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The use of deuterated phenylalanine for the elucidation of the phenylalanine-tyrosine metabolism
CLIKICA
CCA
THE 01;
CHIMICA
277
ACTA
4754
USE THE
H:CH.
OF DEUTERATED
CURTIIJS,
J. A. \‘(jLLRIIN
I.aboratovy of Cli?zical Chrmistvy, (Switxrla~zd) (Rcccivcd
PHENYLALANINE
PHENYLALANINE-TYROSINE
August
The
AND Ii.
Pcdiatvic
FOR
THE
ELUCIDATION
METABOLISM”
BAERLOCHER
Department,
h’indrrsfiital
Zurich,
C’niucvsity
ofZurich
3, 1971)
application
ilz vielo of stable
isotopes,
e.g. deuterium,
for the study
of
metabolic pathways is demonstrated on the example of the phenylalanine-tyrosine metabolism. A healthy child, a patient with phenylketonuria and a patient with hyper phenylalaninemia were loaded with ZOOmg/kg of deuterated phenylalanine. The concentration and deuterium content of the excreted aromatic acids were examined by gas chromatographyandgas chromatography/mass spectrometry. Unlike in the healthy child, no deuterium was incorporated into the metabolites of tyrosine by the patients with phenylketonuria and hyperphenylalaninemia. The nz-hydroxylation of phenylalanine leading to m-OH-phenylacetic acid was blocked during the loading test in phenylketonuric
as well as in hyperphenylalaninemic
patients.
ISTRODUCTION
The use of radioactive isotopes as metabolic tracers in man is not without risk and only in certain cases can it be justified. The application of non-radioactive, stable isotopes, e.g. r5N, 13C or deuterium was rarely used until now because the measurement of such isotopes presented some analytical problemsl. The most suitable instrument for the detection of stable isotopes is the mass spectrometer. The application of mass spectrometric isotope measurements is demonstrated on the example of o-OH-phenylacetic acid (Fig. I), an important excretion product in phenylketonuria (PKU). The formation of derivatives is necessary because the mass spectrum is recorded in combination with a gas chromatograph. After separation, the quantitation of isotope abundance is done on the molecular signal if its amount is high * Partly presented at the 9th Symposium bolism (Leeds, July 1971). Supported by the I’orschung (No. 3.586.71).
Schweizerischer
of the Society Nationalfonds
for the Study zur
of Inborn
Fijrderung
der
Chim.
Acta,
Clin.
Errors
of Meta-
wissenschaftlichen
37 (1972)
277-28.5
CURTIUS
278 i:n (_)
)_, o-OH-PHENYtACETlC
ACID
2!23
238
~
~ 250
100
~EUTERATED
GO-
I ~ 250
Fig. I. Mass spectrum
of o-OH-phenylacetic
acid (GC/MS combination:
LKB 9000).
%A
15-
lo-
5-
j NOT
)
DEUTERATED
Fig. 2, Comparison Cliiz. Chk.
j
240
DEUTERATED
of higher masses of o-OH-phenylacetickcid
Acta, 37
(1972) 277~285
(see Fig.
I).
et 611.
PHENYLALANINE-TYROSINE
enough.
279
METABOLISM
By replacement
of deuterium,
peaks with higher masses are observed
as can
be seen in Fig. 2. The separation and purification of the different compounds is very troublesome and usually results in an inadequate yield. However, since the introduction of the combination gas chromatograph/mass spectrometer a few years ago, this problem has been simplifiedz*3. Complicated biological mixtures can thus be separated and analyzed by this method4. MATERIALS
AND
METHODS
Subjects. A normal child (age: 3 years), a patient with PKU (age: 12 5/12 years) and a patient with hyperphenylalaninemia (age: I 4/12 years) were studied. Collection of urine. During the collection of urine, none of the subjects was on dietary treatment. Urinary samples were collected in different portions (see Figs. IO and
and stored at -zoo. 37% deuterated D,L-phenylalanine: Isocommerz, Leipzig, E.-Germany. Solvents: All solvents were of reagent grade and were redistilled before use. Reference compounds were obtained from Fluka, Switzerland, Merck, W.-Ger-
II)
many and Schuchard,
W.-Germany.
m-Hydroxyphenylhydracrylic
gift of Dr. &I. D. Armstrong. Igfternal standard was ro-undecenoid
acid methyl
acid was a generous
ester (IOO mg in IOO ml py-
ridine). Methylation was carried out with diazomethane according to Voge15. Trimethylsilylation was carried out with the following mixture : pyridine-hexamethyldisilazane-trimethylchlorosilane Creatinine : Urinary creatinine
(IO : 4: I). was determined
according
Extraction procedure and gas chromatography (Complete
to JaW. procedure
see Viillmin
et a1.7. (a) Extraction. Urine was extracted three times with ethyl acetate. The combined ethyl acetate solutions were re-extracted twice with sodium bicarbonate. The combined bicarbonate solutions were adjusted to pH z and re-extracted three times with ethyl acetate. The solution was dried over Na,SO, and concentrated to about 5 ml. After adding the internal standard, the mixture was methylated with diazomethane in ether. The solution was then concentrated with N, at about 40’ to about IOO ~1. The methylated sample was silylated for IO min with 0.5 ml of silylation mixture at room temperature. The precipitate was centrifuged and 2 ~1 of the supernatant were injected into the gas chromatograph. (b) Gas chromatography. A gas chromatograph Aerograph model 1520 with glass columns XE 60 30/o on Chromosorb G, So-IOO mesh (2 mx2.7 mm i.d.) was used. Carrier gas was N, (45 ml/min) and the apparatus was equipped with a flame ionization detector. The temperatures were as follows : column : 5 min (isotherm) IIOO, prog. 4”/min, 200’; injector: 230”; detector: 220'. (c) Gas chromatography/mass spectrometry (GCIM.5). For identification of the individual aromatic acids and for the calculation of the deuterium content a combination GCjMS LKB 9000 was used. Columns and conditions were identical with those described above.
Clin.Chim. Acta, 37 (1972) 277-285
CCRTIUS
280
et al.
Cahclation of the deuterium content The total deuterium content of the individual aromatic acids was calculated by correcting with the isotope peaks of the non-deuterated compound (see, e.g., Biemanns).
1
13
12
II__/
_:
-
1
MIN
30
25
20
15
10
5
0
Fig. 3, Gas-chromatographic separation of a test mixture of aromatic acids as methyl esters/ trimethylsilyl derivatives. I, phenylacetic acid; 2, mandelic acid, 3, phenyllactic acid; 1, o-OHphenylacetic acid; 5, m-OH-phenylacetic acid; 0, fi,-OH-phenylacetic acid; 7, phenylpyruvic acid; 8, nz-OH-phenylhydracrylic acid ; 9, homovanillic acid; IO, p-OH-phenyllactic acid ; I I, vanillylmandelic acid; I 2, $-OH-phenylpyruvic acid ; I 3, hippuric acid. RESULTS
Fig. 3 shows a gas-chromatographic separation of a test mixture of aromatic acids as methyl esters/trimethylsilyl ethers. The gas-chromatographic profile of the urinary aromatic acids in a healthy child is given in Fig. 4. The main metabolites are P-OH-phenylacetic acid, homovanillic acid, P-OHphenyllactic acid, and hippuric acid. The urinary profile of a patient with PKU is presented in Fig. 5. Metabolites at high concentrations are: phenylacetic acid, phenyllactic acid, o-OH-phenylacetic acid, phenylpyruvic acid, but also P-hydroxylated compounds (P-OH-phenyllactic acid, fi-OH-phenylpyruvic acid) and finally hippuric acid are increased. Phenylalani?ze loading tests Three subjects were studied,
a normal
child,
a patient
with I’Kl-
[plasma
PBENYLALANINE-TVROSINE
,
1:
t MIN 20
METANOLIS>I
1
t5
lb
/
5
7
0
Fig. 4. Gas-chromatographic separation of urinary aromatic acids of a normal child (age: 9 months). 2, mandelic acid; 3. phenyllactic acid; 4, o-OH-phenylacetic acid; 5, nz-OH-phenylacetic acid ; 6, p-OH-phenylacetic acid ; 7, phenylpyruvic acicl ; 8, m-OH-phenylhydracrylic acid ; 9, homovanillic acid ; IO, p-OH-phenyllactic acid ; I I, vanillylmandelic acid; 12, $-OH-phenylpyruvic acid; 13, hippuric acid. Fig. 5. Gas chromatographic separation of urinary aromatic acids of a patient with YKI: (age: 14 days; urme was collected just prior to beginnin g of diet). I, phenylacctic acid; 2, mandeiic acid; 3, phenyllactic acid; 4. o-OH-phenylacetic acid; 5. nz-OH-phenylacetic acid; 6, p-OHphenylacetic acid ; 7. phcnylpyruvic acid; 8, m-OH-phenylhydracrylic acid; 9, homovanillic acid; IO, B-OH-phenyllactic acid; II, vanillylmandelic acid; 12, p-OH-phcnylpyruvic acid; 13, hippuric acid.
phenylalanine concentration: 2.5 mg/roo ml), and a patient with ll~pe~l~enylala~linemia (plasma phenylalanine concentration: 7 mg/roo ml). These three subjects were orally loaded with 200 mg/kg body weight of deuterated phenylalanine. Fig. 6 shows the mass spectrum of deuterated phenylalanine. We then studied two different variables: concentration changes of phenylalanine and tyrosine and their metabolites, and secondly, the deuterium distribution pattern among the metabolic pathways. (a) Concentrafiox of rnctabolitcs: In the healthy child. during the loading test the following metabolites are increased: phenylpyruvic acid, phenyllactic acid, o-OH-phenylacetic acid, phenylacetic acid, mandelic acid (first described by Blauo), $-OH-phenylpyruvic acid, p-OH-phenylacetic acid and $-OH-phenyllactic acid.
CURTIUS
282
et (21.
INT. (%)
50 26
65
,,i, 20
I
50
200
Fig. 6. Mass spectrum A similar
of dcutcrated
pattern
phenylalanine
but a higher increase
(GC/MS combination:
of these metabolites
I
I
I
250
I,K’L3 9000).
was found in case
of PKU after phenylalanine load. The unexpectedly high excretion of $-hydroxylated compounds has already been described by other investigatorsl”. (b) Labeling pattcvns: Fig. 7 represents our results after load with deuterated phenylalanine in a healthy child. It should be emphasized that also m-OH-phenylacetic acid, mandelic acid and homovanillic acid are considerably deuterated. In contrast, the situation in case of PKU is different. No deuterium incorporation could be detected in the metabolites of tyrosine; $-OH-phenylpyruvic acid, $-OH-phenyllactic acid, $-OH-phenylacetic acid, and homovanillic acid could not be found deuterated. It is of special interest, that m-OH-phenylacetic acid was not deuterated. Mandelic acid on the other hand was still deuterated to about the same extent as in a healthy child. These results raise some interesting questions with respect to the importance of the various pathways involved (see DISCUSSION). In the patient with hyperphenylalaninemia, the same deuteration pattern was observed as was in the PKU patient. (c) Concentration and deuteration of two typical metabolites. The concentration and deuteration of o-OH-phenylacetic acid (phenylalanine metabolite)and $-OHphenyllactic acid (tyrosine metabolite) are demonstrated in different periods of urine collection after load (Fig. S). During loading with deuterated phenylalanine, the concentratio?z of o-OHphenylacetic acid rose in all three subjects, markedly in the two patients but only slightly in the healthy child. It is remarkable that this metabolite showed the same increase in hyperphenylalaninemia as well as in PKU. The basic value, however, was considerably lower in the hyperphenylalaninemic patient. The marked dcuteratio~z of o-OH-phenylacetic acid could be found in all three subjects. The different phenylalanine tissue pool of the three children makes an interpretation of these results very difficult. In contrary, a different pattern for p-OH-phenyllactic acid was obtained. Its concentration increased in all three subjects just as with the immediate metabolites of phenylalanine, but deuteratiox was found only in the healthy child.
PHENYLALANfNE-TYROSINE
METABOLISM
pg ACl”/mg rsh9
*
CREATlNlNt
DEUTFRIUM
CON I kNT
TOlAL DEUTER:UM
NORMAL _.e___
S.
I. Blau9 was the first to report the presence of mandelic acid in PKlr. We found that mandelic acid is also present in metabolically healthy subjects after a phenylalanine load. The marked labeling of mandelic acid after loading with deuterated
phenylalanine proofs the hypothesis that it is definitely a product of the phenylalanine metabolism. 2. The fact that we were unable to detect any deuteration of hippuric acid suggests that hippuric acid is synthesized mainly from alimentary benzoic arid and not through the pl~et~~lala~~ine~pl~en~lp~ruvic acid pathway. 3. It is well known that in PKU ~-l~~drox~lated metabolites, lactic acid and /+OH-phenylpyruvic acid are elevated’“. In PKU,
e.g. b-OH-phenylno deuterium was
found in p-hydroxylated compounds. This lack of deuteration shows rather unambiguously that the enzyme block between phenylalanine and tyrosine cannot be circumvented by other metabolic connections and would therefore substantiate the hypothesis that a secondary inhibition due to the metabolites of 1~llen~l~~laniIle does take place. 4. In the healthy child, homovanillic acid, the main metsbolite of dopamine was deuterated whereas in PKU this compound showed no deuterium incorporation. In addition, the concentration of homovanillic acid was markedly decreased in patients with PKW. Under this aspect, the cprestion arises once more, whether the lack of tyrosine metabolites, ~:.g. of the biologically active amines and I~omovanillic acid niay be a contributing factor to the damage in brain function. 5. In the healthy subject, Fs-OH-phenylacetic acid was markedly deuterated, but practically not in patients with PKU. It seems that in PKLJ, nz-hydroxylation of phenylacetic acid is blocked. W’e would like to point to the fact that qph-hydroxylation of tyrosine is catalyzed by tyrosine-3-hydroxylast, and that several inhibitors of this enzyme are known, one of which is phenylalaninel’. 6. h patient with hyperphen~7lalaninemia, whose plasma phenglalanine was 7.0 mgjroo ml before load, showed the same deuteration pattern as the patient \vith
PHENYLALANINE-TYROSISE
285
METABOLISM
PKG with a plasma phenylalanine of 25 mg/roo ml. In the former, the concentration of the metabolites and of phenylalanine itself was lower before and during the loading test. The lack of deuteration of tyrosine metabolites makes it obvious that in our patient with hyperphenylalaninemia, no phenylalanine or nearly none is metabolized to tyrosine. The lower phenylalanine concentration and the lower concentration of its metabolites might suggest that another metabolic pathway for phenylalanine degradation exists in patients with hyperphenylalaninemia. The loading tests were carried out with n,L-phenylalanine. Since a number of enzymes react specifically with the D- or L-form of the substrate only, our conclusions are subject to some reservation. Studies with L-phenylalanine are in progress. In addition, a possible alanine and tyrosine In conclusion, may
be a powerful
intestinal bacterial interaction on the metabolites of phenylhas not been taken into account. the present investigation shows that the stable isotope technique instrument
for the study
of metabolic
pathways
in man.
K. SCHOEKHEIMER AND D. RITTEK.UERG, J. Bid. Chew., III (193-j) 163. J, C. HOLMFS AND F. A. &fORELL, A#. Sfxctvoscofiy, II (1957) 86. R. RYHAGE, Anal. C&n., 36 (1964) 9j9. J, L. ~%NKUS, D. CHARLES AND S. C. CHATTORAJ, J.Riol. Chem., 246 (1971) 633. .\.I.VOGEL, Practical Organic Chemistry, Longmans, New \;ork,1964, p. 971. 31.JAFFI~,Z. Ph~~siol. Chum., IO (1886) 399. J. X. VIJLLmN, H. R. BOSSHARD, iU. >~ULLER, S. RAMPISI ASD I-I.-CH. CURTIUS, Z. IiZi?z. Chew. liiin.Biochcnz., 9 (1971) 402. I<.BIEMANS, Xlass SPcctronzc,try~Ovgalzic Chemical Applicatzom, A\lcGmwHill Book CO. Inc., New X'ork,1962. Ii. IJLAV, Clzx. CAiwz.Acta, 27 (1970) 5. .I.&xxea, Biochmistvy ofthr Amino Acids, T-01.II, .1cademic Press,New 170rk, 1965. p. 1069. T. NAG.\TSU, 12.LEVITT ANU S. I~DEITRIEI),.I.Bid.
Chm.,
239 (1964)
2910.
CCA
THE 01;
CHIMICA
277
ACTA
4754
USE THE
H:CH.
OF DEUTERATED
CURTIIJS,
J. A. \‘(jLLRIIN
I.aboratovy of Cli?zical Chrmistvy, (Switxrla~zd) (Rcccivcd
PHENYLALANINE
PHENYLALANINE-TYROSINE
August
The
AND Ii.
Pcdiatvic
FOR
THE
ELUCIDATION
METABOLISM”
BAERLOCHER
Department,
h’indrrsfiital
Zurich,
C’niucvsity
ofZurich
3, 1971)
application
ilz vielo of stable
isotopes,
e.g. deuterium,
for the study
of
metabolic pathways is demonstrated on the example of the phenylalanine-tyrosine metabolism. A healthy child, a patient with phenylketonuria and a patient with hyper phenylalaninemia were loaded with ZOOmg/kg of deuterated phenylalanine. The concentration and deuterium content of the excreted aromatic acids were examined by gas chromatographyandgas chromatography/mass spectrometry. Unlike in the healthy child, no deuterium was incorporated into the metabolites of tyrosine by the patients with phenylketonuria and hyperphenylalaninemia. The nz-hydroxylation of phenylalanine leading to m-OH-phenylacetic acid was blocked during the loading test in phenylketonuric
as well as in hyperphenylalaninemic
patients.
ISTRODUCTION
The use of radioactive isotopes as metabolic tracers in man is not without risk and only in certain cases can it be justified. The application of non-radioactive, stable isotopes, e.g. r5N, 13C or deuterium was rarely used until now because the measurement of such isotopes presented some analytical problemsl. The most suitable instrument for the detection of stable isotopes is the mass spectrometer. The application of mass spectrometric isotope measurements is demonstrated on the example of o-OH-phenylacetic acid (Fig. I), an important excretion product in phenylketonuria (PKU). The formation of derivatives is necessary because the mass spectrum is recorded in combination with a gas chromatograph. After separation, the quantitation of isotope abundance is done on the molecular signal if its amount is high * Partly presented at the 9th Symposium bolism (Leeds, July 1971). Supported by the I’orschung (No. 3.586.71).
Schweizerischer
of the Society Nationalfonds
for the Study zur
of Inborn
Fijrderung
der
Chim.
Acta,
Clin.
Errors
of Meta-
wissenschaftlichen
37 (1972)
277-28.5
CURTIUS
278 i:n (_)
)_, o-OH-PHENYtACETlC
ACID
2!23
238
~
~ 250
100
~EUTERATED
GO-
I ~ 250
Fig. I. Mass spectrum
of o-OH-phenylacetic
acid (GC/MS combination:
LKB 9000).
%A
15-
lo-
5-
j NOT
)
DEUTERATED
Fig. 2, Comparison Cliiz. Chk.
j
240
DEUTERATED
of higher masses of o-OH-phenylacetickcid
Acta, 37
(1972) 277~285
(see Fig.
I).
et 611.
PHENYLALANINE-TYROSINE
enough.
279
METABOLISM
By replacement
of deuterium,
peaks with higher masses are observed
as can
be seen in Fig. 2. The separation and purification of the different compounds is very troublesome and usually results in an inadequate yield. However, since the introduction of the combination gas chromatograph/mass spectrometer a few years ago, this problem has been simplifiedz*3. Complicated biological mixtures can thus be separated and analyzed by this method4. MATERIALS
AND
METHODS
Subjects. A normal child (age: 3 years), a patient with PKU (age: 12 5/12 years) and a patient with hyperphenylalaninemia (age: I 4/12 years) were studied. Collection of urine. During the collection of urine, none of the subjects was on dietary treatment. Urinary samples were collected in different portions (see Figs. IO and
and stored at -zoo. 37% deuterated D,L-phenylalanine: Isocommerz, Leipzig, E.-Germany. Solvents: All solvents were of reagent grade and were redistilled before use. Reference compounds were obtained from Fluka, Switzerland, Merck, W.-Ger-
II)
many and Schuchard,
W.-Germany.
m-Hydroxyphenylhydracrylic
gift of Dr. &I. D. Armstrong. Igfternal standard was ro-undecenoid
acid methyl
acid was a generous
ester (IOO mg in IOO ml py-
ridine). Methylation was carried out with diazomethane according to Voge15. Trimethylsilylation was carried out with the following mixture : pyridine-hexamethyldisilazane-trimethylchlorosilane Creatinine : Urinary creatinine
(IO : 4: I). was determined
according
Extraction procedure and gas chromatography (Complete
to JaW. procedure
see Viillmin
et a1.7. (a) Extraction. Urine was extracted three times with ethyl acetate. The combined ethyl acetate solutions were re-extracted twice with sodium bicarbonate. The combined bicarbonate solutions were adjusted to pH z and re-extracted three times with ethyl acetate. The solution was dried over Na,SO, and concentrated to about 5 ml. After adding the internal standard, the mixture was methylated with diazomethane in ether. The solution was then concentrated with N, at about 40’ to about IOO ~1. The methylated sample was silylated for IO min with 0.5 ml of silylation mixture at room temperature. The precipitate was centrifuged and 2 ~1 of the supernatant were injected into the gas chromatograph. (b) Gas chromatography. A gas chromatograph Aerograph model 1520 with glass columns XE 60 30/o on Chromosorb G, So-IOO mesh (2 mx2.7 mm i.d.) was used. Carrier gas was N, (45 ml/min) and the apparatus was equipped with a flame ionization detector. The temperatures were as follows : column : 5 min (isotherm) IIOO, prog. 4”/min, 200’; injector: 230”; detector: 220'. (c) Gas chromatography/mass spectrometry (GCIM.5). For identification of the individual aromatic acids and for the calculation of the deuterium content a combination GCjMS LKB 9000 was used. Columns and conditions were identical with those described above.
Clin.Chim. Acta, 37 (1972) 277-285
CCRTIUS
280
et al.
Cahclation of the deuterium content The total deuterium content of the individual aromatic acids was calculated by correcting with the isotope peaks of the non-deuterated compound (see, e.g., Biemanns).
1
13
12
II__/
_:
-
1
MIN
30
25
20
15
10
5
0
Fig. 3, Gas-chromatographic separation of a test mixture of aromatic acids as methyl esters/ trimethylsilyl derivatives. I, phenylacetic acid; 2, mandelic acid, 3, phenyllactic acid; 1, o-OHphenylacetic acid; 5, m-OH-phenylacetic acid; 0, fi,-OH-phenylacetic acid; 7, phenylpyruvic acid; 8, nz-OH-phenylhydracrylic acid ; 9, homovanillic acid; IO, p-OH-phenyllactic acid ; I I, vanillylmandelic acid; I 2, $-OH-phenylpyruvic acid ; I 3, hippuric acid. RESULTS
Fig. 3 shows a gas-chromatographic separation of a test mixture of aromatic acids as methyl esters/trimethylsilyl ethers. The gas-chromatographic profile of the urinary aromatic acids in a healthy child is given in Fig. 4. The main metabolites are P-OH-phenylacetic acid, homovanillic acid, P-OHphenyllactic acid, and hippuric acid. The urinary profile of a patient with PKU is presented in Fig. 5. Metabolites at high concentrations are: phenylacetic acid, phenyllactic acid, o-OH-phenylacetic acid, phenylpyruvic acid, but also P-hydroxylated compounds (P-OH-phenyllactic acid, fi-OH-phenylpyruvic acid) and finally hippuric acid are increased. Phenylalani?ze loading tests Three subjects were studied,
a normal
child,
a patient
with I’Kl-
[plasma
PBENYLALANINE-TVROSINE
,
1:
t MIN 20
METANOLIS>I
1
t5
lb
/
5
7
0
Fig. 4. Gas-chromatographic separation of urinary aromatic acids of a normal child (age: 9 months). 2, mandelic acid; 3. phenyllactic acid; 4, o-OH-phenylacetic acid; 5, nz-OH-phenylacetic acid ; 6, p-OH-phenylacetic acid ; 7, phenylpyruvic acicl ; 8, m-OH-phenylhydracrylic acid ; 9, homovanillic acid ; IO, p-OH-phenyllactic acid ; I I, vanillylmandelic acid; 12, $-OH-phenylpyruvic acid; 13, hippuric acid. Fig. 5. Gas chromatographic separation of urinary aromatic acids of a patient with YKI: (age: 14 days; urme was collected just prior to beginnin g of diet). I, phenylacctic acid; 2, mandeiic acid; 3, phenyllactic acid; 4. o-OH-phenylacetic acid; 5. nz-OH-phenylacetic acid; 6, p-OHphenylacetic acid ; 7. phcnylpyruvic acid; 8, m-OH-phenylhydracrylic acid; 9, homovanillic acid; IO, B-OH-phenyllactic acid; II, vanillylmandelic acid; 12, p-OH-phcnylpyruvic acid; 13, hippuric acid.
phenylalanine concentration: 2.5 mg/roo ml), and a patient with ll~pe~l~enylala~linemia (plasma phenylalanine concentration: 7 mg/roo ml). These three subjects were orally loaded with 200 mg/kg body weight of deuterated phenylalanine. Fig. 6 shows the mass spectrum of deuterated phenylalanine. We then studied two different variables: concentration changes of phenylalanine and tyrosine and their metabolites, and secondly, the deuterium distribution pattern among the metabolic pathways. (a) Concentrafiox of rnctabolitcs: In the healthy child. during the loading test the following metabolites are increased: phenylpyruvic acid, phenyllactic acid, o-OH-phenylacetic acid, phenylacetic acid, mandelic acid (first described by Blauo), $-OH-phenylpyruvic acid, p-OH-phenylacetic acid and $-OH-phenyllactic acid.
CURTIUS
282
et (21.
INT. (%)
50 26
65
,,i, 20
I
50
200
Fig. 6. Mass spectrum A similar
of dcutcrated
pattern
phenylalanine
but a higher increase
(GC/MS combination:
of these metabolites
I
I
I
250
I,K’L3 9000).
was found in case
of PKU after phenylalanine load. The unexpectedly high excretion of $-hydroxylated compounds has already been described by other investigatorsl”. (b) Labeling pattcvns: Fig. 7 represents our results after load with deuterated phenylalanine in a healthy child. It should be emphasized that also m-OH-phenylacetic acid, mandelic acid and homovanillic acid are considerably deuterated. In contrast, the situation in case of PKU is different. No deuterium incorporation could be detected in the metabolites of tyrosine; $-OH-phenylpyruvic acid, $-OH-phenyllactic acid, $-OH-phenylacetic acid, and homovanillic acid could not be found deuterated. It is of special interest, that m-OH-phenylacetic acid was not deuterated. Mandelic acid on the other hand was still deuterated to about the same extent as in a healthy child. These results raise some interesting questions with respect to the importance of the various pathways involved (see DISCUSSION). In the patient with hyperphenylalaninemia, the same deuteration pattern was observed as was in the PKU patient. (c) Concentration and deuteration of two typical metabolites. The concentration and deuteration of o-OH-phenylacetic acid (phenylalanine metabolite)and $-OHphenyllactic acid (tyrosine metabolite) are demonstrated in different periods of urine collection after load (Fig. S). During loading with deuterated phenylalanine, the concentratio?z of o-OHphenylacetic acid rose in all three subjects, markedly in the two patients but only slightly in the healthy child. It is remarkable that this metabolite showed the same increase in hyperphenylalaninemia as well as in PKU. The basic value, however, was considerably lower in the hyperphenylalaninemic patient. The marked dcuteratio~z of o-OH-phenylacetic acid could be found in all three subjects. The different phenylalanine tissue pool of the three children makes an interpretation of these results very difficult. In contrary, a different pattern for p-OH-phenyllactic acid was obtained. Its concentration increased in all three subjects just as with the immediate metabolites of phenylalanine, but deuteratiox was found only in the healthy child.
PHENYLALANfNE-TYROSINE
METABOLISM
pg ACl”/mg rsh9
*
CREATlNlNt
DEUTFRIUM
CON I kNT
TOlAL DEUTER:UM
NORMAL _.e___
S.
I. Blau9 was the first to report the presence of mandelic acid in PKlr. We found that mandelic acid is also present in metabolically healthy subjects after a phenylalanine load. The marked labeling of mandelic acid after loading with deuterated
phenylalanine proofs the hypothesis that it is definitely a product of the phenylalanine metabolism. 2. The fact that we were unable to detect any deuteration of hippuric acid suggests that hippuric acid is synthesized mainly from alimentary benzoic arid and not through the pl~et~~lala~~ine~pl~en~lp~ruvic acid pathway. 3. It is well known that in PKU ~-l~~drox~lated metabolites, lactic acid and /+OH-phenylpyruvic acid are elevated’“. In PKU,
e.g. b-OH-phenylno deuterium was
found in p-hydroxylated compounds. This lack of deuteration shows rather unambiguously that the enzyme block between phenylalanine and tyrosine cannot be circumvented by other metabolic connections and would therefore substantiate the hypothesis that a secondary inhibition due to the metabolites of 1~llen~l~~laniIle does take place. 4. In the healthy child, homovanillic acid, the main metsbolite of dopamine was deuterated whereas in PKU this compound showed no deuterium incorporation. In addition, the concentration of homovanillic acid was markedly decreased in patients with PKW. Under this aspect, the cprestion arises once more, whether the lack of tyrosine metabolites, ~:.g. of the biologically active amines and I~omovanillic acid niay be a contributing factor to the damage in brain function. 5. In the healthy subject, Fs-OH-phenylacetic acid was markedly deuterated, but practically not in patients with PKU. It seems that in PKLJ, nz-hydroxylation of phenylacetic acid is blocked. W’e would like to point to the fact that qph-hydroxylation of tyrosine is catalyzed by tyrosine-3-hydroxylast, and that several inhibitors of this enzyme are known, one of which is phenylalaninel’. 6. h patient with hyperphen~7lalaninemia, whose plasma phenglalanine was 7.0 mgjroo ml before load, showed the same deuteration pattern as the patient \vith
PHENYLALANINE-TYROSISE
285
METABOLISM
PKG with a plasma phenylalanine of 25 mg/roo ml. In the former, the concentration of the metabolites and of phenylalanine itself was lower before and during the loading test. The lack of deuteration of tyrosine metabolites makes it obvious that in our patient with hyperphenylalaninemia, no phenylalanine or nearly none is metabolized to tyrosine. The lower phenylalanine concentration and the lower concentration of its metabolites might suggest that another metabolic pathway for phenylalanine degradation exists in patients with hyperphenylalaninemia. The loading tests were carried out with n,L-phenylalanine. Since a number of enzymes react specifically with the D- or L-form of the substrate only, our conclusions are subject to some reservation. Studies with L-phenylalanine are in progress. In addition, a possible alanine and tyrosine In conclusion, may
be a powerful
intestinal bacterial interaction on the metabolites of phenylhas not been taken into account. the present investigation shows that the stable isotope technique instrument
for the study
of metabolic
pathways
in man.
K. SCHOEKHEIMER AND D. RITTEK.UERG, J. Bid. Chew., III (193-j) 163. J, C. HOLMFS AND F. A. &fORELL, A#. Sfxctvoscofiy, II (1957) 86. R. RYHAGE, Anal. C&n., 36 (1964) 9j9. J, L. ~%NKUS, D. CHARLES AND S. C. CHATTORAJ, J.Riol. Chem., 246 (1971) 633. .\.I.VOGEL, Practical Organic Chemistry, Longmans, New \;ork,1964, p. 971. 31.JAFFI~,Z. Ph~~siol. Chum., IO (1886) 399. J. X. VIJLLmN, H. R. BOSSHARD, iU. >~ULLER, S. RAMPISI ASD I-I.-CH. CURTIUS, Z. IiZi?z. Chew. liiin.Biochcnz., 9 (1971) 402. I<.BIEMANS, Xlass SPcctronzc,try~Ovgalzic Chemical Applicatzom, A\lcGmwHill Book CO. Inc., New X'ork,1962. Ii. IJLAV, Clzx. CAiwz.Acta, 27 (1970) 5. .I.&xxea, Biochmistvy ofthr Amino Acids, T-01.II, .1cademic Press,New 170rk, 1965. p. 1069. T. NAG.\TSU, 12.LEVITT ANU S. I~DEITRIEI),.I.Bid.
Chm.,
239 (1964)
2910.