By Jerry Fireman
Boron-doped diamond ECD electrodes operate over a greater potential window without suffering from the high noise resulting from the oxidation of water in the mobile phase.
Huntington’s disease, also known as Huntington’s chorea or chorea major, is a genetic neurological disorder characterized by abnormal body movements (chorea).There is no treatment available today to arrest the progression of Huntington’s disease although some of its symptoms can be alleviated through medication and nutrition. Oklahoma Medical Research Foundation (OMRF) scientists are looking at derivatives of a promising compound, lanthionine ketimine (LK), that have shown the potential to stop damage to nerve cells and reduce inflammation. A critical part of this research involves measuring the bioavailability of various active compounds in the central nervous system.
Electrochemical detectors (ECDs) provide a state-of-the-art method for measuring biological compounds at very low concentrations. But, conventional graphitic and glassy carbon electrodes used with ECDs cannot detect LK and its derivatives because these compounds don’t oxidize or reduce within the potential window that can be used with conventional electrodes. OMRF researchers have overcome this problem by using boron-doped diamond ECD electrodes that can operate over a greater potential window without suffering from the high noise resulting from the oxidation of water in the mobile phase.
Causes of Huntington’s disease
Huntington’s disease is inherited by approximately three to seven per 100,000 people of Western European descent. It is caused by a trinucleotide repeat expansion in the Huntingtin gene that codes for the Huntingtin protein. Both the gene and protein are spelled differently than the disease. The expansion produces an altered form of the Huntingtin protein that causes neuronal cell death in certain areas of the brain. The symptoms of the disease include abnormal body movements and lack of coordination. Mental abilities and behavior may also be affected.
Huntington’s disease is believed to damage nerve cells through inflammation, excess nitric oxide production, and excess glutamate excitotoxicity. The OMRF researchers focused on LK and its derivatives because they have antioxidant, antineuroinflammatory, and neuroprotective properties. Scientists have been conducting laboratory tests with LK, and early data indicates the compound could stop damage to nerve cells and reduce inflammation. Doing so would delay the motor-function deterioration caused by Huntington’s disease.
Reaching brain cells is key to search for treatment
A key challenge in developing a compound for treatment or prevention of this disease is improving the delivery of LK derivatives to target brain cells and particularly improving permeability through the blood-brain barrier. The OMRF researchers are working to improve delivery through derivatization of LK to form LK esters (LKEs) and LK amides (LKAs). For example LK ethyl ester has proven more capable of penetrating cell membranes and reaching intracellular targets of action.
A critical aspect of improving the delivery of LK derivatives involves the ability to accurately measure them at very low concentrations in the brain of animals used in research. Nervous system cells contain many different components that must usually be separated before they can be analyzed and quantitated. High performance liquid chromatography (HPLC) provides an excellent separation tool. Mass spectrometry (MS) can be used to detect the presence of LK derivatives in very small quantities, however, MS is very time consuming and requires expensive equipment and a highly trained operator.
ECD plays a key role
This helps explain why ECD has become such a popular method of measuring biological and clinical molecules. ECD detectors work by applying a voltage between a working and a reference electrode in a flow cell. As molecules pass through the flow cell, those that can be easily oxidized or reduced at the applied potential react at the working electrode, producing a flow of electrons. The detector measures this flow. Only those molecules that will oxidize or reduce at the electrode at the applied potential are detected, thus providing a high degree of selectivity. The inherent sensitivity, selectivity, and wide dynamic range of this technique have caused it to be used extensively in brain research and other areas of medical research.
Up to now, noble metals such as silver and platinum and various forms of carbon have been used as ECD electrodes. In particular, carbon-based electrodes have found extensive use in a broad range of applications. These electrodes are limited in the molecules they can detect because of restrictions in the potential ranges that can be used with these electrodes. These restrictions arise as a result of water electrolysis at conventional electrodes; at positive potentials, water decomposes to form oxygen, and hydrogen forms at negative potentials, leaving a window of only approximately one volt with which to oxidize or reduce analytes. This window is too narrow to identify LK variants.
Boron-doped diamond electrodes extend ECD
“Diamond, in its natural state, is unsuitable for use as an electrode because of its inertness. However, methods have been developed for including metal dopants in diamond films that render the inherently insulting diamond film conductive.
Boron-doped diamond electrodes are typically constructed on a supporting substrate, most often silicon or a metal. The polycrystalline thin film is formed by chemical vapor deposition. At high levels of boron doping, diamond becomes highly conductive, making it a suitable electrode material. A key advantage of boron-doped diamond electrodes is that they open up the potential window to between three and four volts, sufficient to detect LK and its variants, many other thiols and phenolic compounds, as well as a wide array of other analytes that are of interest to researchers.
Michael Granger, Research Scientist at the University of Utah, Salt Lake City, and a pioneer in the development of boron-doped diamonds commented: “There is no one ideal electrode material that is conducive to all electrochemical applications. Boron-doped diamond electrodes appear superior to graphitic carbon electrodes in several areas. BDD electrodes possess a larger working potential window with smaller background currents, longer stability in many electroanalytical environments, are much more corrosion resistant, and are less prone to adsorption than other carbon electrodes. These attributes are a consequence of the extremely high atomic density of the material and the hydrogen terminated surface.”
Methods used at OMRF
Kenneth Hensley, who is leading the OMRF research on lanthionine ketimine, and Kelly Williamson developed the following method for assaying LK and LKE using the boron-doped diamond electrodes. LK and LKE are separated using isocratic reverse-phase HPLC. The HPLC mobile phase is 3% acetonitrile in 50 mM lithium acetate at pH 5.6. Plasma samples are prepared for HPLC by rapid microfiltration through a 7 kDa Pierce Biotechnology spin-filter. Concentrations are determined by comparison of external calibration curves.
Specific spike-recovery samples (LK + LKE spiked into plasma) are assessed with each assay to confirm extraction efficiency. Tissue samples are homogenized in saline plus 0.1% triton X-100, adjusted to 5 mg/mL protein, and centrifuged to remove insoluble matter, and supernatant processed similarly to plasma. Tissue drug concentrations are reported in moles/mg protein and moles/g tissue wet weight.
The HPLC is equipped with a CoulArray coulometric ECD detector from ESA Biosciences, Inc., Chelmsford, Mass. The ESA boron-doped diamond electrodes use a thin-film amperometric cell design with a palladium reference electrode. The boron-doped diamond is deposited on a wafer, then cut to the proper size and shape. The wafer is coated with a conductive backing layer. This electrode chip is then placed into the 5040 analytical cell. Contact with the electrode and sealing against a gasket is made with a pin assembly, which makes continuous contact with the working electrode. The cell is then connected to, and is controlled by, the CoulArray potentiostat.
New electrodes helpthe search for treatment
“We have used boron-doped diamond electrodes for about eight months and have seen no degradation of the electrode or negative issues of any kind,” Hensley said. “We have measured samples in the micromolar range with boron-doped diamond electrodes after administering various proprietary drugs in order to determine the availability of each drug candidate in the central nervous system. We have also had a very positive experience with ESA technical support in helping us get up and running quickly.”
With a grant from the Hereditary Disease Foundation of New York City, the OMRF team is now using the boron-doped diamond electrodes in pre-clinical tests used to determine whether two proprietary LK derivatives slow the progress of Huntington’s disease. The team is testing these compounds on mice, which have been genetically engineered to develop Huntington’s disease. They will look at whether the compounds slow the loss of body control that begins at an early stage in the disease. Hensley will also investigate the compounds’ ability to stop the destruction of neurons and so improve brain function.
“Our goal is to help people with the disease live longer and healthier lives,” Hensley said. “If we could slow the rate of progression of the disease we could turn a 10- to 20-year illness into a 50-year illness. People would receive decades of more effective treatments and watch their children grow into adults.”
About the Author Jerry Fireman is President of Structured Information in Arlington, Massachusetts. He has been writing about biotechnology for 20 years.