- Email: [email protected]
Determination of phenylpyruvic acid in the urine of patients with oligophrenia phenylpyruvica
CLINICACHIMICAACTA
424
DETERMINATION OF PATIENTS
OF PHENYLPYRUVIC WITH
T. P. THE, Paediatric Department,
OLIGOPHRENIA
P. FLEURY
AND
VOL. 2 (1957)
ACID IN THE
URINE
PHENYLPYRUVICA
C. L. J. VINK
University Hospital, Leiden
(The Netherlands)
INTRODUCTION The diagnostic indication for oligophrenia phenylpyruvica is formed by the excretion of phenylpyruvic acid (PPA) in the urine. The quantitative determination of PPA, however, presents certain difficulties. Gravimetric and calorimetric determinations of the 2,4-dinitrophenylhydrazone (PENROSE AND QUASTEL’) are not specific, since other keto-acids of the urine are also determined. This results in increased false values. More specific is the determination of PPA with ferric salts, described by POLONOVSKIet al., a BERRY AND WOOLF~,JERVIS~,KROPP AND LANG5 and KEUP~. POLONOVSKIet al., 2 used a very simple method. They determined PPA in the urine (diluted 50 times) with ferri-ammonium sulfate at a pH between 1 and 7. BERRY AND WOOLF~extracted PPA with ether from an acidified solution, then evaporated the ether in a stream of nitrogen at room temperature, and dissolved the residue in a buffer with a pH of 2.2. After addition of FeCl,, PPA was determined calorimetrically. JERVIS’ described a similar procedure for serum after deproteinizing. The only ways in which this method differs from the others are: the ether is dried with anhydrous sodium sulfate and evaporation is performed in vacua. KROPP AND LANGEstabilized the green reaction product with ferric citrate in order to increase the accuracy of the calorimetric determination. KEUP~ used a paper chromatographic analysis, which is more specific than any method mentioned here. However, it is rather time-consuming for clinical routine work. The purpose of this article is to study the reaction of PPA with FeCl, in order to obtain a simple, routine method in the clinical laboratory. Its specificity will also be discussed. MATERIALS A Beckman spectrophotometer (Model B) is used to determine the concentration of the green reaction product. The calibration curve and the recoveries are determined with the Na salt of PPA (Hoffmann-La Roche, Basle), purity about g6 %. The results are corrected for the 2.1 oh water content of this Na salt. Our experience is that the crystalline PPA is transformed into mandelic acid (odour). We found the uncorrected m.p. of this acid to be 1rS.5” C, which is in agreement with SCHOORL’,who found 120~ C to be the m.p. of dl-mandelic acid. Pb acetate References p. 428
VOL. 2 (1957)
PHENYLPYRUVICACID IN URINE
425
produces sheaves of needle-shaped crystals with mandelic acid. In solution, PPA is unstable, especially in the alkaline range. Buffer solution pH 2.2: glycine, 4.35 g; NaCl, 3.40 g; 0.1 N HCl, 420 ml; plus distilled water to make up to I 1. FeCl, solution: IO % of FeCI, in distilled water. The concentration of PPA was determined in several urines of patients with oligophrenia phenylpyruvica. Fresh urines should be used because of the unstability of PPA. Recoveries of PPA were determined in normal urines. METHODSAND RESULTS
A disadvantage in the determination of PPA with ferric chloride is the dependence of time, temperature, and pH, on the formation and stability of the green reaction component. We get good results at a pH of 2.2; therefore, all the determinations were performed at this pH. The authors, using ferric salt methods, waited an arbitrary interval after mixing (at room temperature) at which the concentration was determined with the use of a calorimeter. POLONOVSKIet al.2 waited about 2 minutes; JERVIS~ exactly 2 minutes, and BERRY AND WOOLF~ between 2 and 33 minutes. We were able to combine the influences of time and temperature in the determination of the concentration. Immediately after the reaction components were mixed, the extinction was followed on the Beckman Spectrophotometer (Model B).
I
1
I
I
2
3
I
4
I
5
I
6
time in minutes
Fig. I. Influence of temperature on the kinetic behaviour of the reaction product of PPA and FeCl,. The results are the mean values of duplicates. The temperatures are the mean values of the beginning and final temperatures. 5 ml buffer pH 2.2 59.4 mg% PPA-Na I ml solution equivalent to: ( 34.8 mg% PPA 0.2 ml FeCls-solution loo/( References p. 4.28
Standard solution 1
5 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Blank 10%
T. P. THE, P. FLEURY, C. L. J. VINK
426
VOL. 2
(1957)
It appeared that the maximum of extinction was not dependent upon the temperature; only the time needed to reach this maximum was dependent on the temperature of the solution. This is illustrated in Fig. I by the extinctions during the progress of the reaction (6 ml of the Na salt of PPA in a buffer of pH 2.2 with 0.2 ml of the FeCl, solution at a mean temperature of 31’, 22’, 16” and IO” C) at wavelengths of 630 mp. It is evident that, independent of the temperature, the maximum of the extinction is comtant. This is of great value in the accurate determination of PPA. A calorimeter and a spectrophotometer that require no adjusting at the zero point during the operation are very suitable for this procedure. The extinction-wavelength curve is determined with the maximum extinctions of identical solutions.
“mm 4000
Fig. 2. Extinction-wavelength
450
500
550
600
630650
X in my curve of the green complex of PPA from maximum extinctions in duplicate.
5 ml buffer pH 2.2 39.4 mg% PPA--Na I ml solution equivalent to 134.8 mg% PPA 0.2 ml FeCl,-solution 10%
Standard solution
and FeCl,.
5 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Dots obtained
Blank 10%
1
Fig. 2 shows two maxima: at 440 and at 630 mp. We preferred the maximum at 630 m,u since the extinction at this wavelength is higher than at 440 mp. In addition, at 440 m,u other (yellow) metabolites in the urine (e.g. phenyllactic acid and other a-hydroxycarboxylic acids) will give interference. All extinctions in this article were therefore read at il.max = 630 m,u. The calibration curve of the green reaction product appeared to be linear. This linearity was measured with extinction coefficients up to about 1.30. Using our method (I ml solution of PPA and 5 ml buffer compared with a blank, cf. Figs. I, 2), the concentration of an unknown sample can be obtained from the following equation : 630m~1 Concentration = 68.3 x Er cm mg PPA per IOO ml solution References p. 428
(I)
PHENYLPYRUVIC
VOL.2 (1957)
ACID IN URINE
427
In analysing the urines of patients with oligophrenia phenylpyruvica it appears that good results are obtained if a dilution of about I : 50 is used. In this dilution, precipitates of phosphates with the reagent are prevented. We used the following method: I ml of fresh urine (diluted IO times with distilled water) is mixed with 5 ml buffer pH 2.2. Then 0.2 ml of FeCl, solution (IO %) is added, mixed, and the maximum
&moo-
10
20
30
Fig. 3. Calibration 5mlbufferpHz.z I ml PPA-sol. (Cont. = 0.88~ x Cont. PPA-Na) 0.2 ml FeCl,-solution 10%
40 So mg PPA in 100 ml solution
curve of PPA. Standard solution
I
3 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Blank 10%
extinction is determined at 630 m,u. It is evident that the concentration of PPA in the urine (total dilution 62 times) is: Concentration = 683 x
630 mP
EI cm mg PPA per
IOO
ml urine
(2)
DISCUSSION
With the method described here, recoveries of 98 yO (mean value) of PPA were obtained in distilled water and in normal urines. This method was compared with the time-consuming one of BERRY AND WOOLF~. Identical results were obtained in standard solutions and in urines of patients with phenylketonuria. Therefore, the simple method lasting less than IO minutes, which we have described here, is preferred. According to the literature, the interference of $-hydroxyphenylpyruvic acid and of homogentisic acid can be disregarded in the methods using ferric saltsapbl @I. Besides, it appeared8 that in patients with oligophrenia phenylpyruvica, the excretion of p-hydroxyphenylpyruvic acid is almost completely dependent on the tyrosine and not on the phenylalanine intake, whereas in our patients, kept on a poor phenylalanine and on a normal tyrosine intake, our calorimetric test was negative. A clear proof of the interference of o-hydroxyphenylpyruvic acid in this test is not known. However, we found that the extinction-wavelength curve of the green References
p. 428
T. P. THE, P. FLEURY,
428
C. L. J. VINK
VOL. 2
(r957j
product in the urine of patients with phenylketonuria has a maximum at the same wavelength as a standard solution of PPA (630 mpj. It appeared that phenyllactic acid gives a yellow reaction product, of which the extinction at 630 m,u is negligible. SUMMARY
A simple, rapid and reproducible method is described for the determination of PPA in the urine of patients with oligophrenia phenylpyruvica. The influence of interfering substances is probably slight.
Description d’une methode simple, reproductible et suffisamment precise que pour pouvoir &tre utilisee en clinique, pour le dosage quantitatif de 1’APP dans l’urine des malades atteints d’oligophrenie phenylpyruvique. ZUSAMMRNFASSUNG
Eine einfache, reproduzierbare und fiir klinische Zwecke hinreichend spezifische Methode zur quantitativen Bestimmung der Phenylbrenztraubensaure in Ham von Kranken mit Oligophrenia phenylpyruvica wird beschrieben. PE 3IOME Omicbmaema
npoc~ot
zs nerico ~ocripous~o~zi~b1i4
onpenenem
M~TOAKonmec=e=oro
HIIKOTIIHOBOtiXEICAOTbI (PPA)
omro~pemieti. B MO9e IIaUEieHTOB,c~pwamw MeTOa o6na.qaeT~OCTaTOsHOfi TO¶HOCTbEO &AH KAUHPeCKHX ~[eA.t!fi.
&xiw&
REFERENCES I L. PENROSE AND J. H. QUASTEL, Biochem. J., 31(1937) 266. 2 M. POLONOVSKI,P.DESGREZAND F.DELBARRE, Bult.soc.chim.biol.,2g(lrg47) 3 J. P. BERRY AND L. J. WOOLF, Nature, 169 (1952) 202.
4 5 6 7 8
A. JERVIS, Proc. SOG. Exptl.Biol. Med., 81 (1952) 7x5. KROPPAND K.LANG, K&n. Wochschr.,33 (1955)482. KEUP, 2. physiol. Chem., 291 (1952)223; Biochem. z., 326 (1954) 14. SCHOORL, Ovganische Analyse, Vol. II, 3rd ed., N.V. Centens Uitgevers R. J. BOSCOTTAND H. BICKEL, Stand. J. CEin. & Lab. Inuest., 5 (1953) 380.
G. K. W. N.
Received
1049.
Mij,
1937. p. 256.
May r3th,
1957
424
DETERMINATION OF PATIENTS
OF PHENYLPYRUVIC WITH
T. P. THE, Paediatric Department,
OLIGOPHRENIA
P. FLEURY
AND
VOL. 2 (1957)
ACID IN THE
URINE
PHENYLPYRUVICA
C. L. J. VINK
University Hospital, Leiden
(The Netherlands)
INTRODUCTION The diagnostic indication for oligophrenia phenylpyruvica is formed by the excretion of phenylpyruvic acid (PPA) in the urine. The quantitative determination of PPA, however, presents certain difficulties. Gravimetric and calorimetric determinations of the 2,4-dinitrophenylhydrazone (PENROSE AND QUASTEL’) are not specific, since other keto-acids of the urine are also determined. This results in increased false values. More specific is the determination of PPA with ferric salts, described by POLONOVSKIet al., a BERRY AND WOOLF~,JERVIS~,KROPP AND LANG5 and KEUP~. POLONOVSKIet al., 2 used a very simple method. They determined PPA in the urine (diluted 50 times) with ferri-ammonium sulfate at a pH between 1 and 7. BERRY AND WOOLF~extracted PPA with ether from an acidified solution, then evaporated the ether in a stream of nitrogen at room temperature, and dissolved the residue in a buffer with a pH of 2.2. After addition of FeCl,, PPA was determined calorimetrically. JERVIS’ described a similar procedure for serum after deproteinizing. The only ways in which this method differs from the others are: the ether is dried with anhydrous sodium sulfate and evaporation is performed in vacua. KROPP AND LANGEstabilized the green reaction product with ferric citrate in order to increase the accuracy of the calorimetric determination. KEUP~ used a paper chromatographic analysis, which is more specific than any method mentioned here. However, it is rather time-consuming for clinical routine work. The purpose of this article is to study the reaction of PPA with FeCl, in order to obtain a simple, routine method in the clinical laboratory. Its specificity will also be discussed. MATERIALS A Beckman spectrophotometer (Model B) is used to determine the concentration of the green reaction product. The calibration curve and the recoveries are determined with the Na salt of PPA (Hoffmann-La Roche, Basle), purity about g6 %. The results are corrected for the 2.1 oh water content of this Na salt. Our experience is that the crystalline PPA is transformed into mandelic acid (odour). We found the uncorrected m.p. of this acid to be 1rS.5” C, which is in agreement with SCHOORL’,who found 120~ C to be the m.p. of dl-mandelic acid. Pb acetate References p. 428
VOL. 2 (1957)
PHENYLPYRUVICACID IN URINE
425
produces sheaves of needle-shaped crystals with mandelic acid. In solution, PPA is unstable, especially in the alkaline range. Buffer solution pH 2.2: glycine, 4.35 g; NaCl, 3.40 g; 0.1 N HCl, 420 ml; plus distilled water to make up to I 1. FeCl, solution: IO % of FeCI, in distilled water. The concentration of PPA was determined in several urines of patients with oligophrenia phenylpyruvica. Fresh urines should be used because of the unstability of PPA. Recoveries of PPA were determined in normal urines. METHODSAND RESULTS
A disadvantage in the determination of PPA with ferric chloride is the dependence of time, temperature, and pH, on the formation and stability of the green reaction component. We get good results at a pH of 2.2; therefore, all the determinations were performed at this pH. The authors, using ferric salt methods, waited an arbitrary interval after mixing (at room temperature) at which the concentration was determined with the use of a calorimeter. POLONOVSKIet al.2 waited about 2 minutes; JERVIS~ exactly 2 minutes, and BERRY AND WOOLF~ between 2 and 33 minutes. We were able to combine the influences of time and temperature in the determination of the concentration. Immediately after the reaction components were mixed, the extinction was followed on the Beckman Spectrophotometer (Model B).
I
1
I
I
2
3
I
4
I
5
I
6
time in minutes
Fig. I. Influence of temperature on the kinetic behaviour of the reaction product of PPA and FeCl,. The results are the mean values of duplicates. The temperatures are the mean values of the beginning and final temperatures. 5 ml buffer pH 2.2 59.4 mg% PPA-Na I ml solution equivalent to: ( 34.8 mg% PPA 0.2 ml FeCls-solution loo/( References p. 4.28
Standard solution 1
5 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Blank 10%
T. P. THE, P. FLEURY, C. L. J. VINK
426
VOL. 2
(1957)
It appeared that the maximum of extinction was not dependent upon the temperature; only the time needed to reach this maximum was dependent on the temperature of the solution. This is illustrated in Fig. I by the extinctions during the progress of the reaction (6 ml of the Na salt of PPA in a buffer of pH 2.2 with 0.2 ml of the FeCl, solution at a mean temperature of 31’, 22’, 16” and IO” C) at wavelengths of 630 mp. It is evident that, independent of the temperature, the maximum of the extinction is comtant. This is of great value in the accurate determination of PPA. A calorimeter and a spectrophotometer that require no adjusting at the zero point during the operation are very suitable for this procedure. The extinction-wavelength curve is determined with the maximum extinctions of identical solutions.
“mm 4000
Fig. 2. Extinction-wavelength
450
500
550
600
630650
X in my curve of the green complex of PPA from maximum extinctions in duplicate.
5 ml buffer pH 2.2 39.4 mg% PPA--Na I ml solution equivalent to 134.8 mg% PPA 0.2 ml FeCl,-solution 10%
Standard solution
and FeCl,.
5 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Dots obtained
Blank 10%
1
Fig. 2 shows two maxima: at 440 and at 630 mp. We preferred the maximum at 630 m,u since the extinction at this wavelength is higher than at 440 mp. In addition, at 440 m,u other (yellow) metabolites in the urine (e.g. phenyllactic acid and other a-hydroxycarboxylic acids) will give interference. All extinctions in this article were therefore read at il.max = 630 m,u. The calibration curve of the green reaction product appeared to be linear. This linearity was measured with extinction coefficients up to about 1.30. Using our method (I ml solution of PPA and 5 ml buffer compared with a blank, cf. Figs. I, 2), the concentration of an unknown sample can be obtained from the following equation : 630m~1 Concentration = 68.3 x Er cm mg PPA per IOO ml solution References p. 428
(I)
PHENYLPYRUVIC
VOL.2 (1957)
ACID IN URINE
427
In analysing the urines of patients with oligophrenia phenylpyruvica it appears that good results are obtained if a dilution of about I : 50 is used. In this dilution, precipitates of phosphates with the reagent are prevented. We used the following method: I ml of fresh urine (diluted IO times with distilled water) is mixed with 5 ml buffer pH 2.2. Then 0.2 ml of FeCl, solution (IO %) is added, mixed, and the maximum
&moo-
10
20
30
Fig. 3. Calibration 5mlbufferpHz.z I ml PPA-sol. (Cont. = 0.88~ x Cont. PPA-Na) 0.2 ml FeCl,-solution 10%
40 So mg PPA in 100 ml solution
curve of PPA. Standard solution
I
3 ml buffer pH 2.2 I ml distilled water 0.2 ml FeCl,-solution
Blank 10%
extinction is determined at 630 m,u. It is evident that the concentration of PPA in the urine (total dilution 62 times) is: Concentration = 683 x
630 mP
EI cm mg PPA per
IOO
ml urine
(2)
DISCUSSION
With the method described here, recoveries of 98 yO (mean value) of PPA were obtained in distilled water and in normal urines. This method was compared with the time-consuming one of BERRY AND WOOLF~. Identical results were obtained in standard solutions and in urines of patients with phenylketonuria. Therefore, the simple method lasting less than IO minutes, which we have described here, is preferred. According to the literature, the interference of $-hydroxyphenylpyruvic acid and of homogentisic acid can be disregarded in the methods using ferric saltsapbl @I. Besides, it appeared8 that in patients with oligophrenia phenylpyruvica, the excretion of p-hydroxyphenylpyruvic acid is almost completely dependent on the tyrosine and not on the phenylalanine intake, whereas in our patients, kept on a poor phenylalanine and on a normal tyrosine intake, our calorimetric test was negative. A clear proof of the interference of o-hydroxyphenylpyruvic acid in this test is not known. However, we found that the extinction-wavelength curve of the green References
p. 428
T. P. THE, P. FLEURY,
428
C. L. J. VINK
VOL. 2
(r957j
product in the urine of patients with phenylketonuria has a maximum at the same wavelength as a standard solution of PPA (630 mpj. It appeared that phenyllactic acid gives a yellow reaction product, of which the extinction at 630 m,u is negligible. SUMMARY
A simple, rapid and reproducible method is described for the determination of PPA in the urine of patients with oligophrenia phenylpyruvica. The influence of interfering substances is probably slight.
Description d’une methode simple, reproductible et suffisamment precise que pour pouvoir &tre utilisee en clinique, pour le dosage quantitatif de 1’APP dans l’urine des malades atteints d’oligophrenie phenylpyruvique. ZUSAMMRNFASSUNG
Eine einfache, reproduzierbare und fiir klinische Zwecke hinreichend spezifische Methode zur quantitativen Bestimmung der Phenylbrenztraubensaure in Ham von Kranken mit Oligophrenia phenylpyruvica wird beschrieben. PE 3IOME Omicbmaema
npoc~ot
zs nerico ~ocripous~o~zi~b1i4
onpenenem
M~TOAKonmec=e=oro
HIIKOTIIHOBOtiXEICAOTbI (PPA)
omro~pemieti. B MO9e IIaUEieHTOB,c~pwamw MeTOa o6na.qaeT~OCTaTOsHOfi TO¶HOCTbEO &AH KAUHPeCKHX ~[eA.t!fi.
&xiw&
REFERENCES I L. PENROSE AND J. H. QUASTEL, Biochem. J., 31(1937) 266. 2 M. POLONOVSKI,P.DESGREZAND F.DELBARRE, Bult.soc.chim.biol.,2g(lrg47) 3 J. P. BERRY AND L. J. WOOLF, Nature, 169 (1952) 202.
4 5 6 7 8
A. JERVIS, Proc. SOG. Exptl.Biol. Med., 81 (1952) 7x5. KROPPAND K.LANG, K&n. Wochschr.,33 (1955)482. KEUP, 2. physiol. Chem., 291 (1952)223; Biochem. z., 326 (1954) 14. SCHOORL, Ovganische Analyse, Vol. II, 3rd ed., N.V. Centens Uitgevers R. J. BOSCOTTAND H. BICKEL, Stand. J. CEin. & Lab. Inuest., 5 (1953) 380.
G. K. W. N.
Received
1049.
Mij,
1937. p. 256.
May r3th,
1957