ 79 (116) 135 ⫾ 57 (113)b

63 ⫾ 14 (60) 55 24.5 ⫾ 3.3 (24.5) 39 48b 21b 3.8 ⫾ 0.5 (3.9)b 189 ⫾ 59 (197) 47 ⫾ 10 (47)b 126 ⫾ 44 (126) 134 ⫾ 86 (127) 132 ⫾ 75 (103)b

HDL, high-density lipoprotein; LDL, low-density lipoprotein. * Data are expressed as percentags for sex, body mass index, smoking, hypertension, and diabetes mellitus or means ⫾ standard deviations (medians) for other measurements. Values within a row with different superscript letters are significantly different (P ⬍ 0.05).

Plasma levels of lipophilic antioxidant vitamins and inflammation markers Absolute amounts of plasma retinol, ␣-carotene, and ␤-carotene were significantly lower in patients with acute ischemic stroke than in control subjects (Table 2). When lipophilic antioxidants were adjusted by plasma cholesterol, only ␣- and ␤-carotene remained significantly lower in the patient group. However, there was no significant difference in the lipophilic antioxidant vitamin plasma levels between patients who had small-artery stroke and those who had large-artery stroke.

Biochemical markers of inflammation assayed, i.e., hsCRP, fibrinogen, ESR, and WBC count, were higher in patients who had large-artery stroke and those who had small-artery stroke than in control subjects (Table 2). In patients as a whole, serum hs-CRP was highly correlated with plasma fibrinogen (r ⫽ 0.48, P ⬍ 0.001), ESR (r ⫽ 0.36, P ⫽ 0.003), and WBC count (r ⫽ 0.33, P ⫽ 0.007). Relations among plasma levels of lipophilic antioxidant vitamins, inflammation markers, and neurologic deficits Table 3 shows that plasma levels of retinol, lycopene, ␣-carotene, and ␤-carotene were, in general, significantly

Table 2 Plasma levels of lipophilic antioxidant vitamins and ratios of lipophilic antioxidant vitamins to cholesterol, and inflammatory markers in control subjects and patients with acute ischemic stroke* Control (n ⫽ 41)

Antioxidant vitamins Retinol (␮mol/L) Retinol /cholesterol (␮mol/mmol) Lycopene (␮mol/L) Lycopene /cholesterol (␮mol/mmol) ␣-Carotene (␮mol/L) ␣-Carotene/cholesterol (␮mol/mmol) ␤-Carotene (␮mol/L) ␤-Carotene/cholesterol (␮mol/mmol) ␣-Tocopherol (␮mol/L) ␣-Tocopherol/cholesterol (␮mol/mmol) ␥-Tocopherol (␮mol/L) ␥-Tocopherol/cholesterol (␮mol/mmol) Inflammation markers Fibrinogen (mg/dL) hs-CRP (mg/L) ESR (mm/h) WBC count (k/␮L)

1.97 ⫾ 0.55 (1.89)a 0.37 ⫾ 0.12 (0.35) 0.13 ⫾ 0.09 (0.09) 0.025 ⫾ 0.019 (0.019) 0.12 ⫾ 0.08 (0.10)a 0.022 ⫾ 0.014 (0.018)a 0.64 ⫾ 0.40 (0.58)a 0.12 ⫾ 0.08 (0.10)a 25.7 ⫾ 6.2 (24.6) 4.76 ⫾ 0.87 (4.77) 2.98 ⫾ 1.53 (2.55) 0.55 ⫾ 0.27 (0.47) 281 ⫾ 44 (278)a 1.6 ⫾ 1.7 (1.1)a 13 ⫾ 10 (10)a 6.0 ⫾ 1.5 (5.8)a

Patients with ischemic stroke Small artery (n ⫽ 35)

Large artery (n ⫽ 33)

1.63 ⫾ 0.44 (1.59)b 0.31 ⫾ 0.09 (0.30) 0.10 ⫾ 0.07 (0.07) 0.019 ⫾ 0.013 (0.013) 0.09 ⫾ 0.08 (0.06)b 0.016 ⫾ 0.013 (0.012)b 0.50 ⫾ 0.41 (0.32)b 0.09 ⫾ 0.07 (0.07)b 24.9 ⫾ 8.2 (22.9) 4.72 ⫾ 1.75 (4.35) 3.05 ⫾ 1.90 (2.57) 0.58 ⫾ 0.35 (0.48)

1.66 ⫾ 0.64 (1.63) 0.34 ⫾ 0.13 (0.36) 0.09 ⫾ 0.07 (0.07) 0.019 ⫾ 0.012 (0.016) 0.07 ⫾ 0.07 (0.05)b 0.016 ⫾ 0.014 (0.012)b 0.34 ⫾ 0.21 (0.36)b 0.07 ⫾ 0.05 (0.07)b 25.1 ⫾ 8.1 (23.9) 5.02 ⫾ 1.25 (4.90) 2.69 ⫾ 1.86 (2.36) 0.52 ⫾ 0.31 (0.46)

337 ⫾ 81 (338)b 6.0 ⫾ 7.0 (3.6)b 21 ⫾ 16 (17)b 7.5 ⫾ 1.9 (6.9)b

339 ⫾ 83 (344)b 8.4 ⫾ 15.4 (5.7)b 21 ⫾ 26 (16)b 8.2 ⫾ 2.7 (8.2)b

ESR, erythrocyte sedimentation rate; hs-CRP, high-sensitity C-reactive protein; WBC, white blood cell * Data are mean ⫾ standard deviation (median). Values within a row with different superscript letters are significantly different (P ⬍ 0.05).

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Table 5 Linear associations between the combined level of plasma ␣-and ␤-carotene and the NIH Stroke Scale*

Table 3 Correlation coefficients of plasma lipophilic antioxidants and inflammation markers Lipophilic antioxidants

hs-CRP

Fibrinogen

WBC count

ESR

Models adjusted for

Estimated slope (95% confidence interval)

P

Retinol Retinol/cholesterol Lycopene Lycopene/cholesterol ␣-Carotene ␣-Carotene/cholesterol ␤-Carotene ␤-Carotene/cholesterol ␣-Tocopherol ␣-Tocopherol/cholesterol ␥-Tocopherol ␥-Tocopherol/cholesterol

⫺0.23* ⫺0.15 ⫺0.28* ⫺0.24* ⫺0.29† ⫺0.23* ⫺0.41‡ ⫺0.37‡ 0.036 0.01 0.09 0.11

⫺0.71 ⫺0.09 ⫺0.14 ⫺0.15 ⫺0.35‡ ⫺0.35‡ ⫺0.26† ⫺0.29† 0.06 0.07 ⫺0.02 0.01

0.18 0.19 ⫺0.05 ⫺0.05 ⫺0.28† ⫺0.26† ⫺0.35‡ ⫺0.36‡ 0.12 0.10 0.09 0.10

⫺0.17 ⫺0.26* ⫺0.07 ⫺0.13 ⫺0.19 ⫺0.23* ⫺0.17 ⫺0.18 0.15 0.09 0.03 ⫺0.03

Age and sex Age, sex, and log hs-CRP Age, sex, log hs-CRP, and stroke type

⫺0.26 (⫺0.017 to ⫺0.50) ⫺0.22 (0.025 to ⫺0.46) ⫺0.16 (0.08 to ⫺0.39)

0.04 0.08 0.19

ESR, erythrocyte sedimentation rate; hs-CRP, high-sensitivity C-reactive protein; WBC, white blood cell * P ⬍ 0.5, † P ⬍ 0.01, ‡ P ⬍ 0.001, bivariate correlation analyzed by Spearman’s rank test.

correlated with hs-CRP, fibrinogen, and WBC count. Levels of ␣- and ␤-carotene were highly correlated with all the above-mentioned inflammation markers. Further, ␣- and ␤-carotene were significantly and negatively correlated with neurologic deficits, as indicated by the score of the NIH Stroke Scale (Table 4). Except for fibrinogen and ESR, the inflammation markers, hs-CRP and WBC, were significantly correlated with scores of the NIH Stroke Scale (Table 4). Table 4 Correlation coefficients of plasma lipophilic antioxidants, inflammation markers, and neurologic deficits as assessed by NIH Stroke Scale, Barthel Index, and Modified Rankin Scale NIH Stroke Scale Lipophilic antioxidants Retinol Retinol/cholesterol Lycopene Lycopene/cholesterol ␣-Carotene ␣-Carotene/cholesterol ␤-Carotene ␤-Carotene/cholesterol ␣-Tocopherol ␣-Tocopherol/cholesterol ␥-Tocopherol ␥-Tocopherol/cholesterol Inflammation markers Fibrinogen hs-CRP ESR WBC count

⫺0.007 0.08 ⫺0.15 ⫺0.12 ⫺0.28* ⫺0.19 ⫺0.27* ⫺0.26* 0.18 0.31* ⫺0.01 0.01 0.17 0.25* 0.12 0.29*

Barthel Index

Modified Rankin Scale

0.16 0.03 0.20 0.16 0.09 0.005 0.13 0.10 ⫺0.13 ⫺0.30* 0.11 0.09

⫺0.07 0.05 ⫺0.22 ⫺0.20 ⫺0.04 0.05 ⫺0.16 ⫺0.14 0.13 0.30* ⫺0.07 ⫺0.02

⫺0.01 ⫺0.17 ⫺0.14 ⫺0.03

0.05 0.22 0.10 0.05

ESR, erythrocyte sedimentation rate; hs-CRP, high-sensitity C-reactive protein; NIH, National Institute of Health; WBC, white blood cell * P ⬍ 0.05, bivariate correlation is analyzed by Spearman’s rank test.

hs-CRP, high-sensitivity; NIH, National Institute of Health * Slope indicates the difference in NIH stroke scale per standard deviation (0.38 ␮mol/L) increase in the combined plasma level of ␣- and ␤-carotenes.

Because ␣-carotene and ␤-carotene were significantly associated with the score of the NIH Stroke Scale, we combined the two carotenes as a new variable (␣/␤-carotene). In linear regression analyses adjusted for age and sex, each standard deviation (0.38 ␮mol/L) increase in the combined level of ␣/␤-carotene was associated with a decrease of 0.26 in score in the NIH Stroke Scale (P ⫽ 0.04; Table 5). After adjustment for plasma hs-CPR (transformed by logarithm) and stroke types, the magnitude of association decreased (P ⫽ 0.08 for adjustment of hs-CRP, P ⫽ 0.19 for adjustment of stroke types; Table 5). In multivariate linear regression analyses, three key variables were analyzed, i.e., ␣/␤-carotene, hs-CRP (transformed by logarithm), and stroke types (large or small artery). We found that the ␣/␤-carotene and hs-CRP levels were not significantly correlated with the score of the NIH Stroke Scale (P ⫽ 0.2 and 0.4, respectively), whereas patients who had large-artery stroke had a 2.8-point increase in the score of the NIH Stroke Scale compared with patients who had small-artery stroke (Table 6).

Discussion In this study we showed that plasma levels of retinol, ␣and ␤-carotene, and cholesterol-adjusted plasma levels of ␣- and ␤-carotenes are significantly lower in patients with acute ischemic stroke than in normal controls, whereas levels of lycopene and ␣- and ␥-tocopherols, with or withTable 6 Multivariate linear regression analysis with the score of the NIH Stroke Scale as dependent variable and with stroke type, combined plasma ␣/␤ carotene level, and logarithmically transformed hs-CRP level as independent variables Variables

␤ regression coefficient (SE)

P

␣/␤-Carotene Log hs-CRP Stroke type*

⫺1.9 (1.5) 3 (1.1) 2.8 (1.2)

0.2 0.4 0.02

hs-CRP, high-sensitivity C-reactive protein; SE, standard error * Stroke type: large artery versus small artery.

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out cholesterol adjustment, are not significantly different. These findings are generally in accord with those of previous reports [6,7]. However, we previously reported that plasma ␣-tocopherol, in addition to carotenoid levels, was lower in patients with ischemic stroke than in controls [7]. The discrepancy between our own studies is unexplained but may suggest that plasma ␣-tocopherol levels in patients who have acute stroke and increased oxidative stress may be affected by other factors such as ascorbate and carotene levels, both of which have been proposed to regenerate ␣-tocopherol by reaction with ␣-tocopheroxyl radicals [28,29]. Although ascorbic acid levels were not measured in our present or previous study [7], this vitamin was shown to be decreased in other studies of acute ischemic stroke in which serum ␣-tocopherol levels in patients were unchanged [30]. Nonetheless, most of these findings support the notion that plasma antioxidants are decreased immediately after acute ischemic stroke. The decrease in carotenes after acute ischemic stroke may be ascribed to several possibilities. One possibility is the consumption of circulating carotenes by increased oxidative stress, which is one of the major pathophysiologic changes after brain insult [2–5,31]. In cerebral ischemia, infiltrating neutrophils can induce one type of nitric oxide synthase, i.e., inducible, to produce toxic amounts of nitric oxide [2–5], which appear to preferably attack ␣- and ␤carotenes over ␣- and ␥-tocopherols in LDL [32]. The second possibility is redistribution of plasma carotene levels. Carotenes are transported in plasma exclusively by lipoproteins (mainly LDL cholesterol) [33]. If redistribution does occur, one would expect concomitant decreases in plasma LDL cholesterol in patients with acute ischemic stroke. However, this appears to be not the case because we found no significant differences in total cholesterol and LDL cholesterol between patient who had acute ischemic stroke and normal controls. A third possibility is that the plasma levels of antioxidant vitamins in patients before stroke insult are already lower than those in controls. Obviously, this cannot be detected by the present study, so we excluded in advance those subjects who had taken any vitamin or herbal supplements. As for the decreased plasma retinol level in patients, we speculate that it may be ascribed to the redistribution of retinol because we found decreased plasma albumin in patients. Albumin, like retinol-binding protein, can easily extravasate from the vascular compartment as a consequence of increased capillary permeability owing to acute inflammation [34]. Like oxidative stress, inflammation is an important event after acute ischemic stroke [2]. Our findings that all inflammation markers measured in this study (fibrinogen, hs-CRP, ESR, and WBC count) are significantly higher in patients with acute ischemic stroke than in healthy controls reemphasize this inflammatory facet of pathology. As we expected, plasma levels of retinol, lycopene, and ␣- and ␤-carotenes were inversely and significantly correlated with most inflammation markers measured in this study. Similar

findings have been observed in other clinical settings. For example, the Nun study showed that significantly lower plasma levels of ␣-carotene, ␤-carotene, and lycopene were associated with increased levels of CRP in a population of Roman Catholic nuns who were 77 to 99 y old [19]. In patients with acute pancreatitis, plasma levels of carotenoids and vitamins A and E are decreased and inversely related to the increase in CRP levels. Moreover, levels of retinol and ␤-carotene in patients with severe acute pancreatitis are significantly lower than those in patients with mild pancreatitis [35]. Although plasma levels of retinol, lycopene, and ␣- and ␤-carotenes were correlated with levels of inflammation markers, only those of ␣- and ␤-carotenes were significantly associated with neurologic deficits at the time of discharge. After adjustment for age and sex, the combined level of ␣/␤-carotene remained significantly correlated with neurologic deficits. Because hs-CRP was also correlated with neurologic deficits, we expected that hs-CRP would be a confounding factor for the correlation between carotenes and neurologic deficits. After further adjusting hs-CRP, we found that the magnitude of association between the level of ␣/␤-carotene and the neurologic deficits decreased. Because neurologic deficits strongly predict the likelihood of a patient’s recovery after stroke [36], plasma levels of carotenes and hs-CRP may predict neurologic outcome in patients with acute ischemic stroke, although we found that hs-CRP and ␣/␤-carotene levels were somewhat less powerful than the large-artery stroke type as predictors of worsened neurologic deficits. In summary, plasma concentrations of ␣- and ␤-carotenes are lower in patients with acute ischemic stroke and are correlated with hs-CRP levels and neurologic deficits. However, the negative association between plasma ␣/␤carotene levels and neurologic deficits is confounded by hs-CRP. Therefore, hs-CRP level should be taken into account when using plasma ␣/␤-carotene levels for predicting neurologic deficits after acute ischemic stroke. Acknowledgments The authors appreciate the laboratory technical assistance of Jenny Sun of the Redox Functional Medicine Laboratory. References [1] De Freitas GR, Bogousslavsky J. Primary stroke prevention. Eur J Neurol 2001;8:1–15. [2] Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999;22:391–7. [3] Zini I, Tomasi A, Grimaldi R, Vannini V, Agnati LF. Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis. Neurosci Lett 1992;138:279 – 82. [4] Globus MY, Busto R, Lin B, Schnippering H, Ginsberg MD. Detection of free radical activity during transient global ischemia and

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