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

doi:10.1016/j.ab.2003.07.013

Analytical Biochemistry

Volume 322, Issue 1, 1 November 2003, Pages 51-59

https://doi.org/10.1016/j.ab.2003.07.013 Get rights and content

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 × 4.6-mm, 5-μm-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, NAD⁺, 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.

Section snippets

Animals and chemicals

All the experiments in which animals were involved were approved by the Institutional Ethical Committees (University of Rome “Tor Vergata” and Catholic University of Rome “Sacro Cuore”), according to international standards and guidelines.

Inborn male Wistar rats of 300–350 g/b.w. were used in this study; they were maintained in a controlled environment and fed with a standard laboratory diet, with water ad libitum. Far-UV HPLC-grade acetonitrile and HPLC-grade chloroform were purchased from

HPLC separation of standard mixture

In Fig. 1 a representative standard separation of the 39 compounds under evaluation is shown. The elution order of phosphorylated compounds, oxypurines, nucleosides, oxidized pyridine coenzymes, ascorbate, and MDA is as previously shown [18], [19]. Adenosine and UMP, IMP and GMP, UDP-GlucNAc and UDP-GalNAc, AMP and GDP-Glc, and UTP and GTP were not fully resolved (Fig. 1B); however, with the aid, when possible, of spectral differences, a reproducible quantization of these compounds could be

Conclusions

In conclusion, we have here shown that tissue homogenization with the present protein precipitating solution, composed of CH₃CN + 10 mM KH₂PO₄, pH 7.40 (3:1; v:v), permits one to obtain final aqueous samples (containing all hydrophilic low-molecular- weight compounds contained in the cell environment) utilizable with the most common analytical techniques and appropriate to the production of reliable and reproducible measurements of redox and energy states under both physiological and pathological

Acknowledgements

This work has been supported in part by research funds of Catholic University of Rome “Sacro Cuore” and Catania University.

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

Abstract

Section snippets

Animals and chemicals

HPLC separation of standard mixture

Conclusions

Acknowledgements

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