- Email: [email protected]
Effect of theophylline on cortisol secretion
Effect of theophytttne secretion
on coftisol
Jeffrey Tulin-Silver, M.D., David E. Schtdngwt, Kenneth P. Mathews, M.D. Ann Arbor. Mich.
M.D., and
Theophylline is thought to improve asthma by increusing intrucellulur cyclic adenosim 3’-5’-monophosphate (CAMP) levels. It has been demonstrated in experimental animals thut elevution of intracellular CAMP in the udrenal eortex causes an increased secretion of corrisol We studied whether therapeutic doses of theophylline given intruvenously and orally to human subjects over 3 days would increase cortisol secretion. A single-blind. 6-duy protocol was employed in Jive normul and jive asthmatic volunteers. Adrenal function wus monitored b) 8 A. M. and 4 P. M. serum cortisol and adrenocorticotropic hormone (ACTH) levels: daily 24-h,, rtrine for urinary-free cortisol (UFF). I7-hJdro.qsteroid.s ( I7-OH). und I7-ketosteroids (I 7-KS ); und ulternute-day cortisol secretoy rutes (FSR) measured b! isotope dilurion after intravenous ’ ‘C-cortisol. Serum theophylline concentration also wus monitored. Results in normal and u.sthmutic subjects were similar. Theophylline caused 11significunt but trunsient increase in L’F‘F and I7-OH excretion. Urine volumes ulso increased significantly, suggesting that the rend ej+iffivt of theophvlline accounted for the increased UFF und I7-OH excretion. FSR increused during the first 24 hr ufter theophylline in eight of nine cuses (p C: 0.05 bv sign test). meun vu1ue.s increusing front 14.2 to 19.3 mg. but this effect had dissipated by day 3 of theophylline odministrution. In contrast to these findings. theophylline bud no efftlct on serum cortisol or ACTH or urinury I7-KS. It is likely that serum cortisol und ACTH remained unchun,qed becurcse the increase in cortisol secretion wus offset by u concomitant increase in cortisol cleurance. It is concluded that theophylline produces u smull. trunsient increase in cortisol secretion and clearunce. and this qfsect is similur in asthmatic and normal subjects.
A biochemical basis for the pharmacologic treatmeat of bronchial asthma has been the increase in intracellular levels of cyclic adenosine 3’-5’-monophasphate (cyclic AMP). Theophylline increases CAMP through its inhibition of cyclic nucleotide phoqhodiesterase. This results in inhibition of bronchial smooth muscle contraction and inhibition of mediator release in asthmatic subjects. 1~the adrenal gland Grahame-Smithet al. showed in 1967l that CAMP was the intracellular mediator of From the Department of Medicine, Sections of Allergy and Endocr.aology. University of Michigan School of Medicine. Supported in part by U.S. Public Health Service Grant No. 2..#0 I -RROOO42. Presented in part at the Thirty-sixth Annual Meeting of the American Academy of Allergy. Atlanta, Ga., Feb.. 1980. Rece*ved for publication April 9, 1980. Accqted for publication Aug. 15. 1980. Rep&t requests to: Kenneth P. Mathews. M.D.. University of ?v%chigan Medical Center, Section of Allergy, D3141 North Olapatient Bldg., Ann Arbor. MI 48109.
oo91-67491811010045+06$00.6010
0
1981
The
C. V. Mosby
the action of adrenocorticotropic hormone (ACTH). In the adrenal cortex, however, increases in CAMP concentrations induced by ACTH always preceded rises in the rate of adrenal steroidogencsis; and adrenal concentration of CAMP remained elevtid while the rate of steroidogenesiswas maintained.’ Espiner et al.* demonstratedalso in sheepthat the ekvation of intracellular CAMP was the necessary physiologic event for increased adrenal stemidogeaesisand cortisol secretion. Increasedintracellular CAMP could be produced by infusions of either ACTH or aminophylline; moreover, the effects on cyclic nuckotide were additive when both compoundswerr! infused simultaneously. This was an expectedfindieg because ACI’H increased CAMP by adenyl cyclase stimulation, whereasaminophylline causedelevated CAMP by inhibition of phosphodiesterase. In the only study published to date involving human subjects,3 aminophyiline given intravenously to six normal women had no effect on plasmacortisol levels; however. serum theophylline concentrations Co.
Vol.
67, No.
1, pp. 45-50
J. ALLERGY
46 Tulin-Silver et al.
CLIN. IMMUNOL. JANUARY 1981
PROTOCOL DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
DAY 6 I
2AM
ENTER STUDY
BAM
ACTH
ACTH
PLACEBO (1.V.l + 15C,,J + 30 + 60 + 90
PLACEBO
PLACEBO
THEO
PLACEBO ACTH
PLACEBO ACTH PF
THEO ACTH
24” U
AMINO hv.)+ 15(C,,,) * 30 + 60 + 90
24’ U
DAY 7
1 PM
THEO I
ACTH PF
BPM
PLACEBO
PLACEBO
THEO
THEO
+
THEO
I
FIG. 1. Study protocol.
were not measured, and therefore it is not known whether therapeutic levels of theophylline were attained. Also, adrenal gland function was determined solely by serum cortisol concentrations, and these measurementswere collected for only 180 min after eachintravenous infusion. Thus it remained to be verified whether theophylline, in monitored quantities or following long-term administration, had a demonstrable effect on cortisol secretion in humans. MATERIALS AND METHODS Subjects A single-blind experiment was run using five normal, healthy volunteers who had no significant past or current history of allergic disease. They had a normal physical examination and a normal biochemical profile (complete blood count, SMA-17, urinalysis, chest x-ray film). For comparison, five additional volunteers were selected whose primary medical problem was extrinsic bronchial asthma. These patients were selected on the basis of a pertinent allergic history without other significant medical problems, a corroborating physical exam, a normal biochemical profile (see above), and skin tests positive for relevant aeroallergens. They had not received any corticosteroid drugs within the past year. The normal subjects ranged in age from 20 to 30 yr and the asthmatics from 19 to 34 yr; in each group, four of five were women. Although two of the asthmatics had recently used theophylline therapeutically, none of the subjects was on any medication for at least 72 hr prior to the start of this study.
Informed consent was obtained from all patients after all attendant risks were explained. Subjects were admitted to the Clinical Research Center for 6 days. At the conclusion of the study, one of the “control” subjects was found to have markedly abnormal baseline adrenal function values, and all of her data was eliminated. Thus, final results compared four normal volunteers with five asthmatic subjects.
Protocol The following protocol was followed over a 6-day period (Fig. 1). Days 1 to 3. Serum collected at 8 A.M. and 4 P.M. daily was assayed for cortisol (PF) and ACTH levels by radioimmunoassay.4* 5 Baseline 24-hr urines were collected with urinary-free cortisol (UFF),4 urinary 17-hydroxysteroids ( 17-OH),6 and urinary 17-ketosteroids (17-KS)’ being determined daily. Completeness of urine collections was verified by daily determinations of creatinine clearance. Placebo capsules identical to capsules containing theophylline were administered orally at 8 A.M., 2 P.M., 8 P.M., and 2 A.M. At 8 A.M. on day 2, a cortisol secretion rate determination (FSR) was initiated by infusing 1 @Zi of L4C-cortisol in 50 ml 5% dextrose in water over 20 min and assessing urinary metabolites by isotope dilution as described by Schteingart et a1.aThis was followed by an additional 50 ml of 5% dextrose in water over 20 min (the latter to simulate the procedure on day 4, as described below). Serum cortisol and ACTH measurements were repeated at 15, 30, 60, and 90 mm after completing the infusions as a control for the measurements on day 4. A control serum
VC.. JME NCI’IBER
67 1
Theophylline
TABLE 1. Urinary -.
corticosteroid
Theophylline (days 4-4 17-Ketosteroids
-. 13.46 f 3.16 10.27 -e 4.42
C1mtr0ls
Asthmatic5
-.
-..
43.70 38.67
Controls
II. Renal
TWLE
-.
effects
of theophylline
-_.
(UFF) (pgl24
-. Corr :&on
1.420
2
1,932
-t 2615
t
329
clearance
4)
2,324 ? 1s4*.; 2,569 .T 31 I$#
l5W
(mllmin)
III % 26 103 -t 7 t-t)
(6Y
(ml124 hr)
I.503
I03 ? 2.6 107 ? 7
of data indicated by like symbols (e.g.. *-*,
lo6 t 28 III -c_ I2
I32 .t 36 II3 ? I.!
indicates p T 0.05. by paired t rest.
theq>hylline* concentration also was measured at 9 A.M. and ’ P.M. DC,VSJ to 6. At 8 A.M. on day 4 the FSR was repeated by intravenously over 20 min. infusing 1 &i of liC-corti~l Immediately thereafter, 7 mglkg aminophylline were infusec over 20 min” ‘I followed by sampling for serum cortis,)l and ACTH at 15, 30, 60, and 90 min in the manner described above. Serum theophylline concentrations were obtained I5 min after the infusion was complete (Cp max) and again 4 hr later (Cp min). These theophylline levels were .lsed to determine subsequent oral theophylline dosage by estimating each patient’s elimination rate constant (Ke) for tb.:ophylline from the following equatior?
(I) T concentration drawn 15 where Cp,,, was the theophylline min &er the aminophylline infusion, Cp,,, the theophylline c;mcentration 4 hr later, and T equalled 4 hr minus the time mtetval between the two levels. Each subject’s theo-.-. *Uni\trrsity of Michigan Pharmacy Laboratory: High-pressure liquid ; hromatography.
Y3.90 *. 57.24*+ xx.95 : 31.71$8
ThO@@ii
Placebo (day 2)
1,565 z I35
Creatinine Controls Asthmatics
hr)
SO.20 + 11.4st 44.28 + 1.5.99s
Theophylline (days 4-6)
1.232 2 l35* 1,783 2 340$
-..
12.98 . 7.89*.; 7.85 .I I .S7$4
indicates p <: 0.05. by paired I test.
Urine volume Controls Asthmatics
‘. 4.97
and placebo
Placebo (days l-3) -..
12.78 -. 3.x 10.0s
+I 5.28
6.83 5 ‘.13i 5.30 -t 2.12gj
56.71 t 36.22 62.58 ZI 28.32
-. Com,?arison of data indicated by like symbols (e.g., *-*. t-t)
13.60 2 4.71 I I.18
(17-OH) (mg124 hr)
free cortisol
2 15.03* k 17.14$
Theophyfline way 4)
MaY 2)
IO.31 2 5.36 6.33 2 2.73 Urinary
-..
Placebo
12.43 -c 3.64 8.93 -t 3.33
6.47 k 2.21* 5.16 -e 1.54$
Asthmatics
47
(17.KS) (mg124 hr)
17-Hydroxystemids
-.. Controls Asthmatics
secretion
excretion
PkebO (days l-3)
-
and cortisol
phylline clearance lated as follows”:
(C1.r). in liters per hour. was then calcu-
Cl,r = (Ke) (V;,)
(21
where Ke was derived from equation 1, and V, was the volume of distribution of theophylline, known from previous studies to be (0.45) (patient’s weight in kilograms).” The amount of theophylline, in milligrams per dose, that each subject would need to take to attain a therapeutic serum concentration was then calculated from the equation”‘: Dose,,,
= CCP, WT) 0-b
(3)
where Cjj was the average Serum tbeophylline concentration desired (I5 pg/ml), and T the time interval between oral theophylline doses (6 hr). This calculated dose was given throughout the remainder of the study. Serum theophylline concentrationsY were measured at 4 P.M. on days 5 and 6. substantiating that therapeutic drug concentrations were maintained.
Statisticrl
an8lysis
Theophylline’s effect on adrenal gland function was determined in four different ways. I. Long-term effects were defined as those effects of
J. ALLERGY
48 Tulin-Silver et al.
TABLE III. 8
A.M.
serum cortisol
Placebo (days l-3)
and ACTH concentrations
Theophylline (days 4-6)
Day 2 (IV placebo) 0”
+15”
Serum cortisol
Controls
17.5 k 2.3
Asthmatics
19.0 rt I.0
CLIN. IMMUNOL. JANUARY 1981
20.2 t 2.9 20.9 ? 5.1
16.2 k 2.5 19.0 2 1.9
+30”
+ 60”
f90”
14.6 k 2.9 13.1 k 2.2
12.5 ‘-t 4.0 11.7 t 2.9
10.3 k 2.8 11.9 k 3.1
(pgldl)
15.2 + 1.3 13.5 2 2.9
Serum ACTH (pglml)
Controls Asthmatics
75.0 2 47.1
66.8 2 25.2
58.8 +- 17.5
62.8 ? 39.5
83.7 k 89.4
61.6 k 38.8
54.5 2 26.5
99.6 2 46.2
91.1 + 35.2
90.0 I 43.1
62.9 ? 20.6
62.3 ? 24.3
64.6 L 25.3
65.1 ? 27.9
theophylline that persistedfor 72 hr. Baseline data collected during the first 3 days of the protocol were comparedwith dataobtained from days 4 to 6. Information from the control subjects was compared with that obtained from the asthmatic patients. Since the time intervals and the distribution of data were similar, statistical significance was determined by the Student’s t test.“’ 2. Transient effects of theophylline were defined as those changes caused by theophylline that may have persistedfor only 24 hr. Accordingly, dataobtained from day 4 (the first day in the protocol that active drug was given) were comparedwith baseline results (days 1 to 3) and with results during the entire interval during which theophylline was administered(days 4 to 6). Data obtained from controls were compared with those obtained from asthmatic subjects. Since the intervals for comparison were different (24 hr vs 72 hr), the test used to measurestatistical significance was the t test for paired observations (paired t test).‘” 3. Short-term effects of theophylline were those effects that the drug might have had on the adrenal gland for the 90 min during which aminophylline was infused. These intravenous infusions produced a series of linear measurements that were most appropriately analyzed by the statistical test of profile analysis.I6 4. All of the above tests used mean values (2 1 SD) to determine statistical significance. Becauseit seemedpossible that changes in absolute quantities might be small, we also decided to employ the sign test,” a statistical analysis measuringpositive and negative deviations from initial values. The sign test was especially appropriatein considering cortisol secretory rates. Because the normal physiologic clearance of i4C-cortisol isotope required 48 hr,8 these secretory rates could be determined only on alternate days. Subsequently, the sign test could comparebaseline adrenal secretory rates (day 2 of the protocol) with results obtained on the first day that theophylline was given (day 4) and with values obtained after more prolonged theophylline administration (day 6).
RESULTS The coefficient of variation for the control measurements (days 1, 2, and 3) was determined for each subject. The mean coefficients of variation for the
nine subjects were lo%, 22%, 34%, 21%, and 16% for the 17-K& 17-OH, UFF, morning serum cortisol, and morning ACTH values, respectively. Table I shows the values obtained for 17-KS. The results were similar whether long-term or transient differences were analyzed. No statistically significant differences were observed. The values obtained for 17-OH and UFF (Table I) illustrate that the only significant differences were between the baseline values (days 1 to 3, day 2) and the transient values (day 4). These differences occurred in both the control and the asthmatic subjects. No significant differences were noted between the control and asthmatic subjects. Table II shows the renal effects of placebo and tbeophylline. Creatinine clearances remained unchanged, but urine volumes were significantly affected and displayed a pattern similar to values obtained for 17-OH and UFF. Because all patients had normal renal function and had unlimited access to water throughout the study, Table II demonstrates that urine volumes increased greatly on day 4 of the protocol. Moreover, Table II shows that this increase had dissipated by the end of the study (days 4 to 6 vs days 1 to 3, not significantly different). Table III shows that values obtained for 8 A.M. serum cortisol and ACTH were not significantly changed whether these numbers were compared for long-term effects (days 1 to 3 vs days 4 to 6) or for short-term changes following intravenous infusion. Serum cortisol and ACTH concentrations at 4 P.M. (not shown) had normal trough values for both control and asthmatic subjects during both placebo and theophylline administration. Fig. 2 shows that the mean FSR increased from a mean value on day 2 of 14.2 to 19.3 mg/24 br on day 4; it subsequently decreased to 17.8 mg/24 hr on day 6. Because the standard deviations were large, these changes were not significant. Looking at each subject’s FSR, however, it can be seen that eight of nine
VOL JME 67 NWBER 1
Theophylline
and cortisol
secretion
49
Day 4 (IV theo~ine)
W.” 0”
+15*
Serum cortisoi I6 1 r 5.5 20 I -e 2.2
15.6 2 4.0 14.3 Lk 3.5
+I#
+90”
16.1 -e 4.4 14.5 k 4.6
14.0 Itr 2‘5 15.0 t 4.2
62.1 L 31.9 75.9 ? 40.1
57.3 t 29.1 77.0 .c_ 39.2
+30 (pgldl)
17.0 f 4.8 14.9 k 4.0 Serum ACTH (pg/ml)
-. 53 i + 15.3 76.1) + 26.3
60.6 _) 29.0 68.3 2 33.8
71.4 2 46.7 64.1 2 45.8
subjects had a higher FSR on day 4 than on day 2. Thk is a significant observation (p < 0.05) by the sign test of trends. By day 6, neither mean values by the paired t test nor direction of change by the sign test was significant. Strum theophylline concentrations were measured throughout the protocol (Fig. l).* Although four of nine. subjects suffered mild to moderate side effects and subsequentrequired slightly decreasedoral theophylline doses, no patient terminated the study because of drug toxicity and no patient missed a prescribed dose of theophylline. Of the 20 serum theophyl‘ine concentrations measuredon days 5 and 6 of the study, 15 values (75%) were within the therapeutic r;mge (10 to 20 ,uglml), and all values were between 8.6 to 27 pg/ml. Theophylline levels on days 4 and 6 were not significantly correlated with cortisol secr&ory values on these same 2 days. DEWJSW3N
M-.)st allergists consider theophylline a major, fir&fine drug for acute and chronic asthma.‘* Its ability t-1 produce bronchodilatation and relaxation of large:airways is dose related,‘O,l1 and it effects these changesvia an increasein intracellular cyclic AMP.lM Becatisethe samesequenceof biochemical changesin the &enal gland initiates cortisol secretion,‘* 2, ly we studied whether theophylline given to human subjects in therapeutic dosescould affect adrenal metabolism. WC found that theophylline had a significant diuretic effect in both control and asthmatic subjects, great-y increasing urine volumes (Table II). As noted earlier, 17-OH and UFF excretion also significantly increased on day 4. Although these metabolites are sensitive indicators of adrenocortical metabolism, they are also affected by changes in renal filtration rates,20I7-KS were not significantly changed during -.. -*Univ: rsity of Michigan Pharmacy Laboratory: High-pressure Iquid ~.hromatography.
,-
,-
I-
)L
FIG. 2. Changes
in cortisol
secretory
rates.
the study period (Table I), and we attribute this to the fact that 17-KS are unaffected by renal filtration mechanismsZoand are only “a rough estimate of the integrity of the adrenal cortex . . giving no information about the gland’s capacity to respond to acute [change]. ‘Q’ Theophylline’s diuretic effect was transient, having dissipated within 72 hr after the drug had been given continuously, suggesting that asthmatics maintained on long-term theophyline therapy neednot be concernedabout dehydration secondaryto this bronchodilator. Theophylline did not cause any short- or long-term changesin cortisol or ACTH concentrations in either
50
Tulin-Silver
J. ALLERGY
et al.
asthmaticor control subjects(Table III). Using profile analysis,16 we compared the slopes of the lines produced by intravenous placebo and intravenous aminophylline administration. The correlation coefficient for these lines equalled 0.82, suggestingthe lines were nearly flat. This substantiatedthat neither cortisol nor ACTH levels varied from control values. FSRs are a precise method of assessingadrenocortical secretion8and also are unaffected by renal filtration ratese20We found that theophylline did cause an increasein baseline FSRsfrom 14.2 to 19.3 mg/24 hr on day 4. Although this mean increase was not significant (p > 0.05), a small increase occurred in nearly all (eight of nine) subjects (p < 0.05 by sign test). This increase was transient, however, and all values and trends had returned toward baseline values by the third day of theophylline administration. In summary, we postulate that theophylline had the following effects on adrenocortical function. Theophylline had a definite diuretic effect as shown by the significant increasesin urine volumes and 17-OH and UFF excretion. We feel that the marked rise of these adrenocortical metabolites through the kidney caused a temporary fall in serum cortisol concentrations, which by negative feedback to the pituitary gland caused a transient rise in ACTH. At the same time theophylline caused a small transient rise in FSR. This increase in adrenocortical secretion temporarily elevated the serum cortisol concentration, which, in turn, by negative feedback to the pituitary caused a fall in ACTH. As a result theophylline’s two effects on serum cortisol and ACTH offset each other. It is concluded that theophylline, in therapeutic quantities, produced a small, transient increase in cortisol secretion and clearance and that these effects were similar in asthmatic and normal subjects. Also, the concentration of theophylline that caused an increased secretion of cortisol in the animal studies noted earlier* was approximately one hundred times (100 X ) that usedto achieve the monitored therapeutic blood concentrationsin our human subjects. Short- or long-term administration of theophylline, in usually recommendeddoses,‘O*11,l8 has no significant overall effect on adrenocortical function. We wish to thank Gary E. Johnson, Pharm.D., and M. Anthony Schork, M.PH., Ph.D., for their professional advice and assistance. REFERENCES
1. Grahame-Smith DC, Butcher RW, Ney RL, SutherlandEW: Adenosine 3’S’-monophosphate as the intracellular mediator
2 I.
3.
4.
5.
CLIN. IMMUNOL. JANUARY 1981
of the action of adrenocorticotropic hormone on the adrenal cortex. J Biol Chem 242:5535, 1967. Espiner EA, Livesey JH, Ross J, Donald RA: Dynamics of cyclic adenosine 3’,5’-monophosphate release during adrenocortical stimulation in viva. Endocrinology 95:838, 1974. Angeli A, et al: Inefficacia Della Teofillina Nel PotenziareLa Risposta Cortisolemica All ’ Iniezione Endovenosa Impulsiva Di Dibutiril Adenosin 3’,5’-Monofostate Ciclico Nell’ Uomo (The ineffectivenessof theophylline in potentiating the cortisol response bolus intravenous injection of dibutyryl adenosine 3’,5’-monophosphate in man). Bolletino, Societa Italiana di Biologia Sperimentale 50~366, 1974. Dash AJ, England BE, Midgley AR, Niswender GD: Specific non-chromatographicradioimmunoassay for human plasma. Steroids 26:647, 1975. Vague PH, Oliver C, Jaquet P, Vague J: Le DosageRadioimmunologique De L’ ACTH Plasmatique. Eur J Clin Biol Res
16483, 1971. 6. Silber CC, Porter RH: The determination of 17,21-dihy-
droxy-20-ketosteroids in urine and plasma. J Biol Chem 210:923, 1954. 1. Chaney AL: Standard II-ketosteroids, in Urine methods of
clinical chemistry, ed. 2. New York, 1958, Academic Press, Inc., p. 79. 8. Schteingart DE, Gregerman RI, Conn JW: A comparison of the characteristics of increased adrenal cortical function in obesity and Cushing’s syndrome. Metabolism 12:484, 1963. 9. Wagner JG: The one compartment open model with constant rate intravenousinfusion, in Fundamentalsof clinical pharmacokinetics, ed. 1. Hamilton, Ill., 1975,Drug Intelligence Publications, Inc., p. 72. 10. Piafsky KM, Ogilvie RI: Dosage of theophylline in bronchial asthma. New Engl J Med 292:1218, 1975. 11. Mitenko PA, Ogilvie RI: Rational intravenous dosesof theophylline. New EngI J Med 289:600, 1973. 12. JenneJW, Wyze E, Rood FS, et al: Phatmacokineticsof theophylline. Application to adjustment of the clinical dose of aminophylline. Clin Pharmacol Ther 13:349, 1972. 13. Slotfeldt ML, JohnsonCE, Grambau GR, Weg JG: Reliability of theophylline clearance in determining chronic oral dosage regimens. Am J Hosp Pharm 34:66, 1979. 14. Sokal RS, Rohlf FJ: Introduction to biostatidcs. San Francisco, 1969,W. H. Freemanand Co. Publishers,pp. 107-109. 15. Edwards AL: Statistical methods, ed. 2. New York, 1967, Holt, Rinehart and Winston, Inc., pp. 215-226. 16. Kshirsajar AH: Multivariate analysis. New York, 1978, Marcel Dekker, Inc., pp. 161-163. 17. Sokal RS, Rohlf FJ: Introduction to biostatistics. San Francisco, 1969,W. H. FreemanandCo. Publishers,pp. 222-223. 18. Ellis EF: Theophylline and Derivatives, in Middleton E, Reed CE, Ellis EF, editors: Allergy: Principles and practice. St. Louis, 1978, The C. V. Mosby Co., p. 434. 19. Williams GH, et al: Diseasesof the adrenal cortex, in Thorn GW, Adams RD, et al, editors: Harrison’s principles of internal medicine. New York, 1977, McGraw-Hill Book Co., p. 524. 20. Peterson RE: Metabolism of adrenal cortical steroids, in Christy NP, editor: The human adrenal cortex. New York, 1971, Harper & Row, Publishers, chap. 4, pp. 87-189. 21. Christy NP: Iatrogenic Cushing’s syndrome, in Christy NP, editor: The human adrenalcortex. New York, 1971, Harper & Row, Publishers, chap. 14, p. 407.
on coftisol
Jeffrey Tulin-Silver, M.D., David E. Schtdngwt, Kenneth P. Mathews, M.D. Ann Arbor. Mich.
M.D., and
Theophylline is thought to improve asthma by increusing intrucellulur cyclic adenosim 3’-5’-monophosphate (CAMP) levels. It has been demonstrated in experimental animals thut elevution of intracellular CAMP in the udrenal eortex causes an increased secretion of corrisol We studied whether therapeutic doses of theophylline given intruvenously and orally to human subjects over 3 days would increase cortisol secretion. A single-blind. 6-duy protocol was employed in Jive normul and jive asthmatic volunteers. Adrenal function wus monitored b) 8 A. M. and 4 P. M. serum cortisol and adrenocorticotropic hormone (ACTH) levels: daily 24-h,, rtrine for urinary-free cortisol (UFF). I7-hJdro.qsteroid.s ( I7-OH). und I7-ketosteroids (I 7-KS ); und ulternute-day cortisol secretoy rutes (FSR) measured b! isotope dilurion after intravenous ’ ‘C-cortisol. Serum theophylline concentration also wus monitored. Results in normal and u.sthmutic subjects were similar. Theophylline caused 11significunt but trunsient increase in L’F‘F and I7-OH excretion. Urine volumes ulso increased significantly, suggesting that the rend ej+iffivt of theophvlline accounted for the increased UFF und I7-OH excretion. FSR increused during the first 24 hr ufter theophylline in eight of nine cuses (p C: 0.05 bv sign test). meun vu1ue.s increusing front 14.2 to 19.3 mg. but this effect had dissipated by day 3 of theophylline odministrution. In contrast to these findings. theophylline bud no efftlct on serum cortisol or ACTH or urinury I7-KS. It is likely that serum cortisol und ACTH remained unchun,qed becurcse the increase in cortisol secretion wus offset by u concomitant increase in cortisol cleurance. It is concluded that theophylline produces u smull. trunsient increase in cortisol secretion and clearunce. and this qfsect is similur in asthmatic and normal subjects.
A biochemical basis for the pharmacologic treatmeat of bronchial asthma has been the increase in intracellular levels of cyclic adenosine 3’-5’-monophasphate (cyclic AMP). Theophylline increases CAMP through its inhibition of cyclic nucleotide phoqhodiesterase. This results in inhibition of bronchial smooth muscle contraction and inhibition of mediator release in asthmatic subjects. 1~the adrenal gland Grahame-Smithet al. showed in 1967l that CAMP was the intracellular mediator of From the Department of Medicine, Sections of Allergy and Endocr.aology. University of Michigan School of Medicine. Supported in part by U.S. Public Health Service Grant No. 2..#0 I -RROOO42. Presented in part at the Thirty-sixth Annual Meeting of the American Academy of Allergy. Atlanta, Ga., Feb.. 1980. Rece*ved for publication April 9, 1980. Accqted for publication Aug. 15. 1980. Rep&t requests to: Kenneth P. Mathews. M.D.. University of ?v%chigan Medical Center, Section of Allergy, D3141 North Olapatient Bldg., Ann Arbor. MI 48109.
oo91-67491811010045+06$00.6010
0
1981
The
C. V. Mosby
the action of adrenocorticotropic hormone (ACTH). In the adrenal cortex, however, increases in CAMP concentrations induced by ACTH always preceded rises in the rate of adrenal steroidogencsis; and adrenal concentration of CAMP remained elevtid while the rate of steroidogenesiswas maintained.’ Espiner et al.* demonstratedalso in sheepthat the ekvation of intracellular CAMP was the necessary physiologic event for increased adrenal stemidogeaesisand cortisol secretion. Increasedintracellular CAMP could be produced by infusions of either ACTH or aminophylline; moreover, the effects on cyclic nuckotide were additive when both compoundswerr! infused simultaneously. This was an expectedfindieg because ACI’H increased CAMP by adenyl cyclase stimulation, whereasaminophylline causedelevated CAMP by inhibition of phosphodiesterase. In the only study published to date involving human subjects,3 aminophyiline given intravenously to six normal women had no effect on plasmacortisol levels; however. serum theophylline concentrations Co.
Vol.
67, No.
1, pp. 45-50
J. ALLERGY
46 Tulin-Silver et al.
CLIN. IMMUNOL. JANUARY 1981
PROTOCOL DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
DAY 6 I
2AM
ENTER STUDY
BAM
ACTH
ACTH
PLACEBO (1.V.l + 15C,,J + 30 + 60 + 90
PLACEBO
PLACEBO
THEO
PLACEBO ACTH
PLACEBO ACTH PF
THEO ACTH
24” U
AMINO hv.)+ 15(C,,,) * 30 + 60 + 90
24’ U
DAY 7
1 PM
THEO I
ACTH PF
BPM
PLACEBO
PLACEBO
THEO
THEO
+
THEO
I
FIG. 1. Study protocol.
were not measured, and therefore it is not known whether therapeutic levels of theophylline were attained. Also, adrenal gland function was determined solely by serum cortisol concentrations, and these measurementswere collected for only 180 min after eachintravenous infusion. Thus it remained to be verified whether theophylline, in monitored quantities or following long-term administration, had a demonstrable effect on cortisol secretion in humans. MATERIALS AND METHODS Subjects A single-blind experiment was run using five normal, healthy volunteers who had no significant past or current history of allergic disease. They had a normal physical examination and a normal biochemical profile (complete blood count, SMA-17, urinalysis, chest x-ray film). For comparison, five additional volunteers were selected whose primary medical problem was extrinsic bronchial asthma. These patients were selected on the basis of a pertinent allergic history without other significant medical problems, a corroborating physical exam, a normal biochemical profile (see above), and skin tests positive for relevant aeroallergens. They had not received any corticosteroid drugs within the past year. The normal subjects ranged in age from 20 to 30 yr and the asthmatics from 19 to 34 yr; in each group, four of five were women. Although two of the asthmatics had recently used theophylline therapeutically, none of the subjects was on any medication for at least 72 hr prior to the start of this study.
Informed consent was obtained from all patients after all attendant risks were explained. Subjects were admitted to the Clinical Research Center for 6 days. At the conclusion of the study, one of the “control” subjects was found to have markedly abnormal baseline adrenal function values, and all of her data was eliminated. Thus, final results compared four normal volunteers with five asthmatic subjects.
Protocol The following protocol was followed over a 6-day period (Fig. 1). Days 1 to 3. Serum collected at 8 A.M. and 4 P.M. daily was assayed for cortisol (PF) and ACTH levels by radioimmunoassay.4* 5 Baseline 24-hr urines were collected with urinary-free cortisol (UFF),4 urinary 17-hydroxysteroids ( 17-OH),6 and urinary 17-ketosteroids (17-KS)’ being determined daily. Completeness of urine collections was verified by daily determinations of creatinine clearance. Placebo capsules identical to capsules containing theophylline were administered orally at 8 A.M., 2 P.M., 8 P.M., and 2 A.M. At 8 A.M. on day 2, a cortisol secretion rate determination (FSR) was initiated by infusing 1 @Zi of L4C-cortisol in 50 ml 5% dextrose in water over 20 min and assessing urinary metabolites by isotope dilution as described by Schteingart et a1.aThis was followed by an additional 50 ml of 5% dextrose in water over 20 min (the latter to simulate the procedure on day 4, as described below). Serum cortisol and ACTH measurements were repeated at 15, 30, 60, and 90 mm after completing the infusions as a control for the measurements on day 4. A control serum
VC.. JME NCI’IBER
67 1
Theophylline
TABLE 1. Urinary -.
corticosteroid
Theophylline (days 4-4 17-Ketosteroids
-. 13.46 f 3.16 10.27 -e 4.42
C1mtr0ls
Asthmatic5
-.
-..
43.70 38.67
Controls
II. Renal
TWLE
-.
effects
of theophylline
-_.
(UFF) (pgl24
-. Corr :&on
1.420
2
1,932
-t 2615
t
329
clearance
4)
2,324 ? 1s4*.; 2,569 .T 31 I$#
l5W
(mllmin)
III % 26 103 -t 7 t-t)
(6Y
(ml124 hr)
I.503
I03 ? 2.6 107 ? 7
of data indicated by like symbols (e.g.. *-*,
lo6 t 28 III -c_ I2
I32 .t 36 II3 ? I.!
indicates p T 0.05. by paired t rest.
theq>hylline* concentration also was measured at 9 A.M. and ’ P.M. DC,VSJ to 6. At 8 A.M. on day 4 the FSR was repeated by intravenously over 20 min. infusing 1 &i of liC-corti~l Immediately thereafter, 7 mglkg aminophylline were infusec over 20 min” ‘I followed by sampling for serum cortis,)l and ACTH at 15, 30, 60, and 90 min in the manner described above. Serum theophylline concentrations were obtained I5 min after the infusion was complete (Cp max) and again 4 hr later (Cp min). These theophylline levels were .lsed to determine subsequent oral theophylline dosage by estimating each patient’s elimination rate constant (Ke) for tb.:ophylline from the following equatior?
(I) T concentration drawn 15 where Cp,,, was the theophylline min &er the aminophylline infusion, Cp,,, the theophylline c;mcentration 4 hr later, and T equalled 4 hr minus the time mtetval between the two levels. Each subject’s theo-.-. *Uni\trrsity of Michigan Pharmacy Laboratory: High-pressure liquid ; hromatography.
Y3.90 *. 57.24*+ xx.95 : 31.71$8
ThO@@ii
Placebo (day 2)
1,565 z I35
Creatinine Controls Asthmatics
hr)
SO.20 + 11.4st 44.28 + 1.5.99s
Theophylline (days 4-6)
1.232 2 l35* 1,783 2 340$
-..
12.98 . 7.89*.; 7.85 .I I .S7$4
indicates p <: 0.05. by paired I test.
Urine volume Controls Asthmatics
‘. 4.97
and placebo
Placebo (days l-3) -..
12.78 -. 3.x 10.0s
+I 5.28
6.83 5 ‘.13i 5.30 -t 2.12gj
56.71 t 36.22 62.58 ZI 28.32
-. Com,?arison of data indicated by like symbols (e.g., *-*. t-t)
13.60 2 4.71 I I.18
(17-OH) (mg124 hr)
free cortisol
2 15.03* k 17.14$
Theophyfline way 4)
MaY 2)
IO.31 2 5.36 6.33 2 2.73 Urinary
-..
Placebo
12.43 -c 3.64 8.93 -t 3.33
6.47 k 2.21* 5.16 -e 1.54$
Asthmatics
47
(17.KS) (mg124 hr)
17-Hydroxystemids
-.. Controls Asthmatics
secretion
excretion
PkebO (days l-3)
-
and cortisol
phylline clearance lated as follows”:
(C1.r). in liters per hour. was then calcu-
Cl,r = (Ke) (V;,)
(21
where Ke was derived from equation 1, and V, was the volume of distribution of theophylline, known from previous studies to be (0.45) (patient’s weight in kilograms).” The amount of theophylline, in milligrams per dose, that each subject would need to take to attain a therapeutic serum concentration was then calculated from the equation”‘: Dose,,,
= CCP, WT) 0-b
(3)
where Cjj was the average Serum tbeophylline concentration desired (I5 pg/ml), and T the time interval between oral theophylline doses (6 hr). This calculated dose was given throughout the remainder of the study. Serum theophylline concentrationsY were measured at 4 P.M. on days 5 and 6. substantiating that therapeutic drug concentrations were maintained.
Statisticrl
an8lysis
Theophylline’s effect on adrenal gland function was determined in four different ways. I. Long-term effects were defined as those effects of
J. ALLERGY
48 Tulin-Silver et al.
TABLE III. 8
A.M.
serum cortisol
Placebo (days l-3)
and ACTH concentrations
Theophylline (days 4-6)
Day 2 (IV placebo) 0”
+15”
Serum cortisol
Controls
17.5 k 2.3
Asthmatics
19.0 rt I.0
CLIN. IMMUNOL. JANUARY 1981
20.2 t 2.9 20.9 ? 5.1
16.2 k 2.5 19.0 2 1.9
+30”
+ 60”
f90”
14.6 k 2.9 13.1 k 2.2
12.5 ‘-t 4.0 11.7 t 2.9
10.3 k 2.8 11.9 k 3.1
(pgldl)
15.2 + 1.3 13.5 2 2.9
Serum ACTH (pglml)
Controls Asthmatics
75.0 2 47.1
66.8 2 25.2
58.8 +- 17.5
62.8 ? 39.5
83.7 k 89.4
61.6 k 38.8
54.5 2 26.5
99.6 2 46.2
91.1 + 35.2
90.0 I 43.1
62.9 ? 20.6
62.3 ? 24.3
64.6 L 25.3
65.1 ? 27.9
theophylline that persistedfor 72 hr. Baseline data collected during the first 3 days of the protocol were comparedwith dataobtained from days 4 to 6. Information from the control subjects was compared with that obtained from the asthmatic patients. Since the time intervals and the distribution of data were similar, statistical significance was determined by the Student’s t test.“’ 2. Transient effects of theophylline were defined as those changes caused by theophylline that may have persistedfor only 24 hr. Accordingly, dataobtained from day 4 (the first day in the protocol that active drug was given) were comparedwith baseline results (days 1 to 3) and with results during the entire interval during which theophylline was administered(days 4 to 6). Data obtained from controls were compared with those obtained from asthmatic subjects. Since the intervals for comparison were different (24 hr vs 72 hr), the test used to measurestatistical significance was the t test for paired observations (paired t test).‘” 3. Short-term effects of theophylline were those effects that the drug might have had on the adrenal gland for the 90 min during which aminophylline was infused. These intravenous infusions produced a series of linear measurements that were most appropriately analyzed by the statistical test of profile analysis.I6 4. All of the above tests used mean values (2 1 SD) to determine statistical significance. Becauseit seemedpossible that changes in absolute quantities might be small, we also decided to employ the sign test,” a statistical analysis measuringpositive and negative deviations from initial values. The sign test was especially appropriatein considering cortisol secretory rates. Because the normal physiologic clearance of i4C-cortisol isotope required 48 hr,8 these secretory rates could be determined only on alternate days. Subsequently, the sign test could comparebaseline adrenal secretory rates (day 2 of the protocol) with results obtained on the first day that theophylline was given (day 4) and with values obtained after more prolonged theophylline administration (day 6).
RESULTS The coefficient of variation for the control measurements (days 1, 2, and 3) was determined for each subject. The mean coefficients of variation for the
nine subjects were lo%, 22%, 34%, 21%, and 16% for the 17-K& 17-OH, UFF, morning serum cortisol, and morning ACTH values, respectively. Table I shows the values obtained for 17-KS. The results were similar whether long-term or transient differences were analyzed. No statistically significant differences were observed. The values obtained for 17-OH and UFF (Table I) illustrate that the only significant differences were between the baseline values (days 1 to 3, day 2) and the transient values (day 4). These differences occurred in both the control and the asthmatic subjects. No significant differences were noted between the control and asthmatic subjects. Table II shows the renal effects of placebo and tbeophylline. Creatinine clearances remained unchanged, but urine volumes were significantly affected and displayed a pattern similar to values obtained for 17-OH and UFF. Because all patients had normal renal function and had unlimited access to water throughout the study, Table II demonstrates that urine volumes increased greatly on day 4 of the protocol. Moreover, Table II shows that this increase had dissipated by the end of the study (days 4 to 6 vs days 1 to 3, not significantly different). Table III shows that values obtained for 8 A.M. serum cortisol and ACTH were not significantly changed whether these numbers were compared for long-term effects (days 1 to 3 vs days 4 to 6) or for short-term changes following intravenous infusion. Serum cortisol and ACTH concentrations at 4 P.M. (not shown) had normal trough values for both control and asthmatic subjects during both placebo and theophylline administration. Fig. 2 shows that the mean FSR increased from a mean value on day 2 of 14.2 to 19.3 mg/24 br on day 4; it subsequently decreased to 17.8 mg/24 hr on day 6. Because the standard deviations were large, these changes were not significant. Looking at each subject’s FSR, however, it can be seen that eight of nine
VOL JME 67 NWBER 1
Theophylline
and cortisol
secretion
49
Day 4 (IV theo~ine)
W.” 0”
+15*
Serum cortisoi I6 1 r 5.5 20 I -e 2.2
15.6 2 4.0 14.3 Lk 3.5
+I#
+90”
16.1 -e 4.4 14.5 k 4.6
14.0 Itr 2‘5 15.0 t 4.2
62.1 L 31.9 75.9 ? 40.1
57.3 t 29.1 77.0 .c_ 39.2
+30 (pgldl)
17.0 f 4.8 14.9 k 4.0 Serum ACTH (pg/ml)
-. 53 i + 15.3 76.1) + 26.3
60.6 _) 29.0 68.3 2 33.8
71.4 2 46.7 64.1 2 45.8
subjects had a higher FSR on day 4 than on day 2. Thk is a significant observation (p < 0.05) by the sign test of trends. By day 6, neither mean values by the paired t test nor direction of change by the sign test was significant. Strum theophylline concentrations were measured throughout the protocol (Fig. l).* Although four of nine. subjects suffered mild to moderate side effects and subsequentrequired slightly decreasedoral theophylline doses, no patient terminated the study because of drug toxicity and no patient missed a prescribed dose of theophylline. Of the 20 serum theophyl‘ine concentrations measuredon days 5 and 6 of the study, 15 values (75%) were within the therapeutic r;mge (10 to 20 ,uglml), and all values were between 8.6 to 27 pg/ml. Theophylline levels on days 4 and 6 were not significantly correlated with cortisol secr&ory values on these same 2 days. DEWJSW3N
M-.)st allergists consider theophylline a major, fir&fine drug for acute and chronic asthma.‘* Its ability t-1 produce bronchodilatation and relaxation of large:airways is dose related,‘O,l1 and it effects these changesvia an increasein intracellular cyclic AMP.lM Becatisethe samesequenceof biochemical changesin the &enal gland initiates cortisol secretion,‘* 2, ly we studied whether theophylline given to human subjects in therapeutic dosescould affect adrenal metabolism. WC found that theophylline had a significant diuretic effect in both control and asthmatic subjects, great-y increasing urine volumes (Table II). As noted earlier, 17-OH and UFF excretion also significantly increased on day 4. Although these metabolites are sensitive indicators of adrenocortical metabolism, they are also affected by changes in renal filtration rates,20I7-KS were not significantly changed during -.. -*Univ: rsity of Michigan Pharmacy Laboratory: High-pressure Iquid ~.hromatography.
,-
,-
I-
)L
FIG. 2. Changes
in cortisol
secretory
rates.
the study period (Table I), and we attribute this to the fact that 17-KS are unaffected by renal filtration mechanismsZoand are only “a rough estimate of the integrity of the adrenal cortex . . giving no information about the gland’s capacity to respond to acute [change]. ‘Q’ Theophylline’s diuretic effect was transient, having dissipated within 72 hr after the drug had been given continuously, suggesting that asthmatics maintained on long-term theophyline therapy neednot be concernedabout dehydration secondaryto this bronchodilator. Theophylline did not cause any short- or long-term changesin cortisol or ACTH concentrations in either
50
Tulin-Silver
J. ALLERGY
et al.
asthmaticor control subjects(Table III). Using profile analysis,16 we compared the slopes of the lines produced by intravenous placebo and intravenous aminophylline administration. The correlation coefficient for these lines equalled 0.82, suggestingthe lines were nearly flat. This substantiatedthat neither cortisol nor ACTH levels varied from control values. FSRs are a precise method of assessingadrenocortical secretion8and also are unaffected by renal filtration ratese20We found that theophylline did cause an increasein baseline FSRsfrom 14.2 to 19.3 mg/24 hr on day 4. Although this mean increase was not significant (p > 0.05), a small increase occurred in nearly all (eight of nine) subjects (p < 0.05 by sign test). This increase was transient, however, and all values and trends had returned toward baseline values by the third day of theophylline administration. In summary, we postulate that theophylline had the following effects on adrenocortical function. Theophylline had a definite diuretic effect as shown by the significant increasesin urine volumes and 17-OH and UFF excretion. We feel that the marked rise of these adrenocortical metabolites through the kidney caused a temporary fall in serum cortisol concentrations, which by negative feedback to the pituitary gland caused a transient rise in ACTH. At the same time theophylline caused a small transient rise in FSR. This increase in adrenocortical secretion temporarily elevated the serum cortisol concentration, which, in turn, by negative feedback to the pituitary caused a fall in ACTH. As a result theophylline’s two effects on serum cortisol and ACTH offset each other. It is concluded that theophylline, in therapeutic quantities, produced a small, transient increase in cortisol secretion and clearance and that these effects were similar in asthmatic and normal subjects. Also, the concentration of theophylline that caused an increased secretion of cortisol in the animal studies noted earlier* was approximately one hundred times (100 X ) that usedto achieve the monitored therapeutic blood concentrationsin our human subjects. Short- or long-term administration of theophylline, in usually recommendeddoses,‘O*11,l8 has no significant overall effect on adrenocortical function. We wish to thank Gary E. Johnson, Pharm.D., and M. Anthony Schork, M.PH., Ph.D., for their professional advice and assistance. REFERENCES
1. Grahame-Smith DC, Butcher RW, Ney RL, SutherlandEW: Adenosine 3’S’-monophosphate as the intracellular mediator
2 I.
3.
4.
5.
CLIN. IMMUNOL. JANUARY 1981
of the action of adrenocorticotropic hormone on the adrenal cortex. J Biol Chem 242:5535, 1967. Espiner EA, Livesey JH, Ross J, Donald RA: Dynamics of cyclic adenosine 3’,5’-monophosphate release during adrenocortical stimulation in viva. Endocrinology 95:838, 1974. Angeli A, et al: Inefficacia Della Teofillina Nel PotenziareLa Risposta Cortisolemica All ’ Iniezione Endovenosa Impulsiva Di Dibutiril Adenosin 3’,5’-Monofostate Ciclico Nell’ Uomo (The ineffectivenessof theophylline in potentiating the cortisol response bolus intravenous injection of dibutyryl adenosine 3’,5’-monophosphate in man). Bolletino, Societa Italiana di Biologia Sperimentale 50~366, 1974. Dash AJ, England BE, Midgley AR, Niswender GD: Specific non-chromatographicradioimmunoassay for human plasma. Steroids 26:647, 1975. Vague PH, Oliver C, Jaquet P, Vague J: Le DosageRadioimmunologique De L’ ACTH Plasmatique. Eur J Clin Biol Res
16483, 1971. 6. Silber CC, Porter RH: The determination of 17,21-dihy-
droxy-20-ketosteroids in urine and plasma. J Biol Chem 210:923, 1954. 1. Chaney AL: Standard II-ketosteroids, in Urine methods of
clinical chemistry, ed. 2. New York, 1958, Academic Press, Inc., p. 79. 8. Schteingart DE, Gregerman RI, Conn JW: A comparison of the characteristics of increased adrenal cortical function in obesity and Cushing’s syndrome. Metabolism 12:484, 1963. 9. Wagner JG: The one compartment open model with constant rate intravenousinfusion, in Fundamentalsof clinical pharmacokinetics, ed. 1. Hamilton, Ill., 1975,Drug Intelligence Publications, Inc., p. 72. 10. Piafsky KM, Ogilvie RI: Dosage of theophylline in bronchial asthma. New Engl J Med 292:1218, 1975. 11. Mitenko PA, Ogilvie RI: Rational intravenous dosesof theophylline. New EngI J Med 289:600, 1973. 12. JenneJW, Wyze E, Rood FS, et al: Phatmacokineticsof theophylline. Application to adjustment of the clinical dose of aminophylline. Clin Pharmacol Ther 13:349, 1972. 13. Slotfeldt ML, JohnsonCE, Grambau GR, Weg JG: Reliability of theophylline clearance in determining chronic oral dosage regimens. Am J Hosp Pharm 34:66, 1979. 14. Sokal RS, Rohlf FJ: Introduction to biostatidcs. San Francisco, 1969,W. H. Freemanand Co. Publishers,pp. 107-109. 15. Edwards AL: Statistical methods, ed. 2. New York, 1967, Holt, Rinehart and Winston, Inc., pp. 215-226. 16. Kshirsajar AH: Multivariate analysis. New York, 1978, Marcel Dekker, Inc., pp. 161-163. 17. Sokal RS, Rohlf FJ: Introduction to biostatistics. San Francisco, 1969,W. H. FreemanandCo. Publishers,pp. 222-223. 18. Ellis EF: Theophylline and Derivatives, in Middleton E, Reed CE, Ellis EF, editors: Allergy: Principles and practice. St. Louis, 1978, The C. V. Mosby Co., p. 434. 19. Williams GH, et al: Diseasesof the adrenal cortex, in Thorn GW, Adams RD, et al, editors: Harrison’s principles of internal medicine. New York, 1977, McGraw-Hill Book Co., p. 524. 20. Peterson RE: Metabolism of adrenal cortical steroids, in Christy NP, editor: The human adrenal cortex. New York, 1971, Harper & Row, Publishers, chap. 4, pp. 87-189. 21. Christy NP: Iatrogenic Cushing’s syndrome, in Christy NP, editor: The human adrenalcortex. New York, 1971, Harper & Row, Publishers, chap. 14, p. 407.