Geroscience Redox Biology Core Services

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. Thus, 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.

We have developed high throughput methods for analysis of redox couples using 35 mg tissue per assay as summarized below. The types of samples amenable to analyses include flash frozen tissue, tissue homogenates, cell pellets, and isolated organelles from invertebrates and vertebrates. Samples are extracted with 5% meta-phosphoric acid (for GSH, GSSG) or 125 mM KOH (for NADPH, NADH, NADP+, NAD+). Compounds are resolved by ion pairing reverse phase HPLC and quantified using electrochemical, fluorescence, or UV/VIS detection. The identities of specific compounds have been confirmed by GC-MS and routine spiking of experimental samples with known quantities of standards is utilized to ensure accurate peak assignment. The limits of detection for each metabolite are in the pmol range.

Glutathione Status: Glutathione peroxidase isoforms consume H2O2 upon oxidation of GSH to GSSG. The ratio of GSH to GSSG, therefore, provides a measure of redox status and antioxidant capacity (Puente et al., 2014).

NADPH/NADP+: Regeneration of GSH and reduced thioredoxin, necessary for continued H2O2 consumption by glutathione peroxidases and peroxiredoxins, respectively requires oxidation of NADPH to NADP+. Thus, the relative levels of NADPH and NADP+ provide an additional index of antioxidant capacity.

NADH/NAD+: The ratio of NADH to NAD+ is intimately linked to the redox couples GSH/GSSG and NADPH/NADP+. 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. In addition, NADH and NADPH can be interconverted by the nicotinamide nucleotide transhydrogenase or NAD+ and NADP+-dependent isoforms of isocitrate dehydrogenase. NAD,NADH, NADP and NADPH are measured as previously described (DeBalsi et al., 2014).

References:

Paul M. Rindler, Angela Cacciola, Michael Kinter, and Luke I. Szweda.  Am J Physiol Heart Circ Physiol.  2016 Nov 1; 311(5): H1091-H1096.

Puente, BN; Kimura, W; Muralidhar, SA; Moon, J; Amatruda, JF; Phelps, KL; Grinsfelder, D; Rothermel, BA; Chen, R; Garcia, JA; Santos, CX; Thet, S; Mori, E; Kinter, M; Rindler, PM; Zacchigna, S; Mukherjee, S; Chen, DJ; Mahmoud, AI; Giacca, M; Rabinovitch, PS; Aroumougame, A; Shah, AM; Szweda, LI; Sadek, HA (2014) The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell, 157, 565-579.

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. 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.

Assay 1:  NADH, & NADPH: $30/sample

Assay 2:  NAD, NADP: $30/sample

Assay 3:  GSH & GSSG: $30/sample

Assay 1 & 2: $50/sample

Assays 1, 2, & 3: $75/sample

F₂-isoprostanes are formed in membranes as a result of free radical attack on arachidonic acid in membrane phospholipids. F₂-isoprostanes are chemically stable end-products of lipid peroxidation and reliable and sensitive markers of lipid peroxidation (Morrow and Roberts, 1999; Roberts and Morrow, 2000; Roberts and Morrow, 1995). Because isoprostanes are produced in every tissue, plasma levels of free F₂-isoprostanes provide a measure of endogenous production of F₂-isoprostanes from all sites in the body, thus providing an excellent marker of whole body oxidative stress levels. Traditional measures of lipid peroxidation, e.g. TBARS or MDA, are unstable and, therefore, difficult to accurately measure. The discovery of F₂-isoprostanes and neuroprostanes as a stable and sensitive marker of lipid peroxidation has been a major improvement over other assays of lipid peroxidation. The sample preparation for the isoprostane assay is critical and in the case of isoprostanes is also very labor intensive. The isoprostane level can be measured in frozen samples of 100 mg of tissue, 1 ml of plasma or 300 μl of urine.

References:

Morrow, J.D., Chen, Y., Brame, C.J., Yang, J., Sanchez, S.C., Xu, J., Zackert, W.E., Awad, J.A. and Roberts II, L.J. (1999). The isoprostanes: unique prostaglandin-like products of free-radical-initiated lipid peroxidation. Drug Metab. Rev. 31, 117-139.

Morrow, J.D. and Roberts II, L.J. (1997). The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36, 1-21.

Roberts II, L.J., Morrow, J.D. (2000). Measurement of F₂-isoprostanes as an index of oxidative stress in vivo. Free Rad. Biol. Med. 28, 505-513.

Ward, W.F., Qi, W., Van Remmen, H., Zackett, W.E., Roberts II, L.J., and Richardson, A. (2005). Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F₂-isoprostane levels. J. Gerontol. A Biol. Sci. Med. Sci.  60, 847-851.

The levels of F₂-isoprostanes (8-iso-PGF2α) are measured using the GC/MS procedure developed by Roberts and Morrow (2000) and described by our group (Ward et al., 2005).

F₂-isoprostanes: $125/sample

Protein Oxidation. Oxidative modifications to proteins can lead to mis-folded proteins that are prone to forming deleterious oligomers or aggregates that can alter cellular homeostasis and contribute to age-related pathologies and to the aging process itself. A predominant form of protein oxidative modification is the formation of carbonyl groups on specific amino acid residues, e.g., lysine, arginine, proline, and threonine. Carbonyl groups can also be formed by the reaction of amino acid residues with aldehydes (malondialdehyde, 4-hydroxy-2-nonenal) and reactive carbonyl derivatives generated through the reaction of reducing sugars or their oxidation products with lysine residues of proteins. The Core uses a modified assay in which oxidized proteins are measured using a fluorescent-based assay to detect protein carbonyls (Chaudhuri et al. 2006). This method is very sensitive and allows quantitative detection of proteins with even very low levels of protein carbonyls. Global changes in oxidized proteins are measured in tissue homogenates treated with fluorescein-5-thiosemicarbazide (FTC) and subjected to electrophoresis on a 12% gel to resolve fluorescence-labeled proteins from FTC. Importantly, samples can also be analyzed by two dimensional gel electrophoresis to identify carbonyl levels of individual proteins. The detailed methods for this analysis can be found in Chaudhuri et al, 2006 and Pierce et al., 2006.

References:

Chaudhuri, A.R., de Waal, E.M., Pierce, A., Van Remmen, H., Ward, W.F., and Richardson, A. (2006). Detection of Protein Carbonyls in Aging Liver Tissue: A Fluorescence-based Proteomic Approach. Mech. Age. Dev., 127, 894-861.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C.(1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

The tissue sample is homogenized in deaerated buffer [20 mM sodium phosphate buffer pH 6.0 containing 0.5 mM MgCl2, 1 mM EDTA and protease cocktail inhibitors (500 µM AEBSF, HCl, 150 nM aprotinin, 0.5 mM EDTA, disodium salt and 1 µM leupeptin hemisulfate)] and centrifuged at 4ºC for 1 hr at 100,000g. The protein concentration in the supernatant was measured by the Bradford assay, and used to measure protein carbonyl groups either before or after exposing the cytosolic extracts to an oxidative stress.

The cytosolic extracts are diluted to 1 mg/ml, and the extracts are then mixed with FTC (1 mM) and incubated at 37ºC for 150 min in the dark. The proteins are precipitated with an equal volume of 20% chilled TCA (v/v) and centrifuged at 16,000g for 5 min at 25ºC. The pellets are then re-suspended and washed five times with 100% ethanol/ethyl acetate (1:1) to remove the unbound FTC. The final pellets were then dissolved in phosphate buffer pH 8.0 containing 0.5 mM MgCl2, 1 mM EDTA and 8M urea. The concentration of the protein in each sample is measured by Bradford assay, and approximately 15-25 µg of protein is subjected to 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After electrophoresis, the image of the fluorescent protein on the gel is captured with the Typhoon 9400 using an excitation wavelength of 488 nm and an emission filter at 520 nm with a 40nm bandpass. The intensity of fluorescence for each lane (from top to bottom of the lane) was calculated using ImageQuant 5.0 (Molecular Dynamics, Amersham) software.

We require snap frozen tissue stored at -80ºC for the protein carbonyl assay.  It is best not to store the tissue for more than a few months before conducting the assay to minimize potential oxidation of DNA during storage. For most tissues, 50 mg of tissue is optimal for analysis but depending on the tissue or sample type,  20 mg is often sufficient.

Protein carbonyls (specific proteins): $275/sample

Protein carbonyls (total): $80/sample

The Core provides investigators with services to measure mitochondrial function (oxidant generation, ATP production, respiration) as well as energy charge (ATP, ADP, and AMP) in fresh tissue samples or isolated mitochondria. This includes in vitro analyses of mitochondrial function in isolated mitochondria, respirometry analysis in fresh tissue using the Oroborus respirometer, as well as measurement of mitochondrial function in cells using the Seahorse XF24 Extracellular Flux Analyzer. While energy status can be measured on flash frozen tissues/cells, assays of mitochondria function must be performed with fresh tissue/cells or freshly-isolated mitochondria.

Additional assays offered as needed (contact Core Leader).

ETC activities. A limited number of users may be interested in measuring electron transport chain activities. Alterations in activity of the electron transport complexes can be measured using BN-PAGE (Schagger, 2001) or spectrophotometrically as we have previously described (Pulliam et al., 2014).

EPR Analysis. Rates of O2. and H2O2 production will be measured using EPR spectroscopy in permeabilized and intact mitochondria. For these studies, we will use a Bruker ESP300 spectrometer operating at 9.45 GHz with 10G field modulation at 100 kHz. The EPR is also equipped with liquid helium cooling, providing capability to measure the oxidation state and composition of protein iron sulfur centers such as exist in aconitase. For details see results from Dr. Van Remmen’s laboratory (Mansouri et al., 2006).

Assays of Mitochondrial Function. We offer investigators the ability to directly measure mitochondrial function in isolated mitochondria, tissue samples and in cultured cells. Alterations in mitochondrial function and increased mitochondrial ROS generation have long been implicated in the reduced capacity in cellular function that occurs with aging. We have optimized an array of assays to measure aspects of mitochondrial function, including ROS generation, mitochondrial respiration, and ATP production. Assays for measuring H2O2 and superoxide anion release, mitochondrial respiration and ATP production from isolated mitochondria are well established. ATP production and H2O2 release are measured in isolated mitochondria using a luciferase/luciferin based system and the fluorogenic probe, Amplex Red (Molecular Probes) respectively. Mitochondrial respiratory function is assessed in isolated mitochondria and permeabilized tissue using the Oroborus O2K high resolution respirometer which allows analysis of mitochondrial respiration without isolation of mitochondria. In addition to studies in isolated mitochondria, we also have the capability to measure oxygen consumption rate (OCR) in cultured cells using the Seahorse Extracellular Flux Analyzer. Many laboratories have used this methodology tomeasure mitochondrial function in cells. The Seahorse Analyzer also contains a probe to measure acidification of the media and to measure the proton production rate (PPR) or rate of extracellular acidification due to lactic acid production during glycolytic energy metabolism. By measuring OCR and PPR simultaneously, we can get a more detailed picture of cellular energetic flux through these two pathways. This technique is a non-invasive and high throughput analysis that allows us to measure the bioenergetic state and physiology of the cell without disruption of the cells. Energy Status. AMP, ADP, and ATP: NADH provides reducing equivalents for electron transport and, as such, levels of NADH and NAD+ are closely linked to mitochondrial function and energy homeostasis. We will measure AMP, ADP, and ATP concentrations to evaluate redox-associated changes in energy charge. Samples are extracted with 125 mM KOH and resolved by ion pairing reverse phase HPLC and quantified using UV/VIS detection.

Assays of Mitochondrial Function. We offer investigators the ability to directly measure mitochondrial function in isolated mitochondria, tissue samples and in cultured cells. Alterations in mitochondrial function and increased mitochondrial ROS generation have long been implicated in the reduced capacity in cellular function that occurs with aging. We have optimized an array of assays to measure aspects of mitochondrial function, including ROS generation, mitochondrial respiration, and ATP production. Assays for measuring H2O2 and superoxide anion release, mitochondrial respiration and ATP production from isolated mitochondria are well established. ATP production and H2O2 release are measured in isolated mitochondria using a luciferase/luciferin based system and the fluorogenic probe, Amplex Red (Molecular Probes) respectively. Mitochondrial respiratory function is assessed in isolated mitochondria and permeabilized tissue using the Oroborus O2K high resolution respirometer which allows analysis of mitochondrial respiration without isolation of mitochondria. In addition to studies in isolated mitochondria, we also have the capability to measure oxygen consumption rate (OCR) in cultured cells using the Seahorse Extracellular Flux Analyzer. Many laboratories have used this methodology tomeasure mitochondrial function in cells. The Seahorse Analyzer also contains a probe to measure acidification of the media and to measure the proton production rate (PPR) or rate of extracellular acidification due to lactic acid production during glycolytic energy metabolism. By measuring OCR and PPR simultaneously, we can get a more detailed picture of cellular energetic flux through these two pathways. This technique is a non-invasive and high throughput analysis that allows us to measure the bioenergetic state and physiology of the cell without disruption of the cells. Energy Status. AMP, ADP, and ATP: NADH provides reducing equivalents for electron transport and, as such, levels of NADH and NAD+ are closely linked to mitochondrial function and energy homeostasis. We will measure AMP, ADP, and ATP concentrations to evaluate redox-associated changes in energy charge. Samples are extracted with 125 mM KOH and resolved by ion pairing reverse phase HPLC and quantified using UV/VIS detection.

Mitochondrial Function Assays: Contact Core Leader for the cost of service