- Email: [email protected]
Determination of creatinine in urine by separation on DEAE-Sephadex and ultraviolet spectrophotometry
.4NALYTICAL
BIOCHEMISTRY
Determination
16,
(1966)
k%.-k!8
of Creatinine
DEAE-Sephadex
and
in Urine
Ultraviolet
by Separation
on
Spectrophotometry
E. McEVOY-BOWE From
the Department of Biochemistry, University of Singapore, Sepoy
Received February
Faculty
of Medicine,
Lines, Singapore
3
8, 1966
Adams, Davis, and Hansen (1) have published a method for the determination of creatinine in urine in which they use Dowex 2 resin to separate the creatinine from other urinary constituents followed by measurement of the compound in a spectrophotometer at 234.5 mp. In the method described here DEAE-Sephadex has been used in place of the Dowex 2 resin. It was found t,hat the use of DEAE-Sephadex for the separation of creatinine in urine possesses two major advantages over the Dowex 2 resin: (1) The Sephadex can be washed completely free of all 235 rnp absorbing material. (2) There was a considerable improvement in the agreement of the light absorption spectra given by the urinary creatinine fractions after separation an the DEAE-Sephadex with that given by a solution of pure creatinine. MATERIALS AND METHODS
Materials. DEAE-Sephadex (A50-Medium) was obtained from Pharmacia, Uppsala, Sweden. Ammonia solution (sp.gr. 0.890-0.900) was obtained from General Chemical and Pharmaceutical Co., Ltd., England. The other chemicals were purchased from British Drug Houses Ltd., England. Determination of Creatinine (Jajfd Reaction). This was carried out by the method of Folin (2). Ammonia Solution at pH 10.4. An, approximate 1 N ammonia solution was prepared by diluting 58 ml of the concentrated ammonia solution to 1 liter. This solution was kept stoppered in the refrigerator. Ten ml of an approximate 0.04 N ammonia solution (ammonia solution l), and 500 ml of an approximate 0.004 N ammonia solution (ammonia soIution 2) were prepared from the stock solution each day. Ammonia solution 2 gave a pH of 10.4 and was the solution used for eluting from t,he DEAE-Sephadex column. The concentration of ammonia solution I should always 1~ tell times the concentration of ammonia solution 2.
154
E.
MCEVOY-ROWE
Standard Creatinine Solution. A solution containing 0.1 gm creatinine in 100 ml of water was prepared and kept in a refrigerator. Standard dilutions for running on the columns were prepared by adding 1 ml of ammonia solution 1 to 1 ml of the standard stock solution and then diluting to 10 ml with water. Standard dilutions for direct measurement in the spectrophotometer were prepared by diluting 1 ml or 0.5 ml of the standard stock solution to 100 ml with ammonia solution 2. Collection of Urine Samples. All ten urine samples were obtained from laboratory staff, and all were voided early in the morning. No attempt was made here to measure 24 hr output. All samples were preserved with toluene and analyzed on the same day. Preparation of the DEAE-Sephadex. About 3 gm of the material was allowed to swell in excess water and the fines removed by decantation. The slurry was then filtered on a Biichner funnel and washed with 50 ml of 0.5 N HCl. In the case of used Sephadex, the slurry was first made acid with 0.5 N HCI, and then filtered on a Biichner funnel. From this point the washing procedure was the same both for new and used Sephadex. The material on the Biichner funnel was then washed successively with 300 ml 0.02 N HCl, 100 ml 0.01 N HCl in 50% (v/v) ethyl alcohol, 300 ml glass-distilled water, 50 ml 0.5 N NaOH, 300 ml glass-distilled water or until the effluent was neutral to litmus paper, and finally washed with ammonia solution 2 until the filtrate was alkaline. The final material was stored as a slurry in ammonia solution 2 until required. Preparation cf Columns. Glass columns having the dimensions 1 X 10 cm were used. Each column had a piece of vinyl tubing attached to the bottom end together with a screw clip. A small piece of high-quality nonabsorbant cotton wool was placed at the bottom of the column as a support for the Sephadex. The column was then almost filled with ammonia solution 2, and a watertight connection was made with the stem of a glass funnel at the top of the glass column by placing a piece of vinyl tubing between the two. The Sephadex slurry was then added slowly to the column through the funnel. The bed was allowed to build up to a height of about 1.5 cm and the screw clip was then opened to allow a slow flow of effluent. The final bed height was 4.5 cm. The glass funnel was then removed and a small piece of nonabsorbant cotton wool was placed on the top of the Sephadex column to protect the surface. Normally a bank of five columns was prepared for simultaneous use. Each column was used for a single run only. Used Sephadex was collected until a convenient amount had accumulated to put through the washing procedure. Spectrophotometry. All measurement’s were carried out in a Bausch h Lomb Spectronic 505 automatic spectrophotometer.
Chromatography of Urinary C,reatinine. One ml of ammonia solution 1 was added to 1 ml or 0.5 ml of urine and the mixture diluted to 10 ml with water; 1 ml of the diluted urine was added to the column and washed in with a few drops of ammonia solution 2. The glass column was almost filled with ammonia solution 2, the glass funnel was then replaced at the top of the column, and excess of ammonia solution 2 was added to the funnel. The effluent was allowed to flow until 10 ml had been collected. The time taken for 10 ml of the effluent to collect was usually about 15 min. The optical density of the effluent, was then measured in the spectrophotometer at 235 rnp. Calculation. The standard creatinine solution, when diluted with ammonia solution 2 to give a concentration of 5 pg/ml, gave an optical density reading of 0.330. The concentration of creatinine in each urine sample was therefore calculated by the equation: (a!b) X 0.1515 = mg creatinine/ml
urine
where a = optical density of the effluent, and b = final volume of urine placed on the column. Recovery Experiments. One-half ml of the standard creatinine solution (1 mg/ml) and 1 ml of ammonia solution 1 were added to 0.5 ml urine and dimted to 10 ml with water. The amount of creatinine originally present in 0.05 ml of urine (final volume of urine placed on the column) was then suhtract.ed from that found in the sample containing added creatinine. RESULTS
AND DISCUSSION
Figure 1 shows the elution curves obtained for a creatinine solution and for urine. It can be seen that the elution peaks coincide and that the creatinine is completely removed from the column in 9.5 ml of effluent. Figure 2 shows the agreement between the light absorption spectra of the effluents from six urine samples and those of creatinine at two different concentrations. It can be seen that the spectrum of one of the effluents is distorted between 218 and 230 m,p. A sample of this particular urine was then subjected to a second run on the DEAE-Sephadex column. In Fig. 3 the absorption spectra obtained from the two effluents are compared. It can be seen that, although the second absorption spectrum gave little or no distortion, the optical density of the two effluents at 235 mp remained unchanged, indicating that the small amounts of impurity in the e&rent from the first chromatographic run did not interfere with the optical density at 235 mp. Up to the present there has been no evidence of significant effects by impurities on the light absorption of the creatinine at 235 mp. A cursory inspection will show that these spectra
1.0
0.4 a -u Ii 0 0.2
0
I 0
I 2
I
I 4
EFFLUENT
I 6
I IO
0 IN
I
ML
FIG. 1. Elution of creatinine and urinary creatinine from a DEAE-Sephadex column: (0) creatinine (0.1 mg placed on column) ; (~0) urinary creatinine (0.2 ml of urine placed on column). Fractionation carried out by a drop counter; mean volume of each fraction 0.48 ml; 3 ml ammonia solution at pH 10.4 added to each fraction before measurement in the spectrophotometer.
210
230
250 WAVELENGTH
270
290
(IT&l)
FIG. 2. Absorption spectra of creatinine and urine eluates from DEAE-Sephadex spectra of creatinine at two levels of concentration (5 and 10 columns : (-) pg/ml) ; (- - -) urine eluates.
DETERRIIXATION
0
I
I
OF
CREATININE
I
I
I
IN
URIKE
I
I
157
I
290
210
FIG. 3. Absorption spect#ra of effluents from two chromatographic same urine sample : (-) first run and (- - -) second run.
runs on the
are not as seriously distorted as the spectra obtained by Adams et al. (1) using Dowex 2 resin. Table 1 shows the creatinine recoveries obtained from ten samples of urine. The mean recovery and its standard deviation are an indication of the accuracy to be expected from the procedure. Table 1 also gives a comparison with the results obtained on the same urines using the Jaffk TABLE II.ESULTS SarI$le
OBTAINED
---,---DEALSephadex
FROM
TEN
Creotininc Jai35 reaction
1 &MPLEs
OF URINE
(MG/ML)
Y. increase of Jsff6 reaction 011 DEAE-Sephadex
1 2 3 4 5 G 7 8 9 10
1.31 1.26 0.81 0.93 0.82 0.92 1.15 0.70 1.76 1 39
1.34 1.49 0.89 1.11 0.92 1.11 1.20 0.85 1.76 1.68
2.3 18.3 9.9 19.4 12.2 20.6 4.4 21.4 0.0 20,s
Mean S.D.
1.11 0.10
1.24 0.10
12.9 8.3
95 92 94 102 94 107 !J7 104 102 104 99.1 5.3
reaction. It can be seen that the Jaff’e reaction gave a mean increase on the DEAE-Sephadex procedure of 12.90/o, whereas Owen, Iggo, Scandrett, and Stewart (3) found, with their urine samples, that the mean creatinine content on determination with Lloyd’s reagent was 6% lower than the total chromogenic content as determined by the method of Folin and Wu (4). However, the disparity between these two figures is probably not significant, in that it is reasonable to expect a wide range of noncreatinine chromogenic material depending upon diet and other factors. The pH value of 10.4 was chosen for the eluting solution because it has been reported that at this pH N-methyl-2-pyridone-5-carboxamide gives a minimum absorption at 233 rn,,u (1). The ionic strength of the solution was kept low in order to obtain maximal retention of urinary constituents on the column. Since most urine samples were diluted at least 100 times in the eluting solution, the pH variation in the effluents was negligible. Table 2 shows the results obtained when a urine sample TABLE
2
EFFECT OF URINARY PH ON PH OF EFFLUENT FROM THE DEAE-SEPHAUEX AND ON OPTICAL DENSITY OF EFFLUENT AT 235 MP (pH
of eluting
Urine pH Effluent pH Optical density
solution
10.4;
initial
4.7 10.4 0.540
pH of sample 5.5 10.4 0.565
of urine
COLUMN,
5.5) 8.9 10.4 0.555
was acidified to a pH of 4.7 with acetic acid, and similarly made alkaline to a pH of 8.4 with ammonia before chromatography. It can be seen that the pH of the effluents from each of these urines was unchanged, and, together with this, there was no significant change in the optical densities at 235 mp. The absorption spectra of these effluents were also unchanged. SUMMARY
A specific method for the determination of creatinine in urine was developed in which DEAE-Sephadex was used to separate creatinine from other urinary constituents. The compound was then measured spectrophotometrically by its absorption at 235 nq at pH 10.4. DEAESephadex was found to be a more suitable material for this type of procedure than the Dowex 2 resin used by Adams, Davis, and Hansen. When ten urine samples were analyzed by both the DEAE-Sephadex procedure and the Jaffb reaction, the results obtained by the Jaff;Q reaction gave a mean increase of 12.9% on those obtained by the DEAESephadex procedure.
.~CKNOWLEDGMENTS I wish assistance
to thank Miss in this work.
Ngan
Swee
May
and
Mr.
Ban
1. ADAMS, 2. FOLIN, 3. OWEN, (1954). 4. FOLJN,
W. S., DAVIS, F. W., AND HANSEN, L. E., Amzl. O., J. Biol. Chewy 17, 469 (1914). J. A., IGGO, B., SCANDRETT, F. J., AND STEWART,
Chin
Kwang
for
technical
REFERENCES
O., AND WLJ, H., J. Biol.
Chewy
38,
81 (1919).
Chem.
34, 845
C. P., Biochem.
(1962). J. 58,
426
BIOCHEMISTRY
Determination
16,
(1966)
k%.-k!8
of Creatinine
DEAE-Sephadex
and
in Urine
Ultraviolet
by Separation
on
Spectrophotometry
E. McEVOY-BOWE From
the Department of Biochemistry, University of Singapore, Sepoy
Received February
Faculty
of Medicine,
Lines, Singapore
3
8, 1966
Adams, Davis, and Hansen (1) have published a method for the determination of creatinine in urine in which they use Dowex 2 resin to separate the creatinine from other urinary constituents followed by measurement of the compound in a spectrophotometer at 234.5 mp. In the method described here DEAE-Sephadex has been used in place of the Dowex 2 resin. It was found t,hat the use of DEAE-Sephadex for the separation of creatinine in urine possesses two major advantages over the Dowex 2 resin: (1) The Sephadex can be washed completely free of all 235 rnp absorbing material. (2) There was a considerable improvement in the agreement of the light absorption spectra given by the urinary creatinine fractions after separation an the DEAE-Sephadex with that given by a solution of pure creatinine. MATERIALS AND METHODS
Materials. DEAE-Sephadex (A50-Medium) was obtained from Pharmacia, Uppsala, Sweden. Ammonia solution (sp.gr. 0.890-0.900) was obtained from General Chemical and Pharmaceutical Co., Ltd., England. The other chemicals were purchased from British Drug Houses Ltd., England. Determination of Creatinine (Jajfd Reaction). This was carried out by the method of Folin (2). Ammonia Solution at pH 10.4. An, approximate 1 N ammonia solution was prepared by diluting 58 ml of the concentrated ammonia solution to 1 liter. This solution was kept stoppered in the refrigerator. Ten ml of an approximate 0.04 N ammonia solution (ammonia solution l), and 500 ml of an approximate 0.004 N ammonia solution (ammonia soIution 2) were prepared from the stock solution each day. Ammonia solution 2 gave a pH of 10.4 and was the solution used for eluting from t,he DEAE-Sephadex column. The concentration of ammonia solution I should always 1~ tell times the concentration of ammonia solution 2.
154
E.
MCEVOY-ROWE
Standard Creatinine Solution. A solution containing 0.1 gm creatinine in 100 ml of water was prepared and kept in a refrigerator. Standard dilutions for running on the columns were prepared by adding 1 ml of ammonia solution 1 to 1 ml of the standard stock solution and then diluting to 10 ml with water. Standard dilutions for direct measurement in the spectrophotometer were prepared by diluting 1 ml or 0.5 ml of the standard stock solution to 100 ml with ammonia solution 2. Collection of Urine Samples. All ten urine samples were obtained from laboratory staff, and all were voided early in the morning. No attempt was made here to measure 24 hr output. All samples were preserved with toluene and analyzed on the same day. Preparation of the DEAE-Sephadex. About 3 gm of the material was allowed to swell in excess water and the fines removed by decantation. The slurry was then filtered on a Biichner funnel and washed with 50 ml of 0.5 N HCl. In the case of used Sephadex, the slurry was first made acid with 0.5 N HCI, and then filtered on a Biichner funnel. From this point the washing procedure was the same both for new and used Sephadex. The material on the Biichner funnel was then washed successively with 300 ml 0.02 N HCl, 100 ml 0.01 N HCl in 50% (v/v) ethyl alcohol, 300 ml glass-distilled water, 50 ml 0.5 N NaOH, 300 ml glass-distilled water or until the effluent was neutral to litmus paper, and finally washed with ammonia solution 2 until the filtrate was alkaline. The final material was stored as a slurry in ammonia solution 2 until required. Preparation cf Columns. Glass columns having the dimensions 1 X 10 cm were used. Each column had a piece of vinyl tubing attached to the bottom end together with a screw clip. A small piece of high-quality nonabsorbant cotton wool was placed at the bottom of the column as a support for the Sephadex. The column was then almost filled with ammonia solution 2, and a watertight connection was made with the stem of a glass funnel at the top of the glass column by placing a piece of vinyl tubing between the two. The Sephadex slurry was then added slowly to the column through the funnel. The bed was allowed to build up to a height of about 1.5 cm and the screw clip was then opened to allow a slow flow of effluent. The final bed height was 4.5 cm. The glass funnel was then removed and a small piece of nonabsorbant cotton wool was placed on the top of the Sephadex column to protect the surface. Normally a bank of five columns was prepared for simultaneous use. Each column was used for a single run only. Used Sephadex was collected until a convenient amount had accumulated to put through the washing procedure. Spectrophotometry. All measurement’s were carried out in a Bausch h Lomb Spectronic 505 automatic spectrophotometer.
Chromatography of Urinary C,reatinine. One ml of ammonia solution 1 was added to 1 ml or 0.5 ml of urine and the mixture diluted to 10 ml with water; 1 ml of the diluted urine was added to the column and washed in with a few drops of ammonia solution 2. The glass column was almost filled with ammonia solution 2, the glass funnel was then replaced at the top of the column, and excess of ammonia solution 2 was added to the funnel. The effluent was allowed to flow until 10 ml had been collected. The time taken for 10 ml of the effluent to collect was usually about 15 min. The optical density of the effluent, was then measured in the spectrophotometer at 235 rnp. Calculation. The standard creatinine solution, when diluted with ammonia solution 2 to give a concentration of 5 pg/ml, gave an optical density reading of 0.330. The concentration of creatinine in each urine sample was therefore calculated by the equation: (a!b) X 0.1515 = mg creatinine/ml
urine
where a = optical density of the effluent, and b = final volume of urine placed on the column. Recovery Experiments. One-half ml of the standard creatinine solution (1 mg/ml) and 1 ml of ammonia solution 1 were added to 0.5 ml urine and dimted to 10 ml with water. The amount of creatinine originally present in 0.05 ml of urine (final volume of urine placed on the column) was then suhtract.ed from that found in the sample containing added creatinine. RESULTS
AND DISCUSSION
Figure 1 shows the elution curves obtained for a creatinine solution and for urine. It can be seen that the elution peaks coincide and that the creatinine is completely removed from the column in 9.5 ml of effluent. Figure 2 shows the agreement between the light absorption spectra of the effluents from six urine samples and those of creatinine at two different concentrations. It can be seen that the spectrum of one of the effluents is distorted between 218 and 230 m,p. A sample of this particular urine was then subjected to a second run on the DEAE-Sephadex column. In Fig. 3 the absorption spectra obtained from the two effluents are compared. It can be seen that, although the second absorption spectrum gave little or no distortion, the optical density of the two effluents at 235 mp remained unchanged, indicating that the small amounts of impurity in the e&rent from the first chromatographic run did not interfere with the optical density at 235 mp. Up to the present there has been no evidence of significant effects by impurities on the light absorption of the creatinine at 235 mp. A cursory inspection will show that these spectra
1.0
0.4 a -u Ii 0 0.2
0
I 0
I 2
I
I 4
EFFLUENT
I 6
I IO
0 IN
I
ML
FIG. 1. Elution of creatinine and urinary creatinine from a DEAE-Sephadex column: (0) creatinine (0.1 mg placed on column) ; (~0) urinary creatinine (0.2 ml of urine placed on column). Fractionation carried out by a drop counter; mean volume of each fraction 0.48 ml; 3 ml ammonia solution at pH 10.4 added to each fraction before measurement in the spectrophotometer.
210
230
250 WAVELENGTH
270
290
(IT&l)
FIG. 2. Absorption spectra of creatinine and urine eluates from DEAE-Sephadex spectra of creatinine at two levels of concentration (5 and 10 columns : (-) pg/ml) ; (- - -) urine eluates.
DETERRIIXATION
0
I
I
OF
CREATININE
I
I
I
IN
URIKE
I
I
157
I
290
210
FIG. 3. Absorption spect#ra of effluents from two chromatographic same urine sample : (-) first run and (- - -) second run.
runs on the
are not as seriously distorted as the spectra obtained by Adams et al. (1) using Dowex 2 resin. Table 1 shows the creatinine recoveries obtained from ten samples of urine. The mean recovery and its standard deviation are an indication of the accuracy to be expected from the procedure. Table 1 also gives a comparison with the results obtained on the same urines using the Jaffk TABLE II.ESULTS SarI$le
OBTAINED
---,---DEALSephadex
FROM
TEN
Creotininc Jai35 reaction
1 &MPLEs
OF URINE
(MG/ML)
Y. increase of Jsff6 reaction 011 DEAE-Sephadex
1 2 3 4 5 G 7 8 9 10
1.31 1.26 0.81 0.93 0.82 0.92 1.15 0.70 1.76 1 39
1.34 1.49 0.89 1.11 0.92 1.11 1.20 0.85 1.76 1.68
2.3 18.3 9.9 19.4 12.2 20.6 4.4 21.4 0.0 20,s
Mean S.D.
1.11 0.10
1.24 0.10
12.9 8.3
95 92 94 102 94 107 !J7 104 102 104 99.1 5.3
reaction. It can be seen that the Jaff’e reaction gave a mean increase on the DEAE-Sephadex procedure of 12.90/o, whereas Owen, Iggo, Scandrett, and Stewart (3) found, with their urine samples, that the mean creatinine content on determination with Lloyd’s reagent was 6% lower than the total chromogenic content as determined by the method of Folin and Wu (4). However, the disparity between these two figures is probably not significant, in that it is reasonable to expect a wide range of noncreatinine chromogenic material depending upon diet and other factors. The pH value of 10.4 was chosen for the eluting solution because it has been reported that at this pH N-methyl-2-pyridone-5-carboxamide gives a minimum absorption at 233 rn,,u (1). The ionic strength of the solution was kept low in order to obtain maximal retention of urinary constituents on the column. Since most urine samples were diluted at least 100 times in the eluting solution, the pH variation in the effluents was negligible. Table 2 shows the results obtained when a urine sample TABLE
2
EFFECT OF URINARY PH ON PH OF EFFLUENT FROM THE DEAE-SEPHAUEX AND ON OPTICAL DENSITY OF EFFLUENT AT 235 MP (pH
of eluting
Urine pH Effluent pH Optical density
solution
10.4;
initial
4.7 10.4 0.540
pH of sample 5.5 10.4 0.565
of urine
COLUMN,
5.5) 8.9 10.4 0.555
was acidified to a pH of 4.7 with acetic acid, and similarly made alkaline to a pH of 8.4 with ammonia before chromatography. It can be seen that the pH of the effluents from each of these urines was unchanged, and, together with this, there was no significant change in the optical densities at 235 mp. The absorption spectra of these effluents were also unchanged. SUMMARY
A specific method for the determination of creatinine in urine was developed in which DEAE-Sephadex was used to separate creatinine from other urinary constituents. The compound was then measured spectrophotometrically by its absorption at 235 nq at pH 10.4. DEAESephadex was found to be a more suitable material for this type of procedure than the Dowex 2 resin used by Adams, Davis, and Hansen. When ten urine samples were analyzed by both the DEAE-Sephadex procedure and the Jaffb reaction, the results obtained by the Jaff;Q reaction gave a mean increase of 12.9% on those obtained by the DEAESephadex procedure.
.~CKNOWLEDGMENTS I wish assistance
to thank Miss in this work.
Ngan
Swee
May
and
Mr.
Ban
1. ADAMS, 2. FOLIN, 3. OWEN, (1954). 4. FOLJN,
W. S., DAVIS, F. W., AND HANSEN, L. E., Amzl. O., J. Biol. Chewy 17, 469 (1914). J. A., IGGO, B., SCANDRETT, F. J., AND STEWART,
Chin
Kwang
for
technical
REFERENCES
O., AND WLJ, H., J. Biol.
Chewy
38,
81 (1919).
Chem.
34, 845
C. P., Biochem.
(1962). J. 58,
426