Additional antianginal efficacy of amiodarone in patients with limiting angina pectoris

Additional antianginal efficacy of amiodarone in patients with limiting angina pectoris Sixty-three patients with stable angina New York Heart Association (NVHA) class Ill and a positive stress test despite triple therapy were randomized to a double-blind protocol, receiving either placebo or amiodarone in a dose of 600 mglday for 10 days, followed by 400 mglday for an additional 10 days, and then by 200 mglday over a total period of 2 months. Comparable bicycle exercise times were observed at baseline in the amiodarone group (6.0 * 1.6 minutes) and in the placebo group (6.0 + 1.6 minutes). With amiodarone, there was a increase in exercise duration of 6.7 * 2.2 minutes versus 6.3 + 2.2 minutes at 1 month and 7.5 f 2.1 minutes versus 6.2 + 1.7 minutes at 2 months (p < 0.05). Also, the amiodarone group had a significant decrease in the double product when compared with the placebo group at 1 month (14,134 & 3,316 versus 17,570 * 4,092 mm Hglmin, p < 0.001) and at 2 months (14,022 + 3,303 and 17,296 + 4,672 mm Hg/min, p < 0.001). The degree of ST segment depression at peak exercise was also significantly reduced. Combination therapy of amiodarone with conventional antianginal therapy is well tolerated and results in a significant improvement in exercise capacity and a mild reduction of symptoms in patients who have continued, limiting angina pectoris with conventional triple therapy. (AM HEART J 1993;125:996.)

Beat J. Meyer, MD, and Franz W. Amann, MD Zurich, Switzerland

Amiodarone, a benzofurane derivative, was initially introduced as an antianginal drug in 1967l and, before its widespread use as an antiarrhythmic agent, it was used extensively in Europe for the treatment of patients with stable, unstable, or variant angina.sm8 Amiodarone decreases myocardial oxygen consumption as a result of mild noncompetitive antagonism of CY-and fi-adrenergic reactions,g-ll resulting in reduced heart rate, reduced contractility, and decreased afterload from systemic vasodilation. In addition, it increases coronary blood supply because of potent coronary vasodilation caused by a direct relaxant effect on coronary smooth muscle cells.12 Thus amiodarone appears to alleviate angina by several mechanisms. Consequently, its antianginal effect might be measurable even in combination therapy. In this short-term, double-blind, placebo-controlled trial, clinical efficacy of additional therapy with amiodarone was demonstrated in a group of patients who continued to be limited by angina pectoris despite conventional medical therapy. From the Cardiology tal Ziirich. Received

for publication

Reprint requests: Beat of Medicine, University Switzerland.

Division. July J. Meyer, Hospital

Department 2, 1992;

of Medicine, accepted

University

Hospi-

Oct. 30, 1992.

MD, Division of Cardiology, Department Ziirich, Rtimistrasse 100, 8091 Ziirich,

1993 by Mosby-Year Book, Copyright 0002.8703/93/$1.00 + .lO 411144058

Inc.

METHODS Inclusion

criteria. The study enrolled men or womenup to 75 years of age with a documented history of chronic stable angina pectoris of New York Heart Association (NYHA) classIII. Their daily activity wasmarkedly limited in spite of antianginal combination therapy; therefore, they were referred to our hospital for evaluation of a revascularization procedure. Patients were eligible only if their individual medication consisted of standard double or triple therapy including a p-blocking agent or calcium channel blocking agent in addition to a long-acting nitrate preparation in standard doses. The diagnosis of coronary artery disease was confirmed by a coronary angiogram revealing 270 “L stenosis of at least one major coronary artery not suitable for a percutaneous transluminal coronary angioplasty procedure, and a positive stress test including angina pectoris or characteristic ST segment depression (2 1.0 mm horizontal or descending ST segment depression persisting 0.08 second after the J point below the isoelectric line) performed under standard antianginal combination therapy. Exclusion criteria. Patients were excluded if they had congestive heart failure caused by severe left ventricular dysfunction (ejection fraction <35 9; ), a history of myocardial infarction occurring less than 3 weeks previously, unstable angina pectoris, arrhythmias requiring antiarrhythmic therapy, and any contraindication to therapy with amiodarone, especially a history of second- or third-degree atrioventricular (AV) block, sick sinus syndrome, or thyroid, liver, or pulmonary disease. Study population. Sixty-three patients were entered into the study and were randomized to amiodarone (A) or

Volume 125, Number 4 American Heati Journal

placebo (P) treatment in a double-blind fashion. Their meanagewas59 +- 9 years. Coronary angiography and left ventricular angiography for biplane assessment of ejection fraction were performed in all patients. Baseline clinical variables and drug treatment of both groups are shown in Table I. None of the listed parameters was significantly different between A or P treated patients at baseline. Study design. Becauseof angina pectoris of limiting degree, patients already were receiving antiischemic double or triple combination therapy consistingof P-blockers,calcium-channel blockers, and long-acting nitrates. After therapy had been optimized, baselinemeasurementswere carried out and patients were randomized to an additional medication wi.th either placeboor amiodaronetablets (200 mg). Initially, 600 mg/day weregiven for 10 days, followed by 400 mglday for an additional 10 days. Finally, patients weremaintained on 200mg/day for the rest of the 2-month study period. The study wasapproved by the ethical committee of the University of Zurich. All patients gave informed consent. Clinical assessment. Symptoms were assessedby an efficacy score and patient treatment preference. Side effects and general well-being were assessed by visual analogscales.Blood pressurewasdetermined usinga random zero sphygomanometer.Heart rate wascounted from the radial pulse after 10 minutes of supine rest and venous blood was tak.en to measureplasmaconcentrations of sodium, potassium, creatinine, aspartate aminotransferase (ASAT), alanine aminotransferase(ALAT), alkaline phosphatase, and -thyroid hormonesat baselineand at followup. A chest x-ray examination wascarried out at baseline and at 2 months. Exercise tes:ting wasperformed on a bicycle ergometerat baselineand at 1 and 2 months during follow-up. Symptom-limited maximal tests were performed approximately 2 to 3 hours after the morning medication. The intraindividual protocol wasidentical on all occasions,generally using steps of 50 W in 3-minute stages.At the initial test, stepwiseincreasesof 25 instead of 50 W could be chosen according to the individual’s physical capacity. Thereafter, the protocol wasnot changed.The test wasterminated for the following end points: angina, hypotension (supine systolic blood pressure2 mm), heart failure, or arrhythmia. In the vast majority of patients the duration of exercise was reflected by the time until the appearanceof angina pectoris, becauseof the entry criteria. Statistical analysis. Baseline comparability between the groups was assessed with t tests for continuous variablesand with chi squaretests for discretevariables. Comparisons of baseline values with follow-up values were made using least squares from an analysis of variance for repeated measures.Probability (p) values <0.05 were considered significant. Unless otherwise stated, values are

mean + standard deviation (SD). RESULTS Effects of study design. Fifty-three patients (43 men and 10 women) completed the study at 2 months and are included in the final evaluation. Ten patients

Meyer and Amunn

997

Table I. Patient characteristics A (n = 32)

Age(yr)

61 + 7

Angina (NYHA) Ejection fraction (5 ) (n = 63) Previous MI (n = 43) Anterior Inferior P-Blockers (n = 57) Atenolol (mg/day) Metoprolol (mg/day) Bisoprolol (mg/day) Calcium blockers

III 59.6 22 12

P (n = 31)

58 t 7 III 58.0 21 10

10

11

28 17 (61 + 26) 9 (144 i 53) 2 (5) 25

29

16 (49 t 13) 9 (190 t 54)

18 (50 + 13) 5 (234 2 80)

28 2

24 4

18 (55 t 29) 8 (100 -f 55)

3 (8 + 3) 23

(n = 48)

Nifedipine Diltiazem Nitrates (n = 58) ISDN Molsidomine A, Amiodarone; Heart

Association

P, Placebo; functional

ISDN, isosorbide dinitrate; NYHA, class; MI, myocardial infarction.

New York

discontinued their participation in the study for the following reasons. In two patients of the amiodaronetreated group and in four patients of the placebo group, urgent coronary artery bypass grafting for unstable angina had to be performed before termination of the study. Three patients (two of the amiodarone group and one of the placebo group) were excluded because of noncompliance. One patient in

the amiodarone group developed marked side effects (bradycardia of 40 beats/min associated with fatigue) requiring discontinuation after 1 month, and one patient developed a rate-dependent conduction distur-

bance within the second month of the study (left bundle branch block at a rate of 80 beats/min). Bicycle exercise testing. Changes in total exercise duration of both groups are shown in Fig. 1. Starting with identical baseline values, mean exercise duration in the amiodarone group increased from 6.0 +- 1.6 to 6.7 f 2.2 minutes at 1 month, and to 7.5 + 2.1

minutes (p < 0.03) at 2 months. In the placebo group, exercise duration remained unchanged (6.0 f 1.8 minutes, 6.3 t- 2.2 minutes, and 6.2 +- 1.7 minutes, respectively). Individual values of total exercise duration of both groups at 2 months compared

mean

with baseline are shown in Fig. 2. In the amiodarone

group, a clear increase of most individual

exercise

durations is seen.

Starting from comparable baseline values (amiodarone 16,876 + 4,458 and placebo 17,377 + 4,481 mm Hg/min), the double product (peak exercise pulse-pressure product) decreased significantly in the amiodarone versus placebo group at 1 month (14,134 f 3,316 versus 17,570 + 4,092, p < 0.001)

998

Meyer and Amann

Exercise

duration

American

at 0, 1 and 2 months

and at 2 months

min

month 0

Double mmHg/min

product

--

month

at 0,l

1

and 2 months

Amiadarone

‘rv

‘wOO

month 2

Placebo =

r

I

p < 0.001

14Ooa

12000

’

month

month

0

ST-depression

at 0,l

1

month

2

and 2 months

mm

in the amiodarone group (Fig. 1). Evaluation of resting and peak exercise systolic and diastolic blood pressures of both groups showed no statistical difference except for a borderline change of resting diastolic blood pressure in the placebo group compared with the amiodarone group at 1 month (Table II). Similarly, resting heart rates were comparable in both groups, whereas exercise heart rates significantly decreased in the amiodaronetreated group at 1 month (p < 0.001) and at 2 months (p < 0.002). Exercise performance was increased in both groups (see Table II). In the amiodarone group, the performed work load increased from 73 + 28 to 92 & 36 W; however, this increase was not statistically significant. Overall symptomatic changes were assessed by an efficacy index. No statistical difference was noted in the subjective assessment of patients in the amiodarone versus placebo group, although there was a trend toward an improvement. in the treatment group. Visual analog scores for frequency of angina1 attacks and for minor side effects were unchanged. Biochemistry. Serum hepatic transaminases and alkaline phosphatase remained normal at 2 months compared with baseline values in the amiodarone and placebo groups. Thyroid function tests changed slightly within the normal range: baseline serum thyroxine (Td) was 104 + 23 nmol/L in the amiodarone group and 110 i: 23 nmol/L in the placebo group. At 2 months, total T4 increased to 126 F 23 nmol/L in the amiodarone group (p < 0.01) whereas it remained unchanged in the placebo group (normal,

Placebo

--

T

Amiodarone

-0.8

(14,022 t 3,303 versus 17,298

? 4,872, p < 0.001; seeFig. 1). The maximum amounts of ST segment depression after 1 month (p < 0.01) and after 2 months (p < 0.02) were also significantly reduced

Placebo

--

1

April 1993 Heart Journal

50 to 160 nmol/L). At baseline, total triiodothyronine (Ts) values in both groups were identical (1.4 nmol/ L). However, at 2 months there was a slight decrease in total Ts levels to 1.2 nmol/L in the amiodarone

group, still remaining within normal limits (0.8 to 3.0 nmol/L). Thyroid-stimulating hormone (TSH) was unchanged, 1.4 t 0.9 and 1.5 i- 0.6 mUL in the amiodarone and placebo groups, respectively. DISCUSSION

This is the first randomized,

1 -2.0 month

0

month

1

Fig. 1. Total exercise duration (upper),

month 2

double product (peak heart rate-systolic blood pressureproduct, middle), and ST segment depression(lower) in patients with refractory angina pectoris treated with additional amiodarone or placebo at baseline and at 1 and 2 months (mean i SEM).

double-blind,

place-

bo-controlled trial to examine the effects of additional short-term treatment with amiodarone in patients with refractory long-standing angina pectoris,

despite

double

or triple

combination

antianginal

therapy consisting of ,B-adrenoceptor and calcium channel antagonists and long-acting nitrates. Our results demonstrate an increase in exercise tolerance,

Volume 125, Number 4 American Heart Journal

Meyer and Amarm 999

II. Rest,ing and peak exercise blood pressure and heart rate at baseline and at patients treated with amiodarone (A) or placebo (P)

Table

Baseline

Rest Heart rate (beats/min) Systolic BP (mm Hg) Diastolic BP (mm Hg) Exercise Heart rate Systolic BP Work load (W) p, Values

at baselme,

at 1 month,

l-

and Z-month follow-up in 53 2 month

1 month

A

P

P

A

P

P

A

P

P

61 * 8 129 + 19 80 + 9

62 +- 9 124 t 16 78 k 8

NS NS NS

56 rt 9 128 + 20 76 I 10

59 k 12 129 * 16 81 + 8

NS NS 0.048

55 * 10 131 t 24 77 + 13

58 t 14 126 k 20 82t12

NS NS NS

106 + 18 160 + 30 73 z!z 28

113 f 19 155 + 24 74 + 34

NS NS NS

91 + 15 153 k 25 83 f 37

111 + 19 157 + 23 86 f 28

0.001 NS NS

92 f 13 152 f 29 92 k 36

110 ~tr 23 156 zt 23 83 k 32

0.002 NS NS

and

at 2 months

represent

differences

between

as expressed by prolonged exercise duration, reduction in the rate-pressure product, and ST segment depression. The symptomatic benefits are modest in this selected group of patients with severe limitation documented by the relatively short baseline exercise duration and by a low double product at the onset of angina. Review of other trials. The antianginal effect of amiodarone was first documented in a clinical trial by Vastesaeger et a1.l in 1967. Its subsequent widespread use and efficacy in treating angina pectoris, as assessed by decreased frequency of angina1 attacks and increased exercise capacity, were reported in two reviews, one by Vacheron13 of 63 studies, and one by Kulbertus et al.* examining 45 studies. Recent comparative trials showed a similar antiischemic effectiveness of amiodarone, diltiazem (180 mg/dl),3 atenolol (100 mg/dl), 5 and long-acting propranolol (160 mg/dl).14 Using a combination of nitrates and amiodarone in a recent Holter study of patients with long-standing angina pectoris, Novo et al.8 demonstrated a reduction of symptomatic and asymptomatic ischemic episodes compared with a treatment phase that used nitrates alone. Potential effects on myocardial oxygen demand and oxygen supply. Amiodarone had a modest brady-

cardic effect, though not significant compared with placebo, with a similar decrease (8 %) in resting heart rate, as reported by Lesbre and E10y.~ During exercise, the pulse pressure product essentially decreased as a result of a lowered heart rate, thus reducing myocardial oxygen consumption, a finding also reported by Varin et al.14 and by Kaski et al.15 In an experimental situation in vitro, amiodarone was found to noncompetitively antagonize the chronotropic responses of rabbit atria to isoproterenol.16 Similarly, the drug inhibited norepinephrine-induced contractions of isolated rat aortic strips.16 These in vitro ef-

groups

A and

P; BP, blood

pressure;

NS,

not statistically

significant.

fects of noncompetitive P- and a-adrenoceptor antagonism are consistent with the effects of amiodarone on cardiovascular and coronary hemodynamics in intact animals and in man.i”> 17-*1 In addition to a decreased demand, an increased myocardial oxygen supply, by improvement of coronary flow reserve, may account for an improved ischemic threshold observed by amiodarone. The direct potent coronary vasodilation properties of amiodarone have been demonstrated in the clinical setting of increased coronary vasomotor tone.22> 23 Furthermore, intracoronary injections of amiodarone in 16 patients with coronary artery disease demonstrated that the drug had potent coronary vasodilator actions.l* The same investigatorsi demonstrated a decrease in coronary vascular resistance and an increase in coronary sinus flow measured by thermodilution, thus confirming the experimental observations of potent coronary and peripheral vasodilation exerted by amiodarone. However, it cannot be judged from our results to what extent coronary vasodilation played a role in the improvement of exercise tolerance in our patients. On the other hand, the efficacy of amiodarone seen in our patients is not fully explained by a reduction of heart rate and blood pressure during peak exercise. Although the pressure product was already significantly reduced after 1 month and did not further decrease thereafter, exercise tolerance further increased at 2 months. Amiodarone, a class III antiarrhythmic agent, is widely used in the treatment of severe supraventricular and ventricular arrhythmias. The antiarrhythmic effect of amiodarone may in part be related to its antiischemic actions in patients with coronary artery disease, as suggested by a recent investigation using continuous ECG monitoring to analyze ventricular arrythmias and ST depression simultaneous1y.s

1000

Meyer and Ammn

mln

American

amiodarone

ii

mln

April 1993 Heart Journal

placebo

12

n=271

(n=24)

IO

10

L-

6

6

6

4

‘r

I

month

2

I

0

month

2

,

month

I

0

month

2

Fig. 2. Effects of short-term administration of amiodarone compared with placebo on individual exercise times in patients receiving the drug for refractory angina pectoris. Note that in the amiodarone group (left), the patient with the most striking fall in exercise duration developed a rate-dependent left bundle branch block at 2 months.

Safety with combination therapy. In the present study, one patient developed severe sinus bradycardia requiring discontinuation of amiodarone. Another patient complained of fatigue and decreased exercise tolerance, as demonstrated by a marked fall of exercise duration at 2 months (Fig. 2) and a ratedependent conduction disturbance. After stopping amiodarone and reducing the @blocking agent temporarily, both patients required no further intervention. It has been reported that in combination with P-blockers and calcium channel blockers, amiodarone, exhibiting mild /3-adrenergic blocking and calcium antagonistic activity, may produce significant and symptomatic bradycardia, sinus arrest, or AV nodal block, particularly in patients with preexisting sick sinus syndrome or impaired AV nodal function, even when these changes are not apparent clinically or on the routine electrocardiogram (ECG).24~ 25 At rest and during exercise, no major reduction in systolic and diastolic blood pressure was observed with amiodarone in combination with the other antiischemic drugs. Thyroid function showed the characteristic changes with amiodarone treatment that are considered to be a drug-induced inhibition in the conversion of peripheral T4 to Ta.26,27 Conclusions. The results observed in this trial are relevant for two major reasons. First, there is an apparent increase in the ischemic threshold, the benefit of an additional low-dose treatment with amiodarone in combination with nifedipineldiltiazem and

P-blockers and long-acting nitrates. Second, this agent, known for multiple adverse effects during long-term administration, was generally well tolerated during the short-term study period in these patients who where unresponsive to conventional medical therapy. However, for prompt detection of cardiac interactions of these agents along with amiodarone, careful follow-up is mandatory. REFERENCES

1. VastesaegerM, 2.

3.

4.

5.

6.

7.

8.

Gillot P, Rasson G. Etude clinique d’une nouvelle medication antiangoreuse. Acta Cardiol1967;22:483-500. Kulbertus HE, Demoulin JC, Bertholet M. Amiodarone: darstellung der antianginijsen Eigenschaften und Analyse der Nebenwirkungen. Herz 1982;7:317-24. Lesbre JP, Eloy JP. An open comparison of amiodarone with diltiazem and glyceryl trinitrate in patients with stable exertional angina. Drugs 1985;29(suppl 3):31-6. Leutenegger A, Liithy E. Eine neue Substanz in der Therapie der Angina pectoris: Amiodarone. Schweiz Med Wochenschr 1968;98:2020-5. Ferraro S, Liguori V, Fazio S, Iacono C, Di Somma S, Petitto M, Galderisi M, Cianfrani M, de Divitis 0. Atenolol and amiodarone: a comparative study of their anti-ischaemic effect. J Int Med Res 1988;16:114-24. Fauchier JP, Cosnay P, Neel Ch, Monpere C, Charbonnier B, Brochier M. Etude comparative du traitement de l’angor de Prinzmetal par betabloqueurs, amiodarone, ou antagonistes calciques. Apropos de 91 cas. Ann Cardiol Angeiol1984;33:56773. Colin WP. Etude a long terme de la tolerance et de l’activite antiangoreuse et antiarrhythmique de l’amiodarone. Ann Cardiol Angeiol 1977;26:247-53. Novo S, Alaimo G, Abrignani MG, Immordino R, Cutietta A, Indovina A, Licata G, Strano A. Effects of low doses of amiodarone on cardiac arrhythmias in patients with chronic

Volume American

125, Number Heart

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Journal

ischaemic heart disease. Em Heart J 1988;9(suppl N):164-8. 9. Charlier R, :Baudine A, Chaillet F. Recherches dans la serie des benzofurannes. XxX11. Mode d’action de l’amiodarone sur le systeme cardiovasculaire. Archs Int Physiol 1967;75:787-808. 10. Charlier R, Deltour G, Baudine A, Chaillet F. Pharmacology of amiodarone, an anti-angina1 drug with a new biological profile. Arzneim Forsch (Drug Res) 1968;18:1408-17. Williams EM. The effect of amiodarone, 11. Singh BN, Vaughan a new anti-angina1 drug, on cardiac muscle. Br J Pharmacol 1970;39:657-67. 12. C8t.e P, Bourassa MG, Delaye J, Janin A, Froment R, David P. Effects of amiodarone on cardiac and coronary hemodynamics and on myocardial metabolism in patients with coronary artery disease. Circulation 1979;59:1165-72. 13. Vacheron A. Utilisation de l’amiodarone dans le traitement de l’insuffisance coronarienne. Bilan des etudes cliniques. Ann Cardiol Angeiol 1979;28:355-9. 14. Varin J, Pelissier C, Legendre M, Delorme G, Dubourg 0, Weiss P, Bourdarias JP. Anti-angina effect of amiodarone versus delayed action propranolol. A double-blind randomized study. Arch Ma1 Coeur 1990;83:1467-73. 15. Kaski JC, Girotti LA, Elizari MV, Lazzari JO, Goldbarg A, Tambussi P,, Rosenbaum MB. Efficacy of amiodarone during long-term treatment of potentially dangerous ventricular arrhythmias in patients with chronic stable ischemic heart disease. AM HEART J 1984;107:648-55. 16. Polster P, Broekhuysen J. The adrenergic antagonism of amiodarone. Biochem Pharmacol 1976;25:131-4. 17. Singh BN, ,Jewitt DE, Downey JM, Kirk E, Sonnenblick EH. Effects of amiodarone on L 8040, novel anti-angina1 and antiarrhythmic drugs, on cardiac and coronary hemodynamics and on cardiac intracellular potentials. Clin Exp Pharmacol Physiol 1976;3:427-42. 18. Gagnol JP, Devos C, Clinet M, Nokin P. Amiodarone. Bio-

19.

20.

21.

22.

23.

24. 25.

26.

27.

1001

chemical aspects and hemodynamic effects. Drugs 1985;29 (suppl 3):1-10. Charlier R. Cardiac actions in the dog of a new antagonist of adrenergic excitation which does not produce competitive blockade of adrenoceptors. Br J Pharmacol 1970;39:668-74. De Boer LWV, Nosta JJ, Kloner RA, Braunwald E. Studies of amiodarone during experimental myocardial infarction: beneficial effects on hemodynamics and infarct size. Circulation 1982;65:508-12. Remme WJ, Van Hoogenhuyze DCA, Krauss XH, Hofman A, Kruyssen DACM, Storm CJ. Acute hemodynamic and antiischemic effects of intravenous amiodarone. Am J Cardiol 1985;55:639-44. Bertholet M, Hastir F, Renier J, Demoulin JC, Kulbertus HE. The value of amiodarone for the treatment of unstable angina. Acta Cardiol 1983;38:503-11. Rutitzky B, Girotti AL, Rosenbaum MB. Efficacy of chronic amiodarone therapy in patients with variant angina pectoris and inhibition of ergonovine coronary constriction. AM HEART J 1982;103:38-43. Wilson JS, Podrid PJ. Side effects from amiodarone. AM HEART d 1991;121:158-71. Lee TH, Friedman PL, Goldman L, Stone PH, Antman EM. Sinus arrest and hypotension with combined amiodarone-diltiazem therapy. AM HEART J 1985;109:163-4. Burger A, Dinichert D, Nicod P, Jenny M, LemarchandBeraud T, Vallotton MB. Effect of amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. J Clin Invest 1971;58:255-9. Zaninovich AA, Bosco SC, Fernandez-Pol AJ. Amiodarone does not affect the distribution and fractional turnover of triiodothyronine from the plasma pool, but only its generation from thyroxine in extrathyroidal tissues. J Clin Endocrinol Metab 1990;70:1721-24.