Plasma antioxidant activity and vascular dementia

Journal of the Neurological Sciences 203 – 204 (2002) 195 – 197 www.elsevier.com/locate/jns

Plasma antioxidant activity and vascular dementia Danuta Ryglewicz *, Maria Rodo, Piotr Krzysztof Kunicki, Malgorzata Bednarska-Makaruk, Alla Graban, Wanda Lojkowska, Hanna Wehr Institute of Psychiatry and Neurology, National Institute of Cardiology, Warsaw, Poland

Abstract Little is known about the role of antioxidant activity in the pathogenesis of stroke-associated neuronal damage and impairment following a stroke. Increased free radical formation together with reduced antioxidant defense may increase neuronal injury. A low concentration of antioxidants such as a-tocopherol may influence the development of post-stroke dementia. The aim of this study was to evaluate the level of a-tocopherol and susceptibility of LDL to oxidation in a group of patients with dementia in comparison to controls. In a group of 68 patients with dementia, according to DSM-IV criteria, 42 with vascular dementia (VaD), 26 with Alzheimer type of dementia (AD) and 46 age-matched persons, with no signs of cognitive disorders (control group), we measured lipids, a-tocopherol and the kinetics of LDL oxidation. The levels of triglycerides (TG) and low-density lipoprotein (LDL) were significantly lower in patients with VaD in comparison to AD patients, but the atherogenic index was similar in both groups. a-Tocopherol was significantly lower in patients with VaD in comparison to patients with AD and controls: 9.9, 12.6 and 12.6 ng/ml, respectively, p < 0.0001. Susceptibility of LDL to oxidation, measured by duration of lag phase did not reveal statistically significant differences between the groups. In patients with VaD, low levels of plasma a-tocopherol were observed, which indicate a reduced antioxidant defense in these subjects. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Vascular dementia; a-Tocopherol; Oxidative stress; Antioxidant defense; Oxidative modification of LDL; Lipids

1. Introduction The causal role of vascular risk factors in different types of dementia has been stressed during the last decade [1]. The sclerosis of small cerebral arteries and arterioles is responsible for the diffused periventricular white matter abnormalities, which are the core lesions for the development of vascular dementia (VaD). Hypercholesterolemia, a wellestablished risk factor for ischemic heart disease, has not been convincingly demonstrated as a factor associated with brain ischemia. Several authors observed, in many ischemicstroke patients, normal or low levels of total cholesterol and LDL cholesterol, although signs of generalized severe atherosclerosis have been diagnosed [2– 4]. This may indicate that not the total level of cholesterol but some forms of LDL may be responsible for the development of cerebral ischemic lesions. Oxidative modification of LDL is considered an important event in atherogenesis [5,6].

* Corresponding author.

The brain appears to be particularly vulnerable to oxidative lipid damage because of its high content of polyunsaturated fatty acids. Lipid peroxidation may alter the fluidity and permeability of neuronal membranes and, thus, cellular functioning, or damage membrane-bound receptors and enzymes [7,8]. The susceptibility of plasma LDL to oxidation can serve as a model of general susceptibility. It can be evaluated by monitoring the kinetics of the change of 234-nm absorption in isolated LDL exposed to in vitro oxidation. The assay measures the amount of produced dienes, which are the early step in polyunsaturated acid oxidation in LDL [6]. The aim of this study was to evaluate the oxidative modification of LDL and a-tocopherol (vitamin E) level in a group of dementia patients.

2. Material and methods The material consists of 68 patients with dementia according to DSM-IV criteria (mean age 69.7 F 7.3). In

0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 2 9 0 - 3

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D. Ryglewicz et al. / Journal of the Neurological Sciences 203 – 204 (2002) 195–197

all cases, CT/MRI examination and neuropsychological tests were carried out. VaD was diagnosed in 42 patients, mean age 71.0 F 6.9 years. All patients, according to NINDS-AIREN criteria, had histories of stroke, focal neurological deficit and focal vascular changes in CT/MRI. Hachinski Ischemia Scale scores were greater than seven. AD, according to NINCDS-ARDRA criteria, was diagnosed in 26 patients, mean age 67 F 8.4 years. All were free of any significant medical illnesses, and had Hachinski Ischemia Scale scores of less than four. The age-matched control group consisted of 46 persons, mean age 67.5 F 6.9 years, with no signs of cognitive dysfunction. Lipids (total cholesterol (TC), triglycerides (TG), lowdensity lipoprotein (LDL), cholesterol (LDL-C), high density lipoprotein (HDL) cholesterol (HDL-C) levels, atherogenic index (TC/HDL-C), kinetics of LDL oxidation and a-tocopherol level were estimated. LDL fraction for oxidation experiments was isolated according to Havel et al. [9]. Blood samples for LDL isolation were collected in 1 mg/ml EDTA tubes and during all the steps of preparation, EDTA was present in a concentration of 0.8 nM. The kinetics of oxidation was measured continuously by monitoring the change in the 234-nm absorption [12]. Three consecutive phases were observed: a lag-phase, during which absorption increases very slowly, a propagation phase with a rapid increase of absorption, and finally a decomposition phase of the products. The lagphase reflects the amount of endogenous antioxidants in LDL and the shorter it is, the greater is their susceptibility to oxidation. Concentration of a-tocopherol in plasma was determined using high-performance liquid chromatography (HPLC). The separation was performed under isocratic conditions on the Supelcosil LC-8-DB (150  4.6 mm, 5 Am) column using methanol –water (95:5, v/v) as a mobile phase with a flow rate of 1.2 ml/min. UV detection was performed at 285 nm. The aliquot of 0.2 ml of plasma, following the addition of 0.2 ml of ethanol with a-tocopherol acetate as internal standard, was extracted using 2 ml of hexane. After centrifugation and freezing at 70 jC in the dark, the dried extract was reconstituted and injected into the HPLC system

Table 1 Mean ( F SD) lipid concentrations, lag-phase duration in patients with vascular dementia (VaD), Alzheimer type dementia (AD) and controls Lipids in serum

Patients with AD, N = 26

Patients with VaD, N = 42

Controls, N = 46

TC (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl) TC/HDL-C Lag phase (min)

226 F 43.7 149 F 38.0* 50.4 F 12.1 134 F 43.3* 4.63 F 1.00 58.9 F 21.6

193 F 47.5* 124 F 42.4* 42.8 F 16.2* 128 F 56.6* 4.94 F 1.84 55.7 F 17.1

215 F 41.4 138 F 38.2 50.0 F 12.3 141 F 65.2 4.47 F 1.06 54.1 F 22.3

* Difference between patients and controls is statistically significant, p < 0.05 (one-way ANOVA).

Table 2 Level of a-tocopherol, mean F SD, in patients with vascular dementia (VaD), Alzheimer type dementia (AD) and controls a-Tocopherol

Patients with AD, N = 26

Patients with VaD, N = 42

Controls, N = 46

a-Tocopherol (Ag/ml) a-Tocopherol/TC (Ag/mg) a-Tocopherol/TC + TG (Ag/ml)

12.8 F 3.42* 6.02 F 2.9

9.9 F 3.48* 5.19 F 1.37*

12.6 F 4.64 5.89 F 1.76

3.67 F 1.32

3.12 F 0.91*

3.55 F 0.89

* Difference between patients and controls is statistically significant, p < 0.05 (one-way ANOVA).

for a-tocopherol analysis. The method was fully validated. No interference from plasma matrix nor from h-, g- and ytocopherol isomers was noted. The yield of recovery was 97.4% and 88.1% for a-tocopherol and internal standard, respectively. The method was calibrated from 0.5 to 50 Ag/ ml with satisfactory intra- and inter-assay precision and an accuracy range of 97 –110%. Lipids were determined by routine enzymatic methods. Data were analyzed by analysis of variance (ANOVA) and nonparametric tests (Mann – Whitney and Kolmogorov – Smirnov) using SPSS 10.0 computer software.

3. Results The levels of TC and LDL-C were significantly lower in patients with VaD in comparison to AD patients, but the atherogenic index was similar in all groups. The lag-phase did not show statistically significant differences between the groups (Table 1). The level of a-tocopherol was significantly lower in patients with VaD in comparison to patients with AD and controls: 9.9, 12.8 and 12.6 Ag/ml, respectively, p < 0.0001. The a-tocopherol/TC ratio and the atocopherol/(TC + TG) ratio were also lower in patients with VaD in comparison to patients with AD (Table 2).

4. Discussion The increased production of free radicals and reactive oxygen species (ROS) leading to oxidative stress appears to play an important role in the pathogenesis of ischemic brain injury [7,10]. ROS are short-lived, and are difficult to measure in biological samples. However, there are indirect indexes that can be used to examine sequela of ROS production, such as the concentration of endogenous antioxidants. The level of a-tocopherol may be one such index. Patients with a low concentration of a-tocopherol, which is a very important antioxidant, may have a higher susceptibility to oxidative stress. Reduced antioxidant defense may increase the signs of post-ischemic injury [11 –13] and may play an important role in the development of subcortical encephalopathy and dementia.

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Our study has shown that patients with VaD had a lower level of a-tocopherol in comparison to AD and controls. This indicates that antioxidant defense was decreased and the susceptibility to oxidative stress, increased. Similarly to other authors, we have found a lower level of TC and LDL-CH in patients with VaD, as compared to AD subjects. Susceptibility of LDL to oxidation was a little higher for patients with VaD as compared to AD, but the difference was not statistically significant. In our previous study, we discovered statistically significant differences between such small groups. The discrepancy between our former and present results may be due to different etiopathogenesis of vascular lesions in the groups of patients with VaD. Higher susceptibility was also revealed in patients who had suffered a stroke, mainly among those with carotid artery stenosis [3]. It is possible that a higher susceptibility of LDL to oxidation occurs in case of atherosclerotic lesions in large arteries and this might not play a significant role in small vessel disease. In patients with subcortical encephalopathy, an increased homocysteine level probably plays a more important role. Our results suggest that a-tocopherol supplementation for patients with dementia could be useful. The suggestion that the antioxidative effect is mediated entirely by atocopherol must, however, be treated with caution. It seems important to provide antioxidant supplementation not only with a-tocopherol but also with various other vitamins, including vitamin C and beta carotene.

Acknowledgements The work was support by the Polish Committee for Scientific Research, grant 4 P05B 095 18.

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