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Tryptophan metabolism in rheumatic disease.

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Tryptophan Metabolism in Rheumatic Disease
By ROBERT
S. PINALS
Five tryptophan metabolites were measured in the urine after an oral tryptophan load in patients with rheumatoid
arthritis, scleroderma, systemic lupus
and a variety of other diseases. An excessive excretion of kynurenine and
3-hydroxykynurenine was a common
but nonspecific occurrence in rheumatic
disease. The excretion did not correlate
well with various clinical parameters.
Inhibition of pyridoxal-dependent hepatic enzymes may be the cause of the
abnormality but the mechanism is
unknown.
Cinque metabolitos de tryptophano esseva mesurate in le urina post cargas oral
de tryptophano administrate a patientes
con arthritis rheumatoidee, scleroderma,
lupus generalisate, e un varietate de
altere morbos. Un excretion excessive de
kynurenina e 3-hydroxykynurenina esseva
un occurrentia commun sed non specific
in casos de morbo rheumatic. Le excretion revelava nulle nette correlation con
ulle del parametros clinic studiate. Inhibition de enzymas hepatic que depende
de pyridoxal es possibilemente le causa
del anormalitate mentionate, sed le
mechanism0 non es clar.
P
REVIOUS STUDIES in patients with rheumatic diseases have demonstrated disturbances in the principal pathway of tryptophan metabolism
(fig. 1).In scleroderma, Price et all found marked increases in excretion of
the kynurenines and lesser increases in other metabolites. Excessive amounts
of 3-hydroxyanthranilic acid, not measured by Price et al., were found to be
excreted in rheumatoid arthritis by McMillan,' Spiera3 and Bett,4 who
also demonstrated increased excretion of kynurenine. In the present study,
five metabolites in the kynurenine pathway were measured after tryptophan
loading in patients with rheumatoid arthritis and other rheumatic diseases,
in patients with a variety of other illnesses and in normal controls.
AND MATERIALS
METHODS
Studies were done on 45 patients with rheumatoid arthritis,5 five with systemic lupus,
four with scleroderma, 16 healthy controls and 45 patients with a variety of other diseases
including, among others, cancer, pneumonia, diabetes, myocardial infarction, cirrhosis,
hepatitis, emphysema, dermatitis and orthopedic conditions. Both the group with rheumatoid arthritis and the group with other diseases had 22 males and 23 females and were of
comparable mean age ( 5 2 and 50, respectively). The patients with scleroderma (mean
age 52) and systemic lupus (mean age 39) were all females. The healthy controls (mean
age 3 7 ) had an equal sex distribution. A loadinq dose of 2.5 Gm. of L-tryptophan was
given to each subject and urine collected for a 24-hour period with toluene as a preservative. No attempt was made to regulate the diet. Salicylates were discontinued 2 days before the collection. Kynurenic acid ( KA ), xanthurenic acid ( XA ), kynurenine ( Kyn), 3-
From the Department of Medicine, Massachusetts General Hospital and Harmrd M e d k d
School, Boston, Mass.
This work was supported by grants A-3564, 2A-5067, and AM-04501 from the United
States Public Health Service.
This is publication No. 368 of the Robert W. Lovett Memorial Unit for the Study of
Diseases Causing Deformities.
662
ARTHRITISA N D RHEUMATISM,VOL. 7, No. 6 (DECEMBER),
1964
<
TRYPTOPHAN METABOLISM IN RHEUMATIC DISEASE
663
~
H
P
H
INDOLEACETIC ACKI
TRYPTOPHAN
SEROTONIN
I
a
fryptaphan
pyrralase
0
s
0
3
H
INDICAN
FORMYL KYNURENINE
OH
~ O C H 2 f H C O O H
kynureninase
ANTHRANILIC ACID
\
fransaminose
-r m*
"'1
KYNURENINE
KYNURENIC ACID
OH
I - H Y DROXYANTHRANILIC
ACID
3-HYDROXYKYNURENINE
X A i k E N I C PICID
0;;;
- ocooHqCoNH
1
b
O4
HOC
COOH
NICOTINIC ACID
CH3
PYR I DONE
ALIPHATIC ACIDS
Fig. 1.-A diagram of tryptophan metabolism in abbreviated form. The main
pathway, in a quantitative sense, is shown with heavy arrows.
hydroxykynurenine ( HKyn) and 3-hydroxyanthranilic acid ( HAA ) were determined by the
method of Coppini, Benassi and Montorsi.22 A sample containing 0.1 to 0.2 ml. of unmodified urine was dried on Whatman # 1 paper and two-dimensional chromatography performed, using butanol-acetic acid-water (4:1:5)in the first phase and water in the second.
Areas containing the previously mentioned metabolites were located under ultraviolet light,
cut out and eluted. Measurement was accomplished by reaction with Ehrlich's reagent
(Kyn), diazotization ( HKyn, HAA, XA) and ultraviolet absorption (KA). Recovery of.
standards added to urine was from 87 to 97 per cent (average 92 per cent) for the various
compounds. Two papers were run for each sample and the two areas for a particular compound eluted together when quantities were small.
RESULTS
The range of values for 24-hour excretion of various tryptophan metabolites after a loading dose is shown in' figures 2-6. Mean values are shown in
table 1. A wide range is apparent in all groups. Serial experiments on the
same individual in several instances yielded similar results. Thus it would
appear that each individual has a characteristic response to tryptophan load-
664
ROBERT S. PINALS
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NORMAL
RHEU MATOlD
ARTHRITIS
SCLERODERMA
SYSTEMIC
LUPUS
OTHER
DISEASES
Fig. 2.-Excretion of 3-hydroxykynurenine (HKyn) in mg./24 hr. after a
tryptophan load in various disease groups. Dots at the bottom of each column in
this and subsequent figures represent failure to detect the compound. For 3-hydroxykynurenine, the lower limit of detectability varies from 3 to 6 mg./24 hr., depending
upon the total urine volume and volume used in chromatography,
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Fig. 3.-Excretion of kynurenine (Kyn) in mg./24 hr. after a tryptophan load
in various disease groups. Lower limit of detectability 2 to 4 mg./24 hr.
ing, not greatly influenced by ordinary variations ini diet and activity. Only
two healthy controls, both females, had HKyn spots; in one of these there
was a family history of arthritis. One was studied a t several points during
the menstrual cycle. Only small variations were seen, similar to those reported previously7; highest excretion of Kyn and HKyn occurred premenstrually, with little change in the other metabolites.
665
TRYPTOPHAN METABOLISM IN RHEUMATIC DISEASE
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Fig. 4.-Excretion of kynurenic acid (KA) in mg./24 hr. after a tryptophan
load in various disease groups. Lower limit of detectability 1.5 to 3 mg./24 hr.
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RHEUMATOID
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Fig. 5.-Excretion of 3-hydroxyanthranilic acid (HAA) in mg./24 hr. after a
tryptophan load in various disease groups, Lower limit of detectability 0.8 to 1.6
mg./24 hr.
The rheumatoid arthritis group had a significantly increased excretion over
the normal group only for HKyn and Kyn ( p < .01). Although the mean
HAA excretion was not significantly elevated, 24 per cent of the patients
with rheumatoid arthritis had greater excretion than any of the normals. Included in the rheumatoid group were six patients with rheumatoid spondylitis; excretion of Kyn and HKyn was high in two and low in four. TWOpatients with psoriasis and arthritis had high values; one with psoriasis alone
had low values. An attempt was made to correlate clinical findings with ex-
ROBERT S. PINALS
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Fig. 6.-Excretion of xanthurenic acid ( X A ) in mg./24 hr. after a tryptophan
load in various disease groups. Lower limit of detectability 0.5 to 1mg./24 hr.
cretion of each metabolite. As in other studies,3J no significant relationship
was found for most parameters, including age, duration of disease, latex
fixation test, presence of nodules, previous treatment and disease activity
in terms of joidt swelling and tenderness. However, there was some correlation between HKyn-Kyn excretion and sedimentation rate ( Rourke-Emstene); those with values below 0.60 mm./min. had a lower HKyn ( p < .01)
and Kyn ( p < .05) excretion.
In scleroderma, excretions tended to be higher than id rheumatoid arthritis. The patients with systemic lupus all had chronic low-grade disease
activity and, therefore, were not representative of the illness in general. The
relatively low values for Kyn and high values for HAA are difficult to explain
and conclusions from the study of such a small group are best avoided.
The group with other diseases differed significantly from the rheumatoid
arthritis group only in having lower HKyn excretion ( p < . O l ) . Among those
with high HKyn values were two patients each with recent myocardial infarcTable 1.-Mean
Normal
Rheumatoid arthritis
Scleroderma
Systemic lupus
Other diseases
Values (mg./24 hr.) for Excretion of Tryptophan
Metabolites in Various Groups
HKyn
Kyn
XA
KA
HAA
2.63
31.2'
85.2'
46.4'
9.72
5.64
33.2*
87.2"
8.70
23.9
4.00
5.04
4.80
5.41
3.03
13.0
14.8
28.5f
7.40
14.1
3.07
5.80
8.52"
10.2*
4.82
uSienificantly elevated over normal at p < .01.
+Significantly elevated over normal at p < .05.
Undetectable quantities were considered as zero for this calculation.
TRYPTOPHAN METABOLISM IN RHEUMATIC DISEASE
667
tion and carcinoma, and one each with megaloblastic anemia, acute leukemia,
pneumococcal pneumonia and sarcoma of bone. The latter had the highest
excretions of HKyn, Kyn, KA and XA in all groups studied.
DISCUSSION
The present study confirms the findings of Price et a1.l in scleroderma and
indicates that a similar abnormality in the kynurenine pathway exists in
rheumatoid arthritis. Bett found excessive Kyn excretion in rheumatoid
arthritis4s8and could reverse the abnormality, as could Price, by the administration of pyridoxine. We have also found this in preliminary studies. However, the pattern of urinary metabolites differs from that seen in pyridoxine
deficiency or antagonism with desoxypyridoxine in that XA excretion is not
significantly e l e ~ a t e dThe
. ~ observations suggest that one or more pyridoxaldependent hepatic enzymes, most particularly kynureninase, may be partially
inhibited in certain disease states. The elevated output of HAA in rheumatoid a r t h r i t i ~ may
~ - ~ have a different basis. No data on the output of this
metabolite after pyridoxine administration are available. Bett4 found that the
difference in excretion of HAA between the control and rheumatoid groups
diminished after tryptophan loading, in contrast to Kyn where loadidg greatly
exaggerated the difference. This would make enzyme inhibition in the pathway at a point beyond HAA an unlikely explanation. Spiera3 has speculated
about mechanisms for increased HAA excretion. Increased diversion of
tryptophan into the knurenine pathway, perhaps through accelerated protein
turnover, is a reasonable possibility.
Derangements in this pathway may be found in non-rheumatic conditions
as shown' by the present study and many others. Various hematologic and
malignant diseases are at times associated with increased excretion of one or
more metabolites.9-l3 Abnormalities have also been reported in schizophrenia,14J5porphyria,15 hepatitis,17 asthma,lRgastrointestinal disorders18 and
in some apparently healthy individuals with a family history of one or
another i l l n e s ~ . *Little
~ ~ ~ information is available on the relationship of the
biochemical abnormality to the clinical state, in terms of remissions and
exacerbations in individual patients and relative severity of disease in different patients. Reversal with pyridoxine has been variable.
Noteworthy in most studies involving tryptophan loading has been the wide
scattering of values for excretion of metabolites, both in healthy and diseased
groups. This may be a reflection of numerous actual or potential variable
factors such as absorption, renal clearance, contribution of endogenous protein turnover to the tryptophan pool and relative activities of the several
enzymes participatidg in the intermediary metabolism of tryptophan. These
latter may be influenced in turn by the availability of pyridoxal and possibly
other members of the vitamin B complex, by hormonal factors and by inhibitors postulated in various disease states. Known hormonal influences
include the induction of tryptophan pyrrolase by corticosteroids,20 the idhibition of kynurenine transaminase by estrogens21 and the alteration of
tryptophan metabolism in pregnancy,6 simulating pyridoxine deficiency but
668
ROBERT S . PINALS
inkcompletely reversed by pyrid~xine.~
The wide range of metabolite excretion
might hide an existing relationship to disease activity or severity. Perhaps
only through serial studies on the same individual during different phases
of his illness could such correlations be demonstrated.
The significance of abnormal tryptophan metabolism in rheumatic and
other diseases is an unsettled matter. An identical defect in many disparate
ilhesses would suggest that the phenomenon is a secondary one, probably
not etiologic. However, present knowledge cannot justify such a conclusion.
In view of the complexity of tryptophan metabolism, alternative explanation&might be found for any abnormal pattern of response to a loading dose.
Further understanding of the mechanisms involved and observation of the
clinical course of individual patients in relation to the biochemical abnormality and its reversal with pyridoxine would be of value.
ACKNOWLEDGMENTS
The author thanks Dr. Hugo Muench for his aid in statistical anlysis and Dr. Stephen
Krane for his advice and guidance.
REFERENCES
supplementation on the urinary ex1. Price, J. M., Brown, R. R., Rukavina, J.
G., Mendelson, C., and Johnson, S.
A. M.: Scleroderma (acrosclerosis),
11. Tryptophan metabolism before and
during
treatment
by
chelation
(EDTA). J. Invest. Dermat. 29:289,
1957.
2. McMillan, M.: The identification of a
fluorescent reducing substance in the
urine of patients with rheumatoid
arthritis. The excretion of 3-hydroxyanthranilic acid in this and other
conditions. J. Clin. Path. 13:140,
1960.
3. Spiera, H.: Excretion of a tryptophan
metabolite in rheumatoid arthritis.
Arth. & Rheumat. 6:364, 1963.
4. Bett, I. M.: Metabolism of tryptophan
in rheumatoid arthritis. Ann. Rheumat. Dis. 21:63, 1962.
5. Ropes, M. W., Bennett, G. A., Cobb,
S., Jacox, R., and Jessar, R. A.: 1958
Revision of diagnostic criteria for
rheumatoid arthritis. Arth. & Rheumat. 2:16, 1959.
6. Wachstein, M., and Lobel, S.: Abnormal tryptophan metabolites in human
pregnancy and their relation to deranged vitamin B6 metabolism. Proc.
SOC. Exper. Biol. & Med. 86:624,
1954.
7. Brown. R. R., Thornton, M. T., and
Price, J . M.: The effect of vitamin
cretion of tryptophan metabolites in
pregnant women. J. Clin. Invest. 40:
617, 1961.
8. Bett, I. M.: Effect of pyridoxine on
tryptophan metabolism in rheumatoid
arthritis. Ann. Rheumat. Dis. 21:388,
1962.
9. Price, J. M.: Disorders of tryptophan
metabolism. Univ. Michigan M. Bnll.
24:461, 1958.
10. Marver, H. S.: Studies on tryptophan
metabolism. I. Urinary tryptophan
metabolites in hypoplastic anemias
and other hematologic dismders. J.
Lab. & Clin. Med. 58:425, 1961.
11. Leppanen, V. V. E., and Oka, M.:
Metabolism of tryptophan in cancer
of various sites. Ann. med. exper. et
biol. Fenniae 41: 123, 1963.
12. Musajo, L., Benassi, C. A,, and Parpajola, A.: Excretion and isolation of
kynurenine and 3-hydroxykynurenine
from human pathologic urine. Clin.
chim. acta 1:229, 1956.
13. Boyland, E., and Williams, D. C.: The
metabolism of tryptophan. I. The
metabolism of tryptophan in patients
suffering from cancer of the bladder.
Biochem. J. 64:578, 1956.
14. Benassi, C . A., Benassi, P., Allegri, G.,
and Ballarin, P.: Tryptophan metabolisin in schizophrenic patients. J.
~~
TRYPTOPHAN METABOLISM IN RHEUMATIC DISEASE
Neurochem. 7:264, 1961.
15. Price, J. M., Brown, R. R., and Peters,
H. A.: Tryptophan metabolism in
porphyria, schizophrenia, and a variety of neurologic and psychiatric
diseases. Neurology 9:456, 1959.
16. Benassi, C. A., Perissinotto, B., and Allegri, G.: The metabolism of tryptophan in patients with bladder cancer
and other urological diseases. Clin.
chim. acta 8:822, 1963.
17. Quagliarello, E., Tancredi, F., Saccone,
C., and Piazza, M.: Interrelation between tryptophan and nicotinic acid
in human viral hepatitis. Nature,
London 194:976, 1962.
18. Knapp, A.: Tryptophanbelastung und
Vitamin-B6-Mangel. I. Die Ausscheidung von Xanthurensaure, Kynurenin,
Trigonellinamid und 5-Hydroxydolessigsaure bei verschiedenen Erkrankungen. Dentsche Gesundh. 16:941,
1961.
19. -:
669
Tryptophanbelastung und VitaminB6-Mangel. 111. Hereditare Faktoren
fur den Ausfall des Tryptophanbelastungs tests. Deutsche Gesundh. 16:
1041, 1961.
20. Knox, W. E.: Two mechanisms which
increase in vivo the liver tryptophan
peroxidase activity: Specific enzyme
adaptation and stimulation of the
pituitary-adrenal system. Brit. J. Exper. Path. 32:462, 1951.
21. Mason, M., and Gullekson, E. H.: Estrogen-enzyme interactions: Inhibition and protection of kynurenine
transaminase by the sulfate esters of
diethylstilbesterol, estradiol, and estrone. J. Biol. Chem. 235: 1312, 1960.
22. Coppini, D., Benassi, C. A., and Montorsi, M.: Quantitative determination
of tryptophan metabolites (via Kynurenine) in biologic fluids. Clin.
Chem. 5:391, 1959.
Robert S . Pinab, M.D., formerly Post-doctoral Fellow, United
States Public Health Service; presently Director, Arthritis Unit,
Lemuel Shuttuck Hospital, Boston, Mass.
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