Biochemical and Mass Spectroscopy Core

Medical College Of Wisconsin, Milwaukee WI

Investigators

Linked publications, trials & patents

Abstract

The Biochemical Core Laboratory of this PPG was originally established 25 years ago as a resource facility to provide a consolidated, highly specialized, well equipped and professionally staffed unit capable of performing a variety of immunoassay and other biochemical assays as required by Program Project Investigators. For the last 20 years, Dr. Roman has served as the director of this facility. Over time, the Core laboratory has also received substantial capital investment from both the Department and MCW to continually upgrade core equipment with state-of the-art technology to extend the range of analyses that can be performed in this laboratory. As will immediately become evident from the following description, no single laboratory supported with R01 grants alone would be able purchase all the equipment or hire the staff that is currently available in this Core laboratory. This centralized facility is critical to the success of each of the proposed projects of this program and the continued support of this facility is is more than justified by the large number of biochemical and analytical determinations required for the proposed studies. Centralizing the personnel and equipment in the Biochemical and Mass Spectroscopy Core allows this laboratory to perform routine biochemical analyses in a more efficient and cost effective manner than if the investigators had setup to perform these analyses within their individual laboratories. Consolidation of all of the analytical work of this program into the Core also increases sample loads to an extent that we can justify automating most assays as we upgrade and replace existing equipment. For example, all 7 of our HPLC systems are now equipped with refrigerated autosamplers (Figure 1) and we converted most of our routine spectrophotometric assays to run on a clinical chemistry analyzer that we modified and reprogrammed to work with the sample volumes we can collect from rats and mice (Figure 2). This has greatly reduced our sample turnaround times and has proven to be highly cost effective by reducing technician time and allowing the work to proceed on a 24hr a day/7 day a week schedule. Moreover, in the last 5 years we have been able to purchase 2 LC/MS/MS systems with departmental, institutional and nonfederal funds (Figures 3 and 4) with refrigerated autosamplers that we now use for state-of-the-art analysis of eicosanoids in a variety of samples and have developed new analytical procedures for the assessment of oxidative stress in tissues by measuring the formation of oxyethidium from dihydroethidium. The centralized Biochemical and Mass Spectroscopy Core will ensure uniformity of assay results between the different laboratories in this program and will maintain complete, computerized and centralized records as well as well-defined quality control guidelines for each assay. New assays will be developed with the Core Staff and Dr. Roman working with specific investigators to meet the ever-changing needs and direction of the program. After an assay is fully documented, it becomes available for use by all the other laboratories within this and other programs. The Core personnel will consult with the investigators and staff in each laboratory regarding the requirements for the isolation and incubation of samples to ensure that the samples are correctly prepared and stored. Samples are submitted to Core B staff for radioimmunoassay (RIA), enzyme-linked immunoassay (EIA), HPLC or LC/MS analysis. The analytical results of each analysis are carefully reviewed and certified by Dr. Roman and/or Lisa Henderson and the staff then provides a detailed electronic copy of the results back to the investigators. Prioritization of sample loads is discussed at weekly meetings between the Core staff and Dr. Roman. These meetings also address issues regarding assay development, documentation and equipment upgrades and repairs. We have found that the experienced staff and collective expertise of the Biochemical and Mass Spectroscopy Core can modify current methods or develop new assays in a more efficient manner than if the investigators had to do so in their individual laboratories. In this regard, the Biochemical Core has been able to adapt to the changing needs of this program and is a prime reason for its continued success. Initially, the Biochemical Core provided RIA measurements of circulating hormones (renin, vasopressin, aldosterone, atrial natriuretic factor and catecholamines) in volumes of plasma suitable for work in large animals. About 10 years ago, these assays were all scaled down to allow for measurements in sample volumes that could be obtained from rats and mice. The Core also developed new assays for second messengers and signaling molecules, including CYP450 eicosanoids, IPS, DAG, cAMP, cGMP, and prostaglandins as the research interests of the Program Investigators evolved. More recently, the core has developed new HPLC based assays for nitric oxide (NO), HPLC based and mass spectroscopy assays to measure the formation of superoxide from the oxidation of dihydroethidium, a microtiter plate assay for measurement of tissue H2O2 and a new fluorescent HPLC assay for measuring tissue levels of biopterin, pterin, BH2 and BH4. The Core Lab staff also modified, optimized and validated commercial ElAs for measuring isoprostane, TXB2, 6-keto-PGI2, cGMP and cAMP. Finally, they developed a new fluorescent microtiter plate assay for measuring urinary microalbumin and LC/MS/MS assays for cytochrome P450 metabolites of arachidonic acid (AA). All of these assays are described in more detail below and have been centralized and standardized in the Biochemical and Mass Spectroscopy Core. In this way, the Core continues to serve as the catalyst for the transfer of new techniques between the laboratories and all research programs served by this centralized facility, without duplication of effort or the need to purchase additional expensive equipment. This aspect of Core B has been, in our minds, one of the keys to the success of research programs in the Department of Physiology and, more recently, throughout MCW as this Core facility continues to expand and serve the needs of other research programs in the renal and cardiovascular fields.