Urinary Catecholamines and the Relationship with Blood Pressure and Pharmacological Therapy

URINARY CATECHOLAMINES AND THE RELATIONSHIP WITH BLOOD

PRESSURE AND PHARMACOLOGICAL THERAPY

Constantinos G. Missouris MD, FRCP,1 Nirmala D Markandu RN,2 Feng J He PhD,2

Maria V Papavasileiou MD,3 Peter Sever PhD,FRCP,FESC,4 Graham A MacGregor

MD, FRCP,2

1Frimley Health, Frimley Trust, Wexham Park Hospital, Slough, UK

2 Wolfson Institute of Preventive Medicine, Queen Mary University of London, UK

3 Sismanoglion Hospital, Athens, Greece

4NHLI, Imperial College, London, UK

Short title: Urinary catecholamines and blood pressure

Total word count: 3,728

Table: 3

Corresponding author

Dr Constantinos Missouris

Frimley Health Frimley Trust, Wexham Park hospital, Slough UK

Telephone: 00(44) 207 0794217

Fax: 00 (44) 207 580 0122

E-mail: [email protected]

1 ABSTRACT

Objectives Our aim was to assess the importance of the sympathetic nervous system as assessed by urinary catecholamine measurement in the aetiology of essential hypertension and the importance of anti-hypertensive therapy in the excretion of urinary catecholamines.

Methods Twenty four hours urinary catecholamine measurement was performed in 1921 subjects who were referred for treatment of hypertension and grouped according to the JNC

VI classification: Of the 655 untreated subjects 59 were normotensive (SBP<140 -DBP<90 mmHg), n=219 stage 1 (SBP 140 -159 or DBP 90-99 mm Hg), n=236 stage 2 (SBP 160-179 or DBP 100-109 mm Hg) and n=141 stage 3 (SBP≥180 or DBP≥110 mm Hg).

Results There was a statistically significant positive relationship between 24 hr urinary nor- epinephrine excretion and the severity of hypertension, such that the higher the blood pressure the higher the urinary nor-epinephrine excretion (mean ± SEM): normotensive group 221±13 nmol/24 hour; stage 1 254±8 nmol/24 hour; stage 2 263±7 nmol/24 hour; and stage 3

296±12 nmol/24 hour (p<0.001). The above relationship remained highly significant when corrected for urinary creatinine, weight, age and gender. No differences were found with urinary epinephrine or dopamine excretion. Urinary nor-epinephrine excretion was increased in those patients taking single drug therapy with either a long acting dihydropyridine calcium antagonist or a β-blocker.

Conclusions Our results demonstrate that in untreated hypertensive patients urinary nor- epinephrine excretion is increased in proportion to the severity of blood pressure rise and also in patients taking a long acting dihydropyridine calcium antagonist or a β-blocker .

Sympathetic over activity, may play a role in the aetiology and maintenance of essential hypertension.

Key words Urinary catecholamines, blood pressure, pharamacological therapy

2 INTRODUCTION

The sympathetic nervous system plays an important role in regulating short term changes in blood pressure. It exerts its effects by secreting catecholamines, nor-epinephrine, epinephrine and dopamine, which are important neurotransmitters and exert cardiovascular and metabolic effects by stimulating adrenergic receptors in a wide variety of cells.

However, the involvement of the sympathetic nervous system in the pathogenesis of established essential hypertension has attracted much interest and controversy. To date, there is considerable discrepancy in the literature as to whether sympathetic nervous system is activated, and whether plasma or urinary catecholamine excretion, possible markers of sympathetic activity are increased in patients with essential hypertension .

In a small number of hypertensive patients the excessive production and release into the circulation of catecholamines by a neoplasm of the chromaffin tissue, such as phaeocromocytoma, characteristically produces paroxysmal or persistent hypertension, and unless identified and treated it carries an adverse prognosis. Fundamental to the detection and diagnosis of this condition is the measurement of plasma or urinary catecholamines and their metabolites.

We set out to evaluate the role of the sympathetic nervous system, as assessed by urinary catecholamines, in a large cohort of subjects undergoing routine investigations for essential hypertension. The present study tested the hypothesis that in the above patients the sympathetic nervous system, as assessed by urinary catecholamine measurement, is activated in proportion to the severity of the blood pressure rise. We also assessed the importance of pharmacological therapy in the levels of urinary catecholamines in patients undergoing screening for phaeochromocytoma.

3 PATIENTS AND METHODS

One thousand nine hundred and twenty seven patients, with essential hypertension, who were electively referred to the Blood Pressure Unit, St. George’s Hospital, London, between

January 1990 and June 1996, from local general practitioners and other hospital physicians for further evaluation and treatment of their blood pressure were studied. A number of these patients who had their blood pressure measured by experienced nurses in our Blood

Pressure Unit were subsequently found to have normal blood pressure readings. 24 hr blood pressure monitoring was not widely available at the time. Criteria for requesting catecholamine measurement included clinical suspicion of phaeochromocytoma, moderate to severe hypertension, variable hypertension, paradoxical response to treatment, failure to respond to treatment or postural drop in blood pressure in untreated patients. Patients also had 24 hr urinary catecholamines measured, particularly as we were routinely measuring urinary sodium and potassium at the same time and could be done on the same 24 hr collection. Patients with phaeochromocytoma, renal artery stenosis and hyperaldosteronism were excluded from the study, so were patients treated with psychotropic drugs or those with a history of alcohol abuse.

Subjects were asked to collect urine over a 24 hour period and advised to avoid food high in amines like bananas for 2-3 days before collection. Urine samples were collected in polyethylene bottles containing 25 ml of 0.6 M hydrochloric acid, stored at 40 C and assayed twice weekly for measurement of nor-epinephrine, epinephrine, dopamine, electrolytes and volume. Urinary catecholamines were extracted on alumina, eluted, and quantified by reverse phase using high-performance liquid chromatography (HPLC) with electrochemical detection. In the UK quality assurance data confirm that urinary

4 norepinephrine has a coefficient of variation 8% , epinephrine 16% and dopamine 8% respectively using the above method.

Supine systolic (SBP) and diastolic (DBP) blood pressure and heart rate were recorded three times, two minutes apart, using a semi-automatic ultrasound sphygmomanometer

(Arteriosonde, Roche) after 5 minutes resting. The patients were grouped according to the severity of hypertension using the JNC VI classification: normotensive SBP<140 & DBP<90 mm Hg; stage 1 SBP 140-159 or DBP 90-99 mm Hg; stage 2 SBP 160-179 or DBP 100-109 mm Hg; stage 3 SBP ≥ 180 or DBP ≥ 110 mm Hg. When systolic and diastolic blood pressures fell into different categories, the higher category was selected to classify the individual’s blood pressure status.

STATISTICS

Results are reported as mean ± SEM. The differences in blood pressure and other continuous variables among different groups were tested by One Way Analysis of

Variance (ANOVA). For the primary outcomes, i.e. urinary catecholamines, general linear models were used to test the difference among groups with adjustment for potential confounding factors. For categorical data (sex and race), χ2 test was used to examine the differences among groups. Univariate and multiple regression analyses were performed to determine how much the blood pressure variability was related to urinary catecholamines and other variables. Logistic models were used to examine the association between urinary catecholamines and the risk of hypertension with adjustment for confounders. All statistical analyses were performed using statistical package for social science (SPSS). A two-tailed p value < 0.05 was considered statistically significant.

5 RESULTS

One thousand nine hundred and twenty seven subjects were studied. Of these 655 (337 male, mean age 47±1 years) were not receiving pharmacological therapy for at least 4 weeks prior to the urinary catecholamine measurement, 552 were on a single antihypertensive drug, and the remaining 718 were on multiple antihypertensive drug treatment.

1. Untreated Group

Of the 655 untreated subjects, 59 were normotensive (SBP<140 & DBP<90 mm Hg), and the remainder were hypertensive according to the JNC VI classification: 219 were in stage 1

(SBP 140-159 or DBP 90-99 mm Hg), 236 were in stage 2 (SBP 160-179 or DBP 100-109 mm Hg), and 141 were in stage 3 (SBP ≥180 or DBP ≥ 110 mm Hg). Their clinical and biochemical indices groups are summarised in Table 1 and Table 2. The mean age was significantly different in the four groups: normotensive 44±2 years, stage 1 43±1 years, stage

2 49±1 years and stage 3 53±1 years (p<0.001). There was also a significant positive relationship between heart rate and the severity of hypertension (p<0.001).

JNC VI Classification and urinary catecholamine excretion

Using the above classification system, we determined that there was a statistically significant positive relationship between 24 hour urinary nor-epinephrine excretion and the severity of hypertension, such that the higher the blood pressure the higher the urinary nor-epinephrine excretion: normotensive group 221±13 nmol/24 hour; stage 1 254±8 nmol/24 hour; stage 2

263±7 nmol/24 hour; and stage 3 296±12 nmol/24 hour (p<0.001). The resting heart rate showed a similar trend. Post Hoc analysis demonstrated that patients with severe hypertension

(stage 3) had significantly higher urinary nor-epinephrine excretion than those subjects with normal or stage 1 blood pressure measurements (p<0.05). The ratio of urinary nor-

6 epinephrine/creatinine showed a similar trend. The above relationships remained highly significant when nor-epinephrine excretion was corrected for urinary creatinine, weight and age, and gender. Furthermore, there was a significant positive correlation between 24 hour urinary nor-epinephrine excretion and mean arterial pressure (p<0.001; r=0.153).The relationship remained significant after adjusting for age, gender, race, body weight, and urinary creatinine excretion (p<0.01, r=0.125). No significant differences were found between

24 hour urinary epinephrine (p=0.77), dopamine (p=0.46) and sodium (p=0.97) among the four blood pressure groups.

We have performed further analyses using logistic regression with adjustment for potential confounding factors. The results showed that an increase of 10 nmol in 24h urinary nor-epinephrine was associated with 7% (OR: 1.06, 95% CI: 1.03-1.11, P=0.002) increase in the risk of hypertension after adjusting for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium, potassium and creatinine excretion (NB:

Hypertension was defined as systolic ≥140 or diastolic ≥90 or on blood pressure treatment). In individuals who were not on any blood pressure treatment, the results were very similar to the above, i.e. an increase of 10 nmol in 24h urinary nor- epinephrine was associated with 6% (OR: 1.06, 95% CI: 1.01-1.10, P=0.011) increase in the risk of hypertension after adjusting for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium, potassium and creatinine excretion.

Multiple regression showed that nor-epinephrine, age, sex, ethnic group, body weight, heart rate, urinary sodium, potassium and creatinine all together explained 17% variability of systolic blood pressure and 7% variability of diastolic blood pressure.

7 Taking all participants together, nor-epinephrine, age, sex, ethnic group, body weight, heart rate, urinary sodium, potassium and creatinine, and blood pressure treatment all together explained 14% variability of systolic blood pressure and 5% variability of diastolic blood pressure. In individuals who were not on any blood pressure treatment, univariate regression analysis showed that nor-epinephrine alone explained 2% variability of systolic blood pressure and 2% variability of diastolic blood pressure.

The association between nor-epinephrine and blood pressure became non-significant after adjusting for confounders, but the whole model was still significant.

2. Effect of treatment on urinary catecholamines

Five hundred and fifty two patients were on treatment with a single antihypertensive drug at the time of urinary catecholamine measurement. Thirty nine were on treatment with a non- dihydropyridine calcium antagonist (25 verapamil, 14 diltiazem), 177 with a long acting dihydropyridine calcium antagonist (125 amlodipine, 52 nifedipine LA preparation), 82 with a slow release dihydropyridine calcium antagonist, 112 with an ACE inhibitor, 87 with a β- blocker, and 55 were on treatment with a diuretic. The clinical and biochemical indices in these treatment groups are summarised in Table 3.

Subjects treated with a long acting dihydropyridine calcium antagonist or a β-blocker had significantly higher urinary nor-epinephrine excretion when compared to untreated patients

(dihydropyridine calcium antagonist and β-blockers vs untreated: 321±12, 312±18 vs 263±5 nmol/24 hours, p<0.001, p<0.05 respectively). After correcting for urinary creatinine excretion, the difference between the dihydropyridine and untreated groups was still highly significant (p<0.001). Using the Bonferroni adjustment, we found that when compared to untreated subjects, patients taking dihydropyridine calcium antagonist (amlodipine, nifedipine

LA) and β-blockers had significantly higher urinary nor-epinephrine levels (amlodipine and

8 nifedipine LA p<0.001, β-blockers p<0.01). Subjects taking short acting calcium antagonists, diuretics and ACE inhibitors had higher urinary nor-epinephrine but the difference was not statistically significant. Subjects on treatment with non-dihydropyridine calcium antagonists had similar values to untreated patients.

Using one way ANOVA, we found a significant difference in age (p<0.001), SBP (p<0.01),

DBP (p<0.01), heart rate (p<0.001), urinary nor-epinephrine (p<0.001), urinary nor- epinephrine/creatinine ratio (p<0.001), and serum nor-epinephrine (p<0.05) among the various groups. There was no significant difference among these groups and urinary epinephrine, dopamine, urinary electrolytes and volume.

DISCUSSION

In our study, one of the largest in the literature, we report that in untreated hypertensive patients the urinary nor-epinephrine excretion is increased in proportion to the severity of blood pressure rise. This relationship remained highly significant when nor-epinephrine excretion was corrected for possible confounders such as urinary creatinine, age and weight.

However, in a substantial proportion of patients, urinary norepinephrine measurements are not raised, thus, suggesting alternative mechanisms may be responsible for the elevated blood pressure. The issue is important because treatments targeting the sympathetic nervous system may not be effective in this group. Furthermore, urinary nor- epinephrine excretion was increased in those patients taking single drug therapy with either a long acting dihydropyridine calcium antagonist or a β-blocker. No differences were found with urinary epinephrine or dopamine excretion.

Sympathetic nerve activity can be assessed directly in humans by means of microelectrodes inserted into superficial nerves , or indirectly, by quantification of plasma nor-epinephrine

9 levels, nor-epinephrine spill over techniques or by assessing urinary catecholamine measurement. The latter circumvents many of the difficulties encountered with invasive techniques, and also takes into account the regional differences in sympathetic outflow that may be present in hypertensive patients. Norepinephrine is synthesized by a series of enzymatic steps in the adrenal medulla and postganglionic neurons of the sympathetic nervous system from amino acid tyrosine. Plasma and urinary norepinephrine are derived primarily from postganglionic neurons with little contribution from the central nervous system or from hormonal release from the adrenal medulla. Due to intervening neuronal and extra neuronal removal processes only a small amount (10%) of norepinephrine released by the sympathetic nerves escapes into the circulation.

Following bilateral adrenalectomy the urinary norepinephrine is reduced by approximately 80% and no change in urinary norepinephrine excretion suggesting that that most of the epinephrine is secreted by the adrenal medulla and that of the norepinephrine by other sources . Furthermore, studies using infusion of radiolabelled catecholamines in patients with renal transplantation have shown that two-thirds of renal norepinephrine and dopamine depend on the presence of renal nerves.

Animal studies have demonstrated that depletion of central and peripheral catecholamine stores could prevent or attenuate the development of hypertension . Furthermore, electrophysiological studies in spontaneously hypertensive rats demonstrated an increased sympathetic neural activity from cervical, splachnic and renal nerves .

Urinary or plasma catecholamine levels serve as an approximation of the activity of the sympathetic nervous system. These measurements, unlike circulating catecholamines, represent the sum of events occurring over a long period of time such as central sympathetic

10 outflow as well as by peripheral mechanisms involved in the release and reuptake into nerve terminals and spill over from different vascular beds.

Patients with essential hypertension have higher levels of nor-epinephrine in the cerebrospinal fluid than normotensive subjects, pointing to possible abnormalities of central monoaminergic neuronal transmission in the pathogenesis of hypertension . Measurement of these levels however, has failed to reveal any consistent abnormality in subjects with essential hypertension . There is general agreement that in some young patients with essential hypertension, plasma catecholamine levels may be raised .

Goldstein reviewed a series of studies comparing plasma catecholamines in patients with essential hypertension and in normotensive controls. Significant elevations in catecholamines were reported in approximately 40% of the above studies . However many of the studies have included only a small number of subjects and it is likely that factors such as age, obesity, salt intake, and the use of antihypertensive drugs may have confounded their results [26, . Our data are in agreement with those reported by Brown MJ et al. 1985 , who found no significant increase in urinary catecholamine excretion in a group of untreated mildly hypertensive subjects and age and sex-matched normotensive controls. However in our study which included a large number of subjects some with more severe hypertension, we found that these untreated patients have significantly higher nor-epinephrine excretion when compared to those subjects with normal and mildly elevated blood pressure.

Finally, our results suggest that urinary nor-epinephrine excretion was increased in those patients taking single drug therapy with either a long acting dihydropyridine calcium antagonist or a β-blocker compared with hypertensive patients not receiving treatment . Care should therefore, be taken in interpreting nor-epinephrine concentrations in hypertensive patients on treatment with the above pharmacological therapy. Therefore pharmacological

11 treatment should be considered when establishing reference values for urinary catecholamines and their metabolites in hypertensive patients undergoing screening for phaeochromocytoma.

Our results demonstrate that in untreated hypertensive patients urinary nor-epinephrine excretion and resting heart rate are increased in proportion to the severity of blood pressure rise. This relationship remained highly significant when nor-epinephrine excretion was corrected for possible confounders such as urinary creatinine, age and weight. This observation supports the hypothesis that, at least in some patients, sympathetic over activity may play an important role in the aetiology and maintenance of essential hypertension.

Acknowledgements

The authors would like to thank Dr Jeff Barron the staff of the Chemical Pathology

Department of St Helier’s Hospital Carshalton for performing the biochemical investigations.

Also we are very grateful to the nurses of the Blood pressure Unit, St George’s Hospital

London for their help with the study.

The authors performed the above study as part of the clinical patient management and no funding was required. Furthermore there were no potential conflicts of interest.

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14 Table 1. Clinical characteristics and urinary catecholamine measurements in untreated individuals investigated for hypertension and grouped according to JNC VI classification (values are mean±SEM).

SBP<140 & SBP 140-159 SBP160-179 or SBP180 or DBP<90 or DBP 90-99 DBP 100-109 DBP110 P value (n=59) (n=219) (n=236) (n=141)

Age (year) 44±2 43±1 49±1 53±1 <0.001

Gender (M/F) 34/25 128/91 107/128 69/72 <0.05#

Race (W/B/A) 34/9/3 127/42/20 110/64/26 75/38/15 0.127#

Body weight (kg) 75.9±2.0 75.3±1.0 78.6±1.0 77.5±1.3 0.132

SBP/DBP (mmHg) 128/78±1.1/0.9 146/91±0.5/0.5 163/99±0.7/0.4 185/110±1.4/0.9 <0.001/<0.001

Heart rate (beat/min) 76±1.8 79±0.8 81±0.9 84±1.4 <0.001

24 hour urine measurements

Nor-epinephrine (nmol) 221±12.5 254±7.7 263±6.9 296±12.1 0.007¶

Nor-epinephrine/creatinine (nmol/mmol) 21±1.08 22±0.83 23±0.68 27±1.20 0.009§

Epinephrine (nmol) 32±3.1 35±1.4 34±1.5 34±1.7 0.275¶

Epinephrine/creatinine (nmol/mmol) 2.9±0.28 3.0±0.12 2.9±0.13 3.1±0.17 0.337§

Dopamine (nmol) 1845±75 1980±54 1919±49 1872±60 0.939¶

Dopamine/creatinine (nmol/mmol) 171±6.4 170±4.6 164±4.9 170±5.3 0.995§

Sodium (mmol) 140±9.0 144±4.7 141±3.9 144±6.6 0.971

Potassium (mmol) 63±3.2 68±2.1 70±1.9 61±2.2 0.023

# by c2 test. ¶ adjusted for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium, potassium and creatinine excretion. § adjusted for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium and potassium excretion.

15 Table 2. 24h urinary nor-epinephrine (mean±1.96xSD) by age group and percentage of hypertensive and normotensive individuals who had nor-epinephrine above the reference range (1.96xSD). Age group <40 40- 50- 60- All participants N 312 274 321 303 Mean Age (years) 33 45 55 67 Nor-epinephrine (nmol/24h) Mean 255 293 300 277 SD 127 149 140 122 Mean+1.96xSD 504 585 574 517 Mean-1.96xSD 6 2 26 37

Hypertensives (systolic≥140 or diastolic≥90 or on BP treatment) N 283 260 317 291 Having nor-epinephrine above upper 1.96SD, N(%) 11 (3.9) 10 (3.8) 11 (3.5) 9 (3.1%)

Normotensives (systolic<140 & diastolic<90) N 29 14 4 12 Having nor-epinephrine above upper 1.96SD, N(%) 0 0 0 0 Untreated participants N 216 157 154 128 Mean Age (years) 32 45 55 67 Nor-epinephrine (nmol/24h) Mean 240 266 280 279 SD 118 114 120 117 Mean+1.96xSD 471 489 516 508 Mean-1.96xSD 9 43 44 50

Hypertensives (systolic≥140 or diastolic≥90) N 187 143 150 116 Having nor-epinephrine above upper 1.96SD, N(%) 5 (2.7) 8 (5.6) 6 (4.0) 4 (3.4)

Normotensives (systolic<140 & diastolic<90) N 29 14 4 12 Having nor-epinephrine above upper 1.96SD, N(%) 0 0 0 0

16 Table 3. Urinary catecholamines in hypertensive patients on no treatment and on different drug therapies (Values are Mean±SEM).

Amlodipne & Nifedipine Verapamil & Beta- ACE Untreated(0) Nifedipine Diuretic(5) SR(2) Diltiazem(3) Blocker(4) Inhibitor(6) LA(1) P value (n=655) (n=177) (n=82) (n=39) (n=87) (n=55) (n=112)

Age (year) 47±1 54±1 53±1 54±2 51±1 50±2 55±1 <0.001

Gender (M/F) 337/316 84/90 37/44 26/13 41/46 42/13 57/54 <0.01#

Race (W/B/A) 346/153/64 82/45/28 39/27/11 13/13/6 49/21/9 31/16/3 62/21/16 0.116

SBP (mmHg) 159±1 160±2 163±2 166±3 153±2 163±3 165±2 0.001

DBP (mmHg) 97±0.5 96±1 96±2 98±2 92±1 99±2 98±1 0.004

Heart rate (beat/min) 80±1 81±1 83±2 75±2 68±1 86±2 82±1 <0.001

Body weight (kg) 77.0±0.6 78.5±1.3 78.5±1.9 77.3±2.3 79.3±1.9 75.2±1.9 78.7±1.6 0.621 Urine

Nor-erpinephrine (nmol/24h) 263.3±4.6 321.2±11.6* 307.4±18.0* 265.7±16.8 311.9±18.3* 287.9±18.6 284.3±13.2* <0.001¶

Nor-epinephrine/creatinine (nmol/mmol) 23.41±0.47 29.84±1.21* 27.23±1.54* 23.70±1.47 27.19±1.72* 29.56±2.27 26.70±1.24 <0.001§

Epinephrine (nmol/24h) 34.2±0.8 32.9±1.6 30.3±2.5 36.2±4.5 35.6±2.5 31.5±3.5 33.2±2.6 0.757¶

Epinephrine/creatinine (nmol/mmol) 2.97±0.08 2.92±0.13 2.79±0.24 3.13±0.34 3.18±0.26 3.31±0.44 2.97±0.23 0.800§

Dopamine (nmol/24h) 1923±29 1886±59 1991±133 1790±122 1911±67 1697±79 1756±68 0.407¶

Dopamine/creatinine (nmol/mmol) 167.8±2.7 171.0±5.4 174.2±11.0 160.7±12.3 169.0±8.1 171.1±8.2 161.0±5.2 0.505§

Sodium (mmol/24h) 142.6±2.7 146.4±5.7 146.7±9.2 149.1±11.4 137.9±6.5 133.3±8.5 131.0±5.9 0.486

Potassium (mmol/24h) 66.7±1.1 71.1±2.2 65.9±3.5 69.5±5.3 70.4±2.5 58.9±3.6 68.2±2.9 0.154

# by c2 test. * P<0.05 versus untreated group. ¶ adjusted for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium, potassium and creatinine excretion. § adjusted for age, sex, ethnic group, body weight, heart rate and 24h urinary sodium and potassium excretion.