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An improved method for separation of urea N and glutamine amide N for 15N metabolic studies
INALTTIC.IL
BIOCHEMISTRY
An Improved
43, l-6
(1971)
Method for Separation of Urea N and GI,utamine Amide N for 15N Metabolic Studies1
R. I’. WILSON2 Department
AND
of Biochemistry, State College, Received
R. A. BLOOMFIELD Mksi.ssippi Mi.ssissippi June
State 39762
University,
25, 1969
In the course of preliminary investigations in our laboratory on the use of the 15N isotope of nitrogen for metabolic studies it became apparent that the present method available for the separation of glutamine amide N and urea N (1) was inadequate for our studies. In this procedure, urea was hydrolyzed by urease, the urea N removed as ammonia by vacuum distillation at a pH above 10, and the glutamine amide N was then released by acid hydrolysis and removed as ammonia by vacuum distillation. The above use of the technique of alkaline diffusion or distillation for the removal and determination of ammonia has been shown to cause alkaline hydrolysis of glutamine amide N (2-4). This problem of alkaline hydrolysis of glutamine amide N became quite apparent during the separation of labeled glutamine amide 15N and, unlabeled urea N by the above methods. Therefore, this investigation was designed to modify the above method in such a manner that urea N could be separated from glutamine amide N without isotopic cont,amination. EXPERIMENTAL
Materials. n-Glutamine amide 15N, 95% enrichment, was obtained from Volk Radiochemical Company, Burbank, California. Urease, type II, 865 units/gm, was obtained from Sigma Chemical Company, St. Louis, Missouri. The Ionac C-101 resin was kindly supplied by Ionac Chemical Company, Birmingham, New Jersey. All other reagents were obt.ained from usual commercial sources. ‘From the Department of Agricultural Chemistry, lumbia. Journal Series No. 5731. Approved by the cultural Experiment Station. * Present address : Department of Biochemistry, State College, Mississippi 39762. 1 @ 1971 by
Academic
Press,
Inc.
University Director of Mississippi
of Missouri, the Missouri State
CoAgri-
University,
2
WILSON
AND
~L~O~FI~LD
Procedurre A. This procedure was carried out essentially as had been previously published (I) : 0.5 ml urease (0.03 gm/lO ml) in 0.5 Jf phosphate buffer, pH 6.5, was added to a 10 ml mixture of urea and glutamine, containing about 2 mg N from each, and incubated at 37” for 30 min. The ammonia was removed by vacuum distillation for 30 min after adjusting the pH of the solution to above 10 with 2 ml saturated borate buffer, pH 10.6, and adding 1 ml 2-octanol as an antifoam agent; the released ammonia was collected in a stanclard HCl solution. After neutralizing the residue from the urea distillation, glutamine amide N was released by adding 1 ml 10 N H,SO, and heating the solution in a boiling water bath for 11 min. The hydrolyzate was neutralized with concentrated NaOH and the ammonia was distilled in the usual fashion after adjusting the pH above 10 with NaOH rather than borate. Procedure 3. This procedure was carried out exactly as above except that concentrated NaOH was used instead of borate buffer to adjust the pH for the removal of the amlnonia after urea hydrolysis. Procedure C. This procedure was developed as a modification of procedure A in order to reduce the possibility of isotopic contamination between the two compounds. The glutamine amide N was released by adding H.,SO, sufficient to make the solution 1 M, followed by heating in a boiling water bath for 11 mins; the solution was then cooled and the pH adjusted to 7.0. The solution containing the released glutamine amide N as ammonia and the unhydrolyzed urea was then passed through a short column containing 2 gm Ionac C-101 resin for a sample containing an estimated 2 mg glutamine amide N. The resin was washed with water and the resulting effluent set aside for urea analysis. The resin was removed from the column and about 10 ml of water added, the pH adjusted to 12, and the ammonia collected in a standard HCl solution by vacuum distillation. The urea was then hydrolyzed by urease as described above. The released ammonia was then collected in the usual manner after adjusting the pH to 12 with concentrated NaOH. 15N ~e~e~~~~u~i~~s. The standard HCl solutions containing the collected ammonia from either urea N or glut,amine amide N were titrated with standard NaOH with the aid of a Beckman Expandomatic pH meter and per cent recovery calculated for each compound. The NH&J was then converted to N, gas under vacuum with NaOBr (5). The NZ gas was assayed for 15N in an isotope ratio mass spectrometer, Nuclide #6-6, RMS-2. RESULTS
AND
DISCUSSION
The Ionac C-101 resin (a modified aluminosilicate cation-exchange resin) was found to be an excellent material for the removal of free
BOLATION
OF
UREA
N
AND
GLUTAMIXE
3
S
ammonia from solutions. It was found that a column containing 1 gm of the resin would remove up to 6 mg NH, N with no detectable ammonia in the effluent. A series of 7 duplicate samples containing from 1-6 mg NH, N were passed through 1 gm samples of the resin, the resin was then removed from the column with about IO ml of water, the pH adjusted to 12 with concentrated KaOH, and the ammonia removed by vacuum distillation for 30 min, resulting in a mean of 93.78 I+ 1.18% recovery. Since there was no detectable ammonia present in the effluent from the columns, it was assumed that the loss of about 7% of the NH, N was a result of incomplete removal of the ammonia from the resin during vacuum distillation. Prolonged vacuum distillation did not consistently correct this loss. Due to the small amount of residual ammonia retained on the resin and the treatment with concentrated NaOH, the resin was discarded following each sample. additional experiments with standard hydrolyzed glutamine solutions indicated that 2 gm of the resin was required to remove the released ammonia. Also, it was found necessary to adjust the pH of the hydrolyzed sample to about 7 before passing it through the resin. Standard solutions containing a mixture of 2 mg unlabeled urea N and 2 mg labeled glutamine amide l”N were prepared and carried through the three procedures described in the experimental ,
of Glutamine Urea
Sample NO. 1 ‘2 :; 4 .i 6 hlean FS.E.
?c recovery 97.44 98.28 106.19 104.22 106.19 101.64
Amide
15N and Vrea
N by Procedure
N
At.om “/;. EN
Glut amine f,!,; changeCL
A amide
N
1:;. recovery
Atom C; L”N
(‘;, change”
0.412 0.478 0.411 0.39% 0.390 0.372
+12.57 +30.60 +12.30 +8.74 +6.56 $1.64
82.46 88.27 89.46 90.16 88.69 90.93
1 ,036 0 880 0 93.i 0 900
-9.04 -2’2.74 -17.91 -20.9%
0.900
-20.98
0.410 0.01.5
+I%.07 4.CJ6
X8.33 1.24
0 .9:20 0.0%
- 18 :3:; 2.45
C1This value represents per cent change unlabeled urea N of 0.366. * This value represents per cent, change labeled glllt,amine amide 15N of 1.139.
from
the
atom
per
cetlt
*jN
trf the
cot~trol
from
the
atom
per
cellt
W
of the
tout roI
WILSON
Separation
Mean k S.E.
Glutamine
Mean * S.E.
No.
B amide
%
%
recovery
change”
07 /O recovery
Atom y0 ‘5N
99.90 100.85 102.75 100.50 103 64 109.48 109.48 113.32
0.488 0.488 0.470 0.421 0.473 0.461 0.530
$33.3J +33.33 f28.42 + 15.03 +29.78 +2.5.96 $44.81
71.15 71.15 70.15 74.95 90.30 73.04 80.71 71.12
1.0.50 1.203 1.263 1.185 1.114 1.084 1.118 1.084
105.24 1.79
0.476 0.012
$30.09 3.40
75.32 2.46
1.138 0.026
% recovery
% change6 -7.81 f5.62 +10.89 +4.04 -2.19 -4.83 -1.84 -4.83 -0.12 2.24
the atom
per
cent
‘5N of the
control
from
the
per
cent
‘“N
control
atom
N
AtOm % ‘SN
N
from
TABLE 3 Amide 16N and Urea
of Glutamine Urea
Sample
N by Procedure
h‘
a This value represents per cent change unlabeled urea N of 0.366. b This value represents per cent change labeled glutamine amide ‘jN of 1.139.
Separation
BLOOMFIELD
TABLE 2 Amide ‘5N and Urea
of Glutamine Urea
Sample No.
AND
of the
N by Procedure
C
Glutamine
amide
% change”
% recovery
AtOm % 15N
N % changeb
78.80 84 55 73.10 88.40 89.34 92.22 91.26 90.30
0.378 0.374 0.367 0.371 0.373 0.359 0.356 0.354
+3.28 +2.19 +0.27 +1.37 +1.91 -1.91 -2.73 -3.28
89.30 92.20 91.30 94.1.5 91.26 97.97 94.14 94.14
1.054 1.153 1.161 1.141 1.088 1 .04.5 1.200 1.110
-7.46 +1.23 +1.93 +O.lS -4.48 -8.25 f5.36 -2.55
86.00 2.40
0.367 0.003
+0.14 0.87
93.06 0.94
1.119 0.019
-1.76 1.73
a This value represents per cent change unlabeled urea N of 0.366. b This value represents per cent change labeled glutamine amide l6N of 1.139.
from
the atom
per
cent
‘5N of the
control
from
the atom
per
cent
15N of t,he control
ISOLATION
OF
UREA
N
AND
GLUTAMINE
N
5
percent l”N of the separated samples compared to the atom per cent ‘“N of control samples. The data from procedure A (Table 1) show a significant amount of mixing of the N from each of the compounds during this separation procedure. The urea N showed an average increase in the atom per cent l”N of 12.07 + 4.0670, which indicates that some of the labeled N from the glutamine amide N was being hydrolyzed and collected along with the urea N, thus increasing the atom per cent 15N in the urea samples. The atom per cent l”N for the glutamine amide N showed an average decrease of 18.33 t 2.45oj0,which indicates that some of the N from the hydrolyzed urea was not removed during the vacuum distillation for urea N, thus diluting out the liiN from the glutamine amide N samples. From the data in Table 2 it is apparent in procedure B that very little of the urea N remained after vacuum distillation to alter the atom per cent liiN for the glutamine amide N sample, a 0.12 5 2.24% decrease. However, during this separation procedure, t.he alkaline hydrolysis of glutamine amide N more than doubled, i.e., the atom per cent ‘“N of urea N incrcasctl 30.09 + 3.40%. Therefore, procedure C was designed to hydrolyze t’he glutamine amide K in the pre~encc of urea and then totally remove the released ammonia before urea hydrolysis. Several urea samples were carried through the glutamine amide N hydrolyzing step with no detectable breakdown or release of ammonia. The Ionac C-101 resin was then used to remove the ammonia released following acid hydrolysis of the glutamine amide N. This couId he clone with no detectable ammonia in the effluent from the resin. The data from this procedure are shown in Table 3. Even though there are some variations in the separate samples, the average atom per cent ‘“N for the urea N and glutamine amide N are wit’hin experimental error. One must consider the accuracy of the mass spectrometer to be about +-1% for these assays. Therefore, it is suggestedthat this procedure gives the best separation of N from these two compounds for *5x incorporation st’udies. It. has also become apparent in this investigation that the purity of the sample3 cannot be predicted by considering the per cent recovery data alone. The per cent recovery data for urea in procedures A and B, however, do indicate that some of the glutamine amide N was being recovered along with the urea N. But this was not evident for the rest of the data. 11~this investigation the per cent recovery is of less importance in the separation procedure than obtaining a pure N, sample from each of the two compounds. The procedures developed in this report can easily be applied to bio-
6
WILSON
AND
BLOOMFIELD
logical samples. We are presently applying this procedure to both blood and tissue samples. For the blood samples, the Ba(OH) ,-ZnSO, protein precipitating reagent is suggested to obtain a neutral extract, followed by concentrating the extract by lyophilization and passing the resulting concentrated sample through an Ionac C-101 resin to remove the free tissue ammonia and hydrolyzed glutamine amide N released during the isolation procedure. For tissue samples, the protein is precipitated with HClO, and removed and the extract neutralized with KOH, the excess KClO, removed, and the resulting neutral protein-free extract treated as above. SUMMARY
An improved method for the separation and isolation of urea N and glutamine amide N for 15N metabolic studies has been developed. This procedure overcomes the problem of alkaline hydrolysis of glutamine amide N encountered in the other methods available. Data are presented which indicate that urea N and glutamine amide N can be separated by this procedure with a minimal isotopic mixing or contamination. Procedures are suggested for application of this method for 15N studies in both blood and tissue samples. ACKNOWLEDGMENTS The authors are grateful to M. A. Putman for his technical assistance and to Dr. R. K. Murmann for performing the ‘“N assays during this investigation. This study was supported by Grant 12540 from the National Institutes of Health. REFERENCES 1. DUDA, 2. BROWN,
G. G., AND HANDLER, P., J. Biol. Chem. 232, 303 (1958). R. H., DUDA, G. D., DORKES, S.. AND HANDLER, P., Arch. Biochem. Biophys. 66, 301 (1957). 3. NATHAN, D. G., AND WARREN, K. S.. Arch. Biochem. Biophys. 81, 377 (1959). 4. REIF, 4. E. Anal. Biochem. 1, 351 (1960). 5. RITTENBERG, D., in “Preparation and Measurement of Isotopic Tracers” (D. W. Wilson, A. 0. C. Nier, and S. P. Reimann, eds.), p. 31. J. E. Edwards, Ann Arbor, 1946.
BIOCHEMISTRY
An Improved
43, l-6
(1971)
Method for Separation of Urea N and GI,utamine Amide N for 15N Metabolic Studies1
R. I’. WILSON2 Department
AND
of Biochemistry, State College, Received
R. A. BLOOMFIELD Mksi.ssippi Mi.ssissippi June
State 39762
University,
25, 1969
In the course of preliminary investigations in our laboratory on the use of the 15N isotope of nitrogen for metabolic studies it became apparent that the present method available for the separation of glutamine amide N and urea N (1) was inadequate for our studies. In this procedure, urea was hydrolyzed by urease, the urea N removed as ammonia by vacuum distillation at a pH above 10, and the glutamine amide N was then released by acid hydrolysis and removed as ammonia by vacuum distillation. The above use of the technique of alkaline diffusion or distillation for the removal and determination of ammonia has been shown to cause alkaline hydrolysis of glutamine amide N (2-4). This problem of alkaline hydrolysis of glutamine amide N became quite apparent during the separation of labeled glutamine amide 15N and, unlabeled urea N by the above methods. Therefore, this investigation was designed to modify the above method in such a manner that urea N could be separated from glutamine amide N without isotopic cont,amination. EXPERIMENTAL
Materials. n-Glutamine amide 15N, 95% enrichment, was obtained from Volk Radiochemical Company, Burbank, California. Urease, type II, 865 units/gm, was obtained from Sigma Chemical Company, St. Louis, Missouri. The Ionac C-101 resin was kindly supplied by Ionac Chemical Company, Birmingham, New Jersey. All other reagents were obt.ained from usual commercial sources. ‘From the Department of Agricultural Chemistry, lumbia. Journal Series No. 5731. Approved by the cultural Experiment Station. * Present address : Department of Biochemistry, State College, Mississippi 39762. 1 @ 1971 by
Academic
Press,
Inc.
University Director of Mississippi
of Missouri, the Missouri State
CoAgri-
University,
2
WILSON
AND
~L~O~FI~LD
Procedurre A. This procedure was carried out essentially as had been previously published (I) : 0.5 ml urease (0.03 gm/lO ml) in 0.5 Jf phosphate buffer, pH 6.5, was added to a 10 ml mixture of urea and glutamine, containing about 2 mg N from each, and incubated at 37” for 30 min. The ammonia was removed by vacuum distillation for 30 min after adjusting the pH of the solution to above 10 with 2 ml saturated borate buffer, pH 10.6, and adding 1 ml 2-octanol as an antifoam agent; the released ammonia was collected in a stanclard HCl solution. After neutralizing the residue from the urea distillation, glutamine amide N was released by adding 1 ml 10 N H,SO, and heating the solution in a boiling water bath for 11 min. The hydrolyzate was neutralized with concentrated NaOH and the ammonia was distilled in the usual fashion after adjusting the pH above 10 with NaOH rather than borate. Procedure 3. This procedure was carried out exactly as above except that concentrated NaOH was used instead of borate buffer to adjust the pH for the removal of the amlnonia after urea hydrolysis. Procedure C. This procedure was developed as a modification of procedure A in order to reduce the possibility of isotopic contamination between the two compounds. The glutamine amide N was released by adding H.,SO, sufficient to make the solution 1 M, followed by heating in a boiling water bath for 11 mins; the solution was then cooled and the pH adjusted to 7.0. The solution containing the released glutamine amide N as ammonia and the unhydrolyzed urea was then passed through a short column containing 2 gm Ionac C-101 resin for a sample containing an estimated 2 mg glutamine amide N. The resin was washed with water and the resulting effluent set aside for urea analysis. The resin was removed from the column and about 10 ml of water added, the pH adjusted to 12, and the ammonia collected in a standard HCl solution by vacuum distillation. The urea was then hydrolyzed by urease as described above. The released ammonia was then collected in the usual manner after adjusting the pH to 12 with concentrated NaOH. 15N ~e~e~~~~u~i~~s. The standard HCl solutions containing the collected ammonia from either urea N or glut,amine amide N were titrated with standard NaOH with the aid of a Beckman Expandomatic pH meter and per cent recovery calculated for each compound. The NH&J was then converted to N, gas under vacuum with NaOBr (5). The NZ gas was assayed for 15N in an isotope ratio mass spectrometer, Nuclide #6-6, RMS-2. RESULTS
AND
DISCUSSION
The Ionac C-101 resin (a modified aluminosilicate cation-exchange resin) was found to be an excellent material for the removal of free
BOLATION
OF
UREA
N
AND
GLUTAMIXE
3
S
ammonia from solutions. It was found that a column containing 1 gm of the resin would remove up to 6 mg NH, N with no detectable ammonia in the effluent. A series of 7 duplicate samples containing from 1-6 mg NH, N were passed through 1 gm samples of the resin, the resin was then removed from the column with about IO ml of water, the pH adjusted to 12 with concentrated KaOH, and the ammonia removed by vacuum distillation for 30 min, resulting in a mean of 93.78 I+ 1.18% recovery. Since there was no detectable ammonia present in the effluent from the columns, it was assumed that the loss of about 7% of the NH, N was a result of incomplete removal of the ammonia from the resin during vacuum distillation. Prolonged vacuum distillation did not consistently correct this loss. Due to the small amount of residual ammonia retained on the resin and the treatment with concentrated NaOH, the resin was discarded following each sample. additional experiments with standard hydrolyzed glutamine solutions indicated that 2 gm of the resin was required to remove the released ammonia. Also, it was found necessary to adjust the pH of the hydrolyzed sample to about 7 before passing it through the resin. Standard solutions containing a mixture of 2 mg unlabeled urea N and 2 mg labeled glutamine amide l”N were prepared and carried through the three procedures described in the experimental ,
of Glutamine Urea
Sample NO. 1 ‘2 :; 4 .i 6 hlean FS.E.
?c recovery 97.44 98.28 106.19 104.22 106.19 101.64
Amide
15N and Vrea
N by Procedure
N
At.om “/;. EN
Glut amine f,!,; changeCL
A amide
N
1:;. recovery
Atom C; L”N
(‘;, change”
0.412 0.478 0.411 0.39% 0.390 0.372
+12.57 +30.60 +12.30 +8.74 +6.56 $1.64
82.46 88.27 89.46 90.16 88.69 90.93
1 ,036 0 880 0 93.i 0 900
-9.04 -2’2.74 -17.91 -20.9%
0.900
-20.98
0.410 0.01.5
+I%.07 4.CJ6
X8.33 1.24
0 .9:20 0.0%
- 18 :3:; 2.45
C1This value represents per cent change unlabeled urea N of 0.366. * This value represents per cent, change labeled glllt,amine amide 15N of 1.139.
from
the
atom
per
cetlt
*jN
trf the
cot~trol
from
the
atom
per
cellt
W
of the
tout roI
WILSON
Separation
Mean k S.E.
Glutamine
Mean * S.E.
No.
B amide
%
%
recovery
change”
07 /O recovery
Atom y0 ‘5N
99.90 100.85 102.75 100.50 103 64 109.48 109.48 113.32
0.488 0.488 0.470 0.421 0.473 0.461 0.530
$33.3J +33.33 f28.42 + 15.03 +29.78 +2.5.96 $44.81
71.15 71.15 70.15 74.95 90.30 73.04 80.71 71.12
1.0.50 1.203 1.263 1.185 1.114 1.084 1.118 1.084
105.24 1.79
0.476 0.012
$30.09 3.40
75.32 2.46
1.138 0.026
% recovery
% change6 -7.81 f5.62 +10.89 +4.04 -2.19 -4.83 -1.84 -4.83 -0.12 2.24
the atom
per
cent
‘5N of the
control
from
the
per
cent
‘“N
control
atom
N
AtOm % ‘SN
N
from
TABLE 3 Amide 16N and Urea
of Glutamine Urea
Sample
N by Procedure
h‘
a This value represents per cent change unlabeled urea N of 0.366. b This value represents per cent change labeled glutamine amide ‘jN of 1.139.
Separation
BLOOMFIELD
TABLE 2 Amide ‘5N and Urea
of Glutamine Urea
Sample No.
AND
of the
N by Procedure
C
Glutamine
amide
% change”
% recovery
AtOm % 15N
N % changeb
78.80 84 55 73.10 88.40 89.34 92.22 91.26 90.30
0.378 0.374 0.367 0.371 0.373 0.359 0.356 0.354
+3.28 +2.19 +0.27 +1.37 +1.91 -1.91 -2.73 -3.28
89.30 92.20 91.30 94.1.5 91.26 97.97 94.14 94.14
1.054 1.153 1.161 1.141 1.088 1 .04.5 1.200 1.110
-7.46 +1.23 +1.93 +O.lS -4.48 -8.25 f5.36 -2.55
86.00 2.40
0.367 0.003
+0.14 0.87
93.06 0.94
1.119 0.019
-1.76 1.73
a This value represents per cent change unlabeled urea N of 0.366. b This value represents per cent change labeled glutamine amide l6N of 1.139.
from
the atom
per
cent
‘5N of the
control
from
the atom
per
cent
15N of t,he control
ISOLATION
OF
UREA
N
AND
GLUTAMINE
N
5
percent l”N of the separated samples compared to the atom per cent ‘“N of control samples. The data from procedure A (Table 1) show a significant amount of mixing of the N from each of the compounds during this separation procedure. The urea N showed an average increase in the atom per cent l”N of 12.07 + 4.0670, which indicates that some of the labeled N from the glutamine amide N was being hydrolyzed and collected along with the urea N, thus increasing the atom per cent 15N in the urea samples. The atom per cent l”N for the glutamine amide N showed an average decrease of 18.33 t 2.45oj0,which indicates that some of the N from the hydrolyzed urea was not removed during the vacuum distillation for urea N, thus diluting out the liiN from the glutamine amide N samples. From the data in Table 2 it is apparent in procedure B that very little of the urea N remained after vacuum distillation to alter the atom per cent liiN for the glutamine amide N sample, a 0.12 5 2.24% decrease. However, during this separation procedure, t.he alkaline hydrolysis of glutamine amide N more than doubled, i.e., the atom per cent ‘“N of urea N incrcasctl 30.09 + 3.40%. Therefore, procedure C was designed to hydrolyze t’he glutamine amide K in the pre~encc of urea and then totally remove the released ammonia before urea hydrolysis. Several urea samples were carried through the glutamine amide N hydrolyzing step with no detectable breakdown or release of ammonia. The Ionac C-101 resin was then used to remove the ammonia released following acid hydrolysis of the glutamine amide N. This couId he clone with no detectable ammonia in the effluent from the resin. The data from this procedure are shown in Table 3. Even though there are some variations in the separate samples, the average atom per cent ‘“N for the urea N and glutamine amide N are wit’hin experimental error. One must consider the accuracy of the mass spectrometer to be about +-1% for these assays. Therefore, it is suggestedthat this procedure gives the best separation of N from these two compounds for *5x incorporation st’udies. It. has also become apparent in this investigation that the purity of the sample3 cannot be predicted by considering the per cent recovery data alone. The per cent recovery data for urea in procedures A and B, however, do indicate that some of the glutamine amide N was being recovered along with the urea N. But this was not evident for the rest of the data. 11~this investigation the per cent recovery is of less importance in the separation procedure than obtaining a pure N, sample from each of the two compounds. The procedures developed in this report can easily be applied to bio-
6
WILSON
AND
BLOOMFIELD
logical samples. We are presently applying this procedure to both blood and tissue samples. For the blood samples, the Ba(OH) ,-ZnSO, protein precipitating reagent is suggested to obtain a neutral extract, followed by concentrating the extract by lyophilization and passing the resulting concentrated sample through an Ionac C-101 resin to remove the free tissue ammonia and hydrolyzed glutamine amide N released during the isolation procedure. For tissue samples, the protein is precipitated with HClO, and removed and the extract neutralized with KOH, the excess KClO, removed, and the resulting neutral protein-free extract treated as above. SUMMARY
An improved method for the separation and isolation of urea N and glutamine amide N for 15N metabolic studies has been developed. This procedure overcomes the problem of alkaline hydrolysis of glutamine amide N encountered in the other methods available. Data are presented which indicate that urea N and glutamine amide N can be separated by this procedure with a minimal isotopic mixing or contamination. Procedures are suggested for application of this method for 15N studies in both blood and tissue samples. ACKNOWLEDGMENTS The authors are grateful to M. A. Putman for his technical assistance and to Dr. R. K. Murmann for performing the ‘“N assays during this investigation. This study was supported by Grant 12540 from the National Institutes of Health. REFERENCES 1. DUDA, 2. BROWN,
G. G., AND HANDLER, P., J. Biol. Chem. 232, 303 (1958). R. H., DUDA, G. D., DORKES, S.. AND HANDLER, P., Arch. Biochem. Biophys. 66, 301 (1957). 3. NATHAN, D. G., AND WARREN, K. S.. Arch. Biochem. Biophys. 81, 377 (1959). 4. REIF, 4. E. Anal. Biochem. 1, 351 (1960). 5. RITTENBERG, D., in “Preparation and Measurement of Isotopic Tracers” (D. W. Wilson, A. 0. C. Nier, and S. P. Reimann, eds.), p. 31. J. E. Edwards, Ann Arbor, 1946.