Acta med. scand. Vol. 197, pp. 303-306, 1975
ACETYLATOR PHENOTYPE AND THE ANTIHYPERTENSIVE RESPONSE TO HYDRALAZINE A. J. Jounela, M. Pasanen and M. J . Mattila From the Departments of’Medicine. Muriu Hospitul. Helsinki. Kemi Centrul Hospitul, Kemi. und the Department of’Phurmacology. University c f Helsinki, Helsinki, Finland
Abstrucr. Twenty-three out-patients with mild or moder-
ate essential hypertension have been treated with a combination of hydralazine (37.5-150 mg daily) and oxprenolol (60 mg daily). Before treatment the patients were phenotyped for polymorphic acetylation by means of the sulphamethazine test: 12 proved to be slow and I I rapid acetylators. A significant correlation was found between daily doses of hydralazine and the plasma hydralazine levels, separately in slow (r =0.480) and in rapid (r =0.580) acetylators. The antihypertensive response to hydralazine correlated well to plasma hydralazine levels. The mean fall of BP in slow acetylators was 33/23 mmHg in supine and 20/18 mmHg in standing position. The corresponding values in rapid acetylators were 22/15 and 21/15 mmHg. The average daily doses of hydralazine needed for these responses were 1.3 mg/kg in slow and I .6 mg/kg in rapid acetylators. To reduce the systolic BP by 20 mmHg, 1.0 mg/kg of hydralazine was needed in slow acetylators; rapid acetylators needed a significantly higher dose of 1.4 mg/kg. During a follow-up of 1 year there have been virtually no side-effects. The results tally with the previous finding of Zacest and Koch-Weser, who demonstrated a similar correlation during the triple-drug regimen. It seems as if hypertensive patients can be succesfully treated with hydralazine and @-blocking drugs without knowledge of the patient’s acetylator phenotype. However, acetylator status is a determinant of tissue levels and long-term toxicity of hydralazine, and patients should be phenotyped because p-blockers may mask the warning side-effects Like isoniazid and sulphamethazine (SMZ), hydralazine (HZ) is acetylated on the polymorphic pattern (2). T h e polymorphism of acetylation correlates t o the activity of liver enzyme acetyltransferase, t h e slow acetylator phenotype is an autosomal recessive trait and there are racial differences with regard to its frequency (2). Slow acetylators a r e liable to generate antinuclear antibodies or systemic lupus during long-term
treatment with H Z (9). This is probably due to a n accumulation of H Z in tissues, since Zacest and Koch-Weser (13) have shown that the same dose given to slow acetylators results in higher plasma H Z levels than in rapid acetylators. I t is known that the antihypertensive response to H Z in rats is related to its concentration in blood and aortic wall (6). We initiated the present trial t o correlate the acetylator status and plasma H Z levels of hypertensive patients to their antihypertensive response to HZ. During our trial, Zacest and Koch-Weser (13) reported that this correlation e xists in a situation where H Z is administered in combination with propranolol and diuretics.
PA TIEN TS A N D METH O D S Patients
Twenty-three out-patients, 12 males and 1 1 females, with established essential hypertension, were admitted to the trial. Their ages ranged from 24 to 58 years (mean 46) and their weights from 61 to 118 kg (mean 80). The patients were accepted for the trial if their diastolic BP, measured during three subsequent visits to the Out-patient Clinic at I-week intervals, was consistently 95 mmHg or more. All cases with a history of angina pectoris, cardiac decompensation, asthma or autoimmune diseases were excluded. After fulfilling these criteria the following tests were obtained in all patients: complete blood count, ESR, serum electrolytes, creatinine and uric acid, urinalysis, 24-hour urinary excretion of metanephrines, LE cells, rheumatoid factor and antinuclear antibodies, ECG, chest X-ray, i.v. pyelography and examination of ocular fundi. On these grounds the patients were classified to fulfill the criteria of grades 1-11 hypertension (WHO). Treatment of blood pressure BP was measured throughout the study in the same room of the Out-patient Clinic by the same trained nurse using
Acta med. scand. 197
304
A. J. Jounela et al.
the same standard mercury sphygmomanometer. Three to six measurements at 5-min intervals in supine and 2-3 measurements in standing position were taken between 4 and 6 p.m. and the three lowest values were averaged. Heart rate was counted from the radial pulse after the last BP recording in each position. Patients were checked in the Out-patient Clinic at weekly intervals. At each visit they were questioned about side-effects and their general condition, BP and heart rate were measured and tablets were counted. At the end of the trial, which lasted 3 months, and one year after starting the treatment, blood count, ESR, urinalysis, serum creatinine, LE cells and antinuclear antibodies were investigated. After the three visits during which the baseline BP was recorded, treatment was started with H Z at a dose of 12.5 mg three times daily. Since our previous experience showed that treatment with HZ alone up to 150 mg daily resulted in therapeutic failure and intolerable side-effects, the treatment with a Padrenoceptor blocking drug oxprenolol (20 mg t.i.d.) was combined with the HZ treatment from the very beginning. The dose of H Z was increased at 1-week intervals by 37.5 mg up to a total daily dose of 150 mg or until the patients were normotensive or until intolerable sideeffects occurred. Chemical measurement of drugs
The polymorphic acetylator phenotype of the patients was determined by using S M Z as a test drug since it is acetylated like isoniazid and HZ (2) and easy to measure. SMZ tablets (10 mg/kg) were given to the patients during their first visit to the Out-patient Clinic. In the morning of the day of their second visit they took a sample of morning urine for a reference, ingested the tablets with a light breakfast, and the 6-hour urine sample was taken in the Out-patient Clinic. The aliquots of urine were stored at
-20°C for several weeks before SMZ and acetyl S M Z were assayed by the chemical Bratton-Marshall procedure (12). This simple measurement of unacetylated and acetylated SMZ in urine is accurate enough to separate slow and rapid acetylators (7). To estimate the plasma HZ, samples of venous blood were drawn during the visits 2-4 hours following the last dose of HZ. Plasma was separated by centrifugation, acidified by adding two drops of 10 N hydrochloric acid, and kept at -20°C for several weeks until assayed according to the spectrophotometric method ( I l ) , which in our hands proved reliable in the concentration range of 0.064 I.Lglml. Statistical analysis
Student's f-test was used for the statistical analysis of the results. RESULTS Acetylator phenotype a n d p l a s m a H Z levels. O n t h e
basis o f present results a n d o u r previous experience
(3), 12 patients of a total of 23 were classified as slow acetylators, their ratios of unacetylated SMZ/total S M Z in urine ranging from 29 to 52%. T h e respective ratios in 11 rapid acetylators ranged from 6 to 19%. T h e plasma H Z levels related to the HZ dosage a r e illustrated in Fig. I . Generally, the plasma levels were higher with increasing doses but t h e highest levels were not reached in rapid acetylators. W h e n the plasma HZ levels are plotted against t h e H Z dosage separately in either phenotype, t h e correla-
0
RAPID ACETVLATORS 0
0
gz
0.8
-
0.0
-
E
1
0 0
0
0
-YN
0
0
5
0.4 -
G 2
0.2
d n
0
-
3
B
0
p'
a
0
0
0
.
0
Fig. I . Correlation between
0
0
daily hydralazine dose and plasma level of the drug in slow rr=O.4801 and in rauid (r =0.580) acetylators.
0
0
0
I 0
0
L 0.5
"
Acta med. scand. 197
"
"
"
" ' ~ 1.o HYDRALAZINE DOSE
~
' 1.5
, mp/kg /day
~
l 20
Acetylator phenotype and antihypertensive response to hydralazine
Table I. Effect of combined hydralazine+oxprenolol on blood pressure ( m m H g , mean + S . E . ) in slow and rapid acetylators Supine BP
Standing BP
tolic
Diastolic
tolic
176f6 143+3
114+4 91 f 2
164&7 115+4 144f5 97k3
167f5 145+4
11Of1
1fj6+8 112f2 145+5 97+3
Sys-
Sys-
Diastolic
Slow acetylators
Before treatment During treatment Rapid aceiylaiors
Before treatment During treatment
95+3
tions are statistically significant (p
ACETYLATOR PHENOTYPE AND THE ANTIHYPERTENSIVE RESPONSE TO HYDRALAZINE A. J. Jounela, M. Pasanen and M. J . Mattila From the Departments of’Medicine. Muriu Hospitul. Helsinki. Kemi Centrul Hospitul, Kemi. und the Department of’Phurmacology. University c f Helsinki, Helsinki, Finland
Abstrucr. Twenty-three out-patients with mild or moder-
ate essential hypertension have been treated with a combination of hydralazine (37.5-150 mg daily) and oxprenolol (60 mg daily). Before treatment the patients were phenotyped for polymorphic acetylation by means of the sulphamethazine test: 12 proved to be slow and I I rapid acetylators. A significant correlation was found between daily doses of hydralazine and the plasma hydralazine levels, separately in slow (r =0.480) and in rapid (r =0.580) acetylators. The antihypertensive response to hydralazine correlated well to plasma hydralazine levels. The mean fall of BP in slow acetylators was 33/23 mmHg in supine and 20/18 mmHg in standing position. The corresponding values in rapid acetylators were 22/15 and 21/15 mmHg. The average daily doses of hydralazine needed for these responses were 1.3 mg/kg in slow and I .6 mg/kg in rapid acetylators. To reduce the systolic BP by 20 mmHg, 1.0 mg/kg of hydralazine was needed in slow acetylators; rapid acetylators needed a significantly higher dose of 1.4 mg/kg. During a follow-up of 1 year there have been virtually no side-effects. The results tally with the previous finding of Zacest and Koch-Weser, who demonstrated a similar correlation during the triple-drug regimen. It seems as if hypertensive patients can be succesfully treated with hydralazine and @-blocking drugs without knowledge of the patient’s acetylator phenotype. However, acetylator status is a determinant of tissue levels and long-term toxicity of hydralazine, and patients should be phenotyped because p-blockers may mask the warning side-effects Like isoniazid and sulphamethazine (SMZ), hydralazine (HZ) is acetylated on the polymorphic pattern (2). T h e polymorphism of acetylation correlates t o the activity of liver enzyme acetyltransferase, t h e slow acetylator phenotype is an autosomal recessive trait and there are racial differences with regard to its frequency (2). Slow acetylators a r e liable to generate antinuclear antibodies or systemic lupus during long-term
treatment with H Z (9). This is probably due to a n accumulation of H Z in tissues, since Zacest and Koch-Weser (13) have shown that the same dose given to slow acetylators results in higher plasma H Z levels than in rapid acetylators. I t is known that the antihypertensive response to H Z in rats is related to its concentration in blood and aortic wall (6). We initiated the present trial t o correlate the acetylator status and plasma H Z levels of hypertensive patients to their antihypertensive response to HZ. During our trial, Zacest and Koch-Weser (13) reported that this correlation e xists in a situation where H Z is administered in combination with propranolol and diuretics.
PA TIEN TS A N D METH O D S Patients
Twenty-three out-patients, 12 males and 1 1 females, with established essential hypertension, were admitted to the trial. Their ages ranged from 24 to 58 years (mean 46) and their weights from 61 to 118 kg (mean 80). The patients were accepted for the trial if their diastolic BP, measured during three subsequent visits to the Out-patient Clinic at I-week intervals, was consistently 95 mmHg or more. All cases with a history of angina pectoris, cardiac decompensation, asthma or autoimmune diseases were excluded. After fulfilling these criteria the following tests were obtained in all patients: complete blood count, ESR, serum electrolytes, creatinine and uric acid, urinalysis, 24-hour urinary excretion of metanephrines, LE cells, rheumatoid factor and antinuclear antibodies, ECG, chest X-ray, i.v. pyelography and examination of ocular fundi. On these grounds the patients were classified to fulfill the criteria of grades 1-11 hypertension (WHO). Treatment of blood pressure BP was measured throughout the study in the same room of the Out-patient Clinic by the same trained nurse using
Acta med. scand. 197
304
A. J. Jounela et al.
the same standard mercury sphygmomanometer. Three to six measurements at 5-min intervals in supine and 2-3 measurements in standing position were taken between 4 and 6 p.m. and the three lowest values were averaged. Heart rate was counted from the radial pulse after the last BP recording in each position. Patients were checked in the Out-patient Clinic at weekly intervals. At each visit they were questioned about side-effects and their general condition, BP and heart rate were measured and tablets were counted. At the end of the trial, which lasted 3 months, and one year after starting the treatment, blood count, ESR, urinalysis, serum creatinine, LE cells and antinuclear antibodies were investigated. After the three visits during which the baseline BP was recorded, treatment was started with H Z at a dose of 12.5 mg three times daily. Since our previous experience showed that treatment with HZ alone up to 150 mg daily resulted in therapeutic failure and intolerable side-effects, the treatment with a Padrenoceptor blocking drug oxprenolol (20 mg t.i.d.) was combined with the HZ treatment from the very beginning. The dose of H Z was increased at 1-week intervals by 37.5 mg up to a total daily dose of 150 mg or until the patients were normotensive or until intolerable sideeffects occurred. Chemical measurement of drugs
The polymorphic acetylator phenotype of the patients was determined by using S M Z as a test drug since it is acetylated like isoniazid and HZ (2) and easy to measure. SMZ tablets (10 mg/kg) were given to the patients during their first visit to the Out-patient Clinic. In the morning of the day of their second visit they took a sample of morning urine for a reference, ingested the tablets with a light breakfast, and the 6-hour urine sample was taken in the Out-patient Clinic. The aliquots of urine were stored at
-20°C for several weeks before SMZ and acetyl S M Z were assayed by the chemical Bratton-Marshall procedure (12). This simple measurement of unacetylated and acetylated SMZ in urine is accurate enough to separate slow and rapid acetylators (7). To estimate the plasma HZ, samples of venous blood were drawn during the visits 2-4 hours following the last dose of HZ. Plasma was separated by centrifugation, acidified by adding two drops of 10 N hydrochloric acid, and kept at -20°C for several weeks until assayed according to the spectrophotometric method ( I l ) , which in our hands proved reliable in the concentration range of 0.064 I.Lglml. Statistical analysis
Student's f-test was used for the statistical analysis of the results. RESULTS Acetylator phenotype a n d p l a s m a H Z levels. O n t h e
basis o f present results a n d o u r previous experience
(3), 12 patients of a total of 23 were classified as slow acetylators, their ratios of unacetylated SMZ/total S M Z in urine ranging from 29 to 52%. T h e respective ratios in 11 rapid acetylators ranged from 6 to 19%. T h e plasma H Z levels related to the HZ dosage a r e illustrated in Fig. I . Generally, the plasma levels were higher with increasing doses but t h e highest levels were not reached in rapid acetylators. W h e n the plasma HZ levels are plotted against t h e H Z dosage separately in either phenotype, t h e correla-
0
RAPID ACETVLATORS 0
0
gz
0.8
-
0.0
-
E
1
0 0
0
0
-YN
0
0
5
0.4 -
G 2
0.2
d n
0
-
3
B
0
p'
a
0
0
0
.
0
Fig. I . Correlation between
0
0
daily hydralazine dose and plasma level of the drug in slow rr=O.4801 and in rauid (r =0.580) acetylators.
0
0
0
I 0
0
L 0.5
"
Acta med. scand. 197
"
"
"
" ' ~ 1.o HYDRALAZINE DOSE
~
' 1.5
, mp/kg /day
~
l 20
Acetylator phenotype and antihypertensive response to hydralazine
Table I. Effect of combined hydralazine+oxprenolol on blood pressure ( m m H g , mean + S . E . ) in slow and rapid acetylators Supine BP
Standing BP
tolic
Diastolic
tolic
176f6 143+3
114+4 91 f 2
164&7 115+4 144f5 97k3
167f5 145+4
11Of1
1fj6+8 112f2 145+5 97+3
Sys-
Sys-
Diastolic
Slow acetylators
Before treatment During treatment Rapid aceiylaiors
Before treatment During treatment
95+3
tions are statistically significant (p
