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A sensitive enzyme linked immunosorbent assay (ELISA) using horseradish peroxidase as a label enzyme and a simple dip-strip test using this ELISA which enable the monitoring of bacitracin (BC) in chicken plasma are described. The anti-BC antiserum which was raised against the antigenic BC-bovine gamma globulin conjugate in rabbit possessed an excellent specificity for BC, exhibiting no cross-reactivity with other antibiotics used as animal feed additives and veterinary drugs. The detection limit of BC by the ELISA was estimated to be 0.1 μg l−1 (equivalent to 1 ng of BC per 1 ml of chicken plasma). The recoveries of BC from chicken plasma ranged from 97% to 103%. The dip-strip using an anti-BC IgG dotted nitrocellulose membrane was prepared and a simple and rapid test for monitoring BC in chicken plasma was developed. Using this test, 50 ng of BC in 1 ml of plasma could be distinguished from that of control (blank) plasma.

The effect on pyrimethamine (PYR) pharmacokinetics of folic acid (FA) which potentiated the PYR teratogenesis was studied in rats. Gavage administration of PYR 1.6 mg/kgBW/day from day 11 to day 15 of gestation did not cause any malformation, but with concomitant dosing of FA 50 mg/kg/day in feed caused malformations in 75% of fetuses. Neither the plasma concentration-time profile nor the plasma protein binding of the gavaged PYR was affected by the concomitant dosing of FA in the non-pregnant rats. The pregnant rats which received the gavaged PYR with or without FA in feed for 5 days (from day 11 to day 15 of gestation) were killed at 4 or 12 hrs after the last dosing of PYR, and their plasma and fetuses were subjected to determination of PYR. The PYR concentration in the maternal plasma was comparable in both groups. The PYR concentration in fetuses of the PYR alone group was significantly higher than that of the PYR with FA group 4 hr after but not 12 hr after the last dosing. These results indicated that the pharmacokinetics of PYR was not affected by the concomitant dosing of FA, suggesting that FA potentiated the PYR teratogenesis by other mechanisms than pharmacokinetic modifications.

Oral administration of pyrimethamine (PYR) 3.6 mg/kgBW/day to the pregnant rats from day 11 to day 15 of gestation caused malformations in all the fetuses, but concomitant intraperitoneal administration of folic acid (ip FA) 50 mg/kg/day inhibited the incidence of malformations by 25%. On the other hand, the same dose of oral FA (in feed) had an opposite effect on PYR teratogenesis; the incidences of malformed fetuses were 0% in the PYR 1.6 group, but 100% in the PYR with oral FA group. The plasma levels of 5-methyltetrahydrofolic acid (5MF), a principal transfer form of folate, were measured after single oral administration of PYR with or without FA (ip or oral) in non-pregnant female rats. The area under the plasma concentration-time curve (AUC) after the concomitant administration of PYR with ip FA was larger than that after PYR alone. This result indicated that the available 5MF for the peripheral tissues was increased by the concomitant administration of ip FA. The AUC after the concomitant administration of PYR with oral FA was smaller than that after PYR alone. These results suggested that ip FA increased the available 5MF to ameliorate, but oral FA decreased the availability to aggravate the PYR teratogenesis.

The relationship between pyrimethamine (PYR) teratogenesis and the plasma level of 5-methyltetrahydrofolic acid (5MF), known as an active form of folate in plasma, was studied in rats using an HPLC-ECD method for 5MF determination. The rat received, for 5 days, in-feed PYR with or without folic acid (FA), which was previously reported as a potentiator of PYR teratogenesis. PYR alone caused a dose-dependent decrease in the plasma 5MF level after the 5 day dosings. A potentiated decrease in the 5MF level was observed after the concomitant dosing of PYR with FA. The concomitant dosing of PYR 1.6 mg/kgBW/day (subteratogenic dose of PYR alone) with FA 50 mg/kg/day, which was 100% teratogenic dose in our previous report, decreased the 5MF level more than that after the dosing of PYR 3.6 mg/kg/day alone (100% teratogenic dose of PYR alone). The sequential changes of the plasma 5MF level after the single oral dosing of PYR 3.6 mg/kg with or without oral FA 50 mg/kg also revealed a strong potentiative effect of oral FA on the 5MF-level-lowering effect of PYR. These results indicate that a decrease in the plasma 5MF level may be related to PYR teratogenesis.

Pyrimethamine (PYR), an inhibitor of dihydrofolate reductase, has been known to induce megaloblastic anemia in human. The dosing of folic acid (PteGlu) or folinic acid (5-formyltetrahydrofolic acid) is recommended for the treatment of PYR-induced anemia, PYR has been also known to be a potent teratogen1,2,3,4. Therefore, we studied the effects of PteGlu and folinic acid on the embryotoxicity of PYR using rats and mice. The plasma level of 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) was also studied. 5-CH3-H4PteGlu is the principal active folate in plasma and its plasma level reflects folate status in the body.