Comparative Bioautography of Folic and Folinic Acids of Erythrocytes and Livers of Normal Ducks and Ducks Infected with Malarial Parasites

Abstract

Since the erythrocytes of ducks infected with P. lophurae contain much more folic and folinic acid than those of uninfected ducks, the Rf values of these growth factors in infected and uninfected red blood cells were compared. Paper chromatography followed by bioautography was used to determine the number of components supporting growth of L. casei and P. cerevisiae. Both normal and infected erythrocytes gave identical bioautographs having two components: (1) with Rf 0.5 active only for L. casei and (2) with Rf 0.7 active both for L. casei and P. cerevisiae. This finding fits the hypothesis that the increased levels of folic and folinic acids result from an altered metabolism of the host red cell rather than from synthesis by the parasites. Livers of infected ducks had less L. casei-active material than those of normal ones, but both showed identical bioautographs. The importance of folic acid and related compounds in the metabolism of malarial parasites is indicated by several facts: (1) the favorable effect of folinic acid on Plasmodium lophurae maintained extracellularly in vitro (Trager, 1958), (2) the favoring effect of para-aminobenzoic acid (Geiman, Anfinsen, McKee, Ormsbee, and Ball, 1946) and folic acid (Glenn and Manwell, 1956) on the development of malarial parasites growing intracellularly in suspensions of intact erythrocytes in vitro, and (3) the powerful antimalarial effect of substances such as pyrimethamine, which seems to act by interfering with the formation of folinic acid (Rollo, 1955; Trager, 1961). Trager (1959) found 20to 50-fold increase in folic acid and 5to 20-fold increase in folinic acid in erythrocytes of 2-month-old ducks infected with P. lophurae. He further found that only one-fifth to one-third of the increased amounts of the two growth factors were within the parasites; the remainder presumably was in the cytoplasm of the infected erythrocytes. It therefore seemed possible that the increased folic and folinic acid were formed Received for publication 10 July 1964. * Supported by Public Health Service Grant 5-T1AI-192. by the host red cells rather than by the parasites themselves. It was of interest to compare the Rf values of these two growth factors from erythrocytes and livers of normal ducks with those from the corresponding tissues of ducks infected with P. lophurae. MATERIALS AND METHODS Two-week-old ducklings were inoculated with P. lophurae in such a way as to give severe and highly synchronous infections (Trager, 1958). On the 5th day of infection blood was taken from infected birds and from corresponding uninfected ones. The red cells were washed, hemolyzed, and incubated 90 min in a diluted buffer with ascorbic acid (Toennies, Bueding, and Phillips, 1956). The hemolysates were deproteinized by autoclaving 1.5 min at 121 C, followed by centrifugation. Extracts from livers of infected and control animals were produced by grinding weighed portions in the buffer-ascorbic acid solutions and were then treated in the same way as hemolysates. The folic and folinic acid contents were determined by microbial assay with L. casei and P. cerevisiae, respectively (Toennies, Frank, and Gallant, 1953). Bioautographic assay Paper chromatography followed by bioautographic visualization of active compounds was used to determine the Rf of the components in liver and red cells which would support the growth of L. casei and P. cerevisiae. The methods used were essentially those of Colon and Moulder (1958) and Sirotnak and co-workers (1963).