Background and aim: The hyper-generation of ROS by impaired mitochondrial ?-oxidation is a cause of enhanced risk of damages and disease progression in fatty liver. In this context we investigated liver autofluorescence (AF) as an innovative and supportive diagnostic tool for the in situ detection of tissue redox-metabolism alteration. Biomolecules naturally present in cells and tissues when excited with light at a suitable wavelength give rise to AF emission, acting as endogenous fluorophores in close dependence on tissue normal or altered morphofunctional conditions. Among the several endogenous fluorophores of liver we focused on NAD(P)Hbound, free and flavins, the coenzymes strictly involved in energetic metabolism, oxidative defense and reductive biosynthesis, as AF intrinsic biosensors of redox-metabolism alteration. Materials and methods: The model of fatty liver with oxidative stress was obtained with a hypercaloric methionine/choline deficient diet (MCD), administered to rats for 1-8 weeks. The AF spectra were collected from bulk tissue in vivo, via fiber optic probe, or ex vivo, from cryostatic tissue sections. A curve fitting analysis was used to estimate the relative contribution of the endogenous fluorophores to the overall emission. Biochemical assays (hepatic lipid peroxidation-TBARS, glutathione (GSH) and ATP) were performed to validate AF results. Results: In vivo data reflect the actual liver metabolic engagement and show a slight and continuous rising in the NAD(P)Hbound/free ratio from the second MCD week, while the redox ratio (flavins/(NADPHtotal + flavins))1 is significantly higher than the control only at the first week. At longer times, the lowering of redox ratio values likely indicates an effort to recover redox-metabolism homehostasis. This condition is achieved despite the real lessening in NAD(P)H binding sites from one MCD week indicated by ex vivo data. In fact, O2 deprivation during tissue removal leads to a NAD(P)H saturation of its binding sites engaged in aerobic respiration, allowing to estimate their actual fraction2. The lowered NAD(P)H binding sites in MCD diet, in turn, are consistent with the decreased mitochondrial activity (ATP) and antioxidant defense engagement (GSH). Conclusions: the AF potential to provide a diagnostic response of liver redox-metabolism alteration is confirmed, as a basis for the development of applications in the biomedical research, i.e. drug response and toxicology, or optimizing of preservation strategies for donor organs for transplantation. (Supported by Fondazione Cariplo, grant n° 2011-0439). 1) Kirkpatrick ND et al., Photochem Photobiol 2005; 2) Croce et al., Photochem Photobiol Sci, 2005.
Autofluorescence and endogenous fluorophores as intrinsic biosensors of liver oxidative stress
GIOVANNI BOTTIROLI;BOTTIROLI G
2014
Abstract
Background and aim: The hyper-generation of ROS by impaired mitochondrial ?-oxidation is a cause of enhanced risk of damages and disease progression in fatty liver. In this context we investigated liver autofluorescence (AF) as an innovative and supportive diagnostic tool for the in situ detection of tissue redox-metabolism alteration. Biomolecules naturally present in cells and tissues when excited with light at a suitable wavelength give rise to AF emission, acting as endogenous fluorophores in close dependence on tissue normal or altered morphofunctional conditions. Among the several endogenous fluorophores of liver we focused on NAD(P)Hbound, free and flavins, the coenzymes strictly involved in energetic metabolism, oxidative defense and reductive biosynthesis, as AF intrinsic biosensors of redox-metabolism alteration. Materials and methods: The model of fatty liver with oxidative stress was obtained with a hypercaloric methionine/choline deficient diet (MCD), administered to rats for 1-8 weeks. The AF spectra were collected from bulk tissue in vivo, via fiber optic probe, or ex vivo, from cryostatic tissue sections. A curve fitting analysis was used to estimate the relative contribution of the endogenous fluorophores to the overall emission. Biochemical assays (hepatic lipid peroxidation-TBARS, glutathione (GSH) and ATP) were performed to validate AF results. Results: In vivo data reflect the actual liver metabolic engagement and show a slight and continuous rising in the NAD(P)Hbound/free ratio from the second MCD week, while the redox ratio (flavins/(NADPHtotal + flavins))1 is significantly higher than the control only at the first week. At longer times, the lowering of redox ratio values likely indicates an effort to recover redox-metabolism homehostasis. This condition is achieved despite the real lessening in NAD(P)H binding sites from one MCD week indicated by ex vivo data. In fact, O2 deprivation during tissue removal leads to a NAD(P)H saturation of its binding sites engaged in aerobic respiration, allowing to estimate their actual fraction2. The lowered NAD(P)H binding sites in MCD diet, in turn, are consistent with the decreased mitochondrial activity (ATP) and antioxidant defense engagement (GSH). Conclusions: the AF potential to provide a diagnostic response of liver redox-metabolism alteration is confirmed, as a basis for the development of applications in the biomedical research, i.e. drug response and toxicology, or optimizing of preservation strategies for donor organs for transplantation. (Supported by Fondazione Cariplo, grant n° 2011-0439). 1) Kirkpatrick ND et al., Photochem Photobiol 2005; 2) Croce et al., Photochem Photobiol Sci, 2005.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
