In the Lab

In the Lab with Dr. Kenneth Hensley, associate member of the Free Radical Biology and Aging Research Foundation, (OMRF), and one of the evaluators of ESA's new electrochemical system featuring the Boron Doped Diamond (BDD) cell.

ESA: Explain briefly the nature of your research. What are some of the challenges you face?

DR. HENSLEY: Our laboratory uses HPLC-electrochemical techniques for three main purposes. First, we analyze animal and human tissue and cell culture extracts for small endogenous antioxidants such as α- and γ-tocopherol and glutathione, as part of our basic science research into redox biochemistry. Second, we analyze animal and human tissue for small molecular biomarkers of oxidative stress including 5-nitro-γ-tocopherol and nitrotyrosine. Third, we synthesize and test small molecule anti-inflammatory agents that we are developing in-house for the treatment of neurological diseases. We are beginning to conduct pharmacokinetics studies in support of this applied research and the BDD is being tasked mainly towards this purpose.

ESA: What interested you in the BDD cell? What problem, if any, were you trying to solve with the BDD? Or were you just interested in BDD as a capability or curiosity?

DR. HENSLEY: Our newest lead compound, invented in our laboratory, is a cyclic thioether that is very difficult to analyze using most electrochemical techniques. We need a technique that can analyze this thioether by oxidizing above 900 mV while minimizing background components from biological matrices. We also need a technique that allows semi-automated, routine analysis of 10s-hundreds of samples. Having had considerable experience and success using HPLC-coulometric detection we decided to attempt the BDD in our drug development project.

ESA: What were you hoping to see with the BDD? Did it meet your expectations? Why or why not?

DR. HENSLEY: We were expecting to effectively and accurately measure the cyclic thioether compounds and its derivative with high sensitivity and selectivity. We also analyzed methionine, reduced glutathione (GSH) and oxidized glutathione (GSSG) during the validation portion of beta-testing phase of the BDD and observed low baseline noise with excellent reproducibility. Yes, the BDD electrode met our expectations and Kelly S. Williamson (a bioanalytical chemist in my lab who routinely uses HPLC coupled with electrochemical detection) stated that this type of detection system (BDD) will enable our laboratory to look at a whole new spectrum of sulfur-based compounds that we previously have not been able to accurately measure in a variety of different sample matrices.

ESA: What capabilities did the BDD bring to your lab that you didn't have before? Why is this important to you?

DR. HENSLEY: A major issue is being able to implement a higher oxidation potential without worry of physically damaging the BDD electrode. This is important because we are able to set the oxidation potential in the 1500-1700 mV range and accurately detect the sulfur-based compounds of interest. However, employing the CoulArray 5600 series electrodes, the user runs the risk (at oxidation potentials greater than 900 mV) of diminished electrode performance and response over subsequent runs, as well as causing permanent cell damage. Other issues consisting of reliability, durability, and overall performance are important variables we considered during and after the beta-testing of the BDD detection system and the BDD electrode met our analytical needs for each specified application.

ESA: How will the BDD impact your research? How will you use the BDD?

DR. HENSLEY: The BDD electrode detection system will enable our laboratory accurately measure thiol-based compounds with increased selectivity and sensitivity. This will expand our knowledge-base and flexibility in performing additional investigational studies regarding drug development in human, animal, and cell culture models via redox biochemistry. It will also help us to answer many of the important questions pertaining to neurological diseases. As previously stated, our laboratory will use the BDD to analyze cyclic thioether compounds and their derivatives, as well as methionine, GSH, and GSSG in different biological models in order to gain additional insight into oxidative metabolism containing sulfur-based compounds.

ESA: What was the best thing about the BDD? Were there any surprises in using the BDD?

DR. HENSLEY: The best thing our laboratory liked was the ease of use, simplicity, and that the BDD electode does not take up a whole lot of bench space. There was only one minor surprise employing the BDD electrode which can be easily corrected. During the beta-testing of the validation phase, temperature plays an important role in peak response when analyzing methionine, GSH, and GSSG. Our laboratory typically analyzes compounds at either ambient temperature or lower. Upon comparision with ESA's results, our peak areas and peak heights were somewhat lower. However, this variation (using the same HPLC conditions) could be explained by analyzing methionine, GSH, and GSSG at 32º C versus ambient temperature, which another beta-test site did obtaining similiar results as ESA.