H. Kyle Webster

Application of simultaneous UV-radioactivity high-performance liquid chromatography to the study of intermediary metabolism : I. Purine nucleotides, nucleosides and bases

Procedures are presented for the continuous-flow, simultaneous measurement of concentration and radioactivity of purine metabolites separated by high-performance liquid chromatography (HPLC). A microprocessor-controlled radioactivity flow detector connected in series to a UV-flow detector provides on-line quantitative monitoring of separated components in the post-column effluent stream. Through use of two HPLC separations--reversed-phase and anion-exchange--quantitation of all major purine nucleotides, nucleosides and bases is possible. The procedures provides a rapid, sensitive, and convenient means for the systematic study of purine metabolism.

Simultaneous Measurement of Quinine and Quinidine in Human Plasma, Whole Blood, and Erythrocytes by High-performance Liquid Chromatography with Fluorescence Detection

A high-performance liquid chromatographic method with fluorescence detection is described for the simultaneous measurement of quinine and quinidine in plasma, whole blood, and erythrocytes. The compounds were separated on an Ultrasphere C18 reversed-phase column (25 cm x 4.6 mm inside diameter, 5 μm particle size) using a mobile phase of acetonitrile/ water/triethylamine (11:88:1, vol/vol) at pH 2.5. The method, simple, accurate, and selective, requires only a single-step liquid-liquid extraction and uses the structurally similar alkaloid, cinchonine, as the internal standard. The commercial impurities, dihydroquinine and dihydroquinidine, and unknown metabolites were well resolved from the parent drugs. The assay is precise, with interassay coefficients of variation 7.0% and an accuracy of 7.3% over a concentration range of 0.125 to 4.0 μg/0.25 ml. The extraction recoveries of the two drugs were similar, averaging 82.9% for quinine and 79.3% for quinidine from the three biological fluids. The clinical application of the method for routine drug monitoring and for estimating the pharmacokinetics of quinine and quinidine in man are discussed.

Chemistry of artemisinin: an overview

The endoperoxide sesquiterpene lactone, artemisinin, and its derivatives have become increasingly important as antimalarial drugs with impressive activity against multidrug resistant forms of Plasmodium falciparum. Artemisinin has a novel structure among known antimalarial compounds with a unique 1,2,4-trioxane ring that is essential for activity. This paper gives an overview of the chemistry of artemisinin and comments on future prospects for artemisinin and its derivatives.

Macrophage activation in vivax malaria: fever is associated with increased levels of neopterin and interferon‐gamma

Summary In order to evaluate the relationship between fever and macrophage activation in vivax malaria, serum inierferon‐gamma (IFN‐y) and urinary neopterin concentrations were determined in That adults with Plasmodlum vivax parasitaemia. Magnitude of fever, after controlling for parasite density, was found to be positively correlated with both IFN‐y (r = 0‐47) and neopterin (r = 0‐57). In the 26 febrile patients, neopterin excretion increased further during the first two days of chemotherapy (P=0‐0002). Mean neopterin values for both groups had fallen to within the normal range by the sixth day post‐trealmem. Thus, the fever of vivax malaria was associated with IFN‐y induced macrophage activation, as reflected by neopterin excretion.

Leishmania mexicana: Purine metabolism in promastigotes, axenic amastigotes, and amastigotes derived from vero cells

Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [14C]formate and [14C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [14C]hypoxanthine, [14C]adenine, and [14C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (greater than or equal to 60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.

Pharmacokinetics of an extended‐dose halofantrine regimen in patients with malaria and in healthy volunteers

The pharmacokinetics and tolerance of a 4.5 gm 7‐day halofantrine loading dose regimen were evaluated in 10 Thai patients with malaria and in 10 noninfected volunteers. Halofantrine peak plasma concentrations and bioavailability on the first day of treatment were significantly lower in patients with malaria than in healthy volunteers. Halofantrine elimination half‐life was significantly shorter in patients with malaria than healthy control subjects (9.5 versus 15.8 days). These data show a distinct effect of acute malaria on the absorption and elimination of the drug. In addition, marked intersubject and intrasubject variability in peak and trough halofantrine levels was observed, indicating variable drug absorption. This dosing regimen was effective and well tolerated, with mild transient diarrhea during the first few days of treatment in both groups. To produce consistently effective drug levels, the currently recommended dosing regimens may be suboptimal. Slow halofantrine elimination raises concern for induction of parasite resistance when the drug is used in endemic areas of the world.

Plasmodium coatneyi-infected rhesus monkeys: a primate modelfor human cerebral malaria

Although several animal models for human cerebral malaria have been proposed in the past, none have shown pathological findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied the pathology of brains of Plasmodium coatneyi (primate malaria parasite)-infected rhesus monkeys. Our study demonstrated parasitized erythrocyte (PRBC) sequestration and cytoadherence of knobs on PRBC to endothelial cells in cerebral microvessels of these monkeys. This is similar to the findings seen in human cerebral malaria. Cerebral microvessels with sequestered PRBC were shown by immunohistochemistry to possess CD36, TSP and ICAM-1. These proteins were not evident in cerebral microvessels of uninfected control monkeys. Our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria.

Haemoglobin-E trait and the clinical course of malaria in Thai soldiers

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Presence of autoimmunity to pancreatic antigens in a patient with fibrocalculous pancreatic diabetes

A case of fibrocalculous pancreatic diabetes (FCPD) is reported for which antibody and cellular immune characteristics were determined. The patient, a Thai woman, had serum islet cell antibodies (ICA) that were detected by both immunoperoxidase staining and an indirect enzyme-linked immunosorbent assay (ELISA). Serum anti-human insulin antibodies were negative by a displacement ELISA. Lymphoproliferation assay against pancreatic antigen prepared from a blood group O cadaveric donor was positive. Increased CD8+ lymphocytes were observed using direct immunofluorescence staining and flow cytometry. CD4+ T lymphocytes, B lymphocytes and NK cells were within normal levels. These findings provide evidence for autoimmunity to pancreatic antigens in a patient with fibrocalculous pancreatic diabetes.

Comparison of antibody responses to the circumsporozoite protein repeat region and to intact sporozoites during acute falciparum malaria

Most acute falciparum malaria patients mount an antibody response to the circumsporozoite (CS) protein which contains a dominant B-cell epitope. In order to investigate whether antibodies against other epitopes on the sporozoite surface may be important during a particular phase of infection or convalescence, we longitudinally studied the antibody responses of 13 Thai patients with acute falciparum malaria. Antibody comparisons were made using intact Plasmodium falciparum sporozoites in an indirect fluorescent antibody test and the recombinant peptide, R32tet32, as capture antigen in an enzyme-linked immunosorbent assay. Antibody response curves derived using the 2 methods were similar, and adsorption with R32tet32 greatly (greater than 95%) diminished anti-sporozoite activity in sera. Thus, peptide constructs containing the CS repeat region epitope, (NANP)n, can be used with confidence to assay anti-sporozoite antibodies, independent of both the time of infection and prior malaria history.