Drug metabolism isolated cells, measurements

If liver cells (hepatocytes) are isolated and grown in culture, drugs are exposed to a similar array of enzymes. Because CYP enzymes are bound to the internal membrane fraction of hepatocytes, the liver can be homogenized and a preparation of vesicles of the hepatocyte endoplasmic reticulum called microsomes can be incubated with drug molecules. This preparation suffers because many of the soluble Phase II conjugation enzymes that are found in the cellular cytoplasm are lost. An alternative method for measuring microsomal metabolism involves isolation of the so-called S9 fraction , which includes the cytosolic soluble conjugation enzymes. 

In this context, the applications of PyMS in biotechnology are of particular interest. The technique has been used to detect and measure the level of metabolites produced in cell culture, such as indole produced by cultured Escherichia coli cells grown in different types of media, and to predict the yield of recombinant protein in culture systems, such as E. coli producing recombinant cytochrome B5 or a-interferon. An analogous application is the use of PyMS to profile drugs and their metabolic breakdown products in serum (e.g., cyclosporin, vancomycin), or to profile microbial isolates for novel drug-producing strains. 

A variety of in vitro assays have been developed that minimize or avoid live animal experimentation. Several capitalize on the sodium channel s affinity for these toxins. Neuronal cell lines lyse in the presence of veratridine, a sodium channel activator, and ouabain, which prevents removal of the excessive sodium ions allowed in by veratridine. In the presence of both these drugs, a sodium channel blocker such as saxitoxin rescues the cells. Cellular viability can then be measured by adding tetrazolium salts that are metabolized by living cells to a colored product. Alternatively, isolated cellular membranes, typically from brain tissue, are used to bind radiolabeled saxitoxin. After incubating receptor and radioligand in the presence of a test sample, any radiolabeled saxitoxin bound to the cell membranes are deposited onto filters by vacuum pressure. Radioactivity from the labeled saxitoxin is then measured with a signal reduction indicating the presence of saxitoxin. 

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