In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice

Free radical species in animals have been measured by X-band ESR spectromet ric method on a block of organs or a portion of homogenized samples. Howeve r, a nondestructive in vivo ESR measurement has been realized by using a re cently developed L-band ESR spectrometry. With this L-band ESR method, we m easured ESR spectra in animals, who received stable nitroxide radicals. L-b and ESR spectra were observed at the upper abdomen of mice as well as at th e heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiper idine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrrolidi ne-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals w ere performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important resul ts were obtained: (1) Free radical clearances were able to analyze by the p harmacokinetic method. (2) The radicals at the head of mice, given 4-hydrox y-TEMPO, were determined quantitatively by a new analytical method using L- band ESR for the first time. (3) The elimination of the radical was found t o be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manne rs. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the dete cted radicals to that of the administered radicals were decreased age-depen dently, but they were independent of the dose of the radicals, suggesting t he age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3- carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collecte d blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results su ggest that the clearance of the nitroxide radical is relevant to the altera tion of the radical in animals following the change of organ distribution a nd metabolism. (7) Both the radical and its corresponding hydroxylamine, wh ich is the reduced form of the radical, were detectable by X-band ESR metho d in the collected urine of mice and rats without and with an oxidizing age nt, respectively. On the basis of the results on L-band ESR spectrometry, the first quantitat ive pharmacokinetic analysis of stable spin probes in animals is proposed.