Received: January 23, 2006
Accepted: July 1, 2006
Ref: Nishimura A, Asano M, Nushida H, Adachi J, Fujiwara S, Ueno Y.  Increased brain 7-hydroperoxycholesterol in Alzheimer type dementia.  Anil Aggrawal's Internet Journal of Forensic Medicine and Toxicology, 2006; Vol. 7, No. 2 (July - December 2006): Published July 1, 2006, (Accessed: 

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Akiyoshi Nishimura

Abstract

Oxidative stress in Alzheimer type dementia (ATD) brains was studied by the analysis of cholesterol-derived hydroperoxide as an index of lipid peroxidation. ATD was diagnosed pathologically using Braak and Braaks’ criteria and human brain tissues were collected at forensic autopsy (control, n=5; ATD, n=7). Tissue samples were obtained from frontal, angular and occipital cortex, frontal, angular and occipital white matter, and hippocampus. The extracted membrane lipids were injected into an octyl column for HPLC with post-column chemiluminescence for detection of two cholesterol-derived hydroperoxides, 7α-(7α-OOH) and 7β-hydroperoxycholest-5-en-3β-ol (7β-OOH). The data showed that the concentrations of 7α-OOH and 7β-OOH in the angular gyrus white matter of the dementia group were the highest and approximately 10-fold greater that corresponding value for the controls. Our present findings suggest that oxidative stress, membrane lipid peroxidation in particular, may reflect the extent of neurodegenerative process in AD, and would also argue in favour of the hypothesis that in AD hippocampal atrophy may contribute to oxidative stress in the posterior associate region.

Keywords

7-hydroperoxycholesterol; Alzheimer type dementia; human brain; HPLC-CL; lipid peroxidation; posterior associate region.

Introduction

The brain in Alzheimer’s disease (AD) is under increased oxidative stress suggesting that free radicals are involved in the pathogenesis of neuron death.¹⁵ Indeed, free radical-induced lipid peroxidation has been evaluated in a number of studies including the analysis of 4-hydroxynonenal (HNE),¹⁶ malondialdehyde (MDA),²² thiobarbituric acid-reactive substances (TBARS),¹³ and F4-isoprostanes²¹ in various AD brain regions compared to control subjects. Little, however, has been done to characterize the oxidized membrane lipids in brains of patients with AD.

We previously developed a method for quantifying cholesterol-derived hydroperoxides using HPLC with chemiluminescent detection (HPLC-CL)¹ and applied this to investigating oxidative stress in a number of models. For example, both paraquat and alcohol administration are well known to produce reactive oxygen species and induce membrane lipid peroxidation. We investigated membrane lipid peroxidation by analyzing the cholesterol-derived hydroperoxides present in the liver and kidneys of rats after administration of a low dose of paraquat.⁴ We found significantly elevated concentrations of 7α-(7α-OOH) and 7β-hydroperoxycholest-5-en-3β-ol (7β-OOH).⁴ Moreover, elevated concentrations 7α-OOH and 7β-OOH were found in human alcoholic fatty liver indicative of augmented lipid peroxidation due to chronic alcohol intake.⁵ We have also identified 7-hydroperoxycholesterol in human liver using liquid chromatography-mass spectrometry² and found elevated 7α-OOH and 7β-OOH concentrations in the plantaris as well as the soleus skeletal muscle of rats after ethanol administration³. Our findings confirm that 7α-OOH and 7β-OOH are good markers of oxidative stress.

The aim of the study reported here was to evaluate whether lipid peroxidation occurred in the membrane fraction of human brain with Alzheimer type dementia (ATD). We measured 7α-OOH and 7β-OOH, then determined whether individual regions brain were particularly susceptible.

Materials and methods

Human Brain tissues were obtained from 12 autopsy cases (7 ATD cases and 5 control cases, postmortem interval < 24hours) in the Department of Legal Medicine, Kobe University School of Medicine, Japan, in compliance with the ethical code of the Ethical Committee of the Japanese Society of Legal Medicine. Demographic data on the subjects are shown in Table 1. There was no significant difference between ATD and control with regard to age, body weight and height, brain weight, brain weight per body weight and postmortem intervals. The control subjects, who had died from causes unrelated to the nervous system, showed no clinical manifestations of dementia or other mental disorders. Historical information of dementia was obtained from the interview to their families. Following macroscopic examination, the frontal lobe (superior frontal gyrus), parietal lobe (post central gyrus) and occipital lobe, hippocampus with entorhinal and transentorhinal areas and adjoining temporal lobe, amygdala, thalamus, substantia nigra and cerebellum were dissected. These tissue blocks were fixed in 10% formalin, embedded in paraffin and sectioned serially at 6 µm. After Gallyus-Braak’s silver impregnation, each case was classified neuropathologically respectively, according to Braak and Braaks’ staging criteria.⁶ Braak and Braaks’ staging for all cases is shown in Table 2. There is a close relation between pathological staging of Braak and Clinical Dementia Rating (CDR)⁹ scale.¹⁰ When the patients reach Braak and Braaks’ stage IV, the severity of neurofibrillary tangles formation is incompatible with CDR of 0.5 or less. Accordingly, we estimated the patient with Braak and Braaks’ stage IV - VI to be ATD in this study, whereas individuals with Braak and Braaks’ stage 0 were listed up as controls.

The right hemisphere was used for all the chemical analysis. Membrane lipids were measured in the frontal cortex (FC), angular gyrus cortex (AGC), occipital cortex (OC), frontal white matter (FW), angular gyrus white matter (AGW), occipital white matter (OW), and hippocampus (H). Total lipids were extracted for the analysis of lipid peroxidation products as follows: Five millilitres of ice-cold chloroform/methanol (2:1, v/v) containing 0.005% (v/v) butylated hydroxytoluene as the antioxidant and 500 pmol β-sitosterol-5α-hydroperoxide as the internal standard was added to approximately 0.1 g of brain tissue, and the mixture homogenized under ice-cold conditions. The homogenate was mixed with 5 ml of chloroform/methanol (2:1, v/v) and 1 ml of distilled water, swirled vigorously for 1 min then centrifuged at 800 g for 20 min. The chloroform layer was aspirated, concentrated in a rotary evaporator, and dried under a nitrogen stream. The subsequent procedure of purification with Sep-Pak (-NH2) was performed as described previously.¹

3,5-Di-tert-butyl-4-hydroxytoluene, luminol (3-aminophthaloylhydrazine) and cytochrome c (from horse heart, type VI) were purchased from Wako Pure Chemical Co. (Osaka, Japan). Cholesterol hydroperoxides, 5α-hydroperoxycholest-6-en-3β-ol (5α-OOH), 7α-OOH, 7β-OOH, as well as β-sitosterol 5α-hydroperoxide (the internal standard) were synthesized as described elsewhere.¹¹