Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity, Impaired Microvascular Function and Increased Endothelial Activation

Tetrahydrobiopterin (BH(4)) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH(4) is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH(2)), which inhibits NOS. Depending on BH(4) availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH(4)/BH(2) ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH(4) deficiency. The primary three biopterin metabolites (BH(4), BH(2) and B(0) [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH(4) was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH(2) was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH(4)/BH(2) ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH(4)/BH(2) ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH(2) in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.