on the colorimetric determination of creatinine by … · carried out in a klett-summerson...

ON THE COLORIMETRIC DETERMINATION OF CREATININE BY THE JAFFE REACTION*

BY ROY W. BONSNES AND HERTHA H. TAUSSKY (From the Department of Obstetrics and Gynecology, Cornell University Medicat College

and the New York Hospital, New York)

(Received for publication, February 5, 1945)

Creatinine has been determined calorimetrically by the Jaffe reaction (1) for many years. Yet there are no published data which indicate why the particular concentrations of reagents are used. Folin (2) in the first paper which describes the determination of creatinine in urine by this reaction simply states: “Als Resultat vielfacher Untersuehungen beztiglich der Reaktion des Kreatinins mit alkalischer Pikrinsiiurelosung hat sich folgendes ergeben . . .” and Shaffer (3) similarly states, “As a result of several hundreds of determinations under different conditions we con- cluded that the optimum conditions are . . .” No further details are given in either instance. Nor does subsequent work indicate what trend might be expected in the color developed if the concentration of picric acid or alkali, the volume, etc., are altered. The following experiments were undertaken, therefore, to obtain such information with particular reference t,o the determination of creatinine in very dilute solution.

EXPERIMENTAL

The color densities were measured throughout with a Klett-Summerson photoelectric calorimeter (4). Filter 54 obtained with the instrument was used. By actual measurement in a Beckman spectrophotometer (5) t,his filter exhibits a maximum absorption at about 525 rnp. This is about the best wave-length at which to measure this color since picric acid absorbs strongly above 510 rnp and the color due to creatinine does not absorb much light above 540 rnp.

The concentrations of creatinine used were such that the readings obtained would fall within the most sensitive range of the photoelectric calorimeter. The volumes of the sample of the creatinine solution and t,he reagents were selected arbitrarily so that the determination could be carried out in a Klett-Summerson calorimeter tube or in Pyrex or other test-tubes which could be adapted to the instrument, and so that ordi- nary volumetric transfer pipettes may be used.

The standard creatinine solutions were prepared fresh daily by the

* This study was aided by a grant from the John and Mary R. Markle Foundation. 581

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582 DETERMIXaTION OF CREATININE

proper dilution of a stock standard containing 0.5 mg. of creatinine per ml. of 0.1 M HCl.

The picric acid solutions were prepared by diluting the saturated stock solution. These dilutions were carried out at 25” =t 1”.

The sodium hydroxide was similarly prepared by diluting a 50 per cent solution by weight to either 10 or 20 per cent. These solutions were then diluted to the desired strength,

E$ect of Varying Concentrations of Pick Acid and Sodium Hydroxide upon Color Developed-The first experiments were carried out at a constant

CONCENTRATlON OF SODIUM HYDROXIDE AWED - %

FIG. 1. Color developed with 20 y of creatinine as a function of the concentration of sodium hydroxide added at the following concentrations of 2 ml. of picric acid: Line 1, saturated picric acid; Lines 2,3, and 4, one-half, one-third, and one-fifth saturated

picric acid respectively; Line 5, average of values.

creatinine concentration as follows: 2 ml. of the picric acid solution and 1 ml. of the sodium hydroxide solution to be studied were added to 2 ml. of a solution containing 20 y of creatinine. The mixture was allowed to stand until the maximum color had developed. A blank determination in which water was used in place of the creatinine solution was prepared simultaneously in all cases. The color developed in both the standard solution and in the reagent blank \vas determined. The zero adjustment of the calorimeter was made with a tube containing distilled water.

The total color developed with different concentrations of picric acid at a constant creatinine concentration is plotted in Fig. 1 as a fun&ion of the concentration of the 1 ml. of sodium hydroxide added. The color


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R. W. BONSNES AND H. H. TAUSSKY 583

obtained (as read with the calorimeter adjusted to zero with distilled water) with 2 ml. of saturated picric acid is described by Line 1. Similarly, the color obtained with 2 ml. of one-half saturated picric acid is shown by Line 2; that with 2 ml. of one-third saturated picric acid by Line 3; and that with 2 ml. of one-fifth saturated picric acid by Line 4. These data seem to indicate that the color is a function of both the concentration of the picric acid and of the alkali. However, if the values obtained at the same concentration of creatinine and of added alkali but at different concentrations of picric acid are corrected for their own reagent blanks, the resulting values are essentially identical. The values so obtained in these

160 c

2 4 &lo b4 0oN%I(&I0N '*,s:."

16 20 22 24

FIG. 2. Total color developed with 30 y of creatinine as a function of the picric acid concentration. Total volume 5 ml.; 1 ml. of 3 per cent sodium hydroxide used in all cases.

experiments have been averaged and are plotted as Line 5 in Fig. 1. The vertical lines indicate the range of values.

These data illustrate that the color obtained in the Jaffe reaction is independent of the concentration of picric acid, at least over a fairly wide range, and that it is dependent upon the concentration of the alkali employed. It is also apparent that as the concentration of the sodium hydroxide is increased the fraction of the total color attributable to creatinine is decreased, until at the higher concentrations of alkali nearly all t,he color is from the reagent blank.

Additional data are presented in Fig. 2 which show that the total color minus the reagent blank is independent, over a wide range, of the picric acid concentration at a constant alkali concentration. Since this graph includes data from experiments in which 3 ml. of creatinine solution and


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584 DETERMINATION OF CREATININE

1 ml. of picric acid were used as well as data from the experiments in which 2 ml. of picric acid were used, the picric acid concentrations are expressed in terms of moles of picric acid.

Rate of Color Development-The rate at which the color develops is inversely proportional to the concentration of both the picric ac?d and the sodium hydroxide; this is shown in Fig. 3.

Proportionality of Color Developed to Coneentration of Creatdnine-The relationship between the color developed and the concentration of creatinine was studied with several different concentrations of picric acid and alkali. As could be predicted from the previous data, the maximum color was obtained with 1 per cent alkali at all concentrations of creatinine.

FIG. 3. The rate of color development as a function of the concentration of the sodium hydroxide added. 20 y of creatinine. Line 1, saturated picric acid added; Line 2, one-third saturated picric acid added; Line 3, one-fifth saturat,ed picric acid added.

Progressively less color was also obt,ained at all concentrat,ions of creatinine as more concentrated alkali was used. These data are shown in Fig. 4 where Lines 1, 2, 3, and 4 were drawn to act as visual guides to the data obtained at 10 to 50 y of creatinine when the alkali concentration (in the 1 ml. of sodium hydroxide added) was 1,2,3, and 4 per cent respectively. The concentration of picric acid used again seems to make little difference, although we have not studied this factor at all concentrations of alkali. However, the dat,a indicated by Line 3 are the average results obtained in experiments in which 1 ml. of five different concentrations (saturated, one-half, one-third, one-fift,h, and one-tenth saturat,ed) of picric acid were used.

It is also apparent from these data that the color developed is not pro-


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R. W. BONSNES AND H. H. T.4USSKY 585

portional to the concentration of creatinine when the total volume is 5 ml. To substantiate t.his qualitative observation we have calculated the approximate molar extinction coefficients from the mean calorimeter readings obtained at creatinine concentrations of from 1 to 10 and at 20, 30, 40, and 50 y per 5 ml. with the creatinine method to be described below. These molar extinction coefficients are shown in Table I. The Lambert-Beer law seems to apply at creatinine concentrations below 10 y per 5 ml. (Fig. 4).

FIG. 4. Color developed as a function of the creatinine concentration. Lines .I, 2,3, and 4 show color obtained with 1,2,3, and 4 per cent NaOI-I, respectively; Lines 5 and 6, result of diluting solutions from Line 3 to 2 and 5 times their original volumes with water; Line 7, t.he Folin-Wu method.

Above this concentration, however, the calculated extinction coefficients show a gradual decrease. The Lamhert-Beer law, t,herefore, does not apply at these concentrations.

However, the proportionality between the color and the concentration can seemingly be improved by diluting the reaction mixture after the color has developed or by allowing the color to develop in a more dilute solution. For example, if the solution obtained in the creatinine method described below is diluted with an equal volume of water (i.e., to a total volume of 10 ml.), the proportionality is improved somewhat. The color developed before dilution, in terms of calorimeter readings, is about the same as t,hat


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586 DETERMINATION OF CREATININE

indicated by Line 3, Fig. 4. The calorimeter readings obtained after the solution is diluted with an equal volume of water are indicated by Line 5. If this reaction mixture is diluted still further to a total volume of 25 ml., the apparent proportionality is still better (Line 6) and the calculated molar extinction coefficients are approximately constant (Table I).

TABLE I

Molar Extinction Coe.ficients (Approximate) *

Creatinine per volume

analyzed

Y micromoles

1 1.8 2 3.5

3 5.3

4 7.1 5 8.9 6 10.6

7 12.4 8 14.2 9 16.9

10 17.7t

20 35.4

30 53.1 40 70.8

50 88.5

-

Creatinine per liter

- -

Molar extinction coefficients, X 101

Undiluted

5.65 5.65 6.00

5.95 5.65 5.65

5.50 5.50 5.40 5.65 5.35

5.05 4.95 4.75

-

I Diluted 1:l

-

6.65 7.90 5.45 6.20 7.35 5.80 6.10 7.40 5.40 6.00 7.50 5.40 5.85 7.25 5.30

1:s Folin method

* The calorimeter reading on the Klett-Summerson instrument multiplied by 2

X 10-s gives a value which approximates D = (2 - log G). If one assumes that the cylindrical tube used in this instrument is approximately equivalent to a solution thickness of 1 cm., D becomes approximately equal to the extinction, Ic, or more

properly k’, where the prime indicates the inclusion of unknown correction factors. This L’ divided by the concentration of creatinine in micromoles per liter is the approximate molar extinction coefficient described.

t This and the subsequent figures in this column must be divided by 6 for the Folin method.

Also, when the color is allowed t,o develop in a more dilute solution, as in the Folin-Wu method for blood (6), the proportionality between the color and the concentration is good (Line 7, Fig. 4) and the molar extinction coefficients are approximately constant (Table I). It is, however, im- mediately apparent that there is a decrease in both total color and in sensitivity when the Lambert-Beer law seems to apply.

Application to Determination of Creatinine and Creatine in Diluted Urine and in Blood Filtrates-We have selected for our own use one procedure from the many possibilities indicated above. The reasons which motivated


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H. \V. BONSNES AKD H. H. TACSSKY 587

this particular selection are presented in the discussion. The values obtained by this procedure have been compared with the values obtained by the Folin methods for the determination of creatinine in urine (7) and in blood filtrates (S).l

The procedure employed by us follolvs: 3 ml. of the unknown solution (diluted urine or blood filtrate) containing up to 50 y of ereatinine are used. To this solution is added 1 ml. of 0.04 III picric acid follo\ved by 1 ml. of standardized 0.75 N (3 per cent) sodium hydroxide. The color is allowed to develop for 15 minutes. It is stable for at least another 30 minutes. The creatinine content of the solution is calculat.ed from a standard curve obtained by averaging the results obtained in six different experiments at 1 to 10 and at 20, 30, 40, and 50 y of creatinine.

Added creatinine may be recovered quant,itat,ively from the urine. Some typical results are shown in Table II.

Creatinine added Creatinine found Creatinine recovered __-__ ___-. _---_

Y Y *er CLm

0 9.5 5 14.4 98

10 19.8 103 20 29.7 101 30 40.0 102

In a series of thirty urines analyzed for creatinine by this procedure the average deviation from the values obtained by the Folin method (7) was -0.4 per cent,, with a maximum range of f10 per cent. However, the majority of the determinations deviated less than =t5 per cent from the value obtained with the Folin method.

Ten whole blood filtrates from patients who might be expected to have normal blood creatininc levels were analyzed by both methods. These bloods contained an average of 1.5 mg. per cent (range 1.4 to 1.7) of creatinine by the Folin method and 1.4 mg. per cent (range 1.2 to 1.5) by the procedure described above.

Creatine is 80 per cent converted to creatinine at these concentrations by heating the creatine-cont.aining solution with picric acid in a boiling water bath for 45 minutes. This percentage conversion is of the same order of magnitude as that obtained in the Folin autoclave method. These observations confirm those of Albanesc and Wnngerin (8) who found tha,t

1 The color density in both these methods was measured with the photoelectric calorimeter. Standard curves were also used in both instances.


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588 DETERMINSTION OF CREATININE

creatine is not converted quantitatively to creatinine by the Folin autoclave method.

The percentage conversion is fairly constant and reaches a maximum value at about 80 per cent conversion when creatine is heated with picric acid in a boiling water bath, as described below. No creatinine is de- stroyed by this procedure. It seems, therefore, that for many purposes the procedure described below might serve as a method for the determination of creatine in dilute solution in that the procedure does not require an autoclave and requires no attention if a constant level water bath is a- vailable.

Creatine in urine is determined by the procedure described above for creatinine after heating 3 ml. of the creatine-containing solution with 1 ml. of 0..04 M picric acid in an unstoppered tube graduated at 5 ml. for 45 minutes in a vigorously boiling water bath.’ After the solution has cooled to room temperature, 1 ml. of alkali is added. The volume is then adjusted to 5 ml. with water. To calculate creatine as creatinine the value obtained by difference in the usual manner is multiplied by 1.25. The values for total creatinine obtained by this method upon dilut,ed urine were essentially the same as those obtained by the Folin autoclave method. If a quantitative conversion is required, the method described by Peters (9) might be applied, or the method described by Benedict (10) can be used before the urine is diluted. These procedures, however, have not been extensively studied by us. No attempt has been made to determine creatine in blood filtrates.

It appears, therefore, that the method described above for the determination of creatinine and creatine yields essentially the same values for creatinine and creatine in dilut,ed urine and for creatinine in blood filtrates as do the Folin methods cited for the determination of these substances. This method has now been in use in our laboratory for the past several months for the routine determination of creatinine.

DISCUSSION

The ideal conditions for the determination of any substance by a colorimetric procedure are those which yield a color which is directly proportional to the concentration of the substance to be determined. The data presented above indicate that this ideal cannot be reached with this particular reaction regardless of the conditions chosen. However, one may approximate this ideal condition over the range of from 10 to 50 y of creatinine if procedures such as are described above (i.e. dilution) or the Folin blood method is used. But, it is doubtful whether dilution of

2 The water bath must be boiling vigorously.


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II. W. DONSNES ASD 11. H. ThUSSKY 589

the reaction mixture actually improves the proportionality. For when these reaction mixtures are diluted 5 times with water, the slope of the line describing the color as a function of the creatinine concentration is decreased to such an extent that the variations which occur probably fall within the resolving power of t.he instrument, since the measurements arc made, perforce, at a constant solution thickness. So what appears to be an improvement in proportionality may be merely an artifact. Further- more, when such procedures are followed, there is a great loss in sensitivity. For instance, when the Folin method is used, a difference of 40 y of creatinine results in a change of 31 scale divisions or 0.78 scale division per microgram. On the other hand, when the procedure described herein is used (assuming a linear relationship), a similar change in creatinine concentration results in a change in 161 scale divisions or approximately 4.0 scale divisions per microgram. It would appear, therefore, that if urines are diluted sufficiently so that the creatinine can be determined by the Folin blood method, as suggested by Peters (9), the low sensitivity of the method might lead to a relatively large error in the result. However, when blood filtrates are analyzed, one must choose between a method with a low sen&ivity or a procedure which has greater sensitivity but which gives lower calorimeter readings.3

The assumption which has been made above that a linear relationship holds in the method described is obviously fallacious. However, it is of interest to calculate the actual percentage error which results if this assumption is made. For these calculations the mean values obtained at 10,20,30,40, and 50 y of creatinine are compared with the values obtained from the best straight lint which fits the data as calculated by the method of least squares. The values which are obtained from the straight line are then 6 per cent high at 10 y, 2 per cent low at 20 y, 1.5 per cent low at 30 y, 1.1 per cent low at 40 y, and 1.4 per cent high at 50 y. If the mean values are compared with the values obtained from the best straight line which fits the data between 10 and 40 y, the values obtained from the straight line are 2 per cent high at 10 y, 2 per cent low at 20 y, 0.75 per cent low at 30 y, and 0.58 per cent high at 40 y. It is possible, therefore, to use a formula4 to calculate the creatinine concentration without introducing a very great error, even though a linear relationship is not found to exist.

3 Similar results have been obtained when the Evelyn photoelectric calorimeter (11) has been used. To use this calorimeter all volumes are doubled. Filter 520 is used at Aperture 6.

4 The mean values, the equations, and the formulas which may be derived from them are not presented because they are relative and not absolute values. It is not expected that identical numerical results can be obtained by others because of the variations which exist in filters.


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590 DETERMIXATION OF CREATININE

However, one must be cognizant of the fact that errors are involved in such a procedure.

The particular method which w-e have outlined above was selected because it fits in best with our present analytical routines. The 0.04 M picric acid and the 0.75 N (3 per cent) alkali are used because the color develops sufficiently rapidly at these concentrations of picric acid and alkali and the blank color is still at a minimum. At the concentration of alkali at which the maximum color develops the rate of color development is too slow even with saturated picrjc acid for our purposes.

It is possible that at the concentration of alkali used more color may be produced from non-creatinine substances such as glucose t.han at lower concentrations of alkali. We have tested this point experimentally wit.1~ respect to glucose. The color remains constant for 30 minutes after the maximum color has been attained with Folin-Wu blood filtrates and in urines to which 1 per cent glucose has been added. There may be an increase in color after 40 minutes. There is no difference in the creatinine values obtained in urine before and after the addition of glucose. The presence of glucose, therefore, does not seem to int.erferc with the determination.

Many questions concerning the mechanism of the Jaffe reaction are raised by the data. We feel, hoiyever, that there is no justification for a discussion of these points until more data are available.

SUMMARY

The effect of varying the concentration of picric acid and of alkali upon the color formed in the Jaffe reaction used for the determination of creatinine has been studied in solutions which c0ntai.n 1 to 50 y of creatinine per 5 ml. The amount of colored creat,inine compound formed as measured by the color developed is independent of the concentration of the picric acid added above a low limiting concentration of picnic acid. The amount of colored creatinine compound formed is greatest at low concentrations of alkali and progressively decreases as the concentration of alkali is increased. The rate at which the color develops is inversely proportional to the concentration of both the picric acid and the sodium hydroxide.

The color formed is not, directly proportional to the concentration of we&nine except at very low concentrations in the 5 ml. of solution employed. By diluting this solution the linearity seems to improve. This apparent improvement, however, is considered an artifact.

A procedure for the determination of creatinine in blood filtrates and in diluted urine is described which is based upon the data obt,ained.


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R. TV. BOKSNES AND H. H. ThUSSKY

BIBLIOGRAPHY

1. Jaffe, X, Z. physiol. Chen~., 10,391 (1886). 2. Folin, O., Z. physiol. Chem., 41,223 (1964). 3. Shaffer, P. A., J. Biol. Chem., 18,525 (1914). 4. Summerson, W. H., J. Biol. Chem., 130,149 (1939). 5. Cary, II. H., and Beckman, A. O., J. Optical Sot. An&., 31,682 (1941). 6. Folin, O., and Wu, H., J. Biol. Chem., 38,81 (1919). 7. Folin, O., J. BioZ. Chem., 1’7,469 (1914). 8. Albanese, A. A., and Wangerin, D. M., Science, 199, 58 (1944). 9. Peters, J. H., J. Biol. Chem., 146, 179 (1942).

10. Benedict, S. R.,J. Biol. Chem.,18,191 (1914). 11. Evelyn, K. A., J. BioZ. Chem., 116.63 (1936).


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Roy W. Bonsnes and Hertha H. TausskyTHE JAFFE REACTION

DETERMINATION OF CREATININE BY ON THE COLORIMETRIC

1945, 158:581-591.J. Biol. Chem.

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