The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one-electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe-S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Decreased Pasteur effect in platelets of aged individuals.

We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

Does coenzyme Q10 play a role in opposing oxidative stress in patients with age-related macular degeneration?

To seek some specific biochemical markers of age-related macular degeneration (AMD), coenzyme Q10 (CoQ10) levels were determined in plasma and platelets from 19 exudative AMD patients and 19 age-matched controls. Lipid peroxidation was followed in plasma in vitro after the addition of a free radical initiator. Most patients had lower plasma CoQ10 content than most controls. Plasma from controls showed greater capacity to oppose the oxidative damage. These results support the concept that free radicals play a pathogenic role in AMD and that CoQ10 may have a protective effect.

Mitochondrial bioenergetics in aging.

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.

Mitochondria, oxidative stress, and antioxidant defences.

Mitochondria are strongly involved in production of reactive oxygen species, considered today as the main pathogenic agent of many diseases. A vicious circle of oxidative stress and damage to cellular structures can lead to either cell death by apoptosis or to a cellular energetic decline and ageing. The early involvement of mitochondria in apoptosis includes expression of pro-apoptotic factors, release of cytochrome c from the inter-membrane space and opening of the permeability transition pore: cytochrome c release appears to precede pore opening. The mitochondrial theory of ageing considers somatic mutations (deletions) of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, Complex I appears to be mostly affected. We have developed the Pasteur effect (enhancement of lactate production by mitochondrial inhibition) as a bio-marker of mitochondrial bioenergetics in human platelets, and found it to be decreased in aged individuals. Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants coenzyme Q is the only one of endogenous biosynthesis; exogenous coenzyme Q, however, may protect cells from oxidative stress in vivo.

Protective effect of exogenous coenzyme Q in rats subjected to partial hepatic ischemia and reperfusion.

In a surgical model of liver ischemia lipid peroxidation occurs, as shown by increase of lipid peroxidation end products, endogenous CoQ9 is oxidized and mitochondrial respiration is lowered; however, pre-treatment of the rats by i.p. injection of CoQ10 for 14 days normalizes the above parameters, presumably by way of the observed high extent of reduction of the incorporated quinone; moreover, liver homogenates of the CoQ10-treated rats are more resistant than those of non-treated rats to oxidative stress induced by an azido free radical initiator. This preliminary study suggests that CoQ10 pre-treatment can be of beneficial effect against oxidative damage during liver surgery transplantation.

Oxidative stress, antioxidant defences and aging.

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.

Mitochondrial complex I defects in aging.

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Coenzyme Q deficiency in mitochondria: kinetic saturation versus physical saturation.

The coenzyme Q (CoQ) concentration in the inner membrane of beef heart mitochondria is not kinetically saturating for NADH oxidation inasmuch as the K(m) of NADH oxidation for endogenous CoQ10 is in the mM range in membrane lipids. Using CoQ1 as an electron acceptor from complex I, we have found additional evidence that the high Km of NADH oxidase for CoQ is not an artifact due to the use of organic solvents in reconstitution studies. We have also obtained experimental evidence that CoQ concentration may be rendered more rate-limiting for NADH oxidation either by a decrease of CoQ content (as in liver regeneration or under an acute oxidative stress), or by a possible increase of the Km for CoQ, as in some mitochondrial diseases and ageing. The possibility of enhancing the rate of NADH oxidation by CoQ therapy is hindered by the fact that the CoQ concentration in mitochondria appears to be regulated by its mixability with the membrane phospholipids. Nevertheless CoQ10 incorporated into heart submitochondrial particles by sonication enhances NADH oxidation (but not succinate oxidation) up to twofold. Nontoxic CoQ homologs and analogs having shorter side-chains with respect to CoQ10 can be incorporated in the mitochondrial membrane without sonication, supporting an enhancement of NADH oxidation rate above 'physiological' values. It is worth investigating whether this approach can have a therapeutical value in vivo in mitochondrial bioenergetic disorders.

Coenzyme Q changes in liver and plasma in the rat after partial hepatectomy.

The levels of coenzyme Q were determined in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ9 content increased in sham-operated rats, whereas it was significantly lower in hepatectomized with respect to non-operated animals. Plasma CoQ9 levels decreased dramatically in hepatectomized animals, but increased strongly in sham-operated in comparison with non-operated rats. The data suggest the possibility of a rate-limiting step in CoQ biosynthesis in hepatectomized animals.

Coenzyme Q depletion in rat plasma after partial hepatectomy.

Coenzyme Q content was monitored in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ9 content increased in sham-operated rats, whilst it was significantly lower in hepatectomized in comparison with non-operated animals at all considered times. On the other hand plasma CoQ9 levels dramatically decreased in hepatectomized animals, while strongly increased in sham-operated in comparison with non-operated rats. The quinone decrease in hepatectomized animals is likely to be due to the attainment of a rate-limiting step in CoQ biosynthesis.

Inhibitor sensitivity of respiratory complex I in human platelets: a possible biomarker of ageing.

NADH-Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19-30 years) individuals and 18 pools from aged ones (66-107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Major changes in complex I activity in mitochondria from aged rats may not be detected by direct assay of NADH:coenzyme Q reductase.

We have investigated the respiratory activities and the concentrations of respiratory chain components of mitochondria isolated from the livers and hearts of two groups of rats aged 6 and 24 months respectively. In comparison with the adult controls (6 months), in aged rats there was a decline in total aerobic NADH oxidation in both tissues; only minor (non-significant) changes, however, were found in NADH:coenzyme Q reductase and cytochrome oxidase activities, and there was no change in ubiquinol-cytochrome c reductase activity. The coenzyme Q levels were slightly decreased in mitochondria from both organs of aged rats. The lowered NADH oxidase activity is not due to the slight decrease observed in the coenzyme Q levels, but is the result of decreased Complex I activity. Since the assay of NADH:coenzyme Q reductase requires quinone analogues, none of which can evoke its maximal turnover [Estornell, Fato, Pallotti and Lenaz (1993) FEBS Lett. 332, 127-131], its activity has been calculated indirectly by taking advantage of the relationship that exists between NADH oxidation and ubiquinol oxidation through the coenzyme Q pool. The results, expressed in this way, show a drastic loss of activity of Complex I in both the heart and the liver of aged animals in comparison with adult controls.

Underevaluation of complex I activity by the direct assay of NADH-coenzyme Q reductase in rat liver mitochondria.

We have shown that the rate of NADH-coenzyme Q reductase in rat liver mitochondria, assayed using the decyl-ubiquinone analog DB, is underevaluated, probably as a result of its low water solubility. In view of drawbacks encountered using other more soluble acceptors in this system, we demonstrate that the most reliable assay of the physiological rate of CoQ reduction by Complex I is the indirect calculation from the total rate of NADH oxidation and the rate of ubiquinol oxidation, using the pool equation of Kröger and Klingenberg [(1973) Eur. J. Biochem. 34, 358-368].

Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat.

We investigated the Coenzyme Q (CoQ) content of different mitochondrial fractions [free mitochondria (FM), synaptic heavy (HM) and light mitochondria (LM)] from three brain areas (cortex, striatum, hippocampus) of rats at different ages. In rats from 2 to 26 months of age, we observed only small differences in total CoQ content (CoQ9 + CoQ10). In FM and LM fractions, values are very similar and appear to be much higher than in HM fractions. The CoQ10/CoQ9 ratios are much higher in brain mitochondria than in other organs, suggesting possible modifications of CoQ biosynthetic pathways in brain; nevertheless they appear to remain constant during ageing. CoQ9 and CoQ10 contents slowly decrease reaching their minimum in rats of 18 months of age, then increase in the older ages. Considering ageing as partially driven by a summation of free radical-mediated processes, we can hypothesize that damage occurring to biological structures in the first half of life might be followed by induction phenomena tending to re-establish the primitive levels of antioxidant molecules.

Protective effect of exogenous coenzyme Q against damage by adriamycin in perfused rat liver.

We have investigated the effect of rat liver perfusion with adriamycin on mitochondrial activities. Although the perfusion treatment per se induces some decline of respiratory activities, adriamycin strongly potentiates this effect; moreover the coenzyme Q9 content of the mitochondrial membrane is significantly lowered by the antibiotic. Coaddition of coenzyme Q10 in the perfusate significantly protects the mitochondria, not only from loss of respiratory activities but also of the endogenous CoQ9 content. Exogenous CoQ10 fails to enhance respiratory activities in control rats, not treated with adriamycin, even though CoQ concentration has been proven not to be kinetically saturating in the respiratory chain under physiological conditions. Thus, the beneficial effect of CoQ10 in the perfusate does not appear to be the result of its role in the respiratory chain but is a consequence of its antioxidant action.

Uptake and distribution of exogenous CoQ in the mitochondrial fraction of perfused rat liver.

The system of perfusing rat livers has been used to evaluate the uptake and incorporation of liposomal CoQ10 into mitochondria. After 90 minutes of perfusion the cells are strongly enriched in CoQ10 up to levels of the same order of magnitude as CoQ9. Heavy and light mitochondrial crude subcellular fractions, low in CoQ10 in control livers, contain high amounts of the quinone after perfusion; yet the purification of these fractions on a metrizamide gradient reveals that the exogenous quinone is mainly associated with the light mitochondrial subfraction, enriched in lysosomes. An increase of the NAD-dependent glutamate-malate oxidase activity is observed in CoQ10 perfused animals. As the total levels of CoQ9 + CoQ10 in these animals are not significantly modified by the CoQ10 incorporated, the observed higher activity is not ascribable to an integration of exogenous quinone into the ubiquinone pool. An antioxidant effect of extramitochondrial CoQ10 on mitochondrial functions is suggested.