Experimental production of a nutritional macrocytic anemia in swine.

1. A deficiency of pteroylglutamic acid has been produced in 32 swine fed a purified diet containing casein and supplemented with seven B vitamins, sulfasuxidine and a folic acid antagonist. The casein was fed at two levels, 10 and 26 per cent. Two types of casein were used: a crude preparation possessing significant "extrinsic factor" activity and a purified casein with little activity. 2. The hematologic manifestations observed were (a) severe macrocytic anemia, (b) leukopenia, due to a proportionately greater reduction in polymorphonuclear than in mononuclear cells, (3) slight thrombocytopenia, and (4) hyperplastic bone marrow with an increase in immature nucleated red cells which resemble the megaloblasts seen in the bone marrow of patients with pernicious anemia. 3. The feeding of a 26 per cent rather than a 10 per cent crude casein diet did not prevent but did delay the onset of the blood changes. Anemia developed most rapidly in the animals receiving 10 per cent purified casein. 4. The group receiving 26 per cent casein developed a greater degree of macrocytosis in the same period of time than did the group receiving 10 per cent casein. In all groups the degree of macrocytosis increased as the duration of the anemia increased. 5. The hematologic manifestations were not delayed nor was their development prevented by the intramuscular administration of 15 U.S.P. units of liver extract every 15 days. 6. The blood and bone marrow returned rapidly to normal following the administration of pteroylglutamic acid, pteroyldiglutamic acid, pteroyltriglutamic and pteroylheptaglutamic acid. Thymine and xanthopterin had little or no activity. Tyrosine, adenine and uracil were inactive. 7. Purified liver extracts and crystalline vitamin B12 were found to possess some hemopoietic activity in several animals but the activity was considerably less than that of the pteroylglutamic acid compounds. 8. The urinary excretion of "tyrosyl" (hydroxphenyl compounds) was not abnormal in the pteroylglutamic acid deficient pigs and was not altered by either pteroylglutamic acid or liver extract therapy. 9. The urinary excretion of allantoin and uric acid did not differ significantly from the normal. Immediately following therapy with pteroylglutamic acid, however, in association with the reticulocytosis and lasting for the same period, there was a marked increase in the excretion of allantoin. 10. The results suggest that both pteroylglutamic acid and a factor in liver extract similar to or identical with vitamin B12 are required for normal hemopoiesis in the pig. ACKNOWLEDGEMENTS The crude methylfolic acid antagonist, xanthopterin, and the pteroylglutamic acid compounds, with the exception of pteroylheptaglutamic acid, were kindly furnished by the Lederle Laboratories, Pearl River, New York, through the courtesy of Dr. T. H. Jukes and Dr. S. M. Hardy. Sulfasuxidine was generously furnished by Sharp & Dohme, Inc., Philadelphia, Pa., through the courtesy of Dr. W. A. Feirer. Pteroylheptaglutamic acid and Natola were supplied by Parke, Davis & Company, Detroit, Mich., through the courtesy of Dr. A. E. Sharp and Dr. J. J. Pfiffner. Biotin was obtained from Hoffmann-LaRoche, Inc., Nutley, N. J., through the courtesy of Dr. E. L. Sevringhaus. The vitamins, with the exception of pteroylglutamic acid and biotin and including vitamin B12 were kindly furnished by Merck and Company, Inc., Rahway, N. J., through the courtesy of Dr. A. Gibson and the late Dr. D. F. Robertson. Experimental liver extracts (No. 1124, 1063, 1066 and 1067) were generously furnished by Armour and Company, Chicago, Illinois through the courtesy of Dr. E. E. Hays. We are indebted to Mrs. Darlene Kehl, Mr. George Trappett, and Mr. Ocie Hadley for technical assistance.