Extrapulmonary Manifestations of Respiratory Disease: With Emphasis on Abnormal Electron (Redox) Metabolism

Symposium on Chronic Respiratory Disease

Extrapulmonary Manifestations of Respiratory Disease With Emphasis on Abnormal Electron (Redox) Metabolism

Eugene D. Robin, MD.,* and James Theodore, MD.**

Patients with respiratory disease commonly demonstrate extrapulmonary manifestations of their disease. These stem largely from abnormal pulmonary gas exchange, resulting in hypoxemia, hypercapnia, and acidosis. In turn these produce abnormal function of nonpulmonary organs and cells. Recently the importance of the lung as a metabolic organ has become apparent. The lung participates in a number of metabolic transformations in which a variety of chemical agents are synthesized, stored, modified, inhibited, or degraded in the lung. 3 • 4.10 These agents may affect the lung locally or may affect extrapulmonary cell or organ function. Since the lung is exposed to the entire cardiac output, it is scarcely surprising that the lung has an important role in the metabolism of a variety of substances important to the whole body economy. Just as abnormalities of renal function can produce abnormal plasma composition so can abnormal pulmonary function produce abnormal plasma composition. These changes in fluid composition ultimately can result in abnormal cellular function. Table 1 lists some of the major known metabolic transformations which occur in the lung. At this time, the changes in these transformations produced by various types of lung diseases are not well defined. Nor are the changes in cell and organ function secondary to changes in lung metabolism well understood. It may be predicted that in the near future, The title of this article was suggested by Dr. Edward Gall, University of Cincinnati Medical School. From the Departments of Medicine and Physiology, Stanford University School of Medicine, S tanford, California ':'Professor of Medicine and Physiology ''''Assistant Professor of Medicine

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Table 1.

Some Metabolic Functions of the Lung

ROLE OF LUNG IN METABOLIC TRANSFORMATION

LOCAL (PULMONARY) EFFECTS

SYSTEMIC (EXTRAPULMONARY) EFFECTS

BIOSYNTHESIS

Normal Lung Cells Secretory antibodies

leA in bronchial secretions, local immunity

Prostaglandins

Varied - e. g., bronchodilatation by prostaglandin E

Kallikreins

Pulmonary hypertension, alveolar duct constriction

Bronchial smooth muscle constriction, increased vascular permeability Antiviral Interferon Breakdown of proteins, tissue Proteolytic enzymes destruction Protection of tissue from Proteolytic enzyme proteolytic injury inhibitors Bronchial smooth muscle Slow reacting substance of anaphylaxis constriction, increased vascular permeability (SRS-A) Surface tension-lowering effects Surfactant in alveoli-stabilizes alveolar size Lung Tumor Cells ACTHoid peptides ADHoid peptides Histamine

Parathormonoid peptides Insulinoid peptides Gonadotropinoid peptides Thyrotropinoid peptides Serotonin (+kinins) Pulmonary vasoconstriction, bronchial constriction Nervous system antimetabolism

Growth hormone Multiple peptide

Spill over into circulation leading to altered immunologic status Varied - e. g., diverse cardiovascular effects such as tachycardia, vasodilatation, facilitate adrenergic transmission to vascular smooth muscle Systemic hypotension, increased vascular permeability Increased vascular permeability Antiviral Breakdown of proteins, tissue destruction

Cushing's syndrome Water retention and hyponatremia Hypercalcemia Hypoglycemia Gynecomastia Hyperthyroidism Carcinoid syndrome (see Kinins) Peripheral neuropathy, cerebellar degeneration, cerebral degeneration, EatonLambert (myasthenic syndrome) Osteoarthropathy and acromegaly Multiple syndromes

CONVERSION AND ACTIVATION

Angiotension I to angiotension II via converting enzyme Bradykinins

See Kallikreins

Vasoconstriction Aldosterone release See Kallikreins

REMOVAL (BINDING, INACTIVATION, DEGRADATION)

Bradykinin See Kallikreins Serotonin See above Prostaglandins E and F See above Histamine See above Norepinephrine

564

See Kallikreins See above See above See above Adrenergic stimulation

565

EXTRAPULMONARY MANIFESTATIONS

major advances will be made and that the chest physician will derive important pathogenetic, diagnostic and therapeutic knowledge from these advances. Hypoxemia, hypercapnia, and acidosis are undoubtedly the most important causes of extrapulmonary manifestations of lung disease. However early detection of the cellular abnormalities produced by these states is difficult. An important variable influenced by these abnormalities which alter general cell function is the oxidation-reduction (redox) state of cells. Oxidation-reduction reactions are a variety of chemical reaction with important biologic and clinical implications. Since both alterations in oxygen metabolism and alterations in acidbase state may affect redox reactions, it is appropriate to regard redox state changes as one type of abnormal cellular function caused by respiratory disease (as well as other diseases). This article will cover the physicochemical basis of redox reactions;6.8 indicate their role in biologic systems; review currently available methodology; review some of the physiologic information currently available; and finally suggest that increasing knowledge and better methods will serve to transform this area into a clinically important area. One useful approach to understanding redox reactions is to compare them with acid-base reactions. Disturbances of acid-base (proton) metabolism are relatively well understood. The basic physicochemical principles, the application of these principles to biologic systems, and the application of biology to clinical problems were well defined as long as 20 years ago. However in the past 10 years methods for measurement of acid-base variables have become simple, well standardized and widely used in medicine. A substantial understanding of the pertubations of acid-base state in various diseases has accumulated. Understanding of acid-base physiology is required by many different clinical specialties, including internal medicine, surgery, pediatrics, anesthesiology, and obstetrics. Specific manipulation of the acid-base disturbances connected with respiratory disease has become an important element of therapeutics. It may be anticipated that a similar train of events will occur with respect to the area of redox reactions.

PHYSICOCHEMICAL BASIS Oxidation-reduction reactions involve the transfer of electrons (e-) from one compound to another just as acid-base reactions involve the transfer of protons (H +) from one compound to another. A reducing agent is a compound which donates electrons in various chemical reactions, just as an acid is a compound which donates protons in water solution. Oxidizing agents are compounds which accept electrons, and are analogous to bases, which are proton acceptors. Oxidizing and reducing agents function as conjugate redox pairs just as acids and bases function as conjugate acid-base pairs: Reduced form (electron donor)

--->

Acid (proton donor)

Electrons + Oxidized form (electron acceptor)

--->

Proton + Base (proton acceptor)

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The flow of electrons from one compound to another involves changes in energy levels and it is desirable to be able to quantitate energy levels of specific redox reactions. In the case of acid-base reactions, the free energy of the system may be measured as an electrical potential as follows: ' 2.3 RT 1oga(H+) E H+= E H++--F-

[1]

where EH+ is the observed potential, E' H+ is the ground state potential; a(H+) is activity (effective concentration) of H+ at temperature = T, R is the universal gas constant, T is the absolute temperature and F is the Faraday. Since -log (H+) = pH and by the Henderson-Hasselbalch equation p

H

=

PKa'

+ 10

. a(Proton acceptor) g a(Proton donor)

[2]

there is a relationship between the electrical equation and the chemical equation. The PKa' of the reaction is that value at which the concentration of proton donor = concentration of proton acceptor . . Note that the acid-base state of a given system can be expressed as a voltage, as a concentration term ((H+) or pH) or less precisely as a concentration ratio of proton acceptor to proton donor. In similar fashion electron donors have characteristic electron pressures (tendencies to give up electrons) and specific electron acceptors have characteristic electron affinities (tendencies to accept electrons). These electron pressures and affinities may also be expressed as an electromotive potential as follows: Eo

=

Eo

+ 2.3 RT 10 nF

g

a(Electron acceptor) a(Electron donor)

in which Eo = the specific observed redox potential. Eo is a standard reduction potential at pH = 7.0, T = 25° C, all concentrations equal to 1 molar, T = absolute temperature, R = gas constant, n = number of electrons being transferred and F = Faraday (96,406 joules X volts-i). When Eo = E' then the concentration of proton acceptor is equal to the concentration of proton donor. Note that the redox state of the system can be expressed as a voltage or less precisely as a concentration ratio of the oxidized to reduced form. Redox potentials express free energy levels. Electrons flow (down hill) from more negative to more positive potentials. Thus quantitation of redox potentials enables the prediction of the direction of flow of electrons from say one redox pair to another. 0

BIOLOGICAL ASPECTS OF REDOX REACTIONS Oxidation-reduction reactions are common and important in biological systems. The functions which are subserved include the provision of

EXTRAPULMONARY MANIFESTATIONS

567

energy, oxidative biosynthesis, biodegradative reactions, and detoxifications. Electron transport often involves certain common electron carriers rather than involving electron flow directly from one substrate to another. Among the most important of these common electron carriers are nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). NAD+ is universally present in cells and accepts electrons from a wide variety of substrates, becoming reduced to NADH (reduced form). Reduced substrate + NAD+ <=' oxidized-substrate + NADH + H+ ~

electrons

Both major energy yielding pathways (anaerobic glycolysis and oxidative phosphorylation) have important steps which are NAD+ dependent. NADP is also universally present in cells and accepts electrons as follows: Reduced substrate+ NADP+ <=' oxidized substrate + NADPH + H+

~~ns

NADP-dependent reactions are involved in the hexose monophosphate shunt and also in the Krebs cycle. Knowledge of the ratio of NAD+ (electron acceptor) to NADH (electron donor) or of the ratio of NADP+ to NADPH in the various cellular or subcellular compartments under a variety of physiologic and pathophysiologic conditions would characterize these systems with respect to redox state. Such data would be analogous to characterizing the acid-base state by knowledge of the pH of a given cellular or body compartment.

MEASUREMENTS OF REDOX STATE Measurements of redox state in vitro in isolated solutions do not present formidable problems. There are several suitable methods. 1. The use of appropriate electrodes. 2. The use of redox indicator dyes, i.e., dyes which have one color in the oxidized form and another color in the reduced form. This is analogous to the use of acid-base indicators for the measurement of pH. 3. Direct measurements of nucleotide redox pairs using fluorometry, changes in pH or by chemical methods. In considering the possibility of such measurements in biologic fluids or under in vivo conditions several questions require clarification: 1. Is it possible to obtain meaningful measurements in plasma or extracellular fluid of redox potential or redox pair ratios? The answer is probably no. There are several reasons. Redox reactions which occur in cells are slow as compared with acid-base reactions.

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Therefore there is an important time lag before these reactions affect extracellular fluid. The distribution of redox reactions is heterogeneous not only from cell to cell but within any cell itself. Even granting valid potential measurements in plasma, it would not be possible to dissect this measurement into its component parts. There is no evidence that there is general regulation of redox state in plasma comparable to the regulatory mechanisms involving the acid-base status of the plasma. Rather, regulation involves the individual metabolic pathways in various cells. Electron transfer reactions are not necessarily in equilibrium with each other. Thus some cell types have enzymes which connect NAD+ and NADP+ (transhydrogenases). But others do not. If we used a redox electrode immersed in plasma to arrive at a value of -x millivolts in plasma, this value could not be meaningfully interpreted in terms of the metabolic state of the organism. Direct measurements of NAD+ and NADH or NADP and NADPH in plasma are subject to many of the same limitations. We may therefore conclude that unlike acid-base measurements simple measurements of redox pairs or redox potential in plasma would not be useful. 2. Is it useful to obtain meaningful direct measurements of NAD+/NADH, NADP+/NADPH in cell mixtures? The answer is probably no. There are several problems besides the technical problems of making such measurements. There is protein binding of electron carriers. Since it is the free (non bound) form which is thermodynamically active, total concentrations do not provide accurate estimates for calculation of redox potential. There is compartmentilization of NAD+ and NADH within cells. Strong evidence exists that the cytosol and mitochondria possess independent nucleotide pools which are not in free equilibrium. As a result, measurements in cellular mixtures are not capable of distinguishing between the amount present in each separate pool. Elegant methods have been worked out by Chance and his coworkers to follow changes in the reduction of NAD+ in intact tissues by spectroscopy or fluorometry. Although these studies have provided valuable data, the data are largely qualitative rather than quantitative. Moreover the changes noted (see below) do not distinguish between free and bound nucleotidesj nor is it possible to distinguish between changes in the cytosol and mitochondrion. 3. Is it possible to develop suitable electrodes or redox dyes for the determination of redox potential? Probably yes, although the technical problems are formidable. To this time no suitable electrodes are available. One approach that is promising is that developed by Krebs and Williamson 12 using substrate redox pairs which are NAD+ and NADP+ dependent. The basis of this approach is as follows: Consider a nucleotide-dependent reaction localized in the cytosol of the cell: Lactate Dehydrogenase Pyruvate + NADH + H+ , ) Lactate + NAD+

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EXTRAPULMONARY MANIFESTATIONS

By the mass action law, [Lactate] [NAD+] = [Pyruvate] [NADH] [H+]

~---'----~~~~=c-c;-

then

[NAD+] [NADH]

K~q

[K;q] [Pyruvate] [H+]

[Lactate]

Determination of [Pyruvate], [Lactate], [H+], and knowledge of the equilibrium constant permits calculation of the cytosol NAD+/NADH ratio. In similar fashion consider a nucleotide reaction limited to the mitochondrial compartment: B-OH Dehydrogenase Acetoacetate + NADH + H+ ( ) Betahydroxybutyrate+ NAD+

By the mass action law, [Betahydroxybutyrate] [NAD+] = K' [Aceto-acetate][NADH][H+] "4

then

[NAD+] [NADH]

[K;q] [Aceto-acetate] [H+]

(BOH)

Determination of [Aceto-acetate], [BOH], [H+], and knowledge of K;q permit calculations of mitochondrial NAD+/NADH. There are several problems with this approach. One major problem is the requirement to measure intracytoplasmic and intramitochondrial [H+]. Except for erythrocytes, there is no entirely acceptable method for making this measurement. However if there are no major changes in [H+] under various clinical circumstances, then the approach can be used to study changes in NAD+/NADH ratio. The mass action law is based on equilibrium conditions. This requires that the enzymes involved in the reactions have sufficiently high activities so that the reactions involved are close to equilibrium. This approach which requires equilibrium is not particularly suitable for looking at rapidly changing events. Despite these problems this approach has provided important data with respect to the redox state of various tissues.

REDOX STATE OF NAD+/NADH IN VARIOUS TISSUES Erythrocyte This cell provides an excellent model for measurements of NAD+/NADH ratios using the Krebs-Williamson approach. The cell may be regarded as having a single compartment with respect to NAD+/NADH since it lacks mitochondria. Methods are available for the direct and accurate measurement of intra-erythrocytic pH. Thus by using measurements of lactate, pyruvate, and red cell pH, it is possible to calculate free NAD+/NADH ratios.

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Measurements of free NAD+/NADH ratios in oxygenated versus nonoxygenated erythrocytes show the following:' . 1. Free NAD+/NADH ratios in oxygenated red cells average about 1,000, a value which corresponds to a redox potential of about-240 millivolts. This value is similar to the value of NAD+/NADH in the cytoplasm of liver cells and alveolar macrophages. 2. Free NAD+/NADH ratios decrease with increasing concentrations of deoxyhemoglobin. This effect is specifically related to the concentration of deoxyhemoglobin since decreases in deoxyhemoglobin produced either by increased oxyhemoglobin or increased carboxyhemoglobin increase NAD+/NADH. 3. Increasing pH decreases erythrocyte NAD+/NADH and decreasing pH increases erythrocyte NAD+/NADH. These studies establish a link between the state of oxygenation of hemoglobin and metabolism in the erythrocyte. How changes in redox state, in turn, modify red cell function will require further work.

Brain Redox changes in brain have been studied with respect to the effects of hypoxia. Lowry et al. performed direct measurements of total N AD+ and NADH in the brains of young mice. (No corrections for binding or compartmentalization). Steady state control values of N AD+ /N ADH were approximately lOO/I. During an 8 minute ischemic period NAD+/NADH decreased largely as a result of a 3 to 4-fold increase in NADH. Total NAD+ concentrations remained fairly high even when there was a brisk rise in the rate of anaerobic glycolysis (Pasteur effect). NADP+/NADPH under control conditions averaged 1.67 and remained constant during ischemia although total NADPH declined. 7 Chance and co-workers measured tissue fluorescence at 340 nm. in brain cortex, in vivo. 2 Increasing fluorescence probably reflects increasing total NADH activity (largely mitochondrial). Tissue fluorescence during anoxia increased more rapidly in brain cortex than in kidney, suggesting greater sensitivity to hypoxia in the former tissue.

Alveolar Macrophages NAD+/NADH dependent substrate analysis has been applied to studies of alveolar macrophage redox state. Lactate/pyruvate ratios have been used to characterize cytoplasmic ratios, and betahydroxybutyrate/aceto acetate ratios have been used to characterize mitochondrial ratios. During exposure to low P0 2 mitochondrial NAD+/NADH falls more rapidly than cytoplasmic ratios. Moreover, after re-exposure to high ambient Po2 's, cytoplasmic NAD+/NADH returns more rapidly toward normal than mitochondrial ratios, suggesting greater vulnerability of the latter to hypoxic injury. Redox changes in both compartments occur before gross evidence of cellular impairment of cell function, such as uptake of high molecular weight dyes or histologic changes. 9 Exposure of alveolar macrophages to the oxidant pollutant N0 2 likewise results in altered redox state of NAD+/NADH. There is an increase in cytoplasmic ratios and a decrease in mitochondrial ratios. These values are returned toward norma! by the use of vitamin E."

EXTRAPULMONARY MANIFESTATIONS

571

Liver Control values of cytoplasmic NAD+/NADH in liver cells are similar to those in erythrocytes and alveolar macrophages. During a period of brief ischemia there is a fall in this ratio, although overall NAD+/NADH (measured enzymatically) does not change. Similar changes in the ratio were found when either lactate/pyruvate or IX glycerophosphate to dihydroxyacetone phosphate were used to calculate the free NAD+/NADH ratios. 5 During starvation and alloxan diabetes there is a decline in cytoplasmic NAD+/NADH. During starvation, mitochondrial NAD+/NADH also falls. However during alloxan diabetes, mitochondrial ratios rise. These studies indicate the sensitivity of liver metabolic pathways to alterations of substrate availability and utilization. 12 The clinical expression of these changes are presumably reflected in several conditions. 1. The development of diabetic ketoacidosis itself is caused by accumulations of betahydroxybutyric and aceto-acetic acid, no doubt associated with abnormalities of mitochondrial redox state. 2. In most patients with diabetic acidosis, approximately 10 per cent of the accumulated anion is represented by lactate, which presumably reflects abnormalities of cytoplasmic redox state. 3. With administration of phenformin, severe lactic acidosis may develop, suggesting major alteration of cytoplasmic redox state.

SUMMARY Changes in redox state provide an early and sensitive indicator of metabolic abnormalities related to changes in O 2 metabolism, acid-base status, and substrate availability and utilization. Accumulated data suggest that characterization of cellular redox state will provide important insight into cellular function analogous to that provided by characterization of acid-base status. As more suitable methodology becomes available, it may be predicted that this area will become of increasing clinical importance. Extrapulmonary manifestations of respiratory disease are common and important. Many of these are caused by hypoxemia, hypercapnia, and acidosis. It is probable that metabolic alterations of the lung in disease independent of changes in gas exchange also results in a variety of extrapulmonary manifestations. Thus this area is of great potential clinical importance. One area of obvious interest involves disturbances in redox (electron) metabolism which are closely related to changes in O 2 , CO 2 and H+ metabolism. We are now in the preclinical phase of understanding these disturbances.

REFERENCES 1. Cassan, S. M., Theodore, J., and Robin, E. D.: Effect of low 0, tensions on red cell glycolysis, NAD"/NADH ratios and transmembrane potential (TMP)- The pasteuroid effect. Clin. Res., 20:574, 1972.

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2. Chance, B., Cohen, P., Jobsis, F., et al.: Intracellular oxidation-reduction state in vivo. Science, 137:499,1962. 3. Harlan, W. R, and Said, S. I.: Selected aspects oflung metabolism. In Bittar, E. E., and Bittar, N. (eds.): The Biological Basis of Medicine. New York, Academic Press, 1969, VoL 6, p.357. 4. Heinemann, H. 0., and Fishman, A. P.: Non-respiratory function of mammalian lung. PhysioL Rev., 49:1,1969. 5. Hohorst, H. J., Kreutz, F. H., and Rein, M.: Steady-state equilibria of DPN-linked reactions and the ox/red state of DPN/DPNH in the cytoplasmic compartment of liver cells in vivo. Biochem. Biophys. Res. Commun., 4:159,1961. 6. Lehninger, A. L.: Biochemistry. New York, Worth Publishers, 1970. 7. Lowry, O. H., Passonneau, J. V., Hasselberger, F. X., et al.: Effect of ischemia on known substrates and co-factors of the glycolytic pathway in brain. J. BioI. Chem., 239:18, 1964. 8. Mahler, H. R, and Cordes, E. H.: Biological Chemistry. New York, Harper and Row, Publishers, 1966. 9. Mintz, S., and Robin, E. D.: Redox state of free nicotinamide-adenine nucleotides in the cytoplasm and mitochondria of alveolar macrophages. J. Clin. Invest., 50:1181,1971. 10. Said, S. 1.: The lung as a metabolic organ. New Eng. J. Med., 279:1330,1968. 11. Simons, J. R, Mintz, S., Freeman, G., et al.: Effect of NO, on redox state of alveolar macrophages. Clin. Res., 20 :582, 1972. 12. Williamson, D. H., Lung, P., and Krebs, H. A.: The redox state of free nicotinamideadenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J., 103 :514, 1967. Stanford University School of Medicine Stanford, California 94305