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Tyrosine and glutamic acid in plasma and urine of patients with altered thyroid function
Tyrosine and Glutamic Acid in Plasma and Urine of Patients with Altered Thyroid Function By Raphael
Belanger,
Nanjappa Chandramohan, and Richard S. Rivlin
The plasma concentrations of glutamic acid and tyrosine were measured in euthyroid, hyperthyroid, and hypothyroid patients. In the group of normal individuals, plasma concentrations Of glutamic acid were more variable than were those of tyrosine. Levels of glutamic acid in plasma were elevated proportionately more than were those of tyrosine in hyperthyroidism, but were normal in hypothyroidism. Oral sodium glutamate tolerance in hyperthyroid patients did not differ from that of normal subjects in the mean increments attained from fasting to peak levels. This contrasts with altered tyrosine tolerance previousiy reoorted in hyperthyroidism. In hypothyroid patients, after ingestion of a load of sodium glutamate the mean increments from fasting to peak levels were slightly greater than normal. Daily variations in the plasma concentrations of glutamic acid were undemonstrable in normal individuals and were not modified by either hyperthy-
Robert
Misbin,
roidism or hypothyroidism. An unexplained elevation in the plasma concentration of glutamic acid at 2 a.m. was observed in several hyperthyroid patients. The urinary excretion of glutamic acid in a 24-hr period was increased nearly tenfold in hyperthyroid patients compared to results obtained in normal subjects. A similar percentage of the total glutamic acid (3137O/0) was excreted during the period from 8 a.m. to 4 p.m. in both normal and hyperthyroid patients. The urinary excretion of tyrosine was also increased in hyperthyroidism; the magnitude of the increase (nearly twofold) was less than that of glutamic acid. These data provide further evidence that in hyperthyroid patients increases in the concentrations of tyrosine and glutamic acid in plasma are consistent findings and demonstrate that the urinary excretion of both of these amino acids is also markedly increased.
I
N HYPERTHYROID PATIENTS, the pattern of amino acid concentrations in plasma undergoes specific changes from that of normal individuals. The levels of tyrosine and glutamic acid are increased without consistent From the Institute of Cancer Research and the Department of Medicine, Francis Delafield Hospital, College of Physicians and Surgeons of Columbia University, New York, N.Y. Received for publication January 13, 1972. Supported by USPHS Grants CA-02332 and AM-15265, Contract u-1562 from the Health Research Council of the City of New York, and by a Grant from the Stella and Charles Guttman Foundation. Raphael Belanger, M.D.: Assistant Professor of Medicine, Uniztersity of Skerbrooke, Sherbrooke, Canada. Formerly, Fellow in Endocrinology, Francis Delafield Hospital, and Assistant in Medicine, College of Physicians and Surgeons of Columbia University, New York, N.Y. Nanjappa Chandramohan, M.D.: Fellow in Medicine, Queens Hospital Center, Jamaica, N.Y. Formerly, Fellow in Endocrinology, Francis Delafield Hospital, and Assistant in Medicine, College of Physicians and Surgeons of Columbia University, New York, N.Y. Robert Misbin, M.D.: Intern in Medicine, Boston University, Boston, MRSS. Formerly, Student, 7ohns Hopkins University School of Medicine, Baltimore, Md. Richard 5. Rivlin, M.D.: Associate Professor of Medicine, ColIege of Physicians and Surgeons of Columbia University, New York, N.Y. Metabolism,
Vol. 21, No. 9 (September),
1972
855
856
BELANGER
ET AL.
alterations in the concentrations of other amino acids or of total a-amino nitrogen. rj2 In experimental animals with hyperthyroid&m, glutamic acid concentrations in plasma are increased proportionately more than are those of any other commonly occurring amino acid. 3 In patients with hypothyroidism, on the other hand, tyrosine concentrations in plasma generally are reduced below normal2 and those of glutamic acid are unchanged.4s5 After ingestion of a tyrosine load, plasma levels of tyrosine are greater than normal in hyperthyroidism and Iower than normal in hypothyroidism.2 The pathogenesis of the elevated plasma level of tyrosine in hyperthyroidism is complex and appears to involve a number of factors, including changes in the turnover rate, hepatic uptake, volume of distribution, and excretion of tyrosine.6,” The pathogenesis of the elevated glutamic acid concentration in plasma has not been defined. The present studies were initiated to compare the magnitude of the changes in tyrosine concentrations in plasma with those of glutamic acid in patients with hyperthyroidism and myxedema and to determine whether oral sodium glutamate tolerance is modified in these patients. In addition, our previous observation that the magnitude of the daily variations in the concentration of tyrosine in plasma is blunted in hypothyroidism but unaffected by hyperthyroidism8 prompted measurements to be made of daily variations in glutamic acid levels in plasma. Finally, these studies were planned to determine whether changes in the urinary excretion of tyrosine and glutamic acid accompany alterations in their plasma concentrations in patients with thyroid disorders. MATERIALS
Fasting Plasma Tyrosine
AND METHODS
and GZutamic Acid Concentrations
Both amino acids were simultaneously measured in 38 euthyroid, 25 hyperthyroid, and nine hypothyroid patients aged 22-79 yrs. The majority of the patients were hospitalized at the time of the investigation. Only in this phase of the study was no attempt made to regulate the quantity of food intake. In all patients, plasma samples were obtained between 8 a.m. and 9 a.m., no food having been permitted from midnight until after the blood samples had been drawn. Diagnoses of hyperthyroidism and myxedema were based upon both clinical and laboratory criteria, the latter including uptake of radioactive iodine by the thyroid gland, serum concentration of thyroxine iodine by column chromatography, serum cholesterol concentration, and resin uptake of 181LIabeled triiodathyronine. Euthyroid patients were freely ambulatory and were either normal controls or patients recovering from a nonendocrine illness such as bronchitis, emphysema, and hypertension, Twelve of the euthyroid patients had been studied previously when they were hospitalized with thyrotoxicosis or myxedema. Each patient was considered to be euthyroid only when clinical and laboratory criteria were both unequivocal.
Diurnal Variations of Plasma Glutamic, Acid Concentrations Measurements were made during two consecutive 24-hr cycles in eight euthyroid, eight hyperthyroid, and six hypothyroid subjects. The patients were predominantly female and ranged in age from 33 to 85 yr. All the patients being studied were on the Metabolic Ward of Francis DeIafieId Hospital for the duration of the study. Each patient received a diet containing a fixed quantity of protein, 1.5 g/kg body weight. The caloric contents of the diets were selected with the objective of minimizing weight changes under the experimental conditions and consisted of approximately 30 Cal/kg body weight for euthyroid subjects, 41 Cal/kg body weight for hyperthyroid patients, and 20-30 Cal/kg
TYROSINE
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ACID AND ALTERED
THYROID
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a57
Each meal contained one-fourth of the total body weight for hypothyroid subjects. amount of protein and was served at 8 a.m., 12 noon, 5 p.m., and 10 p.m. Patients received the diets for z days prior to the onset of the study. The patients were freely ambulatory during the daytime hours but were required to remain in bed from 10 p.m. to 7 a.m. Samples of blood were obtained by venipuncture immediately before each meal, except for lunch, and at 11 a.m., 2 p.m., and 2 a.m. during the two daily cycles.
Oral
Tolerance
of Monosodium
Glufumafe
Studies were performed in 24 euthyroid, 17 hyperthyroid and 8 hypothyroid patients, aged 28-80 yr. Food intake was regulated as described in the previous section. Following an overnight fast, each patieht received orally between 8-9 a.m. a dose of monosodium glutamate (SO mg/kg body weight) dissolved in orange juice. Initial studies using glutamic acid rather than the sodium salt had to be abandoned because of the relative insolubility of the acid. Plasma samples were withdrawn from each patient before ingestion of monosodium glutamate and at 15, 30, 45, 60, 90, 120, and 180 min after loading. Although uptoward symptoms in certain individuals had been reported after ingestion of monosodium glutamate,g none was recorded in any of these patients.
Techniques
of Blood Collection and Storage
Samples of 10 ml heparinized blood were drawn with each venipuncture. Whole blood samples to be analyzed for glutamic acid were kept on ice for no longer than 6 hr before centrifugation in the cold at 2000 g for 10 min. We had previously determined that samples may be kept for at least 12 hr without detectable changes in glutamic acid concentration. After centrifugation, plasma samples were immediately recovered, diluted with 3 volumes of distilled water, and protein precipitated by the addition of 1 voIume of 30% trichloroacetic acid. After a second centrifugation, supernatant solutions were recovered and stored at -20°C until the time of assay. NO samples were stored for more than 3 wk before assays were performed. Under these conditions, glutamic acid concentrations remained stable. Samples of deproteinized plasma rather than whole plasma were stored because of our previous observations that glutamic acid concentrations in frozen whole plasma become falsely high with prolonged storage. The levels of glutamic acid recorded in whole plasma became progressively greater with each week of storage at -ZO”C, and after 1 mo of storage the concentrations measured may be as much as twice those of freshly drawn plasma.
Assay of Glutumic Acid and Tyrosine in PIusma Plasma glutamic acid concentrations were assayed by a modification of the enzymatic fluorometric method of Graham and Aprison .I0 Glassware was prepared for use by boiling in nitric acid. In the assay, 0.2 ml of deproteinized plasma was added to a 1.0 ml mixture of glycine (0.5 M) and hydrazine hydrate buffer (0.013 M), pH 8.6. After additibn of 20 ccl NAD+ (0.01 M), contents were mixed and readings of fluorescence were made in the Aminco-Bowman spectrophotofluorometer. Ten microliters of purified glutamate dehydrogenase, 10 mg/ml, NHs-free (Sigma), were added to each cuvette. Samples were incubated at room temperature for 30 min, and fluorescence was determined again. Glutamic acid concentrations were determined from the differences in the two readings. Plasma samples were assayed in duplicate and were compared to known standards determined in a similar manner. The recovery of added glutamic acid was measured in each experiment and was SS-100% throughout the concentration range employed. Tyrosine concentrations were measured employing a fluorometric method.11 Under the conditions of assay, recovery of added tyrosine was 87-lOO%, and values obtained were similar to those previously reported from this laboratory.2J2 Each analysis was performed in duplicate. The agreement between duplicate measurements of the same sample was determined to be 1.9% for the entire range of plasma concentrations. As in previous reports from this laboratory, 2~2 standard solutions have been prepared that were applicable
to the entire
range of tyrosine
concentrations,
both in fasting
plasma
BELANGER
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ET AL.
and after a tyrosine load. Prior to obtaining fluorescence measurements, dilutions of the fina1 solutions of tyrosine-nitrosonaphthol derivatives from both unknown and standard it has been found simpler to make solutions have’ been made similarly. 18 In practice, appropriate dilutions in the initial plasma samples or in the supernatant solutions following trichloroacetic acid precipitation before performing the entire assay.
Urinary Excretion of Tyrosine and Glutamic Acid The urinary
excretion of tyrosine during a 2&hr period, and during the interval from to 4 p.m., was measured in eight hyperthyroid, ten euthyroid, and three hypothyroid patients. Glutamic acid in urine was simultaneously assayed in samples from each of these patients, as we11 as in that from one additional hyperthyroid and euthyroid patient. Three euthyroid individuals were studied as outpatients, i.e., meals prepared in the metabolic kitchen of the hospital were eaten at home and urine collections were also obtained at home. A11 the remaining patients were hospitalized on the metabolic ward at Francis Delafield Hospital and received diets described above for at feast 2 days prior to the urine collections. Urine samples were placed in iced bottles to which 10 ml of 50% HCl had been added previously. Completed collections were divided into SO-40-ml aliquots and frozen until the time of assay. On the day of assay, urine was thawed and centrifuged at 2000 g for 10 min. chromatographic For the assays of tyrosine and glutamic acid, a simple column procedure was developed that effectively separated the amino acids from other substances that would interfere with their fluorometric determinations. A 20 ml column of 1 cm diameter (Bellco) stoppered with glass wool was filled to a height of 7 cm with Dowex SO resin. The columns were washed and equilibrated successively with 0.05 M NaOH and 0.2 M NaHzP04, pH 2.2. Urine aliquots of 8 ml per sample were mixed with 2 ml of 30% trichloroacetic acid. Following centrifugation as above, 5 ml of the supematant solutions were added to 10 ml of 0.2 M NaHzP04. The pH of the solutions was adjusted to the range of 2.2-2.8. These solutions were then passed through the Dowex column. The columns were washed three times with 5 ml of NaH2P04, pH 2.2, and the eluates were discarded. To recover tyrosine and glutamic acid, 20 ml of 0.2 M NazHP04, pH 9.6, were passed through the column, and 16.0 ml of the eluates collected. The eluates were brought to a final volume of 20.0 ml by the addition of 0.01 N HNOR. Five-milliliter aliquots of the eluates were assayed for tyrosine and 2-ml aliquots were assayed for glutamic acid by methods similar to those used for plasma. Standard solutions of both tyrosine and glutamic acid were subjected to column chromatography in the same manner. Recovery experiments were performed by adding known amounts of each amino acid to urine samples. Under the conditions of assay, recovery of added tyrosine was ~~-10.5% and of gjutamic acid 9o-113%, as compared to known standard solutions similarly treated. 8 a.m.
RESULTS
Fasting Concentrations
of Tyrosine and Glufamic Acid in Plasma
To compare the effects of altered thyroid function on the plasma concentrations of tyrosine and glutamic acid, simultaneous assays of both amino acids were performed in 38 euthyroid, 25 hyperthyroid, and 6 hypothyroid subjects. Results of these assays are shown in Fig. I. As in previous studies,2 plasma tyrosine concentrations were greater than normal in hyperthyroid patients and lower than normal in hypothyroid patients. The concentrations of glutamic acid in normal individuals exhibited a much greater range of values than did the concentrations of tyrosine. In hyperthyroid patients, the concentrations of glutamic acid in plasma were significantly increased (p
TYROSINE
AND GLUTAMIC
859
ACID AND ALTERED THYROID FUNCTION
PLASMA TYROSINEloo PLASM&, ;LUTAMIC (25)
120
(25)
80
80
gMOLES/LltER
0
NORMAL
HYPE& HYPOTHYROID THYROID
NORMAL
WYPER- HYPOTHYROID THYROID
Fig. 1. Fasting concentrations of tyrosine and glutamic acid assayed simultaneously in plasma of euthyroid, hyperthyroid, and hypothyroid patients. Data are presented as mean f 1 SEM. Figures in parentheses refer to number of patients in each group.
in hyperthyroidism appeared to be greater than that of tyrosine in the same individuals. In the hypothyroid patients, glutamic acid concentrations were indistinguishable from those of the euthyroid groupDiurnal Variations
of Glutamic Acid Concentrations
in Plasma
In normal subjects, the concentrations of glutamic acid measured at intervals during 2 consecutive days did not show any consistent changes (Fig. 2). In the group of hyperthyroid patients, plasma concentrations of glutamic acid were higher than normal at all the times studied (p0.05). Studies of Ova1 Sodium Glutamate Tolerance Measurements euthyroid,
of
sodium
and 8 hypothyroid
glutamate tolerance in 17 hyperthyroid, 24 subjects, modeled after earlier studies of tyrosine
BELANGER ET AL.
HYPER77+W?Ol,, (81
120 -
80PLASMA GLUTAMIC ACID (lMDLES/L) 40 -
1
MEALS
4
+
+
I
0 8 AM
12 NOON
I 4 PM
$_
I
I 8 PM
,,,,,, 12 MN
4 AM
8 AM
TIME OF DAY Fig. 2. Variations in plasma concentrations of glutamic acid with time of day in euthyroid, hyperthyroid, and hypothyroid subjects. Data shown are mean -C 1 SEM of values obtained during two successive 24-hr periods. Values are expressed as pmoles/liter plasma. Meals fed are shown as arrows at 8 a.m., 12 noon, 5 p.m., and 10 p.m.
tolerance,2 are shown in Fig. 3. As noted above, fasting levels of glutamic acid were greater in hyperthyroid than in normal subjects. Hyperthyroid and normal patients did not, however, differ significantly (p>0.05) from one another in the mean increments from fasting to peak levels. In hypothyroid subjects, the mean increment between the concentration at zero time and at 30 min (86.9 -C 18.4 pmoles/liter) was significantly greater (p
increased slightly more than in normal subjects following ingestion of a load of sodium glutamate, whereas plasma levels of tyrosine following a tyrosine load increased less than in normal subjects.a Urinary Excretion of Tyrosine and Glutamic Acid Measurements of the urinary excretion of glutamic acid in eight hypothyroid and ten euthyroid individuals are shown in Table 1. Values for glutamic acid excretion by normal subjects obtained by the present methods were
861
TYROSINE AND GLUTAMIC ACID AND ALTERED THYROID FUNCTION
-I 160 t
1
PLASMA GLUTAMIC ACID (vMOLES I L 1
0
’
0
“y ....___.._._ p
4
EUTHYRWO 1241
40 -
1
60
I20
160 0
60
120
160
TIME AFTER GLUTAMIC ACID INGESTION (MINUTES)
Fig. 3. Plasma concentrations of glutamic acid before and at intervals after ingestion of oral load of monosodium glutamate (50 mg/kg body weight) in euthyroid, hyperthyroid, and hypothyroid patients. Data shown are mean + 1 SEM. Figures in parentheses refer to number of patients in each group.
lower than those reported using mechanized column chromatography;14 this difference may be due in part to artifactual conversion of glutamine to glutamic acid during the latter procedure.15 The results reported in ten normal subjects included three in whom no glutamic acid could be detected in the urine by these methods. It is readily apparent from Table 1 that the 24-hr excretion of glutamic acid was increased approximately ten-fold in hyperthyroidism. A similar fraction of the total glutamic acid was excreted in the period from 8 a.m. to 4 p.m. in both hyperthyroid and euthyroid subjects (~-37%). Follow-up studies in six hyperthyroid patients after they had become euthyroid indicated that in five a dramatic decrease in glutamic acid excretion had occurred, and in only one patient was the excretion unchanged. The administration of SO mg/kg sodium glutamate in a single oral dose elevated the plasma concentrations similarly in both euthyroid and hyperthyroid patients (Fig. 3) but had no effect on increasing the urinary excretion of glutamic acid in either group. In three hypothyroid patients the urinary excretion of glutamic acid Table 1. Urinary Excretion of Glutamic Acid in Hyperthyroid and Euthyroid Subjects
Group
Number of Subjects
Hyperthyroid Euthyroid
8 10
Basal Excretion of Glutamic Acid (amoles/ a.m.(pmoles/24 hr) 4 p.m.)
48.24 r 13.29t 4.63 2 1.23
14.79 f 5.17t 1.76 f 0.59
Excretion of Glutamic Acid After a Loading Dose’ (amoles/ a.m.@moles/24 hr) 4 p.m.)
56.93 -e 13.46t 4.36 -L 1.46
13.63 Z!I4.50t 1.07 ?I 0.60
l Monosodium glutamate, 50 mg/kg, ingested at 8 a.m. f Significance of difference between euthyroid and hyperthyroid groups is 0.01 or less.
862
BELANGER
ET AL.
averaged 4.48 pmoles/% hr, which is similar to that in normal individuals. The amino acid chromatographic patterns in congenital hypothyroidism were also observed to be in the normal range by previous workers.r6 Measurements were also made of the urinary excretion of tyrosine in nine hyperthyroid and 11 euthyroid subjects and are given in Table 2. The results for the urinary excretion of tyrosine in normal subjects agreed closely with reported values. 14*r7 The urinary excretion of tyrosine was increased nearly 70% (p
The present study extends the previous observation1 that glutamic acid and tyrosine concentrations are increased in plasma of patients with hyperthyroidism. Sodium glutamate tolerance in hyperthyroid patients is similar to that in normal subjects in that the concentration of glutamic acid returns to the fasting level within 2 hr, and the incremental increases are similar. These observations are in contrast to the prolonged elevation observed in the concentration of tyrosine in plasma in thyrotoxic subjects following ingestion of tyrosine. Tolerance to tryptophan and phenylalanine appears to be normal in hyperthyr0idism.l The finding that plasma levels of glutamic acid are normal in hypothyroid patients and that after a sodium glutamate load plasma levels are increased somewhat more in hypothyroid than in normal subjects is also in contrast to observations concerning tyrosine. In hypothyroid subjects, plasma levels of tyrosine both before and after a tyrosine load are lower than those of normal subjects.” Measurements of the diurnal variations of plasma glutamic acid concentrations indicate no consistent changes during a 24-hr period in normal individuals. Others have noted small daily fluctuations in glutamic acid levels but of much lesser magnitude than those observed for other amino acids, including tyrosine.18730 H yperthyroidism appears to have little effect on the daily rhythm of glutamic acid. The increases observed at 2 a.m. in several of the hyperthyroid patients remain unexplained. Hyperthyroidism also has little effect on the circadian variations of tyrosine levels8 Hypothyroidism does not affect the diurnal variations in glutamic acid concentrations in plasma, Table 2. Urinary Excretion of Tyrosine in Hyperthyroid
and Euthyroid Subjects
Basal Excretion Number of Subjects
Group
Hyperthyroid Euthyroid Significance t Significance l
9 11
(amoles/
hr)
244.1 +- 31.2’ 147.4 ?z 18.6
of Tyrosine (pmoles/ 8 a.m.-4 p.m.)
97.0 -c’22.2t 47.7 -c 7.9
of difference between euthyroid and hyperthyroid group is
TYROSINE
AND GLUTAMIC
ACID AND ALTERED
THYROID
863
FUNCTION
although we have observed previously* that hypothyroidism blunts the daily variations of tyrosine levels. The similarities and differences between the effects of hyperthyroidism and hypothyroidism on tyrosine and glutamic acid concentrations in plasma and urine are summarized in Table 3. Measurements of the urinary excretion of glutamic acid and tyrosine in hyperthyroid patients revealed that glutamic acid excretion is nearly ten times normal. Urinary tyrosine excretion is also increased, as recently reported.15.” The increase in tyrosine excretion (nearly twofold) is of lesser magnitude than that of glutamic acid. Thus, increases in the concentrations of glutamic acid in both plasma and urine appear to be greater than are those of tyrosine in patients with hyperthyroidism. In hyperthyroidism, the increased excretion of tyrosine, glutamic acid, hydroxyproline,“l and perhaps other amino acidsls may possibly contribute to the negative nitrogen balance in this disorder. It is not likely that aminoaciduria could be a major factor in clinical hyperthyroidism, however, because the actual amounts excreted in a 24-hr period are only milligram quantities. The pathogenesis of the alterations in glutamic acid metabolism in hyperthyroidism requires further definition. Several enzymes involved in glutamic acid metabolism are under thyroid hormone control. The activity of hepatic tyrosine-a-ketoglutarate transaminase, which synthesizes glutamic acid from a-ketoglutarate, is increased in hyperthyroidism2”-s4 and could account for a small portion of the increased amounts of glutamic acid measured in plasma and urine. Thyroid hormone treatment also increases the hepatic and renal activity of glutaminase, which catalyzes the deamination of glutamine to glutamic acid.e5 The activity of hepatic glutamate dehydrogenase, which degrades glutamic acid, is decreased by thyroxine in vitro, but is increased by thyroxine treatment in vivo.“” Glutamate dehydrogenase activity in kidney is unaffected by thyroid hormone.s5 These studies in experimental animals suggest that thyroid hormone enhances the enzymatic synthesis of glutamic acid from various precursors. The fact that normal glutamic acid tolerance is observed in hyperthyroid subjects, as shown here, further suggests that Table 3. Comparison
of the Effects of Altered Thyroid and Glutamic Acid Metabolism Hyperthyroidism
Plasma tyrosine concentration Plasma glutamic acid concentration Oral tyrosine tolerance Oral glutamic acid tolerance Daily rhythm of plasma tyrosine Daily rhythm of plasma glutamic acid Basal urinary Basal urinary
excretion excretion
of tyrosine of glutamic
acid
Increased Increased Abnormal* Normal Unchanged Probably unchanged Increased Greatly increased
Function
On Tyrosine
Hypothyroidism
Decreased Normal Abnormal* Probably abnormal* Blunted Unchanged Probably normal Probably normal
or reduced
*After ingestion of a dose of tyrosine, plasma levels of tyrosine are greater than normal in hyperthyroidism, and lower than normal in hypothyroidism. After ingestion of a dose of glutamic acid, plasma glutamic acid concentrations appear to undergo a greater than normal increase in hypothyroidism.
864
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increased synthesis from endogenous substrates, rather than decreased degradation o’r changes in body distribution, is primarily responsible for the increased glutamic acid concentrations in plasma and urine. It is evident from the present study that the increases in plasma and urine concentrations of tyrosine and glutamic acid in patients with hyperthyroidism constitute consistent and reproducible features of this disorder. Regardless of the ultimate diagnostic usefulness s7 that plasma concentrations of one or both amino acids may have in the clinical recognition of thyroid disease, it is important to recognize that specific disturbances in ammo acid metabolism are one of the hallmarks of hyperthyroidism. ACKNOWLEDGMENT The authors are indebted to Mrs. Barbara Maybruch for technical assistance; to Dr. Robert Canfield and Dr. Dezider Grunberger, Columbia University; Dr. David Goodman, Albany Medical College; and Dr. Kenneth Melmon, University of California for valuable suggestions. Miss Anna J. Wilson and Mrs. Edith Hamilton provided dietary and nursing services, respectively, for patients hospitalized on the Metabolic Ward of Francis Delafield Hospital. REFERENCES 1. Melmon, K. L., Rivlin, R., Oates, J. A., and Sjoerdsma, A.: Further studies of plasma tyrosine in patients with altered thyroid function. J. Chn. Endocr. 24:691, 1964. 2. Rivlin, R. S., Melmon, K. L., and Sjoerdsma, A.: An oral tyrosine tolerance test in thyrotoxicosis and myxedema. New Eng. J. Med. 272:1143, 1965. 3. Ness, G. C., Takahashi, T., and Lee, Y.-P.: Thyroid hormones on amino acid and protein metabolism. I. Concentration and composition of free amino acids in blood plasma of the rat. Endocrinology 85:1166, 1969. 4. Foley T. H., Landon, D. R., and Prenton, M. A.: Arterial plasma concentrations and forearm clearances of amino acids in myxedema. J. Chn. Endocr. 26:781, 1966. 5. Nyhan, W. L., Yujnovsky, A. O., and Wehr, R. F.: Amino acids and cell growth. In Cheek, D. B., [Ed.): Human Growth: Body Composition, Cell Growth, Energy and Intelligence. Philadelphia, Lea and Febiger, 1968, pp. 396-416. 6. Rivlin, R. S., and Kaufman, S.: Effects of altered thyroid function in rats upon the formation and distribution of tyrosine. Endocrinology 77:295, 196.5. 7. -, and Asper, S. P. : EditoriaI: Tyrosine and the thyroid hormones. Amer. J. Med. 40~823, 1966.
8. BClanger, R., and Rivlin, R. S.: Daily variations in plasma concentration of tyrosine in thyrotoxicosis and mvxedema. Metabolism 20:384, 1971. 9. Schaumburg, H. H., Byck, R., Gerstl, R., and Mashman, J. H.: Monosodium Lglutamate: Its pharmacology and role in the Science Chinese restaurant syndrome. 163:826, 1969. 10. Graham, L. T., Jr., and Aprison, M. H. : Fluorometric determination of aspartate, glutamate and a-aminobutyrate in nerve tissue using enzymic methods. Anal. Biochem. 15:487,1966. 11. Waalkes, T. I’., and Udenfriend, S.: A fluorometric method for the estimation of tyrosine in plasma and tissues. J. Lab. Clin. Med. 50~733, 1957. 12. Rivlin, R. S., and Melmon, K. L.: Cortisone-provoked depression of plasma tyrosine concentration: Relation to enzyme induction in man, J, Chn. Invest. 44 :1690, 1965. 13. Williams, T., and Besser, G. M.: Effects of treatment on tyrosine tolerance in thyroid disease. A modified tyrosine assay. Clin. Chem. 17:148, 1971. 14. Zinneman, H. H., Johnson, J. J., and Seal, U. 5.: Effect of short-term therapy with cortisol on the urinary excretion of free amino acids. J. Clin. Endocr. 23:996, 1963.
865
TYROSINE AND GLUTAMIC ACID AND ALTERED THYROID FUNCTION 1s. Felts, J. H., and King, J. S.: Enhanced secretion of free amino acids by hyperthyroid patients. Clin. Chem. 17:388, 1971. 16. Gabr, M., El-Gawad, Z. A., and ElBehairy, F. : Chromatographic pattern of serum and urinary amino acids in cretinism. Acta Paediat. Stand. 55:79, 1966. 17. Akisawa, J.: Clinical studies on the intermediary metabolism of tyrosine III. Urinary excretion of intermediary metaboIites of tyrosine in patients with some endocrine diseases and the related disorders. Jap. Arch. Intern. Med. 11:163, 1964. 18. Wurtman, R. J., Rose, C. M., Chou, C., and Larin, F. F.: Daily rhythms in the concentration of various amino acids in human plasma. New Eng. J. Med. 279:171, 1968. 19. Feigin, R. D., Klainer, A. S., and Beisel, W. R.: Factors affecting circadian periodicity of blood amino acids in man. Metabolism X7 ~764, 1968. 20. -, -, and -. : Circadian periodicity of blood amino-acids in adult men. Nature (London) 215:512, 1967. 21. Kivirikko, K. I., Koivusalo, M., Laitinen, O., and Lamburg, B.-A.: Hydroxyproline in the serum and urine of patients with hyperthyroidism. J. Clin. Endocr. 243222, 1964.
22.
Rivlin,
R.
Hepatic tyrosine plasma tyrosine altered thyroid 73:103, 1963.
S.,
and
Levine,
R.
J.:
transaminase activity and concentration in rats with function. Endocrinology
23. Litwack, G. A., Al-Nejjar, Z. H., Sears, M. L., and Ostheimer, G. W.: Timecourse of tyrosine transaminase and phydroxyphenylpyruvate oxidase activities during thyroid administration. Nature (London) 201:1028,1964. 24. Boctor, Harper, A. E.: and thiouracil Proc. Sot. Exp. 25. Grillo,
A. M., Rogers, Q. R., and The influence of thyroxine on rats fed excess tyrosine. Biol. Med. 133 :82x, 1970.
M. A., and Coghe,
M.:
Gluta-
minases in liver and kidney of hypo- and hyperthyroid rats. Enzymologia 33:fascicle 5 :237,1967. 26. Hoch, F. L.: thyroid hormones. 1962.
Biochemical actions of Physiol. Rev. 42:605,
27. Mochizuki, A., and Lee, Y.-P.: Effects of thyroid hormones on amino acid and protein metabolism. II. Glutamate concentration in rat tissues after thyroidectomy and thyroid hormone treatment. Endocrinology 87:816,1970.
Belanger,
Nanjappa Chandramohan, and Richard S. Rivlin
The plasma concentrations of glutamic acid and tyrosine were measured in euthyroid, hyperthyroid, and hypothyroid patients. In the group of normal individuals, plasma concentrations Of glutamic acid were more variable than were those of tyrosine. Levels of glutamic acid in plasma were elevated proportionately more than were those of tyrosine in hyperthyroidism, but were normal in hypothyroidism. Oral sodium glutamate tolerance in hyperthyroid patients did not differ from that of normal subjects in the mean increments attained from fasting to peak levels. This contrasts with altered tyrosine tolerance previousiy reoorted in hyperthyroidism. In hypothyroid patients, after ingestion of a load of sodium glutamate the mean increments from fasting to peak levels were slightly greater than normal. Daily variations in the plasma concentrations of glutamic acid were undemonstrable in normal individuals and were not modified by either hyperthy-
Robert
Misbin,
roidism or hypothyroidism. An unexplained elevation in the plasma concentration of glutamic acid at 2 a.m. was observed in several hyperthyroid patients. The urinary excretion of glutamic acid in a 24-hr period was increased nearly tenfold in hyperthyroid patients compared to results obtained in normal subjects. A similar percentage of the total glutamic acid (3137O/0) was excreted during the period from 8 a.m. to 4 p.m. in both normal and hyperthyroid patients. The urinary excretion of tyrosine was also increased in hyperthyroidism; the magnitude of the increase (nearly twofold) was less than that of glutamic acid. These data provide further evidence that in hyperthyroid patients increases in the concentrations of tyrosine and glutamic acid in plasma are consistent findings and demonstrate that the urinary excretion of both of these amino acids is also markedly increased.
I
N HYPERTHYROID PATIENTS, the pattern of amino acid concentrations in plasma undergoes specific changes from that of normal individuals. The levels of tyrosine and glutamic acid are increased without consistent From the Institute of Cancer Research and the Department of Medicine, Francis Delafield Hospital, College of Physicians and Surgeons of Columbia University, New York, N.Y. Received for publication January 13, 1972. Supported by USPHS Grants CA-02332 and AM-15265, Contract u-1562 from the Health Research Council of the City of New York, and by a Grant from the Stella and Charles Guttman Foundation. Raphael Belanger, M.D.: Assistant Professor of Medicine, Uniztersity of Skerbrooke, Sherbrooke, Canada. Formerly, Fellow in Endocrinology, Francis Delafield Hospital, and Assistant in Medicine, College of Physicians and Surgeons of Columbia University, New York, N.Y. Nanjappa Chandramohan, M.D.: Fellow in Medicine, Queens Hospital Center, Jamaica, N.Y. Formerly, Fellow in Endocrinology, Francis Delafield Hospital, and Assistant in Medicine, College of Physicians and Surgeons of Columbia University, New York, N.Y. Robert Misbin, M.D.: Intern in Medicine, Boston University, Boston, MRSS. Formerly, Student, 7ohns Hopkins University School of Medicine, Baltimore, Md. Richard 5. Rivlin, M.D.: Associate Professor of Medicine, ColIege of Physicians and Surgeons of Columbia University, New York, N.Y. Metabolism,
Vol. 21, No. 9 (September),
1972
855
856
BELANGER
ET AL.
alterations in the concentrations of other amino acids or of total a-amino nitrogen. rj2 In experimental animals with hyperthyroid&m, glutamic acid concentrations in plasma are increased proportionately more than are those of any other commonly occurring amino acid. 3 In patients with hypothyroidism, on the other hand, tyrosine concentrations in plasma generally are reduced below normal2 and those of glutamic acid are unchanged.4s5 After ingestion of a tyrosine load, plasma levels of tyrosine are greater than normal in hyperthyroidism and Iower than normal in hypothyroidism.2 The pathogenesis of the elevated plasma level of tyrosine in hyperthyroidism is complex and appears to involve a number of factors, including changes in the turnover rate, hepatic uptake, volume of distribution, and excretion of tyrosine.6,” The pathogenesis of the elevated glutamic acid concentration in plasma has not been defined. The present studies were initiated to compare the magnitude of the changes in tyrosine concentrations in plasma with those of glutamic acid in patients with hyperthyroidism and myxedema and to determine whether oral sodium glutamate tolerance is modified in these patients. In addition, our previous observation that the magnitude of the daily variations in the concentration of tyrosine in plasma is blunted in hypothyroidism but unaffected by hyperthyroidism8 prompted measurements to be made of daily variations in glutamic acid levels in plasma. Finally, these studies were planned to determine whether changes in the urinary excretion of tyrosine and glutamic acid accompany alterations in their plasma concentrations in patients with thyroid disorders. MATERIALS
Fasting Plasma Tyrosine
AND METHODS
and GZutamic Acid Concentrations
Both amino acids were simultaneously measured in 38 euthyroid, 25 hyperthyroid, and nine hypothyroid patients aged 22-79 yrs. The majority of the patients were hospitalized at the time of the investigation. Only in this phase of the study was no attempt made to regulate the quantity of food intake. In all patients, plasma samples were obtained between 8 a.m. and 9 a.m., no food having been permitted from midnight until after the blood samples had been drawn. Diagnoses of hyperthyroidism and myxedema were based upon both clinical and laboratory criteria, the latter including uptake of radioactive iodine by the thyroid gland, serum concentration of thyroxine iodine by column chromatography, serum cholesterol concentration, and resin uptake of 181LIabeled triiodathyronine. Euthyroid patients were freely ambulatory and were either normal controls or patients recovering from a nonendocrine illness such as bronchitis, emphysema, and hypertension, Twelve of the euthyroid patients had been studied previously when they were hospitalized with thyrotoxicosis or myxedema. Each patient was considered to be euthyroid only when clinical and laboratory criteria were both unequivocal.
Diurnal Variations of Plasma Glutamic, Acid Concentrations Measurements were made during two consecutive 24-hr cycles in eight euthyroid, eight hyperthyroid, and six hypothyroid subjects. The patients were predominantly female and ranged in age from 33 to 85 yr. All the patients being studied were on the Metabolic Ward of Francis DeIafieId Hospital for the duration of the study. Each patient received a diet containing a fixed quantity of protein, 1.5 g/kg body weight. The caloric contents of the diets were selected with the objective of minimizing weight changes under the experimental conditions and consisted of approximately 30 Cal/kg body weight for euthyroid subjects, 41 Cal/kg body weight for hyperthyroid patients, and 20-30 Cal/kg
TYROSINE
AND GLUTAMIC
ACID AND ALTERED
THYROID
FUNCTION
a57
Each meal contained one-fourth of the total body weight for hypothyroid subjects. amount of protein and was served at 8 a.m., 12 noon, 5 p.m., and 10 p.m. Patients received the diets for z days prior to the onset of the study. The patients were freely ambulatory during the daytime hours but were required to remain in bed from 10 p.m. to 7 a.m. Samples of blood were obtained by venipuncture immediately before each meal, except for lunch, and at 11 a.m., 2 p.m., and 2 a.m. during the two daily cycles.
Oral
Tolerance
of Monosodium
Glufumafe
Studies were performed in 24 euthyroid, 17 hyperthyroid and 8 hypothyroid patients, aged 28-80 yr. Food intake was regulated as described in the previous section. Following an overnight fast, each patieht received orally between 8-9 a.m. a dose of monosodium glutamate (SO mg/kg body weight) dissolved in orange juice. Initial studies using glutamic acid rather than the sodium salt had to be abandoned because of the relative insolubility of the acid. Plasma samples were withdrawn from each patient before ingestion of monosodium glutamate and at 15, 30, 45, 60, 90, 120, and 180 min after loading. Although uptoward symptoms in certain individuals had been reported after ingestion of monosodium glutamate,g none was recorded in any of these patients.
Techniques
of Blood Collection and Storage
Samples of 10 ml heparinized blood were drawn with each venipuncture. Whole blood samples to be analyzed for glutamic acid were kept on ice for no longer than 6 hr before centrifugation in the cold at 2000 g for 10 min. We had previously determined that samples may be kept for at least 12 hr without detectable changes in glutamic acid concentration. After centrifugation, plasma samples were immediately recovered, diluted with 3 volumes of distilled water, and protein precipitated by the addition of 1 voIume of 30% trichloroacetic acid. After a second centrifugation, supernatant solutions were recovered and stored at -20°C until the time of assay. NO samples were stored for more than 3 wk before assays were performed. Under these conditions, glutamic acid concentrations remained stable. Samples of deproteinized plasma rather than whole plasma were stored because of our previous observations that glutamic acid concentrations in frozen whole plasma become falsely high with prolonged storage. The levels of glutamic acid recorded in whole plasma became progressively greater with each week of storage at -ZO”C, and after 1 mo of storage the concentrations measured may be as much as twice those of freshly drawn plasma.
Assay of Glutumic Acid and Tyrosine in PIusma Plasma glutamic acid concentrations were assayed by a modification of the enzymatic fluorometric method of Graham and Aprison .I0 Glassware was prepared for use by boiling in nitric acid. In the assay, 0.2 ml of deproteinized plasma was added to a 1.0 ml mixture of glycine (0.5 M) and hydrazine hydrate buffer (0.013 M), pH 8.6. After additibn of 20 ccl NAD+ (0.01 M), contents were mixed and readings of fluorescence were made in the Aminco-Bowman spectrophotofluorometer. Ten microliters of purified glutamate dehydrogenase, 10 mg/ml, NHs-free (Sigma), were added to each cuvette. Samples were incubated at room temperature for 30 min, and fluorescence was determined again. Glutamic acid concentrations were determined from the differences in the two readings. Plasma samples were assayed in duplicate and were compared to known standards determined in a similar manner. The recovery of added glutamic acid was measured in each experiment and was SS-100% throughout the concentration range employed. Tyrosine concentrations were measured employing a fluorometric method.11 Under the conditions of assay, recovery of added tyrosine was 87-lOO%, and values obtained were similar to those previously reported from this laboratory.2J2 Each analysis was performed in duplicate. The agreement between duplicate measurements of the same sample was determined to be 1.9% for the entire range of plasma concentrations. As in previous reports from this laboratory, 2~2 standard solutions have been prepared that were applicable
to the entire
range of tyrosine
concentrations,
both in fasting
plasma
BELANGER
858
ET AL.
and after a tyrosine load. Prior to obtaining fluorescence measurements, dilutions of the fina1 solutions of tyrosine-nitrosonaphthol derivatives from both unknown and standard it has been found simpler to make solutions have’ been made similarly. 18 In practice, appropriate dilutions in the initial plasma samples or in the supernatant solutions following trichloroacetic acid precipitation before performing the entire assay.
Urinary Excretion of Tyrosine and Glutamic Acid The urinary
excretion of tyrosine during a 2&hr period, and during the interval from to 4 p.m., was measured in eight hyperthyroid, ten euthyroid, and three hypothyroid patients. Glutamic acid in urine was simultaneously assayed in samples from each of these patients, as we11 as in that from one additional hyperthyroid and euthyroid patient. Three euthyroid individuals were studied as outpatients, i.e., meals prepared in the metabolic kitchen of the hospital were eaten at home and urine collections were also obtained at home. A11 the remaining patients were hospitalized on the metabolic ward at Francis Delafield Hospital and received diets described above for at feast 2 days prior to the urine collections. Urine samples were placed in iced bottles to which 10 ml of 50% HCl had been added previously. Completed collections were divided into SO-40-ml aliquots and frozen until the time of assay. On the day of assay, urine was thawed and centrifuged at 2000 g for 10 min. chromatographic For the assays of tyrosine and glutamic acid, a simple column procedure was developed that effectively separated the amino acids from other substances that would interfere with their fluorometric determinations. A 20 ml column of 1 cm diameter (Bellco) stoppered with glass wool was filled to a height of 7 cm with Dowex SO resin. The columns were washed and equilibrated successively with 0.05 M NaOH and 0.2 M NaHzP04, pH 2.2. Urine aliquots of 8 ml per sample were mixed with 2 ml of 30% trichloroacetic acid. Following centrifugation as above, 5 ml of the supematant solutions were added to 10 ml of 0.2 M NaHzP04. The pH of the solutions was adjusted to the range of 2.2-2.8. These solutions were then passed through the Dowex column. The columns were washed three times with 5 ml of NaH2P04, pH 2.2, and the eluates were discarded. To recover tyrosine and glutamic acid, 20 ml of 0.2 M NazHP04, pH 9.6, were passed through the column, and 16.0 ml of the eluates collected. The eluates were brought to a final volume of 20.0 ml by the addition of 0.01 N HNOR. Five-milliliter aliquots of the eluates were assayed for tyrosine and 2-ml aliquots were assayed for glutamic acid by methods similar to those used for plasma. Standard solutions of both tyrosine and glutamic acid were subjected to column chromatography in the same manner. Recovery experiments were performed by adding known amounts of each amino acid to urine samples. Under the conditions of assay, recovery of added tyrosine was ~~-10.5% and of gjutamic acid 9o-113%, as compared to known standard solutions similarly treated. 8 a.m.
RESULTS
Fasting Concentrations
of Tyrosine and Glufamic Acid in Plasma
To compare the effects of altered thyroid function on the plasma concentrations of tyrosine and glutamic acid, simultaneous assays of both amino acids were performed in 38 euthyroid, 25 hyperthyroid, and 6 hypothyroid subjects. Results of these assays are shown in Fig. I. As in previous studies,2 plasma tyrosine concentrations were greater than normal in hyperthyroid patients and lower than normal in hypothyroid patients. The concentrations of glutamic acid in normal individuals exhibited a much greater range of values than did the concentrations of tyrosine. In hyperthyroid patients, the concentrations of glutamic acid in plasma were significantly increased (p
TYROSINE
AND GLUTAMIC
859
ACID AND ALTERED THYROID FUNCTION
PLASMA TYROSINEloo PLASM&, ;LUTAMIC (25)
120
(25)
80
80
gMOLES/LltER
0
NORMAL
HYPE& HYPOTHYROID THYROID
NORMAL
WYPER- HYPOTHYROID THYROID
Fig. 1. Fasting concentrations of tyrosine and glutamic acid assayed simultaneously in plasma of euthyroid, hyperthyroid, and hypothyroid patients. Data are presented as mean f 1 SEM. Figures in parentheses refer to number of patients in each group.
in hyperthyroidism appeared to be greater than that of tyrosine in the same individuals. In the hypothyroid patients, glutamic acid concentrations were indistinguishable from those of the euthyroid groupDiurnal Variations
of Glutamic Acid Concentrations
in Plasma
In normal subjects, the concentrations of glutamic acid measured at intervals during 2 consecutive days did not show any consistent changes (Fig. 2). In the group of hyperthyroid patients, plasma concentrations of glutamic acid were higher than normal at all the times studied (p
of
sodium
and 8 hypothyroid
glutamate tolerance in 17 hyperthyroid, 24 subjects, modeled after earlier studies of tyrosine
BELANGER ET AL.
HYPER77+W?Ol,, (81
120 -
80PLASMA GLUTAMIC ACID (lMDLES/L) 40 -
1
MEALS
4
+
+
I
0 8 AM
12 NOON
I 4 PM
$_
I
I 8 PM
,,,,,, 12 MN
4 AM
8 AM
TIME OF DAY Fig. 2. Variations in plasma concentrations of glutamic acid with time of day in euthyroid, hyperthyroid, and hypothyroid subjects. Data shown are mean -C 1 SEM of values obtained during two successive 24-hr periods. Values are expressed as pmoles/liter plasma. Meals fed are shown as arrows at 8 a.m., 12 noon, 5 p.m., and 10 p.m.
tolerance,2 are shown in Fig. 3. As noted above, fasting levels of glutamic acid were greater in hyperthyroid than in normal subjects. Hyperthyroid and normal patients did not, however, differ significantly (p>0.05) from one another in the mean increments from fasting to peak levels. In hypothyroid subjects, the mean increment between the concentration at zero time and at 30 min (86.9 -C 18.4 pmoles/liter) was significantly greater (p
increased slightly more than in normal subjects following ingestion of a load of sodium glutamate, whereas plasma levels of tyrosine following a tyrosine load increased less than in normal subjects.a Urinary Excretion of Tyrosine and Glutamic Acid Measurements of the urinary excretion of glutamic acid in eight hypothyroid and ten euthyroid individuals are shown in Table 1. Values for glutamic acid excretion by normal subjects obtained by the present methods were
861
TYROSINE AND GLUTAMIC ACID AND ALTERED THYROID FUNCTION
-I 160 t
1
PLASMA GLUTAMIC ACID (vMOLES I L 1
0
’
0
“y ....___.._._ p
4
EUTHYRWO 1241
40 -
1
60
I20
160 0
60
120
160
TIME AFTER GLUTAMIC ACID INGESTION (MINUTES)
Fig. 3. Plasma concentrations of glutamic acid before and at intervals after ingestion of oral load of monosodium glutamate (50 mg/kg body weight) in euthyroid, hyperthyroid, and hypothyroid patients. Data shown are mean + 1 SEM. Figures in parentheses refer to number of patients in each group.
lower than those reported using mechanized column chromatography;14 this difference may be due in part to artifactual conversion of glutamine to glutamic acid during the latter procedure.15 The results reported in ten normal subjects included three in whom no glutamic acid could be detected in the urine by these methods. It is readily apparent from Table 1 that the 24-hr excretion of glutamic acid was increased approximately ten-fold in hyperthyroidism. A similar fraction of the total glutamic acid was excreted in the period from 8 a.m. to 4 p.m. in both hyperthyroid and euthyroid subjects (~-37%). Follow-up studies in six hyperthyroid patients after they had become euthyroid indicated that in five a dramatic decrease in glutamic acid excretion had occurred, and in only one patient was the excretion unchanged. The administration of SO mg/kg sodium glutamate in a single oral dose elevated the plasma concentrations similarly in both euthyroid and hyperthyroid patients (Fig. 3) but had no effect on increasing the urinary excretion of glutamic acid in either group. In three hypothyroid patients the urinary excretion of glutamic acid Table 1. Urinary Excretion of Glutamic Acid in Hyperthyroid and Euthyroid Subjects
Group
Number of Subjects
Hyperthyroid Euthyroid
8 10
Basal Excretion of Glutamic Acid (amoles/ a.m.(pmoles/24 hr) 4 p.m.)
48.24 r 13.29t 4.63 2 1.23
14.79 f 5.17t 1.76 f 0.59
Excretion of Glutamic Acid After a Loading Dose’ (amoles/ a.m.@moles/24 hr) 4 p.m.)
56.93 -e 13.46t 4.36 -L 1.46
13.63 Z!I4.50t 1.07 ?I 0.60
l Monosodium glutamate, 50 mg/kg, ingested at 8 a.m. f Significance of difference between euthyroid and hyperthyroid groups is 0.01 or less.
862
BELANGER
ET AL.
averaged 4.48 pmoles/% hr, which is similar to that in normal individuals. The amino acid chromatographic patterns in congenital hypothyroidism were also observed to be in the normal range by previous workers.r6 Measurements were also made of the urinary excretion of tyrosine in nine hyperthyroid and 11 euthyroid subjects and are given in Table 2. The results for the urinary excretion of tyrosine in normal subjects agreed closely with reported values. 14*r7 The urinary excretion of tyrosine was increased nearly 70% (p
The present study extends the previous observation1 that glutamic acid and tyrosine concentrations are increased in plasma of patients with hyperthyroidism. Sodium glutamate tolerance in hyperthyroid patients is similar to that in normal subjects in that the concentration of glutamic acid returns to the fasting level within 2 hr, and the incremental increases are similar. These observations are in contrast to the prolonged elevation observed in the concentration of tyrosine in plasma in thyrotoxic subjects following ingestion of tyrosine. Tolerance to tryptophan and phenylalanine appears to be normal in hyperthyr0idism.l The finding that plasma levels of glutamic acid are normal in hypothyroid patients and that after a sodium glutamate load plasma levels are increased somewhat more in hypothyroid than in normal subjects is also in contrast to observations concerning tyrosine. In hypothyroid subjects, plasma levels of tyrosine both before and after a tyrosine load are lower than those of normal subjects.” Measurements of the diurnal variations of plasma glutamic acid concentrations indicate no consistent changes during a 24-hr period in normal individuals. Others have noted small daily fluctuations in glutamic acid levels but of much lesser magnitude than those observed for other amino acids, including tyrosine.18730 H yperthyroidism appears to have little effect on the daily rhythm of glutamic acid. The increases observed at 2 a.m. in several of the hyperthyroid patients remain unexplained. Hyperthyroidism also has little effect on the circadian variations of tyrosine levels8 Hypothyroidism does not affect the diurnal variations in glutamic acid concentrations in plasma, Table 2. Urinary Excretion of Tyrosine in Hyperthyroid
and Euthyroid Subjects
Basal Excretion Number of Subjects
Group
Hyperthyroid Euthyroid Significance t Significance l
9 11
(amoles/
hr)
244.1 +- 31.2’ 147.4 ?z 18.6
of Tyrosine (pmoles/ 8 a.m.-4 p.m.)
97.0 -c’22.2t 47.7 -c 7.9
of difference between euthyroid and hyperthyroid group is
TYROSINE
AND GLUTAMIC
ACID AND ALTERED
THYROID
863
FUNCTION
although we have observed previously* that hypothyroidism blunts the daily variations of tyrosine levels. The similarities and differences between the effects of hyperthyroidism and hypothyroidism on tyrosine and glutamic acid concentrations in plasma and urine are summarized in Table 3. Measurements of the urinary excretion of glutamic acid and tyrosine in hyperthyroid patients revealed that glutamic acid excretion is nearly ten times normal. Urinary tyrosine excretion is also increased, as recently reported.15.” The increase in tyrosine excretion (nearly twofold) is of lesser magnitude than that of glutamic acid. Thus, increases in the concentrations of glutamic acid in both plasma and urine appear to be greater than are those of tyrosine in patients with hyperthyroidism. In hyperthyroidism, the increased excretion of tyrosine, glutamic acid, hydroxyproline,“l and perhaps other amino acidsls may possibly contribute to the negative nitrogen balance in this disorder. It is not likely that aminoaciduria could be a major factor in clinical hyperthyroidism, however, because the actual amounts excreted in a 24-hr period are only milligram quantities. The pathogenesis of the alterations in glutamic acid metabolism in hyperthyroidism requires further definition. Several enzymes involved in glutamic acid metabolism are under thyroid hormone control. The activity of hepatic tyrosine-a-ketoglutarate transaminase, which synthesizes glutamic acid from a-ketoglutarate, is increased in hyperthyroidism2”-s4 and could account for a small portion of the increased amounts of glutamic acid measured in plasma and urine. Thyroid hormone treatment also increases the hepatic and renal activity of glutaminase, which catalyzes the deamination of glutamine to glutamic acid.e5 The activity of hepatic glutamate dehydrogenase, which degrades glutamic acid, is decreased by thyroxine in vitro, but is increased by thyroxine treatment in vivo.“” Glutamate dehydrogenase activity in kidney is unaffected by thyroid hormone.s5 These studies in experimental animals suggest that thyroid hormone enhances the enzymatic synthesis of glutamic acid from various precursors. The fact that normal glutamic acid tolerance is observed in hyperthyroid subjects, as shown here, further suggests that Table 3. Comparison
of the Effects of Altered Thyroid and Glutamic Acid Metabolism Hyperthyroidism
Plasma tyrosine concentration Plasma glutamic acid concentration Oral tyrosine tolerance Oral glutamic acid tolerance Daily rhythm of plasma tyrosine Daily rhythm of plasma glutamic acid Basal urinary Basal urinary
excretion excretion
of tyrosine of glutamic
acid
Increased Increased Abnormal* Normal Unchanged Probably unchanged Increased Greatly increased
Function
On Tyrosine
Hypothyroidism
Decreased Normal Abnormal* Probably abnormal* Blunted Unchanged Probably normal Probably normal
or reduced
*After ingestion of a dose of tyrosine, plasma levels of tyrosine are greater than normal in hyperthyroidism, and lower than normal in hypothyroidism. After ingestion of a dose of glutamic acid, plasma glutamic acid concentrations appear to undergo a greater than normal increase in hypothyroidism.
864
BELANGER
ET AL.
increased synthesis from endogenous substrates, rather than decreased degradation o’r changes in body distribution, is primarily responsible for the increased glutamic acid concentrations in plasma and urine. It is evident from the present study that the increases in plasma and urine concentrations of tyrosine and glutamic acid in patients with hyperthyroidism constitute consistent and reproducible features of this disorder. Regardless of the ultimate diagnostic usefulness s7 that plasma concentrations of one or both amino acids may have in the clinical recognition of thyroid disease, it is important to recognize that specific disturbances in ammo acid metabolism are one of the hallmarks of hyperthyroidism. ACKNOWLEDGMENT The authors are indebted to Mrs. Barbara Maybruch for technical assistance; to Dr. Robert Canfield and Dr. Dezider Grunberger, Columbia University; Dr. David Goodman, Albany Medical College; and Dr. Kenneth Melmon, University of California for valuable suggestions. Miss Anna J. Wilson and Mrs. Edith Hamilton provided dietary and nursing services, respectively, for patients hospitalized on the Metabolic Ward of Francis Delafield Hospital. REFERENCES 1. Melmon, K. L., Rivlin, R., Oates, J. A., and Sjoerdsma, A.: Further studies of plasma tyrosine in patients with altered thyroid function. J. Chn. Endocr. 24:691, 1964. 2. Rivlin, R. S., Melmon, K. L., and Sjoerdsma, A.: An oral tyrosine tolerance test in thyrotoxicosis and myxedema. New Eng. J. Med. 272:1143, 1965. 3. Ness, G. C., Takahashi, T., and Lee, Y.-P.: Thyroid hormones on amino acid and protein metabolism. I. Concentration and composition of free amino acids in blood plasma of the rat. Endocrinology 85:1166, 1969. 4. Foley T. H., Landon, D. R., and Prenton, M. A.: Arterial plasma concentrations and forearm clearances of amino acids in myxedema. J. Chn. Endocr. 26:781, 1966. 5. Nyhan, W. L., Yujnovsky, A. O., and Wehr, R. F.: Amino acids and cell growth. In Cheek, D. B., [Ed.): Human Growth: Body Composition, Cell Growth, Energy and Intelligence. Philadelphia, Lea and Febiger, 1968, pp. 396-416. 6. Rivlin, R. S., and Kaufman, S.: Effects of altered thyroid function in rats upon the formation and distribution of tyrosine. Endocrinology 77:295, 196.5. 7. -, and Asper, S. P. : EditoriaI: Tyrosine and the thyroid hormones. Amer. J. Med. 40~823, 1966.
8. BClanger, R., and Rivlin, R. S.: Daily variations in plasma concentration of tyrosine in thyrotoxicosis and mvxedema. Metabolism 20:384, 1971. 9. Schaumburg, H. H., Byck, R., Gerstl, R., and Mashman, J. H.: Monosodium Lglutamate: Its pharmacology and role in the Science Chinese restaurant syndrome. 163:826, 1969. 10. Graham, L. T., Jr., and Aprison, M. H. : Fluorometric determination of aspartate, glutamate and a-aminobutyrate in nerve tissue using enzymic methods. Anal. Biochem. 15:487,1966. 11. Waalkes, T. I’., and Udenfriend, S.: A fluorometric method for the estimation of tyrosine in plasma and tissues. J. Lab. Clin. Med. 50~733, 1957. 12. Rivlin, R. S., and Melmon, K. L.: Cortisone-provoked depression of plasma tyrosine concentration: Relation to enzyme induction in man, J, Chn. Invest. 44 :1690, 1965. 13. Williams, T., and Besser, G. M.: Effects of treatment on tyrosine tolerance in thyroid disease. A modified tyrosine assay. Clin. Chem. 17:148, 1971. 14. Zinneman, H. H., Johnson, J. J., and Seal, U. 5.: Effect of short-term therapy with cortisol on the urinary excretion of free amino acids. J. Clin. Endocr. 23:996, 1963.
865
TYROSINE AND GLUTAMIC ACID AND ALTERED THYROID FUNCTION 1s. Felts, J. H., and King, J. S.: Enhanced secretion of free amino acids by hyperthyroid patients. Clin. Chem. 17:388, 1971. 16. Gabr, M., El-Gawad, Z. A., and ElBehairy, F. : Chromatographic pattern of serum and urinary amino acids in cretinism. Acta Paediat. Stand. 55:79, 1966. 17. Akisawa, J.: Clinical studies on the intermediary metabolism of tyrosine III. Urinary excretion of intermediary metaboIites of tyrosine in patients with some endocrine diseases and the related disorders. Jap. Arch. Intern. Med. 11:163, 1964. 18. Wurtman, R. J., Rose, C. M., Chou, C., and Larin, F. F.: Daily rhythms in the concentration of various amino acids in human plasma. New Eng. J. Med. 279:171, 1968. 19. Feigin, R. D., Klainer, A. S., and Beisel, W. R.: Factors affecting circadian periodicity of blood amino acids in man. Metabolism X7 ~764, 1968. 20. -, -, and -. : Circadian periodicity of blood amino-acids in adult men. Nature (London) 215:512, 1967. 21. Kivirikko, K. I., Koivusalo, M., Laitinen, O., and Lamburg, B.-A.: Hydroxyproline in the serum and urine of patients with hyperthyroidism. J. Clin. Endocr. 243222, 1964.
22.
Rivlin,
R.
Hepatic tyrosine plasma tyrosine altered thyroid 73:103, 1963.
S.,
and
Levine,
R.
J.:
transaminase activity and concentration in rats with function. Endocrinology
23. Litwack, G. A., Al-Nejjar, Z. H., Sears, M. L., and Ostheimer, G. W.: Timecourse of tyrosine transaminase and phydroxyphenylpyruvate oxidase activities during thyroid administration. Nature (London) 201:1028,1964. 24. Boctor, Harper, A. E.: and thiouracil Proc. Sot. Exp. 25. Grillo,
A. M., Rogers, Q. R., and The influence of thyroxine on rats fed excess tyrosine. Biol. Med. 133 :82x, 1970.
M. A., and Coghe,
M.:
Gluta-
minases in liver and kidney of hypo- and hyperthyroid rats. Enzymologia 33:fascicle 5 :237,1967. 26. Hoch, F. L.: thyroid hormones. 1962.
Biochemical actions of Physiol. Rev. 42:605,
27. Mochizuki, A., and Lee, Y.-P.: Effects of thyroid hormones on amino acid and protein metabolism. II. Glutamate concentration in rat tissues after thyroidectomy and thyroid hormone treatment. Endocrinology 87:816,1970.