299
Clinica Chimica Acta, 62 (1975) 299-304 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7161
INCREASED ARTIFICIAL
PYROGLUTAMIC DIETS
V.G. OBERHOLZER, Queen Elizabeth (Received
C.B.S. WOOD, T. PALMER*
Hospital
February
ACID LEVELS
for Children,
Hackney
IN PATIENTS
ON
and B. MARY HARRISON
Road, London,
E2 8PS (U.K.)
24, 1975)
Summary Increased plasma and urine levels of pyroglutamic dcid were found in 4 patients being fed the low-lactose food Nutramigen. Pyroglutamic acid was detected and estimated by a variety of methods, and the merits of the techniques used and their application in a screening programme are discussed.
Introduction Pyroglutamic aciduria arising from a presumed metabolic defect has been described in three patients. The first was a 20-year-old man who presented with moderate mental retardation, generalized spastic tetraparesis, and chronic metabolic acidosis [1,2]. The second was a 14-month-old girl with chronic metabolic acidosis but no neurological abnormality [3]. Both had serum pyroglutamic acid (5oxo-proline) levels of 50-65 mg/lOO ml. A younger sister of the second case also developed a metabolic acidosis and excreted large amounts of pyroglutamic acid. Results suggested that both sisters had a defect in the synthesis of glutathione [ 41. We first detected gross urinary amounts of pyroglutamic acid during routine metabolic screening of a 2-month-old boy with gastroenteritis. The pyroglutamic acid levels were much lower than in the 2 patients previously described, there was a marked variation in levels with time, and there were no clinical symptoms which could be attributed to the increased pyroglutamic acid levels. It seemed possible therefore that the pyroglutamic aciduria might be a secondary feature in his case. We also investigated pyroglutamic acid levels in 3 other patients with gastroenteritis who were being given the same dietary treatment, Nutramigen, as our original patient.
* Present
address:
Trent Polytechnic,
Nottingham,
NG1 4BU.
U.K.
300
Methods Storage of specimens All plasma specimens were stored at 40°C prior to analysis, and all urine specimens were stored at -15” C to prevent any breakdown of the glutamine content into pyroglutamic acid on storage [ 51. Chromatography One-way chromatography on Whatman No. 1 paper was performed directly on untreated urine using n-butanollacetic acid/water (100 : 22 : 56, v/v) solvent overnight. Papers were dried and hung over a boiling water bath for 30 minutes to remove all traces of acid solvent. Organic acids were detected using a bromocresol green reagent [6]. Pyroglutamic acid and other compounds containing chlorinatable nitrogen were detected using a starch-iodine reagent after chlorination [7]. A direct estimation of the pyroglutamic acid present was made by visual comparison of the spots obtained using either stain with those from standard solutions. The R, values, under these conditions, of some of the important organic acids found in urine are given in Table I. Two-way chromatography involved the isolation of an organic acid extract using a column of Dowex 1 X 8 resin (200-400 mesh) in the formate form [ 61. The formic acid eluate was taken to dryness in a rotary evaporator at 40” C and the residue dissolved in a small amount of 30% aqueous isopropanol containing ammonia. A 2 ~1 sample was then applied as a spot to a 10 X 10 cm cellulose thin-layer sheet Merck No. 5552 or Schleicher and &hull F 1440, followed by ascending chromatography using pure ethanol/O.88 ammonia / water (150 : 8 : 40, v/v) as first solvent and 2-ethyl-l-butanol/formic acid/ water as the second [8] . The later solvent system was made up by allowing 2-ethyl-1-butanol/98% formic acid/water (40 : 12 : 48, v/v) mixture to equilibrate for 24 hours, then taking 30 ml of the upper layer and mixing with 5 ml 98% formic acid just before use. The separation obtained using 2 ~1 of a mixture of organic acids at concentrations of 1 mg/ml is shown in Fig. 1.
TABLE Rf
I
VALUES
OF
WHATMAN
NO.
ORGANIC 1
PAPER
ACIDS
BY
USING
DESCENDING
n-BUTANOLIACETIC
SOLVENT.
Organic
acid
Rf
value
Hippuric
(dark
in UV)
84-86
Fumaric
(dark
in UV)
80-84
Phenylacetylglutamine
78-81
Succinic
76-79
Methylmalonic
76-81
&hydroxybutyric Lactic
(main
Glycollic
76-79 band)
74-76 62-65
Pyroglutamic
60-64
Glyceric
49-51
(range)
CHROMATOGRAPHY ACID/WATER
OVERNIGHT (100
:
22
:
56.
ON v/v)
AS
301
01
’
’
20
’
’
40
/
’
’
60
Solvent
’
80
’
’
100
2
Fig. 1. Separation of organic acids by two-way thin layer chromatography. Solvent 1, ethanol/O.88 ammonia/water (150 : 8 : 40, v/v); Solvent 2. 2-ethyl-1-butanol/formic acid/water (40 : 12 : 48, v/v, using 30 ml upper layer + 5 ml 98% formic acid). Organic acids: 1, citric; 2, tartaric; 3, malic: 4. malonic; 5, aconitic; 6, glyceric; 7, a-ketoglutaric: 8. succinic; 9, methylmalonic: 10, glycollic; 11, pyroglutamic; 12, glutaric; 13, adipic; 14, fumaric: 15. phenylacetylglutamine: 16, lactic; 17. fl-hydroxybutyric; 18, hippuric: 19, hydrochloric acid.
Detection of organic nitrogen was as above.
acids and of compounds
containing
chlorinatable
Gas-liquid chromatography The standard methods of organic acid analysis by gas-liquid chromatography [9] are unsuitable for pyroglutamic acid determination, since this compound is insoluble in ether. The method of Marstein et al. [lo] was therefore employed. Indirect estimation of pyroglutamic acid Acidified samples of urine or deproteinized plasma were passed through a column of Dowex 50 X 8 resin to remove amino acids. The eluate and washings were pooled, norleucine was added as internal standard, and the pyroglutamic acid present was hydrolysed to glutamic acid by adding HCl to a final concentration of 2 N and maintaining the solution at 100°C for 2 hours [ 111. Glutamic acid levels were determined on an amino acid analyser [ 121. A standard solution of pyroglutamic acid was found to be completely converted to glutamic acid under the conditions used for hydrolysis. Identification of pyroglutamic acid An abnormally high amount of glutamic acid was produced by mild hydrolysis of extracts from plasma and urine specimens from the 4 patients on Nutramigen. This glutamic acid could have been produced from other compounds beside pyroglutamic acid, in particular from phenylacetylglutamine, which has been found in the urine of normal subjects [6] . However all the
302
chromatography results were consistent with the compound present in increased amounts in the urines of these patients being identified as pyroglutamic acid. Investigation of an ultrafiltrate of plasma from a control subject showed that only those fractions corresponding to the glutamine, glutamic acid and pyroglutamic acid positions after one-way chromatography contained more than trace amounts of glutamic acid after mild hydrolysis. A sample of plasma from a patient on Nutramigen gave similar results except that there was a much higher amount of glutamic acid from that fraction corresponding to the pyroglutamic acid position. Finally an attempt was made to extract pyroglutamic acid from the urine of one of the patients. Cations and amino acids were removed on a Dowex 50 X 8 resin and the eluate was washed with ether to remove ether-soluble acids. The aqueous phase was applied to a column of Dowex 1 X 8 resin in the formate form and the organic acids were eluted with 12 N formic acid. The residue obtained after taking to dryness in a rotary evaporator was dissolved in a minimum quantity of methanol, and pyroglutamic acid was crystallized from the solution by the addition of ether. The crystals were washed in ether and dried. The infra-red spectrum of the crystals were consistent with identification as pyroglutamic acid, although 2 extra peaks were found, presumably due to the presence of impurities. From all this it was concluded that increased quantities of pyroglutamic acid were indeed present in the plasma and urine of the patients on Nutramigen. Results Urine levels of pyroglutamic acid, measured indirectly as glutamic acid, and also estimated directly by comparison with standards following paper chromatography, are summarized in Table II. There was reasonable agreement between the values obtained by the two methods for the same specimens. There was also good agreement between the level determined on a single occasion by gas-liquid chromatography, 1700 mg/g creatinine, and the corresponding value of 1840 mg/g creatinine obtained by the indirect method. Thus there was no evidence for the production of significant quantities of glutamic acid from anything other than pyroglutamic acid in the acid fractions of the patients’ urine. Plasma levels of pyroglutamic acid, measured indirectly as glutamic acid following hydrolysis, are also given in Table II. The amounts present were too small to be detected directly by paper chromatography. It can be seen from Table II that all plasma and urine levels of pyroglutamic acid determined on the patients on Nutramigen were higher than those in subjects having normal diets. In a patient whose Nutramigen intake was interrupted while clearance studies and a protein loading test were performed on him, the plasma pyroglutamic acid level fell in 24 hours from 6.5 to 1.6 mg/lOO ml, and the urine pyroglutamic acid level similarly fell from 1650 to 630 mg/g creatinine. Pyroglutamic acid was detected by paper chromatography in a loose stool from one of the patients on Nutramigen, but not in a later firm stool. The pyroglutamic acid contents of the two specimens, measured indirectly as glu-
303
TABLE
II
PYROGLUTAMIC
ACID
LEVELS
(MEAN
AND
RANGE)
IN
PATIENTS
ON
NUTRAMIGEN
AND
IN
CONTROLS
Patients
Plasma
mg/100
(indirect Urine
mg/g
Urine
mg/g
(direct
NO.
No.
cimens
3
6
4
9
4230
(340
-16500
)
5
106
4
8
6500
(500
-24500
)
*
None
screened
by direct
ml
creatinine method) creatinine assessment)
* 2000
cases
spe-
Controls
patients
method)
(indirect
on Nutramigen Mean
(range)
NO.
Mean
(range)
subjects
6.2
chromatography
(
1.5-
14.0)
of untreated
3
0.60
(
0.24-
(28
1.2) -165
)
detected
urine.
tamic acid following hydrolysis, were 1.4 and 0.13 mg/g stool, respectively. A sample of Nutramigen was analysed by the method used for urine prior to two-way chromatography. The extract gave an acid spot with the same Rf values as pyroglutamic acid. By reaction with chlorine followed by a starchpotassium iodide reagent and comparison with a series of standards the concentration of the pyroglutamic acid was estimated to be 600 mg per 100 g Nutramigen powder. An acid spot similar to pyroglutamic acid was also found in a liquid stool from a patient with gastroenteritis following a feed of Pregestimil. Discussion It was concluded that the high pyroglutamic acid levels in the patients being fed Nutramigen were dietary in origin, especially since the levels varied with Nutramigen intake, and pyroglutamic acid was found both in Nutramigen powder and in a loose stool from one of the patients. Nutramigen is a lactosefree food commonly given to patients with gastroenteritis and lactose intolerance. Its nitrogen content consists of hydrolysed casein. Pyroglutamic acid may be formed from glutamic acid on heating, depending on the pH [lo], and thus the pyroglutamic acid in Nutramigen may have been formed during the preparation or subsequent treatment of the hydrolysed casein. If this is so, other foods such as Pregestimil containing hydrolysed casein may well have a raised pyroglutamic acid content. There is no reason to suppose this will affect the value of the foods, but it should be borne in mind during metabolic screening. The presence of similar artifacts lactulose and homocitrulline in milk preparations is well known [ 13,141. Raised levels of pyroglutamic acid have been reported in patients with hepatic coma [6], but there was no evidence of liver dysfunction in any of our patients. This study has shown that the indirect determination of pyroglutamic acid as glutamic acid is a useful technique, although it cannot be employed when significant amounts of phenylacetylglutamine are present. The pyroglutamic acid levels obtained by this method, especially for the control specimens, should be treated with a certain amount of caution in view of the nature of the
304
method and the dangers of contamination by glutamic acid. Nevertheless the control plasma levels are less tha I those given by Wolfersberger and Tabachnik [ 151, and it seems likely that the je authors measured largely pyroglutamic acid produced from glutamine on storage. The value of the paper chromatography method used has also been demonstrated, since if this is capable of detecting a moderate organic aciduria, it will easily detect a more severe one. The butanol-acetic solvent system which is commonly employed for routine metabolic screening is not ideal for organic acid separation. However by staining the half of the paper furthest from the origin for organic acids and retaining the other half for the detection of carbohydrates, a urine specimen can be conveniently screened for these two classes of compounds. No prior preparation is needed, so the scope of the procedure is not limited by such factors as solubility in ether. Once an organic aciduria has been revealed by this simple technique, it can then be investigated in more detail by two-way chromatography or by gas-liquid chromatography. Acknowledgements We are grateful to Dr D. Gompertz, Department of Medicine, smith Hospital, for his assistance with gas-liquid chromatography.
Hammer-
References 1
E.
Jellum,
T.
Kluge.
H.C.
Borreson,
0.
Stokke
and
L. Eldjam,
Stand.
.J. Clin.
Lab.
(1974)
1
Invest..
26
(1970)
327 2
L. Eldiarn.
3
L. Hagenfeldt.
4
A.
Larson,
(1974)
E. Jellum A. R.
5
T. Palmer, J. Nordmann Heinemann,
7
H.N.
Rydon
8
A.R.
Jones,
9
D.
ZetterstrGm,
Gompertz
V.G.
Clin.
Chim.
E. ZetterstrHm. L.
Oberholzer and
Acta.
Acta
Hagenfeldt.
R.M.C.
Dawson,
Oxford
University
T. Palmer.
13
1.S.
Menzies
Heinemann. Gerritsen,
Vol.
R.
Smith,
G.H.
and
and
D.C. Press. Rossiter.
and
J.W.T.
London, S.H.
Vol. Lipton,
edn,
1969.
Nature.
49
(1973)
Paediatr.
Anderson,
311
&and.,
M.G.
and
2nd
W.H. edn,
B. Levin Seakins. 1, 3rd F.M.
Clin.
H. HGrnell,
Pediatr.
Res..
and V.G.
Strong
(1974)
Wolfersberger
and J. Tabachnik,
335 and Electrophoretic
Techniques,
Chim.
40
25
(1953)
(1972)
Acta,
49
K.M.
Jones
Oberholzer,
Clin.
and
394
5 (1973)
389
(eds).
Data
for
Biochemical
Research,
p. 53
in I. Smith edn.
52
922. Chem..
Acta,
Elliott
1969.
Acta.
Chromatographic
(1952) Anal.
Chim.
L. Eldiam,
Chim. (ed.),
1969, and
(ed.).
Sci.
Chromatographic
Mol.
Med.. and
45
(1973)
827
Electrophoretic
Techniques.
p. 310 H.
Waisman,
Biochem.
379 15
63
S. Dreborg
p. 342
169
Skraba,
Clin.
Elliott,
Clin.
in I. Smith
W.J.
Draffan.
E. Jellum
M.A.
B. Levin.
1, 3rd
Dowling
and
S. Marstein,
and
Nordmann,
and P.W.G. E.J.
11 12
R.
London,
10
T.
Stokke,
and
852
6
14
and 0.
Larsson
Experientia.
29
(1973)
346
Biophys.
Res.
Commun.,
4 (1961)
8
Clinica Chimica Acta, 62 (1975) 299-304 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7161
INCREASED ARTIFICIAL
PYROGLUTAMIC DIETS
V.G. OBERHOLZER, Queen Elizabeth (Received
C.B.S. WOOD, T. PALMER*
Hospital
February
ACID LEVELS
for Children,
Hackney
IN PATIENTS
ON
and B. MARY HARRISON
Road, London,
E2 8PS (U.K.)
24, 1975)
Summary Increased plasma and urine levels of pyroglutamic dcid were found in 4 patients being fed the low-lactose food Nutramigen. Pyroglutamic acid was detected and estimated by a variety of methods, and the merits of the techniques used and their application in a screening programme are discussed.
Introduction Pyroglutamic aciduria arising from a presumed metabolic defect has been described in three patients. The first was a 20-year-old man who presented with moderate mental retardation, generalized spastic tetraparesis, and chronic metabolic acidosis [1,2]. The second was a 14-month-old girl with chronic metabolic acidosis but no neurological abnormality [3]. Both had serum pyroglutamic acid (5oxo-proline) levels of 50-65 mg/lOO ml. A younger sister of the second case also developed a metabolic acidosis and excreted large amounts of pyroglutamic acid. Results suggested that both sisters had a defect in the synthesis of glutathione [ 41. We first detected gross urinary amounts of pyroglutamic acid during routine metabolic screening of a 2-month-old boy with gastroenteritis. The pyroglutamic acid levels were much lower than in the 2 patients previously described, there was a marked variation in levels with time, and there were no clinical symptoms which could be attributed to the increased pyroglutamic acid levels. It seemed possible therefore that the pyroglutamic aciduria might be a secondary feature in his case. We also investigated pyroglutamic acid levels in 3 other patients with gastroenteritis who were being given the same dietary treatment, Nutramigen, as our original patient.
* Present
address:
Trent Polytechnic,
Nottingham,
NG1 4BU.
U.K.
300
Methods Storage of specimens All plasma specimens were stored at 40°C prior to analysis, and all urine specimens were stored at -15” C to prevent any breakdown of the glutamine content into pyroglutamic acid on storage [ 51. Chromatography One-way chromatography on Whatman No. 1 paper was performed directly on untreated urine using n-butanollacetic acid/water (100 : 22 : 56, v/v) solvent overnight. Papers were dried and hung over a boiling water bath for 30 minutes to remove all traces of acid solvent. Organic acids were detected using a bromocresol green reagent [6]. Pyroglutamic acid and other compounds containing chlorinatable nitrogen were detected using a starch-iodine reagent after chlorination [7]. A direct estimation of the pyroglutamic acid present was made by visual comparison of the spots obtained using either stain with those from standard solutions. The R, values, under these conditions, of some of the important organic acids found in urine are given in Table I. Two-way chromatography involved the isolation of an organic acid extract using a column of Dowex 1 X 8 resin (200-400 mesh) in the formate form [ 61. The formic acid eluate was taken to dryness in a rotary evaporator at 40” C and the residue dissolved in a small amount of 30% aqueous isopropanol containing ammonia. A 2 ~1 sample was then applied as a spot to a 10 X 10 cm cellulose thin-layer sheet Merck No. 5552 or Schleicher and &hull F 1440, followed by ascending chromatography using pure ethanol/O.88 ammonia / water (150 : 8 : 40, v/v) as first solvent and 2-ethyl-l-butanol/formic acid/ water as the second [8] . The later solvent system was made up by allowing 2-ethyl-1-butanol/98% formic acid/water (40 : 12 : 48, v/v) mixture to equilibrate for 24 hours, then taking 30 ml of the upper layer and mixing with 5 ml 98% formic acid just before use. The separation obtained using 2 ~1 of a mixture of organic acids at concentrations of 1 mg/ml is shown in Fig. 1.
TABLE Rf
I
VALUES
OF
WHATMAN
NO.
ORGANIC 1
PAPER
ACIDS
BY
USING
DESCENDING
n-BUTANOLIACETIC
SOLVENT.
Organic
acid
Rf
value
Hippuric
(dark
in UV)
84-86
Fumaric
(dark
in UV)
80-84
Phenylacetylglutamine
78-81
Succinic
76-79
Methylmalonic
76-81
&hydroxybutyric Lactic
(main
Glycollic
76-79 band)
74-76 62-65
Pyroglutamic
60-64
Glyceric
49-51
(range)
CHROMATOGRAPHY ACID/WATER
OVERNIGHT (100
:
22
:
56.
ON v/v)
AS
301
01
’
’
20
’
’
40
/
’
’
60
Solvent
’
80
’
’
100
2
Fig. 1. Separation of organic acids by two-way thin layer chromatography. Solvent 1, ethanol/O.88 ammonia/water (150 : 8 : 40, v/v); Solvent 2. 2-ethyl-1-butanol/formic acid/water (40 : 12 : 48, v/v, using 30 ml upper layer + 5 ml 98% formic acid). Organic acids: 1, citric; 2, tartaric; 3, malic: 4. malonic; 5, aconitic; 6, glyceric; 7, a-ketoglutaric: 8. succinic; 9, methylmalonic: 10, glycollic; 11, pyroglutamic; 12, glutaric; 13, adipic; 14, fumaric: 15. phenylacetylglutamine: 16, lactic; 17. fl-hydroxybutyric; 18, hippuric: 19, hydrochloric acid.
Detection of organic nitrogen was as above.
acids and of compounds
containing
chlorinatable
Gas-liquid chromatography The standard methods of organic acid analysis by gas-liquid chromatography [9] are unsuitable for pyroglutamic acid determination, since this compound is insoluble in ether. The method of Marstein et al. [lo] was therefore employed. Indirect estimation of pyroglutamic acid Acidified samples of urine or deproteinized plasma were passed through a column of Dowex 50 X 8 resin to remove amino acids. The eluate and washings were pooled, norleucine was added as internal standard, and the pyroglutamic acid present was hydrolysed to glutamic acid by adding HCl to a final concentration of 2 N and maintaining the solution at 100°C for 2 hours [ 111. Glutamic acid levels were determined on an amino acid analyser [ 121. A standard solution of pyroglutamic acid was found to be completely converted to glutamic acid under the conditions used for hydrolysis. Identification of pyroglutamic acid An abnormally high amount of glutamic acid was produced by mild hydrolysis of extracts from plasma and urine specimens from the 4 patients on Nutramigen. This glutamic acid could have been produced from other compounds beside pyroglutamic acid, in particular from phenylacetylglutamine, which has been found in the urine of normal subjects [6] . However all the
302
chromatography results were consistent with the compound present in increased amounts in the urines of these patients being identified as pyroglutamic acid. Investigation of an ultrafiltrate of plasma from a control subject showed that only those fractions corresponding to the glutamine, glutamic acid and pyroglutamic acid positions after one-way chromatography contained more than trace amounts of glutamic acid after mild hydrolysis. A sample of plasma from a patient on Nutramigen gave similar results except that there was a much higher amount of glutamic acid from that fraction corresponding to the pyroglutamic acid position. Finally an attempt was made to extract pyroglutamic acid from the urine of one of the patients. Cations and amino acids were removed on a Dowex 50 X 8 resin and the eluate was washed with ether to remove ether-soluble acids. The aqueous phase was applied to a column of Dowex 1 X 8 resin in the formate form and the organic acids were eluted with 12 N formic acid. The residue obtained after taking to dryness in a rotary evaporator was dissolved in a minimum quantity of methanol, and pyroglutamic acid was crystallized from the solution by the addition of ether. The crystals were washed in ether and dried. The infra-red spectrum of the crystals were consistent with identification as pyroglutamic acid, although 2 extra peaks were found, presumably due to the presence of impurities. From all this it was concluded that increased quantities of pyroglutamic acid were indeed present in the plasma and urine of the patients on Nutramigen. Results Urine levels of pyroglutamic acid, measured indirectly as glutamic acid, and also estimated directly by comparison with standards following paper chromatography, are summarized in Table II. There was reasonable agreement between the values obtained by the two methods for the same specimens. There was also good agreement between the level determined on a single occasion by gas-liquid chromatography, 1700 mg/g creatinine, and the corresponding value of 1840 mg/g creatinine obtained by the indirect method. Thus there was no evidence for the production of significant quantities of glutamic acid from anything other than pyroglutamic acid in the acid fractions of the patients’ urine. Plasma levels of pyroglutamic acid, measured indirectly as glutamic acid following hydrolysis, are also given in Table II. The amounts present were too small to be detected directly by paper chromatography. It can be seen from Table II that all plasma and urine levels of pyroglutamic acid determined on the patients on Nutramigen were higher than those in subjects having normal diets. In a patient whose Nutramigen intake was interrupted while clearance studies and a protein loading test were performed on him, the plasma pyroglutamic acid level fell in 24 hours from 6.5 to 1.6 mg/lOO ml, and the urine pyroglutamic acid level similarly fell from 1650 to 630 mg/g creatinine. Pyroglutamic acid was detected by paper chromatography in a loose stool from one of the patients on Nutramigen, but not in a later firm stool. The pyroglutamic acid contents of the two specimens, measured indirectly as glu-
303
TABLE
II
PYROGLUTAMIC
ACID
LEVELS
(MEAN
AND
RANGE)
IN
PATIENTS
ON
NUTRAMIGEN
AND
IN
CONTROLS
Patients
Plasma
mg/100
(indirect Urine
mg/g
Urine
mg/g
(direct
NO.
No.
cimens
3
6
4
9
4230
(340
-16500
)
5
106
4
8
6500
(500
-24500
)
*
None
screened
by direct
ml
creatinine method) creatinine assessment)
* 2000
cases
spe-
Controls
patients
method)
(indirect
on Nutramigen Mean
(range)
NO.
Mean
(range)
subjects
6.2
chromatography
(
1.5-
14.0)
of untreated
3
0.60
(
0.24-
(28
1.2) -165
)
detected
urine.
tamic acid following hydrolysis, were 1.4 and 0.13 mg/g stool, respectively. A sample of Nutramigen was analysed by the method used for urine prior to two-way chromatography. The extract gave an acid spot with the same Rf values as pyroglutamic acid. By reaction with chlorine followed by a starchpotassium iodide reagent and comparison with a series of standards the concentration of the pyroglutamic acid was estimated to be 600 mg per 100 g Nutramigen powder. An acid spot similar to pyroglutamic acid was also found in a liquid stool from a patient with gastroenteritis following a feed of Pregestimil. Discussion It was concluded that the high pyroglutamic acid levels in the patients being fed Nutramigen were dietary in origin, especially since the levels varied with Nutramigen intake, and pyroglutamic acid was found both in Nutramigen powder and in a loose stool from one of the patients. Nutramigen is a lactosefree food commonly given to patients with gastroenteritis and lactose intolerance. Its nitrogen content consists of hydrolysed casein. Pyroglutamic acid may be formed from glutamic acid on heating, depending on the pH [lo], and thus the pyroglutamic acid in Nutramigen may have been formed during the preparation or subsequent treatment of the hydrolysed casein. If this is so, other foods such as Pregestimil containing hydrolysed casein may well have a raised pyroglutamic acid content. There is no reason to suppose this will affect the value of the foods, but it should be borne in mind during metabolic screening. The presence of similar artifacts lactulose and homocitrulline in milk preparations is well known [ 13,141. Raised levels of pyroglutamic acid have been reported in patients with hepatic coma [6], but there was no evidence of liver dysfunction in any of our patients. This study has shown that the indirect determination of pyroglutamic acid as glutamic acid is a useful technique, although it cannot be employed when significant amounts of phenylacetylglutamine are present. The pyroglutamic acid levels obtained by this method, especially for the control specimens, should be treated with a certain amount of caution in view of the nature of the
304
method and the dangers of contamination by glutamic acid. Nevertheless the control plasma levels are less tha I those given by Wolfersberger and Tabachnik [ 151, and it seems likely that the je authors measured largely pyroglutamic acid produced from glutamine on storage. The value of the paper chromatography method used has also been demonstrated, since if this is capable of detecting a moderate organic aciduria, it will easily detect a more severe one. The butanol-acetic solvent system which is commonly employed for routine metabolic screening is not ideal for organic acid separation. However by staining the half of the paper furthest from the origin for organic acids and retaining the other half for the detection of carbohydrates, a urine specimen can be conveniently screened for these two classes of compounds. No prior preparation is needed, so the scope of the procedure is not limited by such factors as solubility in ether. Once an organic aciduria has been revealed by this simple technique, it can then be investigated in more detail by two-way chromatography or by gas-liquid chromatography. Acknowledgements We are grateful to Dr D. Gompertz, Department of Medicine, smith Hospital, for his assistance with gas-liquid chromatography.
Hammer-
References 1
E.
Jellum,
T.
Kluge.
H.C.
Borreson,
0.
Stokke
and
L. Eldjam,
Stand.
.J. Clin.
Lab.
(1974)
1
Invest..
26
(1970)
327 2
L. Eldiarn.
3
L. Hagenfeldt.
4
A.
Larson,
(1974)
E. Jellum A. R.
5
T. Palmer, J. Nordmann Heinemann,
7
H.N.
Rydon
8
A.R.
Jones,
9
D.
ZetterstrGm,
Gompertz
V.G.
Clin.
Chim.
E. ZetterstrHm. L.
Oberholzer and
Acta.
Acta
Hagenfeldt.
R.M.C.
Dawson,
Oxford
University
T. Palmer.
13
1.S.
Menzies
Heinemann. Gerritsen,
Vol.
R.
Smith,
G.H.
and
and
D.C. Press. Rossiter.
and
J.W.T.
London, S.H.
Vol. Lipton,
edn,
1969.
Nature.
49
(1973)
Paediatr.
Anderson,
311
&and.,
M.G.
and
2nd
W.H. edn,
B. Levin Seakins. 1, 3rd F.M.
Clin.
H. HGrnell,
Pediatr.
Res..
and V.G.
Strong
(1974)
Wolfersberger
and J. Tabachnik,
335 and Electrophoretic
Techniques,
Chim.
40
25
(1953)
(1972)
Acta,
49
K.M.
Jones
Oberholzer,
Clin.
and
394
5 (1973)
389
(eds).
Data
for
Biochemical
Research,
p. 53
in I. Smith edn.
52
922. Chem..
Acta,
Elliott
1969.
Acta.
Chromatographic
(1952) Anal.
Chim.
L. Eldiam,
Chim. (ed.),
1969, and
(ed.).
Sci.
Chromatographic
Mol.
Med.. and
45
(1973)
827
Electrophoretic
Techniques.
p. 310 H.
Waisman,
Biochem.
379 15
63
S. Dreborg
p. 342
169
Skraba,
Clin.
Elliott,
Clin.
in I. Smith
W.J.
Draffan.
E. Jellum
M.A.
B. Levin.
1, 3rd
Dowling
and
S. Marstein,
and
Nordmann,
and P.W.G. E.J.
11 12
R.
London,
10
T.
Stokke,
and
852
6
14
and 0.
Larsson
Experientia.
29
(1973)
346
Biophys.
Res.
Commun.,
4 (1961)
8
