Harry P. Broquist

Prevention of Chemotherapeutic Effects of 4-Amino-N10-Methyl-Pteroyl-glutamic Acid on Mouse Leukemia by Citrovorum Factor.

Summary The effect of 4-amino-N10-methyl-PGA in prolonging the survival time of mice with transplanted leukemia AK4 can be blocked almost completely by prior administration of 1/3 to 2/3 as much by dry weight of a preparation of citrovorum factor. Thus, citrovorum factor is at least 12 to 24 times as active as pteroylglutamic acid in preventing the antileukemic effect of 4-amino-N10-methyl-PGA.

Pteridines and the Nutrition of the Protozoon Crithidia fasciculata.

Summary The Trypanosomid flagellate Crithidia fasciculata was found to have a growth requirement for certain 6-substituted pteridines. The most active compound studied was 2-amino-4-hydroxy-6 (1′, 2′-dihydroxy-propyl) pteridine (“biopterin”) which was isolated from human urine. A requirement for both pteroylglutamic acid and an appropriate pteridine was demonstrated for the organism.

Evidence for occurrence of 10-formyltetrahydrofolic acid in human urine.

Summary Microbiological studies with Leuconostoc citrovorum, ATCC 8081 (Pediococcus cerevisiae) showed that a heat labile form of citrovorum factor (HLCF) occurs in the urine of normal adults after an oral dose of folic and ascorbic acids. Anhydroleucovorin (AHL) resembled HLCF in that AHL was active for growth of L. citrovorum (13% as active as leucovorin) when acid solutions were assayed aseptically, but neutral solutions of AHL were low in microbiological activity unless ascorbic acid was present. HLCF was purified by large scale paper chromatography which permitted a study of its chemical properties. The chemical relationships between 5CHOTHFA, AHL, and 10-CHOTHFA were discussed with regard to the chemical properties that were demonstrated for HLCF from which it was concluded that HLCF is probably 10CHOTHFA.

Evidence for the involvement of folic acid in histidine synthesis in microorganisms.

When Torula cremoris was cultured in a simple medium in which the nitrogen source was principally ammonium sulfate, the organism was readily inhibited by aminopterin, a folic acid antagonist. The growth inhibition by aminopterin was overcome in a noncompetitive fashion by the stepwise addition of methionine, adenine, and histidine. Histidine was ineffective in counteracting aminopterin toxicity in the absence of adenine. When Lactobacillus arabinosus was cultured in a synthetic medium containing ample purine bases but deficient in histidine, aminopterin was found to be more toxic under these conditions than in a medium to which histidine was added. It was concluded from the above experiments that aminopterin blocks the synthesis of histidine in these microorganisms, and implies that folic acid coenzyme is concerned in the biosynthesis of histidine.

Certain relationships between pteroylglutamic acid, citrovorum factor, and cortisone.

Summary 1. Cortisone was found to replace pteroylglutamic acid and citrovorum factor for growth of Streptococcus faecalis and Leuconostoc citrovorum, respectively. Cortisone reversed the growth inhibition of Le. citrovorum produced by toxic amounts of Aminopterin. 2. Cortisone in vitro stimulated the oxidation of tyrosine by liver homogenates from rats fed a purified diet containing 1%percnt; succinylsulfathiazole with or without PGA.

Some Observations on Growth Factors Required by Leuconostoc citrovorum.

Summary 1. Leaconostoc citrovorum was found to respond to a growth-promoting factor in the concentrated liver extract, but this organism did not respond to vitamin B12. The alkali-stable nature of the “citrovorum factor” further contrasts it with vitamin B12. 2. Two fractions were separated from liver extract by paper strip chromatography; one of these fractions was presumably thymidine and promoted growth of Lactobacillus leichmannii and Leuconostoc citrovorum. The other fraction was inactive for L. leichmannii but active for L. citrovorum. 3. Although thymidine or high levels of pteroylglutamic acid (PGA) when tested singly were only partially effective in promoting growth of L. citrovorum, the simultaneous addition of thymidine plus PGA produced marked growth of the organism. This finding suggests a functional relationship between thymidine, PGA and the “citrovorum factor.”

Further observations on the microbiological assay for vitamin B12 using Lactobacillus leichmannii.

Summary 1. A comparison was made of vit. B12, B12b, B12c and “ammino-cobalamin” in the microbiological assay with Lactobacillus leichmannii using aseptic or non-aseptic assay technic. The growth response of L. leichmannii was significantly less when vit. B12 was added aseptically to the medium as compared to when vit. B12 was autoclaved in the medium. This effect was not noted for vit. B12b, B12c and “ammino-cobalamin.” 2. By means of paper strip chromatography it was demonstrated that when vit. B12 is autoclaved with thiomalic acid, vit. B12 is transformed into a new compound which is distinct from vit. B12b. The vit. B12-thiomalic acid reaction product is more potent microbiologically than vit. B12 when the compounds are compared by aseptic assay technic. 3. The significance of these results in the interpretation of the microbiological assay of the vit. B12 content of natural materials when vit. B12

CHAPTER 24 – Biogenesis and Metabolism of Folic Acid and Vitamin B12

Publisher Summary This chapter explores the biogenesis and metabolism of folic acid and vitamin B12. The biosynthesis of folic acid by microorganisms the aromatic ring of the p-aminobenzoic acid group is formed from carbohydrate via shikimic acid. Glutamic acid is possibly added after p-aminobenzoic acid has combined with the pteridine group of the molecule. The close biogenetic relationship that appears to exist between folic acid and riboflavin is further borne out by a recent study of Clapper and Meade who found that when the amount of folic acid required for growth of a strain of Streptococcus faecalis was limiting, the amount of riboflavin synthesized by the organism was depressed. This relationship also held in nonproliferating cells. Folic acid may be degraded to a pteridine that can be utilized for riboflavin synthesis. It may be possible that the folic acid component of the various coenzymes involved in single-carbon transfer reactions occurs in combination with additional amino acids attached by peptide linkage to the glutamic acid group.

Biological activity of a derivative of the citrovorum factor.

Summary Exposure of citrovorum factor (leucovorin) solutions to pH 2 for 24 hours at 25 °C resulted in inactivation of L. citrovorum activity but produced no alteration in the activity for S. faecalis. The acid-treated leucovorin was about one-tenth as active as leucovorin in reversing Aminopterin toxicity in mice. When administered to patients with pernicious anemia in relapse the acid-treated leucovorin was less active than leucovorin.

Effect of Certain Carbohydrates on Nutritional Requirements of Lactobacillus pentosus

Summary 1. L. pentosus, Strain 124-2, will grow on a synthetic medium containing L-arabinose as the carbohydrate source provided a small amount of glucose is added. 2. The supplement of glucose can be replaced by a liver extract. The active principle in the liver extract could not be separated by ion exchange on synthetic resins, charcoal adsorption or paper chromatography from the sugar present in the liver extract. The total activity in the liver extract could be accounted for as the sugar content. Glucosazone was isolated from an active fraction of liver extract.