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Tyrosine metabolism in cystic fibrosis
166
CLINICA CHIMICA ACTA
TYROSINE
METABOLISM
IN CYSTIC
FIBROSIS
R. ROBINSON
SUMMARY
Children with cystic fibrosis excrete in the urine excessive amounts of p-hydroxyphenylacetic acid (PHPA) , and p-hydroxyphenyllactic acid (PHPL) . These findings are in accord with the hypothesis that there is a block in tyrosine catabolism in these patients due to a deficiency of P-hydroxyphenylpyruvate hydroxylase activity. There is no evidence that this deficiency is caused by a lack of ascorbic acid.
Children with cystic fibrosis have abnormally high serum tyrosine values l. Tyrosine could accumulate if a block occurred in one of the steps by which it is broken down. The serum tyrosine level might then be expected to rise to abnormal levels. Tyrosine is broken down in the liver. The catabolic pathways are well known. The metabolites formed are excreted in the urine. If there were a block in the catabolic pathway, the excretion of metabolites in the urine would be abnormal, and it might be possible to deduce the site of the block from the pattern of excretion of these metabolites. Urine specimens from children with cystic fibrosis have been examined for the phenolic acid metabolites of tyrosine to test the hypothesis that there might be a block in tyrosine catabolism in these patients. MATERIAL
AND
METHODS
Urine specimens were obtained from 17 patients with cystic fibrosis. The patients all attended an out-patient’s clinic and the specimens had been collected at home by the parent before attending the clinic. On being received, the specimens were acidified with a few drops of concentrated hydrochloric acid (to pH approximately 4). They were then stored in a refrigerator at 4” until they were examined. This may have caused the loss of some p-hydroxyphenylpyruvic acid. The creatinine content of the urine was determined by either the standard Technicon AutoAnalyzer method or by the method of Bonsnes and TausskyZ. The aromatic metabolites of tyrosine were extracted by the method of Hill et d3: 3 ml of acidified urine was pipetted into a flask and 6 g of anhydrous sodium Cdin.Chim. Acta, ~4 (x966)
166-170
TYROSINE
sulphate acetate
167
METABOLISM
added. added.
The cake
of sodium
sulphate
The flask was then stoppered
was broken and placed
up and 15 ml of ethyl in the refrigerator
for an
hour. During this time the flask was occasionally taken out and the cake of sodium sulphate again broken up if necessary, using a glass rod. An aliquot of the ethyl acetate extract equivalent to the volume of urine containing I mg of creatinine was removed. This was evaporated to dryness under reduced pressure. The extract was applied to a IO-inch square of Whatman No. I filter paper to form a spot less than I cm in diameter at a distance of about an inch from two adjacent sides of the paper. The transference of the extract to the paper was made with small amounts mitted
of ethyl
acetate.
The extracted
phenolic
acids were than
to two-dimensional chromatographic separation. The first solvent system was isopropanol-0.880 ammonia-water
lowed by n-butanol-pyridine-water
(70 : 15 :15). The chromatograms
sub-
(80 :IO : IO) fol-
were allowed to
develop for about 16 h in each solvent system. After drying, the chromatograms were sprayed with 10% sodium carbonate solution and allowed to dry again. They were then sprayed with diazotised sulphanilic acid (the Van den Bergh reagent). Using this technique, $-hydroxyphenyllactic acid (PHPL) is scarcely detectable in normal urine; p-hydroxyphenylacetic acid (PHPA) can be detected in small amounts almost invariably. Specimens of urine were examined from four patients before and after hydrolysing by boiling with 15% (w /v ) o f cont. HCl. One cannot rely on the accuracy of timed collections
of urine obtained
from children in their homes. The chromatograms
were therefore prepared from the volume of urine containing I mg of creatinine. In comparing one urine with another one, the basis of an equal creatinine content is not ideal, but it is probably
the best method
available
at present.
However,
must be recognised that creatinine excretion is more variable in children adults. For this reason no attempt was made to determine quantitatively cretion of any of the tyrosine metabolites.
it
than in the ex-
The chromatograms were compared visually with chromatograms prepared from the urine of normal children. Where there was no obvious difference from normal in the rate of excretion of a particular metabolite, this is indicated in Table I by “N”. When the excretion indicated by “+“.
of a metabolite
was obviously
much greater
than normal
this is
The effect of ascorbic acid administration was examined on 4 patients. A control specimen of urine was collected and then the patient was given IOO mg of ascorbic acid by mouth twice a day for two weeks. At the end of this time, a second specimen of urine was collected for examination. Two patients’ urines were also examined after they had been taking
ascorbic
acid for four weeks.
RESULTS
These are summarised in Table I. Individual patients’ excretion of both PHPL and PHPA seemed to vary only slightly from day to day. Most of the patients’ urines were examined on several occasions but only the result on the first specimen is shown in the table, except for the patients who were treated with ascorbic acid. All the patients excreted excessive amounts of PHPA; 6 of the 17 patients also excreted increased amounts of PHPL. Clin. China. Acta,
14 (1966) 166-170
168 TABLE
R.ROBINSON I
EXCRETION _____.~
OF
TYROSINE
METABOLITES
BY
PATIENTS
WITH
CYSTIC
Patient
PHPL* excreted PHPA ~~______ ~~ --~
1. Control
-t + N N iN R: N N N N N
I. 2. 2. 2. 3. 3. 4. 4. 4. 5. 6. ;. 8. 9.
after 2 Control after 2 after 4 Control after z Control after 2 after 4
weeks on ascorbic
acid
weeks on ascorbic weeks on ascorbic
acid acid
weeks on ascorbic acid weeks on ascorbic weeks on ascorbic
acid acid
N’ N
IO.
N
II.
T
12.
N
13. 14. ‘5. 16. ‘7.
?j i!_
FIBROSIS
* * excwted
.~~
i1. ; + $-L t + i I T + A
iN
*PHPL = p-hydroxyphenyllactic acid. **PHPA = P-hydroxyphenylacetic acid. N indicates excretion is normal. + indicates excretion is abnormally high.
It is known that ascorbic acid deficiency inhibits the breakdown of tyrosine4. The possibility that our patients were excreting increased amounts of PHPL and PHPA because they were ascorbic acid-deficient was tested by examining their urine before and after they had been saturated with ascorbic acid as described above. In no case did the excretion of either PHPL or PHPA fall after the patient had been saturated with ascorbic acid; one patient excreted rather more PHPL after he had been saturated with ascorbic acid than he did before. Four specimens of urine were examined before and after acid hydrolysis. More PHPL and PHPA were found in the hydrolysed than the unhydrolysed specimens. This indicates that both acids are excreted to some extent in conjugated form. The hydrolysed urines were also examined for the hydantoin of p-hydroxyphenylpyruvic acid. This was identified but it was not possible to say whether it was present in increased amounts, as the urines were not examined immediately after they were collected and p-hydroxyphenylpyruvic acid is not stable. DISCUSSIOlr;
The outstanding findings were that all the patients with cystic fibrosis excreted excessive amounts of PHPA and 6 of the 17 patients also excreted raised amounts of PHPL. These results would be expected if the patients suffered from a deficiency of p-hydroxyphenylpyruvate hydroxylase activity. Reaction (2) (Fig. I) would then be blocked, p-hydroxyphenylpyruvic acid would accumulate, and the pathways leading Clin. Chim. Acta,
14 (1966) 166-170
TYROSINE
METABOLISM
1%
to the formation of PHPL and PHPA would be utilized to a much greater extent than in normal individuals. The cystic fibrosis patients bear some resemblance to the case of tyrosinosis described by Halvorsen and Gjessir@. However, it is evident that their patient excreted very much larger amounts of PHPL and PHPA than our cystic fibrosis patients. Tanaguchi and Gjessing8 examined liver and kidney tissue from a patient with tyrosinosis and found that p-hydroxyphenylpyruvate hydroxylase activity was absent from both tissues.
p-hydroxyphenylacetic
acid
t (5)
(==J’yy==JyH HO Tyrosine
Hflo; HO p-hydroxyphenylpyruvic
H4”o;
OH 2-5 dihydroxyphenylpyruvic acid
(6) 11 acid
OH Homogentisic
~~“ption
acid
CH2CH(OH)COOH
/ 0\ HO p-hydrcxyphenyllactic Fig.
I.
Catabolism
acid
of tyrosine.
It seems likely that patients with cystic fibrosis suffer from a lack of some essential co-factor rather than from complete absence of the enzyme. It is known that $-hydroxyphenylpyruvate hydroxylase requires ascorbic acid as a co-factor 4. However, when four of our patients were saturated with ascorbic acid, the amounts of PHPL and PHPA they excreted remained unchanged. A lack of dietary ascorbic acid did not therefore cause the increased excretion of phenolic acids. The pattern of excretion of tyrosine metabolites in the patients with cystic fibrosis resembled that seen in patients with parenchymatous liver damage7. However, though liver damage occurs in patients with cystic fibrosis, it is not believed to be so universal as the urinary phenolic acid results would suggest. It may be concluded that patients with cystic fibrosis metabolise tyrosine abnormally. The abnormality seems to be due to a deficiency in $-hydroxyphenylpyruvate hydroxylase activity, the cause of which is at present unknown. Halvorsen and Gjessing& considered that a high level of circulating tyrosine and its metabolites might have a toxic effect on renal tubular cells and possibly on liver cells. They found that their patient with tyrosinosis benefited from a diet low Gin.
Chim. Acta,
14 (1966)
166-170
R. ROBINSON
170
in tyrosine and phenylalanine. It might be worthwhile carrying out a trial of such a diet on patients with cystic fibrosis. ACKNOWLEDGEMEKTS
I am indebted to Professor D. V. Hubble for permission to study patients under his care, and to Dr A. M. H. MacMahon and the late Harold B. Salt for organising the collection of specimens. REFERENCES I 2 3 4 5 6 7
T. R. G. S. S. K. R.
Clin.
1. BONHAM, R. ROBINSON AND A. M. H. MACMAHON, Lancet, ii (1965) -, 1188 W. BONSNES AND H. H. TAUSSKY, J. Biol. Cham., 158 (1945) 581. A. HILL, J, RATCLIFFE AND P. SMITH, Chem & Ind. (London), (1959) 399 Z. LEVINE, M. DANN AND E. MARPLES, J. Clin. Invrst., 22 (1943) 551. HALVORSEN AND L. R. GJESSING, Brif. Med. J., ii (1964) 1171. TANAGIJCHI AND L. R. GJESSING,&it. Med. J., i (1965) 968. ROBINSON, Nature, 194 (1962) 879. Chim.
Acta,
14 (1966) 166-170
CLINICA CHIMICA ACTA
TYROSINE
METABOLISM
IN CYSTIC
FIBROSIS
R. ROBINSON
SUMMARY
Children with cystic fibrosis excrete in the urine excessive amounts of p-hydroxyphenylacetic acid (PHPA) , and p-hydroxyphenyllactic acid (PHPL) . These findings are in accord with the hypothesis that there is a block in tyrosine catabolism in these patients due to a deficiency of P-hydroxyphenylpyruvate hydroxylase activity. There is no evidence that this deficiency is caused by a lack of ascorbic acid.
Children with cystic fibrosis have abnormally high serum tyrosine values l. Tyrosine could accumulate if a block occurred in one of the steps by which it is broken down. The serum tyrosine level might then be expected to rise to abnormal levels. Tyrosine is broken down in the liver. The catabolic pathways are well known. The metabolites formed are excreted in the urine. If there were a block in the catabolic pathway, the excretion of metabolites in the urine would be abnormal, and it might be possible to deduce the site of the block from the pattern of excretion of these metabolites. Urine specimens from children with cystic fibrosis have been examined for the phenolic acid metabolites of tyrosine to test the hypothesis that there might be a block in tyrosine catabolism in these patients. MATERIAL
AND
METHODS
Urine specimens were obtained from 17 patients with cystic fibrosis. The patients all attended an out-patient’s clinic and the specimens had been collected at home by the parent before attending the clinic. On being received, the specimens were acidified with a few drops of concentrated hydrochloric acid (to pH approximately 4). They were then stored in a refrigerator at 4” until they were examined. This may have caused the loss of some p-hydroxyphenylpyruvic acid. The creatinine content of the urine was determined by either the standard Technicon AutoAnalyzer method or by the method of Bonsnes and TausskyZ. The aromatic metabolites of tyrosine were extracted by the method of Hill et d3: 3 ml of acidified urine was pipetted into a flask and 6 g of anhydrous sodium Cdin.Chim. Acta, ~4 (x966)
166-170
TYROSINE
sulphate acetate
167
METABOLISM
added. added.
The cake
of sodium
sulphate
The flask was then stoppered
was broken and placed
up and 15 ml of ethyl in the refrigerator
for an
hour. During this time the flask was occasionally taken out and the cake of sodium sulphate again broken up if necessary, using a glass rod. An aliquot of the ethyl acetate extract equivalent to the volume of urine containing I mg of creatinine was removed. This was evaporated to dryness under reduced pressure. The extract was applied to a IO-inch square of Whatman No. I filter paper to form a spot less than I cm in diameter at a distance of about an inch from two adjacent sides of the paper. The transference of the extract to the paper was made with small amounts mitted
of ethyl
acetate.
The extracted
phenolic
acids were than
to two-dimensional chromatographic separation. The first solvent system was isopropanol-0.880 ammonia-water
lowed by n-butanol-pyridine-water
(70 : 15 :15). The chromatograms
sub-
(80 :IO : IO) fol-
were allowed to
develop for about 16 h in each solvent system. After drying, the chromatograms were sprayed with 10% sodium carbonate solution and allowed to dry again. They were then sprayed with diazotised sulphanilic acid (the Van den Bergh reagent). Using this technique, $-hydroxyphenyllactic acid (PHPL) is scarcely detectable in normal urine; p-hydroxyphenylacetic acid (PHPA) can be detected in small amounts almost invariably. Specimens of urine were examined from four patients before and after hydrolysing by boiling with 15% (w /v ) o f cont. HCl. One cannot rely on the accuracy of timed collections
of urine obtained
from children in their homes. The chromatograms
were therefore prepared from the volume of urine containing I mg of creatinine. In comparing one urine with another one, the basis of an equal creatinine content is not ideal, but it is probably
the best method
available
at present.
However,
must be recognised that creatinine excretion is more variable in children adults. For this reason no attempt was made to determine quantitatively cretion of any of the tyrosine metabolites.
it
than in the ex-
The chromatograms were compared visually with chromatograms prepared from the urine of normal children. Where there was no obvious difference from normal in the rate of excretion of a particular metabolite, this is indicated in Table I by “N”. When the excretion indicated by “+“.
of a metabolite
was obviously
much greater
than normal
this is
The effect of ascorbic acid administration was examined on 4 patients. A control specimen of urine was collected and then the patient was given IOO mg of ascorbic acid by mouth twice a day for two weeks. At the end of this time, a second specimen of urine was collected for examination. Two patients’ urines were also examined after they had been taking
ascorbic
acid for four weeks.
RESULTS
These are summarised in Table I. Individual patients’ excretion of both PHPL and PHPA seemed to vary only slightly from day to day. Most of the patients’ urines were examined on several occasions but only the result on the first specimen is shown in the table, except for the patients who were treated with ascorbic acid. All the patients excreted excessive amounts of PHPA; 6 of the 17 patients also excreted increased amounts of PHPL. Clin. China. Acta,
14 (1966) 166-170
168 TABLE
R.ROBINSON I
EXCRETION _____.~
OF
TYROSINE
METABOLITES
BY
PATIENTS
WITH
CYSTIC
Patient
PHPL* excreted PHPA ~~______ ~~ --~
1. Control
-t + N N iN R: N N N N N
I. 2. 2. 2. 3. 3. 4. 4. 4. 5. 6. ;. 8. 9.
after 2 Control after 2 after 4 Control after z Control after 2 after 4
weeks on ascorbic
acid
weeks on ascorbic weeks on ascorbic
acid acid
weeks on ascorbic acid weeks on ascorbic weeks on ascorbic
acid acid
N’ N
IO.
N
II.
T
12.
N
13. 14. ‘5. 16. ‘7.
?j i!_
FIBROSIS
* * excwted
.~~
i1. ; + $-L t + i I T + A
iN
*PHPL = p-hydroxyphenyllactic acid. **PHPA = P-hydroxyphenylacetic acid. N indicates excretion is normal. + indicates excretion is abnormally high.
It is known that ascorbic acid deficiency inhibits the breakdown of tyrosine4. The possibility that our patients were excreting increased amounts of PHPL and PHPA because they were ascorbic acid-deficient was tested by examining their urine before and after they had been saturated with ascorbic acid as described above. In no case did the excretion of either PHPL or PHPA fall after the patient had been saturated with ascorbic acid; one patient excreted rather more PHPL after he had been saturated with ascorbic acid than he did before. Four specimens of urine were examined before and after acid hydrolysis. More PHPL and PHPA were found in the hydrolysed than the unhydrolysed specimens. This indicates that both acids are excreted to some extent in conjugated form. The hydrolysed urines were also examined for the hydantoin of p-hydroxyphenylpyruvic acid. This was identified but it was not possible to say whether it was present in increased amounts, as the urines were not examined immediately after they were collected and p-hydroxyphenylpyruvic acid is not stable. DISCUSSIOlr;
The outstanding findings were that all the patients with cystic fibrosis excreted excessive amounts of PHPA and 6 of the 17 patients also excreted raised amounts of PHPL. These results would be expected if the patients suffered from a deficiency of p-hydroxyphenylpyruvate hydroxylase activity. Reaction (2) (Fig. I) would then be blocked, p-hydroxyphenylpyruvic acid would accumulate, and the pathways leading Clin. Chim. Acta,
14 (1966) 166-170
TYROSINE
METABOLISM
1%
to the formation of PHPL and PHPA would be utilized to a much greater extent than in normal individuals. The cystic fibrosis patients bear some resemblance to the case of tyrosinosis described by Halvorsen and Gjessir@. However, it is evident that their patient excreted very much larger amounts of PHPL and PHPA than our cystic fibrosis patients. Tanaguchi and Gjessing8 examined liver and kidney tissue from a patient with tyrosinosis and found that p-hydroxyphenylpyruvate hydroxylase activity was absent from both tissues.
p-hydroxyphenylacetic
acid
t (5)
(==J’yy==JyH HO Tyrosine
Hflo; HO p-hydroxyphenylpyruvic
H4”o;
OH 2-5 dihydroxyphenylpyruvic acid
(6) 11 acid
OH Homogentisic
~~“ption
acid
CH2CH(OH)COOH
/ 0\ HO p-hydrcxyphenyllactic Fig.
I.
Catabolism
acid
of tyrosine.
It seems likely that patients with cystic fibrosis suffer from a lack of some essential co-factor rather than from complete absence of the enzyme. It is known that $-hydroxyphenylpyruvate hydroxylase requires ascorbic acid as a co-factor 4. However, when four of our patients were saturated with ascorbic acid, the amounts of PHPL and PHPA they excreted remained unchanged. A lack of dietary ascorbic acid did not therefore cause the increased excretion of phenolic acids. The pattern of excretion of tyrosine metabolites in the patients with cystic fibrosis resembled that seen in patients with parenchymatous liver damage7. However, though liver damage occurs in patients with cystic fibrosis, it is not believed to be so universal as the urinary phenolic acid results would suggest. It may be concluded that patients with cystic fibrosis metabolise tyrosine abnormally. The abnormality seems to be due to a deficiency in $-hydroxyphenylpyruvate hydroxylase activity, the cause of which is at present unknown. Halvorsen and Gjessing& considered that a high level of circulating tyrosine and its metabolites might have a toxic effect on renal tubular cells and possibly on liver cells. They found that their patient with tyrosinosis benefited from a diet low Gin.
Chim. Acta,
14 (1966)
166-170
R. ROBINSON
170
in tyrosine and phenylalanine. It might be worthwhile carrying out a trial of such a diet on patients with cystic fibrosis. ACKNOWLEDGEMEKTS
I am indebted to Professor D. V. Hubble for permission to study patients under his care, and to Dr A. M. H. MacMahon and the late Harold B. Salt for organising the collection of specimens. REFERENCES I 2 3 4 5 6 7
T. R. G. S. S. K. R.
Clin.
1. BONHAM, R. ROBINSON AND A. M. H. MACMAHON, Lancet, ii (1965) -, 1188 W. BONSNES AND H. H. TAUSSKY, J. Biol. Cham., 158 (1945) 581. A. HILL, J, RATCLIFFE AND P. SMITH, Chem & Ind. (London), (1959) 399 Z. LEVINE, M. DANN AND E. MARPLES, J. Clin. Invrst., 22 (1943) 551. HALVORSEN AND L. R. GJESSING, Brif. Med. J., ii (1964) 1171. TANAGIJCHI AND L. R. GJESSING,&it. Med. J., i (1965) 968. ROBINSON, Nature, 194 (1962) 879. Chim.
Acta,
14 (1966) 166-170