http://www.healthcare.uiowa.edu/corefacilities/esr/publications/buettnerpubs/pdf/FRBM-2001-30-1191-FQS-Redox.pdf

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.

Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH-generating system in proportion to protein concentrations as determined by three independent methods: ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 μmol H2O2/g microsomal protein per minute. The oxygen concentration for half-maximal rates was 50 μM. It is suggested that NADPH-dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P-450.



Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol-oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation: viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH- or an H2O2-generating system exhibits identical characteristics as follows: (a) ethanol concentration for half-maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half-maximal effect: 150 μM); (d) inhibition by catalase inhibitors, e.g. azide (half-maximal effect: 50 μM), with identical azide insensitive rates; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH-dependent acetaldehyde production is suppressed in the presence of an active H2O2-utilizing system.



Thus, it is concluded that the NADPH-dependent microsomal ethanol-oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb01711.x

Hepatic Microsomal Ethanol Oxidation

Significance: The redox code is a set of principles that defines the positioning of the nicotinamide adenine dinucleotide (NAD, NADP) and thiol/disulfide and other redox systems as well as the thiol redox proteome in space and time in biological systems. The code is richly elaborated in an oxygen-dependent life, where activation/deactivation cycles involving O2 and H2O2 contribute to spatiotemporal organization for differentiation, development, and adaptation to the environment. Disruption of this organizational structure during oxidative stress represents a fundamental mechanism in system failure and disease. Recent Advances: Methodology in assessing components of the redox code under physiological conditions has progressed, permitting insight into spatiotemporal organization and allowing for identification of redox partners in redox proteomics and redox metabolomics. Critical Issues: Complexity of redox networks and redox regulation is being revealed step by step, yet much still needs to be lea...

The Redox Code

https://yellen.hms.harvard.edu/reprints/2011_Hung_Tantama_Yellen-Cell_Metab.pdf

Imaging Cytosolic NADH-NAD+ Redox State with a Genetically Encoded Fluorescent Biosensor

Renal ischemia: a new perspective.

1
The extracellular ratio [lactate]/[pyruvate] is used as a means to poise the cytosolic NADH/NAD+ system. This is achieved by stepwise changes of [lactate]/[pyruvate] in the perfusate in an open system. Fluorescence and absorbance at NADH-specific wavelengths is recorded simultaneously with single or dual wavelength methods. Fluorescence excitation and absorbance difference spectra are obtained with the help of an instrument computer. The readout is calibrated by infusion of internal standards, permitting an estimation of the enhancement of quantum yield of NADH-specific fluorescence.

2
Mathematical treatment on the basis of the mass action law permits extrapolation to fully reduced and fully oxidized cytosolic bound NADH. In addition, the apparent equilibrium constant for bound NADH/NAD+ and lactate/pyruvate is obtained (0.022 to 0.071), corresponding to a midpoint potential of bound NADH of – 250 to – 265 mV. Accordingly, the ratio of dissociation constants, KNAD+/KNADH, relevant for the cytosolic binding sites, is 200–700. Cytosolic NADH-binding capacity is estimated to be around 80 nmol/g liver wet weight. Under normoxic conditions at [lactate]/[pyruvate] of 10 in the perfusate, the ratio of contents of cytosolic bound NADH/free NADH is 100.

3
The existence of near-equilibrium conditions in the system is suggested by measurement of net H+ transport coupled to lactate import, as well as by determination of an intracellular dependent indicator metabolite couple, α-glycerophosphate/dihydroxyacetone phosphate. Problems regarding the validity of tissue contents of lactate/pyruvate as indicator for the cytosolic NADH/NAD+ potential are discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb01840.x

State of Oxidation-Reduction and State of Binding in the Cytosolic NADH-System as Disclosed by Equilibration with Extracellular Lactate/Pyruvate in Hemoglobin-Free Perfused Rat Liver

1
The interactions of a ‘Type I' compound, hexobarbital, and of a ‘Type II’ compound, metyrapone, with the mixed function oxidase were demonstrated by spectrophotometric techniques in hemoglobin-free perfused livers from phenobarbital-treated rats. The binding signal for hexobarbital occurred when cytochrome P-450 was mainly oxidized (normoxic perfusion) or reduced (anoxic perfusion). The binding signal disappeared as hexobarbital was metabolized.

2
Functional aspects were demonstrated by simultaneous readings of (a) the reduced cytochrome P-450-CO complex, providing an indication of the degree of reduction of cytochrome P-450; (b) surface fluorescence (366 > 420 nm); and (c) oxygen uptake.

The steady state level of reduced P-450-CO was increased when substrate was metabolized. Surface fluorescence intensity decreased following hexobarbital addition. This decrease was explained partly by a decrease of the tissue level of NADPH, and partly also by interference of the P-450-hexobarbital complex at the excitation wavelength. Extra oxygen uptake was 2.2–2.4 moles per mole of hexobarbital. Half-maximal concentration of hexobarbital was 59 and 75 μM for extra oxygen uptake and binding to the oxidase, respectively, in agreement with data for enzymatic activity and binding from isolated liver microsomes.

The parameters (a, b, c) returned to their original level as hexobarbital was metabolized, similar to findings with the steroid 21-hydroxylase system of isolated adrenocortical microsomes.

3
The overall degree of reduction of the NADPH system, NADPH/NADPH + NADP+, decreased from 0.8 to 0.7 in livers from phenobarbital-treated rats when hexobarbital was added. In contrast, the free cytosolic NADH system did not undergo redox changes as indicated by a constant ratio of lactate/pyruvate in the perfusate.

4
Mitochondrial NADH dehydrogenase was shielded against the barbiturate by the endoplasmic reticulum as a consequence of phenobarbital induction.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1970.tb01037.x

Interaction of mixed function oxidase with its substrates and associated redox transitions of cytochrome P-450 and pyridine nucleotides in perfused rat liver.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2869%2980008-6

Reduction kinetics and content of cytochrome P-450 by application of dual wavelength techniques to hemoglobin-free perfused rat liver

Eine Methode zur Differentialspektrophotometrie durch Modulation eines Monochromators wird beschrieben Der Monochromator oscilliert mit variabler Bandbreite zwischen λ1 und (λ1+Δλ) Wenn (λ1+Δλ) die Wellenlange des Absorptionsmaximums ist, erscheint am Photo multiplierausgang einer Sinuswelle, deren Amplitude der Extinktions differenz zwischen λ1 und (λ1+Δλ) proportional ist Die Schaltung zur ultralinearen Messung dieser Amplitude ist angegeben und ihre Anwendung mit dem Zweistrahlphotometer „Rapidspektroskop“ gezeigt Die Empfindlichkeit wird durch die Kombination um den Faktor 10 bis 100 gesteigert Der Rauschpegel ist bei der Monochromator-Modulationstechnik etwa 10−4 E

Ein Gerät zur höchstempfindlichen Differentialspektrophotometrie mit dem Rapidspektroskop

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2870%2980291-5

Bolko Brauser

Papers

State of Oxidation-Reduction and State of Binding in the Cytosolic NADH-System as Disclosed by Equilibration with Extracellular Lactate/Pyruvate in Hemoglobin-Free Perfused Rat Liver

Interaction of mixed function oxidase with its substrates and associated redox transitions of cytochrome P-450 and pyridine nucleotides in perfused rat liver.

Reduction kinetics and content of cytochrome P-450 by application of dual wavelength techniques to hemoglobin-free perfused rat liver

Ein Gerät zur höchstempfindlichen Differentialspektrophotometrie mit dem Rapidspektroskop

Redox transitions of cytochromes and pyridine nucleotides upon stimulation of an isolated rat ganglion