Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

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

Giuseppe Lazzarino1, Angela Maria Amorini, Giovanna Fazzina

  • 1Laboratory of Biochemistry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. lazzarig@mbox.unict.it

Analytical Biochemistry
|January 7, 2004
PubMed
Summary

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Glutamate accumulation in myelofibrosis microenvironment rewires mesenchymal stromal cells metabolic and epigenetic profiles.

Cell communication and signaling : CCS·2026
Same author

HMC3 revealed: how much do these "Microglia" really tell us?

Frontiers in immunology·2026
Same author

PARP1 rewires neuroinflammatory and redox metabolism associated with reactive neuroglia in neuropathic pain.

Redox biology·2026
Same author

Memantine prevents acute stress-induced memory deficits by reversing sex-dependent pathophysiological glutamatergic alterations in the dorsal hippocampus.

British journal of pharmacology·2026
Same author

Effect of electronic cigarette aerosols on cisplatin resistance in head and neck cancer cells: a collaborative replication study.

BMC cancer·2026
Same author

Carnosine protects human microglia against Aβ oligomers through a multimodal mechanism of action: inhibition of oxidative stress, rescue of cellular energy status, and enhancement of phagocytosis.

Frontiers in immunology·2026

This study introduces a streamlined method for preparing biological samples to analyze both redox and energy states in a single step. Traditional methods often require multiple extractions, which can alter sensitive compounds like NADH and NADPH. The new approach uses ice-cold, nitrogen-saturated solvents to deproteinize samples without oxidation. After removing organic solvents with chloroform, the remaining solution is analyzed via HPLC using a specialized column. This allows the simultaneous measurement of 39 compounds, including glutathione, NAD+/NADH, and energy metabolites. The method was tested on rat tissues and found to preserve metabolite integrity. It was also applied to rats with head trauma to study metabolic changes. The researchers propose that this protocol improves accuracy and reduces variability in metabolic studies.

Area of Science:

  • Biochemical analysis techniques in cellular metabolism
  • Neurochemical profiling in trauma research
  • Analytical chemistry in biological sample preparation

Background:

Current methods for analyzing cellular redox and energy states often require multiple sample preparations, increasing variability and resource use. Prior research has shown that traditional deproteinization techniques can lead to oxidation of sensitive metabolites like NADH and NADPH. This gap motivated the development of a streamlined method that preserves redox-sensitive compounds. It was already known that glutathione ratios and NAD+/NADH balances are critical for assessing cellular stress. However, no prior work had resolved how to measure these alongside energy metabolites in a single preparation. This paper's contribution is a unified sample processing method that avoids oxidation and solvent interference. The method allows for simultaneous quantification of 39 compounds, including redox and energy markers. This approach addresses the challenge of maintaining compound stability during extraction. It provides a solution for researchers needing comprehensive metabolic profiling from limited sample volumes.

Keywords:
metabolic profilingion-pairing HPLCbiological sample preparationredox state analysis

Frequently Asked Questions

The method uses ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 to deproteinize samples without oxidation.

By using nonoxidizing conditions during deproteinization and chloroform washing to remove interfering substances.

It enables ion-pairing HPLC separation of 39 compounds, including redox and energy metabolites.

It acts as an ion-pairing reagent to facilitate the HPLC separation of charged metabolites.

It was tested on rats with moderate closed-head trauma to quantify changes in redox and energy markers.

Related Experiment Videos

Purpose Of The Study:

The study aimed to develop a single-sample preparation protocol for analyzing multiple biochemical markers of cellular redox and energy states. The specific problem addressed is the need for a reliable, non-oxidizing extraction method that preserves sensitive metabolites. The motivation stems from the limitations of current multi-step procedures that risk altering compound ratios. The researchers propose a method that enables simultaneous ion-pairing HPLC analysis of 39 compounds. This includes redox indicators like GSH, GSSG, and NAD+ as well as energy metabolites like oxypurines. The goal is to reduce variability and resource use by eliminating the need for separate extractions. The method was tested on rat tissue extracts to validate its applicability. The study also aimed to demonstrate the method's utility in pathological conditions like closed-head trauma.

Main Methods:

The method involves tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. This step ensures fast deproteinization under nonoxidizing conditions. After homogenization, samples are centrifuged to pellet proteins. The supernatant is washed three times with HPLC-grade chloroform to remove organic solvents and lipid-soluble substances. The remaining aqueous phase is suitable for ion-pairing HPLC analysis. A Kromasil C-18 column with tetrabutylammonium hydroxide is used for separation. The column dimensions are 250 x 4.6 mm with 5-microm particles. This setup enables the detection of 39 compounds in a single run. The method was applied to rat tissues to test its effectiveness in preserving metabolite integrity.

Main Results:

The method successfully preserved redox-sensitive compounds like NADH and NADPH without oxidation. It enabled the simultaneous determination of 39 compounds, including GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH. The aqueous phase obtained after chloroform washing was free of interfering substances. This allowed accurate ion-pairing HPLC analysis of redox and energy markers. The method was tested on rat tissues and showed consistent recovery of metabolites. It was also applied to rats with moderate closed-head trauma to assess metabolic changes. The procedure demonstrated high reproducibility across tissue types. The method's simplicity and reliability make it suitable for routine use in metabolic studies.

Conclusions:

The authors propose that this single-sample preparation method is reliable for redox and energy state analysis. It reduces the need for multiple extractions and preserves metabolite integrity. The method's application in trauma studies shows its utility in pathological conditions. It was already known that redox and energy markers are sensitive to sample preparation. The researchers suggest that this protocol improves accuracy by avoiding oxidation and solvent interference. The method's ability to measure 39 compounds in a single run is a key advantage. It was already known that traditional methods risk altering compound ratios. The study confirms that this approach maintains the stability of redox-sensitive metabolites.

The researchers propose that this protocol improves accuracy by avoiding oxidation and solvent interference.