Global metabolic responses of mice to Trypanosoma brucei brucei infection.

Human African trypanosomiasis (HAT) is transmitted by tsetse flies and, if untreated, is fatal. Treatment depends on infection stage, and early diagnosis is crucial for effective disease management. The systemic host biochemical changes induced by HAT that enable biomarker discovery or relate to therapeutic outcome are largely unknown. We have characterized the multivariate temporal responses of mice to Trypanosoma brucei brucei infection, using (1)H nuclear magnetic resonance (NMR) spectroscopic metabolic phenotyping of urine and plasma. Marked alterations in plasma metabolic profiles were detected already 1 day postinfection. Elevated plasma concentrations of lactate, branched chain amino acids, and acetylglycoprotein fragments were noted. T. brucei brucei-infected mice also had an imbalance of plasma alanine and valine, consistent with differential gluconeogenesis (parasite)-ketogenesis (host) pathway counterflux, involving stimulated host glycolysis, ketogenesis, and enhanced lipid oxidation in the host. Histopathologic evidence of T. brucei brucei-induced extramedullary hepatic hemopoiesis, renal interstitial nephritis, and a provoked inflammatory response was also noted. Metabolic disturbance of gut microbiotal activity was associated with infection, as indicated by changes in the urinary concentrations of the microbial co-metabolites, including hippurate. Concluding, parasite infection results in multiple systemic biochemical effects in the host and disturbance of the symbiotic gut microbial metabolic interactions. Investigation of these transgenomic metabolic alterations may underpin the development of new diagnostic criteria and metrics of therapeutic efficacy.

Attempt to correlate urine arsenic excretion with clinical course during melarsoprol therapy of patients with Rhodesian trypanosomiasis.

This study enrolled 28 CNS-involved patients with Trypanosoma brucei rhodesiense at the Kenya Trypanosomiasis Research Institute (Alupe, Kenya) to examine treatment efficacy and toxicity of melarsoprol in relation to renal excretion/dose relationships. This study complied with World Health Organization treatment recommendations, initially treating with suramin followed by three courses of melarsoprol. Traced study patients had a relapse rate of 4.1%. The toxicity and crude death rate was 7.1%. Total urine arsenic output was measured between 24 and 48 hr after the last dose for each course. The range of means of total urine arsenic output between the three treatment courses was 356-511 micrograms. There was no correlation comparing melarsoprol dose, estimated creatine clearance, or urine arsenic output. Urinary pharmacokinetic parameters are not predictive of toxicity or therapeutic efficacy.

Changes in albumin levels in blood and urine of Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Serum albumin and glucose concentrations and urinary excretion of alpha-keto acids and proteins were determined in samples obtained throughout a chronic Trypanosoma brucei gambiense infection in Microtus montanus. An increase in urinary excretion of alpha-keto acids and proteins during the terminal stage of disease was accompanied by a decrease in serum glucose concentration. This terminal hypoglycemia reflected a depletion of liver glycogen in most animals. In contrast (and the major focus of this study) serum albumin concentration was decreased by the second week of infection and in the final sample obtained was less than 50% of that measured in preinfection samples. Female animals survived approximately 1 wk longer than males and were less susceptible during the acute phase of disease. This relative resistance was most likely due to the fact that female animals were relatively more efficient in limiting parasitemia during the first week of infection. The similarity between humans and voles in terms of protein and alpha-keto acid excretion and changes in serum concentrations of glucose and albumin during trypanosome infection further validate the use of Microtus as an experimental model for trypanosomiasis in humans.

Increased excretion of aromatic amino acid catabolites in animals infected with Trypanosoma brucei evansi.

Aromatic amino acid catabolism by Trypanosoma brucei evansi was investigated in vivo using C3HeB/FeJ mice. The major catabolites detected by gas chromatography in the urines of infected animals were phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, and indole-3-pyruvic acid. Identity of each compound was confirmed by gas chromatography-mass spectrometry. Concentrations of catabolites in urine of infected mice were correlated with parasitemia and returned to normal following suramin treatment. Other aromatic amino acid metabolites, including indole-3-acetic acid, indole-3-lactic acid, and 4-hydroxyphenyllactic acid, were detected in urine from infected animals by gas chromatography mass spectrometry, although quantities were too low to be quantified reproducibly. Both phenylpyruvic acid and 4-hydroxyphenylpyruvic acid were also detected in urine of dogs and donkeys experimentally infected in Egypt with a recent field isolate of T. b. evansi. Tryptophan metabolites could not be assayed in dog and urine samples because formalin, which degraded the indole acids, had to be added before the samples could be imported into the U.S. Finally, concentrations of urinary catabolites during infection were correlated with the tyrosine aminotransferase activity in infected mouse sera.

Metabonomic investigation of single and multiple strain Trypanosoma brucei brucei infections.

Although co-infections are common and can have important epidemiologic and evolutionary consequences, studies exploring biochemical effects of multiple-strain infections remain scarce. We studied metabolic responses of NMRI mice to Trypanosoma brucei brucei single (STIB777AE-Green1 or STIB246BA-Red1) and co-infections using a (1)H nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling strategy. All T. b. brucei infections caused an alteration in urinary biochemical composition by day 4 postinfection, characterized by increased concentrations of 2-oxoisocaproate, D-3-hydroxybutyrate, lactate, 4-hydroxyphenylacetate, phenylpyruvate, and 4-hydroxyphenylpyruvate, and decreased levels of hippurate. Although there were no marked differences in metabolic signatures observed in the mouse infected with a single or dual strain of T. b. brucei, there was a slower metabolic response in mice infected with T. b. brucei green strain compared with mice infected with either the red strain or both strains concurrently. Pyruvate, phenylpyruvate, and hippurate were correlated with parasitemia, which might be useful in monitoring responses to therapeutic interventions.

Increased urinary excretion of aromatic amino acid catabolites by Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Microtus montanus infected with Trypanosoma brucei gambiense for 16 and 21 days excreted significantly greater quantities of several aromatic amino acid catabolites when compared to uninfected control animals. Very large quantities of three aromatic alpha-keto acids (alpha-oxocarboxylic acids), phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid, were excreted by infected animals. Increased excretion of indole-3-lactic acid and indole-3-acetic acid was also detected. Gas chromatographic-mass spectral analysis of the trimethylsilyl derivatives of phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid confirms the identity of the aromatic alpha-keto acids elevated during infection. The marked alpha-keto aciduria indicates that a large disturbance exists in aromatic amino acid metabolism in this chronic animal model of African trypanosomiasis. The disturbance may contribute to the pathogenesis of the disease. The increased catabolite concentrations may also prove to be useful diagnostically and prognostically.

Fibrinogen and fibrinogen/fibrin degradation products in the urine of rabbits infected with Trypanosoma (trypanozoon) brucei.

Studies have been carried out on the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei to determine whether fibrinogen or fibrinogen/fibrin degradation products (FDP) could be detected. No fibrinogen was found but during the last two weeks of this 7-week infection low levels of FDP were present in the urine which did not exceed 5 microgram/ml. Rabbit urine was shown to contain a potent proteolytic enzyme capable of breaking down rabbit fibrinogen and both early and late FDP were present in the cleavage products. No deposits of fibrin were detected in the kidney, but casts were present in the urine suggesting renal damage. The most likely explanation of the urinary FDP is that either an increase in the glomerular permeability occurs allowing filtration of plasma FDP or a local fibrinogenolysis in the kidney tubules.

Multiple alpha-keto aciduria in Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Microtus montanus chronically infected with a monomorphic strain of Trypanosoma brucei gambiense excreted in urine greatly elevated quantities of not only the aromatic alpha-keto acids, phenylpyruvic and 4-hydroxyphenylpyruvic acids, but also two aliphatic alpha-keto acids, pyruvic and alpha-ketoglutaric acids. Elevated keto acid excretion began approximately midway through infection and quantities remained elevated until death. Daily keto acid excretion did not correlate with daily parasitemia. Thus, a large metabolic disturbance exists in laboratory animals infected with African trypanosomes. The multiple alpha-keto aciduria potentially contributes to the pathogenesis of chronic African trypanosomiasis.

Cations in body fluids of Trypanosoma brucei in infected rabbits.

Serum copper, magnesium, zinc, calcium and ionized calcium (Ca++) concentrations were compared in 6 rabbits infected with Trypanosoma brucei brucei and 5 uninfected rabbits. There was a significant depletion of Mg and Zn and a significant increase in Cu from about day 10 of infection to the end. There was no change in plasma total calcium or free diffusible calcium. There was a development of kidney damage as shown clinically by proteinuria and urinary loss of magnesium and zinc, and histologically by the observation of hypercellularity in the glomeruli and tubular degeneration. Our findings thus indicate that trypanosomiasis causes kidney damage which may be responsible for the depletion of the cations seen in the study. Some of the clinical manifestations associated with African trypanosomiasis such as convulsions, anaemia, electrocardiographic changes and splenomegaly may therefore be related to these cation changes.

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy.

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 mumol/l and 3-6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at -70 degrees C. Plasma samples can be stored at -20 degrees C for up to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.