Tetrahydrobiopterin Supplementation Improves Phenylalanine Metabolism in a Murine Model of Severe Malaria

Tetrahydrobiopterin (BH4) is an essential cofactor for both phenylalanine hydroxylase and nitric oxide synthase. Patients with severe malaria have low urinary BH4, elevated plasma phenylalanine, and impaired endothelium-dependent vasodilation, suggesting that BH4 depletion may limit phenylalanine metabolism and nitric oxide synthesis. We infected C57BL/6 mice with <i>Plasmodium berghei</i> ANKA to characterize BH4 availability and to investigate the effects of BH4 supplementation. <i>P. berghei</i> ANKA infection lowered BH4 in plasma, erythrocytes, and brain tissue but raised it in aorta and liver tissue. The ratio of BH4 to 7,8-BH2 (the major product of BH4 oxidation) was decreased in plasma, erythrocytes, and brain tissue, suggesting that oxidation contributes to BH4 depletion. The continuous infusion of sepiapterin (a BH4 precursor) and citrulline (an arginine precursor) raised the concentrations of BH4 and arginine in both blood and tissue compartments. The restoration of systemic BH4 and arginine availability in infected mice produced only a minor improvement in whole blood nitrite concentrations, a biomarker of NO synthesis, and failed to prevent the onset of severe disease symptoms. However, sepiapterin and citrulline infusion reduced the ratio of phenylalanine to tyrosine in plasma, aortic tissue, and brain tissue. In summary, BH4 depletion in <i>P. berghei</i> infection may compromise both nitric oxide synthesis and phenylalanine metabolism; however, these findings require further investigation in human patients with severe malaria

Effect of IL-10 and IL-6 on the regulation of hepcidin in (a) primary macrophages and (b) HepG2 cells. Data show fold-increase relative to mRNA in media control.

<p>Error bars indicate standard error of the mean. Bar plots show data from at least 3 independent experiments. ** <i>P</i><0.01, ***<i>P</i><0.001 (Mann-Whitney test, compared with Media control).</p

Proposed model of hepcidin in malaria infection.

<p>The regulation of hepcidin in response to infection may vary with cell type. A major response to infection occurs in hepatocytes in response to IL-6. However, our observations support the role of IL-10 in primary macrophages. Availability of iron to erythroid developing cells ultimately depends on macrophages and thus the high concentration of IL-10 may play a key regulatory role. Indeed, actively dividing cells like those found in the bone marrow are more susceptible to oxidative damage. In this context, both the direct anti-inflammatory effect of IL-10 and its indirect effect on iron restriction through the up-regulation of hepcidin may be beneficial.</p

Baseline clinical characteristics of the study population.

<p>Values are presented as median [interquartile range]. ADMA: asymmetric dimethylarginine, sVCAM: soluble vascular cell adhesion molecule, PfHRP2: <i>P</i>. <i>falciparum</i> histidine-rich protein 2.</p><p>^a p < 0.001 compared to healthy Gambian children by Mann-Whitney test.</p><p>^b p < 0.001 compared to uncomplicated malaria by Mann-Whitney test.</p><p>Baseline clinical characteristics of the study population.</p

DDAH regulates NO synthesis via ADMA metabolism.

<p>Protein arginine methyltransferases (PRMTs) methylate arginine (Arg) residues on proteins to form asymmetric dimethylarginine (ADMA). Proteolysis releases free ADMA that inhibits nitric oxide synthase (NOS). Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes free ADMA to citrulline (Cit) that can be recycled to arginine. Inactivation of DDAH leads to accumulation of ADMA, inhibition of endothelial NO synthesis, and endothelial dysfunction.</p

The ADMA/arginine ratio is acutely elevated in African children with severe malaria.

<p>ADMA and arginine concentrations were measured in plasma samples collected at the time of presentation (Day 0) and at follow-up visits 28 days later (Day 28) in children with WHO-defined uncomplicated malaria or severe malaria. Healthy Gambian children served as a reference group. Wilcoxon test was used for pair-wise comparison of admission and day 28 mesurements within individuals (47 paired observations from patients with severe malaria; 65 paired observations from patients with uncomplicated malaria). Mann-Whitney test was used to compare patients with severe malaria (n = 81) versus uncomplicated malaria (n = 75) and to compare patients with uncomplicated malaria versus healthy children (n = 31). Each horizontal line depicts the group median. **** p < 0.0001; ns p > 0.05.</p

Multiple linear regression analysis of the relationships between ADMA and arginine and hemoglobin, HRP2, sVCAM, or lactate.

<p>ADMA, Arg, HRP2, and sVCAM were natural log-transformed. Lactate was square root-transformed. ADMA and arginine were explanatory variables in four separate linear models predicting hemoglobin, HRP2, sVCAM, or Lactate.</p><p>Multiple linear regression analysis of the relationships between ADMA and arginine and hemoglobin, HRP2, sVCAM, or lactate.</p

Correlation of ADMA with biomarkers of anemia, hemolysis, parasite biomass, endothelial activity, and tissue perfusion among children with severe malaria.

<p>ADMA, Arg, ADMA/Arg, HRP2 and sVCAM were natural log-transformed. Hemoglobin was normally distributed and was not transformed. Lactate was square root-transformed. Haptoglobin could not be transformed to a normally distributed variable. All correlations were calculated using Pearson’s method, except for correlations with haptoglobin which were calculated using Spearman’s method. A plot of each correlation is presented in the supplement (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005119#ppat.1005119.s005" target="_blank">S3 Fig</a>).</p><p>Correlation of ADMA with biomarkers of anemia, hemolysis, parasite biomass, endothelial activity, and tissue perfusion among children with severe malaria.</p

ADMA and arginine concentrations in plasma.

<p>Values are presented as median [interquartile range]. ADMA: asymmetric dimethylarginine. Day 0 was the day of initial presentation to clinic or hospital.</p><p>^a p < 0.0001 compared to healthy Gambian children by Mann-Whitney test.</p><p>^b p < 0.0001 compared to uncomplicated malaria by Mann-Whitney test.</p><p>^c p < 0.0001 compared to admission by Wilcoxon matched-pairs signed-rank test.</p><p>ADMA and arginine concentrations in plasma.</p