Sensitive and specific method for the simultaneous determination of natural and synthetic catecholamines and 3,4-dihydroxyphenylglycol in microdialysis samples

The relatively new technique of microdialysis provides new possibilities for investigating in vivo the functioning of the sympathetic nervous system. The small sample volumes obtained, however, are a great challenge for analytical chemists. We report here a HPLC method for measuring in one run both natural and synthetic catecholamines [dopamine, (nor)epinephrine, -methylnorepinephrine, isoproterenol and epinine] and the intraneuronal metabolite 3,4-dihydroxyphenylglycol in small microdialysis samples after derivatization with the fluorogenic agent 1,2-diphenylethylenediamine. No prior clean-up step is necessary. N-Ethylmaleimide is necessary for preventing an inhibitory action on derivatization occurring in in vivo microdialysis samples. The method can handle large numbers of samples, is sensitive (on-column detection limits 30 to 200 fg) and reproducible (RSD 1 to 7%). Recovery characteristics of the commercial microdialysis probe used (CMA/20) were extensively investigated both in vitro and in vivo at various perfusion rates; for practical purposes a rate of 2 μl/min and sampling at 10-min intervals was found to be workable and to give good and reproducible recoveries (50 to 70%).     

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish

Following the intestinal absorption of dietary benzo[a]pyrene (BP) by the killifish, this compound becomes incorporated along with dietary triglycerides into membrane-bound fat vacuoles within the intestinal epithelial cell (1985. J. Lipid Res. 26: 428-434). These vacuoles, arising from the smooth endoplasmic reticulum, are important transient structures involved in both the uptake and metabolism of dietary BP and, presumably, other lipophilic toxicants as well. In the present study we used subcellular fractions isolated from the intestines of spot (Leiostomus xanthurus), a teleost fish, to study factors that influence the metabolism of BP in a fat vacuole/microsomal system. Triglyceride-solubilized BP is capable of diffusion from fat vacuoles to microsomal enzymes. Increases in the concentration of fat vacuoles decrease the availability of BP to microsomal BP hydroxylase. The effect of fat vacuoles on the activity of BP hydroxylase becomes more pronounced as the concentration of BP in our test system decreases. Addition of cytosolic glutathione transferases to the fat vacuole/microsomal system enhances the activity of BP hydroxylase. Examination of binding of 3H-labeled BP to killifish (Fundulus heteroclitus) intestinal cytosolic proteins in vivo indicated that a large fraction of the radioactivity was associated within glutathione transferase. These results suggest that dietary fat inhibits metabolism of low levels of BP in the intestine. A consequence of this would be greater exposure of peripheral tissues to dietary carcinogens.