ANALYTICAL BIOCHEMISTRY 70, 377-380 (1976)
A Sensitive Assay for Allantoin JOHN ABRAHAM, FIORINDO A . SIMEONE, AND ROBERT W . HOPKINS
Division of Biological and Medical Sciences of Brown University, and the Department of Surgery, The Miriam Hospital, Providence, Rhode Island Received January l, 1975; accepted September 19, 1975 Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 ~g per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.
Several reports (1-9) have been published on the determination of allantoin. Most of these methods have been based on the application of the Rimini- Schryver color reaction for formaldehyde to the aldehyde group of glyoxylic acid which is formed by the hydrolysis of allantoin. Several compounds, including uric acid, ergothioneine, and glucose may interfere with the assay. Therefore, Christman et al. (5) have pointed out the desirability of the quantitative separation of allantoin from other constituents of blood prior to the analysis. Cline et al. (10) have investigated the utilization of the color reaction between p-dimethylaminobenzaldehyde and allantoin on paper chromatograms for the quantitation of allantoin. Based on this, Zelck (11) has reported a procedure to determine concentrations of allantoin in urine. Though the method was specific, the sensitivity of the assay was such that the lowest amount of allantoin that could be determined was 20/xg (200 mg/100 ml). The reported values (5) for the concentration of allantoin in the blood of various animals range between 1 and 3 rag/100 ml. This work was undertaken to develop a method which would be specific and sensitive enough to measure the concentration of allantoin in serum and lymph of mongrel dogs in studies of hemorrhagic shock.
METHODS Reagents and materials. Thin-layer chromatography (TLC) plates. Flexible cellulose-coated plates, 20 x 20 cm (J. T. Baker Chemical Co.) are used. Allantoin standard. A stock solution containing 1.0 mg/ml is prepared by dissolving allantoin (Nutritional Biochemical Corporation) in distilled 377 Copyright © 1976by AcademicPress. Inc. All rights of reproductionin any formreserved.
378
ABRAHAM, S I M E O N E AND HOPKINS
water heated to 50°C. Working standards are prepared by the proper dilution of the stock solution. It is desirable to use a fresh standard because of the possibility of decomposition of allantoin in neutral or acidic media (3). Spraying solution. This is p r e p a r e d by dissolving 1.0 g p-dimethylaminobenzaldehyde (certified, Fisher Scientific Company) in 3 ml of concentrated hydrochloric acid and then diluting to 15 ml with 1-Butanol (certified, Fisher Scientific Company). This solution is sufficient to spray two TLC plates and is prepared fresh every day. Apparatus. The Photovolt TLC densitometer Model 530 equipped with an integrator is used to scan the plates. Procedure. Large amounts of protein in serum and lymph causes tailing of the spots when the TLC plate is developed. Most of the proteins are removed by adding 0.4 ml of serum or lymph to 0.2 ml of 1.0 N perchloric acid and centrifuging. Urine samples are diluted at least 100 times and used without deproteinization. TLC analysis ofallantoin. Ten-microliter aliquots of the solutions to be analyzed are spotted with 10/xl disposable pipets on a thin-layer plate. In addition, 10-/zl aliquots of standard solutions, which are to be compared with the samples, are spotted on the same plate. The plate is developed using a mobile phase of 1.0 N acetic acid:ethanol:l-Butanol, 2:2:7 by volume. The length of the development is approximately 17 cm, which requires about 2.5 hr. After the development, the TLC plate is air-dried until the solvent has evaporated. It is then dried in an oven at 75°C for 15 min. The plate is then sprayed with a solution ofp-dimethylaminobenzaldehyde,dried in air for 5 min, and again dried for another 5 min in an oven at 75°C. The plate is then allowed to equilibrate for 25 min in a closed chamber. The color densities of the spots are read in a densitometer using the 445 nm filter. By comparing the densities of spots of the sample solutions with those of the standards, the concentration of allantoin in the solution can be calculated. This may be converted to the concentration of allantoin in serum, lymph, or urine by using the appropriate dilution factor. Results. The Rs value of allantoin in the solvent system described above was 0.23. The reproducibility of the method has been ascertained by determining allantoin in different standard solutions. The results of these determinations are given in Table 1. In order to determine the recovery of the added allantoin, known amounts of allantoin were added to dog's serum and the total allantoin content was determined. The results are given in Table 2. The average recovery of added allantoin was 93.2%.
DISCUSSION As shown in Table 1, amounts as small as 0.1/xg (0.5 mg/100 ml) can be accurately determined by this procedure. In the method described by
379
ASSAY F O R A L L A N T O I N TABLE 1 ANALYSIS OF STANDARD ALLANTO1N SOLUTIONS
Volume of solution in each spot (p,I) 20 10 10 10
Concentration of allantoin given (mg/100 ml) 0.5 1.0 3.0 4.0
Concentration of allantoin found a (mg/100 ml)
(0.10) r' (0.10) (0.30) (0.40)
0.45 0.9 3.2 4.1
_+ 0.004 _+ 0.009 -+ 0.12 _+ 0.32
(0.09) (0.09) (0.32) (0.41)
Each result is average of seven determinations, and the error is expressed as standard erFor.
Values in parentheses are the amounts of allantoin (/~g) in each spot.
Zelck (11), amounts smaller than 20 /xg (200 mg/100 ml) could not be assayed. Thus, the technique described has improved the sensitivity of the assay about 400-fold. The color produced is found to obey Beer's law in the range 0.1 to 1.0/zg of allantoin. Deviation from Beer's law is observed when the amount of allantoin increased above 1.0 tzg per spot. Under those circumstances the sample should be appropriately diluted. The conditions and precautions necessary for the increased sensitivity are discussed below. As has been demonstrated by Cline (10), we observed that the color produced in the reaction between allantoin and p-dimethylaminobenzaldehyde is dependent on the amount of acid and p-dimethylaminobenzaldehyde in the spray. Of the several concentrations ~Aed, the amount of hydrochloric acid andp-dimethylaminobenzaldehyde recommended in our procedure was found to produce the best results. The solvent in the spray also affects the color development. 1-Butanol was found to be superior to other solvents, including methanol, chloroform, ether, and acetone. The intensity of the color reaction on the plate is also influenced by the TABLE 2 RECOVERY OF ALLANTOIN A D D E D TO SERUM
Volume of serum taken (ml)
Amount of allantoin added to serum (/xg)
0.40 0.40 0.40 0.40
-10.0 20.0 30.0
Total allantoin found a (/xg) 16.2b 24.9 32.3 44.1
+ 0.85 _+ 0.57 _+ 0.76 _+ 1.2
Recovery (%) -95.0 89.2 95.5
~' Each result is an average of four determinations. b This is the amount of allantoin found in 0.4 ml serum of a mongrel dog, and corresponds to a concentration of 4.05 mg/100 ml.
380
ABRAHAM, SIMEONE AND HOPKINS
relative humidity of the atmosphere. Elaborate arrangements would be required to keep the humidity low and constant. The effect of this variable is minimized by running standards simultaneously with the unknown in each TLC plate. Other factors that influenced the color density are the drying conditions after the spraying. Drying the plate at high temperature for long periods produced high background readings. The best results are obtained when the plate is heated at 75°C for 5 min. When the plate is left exposed to air after the spraying and drying, the background readings increased, probably due to the slow air-oxidation of the reagent p-dimethylaminobenzaldehyde. This effect is minimized by storing the plate in a closed chamber until it is read. Under these circumstances, the color was stable for at least 1 hr. The sensitivity of the present assay should permit the determination of allantoin in any biological fluid in which the concentration is above 1.5 mg%. However, when allantoin concentration is lower than 1.5 rag%, the volume of the sample spotted on the plate must be increased to 20/xl. No attempt was made to increase further the volume of solution in each spot. Since allantoin is separated by TLC before the color reaction, interference from uric acid, urea, and other amido compounds is eliminated. The method is specific and selective. This assay is applicable to any fluids containing allantoin, provided the concentration of all other substances would not cause tailing of the spots when TLC is developed. Several samples can be analyzed in a relatively short time. This method is suited for the routine determination of allantoin. We have applied this method to the measurement of allantoin in blood, thoracic duct lymph, and urine of dogs subjected to a period of oligemia (12).
ACKNOWLEDGMENTS This work was supported by The Miriam Hospital and by PHS Research Grant No. GM 19930 from the National Institutes of Health.
REFERENCES 1. Christman, A. A. (1926) J. Biol. Chem. 70, 173-191. 2. Fosse, R., Brunel, A., de Graeve, P., Thomas, P. E., and Sarazin, J. (1930) C. R. Acad. Sci. 191, 1153-1155. 3. Young, E. G., and Conway, C. F. (1942)J. Biol. Chem. 142, 839-853. 4. Young, E. G., Macpherson, C. C., Wentworth, H. P., and Hawkins, W. W. (1944) J. Biol. Chem. 152, 245-253. 5. Christman, A. A., Foster, P. W., and Esterer, M. B. (1944)J. Biol. Chem. 155, 161 - 17 I. 6. Domnas, A. ( l % l ) J . Biochem. 511,46-51. 7. Stahl, A., Schang, A. M., Brogard, J. M., and Coumaros, C. (1970)Ann. Biol. Clin. 28, 377-385. 8. Willemot, J., and Parry, G. (1972)Ann. Pharm. 311, 389-390. 9. Nirmala, J., and Sastry, K. S. (1972)Anal. Biochem. 47, 218-227. 10. Cline, R. E., and Fink, R. M. (1956) Anal. Chem. 28, 47-52. 11. Zelck, U. (1%3) Biochemische Zeitschrift 337, 525-530. 12. Simeone, F. A., Abraham, J., Hopkins, R. W., and Damewood, C. A. (1975)J. Surg. Res. 19, 373-380.
A Sensitive Assay for Allantoin JOHN ABRAHAM, FIORINDO A . SIMEONE, AND ROBERT W . HOPKINS
Division of Biological and Medical Sciences of Brown University, and the Department of Surgery, The Miriam Hospital, Providence, Rhode Island Received January l, 1975; accepted September 19, 1975 Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 ~g per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.
Several reports (1-9) have been published on the determination of allantoin. Most of these methods have been based on the application of the Rimini- Schryver color reaction for formaldehyde to the aldehyde group of glyoxylic acid which is formed by the hydrolysis of allantoin. Several compounds, including uric acid, ergothioneine, and glucose may interfere with the assay. Therefore, Christman et al. (5) have pointed out the desirability of the quantitative separation of allantoin from other constituents of blood prior to the analysis. Cline et al. (10) have investigated the utilization of the color reaction between p-dimethylaminobenzaldehyde and allantoin on paper chromatograms for the quantitation of allantoin. Based on this, Zelck (11) has reported a procedure to determine concentrations of allantoin in urine. Though the method was specific, the sensitivity of the assay was such that the lowest amount of allantoin that could be determined was 20/xg (200 mg/100 ml). The reported values (5) for the concentration of allantoin in the blood of various animals range between 1 and 3 rag/100 ml. This work was undertaken to develop a method which would be specific and sensitive enough to measure the concentration of allantoin in serum and lymph of mongrel dogs in studies of hemorrhagic shock.
METHODS Reagents and materials. Thin-layer chromatography (TLC) plates. Flexible cellulose-coated plates, 20 x 20 cm (J. T. Baker Chemical Co.) are used. Allantoin standard. A stock solution containing 1.0 mg/ml is prepared by dissolving allantoin (Nutritional Biochemical Corporation) in distilled 377 Copyright © 1976by AcademicPress. Inc. All rights of reproductionin any formreserved.
378
ABRAHAM, S I M E O N E AND HOPKINS
water heated to 50°C. Working standards are prepared by the proper dilution of the stock solution. It is desirable to use a fresh standard because of the possibility of decomposition of allantoin in neutral or acidic media (3). Spraying solution. This is p r e p a r e d by dissolving 1.0 g p-dimethylaminobenzaldehyde (certified, Fisher Scientific Company) in 3 ml of concentrated hydrochloric acid and then diluting to 15 ml with 1-Butanol (certified, Fisher Scientific Company). This solution is sufficient to spray two TLC plates and is prepared fresh every day. Apparatus. The Photovolt TLC densitometer Model 530 equipped with an integrator is used to scan the plates. Procedure. Large amounts of protein in serum and lymph causes tailing of the spots when the TLC plate is developed. Most of the proteins are removed by adding 0.4 ml of serum or lymph to 0.2 ml of 1.0 N perchloric acid and centrifuging. Urine samples are diluted at least 100 times and used without deproteinization. TLC analysis ofallantoin. Ten-microliter aliquots of the solutions to be analyzed are spotted with 10/xl disposable pipets on a thin-layer plate. In addition, 10-/zl aliquots of standard solutions, which are to be compared with the samples, are spotted on the same plate. The plate is developed using a mobile phase of 1.0 N acetic acid:ethanol:l-Butanol, 2:2:7 by volume. The length of the development is approximately 17 cm, which requires about 2.5 hr. After the development, the TLC plate is air-dried until the solvent has evaporated. It is then dried in an oven at 75°C for 15 min. The plate is then sprayed with a solution ofp-dimethylaminobenzaldehyde,dried in air for 5 min, and again dried for another 5 min in an oven at 75°C. The plate is then allowed to equilibrate for 25 min in a closed chamber. The color densities of the spots are read in a densitometer using the 445 nm filter. By comparing the densities of spots of the sample solutions with those of the standards, the concentration of allantoin in the solution can be calculated. This may be converted to the concentration of allantoin in serum, lymph, or urine by using the appropriate dilution factor. Results. The Rs value of allantoin in the solvent system described above was 0.23. The reproducibility of the method has been ascertained by determining allantoin in different standard solutions. The results of these determinations are given in Table 1. In order to determine the recovery of the added allantoin, known amounts of allantoin were added to dog's serum and the total allantoin content was determined. The results are given in Table 2. The average recovery of added allantoin was 93.2%.
DISCUSSION As shown in Table 1, amounts as small as 0.1/xg (0.5 mg/100 ml) can be accurately determined by this procedure. In the method described by
379
ASSAY F O R A L L A N T O I N TABLE 1 ANALYSIS OF STANDARD ALLANTO1N SOLUTIONS
Volume of solution in each spot (p,I) 20 10 10 10
Concentration of allantoin given (mg/100 ml) 0.5 1.0 3.0 4.0
Concentration of allantoin found a (mg/100 ml)
(0.10) r' (0.10) (0.30) (0.40)
0.45 0.9 3.2 4.1
_+ 0.004 _+ 0.009 -+ 0.12 _+ 0.32
(0.09) (0.09) (0.32) (0.41)
Each result is average of seven determinations, and the error is expressed as standard erFor.
Values in parentheses are the amounts of allantoin (/~g) in each spot.
Zelck (11), amounts smaller than 20 /xg (200 mg/100 ml) could not be assayed. Thus, the technique described has improved the sensitivity of the assay about 400-fold. The color produced is found to obey Beer's law in the range 0.1 to 1.0/zg of allantoin. Deviation from Beer's law is observed when the amount of allantoin increased above 1.0 tzg per spot. Under those circumstances the sample should be appropriately diluted. The conditions and precautions necessary for the increased sensitivity are discussed below. As has been demonstrated by Cline (10), we observed that the color produced in the reaction between allantoin and p-dimethylaminobenzaldehyde is dependent on the amount of acid and p-dimethylaminobenzaldehyde in the spray. Of the several concentrations ~Aed, the amount of hydrochloric acid andp-dimethylaminobenzaldehyde recommended in our procedure was found to produce the best results. The solvent in the spray also affects the color development. 1-Butanol was found to be superior to other solvents, including methanol, chloroform, ether, and acetone. The intensity of the color reaction on the plate is also influenced by the TABLE 2 RECOVERY OF ALLANTOIN A D D E D TO SERUM
Volume of serum taken (ml)
Amount of allantoin added to serum (/xg)
0.40 0.40 0.40 0.40
-10.0 20.0 30.0
Total allantoin found a (/xg) 16.2b 24.9 32.3 44.1
+ 0.85 _+ 0.57 _+ 0.76 _+ 1.2
Recovery (%) -95.0 89.2 95.5
~' Each result is an average of four determinations. b This is the amount of allantoin found in 0.4 ml serum of a mongrel dog, and corresponds to a concentration of 4.05 mg/100 ml.
380
ABRAHAM, SIMEONE AND HOPKINS
relative humidity of the atmosphere. Elaborate arrangements would be required to keep the humidity low and constant. The effect of this variable is minimized by running standards simultaneously with the unknown in each TLC plate. Other factors that influenced the color density are the drying conditions after the spraying. Drying the plate at high temperature for long periods produced high background readings. The best results are obtained when the plate is heated at 75°C for 5 min. When the plate is left exposed to air after the spraying and drying, the background readings increased, probably due to the slow air-oxidation of the reagent p-dimethylaminobenzaldehyde. This effect is minimized by storing the plate in a closed chamber until it is read. Under these circumstances, the color was stable for at least 1 hr. The sensitivity of the present assay should permit the determination of allantoin in any biological fluid in which the concentration is above 1.5 mg%. However, when allantoin concentration is lower than 1.5 rag%, the volume of the sample spotted on the plate must be increased to 20/xl. No attempt was made to increase further the volume of solution in each spot. Since allantoin is separated by TLC before the color reaction, interference from uric acid, urea, and other amido compounds is eliminated. The method is specific and selective. This assay is applicable to any fluids containing allantoin, provided the concentration of all other substances would not cause tailing of the spots when TLC is developed. Several samples can be analyzed in a relatively short time. This method is suited for the routine determination of allantoin. We have applied this method to the measurement of allantoin in blood, thoracic duct lymph, and urine of dogs subjected to a period of oligemia (12).
ACKNOWLEDGMENTS This work was supported by The Miriam Hospital and by PHS Research Grant No. GM 19930 from the National Institutes of Health.
REFERENCES 1. Christman, A. A. (1926) J. Biol. Chem. 70, 173-191. 2. Fosse, R., Brunel, A., de Graeve, P., Thomas, P. E., and Sarazin, J. (1930) C. R. Acad. Sci. 191, 1153-1155. 3. Young, E. G., and Conway, C. F. (1942)J. Biol. Chem. 142, 839-853. 4. Young, E. G., Macpherson, C. C., Wentworth, H. P., and Hawkins, W. W. (1944) J. Biol. Chem. 152, 245-253. 5. Christman, A. A., Foster, P. W., and Esterer, M. B. (1944)J. Biol. Chem. 155, 161 - 17 I. 6. Domnas, A. ( l % l ) J . Biochem. 511,46-51. 7. Stahl, A., Schang, A. M., Brogard, J. M., and Coumaros, C. (1970)Ann. Biol. Clin. 28, 377-385. 8. Willemot, J., and Parry, G. (1972)Ann. Pharm. 311, 389-390. 9. Nirmala, J., and Sastry, K. S. (1972)Anal. Biochem. 47, 218-227. 10. Cline, R. E., and Fink, R. M. (1956) Anal. Chem. 28, 47-52. 11. Zelck, U. (1%3) Biochemische Zeitschrift 337, 525-530. 12. Simeone, F. A., Abraham, J., Hopkins, R. W., and Damewood, C. A. (1975)J. Surg. Res. 19, 373-380.
