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Zinc and beta-hematin inhibition

November 25, 2015 - 07:52 -- Pierre Lutgen

Our partners at the Al Quds University in Palestine have found that a zinc-arginine complex strongly inhibits beta-hematin crystallization, like quinine does, but that zinc or arginine alone are not effective. Arginine and zinc play an important role in the human physiology. The plants from the Artemisia family are rich in these constituents which play probably a key role against malaria and other diseases. They easily form a complex in a large range of reagent concentrations (E Bottari et al., Monatshefte Chemie 2014, 145, 1707-1714). Researchers have also reported the formation of a complex between zinc and naproxen (H. Abu Ali et al. European Journal of Medicinal Chemistry 89 (2015) 67-76). Zinc complexes between zinc and amino acids including arginine are used for the treatment of elevated copper toxicities (US Patent 8247398 B2). Chelation of zinc with quercetin and luteolin has been studied recently in Greece (A Primikyri et al., J Phys Chem, 2015, 119, 83-95).

In vitro Plasmodium growth is slower in thalassemic erythrocytes. A 75 % reduction was observed in comparison with normal erythrocytes. Thalassemic erythrocytes contain about a two-fold higher concentration of Zn-protoporphyrin than normal erythrocytes (J Martiney et al., Molecular Medicine, 1996, 2, 236-246). The same mechanism was studied in Indonesia (JK Iyer et al. Molecular Medicine, 2003, 5-8, 175-182). Erythrocyte zinc-protoporphyrin, normally present at 0.5 µM can elevate 10-fold in anemias related to thalassemia, iron deficiency, hemolysis, inflammatory disease. Zinc-protoporphyrin inhibits heme crystallization in a mechanism similar to the antimalrial quinolines. Zinc-protoporphyrin does not form crystals alone. Inhibition of heme crystal formation occurs with an inhibitory concentration IC50 of 5µM. These authors have demonstrated that zinc-protoporphyrin binds to hemozoin crystals in a saturable way. When amounts of bioavailable iron in the reticulocyte are low, zinc is inserted into the protoporphyrin in lieu of iron. Some studies indeed have reported protection from severe malaria disease by iron deficiency and exarcerbation of malaria disease by iron repletion. Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury and neurological deficits (Y Gong et al., Acta Neurochir Suppl 2006, 96, 232-6).

Zinc deficiency is common in less developed countries, probably because dietary factors influence zinc absorption. Phytate which is present in staple foods like cereals, corn and rice, has a strong negative effect on zinc absorption from composite meals. Iron can have a negative effect on zinc absorption. Amino acids, such as histidine and methionine are known to have a positive effect on zinc absorption. Saponins have no effect (S Southon et al., Brit J Nutrition. 1988, 59, 389-396). Zn absorption was significantly less in rats fed on chlorogenic acid or caffeic acid than in the control group (C Coudray et al., 1998, 80, 575-84). There is an emerging realization that physiological processes controlling zinc availability and uptake are important in both immune function and pathogen virulence.

Zinc has a pivotal role in the entire immune system fostering resistance to infections by virus, fungi and bacteria. This might be one of the reasons why human milk is rich in zinc. Zinc supplementation improves the cellular immune status, raises CD4 and the CD4/CD8 ratio (Sazawal et al., Indian Peediatr, 11997, 34, 589-97). Malaria is associated with a rapid decline in CD4 cell count (J Mermin et al., JAIDS, 2006, 41, 129-130. After stress or trauma zinc concentration decreases (LM Gaetke et al., Am J Physiol 1997, 272, E952-6). In the early response to pathogenic infection, host cytokine release leads to an acute inflammatory response that includes a systemic lowering of plasma zinc levels or hypozincemia. This is accomplished in great part by the metal chelating protein, metallothionein. These host responses decrease serum zinc levels by up to 80%. IL-6 plays a key role (J Luzzi PNAS, 2005 102, 6843-48). The University of Louvain has shown that the Artemisia annua from Luxembourg poor in artemisinin causes the strongest IL-6 and IL-8 inhibition when compared to teas from other origins (PM de Magalhaes et al., Food Chemistry 2012, 134, 864-871).

Plasma zinc concentrations are an imperfect measure of zinc nutritional status. Plasma zinc represents only a fraction of total body zinc, and alternative measures of zinc status such as platelet, lymphocyte, or tissue zinc are not well-suited for large field trials in developing countries (36). Measurement of metallothionein levels is a potentially more sensitive alternative to plasma zinc for the assessment of zinc status. Metallothionein production is induced by available zinc (37,38). Both human and animal studies have demonstrated that production of metallothionein significantly increased after dietary zinc supplementation. Many pathogens require zinc for virulence and for adherence to the mammalian cells. Over 250 enzymes and transcription factors annotated in the Plasmodium falciparum genome require tighly bound zinc ions By XFM (X-ray fluorescence microprobe) it was demonstrated that the Plasmodium parasite induces a striking influx of zinc into the host cell and then drives a developmentally important compartmentalization of this element in parallel with iron redistribution and hemozoin formation. The parasite-driven fluxes of weakly bound zinc are essential to pathogenicity (RG Marvin et al., Chemistry & Biology 2012, 19, 731-741). The authors find that the Plasmodium falciparum parasite requires acquisition of 30 million zinc atoms before host cell rupture, corresponding to a 400% increase in total zinc concentration. Zinc accumulates in a free available form in parasitophorous compartments outside the food vacuole, including mitochondria. Restriction of zinc availability via small molecule treatment causes a drop in mitochondrial membrane potential and severely inhibits parasite growth. The XFM maps show both free and losely bound metal ions and metal ions that are tightly bound to proteins. In the case of malaria distinct compartmentalization of iron and zinc occurs within late stages of the parasite life cycle. The localized iron corresponds with the site of hemozoin crystal formation.

All this appears to be in contradiction with the fact that plasma zinc concentrations are depressed during the acute phase response in children, as was observed on a cohort of 689 children from Ghana, Tanzania, Zambia (C Duggan et al, J Nutr 2005, 135, 802-7). The supplementation of zinc on morbidity due to Plasmodium falciparum was studied in Papua New Guinea. It resulted in a reduction up to 69% of P falciparum health-center based episodes.  It is of interest that the effects of Zn on morbidity were not reflected in malariametric indicators, as for exemple parasite density or spleen enlargment  (AH Shankar et al., Am J Trop Med Hyg2000, 62-6, 663-669). The average zinc concentration in infected host cells containing a schizont-stage parasite was found to be 4-fold higher than in uninfected cells and localized zinc concentrations exceeded average zinc levels within the erythrocyte by over 20-fold (RG Marvin op.cit.). The same authors showed that the zinc chelator TPEN was highly active against the Plasmodium parasite at micromolar concentrations but the addition of two molar equivalents of zinc relative to TPEN rescued parasite growth. Dipicolinic acid (DPA) like other chelators was also found to inhibit the intracellular developement of the parasite with an ED50 of 1 mM (H Ginsburg et al., Biochim Biophys Acta 1986, 886, 337-44). These data indicate that if enough chelator is available survival of the parasite is no longer possible.

Pierre Lutgen and Mutaz Akkawi