Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions.
The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.
Histochemistry and Cytometry Section, IGM-CNR, Department of Animal Biology, Piazza Botta, 10, 27100 Pavia, Italy. leta@igm.cnr.it.
Anna Cleta
AC
Croce
Andrea
A
Ferrigno
Mariapia
M
Vairetti
Roberta
R
Bertone
Isabel
I
Freitas
Giovanni
G
Bottiroli
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D005227:Fatty Acids / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D005453:Fluorescence; D022781:Hepatocytes / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D009249:NADP / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014801:Vitamin A / Q000378:metabolism
D005227:Fatty Acids; D005415:Flavins; D014801:Vitamin A; D009249:NADP