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On the determination of available lysine in casein and rapeseed protein concentrates using 2,4,6-trinitrobenzenesulphonic acid (TNBS) as a reagent for free epsilon amino group of lysine
ANALYTICAL BIOCHEMISTRY "]0, 434-439 (1976)
On the Determination of Available Lysine in Casein and Rapeseed Protein Concentrates Using 2,4,6-Trinitrobenzenesulphonic Acid (TNBS) as a Reagent for Free Epsilon Amino Group of Lysine ANDERS E K L U N D
Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, P.O. Box 575, S-751 23 Uppsala, Sweden Received March 25, 1975; accepted September 2, 1975 The determination of available lysine was studied using 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for free epsilon amino group of lysine. By establishing optimal conditions for the hydrolysis of TNP-labelled protein, a modification of a previously described TNBS-method was proposed. The changes reduced the analytical variations substantially from the original method and made it possible to obtain in a simple and rapid way quite reproducible values for the contents of available lysine in casein and rapeseed protein concentrates.
Proteins of vegetable origin constitute a major part of the protein consumed by inhabitants of developing countries. These proteins very often contain rather low amounts of lysine, which is a very important dietary factor. This is one of the reasons why the quantitative determination of lysine has attracted so much interest in the nutritional field. Determinations of lysine contents are also vitally important in the quality control of animal proteins, for example in the dairy and fishery industries. Consequently, simple and rapid assay methods for lysine would be of the greatest value, e.g., to evaluate the nutriment value of food mixtures, in plant-breeding to look for higher levels of lysine in new varieties of protein-rich plants, and in industrial food processing to control the protein quality of the product. Today lysine is commonly analysed in an automatic amino acid analyser, mainly according to the technique reported by Spackman et al. (1). Even if the procedure, due to a high level of automation, is rather convenient, the equipment required for these analyses is rather expensive and may not be accessible in every case, especially not in developing countries. The lysine value obtained is a total value and may include a fraction of lysine which has been changed by different chemical reactions, such as the Maillard reaction (2), to become biologically unavailable. 434 Copyright © 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.
D E T E R M I N A T I O N OF AVAILABLE L Y S I N E
435
In nutritional work the determination of available iysine is often more important than the analysis of total lysine. It is well established that lysine must have a free epsilon amino group to be biologically available. At present methods similar to that of Carpenter et al. (3-5), involving the use of 1-fluoro-2,4-dinitrobenzene (FDNB) as a reagent for determining the free epsilon amino groups of lysine, are generally recommended for the analysis of available lysine (6). However, these methods are comparatively laborious and time consuming for the routine analysis of proteins. A few years ago Kakade and Liener presented a method by which the amount of available lysine was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS) which specifically reacts with primary amino groups (7). The method was designed on a micro level dealing with 1-10 mg samples. Ousterhout and Wood (8) adopted a slightly modified TNBS-method for determination of available lysine in fish meals and used a sample size of 100 mg. When we tried to use the procedure of Kakade and Liener for measuring the contents of available lysine in protein foodstuffs, we obtained satisfactory results with some materials but found it rather difficult to obtain reproducible values with some other materials. A poor reproducibility of the method of Kakade and Liener has also been reported by Hall et al. (9). However, since the method was very rapid and quite simple we tried to increase the accuracy of the method by increasing the sample size as well as by chosing optimal conditions for the hydrolysis of the proteins after coupling to TNBS. In the present paper a slightly modified TNBS-method is described. Comparisons are made between analyses of the contents of available lysine in casein and rapeseed protein concentrates performed by the new procedure, by the method of Kakade and Liener (7) and by the FDNB-method of Rao et al. (10). MATERIALS AND METHODS
The chemicals used were of analytical grade. "Hammarsten" casein was delivered from E. Merck A G, Darmstadt. Rapeseed protein concentrates (Brassica n a p u s L.) were prepared by a procedure described previously (11). Prior to analysis the protein concentrates were defatted by extraction with diethyl ether in a Soxhlet apparatus. TNBS was purchased from Sigma Chemical Co., St. Louis, Missouri, N~-TNP-L-lysine was delivered from Nutritional Biochemical Corp., Cleveland, Ohio. Preliminary experiments were carried out to establish the optimal conditions for determining the amount of available lysine in casein. In order to decrease the sampling error the sample weights were increased in comparison with those recommended by Kakade and Liener (7). The following procedure was adopted: The fat-free sample was finely ground in a cyclone sample mill (Tecator AB, Helsingborg) to pass a B.S. 70-mesh sieve. This means that the particle size of the powder did not exceed 210
436
ANDERS E K L U N D
/xm. To 30 mg of this sample in a 50 ml flat-bottomed Pyrex flask equipped with an extralong neck, 5 ml of 0.48 M NaHCO3, pH 8.5, were added. The flasks were placed in a constant-temperature shaking bath at 40°C for 10 min prior to the addition of 5 ml of 34 mM TNBS-solution. After an incubation period of 2 hr at 40°C, 15 ml of concentrated HC1 were added and the flasks were sealed. With some samples the flasks were evacuated before they were sealed. The hydrolysis was carried out at 110°C for 90 min in an Electrolux drying oven, model no. A 28482, equipped with a fan. In the preliminary experiments some samples were hydrolyzed at various temperatures between 90-120°C for 60 min, and other samples were hydrolyzed at I10°C for different times ranging from 30 to 120 min. After cooling to room temperature, the hydrolyzates were filtered (Munktell's Swedish Filter Paper no. 2OH, Grycksbo Pappersbruk AB, Sweden) and diluted to 200 ml with distilled water. Ten milliliters were transferred to a glass-stoppered test tube and extracted four times with 10 ml of diethyl ether in order to remove TNP-N-terminal amino acids or peptides and picric acid produced during reaction. Residual ether was evaporated by placing the tubes in a hot shaking-bath until no smell of ether could be detected. The optical density of the aqueous solution was measured at 346 nm against a blank prepared in the same way except that the concentrated HCI was added to the protein suspension prior to the addition of the TNBS reagent. The amounts of N'-TNP-lysine were calculated from a standard curve ofN'-TN P-L-lysine subjected to the same procedure as the protein samples. According to Ousterhout and Wood (8) and Hall et al. (9) the standard curve can also be prepared from pure DL-lysine carried through the procedure. For comparison, the contents of available lysine were determined by the TNBS-method of Kakade and Liener (7) as well as by the FDNB-method of Rao et al. (10) modified according to Blom et al. (12) so that 1.00 g of 2,4-dinitrophenol was added just prior to hydrolysis of the FDNB-treated sample. With the former method (7) the hydrolysis was performed by autoclaving the samples in tubes covered with inverted glass vials at 120°C (15-17 psi) for 1 hr. A 10 1 CERTOclav (Gruber & Kaja, Vienna) was used for this purpose. Hydrolyzates (6 M HCI; 110°C for 20 and 70 hr) for the analysis of total lysine content were obtained as previously described (13). Total lysine was determined according to Spackman et al. (1). Nitrogen was analysed by a modified Kjeldahl procedure using a Tecator Digestion System model 40 and a modified steam-distillation apparatus, according to Parnas and Wagner, with automatic regulation of steam pressure, model Btichi (14).
RESULTS AND DISCUSSION Figure 1 shows the effect of hydrolysis temperature on the yield of N~-TNP-lysine from casein, expressed as milligrams of lysine per gram of
DETERMINATION OF AVAILABLE LYSINE
550!
437
5
500
~
L
TEMPERATURE, °C
FIG. 1. Effects of hydrolysis temperature on the analytical results obtained for the available lysine content of casein. Hydrolysis time was 60 min. The bars represent means _+ standard deviations (15). Figures over the bars indicate the number of single analyses. • = Triplicate determinations carried out on samples hydrolyzed in flasks sealed without evacuation. • = Triplicate determinations carried out on samples hydrolyzed in flasks evacuated before sealing. • and • for each temperature were operated at the same time.
nitrogen, as obtained by the present modification of the TNBS-method. Somewhat higher values were obtained when the hydrolysis was performed at 110°C compared with the other temperatures used. Evacuation of the hydrolysis flasks before sealing did not give better yields of lysine than sealing without vacuum. In another series of experiments the temperature during hydrolysis was maintained at 110°C while the time of hydrolysis was changed from 30 to 120 min (Fig. 2). The optimal yield oflysine was obtained when the time of hydrolysis was 75 to 90 min. Hydrolysis for longer periods of time seemed to lower the values. Therefore, in the procedure that was finally adopted the hydrolysis was performed at 110°C for 90 min. Table 1 shows the amounts of available lysine in casein and rapeseed protein concentrates determined by the present modification of the TNBS-method and the corresponding values obtained according to the method described by Kakade and Liener. Throughout, we obtained higher values with the modified method, and the degree of variability between repeated analyses of the same material was lower compared to the variability obtained by the Kakade and Liener method. The accuracy of the
~ 600 500
ca
30~ ~r
J0
'
so
. . 90. .
~2g
HYDROLYSIS T~ME mm
FtG. 2. Effect of hydrolysis time on the analytical results obtained for the available lysine content of casein. Hydrolysis temperature was 110°C. The bars represent means + standard deviations. Figures over the bars indicate the number of single analyses.
438
ANDERS EKLUND TABLE 1
AVAILABLE LYSlNE CONTENTS OF CASEIN AND RAPESEED PROTEIN CONCENTRATES DETERMINED ACCORDING TO THREE DIFFERENT METHODSa
Available lysine (mg/gof N) Protein material Casein
Kakade and Liener (7) method
Rao et al. Present (10) method method
Total lysine (1) (mg/g of N)
385 -+ 191 n=7
480 -+ n=4
6
534 _-x- 13 n=ll
517 n=2
263 --- 99
320 - 22
347 -+ 23
374
cv. Panther)
n = 6
n = 8
n = 19
n = 2
Rapeseed protein concentrate
307 --- 40
--
355 --- 24
371
n = 21
n = 2
Rapeseed protein concentrate
(Brassica napus,
(Brass&a napus, cv. Guile)
n = 61
a For comparison, analytical data of the total lysine content is given. Mean values ___standard deviationsof n single determinations. modified method seem to be as high as that of the much more difficult and lengthy FDNB-method. The values for available lysine obtained by the present method seem to correlate quite well with the amounts of total lysine (Table 1). Compared with the previous TNBS-method the proposed changes in procedure are as follows: (i) a larger sample size, 30 mg, is used; (ii) the hydrolysis is performed in sealed flasks instead of the samples being autoclaved in tubes covered with inverted glass vials; and (iii) optimal conditions for the hydrolysis of the TNP-labelled protein have been established. In the present studies a sample size of 30 mg seemed to be compatible with a good reproducibility. With some proteins, however, an even larger sample size might be required to obtain satisfactory results. The use of sealed flasks during hydrolysis eliminated the strong corrosive action of the hydrochloric acid upon the metal autoclave, a serious complication which has also been reported by Hall e t al. (9). The advantages obtained by using TNBS instead of FDNB for the determination of available lysine, such as simplicity, ease of handling, and rapidity, were discussed by Kakade and Liener (7) and should also be valid for the present modified procedure. We hope that the present modification could be successfully applied in the determination of available lysine in those cases where the method proposed by Kakade and Liener does not give satisfactory results.
DETERMINATION OF AVAILABLE LYSINE
439
ACKNOWLEDGMENTS This investigation was supported by grants from the Tri-Centennial Fund of the Bank of Sweden (project 67/54) and the Swedish Nutrition Foundation. The technical assistance of Miss K. Johansson is gratefully acknowledged,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15.
Spackman, D. H., Stein, W. H., and Moore, S. (1958)Anal. Chem. 30, 1190. Bjarnason, B., and Carpenter, K. J. (1970) Brit. J. Nutr. 24, 313. Carpenter, K. J. (1960) Biochem. J. 77, 604. Boyne, A. W., Carpenter, K. J., and Woodham, A.A. (1961) J. Sci. Food Agric. 12, 832. Carpenter, K. J., and March, B. E. (1961) Brit. J. Nutr. 15, 403. Anonymous (1974) P A G Bulletin 4: 3, 17. Kakade, M. L., and Liener, I. E. (1969)Anal. Biochem. 27, 273. Ousterhout, L. E., and Wood, E. M. (1970)Poult. Sci. 49, 1423. Hall, R. J., Tfinder, N., and Givens, D. I. (1973) Analyst 98, 673. Rao, S. R., Carter, F. L., and Frampton, V. L. (1963)Anal. Chem. 35, 1927. Agren, G., and Eklund, A. (1972)J. Sci. FoodAgric. 23, 1457. Blom, L., Hendricks, P., and Caris, J. (1967)Anal. Biochem. 21, 382. Agren, G., and Lied6n, S. A. (1968)Acta Chem. Scand. 22, 1981. Anonymous (1971) Manual: Tecator System for Determination of Nitrogen. Tecator AB, Helsingborg, Sweden. Snedecor, G. W., and Cochran, W. G. (1967) Statistical Methods, 6th ed., Iowa State University Press, Ames, Iowa.
On the Determination of Available Lysine in Casein and Rapeseed Protein Concentrates Using 2,4,6-Trinitrobenzenesulphonic Acid (TNBS) as a Reagent for Free Epsilon Amino Group of Lysine ANDERS E K L U N D
Institute of Medical and Physiological Chemistry, Biomedical Centre, University of Uppsala, P.O. Box 575, S-751 23 Uppsala, Sweden Received March 25, 1975; accepted September 2, 1975 The determination of available lysine was studied using 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for free epsilon amino group of lysine. By establishing optimal conditions for the hydrolysis of TNP-labelled protein, a modification of a previously described TNBS-method was proposed. The changes reduced the analytical variations substantially from the original method and made it possible to obtain in a simple and rapid way quite reproducible values for the contents of available lysine in casein and rapeseed protein concentrates.
Proteins of vegetable origin constitute a major part of the protein consumed by inhabitants of developing countries. These proteins very often contain rather low amounts of lysine, which is a very important dietary factor. This is one of the reasons why the quantitative determination of lysine has attracted so much interest in the nutritional field. Determinations of lysine contents are also vitally important in the quality control of animal proteins, for example in the dairy and fishery industries. Consequently, simple and rapid assay methods for lysine would be of the greatest value, e.g., to evaluate the nutriment value of food mixtures, in plant-breeding to look for higher levels of lysine in new varieties of protein-rich plants, and in industrial food processing to control the protein quality of the product. Today lysine is commonly analysed in an automatic amino acid analyser, mainly according to the technique reported by Spackman et al. (1). Even if the procedure, due to a high level of automation, is rather convenient, the equipment required for these analyses is rather expensive and may not be accessible in every case, especially not in developing countries. The lysine value obtained is a total value and may include a fraction of lysine which has been changed by different chemical reactions, such as the Maillard reaction (2), to become biologically unavailable. 434 Copyright © 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.
D E T E R M I N A T I O N OF AVAILABLE L Y S I N E
435
In nutritional work the determination of available iysine is often more important than the analysis of total lysine. It is well established that lysine must have a free epsilon amino group to be biologically available. At present methods similar to that of Carpenter et al. (3-5), involving the use of 1-fluoro-2,4-dinitrobenzene (FDNB) as a reagent for determining the free epsilon amino groups of lysine, are generally recommended for the analysis of available lysine (6). However, these methods are comparatively laborious and time consuming for the routine analysis of proteins. A few years ago Kakade and Liener presented a method by which the amount of available lysine was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS) which specifically reacts with primary amino groups (7). The method was designed on a micro level dealing with 1-10 mg samples. Ousterhout and Wood (8) adopted a slightly modified TNBS-method for determination of available lysine in fish meals and used a sample size of 100 mg. When we tried to use the procedure of Kakade and Liener for measuring the contents of available lysine in protein foodstuffs, we obtained satisfactory results with some materials but found it rather difficult to obtain reproducible values with some other materials. A poor reproducibility of the method of Kakade and Liener has also been reported by Hall et al. (9). However, since the method was very rapid and quite simple we tried to increase the accuracy of the method by increasing the sample size as well as by chosing optimal conditions for the hydrolysis of the proteins after coupling to TNBS. In the present paper a slightly modified TNBS-method is described. Comparisons are made between analyses of the contents of available lysine in casein and rapeseed protein concentrates performed by the new procedure, by the method of Kakade and Liener (7) and by the FDNB-method of Rao et al. (10). MATERIALS AND METHODS
The chemicals used were of analytical grade. "Hammarsten" casein was delivered from E. Merck A G, Darmstadt. Rapeseed protein concentrates (Brassica n a p u s L.) were prepared by a procedure described previously (11). Prior to analysis the protein concentrates were defatted by extraction with diethyl ether in a Soxhlet apparatus. TNBS was purchased from Sigma Chemical Co., St. Louis, Missouri, N~-TNP-L-lysine was delivered from Nutritional Biochemical Corp., Cleveland, Ohio. Preliminary experiments were carried out to establish the optimal conditions for determining the amount of available lysine in casein. In order to decrease the sampling error the sample weights were increased in comparison with those recommended by Kakade and Liener (7). The following procedure was adopted: The fat-free sample was finely ground in a cyclone sample mill (Tecator AB, Helsingborg) to pass a B.S. 70-mesh sieve. This means that the particle size of the powder did not exceed 210
436
ANDERS E K L U N D
/xm. To 30 mg of this sample in a 50 ml flat-bottomed Pyrex flask equipped with an extralong neck, 5 ml of 0.48 M NaHCO3, pH 8.5, were added. The flasks were placed in a constant-temperature shaking bath at 40°C for 10 min prior to the addition of 5 ml of 34 mM TNBS-solution. After an incubation period of 2 hr at 40°C, 15 ml of concentrated HC1 were added and the flasks were sealed. With some samples the flasks were evacuated before they were sealed. The hydrolysis was carried out at 110°C for 90 min in an Electrolux drying oven, model no. A 28482, equipped with a fan. In the preliminary experiments some samples were hydrolyzed at various temperatures between 90-120°C for 60 min, and other samples were hydrolyzed at I10°C for different times ranging from 30 to 120 min. After cooling to room temperature, the hydrolyzates were filtered (Munktell's Swedish Filter Paper no. 2OH, Grycksbo Pappersbruk AB, Sweden) and diluted to 200 ml with distilled water. Ten milliliters were transferred to a glass-stoppered test tube and extracted four times with 10 ml of diethyl ether in order to remove TNP-N-terminal amino acids or peptides and picric acid produced during reaction. Residual ether was evaporated by placing the tubes in a hot shaking-bath until no smell of ether could be detected. The optical density of the aqueous solution was measured at 346 nm against a blank prepared in the same way except that the concentrated HCI was added to the protein suspension prior to the addition of the TNBS reagent. The amounts of N'-TNP-lysine were calculated from a standard curve ofN'-TN P-L-lysine subjected to the same procedure as the protein samples. According to Ousterhout and Wood (8) and Hall et al. (9) the standard curve can also be prepared from pure DL-lysine carried through the procedure. For comparison, the contents of available lysine were determined by the TNBS-method of Kakade and Liener (7) as well as by the FDNB-method of Rao et al. (10) modified according to Blom et al. (12) so that 1.00 g of 2,4-dinitrophenol was added just prior to hydrolysis of the FDNB-treated sample. With the former method (7) the hydrolysis was performed by autoclaving the samples in tubes covered with inverted glass vials at 120°C (15-17 psi) for 1 hr. A 10 1 CERTOclav (Gruber & Kaja, Vienna) was used for this purpose. Hydrolyzates (6 M HCI; 110°C for 20 and 70 hr) for the analysis of total lysine content were obtained as previously described (13). Total lysine was determined according to Spackman et al. (1). Nitrogen was analysed by a modified Kjeldahl procedure using a Tecator Digestion System model 40 and a modified steam-distillation apparatus, according to Parnas and Wagner, with automatic regulation of steam pressure, model Btichi (14).
RESULTS AND DISCUSSION Figure 1 shows the effect of hydrolysis temperature on the yield of N~-TNP-lysine from casein, expressed as milligrams of lysine per gram of
DETERMINATION OF AVAILABLE LYSINE
550!
437
5
500
~
L
TEMPERATURE, °C
FIG. 1. Effects of hydrolysis temperature on the analytical results obtained for the available lysine content of casein. Hydrolysis time was 60 min. The bars represent means _+ standard deviations (15). Figures over the bars indicate the number of single analyses. • = Triplicate determinations carried out on samples hydrolyzed in flasks sealed without evacuation. • = Triplicate determinations carried out on samples hydrolyzed in flasks evacuated before sealing. • and • for each temperature were operated at the same time.
nitrogen, as obtained by the present modification of the TNBS-method. Somewhat higher values were obtained when the hydrolysis was performed at 110°C compared with the other temperatures used. Evacuation of the hydrolysis flasks before sealing did not give better yields of lysine than sealing without vacuum. In another series of experiments the temperature during hydrolysis was maintained at 110°C while the time of hydrolysis was changed from 30 to 120 min (Fig. 2). The optimal yield oflysine was obtained when the time of hydrolysis was 75 to 90 min. Hydrolysis for longer periods of time seemed to lower the values. Therefore, in the procedure that was finally adopted the hydrolysis was performed at 110°C for 90 min. Table 1 shows the amounts of available lysine in casein and rapeseed protein concentrates determined by the present modification of the TNBS-method and the corresponding values obtained according to the method described by Kakade and Liener. Throughout, we obtained higher values with the modified method, and the degree of variability between repeated analyses of the same material was lower compared to the variability obtained by the Kakade and Liener method. The accuracy of the
~ 600 500
ca
30~ ~r
J0
'
so
. . 90. .
~2g
HYDROLYSIS T~ME mm
FtG. 2. Effect of hydrolysis time on the analytical results obtained for the available lysine content of casein. Hydrolysis temperature was 110°C. The bars represent means + standard deviations. Figures over the bars indicate the number of single analyses.
438
ANDERS EKLUND TABLE 1
AVAILABLE LYSlNE CONTENTS OF CASEIN AND RAPESEED PROTEIN CONCENTRATES DETERMINED ACCORDING TO THREE DIFFERENT METHODSa
Available lysine (mg/gof N) Protein material Casein
Kakade and Liener (7) method
Rao et al. Present (10) method method
Total lysine (1) (mg/g of N)
385 -+ 191 n=7
480 -+ n=4
6
534 _-x- 13 n=ll
517 n=2
263 --- 99
320 - 22
347 -+ 23
374
cv. Panther)
n = 6
n = 8
n = 19
n = 2
Rapeseed protein concentrate
307 --- 40
--
355 --- 24
371
n = 21
n = 2
Rapeseed protein concentrate
(Brassica napus,
(Brass&a napus, cv. Guile)
n = 61
a For comparison, analytical data of the total lysine content is given. Mean values ___standard deviationsof n single determinations. modified method seem to be as high as that of the much more difficult and lengthy FDNB-method. The values for available lysine obtained by the present method seem to correlate quite well with the amounts of total lysine (Table 1). Compared with the previous TNBS-method the proposed changes in procedure are as follows: (i) a larger sample size, 30 mg, is used; (ii) the hydrolysis is performed in sealed flasks instead of the samples being autoclaved in tubes covered with inverted glass vials; and (iii) optimal conditions for the hydrolysis of the TNP-labelled protein have been established. In the present studies a sample size of 30 mg seemed to be compatible with a good reproducibility. With some proteins, however, an even larger sample size might be required to obtain satisfactory results. The use of sealed flasks during hydrolysis eliminated the strong corrosive action of the hydrochloric acid upon the metal autoclave, a serious complication which has also been reported by Hall e t al. (9). The advantages obtained by using TNBS instead of FDNB for the determination of available lysine, such as simplicity, ease of handling, and rapidity, were discussed by Kakade and Liener (7) and should also be valid for the present modified procedure. We hope that the present modification could be successfully applied in the determination of available lysine in those cases where the method proposed by Kakade and Liener does not give satisfactory results.
DETERMINATION OF AVAILABLE LYSINE
439
ACKNOWLEDGMENTS This investigation was supported by grants from the Tri-Centennial Fund of the Bank of Sweden (project 67/54) and the Swedish Nutrition Foundation. The technical assistance of Miss K. Johansson is gratefully acknowledged,
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15.
Spackman, D. H., Stein, W. H., and Moore, S. (1958)Anal. Chem. 30, 1190. Bjarnason, B., and Carpenter, K. J. (1970) Brit. J. Nutr. 24, 313. Carpenter, K. J. (1960) Biochem. J. 77, 604. Boyne, A. W., Carpenter, K. J., and Woodham, A.A. (1961) J. Sci. Food Agric. 12, 832. Carpenter, K. J., and March, B. E. (1961) Brit. J. Nutr. 15, 403. Anonymous (1974) P A G Bulletin 4: 3, 17. Kakade, M. L., and Liener, I. E. (1969)Anal. Biochem. 27, 273. Ousterhout, L. E., and Wood, E. M. (1970)Poult. Sci. 49, 1423. Hall, R. J., Tfinder, N., and Givens, D. I. (1973) Analyst 98, 673. Rao, S. R., Carter, F. L., and Frampton, V. L. (1963)Anal. Chem. 35, 1927. Agren, G., and Eklund, A. (1972)J. Sci. FoodAgric. 23, 1457. Blom, L., Hendricks, P., and Caris, J. (1967)Anal. Biochem. 21, 382. Agren, G., and Lied6n, S. A. (1968)Acta Chem. Scand. 22, 1981. Anonymous (1971) Manual: Tecator System for Determination of Nitrogen. Tecator AB, Helsingborg, Sweden. Snedecor, G. W., and Cochran, W. G. (1967) Statistical Methods, 6th ed., Iowa State University Press, Ames, Iowa.