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Cystinuria with hyperuricemia and methioninuria
Case Reports Cystinuria with Hyperuricemia
and
Met hioninuria* Biochemical J.
KING,
STANTON
JR.,
Study of a Case
PH.D.t
Winston-Salem,
and ARTHUR
WAINER,
PH.D.
North Carolina
An otherwise typical cystinuric subject manifested several seemingly innocuous additional biochemical abnormalities: hyperuricemia, methioninuria and several serum amino acids in abnormal amounts. In contrast to findings reported in classic cystinuria, urinary cystine was decreased by homocysteine loading and temporarily decreased, followed by only a small increase after methionine loading. The response to taurine loading was atypical. Pyridoxine hydrochloride administration tended to correct some of the serum amino acid aberrations. Radiomethionine and radiotaurine administration revealed no absolute defect in the pathway between methionine and the metabolites of taurine. The response to penicillamine treatment was like that of the usual cystinuric patient. During methionine loading, urinary methionine was decreased, despite elevated serum levels, indicating that the methioninuria is not the result of a tubular defect.
T
HIS report cystine
uricemia
and
plications other
cases
not
of in
patients
of cystinuria
during
with or
Hambraeus
were their
made short
are recorded
by
The either
laboratory
by
which
hospitalization
seen
cystinuria
this
reported
servations
a case
accompanied
methioninuria.
were
examined cases
describes lithiasis
on
latter in
the
comtwelve
cystine the
stones
fifty-three
[7].
Some
this
and
periods
cept for congenital absence of the right and left upper lateral incisors. At age twenty-four a stone, thought to be composed of uric acid, was removed from her right kidney. No further urologic disorder was manifested until February 1963 when a 1.5 cm. radiolucent stone, also considered to be a uric acid stone, was removed from the right kidney pelvis. The left kidney was normal. An alkalinizing diet was instituted but in January 1964 it was again necessary to remove a 1.5 cm. stone, presumed to be composed of uric acid, from the left kidney pelvis. Adherence to the alkalinizing diet was encouraged. In October 1965 a 1 cm. and a 5 mm. stone were seen on a roentgenogram and, because of rapid enlargement, were removed in January 1966. The larger (2 gm.) stone was competently analyzed and found to be composed of 95 per cent cystine and 5 per cent apatite. At this time the stones recovered in 1958 and 1964 were also analyzed and found to consist of cystine. Serum uric acid, measured elsewhere on several occasions during the preceding two years, had been
and hyper-
obother
of elective
here.
C&&SE HISTORY Mrs. J.D.M. (NCBH No. 43 02 45) was referred to the North Carolina Baptist Hospital in March 1966. She was a thirty-two year old white woman, intelligent and cooperative, the mother of two normal children, eight and eleven years of age. Except for her urologic difficulties she was in good health. There was no history of abortion or family history of urolithiasis. Physical examination was unrevealing ex-
* From the Departments of Urology and Biochemistry, The Bowman Gray School of Medicine of Wake Forest College, Winston-Salem, North Carolina. This work was supported by Grants AM-08050 and 07233 from the U.S. Public Health Service, and by the John A. Hartford Foundation. Manuscript received July 19, 1966. t Recipient of a Research Career Development Award from National Institutes of Health. VOL.
43,
JULY
1967
125
Cystinuria,
126
TABLE ABERRANT
Hyperuricemia I
AMINO
ACIDS IN CONTROL
Amino Acid
Subject
TatI Cys Met Cys-hornocys 0-n Lys (+ I-MeHis) Arg
and
URINE
9.3 752 96t 110 271 1,960 431
(M~./DAY)
Normal * 87 6 5 <2 2 180 4
* Average for normal women, from Soupart [24]. t 139 three days after going off penicillamine.
reported to be between 5.5 and 9.0 mg. per cent. Urinary uric acid excretion was 550 mg. per day. On the present admission, the serum and urine uric acid [Z] were 8.2 mg. per cent and 520 mg. per day, respectively. For adult women, the corresponding normal values in our clinical laboratory are 4.95 + 0.11 (S.D.) and 412 f 69, respectively, in agreement with others [3,4]. Results of other routine clinical laboratory studies were normal, as was serum immunoelectrophoresis. BIOCHEMICAL
Methioninuria-King,
Urine was collected at zero, four, eight and twelve hours, and plasma at one and a half hours after the dose. There were significant changes in the urinary aminograms (Table II). The excretion rate for methionine decreased sharply; cystine excretion decreased during the first four hours and was followed by a small increase. Taurine excretion increased at first, then declined to its control value. Homocysteine-cysteine mixed disulfide excretion gradually increased to three times the control value. A peak corresponding to methionine sulfoxide (which appears almost coincidentally with aspartic acid) was increased in the aminogram of the four to eight hour urine [7], as were peaks corresponding to cystathionine and peaks No. 49,60 and 62 [8]; the latter are unidentified, 0
D
STUDIES
A control twenty-four hour urine and a nonfasting plasma sample, promptly deproteinized, were examined with the amino acid analyzer. A typical cystinuric urinary aminogram [I] was obtained (Table I) except that an abnormally high peak eluting at the methionine position and equivalent to 96 mg. methionine per day was found (normal: 3 to 12 mg.) (/3-Aminoisobutyric acid was elevated two- to threefold, but this was true also of the patient’s mother, sister, both children and maternal grandmother and thus is apparently familial and nonspecific [5].) Serum methionine was 52 micromoles (PM) per L., approximately twice normal. The patient was given 0.5 PC. of S35-taurine per kg. body weight, for later estimation of taurine metabolites [6]. On the four following days, 140 mg. taurine was given each morning and evening (about 5 mg. per kg. per day). The urinary response is illustrated in Figure 1, together with the response of normal subjects and that of treated and untreated cystinuric subjects. On the morning after completing the taurine load, breakfast was omitted and a control plasma sample obtained. Five grams of Lmethionine was given orally, in a single dose.
Wainer
0
I
I11,, , , , 3
0
++01234 A
8
(
,
,
,
,
E
6
7
8.
c Days on taurine load (280 mglday)
FIG. 1. Urinary taurine response to oral taurine load in normal subjects (I, average of four), cystinuric subjects who had been receiving penicillamine treatment until day before beginning of test (II, average of two), or not on treatment (III, average of two subjects), as compared with the present case (IV). All averages are fairly representative of individual values. At A, pretreatment value for classic cystinuric subjects; B, value after several weeks of penicillamine treatment (discontinued day or two before taurine load test begin). At C, penicillamine treatment begun in present case, taurine administration discontinued. AMERICAN
JOURNAL
OF
MEDICINE
Cystinuria,
Hyperuricemia
TABLE CHANGES
SEEN IN SOME URINARY
AMINO AND
ACIDS
AFTER
CLASSIC
ORAL
Taurine
II
ADMINISTRATION
CYSTINURIC
127
Wuiner
and Methioninuria-King,
(B)
PATIENTS
OF
5
GM.
METHIONINE
IN METHIONINURIC
(A)
(MG./HR.)
CysteineHomocysteine
Methionine
Cystine
Time of Determination
A
B
A
B
A
B
A
B
Control O-4 hr. 4-8 hr.1 8-12 hr.
0.38 1.70 1.09 0.62
7.12* 4.66 3.09 3.71
31.30 22.15 25.60 36.65
29.70 24.85 32.00 35.10
4.00 0.63 1.04 0.56
0.34 1.06 5.59 0.42
4.58 5.59 15.75 18.30
2.83 3.70 9.31 10.52
* See text. t Also, peaks corresponding to methionine sulfoxide, cystathionine and peaks No. 49, 60, 62 [8] increased in subject A; only cystathionine in subject B.
but methionine oligopeptides commonly emerge in this interval [9]; the latter two in fact happen to correspond in elution behavior to methionylmethionine and methionylmethionylmethionine. Moreover, these unidentified peaks were removed by acid hydrolysis of the urine. Homoserine, S-(1,2_dicarboxyethyl) cysteine [ 701, a-aminobutyric acid [7] and serine peaks in these urines were normal. The peak corresponding to homocystine was clearly elevated, but not measurable because of coincidence of other compounds [8]. The plasma methionine level at one and a half hours was tenfold the control value, indicating good absorption. For comparison, Table II also shows the results obtained in the same way with a second cystinuric subject, who was “classic” except for the fact that he consistently excreted normal amounts of taurine; this is the only cystinuric patient we have seen who does so.
Abnormalities in the previously mentioned plasma samples are shown in Table III. Because the column was operated at 60’~. some of the glutamic acid was artifactually derived from glutamine; unaltered glutamine was not separated from serine. Glutamic acid and cystine were subnormal, whereas threonine, serine and alanine were high. o-Penicillamine (Cupriminea; Merck, Sharp and Dohme) was next administered in four 500 mg. doses daily [ 7 I]. The response of urinary taurine to penicillamine dosage was typical of classic cystinuria (Fig. 1). After dosage for four days, the twenty-four hour urine contained 222 mg. cystine, 222 mg. methionine and 30 mg. homocysteine-cysteine. Values for the other amino acids were unchanged from the control value. Methionine could be measured after treating 3 ml. of this urine (adjusted to pH 7.8) with 0.2 ml. 2-mercaptoethanol, allowing
TABLE ABERRANT
PLASMA
AMINO
ACIDS
AND
EFFECTS
OF METHIONINE
III LOAD
AND
OF PYRIDOXINE
HYDROCHLORIDE
LOAD
Bg 1% Hours After Methionine
and Penicillamine
13
231
61
f
sert
Cys/2 28
30 52
* Values obtained in this laboratory. t 1 Obscured by cysteine-penicillamine J Value unreliable; plasma not promptly deproteinized. VOL.
43,
JULY
1967
89 jz 31 f 36 f 29 f 58 * 133 zt 25 23xlz 5
128
Cystinuria,
z k v) $ Q $ 0
0.6
:
0.2
Hyperuricemia
and Methioninuria-King,
Wainer
0.4 0.3
0
FIG. 2. Portion of urine aminogram, showing (above) chromatographic peaks for cystine (C), penicillamine-cysteine (PC), methionine (M) and homocysteinecysteine (HC) and the corresponding radioactivity detected (below) after administration of S”-methionine. The specific activity of the cystine was 20 per cent that of the methionine. The sample used was collected two to four hours after the administration of Sz-methionine.
the mixture to stand for ten minutes and diluting it with pH 2.88 citrate starting buffer for sampling. This treatment removed penicillaminecysteine mixed disulfide, which under the conditions used here [8] emerges from the column coincidentally with methionine, as well as penicillamine-penicillamine disulfide, homocysteine-cysteine mixed disulfide, homocysteine, cystine and peak No. 60. Excess mercaptoethanol emerged just after urea (seventy-two minutes). cysteine” The “total excretion (cysteine in cystine and penicillamine-cysteine) was then calculated; it was 77 per cent of the control value. The total recovery of penicillamine was 34.2 per cent. These figures are in good agreement with values obtained in classic cystinuric patients [ 771. The patient was then discharged, but after a total of six days on penicillamine a minor allergic rash developed from the drug, the administration of which was stopped; she was readmitted one week after discharge. An allergist began a graduated desensitizing course of penicillamine, starting at 25 mg. four times a day. The urinary aminogram was similar to the control, except for a relatively small penicil-
lamine-cysteine peak. At this time the taurine nonsulfate metabolite radioactivity [6] was severalfold that of the urinary taurine, which amounted to 14 mg. per day. On the following morning 100 bc. of S35methionine (plus 20 mg. cold carrier methionine) was given orally and urine specimens were collected every two hours throughout the day, plus an overnight specimen. Representative results of radiochromatography of the specimens are shown in Figure 2, which demonstrates active synthesis of S35-cystine from the administered S35-methionine. On the following day, 2 gm. m-homocysteine thiolactone hydrochloride was given in three equal doses, and the corresponding twenty-four hour urine was collected and examined chromatographically to determine whether the patient could substitute this compound for penicillamine, should she remain sensitive to the latter, and also to determine the response to homocysteine loading. The following pertinent values were found (milligrams per day): taurine 20, cystine 557 and homocysteine-cysteine 337 ; homocystine was elevated but technically not measurable. Homocysteine emerges between AMERICAN
JOURNAL
OF
MEDICINE
Cystinuria,
Hyperuricemia
and Methioninuria-King,
cystine and methionine, and was not seen here (about two-thirds of a 1.59 PM sample of homocysteine free base, run separately, appeared as homocystine, the time interval between the two being similar to that for cysteine and cystine). The “total cysteine” excretion was thus 752 mg., a value equal to the control value; nonetheless, cystine excretion was lowered 200 mg. in one day of treatment. Apparently for dietary reasons, the pH of the patient’s urine was generally between 6 and 7 during this study, facilitating the formation of mixed disulfide. The same dosage was used for four days with a second cystinuric subject (the same person mentioned previously) without altering his output of cystine, although his cysteine-homocysteine excretion increased from 67 to 340 to 400 mg. per day. On discharge six days after her readmission the patient was tolerating and continued to tolerate therapeutic doses of penicillamine. At home she took 50 mg. of pyridoxine hydrochloride each day for the next eight days and on the last day submitted a plasma specimen. The purpose was to determine what effect, if any, this would have on the abnormally high serum threonine and serine levels; it seemed possible that the pyridoxal phosphate-dependent “dehydrases” and desulfhydrases responsible for conversion of certain amino acids to their corresponding keto-acids [ 721 might be deficient and that this deficiency could be corrected by the administration of relatively large doses of pyridoxine, by analogy with certain other disorders [ 73- 751 in which the affinity of apoenzyme for coenzyme is apparently low and which are correctable with relatively massive doses of vitamin Bc [ 761. The plasma threonine was lower and the serine plus glutamine and glutamic acid peaks were within the normal range after this regimen (Table III). Two relatively large peaks were present in the phenylalanine and homocystine position, not actually identified, but respectively equivalent to 262 PM phenylalanine per L. (normal: 48) and 341 PM homocystine 2 per L. (normal: 0). None of the amino acids was quantitatively abnormal in plasma from another cystinuric subject who had been taking 1.5 gm. penicillamine and 25 mg. pyridoxine hydrochloride per day for several months. Morning specimens of urine from the patient’s parents, two children, sister and three of her four grandparents were chromatographed. VOL.
43,
JULY
1967
Wainer
129
All gave a normal pattern (except for flaminoisobutyric acid, previously mentioned, and in the case of the sister a high output of glutamine, accompanied by a urine pH of 7.1). COMMENTS
In the patient here described there was an atypical response to taurine loading, and also a decrease in cystine excretion after both methionine loading (during the first hours) and homocysteine loading, both of which reportedly increase cystine excretion in classic cystinuria [ 77-201. The decreased plasma serine after methionine loading probably reflects its increased use in cystathionine synthesis. The patient is capable of rapidly making cysteine from methionine [27] at a rate quite comparable to that for another cystinuric subject similarly studied. Her (nonloaded) production and further metabolism of taurine is also typical of cystinuria. Much of the urinary cystine evidently derives from methionine, rather than from ingested cystine [ 77,791; there is no radiochemical evidence here of impediments in any of the metabolic steps between the two. The serum serine, threonine and glutamic acid abnormalities were evidently rectified to some extent by the administration of pyridoxine hydrochloride, although this did not benefit the patient in any clinically obvious way. The cystinuria was of the completely recessive type I [22,23]. The studies on the urine of family members provided no evidence for or against heritability of the methioninuria. No basis for the increased urinary methionine has been found. Acknowledgment: We thank Drs. W. H. Boyce and George Drach for clinical cooperation and Dr. S. Mizuhara, Okayama University Medical School, for an authentic sample of S-(1,2-dicarboxyethyl) cysteine. REFERENCES
1. (a) HAMBRAEUS, L. Cystinuria in Sweden. x. Quantitative study of the urinary amino acid excretion in cystinurics. Acta SIX. med. upsalien., 69: 1, 1964. (b) HAMBRAEUS, L. Personal communication. 2. BITTNER, D. L., HALL, S. G. and MCCLEARY, M. L. A method for determination of uric acid, using the cupric phenanthroline indicator system. Am. J. Clin. Path., 40: 423, 1963. 3. RYCKEWAERT, A., DRY, J., PAOLAGGI, F. and LELLOUGH, J. Taux de I’uricCmie dans la population
130
4.
5. 6.
7. 8.
9.
10.
11. 12.
13.
14.
Cystinuria,
Hyperuricemia
and Methioninuria-King,
g&n&ale. Quelques corr&tions de I’uricCmie. Reu. Franc. itad. clin. et biol., 11 : 72, 1966. MIKKELS~EIN,W. M., DODGE, H. J., EPSTEIN,F. H., VALXENBURG, H. and DUFF, I. F. Serum mate levels in a normal population sample. J. Lab. & Clin. Med., 60: 999, 1962. MEISTER, A. Biochemistry of the Amino Acids, vol. 2, p. 1035. New York, 1965. Academic Press. WAINER, A., KING, J. S., JR., GOODMAN, H. 0. and THOMAS, J. J. Sss taurine metabolism in normal and mongoloid individuals. Proc. SW. Expm. Biol. &? Med., 121: 212, 1966. DENT, C. E. Methionine metabolism and alphaaminobutyric acid. Science, 105: 335, 1947. KING, J. S. JR. Observations on the ninhydrinpositive substances in human urine. Clin. chim. acta, 9: 441, 1964. KING, J. S. JR. and WAINER, A. Column chromatography of some methionine peptides. J. Chromatography, 24: 215, 1966. KUWAKI, T. and MIZUHARA, S. S-(1,2-dicarboxyethyl)-cysteine in urine and kidney. Biochim. et biophys. acta, 115: 491, 1966. KING, J. S. JR. and BOYCE, W. H. Effect of penicillamine on cystinuria. Inuest. Ural., 2: 595, 1965. MEISTER, A. Biochemistry of the Amino Acids, vol. 2, pp. 668, 679, 793. New York, 1965. Academic PreSS. GERSCHOFF,S. N., MAYER, A. L. and KULCZYCKI, L. L. Effect of pyridoxine administration on the urinary excretion of oxalic acid, pyridoxine, and related compounds in mongoloids and nonmongoloids. Am. J. Clin. Nutrition, 7: 76, 1959. HACBERG, B., HAMFELT, A. and HANSSON, 0. Tryptophan-tolerance tests and vitamin Bs (pyri-
15.
16. 17.
18.
19.
20.
21.
22. 23.
24.
Wainer
doxine) metabolism in epileptic children. Develop. Med. G3 Child Neural., 7: 50, 1965. HEELEY, A. F. and ROBERTS, G. E. Tryptophan metabolism in psychotic children. Develop. Med. @ Child Neural., 7: 46, 1965. SCRIVNER,C. R. Vitamin Bs-dependency and infantile convulsions. Pediatrics, 26: 62, 1960. BRAND, E., CAHILL, G. F. and HARRIS, M. M. Cystinuria. II. The metabolism of cystine, cysteine, methionine and glutathione. J. Biol. Chem., 109: 69, 1935, BRAND, E., CAHILL, G. F. and BLOCK, R. J. Cystinuria. Iv. The metabolism of homocysteine and homocystine. J. Biol. Chem., 110: 399, 1935. LEWIS, H. B., BROCVN,B. H. and WHITE, F. R. The metabolism of sulfur. XXIII. The influence of the ingestion of cystine, cysteine, and methionine on the excretion of cystine in cystinuria. J. Biol. Chem., 114: 171,1936. SMITH, D. R., KOLB, F. 0. and HARPER, H. A. The management of cystinuria and cystine-stone disease. J. Ural., 81: 61, 1959. REED, L. J., CAVALLINI, D., PLUM, F., RACHELE, J. R. and DU VIGNEAUD, V. The conversion of methionine to cystine in a human cystinuric. J. Biol. Chem., 180: 783, 1949. HARRIS, H. and ROBSON, E. B. Cystinuria. ‘4m. J. Med., 22: 774, 1957. ROSENBERG,L. E., DOWNING,S., DURANT, J. L. and SEGAL, S. Cystinuria: biochemical evidence for three genetically distinct diseases. J. Gin. Znuest., 45: 365, 1966. SOUPART, P. Urinary excretion of free amino acids in normal adult men and women. Clin. chim. acta, 4: 265, 1959.
AMERICAN
JOURNAL
OF
MEDICINE
and
Met hioninuria* Biochemical J.
KING,
STANTON
JR.,
Study of a Case
PH.D.t
Winston-Salem,
and ARTHUR
WAINER,
PH.D.
North Carolina
An otherwise typical cystinuric subject manifested several seemingly innocuous additional biochemical abnormalities: hyperuricemia, methioninuria and several serum amino acids in abnormal amounts. In contrast to findings reported in classic cystinuria, urinary cystine was decreased by homocysteine loading and temporarily decreased, followed by only a small increase after methionine loading. The response to taurine loading was atypical. Pyridoxine hydrochloride administration tended to correct some of the serum amino acid aberrations. Radiomethionine and radiotaurine administration revealed no absolute defect in the pathway between methionine and the metabolites of taurine. The response to penicillamine treatment was like that of the usual cystinuric patient. During methionine loading, urinary methionine was decreased, despite elevated serum levels, indicating that the methioninuria is not the result of a tubular defect.
T
HIS report cystine
uricemia
and
plications other
cases
not
of in
patients
of cystinuria
during
with or
Hambraeus
were their
made short
are recorded
by
The either
laboratory
by
which
hospitalization
seen
cystinuria
this
reported
servations
a case
accompanied
methioninuria.
were
examined cases
describes lithiasis
on
latter in
the
comtwelve
cystine the
stones
fifty-three
[7].
Some
this
and
periods
cept for congenital absence of the right and left upper lateral incisors. At age twenty-four a stone, thought to be composed of uric acid, was removed from her right kidney. No further urologic disorder was manifested until February 1963 when a 1.5 cm. radiolucent stone, also considered to be a uric acid stone, was removed from the right kidney pelvis. The left kidney was normal. An alkalinizing diet was instituted but in January 1964 it was again necessary to remove a 1.5 cm. stone, presumed to be composed of uric acid, from the left kidney pelvis. Adherence to the alkalinizing diet was encouraged. In October 1965 a 1 cm. and a 5 mm. stone were seen on a roentgenogram and, because of rapid enlargement, were removed in January 1966. The larger (2 gm.) stone was competently analyzed and found to be composed of 95 per cent cystine and 5 per cent apatite. At this time the stones recovered in 1958 and 1964 were also analyzed and found to consist of cystine. Serum uric acid, measured elsewhere on several occasions during the preceding two years, had been
and hyper-
obother
of elective
here.
C&&SE HISTORY Mrs. J.D.M. (NCBH No. 43 02 45) was referred to the North Carolina Baptist Hospital in March 1966. She was a thirty-two year old white woman, intelligent and cooperative, the mother of two normal children, eight and eleven years of age. Except for her urologic difficulties she was in good health. There was no history of abortion or family history of urolithiasis. Physical examination was unrevealing ex-
* From the Departments of Urology and Biochemistry, The Bowman Gray School of Medicine of Wake Forest College, Winston-Salem, North Carolina. This work was supported by Grants AM-08050 and 07233 from the U.S. Public Health Service, and by the John A. Hartford Foundation. Manuscript received July 19, 1966. t Recipient of a Research Career Development Award from National Institutes of Health. VOL.
43,
JULY
1967
125
Cystinuria,
126
TABLE ABERRANT
Hyperuricemia I
AMINO
ACIDS IN CONTROL
Amino Acid
Subject
TatI Cys Met Cys-hornocys 0-n Lys (+ I-MeHis) Arg
and
URINE
9.3 752 96t 110 271 1,960 431
(M~./DAY)
Normal * 87 6 5 <2 2 180 4
* Average for normal women, from Soupart [24]. t 139 three days after going off penicillamine.
reported to be between 5.5 and 9.0 mg. per cent. Urinary uric acid excretion was 550 mg. per day. On the present admission, the serum and urine uric acid [Z] were 8.2 mg. per cent and 520 mg. per day, respectively. For adult women, the corresponding normal values in our clinical laboratory are 4.95 + 0.11 (S.D.) and 412 f 69, respectively, in agreement with others [3,4]. Results of other routine clinical laboratory studies were normal, as was serum immunoelectrophoresis. BIOCHEMICAL
Methioninuria-King,
Urine was collected at zero, four, eight and twelve hours, and plasma at one and a half hours after the dose. There were significant changes in the urinary aminograms (Table II). The excretion rate for methionine decreased sharply; cystine excretion decreased during the first four hours and was followed by a small increase. Taurine excretion increased at first, then declined to its control value. Homocysteine-cysteine mixed disulfide excretion gradually increased to three times the control value. A peak corresponding to methionine sulfoxide (which appears almost coincidentally with aspartic acid) was increased in the aminogram of the four to eight hour urine [7], as were peaks corresponding to cystathionine and peaks No. 49,60 and 62 [8]; the latter are unidentified, 0
D
STUDIES
A control twenty-four hour urine and a nonfasting plasma sample, promptly deproteinized, were examined with the amino acid analyzer. A typical cystinuric urinary aminogram [I] was obtained (Table I) except that an abnormally high peak eluting at the methionine position and equivalent to 96 mg. methionine per day was found (normal: 3 to 12 mg.) (/3-Aminoisobutyric acid was elevated two- to threefold, but this was true also of the patient’s mother, sister, both children and maternal grandmother and thus is apparently familial and nonspecific [5].) Serum methionine was 52 micromoles (PM) per L., approximately twice normal. The patient was given 0.5 PC. of S35-taurine per kg. body weight, for later estimation of taurine metabolites [6]. On the four following days, 140 mg. taurine was given each morning and evening (about 5 mg. per kg. per day). The urinary response is illustrated in Figure 1, together with the response of normal subjects and that of treated and untreated cystinuric subjects. On the morning after completing the taurine load, breakfast was omitted and a control plasma sample obtained. Five grams of Lmethionine was given orally, in a single dose.
Wainer
0
I
I11,, , , , 3
0
++01234 A
8
(
,
,
,
,
E
6
7
8.
c Days on taurine load (280 mglday)
FIG. 1. Urinary taurine response to oral taurine load in normal subjects (I, average of four), cystinuric subjects who had been receiving penicillamine treatment until day before beginning of test (II, average of two), or not on treatment (III, average of two subjects), as compared with the present case (IV). All averages are fairly representative of individual values. At A, pretreatment value for classic cystinuric subjects; B, value after several weeks of penicillamine treatment (discontinued day or two before taurine load test begin). At C, penicillamine treatment begun in present case, taurine administration discontinued. AMERICAN
JOURNAL
OF
MEDICINE
Cystinuria,
Hyperuricemia
TABLE CHANGES
SEEN IN SOME URINARY
AMINO AND
ACIDS
AFTER
CLASSIC
ORAL
Taurine
II
ADMINISTRATION
CYSTINURIC
127
Wuiner
and Methioninuria-King,
(B)
PATIENTS
OF
5
GM.
METHIONINE
IN METHIONINURIC
(A)
(MG./HR.)
CysteineHomocysteine
Methionine
Cystine
Time of Determination
A
B
A
B
A
B
A
B
Control O-4 hr. 4-8 hr.1 8-12 hr.
0.38 1.70 1.09 0.62
7.12* 4.66 3.09 3.71
31.30 22.15 25.60 36.65
29.70 24.85 32.00 35.10
4.00 0.63 1.04 0.56
0.34 1.06 5.59 0.42
4.58 5.59 15.75 18.30
2.83 3.70 9.31 10.52
* See text. t Also, peaks corresponding to methionine sulfoxide, cystathionine and peaks No. 49, 60, 62 [8] increased in subject A; only cystathionine in subject B.
but methionine oligopeptides commonly emerge in this interval [9]; the latter two in fact happen to correspond in elution behavior to methionylmethionine and methionylmethionylmethionine. Moreover, these unidentified peaks were removed by acid hydrolysis of the urine. Homoserine, S-(1,2_dicarboxyethyl) cysteine [ 701, a-aminobutyric acid [7] and serine peaks in these urines were normal. The peak corresponding to homocystine was clearly elevated, but not measurable because of coincidence of other compounds [8]. The plasma methionine level at one and a half hours was tenfold the control value, indicating good absorption. For comparison, Table II also shows the results obtained in the same way with a second cystinuric subject, who was “classic” except for the fact that he consistently excreted normal amounts of taurine; this is the only cystinuric patient we have seen who does so.
Abnormalities in the previously mentioned plasma samples are shown in Table III. Because the column was operated at 60’~. some of the glutamic acid was artifactually derived from glutamine; unaltered glutamine was not separated from serine. Glutamic acid and cystine were subnormal, whereas threonine, serine and alanine were high. o-Penicillamine (Cupriminea; Merck, Sharp and Dohme) was next administered in four 500 mg. doses daily [ 7 I]. The response of urinary taurine to penicillamine dosage was typical of classic cystinuria (Fig. 1). After dosage for four days, the twenty-four hour urine contained 222 mg. cystine, 222 mg. methionine and 30 mg. homocysteine-cysteine. Values for the other amino acids were unchanged from the control value. Methionine could be measured after treating 3 ml. of this urine (adjusted to pH 7.8) with 0.2 ml. 2-mercaptoethanol, allowing
TABLE ABERRANT
PLASMA
AMINO
ACIDS
AND
EFFECTS
OF METHIONINE
III LOAD
AND
OF PYRIDOXINE
HYDROCHLORIDE
LOAD
Bg 1% Hours After Methionine
and Penicillamine
13
231
61
f
sert
Cys/2 28
30 52
* Values obtained in this laboratory. t 1 Obscured by cysteine-penicillamine J Value unreliable; plasma not promptly deproteinized. VOL.
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JULY
1967
89 jz 31 f 36 f 29 f 58 * 133 zt 25 23xlz 5
128
Cystinuria,
z k v) $ Q $ 0
0.6
:
0.2
Hyperuricemia
and Methioninuria-King,
Wainer
0.4 0.3
0
FIG. 2. Portion of urine aminogram, showing (above) chromatographic peaks for cystine (C), penicillamine-cysteine (PC), methionine (M) and homocysteinecysteine (HC) and the corresponding radioactivity detected (below) after administration of S”-methionine. The specific activity of the cystine was 20 per cent that of the methionine. The sample used was collected two to four hours after the administration of Sz-methionine.
the mixture to stand for ten minutes and diluting it with pH 2.88 citrate starting buffer for sampling. This treatment removed penicillaminecysteine mixed disulfide, which under the conditions used here [8] emerges from the column coincidentally with methionine, as well as penicillamine-penicillamine disulfide, homocysteine-cysteine mixed disulfide, homocysteine, cystine and peak No. 60. Excess mercaptoethanol emerged just after urea (seventy-two minutes). cysteine” The “total excretion (cysteine in cystine and penicillamine-cysteine) was then calculated; it was 77 per cent of the control value. The total recovery of penicillamine was 34.2 per cent. These figures are in good agreement with values obtained in classic cystinuric patients [ 771. The patient was then discharged, but after a total of six days on penicillamine a minor allergic rash developed from the drug, the administration of which was stopped; she was readmitted one week after discharge. An allergist began a graduated desensitizing course of penicillamine, starting at 25 mg. four times a day. The urinary aminogram was similar to the control, except for a relatively small penicil-
lamine-cysteine peak. At this time the taurine nonsulfate metabolite radioactivity [6] was severalfold that of the urinary taurine, which amounted to 14 mg. per day. On the following morning 100 bc. of S35methionine (plus 20 mg. cold carrier methionine) was given orally and urine specimens were collected every two hours throughout the day, plus an overnight specimen. Representative results of radiochromatography of the specimens are shown in Figure 2, which demonstrates active synthesis of S35-cystine from the administered S35-methionine. On the following day, 2 gm. m-homocysteine thiolactone hydrochloride was given in three equal doses, and the corresponding twenty-four hour urine was collected and examined chromatographically to determine whether the patient could substitute this compound for penicillamine, should she remain sensitive to the latter, and also to determine the response to homocysteine loading. The following pertinent values were found (milligrams per day): taurine 20, cystine 557 and homocysteine-cysteine 337 ; homocystine was elevated but technically not measurable. Homocysteine emerges between AMERICAN
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Cystinuria,
Hyperuricemia
and Methioninuria-King,
cystine and methionine, and was not seen here (about two-thirds of a 1.59 PM sample of homocysteine free base, run separately, appeared as homocystine, the time interval between the two being similar to that for cysteine and cystine). The “total cysteine” excretion was thus 752 mg., a value equal to the control value; nonetheless, cystine excretion was lowered 200 mg. in one day of treatment. Apparently for dietary reasons, the pH of the patient’s urine was generally between 6 and 7 during this study, facilitating the formation of mixed disulfide. The same dosage was used for four days with a second cystinuric subject (the same person mentioned previously) without altering his output of cystine, although his cysteine-homocysteine excretion increased from 67 to 340 to 400 mg. per day. On discharge six days after her readmission the patient was tolerating and continued to tolerate therapeutic doses of penicillamine. At home she took 50 mg. of pyridoxine hydrochloride each day for the next eight days and on the last day submitted a plasma specimen. The purpose was to determine what effect, if any, this would have on the abnormally high serum threonine and serine levels; it seemed possible that the pyridoxal phosphate-dependent “dehydrases” and desulfhydrases responsible for conversion of certain amino acids to their corresponding keto-acids [ 721 might be deficient and that this deficiency could be corrected by the administration of relatively large doses of pyridoxine, by analogy with certain other disorders [ 73- 751 in which the affinity of apoenzyme for coenzyme is apparently low and which are correctable with relatively massive doses of vitamin Bc [ 761. The plasma threonine was lower and the serine plus glutamine and glutamic acid peaks were within the normal range after this regimen (Table III). Two relatively large peaks were present in the phenylalanine and homocystine position, not actually identified, but respectively equivalent to 262 PM phenylalanine per L. (normal: 48) and 341 PM homocystine 2 per L. (normal: 0). None of the amino acids was quantitatively abnormal in plasma from another cystinuric subject who had been taking 1.5 gm. penicillamine and 25 mg. pyridoxine hydrochloride per day for several months. Morning specimens of urine from the patient’s parents, two children, sister and three of her four grandparents were chromatographed. VOL.
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All gave a normal pattern (except for flaminoisobutyric acid, previously mentioned, and in the case of the sister a high output of glutamine, accompanied by a urine pH of 7.1). COMMENTS
In the patient here described there was an atypical response to taurine loading, and also a decrease in cystine excretion after both methionine loading (during the first hours) and homocysteine loading, both of which reportedly increase cystine excretion in classic cystinuria [ 77-201. The decreased plasma serine after methionine loading probably reflects its increased use in cystathionine synthesis. The patient is capable of rapidly making cysteine from methionine [27] at a rate quite comparable to that for another cystinuric subject similarly studied. Her (nonloaded) production and further metabolism of taurine is also typical of cystinuria. Much of the urinary cystine evidently derives from methionine, rather than from ingested cystine [ 77,791; there is no radiochemical evidence here of impediments in any of the metabolic steps between the two. The serum serine, threonine and glutamic acid abnormalities were evidently rectified to some extent by the administration of pyridoxine hydrochloride, although this did not benefit the patient in any clinically obvious way. The cystinuria was of the completely recessive type I [22,23]. The studies on the urine of family members provided no evidence for or against heritability of the methioninuria. No basis for the increased urinary methionine has been found. Acknowledgment: We thank Drs. W. H. Boyce and George Drach for clinical cooperation and Dr. S. Mizuhara, Okayama University Medical School, for an authentic sample of S-(1,2-dicarboxyethyl) cysteine. REFERENCES
1. (a) HAMBRAEUS, L. Cystinuria in Sweden. x. Quantitative study of the urinary amino acid excretion in cystinurics. Acta SIX. med. upsalien., 69: 1, 1964. (b) HAMBRAEUS, L. Personal communication. 2. BITTNER, D. L., HALL, S. G. and MCCLEARY, M. L. A method for determination of uric acid, using the cupric phenanthroline indicator system. Am. J. Clin. Path., 40: 423, 1963. 3. RYCKEWAERT, A., DRY, J., PAOLAGGI, F. and LELLOUGH, J. Taux de I’uricCmie dans la population
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doxine) metabolism in epileptic children. Develop. Med. G3 Child Neural., 7: 50, 1965. HEELEY, A. F. and ROBERTS, G. E. Tryptophan metabolism in psychotic children. Develop. Med. @ Child Neural., 7: 46, 1965. SCRIVNER,C. R. Vitamin Bs-dependency and infantile convulsions. Pediatrics, 26: 62, 1960. BRAND, E., CAHILL, G. F. and HARRIS, M. M. Cystinuria. II. The metabolism of cystine, cysteine, methionine and glutathione. J. Biol. Chem., 109: 69, 1935, BRAND, E., CAHILL, G. F. and BLOCK, R. J. Cystinuria. Iv. The metabolism of homocysteine and homocystine. J. Biol. Chem., 110: 399, 1935. LEWIS, H. B., BROCVN,B. H. and WHITE, F. R. The metabolism of sulfur. XXIII. The influence of the ingestion of cystine, cysteine, and methionine on the excretion of cystine in cystinuria. J. Biol. Chem., 114: 171,1936. SMITH, D. R., KOLB, F. 0. and HARPER, H. A. The management of cystinuria and cystine-stone disease. J. Ural., 81: 61, 1959. REED, L. J., CAVALLINI, D., PLUM, F., RACHELE, J. R. and DU VIGNEAUD, V. The conversion of methionine to cystine in a human cystinuric. J. Biol. Chem., 180: 783, 1949. HARRIS, H. and ROBSON, E. B. Cystinuria. ‘4m. J. Med., 22: 774, 1957. ROSENBERG,L. E., DOWNING,S., DURANT, J. L. and SEGAL, S. Cystinuria: biochemical evidence for three genetically distinct diseases. J. Gin. Znuest., 45: 365, 1966. SOUPART, P. Urinary excretion of free amino acids in normal adult men and women. Clin. chim. acta, 4: 265, 1959.
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