Bioassay techniques Subject

Although a potential advantage of inhaled antibiotic therapy is the achievement of high concentrations in the sputum, there is substantial variability reported, which may reflect differences in collection and bioassay techniques. There is no clear relationship between systemic and inhaled doses of individual agents. Currently, the decisions about inhaled doses should be made on data specific for an individual agent. In trials with the commercially available inhaled tobramycin product, sputum concentrations of 1200 pg per mL were measured 10 minutes after the dose. Measured concentrations exceeded 25 times the minimum inhibitory concentration (MIC) for the most resistant isolate in 95% of subjects evaluated [7]. 

Complicated biological systems (bioassays) at trace element concentration levels typical for offshore waters, are subject to serious danger of contamination. Without extreme precautions e.g. Carpenter and Lively (1980) and Fitzwater et al. (1982) found the toxic effect (inhibition of primary production) of contamination by the incubation bottles. Effects of adsorption to walls and particulate matter (sediment) should not be underestimated. Use of clean lab techniques and regular check of the trace element concentrations throughout the (biological) experiments is necessary to get an indication of the actual concentration and possible distribution of the different elements. Depending on the type of experiments it could be possible that other parametes should be known or even controlled pE, PO2, ionic strength, temperature, DOC etc. 

The usual technique used is the bioassay in mice developed by Yasumoto that we described in the European Regulations. One may also use the bioassay in rats developed by Kat [74]. The bioassay in mice is the most widely used because it is more sensitive (OA 4 pg) than exposure by oral ingestion in rats (OA 10 pg), but the bioassay in mice is subject to false positives by interference of nonphycotoxic components, and it is more expensive since mice die or are disposed of, while rats can be used repeatedly, although reading the test in them depends on the subjective examination of feces. 

Because of the low cost of this bioassay test ( 500-1,000/chemical) relative to the often unpredictable and always more costly demands of chemical analysis, most chemicals are bioassayed by Salmonella before they are chemically analyzed. Plans call for 300 chemicals to be tested in the microbial system in FY-80 with a eventual goal of 1,000 bioassays of new chemicals a year by 1983. Test chemicals producing positive, ambiguous and selected negative bioassay results are then subjected to comprehensive chemical analysis. An important aspect is that the separation technique used in the chemical analysis must insure ... 

After separation, the resultant fractions are retested and the positive fractions are further fractionated or subjected to complete component identification and quantitation using sophisticated chromatographic and spectroscopic techniques, GC/MS (El and Cl), HPLC, NMR, IR, etc. Each component of the final active fraction is then bioassayed separately to determine the chemical(s) responsible for the original observed biological activity. It may also become necessary to synthesize the isolated active chemical for confirmation purposes. 

If one wants to determine prostaglandins at nanogram level, three different methods are recommended bioassay, radioimmunoassay (RIA) and gas chromatography-mass spectroscopy (GC-MS). These methods will be discussed at this workshop by several investigators and the advantages and disadvantages of each of these techniques will be the subject of a debate. It is clear that having all assays available would be the most ideal situation but for practical reasons restrictions are often necessary. At our laboratory, bioassay and GC-MS are applied for various purposes and facilities and some experience with RIA are available. 

Most behavioral bioassays reviewed here offer the subjects a small number (usually two) choices, then the frequency of choices is recorded and analyzed with relatively simple statistical techniques. The purpose of such assays usually is to determine biological activity compared to a blank or solvent control quantitative rankings of activity are better developed using bioassays with multiple choices. 

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