A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-mum-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAW, NADH, NADP(+), NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma. (C) 2003 Elsevier Inc. All rights reserved.

Lazzarino, G., Amorini, A.m., Fazzina, G., Vagnozzi, R., Signoretti, S., Donzelli, S., et al. (2003). Single-sample preparation for simultaneous cellular redox and energy state determination. ANALYTICAL BIOCHEMISTRY, 322(1), 51-59 [10.1016/j.ab.2003.07.013].

Single-sample preparation for simultaneous cellular redox and energy state determination

VAGNOZZI, ROBERTO;
2003-01-01

Abstract

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-mum-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAW, NADH, NADP(+), NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma. (C) 2003 Elsevier Inc. All rights reserved.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore MED/27 - Neurochirurgia
English
Energy state; GSH and GSSG; HPLC; Metabolite analyses; Organic tissue deproteinization; Redox state; Reduced and oxidized nicotinic coenzymes
Lazzarino, G., Amorini, A.m., Fazzina, G., Vagnozzi, R., Signoretti, S., Donzelli, S., et al. (2003). Single-sample preparation for simultaneous cellular redox and energy state determination. ANALYTICAL BIOCHEMISTRY, 322(1), 51-59 [10.1016/j.ab.2003.07.013].
Lazzarino, G; Amorini, Am; Fazzina, G; Vagnozzi, R; Signoretti, S; Donzelli, S; Di Stasio, E; Giardina, B; Tavazzi, B
Articolo su rivista
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/49568
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