The world's scientific and social network for malaria professionals
Subscribe to free Newsletter | 10054 malaria professionals are enjoying the free benefits of MalariaWorld today

Bicarbonates, hydrogen peroxide and malaria.

August 13, 2015 - 15:28 -- Pierre Lutgen

Polyphenols are a large group of antioxidants naturally known for their protective effect against oxygen metabolites, acting as free radical scavengers. In contrast to the beneficial effects it has recently been reported that some polyphenols may promote oxidative damage. These harmful effects are suspected to result from a pro-oxidant action.

In 2012 Fr Van der Kooy from Leiden University had noticed that he obtained different results in his trials with infusions prepared with fresh distilled water and distilled water in an open vessel where it had been able to absorb carbon dioxide (personal communication). A Japanese group found that 7 out of 14 flavonoids generated hydrogen peroxide in an acetate buffer in the order of myricetin > baicalein > quercetin > epicachetin >catechin >fisetin >dihyydroxyflavone (YH Miura et al., Biol Phar Bull, 1998, 21, 93-96). Addition of polyphenol rich beverages like coffee or tea to commonly used cell culture media generate substantial amounts of hydrogen peroxide (M Akagawa et al., Biosci Biotechnol Biochem, 2003, 67, 2632-40), (LH Long et al., Biochem Biophys Res Commun, 2000, 273, 50-53). Increased urinary hydrogen peroxide levels are caused by coffee or tea drinking. It increased 6-fold in urine collected 2 h after drinking a cup of coffee (K Hiramoto et al., Biol Pharm Bull. 2002, 25, 1467-71).

BICARBONATE AND HYDROGEN PEROXIDE

The results obtained by Mutaz Akkawi at the Al Quds University on the promotion of the inhibitory effect of sodium.bicarbonate on beta-hematin by Artemisia infusions (S Jaber et al., J Pharm Pharmacol, 2015, 3, 63-72) and a similar effect discovered for bicarbonate and Cymbopogon citratus (personal communication), raise many questions.

The answer eventually is described in a paper from Taiwan (NC Yang et al., Biosci Biotechnol Biochem 2010, 74, 63-68). Polyphenols can oxidize in the culture medium BME (Basal Media Eagle which is principaly used for diploid or primary cell cultures) to geenerate hydrogen peroxide. Generally sodium bicarbonate is used during preparation of the medium for trials to raise and stabilize pH. The authors worked with resveratrol and the gallate EGCG. With BME containing bicarbonate there is a rapid and almost total degradation of the two polyphenols. But when they replace bicarbonate by a mixture of vitamins, or other inorganic salts or amino acids no degragadation occurs. There is no degradation in butanol or butanol saturated with bicarbonate. Milli-Q water has no effect on degradation, nor sodium phosphate nor glucose added to this water, but the addition of 2,2 g/L of sodium bicarbonate to water leads to a rapid and total degradation of these polyphenols. The degradation goes along with the production of hydrogen peroxide. This generation is proprtional to the concentrations of bicarbonate in water.

HYDROGEN PEROXIDE AND HEMOZOIN

Hydrogen peroxide is able to degrade hemozoin (M Chen et al, Molec and Biochem Parasitol 2001, 113, 1-8). All Plasmodium species produce a brown birefringent crystal known as malarial pigment or hemozoin. Hydrogen peroxide as a test reagent can distinguish the hemozoins by different concentrations needed to degrade half of the crystals. These differences have been studied for P falciparum. P malariae, P ovale, P knowlesi, P yoelli et others (GS Noland et al.,Mol Biochem Parasitol 2003, 130, 91-99).

The involvement of H2O2 in the mechanism of action of chloroquine is supported by the fact that it enhances the activity against Plasmodium. Chloroquine by itself is a promoter of oxidative stress. The potentiation of chloroquine activity by the peroxidase-hydrogen peroxide system was already described 25 years ago (K Malhotra et al., Antimicrob Agents and Chemother 1990, 34, 1981-1985). Peroxide mediated oxidation of the porphyrin ring leads to its opening and subsequent breakdown). Inhibition of detoxification of ferriprotoporphyrin by hydrogen peroxide could account for the effectiveness of chloroquine in malaria (CD Fitch et al., Life Sciences 25 :42, 1988). Hydrogen peroxidee also shows synergism with other antimalarials or antibiotics (S Utaida eet al., Southeast Asian J Prop Med Public Health, 2014, 45, 1-5). Hydrogen peroxide and hydroxyl radicals which are generated by macrophages in a respiratory burst at the beginning of an infection have also been shown to kill protozoa such as Toxoplasma and Leishmania (H Dockrell et al., Infection and Immunity 1984, 43, 451-6). A thesis from Brazil found that the extract of sodium bicarbonate + A annua compared to artemisinin in vitro was the most effective with an LC50 of 0,067 mg/L on larval hatchability and development of gastrointestinal nematodes (AC Cala, Thesis Univ Estadial Paulista, 2010). In a more recent paper the same research team found that the LC99 for egg hatching of these gastrointestinal nematodes in sheep was 66.49 mg/L for extracts obtained with distilled water but only 1.40 for extracts obtained with 0,1% of bicarbonate in water (A Cala et al., Parasitol Res 07 May 2014).

The susceptibility to oxidant mediated killing of Plasmodium falciparum was assessed by exposure to hydrogen peroxide. The parasites were most susceptible during maturation, although a reduction in parasite invasion was also observed (S Kamchonwongpaisan et al., Parasitology, 1989, 99, 171-4). In another study, Plasmodium yoelii and Plasmodium berghei were killed in vitro by hydrogen peroxide at concentrations as low as 10¯⁵ M, in the nanomolar range as for artemisinin. Higher concentrations were required In the presence of added normal erythrocytes. Injection of hydrogen peroxide in vivo significantly reduced P yoelii parasitemia but has less effect on P berghei (M Hazel et al., Infection and Immunity, 1983, 39, 456-459). This same effect is described in another paper (IA Clark et al., Clin Exp Immunol, 1984, 56, 524-530). Intravenous injection of t-butyl hydrperoxide rapidly killed Plasmodium vinckei in mice and caused haemolysis. The injections of the hydroperoxide caused parasites to disintegrate inside erythrocytes The same dose seemed harmless to unparasitized mice. Many synthetic peroxides, like trioxolanes, have been developed as antimalarials.

Although the deleterious effects of H2O2-induced oxidative stress on malaria parasite viability are well established, and there is increasing evidence that some antimalarials exert their effect, at least in part, through oxidative mechanisms, the mechanisms by which oxidative stress disrupt parasite function are not fully understood. A recent stuy (D van Schalkwyk et al., PlosOne, 2013, DOI 10.1371) has shown that the oxidising agent H2O2, at concentrations comparable to those to which the parasite may be exposed in vivo causes a decrease in parasite ATP levels and a profound disruption of intracellular pH regulation: an acidification of the parasite cytosol and an alkalinisation of the DV.. A number of processes operate in the food vacuole to decompose the hydrogen peroxide that is produced in parasitized erythrocytes as consequence of the conversion of oxyhemoglobin to methemoglobin in schizonts. No increase in hydrogen peroxide over that observed in uninfected erythrocytes could be detected at the ring stage when host cell digestion is absent (H Atamna et al., Mol Biochem Parasitol 1993, 61, 231-41). Plasmodium falciparum also imports the human protein peroxiredoxin into its cytosol as an enzymatic scavenger for peroxide detoxification (S Koncarevic et al., PNAS, 2009, 106.22).

Artemisia plants are rich in polyphenols. This may partially explain their antimalarial properties by the generation of hydrogen peroxide. Our body constantly produces hydrogen peroxide. Peroximoses in white blood cells break down organic molecules and generate hydrogen peeroxide as by-product. Lets not forget that the pancreas generates a lot of sodium bicarbonate which may react with polyphenols. Normal bicarbonate concentration is serum is approximately 25 mM. The natural supply of hydrogen peroxide in the body may be exhausted during a viral attack. A supply of hydrogen peroxide from external sources might be beneficial in this case to kill the viruses which are anaerobic.

If hydrogen peroxide really has a killing effect on Plasmodium, it is important to avoid the consumprion of substances which inhibit the peroxide production. It was shown that Vitamin C prevents the generation of hydrogen peroxide in caco-2 cells. (SC Roques et al., Free Radic Res, 2002, 36, 593-9). Prudence is thus recommended for the consumption of ascorbic acid alongside Artemisia annua tea.

ANTI-OXIDANTS : FRIEND OR FOE

Many studies have found a lack of effect of antioxidant supplementation in regard to health promotion in humans. Moreover , this supplementation has been linked to increased incidence of a number of diseases. An excellent review paper was published in Germany, with the title « Extending life span by increasing oxidative stress (M Ristow et al., Free Radical Biology 2011, 51, 327-336). Vitamin C blocks the molecular mediators of endogenous ROS defense capacity after physical exercise. They also suggest that a so-called adaptative response may explain how increased ROS formation culminates in promotion of health and life span. Low doses of ROS seem to exert such effect, whereas higher doses are unquestionably detrimental, following the concept of hormesis.

But the hormesis curve may be different for parasites and humans.

The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. It does it by expelling from the erythrocyte the NO producing arginine and importing antioxidant enzymes. Recent findings have shown that in addition to these redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage. To kill Plasmodium we need pro-oxidants like artemisinin, chloroquine, ROS, hydrogen peroxide and not anti-oxidants like vitamins or flavonoids

For humans a meta-analysis published by the University of York states that for cardiovascular diseases Vitamin E cannot be recommended and beta carotene should be actively discouraged. Another systematic review by JAMA (2007, 297, 842-57) showed that Vitamin A and E significantly increased mortality.

According to the Wall Street Journal the Vitamin business in the US has grown to 15 billion dollars.