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Arginine, a deadly weapon against gametocytes.

July 5, 2015 - 20:26 -- Pierre Lutgen

Amino-acids in Artemisia annua have barely been studied. The analytical data published by EA Brisibe and J Ferreira date back to 2009 (Food Chemistry, 2009, 115, 1240-1246). Their role in malaria infections has been ignored, except for a study published in Japan (DT Uyen et al., Biol Pharm Bull. 2008, 31, 1483-1488). To gain insight into the mechanism of malarial haemozoin formation, they examined the effects of amino acids on beta hematin formation in vitro. Surprisingly some of these amino acids like arginine, histidine, lysine showed a significant inhibition. In contrast beta-hematin formation was enhanced by leucine, isoleucine, valine and methionine.

A very recent paper from China (Li Zheng et al., Parasites & Vectors, 2015, 8 :326) shows that arginine supplementation not only reduced parasitemia and prolonged survival of malaria infected mice by raising the NO produced endogenously, but also reduced the numbers of zygotes and oocysts in mosquitoes fed on infected blood containing arginine. This confirms earlier results from China. Nitric oxide inhibits the development of Plasmodium yoelii gametocytes into gametes (Ya-Ming Cao et al., Parasitol Internat. 1998, 47, 157-166). It inhibits the exflagellation of male gametocytes (YJ Liu et al., Zhongguo Ji Sheng…, 2007, 25, 206-8).

In the fight against malaria most efforts have concentrated on the elimination of asexual parasites and the symptoms they cause : fever, nausea, inflammation, coma…. The fight against the sexual forms, i.e. gametocytes has been neglected. It has occasionnally been observed that certain drugs like primaquine have a gametocydal effect. But primaquine is very toxic for people with the G6DP deficiency. Chloroquine and artemisinine only affect young gametocytes but have no effect on mature gametocytes. Sulfadoxine-pyrimethamine even increases the number of gametocytes. Anyway these pharmaceuticaal drugs are only administered during three days, and mature gametocytes develop on day ~14 of the infection.

Among all amino acids arginine has the specific ability to enhance NO production and inactivation of gametocytes involves reactive nitrogen species (T Naotunne et al., Immunology, 1993, 78, 555-562). Nitric oxide is a known mediator of parasite killing by WBC as was shown when the production of NO by arginine is blocked by an inhibitor.

The digestion of hemoglobin by Plasmodium falciparum leads to an excess of amino acids which rise to high extracellular levels during the trophozoite stage. Alanine, valine, histidine are excreted as waste from the infected erythrocyte. The most dramatic relative change in extracellular levels occur for ornithine and citrulline which accumulate and are derived from arginine, via the arginase pathway. Arginine levels are depleted to almost undetectable levels over 40 hours during the trophozoite stage. The ornithin produced accounts for the majority of depeleted arginine. (K Olzewski et al., Cell Host & Microbe, 2009, 5, 191-199). This is intriguing because several studies have demonstrated a strong correlation between reduced arginine levels and advanced malaria in both child and adult malaria patients. The model proposed by the authors is that the parasite modulates the activity of the host enzymes iNOS and arginase. iNOS is used by the host immune system to generate antimicrobial NO radicals from arginine. Depletion of the plasma arginine pool may suppress this immune response by removing the substrate for NO production. The parasite does almost everything it can to eliminate arginine which is going to kill it.

NO down-regulates endothelial inflammation and has a vasodilatory capacity and in this way reduces the adhesion of infected erythrocytes in small vessels which is leading to severe and cerebral malaria and further development into gametocytes by escaping their renal destruction. It also binds to hemoglobin to prevent the generation of free heme (I Gramaglia et al., Nat Med 2006 12 1477-1422).

There is substantial evidence linking hypoargininemia to malarial infection and progression to cerebral malaria (I Gamaglia et al., Nat Med 2006, 12, 1417-22). A study (Lopansri et al, The Lancet, 2003 361, 676-78) measured concentrations of arginine in cryopreserved plasma samples from Tanzanian children. The concentrations were low in individuals with cerebral malaria, intermediate in those with uncomplicated malaria and within the normal range in healthy controls.

It was also found that reduced erythrocyte deformability is associated with hypoargininemia and low nitric oxide ; it starts on day zero of infection and returns to normal values only at full convalescence on day 28. Deformability can be restored by arginine supplementation (J Rey et al., Scientific Reports, 2013, 4, no 3767).

Arginine is also a crucial amino acid for macrophage activation, and influences other arms of host immune responses, including T and B cells. In Leishmania infection the parasite desactivates macrophages. Arginine however will trigger iNOS activity and promote parasite killing in macrophages. This highlights the importance of arginine in iNOS mediated control of leishmaniasis (N Wanasen, Immunol Res 2008, 41, 15-25). NO also kills the trophozoites of Giardia lamblia.

Hypoargininemia is a hallmark of pathogenic inflammations, bacterial sepsis or traumatic insult. iNOS mediated production of NO occurs also in human tuberculosis and leprosy ; NO can kill mycobacteria, and arginine leads to clinical improvement in TB patients (Th Schön, Thesis, Linköping University, 2002, No 749).

Arginine supplementation will restore and increase NO levels. Arginine capsules can be found in drugstores but it is probably easier for most people in Africa to rely on nutrition rich in arginine ; meat, fishes, eggs, some vegetables like garlic, and nuts like avocados. Peanuts are a common staple in many countries and are very rich in in arginine. This may explain why Artemisia annua powder in peanut butter has shown excellent therapeutic results in several of our trials. But even at the higher levels of supplementation in these combinations we are far from toxic levels. The safety profile of arginine iv-infusion in moderately severe malaria was studied in Australia. 12 g over 30 minutes are well tolerated and no clinically important changes in hemodynamic or biochemical factors were noticed (TW Yeo et al., PLOSone, 2008, 3-6, e2347). At DUKE US a clinical trial was also run studying the arginine kinetics and metabolism in children with severe falciparum malaria (H Zhang et al., Poster). It confirmed a significative inverse association between malaria severity and NO production in the NOS pathway. Further research is necessary to improve our understanding of the complex mechanisms that regulate NO’s role and further clinical trials are recommended to study the impact of arginine. For that reason the sales of arginine capsules on drugstore shelves with indiscriminate dosage appears inappropriate in our eyes. Arginine , iNOS and NO indeed have a Janus face and at excessive levels may cause more harm than good and even cancer.

Let’s not forget that mother’s milk especially colostrum is also rich in argininine. This may contribute to the immunity of newborns against malaria in the first weeks or months. In colostrum (1-3 days postpartum) nitrite concentrations are much higher than in mature milk (0.08 mg/100mL versus 0.001). Medicinal herbs contain on the average higher amounts of nitrate and nitrite than fruits or vegetables : a mean nitrate concentration of 1.240 mg/kg fresh weight versus 336 mg/kg in fruits and vegetables (EFSA Journal, 2008, 689, 1-79).The nitrate-nitrite-nitrite oxide pathway has been shown to exist in many alternative herbal medicines or dietary supplements.

Artemisia plants are very rich in arginine. A recent study from Ukraine (O Ochkur et al., Pharm Innovat J. 2013, 2, 64-67) has analyzed the amino acid content in some 8 plants of this subgenus and found that they are all 5 to 10 times richer in arginine than other herbs or vegetables, with A annua topranking. This confirms the result of J Ferreira quoted in the introduction of this document.

The prophylactic properties of regular drinking of Artemisia tea have been noticed in many countries and documented in Uganda (PE Ogwang et al., Trop J Pharmaceut Res « 012, 13, 445-453). Maybe the same practice will reduce the gamecyte load in asymptomatic individuals and block transmisssion. That is what we want to find out in clinical trials with African partners.