Redox Couples And Energy Charge 


Changes in redox status can have a major impact on cellular and physiologic processes and are believed to underlie many functional decrements associated with the aging process.  Cellular energy production and survival depends upon a series of oxidation/reduction reactions.  These reactions can give rise to free radical and pro-oxidant species that can act as regulatory molecules and induce oxidative damage.  As such, the redox potential of a cell is a reflection of multiple interacting molecules and biological processes that influence both oxidant production and removal and ultimately cellular homeostasis. Thus, accurate assessment of driving forces for and consequences of altered redox status requires coordinated evaluation of numerous contributing molecules, pathways, and systems.

Superoxide anion (O2 .), produced through the one-electron reduction of O2, is rapidly reduced to H2O2 by superoxide dismutase.  Removal of H2O2 is catalyzed by catalase and various isoforms of glutathione peroxidase and peroxiredoxin.  Glutathione peroxidase and peroxiredoxin require the oxidation of GSH to GSSG and sulfhydryl residues on thioredoxin, respectively.  Subsequent reduction and regeneration of GSH and reduced thioredoxin requires oxidation of NADPH to NADP+.  Importantly, the ratio of NADH to NAD+ is intimately linked to these well known antioxidant redox couples.  NADH is the primary carrier of electrons derived from the oxidation of glucose and fatty acids and the relative ratio of NADH to NAD+ is a determinant of free radical production.  Thus, the ratios of GSH to GSSG, reduced to oxidized thioredoxin (-SH/S-S), NADPH to NADP+, and NADH to NAD+ provide important indices of organ, cellular, and organelle redox status and the potential for oxidative stress and damage.  Ultimately, changes in the redox potential are influenced by and impact the energetic status of the cell best reflected by the energy charge ([ATP + 1/2ADP] / [ATP + ADP + AMP]).

Experimental Measures

Metabolites are resolved by reverse phase ion pairing HPLC and detected and quantified using electrochemical analysis (GSH and GSSG) and fluorescence (NADH and NADPH) and UV/VIS (NAD+, NADP+, CoASH, and acetyl-CoA) spectroscopy as we have described previously (see below).  The identities of relevant compounds are confirmed by GC-MS and routine spiking of experimental samples with known quantities of standards to ensure accurate peak assignment.  A major advantage conferred by these methods is the ability to analyze all redox couples of interest using biological samples containing less than 0.25 mg of protein and two extraction protocols.  The types of samples amenable to analyses include isolated organelles, protein homogenates, and flash frozen tissue or cells from invertebrates and vertebrates.  Samples are prepared for analysis of GSH, GSSG, NADP+, and NAD+ by extraction with 5% meta-phosphoric acid.  Importantly, extraction with 5% meta-phosphoric acid yields protein pellets that can be further probed by non-reducing gel electrophoresis followed by Western blot analysis for the relative level of oxidized to reduced thioredoxin.  A separate extraction with 125 mM KOH enables analysis of NADPH and NADH as well as AMP, ADP, and ATP.  The limits of detection for the various compounds are:  

  1. 1 pmol in 1 and 25 µg tissue for GSH and GSSG, respectively; 
  2. 5 pmol in 5 – 50 µg tissue for NADPH and NADH; 
  3. 100 pmol in 50 µg tissue for NADP+ and NAD+; and 
  4. 100 pmol in 50 µg tissue for AMP, ADP, and ATP. 
These values are given for heart tissue, however metabolite concentrations vary less than 1-fold between tissues and would therefore have similar detection limits.


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Sample Preparation Guidelines

Tissue and cells must be rapidly frozen and pulverized in liquid nitrogen and shipped on dry ice.  It is critical to avoid ischemic periods either during euthanasia or tissue isolation.  This is particularly critical in measuring energy charge as brief ischemic durations result in the rapid consumption of ATP and a drop in the energy charge.  Ischemia can also change the relative ratio of various redox couples preventing accurate assessment of in vivo steady state concentrations.  It is therefore important to euthanize by means that prevents loss of blood flow (e.g. CO2) and to flash freeze tissue for shipment.