Several cellular biomarkers are used to measure the extent and effects of blood Lead (BL) on various biochemical systems. This study explored the levels of Free erythrocyte porphyrins (FEP) as a biomarker of heme biosynthesis disorder among the study participants. The heme biosynthesis process is inactivated by either iron deficiency, failed iron regulatory system or inhibited δ-aminolevulinic acid dehydratase (δ-ALAD) enzyme [31].
Free erythrocyte porphyrin levels of ˃50µg/ dL are important indicator of heme biosynthesis disorder. Common causes of this disorder include iron deficiency due to malnutrition and poor iron absorption, elevated hepcidin levels, and ALAD inhibition by blood Lead [16-18,20, 22, 29]. Findings of this study (Table 1) indicate that 88.2% (n=175) of all study participants were moderately anemic Hb ˃5g/dL < 10g/dL. Of the 175 participants, 111 (56.6%) had normal functioning biosynthesis system (FEP levels ˃50µg/ dL), although with moderate anemia. Therefore, the observed low levels of Hb, cannot be associated with iron deficiency. The candid cofounding factors for the observed low Hb levels therefore are oxidative stress and accumulated erythrocytic pyrimidine nucleotides. Oxidative may be caused by both BL and parasite density while the accumulated erythrocytic pyrimidine nucleotides are caused by inhibition of pyrimidine 5 nucleotidase enzyme (P5N) by BL. Enhanced oxidative challenges brought about by both parasites and Lead ions induces eryptosis, while the accumulated nucleotides cause cellular hemolysis. The study on oxidative challenges and pyrimidine nucleotide levels during both malaria infection and Lead toxicity was outside the scope of this work.
Considering the group of 39 participants (Table 1) with parasite density (PD) (1.2-2.2×103 parasites/µL of blood), no detectible BLL, perfectly functioning heme biosynthesis system FEP ˃10µg/ dL, and no anemia (mean Hb=10.4 g/ dL). It is likely that low PD by its own may not induce anemia especially to people living in malaria endemic areas. However, coexistence with another confounding factor like BL heightens the anemia pathogenesis. We can therefore hypothesize that progression to a severe anemia status is multifactorial and is exacerbated by having both malaria infection and blood Lead simultaneously.
Table-1, further shows a 4% (n=8) severe anemic group of participants (Hb < 5g/dL), with a fairly functioning heme synthesis system (FEP = 36.2 µg/ dL) still under 50µg/ dL but with elevated PD. This gives an indication that the rate at which red blood cells (RBCs) were being destroyed by parasites surpassed their rate of synthesis hence the observed severe anemia status. Indiscriminate destruction of RBCs cause imbalances in the mediators of inflammation, Interleukin 6 cytokines (IL6) levels and this may affect the hepcidin expression [32, 33]. Interferon (IFN)-γ) known to induce the production of TRAIL (TNF-related apoptosis-inducing ligand) is also activated by elevated PD (34). Again, the reported BLL (9.7 µg/ dL) of this group was high enough to induce oxidative stress. As well as inhibition of P5N enzyme hence eryptosis and hemolysis respectively, likely associated with the observed low Hb status. It is further observed (Table 1) that 16/198 participants that had low PD and elevated BLL were anemic (Hb=7.0g/dL) and suffered from iron deficiency anemia as indicated by the heme biosynthesis disorder (FEP ˃50µg/ dL). The group’s low mean Hb levels could be attributed to elevated PD, BLL and other cofounding factors like iron deficiency due to malnutrition, inhibited iron absorption and inhibited (δ-ALAD) enzyme activity.
This study reports a lower mean BLL (=8.6µg/dL) compared to literature [35-37]. Furthermore, an uneven distribution of BLL among the study participants was observed. This is attributed to the fact that BL binds the RBC’s enzyme δ-ALAD which is polymorphic with two major alleles; δ-ALAD-1 and δ-ALAD-2 that differ in electronegativity [38]. Having high frequency of δ-ALAD-1 translates into higher BL burden than those with δ-ALAD-2 [39].
A moderate interaction between the three variables (FEP/Hb, Parasite density and hemoglobin levels) after a multivariate analysis (Table 2) is reported. In addition, parasite density and BLL significantly correlated (r =0.377, P = ˂0.001) to affect the anemia status of the host Table 2. Regression analysis (Pearson’s) models show that FEP: Hb µg /g ratio increases exponentially with the blood Pb while Hb decreased with increasing FEP (Fig. 1). Again, a negative and strong Pearson correlation between FEP: Hb µg /g and Hb levels (Table 2 and Fig.1) as compared to that of parasite density and hemoglobin levels (Fig.2) were observed. This seem to concur with the argument that the etiology of severe anemia is multifactorial and therefore, there is need to study each of these contributing factors.
It can therefore be speculated that progression to severe anemia during malaria infection and Lead toxicity involves destruction of erythrocytes, inhibition of heme synthesis inhibition (ineffective erythropoiesis) and interference of hepcidin iron regulatory system. The increased erythrocytic clearance due to extrinsic and intrinsic challenges increase the susceptibility to phagocytosis and hence anemia. Reduced serum iron (substrate) availability further complicates the heme synthesis mechanism by disturbing the enzyme /substrate enzymatic reaction equation.
However, this study did not find a direct relationship between high blood Lead concentrations with parasite density as previously reported. It is likely that as BLL increases, hepcidin expression is upregulated decreasing the available serum iron important for parasite survival. Blood Lead levels and malaria parasites (Table 2 and Fig.3) in the study population seems to support the argument that BLL had little or no direct effect on parasite density levels.
Children who participated in the study had no co-morbid diseases like HIV, sickle cell traits which are known confounding factors of anemia pathogenesis. This study therefore represents the first reported significant association between malaria and Lead poisoning during the anemia pathogenesis among the pediatric population in Uganda.
Study limitations
This study did not asses the nutritional status of the participants which is an important confounding factor. We therefore recommend that future studies incorporate nutritional status of participants for a better understanding of the relationship between blood Lead levels and anemia during malaria infection.