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A sensitive and convenient micromethod for estimation of urea, citrulline, and carbamyl derivatives
ANALYTICAL
BIOCHEMISTRY
A Sensitive of Urea, DONALD
16,
and
200-205 (1966)
Convenient
Citrulline,
Micromethod and
HUNNINGHAKE
From the Department
Carbamyl AND
University Kansas City, Kansas
Estimation
Derivatives
SANTIAGO
of Biochemistry,
Received March
for
GRISOLIA
of Kansas Medical
Center,
2, 1966
Most calorimetric methods presently employed for the estimation of urea, citrulline, and related compounds are based on Fearon’s method (1). Of these, the more commonly used modification and method is that of Archibald (2). The important metabolic roles of carbamyl derivatives have prompted modifications of the use of the diacetyl monoxime reagent to extend its sensitivity particularly for the estimation of carbamyl aspartate. Of these modifications, the most sensitive is that of Gerhart and Pardee (3). While this and other methods are sensitive, they have the disadvantage of being lengthy and difficult to reproduce due to the instability of the chromogen formed. Moreover, many procedures including that of Archibald do not obey Beer’s law, so that standard curves are required. Thus it appeared to be of interest to develop a reasonably reproducible and sensitive method for ureido compounds which preferably obeys Beer’s law. Such a method is presented here; this simple, sensitive, and rapid procedure is applicable to urea, carbamyl compounds, and derivatives thereof. MATERIALS
37’0 diacetyl monoxime (2,3-butanedione 2-oxime, Eastman Organic Chemicals), stored at 4O in a dark plastic container. Concentrated H,SO, (analytical grade). H,S04-FeCl, mixture (1 ml of 0.1 M FeCl, is added to 99 ml of concentrated H,SO,) . Semidine (N-phenyl-p-phenylenediamine*HCI, Eastman Organic Chemicals). A saturated solution of semidine is prepared as follows: 200 mg of semidine is mixed with 10 ml of 95% ethanol (to facilitate solution since semidine is more soluble in ethanol than in water), and then diluted to 100 ml with deionized water. After mixing, any undissolved semidine is filtered off. This solution should be stored at about 4’, and is stable for at least 6 weeks. 200
ESTIMATION
OF
CARBAMYL
201
DERIVATIVES
PROCEDURE
To a 1.0 ml sample (in a 12-20 ml test tube) are added 0.5 ml of diacetyl monoxime, 0.5 ml of H&30,, and 0.5 ml of semidine. The additions should be made in that order and then the tube contents mixed thoroughly. The samples are heated for 10 min at 90” in a constanttemperature water bath and then cooled for 5-10 min at room temperature. One ml of the H&30,-FeCl, mixture is then added with care taken to direct the mixture toward the center of the tube. After mixing and at least 5 min of cooling at room temperature, the samples are read in a Beckman DU spectrophotometer with cells of 1 cm light path. All samples are read against a reagent blank. The red color produced is stable; it does not change significantly over a 1 hr period. After heating, only a small amount of color is produced, but, after addition of the H,SOa-FeCl,, an intense red color is obtained immediately. The reagent blank has a bluish green color that changes to a more intense green after addition of the H2S04-FeCI,. RESULTS
AND DISCUSSION
A typical standard curve for urea is illustrated in Fig. 1. A linear curve is obtained in the range (0.01-0.10 pmole; although not shown, only minimal deviation of the curve is noted between 0.10 and 0.20 pmole. Figure 2 illustrates the standard curve obtained for citrulline. A linear
0.05 0.01 0.02 p MOLE OF UREA
0. IO
FIG. 1. Standard curve obtained for urea, under the standard conditions in the text. Absorbancy was read at 545 mp.
described
202
HUNNINGHAKE
AND
GRISOLIA
I .200 0 I .ooo $ 5
.800-
g
.600
zl a
.400-
/
-
0
/
/ .2ooc
,f
I/
, 0.02
I
0.04 p MOLE
FIG. scribed
2. Standard in the text.
I
0.10 OF CITRULLINE
curve obtained for citrulline under Absorbancy was read at 550 rnp.
the
0.20 standard
conditions
de-
,800 0’ 5 c$ .600 0
0.10
0.20 pMOLE
FIG. 3. Standard curve obtained described in the text. Absorbancy
0.50 OF CARBAMYL for carbamyl aspartate was read at 550 rnp.
0.75 ASPARTATE under
standard
1.0 conditions
ESTIMATION
OF
CARBAMYL
203
DERIVATIVES
curve between 0.02 and 0.20 pmole is obtained. Figure 3 illustrates the standard curve for carbamyl aspartate. The sensitivity of the method is in the range of 0.10 to 1.0 pmole. Typical absorption spectra for citrulline and urea are illustrated in Fig. 4. Table 1 illustrates the relative chromogenicity of a number of reagents. It should be noted that, under the conditions used, dihydrouracil yields
A 0
A+ / \
l.lOO-
/I
CITRULLINE UREA
‘\
, 520
540 560 WAVE’LENGTH
580
600
FIG. 4. Absorption spectra of citrulline and urea. The standard conditions described in the text were used, cxccpt that absorbancy was measured at the indicated wavelengths. Urea (0.1 pmole) and citrulline (0.2 ,umole) were used.
T.4BLE COMPARATIVE
CHROMOGENICITY
1 OF
SEVERAL
COMPOUNDS
(0.2 pmole of the indicated compounds was assayed under the standard conditions outlined in the text) Compound
Urea Citrulline Carbamyi aspartate Dihydrothymine Dihydrouracil Carbamyl @alanine Uracil Dihydroorotate Arginine Alloxan 0 Under strongly acidic conditions, carbamyl derivative (see text).
Absorbancy
at 550 III,,
1.7s 1.07 0.217 0.119 0.570 0.980 0.040 0.025 0.020 0.026 the ring of dihydrouracil
Relative
chromogenieity
1.0 0.60 0.13 0.07 0.3”” 0.55 0.02 0.01 0.01 0.01 can be opened to its
204
HUNNINGHAKE
AND
GRISOLIA
about one-half the color given by carbamyl p-alanine. The stability of dihydrouracil to acid depends greatly on the acid and conditions used. For example, under the conditions of Archibald (2) as modified by Grisolia (4)) changing the H,PO,-H,SO, mixture (from 3 to 1 to 1 to 3) opens the dihydrouracil ring readily so that no difference in chromogenicity is then noted with diacetyl monoxime between dihydropyrimidines and their related carbamyl amino acids. That the H+ effect is due to ring opening was shown by absorbancy measurements at 220 no (opening of the ring causes a decrease in absorbancy) . This method is straightforward, simple, and reasonably sensitive; also, it could be made more sensitive by changing volumes, etc. All the reagents have proved to be stable; no changes have been noted after six weeks of storage. The chromogenicity is entirely reproducible both from day to day and within the range of the standard curves presented. No variations larger than those to be expected from the sensitivity and/or accuracy of the equipment used were detected. The concentration of semidine recommended is optimal. Reducing it to one-half, while decreasing the green blank color, decreases chromogenicity approximately 40% ; doubling the amount of semidine reduces chromogenicity (about 30%). The semidine and diacetyl monoxime can be mixed beforehand and added as such. However, one cannot mix these reagents with the H,SO,. The FeCI,-H&SO, can be reduced to one-half with only moderate reduction of chromogenicity; doubling the amount reduces the color markedly. While heating may be carried out in a boiling water bath with similar results to those obtained at 90”, we found it more convenient to maintain an unattended constant-temperature water bath; also, it is not necessary to cap the tubes as is commonly done when using boiling water baths. Cooling and the mode of addition of Fe&-H,SO, are most important addition cannot be done without some cooling of the tubes or chromogenicity decreases, and they cannot be cooled below a certain temperature. For example, cooling the tubes in a water bath at either 25’ or 45’ followed by addition of the FeCI,-H*SO, reagent (when maintaining the tubes in the water bath) reduced chromogenicity. Identical samples were cooled, one at room temperature for 5 min, at which time the sample temperature was 54” ; it raised to 82’ upon addition of the Fe&H2S04 reagent. The other sample was cooled in a water bath, its temperature decreasing to 30” ; this was raised only to 52” upon addition of the FeCl,-HzOl reagent; chromogenicity was reduced about 30%. Nevertheless, if the sample cooled in the water bath was then mixed with the H2S04-FeCI, mixture out of the water bath there was an increase of some 35” and the extent of color was then satisfactory. From these and other experiments it appears that the samples should be at 30” to 60’ before
ESTIMATION
OF
CARBAMYL
DERIVATIVES
205
completion with the H2S04-FeCI, mixture and that the temperature should then be raised above 60” but not higher than 85”. Recovery of added urea to protein solutions was carried out. Deproteinization was carried out with 10% HClO, (v/v) alcohol (4 vol) and trichloroacetic acid. The latter reagent was not satisfactory since turbidity develops which interferes with color measurements. The recoveries were satisfactory, e.g., over 90% was the minimum and usually there was complete recovery in the range of 1 to 10 mg protein and with as low as 0.02 pmole urea. The following proteins were used; crystalline triosephosphate dehydrogenase, mammalian ornithine, transcarbamylase, crystalline carbamate kinase, and egg albumin. All proteins tested showed negligible chromogenicity. Indeed, addition of 1 mg of these proteins to the assay mixtures showed negligible chromogenicity. The method could be extended to many other carbamyl compounds, even to carbamyl phosphate, after its conversion to urea (5). SH reagents interfere with color development in other methods, e.g., in the Archibald procedure (2). With the present method there was negligible inhibition with addition of 1 ,umole of either cysteine or mercaptoethanol per tube; 5 pmoles produced a 33% inhibition. SUMMARY
A simple, rapid, reproducible method for the millimicromolar molar estimation of urea, citrulline, carbamyl aspartate, compounds is presented. The method obeys Beer’s law.
to microand related
ACKNOWLEDGMENTS One of us (D.H.) is a trainee under U.S.P.H.S. Training Grant number ITI GM 1342. This work has been supported also in part by U.S.P.H.S. grant number AM-01855. REFERENCES J. 33,992 (1939). Chem. 156, 121 (1944). 3. GERHART, J. C., AND PARDEE, A. B., J. Biol. Chem. Z37,&91 (1962). 4. GRISOLM, S., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 2, p. 350, Academic Press, New York, 1955. 5. ALLEN, C. M., JR., AND JONES, M. E., Biochemistry 3, 1239 (1964). 1. FEARON, W. R., Biochem. 2. ARCHIBALD, R. M., J. Biol.
BIOCHEMISTRY
A Sensitive of Urea, DONALD
16,
and
200-205 (1966)
Convenient
Citrulline,
Micromethod and
HUNNINGHAKE
From the Department
Carbamyl AND
University Kansas City, Kansas
Estimation
Derivatives
SANTIAGO
of Biochemistry,
Received March
for
GRISOLIA
of Kansas Medical
Center,
2, 1966
Most calorimetric methods presently employed for the estimation of urea, citrulline, and related compounds are based on Fearon’s method (1). Of these, the more commonly used modification and method is that of Archibald (2). The important metabolic roles of carbamyl derivatives have prompted modifications of the use of the diacetyl monoxime reagent to extend its sensitivity particularly for the estimation of carbamyl aspartate. Of these modifications, the most sensitive is that of Gerhart and Pardee (3). While this and other methods are sensitive, they have the disadvantage of being lengthy and difficult to reproduce due to the instability of the chromogen formed. Moreover, many procedures including that of Archibald do not obey Beer’s law, so that standard curves are required. Thus it appeared to be of interest to develop a reasonably reproducible and sensitive method for ureido compounds which preferably obeys Beer’s law. Such a method is presented here; this simple, sensitive, and rapid procedure is applicable to urea, carbamyl compounds, and derivatives thereof. MATERIALS
37’0 diacetyl monoxime (2,3-butanedione 2-oxime, Eastman Organic Chemicals), stored at 4O in a dark plastic container. Concentrated H,SO, (analytical grade). H,S04-FeCl, mixture (1 ml of 0.1 M FeCl, is added to 99 ml of concentrated H,SO,) . Semidine (N-phenyl-p-phenylenediamine*HCI, Eastman Organic Chemicals). A saturated solution of semidine is prepared as follows: 200 mg of semidine is mixed with 10 ml of 95% ethanol (to facilitate solution since semidine is more soluble in ethanol than in water), and then diluted to 100 ml with deionized water. After mixing, any undissolved semidine is filtered off. This solution should be stored at about 4’, and is stable for at least 6 weeks. 200
ESTIMATION
OF
CARBAMYL
201
DERIVATIVES
PROCEDURE
To a 1.0 ml sample (in a 12-20 ml test tube) are added 0.5 ml of diacetyl monoxime, 0.5 ml of H&30,, and 0.5 ml of semidine. The additions should be made in that order and then the tube contents mixed thoroughly. The samples are heated for 10 min at 90” in a constanttemperature water bath and then cooled for 5-10 min at room temperature. One ml of the H&30,-FeCl, mixture is then added with care taken to direct the mixture toward the center of the tube. After mixing and at least 5 min of cooling at room temperature, the samples are read in a Beckman DU spectrophotometer with cells of 1 cm light path. All samples are read against a reagent blank. The red color produced is stable; it does not change significantly over a 1 hr period. After heating, only a small amount of color is produced, but, after addition of the H,SOa-FeCl,, an intense red color is obtained immediately. The reagent blank has a bluish green color that changes to a more intense green after addition of the H2S04-FeCI,. RESULTS
AND DISCUSSION
A typical standard curve for urea is illustrated in Fig. 1. A linear curve is obtained in the range (0.01-0.10 pmole; although not shown, only minimal deviation of the curve is noted between 0.10 and 0.20 pmole. Figure 2 illustrates the standard curve obtained for citrulline. A linear
0.05 0.01 0.02 p MOLE OF UREA
0. IO
FIG. 1. Standard curve obtained for urea, under the standard conditions in the text. Absorbancy was read at 545 mp.
described
202
HUNNINGHAKE
AND
GRISOLIA
I .200 0 I .ooo $ 5
.800-
g
.600
zl a
.400-
/
-
0
/
/ .2ooc
,f
I/
, 0.02
I
0.04 p MOLE
FIG. scribed
2. Standard in the text.
I
0.10 OF CITRULLINE
curve obtained for citrulline under Absorbancy was read at 550 rnp.
the
0.20 standard
conditions
de-
,800 0’ 5 c$ .600 0
0.10
0.20 pMOLE
FIG. 3. Standard curve obtained described in the text. Absorbancy
0.50 OF CARBAMYL for carbamyl aspartate was read at 550 rnp.
0.75 ASPARTATE under
standard
1.0 conditions
ESTIMATION
OF
CARBAMYL
203
DERIVATIVES
curve between 0.02 and 0.20 pmole is obtained. Figure 3 illustrates the standard curve for carbamyl aspartate. The sensitivity of the method is in the range of 0.10 to 1.0 pmole. Typical absorption spectra for citrulline and urea are illustrated in Fig. 4. Table 1 illustrates the relative chromogenicity of a number of reagents. It should be noted that, under the conditions used, dihydrouracil yields
A 0
A+ / \
l.lOO-
/I
CITRULLINE UREA
‘\
, 520
540 560 WAVE’LENGTH
580
600
FIG. 4. Absorption spectra of citrulline and urea. The standard conditions described in the text were used, cxccpt that absorbancy was measured at the indicated wavelengths. Urea (0.1 pmole) and citrulline (0.2 ,umole) were used.
T.4BLE COMPARATIVE
CHROMOGENICITY
1 OF
SEVERAL
COMPOUNDS
(0.2 pmole of the indicated compounds was assayed under the standard conditions outlined in the text) Compound
Urea Citrulline Carbamyi aspartate Dihydrothymine Dihydrouracil Carbamyl @alanine Uracil Dihydroorotate Arginine Alloxan 0 Under strongly acidic conditions, carbamyl derivative (see text).
Absorbancy
at 550 III,,
1.7s 1.07 0.217 0.119 0.570 0.980 0.040 0.025 0.020 0.026 the ring of dihydrouracil
Relative
chromogenieity
1.0 0.60 0.13 0.07 0.3”” 0.55 0.02 0.01 0.01 0.01 can be opened to its
204
HUNNINGHAKE
AND
GRISOLIA
about one-half the color given by carbamyl p-alanine. The stability of dihydrouracil to acid depends greatly on the acid and conditions used. For example, under the conditions of Archibald (2) as modified by Grisolia (4)) changing the H,PO,-H,SO, mixture (from 3 to 1 to 1 to 3) opens the dihydrouracil ring readily so that no difference in chromogenicity is then noted with diacetyl monoxime between dihydropyrimidines and their related carbamyl amino acids. That the H+ effect is due to ring opening was shown by absorbancy measurements at 220 no (opening of the ring causes a decrease in absorbancy) . This method is straightforward, simple, and reasonably sensitive; also, it could be made more sensitive by changing volumes, etc. All the reagents have proved to be stable; no changes have been noted after six weeks of storage. The chromogenicity is entirely reproducible both from day to day and within the range of the standard curves presented. No variations larger than those to be expected from the sensitivity and/or accuracy of the equipment used were detected. The concentration of semidine recommended is optimal. Reducing it to one-half, while decreasing the green blank color, decreases chromogenicity approximately 40% ; doubling the amount of semidine reduces chromogenicity (about 30%). The semidine and diacetyl monoxime can be mixed beforehand and added as such. However, one cannot mix these reagents with the H,SO,. The FeCI,-H&SO, can be reduced to one-half with only moderate reduction of chromogenicity; doubling the amount reduces the color markedly. While heating may be carried out in a boiling water bath with similar results to those obtained at 90”, we found it more convenient to maintain an unattended constant-temperature water bath; also, it is not necessary to cap the tubes as is commonly done when using boiling water baths. Cooling and the mode of addition of Fe&-H,SO, are most important addition cannot be done without some cooling of the tubes or chromogenicity decreases, and they cannot be cooled below a certain temperature. For example, cooling the tubes in a water bath at either 25’ or 45’ followed by addition of the FeCI,-H*SO, reagent (when maintaining the tubes in the water bath) reduced chromogenicity. Identical samples were cooled, one at room temperature for 5 min, at which time the sample temperature was 54” ; it raised to 82’ upon addition of the Fe&H2S04 reagent. The other sample was cooled in a water bath, its temperature decreasing to 30” ; this was raised only to 52” upon addition of the FeCl,-HzOl reagent; chromogenicity was reduced about 30%. Nevertheless, if the sample cooled in the water bath was then mixed with the H2S04-FeCI, mixture out of the water bath there was an increase of some 35” and the extent of color was then satisfactory. From these and other experiments it appears that the samples should be at 30” to 60’ before
ESTIMATION
OF
CARBAMYL
DERIVATIVES
205
completion with the H2S04-FeCI, mixture and that the temperature should then be raised above 60” but not higher than 85”. Recovery of added urea to protein solutions was carried out. Deproteinization was carried out with 10% HClO, (v/v) alcohol (4 vol) and trichloroacetic acid. The latter reagent was not satisfactory since turbidity develops which interferes with color measurements. The recoveries were satisfactory, e.g., over 90% was the minimum and usually there was complete recovery in the range of 1 to 10 mg protein and with as low as 0.02 pmole urea. The following proteins were used; crystalline triosephosphate dehydrogenase, mammalian ornithine, transcarbamylase, crystalline carbamate kinase, and egg albumin. All proteins tested showed negligible chromogenicity. Indeed, addition of 1 mg of these proteins to the assay mixtures showed negligible chromogenicity. The method could be extended to many other carbamyl compounds, even to carbamyl phosphate, after its conversion to urea (5). SH reagents interfere with color development in other methods, e.g., in the Archibald procedure (2). With the present method there was negligible inhibition with addition of 1 ,umole of either cysteine or mercaptoethanol per tube; 5 pmoles produced a 33% inhibition. SUMMARY
A simple, rapid, reproducible method for the millimicromolar molar estimation of urea, citrulline, carbamyl aspartate, compounds is presented. The method obeys Beer’s law.
to microand related
ACKNOWLEDGMENTS One of us (D.H.) is a trainee under U.S.P.H.S. Training Grant number ITI GM 1342. This work has been supported also in part by U.S.P.H.S. grant number AM-01855. REFERENCES J. 33,992 (1939). Chem. 156, 121 (1944). 3. GERHART, J. C., AND PARDEE, A. B., J. Biol. Chem. Z37,&91 (1962). 4. GRISOLM, S., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 2, p. 350, Academic Press, New York, 1955. 5. ALLEN, C. M., JR., AND JONES, M. E., Biochemistry 3, 1239 (1964). 1. FEARON, W. R., Biochem. 2. ARCHIBALD, R. M., J. Biol.