The Canadian Brian John Bandy took the opposite path to many Brazilian researchers: he left his country on completing his doctorate to pursue his career here. He came in 1996, took a post-doctorate at the Chemistry Institute of the University of São Paulo (IQ-USP) and returned to Canada the following year. Once More attracted by Brazil, he came again in 1998 for a second stay at the same institute. During these peregrinations he completed a study that increased the importance of a group of substances, the bioflavonoids, that had previously been seen just as vitamins. Bandy showed that they are much more than that: they protect animal cells from aging and premature death. The article he signed with his supervisor, Etelvino Bechara, was on the cover of the August issue of the Journal of Bioenergetics and Biomembranes, of the United States.
The results attest for the first time to the essential role of bioflavonoids in energy-producing reactions within the cell and suggest how vitamins act with them to the benefit of the organism. Found in plant extracts and abundant, for example, in green tea (Camellia sinensis) and in red wine, flavonoids – called bioflavonoids when seen as nutrients – act together with vitamin C or ascorbic acid, also found in fruit and green vegetables. It has been known for some years that ascorbic acid in isolation can even be toxic, although indirectly, because it promotes reactions that release damaging substances to cells – a warning to those that take excessive doses of vitamin C and think they need nothing else, neither fruit nor green vegetables.
Using techniques that monitor the transfer of electrons, the alterations to the shape of the protein molecules called cytochromes and the formation and disappearance of free radicals, Brian and Bechara detailed these interaction mechanisms, which derive from a property of the bioflavonoid molecules: double chemical affinity, both through the water and through the mitochondrial membrane – a cell compartment in which chemical reactions responsible for energy production occur. The bioflavonoids – at least five of the 20 tested – probably remain partially involved with the mitochondrion and partially outside it. It is an ideal position for catching electrons of the ascorbate which have affinity only through the water (they remain distant from the membrane), conducting them inside the membranes of dozens of mitochondria contained in each animal cell – a human liver cell, for example, has 1,000 to 2,000 mitochondria.
Having overcome the membrane, the electrons reach the destination of benefit to those that wish to postpone old age: the cytochromes c, proteins that play a role in the reactions in cellular respiration – leading to the production of energy for the cell. Together, the cytochromes arered and look like blood because of the iron atoms, which form a branch of the molecule, what is called the group heme. It is due to the cytochromes that the mitochondria are normally dark beige in color. Another peculiarity is that, sometimes the mitochondria gives off light, as a result of the reaction of the oxygen with an excited electron, during the reactions in cellular respiration.
In this process, one of the compounds formed is hydrogen peroxide (oxygenated water, H2O2). Reacting with the c cytochrome, the H2O2 gives rise to hydroxyl radicals. These are the danger. The hydroxyls, one of the types of the so-called free radicals, break the heme and remove the iron from the cytochrome, with consequences that are not good, from the standpoint of the functioning of the organism: without the iron, the cytochrome loses not just the characteristic red color, but also the capacity to act in cellular respiration. It is worse still, when the radicals cause the mitochondria to swell and break. And, free inside the cell, the cytochromes c trigger the process of cell destruction, the so-called apoptosis.
Neither Bandy nor Bechara intended to study bioflavonoids in this role. At the kickoff of the work, Bandy proved that the ascorbate alone protects the cytochrome c from the attack of the peroxides, an effect not previously well characterized. But it was a limited effect. In a situation very close to reality – with liposomes, a kind of artificial membrane made with the most common molecules in the membrane of the mitochondria – did not work. This result is explained by the fact that the ascorbate has affinity only to water – in practice it is distant from the membrane.
Eager to discover the mechanisms and the substances that prevent the destruction of the cytochrome c, Bandy decided to test the bioflavonoid, which, as he knew, had an affinity to the membrane. There was only one on the shelf; epicatekin He tested it and it worked. “It was pure chance”, he says. “There are hundreds of bioflavonoids and they do not all work like that”. The discovery would have come to nothing , for example, he had used naringinine or hesperetin, two of the around 20 bioflavonoids that he tested later and seem not to care for the ascorbate.
Until then, the bioflavonoids were only known as antioxidants, which counteract the effect of the free radicals. “No situation in which they were really essential to the cellular processes had thus far been described”. Of the joint reactions, the classic case was vitamin P, which together with vitamin C, prevents the blood vessels being permeable and thus prevents scurvy – an illness that causes bleeding of the gums and serious general hemorrhages. It was typical of travelers in the 16th century, who remained for months on the high-seas without eating fruit or fresh vegetables. It was deadly. There are accounts of where, of 400 people on board, only ten survived. The Hungarian Albert Szent-Gyorgyi (1893-1986, Nobel Prize for Medicine in 1937) discovered that vitamin C, which he himself had discovered in 1928, did not work alone in preventing the blood vessels from breaking; something more was needed, vitamin P.
The results that Bandy achieved also had practical implications. In treating patients that survived a heart attack or during heart surgery, for example, ascorbate seems not to offer the least protection to the mitochondria of the heart cells against the attack of peroxides. Perhaps it works with certain bioflavonoids”, he thought. In principle, his work could also help to delay the onset of aging, since it suggests a combination of nutrients that would be efficient in minimizing cell damage – the so-called oxidative stress, the result of the respiration process taking place in the mitochondria.
The research goes on. Recently Bandy found the first evidence of what we could call the bad side of ascorbate. On its own, even in low concentrations, it can cause the mitochondria to swell, the first step in the death of a cell. “Ascorbate on its own is dangerous”, he comments, before recounting another novelty, also subject to deeper checking: there are bioflavonoids able to block the action of the ascorbate. Encouraged by fact that his postdoctoral has been renewed until September next year in order to enable him to explore this other side of the cooperation between the ascorbate and the bioflavonoids, he is readying himself to deal with isolated mitochondria, still closer to the real organisms.
At 44 years of age, Bandy is not likely to return any time soon to his own country for good. Bechara is helping him find a place at a Brazilian university. They met in 1995, at a congress on free radicals in Pasadena, in the United States. Bandy had completed his doctorate at the Simon Fraser University of Burnaby, Canada. In his thesis he had shown how athletes spend energy and produce more mitochondria. Bechara was impressed by the fact that Bandy had written an article – on the role of mitochondria in oxidative stress – which had already become an international reference.
Bechara, who at that time was already studying the aminolevulinic acid , one of the sources of free radicals that attack mitochondria, saw the new doctor as an excellent partner. Since then, he has watched Bandy’s development and his liking for Brazil: the Canadian, who already liked fishing and long walks through the forest, did extra courses at the Chemistry Institute and nowadays he feels at ease dancing samba and playing soccer.
Bechara as supervisor was also engaged in the task of retaining promising researchers. Before Bandy, he brought the Italian Paolo Di Mascio, a recently qualified doctor in Dusseldorf, Germany. Mascio stayed for two years, did a competitive examination and is now a lecturer at USP’s Chemistry Institute.
The Interaction of Ascorbate, Bioflavonoids and Biofactors Redox in induced c Cytochromes, Peroxidative Stress and Apoptosis (nº 97/07865-2); Modality Postgraduate grant in the country; Coordinator Etelvino José Henriques Bechara – USP’s Chemistry Institute; Investment R$ 96.000,00