In vivo analysis of hepatic NADH fluorescence. Methodological approach to exclude Ito-cell vitamin A-derived autofluorescence.
Assessment of hepatic surface NADH fluorescence is complicated by questions concerning the relative contribution of Ito cell-associated vitamin A autofluorescence, which cannot be distinguished from each other using ultraviolet epi-illuminated microfluorographs. However, as presented herein, vitamin A autofluorescence can easily be eliminated due to its rapid photobleaching property. In line with in vitro studies on liver perfusion reported by Suematsu and coworkers (1993b), we assessed NADH fluorescence in liver surface regions, which were epi-illuminated for elimination of vitamin A autofluorescence and were allowed to recover for 20 min prior to onset of the experiment. To assess whether this procedure is stringent for accurate estimation of NADH fluorescence, we studied NADH fluorescence in non prebleached surface regions after a 30 sec and 60 sec epi-illumination period. Epi-illumination of these hepatic surface regions for more than 20 sec guaranteed also complete elimination of vitamin A autofluorescence (Vollmar et al., 1996) and further allowed to calculate the subsequent loss of NADH fluorescence due to a potential photobleaching effect after 30 sec and 60 sec of continuous light exposure. Corresponding data of the present study reveal a constant loss of NADH fluorescence of approximately 20% due to its photobleaching property over the time period from 20-60 sec of epi-illumination. Linear regression analysis of the kinetics of NADH fluorescence upon continuous light exposure allowed the calculation of the initial NADH fluorescence, excluding the interference with the Ito cell vitamin A-derived autofluorescence. The concurrency of this mathematically assessed value for the initial (non-bleached) NADH fluorescence within normal hepatic livers with the recovered value of NADH fluorescence allows the conclusion that the procedure for elimination of vitamin A autofluorescence, including ultraviolet epi-illumination with a subsequent NADH recovery period of at least 20 min, is not a prerequisite for the reliable assessment of intraindividual changes of hepatic NADH fluorescence in vivo. Thus, valid assessment of hepatic NADH fluorescence in vivo can be performed by analyzing initial NADH fluorescence bleaching kinetics, and does not necessarily require the 20 min recovery period after bleaching of the Ito cell vitamin A-derived autofluorescence.
Burkhardt
M
M
Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany.
Vollmar
B
B
Menger
M D
MD
D000818:Animals; D016477:Artifacts; D008099:Liver / Q000166:cytology* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D014801:Vitamin A
D009243:NAD; D014801:Vitamin A