THE DIRECT DETERMINATION OF VALINE AND LEUCINE IN FRESH ANIMAL TISSUES*

Transcription

1 THE DIRECT DETERMINATION OF VALINE AND LEUCINE IN FRESH ANIMAL TISSUES* BY B. S. SCHWEIGERT, J. M. McINTIRE, C. A. ELVEHJEM, AND F. M. STRONG (From the Departmerit of Biochemistry, College of Agriculture, University of Wisconsin, Madison) (Received for publication, May 4, 944) Very limited information is available at the present time on the quantit,ative amounts of the essential aliphatic amino acids in various dietary constituents. Since the reports of Rose et al. (, ) on the qualitative requirements for eight amino acids, there has been much interest in the reinvestigation of the amino acid composition of proteins. Chemical methods have not usually been satisfactory for determining the amino acid content of foods directly, since the fat and carbohydrate present interfere with the chemical methods employed. The use of Lactobacillus arabinosus in our laboratory as a test organism for vitamin analyses prompted us to investigate the adaptability of this organism to the amino acid analysis of foods. Methods of hydrolysis of animal tissues have been studied and it has been found that the leucine and valine content of the hydrolysates can be determined directly. EXPERIMENTAL The composition of the basal medium and the amounts of each constituent per tube are shown in Table I. Glucose, sodium acetate, and the amino acids are weighed out separately for each series of determinations. The other ingredients of the medium are kept as stock solutions preserved under toluene, in a refrigerator, when not in use. The following solutions are prepared as suggested by Snell and Wright (3): adenine, guanine, and uracil solution, riboflavin solution, and inorganic salts, Solutions A and B. The biotin (free acid) solution is adjusted with distilled water to a concentration of 0. y per cc. The thiamine, calcium pantothenate, and pyridoxine solution is prepared by dissolving the vitamins in distilled water and diluting to a concentration of 00 y of each per cc. of solution. Nicotinic acid and p-aminobenzoic acid solutions are prepared in a similar manner. Stock solutions of leucine and valine standards are prepared separately at a concentration of mg. per cc. in distilled water. The procedure for preparing the basal medium for leucine or valine assay Downloaded from by guest on January 3, 09 + Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the National Live Stock and Meat Board. 83

2 84 DETERMINATION OF VALINE AND LEUCINE is as follows: Approximately 00 cc. of distilled water are added to the correct amounts of the amino acids, the mixture is warmed, and a small amount of N hydrochloric acid is added to effect solution. After this solution is cooled, appropriate amounts of each of the other constituents are added. After neutralization (bromothymol blue indicator) the entire medium is adjusted to volume, for example to 500 cc. for 00 tubes, so that 5 cc. of medium are added to each assay tube. For the construction of the standard curve 0, 0.5,.0,.5,.0,.5, 3.0, 4.0, and 5.0 cc. of amino acid standard solution are used per tube. Triplicate determinations are TABLE Basal Medium for Saline and Leucine Assay with Lactobacillus arabincsus Constituent Glucose... Sodium acetate.... Thiamine chlsride... Ca pantothenate.... Pyridoxine.... Riboflavin.... Nicotinic acid... p-aminobensoic acid... Biotin... Adenine, guanine, uracil..... Inorganic salts.... Amount per tube P Y See text I constituent l(f)-glutamic acid... I-Asparagine.... I(+)-Lysine monohydrochloride... dl-threonine... dl-valine*... dl-isoleucine... dl-a-alanine... I(-)-Cystine.... l(-)-leucine*.... dl-methionine.... dl-phenylalanine.... Z(+)-Arginine monohydrochloride... I(+)-Histidine monohydrochloride... E(-)-Tyrosine... I(-)-Tryptophane... The amino acids were purchased from Merck and Company, Inc. * Appropriate amino acid omitted from the basal medium. - Amount per tube _- - w. 4 4 I Downloaded from by guest on January 3, 09 made at the six lower levels and duplicate determinations at the three higher levels of amino acid. Assay tubes for these standards are prepared each time from the stock solutions described above, these being prepared at a concentration of 5 y per cc. for l-leucine and 50 y per cc. for dl-valine. Duplicate tubes at three levels of hydrolysate concentration are used routinely. The volume of medium in each tube is adjusted to 0 cc. by the addition of distilled water. The tubes are plugged with cotton, autoclaved for 0 to 5 minutes at 5 pounds pressure, and cooled.

3 SCHWEIGERT, MCINTIRE, ELVEHJEM, AND STRONG 85 To prepare the inoculum, a transfer of Lactobadlus ambinosus is made from the stock culture (grown on yeast-dextrose-agar) to a tube of previously prepared medium. After incubation for 8 to 36 hoursat 37, the cells are centrifuged down and the liquid is decanted. The cells are suspended in approximately 0 cc. of isotonic saline solution, and the assay tubes are inoculated aseptically with a drop of the saline suspension. The tubes are incubated at 37 for 7 hours and the growth response is measured by titrating the entire contents of each tube with 0. N sodium hydroxide, bromothymol blue being employed as the indicator. Typical standard curves for leucine and valine are shown in Figs. and. The animal tissues used for amino acid analysis were prepared by thorough grinding and mixing to assure homogeneity. The samples were 0 40 M&MS di-valine MICROGRAMS I- LEUUNE FIG. FIG. FIG.. Typical standard curve obtained for dl-valine. FIG.. Typical standard curve obtained for Z-leucine. then stored in dark colored bottles in a cold room at -4. The percentage of nitrogen in the samples was determined in duplicate by the Kjeldahl method and the protein content of the tissues was calculated by multiplying by 6.5. A detailed study of hydrolysis methods was carried out to establish a suitable procedure to give maximum liberation of the amino acids. The effect of acid concentration and length of time of hydrolysis was investigated. In each case a gm. sample of fresh tissue was hydrolyzed with 5 cc. of reagent. After hydrolysis the samples were adjusted to a known volume, filtered, and a suitable aliquot was neutralized and diluted to 00 cc. preparatory to amino acid analysis. Treatment with hydrochloric acid resulted in more rapid hydrolysis than with sulfuric acid at the same normality, a result which is in agreement Downloaded from by guest on January 3, 09

4 86 DETERMINATION OF VALINE AND LEUCINE with the work of Vickery (4) on purified proteins. The effect of time of hydrolysis on the amounts of leucine and valine liberated is shown in Fig. 3. The following hydrolysis procedures were found to give satisfactory results: autoclaving at 5 pounds pressure per sq. in. for 5 to 0 hours with N hydrochloric acid, refluxing for 4 hours with or 4 N hydrochloric TIME OF HYDROLYSIS CHOURSI FIG. 3. Effect of time of hydrolysis of a sample of fresh veal tissue auto&wed with N HCl at 5 pounds pressure per sq. in. Curve, the amount of leucine liberated, expressed as the per cent of leucine in the fresh tissue; Curve, the amount of valine liberated, expressed as the per cent of valine in the fresh tissue. TABLE Leucine and Valine Content of Fresh Muscle and Organ Tissue and Muscle and Organ Tissue Protein All values are given in per cent. Tissue Pork... Veal.... Lamb.... Beef.... liver.... kidney.... heart... brain... ( tongue... No. of samples II Protein Valine in fresh tissue Valine in protein Leucine in fresh tissue Leucine in protein Downloaded from by guest on January 3, 09 acid, and refluxing for 4 hours with 5 N sulfuric acid. Autoclaving for 5 hours, with N hydrochloric acid was used for all subsequent assays. Shorter periods of refluxing were not investigated. Recoveries of added leucine and valine were employed to check the stability of these amino acids during the hydrolysis procedures.

5 SCHWEIGERT, MCINTIRE, ELVEHJEM, AND STRONG 87 The percentage of Z-leucine and I-valine in several samples of pork, veal, lamb, and beef tissues has been determined. The results in each case are calculated on the basis of 00 per cent activity for the I isomer and 0 per cent for the d isomer; i.e., 50 per cent for the dl mixture. These results are shown in Table II. DISCUSSION The constituents of the basal medium, except for the amino acids, are similar to those used by Snell and Wright (3) save for an increase in the glucose and sodium acetate content (Krehl et al. (5)). Adenine, guanine, uracil, and the inorganic salts are added in the concentrations suggested by Snell and Wright, but the vitamin levels have been increased in some cases. The amino acid mixture is similar to that reported by Shankman (6). No attempt was made to establish the exact levels of all of the amino acids which are necessary to support maximum growth. It will be noted that slight differences occur in the amino acid composition of the basal media used by various workers. Variations in the purity of the amino acids used, or differences in strains of Lactobacillus arabinosus, may account for differences in the amino acid requirements reported for this organism. The maximum growth obtained with Lactobacillus arabinosus on the synthetic medium used in this work is similar to that obtained on a casein hydrolysate when tryptophane and cystine are added. The curves obtained for leucine and valine are similar to those reported by Kuiken cd al. (7). Similar microbiological procedures have been reported by Shankman (6), Hegsted (8), and McMahan and Snell (9). Serine and proline were added to the basal medium and no increase in titrations was observed. These amino acids were accordingly omitted from the basal mediu m. Excellent checks were obtained at different levels of sample regardless of the portion of the standard curve represented by the titrations obtained. A few represent,ative assays are shown in Table III. Duplicate analyses usually checked within 5 per cent and all values were checked within 0 per cent. Recoveries of valine and leucine were carried out with the earlier experimental work on hydrolysis and occasionally with later assays. Twenty determinations of the recovery of valine averaged 0 per cent; range, 87 to per cent and twelve for the recovery of leucine averaged 03 per cent; range 94 to 8 per cent. In preliminary work a dl-leucine standard was used. Comparisons of different samples of dl-leucine showed that a variation in activity was obtained. A sample of I-leucine and three different dl-leucine standards were compared; the dl isomers were found to have from 33 to 4 per cent activity rather than the theoretical 50 per cent activity. Since three samples of dl-leucine exhibited less than 50 per cent activity as compared Downloaded from by guest on January 3, 09

6 88 DETERMINATION OF VALINE AND LEUCINE to the isomer, each sample was tested for moisture by drying it over phosphorus pentoxide for 6 hours at loo, and 0. mm. pressure. No detectable loss in weight occurred. The isomer and a new dl-leucine sample gave identical standard curves; therefore, no further attempt was made to find out why the older CL! isomers were less than 50 per cent as active as the isomer. It is very likely that they were contaminated with other amino acids, as has been observed by Hegsted and Wardwell (0). A dl-valine standard was used throughout the experimental work and three dl-valine standards were found to possess the same activity for Sample Beef round Average.. Beef liver Average.. Hydrolysate per tube TABLE III Detailed Analysis of Animal Tissues T Ti$;t: N&I) d. fd Z-V&X Xculated I-valine per tube I Valine in SWIlple 7 per cent ml ml I-Leucine :aculated I-h&e per tube Leucine in sample 7 per cent Downloaded from by guest on January 3, 09 Lactobacillus arabinosus. A sample of I-valineland the dl standard were compared and the dl isomer was found to have 50 per cent activity; therefore, all values for I-valine were calculated on the basis of 50 per cent activity for the dl mixture. These observations suggest that dl isomers, when used as standards, should be checked against the pure isomer whenever possible in experimental work of this type. The results in Fig. 3 show that very rapid increases in the amounts of leucine and valme liberated occur during the first hours of hydrolysis under the conditions used. Slight additional increases were observed in Kindly supplied by Merck and Company, Inc.

7 SCHWJXGERT, MCINTIRE, ELVEHJEM, AND STRONG 89 the period from hours to 5 hours, but no further change occurred in 0 hours of autoclaving. Similar hydrolysis studies have been reported with purified protein hydrolysates by Hess and Sullivan (ll), Vickery (4), and McMahan and Snell (9). Kuiken et al. (7) and Hegsted (8) refluxed protein samples with 5 N sulfuric acid and 6 N hydrochloric acid, respectively, for 4 hours. No mention was made as to criteria for completeness of hydrolysis, but these procedures should give satisfactory results. The adaptability of microbiological methods to the determination of amino acids in fresh tissues greatly simplifies amino acid determinations. Furthermore, the results are obtained on the foods directly, and the amounts of leucine and valine in the crude material need not be calculated from amino acid values obtained on isolated proteins as has customarily been done when chemical methods of analysis are employed. The possibility that some amino acids are lost when the water-soluble nitrogenous constituents are discarded is also eliminated. The water-soluble fraction obtained in the preparation of meat protein has been estimated by Beach et al. () to contain from 8 to 4 per cent of the total nitrogen. Preliminary results indicate that satisfactory results are obtained when leucine and valine are determined directly on cereal and legume hydrolysates. Hydrolysis of the crude material may not be applicable to the satisfactory determination of some of the other amino acids. The necessity of studies of the conditions of hydrolysis used in developing adequate procedures for the determination of amino acids in crude materials cannot be overemphasized. The values obtained for valme are somewhat higher than those reported by Block and Bolling (3), who employed chemical methods of determination. They report 0.7 per cent valine in fresh meat (calculated from protein analysis) and 3.4 per cent valine in the muscle.protein, whereas we found an average of 0.93 per cent valine in fresh beef, veal, lamb, and pork muscle and 5. per cent in the muscle protein. The amount of leucine reported by Block and Bolling was.4 per cent in fresh meat and. per cent in the muscle protein. In this case our results were lower, since an average of.4 per cent leucine was found in the muscle tissue and 7.6 per cent in the muscle protein. It can readily be seen (Table II) that the percentage of valine and leucine in the fresh tissues varies with the protein content, but the amino acid composition of the protein is relatively constant. This has also been shown to be true for several other amino acids by Beach et al. (). Purified casein was also investigated and Table IV presents a comparison of the results together with values from the literature. Since more extensive information is available for casein than for most other proteins, a critical comparison can be made of the results obtained by several workers. Downloaded from by guest on January 3, 09

8 90 DETERMINATION OF VALINE AND LEUCINE It is recognized that the analyses were made on different samples of casein; however, the wide variations emphasize the importance of proper standardization of hydrolysis procedures and methods of amino acid analysis. TABLE Leucine and Valine Content of Casein IV Amino acid Method of analysis Results Reference Leucine I Valine I I I Chemical Microbiological Chemical Microbiological I I SUMMARY per cmt Foreman (4) Van Slyke (5) Abderhalden (6) Block and Boiling (7) Present work Kuiken et al. (7) Hegsted (8) Foreman (4) Block and Bolling (7) Present work Kuiken et al. (7) McMahan and Snell (9) Hegsted (8) Satisfactory standard curves are obtained for leucine and valine on a synthetic amino acid medium when Lactobadlus arabinosus is used as the test organism. Results of hydrolysis studies show that fresh muscle tissues can be hydrolyzed directly for leucine and valine analysis without preliminary removal of fat, moisture, and water-soluble constituents. Satisfactory hydrolysis of animal tissues was obtained by autoclaving with N HCl for 5 to 0 hours. Fresh muscle tissues contain an average of 0.93 per cent valine and.4 per cent leucine. Muscle and organ tissue proteins contain an average of 5.3 per cent valine and 7.7 per cent leucine. Downloaded from by guest on January 3, 09 BIBLIOGRAPHY. Rose, W. C., Haines, W. J., and Johnson, J. E., J. Biol. Chem., 46, 683 (94).. Rose, W. C., Haines, W. J., Johnson, J. E., and Warner, D. T., J. BioZ. Chem., 48, 457 (943). 3. Snell, E. E., and Wright, L. D., J. Biol. Chem., 39, 675 (94). 4. Vickery, H. B., J. Biol. Chem., 53, 495 (9). 5. Krehl, W. A., Strong, F. M., and Elvehjem, C. A., Znd. and Eng. Chem., Anal. Ed.,, 346 (943). 6. Shankman, S., J. BioE. Chem., 60, 305 (943).

9 tx!hweigfxtt, MCINTIRE, ELVEHJEM, AND STRONG 9 7. Kuiken, K. A., Norman, W. H., Lyman, C. M., Hale, F., and Blotter, L., J. Biol. Chem., 6, 65 (943). 8. Hegsted, D. M., J. Biol. Chem., 6, 93 (944). 9. McMahan, J. R., and Snell, E. E., J. Biol. Chem., 6, 83 (944). 0. Hegsted, D. M., and Wardwell, E. D., J. Biol. Chem., 63, 67 (944).. Hess, W. C., and Sullivan, M. X., Arch. Biochem., 3, 53 (943).. Beach, E. F., Munks, B., and Robinson, A., J. Biol. Chem., 43, 43 (943). 3. Block, R. J., and Bolling, D., Arch. Biochem., 3, 7 (943). 4. Foreman, F. W., Biochem..I., 3, 378 (99). 5. Van Slyke, D. D., Ber. them. Ges., 43, 370 (90). 6. Abderhalden, E.,. physiol. Chem., 44, 3 (906). 7. Block, R. J., and Bolling, D., The determination of the amino acids, Minneapolis, revised edition (940). Downloaded from by guest on January 3, 09

10 THE DIRECT DETERMINATION OF VALINE AND LEUCINE IN FRESH ANIMAL TISSUES B. S. Schweigert, J. M. McIntire, C. A. Elvehjem and F. M. Strong J. Biol. Chem. 944, 55:83-9. 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- Downloaded from by guest on January 3, 09