ANTIOXIDANT CAPACITY IN THE LIPOPHILIC FRACTION OF ALZHEIMER’S BRAIN TISSUES

Th e aim of this study was to investigate the antioxidant capacity (AC) in the lipophilic fraction of postmortem motorcortex (MC), nucleus caudatus (NC) and gyrus temporalis (GT) from controls (C) and Alzheimer’s disease (AD) patients. Th e initial samples consisted of  human brain tissues of AD and C. AC of the diff erent region of human brain were measured by using the fl uorescent method of the oxygen radical absorbance capacity (ORAC). Peroxyl and hydroxyl radical generators were used in the analysis. All ORAC analysis were carried out on the Perkin-Elmer spectrofl uorometer LS  with fl uorescent fi lters, Ex:  nm; Em:  nm. Final results were calculated using the diff erences between area under the quenching curve of fl uorescein (FL), blank and analyzed biological samples. AC against peroxyl radicals (ORAC-ROO°) of lipophilic fraction in MC of AD was statistically signifi cantly lower in comparison with MC of C (p < ,). No changes in the AC against hydroxyl radicals (ORAC°OH) of lipophilic fraction of AD were found in comparison with C. Reduction of total protein in GT of AD (p < ,) was found. Th e results showed that in the MC of AD brain the balance between production of free radicals and the neutralization by a complex antioxidant system is disturbed. Th e manual fl uorescent method for AC measurements proved to be suffi ciently appropriate and sensitive for the AC measurements of lipophilic fraction of postmortem brain tissues from diff erent patological conditions.


Introduction
Damage and progressive cell death that occurs in neurodegenerative disorders such as Alzheimer's disease (AD) has been associated with activities of diff erent free radicals (FR). However, there is growing evidence for a cascade of multiple deleterious factors, including oxidative stress, lipid peroxidation and altered iron metabolism leading to excess FR production, mitochondrial dysfunction and disturbed calcium homeostasis, which in turn result in cytosceletal damage and cell death in AD. Oxidative stressors induce an imbalance between oxidants and antioxidants in favor of the former leading to the oxidative damage of the molecules like deoxyribonucleic acid, lipids and proteins (, ). Furthermore, brain aging in AD patients is accompanied by the alteration of several neurotransmitter systems with a pronounced defi cit in the cholinergic system and abnormal accumulation of amyloid-ß peptides and hyperphosphorylated tau. Nucleus basalis Maynert, hypocampus and cortex are mostly aff ected area in AD brain. Th e present study was aimed to investigate the AC in the lipophilic fraction of diff erent brain regions, by means of ORAC method involving two diff erent FR generators (, , , , , , ), modifi ed by Sofi c et al. (). ORAC method has an advantage over other assays, because this method utilizes an area-undercurve technique and thus combines both inhibition time and inhibition degree of FR action by an antioxidant into a single quantity. Fluorescein (FL) was used as a target of FR attack, with ,`-azobis (-amidino-propane) dihydrochloride (AAPH) as a peroxyl radical (ROO o ) generator, and hydrogen peroxide with cupric sulfate penta hydrate as an hydroxyl radical (OH o ) generator. ROO o is a common FR found in the body and used in the antioxidant activity assays (,,). It is slightly less reactive than OH o and thus possesses an "extended" half-life of seconds instead of nanoseconds. In original developed automated method beta -phycoerythrin (ß-PE), a protein from Porphyridium cruentum, was used as a target of FR attack (,,,,,,). An improved ORAC method has been developed and validated using FL as the photo sensor. Ou et al., () demonstrated that FL is superior to ß-PE.
In clinical studies where analysis of antioxidant status is important, ORAC method have been used to evaluate the hydrophilic and lipophilic antioxidants in postmortem brain tissue.

Patients and Controls
Th e initial sample of total  subjects consisted of  controls (C) brains and  Alzheimer's disease (AD) brains. Motorcortex (MC), gyrus temporalis (GT) and nucleus caudatus (NC) were collected and stored at -°C until analyzed. Brains were matched for age (C , ± , years; AD , ± ,) and postmortem time (C  ±  hours; AD , ± ,). Control brains did not show any abnormal histopathological changes. Th e death of control subjects was mainly caused by cardiac and pulmonary arrest and by diff erent tumours. Neuropathological diagnosis was based on histological examination of characteristic Alzheimer's degeneration, the number of senile plaques and neurofi brillary tangles were determined according to the graduation of Khachaturian (). AD patients died from cardiac and pulmonary deficits. All patients, before death, were underwent psychopharmaceutic therapy and received antibiotics.

Sample preparation
Th e crude tissue extracts from postmortem brain were prepared by homogenizing the tissues in a  mM phosphate buffer pH = , ( ml buffer per gram of tissue). μl of brain tissue homogenate was transferred to a glass tube, μl of ethanol and μl of water was added and mixed, and then μl of hexane was added, followed by mixing. Th e mixture was left to sit for - min or until two layers appeared, followed by centrifugation for  min at  rpm. Th e hexane layer was removed and added to a separate amber tube. An additional μl of hexane was added to the original tube, mixed, left to settle for min, and then centrifuged for  min at  rpm. Th e hexane layer was removed and combined with first extract. μl of , M perchloric acid was added in the combined hexane extract to precipitate the protein. Th e sample was then centrifuged for  min at  rpm. From the supernatant,  μl was added to μl of phosphate buffer, mixed and used for the further measurement. The manual antioxidant radical absorbance capacity (ORAC) assay Manual ORAC analysis were performed on a Perkin Elmer spectrofl uorometer LS  with a fl uorescent fi lters (Ex:  nm; Em:  nm). In the fi nal assay mix-ture ( ml total volume) FL (, nM) was used as a target of FR attack, with AAPH ( mM) as a peroxyl radical generator (ORAC-ROO° assay), and HO-Cu+ (HO , ; CuSO x HO , mM) as mainly a hydroxyl radical generator (ORAC-OH° assay). Th e spectrofl uorometer was programmed to record the fl uorescence of FL every  min after AAPH, or HO -Cu+ were added for as long as  min and the samples were thermostated at   C (KP -D Lauda, Lauda Koenigshofen). All fluorescence measurements were expressed relative to the initial reading. Final results were calculated using the differences of areas under the FL decay curves between the blank and a sample and expressed as a μmol per g of fresh brain tissue. Biochemical analysis Colorimetric determination of total protein was based on the principle of the Biuret reaction using bovine serum albumin as a standard. Cholesterol and triglycerides were measured using comercially available enzymatic kits, which are based on the spectrophotometric determination.

Statistical analysis
For statistical comparisons Student's test was performed.

Results
Th e ORAC-ROO˙ and ORAC-OH. values from lipophilic NC, GT and MC fractions of C and AD are shown in Table  and Table .
In the MC of AD patients antioxidant capacity against peroxyl radical was signifi cantly lower than in the MC of C, p < , by Student's t-test (Table ).
No changes in the antioxidant capacity were found in brain tissues of AD in comparison with C, when hydroxyl radical generator was used in the assay (Table ).
Total protein, cholesterol and triglycerides in diff erent brain regions of C and AD are presented in Table .
Reduction of total protein in GT of AD, p < , was found. No changes of cholesterol and triglycerides were found in brain tissues of AD in comparison with C (Table ).
AC measured with diff erent FR generators have diff erent numerical values. Because of this fact it is necessary for estimation of antioxidant status of some sample to determine antioxidant score, which is a sum of ORAC-ROO o and ORAC-OH o in hydrophilic fraction and in lipophilic fraction. In this study, AC of lipophilic brain antioxidants was measured. Extracting the lipophilic components with hexane before analysis of the hydrophilic antioxidant fraction did not signifi cantly alter the AC of the hydrophilic fraction (). Th us, it appeared that there was no carry over of the lipophilic components into the aqueous compartment, and one could obtain the same hydrophilic ORAC value, whether the samples were fi rst extracted with hexane or not. Regarding our previously published data of decreased AC in hydrophilic fraction of diff erent AD brain regions (), AC in lipophilic fraction was about  -  of AC measured in hydrophilic fraction. Protein content, which may be regarded an index of cell loss or atrophy () was reduced in GT of AD brains in comparison with C. However, no diff erences between AD and C were found with respect to the cholesterol and triglycerides investigated. Our results suggested that lipophilic antioxidant defense system in brain is region-dependently disturbed.

Conclusion
-Lipophilic fractions ORAC-ROO o in MC of AD was statistically significantly lower in comparison with MC of C (p < ,).
-No changes in the AD lipophilic fraction ORAC-°OH were found in comparison with C.
-Reduction of total protein in GT of AD (p < ,) in comparison with C was found.
-No changes of cholesterol and triglycerides were found in brain tissues of AD in comparison with C.
-Th e results showed that in the MC of AD brain the balance between production of FR and the neutralization by a complex lipophilic antioxidant system is disturbed.
-Th e manual fl uorescent method for AC measurements proved to be suffi ciently appropriate and sensitive for the AC measurements of lipophilic fraction of postmortem brain tissues.