Original article
Oxidative stress and erythrocyte acetylcholinesterase (AChE) in hypertensive and ischemic patients of both acute and chronic stages

https://doi.org/10.1016/j.biopha.2007.10.002Get rights and content

Abstract

Ischemic stroke is a leading cause of mortality and disability particularly in the elderly. Hypertension is the most important risk factor in strokes, representing roughly 70% of all cases. Oxidative stress is believed to be one of the mechanisms taking part in neuronal damage in stroke. It is well documented that cholinergic system plays a key role in normal brain functions and in memory disturbances of several pathological processes, such as in cerebral blood flow regulation. This study investigated the oxidative status and acetylcholinesterase (AChE) activity in whole blood in patients diagnosed with acute and chronic stages of ischemia, as well as with hypertension. Malondialdehyde (MDA) levels and protein carbonylation content showed increased levels both in the acute ischemic groups and in the hypertensive group, when compared to the control. Catalase activity and reduced glutathione (GSH) levels in the acute group were also higher than in the hypertensive, chronic ischemic and control groups (p < 0.05). The activity of AChE in acute ischemic patients was significantly higher than that presented by the control, hypertensive and chronic ischemic patients (p < 0.05). The hypertensive group presented AChE activity significantly lower than control and chronic groups. In spite of having a defined location the ischemic event results in a systemic disorder that induces changes, which can be detected by measuring the peripheral markers of oxidative stress and AChE activity in erytrocytes.

Introduction

The central nervous system (CNS) has a high rate of oxygen metabolism and, because of its strict aerobic glucose metabolism it is totally dependent on an adequate arterial blood flow [1]. If this flow is disrupted for even a short period of time the result is cell damage or death. There are many causes for this disruption and these are collectively referred to as stroke [2]. Ischemic stroke is one of the leading causes of death worldwide, representing a huge public health concern caused by its high morbidity and long-term disability [3]. The majority of the cases it is not fatal, and the ones that survive are at a high risk of subsequent vascular complications and new vascular accidents [4]. Cerebral ischemia results in a number of hemodynamic, biochemical, and neurophysiologic alterations that can be linked clinically to behavioral and pathologic disturbances [5].

Risk factors for ischemic strokes are multiple and combined, e.g. age, hypertension (HT), cigarette smoking, atrial fibrillation, hyperlipidemia and diabetes mellitus (DM) [3]. In recent years, the identification of molecules contributing to neuronal death, particularly apoptosis has thrown light on the pathogenesis of brain damage after ischemic stroke [1], [4]. During cerebral ischemia and reperfusion in particular, a number of events predispose the brain to form ROS (Reactive Oxygen Species), such as decrease in adenosine triphosphate (ATP) levels, loss of Ca2+ homeostasis, excitotoxicity, alteration of arachidonic acid metabolism, mitochondrial dysfunction, acidosis and edema [1], [6]. Oxidative stress is believed to be one of the mechanisms taking part in neuronal damage in stroke [7]. These species lead to the oxidative damage of cellular macromolecules, such as lipids, proteins and nucleic acids [8]. Moreover, ROS have multiple effects on vascular cells, which can alter acute regulation of vascular tone and may also have more effects within the vessel wall by influencing inflammation, permeability and vascular structure [9]. Accumulating evidence has suggested that ischemia-induced inflammation plays a crucial role in the acute phase after stroke. 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], [11]. Some biological substances that may be potential peripheral markers in stroke have been investigated, since it is difficult to directly measure free radical and oxidized molecules in the human brain [11]. Significant increases in lipoperoxidation products or decreases of some antioxidants in plasma have been reported in stroke patients, and the presence of oxidative stress in stroke has been judged by these indices [4]. One of the well-known lipoperoxidation products is malondialdehyde (MDA) [4], [11].

Proteins are considered to be the most susceptible target for oxidative modification. Because of the role of such molecules as enzymatic catalysts, their damage may impair their activity [12]. Protein carbonyl content is currently one of the most general indicators and commonly used markers of protein oxidation [13].

A sensitive balance between the generation and neutralization of oxidants by different intra- and extracellular defense mechanisms helps to protect vital cell components [14]. Circulating scavenging antioxidants with a high redox potential, such as reduced glutathione (GSH), as well as intracellular antioxidant enzymes, such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase, maintain this equilibrium [15]. The antioxidant activity of plasma and erythrocytes may be an important factor providing protection against neurological damage caused by stroke-associated oxidative stress [16].

Furthermore, dementia has become a major public health issue due to the increase of elderly people in the population. Apart from Alzheimer's disease (AD), vascular dementia (VaD) is the second largest cause of dementia in the elderly, representing 15–20% of all cases worldwide [17]. VaD is a common clinical syndrome of intellectual decline and results from ischemic or hemorrhagic cerebrovascular disease (CVD), as well as from hypoperfusive ischemic cerebral injury resulting from cardiovascular and circulatory disorders [18]. There is growing evidence that the cholinergic system is involved in VaD [19]. In fact, deficits in cholinergic markers have been documented in human cases of VaD, regardless of any concomitant AD pathology [19], [20]. Cholinergic mechanisms play a role in the modulation of regional cerebral blood flow [21]. Literature data have demonstrated that one of the main mechanisms responsible for appropriate cholinergic function is performed by the acetylcholinesterase (AChE) enzyme [22]. Moreover, high levels of AChE activity are found in non-neuronal tissues, such as erythrocytes, platelets and lymphocytes [23]. Red blood cell acetylcholinesterase (RBC-AChE) is easily obtained from humans and presents a structure and mechanistic property similar to brain synapse AChE [24], [25].

Therefore, our study aimed to verify lipid peroxidation, using the thiobarbituric acid reactive substances (TBARS) content determination; protein oxidative profile, using protein carbonyl assay; antioxidant defense, using catalase and reduced glutathione levels as parameters; and evaluate the activity of AChE in the red blood cells. The study was carried out with patients who had been diagnosed with cerebrovascular ischemic accident, both acute and convalescent, as well as another group composed of hypertensive subjects. It is important to note that we judged the combined analysis of the groups mentioned above to be essential, as hypertension is the most important risk factor in strokes.

Section snippets

Patient selection

For this study we selected 10 patients with clinical symptoms of ischemia, admitted within a few hours after onset of neurologic deficit to the emergency room of the Federal University of Santa Maria Hospital. On admission, all patients underwent full physical and neurological examinations. Computed tomography (CT) of the brain was performed to exclude intracranial hemorrhage. Blood samples were taken until 7 days after diagnosis.

The convalescent ischemic group constituted 29 outpatients from

Lipid peroxidation

The hypertensive group demonstrated increased MDA content when compared to the control. However, it was statistically lower when compared to acute or chronic ischemia groups (p < 0.05) (Fig. 1). No significant difference was found between the acute and chronic patients.

Protein carbonyl content

Protein oxidation, determined by protein carbonyl content in serum samples, is shown in Fig. 2. In spite of the fact that most of the subjects in the chronic stage had been hypertensive beforehand, the hypertensive group presented

Discussion

The brain is vulnerable to oxidative stress due to its high amount of polyunsaturated fatty acids, low repair mechanism capacity and non-replicating nature of its neuronal cells [2]. ROS may contribute to brain injury by directly attacking macromolecules, including proteins, lipids and DNA or indirectly by affecting cellular signaling pathways and genes [8].

Lipid damage induces the phenomenon known as lipoperoxidation, which culminates in MDA formation. Peroxidation of lipids can disrupt the

Conclusion

This study showed that ischemic stroke, despite being located in the brain, results in systemic disorders which cause biochemical changes, and may be detected peripherally by the determination of oxidative stress markers and activity of RBC-AChE. In the acute phase, free radicals and ROS are likely to be directly involved in biological cell damage. The erythrocyte AChE seems to be altered in vascular disorders, being increased in acute stage and decreased in the hypertensive group. Finally, we

Acknowledgments

The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS).

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