Discovery of metabolic alterations in the serum of patients infected with Plasmodium spp. by high-resolution metabolomics.

INTRODUCTION: Despite the advances in diagnosis and treatment, malaria has still not been eradicated. Metabolic interactions between the host and Plasmodium may present novel targets for malaria control, but such interactions are yet to be deciphered. An exploration of metabolic interactions between humans and two Plasmodium species by high-resolution metabolomics may provide fundamental insights that can aid the development of a new strategy for the control of malaria. OBJECTIVES: This study aimed at exploring the metabolic changes in the sera of patients infected with Plasmodium falciparum and Plasmodium vivax. METHODS: Uni- and multivariate metabolomic analyses were performed on the sera of four groups of patients, namely normal control (N, n = 100), P. falciparum-infected patients (PF, n = 21), P. vivax-infected patients (PV, n = 74), and non-malarial pyretic patients (Pyr, n = 25). RESULTS: Univariate and multivariate analyses of N, PF, and PV groups showed differential metabolic phenotypes and subsequent comparisons in pairs revealed significant features. Pathway enrichment test with significant features showed the affected pathways, namely glycolysis/gluconeogenesis for PF and retinol metabolism for PV. The metabolites belonging to the affected pathways included significantly low 2,3-diphosphoglycerate and glyceraldehyde-3-phosphate in the sera of PF. The sera of PV had significantly low levels of retinol but high levels of retinoic acid. CONCLUSION: Our study reveals metabolic alterations induced by Plasmodium spp. in human serum and would serve as a milestone in the development of novel anti-malarial strategies.

Carbohydrate metabolism in erythrocytes of copper deficient rats.

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.

Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency.

BACKGROUND: Recently, several patients with abnormal polyol profiles in body fluids have been reported, but the origins of these polyols are unknown. We hypothesized that they are derived from sugar phosphate intermediates of the pentose phosphate pathway (PPP), and we developed a semiquantitative method for profiling of pentose phosphate pathway intermediates. METHODS: Sugar phosphates in blood spots were simultaneously analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C(18) HPLC column. The tandem mass spectrometer was operated in the multiple-reaction monitoring mode. Enzymatically prepared D-[(13)C(6)]glucose 6-phosphate was used as internal standard. The method was used to study sugar phosphates abnormalities in a patient affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). RESULTS: In control blood spots, dihydroxyacetone phosphate, pentulose 5-phosphates, pentose 5-phosphates, hexose 6-phosphates, and sedoheptulose 7-phosphate were detected. Detection limits ranged from approximately 100 to approximately 500 nmol/L. Glyceraldehyde 3-phosphate and erythrose 4-phosphate were undetectable. Intra- and interassay imprecision (CVs) were 10-17% and 12-21%, respectively. In blood from the TALDO1-deficient patient, sedoheptulose 7-phosphate was increased. CONCLUSIONS: The new method allows investigation of patients in whom a defect in the PPP is suspected. Measurements of sugar phosphate intermediates of the PPP may provide new insights into metabolic defects underlying the accumulating polyols.

Hereditary deficiency of lactate dehydrogenase H-subunit.

We report herein the fifth family of hereditary deficiency of lactate dehydrogenase (LDH) H-subunit with an autosomal recessive inheritance including two cases of complete deficiency. Their LDH activities were low both in the serum and in the red blood cells (RBC). Electrophoretic analysis revealed that the patients with the complete deficiency had only the LDH5 isozyme. The complete deficiency was associated with marked elevation of fructose-1, 6-diphosphate (FDP) and dihydroxyacetonephosphate (DHAP) and a less marked rise in glyceraldehyde-3-phosphate (GA3P) among glycolytic intermediates in the RBC. Furthermore, hemolysis was observed in the present cases, but this finding was not included in the other reports.

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.

Red cell metabolic alterations in postnatal life in term infants: glycolytic intermediates and adenosine triphosphate.

Red cell glycolytic intermediates and adenosinetriphosphate were evaluated in term infants from birth to on year of age and compared to values obtained from normal adults and subjects wit a population of a similar mean cell age. The concentration of glycolytic intermediates, with the exception of phosphoenolypyruvate were elevated at birth when compared to normal subjects, consistent with a young mean red cell population. The mean levels of red cell glucose-6-phosphate, fructose-6-phosphate, and "total triose phosphate" were elevated on days 1 and 4 of life when compared to both red cells from normal adults and subjects with a similar young mean red cell age. Glucose-6-phosphate steadily increased in concentration, peaked at 3 to 4 wk of age, and then progressively decreased in value. Total triose phosphate declined to a mildly elevated concentration by 3 to 4 wk of age. The mean concentrations of 2,3-diphosphoglycerate and adenosine triphosphate were normal on day 1, increased on day 4, and then declined by 3 to 4 wk to normal values, until 5 to 6 months when both increased. The mean phosphoenolpyruvate concentration was decreased on day 1 of age when compared to red cells of a similar mean age, but this decrease was not significant (P greater than 0.05). The mean concentrations of 3-phosphoglycerate increased at 3 to 4 wk of age and remained elevated for cell age at 11 to 12 months but this increase was no statistically significant (P greater than 0.05). 3-Phosphoglycerate levels did not change significantly throughout the first year of life. At one year of age, all red glycolytic intermediates and adenosine triphosphate were elevated when compared to red cells from normal adults, but were comparable to those observed in subjects with a red cell population of a similar mean cell age, consistent with the persistence of a young red cell population throughout the first year of life.