SESSION 2: EMERGING TECHNOLOGY FOR THE STUDY OF CELLULAR SULFHYDRYLS What Methods Are Available for Clinical Evaluation of Thiol Status? Martha H. Stipanuk, Cornell University How Can We Evaluate Redox in Whole Cells: State of the Art and Novel Approaches Martin A. Philbert, University of Michigan Martha H. Stipanuk, Ph.D., Professor, Division of Nutritional Sciences, Cornell University, Ithaca, New York, presented information on the methods available today for clinical evaluation of thiol status, capabilities and deficiencies. Martin A. Philbert, Ph.D., Associate Professor of Toxicology and Associate Chair, Department of Environment Health Sciences, University of Michigan, Ann Arbor, presented information on novel approaches to evaluate redox in whole cells, focusing on nanotechnologies under development. The study of sulfhydryl switches demands that we have good tools for the estimation of redox-sensitive components. Blood is the most readily available tissue for clinical study of thiol status. There are several key technical concerns in the measurement of redox molecules, as well as concerns over interpretation of the relevance of plasma readings to report on intracellular thiol biochemistry. Key to accurate estimation of the oxidation status of redox couples is the use of iodoacetate (slow), monobromobimane or N-ethylmaleimide to bind free SH groups, thus arresting oxidation and inhibiting re-equilibration. There may be additional plasma components that interfere with the sulfhydryl status of a sample, including hemolysis (erythrocyte glutathione levels are far greater than plasma levels), ă-glutamyl transpeptidase and free metals. A useful technique is to derivatize the amino group rather than the sulfhydryl group, since this allows concomitant measurement of the reduced and oxidized couples of cysteine and glutathione. Cysteine (250 ́mol/L) constitutes greater than 80% of plasma thiols, whereas glutathione and thioredoxin become more important intracellular players, in the cytosol and nucleus, respectively. During this session, a picture of the relationship between the extracellular and intracellular environments started to emerge: extracellularly, the cys/ cySS (cytsteine/ cystine) couple is predominant, and typically more oxidized than the GSH/ GSSG couple. Intracelluarly, the now predominant GSH/GSSG couple is always more reduced than in the extracellular compartment. Yet even given these many differences between plasma and intracellular thiol status, measurement of plasma redox couples is not without merit, since changes in plasma redox are reported with altered physiological states. For example, chemotherapy is associated with oxidation of the plasma GSH/GSSG couple, and smokers have been reported to have a significantly greater oxidized plasma cys/cySS couple than either non-smokers or former smokers. Even aging alone is seen to cause a shift in the plasma cys/cySS ratio to a more oxidized state, although interestingly this becomes evident at a younger age than any shift in the plasma GSH/GSSG ratio, which is not evident until after about 45 years of age. In considering measurement of tissue glutathione, one runs into the problem that different cell types, or even similar cell types at different physiological stages, may have very different glutathione levels or redox states, requiring separate measurement. One way to overcome this is to study a monoculture. However, this also is confounded by the fact that typically cell culture is carried out under air (21% oxygen). Whereas this is not too different than the environment of a lung cell (~16%), it differs considerably from the 5 – 9% oxygen more common for other tissues within the body. Culture under hypoxic (or whole-body normoxic) conditions is possible, yet still there is a concern that the redox status is altered as soon as one disrupts the normal physiology of the cell. One way to overcome this may be to turn to nanotechnology, placing redox sensors within the living cell. Fluorescent dyes have been used as indicators of various metabolites (e.g., calcium) within living cells. However, the use of fluorescent dyes for evaluation of redox is problematic, since frequently they bind to the cellular components under study, altering the environment that they were intended to evaluate. Furthermore, the sensitivity of fluorescent dyes to reflect thiol status within hydrophobic and hydrophilic environments within the cell may be very different, providing non-interpretable data when mixtures are viewed as one dataset. Other parameters that may similarly cause these dyes to provide unequal responses are subcellular changes in pH, ion concentration, membrane potential, or simply lack of diffusion allowing compartmentalization. Dr. Philbert and colleagues are developing a series of micro-probes termed Probes Encapsulated by Biologically-Localized Embedding (PEBBLEs). These sensors appear to be sufficiently sensitive to be used at concentrations that do not perturb the natural redox by their presence, and the matrix can be altered to meet the needs of the study environment (i.e., hydrophobicity etc.). PEBBLEs are also under development to act as biosensors for a number of other cellular endpoints, unrelated to redox. A series of sensors that signal at different redox potentials could, in real time, show how various compartments of the cell fluctuate back and forth through redox changes, or show how tumor and normal cells vary during passage through the cell cycle. August, 2003 NIH Meeting Remember we are NOT Doctors and have NO medical training. This site is like an Encylopedia - there are many pages, many links on many topics. Support our work with any size DONATION - see left side of any page - for how to donate. You can help raise awareness of CAM.