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Hemolytic anemia and antimalarials

June 24, 2013 - 14:58 -- Pierre Lutgen

Authors: Constant Kansango Tchandema and Pierre Lutgen

G6DP (glucose-6-phospate dehydrogenase) deficiency is a genetic disease which may lead to hemolysis.

This deficiency has a strong link with malaria. In fact it was discovered only 60 years ago when American doctors (Science 1956, 124, 484-5) were confronted with an unusual hemolytic reaction that occurred in ethnic Black individuals following the administration of primaquine. Most antimalarials have a hemolytic effect. The strongest are known for quinine, chloroquine and primaquine. At high doses Fansidar® and Coartem® also cause significant hemolysis ( F C Anaba et al., Pak J Pharm Sci 25:4 2012, 851-55). A recent study in Nigeria ( PC Chikezie et al., Iranian J of Blood and Cancer, 2009, 4, 151-157) compares the effect of five antimalarial drugs on methemoglobin concentrations in erythrocytes. The five drugs showed a concentration dependent elevation of plasma methemoglobin. Coartem® showed the highest propensity. Another study by the same authors (Afr J Biochem Res. 4:3 57-64, 2010) ascertained the osmotic fragility of erythrocytes after administration of the same 5 antimalarial drugs (Fansidar-SP, Halofantrine, Quinine, Chloroquine, Coartem). All generated a rapid accumulation of ROS overwhelming the antioxidant defense capacities of the erythrocytes and caused depletion of erythrocyte glutathione concentration. Again the most aggressive effect on the average was noticed for Coartem®. Dihydroartemisinin is known to cause embryonic erythrocyte depletion and a significant delay in erythroid differentiation ( S Finaurini et al., Toxicology 276, 2010, 128-134). During 2010-2012, a total of 19 cases of delayed hemolytic anemia after treatment of severe malaria with artesunate were published in the peer-reviewed medical literature.

All this may explain to same extent the frequent hemoglobinuria and blackwater fever noticed in malaria patients.

G6DP is a housekeeping enzyme which performs vital functions within all cells of the body. In the erythrocyte it has a particularly important role. It generates reduced glutathione (GSH), the main defense against oxidative stress in the red blood cell. Uncompensated oxidant stress in the erythrocyte results in the oxidation of hemoglobin, membrane damage and hemolysis. This produces a significant degree of morbidity and mortality in G6DP deficient individuals. But at the same time G6DP deficiency protects against P falciparum. Increased vulnerability of the G6DP deficient erythrocyte to oxidant stress is the mechanism underlying the relative protection against Plasmodium falciparum parasitization. A study from Colombia shows that in the case of treatment failure with amodiaquine the glutathione level in the erythrocytes in higher than in the case of adequate clinical response. ( L Zuluaga et al., Malaria Journal, 2007, 6-47) It had been shown in previous studies that chloroquine resistance is associated with a significant increase in G6DP and GSH.

LS Greene (Yearbook of Physical Anthropology 36:153-175, 1993) has made some 20 years ago a survey of all the studies which have evaluated this protective effect against malaria which is described and confirmed in a large number of studies. An increase in GSH has been observed in infected erythrocytes, and this GSH is of parasitic origin, produced by the parasite for its own protection against oxidative stress. In G6DP deficient individuals Plasmodium falciparum the production of GSH is insufficient to protect the parasite against oxidative death and the erythrocyte against hemolysis.

Fava beans are likely to produce hemolysis. Many studies have shown that quinine, primaquine, chloroquine also may lead to hemolysis. The antimalarial activity of quinine and compounds vicine and convicine present in broad beans eventually follow the same path. In the gastrointestinal tract they are hydrolyzed to form unstable pyrimidine aglycones. They reduce red blood cell GSH and generate hydrogen peroxides and free radical species.

More worrisome in this context is the recommendation by WHO to use Fansidar (sulfadoxine-pyrimethamine) as Intermittent Preventive Treatment in pregnancy. This may lead to anemia and more particularly megaloblastic anemia which is detrimental for mother and foetus. In no case should it be used for G6PD mothers which anyway have a higher protection against malaria ( FP Mockenhaupt et al., Trop Med Int Health 2003, 8:2, 118-24) and where the use of Fansidar might have dramatic effects. In malaria endemic areas in Africa up to 30% of the population carry the G6PD deficiency. According to US-FDA there are no adequate and well-controlled studies in pregnant women for Fansidar. However, due to the teratogenic effect shown in animals pyrimethamine-sulfadoxine , Fansidar therapy should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus. And it is recommended that women of child bearing potential who are traveling to areas where malaria is endemic should be warned against becoming pregnant, and should be advised to practice contraception during prophylaxis with Fansidar and for three months after the last dose. That Fansidar remains recommended by WHO for African women appears very questionable when several studies have shown that the resistance to Fansidar reaches 60% in pregnant women (BL Nahlen et al., Lancet, 1989, 2, 830-834) and that this is known since 20 years. In areas where Plasmodium falciparum has been endemic , there is an increase in G6DP deficient individuals. This has been recently verified by several authors (R Howes et al., PLOS Medicine, 2012 9:11).

In sub-Saharan Africa G6DP deficiency may reach 30%, as a result of a natural selection process or another mechanism which has not been elucidated. . Some earlier studies have shown that the regulation of G6DP only occurs in liver and adipose tissue (L.P. Stabile et al., J of Lipid Research, 1998, 39, 1951-63). It is regulated by both nutritional and hormonal stimuli. For example, when after a period of starvation in rodents G6DP activity decreases 80% in mice and rats consuming a high-fat diet versus those fed a low-fat diet. But this inhibition of G6PD is only induced by polyunsaturated fatty acids (PUFAs), not by monounsaturated or saturated fatty acids. The most efficient PUFA to this effect is arachidonic acid, present in Artemisia plants and not in other ones. Another study showed that high glucose impairs G6PD activity leading to increased oxidative stress (Z Zhang et al., The FASEB J, 2010, 24, 1487-1505).Experimental data show that hyperglycaemia can reduce expression of the G6PD gene and activity of the enzyme (A Heyman et al., Diabetes Care 2012, 35. E58; C.Carette et al., Diabetes and Metabolism, 2011, 37, 79-82).  

Could this explain why the effectiveness of artemisinin is enhanced by olive oil, peanut oil, palm oil?All available data indicate that the hepatic G6PD gene is transcriptionally regulated and that this effect has a half-life of several days or weeks and could thus explain some prophylactic activities of nutritional compounds. It would of course be interesting to find natural substances which inhibit G6DPand GSH and in this way act as prophylactics against malaria. It is for example the case for gallated catechins ( ES Shin et al., Bioorg Med Chem 2008 1:16 3580-6) or scopoletin from cassava or saponins of Panax notoginseng ( Z Yang et al., Zhongguo Zhong Yao Za Zhi, 2011, 36:17, 2413-17). Paracetamol also reduces GSH.

The challenge seems to be to find the right balance between prophylaxis and therapy or the right balance of antioxidant levels at different stages of the malaria infection. A high oxidative stress as a result of low G6DP will be detrimental for the survival of young parasites, but if this stress becomes too high in the case of severe malaria or after consumption of nutritional and antimalarial substances which enhance oxidative stress hemolytic anemia and death may follow. Artemisia annua tea could present the right balance of compounds to cure malaria. The oxidative stress due to artemisinin which kills the parasite at a young stage is modulated by flavonoids, scopoletin, saponins. Scopoletin is one of the strongest antioxidants. In many aspects Artemisia annua from Luxembourg has the strongest anti-inflammatory effect (PM de Magalhaes et al., Food Chemistry, 134, 2012, 864-871).. It is poor in artemisinin but relatively rich in scopoletin and saponins. All this pleads for consumption of the herb in the form of leaf powder in capsules rather than solvent extracts which do not dissolve some of the constituents. Quercetin, azadirachtin, luteolin, thujone and resveratrol present in other plants also increase glutathione and reduce the oxidative stress.

At this stage it is important to note that no hematolytic or hematological problem were ever reported in the scientific literature for Artemisia annua infusions used to cure or prevent malaria.