- Email: [email protected]
The investigation of aromatic acids in phenylketonuria, alkaptonuria and tyrosinosis using gas-liquid chromatography
CLINICA CHIMICA ACTA
455
CCA 4879
THE
INVESTIGATION
ALKAI’TONURIA
OF AROMATIC
AND TYROSINOSIS
ACIDS IN PHENYLKETONURIA, USING
GAS-LIQUID
CHRO~I~4TOGR‘4~HY
SUMMARS
A rapid and accurate method for the measurement of six aromatic acids which are excreted in phenylketonuria has been developed. The same procedure was applied to urine from patients with alkaptonuria and tyrosinosis. The urinary aromatic acid profiles can be used to confirm the diagnosis and to monitor dietary treatment. The regular excretion of mandelic acid in phenylketonuria is confirmed in this study and its excretion in eight cases of uncontrolled phen~lketonuria is reported. The “finding of a second peak.in an alkaptonuric urinary profile is reported but this has not been identified.
INTRODUCTION
The inborn errors of metabolism, phenylketonuria, alkaptonuria, and tyrosinosis are characterized by the urinary excretion of aromatic acid metabolites in excessive quantities. A variety of methods have been described for the identification and measurement of the aromatic acids. These have included wet chemistry methodslF2 and a variety of chromatographic techniques3-“. The aromatic acid profiles are characteristic of a specific metabolic defect and may be used to confirm the diagnosis obtained from amino acid studies and other investigations. In addition, quantitative measurement of individual urinary aromatic acids provides important information as to the fate of ingested amino acids in diseases such as phenylketonuria where there is a block in the metabolic pathway involving the particular acid. This paper describes a rapid gas-liquid chromatographic method for the measurement of urinary aromatic acids associated with phenylketonuria, alkaptonuria, and tyrosinosis.
456 METHODS
AND
MATERIALS
Whenever possible, 24-h urine samples were collected, but in cases where this was not possible, random urine specimens were collected. Specimens were stored at prior to analysis.
-20’
An ilerograph
HY-FI
Xodel
6ooD gas chromatograph
with
a single
column
and flame ionization detector was used. Two 6-ft. x 0.125 in. i.d. stainless steetcolumns were packed with 5oj SE-30 and 8% SE 52 60-80 mesh acid-washed, HMDS-treated Chromosorb
W.
Extraction ?f aromatic acids One ml of urine was acidified
by the addition
of two drops of concentrated
l~ydroclllo~-ic acid. The addition of 2 ml of saturated sodium chloride solution was followed by extraction with 3 x 3 ml volumes of ethyl acetate. The sample was mixed and the interface emulsion broken by centrifugation. The ethyl acetate extracts were evaporated to dryness using a vacuum manifold apparatus, a single unit of which is shown in Fig. I. The units were connected in series so that 6 extracts could be dried simultaneousl~r, The number could be extended to include as many units as desired. The total time for extraction Preparation of derivatives J’olatile derivatives
of 6 samples was 45 min.
joy gas-lipid chrornatogra$hy (GLC) of the extracted aromatic acids were prepared by the addi-
tion of ISO ,ul of X,0-bis-(trimethylsilyI)-acetamide (BSA). The appropriate internal standard was added and the volume made up to joo ,ul with dioxane. The reaction
TO
NEXT
- - _
-
UNIT VACCUM
MANIFOLD
SERUM
19
_.-_, .*.., .. .. . ..,.. ..... ... .. **... ..**.
TEST
CAP
6A6E
NEEDLE
TUBE EXTRACT
CONTAINING
TO
VACCUM
MEASUREMEST
OF
AROMATIC
ACIDS
BY
c;Lc
457
was allowed to proceed at room temperature was kept sealed until samples were withdrawn Gas-liquid
for 15 min in a stoppered for analysis.
tube which
chrontatogra$hy
Five-$ aliquots of the reaction mixture were chromatographed with temperature programming. The flame ionization detector was maintained at 240°, the helium flow rate was 35 ml/mm, and the range and attenuation were varied according to the concentration of the aromatic acids present in the extract. Identification of each aromatic acid in the urine extracts was achieved by co-chromatography with the TMS derivatives of authentic compounds on both columns. Peak
awa deterwtixation
All peak areas were measured by triangulation. The peak area ratio of each acid to internal standard was plotted against the corresponding weight of standard compound. Each calibration curve was straight line with zero intercept. RESULTS
The profile obtained for six authentic aromatic acids is shown in Fig. 2 A. The separation of the peaks makes accurate measurement of each acid possible. Fig. 2 B shows the aromatic acid excretion pattern of a normal individual, typical of the profiles obtained from all normal subjects studied. Fig. 3 shows the aromatic acid excretion pattern of 2 untreated phenylketonuric patients. The profiles obtained from a phenylketonuric patient while on normal diet and during therapy with a low phenylalanine diet are shown in Fig. 4.
A
1
0
5
IL IO
15
20
25
30
35
40
45
50
30
35
40
45
50
minutes
0
5
10
15
20
25 minutes
Fig. 2. (A) Separation of mixture of authentic aromatic acids. I, phenylacetic acid: 2, maudelic acid; 3, o-hydroxyphenylacetic acid; 4, ,%phenyllactic acid; 5. p-hydroxyphenylacetic acid; 6, Bphenylpyruvic acid; 7. hippuric acid; IS, internal standard. (B) -4romatic acid profile of normal human urine.
Clis.
Chim.
Acta.
37 (1972)
455-462
HILL
IO
5
IS
20
25
30
35
40
45
50
30
35
40
45
50
et al.
minutes
t 0
5
15
10
20
25 minutes
Fig. 3. (A) and (B) Aromatic Legend as for Fig. 2. TABLE
1
COKCENTRATIOX KETONURIA (mg/g
OF OS
USCOXJUGATED
NORMAL
AND
AROMATIC
ACIDS
PHESYLALASISE-RESTRICTED
IN
CRIXE
SPECIMESS
OF
12
CASES
017 I’HEXYL~
DIETS
crcatinine)
Subject
Phrwvlacrtic acid
.ZfandPlzc ucld
Normal diet P.S. 213 l’.H. 62 E.1’. 73 K.J. 72 Ii.Ii. 60 E.L. Sj \..I>. C.A.
Restricted I1.K. 11,s. I,.L,.
_~
acid profiles of urine from two cases of untreated phcn~lketonuri~1.
V.H.
IS 15 3’ 38 3-l '4 39
ii
7’
I.35
phcnplalanine 33
diet 0 '3
3.3
2,
IL0
0.6
2.0
The most significant difference is the disappearance of the peaks representing phenylacetic acid, mandelic acid, o-hydroxyphenylacetic acid, phenyllactic acid and phenylpyruvic acid during treatment. The excretion of o-hydroxyphenplacetic acid has been widely used as a diagnostic aid for the confirmation of phenylketonuria2,5. The values reported for the six aromatic acids excreted in phenylketonuria may be obtained from the same analysis. The values for 8 patients with uncontrolled phenylketonuria, and 4 patients receiving a phenylalanine-restricted diet are shown in Table I. Clip. Chiln.
.4&a,
37 (1971)
.+jj-+hL
MEASUREMENT
OF AROMATIC
ACIDS BY
GUI
450
Profiles of urine extracts from a patient with alkaptonuria and a normal subject are shown in Fig. 5. Homogentisic acid is present in abnormal amounts in urine from alkaptonurics, whereas none is found in extracts of normal urine. The amounts of ho~noge~ltisic acid in the alkaptonuric urine was found to be 5.1 g/z4 h. A second
L 0
5
IO
I.5
20
25
30
3.5
40
45
50
30
35
40
45
50
minutes
r
0
5
IO
1s
20
25 minutes
Fig. ‘4. (4) Aromatic acid profile of urine from a case of untreated phenylketonuria and (B), profile of same patient during therapy with a low phenylalanine diet. Legend as for Fig. 2.
Fig. 5. Aromatic acid profiles of urine from (X) normal ria. HGA, homogentisic acid; IS, internal standard.
individual
and (B) patient
Clin. Chiwz. Acta,
with alkaptonu-
37 (1972) 45.=,-162
460
HILL
peak was found to appear with a retention unidentified.
tit al.
time of 6 min. As yet this peak remains
Fig. 6 shows the pattern obtained from the urine of a patient with tyrosinosis” as compared with a normal subject. The two aromatic acids, P-llydroxvphenvlacetic acid and $-hydroxyphenyllactic acid are grossly elevated. No fi-hpdroxyphenylpyruvic acid was observed in urinary profiles of this patient. DISCUSSION
In order to use urinary
aromatic
acid profiles obtained
by GLC to confirm
a
diagnosis and to monitor the effect of dietary treatment of inborn errors of metabolism, a rapid method of extraction and analyses must be available. The rapid evaporation of samples using the vacuum manifold apparatus makes such monitoring feasible. The total time required for a single sample is under z 11. The analysis allowed ecxellent separation and quantitative measurement of 0 unconjugated aromatic acids in phenylketonuria. The concentration of unconjugated phenylacetic acid in untreated phenylketonuria was observed to range from 60 to 213 mg/g creatinine as shown in Table I. Quantitative measurements of mandelic acid have not previously been reported in uncontrolled phenylketonuria. The excretion was found to range from 15 to 71 mg/g creatinine. In normal individuals the value was less than I mg/g creatinine.
0
5
IO
15
20
5
IO
IS
20
25
30
35
40
45
25
30
35
40
45
minutes
Fig. 6. Aromatic acid profiles of (A) patient for Fig. 2; 8, P-hydrosyphenyllactic acid. Clin. Chim.
A&,
37 (1972)
4.jjp461
with tyrosinosis
and (B) normal
indixidual.
Legend as
MEASUREMENT
OI: AROMATIC
ACIDS BY
Ortho-hydroxyphenyla~etic nosis of phenylketonuria.
461
GLC
acid has been of primary
The separation
importance
of the peak corresponding
in the diagto o-hydroxy-
phenylacetic acid from phenyllactic acid is not easily accomplished. However, in all cases the slight overlapping of these peaks did not interfere with peak area measurement. A range of 63 to 219 mg/g creatinine was found in 8 cases of untreated phenylketonuria. The excretion of phenylla~ti~ acid is grossly elevated in these cases. Altl~ougl~ it is present in small amounts in normal individuals, the increased excretion in uncontrolled phenylketonuria allows this peak to be identified readily in urinary profiles. The observed range in this study was 164 to 1965 mg/g creatinine (Table I) for untreated patients. Phenylpyruvic acid is excreted in abnormal amounts in uncontrolled phenylketonuria. None was found in normal subjects. The excretion of this compound ranged from 226 to 1910 mg/g creatinine (Table I). .Uthough was determined
for completeness.
p-hydroxyphenylacetic acid is not increased in phenylketonuria, it The range of values observed for all subjects shows
good agreement
with those reported by Williams
and Sweeley7 for normal individuals.
The values reported for phenylacetic acid, ~-l~ydroxyphen~la~eti~ acid, phenyllactic acid and phenylpyruvic acid are in good agreement with the results of other workers4,8-10. The urinary excretion of homogentisic acid in alkaptonuria is grossly elevated. Sadilekll reported that the average urinary excretion of homogentisic acid in these patients was 5 g/24 11. The value of 5.1 g/z4 h for the patient in this study is in good agreement . The urinary profile of the patient with alkaptonuria showed a second major peak with a retention time of 6 min. Identification of the peak was attempted by cochromatography with a variety of aromatic acids witout success. The possibility of the peak being due to an artefact cannot be excluded. The profile obtained from the urinary extract from a patient with tyrosinosis is readily distinguished from those characteristic of phenylketonuria and alkaptonuria. The two elevated phenolic acids, fi-hydroxyphenylacetic acid and $-hydroxyphenyllactic acid, are widely separated. This patient did not excrete excessive amounts of p-hydroxyphenylpyruvic acid and probably represents a clinical and biochemical variant”.
Grants
This study was assisted (Project Nos. 607-7-143
gram, Canada. The authors
by funds provided by the Public Health Research and 635-z-2) of the National Health Grants Pro-
wish to thank
Dr. J. A. Trew for his helpful advice.
IIEFERBNCES I 1 3 1
G. A. &!I. Il. M. D. IT. E.
JERVIS, PYOC. Sot. Exp.
Biol.
Med.,
83 (1950)
75.
ARMSTRONG,
K. N. F. SHR'~~; AND Ii.S. ROIUNSON, j.Riol. Chcm.,
ARMSTROIL.G,
IS.
213 (Igjj) 797. N. F. SXIAIVAND P. E. WALL, J. Biol.Chew., 218 (1956) 293. HOFFMAX AND K. hf. GOOIXNG, Anal. Biochem., 31 (1969) 471. 5 Ii.BLAU, Clin.Chim. Acfa, 27 (1970) 5.
HILL
462 6 A. HILL AND W. A. ZALESKI, Clin. Biochewz.,
ct cd.
in press (1972). 7 C. hl. WILLIAMS AND C. C. SWEELEY, Gas Chromatography of Lrrinary Aromatic Acids, in H. A. SZYMANSKI (Ed.), Biochemical Applications of Gas Chvowatography, Plcnum Press, New York, 1964, p. 225. 8 M. D. ARMSTRONG Ax" K. L. Low, Pvoc. Sot. Exp. Bml. ATled., 9.+r (19.57) T~L. 9 C. 21. WILLIAMS, G. E. DORIS AND &I. (;REER, Z~?d.BiOChEnZ., 6 (1963) 468. IO J. IN. VAVICH AKD R. R. HowELL, j. Lab. CZz$t.AWr~d..77 (1971) I.59. II L. SADILEK, Sprcialia, 15 (1966) 411. Clin. Chiwz. Acta,
37 (1972)
455-462
455
CCA 4879
THE
INVESTIGATION
ALKAI’TONURIA
OF AROMATIC
AND TYROSINOSIS
ACIDS IN PHENYLKETONURIA, USING
GAS-LIQUID
CHRO~I~4TOGR‘4~HY
SUMMARS
A rapid and accurate method for the measurement of six aromatic acids which are excreted in phenylketonuria has been developed. The same procedure was applied to urine from patients with alkaptonuria and tyrosinosis. The urinary aromatic acid profiles can be used to confirm the diagnosis and to monitor dietary treatment. The regular excretion of mandelic acid in phenylketonuria is confirmed in this study and its excretion in eight cases of uncontrolled phen~lketonuria is reported. The “finding of a second peak.in an alkaptonuric urinary profile is reported but this has not been identified.
INTRODUCTION
The inborn errors of metabolism, phenylketonuria, alkaptonuria, and tyrosinosis are characterized by the urinary excretion of aromatic acid metabolites in excessive quantities. A variety of methods have been described for the identification and measurement of the aromatic acids. These have included wet chemistry methodslF2 and a variety of chromatographic techniques3-“. The aromatic acid profiles are characteristic of a specific metabolic defect and may be used to confirm the diagnosis obtained from amino acid studies and other investigations. In addition, quantitative measurement of individual urinary aromatic acids provides important information as to the fate of ingested amino acids in diseases such as phenylketonuria where there is a block in the metabolic pathway involving the particular acid. This paper describes a rapid gas-liquid chromatographic method for the measurement of urinary aromatic acids associated with phenylketonuria, alkaptonuria, and tyrosinosis.
456 METHODS
AND
MATERIALS
Whenever possible, 24-h urine samples were collected, but in cases where this was not possible, random urine specimens were collected. Specimens were stored at prior to analysis.
-20’
An ilerograph
HY-FI
Xodel
6ooD gas chromatograph
with
a single
column
and flame ionization detector was used. Two 6-ft. x 0.125 in. i.d. stainless steetcolumns were packed with 5oj SE-30 and 8% SE 52 60-80 mesh acid-washed, HMDS-treated Chromosorb
W.
Extraction ?f aromatic acids One ml of urine was acidified
by the addition
of two drops of concentrated
l~ydroclllo~-ic acid. The addition of 2 ml of saturated sodium chloride solution was followed by extraction with 3 x 3 ml volumes of ethyl acetate. The sample was mixed and the interface emulsion broken by centrifugation. The ethyl acetate extracts were evaporated to dryness using a vacuum manifold apparatus, a single unit of which is shown in Fig. I. The units were connected in series so that 6 extracts could be dried simultaneousl~r, The number could be extended to include as many units as desired. The total time for extraction Preparation of derivatives J’olatile derivatives
of 6 samples was 45 min.
joy gas-lipid chrornatogra$hy (GLC) of the extracted aromatic acids were prepared by the addi-
tion of ISO ,ul of X,0-bis-(trimethylsilyI)-acetamide (BSA). The appropriate internal standard was added and the volume made up to joo ,ul with dioxane. The reaction
TO
NEXT
- - _
-
UNIT VACCUM
MANIFOLD
SERUM
19
_.-_, .*.., .. .. . ..,.. ..... ... .. **... ..**.
TEST
CAP
6A6E
NEEDLE
TUBE EXTRACT
CONTAINING
TO
VACCUM
MEASUREMEST
OF
AROMATIC
ACIDS
BY
c;Lc
457
was allowed to proceed at room temperature was kept sealed until samples were withdrawn Gas-liquid
for 15 min in a stoppered for analysis.
tube which
chrontatogra$hy
Five-$ aliquots of the reaction mixture were chromatographed with temperature programming. The flame ionization detector was maintained at 240°, the helium flow rate was 35 ml/mm, and the range and attenuation were varied according to the concentration of the aromatic acids present in the extract. Identification of each aromatic acid in the urine extracts was achieved by co-chromatography with the TMS derivatives of authentic compounds on both columns. Peak
awa deterwtixation
All peak areas were measured by triangulation. The peak area ratio of each acid to internal standard was plotted against the corresponding weight of standard compound. Each calibration curve was straight line with zero intercept. RESULTS
The profile obtained for six authentic aromatic acids is shown in Fig. 2 A. The separation of the peaks makes accurate measurement of each acid possible. Fig. 2 B shows the aromatic acid excretion pattern of a normal individual, typical of the profiles obtained from all normal subjects studied. Fig. 3 shows the aromatic acid excretion pattern of 2 untreated phenylketonuric patients. The profiles obtained from a phenylketonuric patient while on normal diet and during therapy with a low phenylalanine diet are shown in Fig. 4.
A
1
0
5
IL IO
15
20
25
30
35
40
45
50
30
35
40
45
50
minutes
0
5
10
15
20
25 minutes
Fig. 2. (A) Separation of mixture of authentic aromatic acids. I, phenylacetic acid: 2, maudelic acid; 3, o-hydroxyphenylacetic acid; 4, ,%phenyllactic acid; 5. p-hydroxyphenylacetic acid; 6, Bphenylpyruvic acid; 7. hippuric acid; IS, internal standard. (B) -4romatic acid profile of normal human urine.
Clis.
Chim.
Acta.
37 (1972)
455-462
HILL
IO
5
IS
20
25
30
35
40
45
50
30
35
40
45
50
et al.
minutes
t 0
5
15
10
20
25 minutes
Fig. 3. (A) and (B) Aromatic Legend as for Fig. 2. TABLE
1
COKCENTRATIOX KETONURIA (mg/g
OF OS
USCOXJUGATED
NORMAL
AND
AROMATIC
ACIDS
PHESYLALASISE-RESTRICTED
IN
CRIXE
SPECIMESS
OF
12
CASES
017 I’HEXYL~
DIETS
crcatinine)
Subject
Phrwvlacrtic acid
.ZfandPlzc ucld
Normal diet P.S. 213 l’.H. 62 E.1’. 73 K.J. 72 Ii.Ii. 60 E.L. Sj \..I>. C.A.
Restricted I1.K. 11,s. I,.L,.
_~
acid profiles of urine from two cases of untreated phcn~lketonuri~1.
V.H.
IS 15 3’ 38 3-l '4 39
ii
7’
I.35
phcnplalanine 33
diet 0 '3
3.3
2,
IL0
0.6
2.0
The most significant difference is the disappearance of the peaks representing phenylacetic acid, mandelic acid, o-hydroxyphenylacetic acid, phenyllactic acid and phenylpyruvic acid during treatment. The excretion of o-hydroxyphenplacetic acid has been widely used as a diagnostic aid for the confirmation of phenylketonuria2,5. The values reported for the six aromatic acids excreted in phenylketonuria may be obtained from the same analysis. The values for 8 patients with uncontrolled phenylketonuria, and 4 patients receiving a phenylalanine-restricted diet are shown in Table I. Clip. Chiln.
.4&a,
37 (1971)
.+jj-+hL
MEASUREMENT
OF AROMATIC
ACIDS BY
GUI
450
Profiles of urine extracts from a patient with alkaptonuria and a normal subject are shown in Fig. 5. Homogentisic acid is present in abnormal amounts in urine from alkaptonurics, whereas none is found in extracts of normal urine. The amounts of ho~noge~ltisic acid in the alkaptonuric urine was found to be 5.1 g/z4 h. A second
L 0
5
IO
I.5
20
25
30
3.5
40
45
50
30
35
40
45
50
minutes
r
0
5
IO
1s
20
25 minutes
Fig. ‘4. (4) Aromatic acid profile of urine from a case of untreated phenylketonuria and (B), profile of same patient during therapy with a low phenylalanine diet. Legend as for Fig. 2.
Fig. 5. Aromatic acid profiles of urine from (X) normal ria. HGA, homogentisic acid; IS, internal standard.
individual
and (B) patient
Clin. Chiwz. Acta,
with alkaptonu-
37 (1972) 45.=,-162
460
HILL
peak was found to appear with a retention unidentified.
tit al.
time of 6 min. As yet this peak remains
Fig. 6 shows the pattern obtained from the urine of a patient with tyrosinosis” as compared with a normal subject. The two aromatic acids, P-llydroxvphenvlacetic acid and $-hydroxyphenyllactic acid are grossly elevated. No fi-hpdroxyphenylpyruvic acid was observed in urinary profiles of this patient. DISCUSSION
In order to use urinary
aromatic
acid profiles obtained
by GLC to confirm
a
diagnosis and to monitor the effect of dietary treatment of inborn errors of metabolism, a rapid method of extraction and analyses must be available. The rapid evaporation of samples using the vacuum manifold apparatus makes such monitoring feasible. The total time required for a single sample is under z 11. The analysis allowed ecxellent separation and quantitative measurement of 0 unconjugated aromatic acids in phenylketonuria. The concentration of unconjugated phenylacetic acid in untreated phenylketonuria was observed to range from 60 to 213 mg/g creatinine as shown in Table I. Quantitative measurements of mandelic acid have not previously been reported in uncontrolled phenylketonuria. The excretion was found to range from 15 to 71 mg/g creatinine. In normal individuals the value was less than I mg/g creatinine.
0
5
IO
15
20
5
IO
IS
20
25
30
35
40
45
25
30
35
40
45
minutes
Fig. 6. Aromatic acid profiles of (A) patient for Fig. 2; 8, P-hydrosyphenyllactic acid. Clin. Chim.
A&,
37 (1972)
4.jjp461
with tyrosinosis
and (B) normal
indixidual.
Legend as
MEASUREMENT
OI: AROMATIC
ACIDS BY
Ortho-hydroxyphenyla~etic nosis of phenylketonuria.
461
GLC
acid has been of primary
The separation
importance
of the peak corresponding
in the diagto o-hydroxy-
phenylacetic acid from phenyllactic acid is not easily accomplished. However, in all cases the slight overlapping of these peaks did not interfere with peak area measurement. A range of 63 to 219 mg/g creatinine was found in 8 cases of untreated phenylketonuria. The excretion of phenylla~ti~ acid is grossly elevated in these cases. Altl~ougl~ it is present in small amounts in normal individuals, the increased excretion in uncontrolled phenylketonuria allows this peak to be identified readily in urinary profiles. The observed range in this study was 164 to 1965 mg/g creatinine (Table I) for untreated patients. Phenylpyruvic acid is excreted in abnormal amounts in uncontrolled phenylketonuria. None was found in normal subjects. The excretion of this compound ranged from 226 to 1910 mg/g creatinine (Table I). .Uthough was determined
for completeness.
p-hydroxyphenylacetic acid is not increased in phenylketonuria, it The range of values observed for all subjects shows
good agreement
with those reported by Williams
and Sweeley7 for normal individuals.
The values reported for phenylacetic acid, ~-l~ydroxyphen~la~eti~ acid, phenyllactic acid and phenylpyruvic acid are in good agreement with the results of other workers4,8-10. The urinary excretion of homogentisic acid in alkaptonuria is grossly elevated. Sadilekll reported that the average urinary excretion of homogentisic acid in these patients was 5 g/24 11. The value of 5.1 g/z4 h for the patient in this study is in good agreement . The urinary profile of the patient with alkaptonuria showed a second major peak with a retention time of 6 min. Identification of the peak was attempted by cochromatography with a variety of aromatic acids witout success. The possibility of the peak being due to an artefact cannot be excluded. The profile obtained from the urinary extract from a patient with tyrosinosis is readily distinguished from those characteristic of phenylketonuria and alkaptonuria. The two elevated phenolic acids, fi-hydroxyphenylacetic acid and $-hydroxyphenyllactic acid, are widely separated. This patient did not excrete excessive amounts of p-hydroxyphenylpyruvic acid and probably represents a clinical and biochemical variant”.
Grants
This study was assisted (Project Nos. 607-7-143
gram, Canada. The authors
by funds provided by the Public Health Research and 635-z-2) of the National Health Grants Pro-
wish to thank
Dr. J. A. Trew for his helpful advice.
IIEFERBNCES I 1 3 1
G. A. &!I. Il. M. D. IT. E.
JERVIS, PYOC. Sot. Exp.
Biol.
Med.,
83 (1950)
75.
ARMSTRONG,
K. N. F. SHR'~~; AND Ii.S. ROIUNSON, j.Riol. Chcm.,
ARMSTROIL.G,
IS.
213 (Igjj) 797. N. F. SXIAIVAND P. E. WALL, J. Biol.Chew., 218 (1956) 293. HOFFMAX AND K. hf. GOOIXNG, Anal. Biochem., 31 (1969) 471. 5 Ii.BLAU, Clin.Chim. Acfa, 27 (1970) 5.
HILL
462 6 A. HILL AND W. A. ZALESKI, Clin. Biochewz.,
ct cd.
in press (1972). 7 C. hl. WILLIAMS AND C. C. SWEELEY, Gas Chromatography of Lrrinary Aromatic Acids, in H. A. SZYMANSKI (Ed.), Biochemical Applications of Gas Chvowatography, Plcnum Press, New York, 1964, p. 225. 8 M. D. ARMSTRONG Ax" K. L. Low, Pvoc. Sot. Exp. Bml. ATled., 9.+r (19.57) T~L. 9 C. 21. WILLIAMS, G. E. DORIS AND &I. (;REER, Z~?d.BiOChEnZ., 6 (1963) 468. IO J. IN. VAVICH AKD R. R. HowELL, j. Lab. CZz$t.AWr~d..77 (1971) I.59. II L. SADILEK, Sprcialia, 15 (1966) 411. Clin. Chiwz. Acta,
37 (1972)
455-462