Novel Instrumentation For Direct On-Line Monitoring of Biological Samples in Real-Time With High-Speed Gas Chromatography

Eastern Michigan University, Ypsilanti MI

Investigators

Abstract

Lipid peroxides or their products have recently been cited as biological second messengers in signal transduction pathways that induce cytoprotective enzymes, stimulate protein kinases, mediate programmed cell death, and stimulate neutrophil adhesion and smooth muscle cell proliferation in a manner similar to platelet activating factor. Identification and quantification of lipid peroxidation products will enhance understanding of the enzymology, biochemistry, and biodisposition of these biological mediators. The current detection methods for these products in cells or subcellular fractions include spectrophotometric assays and liquid chromatography, though none are capable of generating data in real-time. Instrumentation and methods to monitor biological processes in tissue cultures in real-time will be developed in this project. Two full-time graduate students will assist in the research and implementation process. The instrument will incorporate a cell incubation device with a cryofocusing inlet suitable for use with high-speed gas chromatography (HSGC), and will be compatible with various platforms. Cells will be cultured in a 37 degrees C oven in standard tissue culture flasks fitted with specialized caps. The caps serve to isolate the products within a flask while providing air and CO2. Direct monitoring will be accomplished by concentrating gaseous products on a cryofocusing trap for injection into a HSGC system. The long-term goal of this project is to develop an integrated, modular incubator/inlet that will provide new capabilities for monitoring biological systems in real-time. The instrument could be used to study many systems, although this work will focus on the characterization of lipid peroxidation products. For inlet design testing and proof of concept work, lipid peroxidation will be stimulated in the isolated microsomal fraction from rat liver, which generates relatively large amounts of products. Detection will be accomplished with time of flight (TOF) mass spectrometry. The high scanning rates of TOF instruments are compatible with high speed GC, and the second dimension of data facilitates positive identification of peroxidation products. This is a decided advantage for complex and dynamic biological systems. A specialized incubator will be constructed in the second phase of the project for use with cell cultures. Final valving configurations will be tested and optimized. The ruggedness of the instrument will be examined in detail, in order to develop an instrument suitable for patent application and marketing.