Tetrahydrobiopterin (BH4) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH4 is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH2), which inhibits NOS. Depending on BH4 availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH4/BH2 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 BH4 deficiency. The primary three biopterin metabolites (BH4, BH2 and B0 [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 BH4 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, BH2 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 BH4/BH2 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 BH4/BH2 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 BH2 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.
Vascular nitric oxide (NO) bioavailability is decreased in severe falciparum malaria and associated with microvascular dysfunction and increased endothelial activation. Nitric oxide synthase (NOS) requires tetrahydrobiopterin (BH4) as a co-factor to convert L-arginine to NO, but when BH4 is low, NOS is “uncoupled” and produces superoxide instead of NO. In conditions of increased oxidative stress, BH4 is converted to dihydrobiopterin (BH2) and biopterin (B0): the resulting BH2 competes with remaining BH4 as a competitive inhibitor of NOS, further decreasing NO production. We measured BH4 and BH2 in the urine of adults with severe and uncomplicated falciparum malaria and compared results to those of controls or those with sepsis. There was a significant decrease in urinary BH4 and increase in BH2 in severe malaria compared to uncomplicated malaria, sepsis, and controls, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4. The BH4/BH2 ratio was associated with increased risk of severe disease, endothelial activation and microvascular dysfunction, likely through impaired NOS function. This additional mechanism of decreased NO in severe malaria suggests that trials evaluating use of adjunctive L-arginine to increase NO in severe malaria may require concurrent therapy to regenerate BH4.