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Intramural Researchers Streamline Free Radical Investigation

By Eddy Ball
September 2006

In a recently published study, two NIEHS researchers demonstrated the performance of a creative shortcut for medical investigators to open new possibilities in their research of the "dark side" of oxygen utilization by the human body. Biologist Marilyn Ehrenshaft and physical chemist Ronald Mason have developed an elegantly simple and "clean" methodology, known as immuno-spin trapping, that offers researchers in free radical biology important advantages over the methodology currently employed. As a result, this achievement could open the way in an effort to understand why the body turns against its own products in autoimmune responses involving the thyroid gland.

Because laboratories typically have the necessary equipment and trained personnel on hand, immuno-spin trapping permits many more investigators to engage in this kind of research. The sensitivity of the methodology also substantially impacts the costs of studying protein radical formation on thyroid peroxidase (TPO) by reducing the amount of compounds required in experiments. The more sensitive detection of protein-derived radicals allows many more scientists to perform bench top experiments that more closely approximate the biological environment in thyroid metabolism in the human body.

The study appeared in the August 1, 2006 issue of Free Radical Biology and Medicine. In it the investigators employed enzyme-linked immunosorbent assay (ELISA), gel electrophoresis and Western blotting, relatively economical testing platforms widely used and easily performed in most laboratories, in the place of direct-electron or spin-trapping electron spin resonance (ESR). The former, more accessible, methodology known as immuno­ spin trapping, was developed previously in Mason's laboratory and has been subsequently employed in the Mason lab as well as others. The use of immuno-spin trapping, instead of ESR, reduces the costs of investigating protein radical formation dramatically, as much as ten-fold or more. It also eliminates the need for laboratories to employ highly specialized chemists to perform ESR analysis for free radical research, helping to make this kind of research more practical for investigators in a variety of disciplines.

By boosting sensitivity so remarkably (100 to 1000 times), the new methodology also impacts what are, for many labs, prohibitive costs of the principal compounds used in this type of research. According to the researchers, "The advent of immuno-spin trapping...reduces the amount of protein needed for radical detection experiments from milligram to microgram quantities" (423). As a result, investigators can now perform far more wide-ranging analyses on the very expensive, but very important, human enzyme, thyroid peroxidase.

The methodology also permits researchers to replicate -in the more controlled setting available in laboratory experiments -conditions that more closely resemble what actually occurs in human beings. According to Mason, "The methodology and its exquisite sensitivity [allowed us to use] concentrations of hydrogen-peroxide low enough to make them physiologically plausible, which is not usually the case. Many hydrogen-peroxide experiments are done with a thousand times more hydrogen-peroxide." By using biologically plausible levels of compounds researchers can eliminate one of the big "ifs" that hinder accurate descriptions of the importance of their work.

In the same issue of the journal, the breakthrough study was recognized in commentary written by Society for Free Radical Biology and Medicine President-Elect Rafael Radi, M.D., Ph.D., a member of the Faculty of Medicine at Universidad de la Republica - Montevideo, Uruguay and International Research Scholar of the Howard Hughes Medical Institute. The methodology developed at NIEHS, Radi observed, "is proving to be a potent, sensitive, and accessible method to detect low levels (e.g., greater than nanomolar) of protein-derived radicals produced in vitro and potentially, and yet to be established, in vivo."

With their original research, Ehrenshaft and Mason have demonstrated very poignantly the values of integrated research methods, divergent thinking and the practical application of the scientific principle of "parsimony,"

seeking the least complicated approach to a phenomenon. Their methodological breakthrough may prove to be a critical step in the long process ahead of discovering how to prevent, delay or treat autoimmune thyroid disorders, such as Hashimoto's disease, pointing the way to effective clinical interventions.

Citations: Ehrenshaft M, Mason RP. 2006. Protein radical formation on thyroid peroxidase during turnover. Free Radic Biol Med 41:422-430.

Radi R. 2006. Immuno-spin trapping: A breakthrough for the sensitive detection of protein-derived radicals, a commentary on "Protein radical formation on thyroid peroxidase during turnover." Free Radic Biol Med 41:416-17.



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