- Email: [email protected]
A new method for allantoin determination and its application in allantoin determination in Agrostemma githago L. seed
ANALYTICAL
BIOCHEMISTRY
91, 304-308 (1978)
A New Method for Allantoin Determination Application in Allantoin Determination Agrostemma githago L. Seed MILADIN Institute
M. VRBASKI, for Biological
BOJANA
Research.
GRUJIC-INJAC,
of Science,
Faculty
and its in
AND DANICA 11000 Belgrade,
GAJIC
Yugoslavia
Received March 30. 1978 We have established several optimal conditions for qualitative and quantitative allantoin determination by applying Ehrlich’s reagent. The limit of detection for allantoin determination amounts to 5 x 10m6mM. Allantoin is determined quantitatively by measuring the absorbance at 440 nm (from 300 to 1000 &ml). The color of the complex becomes stable by standing for IO min at room temperature. We have used these conditions for allantoin determination in Agrostrmma githago seed.
In higher plants allantoin is an important product of nucleoprotein catabolism. In addition, allantoin has a very important role as a urinary indicator in purine catabolism. Several methods for allantoin determination are known. Most of the methods are based on its determination via alantoic acid (l-3). In this method, allantoin is converted to alantoic acid by mild alkaline hydrolysis, followed by acid hydrolysis of the latter to urea plus glyoxylic acid. Allantoin can also be isolated from higher plants such as Aesculus hippocastanum (4) and corn cockle (as the representative of weed species) (5,6). Allantoin determination according to this method is based on the reaction of alantoic acid with 2,4-dinitrophenylhydrazine in hydrochloric acid. Some authors used p-dimethylaminobenzaldehyde for quantitative allantoin determination (7,8). Abraham ef al. (9) separated allantoin by thinlayer chromatography and spraying with an acidic solution of p-dimethylaminobenzaldehyde. This method can be utilized for the estimation of allantoin in serum, lymph, and urine. We have applied Ehrlich’s reagent for allantoin determination in plant material. MATERIALS
AND METHODS
p-Dimethylaminobenzaldehyde of analytical grade (Serva, Heidelberg) was used in the experiment and absorbance was read on a Beckman DU-2 0003-2697/78/0911-0304$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
304
312
.I. GABARRO
S is the surface of the Gaussian curve and is proportional to the length of the segment of the molecule contributing to this mode. AT, the mean width of the function, which according to certain authors (13) would be related to the standard deviation about the mean value of base composition inside the thermosome. The main concern of this paper is the extraction of the physical information underlying the melting curves, that is, transformation of the rough data as they come out from the apparatus into a sum of well-characterized Gaussian functions. In order to carry out this process we must go through the following steps: (a) The experimental equipment yields the optical density as a function of the temperature (Fig. la): however, as already mentioned, the derivative of the optical density dOD/dT vs T is a better representation, both for the visualization and the parameterization of the modes. Thus we must calculate the derivative dOD/dT of the data yielded by the measuring device. In addition, to analyze the data by means of the finite Fourier transform as we will show later, a new set of points with abscissas at equally spaced intervals must be obtained by interpolation from the experimental data set. (b) As we can observe from Fig. lb, noise due to experimental conditions is superposed to the signal. This noise can be properly eliminated by convolving the data set with an appropriate numerical filter, obtained as the solution of a least-squares integral equation. (c) A powerful algorithm, related to the Fourier transform is used to determine the number of thermosomes being present on the sample, as well as their approximate mean melting temperature T,. (d) In order to obtain the complete set of parameters concerning the thermosomes (surface S, mean width AT, and the exact mean melting temperature T,) a curve-fitting procedure is used in a final step. INTERPOLATION
OF THE DATA
As stated above we have a set of N experimental points characterized by two coordinates: the temperature T on the abscissa and the optical density OD in the ordinate. We want to transform {Ti,ODi} (i = 1, . . . , N), into a new set of N’ points { Ti’,(dOD/dT)i’} (i = 1, . . . , N I), where dODldT is the derivative of the optical density (hereafter we will refer to dOD/dT by the symbol Y) and Ti+l’ - Ti’ = 6T (6T is a constant which is usually chosen as the mean interval between consecutive temperatures, ST = (Tly - T,)I(N - 1); we also have T,’ = T1, and N’ = [(T,%, - T&ST] + 1). In obtaining this new set of points, we follow an algorithm devised by Akima (4), in which we interpolate a third-degree polynomial between two consecutive points. The polynomial passing through points i and i + 1 is:
VRBASKI.
306
GRUJIC-INJAC
AND GAJIC
standing for 10 min at 80°C. Glass plates 200 x 200 mm and 180 x 300 mm as well as silica gel (SiO, x G) (according to Stahl, Merck product) were used for thin-layer chromatography. RESULTS AND DtSCUSSiON
The limit of detection for qualitative allantoin determination on thinlayer chromatography with the application of Ehrlich’s reagent is 5 x lop6 mM. The absorbance maximum of Ehrlich’s reagent and that of the Ehrlich’s reagent + allantoin complex are very close when distilled water (380-400 nm) is used as a reference solution. However, when Ehrlich’s reagent is used as a reference solution. However, when Ehrlich’s reagent is used as a reference solution the maximum for Ehrlich’s reagent + allantoin complex is moved to 440 nm (Fig. I). The absorbance at 440 nm (Fig. 2) reveals that the color of the complex is stable over the interval from 10 to 30 min, after which it becomes slightly unstable up to 60th min. This fact indicates that after 10 min spectrophotometric measurements already may be performed. It was found that the stability of the color of the Ehrlich’s reagent + ailantoin complex is temperature dependent, i.e., the complex is stable over the temperature range from 15 to 40°C. The complex becomes unstable at temperatures over 40” and readings of absorbance do not give adequate results (Fig. 3). The results obtained concerning limits of detection, in which the Lamber-Beer law is applicable, show that allantoin can be measured, un-
1. to 1 lx?0 ,io.w 0 v 0.60 2 & 2 0.70 060 --
0
‘---‘-.-.-.
!O
20
30
LO
50
60
~/ME(minufesl
FIG. 2. The stability of the atlantain + Ehrlich’s reagent complex and its dependence upon time. The coior is stable over the interval from 10 to 30 min.
ALLANTOIN
DETERMINATION
307
.I__.
. l
o
10
15 18
* l
l
22 25 2830
35
*
40
SO
60
T
FIG. 3. The stability of the allantoin + Ehrlich’s reagent complex and its dependence upon temperature. The complex is stable over the temperature range from 15 to 40°C.
der the conditions specified above, in concentrations from 300 to 1000 pg/rnl (Fig. 4). The reproducibility of this method ranges from 95 to 98.9% the average reproducibility being 96.95%. The results obtained also reveal that allantoin can be determined by one relatively rapid method, i.e.. by applying Ehrlich’s reagent, reading the
130. 120. IlOIOO-
$090. r: v t,
z
080?
O?O060.
2 050-
0.0
0 10
020
030
OLO
050
060
070
080
090
I. 00 f-rig/ml
C~~CE~~~~liO~
FIG. 4. Quantitative binding of allantoin with Ehriich’s reagent
308
VRBASKI.
GRUJIt-INJAC
AND GAJIt
absorbance at 440 nm; and achieving the stability of the complex. Our micro-method was used for allantoin measurement in corn cockle seed, and it is probably also applicable, with certain modifications, to the allantoin determination in other plant species, which would be useful due to its role in the catabolism of some organic compounds of living organisms. REFERENCES 1. Young, E. G., and Conway. C. F. (1942) J. Biol. Chem. 142, 839. 2. Pentz, E. I. (1969) Anal. Biochem. 27, 333. 3. Botchers. R. (1977) Anal. Biochem. 79, 612. 4. Schultze. E., and Barbieri. J. (1881) Ber. 14, 1602. 5. Foss, R., and Hieulle. A. (1924) Camp. Rend. 179, 636. 6. Foss. R.. Brunei, A.. and Zhomas, P. E. (1931) Camp. Rend. 192, 1615. 7. Christman, A. A., Foster, W. P., and Esterer. B. M.. (1944) J. Biol. Chem. 155, 161. 8. Zimmerman, A. (1956) Natunc,issensc,ha~en 43, 399. 9. Abraham. J., Simeone. F. A., and Hopkins, R. W. (1976) AIla/. B&hem. 70, 377. 10. Fink, K., Cline, R. E.. and Fink, R. M. (1963) Anal. Chem. 35, 389. 11. VrbaSki, M. M. (1974) Master thesis, Faculty of Science, University of Belgrade.
BIOCHEMISTRY
91, 304-308 (1978)
A New Method for Allantoin Determination Application in Allantoin Determination Agrostemma githago L. Seed MILADIN Institute
M. VRBASKI, for Biological
BOJANA
Research.
GRUJIC-INJAC,
of Science,
Faculty
and its in
AND DANICA 11000 Belgrade,
GAJIC
Yugoslavia
Received March 30. 1978 We have established several optimal conditions for qualitative and quantitative allantoin determination by applying Ehrlich’s reagent. The limit of detection for allantoin determination amounts to 5 x 10m6mM. Allantoin is determined quantitatively by measuring the absorbance at 440 nm (from 300 to 1000 &ml). The color of the complex becomes stable by standing for IO min at room temperature. We have used these conditions for allantoin determination in Agrostrmma githago seed.
In higher plants allantoin is an important product of nucleoprotein catabolism. In addition, allantoin has a very important role as a urinary indicator in purine catabolism. Several methods for allantoin determination are known. Most of the methods are based on its determination via alantoic acid (l-3). In this method, allantoin is converted to alantoic acid by mild alkaline hydrolysis, followed by acid hydrolysis of the latter to urea plus glyoxylic acid. Allantoin can also be isolated from higher plants such as Aesculus hippocastanum (4) and corn cockle (as the representative of weed species) (5,6). Allantoin determination according to this method is based on the reaction of alantoic acid with 2,4-dinitrophenylhydrazine in hydrochloric acid. Some authors used p-dimethylaminobenzaldehyde for quantitative allantoin determination (7,8). Abraham ef al. (9) separated allantoin by thinlayer chromatography and spraying with an acidic solution of p-dimethylaminobenzaldehyde. This method can be utilized for the estimation of allantoin in serum, lymph, and urine. We have applied Ehrlich’s reagent for allantoin determination in plant material. MATERIALS
AND METHODS
p-Dimethylaminobenzaldehyde of analytical grade (Serva, Heidelberg) was used in the experiment and absorbance was read on a Beckman DU-2 0003-2697/78/0911-0304$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
304
312
.I. GABARRO
S is the surface of the Gaussian curve and is proportional to the length of the segment of the molecule contributing to this mode. AT, the mean width of the function, which according to certain authors (13) would be related to the standard deviation about the mean value of base composition inside the thermosome. The main concern of this paper is the extraction of the physical information underlying the melting curves, that is, transformation of the rough data as they come out from the apparatus into a sum of well-characterized Gaussian functions. In order to carry out this process we must go through the following steps: (a) The experimental equipment yields the optical density as a function of the temperature (Fig. la): however, as already mentioned, the derivative of the optical density dOD/dT vs T is a better representation, both for the visualization and the parameterization of the modes. Thus we must calculate the derivative dOD/dT of the data yielded by the measuring device. In addition, to analyze the data by means of the finite Fourier transform as we will show later, a new set of points with abscissas at equally spaced intervals must be obtained by interpolation from the experimental data set. (b) As we can observe from Fig. lb, noise due to experimental conditions is superposed to the signal. This noise can be properly eliminated by convolving the data set with an appropriate numerical filter, obtained as the solution of a least-squares integral equation. (c) A powerful algorithm, related to the Fourier transform is used to determine the number of thermosomes being present on the sample, as well as their approximate mean melting temperature T,. (d) In order to obtain the complete set of parameters concerning the thermosomes (surface S, mean width AT, and the exact mean melting temperature T,) a curve-fitting procedure is used in a final step. INTERPOLATION
OF THE DATA
As stated above we have a set of N experimental points characterized by two coordinates: the temperature T on the abscissa and the optical density OD in the ordinate. We want to transform {Ti,ODi} (i = 1, . . . , N), into a new set of N’ points { Ti’,(dOD/dT)i’} (i = 1, . . . , N I), where dODldT is the derivative of the optical density (hereafter we will refer to dOD/dT by the symbol Y) and Ti+l’ - Ti’ = 6T (6T is a constant which is usually chosen as the mean interval between consecutive temperatures, ST = (Tly - T,)I(N - 1); we also have T,’ = T1, and N’ = [(T,%, - T&ST] + 1). In obtaining this new set of points, we follow an algorithm devised by Akima (4), in which we interpolate a third-degree polynomial between two consecutive points. The polynomial passing through points i and i + 1 is:
VRBASKI.
306
GRUJIC-INJAC
AND GAJIC
standing for 10 min at 80°C. Glass plates 200 x 200 mm and 180 x 300 mm as well as silica gel (SiO, x G) (according to Stahl, Merck product) were used for thin-layer chromatography. RESULTS AND DtSCUSSiON
The limit of detection for qualitative allantoin determination on thinlayer chromatography with the application of Ehrlich’s reagent is 5 x lop6 mM. The absorbance maximum of Ehrlich’s reagent and that of the Ehrlich’s reagent + allantoin complex are very close when distilled water (380-400 nm) is used as a reference solution. However, when Ehrlich’s reagent is used as a reference solution. However, when Ehrlich’s reagent is used as a reference solution the maximum for Ehrlich’s reagent + allantoin complex is moved to 440 nm (Fig. I). The absorbance at 440 nm (Fig. 2) reveals that the color of the complex is stable over the interval from 10 to 30 min, after which it becomes slightly unstable up to 60th min. This fact indicates that after 10 min spectrophotometric measurements already may be performed. It was found that the stability of the color of the Ehrlich’s reagent + ailantoin complex is temperature dependent, i.e., the complex is stable over the temperature range from 15 to 40°C. The complex becomes unstable at temperatures over 40” and readings of absorbance do not give adequate results (Fig. 3). The results obtained concerning limits of detection, in which the Lamber-Beer law is applicable, show that allantoin can be measured, un-
1. to 1 lx?0 ,io.w 0 v 0.60 2 & 2 0.70 060 --
0
‘---‘-.-.-.
!O
20
30
LO
50
60
~/ME(minufesl
FIG. 2. The stability of the atlantain + Ehrlich’s reagent complex and its dependence upon time. The coior is stable over the interval from 10 to 30 min.
ALLANTOIN
DETERMINATION
307
.I__.
. l
o
10
15 18
* l
l
22 25 2830
35
*
40
SO
60
T
FIG. 3. The stability of the allantoin + Ehrlich’s reagent complex and its dependence upon temperature. The complex is stable over the temperature range from 15 to 40°C.
der the conditions specified above, in concentrations from 300 to 1000 pg/rnl (Fig. 4). The reproducibility of this method ranges from 95 to 98.9% the average reproducibility being 96.95%. The results obtained also reveal that allantoin can be determined by one relatively rapid method, i.e.. by applying Ehrlich’s reagent, reading the
130. 120. IlOIOO-
$090. r: v t,
z
080?
O?O060.
2 050-
0.0
0 10
020
030
OLO
050
060
070
080
090
I. 00 f-rig/ml
C~~CE~~~~liO~
FIG. 4. Quantitative binding of allantoin with Ehriich’s reagent
308
VRBASKI.
GRUJIt-INJAC
AND GAJIt
absorbance at 440 nm; and achieving the stability of the complex. Our micro-method was used for allantoin measurement in corn cockle seed, and it is probably also applicable, with certain modifications, to the allantoin determination in other plant species, which would be useful due to its role in the catabolism of some organic compounds of living organisms. REFERENCES 1. Young, E. G., and Conway. C. F. (1942) J. Biol. Chem. 142, 839. 2. Pentz, E. I. (1969) Anal. Biochem. 27, 333. 3. Botchers. R. (1977) Anal. Biochem. 79, 612. 4. Schultze. E., and Barbieri. J. (1881) Ber. 14, 1602. 5. Foss, R., and Hieulle. A. (1924) Camp. Rend. 179, 636. 6. Foss. R.. Brunei, A.. and Zhomas, P. E. (1931) Camp. Rend. 192, 1615. 7. Christman, A. A., Foster, W. P., and Esterer. B. M.. (1944) J. Biol. Chem. 155, 161. 8. Zimmerman, A. (1956) Natunc,issensc,ha~en 43, 399. 9. Abraham. J., Simeone. F. A., and Hopkins, R. W. (1976) AIla/. B&hem. 70, 377. 10. Fink, K., Cline, R. E.. and Fink, R. M. (1963) Anal. Chem. 35, 389. 11. VrbaSki, M. M. (1974) Master thesis, Faculty of Science, University of Belgrade.