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Studies on creatine determination by α-naphthol-diacetyl reaction
ANALYTICAL
BIOCEFNISTBY
Studies
40,
18-28
on
(1971)
Creatine
Determination
by cr-Naphthol-Diacetyl
Reaction
TAO WONG Department
of State
Chemktrg, College,
Mississippi Mississippi
State 39762
University,
Received July 1, 1970
Based upon the observation by Barritt (1) that a-naphthol enhances color formation in the creatine-diacetyl reaction, Eggleton et ~2. (2) in 1943 described a procedure for the determination of creatine. This procedure and its modifications have been extensively used for the determination of this compound. During the course of Istudying the determination of phosphocreatine (3), we were puzzled by the controversy in the literature as to whether or not other guanidino compounds such as arginine, etc. also yield a significant amount of color with the a-naphthol-diacetyl reaction, and, thus, constitute a source of error for the determination of creatine (1,2,&6). In this communication we wish to offer explanations for this disagreement and to present the conditions that minimize error in the creatine estimation if the prior isolation of creatine is not carried out (7). When Barritt first introduced the cr-naphthol-diacetyl reaction, his solution contained 10% alcohol. However, in the procedure described by Eggleton et al. the alcohol is omitted entirely. In 1956, Rosenberg et al. (8) recommended a procedure for the determination of arginine which is essentially a slight modification of the Barritt procedure. One minor difference is that 10% ethanol is replaced by 20% n-propanol. We have therefore studied the effects of alcohol and found that the effects are multiple, profound, and diverse. PROCEDURES
Experiments in the Absence of Alcohol. The Eggleton et al. (2) procedure or those slightly modified as specified in the text were used. Effects Produced by Addition of Alcohol. The Rosenberg et al. (8) procedure or those slightly modified as specified in the text were used. The reason for the switch to the Rosenberg et al. procedure was that Na2C03 in the Eggleton et al. procedure interfered with the ,alcohol additions in many cases. 18
DEXEFMINATION
OF
19
CREATINE
The readings of absorbance due to the red color formed in all procedures used were recorded either by a Coleman Junior or by a Perkin-Elmer 202 spectrophotometer. The scanning speed of the Perkin-Elmer 202 was set at the fast scale, which takes 2 min to cover the range from 350 to 750 rnp. Such a speed is fast enough to observe the phenomena described in experimental section IC below. The wavelength of the Perkin-Elmer 202 was calibrated with a holmium oxide filter for each reading. MATERIALS
The sources of all chemicals used have been previously
described (3).
EXPERIMENTS
I. Experiments
in the Absence of Alcohol
In determining creatine by the a-naphthol-diacetyl method, it is controversial whether the presence of arginine or other guanidino compounds also yields a significant amount of color and, thus, constitutes a source of error (see Table 1). The value of “ratio,” which is defined as the Disagreement
TABLE 1 in the Literature over Amount of Color Produced by Various Guanidino Compounds
Extinction coefficient of arginine and glycocyamine
Extinction coefficient of guanidine
extinction coefficient of creatine
extinction coefficient of creatine
1/g l/10 l/5-1/6 l/2.5-1/3 About 1
l/9 l/10 l/10
About 1
Alcohol
Ref.
-
2 4 5
-
10% ethanol
Table
1 of ref.
6
1
molar extinction coefficient of a guanidino compound to that of creatine, is influenced by various experimental conditions which include: (A) time, (B) temperature, (C) wavelength, and (D) addition of alcohol. (A) Time. Variation with time of the optical densities of color complexes at 530 rnp produced by creatine, arginine, glycocyamine, and guanidine at 25” is shown in Fig. 1. Creatine uniquely produced a very high color intensity that attained its maximal intensity much earlier than the others. At 25”, it took less than 15 min for creatine to attain its maximal color intensity, while 75 min was required for arginine and
20
TAO
WONG
___-
_-
---
CREATINE
..,._...,....“.........~
_____._._._.
ARGININE
-
m.~~~cY.aIN~
GOANIDINE
Fm. 1. Optical density of red color complex formed by creatine, arginine, glycocyamine, and guanidine at 530 m,u vs time at 250°C. Each tube contains 0.4 eole guanidino compounds.
guanidine, snd 2 hr or more for glycocyamine. As a result, the “ratio” at 25” was small at the beginning, but increased continuously and subsequently leveled off, as shown by Figs. 2 and 3. (B) Temperature. The a-naphthol-diacetyl reaction usually was conducted at room temperature. Occasionally a low temperature was employed (10). The effect on the “ratio” due to temperature was investigated, and the results are shown in Figs. 2 and 3. Generally speaking, lowering the temperature retarded all reaction rates, but not in the
DETERMINATION
c--a 4 Q
* i
OF
EXTINCTIO;; EXTINCTIOU
COSFZICIEi:T COEFFICIENT
OF ARGIIIKE OP CRFATINE
EXTIHCTION ~XTINCTIOB
CCmPFICIE::T COBF-
OF GUA:IIDINE 0F c RE AT 1:s ->
/
/’
I/ i
0
//
I
/
30
21
CREATINE
,a'
"."'
_---.-._
253c
..,. .... . . ..& ."'.. * ,,,.,.
.. ..A "
60
90
-.A .fi
0 25 c 15%
120
TIME IN EllNUTES
FIG. 2. “Ratio” of extinction creatine at 530 mp vs time.
coefficient of arginine
and of guanidine
to that of
came proportion for different guanidino compounds. A lower temperature delayed the reactions of arginine, glycocyamine, and guanidine to a greater extent than that of creatine. Thus at 15” creatine reached its maximal color intensity at about 35 min, when the “ratio” for glycocyamine was 1/T. On the other hand, at 0” creatine reached its maximal color intensity at about 2.25 hr, but the “ratio” for glycocyamine at this moment was only 1/1s. (It was difl!cult to obtain the “ratio” for the temperature of 25” at 15 min because it changed too quickly.) Therefore, the “ratios” diminish for at least the first 2 hr if a lower temperature is employed. It was found with experiments at 0” that 1% cr-naphthol was frozen in stock alkali. This diBiculty was circumvented by using 3 vol of 1/37o a-naphthol in diluted (1 + 3 dilution) stock alkali.
TAO
FIG.
3. “Ratio”
of extinction
WONG
coefficient
of glycocyamine
to that of creatine,
vs time.
(Cl Wavelength. Eggleton et al. first reported that the red color caused by creatine has maximal absorption at 525 rn+ There was no report on the wavelength of maximal absorption by other guanidino compounds. During the course of studying the wavelengths of maximal absorption, the following phenomena were observed at room temperature. The wavelength of maximal absorption (hmax) of the color formed by creatine was not stationary but changed for at least the first 2 hr at room temperature. Soon after the appearance of the color, &,a shifted continuously but slowly, and slightly toward ultraviolet. After reaching 528 e 1 rnp, the X,,, shifted back continuously, slowly and slightly toward the infrared. The time to reach 528 ‘k 1 rn,u coincided with the time to achieve maximal intensity of the color development. Thus, at 23”, Amax reached 532 Z!Z 1 rnp after 5 min. It reached 528 C 1 rnp after 20 min when the color attained its maximal intensity at 18 -+ 2 min. The location was at 534 zk 1 mp after 66 min. Although the magnitude of
DETERMINATION
OF
CREATINE
23
the shifting was not large, this phenomenon may partly explain the many different wavelengths employed in creatine determination. In the literature, various values of the wavelength for creatine determination have been used: 520 rnp (4,6,10-12), 525 rnp (13-15), 530 mp (2,3,16), 533 rnp (17), 535 rnp (18)) and 546 rnp (19,20). The absorption curves at various time interwined at or near 570 rnp. The advantage of employing 570 rnp is recorded in the following discussion. The red color formed by arginine, glycocyamine, and guanidine had its maximal absorption at 523 _t 1 rnp, 522 4 1 rnp, and 525 t_ 1 rnp, respectively, when color intensities were at their maximal values. The locations of x,,, values subsequently shifted, like that of creatine, slowly, slightly, and continuously toward infrared. However, we were unable to decide whether or not there was a shift during the early period. Using different wavelengths for the estimation of creatine in the presence of other guanidino compounds should yield different “ratio” values because their maximal absorptions are different. Two extremes, 520 rn,p and 546 rnp, were selected to test the effect on the “ratio,” and the results are shown by Fig. 3. The “ratio,” as expected, is indeed different if a different wavelength is employed. (D) Ad&tion of Alcohol. See II below. II. Efects Produced by Addition of Alcohol Ethanol and n-propanol were used for these studies. Their concentrations are shown in Table 2. All experiments were carried out at room temperature. The effects caused by addition of various concentrations of ethanol or n-propanol are as follows: (A) It stabilizes the location of A,,,, the absorption peak. (B) It relocates the position of h,,, -the higher the alcohol concentration, the more the absorption peak ‘shifts toward the infrared. Besides, addition of alcohol, especially at high concentration, alters the shapes of the curves, as shown by Fig. 4. The wavelength locations of x maX of creatine, arginine, glycocyamine, and guanidine in various concentrations of alcohol are in Table 2. (C) It accelerates the color formation. The time to reach the maximal color intensity was about one-half or less of the time when no alcohol is added. In many cases, the time was only a fraction of that without alcohol. (D) It alters the molar extinction coefficients. With arginine, guanidine, or glycocyamine, the color was nearly always intensified by addition of alcohol In most cases, the molar extinction coefficients increased twoor three-fold. With creatine, the molar extinction coefficients decreased. The relative extinction coefficients of these four guanidino compounds
TAO
WONG
M -t-l iyQ%
m +I
DETERMINATION
OF
25
CREATINE
FIO. 4. Absorption curves of colored solutions formed by creatine in of 0% (270c), 20% (26°c), 40% (25”C), 60% WC), and 80% (26°C) Each tube contained 0.4 rmole creatine.
Pre=nce
ethanol.
in various concentrations of ethanol and n-propanol using the wavelength of the respective maximal absorption are given in Table 2. DISCUSSION
Two nonenzymic methods are generally employed for estimating creatine in biological samples. They are (a) the Jaff& reaction after conversion of creatine into creatinine (21,22) and (b) the a-naphtholdiacetyl reaction. For many reasons, method (a) is subject to gross errors (5,14). The errors, in our opinion, may reach as high as 2907’0 or even higher when the method is applied to urine which contains a very large amount of creatine. Method (b) is not ideal either, as evidenced by the numerous modifications suggested. This may be attributed partly to insufficient understanding of the nature of the reaction. A thorough description of the method and the various factors which may lead to erroneous results was given by Kanig (19). It is hoped that the present investigation will lead to a further improvement of the method. Of six wavelengths employed in the a-naphthol-diacetyl reaction, the use of 520 rnp and 525 mp has a disadvantage only. Using one of the other four wavelengths has both an advantage and a disadvantage. For
26
TAO WONG
color stability, especially at room temperature, the desirability is in the following order: 546 rnp > 535 rnp > 533 mp > 530 rnp. For sensitivity, the order is reversed. Therefore, one may select any one of these four wavelengths depending on the particular purpose one wishes to achieve. If the primary concern is stability of color, the use of 570 rnp is recommended; however, the intensity of color at this wavelength is only about half of that at 530 rnp (9). Temperature is an important factor when creatine is estimated in the presence of arginine, glycocyamine, and guanidine, and 0” is recommended. It was found that using 0” also has an advantage over room temperature when creatine is estimated in the presence of creatinine. The conversion of creatinine into creatine is influenced by temperature (Fig. 5 of ref. 19). Its hydrolysis rate diminishes significantly as the temperature decreases. As a result, the “ratio” for creatine at 0” is smaller than that at room temperature. Thus at 27” the “ratio” is about 0.025 after 15 min, whereas at 0” it is 0.011 after 2.25 hr. For determination of creatine in the absence of other guanidino compounds, operation at 0” is not necessary. It was found that working at room temperature is satisfactory provided the room temperature is below 25”. In our laboratory when room temperature was above 25”, 15” was used with the aid of a water bath. The advantages of 15” in comparison with a higher temperature are more stable color and a slight increase in sensitivity. Time is also an important factor. In the presence of other guanidino compounds at 0” readings are to be taken before the color is fully developed. In the absence of other guanidino compounds at room temperature, readings are to be taken immediately after the color is fully developed. Following are procedures for the estimation of urinary creatine using the temperature of 0” for all solutions and operations. Up to 0.4 pmole of creatine solution (0.1 ml of urine) is delivered to a 10 ml graduated tube. Then 5 ml of freshly prepared 0.57 o cu-naphthol in alkali solution and 1 ml of 0.05% diacetyl are added. This is diluted by water to make a 10 ml volume. After mixing, the solution is left in an ice bath for 1.75 hr with occasional shakings. The red color is determined photometrically at 530 mp. An alkali solution is prepared by dissolving 20 gm of NaOH and 55 gm of anhydrous Na,C03 in 1 liter of water. In 1948, Ennor and Stocken (23) found that --SH compounds such as glutathione inhibit the cu-naphthol-diacetyl reaction. For the estimation of creatine in biological tissues other than urine, serum, and skeletal muscle, the addition of p-chloromercuribenzoate (PCMB) is necessary to remove the inhibition unless prior isolation of creatine is carried out (6).
DEX’EBMINATION
OF
CBEATINE
27
Many experiments were conducted by us to discover the eventual effects of PCMB on the a-naphthol-diacetyl reaction. No significant difference in results between using and omitting PCMB was found. Thus, the addition of PCMB has no effect on h,,, for creatine. This is also true for glycocyamine. No change in “ratio” for glycocyamine was found at room temperature and lower temperatures. It was after the completion of experiments and the writing of this manuscript that the article by Gundlach et al. (24) was brought to our attention. They proposed that creatine be estimated by the cy-naphtholdiacetyl reaction at 7.5” using an automatic analyzer. According to our experience the temperature can be brought down further to 0” for better results. SUMMARY
1. For estimation of creatine in the presence of other guanidino compounds the following factors are important: (a) time, (b) temperature, (c) wavelength, and (d) addition of alcohol. 2. In the absence of alcohol, creatine, arginine, glycocyamine, and guanidine all react with a-naphthol-diacetyl in alkali to form a redcolored solution. Creatine uniquely forms an intensive color in a short time. Lowering the temperature retards all reaction rates but not at the same proportion; it delays the reactions with arginine, glycocyamine, and guanidine to a greater extent than that of creatine. 3. An improved procedure for the estimation of creatine in the presence of other guanidino compounds is presented. ACKNOWLEDGMENTS The author wishes to express his gratitude to Dean L. C. Behr and Dr. D. W. Emerich for their encouragement throughout the investigation. He also thanks the Biological and Physical Science Research Institute, Mississippi State University, State College, Mississippi, for the financial support. REFERENCES M. M., J. Path. Bact. 42, 441 (1936). P., ELSDEN, S. R., GOUOH, N., Biochem. J. 37, 526 (1943). WONQ, T., Anal. Biochem. 27, 218 (1969). MELVILLE, R. S., AND HUMMEL, J. P., J. Bid. Chem. 191, 333 (1951). F~AAFLAUB, J., AND ABELIN, I., B&hem. 2. 321, 153 (1950). EDEN, E., H~ISON, D. D., AND LINNANE, A. W., Azcstralian J. Exptl. Bid. 35 333 (1954). LAUBER, V. K., 2. Klin. Chem. 4, 119 (1966). RBSENBEUQ, H., ENNOR, A. H., AND MORRISON, J. F., Biochem. J. 63, 153 (1956). WONO, T., AND WENTHORTH, S. D., unpublished observation (1968). BONAS, J. E., COHEN, B. D., AND NATELSON, S., Microchem. J. 7, 63 (1963). GEREIEZS, G. B., GERBER, G., AND ALTMAN, K. I., And. Chem. 33, 852 (1901).
1. BARRITT, 2. EQGLETON,
3. 4. 5. 6.
7. 8.
9.
19. 11.
28
TAO
WONG
12. GEIFFITHS, W. J., Cl&. C/&n. Acta 9, 210 (1964). 13. DUBNOFF, J. W., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 635. Academic Press, New York, 1957. 14. ANDERSON, D. R., WILLXAMS, C. M., KRISE, G. M., AND DOWBEN, R. M., Biochem. J. 67, 258 (1957). 15. KUBOHARA, S. S., Cl&. Chsm. 7, 384 (1961). 16. ABELIN, I., AND RAAFLWB, J., Biochem. 2. 323, 382 (1952). 17. MENANCW, A., TEMPE, J., AND Mom, G., Phyd. Veg. 4 (4), 317-22 (19661, Chem. Abstr. 66, 92271~ (1967). 18. SAKAQUCHI, M., E~JITA, M., AND SHIMIZU, W., Nippon Suisan Gakkaishi 29, 5314 (19631, Chem. Abstr. 61, 4609e (1964). 19. KANIQ, K., Z. Physid. Chem. 366, 247 (1957). 20. THORN, W., ISSELHABD, W., AND IRMSCHER, K., B&hem. 2. 330, 385 (1958). 21. FOLIN, O., J. Bid. Chem. 17, 469 (1914). 22. TAUSSKY, H. H., in “Standard Methods of Clinical Chemistry” (D. Seligson, ed.), Vol. HI, p. 99. Academic Press, New York, 1961. 23. ENNOR, A. H., AND STOCKEN, L. A., B&hem. .I. 42, 557 (1948). 24. GUNDLACH, G., HOPPEF~~EYLER, G., AND JOHANN, H., Z. Klin. Chem. K&L Biothem. 1968, 6(5), 415-18, Chem. Abstr. 70, 44737~ (1969).
BIOCEFNISTBY
Studies
40,
18-28
on
(1971)
Creatine
Determination
by cr-Naphthol-Diacetyl
Reaction
TAO WONG Department
of State
Chemktrg, College,
Mississippi Mississippi
State 39762
University,
Received July 1, 1970
Based upon the observation by Barritt (1) that a-naphthol enhances color formation in the creatine-diacetyl reaction, Eggleton et ~2. (2) in 1943 described a procedure for the determination of creatine. This procedure and its modifications have been extensively used for the determination of this compound. During the course of Istudying the determination of phosphocreatine (3), we were puzzled by the controversy in the literature as to whether or not other guanidino compounds such as arginine, etc. also yield a significant amount of color with the a-naphthol-diacetyl reaction, and, thus, constitute a source of error for the determination of creatine (1,2,&6). In this communication we wish to offer explanations for this disagreement and to present the conditions that minimize error in the creatine estimation if the prior isolation of creatine is not carried out (7). When Barritt first introduced the cr-naphthol-diacetyl reaction, his solution contained 10% alcohol. However, in the procedure described by Eggleton et al. the alcohol is omitted entirely. In 1956, Rosenberg et al. (8) recommended a procedure for the determination of arginine which is essentially a slight modification of the Barritt procedure. One minor difference is that 10% ethanol is replaced by 20% n-propanol. We have therefore studied the effects of alcohol and found that the effects are multiple, profound, and diverse. PROCEDURES
Experiments in the Absence of Alcohol. The Eggleton et al. (2) procedure or those slightly modified as specified in the text were used. Effects Produced by Addition of Alcohol. The Rosenberg et al. (8) procedure or those slightly modified as specified in the text were used. The reason for the switch to the Rosenberg et al. procedure was that Na2C03 in the Eggleton et al. procedure interfered with the ,alcohol additions in many cases. 18
DEXEFMINATION
OF
19
CREATINE
The readings of absorbance due to the red color formed in all procedures used were recorded either by a Coleman Junior or by a Perkin-Elmer 202 spectrophotometer. The scanning speed of the Perkin-Elmer 202 was set at the fast scale, which takes 2 min to cover the range from 350 to 750 rnp. Such a speed is fast enough to observe the phenomena described in experimental section IC below. The wavelength of the Perkin-Elmer 202 was calibrated with a holmium oxide filter for each reading. MATERIALS
The sources of all chemicals used have been previously
described (3).
EXPERIMENTS
I. Experiments
in the Absence of Alcohol
In determining creatine by the a-naphthol-diacetyl method, it is controversial whether the presence of arginine or other guanidino compounds also yields a significant amount of color and, thus, constitutes a source of error (see Table 1). The value of “ratio,” which is defined as the Disagreement
TABLE 1 in the Literature over Amount of Color Produced by Various Guanidino Compounds
Extinction coefficient of arginine and glycocyamine
Extinction coefficient of guanidine
extinction coefficient of creatine
extinction coefficient of creatine
1/g l/10 l/5-1/6 l/2.5-1/3 About 1
l/9 l/10 l/10
About 1
Alcohol
Ref.
-
2 4 5
-
10% ethanol
Table
1 of ref.
6
1
molar extinction coefficient of a guanidino compound to that of creatine, is influenced by various experimental conditions which include: (A) time, (B) temperature, (C) wavelength, and (D) addition of alcohol. (A) Time. Variation with time of the optical densities of color complexes at 530 rnp produced by creatine, arginine, glycocyamine, and guanidine at 25” is shown in Fig. 1. Creatine uniquely produced a very high color intensity that attained its maximal intensity much earlier than the others. At 25”, it took less than 15 min for creatine to attain its maximal color intensity, while 75 min was required for arginine and
20
TAO
WONG
___-
_-
---
CREATINE
..,._...,....“.........~
_____._._._.
ARGININE
-
m.~~~cY.aIN~
GOANIDINE
Fm. 1. Optical density of red color complex formed by creatine, arginine, glycocyamine, and guanidine at 530 m,u vs time at 250°C. Each tube contains 0.4 eole guanidino compounds.
guanidine, snd 2 hr or more for glycocyamine. As a result, the “ratio” at 25” was small at the beginning, but increased continuously and subsequently leveled off, as shown by Figs. 2 and 3. (B) Temperature. The a-naphthol-diacetyl reaction usually was conducted at room temperature. Occasionally a low temperature was employed (10). The effect on the “ratio” due to temperature was investigated, and the results are shown in Figs. 2 and 3. Generally speaking, lowering the temperature retarded all reaction rates, but not in the
DETERMINATION
c--a 4 Q
* i
OF
EXTINCTIO;; EXTINCTIOU
COSFZICIEi:T COEFFICIENT
OF ARGIIIKE OP CRFATINE
EXTIHCTION ~XTINCTIOB
CCmPFICIE::T COBF-
OF GUA:IIDINE 0F c RE AT 1:s ->
/
/’
I/ i
0
//
I
/
30
21
CREATINE
,a'
"."'
_---.-._
253c
..,. .... . . ..& ."'.. * ,,,.,.
.. ..A "
60
90
-.A .fi
0 25 c 15%
120
TIME IN EllNUTES
FIG. 2. “Ratio” of extinction creatine at 530 mp vs time.
coefficient of arginine
and of guanidine
to that of
came proportion for different guanidino compounds. A lower temperature delayed the reactions of arginine, glycocyamine, and guanidine to a greater extent than that of creatine. Thus at 15” creatine reached its maximal color intensity at about 35 min, when the “ratio” for glycocyamine was 1/T. On the other hand, at 0” creatine reached its maximal color intensity at about 2.25 hr, but the “ratio” for glycocyamine at this moment was only 1/1s. (It was difl!cult to obtain the “ratio” for the temperature of 25” at 15 min because it changed too quickly.) Therefore, the “ratios” diminish for at least the first 2 hr if a lower temperature is employed. It was found with experiments at 0” that 1% cr-naphthol was frozen in stock alkali. This diBiculty was circumvented by using 3 vol of 1/37o a-naphthol in diluted (1 + 3 dilution) stock alkali.
TAO
FIG.
3. “Ratio”
of extinction
WONG
coefficient
of glycocyamine
to that of creatine,
vs time.
(Cl Wavelength. Eggleton et al. first reported that the red color caused by creatine has maximal absorption at 525 rn+ There was no report on the wavelength of maximal absorption by other guanidino compounds. During the course of studying the wavelengths of maximal absorption, the following phenomena were observed at room temperature. The wavelength of maximal absorption (hmax) of the color formed by creatine was not stationary but changed for at least the first 2 hr at room temperature. Soon after the appearance of the color, &,a shifted continuously but slowly, and slightly toward ultraviolet. After reaching 528 e 1 rnp, the X,,, shifted back continuously, slowly and slightly toward the infrared. The time to reach 528 ‘k 1 rn,u coincided with the time to achieve maximal intensity of the color development. Thus, at 23”, Amax reached 532 Z!Z 1 rnp after 5 min. It reached 528 C 1 rnp after 20 min when the color attained its maximal intensity at 18 -+ 2 min. The location was at 534 zk 1 mp after 66 min. Although the magnitude of
DETERMINATION
OF
CREATINE
23
the shifting was not large, this phenomenon may partly explain the many different wavelengths employed in creatine determination. In the literature, various values of the wavelength for creatine determination have been used: 520 rnp (4,6,10-12), 525 rnp (13-15), 530 mp (2,3,16), 533 rnp (17), 535 rnp (18)) and 546 rnp (19,20). The absorption curves at various time interwined at or near 570 rnp. The advantage of employing 570 rnp is recorded in the following discussion. The red color formed by arginine, glycocyamine, and guanidine had its maximal absorption at 523 _t 1 rnp, 522 4 1 rnp, and 525 t_ 1 rnp, respectively, when color intensities were at their maximal values. The locations of x,,, values subsequently shifted, like that of creatine, slowly, slightly, and continuously toward infrared. However, we were unable to decide whether or not there was a shift during the early period. Using different wavelengths for the estimation of creatine in the presence of other guanidino compounds should yield different “ratio” values because their maximal absorptions are different. Two extremes, 520 rn,p and 546 rnp, were selected to test the effect on the “ratio,” and the results are shown by Fig. 3. The “ratio,” as expected, is indeed different if a different wavelength is employed. (D) Ad&tion of Alcohol. See II below. II. Efects Produced by Addition of Alcohol Ethanol and n-propanol were used for these studies. Their concentrations are shown in Table 2. All experiments were carried out at room temperature. The effects caused by addition of various concentrations of ethanol or n-propanol are as follows: (A) It stabilizes the location of A,,,, the absorption peak. (B) It relocates the position of h,,, -the higher the alcohol concentration, the more the absorption peak ‘shifts toward the infrared. Besides, addition of alcohol, especially at high concentration, alters the shapes of the curves, as shown by Fig. 4. The wavelength locations of x maX of creatine, arginine, glycocyamine, and guanidine in various concentrations of alcohol are in Table 2. (C) It accelerates the color formation. The time to reach the maximal color intensity was about one-half or less of the time when no alcohol is added. In many cases, the time was only a fraction of that without alcohol. (D) It alters the molar extinction coefficients. With arginine, guanidine, or glycocyamine, the color was nearly always intensified by addition of alcohol In most cases, the molar extinction coefficients increased twoor three-fold. With creatine, the molar extinction coefficients decreased. The relative extinction coefficients of these four guanidino compounds
TAO
WONG
M -t-l iyQ%
m +I
DETERMINATION
OF
25
CREATINE
FIO. 4. Absorption curves of colored solutions formed by creatine in of 0% (270c), 20% (26°c), 40% (25”C), 60% WC), and 80% (26°C) Each tube contained 0.4 rmole creatine.
Pre=nce
ethanol.
in various concentrations of ethanol and n-propanol using the wavelength of the respective maximal absorption are given in Table 2. DISCUSSION
Two nonenzymic methods are generally employed for estimating creatine in biological samples. They are (a) the Jaff& reaction after conversion of creatine into creatinine (21,22) and (b) the a-naphtholdiacetyl reaction. For many reasons, method (a) is subject to gross errors (5,14). The errors, in our opinion, may reach as high as 2907’0 or even higher when the method is applied to urine which contains a very large amount of creatine. Method (b) is not ideal either, as evidenced by the numerous modifications suggested. This may be attributed partly to insufficient understanding of the nature of the reaction. A thorough description of the method and the various factors which may lead to erroneous results was given by Kanig (19). It is hoped that the present investigation will lead to a further improvement of the method. Of six wavelengths employed in the a-naphthol-diacetyl reaction, the use of 520 rnp and 525 mp has a disadvantage only. Using one of the other four wavelengths has both an advantage and a disadvantage. For
26
TAO WONG
color stability, especially at room temperature, the desirability is in the following order: 546 rnp > 535 rnp > 533 mp > 530 rnp. For sensitivity, the order is reversed. Therefore, one may select any one of these four wavelengths depending on the particular purpose one wishes to achieve. If the primary concern is stability of color, the use of 570 rnp is recommended; however, the intensity of color at this wavelength is only about half of that at 530 rnp (9). Temperature is an important factor when creatine is estimated in the presence of arginine, glycocyamine, and guanidine, and 0” is recommended. It was found that using 0” also has an advantage over room temperature when creatine is estimated in the presence of creatinine. The conversion of creatinine into creatine is influenced by temperature (Fig. 5 of ref. 19). Its hydrolysis rate diminishes significantly as the temperature decreases. As a result, the “ratio” for creatine at 0” is smaller than that at room temperature. Thus at 27” the “ratio” is about 0.025 after 15 min, whereas at 0” it is 0.011 after 2.25 hr. For determination of creatine in the absence of other guanidino compounds, operation at 0” is not necessary. It was found that working at room temperature is satisfactory provided the room temperature is below 25”. In our laboratory when room temperature was above 25”, 15” was used with the aid of a water bath. The advantages of 15” in comparison with a higher temperature are more stable color and a slight increase in sensitivity. Time is also an important factor. In the presence of other guanidino compounds at 0” readings are to be taken before the color is fully developed. In the absence of other guanidino compounds at room temperature, readings are to be taken immediately after the color is fully developed. Following are procedures for the estimation of urinary creatine using the temperature of 0” for all solutions and operations. Up to 0.4 pmole of creatine solution (0.1 ml of urine) is delivered to a 10 ml graduated tube. Then 5 ml of freshly prepared 0.57 o cu-naphthol in alkali solution and 1 ml of 0.05% diacetyl are added. This is diluted by water to make a 10 ml volume. After mixing, the solution is left in an ice bath for 1.75 hr with occasional shakings. The red color is determined photometrically at 530 mp. An alkali solution is prepared by dissolving 20 gm of NaOH and 55 gm of anhydrous Na,C03 in 1 liter of water. In 1948, Ennor and Stocken (23) found that --SH compounds such as glutathione inhibit the cu-naphthol-diacetyl reaction. For the estimation of creatine in biological tissues other than urine, serum, and skeletal muscle, the addition of p-chloromercuribenzoate (PCMB) is necessary to remove the inhibition unless prior isolation of creatine is carried out (6).
DEX’EBMINATION
OF
CBEATINE
27
Many experiments were conducted by us to discover the eventual effects of PCMB on the a-naphthol-diacetyl reaction. No significant difference in results between using and omitting PCMB was found. Thus, the addition of PCMB has no effect on h,,, for creatine. This is also true for glycocyamine. No change in “ratio” for glycocyamine was found at room temperature and lower temperatures. It was after the completion of experiments and the writing of this manuscript that the article by Gundlach et al. (24) was brought to our attention. They proposed that creatine be estimated by the cy-naphtholdiacetyl reaction at 7.5” using an automatic analyzer. According to our experience the temperature can be brought down further to 0” for better results. SUMMARY
1. For estimation of creatine in the presence of other guanidino compounds the following factors are important: (a) time, (b) temperature, (c) wavelength, and (d) addition of alcohol. 2. In the absence of alcohol, creatine, arginine, glycocyamine, and guanidine all react with a-naphthol-diacetyl in alkali to form a redcolored solution. Creatine uniquely forms an intensive color in a short time. Lowering the temperature retards all reaction rates but not at the same proportion; it delays the reactions with arginine, glycocyamine, and guanidine to a greater extent than that of creatine. 3. An improved procedure for the estimation of creatine in the presence of other guanidino compounds is presented. ACKNOWLEDGMENTS The author wishes to express his gratitude to Dean L. C. Behr and Dr. D. W. Emerich for their encouragement throughout the investigation. He also thanks the Biological and Physical Science Research Institute, Mississippi State University, State College, Mississippi, for the financial support. REFERENCES M. M., J. Path. Bact. 42, 441 (1936). P., ELSDEN, S. R., GOUOH, N., Biochem. J. 37, 526 (1943). WONQ, T., Anal. Biochem. 27, 218 (1969). MELVILLE, R. S., AND HUMMEL, J. P., J. Bid. Chem. 191, 333 (1951). F~AAFLAUB, J., AND ABELIN, I., B&hem. 2. 321, 153 (1950). EDEN, E., H~ISON, D. D., AND LINNANE, A. W., Azcstralian J. Exptl. Bid. 35 333 (1954). LAUBER, V. K., 2. Klin. Chem. 4, 119 (1966). RBSENBEUQ, H., ENNOR, A. H., AND MORRISON, J. F., Biochem. J. 63, 153 (1956). WONO, T., AND WENTHORTH, S. D., unpublished observation (1968). BONAS, J. E., COHEN, B. D., AND NATELSON, S., Microchem. J. 7, 63 (1963). GEREIEZS, G. B., GERBER, G., AND ALTMAN, K. I., And. Chem. 33, 852 (1901).
1. BARRITT, 2. EQGLETON,
3. 4. 5. 6.
7. 8.
9.
19. 11.
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12. GEIFFITHS, W. J., Cl&. C/&n. Acta 9, 210 (1964). 13. DUBNOFF, J. W., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 635. Academic Press, New York, 1957. 14. ANDERSON, D. R., WILLXAMS, C. M., KRISE, G. M., AND DOWBEN, R. M., Biochem. J. 67, 258 (1957). 15. KUBOHARA, S. S., Cl&. Chsm. 7, 384 (1961). 16. ABELIN, I., AND RAAFLWB, J., Biochem. 2. 323, 382 (1952). 17. MENANCW, A., TEMPE, J., AND Mom, G., Phyd. Veg. 4 (4), 317-22 (19661, Chem. Abstr. 66, 92271~ (1967). 18. SAKAQUCHI, M., E~JITA, M., AND SHIMIZU, W., Nippon Suisan Gakkaishi 29, 5314 (19631, Chem. Abstr. 61, 4609e (1964). 19. KANIQ, K., Z. Physid. Chem. 366, 247 (1957). 20. THORN, W., ISSELHABD, W., AND IRMSCHER, K., B&hem. 2. 330, 385 (1958). 21. FOLIN, O., J. Bid. Chem. 17, 469 (1914). 22. TAUSSKY, H. H., in “Standard Methods of Clinical Chemistry” (D. Seligson, ed.), Vol. HI, p. 99. Academic Press, New York, 1961. 23. ENNOR, A. H., AND STOCKEN, L. A., B&hem. .I. 42, 557 (1948). 24. GUNDLACH, G., HOPPEF~~EYLER, G., AND JOHANN, H., Z. Klin. Chem. K&L Biothem. 1968, 6(5), 415-18, Chem. Abstr. 70, 44737~ (1969).