Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy*♦

Abstract

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism. Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism. The role of the intrinsic fluorophore NADH as the principal electron donor in glycolytic and oxidative energy metabolism makes it a convenient non-invasive fluorescent probe of metabolic state (1Chance B. Jobsis F. Schoener B. Cohen P. Science. 1962; 137: 499-508Crossref PubMed Scopus (631) Google Scholar, 2Chance B. Williams G.R. Adv. Enzymol. Relat. Subj. Biochem. 1956; 17: 65-134PubMed Google Scholar). Traditionally, fluorimetric studies of metabolic dynamics have characterized metabolic states by the total NADH concentration. However, Williams et al. (3Williamson D.H. Lund P. Krebs H.A. Biochem. J. 1967; 103: 514-527Crossref PubMed Scopus (1279) Google Scholar) pointed out that the reaction velocity of a given intracellular NADH-linked dehydrogenase depends on the concentration of locally available NADH, i.e. the local concentration of free NADH. Given this thermodynamic importance of free NADH, considerable research has been done to discriminate the intracellular free fraction of NADH. Analytical chemistry techniques have provided the most accurate and detailed information about intracellular free and total NADH. Pyridine nucleotide extraction (4Klaidman L.K. Leung A.C. Adams J.D. Anal. Biochem. 1995; 228: 312-317Crossref PubMed Scopus (126) Google Scholar, 5Garofalo O. Cox D.W.G. Bachelard H.S. J. Neurochem. 1988; 51: 172-176Crossref PubMed Scopus (46) Google Scholar) gives an absolute measure of the total tissue NAD+ and NADH concentrations, while the metabolite indicator method (3Williamson D.H. Lund P. Krebs H.A. Biochem. J. 1967; 103: 514-527Crossref PubMed Scopus (1279) Google Scholar, 6Bucher T. Brauser B. Conze A. Klein F. Langguth O. Sies H. Eur. J. Biochem. 1972; 27: 301-371Crossref PubMed Scopus (134) Google Scholar, 7Merrill D.K. Guynn R.W. J. Neurochem. 1976; 27: 459-464Crossref PubMed Scopus (16) Google Scholar, 8Merrill D.K. Guynn R.W. Brain Res. 1982; 239: 71-80Crossref PubMed Scopus (13) Google Scholar) has been used to infer both the cytoplasmic and mitochondrial free NAD+/NADH ratio from the concentrations of specific cytoplasmic and mitochondrial redox couples. However, these techniques entail destroying the tissue, thereby restricting the study of metabolic dynamics to single shot measurements. Furthermore, these techniques are also intrinsically incapable of resolving spatial variations in the free/bound state ratios. In contrast, fluorescence spectroscopic techniques (9Chance B. Baltscheffsky H. J. Biol. Chem. 1958; 233: 736-739Abstract Full Text PDF PubMed Google Scholar, 10Salmon J.M. Kohen E. Viallet P. Hirschberg J.G. Wouters A.W. Kohen C. Thorell B. Photochem. Photobiol. 1982; 36: 585-593Crossref PubMed Scopus (86) Google Scholar, 11, Velick, S. F. (1961) in A Symposium on Light and Life (McElroy, W. D., and Glass, B., eds) pp. 108-143, The Johns Hopkins Press, Baltimore, MDGoogle Scholar, 12Gafni A. Brand L. Biochemistry. 1976; 15: 3165-3171Crossref PubMed Scopus (115) Google Scholar, 13Paul R.J. Schneckenburger H. Naturwissenschaften. 1996; 83: 32-35Crossref PubMed Scopus (59) Google Scholar, 14Wakita M. Nishimura G. Tamura M. J. Biochem. 1995; 118: 1151-1160Crossref PubMed Scopus (104) Google Scholar, 15Hones G. Hones J. Hauser M. Biol. Chem. Hoppe-Seyler. 1986; 367: 103-108Crossref PubMed Scopus (9) Google Scholar) are non-destructive and readily extendable to an imaging modality to address spatial heterogeneity. These techniques are limited, however, by the ambiguous distinction between free and bound NADH fluorescence. Binding-induced shifts of the emission spectrum (up to ∼20 nm) (9Chance B. Baltscheffsky H. J. Biol. Chem. 1958; 233: 736-739Abstract Full Text PDF PubMed Google Scholar, 10Salmon J.M. Kohen E. Viallet P. Hirschberg J.G. Wouters A.W. Kohen C. Thorell B. Photochem. Photobiol. 1982; 36: 585-593Crossref PubMed Scopus (86) Google Scholar, 11, Velick, S. F. (1961) in A Symposium on Light and Life (McElroy, W. D., and Glass, B., eds) pp. 108-143, The Johns Hopkins Press, Baltimore, MDGoogle Scholar) are small compared with the width of the NADH spectrum (∼150 nm). Fluorescence lifetime is a more sensitive probe of NADH binding because it is enhanced significantly (up to 10 times) (12Gafni A. Brand L. Biochemistry. 1976; 15: 3165-3171Crossref PubMed Scopus (115) Google Scholar, 13Paul R.J. Schneckenburger H. Naturwissenschaften. 1996; 83: 32-35Crossref PubMed Scopus (59) Google Scholar, 14Wakita M. Nishimura G. Tamura M. J. Biochem. 1995; 118: 1151-1160Crossref PubMed Scopus (104) Google Scholar). However, the fluorescence decay of bound NADH is usually multi exponential with shorter components that can be comparable with the decay time of free NADH (12Gafni A. Brand L. Biochemistry. 1976; 15: 3165-3171Crossref PubMed Scopus (115) Google Scholar). This makes it difficult to attribute a fast fluorescence decay component in tissue to free or bound NADH. Since intracellular NADH exists in dynamic pools of both the free form and bound to many different dehydrogenases, the integrated cellular spectra are an uncertain combination of many different spectral influences. What is required is a fluorescence signature that can unambiguously discriminate between free and bound intracellular NADH. Here we show that global analysis of time resolved fluorescence and anisotropy decays (16Knutson J.R. Beechem J.M. Brand L. Chem. Phys. Lett. 1983; 102: 501-507Crossref Scopus (540) Google Scholar, 17Lakowicz J.R. Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New Scholar) provides information on the state dynamics and of intracellular NADH. of NADH to an times) increase in the anisotropy decay time G. Hones J. Hauser M. Biol. Chem. Hoppe-Seyler. 1986; 367: 103-108Crossref PubMed Scopus (9) Google the in the of the free NADH and the binding offers the most sensitive discrimination of free and enzyme bound NADH. Furthermore, the in the lifetime of NADH in the intracellular environment provides a measure of the of fluorescence for changes in the fluorescence we can infer changes in the NADH concentration as a of metabolic a of metabolic state changes in the total concentration of NADH and free and bound thereby the between and The for and from has been Science. PubMed Scopus Google Scholar, M. A.C. PubMed Scopus Google Scholar). these imaging that the fluorescence response of to hypoxia a state in Science. PubMed Scopus Google Scholar). used mitochondrial with a of over the a of to the in the state for 10 to spectra in the The to for 10 to spectra in the of the metabolic spectra more with for The spectra with to the spectra a metabolic transition and the of time resolved fluorescence has been Science. PubMed Scopus Google Scholar, Chem. Phys. Scopus Google Scholar) (3Williamson D.H. Lund P. Krebs H.A. Biochem. J. 1967; 103: 514-527Crossref PubMed Scopus (1279) Google Scholar). A and emission nm) used to the NADH fluorescence from the the intracellular fluorescence of both a NADH and a of emission the of the fluorescence to than The emission also a from intracellular that is to NADH. the low concentration of in the and in energy metabolism makes this fluorescence a and The of both time resolved fluorescence spectra and anisotropy decays time single on have been in Chem. Phys. Scopus Google we be fluorescence tissue the with to the of the and the a as to effectively the in a in the This by the over an the of the of the of fluorescence the This to tissue the of the that be The of the fluorescence by a of and components These are with to the of the In the this these fluorescence components are an the and the fluorescence for these fluorescence decay we can the total fluorescence decay also as the fluorescence and the anisotropy decay by and the is the to the preferentially over the The total fluorescence decay is a measure of the of the The anisotropy of the fluorescence is the to the fluorescence. 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P. A. T. M. Scopus Google Scholar). this to between free and bound NADH as as to the viscosity of the intracellular a single exponential anisotropy decay the decay of a free more decays are also associated anisotropy is the form of fluorescence anisotropy from a of fluorophore fluorophore fluorescence and fluorescence anisotropy The fluorescence anisotropy decay from the is the average of the fluorescence anisotropy decays of the by fluorescence the anisotropy decay with the associated anisotropy a time with lifetime decay component as by B. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Chem. Phys. Lett. 1996; Scopus Google Scholar). has of total decay and of anisotropy decay The analysis of anisotropy decay is more difficult than a simple provided by most analysis it to to these The this analysis is a form of global analysis for associated anisotropy that the fluorescence decays are the and components of the fluorescence can the anisotropy decay In however, we we the and the is the and the is the the between the and fluorescence the and are of Furthermore, in the functional form of is more than a simple of exponential The most form of both the and is to that are to for and these with a we a for both these is the of in the is the of and is the can be for the with a different the we the of in the functional form of the in and the the and the can be calculated from the and as while the Furthermore, by the functional form of the and decays and we that is a of between the and the and time of the total fluorescence i.e. the the the anisotropy decay are specific to the The that are between the and decays are as global The of global to a as global analysis (16Knutson J.R. Beechem J.M. Brand L. Chem. Phys. Lett. 1983; 102: 501-507Crossref Scopus (540) Google Scholar, 17Lakowicz J.R. Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New Scholar). This or more that have a of and of with A measure of global of be as the global is the of global The is in by a global we can and and the that have the in both The global provides with both the total decay and the anisotropy decay a single with an on the This global by a of to have fluorescence and anisotropy decays with a lifetime of a time of and a anisotropy of These are in with in J. J. PubMed Scopus Google Scholar, M. Chem. PubMed Scopus Google Scholar). Fluorescence resolved fluorescence decays from the of the of This of with to a the intrinsic fluorescence in by an average of with the integrated fluorescence to with a due to mitochondrial metabolism Brain Res. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). This response The intrinsic fluorescence from the of both a NADH and a that to However, of emission that the from the NADH to the fluorescence than Fluorescence decays and and used to the total fluorescence decay and the anisotropy total fluorescence and anisotropy decay a global analysis to the both the state lifetime and the The average of the total fluorescence decays both and are in The tissue fluorescence is to have exponential decay components these and or concentrations are in The are decay of the state of species. The of the are the concentrations of the species. In response to is a decrease in the time The however, are in are in the of the lifetime in of the shorter lifetime species. The of these changes is a decrease in the average lifetime from to the total fluorescence decay we can a of the changes in the absolute concentrations of the are calculated to and The increase in the total concentration of NADH hypoxia calculated to be is than the in the total fluorescence due to the decrease in the fluorescence average The are due to in the integrated fluorescence response to hypoxia and to in the the to the fluorescence from normoxia can be calculated to and that while has the concentration it the to the total fluorescence due to of the fluorescence lifetime in a new The total fluorescence decay a the response due to from the tissue that by This increase in has been and is it is due to mitochondrial swelling M. J. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the fluorescent we calculated the anisotropy decays from the intrinsic tissue and fluorescence anisotropy decays a by a and a This form is of fluorescence anisotropy from a of fluorophore with fluorescence decay and fluorescence anisotropy decay and is as associated anisotropy B. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Chem. Phys. Lett. 1996; Scopus Google Scholar). the associated anisotropy in we the time and anisotropy of of the total fluorescence decay and thereby characterized the of NADH as a of metabolic The anisotropy by the are in the lifetime has a fast time of normoxia that hypoxia to In contrast, lifetime are with that are to the tissue metabolic The of the are also to metabolic a decrease of the anisotropy from to an of than time of anisotropy in a new of NADH in of the intracellular fluorescence can be used to by the of NADH in NADH exists in and and measure the total fluorescence decay to be with and an average lifetime of The lifetime components are comparable with in and are to from the and (12Gafni A. Brand L. Biochemistry. 1976; 15: 3165-3171Crossref PubMed Scopus (115) Google Scholar, F. P. A. T. M. Scopus Google Scholar). The fluorescence anisotropy decay to be with a time of and an anisotropy of The dynamics are with by et al. F. P. A. T. M. Scopus Google Scholar) that the NADH time a on viscosity and on the by et al. F. P. A. T. M. Scopus Google we a time for free NADH of the of tissue in NADH fluorescence and anisotropy are sensitive to enzyme an we the Krebs enzyme mitochondrial dehydrogenase NADH a concentration of binding available NADH is In this the fluorescence decay is with components and an average lifetime of The anisotropy decays as a with an anisotropy of and a time of The single exponential anisotropy decay that the NADH fluorophore is bound the a concentration of binding is a of free and bound NADH a fluorescence anisotropy decay with the form of associated anisotropy The anisotropy of this by the and lifetime decay and the for free and bound NADH The of free and bound NADH however, as a and by the global to be The of the global of free/bound NADH by the free/bound ratio the average lifetime of the This method a free/bound NADH ratio of in with the from the global The of the anisotropy decay of the to that in tissue is of free and bound NADH in tissue. This the of global lifetime and associated anisotropy to discriminate and intracellular free and bound NADH. of and the lifetime and anisotropy of NADH in to in tissue to the fluorescent of intracellular NADH. This and the of the hypoxia are in The lifetime has a relatively fast time than that of NADH tissue This is with this free NADH in a The lifetime of is comparable with the lifetime of free NADH shorter than the lifetime of free NADH. the lifetime and anisotropy for that it is free NADH in a has a lifetime that is with NADH or NADH. time however, is of than that of free NADH, that this is also bound NADH. This a decrease in anisotropy an enhanced of fast a fast of the an or a specific in the binding increases the between the and emission the is we can this as enzyme bound on the of The lifetime and are on the of and are than the of or NADH. the enhanced lifetime and that these are NADH bound to The of as enzyme bound that are different binding NADH. In are to be many different binding NADH, and fluorescence is as components (3Williamson D.H. Lund P. Krebs H.A. Biochem. J. 1967; 103: 514-527Crossref PubMed Scopus (1279) Google Scholar, B. Baltscheffsky H. J. Biol. Chem. 1958; 233: 736-739Abstract Full Text PDF PubMed Google Scholar, 11, Velick, S. F. (1961) in A Symposium on Light and Life (McElroy, W. D., and Glass, B., eds) pp. 108-143, The Johns Hopkins Press, Baltimore, MDGoogle Scholar, 12Gafni A. Brand L. Biochemistry. 1976; 15: 3165-3171Crossref PubMed Scopus (115) Google Scholar, J.D. J. Biol. Chem. Full Text PDF PubMed Google of the of the fluorescence a of state and in a new is that we the fluorescence decay component of free NADH in However, it is difficult to this an to decay or a small intracellular concentration of free NADH. the of these the concentration of free NADH is given by the of while the total concentration of bound NADH is given by the of the of the bound The ratio of the free/bound NADH can be calculated to be normoxia and hypoxia the ratio of free NADH to the total concentration of bound NADH, it the of the enzyme bound a of NADH different while the free NADH concentration to to the total concentration of bound NADH, bound changes with a of binding and concentrations in the intracellular the as free NADH, we can time with the for free NADH by et al. F. P. A. T. M. Scopus Google Scholar) to the local intracellular In this the local viscosity by free intracellular NADH is calculated to be to normoxia and hypoxia to to A of intrinsic are in NADH, and are of concentration to a fluorescence the can be is of NADH and A. A. Photochem. Photobiol. Scopus (86) Google Scholar). is to the of these to the intracellular fluorescence. The concentrations of have been by in (4Klaidman L.K. Leung A.C. Adams J.D. Anal. Biochem. 1995; 228: 312-317Crossref PubMed Scopus (126) Google Scholar). that the total concentration of and is than that of and by a of The concentration of the fluorescent NADH to be than that of the fluorescent Furthermore, the of mitochondrial NADH due to environmental has been to be a of than that of J.M. J. Biol. Chem. 1962; Full Text PDF Google Scholar). This that the of to the intrinsic while a small fraction of the NADH NADH, the ratio of the fluorescent to form is the of However, NADH and are in NADH to energy metabolism and to M. T. Biochem. PubMed Scopus Google Scholar). has been that the of NADH and in E. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, tissue of nucleotide in and have that the fluorescence changes the transition are by changes in NADH with a from B. J.R. Schoener B. Biochem. Scholar). a fluorescence that is with to metabolic the of a metabolically from intrinsic fluorescence can be used to the conformation and environment of intracellular NADH. binding of NADH has been characterized by a of fluorescence techniques that are sensitive to The is the of the NADH emission is than The lifetime of NADH is more sensitive to enzyme binding and can be enhanced by a of Fluorescence anisotropy offers an to enzyme binding due to the in between NADH and the that mitochondrial dehydrogenase has a of binding increases the time of NADH by more than an of the fluorophore effectively on time of the intracellular NADH offers the most sensitive fluorescence probe of the free bound on measurements of we that intracellular NADH exists in both free and bound in comparable concentrations However, it is the bound NADH that the total tissue fluorescence due to enhanced This of the bound NADH fluorescence has of the resolved tissue fluorescence spectra to that tissue NADH is enzyme bound (9Chance B. Baltscheffsky H. J. Biol. Chem. 1958; 233: 736-739Abstract Full Text PDF PubMed Google Scholar). In contrast, that free NADH is in in concentration in fluorescence. The ratio of free NADH to enzyme bound of NADH is to significantly while the ratio of free NADH to total bound NADH This a of NADH enzyme binding is a increase in the concentration of free NADH and the concentration of NADH bound to that low lifetime The concentration of NADH bound to causing lifetime increases to a The of this of NADH binding is a decrease in the average lifetime of the intrinsic causing the fluorescence increase to underestimate the calculated concentration increase Since the ratio of free to bound NADH this calculated concentration increase to both free and bound pools of NADH. In of NADH from that the total NADH concentration by hypoxia O. Cox D.W.G. Bachelard H.S. J. Neurochem. 1988; 51: 172-176Crossref PubMed Scopus (46) Google Scholar). indicator a increase in the free mitochondrial NADH D.K. Guynn R.W. Brain Res. 1982; 239: 71-80Crossref PubMed Scopus (13) Google Scholar) in These concentration increases are to calculated concentration increase than to fluorescence increase. However, a be between and the of the metabolic indicator method because NADH increases are the of hypoxia as as the of the both of from In fluorescence changes to concentration we have that changes in the are to changes in the This is the The depends on the of and can be as a of the fluorescence and spectral of the as by the J. Chem. Phys. 1962; Scopus Google Scholar). that the of the NADH of the intrinsic fluorescence changes hypoxia that the average of intracellular NADH is significantly However, this is as we the of the spectral have that the local of NADH enzyme binding in an However, the environment of an enzyme bound of the binding of the enzyme as as the the enzyme can the of the fluorophore S. Brand L. J. Phys. Chem. B. Scopus Google Scholar). the of the on a in tissue an in this The dynamics of intracellular free NADH a relatively low viscosity This is because fast and changes on that are and sensitive to the viscosity and as the P. J. P. S. Biochem. PubMed Scopus Google Scholar). comparable with that of have been in a of and P. J. P. S. Biochem. PubMed Scopus Google Scholar, A. B. J. Biol. PubMed Scopus Google Scholar, J.R. J. Biol. PubMed Scopus Google Scholar, A. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The decrease in the intracellular viscosity hypoxia a of the intracellular environment by the osmotic swelling of the and mitochondria. hypoxia is to osmotic swelling and an increase in M. J. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). This increase in is to be due to mitochondrial swelling M. J. PubMed Scopus Google Scholar) in response to hypoxia is that mitochondrial swelling increases the of and the that is a between the of and of the Given that we also an increase in it is that swelling and the is the intracellular have that the response of NADH to metabolic is more than a simple concentration increase. in the concentration also in the conformation and the environment of intracellular NADH. have a to discriminate and these a more of the dynamics of the intracellular NADH concentration and free and bound is a to this time resolved probe to an imaging modality to the spatial of the NADH concentration and conformation, both on an and also on a has been a in the of intrinsic NADH fluorescence as a probe of a of cellular and tissue from Science. PubMed Scopus Google Scholar) to Science. Google Scholar) to enzyme F. H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The fluorescence of NADH concentration and conformation to the role of NADH in these and to an accurate of NADH and cellular energy metabolism in S. and W. for many and Williams for Global of and NADH of is the principal electron donor in metabolism and of the most in the The that the is a fluorophore makes it an non-invasive fluorescent probe of metabolic However, free and NADH has been chemistry techniques entail destroying the tissue and to and fluorescent techniques between free and NADH. PDF