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The recovery of amino acids from acid hydrolysates of whole urine from patients with thermal burns
CLINJCA
THE
CHIMICA
421
ACTA
RECOVERY
WHOLE
URINE
FRANCES
L. ESTES
OF AMINO FROM
ACIDS
PATIENTS
AND ROBERT
FROM WITH
ACID
HYDROLYSATES
THERMAL
OF
BURNS
L. WETZEL
The University of Texas, Medical Bvanch, Galveston. Texas (U.S.A.) (Receixwl
September
2 Ist,
1966)
SUMMARY
Whole urine samples, hydrolyzed with 6 N HCl at relationship
between
For maximum proline,
obtained from for varying
the hydrolysis
recovery
and glutamic
patients after a thermal burn, were periods of time. There was no uniform
100’
time and the recovery
the hydrolysis
times
acid to 24 h for valine,
varied
of any of the amino acids.
from 3 h, for aspartic
isoleucine,
acid,
leucine and phenylalanine.
The maximum recoveries of the basic amino acids were achieved with hydrolysis times of 12 h or less. Determination of the basic amino acids was complicated by the formation of considerable ammonia, in the sample after 46 h hydrolysis.
which accounted
for nearly 50% of the nitrogen
The observations indicate the diverse nature of the peptide components of urine, and delineate the difficulties involved in determining the total concentration of any of the amino acids.
INTRODUCTION
For the determination HCl for IS h under reflux at
of combined amino acids in urine, hydrolysis with 6 N (ref. I), and for 20-24 h in sealed tubes at 105-110”
125’
(ref. 2) are representative of the conditions used. The amount of the individual amino acids found in whole urine obtained from burn patients after hydrolysis with 6 N HCl for 20 h in sealed tubes at 100~ was less than the sum of those found in separated fractions of the urine hydrolyzed under the same conditions for 8 h (ref. 3). For aspartic acid, glutamic acid, and lysine one of the fractions accounted for more than was recovered from the whole urine. These observations suggested that the effect of hydrolysis time on whole urine samples should be examined. The amount of each amino acid recovered from a peptide or a protein is dependent on the hydrolysis conditions used. For individual proteins, samples hydrolyzed for varying periods show the yield of the amino acid to be a function of time. This permits an extrapolation of the values, either to zero or to infinite time, to correct for Supported
by U. S. Public
Health
Research
Grant
GM 10903.
Clin. Chim. Ada,
15 (1967) 421-427
422
ESTES, WETZEL
the effect of hydrolysis
time on the destruction
or release of amino acids. The extent
of the destruction, however, is a function of the composition of the protein concentration in the hydrolysis mixture, as well as the hydrolysis conditions. Although
they cannot
of urine samples
be regarded
from burn patients
in the investigation
of materials
as typical,
should indicate
examination
and its
of the hydrolysis
some of the problems
involved
to be found in urine. It could not be expected
that
the hydrolytic recovery of any of the amino acids would be linear with time since more than one peptide sequence must be assumed to be present, and the amino acids in the unhydrolyzed
urine cannot
be assumed
to be unreactive.
Furthermore,
the
hydrolysis of these samples was complicated by the presence of large amounts of carbohydrate materials, and material(s) which yields considerable ammonia even on mild hydrolysis. Previous observations had indicated that for 18-h hydrolysis larger amounts of both acidic and basic amino acids were recovered with 6 N HCl than with 3 N HCl even though observations
the ammonia
should be examined. limited
formation
with urine fractions
was considerably
had indicated
Since it was not possible to examine
to hydrolysis
greater.
that hydrolysis
Likewise,
previous
times of less than 18 h
all conditions,
this study was
with 6 N HCl at 100“.
METHOD
Twenty-four night and filtered.
urine samples were acidified to pH 2 with HC.1, refrigerated overFor the aliquots of the sample to be used in the determination of
the basic amino acids, ammonia was removed by neutralizing
the aliquot to pH IO-II
and desiccating over solid NaOH under reduced pressure. After restoring the volume and reacidification, the samples were hydrolyzed with 6 N HCI in tubes seaIed under nitrogen,
at 100~ for the indicated
times.
Immediately
after hydrolysis
the samples
were removed from the tubes and the HCl removed by rotary evaporation. For analysis, the samples were dissolved in pH 2.2 citrate buffer (0.2 1v) to a final volume of 5.0 ml. For those hydrolysates in which the removal of ammonia was required for the determination of the basic amino acids, the samples were dissolved in water, adjusted to pH II with sodium hydroxide and the anlmonia removed under reduced pressure. The dry sample was taken up in 5.0 ml of pH 2.2 citrate buffer (0.2 N). The amino acids were determined, on the Beckman Spinco Amino
dcid
Analyzer Model 12oB, by the method of Spackman et aL4. For each sample appropriate aliquots (0.5 to 2.0 ml) were placed on the column. The acidic and neutral amino acids were determined on the 150 x 0.9 cm column, the temperature was raised from 30 to 56” and the buffer changed from pH 3.28 citrate buffer (0.2 N) to pH 4.25 citrate buffer (0.2 N) after 307.5 ml (IO I/* h) of eluant flow. The basic amino acids were determined on the 50 x 0.9 cm column with the temperature change after 340 ml (II '/3 h) of pH 4.26 citrate buffer (0.38 L%‘).Ammonia was determined on the 15 x 0.9 cm column with an appropriate dilution of the sample, usually I :IOO, using pH 5.~8 citrate buffer (0.35 N) at 56”. Total nitrogen was determined by the classical Dumas method utilizing a Colman Nitrogen Analyzer.
Clin. Chim.
Acta,
15
(1967)421-+z7
RECOVERY OF AMINO ACIDS IN PATIENTS WITH THERMAL BURNS
423
RESULTS AND DISCUSSION
For all hydrolysis times considerable humin was present. When the humin was removed by centrifugation before the analysis, lower values for the amino acid concentrations were obtained. Apparently some of the amino acids were washed out of the humin on the column. All values shown were determined without removal of the humin. The observed values for the urea, creatinine, and ammonia recovered have been shown in Table I, together with the amino acids. The data has been reported in micromoles per milliliter of urine, so that the differences in recoveries of the amino acids at the various hydrolysis times would not be unduly magnified. For the sample in which each could be evaluated, the glycine concentration appeared to be approximately ten times that of alanine. The large amounts involved, however, did not permit good separation of the peaks for these amino acids. Although dilution of the sample permitted better evaluation of the glycine, the resulting alanine peak was not regarded as yielding more than an approximation of the amount present. TABLE
I
THE EFFBCT OF HYDROLYSIS TIME ON THE RECOVERY OF AntINo r\CIDSFROM POST BURN URINE*
(Micromoles per ml) Hydrolysis 0
Taurine Hydroxyprolinc Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alaninc Valine lsoleucine Leucine Tyrosine Phenylalanine P-Alanine
PBNC*** I.704 PBNC PBNC Tt 0.218
0.240 0.204 0.166 PBNC
Hydroxylysine Ornithinc Lysine I-Methvlhistidine Histidine 3-Methylhistidine Arginine
0.058 0.252 0.188 1.098 0.482
Total, //moles recovered Urea Creatinine Ammonia * ** *** f
6
0.916 0.878
I.092 o.gor 2.218 I.*04 1.706 2.076 3.350 r9.770
2.550
1.7*0 2.272 4.238 20.632 0.388 0.410 0.702 0.744 0.622 0.132
0.497 PBNC 0.922 0.658 0.766 0.226
36.204
Total, /smoles recovered
0.182 o. 106 2.996 I.504 1.646 0.832 0.278
0.258 0.162 PBNC PBNC I.634 1.882 0.458
7.544 7.969 0.278
Extrapolation
time in hours 3
8.900 0.446 33.680
8.867 0.790 z5.037
I2
24
48
7.677
0.760 0.052 1.607 0.602 0.972 1.670 3.002 12.955
2.415 0.496 0.526 0.888 0.626 0.454 0.116
0.927 0.602 1.062 0.902 0.817 0.180
0.477 0.312 0.577 0.392 0.367 0.100
1.4 (24) 0.9 (48)
47.136
26.405
0.384 0.200 1.178 0.317 1.506 0.509 0.360
0.121 0.133 0.650 0.180 0.235 0.238 0.296
0.135 0.197
0.65 (24)
4.464
I.853
8.750 0.356 405.8
2.935 0.097 456.0
1.036 0.760 2.136 0.860 I.470 2.116 3.980 22.507
0.347
0.125 2.332 I .97o 2.657 I .660 2.212
I.4 (24)** 3.2 (48)
I.385 0.397 0.410
0.060 0.285 510.7
14.3 (24)
Seven days after 34% burn (177; 3”). Total volume 1800 ml (1.59”/& N). Bracket number indicates maximum time used for the extrapolation. PBNC = Present but not calculable, usually because the peak was distorted. T = Trace. Clin. Chim. Acta,
15 (1967) 421-427
424
ESTES, WETZEL
The appearance
of the peak suggested
the possibility
been eluted along with the alanine. The diffused conspicuous in the unhydrolyzed samples.
that other material
glycine-alanine
may have
peaks were most
Although the maximuln recoveries for threonine, serine, valine, leucine and phenylalanine were observed after 24 h hydrolysis, the total amino acids recovered were 44% less than with 12 h hydrolysis. concentration
Most conspicuous
was the decrease in glycine
at 24 h. For the basic amino acids maximum
recoveries
were obtained
with no more than 12 h hydrolysis. For most of the amino acids included in Table I, more than one maximum
was
observed. This was most apparent in the case of serine, which is known to break down rapidlyb. Since the yield of the amino acids was not a direct function of time, determination of the amount initially present by extrapolation became a questionable procedure.
In general,
the amino acids appeared
to fall into two groups;
those which
disappeared rapidly between 12 and 24 h and those which disappeared most rapidly between 24 and 48 h hydroIysis. Extrapolated values for representatives of each group have been included
in Table
I. These values are probably
a better
indication
of the rate of breakdown between the times in question than of the initial values. The effect of extrapolating from selected hydrolysis times is most conspicuous
in
the case of lysine in which the maximum observed recovery was after 3 h hydrolysis. Extrapolation using the observed amounts recovered at 12 and 24 h hydrolysis would indicate that 1.6 ymole was initially present whereas extrapolation using the values for 3 and 12 h hydrolysis, 3.6 pmoles would have seemed to be present initially. The non-linear effect of hydrolysis time on the recovery of each of the amino acids does not justify extrapolation of the concentrations to a zero time value and suggested that each of them was involved in more than one peptide sequence. This also serves to indicate
the limits of reliability
of comparison
among the amino acids
for any hydrolysis time. The maximum recovery of proline and of ll~drox~-pr~~lil~e was obtained after 3 and 6 h hydrolysis respectively. Since the concentration of hydroxyproline concentrations concentrations
decreased more rapidly than did that of proline, the ratio of their varied as a function of hydrolysis time. A comparison of the relative of glutamic and aspartic acids showed a similar but more erratic
variation. These observations preclude more than relative comparison of concentrations of the individual amino acids. The differences in apparent rate of hydrolysis suggest an effect of initial concentration on the rate of the reaction. This was evident even from the hydrolysis of urea, which is known to be a first order reaction independent of acicl concentration, and is assumed to be the velocity of the isomeric change of urea to ammonium cyanate. The effect of hydrolysis time on the amount of urea recovered has been shown in Fig. I. The increase in urea concentration, and the maintenance of that concentration for hydrolysis times of 3, 6, and 12 h indicated urea formation, possibly from a breakdown of other materials. The clifferences in the amounts of arginine found for these hydrolysis times does not indicate that the breakdown of arginine could account for the urea formed. I!?+~analogy this would question the contribution of the guanido derivatives of fatty acids found in urine, such as guanido-valtric acid” and guanidoisobutyric acid7 which do not produce color with ninhydrin. The high concentration of urea present in the sample and its more rapid disClin. Chin.
Ada,
15 (1907)
421-427
RECOVERY
OF AMINO ACIDS IN PATIENTS
WITH THERMAL
BURNS
425
appearance between 12 and 24 h as compared to the rate of disappearance between 24 and 48 h suggested that the cyanate formed may be reacting with other components in the system. The reaction of cyanate present in aqueous urea, with amino acids was reported by Stark et aL8, and the formation of N-substituted hydantoins from cyanate with carbamyl- and acetyl-amino acids at pH 7.0 has been examineas. For recovery of the amino acids from the hydantoins hydrolysis in 6 N HCl at 100' for 22 h was used. The conditions for formation and for the recovery of the amino acids would indicate that the formation of hydantoin derivatives would not be a complicating factor in the recovery of the amino acids from urine containing large amounts of urea.
8-
2-
0 ]
0
I
6
I
12
I
18
I
24
I
30
I
36
I
42
I
48h
Hydrolysis Time
Fig. I. Effect
of time on the concentration
of urea in hydrolpsates
of urine
Usually the formation of ammonia in the hydrolysates is regarded as indication of destruction of serine and threonine as well as the amides of glutamic and aspartic acids. In keeping with the conditions used for determining the amide nitrogen from glutaminyl and asparaginyl peptides 10, the ammonia formed from 3 and 6 h hydrolysis could be from such sources. After 12 h hydrolysis, however, more than IO times as much ammonia was formed. This concentration cannot be accounted for by the destruction of the amino acids including those not indicated in Table I and serves to indicate the presence of nitrcgen-containing material which does not yield color with ninhydrin. It would be expected that the conditions of hydrolysis would lead to ammonia formation from urea; however, the marked increase in ammonia formation preceeded the decrease in urea concentration. Likewise, the increase in ammonia was not accompanied by a comparable increase in the amounts of glutamic or of aspartic acid. These considerations suggested that nucleotide material may be contributing to both the NH, and urea. After 48 h hydrolysis ammonia accounted for almost 509; of the total N present in the sample. Cli,z. Chinz.
Ada,
15 (1967) e+~‘-427
426
ESTES,
Although
some difficulties
with ammonia
were encountered
WETZEL
in the determina-
tion of the basic amino acids for the 3 h and 6 h hydrolysis samples, removal of NH, was essential for determination of the basic amino acids for all of the other samples. Conventionally the hydrolysate placed in a desiccator and evacuated
or whole urine is brought to pH II, the sample or aspirated for the required length of time.
For some of the hydrolysates this did not remove the ammonia sufficiently to reveal the presence of lysine. For the removal of the NH, to an amount which did not interfere
with the evaluation
evaporated
on the rotary
of adjacent
evaporator,
peaks the samples
were taken
to pH
diluted with water, again taken to pH
the volume reduced. After two or more such treatments, the aqueous mixture basic. The sample was neutralized, the volume reduced and the sample in pH 2.2 citrate buffer. The effects of these treatments amino acids are shown in Table II.
4 +xz+zoacid
LkSiCCUtiO~
ruapovation
remained taken up
of the basic
Desiccatzon
Rotary
evaporation ______
Hydroxyl!-sine Ornithine Lysine 1.Methylhistidinc
0.384 0.200 I.178 0.3’7
O.Ogj
0
0.I3.i
0.097 0.667 0.126
0 0 0
0.197 Obscured Obscured
Histidinc 3.Methrlhistidine Creatinine Arginine
1.506
0.917 0.362
1.347 0.270
1.385 0.397
NH, NH,
remainmg origmally present ~~____
It is apparent
0.509 0.356 1.480
0.212
0
0.285
0.360
O.jO
O.zfIO
4.170
2.6gj
405.8
~
from this table
II,
and
48-h hydvolysis
~2_hlzydrolyzs Kotary
on the analyses
II
that for the 12-h hydrolysis
33.952 510.7
sample
better
re-
coveries were achieved with the rotary evaporation method while for the 48-h samples the converse treatment gave better recoveries. Further work is required to determine the factors involved in the effect of ammonia removal, and the means of doing so on the basic amino acids. Hence, the confidence limits for the analysis of the basic amino acids have not been established. The values given in Table I are the maximum values observed for the hydrolysis times involved. The loss of the basic amino acids during the removal of ammonia by repeated treatment with alkali and evaporation would serve to indicate that some loss would be involved in a single treatment of this type. Until the losses are evaluated the recovery of the basic amino acids and their concentration in the samples must be regarded as comparative. The diverse values for the basic amino acids with the removal of ammonia by the addition of a base led to consideration of the effect of a base on amino acids. The instability of cystine in presence of a base is well known. Losses in lysine however, are usually attributed to the carbohydrate material present. Rohak” observed the formation of
ACta, 15
42'-427
RECOVERY
OF AMINO
ACIDS
from alkali treatment
IN PATIENTS
of ribonuclease
to involve c@ elimination
WITH
THERMAL
427
BURNS
A. The formation
of this material
of cystine residues to form dehydroalanine
with the epsilon-N of the lysine. There was no evidence amino acids, and the chromatograms had no indication
appeared
which condensed
for the reaction from free of peaks which might be
related
to lysinoalanine. Because amino acid concentrations are often expressed in relationship to creatinine content, the creatinine concentration in the samples as determined along
with the basic amino acids has been included. The variations of the creatinine concentration of acid hydrolysis
as well as the addition
are undoubtedly
of the base for ammonia
acid solution any creatine present would have been converted in neutral or basic solutions an equilibrium mixture of these obtained12.
Since ammonia
acids in the unhydrolyzed
removal
was required
an expression
removal.
for the analysis
of the basic amino
urine, it, too, may not be a true measure
creatinine present in the original sample. The observations on these samples differed from thoseof normalurine with 6 IL’ HCl, for 18 h at 125’ sine concentration was twice hydrolyzed hydrolysis contained
urine. Likewise,
Thus, in
to creatinine while materials would be of the actual hydrolyzed
reported by Stein’. For the normal urine, the tyrothat of phenylalanine for both unhydrolyzed and
as
the normal samples contained
only traces of proline before
and very small amounts after hydrolysis. By contrast, these samples a large amount even before hydrolysis. Furthermore, the considerable
increase in the concentration of isoleucine and leucine with hydrolysis was not observed on samples of urine from normal individuals. For all the hydrolysis times observed, the concentrations of each of the amino acids were at least twice that reported for the normal urine. At least in part, these differences may be related to the sources of the samples. The apparent
maximum
recovery
and the marked decrease in concentration
of hydroxylysine
with only
with 24 h hydrolysis
12
h hydrolysis
may be a contributing
factor in the limited number of reports regarding its presence in urine. KEFEKENCES
I W. H. STEIN,J.B~OZ. Chenz.,ZOI (1953) 45. 2 Ii.TOCZKO, I. SZUMIEL AND J. MANICKI, C&n. Chim. Ada, 3 F. L. ESTES, Biochim. Biophys. Acta, IOI (1965) 369. 4 D. H. SPACKMAN,W.H.STEIN AND S. MOORE, Anal.Chem.,
II (1965) 501.
30 (1958) 1190. 5 M. W. RESS, Biochem. J., 40 (r946) 632. 6 F. IRREVERRE, R.L.EvANs, A. R. HAYDEN AND R.SILBER,N&AYP, 180 (1957) 704 7 N. THOAI AND G. LACOME. B&him. Biophys. Acta, 29 (1958) 437. 8 G. R. STARK,~. H. STEIN AND S.MOORE,~. Biol.Chem., 235 (1960) 3177. 9 G. R. STARK, Biochemistry, 4 (1965) 1030, 2363. IO A. C. CHIBNALL, J. L.MANGAN AND M. W. REES, Biochem. J., 68 (1958) III. II 2. BOHAK, J. Bid. Chem., 239 (1964) 2878. 12 G. EDGAR AND H. E. SHIVER, J. Am. Chem. Sac., 47 (1925) 1179. Clin.
Chim.
Ada,
15 (1967) 421-427
THE
CHIMICA
421
ACTA
RECOVERY
WHOLE
URINE
FRANCES
L. ESTES
OF AMINO FROM
ACIDS
PATIENTS
AND ROBERT
FROM WITH
ACID
HYDROLYSATES
THERMAL
OF
BURNS
L. WETZEL
The University of Texas, Medical Bvanch, Galveston. Texas (U.S.A.) (Receixwl
September
2 Ist,
1966)
SUMMARY
Whole urine samples, hydrolyzed with 6 N HCl at relationship
between
For maximum proline,
obtained from for varying
the hydrolysis
recovery
and glutamic
patients after a thermal burn, were periods of time. There was no uniform
100’
time and the recovery
the hydrolysis
times
acid to 24 h for valine,
varied
of any of the amino acids.
from 3 h, for aspartic
isoleucine,
acid,
leucine and phenylalanine.
The maximum recoveries of the basic amino acids were achieved with hydrolysis times of 12 h or less. Determination of the basic amino acids was complicated by the formation of considerable ammonia, in the sample after 46 h hydrolysis.
which accounted
for nearly 50% of the nitrogen
The observations indicate the diverse nature of the peptide components of urine, and delineate the difficulties involved in determining the total concentration of any of the amino acids.
INTRODUCTION
For the determination HCl for IS h under reflux at
of combined amino acids in urine, hydrolysis with 6 N (ref. I), and for 20-24 h in sealed tubes at 105-110”
125’
(ref. 2) are representative of the conditions used. The amount of the individual amino acids found in whole urine obtained from burn patients after hydrolysis with 6 N HCl for 20 h in sealed tubes at 100~ was less than the sum of those found in separated fractions of the urine hydrolyzed under the same conditions for 8 h (ref. 3). For aspartic acid, glutamic acid, and lysine one of the fractions accounted for more than was recovered from the whole urine. These observations suggested that the effect of hydrolysis time on whole urine samples should be examined. The amount of each amino acid recovered from a peptide or a protein is dependent on the hydrolysis conditions used. For individual proteins, samples hydrolyzed for varying periods show the yield of the amino acid to be a function of time. This permits an extrapolation of the values, either to zero or to infinite time, to correct for Supported
by U. S. Public
Health
Research
Grant
GM 10903.
Clin. Chim. Ada,
15 (1967) 421-427
422
ESTES, WETZEL
the effect of hydrolysis
time on the destruction
or release of amino acids. The extent
of the destruction, however, is a function of the composition of the protein concentration in the hydrolysis mixture, as well as the hydrolysis conditions. Although
they cannot
of urine samples
be regarded
from burn patients
in the investigation
of materials
as typical,
should indicate
examination
and its
of the hydrolysis
some of the problems
involved
to be found in urine. It could not be expected
that
the hydrolytic recovery of any of the amino acids would be linear with time since more than one peptide sequence must be assumed to be present, and the amino acids in the unhydrolyzed
urine cannot
be assumed
to be unreactive.
Furthermore,
the
hydrolysis of these samples was complicated by the presence of large amounts of carbohydrate materials, and material(s) which yields considerable ammonia even on mild hydrolysis. Previous observations had indicated that for 18-h hydrolysis larger amounts of both acidic and basic amino acids were recovered with 6 N HCl than with 3 N HCl even though observations
the ammonia
should be examined. limited
formation
with urine fractions
was considerably
had indicated
Since it was not possible to examine
to hydrolysis
greater.
that hydrolysis
Likewise,
previous
times of less than 18 h
all conditions,
this study was
with 6 N HCl at 100“.
METHOD
Twenty-four night and filtered.
urine samples were acidified to pH 2 with HC.1, refrigerated overFor the aliquots of the sample to be used in the determination of
the basic amino acids, ammonia was removed by neutralizing
the aliquot to pH IO-II
and desiccating over solid NaOH under reduced pressure. After restoring the volume and reacidification, the samples were hydrolyzed with 6 N HCI in tubes seaIed under nitrogen,
at 100~ for the indicated
times.
Immediately
after hydrolysis
the samples
were removed from the tubes and the HCl removed by rotary evaporation. For analysis, the samples were dissolved in pH 2.2 citrate buffer (0.2 1v) to a final volume of 5.0 ml. For those hydrolysates in which the removal of ammonia was required for the determination of the basic amino acids, the samples were dissolved in water, adjusted to pH II with sodium hydroxide and the anlmonia removed under reduced pressure. The dry sample was taken up in 5.0 ml of pH 2.2 citrate buffer (0.2 N). The amino acids were determined, on the Beckman Spinco Amino
dcid
Analyzer Model 12oB, by the method of Spackman et aL4. For each sample appropriate aliquots (0.5 to 2.0 ml) were placed on the column. The acidic and neutral amino acids were determined on the 150 x 0.9 cm column, the temperature was raised from 30 to 56” and the buffer changed from pH 3.28 citrate buffer (0.2 N) to pH 4.25 citrate buffer (0.2 N) after 307.5 ml (IO I/* h) of eluant flow. The basic amino acids were determined on the 50 x 0.9 cm column with the temperature change after 340 ml (II '/3 h) of pH 4.26 citrate buffer (0.38 L%‘).Ammonia was determined on the 15 x 0.9 cm column with an appropriate dilution of the sample, usually I :IOO, using pH 5.~8 citrate buffer (0.35 N) at 56”. Total nitrogen was determined by the classical Dumas method utilizing a Colman Nitrogen Analyzer.
Clin. Chim.
Acta,
15
(1967)421-+z7
RECOVERY OF AMINO ACIDS IN PATIENTS WITH THERMAL BURNS
423
RESULTS AND DISCUSSION
For all hydrolysis times considerable humin was present. When the humin was removed by centrifugation before the analysis, lower values for the amino acid concentrations were obtained. Apparently some of the amino acids were washed out of the humin on the column. All values shown were determined without removal of the humin. The observed values for the urea, creatinine, and ammonia recovered have been shown in Table I, together with the amino acids. The data has been reported in micromoles per milliliter of urine, so that the differences in recoveries of the amino acids at the various hydrolysis times would not be unduly magnified. For the sample in which each could be evaluated, the glycine concentration appeared to be approximately ten times that of alanine. The large amounts involved, however, did not permit good separation of the peaks for these amino acids. Although dilution of the sample permitted better evaluation of the glycine, the resulting alanine peak was not regarded as yielding more than an approximation of the amount present. TABLE
I
THE EFFBCT OF HYDROLYSIS TIME ON THE RECOVERY OF AntINo r\CIDSFROM POST BURN URINE*
(Micromoles per ml) Hydrolysis 0
Taurine Hydroxyprolinc Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alaninc Valine lsoleucine Leucine Tyrosine Phenylalanine P-Alanine
PBNC*** I.704 PBNC PBNC Tt 0.218
0.240 0.204 0.166 PBNC
Hydroxylysine Ornithinc Lysine I-Methvlhistidine Histidine 3-Methylhistidine Arginine
0.058 0.252 0.188 1.098 0.482
Total, //moles recovered Urea Creatinine Ammonia * ** *** f
6
0.916 0.878
I.092 o.gor 2.218 I.*04 1.706 2.076 3.350 r9.770
2.550
1.7*0 2.272 4.238 20.632 0.388 0.410 0.702 0.744 0.622 0.132
0.497 PBNC 0.922 0.658 0.766 0.226
36.204
Total, /smoles recovered
0.182 o. 106 2.996 I.504 1.646 0.832 0.278
0.258 0.162 PBNC PBNC I.634 1.882 0.458
7.544 7.969 0.278
Extrapolation
time in hours 3
8.900 0.446 33.680
8.867 0.790 z5.037
I2
24
48
7.677
0.760 0.052 1.607 0.602 0.972 1.670 3.002 12.955
2.415 0.496 0.526 0.888 0.626 0.454 0.116
0.927 0.602 1.062 0.902 0.817 0.180
0.477 0.312 0.577 0.392 0.367 0.100
1.4 (24) 0.9 (48)
47.136
26.405
0.384 0.200 1.178 0.317 1.506 0.509 0.360
0.121 0.133 0.650 0.180 0.235 0.238 0.296
0.135 0.197
0.65 (24)
4.464
I.853
8.750 0.356 405.8
2.935 0.097 456.0
1.036 0.760 2.136 0.860 I.470 2.116 3.980 22.507
0.347
0.125 2.332 I .97o 2.657 I .660 2.212
I.4 (24)** 3.2 (48)
I.385 0.397 0.410
0.060 0.285 510.7
14.3 (24)
Seven days after 34% burn (177; 3”). Total volume 1800 ml (1.59”/& N). Bracket number indicates maximum time used for the extrapolation. PBNC = Present but not calculable, usually because the peak was distorted. T = Trace. Clin. Chim. Acta,
15 (1967) 421-427
424
ESTES, WETZEL
The appearance
of the peak suggested
the possibility
been eluted along with the alanine. The diffused conspicuous in the unhydrolyzed samples.
that other material
glycine-alanine
may have
peaks were most
Although the maximuln recoveries for threonine, serine, valine, leucine and phenylalanine were observed after 24 h hydrolysis, the total amino acids recovered were 44% less than with 12 h hydrolysis. concentration
Most conspicuous
was the decrease in glycine
at 24 h. For the basic amino acids maximum
recoveries
were obtained
with no more than 12 h hydrolysis. For most of the amino acids included in Table I, more than one maximum
was
observed. This was most apparent in the case of serine, which is known to break down rapidlyb. Since the yield of the amino acids was not a direct function of time, determination of the amount initially present by extrapolation became a questionable procedure.
In general,
the amino acids appeared
to fall into two groups;
those which
disappeared rapidly between 12 and 24 h and those which disappeared most rapidly between 24 and 48 h hydroIysis. Extrapolated values for representatives of each group have been included
in Table
I. These values are probably
a better
indication
of the rate of breakdown between the times in question than of the initial values. The effect of extrapolating from selected hydrolysis times is most conspicuous
in
the case of lysine in which the maximum observed recovery was after 3 h hydrolysis. Extrapolation using the observed amounts recovered at 12 and 24 h hydrolysis would indicate that 1.6 ymole was initially present whereas extrapolation using the values for 3 and 12 h hydrolysis, 3.6 pmoles would have seemed to be present initially. The non-linear effect of hydrolysis time on the recovery of each of the amino acids does not justify extrapolation of the concentrations to a zero time value and suggested that each of them was involved in more than one peptide sequence. This also serves to indicate
the limits of reliability
of comparison
among the amino acids
for any hydrolysis time. The maximum recovery of proline and of ll~drox~-pr~~lil~e was obtained after 3 and 6 h hydrolysis respectively. Since the concentration of hydroxyproline concentrations concentrations
decreased more rapidly than did that of proline, the ratio of their varied as a function of hydrolysis time. A comparison of the relative of glutamic and aspartic acids showed a similar but more erratic
variation. These observations preclude more than relative comparison of concentrations of the individual amino acids. The differences in apparent rate of hydrolysis suggest an effect of initial concentration on the rate of the reaction. This was evident even from the hydrolysis of urea, which is known to be a first order reaction independent of acicl concentration, and is assumed to be the velocity of the isomeric change of urea to ammonium cyanate. The effect of hydrolysis time on the amount of urea recovered has been shown in Fig. I. The increase in urea concentration, and the maintenance of that concentration for hydrolysis times of 3, 6, and 12 h indicated urea formation, possibly from a breakdown of other materials. The clifferences in the amounts of arginine found for these hydrolysis times does not indicate that the breakdown of arginine could account for the urea formed. I!?+~analogy this would question the contribution of the guanido derivatives of fatty acids found in urine, such as guanido-valtric acid” and guanidoisobutyric acid7 which do not produce color with ninhydrin. The high concentration of urea present in the sample and its more rapid disClin. Chin.
Ada,
15 (1907)
421-427
RECOVERY
OF AMINO ACIDS IN PATIENTS
WITH THERMAL
BURNS
425
appearance between 12 and 24 h as compared to the rate of disappearance between 24 and 48 h suggested that the cyanate formed may be reacting with other components in the system. The reaction of cyanate present in aqueous urea, with amino acids was reported by Stark et aL8, and the formation of N-substituted hydantoins from cyanate with carbamyl- and acetyl-amino acids at pH 7.0 has been examineas. For recovery of the amino acids from the hydantoins hydrolysis in 6 N HCl at 100' for 22 h was used. The conditions for formation and for the recovery of the amino acids would indicate that the formation of hydantoin derivatives would not be a complicating factor in the recovery of the amino acids from urine containing large amounts of urea.
8-
2-
0 ]
0
I
6
I
12
I
18
I
24
I
30
I
36
I
42
I
48h
Hydrolysis Time
Fig. I. Effect
of time on the concentration
of urea in hydrolpsates
of urine
Usually the formation of ammonia in the hydrolysates is regarded as indication of destruction of serine and threonine as well as the amides of glutamic and aspartic acids. In keeping with the conditions used for determining the amide nitrogen from glutaminyl and asparaginyl peptides 10, the ammonia formed from 3 and 6 h hydrolysis could be from such sources. After 12 h hydrolysis, however, more than IO times as much ammonia was formed. This concentration cannot be accounted for by the destruction of the amino acids including those not indicated in Table I and serves to indicate the presence of nitrcgen-containing material which does not yield color with ninhydrin. It would be expected that the conditions of hydrolysis would lead to ammonia formation from urea; however, the marked increase in ammonia formation preceeded the decrease in urea concentration. Likewise, the increase in ammonia was not accompanied by a comparable increase in the amounts of glutamic or of aspartic acid. These considerations suggested that nucleotide material may be contributing to both the NH, and urea. After 48 h hydrolysis ammonia accounted for almost 509; of the total N present in the sample. Cli,z. Chinz.
Ada,
15 (1967) e+~‘-427
426
ESTES,
Although
some difficulties
with ammonia
were encountered
WETZEL
in the determina-
tion of the basic amino acids for the 3 h and 6 h hydrolysis samples, removal of NH, was essential for determination of the basic amino acids for all of the other samples. Conventionally the hydrolysate placed in a desiccator and evacuated
or whole urine is brought to pH II, the sample or aspirated for the required length of time.
For some of the hydrolysates this did not remove the ammonia sufficiently to reveal the presence of lysine. For the removal of the NH, to an amount which did not interfere
with the evaluation
evaporated
on the rotary
of adjacent
evaporator,
peaks the samples
were taken
to pH
diluted with water, again taken to pH
the volume reduced. After two or more such treatments, the aqueous mixture basic. The sample was neutralized, the volume reduced and the sample in pH 2.2 citrate buffer. The effects of these treatments amino acids are shown in Table II.
4 +xz+zoacid
LkSiCCUtiO~
ruapovation
remained taken up
of the basic
Desiccatzon
Rotary
evaporation ______
Hydroxyl!-sine Ornithine Lysine 1.Methylhistidinc
0.384 0.200 I.178 0.3’7
O.Ogj
0
0.I3.i
0.097 0.667 0.126
0 0 0
0.197 Obscured Obscured
Histidinc 3.Methrlhistidine Creatinine Arginine
1.506
0.917 0.362
1.347 0.270
1.385 0.397
NH, NH,
remainmg origmally present ~~____
It is apparent
0.509 0.356 1.480
0.212
0
0.285
0.360
O.jO
O.zfIO
4.170
2.6gj
405.8
~
from this table
II,
and
48-h hydvolysis
~2_hlzydrolyzs Kotary
on the analyses
II
that for the 12-h hydrolysis
33.952 510.7
sample
better
re-
coveries were achieved with the rotary evaporation method while for the 48-h samples the converse treatment gave better recoveries. Further work is required to determine the factors involved in the effect of ammonia removal, and the means of doing so on the basic amino acids. Hence, the confidence limits for the analysis of the basic amino acids have not been established. The values given in Table I are the maximum values observed for the hydrolysis times involved. The loss of the basic amino acids during the removal of ammonia by repeated treatment with alkali and evaporation would serve to indicate that some loss would be involved in a single treatment of this type. Until the losses are evaluated the recovery of the basic amino acids and their concentration in the samples must be regarded as comparative. The diverse values for the basic amino acids with the removal of ammonia by the addition of a base led to consideration of the effect of a base on amino acids. The instability of cystine in presence of a base is well known. Losses in lysine however, are usually attributed to the carbohydrate material present. Rohak” observed the formation of
ACta, 15
42'-427
RECOVERY
OF AMINO
ACIDS
from alkali treatment
IN PATIENTS
of ribonuclease
to involve c@ elimination
WITH
THERMAL
427
BURNS
A. The formation
of this material
of cystine residues to form dehydroalanine
with the epsilon-N of the lysine. There was no evidence amino acids, and the chromatograms had no indication
appeared
which condensed
for the reaction from free of peaks which might be
related
to lysinoalanine. Because amino acid concentrations are often expressed in relationship to creatinine content, the creatinine concentration in the samples as determined along
with the basic amino acids has been included. The variations of the creatinine concentration of acid hydrolysis
as well as the addition
are undoubtedly
of the base for ammonia
acid solution any creatine present would have been converted in neutral or basic solutions an equilibrium mixture of these obtained12.
Since ammonia
acids in the unhydrolyzed
removal
was required
an expression
removal.
for the analysis
of the basic amino
urine, it, too, may not be a true measure
creatinine present in the original sample. The observations on these samples differed from thoseof normalurine with 6 IL’ HCl, for 18 h at 125’ sine concentration was twice hydrolyzed hydrolysis contained
urine. Likewise,
Thus, in
to creatinine while materials would be of the actual hydrolyzed
reported by Stein’. For the normal urine, the tyrothat of phenylalanine for both unhydrolyzed and
as
the normal samples contained
only traces of proline before
and very small amounts after hydrolysis. By contrast, these samples a large amount even before hydrolysis. Furthermore, the considerable
increase in the concentration of isoleucine and leucine with hydrolysis was not observed on samples of urine from normal individuals. For all the hydrolysis times observed, the concentrations of each of the amino acids were at least twice that reported for the normal urine. At least in part, these differences may be related to the sources of the samples. The apparent
maximum
recovery
and the marked decrease in concentration
of hydroxylysine
with only
with 24 h hydrolysis
12
h hydrolysis
may be a contributing
factor in the limited number of reports regarding its presence in urine. KEFEKENCES
I W. H. STEIN,J.B~OZ. Chenz.,ZOI (1953) 45. 2 Ii.TOCZKO, I. SZUMIEL AND J. MANICKI, C&n. Chim. Ada, 3 F. L. ESTES, Biochim. Biophys. Acta, IOI (1965) 369. 4 D. H. SPACKMAN,W.H.STEIN AND S. MOORE, Anal.Chem.,
II (1965) 501.
30 (1958) 1190. 5 M. W. RESS, Biochem. J., 40 (r946) 632. 6 F. IRREVERRE, R.L.EvANs, A. R. HAYDEN AND R.SILBER,N&AYP, 180 (1957) 704 7 N. THOAI AND G. LACOME. B&him. Biophys. Acta, 29 (1958) 437. 8 G. R. STARK,~. H. STEIN AND S.MOORE,~. Biol.Chem., 235 (1960) 3177. 9 G. R. STARK, Biochemistry, 4 (1965) 1030, 2363. IO A. C. CHIBNALL, J. L.MANGAN AND M. W. REES, Biochem. J., 68 (1958) III. II 2. BOHAK, J. Bid. Chem., 239 (1964) 2878. 12 G. EDGAR AND H. E. SHIVER, J. Am. Chem. Sac., 47 (1925) 1179. Clin.
Chim.
Ada,
15 (1967) 421-427