The detection of age-, gender-, and region-specific changes in mouse brain tocopherol levels via the application of different validated HPLC methods

Abstract

The aging process clearly increases the demand for antioxidant protection, especially in the brain, involving that provided by α-tocopherol (αT). However, little is known about the age-related changes in brain αT levels and the influencing effect of gender on it, in human or murine samples as well. Accordingly, the aim of the current study was to detect age-, gender- and region-specific changes in αT concentrations in mouse brain tissue and to assess the influencing effect of plasma αT levels on it. Female and male C57BL/6 mice at the ages of 6, 16 and 66 weeks (n = 9 in each group) were applied. αT levels were determined with high performance liquid chromatography (HPLC) from the striatum, cortex, hippocampus, cerebellum, brainstem and from plasma samples. A detailed validation process was carried out for the applied HPLC method as well. The results demonstrated that brain αT levels significantly increased in the striatum, cortex, and hippocampus with aging in both genders, but in a more pronounced way in females with an increasing magnitude of this difference. In case of the cerebellum, a moderate elevation could be detected only in females, whereas in case of the brainstem there was no significant change in αT level. With regard to plasma samples, no clear trend could be identified. The current study is the first to present age-dependent gender-specific changes in αT level in certain brain regions of the C57Bl/6 mouse strain, and may provide meaningful information for future therapeutic studies targeting aging-related processes.

The assessment of possible gender-related effect of endogenous striatal alpha-tocopherol level on MPTP neurotoxicity in mice

“…The reagents for αT high performance liquid chromatography (HPLC) measurement has already been reported [ 2 ], and besides those, we used the following chemicals in this study: disodium-ethylenediaminetetraacetate dihydrate (Na 2 EDTA∗2H 2 O; Lach-Ner s.r.o, Neratovice, Czech Republic), sodium-metabisulfite (Na 2 S 2 O 5 ; Fluka, Budapest, Hungary, Honeywell Group), sodium-dihydrogenphosphate (NaH 2 PO 4 ; Reanal Laboratory Chemicals, Budapest, Hungary), HPLC purity absolute ethanol (EtOH) and acetonitrile (ACN; VWR International, Radnar, PA, USA), MPTP hydrochloride (MedChemExpress, Monmouth Junction, NJ, USA) and the following substances were obtained from Sigma-Aldrich (Saint Louis, MO, USA): perchloric acid (HClO 4 ), phosphoric acid (H 3 PO 4 ), sodium hydroxide (NaOH), buthyl-hydroxy-toluol (BHT), sodium-octyl-sulphate (NaOS), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxybenzylamine hydrobromide (DHBA∗HBr, internal standard (IS)), homovanillic acid (HVA), dopamine hydrochloride (DA∗HCl) and isoproterenol hydrochloride (IPR∗HCl, IS).…”

“…injection, all the animals were deeply anesthetized with isoflurane (Forane®; Abbott Laboratories Hungary Ltd., Budapest, Hungary). Sample collection and preparation was similar as described previously [ 2 ]. Briefly, plasma and halved striatal samples were collected for the determination of αT and catecholamine concentrations.…”

“…For the quantification of αT and the IS (rac-tocol) a previously published method was used for both mouse plasma samples (applying diode-array detector (DAD)) [ 2 , 7 ] and mouse brain samples (fluorescence detector (FLD)) [ 2 ]. For the analysis Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) was used under isocratic conditions.…”

“…Although neurodegenerative disorders, including Parkinson's disease (PD), may differ in clinical and pathological characteristics, their pathomechanisms may involve some common features, such as glutamate excitotoxicity, mitochondrial dysfunction and reduced antioxidant capacity [ 1 ]. Regarding the antioxidant aspects, strong evidence suggest that α- and γ-tocopherols have important role in antioxidant protection in the central nervous system (CNS) due to its lipid-rich structure [ 2 , 3 , 4 , 5 ]. Numerous studies assessed age- and gender-related differences in α-tocopherol (αT) levels in serum/plasma samples of healthy individuals [ 6 , 7 ] and furthermore, the influencing effect of dietary tocopherol intake on PD-related parameters, including those studies that focused on the achievement of neuroprotection via the administration of exogenous αT [ 1 , 3 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ].…”

“…These conflicting findings may be partially explained by the differences in the MPTP treatment regimen as well. Our previous study demonstrated that a female-predominant increase in endogenous striatal αT level evolves with aging, providing significant differences between genders already at 16 weeks of age [ 2 ].…”