Decreased Rate of Plasma Arginine Appearance in Murine Malaria May Explain Hypoargininemia in Children With Cerebral Malaria

Abstract

Background: Plasmodium infection depletes arginine, the substrate for nitric oxide synthesis, and impairs endothelium-dependent vasodilation. Increased conversion of arginine to ornithine by parasites or host arginase is a proposed mechanism of arginine depletion.

Methods: We used high-performance liquid chromatography to measure plasma arginine, ornithine, and citrulline levels in Malawian children with cerebral malaria and in mice infected with Plasmodium berghei ANKA with or without the arginase gene. Heavy isotope-labeled tracers measured by quadrupole time-of-flight liquid chromatography-mass spectrometry were used to quantify the in vivo rate of appearance and interconversion of plasma arginine, ornithine, and citrulline in infected mice.

Results: Children with cerebral malaria and P. berghei-infected mice demonstrated depletion of plasma arginine, ornithine, and citrulline. Knock out of Plasmodium arginase did not alter arginine depletion in infected mice. Metabolic tracer analysis demonstrated that plasma arginase flux was unchanged by P. berghei infection. Instead, infected mice exhibited decreased rates of plasma arginine, ornithine, and citrulline appearance and decreased conversion of plasma citrulline to arginine. Notably, plasma arginine use by nitric oxide synthase was decreased in infected mice.

Conclusions: Simultaneous arginine and ornithine depletion in malaria parasite-infected children cannot be fully explained by plasma arginase activity. Our mouse model studies suggest that plasma arginine depletion is driven primarily by a decreased rate of appearance.

Keywords: endothelium; host response; metabolism; nitric oxide; plasmodium; urea cycle.

Figure 1.

Arginine, ornithine, and citrulline were acutely depleted in Malawian children with malaria. Amino acid concentrations were measured in plasma samples obtained at admission and at follow-up visits 28 days later from patients with cerebral malaria. Patients with uncomplicated malaria and healthy Malawian children were also studied. The Mann–Whitney test was used to compare 29 patients with cerebral malaria to 30 with uncomplicated malaria or 27 healthy controls. The Wilcoxon test was used for pair-wise comparison of admission and recovery values in patients with cerebral malaria (15 observations). *P < .05, **P < .01, and ***P < .001. Abbreviation: NS, not significant (P > .05).

Figure 2.

Depletion of plasma arginine, ornithine, and citrulline during Plasmodium berghei infection. A, Amino acids were measured by high-performance liquid chromatography in plasma from mice culled on days 2, 4, or 6 after inoculation with P. berghei ANKA. Data represent results from 3 independent experiments (uninfected, n = 24; infected, day 2, n = 20; infected, day 4, n = 21; and infected, day 6, n = 20). B, Plasma amino acids were assessed on day 6 after inoculation from mice infected with wild-type (WT) or arginase knockout (argKO) P. berghei ANKA parasites. Data represent results from 10 mice per group. C, Uninfected controls not subjected to dietary restriction were provided with ad libitum access to food. Uninfected mice subjected to dietary restriction received only as much food as was consumed each day by a paired mouse infected with P. berghei ANKA. Data are pooled from 6 experiments, with 22 uninfected controls with ad libitum access to food and 19 pairs of infected mice and uninfected mice subjected to dietary restriction. Box plots depict median values and interquartile ranges. Whiskers extend to the highest and lowest values within 1.5 times the 75th and 25th percentiles, and values outside this range were plotted as individual points (by the Tukey method). *P < .05, **P < .01, and ***P < .001, by the Kruskal–Wallis test, followed by the Dunn multiple comparisons test (A), the Mann–Whitney test (B and C; uninfected with vs uninfected without dietary restriction), or the Wilcoxon matched pairs signed rank test (C; uninfected with dietary restriction vs infected). Abbreviation: NS, not significant (P > .05).

Figure 3.

Metabolic flux of plasma arginine (Arg), ornithine (Orn), and citrulline (Cit). Rates of appearance and interconversion of plasma Arg, Orn, and Cit were calculated in from plasma enrichment of continuously infused heavy isotope–labeled metabolic tracers, as measured by quadrupole time-of-flight liquid chromatography/mass spectrometry. Uninfected mice subjected to dietary restriction received only as much food as was consumed each day by a paired mouse infected with Plasmodium berghei ANKA. Uninfected controls not subjected to dietary restriction were provided with ad libitum access to food. Data are pooled from 3 experiments, with 10 uninfected controls with ad libitum access to food, and 10 pairs of infected mice and uninfected mice subjected to dietary restriction. Tracer infusions were performed on day 6 after inoculation. Box plots depict median values, interquartile ranges, and ranges. Uninfected controls with or without dietary restriction were compared by the Mann–Whitney test; uninfected controls with dietary restriction were compared to infected mice by the Wilcoxon matched pairs signed rank test. All units in µmol × kg⁻¹ × hour⁻¹. *P < .05, **P < .01, and ***P < .001. Abbreviations: NOS, nitric oxide synthase; NS, not significant (P > .05).