Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions

Abstract

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.

Epidermal Growth Factor-induced Vacuolar (H+)-ATPase Assembly

“…The cell selection criteria were the same as described above. In the course of these studies we encountered cells in which there was notable autofluorescence as previously observed by others (31). Autofluorescence is visible in all channels but is much weaker in the infrared channel.…”

Optimizing normoxic conditions in liver devices using enhanced gel matrices

“…NADH is highly autofluorescent when illuminated with UV light (366 nm), but its oxidized form, NAD þ , is not. This is why the fluorescence intensity of NADH is widely used to assess the hepatocellular metabolic status (Barlow and Chance, 1976;Croce et al, 2004;ObiTabot and Hanrahan, 1993;Paxian et al, 2004). In our study, changes in the NADH fluorescence intensity were quantified after 15 min of exposure to single gas supply.…”

“…A decreased O 2 tension is paralleled by decreases in the concentration ratio of nicotinamide adenine dinucleotide (NAD) compared to its reduced form (NADH). As such, the autofluorescence of NADH has been widely used as an indicator of cellular metabolic status to elucidate the states of both hepatic tissue and cells under hypoxic conditions (Chandel and Budinger, 1997;Croce et al, 2004;Obi-Tabot and Hanrahan, 1993;Paxian et al, 2004).…”

CO2 Permeability of Rat Hepatocytes and Relation of CO2 Permeability to CO2 Production

“…This showed that non-vital cardiomyocytes no longer possess CA activity and thus do not contribute to the mass spectrometric signal. In the case of hepatocytes, we were not able to use DNSA staining because of the very high auto-fluorescence of hepatocytes [19]. For hepatocytes, we use an alternative approach.…”