DT-Diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function.

The activity of purified DT-diaphorase in the reduction of ubiquinone homologues of different side-chain length incorporated in uni- and multilamellar vesicles was determined. The direct relationship between the reduced state of ubiquinones and the inhibition of lipid autoxidation induced by thermolabile azocompounds was also demonstrated. Results demonstrate that DT-diaphorase is able to generate and to maintain the reduced, antioxidant form of ubiquinones in both types of vesicles. Furthermore, the results reported herein show that, in the presence of nicotinamide adenine dinucleotide (NADH) and DT-diaphorase, ubiquinol-containing multilamellar vesicles exposed to a lipophilic azocompound did not undergo lipid peroxidation, whereas in vesicles lacking either NADH or DT-diaphorase, thiobarbituric acid reactive substances (TBARS) formation occurred. It is suggested that DT-diaphorase may be responsible for maintaining the reduced state of ubiquinones in various nonmitochondrial cellular membranes.

The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.

The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

The role of ascorbate in antioxidant protection of biomembranes: interaction with vitamin E and coenzyme Q.

One of the vital roles of ascorbic acid (vitamin C) is to act as an antioxidant to protect cellular components from free radical damage. Ascorbic acid has been shown to scavenge free radicals directly in the aqueous phases of cells and the circulatory system. Ascorbic acid has also been proven to protect membrane and other hydrophobic compartments from such damage by regenerating the antioxidant form of vitamin E. In addition, reduced coenzyme Q, also a resident of hydrophobic compartments, interacts with vitamin E to regenerate its antioxidant form. The mechanism of vitamin C antioxidant function, the myriad of pathologies resulting from its clinical deficiency, and the many health benefits it provides, are reviewed.

The relative essentiality of the antioxidative function of coenzyme Q--the interactive role of DT-diaphorase.

This paper will address two aspects regarding the antioxidative role of coenzyme Q (CoQ): (1) Is the antioxidant function of CoQ primary or secondary (coincidental), i.e. was this molecule selected during evolution to function primarily as an essential functional component of the mitochondrial electron transfer chain and oxidative phosphorylation processes, is its antioxidative capability merely a coincidence of its hydroquinone structure, or was its synthetic enzyme sequence selected on the basis of the advantage to the evolving organism of both functions of CoQ? (2) What is the mechanism whereby the hydroquinone (antioxidant) form of CoQ (CoQH2) is maintained in high proportion in the various and many membranes in which it resides, and in which an obvious electron transfer mechanism to reduce it is not present? The essentiality of the antioxidative role of CoQH2 will be explored and compared to other primary and secondary antioxidants. Recent evidence implicating the two-electron quinone reductase, DT-diaphorase, in the maintenance of the reduced, antioxidant state of CoQ during the oxidative stress of exhaustive exercise will be presented, and a hypothesis concerning the evolutionary significance of DT-diaphorase will be offered.

An analysis of the role of coenzyme Q in free radical generation and as an antioxidant.

The vital role of coenzyme Q in mitochondrial electron transfer and its regulation, and in energy conservation, is well established. However, the role of coenzyme Q in free oxyradical formation and as an antioxidant remains controversial. Demonstration of the existence of the semiquinone form of coenzyme Q during electron transport, coupled with recent evidence that hydrogen peroxide (but not molecular oxygen) may act as an oxidant of the semiquinone, suggests that the highly reactive OH. radical may be formed from the semiquinone. On the other hand, data exist implicating the Fe-S species as the source of electron transfer chain, free radical production. Additional data exist suggesting instead that the unpaired electron of the coenzyme Q semiquinone most likely dismutases superoxide radicals. These concepts and those arising from observations at several levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, supporting the concept of reduced coenzyme Q as an antioxidant, will be presented. The results of recent studies on the interaction between the two-electron quinone reductase--DT diaphorase and coenzyme Q10 will be presented. The possibility that superoxide dismutase may interact with reduced coenzyme Q, in conjunction with DT diaphorase inhibiting its autoxidation, will be described. The regulation of cellular coenzyme Q concentrations during oxidative stress accompanying aerobic exercise, resulting in increased protection from free radical damage, will also be presented.

The participation of coenzyme Q in free radical production and antioxidation.

Published experimental data pertaining to the participation of coenzyme Q as a site of free radical formation in the mitochondrial electron transfer chain and the conditions required for free radical production have been reviewed critically. The evidence suggests that a component from each of the mitochondrial NADH-coenzyme Q, succinate-coenzyme Q, and coenzyme QH2-cytochrome c reductases (complexes I, II, and III), most likely a nonheme iron-sulfur protein of each complex, is involved in free radical formation. Although the semiquinone form of coenzyme Q may be formed during electron transport, its unpaired electron most likely serves to aid in the dismutation of superoxide radicals instead of participating in free radical formation. Results of studies with electron transfer chain inhibitors make the conclusion dubious that coenzyme Q is a major free radical generator under normal physiological conditions but may be involved in superoxide radical formation during ischemia and subsequent reperfusion. Experiments at various levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, support the view that coenzyme Q, mainly in its reduced state, may act as an antioxidant protecting a number of cellular membranes from free radical damage.

The anticancer enzyme DT diaphorase is induced selectively in liver during ascites hepatoma growth.

DT diaphorase catalyzes the transfer of two electrons to quinones to form relatively stable hydroquinones, thus protecting cells from damage by semiquinone production and subsequent superoxide radical formation. A rapid and substantial increase in the activity of DT diaphorase occurs in the cytosolic and microsomal fractions of livers of rats with Zajdela ascites hepatoma under conditions which generally depress the activity of other xenobiotic-metabolizing enzymes. The increase is time-dependent, parallels the increase in the specific activity of DT diaphorase of the growing hepatoma cells, and is limited to the liver. Treatment of rats with hepatoma cytosol results in a rapid increase in liver cytosolic DT diaphorase activity in a dose-dependent manner.

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems.

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.

Protein synthesis in skeletal muscle from normal and diabetic rats following increased contractile activity in situ.

Protein synthesis was measured in rat skeletal muscle after one hour of heavy work. Direct, supramaximal electrical stimulation in situ under anesthesia resulted in an increase in the in vitro rate of protein synthesis in the soleus and gastrocnemius muscles during the next two hours. Most of the increase was located in the nuclear-connective tissue fraction. No increase was observed in the actomyosin, microsomal or soluble fractions. The same pattern was also observed in the flacid contralateral soleus. Passive stretch of the same muscle group did not result in such changes in protein synthesis. The response was obliterated by rendering the rat diabetic. The observed increase in protein synthesis may represent the initial stages of the adaptive response to increases in muscular activity.

Tissue coenzyme Q (ubiquinone) and protein concentrations over the life span of the laboratory rat.

The coenzyme Q (ubiquinone) concentrations of a number of tissues have been determined over the life span of the male laboratory rat. Coenzyme Q increased between 2 and 18 months and decreased significantly at 25 months in the heart and kidney, and the gastrocnemius, oblique and deep aspect (red) vastus lateralis muscles. The coenzyme Q concentration of liver increased over the life span, while it remained relatively constant in brain, lung, and the superficial aspect (white) of the vastus lateralis muscle. Data are also included for organ weights and protein contents of tissues over the life span. The various roles of coenzyme Q in cellular electron transfer and its regulation, energy conservation in oxidative phosphorylation, and its clinical efficacy in diseases of energy metabolism are discussed. It is hypothesized that coenzyme Q serves as a free radical quencher in the mitochondrion, a major site of free radical formation, in addition to its other roles in cellular energy metabolism, and that its cellular diminution may contribute to the loss of cellular function accompanying ageing.

The effect of endurance exercise on bone dimensions, collagen, and calcium in the aged male rat.

Sixteen weeks of a relatively mild running program, started at 22 months of age, lowered the body weights of 26-month-old male rats to the level of 9-month-old rats and increased the weights and the collagen densities of hind limb bones to levels greater than those of 9-, 22-, and 26-month-old sedentary rats. The densities (g/cm3) and the calcium densities (mg/cm3) of the hind limb bones decreased with age and were restored to the 9-month level by training the elderly rats to run. These data suggest that exercise is capable of inducing a compensation for, or a reversal of, age-associated bone loss (osteoporosis) and restoring the bone mineral content in aged rats to the level of those of mature young adult animals.

Elevation of tissue coenzyme Q (ubiquinone) and cytochrome c concentrations by endurance exercise in the rat.

Six months of enforced and voluntary endurance training of young female Wistar rats resulted in significant decreases of body weight and gastrocnemius muscle wet weight and protein content, and increases in heart weight and protein content, and liver protein content. The coenzyme Q and cytochrome c concentrations of cardiac, gastrocnemius, and deep red region of the vastus lateralis muscles were increased, while small or nonsignificant trends toward increases in cytochrome c and coenzyme Q were seen in kidney, brain, lung, liver, internal + external oblique muscles, and the superficial white region of the vastus lateralis muscle. These results are discussed with regard to several roles for coenzyme Q in cellular function.

The influence of age and endurance exercise on the myoglobin concentration of skeletal muscle of the rat.

The myoglobin concentrations of gastrocnemius muscles of 2-, 5-, 9-, and 25-month-old sedentary, 25-month-old weight-restricted, and 25-month-old endurance-trained (treadmill running) Sprague-Dawley male rats were determined. The concentration of myoglobin was greater in the 5-month-old than in the 2-month-old rat and was lower in the 9- than in the 5-month-old rat. The gastrocnemius myoglobin concentration was the same for 9- and 25-month-old animals. Four months of endurance training, starting at 21 months of age, resulted in gastrocnemius myoglobin concentrations greater than either that of the 25-month-old weight-matched, calorie-restricted control or the 25-month-old sedentary control groups. The gastrocnemius myoglobin concentration of the elderly trained group was intermediate between that of the 5- and 9-month-old animals. These results are consistent with the known loss of oxidative capacity of skeletal muscle with age and its restoration with endurance exercise.

Exercise-induced reversal of age-related declines of oxidative reactions, mitochondrial yield, and flavins in skeletal muscle of the rat.

The ability of gastrocnemius muscle homogenates to catalyze the oxidation of succinate, glutamate + malate, pyruvate + malate, palmitoyl-coenzyme A, decanoylcarnitine and palmitoylcarnitine in the presence of ADP decreased by approximately 32% in sedentary male Sprague-Dawley rats between the ages of 9 and 25 months. Following 21 weeks of treadmill training (running), such homogenates from 25-month-old animals catalyzed oxidations 55% more rapidly than those from 25-month-old sedentary rats, and 17% faster than those from 9-month-old sedentary rats. Total and peptide-bound flavin of gastrocnemius muscles also declined between 9 and 25 months of age and were elevated in the 25-month-old endurance trained rats to levels greater than both 9- and 25-month-old sedentary animals. The yield of protein in the mitochondrial fraction from the quadriceps femoris muscle decreased between 9 and 25 months and was restored to the 9-month level by endurance training. The kinetic characteristics of the isolated mitochondria were not influenced by age or exercise. These data indicate that 2-year-old rats retain the capacity to increase skeletal muscle oxidative capacity and mitochondrial population density in response to endurance training.

Myocardial protein synthesis during aging and endurance exercise in rats.

Rates of protein synthesis, and RNA and cytochrome c concentrations, were assayed in hearts of 9- and 25-month-old sedentary rats and 25-month-old rats trained to run 5 days/week for 21 weeks. Isolated working hearts were perfused with modified Krebs-Henseleit bicarbonate buffer labelled with [14C]phenylalanine. Protein synthesis rate decreased with age, as did RNA content and efficiency of protein synthesis, an indication of the activity per ribosome. None of these three parameters were altered by endurance training. Cytochrome c concentration, which also decreased significantly between 9 and 25 months, was increased in the 25-month-old endurance trained heart to the level of the 9-month-old sedentary heart. We conclude that the depressed protein synthetic system of aged animals is less of a liability in the endurance trained than in the sedentary animal as a consequence of the associated improvement in functional capacity that serves to minimize homeostatic disequilibrium in response to environmental challenges.

Myocardial adaptations to endurance exercise in aged rats.

Isolated perfused working hearts of 25-mo-old male Sprague-Dawley rats trained to run for 16 wk were compared with hearts from 9- and 25-mo-old sedentary animals. Under low work load conditions, systolic and diastolic aortic pressures, aortic flow, and oxygen consumption of the three groups were similar. Under high work load, systolic pressure of trained old and 9-mo groups were higher than the 25-mo sedentary values, but diastolic pressures were similar. At a systolic pressure of 150 mmHg, coronary flow of the old trained heart was higher than that of the age-matched controls, although not equal to the 9-mo sedentary group. The oxygen consumption of the intact hearts under the latter conditions follows the same quantitative trend. Left ventricular cytochrome c concentrations and rates of oxidation of glutamate-malate, palmitoylcarnitine, and succinate were increased in the older rats by training but not to the level of the 9-mo old. These data indicate that appropriate exercise in aged animals improves myocardial function and aerobic energy metabolism.

A rapid biuret assay for protein of whole fatty tissues.

A rapid biuret procedure is described which avoids the turbidity that occurs with protein analysis of intact fatty tissues. Recovery is complete and absorbancy linear with both concentration of the soluble crystalline serum albumin standard and the volume of homogenate of a variety of tissues. This method has been used successfully for the determination of protein concentrations of homogenates of whole rat heart, liver, kidney, brain, lung, and the following muscles: gastrocnemius, interior and exterior obliques, red and white vastus lateralis, and soleus.

Effects of age and cardiac work in vitro on mitochondrial oxidative phosphorylation and (3H)-leucine incorporation.

Mitochondria isolated from in vitro perfused rat heart preparations were used to study the combined effects of age and physical stress. Age-related declines in oxidative phosphorylation catalyzed by mitochondria from nonperfused hearts were not observed. Low work load perfusion resulted in decreased respiration by mitochondria from 24-month-old hearts (p less than .01) but not 10-month old hearts, while high work load perfusion resulted in decreased respiration in both ages. However, the decrease by the 24-month-old hearts was significantly greater than those in the younger hearts (p less than .01). Compared to age-matched low work load hearts, 5- and 10-month-old high work load hearts increased mitochondrial protein synthesis by 86% and 93%, respectively, 15-month-old hearts increased by 60%, and 24-month-old hearts by 13%. The results of this study provide evidence that the ability of the heart to respond appropriately so as to adapt to stress decreases with age, becoming apparent in the laboratory rat between 10 and 15 months of age.