The successful degradation and synthesis of the accessory food factor variously known as vitamin Be, L. casei factor, S. lactis R factor, folic acid and, finally, pteroylglutamic acid (Angier et al., 1946) has not only given rise to more exact work on the significance of this vitamin in anaemia of different kinds, hut has also enabled a more precise standardization of the microbiological assay of the factor to be made. Such assay, of course, involves the heat sterilization of the standard test solutions containing folic acid and, as little is known of the thermal stability of this vitamin, this study was undertaken. The first step was to establish the relation between pll and recovery ol" adiletl folic acid at temperatures of 100° and 121° C. (15 Ib. steam pressure), concurrently investigating the effect of various buffer solutions. The second step was to study the kinetics of the thermal destruction demonstrated. EXPERIMENTAL. MATERIALS. Folic acid 3torJ: solution.Synthetic pteroylglutamic acid ("Folvitc" ot Lederle Laljoratories) was used. 10 mg. were dissolved in distilled water by steaming and made up to 2 litres. Later a 50 p.c. water-alcohol mixture was used to dissolve Ihe "Folvite". 1 ml. ot atock solution contained 5jig. of *'Folvite". Buffer Solutions.(a) Sodium Acetate-Bydrochlori^; Acid. AIM sulution of sodium acetate was prepared in distilled water. For most experiments 10 ml. were diluted to about 40 ml. and the pH adjusted to the desired value with 6 N HCl. The volume was then miade up to 50 ml. In preparing larger volumes 50 ml. of acetate after pH adjustment were made up to 250 ml.(b) Sodium Phoaphatc-Cilric Acid. Solutions of 0*1 M citric acid and 0-2 M disodium hydrogen phosphate were prepared. Varying volumes of tho one were made up to 50 ml. with the other to yield the desired pH. Any final adjustment was made with 6 N HCl or 10 p.c. NaOH.(c) PhoNphate-Phthalatc. 10 ml. of a solution 0-5 M with respect to both potassium dihydrogen phosphate and potassium hydrogen phthalate were diluted to about 45 ml., the pH adjusted to the desired value with NnOII solution and the volume made up to 50 ml.(d) Glycine-Sadluvi Hydroxide. A solution of glycine (0-1 M) was prepared in 0-1 N NaCl solution and varying volumes as required were made up to 50 ml. with 0*1 N NaOH,
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,
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