Comparison of dark-field and bright-field incident illumination for in vivo measurements of reduced pyridine nucleotides

Bright-field and dark-field illumination techniques for in vivo measurements of reduced pyridine nucleotide fluorescence were compared in 15 rats during periods of normocapnia, hypocapnia, hypercapnia, and anoxia. Parameters investigated included fluorescence, cortical reflectance, cortical blood flow, and electroencephalograms. In normal brain, with preserved autoregulation, reduced pyridine nucleotide fluorescence was constant through a wide range in P_a(CO₂), cortical blood flow, and cerebral blood volume in animals studied using vertical illumination (bright-field) techniques. There was a marked increase in reduced pyridine nucleotide fluorescence at death from anoxia. Artifacts were reduced by monochromators for excitation, emission, and reflected light; low-intensity vertical excitation energy and high-sensitivity recording instrumentation; and a small avascular (123 μm) field. Potential sources of error include photodecomposition, hemoglobin interference from absorption and reflectance, and light scattering. Vertical excitation techniques using a small field appeared to give more reliable and reproducible results than circumferential techniques using a larger field of observation.     

(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.

In vivo analysis of the metabolic state of tissue by means of reduced nicotinamide adenine dinucleotide (NADH) fluorimetry is disturbed by tissue movements and by hemodynamic and oximetric effects. These factors cause changes in the absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to compensate measured NADH fluorescence intensities for these effects. In this paper we show on theoretical grounds that the ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compensatory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflectance images, using a CCD camera with a biprism configuration. The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood-perfused myocardium.     

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.

The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.     

Oxygen and glucose withdrawal on portal veins: NADH fluorescence and spontaneous activity

Parallel with the spontaneous mechanical activity, changes in steady-state levels of reduced pyridine nucleotides (PN) of rat portal veins were recorded by continuous monitoring of surface fluorescence. These data were correlated with changes in tissue concentrations of glucose, lactate and ATP when subjected to long-term anoxia and/or glucose depletion. In response to anoxia and addition of glucose the fluorescence intensity excited at 366 nm increased, representing a reduction of PN. This was accompanied by a higher tissue lactate content. Spontaneous contraction force decreased independently of accelerated glycolysis, which did not compensate for eliminated oxidative metabolism. The frequency of spontaneous contractions changed time-dependent. When O2 was added after more than 3 h of anoxia the spontaneous activity was restituted and PN were re-oxidized. Contraction relaxation cycles of spontaneous activity were accompanied by phasic changes of PN fluorescence clearly detectable in vessels supplied with glucose only. Suppressive effects of O2 and/or glucose depletion upon spontaneous contractile activity seem to be mediated more by alterations of cell membrane properties than by changes in energy metabolism.     

Oscillations of redox states in synchronously dividing cultures of Acanthamoeba castellanii and Schizosaccharomyces pombe.

The redox state of the mitochondria of Acanthamoeba castellanii and Schizosaccharomyces pombe was assessed with a flying-spot fluorometer (Chance et al. 1978. Am. J. Physiol. 235:H 809) that provides excitation appropriate for oxidized flavoprotein or reduced pyridine nucleotide. Fluorescence signals could be resolved from the thin films of cultures that were only one cell deep. In both organisms anoxia was associated with an increased pyridine nucleotide and decreased flavoprotein fluorescence. The addition of mitochondrial uncoupling agents increased the flavoprotein fluorescence and the fluorometer was able to resolve uncoupler-sensitive and uncoupler-insensitive fractions of S. pombe cultures. In both synchronous and asynchronous cultures of A. castellanii and S. pombe the mitochondrial redox state oscillates with a period of 4.5 +/- 1.0 min. Oscillations with much longer period, of the order of an hour, are observed in synchronous cultures and these oscillations correlate with similar oscillations in respiratory rate, uncoupler sensitivity, and adenine nucleotide pool sizes. The results are consistent with the hypothesis that synchronous cultures of A. castellanii and S. pombe oscillate between the ADP-limited (state 4) and ADP-sufficient (state 3) respiratory states, i.e., exhibit in vivo respiratory control.     

Characteristics of fluoroprobes for measuring intracellular pH

We evaluated four different fluoroprobes to determine their capabilities and limitations in measuring intracellular pH by the fluorescent indicator technique. In vitro, carboxyfluorescein, dimethylcarboxyfluorescein, biscarboxyethyl carboxyfluorescein, and 4-methylumbelliferone (4MU) all showed comparably intense fluorescence and excellent pH sensitivity near their respective pKa values. Major differences were found between 4MU and the fluoresceins in terms of protein binding, concentration effects, bleach rates, and the retention time within cells. Both fluorescence and a fluorescence ratio at pH-sensitive/pH-insensitive excitation wavelengths increased with pH for all compounds, and the ratio completely corrected for large changes in the excitation light intensity. In contrast, the ratio showed large artifactual changes as dye concentration increased because of self-quenching effects and spectral shifts. Protein interactions likewise caused spectral shift and ratio aberrancies, but calcium, magnesium, and oxygen had no effect on the fluorescence ratios. We conclude that measurements of cell pH by fluorescence techniques are subject to artifacts induced by self-quenching and protein binding. Use of the fluorescence ratio technique does not necessarily correct for these artifacts, and in particular the ratio technique does not correct for changes in fluoroprobe concentration. Because the major artifacts cause the ratios for 4MU and for the fluoresceins to move in opposite directions, an experimental maneuver can be shown to cause a true change in pH if the fluorescence and ratios change in the same direction for these two classes of fluoroprobes.     

Photokinetic assay of NADH and NADPH in microdissected tissue samples

A high sensitivity is reached in photokinetic assay of reduced pyridine nucleotides with extracts of Achromobacter fischerii. When occurring together, NADH and NADPH are difficult to measure individually, since both initiate light emission. This problem has been solved by selective enzymic oxidation of either nucleotide before the measurement of the other. Assay of the reduced nucleotides in extracts of microdissected samples from pancreatic islets, exocrine pancreas, and liver demonstrates the applicability of the methods.     

Calcium-dependent activation of mitochondrial metabolism in mammalian cells

Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca2+-sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism.     

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.     

In vivo interactions between photosynthesis,mitorespiration, and chlororespiration in Chlamydomonas reinhardtii

Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.     

Spectral and biological changes induced in nicotinic acid and related compounds by ultraviolet light

1. Irradiation of nicotinic acid, nicotinamide, nicotinamide N-oxide, N'-methyl-4-pyridone-3-carboxamide, reduced nicotinamide-adenine dinucleotide and pyridine with ultraviolet light at 253.7mmu leads to striking spectral changes. 2. Nicotinic acid and nicotinamide are broken down to photosensitive intermediates which in turn undergo photodecomposition. 3. A major photoproduct of [7-(14)C]nicotinic acid is radioactive and absorbs ultraviolet light, but is inactive as a growth factor for Candida pseudotropicalis. 4. Irradiation of nicotinamide gives rise to small quantities of a biologically active photoproduct having the same R(F) as nicotinic acid. A second photoproduct is also formed, but its identity has not yet been established. 5. Irradiation of nicotinamide N-oxide leads to the formation of several photoproducts, one of which has the same R(F) as nicotinamide, absorbs ultraviolet light, and is biologically active. 6. Evidence is presented that irradiation of ethanolic solutions of N'-methyl-4-pyridone-3-carboxamide gives rise to acetaldehyde. 7. Irradiation of reduced nicotinamide-adenine dinucleotide in the presence of acetaldehyde leads to the formation of oxidized nicotinamide-adenine dinucleotide, which in turn can break down to nucleotide and/or nucleoside (depending on the conditions of the reaction). 8. The quantum yields of photolysis and the molar photosensitivities have been determined for N'-methyl-4-pyridone-3-carboxamide and nicotinamide N-oxide. 9. The possible biological significance of these photoreactions is discussed in relation to photosynthesis, visual-pigment metabolism and ultraviolet-light-induced cell damage. 10. A four-step theory is presented for the biochemical evolution of oxidation-reduction systems, involving photoactivated transformations of pyridine derivatives.     

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.     

Role of Pyridine Nucleotides in the Control of Respiration in Ultraviolet-Irradiated Escherichia coli B/r Cells

Escherichia coli B/r cells grown on glycerol-containing medium and irradiated with ultraviolet light to about 1% survival respire for about 1 hr and then cease completely for several hours. The results of studies on cell-free respiration and analyses of pyridine nucleotide levels at various times after ultraviolet irradiation show that the cessation of respiration is associated with two changes—loss of glycerol kinase activity and complete disappearance of pyridine nucleotides. Under other cultural conditions in which respiratory inhibition is less complete and more transitory, the losses of pyridine nucleotides are smaller and the rises which follow are correlated with increases in respiratory activity.     

Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells.

The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.     

Role of DNA Sequences in Genetic Recombination in the Iso-1-Cytochrome c Gene of Yeast. I. Discrepancies between Physical Distances and Genetic Distances Determined by Five Mapping Procedures

Recombination rates have been examined in two-point crosses of various defined cyc1 mutants using five mapping methods. Nucleotide sequences of mutant codons were identified in previous studies from alterations in functional iso-1-cytochromes c produced by intragenic revertants. Heteroallelic diploids were analyzed for rates of mitotic recombination that occurred spontaneously and that were induced with x-rays, ultraviolet light and the near-ultraviolet light emitted by sunlamps, as well as rates of meiotic recombination that occur after sporulation. Frequencies of both mitotic and meiotic recombination do not necessarily correspond with physical distances separating altered nucleotides. The most extreme discrepancy involved two adjacent intervals of thirteen basepairs which differed approximately thirty-fold in their spontaneous and X-ray-induced recombination rates. Marked disproportions between genetic and physical distances appear to be due to the interaction of the two nucleotide sequences in the heteroallelic combination and not to the sequences of the mutant codons alone. Recombination values that were obtained by all five methods could not be used to establish to correct order of mutant sitesmrelationships of the recombination rates for the various pairwise crosses are different after mitosis from those after meiosis, suggesting that these two recombinational processes are to some extent different in their dependence on particular nucleotide configurations. On the other hand, the relationships of the rates induced by UV-, sunlamp- and X-irradiation were identical or very similar. In addition to the intrinsic properties of the alleles affecting frequencies of mitotic and meiotic recombination rates, two- to threefold variations in recombination rates could be attributed to genetic backgrounds.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Linear correlation between acetoacetate/beta-hydroxybutyrate in arterial blood and oxidized flavoprotein/reduced pyridine nucleotide in freeze-trapped human liver tissue.

A comparison of two methods of measuring liver mitochondrial redox state demonstrated that a linear correlation exists between acetoacetate/beta-hydroxybutyrate ratio in arterial blood (arterial ketone body ratio; AKBR) and oxidized flavoprotein/reduced pyridine nucleotide in human liver tissue (FP/PN) as measured by tissue fluorescence spectroscopy, such that [FP/PN] = 0.64 + 0.49 x [AKBR] (r = 0.84, P less than 0.001). This result supports the validity of AKBR as a method of measuring the hepatic mitochondrial redox state of pyridine nucleotide using arterial blood.     

A phenomenon of photoenhancement of acridine orange-induced fluorescence.

It is a common experience that, with exposure to exciting radiation of the fluorescence microscope, the acridine orange-induced red fluorescence of the nucleus, produced by Feulgen hydrolysis, fades with a concomitant shift to green. The present investigation reports a phenomenon of photoenhancement observed in the hydrolyzed cytoplasm where pale green fluorescence increases in intensity with exposure to exciting radiation. The phenomenon has been noticed in Rhizobium, Oscillatoria, tomato root tip cells and human buccal epithelial cells. It is tentatively concluded that the gain in fluorescence yield is due to certain conformational changes of the acridine orange-protein complex induced by ultraviolet light flux.     

Metabolic control reactions of the intact urinary bladder of the toad

Metabolic control reactions have been studied in the intact toad bladder by means of fluorescence spectrophotometric measurement of reduced pyridine nucleotide and by measurement of respiration with the platinum electrode. substrates such as pyruvate and succinate lead to prompt increases in reduction level of pyridine nucleotide with only slight acceleration of respiration. major metabolic control is exerted by adp, which depletes the intact bladder of reduced pyridine nucleotide and accelerates respiration. respiratory control ratios, as for isolated mitochondria, depend upon the substrate being metabolized. a significant fraction of added adp appears to gain entry into the intact toad bladder and is converted to atp, anaerobiosis and amobarbital lead to increased levels of reduction of pyridine nucleotide. the spectroscopic and metabolic properties of the reduced pyridine nucleotide being studied identify it with that fraction of dpnh which is bound at one of the energy conservation sites linking phosphorylation reactions with electron transfer.