ANALYTICAL
BIOCHEMISTRY
88,
598-604 (1978)
A Ninhydrin-Orthophthalaldehyde Reagent for the Determination of NT-Methylhistidine LEIGH Department
C. WARD
of Biochemistry. University St. Lucia, Queensland, Australia,
of Queensland, 4067
Received October 18, 1977; accepted January 27, 1978 A ninhydrin-orthophthalaldehyde reagent for the determination of NV-methylhistidine has been developed. The sensitivity of the reagent is similar to that of ninhydrin. Since most amino acids are not detected by the reagent, this allows the determination of Nr-methylhistidine in the presence of high concentrations of other amino acids. The reagent has been successfully used for the analysis of iV’-methylhistidine in human urine.
The measurement of the excretion of N’-methylhistidine [His(rMe)] has been proposed as a method for determining the rate of myofibrillar protein catabolism (1). The procedure requires the accurate quantification of His(rMe) in myofibrillar proteins and in urine, in which the concentration of His(TMe) is very low relative to that of other amino acids. A number of methods using ion exchange chromatography for the analysis of methylamino acids have been described (2-10). All of these procedures use ninhydrin for the colourimetric determination of His(7Me). Unfortunately the high concentrations of other amino acids in the column eluate yield an intense reaction with ninhydrin. To avoid this problem, preliminary ion exchange separations to remove the neutral and acidic amino acids have been used (3,4); however, it may still be necessary to avoid introducing the ninhydrin reagent for the first hour of the analysis to prevent reaction with remaining neutral and acidic amino acids (4). Orthophthalaldehyde (OPT) has been used for the fluorimetric detection of amino acids (1 l- 13) and has been used in conjunction with ninhydrin for the detection of imidazole compounds, including His(TMe), on thinlayer chromatograms (14,15). The present report describes the development of a combined ninhydrinOPT reagent for the colourimetric detection of His(TMe) which overcomes the disadvantages of ninhydrin and does not require a fluorimeter, which is essential when OPT alone is used. 0003-2697/78/0882-0598$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
598
NINHYDRIN-ORTHOPHTHALALDEHYDE
EXPERIMENTAL
REAGENT
599
AND RESULTS
Equipment. Spectrophotometric measurements were performed using either a Unicam SP800 (Pye Unicam Ltd., Cambridge) or an Hitachi 100-60 (Hitachi Ltd., Tokyo) recording spectrophotometer fitted with temperature-controlled cuvette holders. Glass cuvettes of IO-mm light path were used. Amino acid analysis was performed with a modified Technicon NC-l amino acid analyser (Technicon Chromatography Corp., New York). Separation of the basic amino acids was performed on a 300 x 6-mm glass column packed with chromobeads A (Technicon) cation exchange resin which was eluted at 30°C with 0.4 M sodium citrate buffer, pH 4.19 (16). Reagents. Amino acids and OPT were obtained from Sigma Chemical Co. Ltd. (St. Louis, Missouri). Other reagents were purchased from B.D.H. Chemicals Ltd. (Poole, U.K.). Wherever possible, reagent grade chemicals were used. Ninhydrin reagent was prepared according to Spackman et al. (17) using titanium trichloride as the reducing agent (18). The reagent was stored under nitrogen in a dark bottle. Preliminary observations. Preliminary experiments demonstrated that solutions of His(rMe) would react slowly with a solution of ninhydrin containing OPT to yield a yellow solution. This yellow solution exhibited a broad absorbance peak with an absorption maximum at 420 nm. The colour yield of the reaction was approximately one third that of the conventional ninhydrin reaction. Adding sodium hydroxide to this reaction mixture yielded a solution which absorbed maximally at 490 nm, the colour yield being similar to that obtained with ninhydrin alone. Since these initial observations indicated the possibility of developing a reagent for the detection of His(rMe) further investigations were performed to define more precisely the assay conditions. Procedure for spectrophotometric assay of His(rMe). A 2.0-ml aliquot of amino acid solution in citrate buffer was added to 1.0 ml of ninhydrin reagent containing 5 g/litre OPT. The solution was mixed thoroughly and allowed to react for 10 min at 40°C. To this solution was added 1.O ml of 2 M sodium hydroxide, and the solution was mixed thoroughly and allowed to cool at room temperature for 5 min. Absorbance was measured at 490 nm. Effect of temperature and OPT concentration. To 2.0-ml aliquots of 0.15 mM His(TMe) in citrate buffer were added increasing volumes of ninhydrin reagent containing OPT at a concentration of 10 g/litre. Ninhydrin reagent not containing OPT was added to yield a total volume of 3.0 ml, and OPT concentrations in the range of 1 to 10 g/litre of reagent. Blank samples were prepared containing no amino acid. Replicate blanks and samples for each OPT concentration were maintained at 20, 30, and 40°C in the heated
600
LEIGH
C. WARD
cuvette holder of the spectrophotometer. The increase in absorbance with time was measured against water at 420 nm. The time taken for maximum colour development decreased as the reaction temperature and the concentration of OPT were increased (Fig. 1). Colour yield was unaffected either by the concentration of OPT or by the temperature of reaction. Increase in the reaction temperature from 20 to 40°C caused a concomitant increase from 0.11 to 0.14 in the absorbance of the blank, which was not measurably affected by the concentration of OPT. Blanks and samples exhibited essentially identical absorbances at 570 nm. Reagent stability. No measurable change in sensitivity or increase in blank absorbance of the reagent was observed upon storage at room temperature for up to 3 weeks, provided that the reagent was stored under nitrogen in a dark bottle. Linearity and sensitivity of reaction. A calibration curve for His(rMe), prepared with standard His(rMe) solutions, was found to be linear in the range of 0 to 0.15 pmol His(rMe)/ml of assay mixture (Fig. 2). Blank absorbance was 0.081 + 0.002 (8) units. Absorbance could be measured at any wavelength between 470 and 520 nm with some loss in sensitivity but with maintenance of linearity of the colour yield. The absorbance did not change measurably within 30 min when solutions were allowed to stand at room temperature in the light. Colour yield expressed as absorbance of a 1.O M solution of His(rMe) for a IO-cm light path was 11.5 x lo3 litre mol-l cm-‘. The equivalent colour yield for reaction with ninhydrin, for 15 min at 100°C and absorbance measured at 570 nm, was 10.6 x lo3 litre mol-’ cm-‘.
r 0
I 0.2 0.4 OPT concentration
FIG. 1. The effect of temperature ment. For assay conditions see text.
and OFT
0.6 (g 100 ml-l) concentration
0.8
upon
1.0
time of colour
develop-
601
NINHYDRIN-ORTHOPHTHALALDEHYDEREAGENT 1.81.6.
ais
concentration
(pm1
ml-'
assay
mixture1
FIG. 2. Calibration curve for His(7Me). For assay conditions see text. The mean + SEM (8) is shown.
Precision of the procedure. The precision of the procedure, estimated from 40 determinations of His(rMe) solutions of various concentrations between 0.03 and 0.15 kmol ml-’ of assay mixture, was plus or minus 3.2% (2 SEM). Specificity. Most common amino acids found in physiological fluids and a number of other amino compounds were assayed at a concentration of 1.O pmol/ml of assay mixture. Colour formation was observed for a number of substances, particularly glutamine, histidine, ammonia, and arginine (Table 1). Since the reagent was developed as an alternative to ninhydrin for estimating His(rMe) isolated by ion exchange chromatography, however, this degree of interference was not of primary concern, as discussed below. Amino acid analysis. The following procedure was based upon the manual method described above. The column eluate (flow rate of 0.7 ml/mine’) was mixed with a stream of ninhydrin-OPT reagent. (0.35 ml min-‘) and allowed to react at 40°C for 10 min in a coil of Teflon tubing (0.9 mm i.d.). The liquid flow from the reaction coil was mixed with a flowing stream of 2.0 M sodium hydroxide (0.35 mYmin) and allowed to cool for 5 min in a second coil of Teflon tubing maintained at room temperature. The absorbance of the solution was measured at 505 and 520 nm in separate colourimeters fitted with 20- and 14-mm path-length flow cells, respectively. Absorbance was recorded on a linear chart recorder with scale expansion facility. In Fig. 3 is shown the chromatogram obtained for 1.0 ml of human urine desalted and treated with histidine decarboxylase, as described previously (19). Only small peaks are observed for the neutral and acidic
602
LEIGH C. WARD
Neutral
-7
h acidic acids
amino
L
/
0"
110 Time
180
(min)
FIG. 3. Chromatogram obtained from the analysis of 1.O ml of human urine. Amino acids were detected with ninhydrin-OPT reagent. The trace records the absorbance measured at 505 nm. A fivefold scale expansion was used equivalent to full scale deflection of 0.2 absorbance units. For detailed assay conditions see the text.
amino acids. A large ammonia peak is present, reflecting the ammonia elution used in desalting the urine samples. No lysine peak is observable, and the peak representing histidine is much smaller than that observed in the corresponding experiment with ninhydrin reagent. Although the ninhydrin-OPT reagent is less sensitive to histidine than is ninhydrin, the high concentration of histidine relative to His(TMe) in urine may result in a significant degree of overlap between the respective peaks. It is recommended, in these cases, that samples are treated with histidine decarboxylase in order to decrease the concentration of histidine. A clearly defined His(TMe) peak is observable in Fig. 3, and a comparison of the peak area with those of His(TMe) standards indicated a urinary concentration of 61 PM and a daily excretion of 205 pmol. This is in good agreement with previously published data (20). CONCLUSIONS
Ninhydrin has long been used as the detection reagent for amino acid analysis because of its high sensitivity to a-amino groups. This sensitivity, however, yields an unacceptable degree of reaction with amino acids present in excess when the determination of His(TMe) is to be performed. The ninhydrin-OPT reagent described in this paper reacts readily with His(TMe) to form a yellow compound, which under alkaline conditions
NINHYDRIN-ORTHOPHTHALALDEHYDE TABLE COLOURYIELDS
OF AMINO
Amino
COMFQUNDS
UFQN
1 REACTION
WITH NINHYDRIN-OPT
REAGENT
Absorbance at 490 nm relative to absorbance of His(rMe)
compound
100 ND” 2.1 0.9 13.2 0.3 4.4 6.8 2.1 ND 1.0 1.0 1.0 ND ND 3.0 2.0 22.0 1.5 3.5 1.2 4.0 ND ND ND 51.0 ND 56.0
NT-methylhistidine Aspartic acid Threonine Serine Glutamine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Tyrosine Omithine Lysine Histidine N,-methylhistidine Hydroxyproline Taurine Citrulline Anserine Imidazole Histamine Arginine Urea Ammonia ” ND.
603
REAGENT
not detectable.
turns red-brown, the reaction being both sensitive and quantitative. This procedure is readily adapted to automated amino acid analysis, and the reagent should also find use in the detection of His(TMe) on paper or thinlayer chromatograms. ACKNOWLEDGMENTS Dr.
The author wishes to thank Professor B. Zerner for the loan of the amino D. Winzor for helpful comments during manuscript preparation.
acid analyser
REFERENCES 1. Armstrong. M. D., and Stave, 2. Deibler, G. E., and Martenson,
U. (1973) Metabolism R. E. (!973) J. Biul.
22,
C&m.
549-560.
248, 2387-2391.
and
604
LEIGH
C. WARD
3. Haverberg, L. N., Munro, H. N., and Young, U. R. (1974)Biochim. Biophys. Acta 371, 226-237. 4. Kakimoto, Y., Matsuoka, Y., Miyake, M., and Konishi, H. (1971) J. Neurochem. 24, 893-902.
5. Long, C. L., and Geiger, J. W. (1975) Biochem. Med. 12, 267-273. 6. Markiw, R. T. (1975) Biochem. Med. 13, 23-27. 7. Zarkadas. C. G. (1975) Canad. J. Biochem. 53, 96-101. 8. Palthy, A., Tyihak, E., Ferenczi, S.. Eckhardt, S.. Kralovansky, J., Lapis, K., and Szende, B. (1975) Proc. Annu. Meet. Hung, Biochem. Sot. 15, 57-58. 9. Waliszewski, K., and Skupin, J. (1976) Bull. Acad. Pol. Sci. 24, 377-380. 10. Helm, R., Vancikoua, Macek, K., and Deyl, Z. (1977) 1. Chromatogr. 133, 390-395. Il. Roth, M. (1971) Anal. Chem. 43, 880-882. 12. Roth, M., and Hampai, A. (1973) J. Chromatogr. 83, 353-356. 13. Benson, J. R., and Hare, P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619-622. 14. Turner, T. D., and Wightman. S. L. (1968) J. Chromatogr. 32, 315-322. 15. Lee, S-C., and Yin, S-J. (1976) J. Chromatogr. 129, 482-487. 16. Atkin, G. E., and Ferdinand, W. (1970) Ana/. Biochem. 38, 313-329. 17. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. 18. James, L. B. (1971) 1. Chromatogr. 59, 178-180. 19. Ward, L. C. (1976) “Muscle Protein Metabolism,” Ph.D. thesis, p. 10, University of Nottingham, Nottingham, U.K. 20. Wannemacher. R. W.. Jr., Dinterman, R. E., Pekarek, R. S., Bartelloni, P. J., and Beisel. W. R. (1975) Amer. J. Clin. Nutr. 28, 110-l 18.
BIOCHEMISTRY
88,
598-604 (1978)
A Ninhydrin-Orthophthalaldehyde Reagent for the Determination of NT-Methylhistidine LEIGH Department
C. WARD
of Biochemistry. University St. Lucia, Queensland, Australia,
of Queensland, 4067
Received October 18, 1977; accepted January 27, 1978 A ninhydrin-orthophthalaldehyde reagent for the determination of NV-methylhistidine has been developed. The sensitivity of the reagent is similar to that of ninhydrin. Since most amino acids are not detected by the reagent, this allows the determination of Nr-methylhistidine in the presence of high concentrations of other amino acids. The reagent has been successfully used for the analysis of iV’-methylhistidine in human urine.
The measurement of the excretion of N’-methylhistidine [His(rMe)] has been proposed as a method for determining the rate of myofibrillar protein catabolism (1). The procedure requires the accurate quantification of His(rMe) in myofibrillar proteins and in urine, in which the concentration of His(TMe) is very low relative to that of other amino acids. A number of methods using ion exchange chromatography for the analysis of methylamino acids have been described (2-10). All of these procedures use ninhydrin for the colourimetric determination of His(7Me). Unfortunately the high concentrations of other amino acids in the column eluate yield an intense reaction with ninhydrin. To avoid this problem, preliminary ion exchange separations to remove the neutral and acidic amino acids have been used (3,4); however, it may still be necessary to avoid introducing the ninhydrin reagent for the first hour of the analysis to prevent reaction with remaining neutral and acidic amino acids (4). Orthophthalaldehyde (OPT) has been used for the fluorimetric detection of amino acids (1 l- 13) and has been used in conjunction with ninhydrin for the detection of imidazole compounds, including His(TMe), on thinlayer chromatograms (14,15). The present report describes the development of a combined ninhydrinOPT reagent for the colourimetric detection of His(TMe) which overcomes the disadvantages of ninhydrin and does not require a fluorimeter, which is essential when OPT alone is used. 0003-2697/78/0882-0598$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
598
NINHYDRIN-ORTHOPHTHALALDEHYDE
EXPERIMENTAL
REAGENT
599
AND RESULTS
Equipment. Spectrophotometric measurements were performed using either a Unicam SP800 (Pye Unicam Ltd., Cambridge) or an Hitachi 100-60 (Hitachi Ltd., Tokyo) recording spectrophotometer fitted with temperature-controlled cuvette holders. Glass cuvettes of IO-mm light path were used. Amino acid analysis was performed with a modified Technicon NC-l amino acid analyser (Technicon Chromatography Corp., New York). Separation of the basic amino acids was performed on a 300 x 6-mm glass column packed with chromobeads A (Technicon) cation exchange resin which was eluted at 30°C with 0.4 M sodium citrate buffer, pH 4.19 (16). Reagents. Amino acids and OPT were obtained from Sigma Chemical Co. Ltd. (St. Louis, Missouri). Other reagents were purchased from B.D.H. Chemicals Ltd. (Poole, U.K.). Wherever possible, reagent grade chemicals were used. Ninhydrin reagent was prepared according to Spackman et al. (17) using titanium trichloride as the reducing agent (18). The reagent was stored under nitrogen in a dark bottle. Preliminary observations. Preliminary experiments demonstrated that solutions of His(rMe) would react slowly with a solution of ninhydrin containing OPT to yield a yellow solution. This yellow solution exhibited a broad absorbance peak with an absorption maximum at 420 nm. The colour yield of the reaction was approximately one third that of the conventional ninhydrin reaction. Adding sodium hydroxide to this reaction mixture yielded a solution which absorbed maximally at 490 nm, the colour yield being similar to that obtained with ninhydrin alone. Since these initial observations indicated the possibility of developing a reagent for the detection of His(rMe) further investigations were performed to define more precisely the assay conditions. Procedure for spectrophotometric assay of His(rMe). A 2.0-ml aliquot of amino acid solution in citrate buffer was added to 1.0 ml of ninhydrin reagent containing 5 g/litre OPT. The solution was mixed thoroughly and allowed to react for 10 min at 40°C. To this solution was added 1.O ml of 2 M sodium hydroxide, and the solution was mixed thoroughly and allowed to cool at room temperature for 5 min. Absorbance was measured at 490 nm. Effect of temperature and OPT concentration. To 2.0-ml aliquots of 0.15 mM His(TMe) in citrate buffer were added increasing volumes of ninhydrin reagent containing OPT at a concentration of 10 g/litre. Ninhydrin reagent not containing OPT was added to yield a total volume of 3.0 ml, and OPT concentrations in the range of 1 to 10 g/litre of reagent. Blank samples were prepared containing no amino acid. Replicate blanks and samples for each OPT concentration were maintained at 20, 30, and 40°C in the heated
600
LEIGH
C. WARD
cuvette holder of the spectrophotometer. The increase in absorbance with time was measured against water at 420 nm. The time taken for maximum colour development decreased as the reaction temperature and the concentration of OPT were increased (Fig. 1). Colour yield was unaffected either by the concentration of OPT or by the temperature of reaction. Increase in the reaction temperature from 20 to 40°C caused a concomitant increase from 0.11 to 0.14 in the absorbance of the blank, which was not measurably affected by the concentration of OPT. Blanks and samples exhibited essentially identical absorbances at 570 nm. Reagent stability. No measurable change in sensitivity or increase in blank absorbance of the reagent was observed upon storage at room temperature for up to 3 weeks, provided that the reagent was stored under nitrogen in a dark bottle. Linearity and sensitivity of reaction. A calibration curve for His(rMe), prepared with standard His(rMe) solutions, was found to be linear in the range of 0 to 0.15 pmol His(rMe)/ml of assay mixture (Fig. 2). Blank absorbance was 0.081 + 0.002 (8) units. Absorbance could be measured at any wavelength between 470 and 520 nm with some loss in sensitivity but with maintenance of linearity of the colour yield. The absorbance did not change measurably within 30 min when solutions were allowed to stand at room temperature in the light. Colour yield expressed as absorbance of a 1.O M solution of His(rMe) for a IO-cm light path was 11.5 x lo3 litre mol-l cm-‘. The equivalent colour yield for reaction with ninhydrin, for 15 min at 100°C and absorbance measured at 570 nm, was 10.6 x lo3 litre mol-’ cm-‘.
r 0
I 0.2 0.4 OPT concentration
FIG. 1. The effect of temperature ment. For assay conditions see text.
and OFT
0.6 (g 100 ml-l) concentration
0.8
upon
1.0
time of colour
develop-
601
NINHYDRIN-ORTHOPHTHALALDEHYDEREAGENT 1.81.6.
ais
concentration
(pm1
ml-'
assay
mixture1
FIG. 2. Calibration curve for His(7Me). For assay conditions see text. The mean + SEM (8) is shown.
Precision of the procedure. The precision of the procedure, estimated from 40 determinations of His(rMe) solutions of various concentrations between 0.03 and 0.15 kmol ml-’ of assay mixture, was plus or minus 3.2% (2 SEM). Specificity. Most common amino acids found in physiological fluids and a number of other amino compounds were assayed at a concentration of 1.O pmol/ml of assay mixture. Colour formation was observed for a number of substances, particularly glutamine, histidine, ammonia, and arginine (Table 1). Since the reagent was developed as an alternative to ninhydrin for estimating His(rMe) isolated by ion exchange chromatography, however, this degree of interference was not of primary concern, as discussed below. Amino acid analysis. The following procedure was based upon the manual method described above. The column eluate (flow rate of 0.7 ml/mine’) was mixed with a stream of ninhydrin-OPT reagent. (0.35 ml min-‘) and allowed to react at 40°C for 10 min in a coil of Teflon tubing (0.9 mm i.d.). The liquid flow from the reaction coil was mixed with a flowing stream of 2.0 M sodium hydroxide (0.35 mYmin) and allowed to cool for 5 min in a second coil of Teflon tubing maintained at room temperature. The absorbance of the solution was measured at 505 and 520 nm in separate colourimeters fitted with 20- and 14-mm path-length flow cells, respectively. Absorbance was recorded on a linear chart recorder with scale expansion facility. In Fig. 3 is shown the chromatogram obtained for 1.0 ml of human urine desalted and treated with histidine decarboxylase, as described previously (19). Only small peaks are observed for the neutral and acidic
602
LEIGH C. WARD
Neutral
-7
h acidic acids
amino
L
/
0"
110 Time
180
(min)
FIG. 3. Chromatogram obtained from the analysis of 1.O ml of human urine. Amino acids were detected with ninhydrin-OPT reagent. The trace records the absorbance measured at 505 nm. A fivefold scale expansion was used equivalent to full scale deflection of 0.2 absorbance units. For detailed assay conditions see the text.
amino acids. A large ammonia peak is present, reflecting the ammonia elution used in desalting the urine samples. No lysine peak is observable, and the peak representing histidine is much smaller than that observed in the corresponding experiment with ninhydrin reagent. Although the ninhydrin-OPT reagent is less sensitive to histidine than is ninhydrin, the high concentration of histidine relative to His(TMe) in urine may result in a significant degree of overlap between the respective peaks. It is recommended, in these cases, that samples are treated with histidine decarboxylase in order to decrease the concentration of histidine. A clearly defined His(TMe) peak is observable in Fig. 3, and a comparison of the peak area with those of His(TMe) standards indicated a urinary concentration of 61 PM and a daily excretion of 205 pmol. This is in good agreement with previously published data (20). CONCLUSIONS
Ninhydrin has long been used as the detection reagent for amino acid analysis because of its high sensitivity to a-amino groups. This sensitivity, however, yields an unacceptable degree of reaction with amino acids present in excess when the determination of His(TMe) is to be performed. The ninhydrin-OPT reagent described in this paper reacts readily with His(TMe) to form a yellow compound, which under alkaline conditions
NINHYDRIN-ORTHOPHTHALALDEHYDE TABLE COLOURYIELDS
OF AMINO
Amino
COMFQUNDS
UFQN
1 REACTION
WITH NINHYDRIN-OPT
REAGENT
Absorbance at 490 nm relative to absorbance of His(rMe)
compound
100 ND” 2.1 0.9 13.2 0.3 4.4 6.8 2.1 ND 1.0 1.0 1.0 ND ND 3.0 2.0 22.0 1.5 3.5 1.2 4.0 ND ND ND 51.0 ND 56.0
NT-methylhistidine Aspartic acid Threonine Serine Glutamine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Tyrosine Omithine Lysine Histidine N,-methylhistidine Hydroxyproline Taurine Citrulline Anserine Imidazole Histamine Arginine Urea Ammonia ” ND.
603
REAGENT
not detectable.
turns red-brown, the reaction being both sensitive and quantitative. This procedure is readily adapted to automated amino acid analysis, and the reagent should also find use in the detection of His(TMe) on paper or thinlayer chromatograms. ACKNOWLEDGMENTS Dr.
The author wishes to thank Professor B. Zerner for the loan of the amino D. Winzor for helpful comments during manuscript preparation.
acid analyser
REFERENCES 1. Armstrong. M. D., and Stave, 2. Deibler, G. E., and Martenson,
U. (1973) Metabolism R. E. (!973) J. Biul.
22,
C&m.
549-560.
248, 2387-2391.
and
604
LEIGH
C. WARD
3. Haverberg, L. N., Munro, H. N., and Young, U. R. (1974)Biochim. Biophys. Acta 371, 226-237. 4. Kakimoto, Y., Matsuoka, Y., Miyake, M., and Konishi, H. (1971) J. Neurochem. 24, 893-902.
5. Long, C. L., and Geiger, J. W. (1975) Biochem. Med. 12, 267-273. 6. Markiw, R. T. (1975) Biochem. Med. 13, 23-27. 7. Zarkadas. C. G. (1975) Canad. J. Biochem. 53, 96-101. 8. Palthy, A., Tyihak, E., Ferenczi, S.. Eckhardt, S.. Kralovansky, J., Lapis, K., and Szende, B. (1975) Proc. Annu. Meet. Hung, Biochem. Sot. 15, 57-58. 9. Waliszewski, K., and Skupin, J. (1976) Bull. Acad. Pol. Sci. 24, 377-380. 10. Helm, R., Vancikoua, Macek, K., and Deyl, Z. (1977) 1. Chromatogr. 133, 390-395. Il. Roth, M. (1971) Anal. Chem. 43, 880-882. 12. Roth, M., and Hampai, A. (1973) J. Chromatogr. 83, 353-356. 13. Benson, J. R., and Hare, P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619-622. 14. Turner, T. D., and Wightman. S. L. (1968) J. Chromatogr. 32, 315-322. 15. Lee, S-C., and Yin, S-J. (1976) J. Chromatogr. 129, 482-487. 16. Atkin, G. E., and Ferdinand, W. (1970) Ana/. Biochem. 38, 313-329. 17. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. 18. James, L. B. (1971) 1. Chromatogr. 59, 178-180. 19. Ward, L. C. (1976) “Muscle Protein Metabolism,” Ph.D. thesis, p. 10, University of Nottingham, Nottingham, U.K. 20. Wannemacher. R. W.. Jr., Dinterman, R. E., Pekarek, R. S., Bartelloni, P. J., and Beisel. W. R. (1975) Amer. J. Clin. Nutr. 28, 110-l 18.
