PubMed · 3202921
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J A Thompson, R D Wells. 1988-07-07. HPLC in nucleic acids research.. https://doi.org/10.1038/334087a0
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UNLABELLED: Genetically modified mammalian cells that express the cytosine deaminase (CD) gene are able to convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). PET with 18F-5-FC may be used for in vivo measurement of CD activity in genetically modified tumors. METHODS: A human glioblastoma cell line was stably transfected with the Escherichia coli CD gene. After incubation of lysates of CD-expressing cells and control cells with 3H-5-FC high-performance liquid chromatography (HPLC) was performed. The uptake of 5-FC was measured after various incubation times using therapeutic amounts of 5-FC. In addition, saturation and competition experiments with 5-FC and 5-FU were performed. Finally, the efflux was measured. RESULTS: We found that 3H-5-FU was produced in CD-expressing cells, whereas in the control cells only 3H-5-FC was detected. Moreover, significant amounts of 5-FU were found in the medium of cultured cells, which may account for the bystander effect observed in previous experiments. However, uptake studies revealed a moderate and nonsaturable accumulation of radioactivity in the tumor cells, suggesting that 5-FC enters the cells only through diffusion. Although a significant difference in 5-FC uptake was seen between CD-positive and control cells after 48 hr of incubation, no difference was observed after 2 hr of incubation. Furthermore, a rapid efflux could be demonstrated. CONCLUSION: 5-Fluorocytosine transport may be a limiting factor for this therapeutic procedure. Quantitation with PET has to rely more on dynamic studies and modeling, including HPLC analysis of the plasma, than on nonmodeling approaches.
Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.
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