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Theophylline increases serum uric acid levels
Theophylline
increases serum uric acid levels
Yutaka Morita, M.D., Ph.D., Yutaro Nishida, M.D., Ph.D., Naoyuki Kamatani, M.D., Ph.D., and Terumasa Miyamoto, Tokyo,
M.D., Ph.D.
Japan
The present study was undertaken to investigate the effect of theophylline on serum uric acid and then to elucidate the mechanisms of action of theophylline as a cause of hyperuricemia. There was a signiJcant increase of serum uric acid levels in male asthmatic patients who received theophylline compared to male control subjects without theophylline (6.3 ? 0.4 mglml, mean ? SEM, versus 4.3 2 0.2 mgiml, p < 0.01). A signi$cant correlation of serum levels of uric acid and theophylline was demonstrated in asthmatic patients who received 200 to 400 mg sustained-release theophylline (male group, r = 0.480, p < 0.001; female group, r = 0.398, p < 0.01). Intravenous administration of aminophylline in three healthy adult male patients did not inhibit uric acid clearance, suggesting that inhibition of excretion of uric acid by theophylline is unlikely. Theophylline slightly inhibited hypoxanthine guanine phosphoribosyltransferase activity in human etythrocyte lysates at concentrations over 5 mM that is considerably more than therapeutic concentrations of theophylline as determined by the conversion of [‘%]hypoxanthine to [Y]inosinic acid. Theophylline caused a moderate inhibition of [14C lhypoxanthine uptake by K-562 cells (-50%) at IOmM that is over 100 times as high as those achieved clinically. Further studies remain to be performed to elucidate the exact mechanisms of theophylline-induced hyperuricemia. (J ALLERGY CLIN IMMUNOL 74: 707-12, I984 .)
Theophylline has been widely used as a bronchodilator in the treatment of patients with obstructive airway diseases including bronchial asthma.’ Recently, it has been reported that theophylline causes hyperuricemia in asthmatic patients.2 The purpose of the present study was to confirm this observation in asthmatic patients receiving theophylline orally and to investigate the mechanisms of theophylline-induced hyperuricemia. MATERIAL Subjects
AND METHODS
Thirty-five patients with allergic diseases including bronchial asthma and six normal individuals were divided into four groups according to sex and the use of theophylline. The group receiving theophylline and the control group were matched in average ages (male, 35 yr; female, 35 yr). Sera from the subjects were stored at -20” C until measurement of uric acid levels.
From the Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Received for publication Aug. 8, 1983. Accepted for publication April 24, 1984. Reprint requests: Yutaka Morita, M.D., Ph.D., Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Abbreviations
used
HGPRTase: Hypoxanthine IMP:
guanine phosphoribosyl-
transferase Inosinic acid
In another study, 112 asthmatic patients (51 male, 61 female) received sustained-release theophylline 200 to 400 mg every 12 hr. Sera from these patients were also stored at -20” C for the determination of uric acid and theophylline Two patients with hypertension and hyperthyroidism, respectively, were included in these studies. Renal function tests were not performed except urinalysis and blood chemistry examination in these studies. However, it did not appear that subjects included in the studies had serious renal function abnormalities because none of them had proteinuria, increased levels of blood urea nitrogen, and serum creatinine. No one took any diuretics. Some patients receiving theophylline were on regular daily p2 agonists such as
salbutamol, terbutaline, tulobuterol, and procaterol hydrochloride.
Determination
of serum
uric acid levels
Uric acid was determined by the enzymatic method described by Liddle et al.’ This method is specific for uric acid and is not influenced by its methylated derivatives such as 707
708
Morita
iiiitRGY
et al
CLiil !MMUNui. NOVEMBER 1984
.
ij .. 0’
; L ..3 i
I control Group
I
I Theophyllina
FIG. 1. Uric acid levels were determined sera from individuals who received males, 11 females) and those without males, 9 females).
I-methyluric acid.’
acid, 3-methyluric
Measutement
Determination
and compared theophylline theophylline
in (12 (9
was determined method.”
levels
by a high-pressure
of the uric acid clearance
Three healthy adult male subjects were injected with 250 mg of aminophylline intravenously over 5 min, and venous blood samples were obtained by venipuncture before and after the start of the injection at 30, 90, and 150 min. The urine was collected before the injection and at a 60-minute interval after the injection. lJric acid in the sera and urine was determined, and uric acid clearance was calculated by the following formula: uric acid clearance = uric acid concentration in urine x urine volume/serum uric acid concentration. Uric acid clearance was determined twice before the aminophylline injection and three times after the injection, and the mean values were obtained.
Measurement activity
I IO
01 Serum
Theophylline
1 20 Levels
(pg/ml)
FIG. 2. Correlation of serum levels of uric acid and theophylline. Sera from 51 male asthmatic patients who received oral sustained-release theophylline 200 to 400 mg every 12 hr were evaluated for uric acid and theophylline levels.
acid, and 1,3-dimethyluric
of serum theophylline
Serum theophylline liquid chromatography
Group
of erythrocyte
H@WTase
HGPRTase activity was determined by measuring the conversion of [“C]IMP from [‘“C]hypoxanthine. The assay mixture contained 800pM of [Y]hypoxanthine (20.7 mCiimmo1, Radiochemical Centre, Amersham, England) I
2mM of 5-phosphoribosyl I-pyrophosphate in 100 ~1 of 1OOmM Tris HCl buffer (pH 7.4) containing 1OmM of MgCl,. The reaction was initiated by the addition of 25 prl of the erythrocyte lysates to the assay mixture in the presence or absence of indicated concentrations of theophylline, continued at 37” C for 30 min, and terminated by the addition of 25 yl of 1OOmM ethylenediaminetetraacetic acid. An aliquot of 25 ~1 was spotted on Eastman Kodak thin-layer plates and developed with a solution of butanol. methanof, water, and 25% NH?OH (60: 20: 20: 1, v/v). The spot for IMP was scraped off and counted for radioactivity by a scintillation counter.
Measurement
of [14Clhypoxanthine
uptake
Human erythroleukemia cells K-562 were subcultured in RPM1 1640 containing 10% dialyzed heat-inactivated fetal calf serum. 100 U/ml of penicillin, and 100 @g/ml of streptomycin under 5% CO? at 37’ C. In the presence or absence of indicated concentrations of theophylline, IO” of K-562 cells were incubated with IO nmol of [“C]hypoxanthine for 24 hr at 37O C. Cells were trapped on the glass fiber filter papers, and their radioactivities were measured. In the time-course experiments, K-562 cells were incubated with 5mM of theophylline. were
VOLUME 7~1 NUMBER 5
Theophylline
* c
lm
.
0
Male
Female
increases
uric
acid
levels
709
.V D( 0.05
L
rr p ( 0.0 I
.
r
1
. . . . .
. 8
. l
.
r = 0,398 p ( 0.002
I -
I 0’
L IO Theophylline
I 20 Levels (pg/ml)
o-5 5-10 Serum Theophylline
O-15
l5-
Levels (ug/ml)
FIG. 3. Correlatilon of serum levels of uric acid and theophylline. Sera from 61 female asthmatic patients who received oral sustained-release theophylline 200 to 400 mg every 12 hr were evaluated for uric acid and theophylline levels.
FIG. 4. Relationship between levels in sera from asthmatic sustained-release theophylline. compared to uric acid levels of theophylline concentrations groups is indicated by the use p < 0.01).
harvested at indicated times, were trapped on the papers, and their radioactivities were measured.
Correlation theophylline
Statistical
Uric acid and theophylline levels were determined in sera from asthmatic patients who received orally 200 to 400 mg of sustained-release theophylline every 12 hr. A significant correlation of serum levels of uric acid and theophylline in male asthmatic patients was demonstrated (Fig. 2, r = 0.480, p < 0.001). Similarly, there was a correlation between two parameters in the female group (Fig. 3, r = 0.398, p < 0.002). The regression analysis also revealed a positive correlation between the serum uric acid and theophylline levels in both male groups and female groups (p < 0.01). Serum uric acid levels were divided into four groups according to the theophylline concentrations, and mean + SEM was calculated in each group. As demonstrated in Fig. 4 there was a significant elevation of serum uric acid in male groups with serum theophylline levels of 10 to 15 and 15 lug/ml, respectively, compared to that with theophylline levels of 0 to 5 pg/ml (p < 0.05, p < 0.01, respectively). In female groups, a significant difference was demon-
Serum
analysis
An analysis of variance and a regression analysis were used to analyze the data. A statistical difference was evaluated by the student’s t test and Bonferroni’s test. RESULTS Comparison of serum uric acid levels of individuals who received theophylline and those without theophylline Uric acid levels were determined in sera from 12 asthmatic patients who received theophylline for treatment and from nine control subjects without the drug. The uric acid level value in sera from male asthmatic patients who received theophylline was 6.3 k 0.4 (mean 4 SEM) mg/dl, whereas the value in sera from male control subjects was 4.3 -+ 0.2 mg/dl (Fig. 1). There was a significant difference between the two values (p < 0.01). In contrast, a significant difference was not demonstrated in sera from the female group (Fig. 1, theophylline group, 4.5 t 0.2 mg/dl; control group, 3.9 2 0.4 mg/dl).
of serum
uric acid and theophylline patients who received oral Statistical significance patients with 0 to 5 ,uglml of in both male and female of asterisks (*,p < 0.05; **,
levels of uric acid and
710
Morita
.I ?,i.LERLY C~lh iMMLJl%!:b NOVEMBER 13e i
et al
TABLE
1. Effect
of aminuphylllne
ui intravenous on
uric
admir>isttd’liOrl acid
clearance
Uric
acid clearance Imllmin)
Aminophylline -~-
Theophylline
Concentration
(rnbl)
FIG. 5. The effect of theophyiline on HGPRTase activity human erythrocyte lysates was determined by measuring the production of [Y]IMP from [Ylhypoxanthine.
In
strated between groups with theophylline levels of 0 to 5 and > 15 pgiml (p < 0.05). Effect of theophylline
on uric acid clearance
Increased levels of uric acid in male patients who received theophylline and a correlation between serum levels of uric acid and theophylline observed in subjects who received sustained-release theophylline could be due to the inhibitory effect of theophylline on uric acid clearance. Therefore, effect of aminophylline on uric acid clearance was evaluated by the intravenous administration of 250 mg of aminophylline in three healthy adult males. As demonstrated in Table I, aminophylline did not produce an inhibition of uric acid clearance. Effect of theophylline HGPRTase activity
on erythrocyte
The action of theophylline causing the increase of serum uric acid could be explained from the inhibitory effect of this drug on HGPRTase. Therefore, HGPRTase activity in erythrocyte lysates was determined with different concentrations of theophylline or buffer. As demonstrated in Fig. 5, theophylline con.centrations over 5mM slightly inhibited erythrocyte HGPRTase activity. Effect of theophyiline uptake in K-562 eels
on [W]hhypoxamthine
It could be possible that theophylline affects the use of purines in cells. Therefore, effect of theophylline on [‘“Clhypoxanthine uptake in K-562 cells was in-
4J-.:I xx .i.P
: -i ’
X.8 + !.(I
9.X _ / 7
:i 8 ” i
vestigated. Fig. 6 indicates that relatively high concentrations of theophylline cause a moderate suppression of [‘“C]hypoxanthine uptake c--,-50%) in K-562 cells. There was the increase of inhibition with 5mM theophylline with incubation time being increased (Fig. 7).
Theophylline is an effective bronchodilator used m the treatment of obstructive airway diseases including bronchial asthma.’ Although the exact mechanisms of action for theophylline to cause bronchodilation are not completely understood, several mechanisms of action have been proposed that include the elevation of cyclic AMP levels through the inhibition of phosphodiesterase and blockade of adenosine receptors .‘j In this study, we demonstrated that there were elevated levels of serum uric acid in the male asthmatic group receiving theophylline compared to the male control group without theophylline (Fig. 3). In contrast, a significant difference was not observed between female groups with and without theophylline probably caused by relatively small numbers of hubjecta. The phosphvtungstate method for uric acid measurement has been demonstrated to be affected by theophylline metabolites, and the ingestion of theophylline has been demonstrated to cause falsely elevated uric acid levels in urine when this test was used for uric acid determination.’ However, the uricase method used in this study is specific for uric acid and not influenced by theophylline metabolites.’ Therefore, our observation that theophylline induces hyperuricemia is on the basis of true increase in uric acid levels. HarterX reported that mean serum uric acid levels in 60 asthmatic subjects were 1.O mg percent higher than levels of normal subjects. He suggested that ephedrine and isoproterenol may have been responsible for the hyperuricemia observed, although most of the pa-
VOLUME 74 NUMBER !j
tients received aminophylline and phenobarbital as well. It has been reported that intravenous administrations of isoproterenol and epinephrine strikingly elevated uric acid levels through P-adrenergic stimulation in the rat.” In our study some patients receiving theophylline were on & agonists. Therefore, it is not possible to exclude the possibility that catecholamines may play a role in hyperuricemia observed in addition to theophylline. In the trial in which sustained-release theophylline was administered to asthmatic patients, there was a significant correlation of serum concentrations of uric acid and theophylline in both male and female groups (Figs. 2!, 3, and 4). In accordance with this result, Yosselson-Superstine et al.‘O demonstrated that there was a positive correlation between serum theophylline and uric acid levels in two of three subjects receiving a single oral theophylline dose. As a next step we investigated the possible mechanisms of action of theophylline as a cause of hyperuricemiii. First, the effect of theophylline on uric acid clearance was evaluated. Theophylline did not inhibit uric acid clearance, suggesting that hyperuricemia induced by theophylline through inhibition of uric acid excretion is unlikely. HGPRTase salvages hypoxanthine or guanine in vertebrates for reuse in nucleotide and nucleic acid biosynthesis.” Deficiency in this enzyme results in accumulation of guanine and hypoxanthine, further degradation to uric acid, and then increased levels of serum uric acid. This condition is known as the Lesch-Nyhan syndrome characterized by mental retardation and self-mutilation.‘” It was reported that rats receiving high doses of caffeine demonstrated Lesch-Nyhan syndrome-like behavior with a decreased HGPRTase activity.‘” In the same study theophylline ingestion was also proved to cause the similar behavior. Furthermore, inhibition of HGPRTase activity by caffeine and theophylline has been described. I-* Therefore, we examined the effect of theophylline on HGPRTase activity in human erythrocytes. There was a slight inhibition at theophylline concentrations over 5mM that is considerably higher than therapeutic concentrations of theophylline (0.05mM to O.lmM). Therefore, increased serum uric acid levels induced by theophylline could not be ascribed to its effect in inhibiting HGPRTase. Uric acid is synthesized from hypoxanthine or guanine through the formation of xanthine. The inhibition of use of hypoxanthine or guanine by cells can cause hyperuricemia. Theophylline inhibited hypoxanthine uptake in K-562 cells by -50% at 1OmM that is more than 100 times as high as those achieved
Theophylline
+ 5t
1 0
I I
increases
uric
acid
levels
711
1 1 I 2 5 IO Theophylline Concentration (mM1
FIG. 6. Effect of theophylline on [Ylhypoxanthine uptake in K-562 cells when 1050f K-562 cells were incubated with 10 nmol of [‘Ylhypoxanthine (20.7 mCi/mmol) in the presence and absence of indicated concentrations of theophylline for 24 hr at 37” C. Cells were trapped on the glass fiber filter papers, and their radioactivities were measured.
Time
(min)
FIG. 7. Time course of inhibition of [Ylhypoxanthine take by theophylline when K-562 cells were with 5mM of theophylline and were harvested cated times. Cells were trapped on the papers, radioactivities were measured.
upincubated at indiand their
clinically. Thus theophylline-induced hyperuricemia could not be explained from this mechanism. Theophylline is metabolized by the action of varieties of enzymes to metabolites that are excreted into the urine. Jenne et al. lJ detected theophylline, 3-methylxanthine, 1,3-dimethyluric acid, and 1-methyluric acid in the urines from patients who received 800 or 1200 mg of aminophylline in 24 hr. Uric acid could be generated from 1-methyluric acid through l-demethylation. However, this pathway is not known at present. Recently, stimulation of release of adrenal cate-
712
Morita
.! ALLERGY CLiit ,iwvvJ?u” WJVEMBER 1984
et al
cholammes by theophylline has been proposed as an additional mechanism of action. i’i Plasma epinephrine and norepinephrine levels were significantly increased at 3 hr after administration of theophylline or placebo therapy in a theophylline-treated group compared to a placebo-treated group. As already mentioned. intra venous administrations of isoproterenol and epineph,. rine were demonstrated to elevate uric acid levels 111 the rat ” Thus it may be possible to speculate that theophylline causes hyperuricemia via stimulation of release of adrenal catecholamincs. Finally, volume contraction caused by theophylline may lead to hyperuricemia as this mechanism is suggested as one of the causes of diuretic-induced hyperuricemia. Ii In conclusion there was a srgniticant Increase 01‘ serum uric acid levels in male asthmatic patients who received theophylline compared to male control subjects without theophylline. A significant correlation of serum levels of uric acid and theophylline was demonstrated in asthmatic patients who received 200 to 400 mg sustained-release theophylline. Further studies remain to be done in order to elucidate the exact mechanisms of theophylline-induced hyperuricemia. We thank
Miss
Emiko
Nagase
for typing
4.
\.
6.
-‘.
X. 9 10.
11 i 2.
13.
the manuscript, 1J.
REFERENCES 1. Ellis EF: Theophylline and derivatives. In Middle&on E Jr, Reed CE. Ellis EF, editors: Allergy: principles and practice. St. Louis. 1978, The CV Mosby Co, p 434 2. Kobayashi K, Noguchi H, Narimatsu H, Yokokawa T. Nishida M, Tsubomizu T, Odagiri T, Negishi M, Adachi M, Nakajima H. Shibuya T, Ide H, Takahashi T: Studies on elevated serum uric acid levels in patients with bronchial asthma. Jpn J Allergol 30:877. 1981 3. Liddle L, Seegmiller JE, Laster L: The enzymatic spec-
15
16.
lrophotometric method ior drtermmatn~n ill urri‘ *.!r! 6 i.,i! Clin Med 54:903. 1959 Buchanan OH. Block WD. Christman AA- The metabohsm ,:; the methylated purines. 1 The enzymatic determination of ur!nary uric acid. J Biol Chem 157:181. 1945 Adams RE. Vandemark Fl.. Schmidt GJ: Murc sens~tr~c high pressure lrquid-chromatographic determination of the I)phylline m serum Clin Chern ?21903. 1976 Rail TM;: Central nervous system stimulants: the xanthines ,I. ~;oodman IS. Gilnran -2. editors: Pharmacological haris o1 !hcrapeutic<, etl 0 New Yark, !98 1. Macmillan Publishing i::x. inc. pp 59i.lli!T Buchanan OH. C’hri,tman AA, Block WI): f-he tnttdbolism %)t the methylated purines. II. Llric acid excretion following the ingestion of caffeine. theophylline. and theobromine. J Rio! Chem 147 1X9. 1945 Harter SC;. Serum uric acid levels m patients with bronchia: asthma. i At.1 t~:~\ 4?:88. 1968 Sumi T. Umeda P Adrenergic regulation or the plasma ic\Ci:, rif purine rnetabolitea in the rat. Eur J Pharmacol 46:243. 1977 Yosselson-Superstine S. Granit D, Superstine E: Theophylhne interference with the phosphotungstate uric acid test. Am ! Hosp Pharm 37: 1522. 19gO lrhninger AL Biochemistry. cd L New York, 1377, Worth Publishers in<,. pp 729-747 I.csch M. Nyhan WL A familial disorder 01 uric acid metabo lism and central nervou\ system function. Am J Med 36.561 i Yh4 !%rrer 1, C~oslell M, Grtsoha S: Lesch-Nyhan syndrome-&kc behavior in rats lrom caffeine ingestion: changes in HGPRTasr ALtivity, urea, 4nJ some nitrogen metabolism enzymes. FEBS I.ctt 141.1’5I ,. 101(3 Nolan LL, Kidder GW: Caffeine: its action on purme metabohzing emymes Biochem Biophys Res Commun 91:253, 1979 lenne JW, Nagasawa HT. Thompson RD: Relationship of urinary metabolites of theophylline to serum theophylline levels Clin Pharmacoi Ther 19:375, 1976 Higbee MD, Kumar M. Galant SP: Stimulation of endogenous Latecholamine release by theophylline: a proposed additional mechanism of action for theophylline effects. J ALLERGY CI.IN i\l>lUNCii.
17
70:?7?
19x2
Steele TH, Oppenheimer S. bactors affecting urate excretion iollowmg diuretic administration in man. Am J Med 47:564. 1%hO
increases serum uric acid levels
Yutaka Morita, M.D., Ph.D., Yutaro Nishida, M.D., Ph.D., Naoyuki Kamatani, M.D., Ph.D., and Terumasa Miyamoto, Tokyo,
M.D., Ph.D.
Japan
The present study was undertaken to investigate the effect of theophylline on serum uric acid and then to elucidate the mechanisms of action of theophylline as a cause of hyperuricemia. There was a signiJcant increase of serum uric acid levels in male asthmatic patients who received theophylline compared to male control subjects without theophylline (6.3 ? 0.4 mglml, mean ? SEM, versus 4.3 2 0.2 mgiml, p < 0.01). A signi$cant correlation of serum levels of uric acid and theophylline was demonstrated in asthmatic patients who received 200 to 400 mg sustained-release theophylline (male group, r = 0.480, p < 0.001; female group, r = 0.398, p < 0.01). Intravenous administration of aminophylline in three healthy adult male patients did not inhibit uric acid clearance, suggesting that inhibition of excretion of uric acid by theophylline is unlikely. Theophylline slightly inhibited hypoxanthine guanine phosphoribosyltransferase activity in human etythrocyte lysates at concentrations over 5 mM that is considerably more than therapeutic concentrations of theophylline as determined by the conversion of [‘%]hypoxanthine to [Y]inosinic acid. Theophylline caused a moderate inhibition of [14C lhypoxanthine uptake by K-562 cells (-50%) at IOmM that is over 100 times as high as those achieved clinically. Further studies remain to be performed to elucidate the exact mechanisms of theophylline-induced hyperuricemia. (J ALLERGY CLIN IMMUNOL 74: 707-12, I984 .)
Theophylline has been widely used as a bronchodilator in the treatment of patients with obstructive airway diseases including bronchial asthma.’ Recently, it has been reported that theophylline causes hyperuricemia in asthmatic patients.2 The purpose of the present study was to confirm this observation in asthmatic patients receiving theophylline orally and to investigate the mechanisms of theophylline-induced hyperuricemia. MATERIAL Subjects
AND METHODS
Thirty-five patients with allergic diseases including bronchial asthma and six normal individuals were divided into four groups according to sex and the use of theophylline. The group receiving theophylline and the control group were matched in average ages (male, 35 yr; female, 35 yr). Sera from the subjects were stored at -20” C until measurement of uric acid levels.
From the Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Received for publication Aug. 8, 1983. Accepted for publication April 24, 1984. Reprint requests: Yutaka Morita, M.D., Ph.D., Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Abbreviations
used
HGPRTase: Hypoxanthine IMP:
guanine phosphoribosyl-
transferase Inosinic acid
In another study, 112 asthmatic patients (51 male, 61 female) received sustained-release theophylline 200 to 400 mg every 12 hr. Sera from these patients were also stored at -20” C for the determination of uric acid and theophylline Two patients with hypertension and hyperthyroidism, respectively, were included in these studies. Renal function tests were not performed except urinalysis and blood chemistry examination in these studies. However, it did not appear that subjects included in the studies had serious renal function abnormalities because none of them had proteinuria, increased levels of blood urea nitrogen, and serum creatinine. No one took any diuretics. Some patients receiving theophylline were on regular daily p2 agonists such as
salbutamol, terbutaline, tulobuterol, and procaterol hydrochloride.
Determination
of serum
uric acid levels
Uric acid was determined by the enzymatic method described by Liddle et al.’ This method is specific for uric acid and is not influenced by its methylated derivatives such as 707
708
Morita
iiiitRGY
et al
CLiil !MMUNui. NOVEMBER 1984
.
ij .. 0’
; L ..3 i
I control Group
I
I Theophyllina
FIG. 1. Uric acid levels were determined sera from individuals who received males, 11 females) and those without males, 9 females).
I-methyluric acid.’
acid, 3-methyluric
Measutement
Determination
and compared theophylline theophylline
in (12 (9
was determined method.”
levels
by a high-pressure
of the uric acid clearance
Three healthy adult male subjects were injected with 250 mg of aminophylline intravenously over 5 min, and venous blood samples were obtained by venipuncture before and after the start of the injection at 30, 90, and 150 min. The urine was collected before the injection and at a 60-minute interval after the injection. lJric acid in the sera and urine was determined, and uric acid clearance was calculated by the following formula: uric acid clearance = uric acid concentration in urine x urine volume/serum uric acid concentration. Uric acid clearance was determined twice before the aminophylline injection and three times after the injection, and the mean values were obtained.
Measurement activity
I IO
01 Serum
Theophylline
1 20 Levels
(pg/ml)
FIG. 2. Correlation of serum levels of uric acid and theophylline. Sera from 51 male asthmatic patients who received oral sustained-release theophylline 200 to 400 mg every 12 hr were evaluated for uric acid and theophylline levels.
acid, and 1,3-dimethyluric
of serum theophylline
Serum theophylline liquid chromatography
Group
of erythrocyte
H@WTase
HGPRTase activity was determined by measuring the conversion of [“C]IMP from [‘“C]hypoxanthine. The assay mixture contained 800pM of [Y]hypoxanthine (20.7 mCiimmo1, Radiochemical Centre, Amersham, England) I
2mM of 5-phosphoribosyl I-pyrophosphate in 100 ~1 of 1OOmM Tris HCl buffer (pH 7.4) containing 1OmM of MgCl,. The reaction was initiated by the addition of 25 prl of the erythrocyte lysates to the assay mixture in the presence or absence of indicated concentrations of theophylline, continued at 37” C for 30 min, and terminated by the addition of 25 yl of 1OOmM ethylenediaminetetraacetic acid. An aliquot of 25 ~1 was spotted on Eastman Kodak thin-layer plates and developed with a solution of butanol. methanof, water, and 25% NH?OH (60: 20: 20: 1, v/v). The spot for IMP was scraped off and counted for radioactivity by a scintillation counter.
Measurement
of [14Clhypoxanthine
uptake
Human erythroleukemia cells K-562 were subcultured in RPM1 1640 containing 10% dialyzed heat-inactivated fetal calf serum. 100 U/ml of penicillin, and 100 @g/ml of streptomycin under 5% CO? at 37’ C. In the presence or absence of indicated concentrations of theophylline, IO” of K-562 cells were incubated with IO nmol of [“C]hypoxanthine for 24 hr at 37O C. Cells were trapped on the glass fiber filter papers, and their radioactivities were measured. In the time-course experiments, K-562 cells were incubated with 5mM of theophylline. were
VOLUME 7~1 NUMBER 5
Theophylline
* c
lm
.
0
Male
Female
increases
uric
acid
levels
709
.V D( 0.05
L
rr p ( 0.0 I
.
r
1
. . . . .
. 8
. l
.
r = 0,398 p ( 0.002
I -
I 0’
L IO Theophylline
I 20 Levels (pg/ml)
o-5 5-10 Serum Theophylline
O-15
l5-
Levels (ug/ml)
FIG. 3. Correlatilon of serum levels of uric acid and theophylline. Sera from 61 female asthmatic patients who received oral sustained-release theophylline 200 to 400 mg every 12 hr were evaluated for uric acid and theophylline levels.
FIG. 4. Relationship between levels in sera from asthmatic sustained-release theophylline. compared to uric acid levels of theophylline concentrations groups is indicated by the use p < 0.01).
harvested at indicated times, were trapped on the papers, and their radioactivities were measured.
Correlation theophylline
Statistical
Uric acid and theophylline levels were determined in sera from asthmatic patients who received orally 200 to 400 mg of sustained-release theophylline every 12 hr. A significant correlation of serum levels of uric acid and theophylline in male asthmatic patients was demonstrated (Fig. 2, r = 0.480, p < 0.001). Similarly, there was a correlation between two parameters in the female group (Fig. 3, r = 0.398, p < 0.002). The regression analysis also revealed a positive correlation between the serum uric acid and theophylline levels in both male groups and female groups (p < 0.01). Serum uric acid levels were divided into four groups according to the theophylline concentrations, and mean + SEM was calculated in each group. As demonstrated in Fig. 4 there was a significant elevation of serum uric acid in male groups with serum theophylline levels of 10 to 15 and 15 lug/ml, respectively, compared to that with theophylline levels of 0 to 5 pg/ml (p < 0.05, p < 0.01, respectively). In female groups, a significant difference was demon-
Serum
analysis
An analysis of variance and a regression analysis were used to analyze the data. A statistical difference was evaluated by the student’s t test and Bonferroni’s test. RESULTS Comparison of serum uric acid levels of individuals who received theophylline and those without theophylline Uric acid levels were determined in sera from 12 asthmatic patients who received theophylline for treatment and from nine control subjects without the drug. The uric acid level value in sera from male asthmatic patients who received theophylline was 6.3 k 0.4 (mean 4 SEM) mg/dl, whereas the value in sera from male control subjects was 4.3 -+ 0.2 mg/dl (Fig. 1). There was a significant difference between the two values (p < 0.01). In contrast, a significant difference was not demonstrated in sera from the female group (Fig. 1, theophylline group, 4.5 t 0.2 mg/dl; control group, 3.9 2 0.4 mg/dl).
of serum
uric acid and theophylline patients who received oral Statistical significance patients with 0 to 5 ,uglml of in both male and female of asterisks (*,p < 0.05; **,
levels of uric acid and
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.I ?,i.LERLY C~lh iMMLJl%!:b NOVEMBER 13e i
et al
TABLE
1. Effect
of aminuphylllne
ui intravenous on
uric
admir>isttd’liOrl acid
clearance
Uric
acid clearance Imllmin)
Aminophylline -~-
Theophylline
Concentration
(rnbl)
FIG. 5. The effect of theophyiline on HGPRTase activity human erythrocyte lysates was determined by measuring the production of [Y]IMP from [Ylhypoxanthine.
In
strated between groups with theophylline levels of 0 to 5 and > 15 pgiml (p < 0.05). Effect of theophylline
on uric acid clearance
Increased levels of uric acid in male patients who received theophylline and a correlation between serum levels of uric acid and theophylline observed in subjects who received sustained-release theophylline could be due to the inhibitory effect of theophylline on uric acid clearance. Therefore, effect of aminophylline on uric acid clearance was evaluated by the intravenous administration of 250 mg of aminophylline in three healthy adult males. As demonstrated in Table I, aminophylline did not produce an inhibition of uric acid clearance. Effect of theophylline HGPRTase activity
on erythrocyte
The action of theophylline causing the increase of serum uric acid could be explained from the inhibitory effect of this drug on HGPRTase. Therefore, HGPRTase activity in erythrocyte lysates was determined with different concentrations of theophylline or buffer. As demonstrated in Fig. 5, theophylline con.centrations over 5mM slightly inhibited erythrocyte HGPRTase activity. Effect of theophyiline uptake in K-562 eels
on [W]hhypoxamthine
It could be possible that theophylline affects the use of purines in cells. Therefore, effect of theophylline on [‘“Clhypoxanthine uptake in K-562 cells was in-
4J-.:I xx .i.P
: -i ’
X.8 + !.(I
9.X _ / 7
:i 8 ” i
vestigated. Fig. 6 indicates that relatively high concentrations of theophylline cause a moderate suppression of [‘“C]hypoxanthine uptake c--,-50%) in K-562 cells. There was the increase of inhibition with 5mM theophylline with incubation time being increased (Fig. 7).
Theophylline is an effective bronchodilator used m the treatment of obstructive airway diseases including bronchial asthma.’ Although the exact mechanisms of action for theophylline to cause bronchodilation are not completely understood, several mechanisms of action have been proposed that include the elevation of cyclic AMP levels through the inhibition of phosphodiesterase and blockade of adenosine receptors .‘j In this study, we demonstrated that there were elevated levels of serum uric acid in the male asthmatic group receiving theophylline compared to the male control group without theophylline (Fig. 3). In contrast, a significant difference was not observed between female groups with and without theophylline probably caused by relatively small numbers of hubjecta. The phosphvtungstate method for uric acid measurement has been demonstrated to be affected by theophylline metabolites, and the ingestion of theophylline has been demonstrated to cause falsely elevated uric acid levels in urine when this test was used for uric acid determination.’ However, the uricase method used in this study is specific for uric acid and not influenced by theophylline metabolites.’ Therefore, our observation that theophylline induces hyperuricemia is on the basis of true increase in uric acid levels. HarterX reported that mean serum uric acid levels in 60 asthmatic subjects were 1.O mg percent higher than levels of normal subjects. He suggested that ephedrine and isoproterenol may have been responsible for the hyperuricemia observed, although most of the pa-
VOLUME 74 NUMBER !j
tients received aminophylline and phenobarbital as well. It has been reported that intravenous administrations of isoproterenol and epinephrine strikingly elevated uric acid levels through P-adrenergic stimulation in the rat.” In our study some patients receiving theophylline were on & agonists. Therefore, it is not possible to exclude the possibility that catecholamines may play a role in hyperuricemia observed in addition to theophylline. In the trial in which sustained-release theophylline was administered to asthmatic patients, there was a significant correlation of serum concentrations of uric acid and theophylline in both male and female groups (Figs. 2!, 3, and 4). In accordance with this result, Yosselson-Superstine et al.‘O demonstrated that there was a positive correlation between serum theophylline and uric acid levels in two of three subjects receiving a single oral theophylline dose. As a next step we investigated the possible mechanisms of action of theophylline as a cause of hyperuricemiii. First, the effect of theophylline on uric acid clearance was evaluated. Theophylline did not inhibit uric acid clearance, suggesting that hyperuricemia induced by theophylline through inhibition of uric acid excretion is unlikely. HGPRTase salvages hypoxanthine or guanine in vertebrates for reuse in nucleotide and nucleic acid biosynthesis.” Deficiency in this enzyme results in accumulation of guanine and hypoxanthine, further degradation to uric acid, and then increased levels of serum uric acid. This condition is known as the Lesch-Nyhan syndrome characterized by mental retardation and self-mutilation.‘” It was reported that rats receiving high doses of caffeine demonstrated Lesch-Nyhan syndrome-like behavior with a decreased HGPRTase activity.‘” In the same study theophylline ingestion was also proved to cause the similar behavior. Furthermore, inhibition of HGPRTase activity by caffeine and theophylline has been described. I-* Therefore, we examined the effect of theophylline on HGPRTase activity in human erythrocytes. There was a slight inhibition at theophylline concentrations over 5mM that is considerably higher than therapeutic concentrations of theophylline (0.05mM to O.lmM). Therefore, increased serum uric acid levels induced by theophylline could not be ascribed to its effect in inhibiting HGPRTase. Uric acid is synthesized from hypoxanthine or guanine through the formation of xanthine. The inhibition of use of hypoxanthine or guanine by cells can cause hyperuricemia. Theophylline inhibited hypoxanthine uptake in K-562 cells by -50% at 1OmM that is more than 100 times as high as those achieved
Theophylline
+ 5t
1 0
I I
increases
uric
acid
levels
711
1 1 I 2 5 IO Theophylline Concentration (mM1
FIG. 6. Effect of theophylline on [Ylhypoxanthine uptake in K-562 cells when 1050f K-562 cells were incubated with 10 nmol of [‘Ylhypoxanthine (20.7 mCi/mmol) in the presence and absence of indicated concentrations of theophylline for 24 hr at 37” C. Cells were trapped on the glass fiber filter papers, and their radioactivities were measured.
Time
(min)
FIG. 7. Time course of inhibition of [Ylhypoxanthine take by theophylline when K-562 cells were with 5mM of theophylline and were harvested cated times. Cells were trapped on the papers, radioactivities were measured.
upincubated at indiand their
clinically. Thus theophylline-induced hyperuricemia could not be explained from this mechanism. Theophylline is metabolized by the action of varieties of enzymes to metabolites that are excreted into the urine. Jenne et al. lJ detected theophylline, 3-methylxanthine, 1,3-dimethyluric acid, and 1-methyluric acid in the urines from patients who received 800 or 1200 mg of aminophylline in 24 hr. Uric acid could be generated from 1-methyluric acid through l-demethylation. However, this pathway is not known at present. Recently, stimulation of release of adrenal cate-
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Morita
.! ALLERGY CLiit ,iwvvJ?u” WJVEMBER 1984
et al
cholammes by theophylline has been proposed as an additional mechanism of action. i’i Plasma epinephrine and norepinephrine levels were significantly increased at 3 hr after administration of theophylline or placebo therapy in a theophylline-treated group compared to a placebo-treated group. As already mentioned. intra venous administrations of isoproterenol and epineph,. rine were demonstrated to elevate uric acid levels 111 the rat ” Thus it may be possible to speculate that theophylline causes hyperuricemia via stimulation of release of adrenal catecholamincs. Finally, volume contraction caused by theophylline may lead to hyperuricemia as this mechanism is suggested as one of the causes of diuretic-induced hyperuricemia. Ii In conclusion there was a srgniticant Increase 01‘ serum uric acid levels in male asthmatic patients who received theophylline compared to male control subjects without theophylline. A significant correlation of serum levels of uric acid and theophylline was demonstrated in asthmatic patients who received 200 to 400 mg sustained-release theophylline. Further studies remain to be done in order to elucidate the exact mechanisms of theophylline-induced hyperuricemia. We thank
Miss
Emiko
Nagase
for typing
4.
\.
6.
-‘.
X. 9 10.
11 i 2.
13.
the manuscript, 1J.
REFERENCES 1. Ellis EF: Theophylline and derivatives. In Middle&on E Jr, Reed CE. Ellis EF, editors: Allergy: principles and practice. St. Louis. 1978, The CV Mosby Co, p 434 2. Kobayashi K, Noguchi H, Narimatsu H, Yokokawa T. Nishida M, Tsubomizu T, Odagiri T, Negishi M, Adachi M, Nakajima H. Shibuya T, Ide H, Takahashi T: Studies on elevated serum uric acid levels in patients with bronchial asthma. Jpn J Allergol 30:877. 1981 3. Liddle L, Seegmiller JE, Laster L: The enzymatic spec-
15
16.
lrophotometric method ior drtermmatn~n ill urri‘ *.!r! 6 i.,i! Clin Med 54:903. 1959 Buchanan OH. Block WD. Christman AA- The metabohsm ,:; the methylated purines. 1 The enzymatic determination of ur!nary uric acid. J Biol Chem 157:181. 1945 Adams RE. Vandemark Fl.. Schmidt GJ: Murc sens~tr~c high pressure lrquid-chromatographic determination of the I)phylline m serum Clin Chern ?21903. 1976 Rail TM;: Central nervous system stimulants: the xanthines ,I. ~;oodman IS. Gilnran -2. editors: Pharmacological haris o1 !hcrapeutic<, etl 0 New Yark, !98 1. Macmillan Publishing i::x. inc. pp 59i.lli!T Buchanan OH. C’hri,tman AA, Block WI): f-he tnttdbolism %)t the methylated purines. II. Llric acid excretion following the ingestion of caffeine. theophylline. and theobromine. J Rio! Chem 147 1X9. 1945 Harter SC;. Serum uric acid levels m patients with bronchia: asthma. i At.1 t~:~\ 4?:88. 1968 Sumi T. Umeda P Adrenergic regulation or the plasma ic\Ci:, rif purine rnetabolitea in the rat. Eur J Pharmacol 46:243. 1977 Yosselson-Superstine S. Granit D, Superstine E: Theophylhne interference with the phosphotungstate uric acid test. Am ! Hosp Pharm 37: 1522. 19gO lrhninger AL Biochemistry. cd L New York, 1377, Worth Publishers in<,. pp 729-747 I.csch M. Nyhan WL A familial disorder 01 uric acid metabo lism and central nervou\ system function. Am J Med 36.561 i Yh4 !%rrer 1, C~oslell M, Grtsoha S: Lesch-Nyhan syndrome-&kc behavior in rats lrom caffeine ingestion: changes in HGPRTasr ALtivity, urea, 4nJ some nitrogen metabolism enzymes. FEBS I.ctt 141.1’5I ,. 101(3 Nolan LL, Kidder GW: Caffeine: its action on purme metabohzing emymes Biochem Biophys Res Commun 91:253, 1979 lenne JW, Nagasawa HT. Thompson RD: Relationship of urinary metabolites of theophylline to serum theophylline levels Clin Pharmacoi Ther 19:375, 1976 Higbee MD, Kumar M. Galant SP: Stimulation of endogenous Latecholamine release by theophylline: a proposed additional mechanism of action for theophylline effects. J ALLERGY CI.IN i\l>lUNCii.
17
70:?7?
19x2
Steele TH, Oppenheimer S. bactors affecting urate excretion iollowmg diuretic administration in man. Am J Med 47:564. 1%hO