The Microbiological Assay of Folic Acid

Abstract

Over the past few years, methods of assay of foUc acid and related compounds have been put forward by various woi-kers. Until recently, however, when the structure of fotic acid was determined, juid its synthesis accomplished (Angier (( al., 194;)) there was no absolute standard, and workera were using their own particular preparations as .standards. Snell and Petcr.oon (1940) published a method of asBuy, using L. casfi e as test organism, and, as standariJ, a faetor proparoil from ?o!iibilized liver. Their basal mediuin contained optimum amounts of all the known rr?4uiremeiits of the organism-acid hydrolysed casein, sodium acetate, glucose, tryptophnne. cystinc, riboflavin, niacin, calcium pantothenate, and mineral salts. Rtsults werr calculated from titration of the acid produced after incubation at 37° (,'. for two days.Mitchell and Snell (1941) then iisod nn assay method with cryatalline Vitamin Be (a Parkc, Davis product) ns standard, the basa! medium being similar to ihat of Snell and Peterson. Imt with the addition of more B vitamins. Mitchell, Snell and Williame (1941) later modified the basal medium previously used by the addition nf purincs, and measured the response turhidimetrically.In tlie same year, Williama (1941) published assay methods for some of the B vitamins, including folie acid. In his method for folic acid, lie used hia own preparatirtn of folic acid aa a standard. The basal medium contained liydrolysed casein, cystme, tryptophiine, sodium acetate, glucose, purines, mineral salts, and six vitnniins of the B complex-pneurin, riboflavin, pyridoxine, niacin, calcium pnntothciuite, and hlotin. Response was measured turbidimetrically after sixteen hours' incubation at 3(1" C. The same autliov (Williams, 1942) then introduced another assay method, this time using L. caKci e with crystalline vitamin Be as standard and, later, Burkholder et al. (1945) used essentially this method, with the addition to the basal medium of asparagine, glutamine, and p-aminnbenzoie acid, the response being measured by titTation of the acid produced after thre? days' incubation at 37° C.Landy and Dicken (1942) published a general method of assay for six B vitamins, including folic acid, using L. casei t and a folic acid preparation supplied by R. .J. Williams. The basal medium included asparagine, and is given in detail later. After three day' incubation at 37-5° C, the aeid produced was determined by titration. LuPkey ct al. (1944) found that the niethud of Mitchell and Snell {loc. cit.) was unreliable when other than liver preparations were being assayed. They studied the effect of the various vitamins, purines, and salts on the organism, and fubsequently modified the basal medium to give more consistent result.^. This medium is sliown 'n\ Table 1.Hesulta were eateulflted from the response, measured turbidimetrically. The organiam nsed wn!: S. Uicti.t R, and the standard wns a serii-s of duplicate tubes containing 0-300jug. of solubilized liver factor,