STUDIES ON THE LIBERATION OF COMPOUNDS IN THE FOLIC ACID GROUP*

Transcription

1 STUDIES ON THE LIBERATION OF COMPOUNDS IN THE FOLIC ACID GROUP* BY T. D. LUCKEY, G. M. BRIGGS, JR.,? I. It. MOORE, C. A. ELVEHJEM, AND I!:. B. HART (Prom the Department of Biochemistry, College of Agricultuse, University of Wisconsin, Madison) (Rcccived for publication, August 4, 1945) The apparent folic acid 1 activity of a food material is dependent upon its content of any one, or a combination, of several compounds (vitamin B, (4) or the liver Lactobacillus casci factor (5), the yeast L. casei factor (5), the third L. casei factor (G), SLR factor (7), and thymine (1, 8)) and the treatment the material has undergone previous.to assay. Bound forms of these compounds have been recognized repeatedly since the early work of Hutchings, Bohonos, and Peterson (9), who suggested that the norit eluatc factor (another name for A. cnsei factor) was generally found associated with proteins. After taka-diastase digestion was shown to be a good method for liberating bound folic acid by Cheldelin et al. (lo), we used this procedure to help distinguish folic acid from vitamins B1o and Bll. We had previously used dilute ammonia under pressure to hydrolyze any ester of folic acid which might have been formed during alcohol separation procedures (11). Recently we showed that treatment. with dilute acid or base increased the apparent folic acid content of certain materials (12); Binkley et al. (13) have used a specific enzyme to liberate vitamin B, from its conjugate in yeast and the Arkansas workers have used purified enzyme preparations to determine preformed and potential folic acid (14, 15). Stokes has shown that 0.05 N HCl and 0.05 N NaOH liberate a thymine-like compound from bacterial cells (16). * Published with the approval of the Director of the Wisconsin Agricultural Esperiment Station. Supported in part by grants from the Wisconsin Alumni Research Foundation and Swift and Company. We wish to thank Parke, Davis and Company, Detroit, Michigan, for vitamin B,; 1Ierck and Company, Inc., Rahway, New Jersey, for crystalline vitamins; Miss Anita Ziegenhagen and Mrs. Lillian Alberty for technical assistance. t Present address, Department of Poultry Husbandry, University of Maryland, College Park, Maryland. 1 Folic acid is a term used to designate a substance necessary for the growth of Streptococcus jaeealis R or Lactobacillus casei on certain defined media (1,2). (Streptococcus faecalis R was previously called Streptococcus Zactis R (3), American Type Culture Collection, No ) 395

2 396 LIBERATION OF FOLIC ACID Since the folic acid content of various materials is of particular interest to us in our attempt to evaluate this group of compounds in chick nutrition, we undertook further studies to determine the best method for liberating these active compounds. EXPERIMENTAL The assay methods with Streptococcus fuecalis R and Lactobacillus casei are those reported previously (1) with the exception that the incubation TABLE Folic Acid -Deficient Media for Streptococcus faecalis and Lactobacillus casei I Constituent Glucose... Casein (acid-hydrolyzed)... Dipotassium phosphate.... Sodiumacetate... Speakman s Salts I3 (17).... I-Tryptophane*.... I-Cystine... Asparagine... Adenine sulfate... Guanine hydrochloride.... Xanthine... Uracil... Thiamine hydrochloride.... Riboflavin... Nicotinic acid.... Pyridoxine hydrochloridet... Calcium pantothenate.... Biotin (free acid)... p-aminobenzoic acid.... Water to... s. faecalis L. casei 1 gm. 2 gm (I ( ml. 30 mg. 10 mg < I CL 1 20 Y 20 Y 20 ( 20 < I li 10.I 50 ml. 50 ml. * When I-tryptophaneis unavailable, twice this amount of dl-tryptophane isused. t Autoclaving this large amount of pyridoxine in the media gives adequate amounts of pseudopyridoxine. Since pyridoxal and pyridoxamine are becoming available (Merck and Company, Inc.), we now use 0.2 y of pyridoxamine per ten tubes. Amount per 10 tubes period leas generally extended to 20 hours as a matter of convenience. The composition of t#he media employed is given in Table I. Turbidimetric methods were used for clear samples and ph curves were run in the case of turbid samples. The final activity in all cases is reported as micrograms of vitamin B, (a standard having a potency of SO,OOO) ; this does not imply that all the activity in the sample was due to vitamin B,. Each value re-

3 LUCKEY, BRIGGS, MOORE, ELVEHJEM, AND HART 397 ported is the average of the results of one to ten separate assays, each consisting of four tubes containing graded levels of the sample. Procedures Cellular samples were homogenized in the Waring blendor for 5 minutes before being treated. Taka-diastase Treatment (Adapted from Method of Cheldelin et al. (lo))- 20 mg. of taka-diastase in 1 ml. of 1 per cent sodium acetate buffer were added to 1 gm. of sample in 8 ml. of buffer at ph 4.5 to 4.7 and incubated under toluene for 24 hours at 37. The solution was then neutralized and assayed directly. Treatment with, Laskowski Enzyme Obtained from Chicken Pancreas- This procedure is based on the recommendations of M. Laskowski (private communication). 1 unit2 of enzyme was added to 20 ml. of a solution containing either 100 mg. of a crude material or 20 mg. of a concentrate and incubated at ph 8 (no buffer) for 24 hours at 37 under toluene. The solution was assayed directly. Treatment with Other Enzymes mg. of the enzyme to be studied was added to 3 mg. of liver preparation, No. 214H (12) (equivalent to 5 gm. of starting material), in 25 ml. of 1 per cent buffer solution and incubated under toluene at 37 for 24 hours. The solution was then neutralized, autoclaved 15 minutes at 15 pounds pressure, and assayed. Acid Hydrolysis-100 mg. of a concentrate or 1 gm. of a natural material was autoclaved in 20 ml. of 1 per cent acetate (sodium) buffer (ph 4.0) for 12 hours at 15 pounds pressure, neutralized, and assayed. Alkaline Hydrolysis-100 mg. of a concentrate or 1 gm. of a natural material was autoclaved in 20 ml. of either 2 N base for 30 minutes or 0.1 N potassium hydroxide for 1 hour. The solution was then neutralized and assayed. Autolysis--100 mg. of sample were placed in 20 ml. of water and incubated under toluene for 24 hours at 37. Results A preliminary report (12) on studies with liver Preparation 214H and yeast indicated that treatment with mild acid (ph 3 or 4) or base (2 N) gave the greatest increase in folic acid activity. Taka-diastase hydrolysis (ph 4.5) produced no greater increase in the apparent vitamin B, content than the same treatment without taka-diastase. Preparation 214H was treated with many available enzymes and the 2 1 unit is that amount of enzyme which will produce the equivalent of 0.5 y of vitamin B, (potency 80,000) per hour for 4 hours when incubated in 11 ml. at 37 with 200 mg. of Difco yeast extract (15).

4 398 LIBERATION OF FOLIC ACID results (Table II) indicate that none of them gave a value higher than that obtained after acid hydrolysis. Further study showed t,hat heat (Fig. 1, A) aids grea.tly in the nonenzymatic liberation of compounds in the folic acid group. Temperatures higher than 122 were not tried, since they seemed impractical. The lower values obtained with concentrations of sodium hydroxide above 1 N (Fig. 1, B) may be due to specific destruction by sodium ions or possibly a toxicity to the bacteria. This effect, is not noted with the concentrations of potassium hydroxide used. TABLE Efect of Enzyme Treatment on Apparent Vitamin B, Content of Liver Preparation 214H* NOllC Autoclaving, Clarase Papain Pepsin Mylase P Pancreatin Ficin 5-Nucleotidase Treatment PH Bllffe1 12 hrs. + hyaluronidase- II Acetate Chloride Acetate Carbonate Acetate Phosphate Vitamin streptoco lls faecalis Bc per gm. LWlObacillw casei Y Y * For the procedure see Treatment with other enzymes. t Obtained from horse semen through the coop,cration of Dr. li. A. Lardy. When the non-enzymatic liberation of activity was st.udied with respect to time (Fig. 1, C), it was found that autoclaving at ph 2 soon caused more destruction than liberation, while autoclaving at ph 3 for 1 to 6 hours gave acceptable results and ph 4 for G hours appeared to be t,he best method. Autoclaving the sample in 2 N base for half an hour gave high values; longer periods were not effective. Again it was noted that sodium hydroxide was less desirable than potassium hydroxide Further studies of the effect of time on the liberation of activity by autoclaving a vitamin Blo and Bn concentrate (Fig. 1, D) and Difco yeast extract (Fig. 1, E) indicated that autoclaving at ph 4 for 12 hours gave excellent results. Various other alkaline treatments gave values lower than those indicated. Consequently autoclaving at ph 4 for 12 hours was used routinely on more than 50 liver preparations. It is of interest to note that

5 LUCKEY, BBIGGS, MOOI~E:, ELVEHJEM, AND HART 399 Ye. A C e-----ha NKoH IN NOi4 --.-PHZ Y p. %F D */*\0- -PH4 i-x- -PHS I5 - FIG. 1. Studies on the non-enzymatic liberation of vitamin I& activity from liver preparations and Difco yeast cstract,. A, effect of t,emperature on the response of Streptococms j uecazis to Preparation 214H treated at) ph 3 for 4 hours; B, effect, of III+] on its response after autoclaving 4 hours at 122 ; C, effect, of t,irne on its response after autoclaving at 122 ; D, effect of time on the response of S. fuecalis to Preparation 215 after autoclaving at 122 ; B, effect, of time on the response of S. Juecnlis to Difco yeast, cstract after autoclaving at 122 ; F, effect of concnnt.mfiion on the response measured net.er autoclaving at 122 for 12 hours at pii 4.

6 ~- L.C. s. f : O.OO! TABLE III E$ect of Dijferent Hydrolysis Methods on Apparent Vitamin B, Content of Materials The values are given in micrograms of vitamin 13, per gm. Sample 1. Ham (fresh).. 2. Beef round Chick kidney liver. 5. Pork liver powder. _ 6. Yeast (Fleischmann) 7. (Anheuser- Busch) Yeast extract (Difco). 9. Grass juice powder Milk (skim) Beet Wheat. 13. Orange juice. 14. Preparation 213BS Lactobacillus casei factor... No treatment s. f.t L. c.i I < ,000 40, : < ph 4, 12 hrs., 15 pounds pres- S E s. f O.OOf :0.04 < s. f o : Enzyme* - - O.O! t : : T 0.1 N KOH, 1 hr., 15 pounds * Obtained from M. Laskowski (see the text). 7 S. f. = Streptococcus faecalis R; L. c. = LactobacilEus casei. 1 Preparation 213B was obtained by the procedure used for Preparation 233D (18). S Prenaration 215 was made in the manner described for the Super Filtrol eluate (18). d from by guest on October 8, L. c. 0.0: 0.0: <o.ot N KOH, $ hr., 15 pounds - s. f. _ <O.Ol <o.o: 0.2( <O.OL 20 <2 <0.004 < (0.04 < : L < : < :0.04 I -

7 LUCKEY, BRIGGS, MOORE, ELVEHJEM, AND HART 401 values with Lactobacillus casei were consistently higher than those with Streptococcus fuecalis, although the values for acid-treated liver preparations showed a greater increase above their original value in the case of X. faecalis than in the case of L. casei. In fact more values decreased than increased for L. casei, while 60 per cent of the S. faecalis values increased more than 2 times. Tb concentration of dry matter in the acid hydrolysis mixture was found to be of significance for values obtained with both Streptococcus faecalis and Lactobacillus casei (Fig. 1, F). The optimum concentration for Difco yeast extract appears to be around 10 to 100 mg. per 20 ml. Through the courtesy of M. Laskowski we were able to test one of his enzyme preparations. When the results obtained with this enzyme were compared with results of other treatments, it was found (Table III) that the Laskowski enzyme gave higher values for some materials (see Samples TABLE IV Variation in Assay of Difco Yeast Extract Treatment Vitamin B, content Sl7eplococcus Lactobacillus faecalis R casei No. of assays r Per sm. Y Per sm. None... I- 3 I ph 3, 4 hrs., autoclaved ( 4, 12 ( ( l 10 9 Laskowski enzyme, 24 hrs and 14), while acid treatment gave higher values for others (Samples 6, 15, and 16). Other materials gave no increase with either acid or enzyme (Samples 9 and 10). It is of considerable interest to note that the third Lactobacillus casei factor gave an increase in activity upon acid hydrolysis, This result substantiates the report of Dayet al. (19). The data also indicate that treatment with 0.1 N potassium hydroxide gave poor results in most cases and 2 N potassium hydroxide treatment was not a good general procedure. Autolysis worked well for samples such as fresh liver, but poorly for yeast extract or ham. Combinations of various treatments, such as acid or base preceded or followed with enzyme treatment, gave lower results than a single treatment. Although Difco yeast extract is an excellent source of potential vitamin B, activity, values obtained with it by various treatments (particularly the Lactobacillus casei values) are so variable that its vitamin B, content is difficult to determine (Table IV). This may be due to a delicate balance between liberation and destruction.

8 402 LIBERrlTION OF FOLIC ACID Incubation of a sample of Anheuser-Busch yeast with xanthopterin, according to the method of Wright and Welch (20), gave no higher values than incubation of the yeast without the xanthopterin. The increase observed may be attributed to the dilute acid (ph 4.5) used. Therefore, conclusions drawn from uncontrolled experiments showing the conversion of xanthopterin to folic acid (21, 22) are open to question. Wright e2 al. have recently shown (23) that xanthopterin or large amounts of neutral salts added to rat liver during incubation cause a definite increase in the folic acid content. This effect was attributed to a protective action against the destruction of folic acid after its liberation. DISCUSSION Although preliminary studies indicated t,hat taka-diastase treatment was of little value for liberating compounds in the folic acid group from such materials as yeast, liver fraction I,, or certain preparations made from them, later work indicates that taka-diastase treatment is generally superior to other methods of liberation for samples such as meat, grains, fruit, or vegetables. Evidently samples of yeast or liver require special treatment in order to determine the maximum apparent vitamin 13, content. The fact that different treatments are rcquircd to give maximum values for different materials may indicate that the compounds in the folic acid group are bound in natural materials by different chemical unions. However, since dilute acid, dilute base, 2 N base, or taka-diastase all gave some liberation of folic acid activity, it seems probable that most of the liberation is due to hydrolysis. Since the standard crystalline vitamin 13, was found to undergo some destruction when autoclaved in dilute acid or base for 4 or 12 hours, the methods used here probably determine the optimum balance between liberation and destruction. Thus it is seen that hydrolysis at ph 3 or in 2 N base gave decreasing values after a ccrtnin lime limit. It was also shown that liberat,ion at ph 2 was impractical, probably because more destruction then liberation occurred, and pii 5 or ph G did not give conditions drastic enough for masimum liberat ion of the activity. It was assumed that these methods involve only a liberation of bound forms of compounds in the folic acid group. That this is not entirely true may be seen from the increased activity obtained with crystalline third Lactobacillus casei factor; thus it, is possible that one compound in the group may be changed into nnot,hor more active compound. No one method can be prescribed to attain maximum folic acid v&es in all l,yprs of InatcriiLls. I akn-tliastnsc dig&ion gives maximum

9 LUCKEY, BRIGGS, MOORE, ELVEHJEM, AND HART 403 values for many foodstuffs. The Laskowski enzyme, prepared from chicken pancreas (15), works well on certain materials such as Difco yeast extract. Autoclaving at pei 4 for 12 hours under 15 pounds pressure gives the highest folic acid values for certain liver preparations. BIBLIOGRAPHY 1. Luckey, T. D., Briggs, G. M., Jr., and Elvehjem, C. A., J. Biol. Chem., 162, 157 (1944). 2. Teply, 1,. J., and Elvehjem, C. A., J. Biol. Chem., 167, 303 (1945). 3. Gunsalus, I. C., Niven, C. F., Jr., and Sherman, J. M., J. Bact., 48, 611 (1944). 4. Pfiffner, J. J., Binkley, S. B., Bloom, E. S., Brown, R. A., Bird, 0. D., Emmett, A. D., Hogan, A. G., and O Dell, B. L., Science, 97, 404 (1943). 5. Stokstad, E. L. R., J. Biol. Chem., 149,.573 (1943). 6. Hutchings, B. L., Stokstad, E. L. R., Bohonos, N., and Slobodkin, N. H., Science, 99, 371 (1944). 7. Keresztesy, J. C., Rickes, E. L., and Stokes, J. L., Science, 97, 465 (1943). 8. Stokstad, E. L. R., J. Biol. Chem., 139, 475 (1941). 9. Hutchings, B. L., Bohonos, N., and Peterson, W. H., J. Biol. Chem., 141, 521 (1941). 10. Cheldelin, V. H., Eppright, M. A., Snell, E. E., and Guirard, B. M., Univ. Tezas Pub., No. 4237, 32 (1942). 11. Briggs, G. M., Jr., Luckey, T. D., Elvehjem, C. A., and Hart, E. B., J. Biol. Chem., 148, 163 (1943). 12. Briggs, G. M., Jr., Luckey, T. D., Elvehjem, C. A., and Hart, E. B., J. BioZ. Chem., 166, 687 (1944). 13. Binkley, S. B., Bird, 0. D., Bloom, E. S., Brown, R. A., Calkins, D. G., Campbell, C. J., Emmett, A. D., and Pfiffner, J. J., Science, 100, 36 (1944). 14. Mims, V., Totter, J. R., and Day, P. L., J. BioZ. Chem., 166, 401 (1944). 16. Laskowski, M., Mims, V., and Day, I. I,., J. BioZ. Chem., 167, 731 (1945). 16. Stokes, J. L., J. Bat., 48, 201 (1944). 17. Spcakman, II. B., J. BioZ. Chem., 68, 395 ( l). 18. Briggs, G. M., Jr., Luckcy, T. D., Elvchjcm, C. A., and Hart, E. B., J. Biol. Chem., 158, 303 (1945). 19. Day, I. L., Mims, V., Totter, J. R., Stokstad, E. L. R., Hutchings, B. L., and Sloane, N. H., J. BioZ. Chem., 167, 423 (1945). 20. Wright, L. D., and Welch, A. D., Science, 98, 179 (1943). 21. Wright, L. D., and Welch, A. D., Am. J. Med. SC., 206, 128 (1943). 22. Tot.ter, J. R., Mims, V., and Day, I. L., Science, 199, 223 (1944). 23. Wright, L. D., Skcggs, 1-I. IL., and Welch, A. I)., Arch. Biochem., 6, 15 (1945).

10 STUDIES ON THE LIBERATION OF COMPOUNDS IN THE FOLIC ACID GROUP T. D. Luckey, G. M. Briggs, Jr., P. R. Moore, C. A. Elvehjem and E. B. Hart J. Biol. Chem. 1945, 161: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1