Physiology& Behavior, Vol. 49, pp. 685-690. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

Plasma Catecholamine Responses During a Personalized Physical Stress as a Dynamic Characterization of Essential Hypertension C. T O S T I - C R O C E , C. L U C A R E L L I , .1 P. B E T r O , * A. F L O R I D I , t R. R I N A L D I , A. S A L V A T I , F. T A G G I * A N D F. S C I A R R A

Istituto di Clinica Medica Generale V, Universit~ "La Sapienza," Roma, Italy *lstituto Superiore di Sanitgt Roma, Italy "?Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universitgt di Perugia, Italy R e c e i v e d 11 June 1990 TOSTI-CROCE, C., C. LUCARELLI, P. BETrO, A. FLORIDI, R. RINALDI, A. SALVATI, F. TAGGI AND F. SCIARRA.

Plasma catecholamine responses during a personalized physical stress as a dynamic characterization of essential hypertension. PHYSIOL BEHAV 49(4) 685-690, 1991.--High performance liquid chromatography (HPLC) with electrochemical detection proves to be a reliable method for determination of plasma cateeholamines (CA) to assess the possible role of the sympathetic nervous system (SNS) in essential hypertension (EH). The present investigation in a group of 15 normotensive (N) and 13 stable EH patients, homogeneous for age and duration of hypertension, was carded out without treatment in the supine position, up-right position and during a personalized bicycle exercise. Mean blood pressure, mean heart rate, plasma renin activity and plasma aldosterone were also evaluated at the various exertion phases. Norepinephrine (NE) and epinephrine (E) showed a progressive increase in N and in EH patients, reaching the highest values at maximum effort. However, EH patients showed higher E plasma levels than N before maximum effort. Dopamine (DA) reached the highest values in N at maximum effort and in EH patients at recovery time. These findings allow us to foresee the possibility of a better characterization of the SNS role in EH. Epinephrine

Norepinepbxine

Dopamine

Physical exercise

THE possibility that the sympathetic nervous system (SNS) may play an important role in the pathogenesis of essential hypertension (EH) is supported by the finding that most patients benefit from drug treatment blocking adrenergic neuronal activity or depressing sympathetic tone of the central nervous system (37). However, evidence of a direct adrenergic role is observed only in rare cases of chromaffin cell tumors, producing high quantities of catecholamines (CA) (7). The difficulties encountered in the study of SNS involvement in EH pathogenesis is, in part, due to the lack of valuable indices for quantifying adrenergic activity. Urinary CA and their metabolites do not reflect the rapid variations in the SNS activity. On the contrary, measurement of plasma CA levels serves as a convenient index of SNS activity, despite certain limitations; "altered metabolism of norepinephrine (NE), for example, accelerated neuronal uptake in depression (12), may modify the influence of release rate on plasma levels. Peripheral levels cannot accurately reflect synaptic cleft levels of NE, but simply 'spillover' (13). Venous plasma levels reflect contribution

Essential hypertension

from many vascular beds, including the one closest to the sampling site (4); thus, specific regional alterations of NE production may be obscured. Epinephrine (E), secreted mostly by adrenal medulla, acts more like a conventional hormone, with plasma levels reflecting production under most circumstances. Despite all the above problems, peripheral plasma CA levels represent the most useful and widely available index of SNS activity" (24). In fact, experimental evidence shows a good correlation between plasma CA levels and general and regional SNS activity (11, 19, 23). Lastly, some studies (21, 24, 29) demonstrated a good correlation between venous and arterial plasma CA concentrations. The low levels of circulating CA may be easily detected by means of reliable and sensitive techniques, such as the radioenzymatic (5,27) method or high performance liquid chromatography (HPLC) (15,22). The great variability in plasma CA levels in relation to physiological and environmental factors imposes a strict study protocol, taking into consideration age, position, time of day, diet and duration of hypertension (3, 8, 17, 32, 34-36); the

1Requests for reprints should be addressed to Dr. Claudio Lucarelli, Laboratorio di Biochimica Clinica, Istituto Superiore di SanitY, Viale Regina Elena 299, 00161 Roma, Italy.

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TOSTI-CROCE ET AL.

different results obtained by various investigators may be partly attributed to the heterogeneity of the population studied (20). The aim of the present investigation was to assess possible differences in the behaviour of plasma E, NE and dopamine (DA) not only in supine position but also following postural changes and bicycle exercise in a homogeneous group of normotensive (N) and EH subjects. METHOD

Materials Norepinephrine (NE), epinephrine (E), dopamine (DA) and N-methyldopamine (NMDA, international standard) were purchased from Sigma (St. Louis, MO). 1-Octane sulfonic acid sodium salt (OSA) and ethylene glycol-0, 0'-bis (2-aminoethyl)N,N,N',N'-tetraacetic acid (EGTA) were purchased from Fluka (Buchs, Switzerland). Methanol (HPLC grade) and all other reagent grade chemicals were obtained from C. Erba (Milan, Italy), CM C-25 Sephadex from Pharmacia (Uppsala, Sweden), acid alumina AG-4 from Bio-Rad Labs (Richmond, CA). All solvents used in the HPLC system were solubilized with distilled water treated with a Milli-Q system (Millipore, Millford, MA).

Subjects The investigation was carried out in 15 N male volunteers, recruited amongst the personnel of the Institute of Clinical Medicine, aged 20-52 years (mean 32.4---2.8 SE) and 13 stable EH patients aged 22-54 years (mean 35.5 - 3.5 SE), with a diastolic blood pressure of >100 mmHg of 1-2 years duration. Patients were submitted to a normal sodium diet (120 mEq Na ÷ , 70 mEq K +) 10 days before the study. Treatment, if already begun, was interrupted at least 20 days before the test. Diagnosis was based on physical examination, renal function tests, serum electrolyte levels, urinary catecholamine excretion, urography and renography. Personalized Bicycle Exercise and Sampling Procedure All subjects underwent double exercise stress tests on a bicycle. The first test for adaptation was a typical graded bicycle test increasing effort at the rate of 25 watts (W) every 2 minutes until the patient was overcome by muscular exhaustion when the maximum heart rate was recorded. The second exercise stress test was performed after 48 h, increasing 25 W every 2 minutes for the first 2 steps, then 25 W/min reaching 85% of the maximum heart rate obtained during the first test. The work load then remained constant until physical exhaustion occurred. A Teflon cannula was placed in the left antecubital vein before the second test; blood samples were drawn for E, NE, DA, aldosterone (A) and plasma renin activity (PRA) determination after 45 min in supine position (phase 1), 5 min in up-right position (phase 2), 10 min resting on bicycle (phase 3), 5 min of exercise (phase 4), at maximum effort (phase 5) and 30 rain after completion of exercise (phase 6). Electrocardiogram, blood pressure and heart rate were continuously recorded. Blood samples were immediately transferred in tubes containing 20 Ixl of a solution containing 60 mg/ml EGTA (as anticoagulant) and 90 mg/ml reduced glutathione (as antioxidant), adjusted to pH 7 with NaOH 0.1 M. Samples were centrifuged at 4°C and plasma was removed and stored at -80°C until assay.

The HPLC apparatus consisted of a Series 410 Liquid Chromatograph Perkin-Elmer (Norwalk, CT) and a model 7125 Rheodyne injector (Cotati, CA) with a 100-p~lsample loop. The column was a reversed-phase C18 Perkin-Elmer (15 cm x 4.6 mm), 5 p~m particle size (Norwalk, CT). The electrochemical detection system Coulochem 5100 A (ESA, Bedford, MA) had a 5011 A analytical cell. The potentials were + 0.25 V for the first electrode and - 0 . 3 5 V for the second. The detector gain was set at 6000 for the first electrode and 20,000 for the second, with fullscale sensitivity of 1.7 nA and 0.5 nA, respectively. Chromatograms were analyzed with a Chemresearch Chromatographic Data Management computer (ISCO, Lincoln, NE) monitoring both detector signals. The mobile phase consisted of 0.013 M sodium acetate with 100 mg/1 OSA (ion-pairing reagent), 20 rag/1 disodium EDTA and 15% methanol (organic modifier) (pH 2.82). The isocratic elution of the compounds was carried out at room temperature with flow rate of 0.8 ml/min. Sample Pretreatment Samples were allowed to thaw at room temperature and a plasma aliquot (1 ml) was spiked with 50 Ixl of internal standard solution (NMDA, 80 ng/ml). Deproteinization was performed by adding 3 volumes of ice-cold methanol containing 2% of 0.5 M perchloric acid and by centrifugation (4000 g × 3 min, 4°C). For the isolation of catecholamines (NE, E, DA) 3 ml of 0.1 M phosphate buffer (pH 7) were added to the supernatant and the mixture was poured onto a CM C-25 Sephadex column (2 × 0.5 cm i.d.). Before use, the column was washed with 5 ml of 0.1 M hydrochloric acid and 10 ml of distilled water and buffered with 10 ml 0.1 M phosphate (pH 7). After the sample had passed through, the column was washed with 5 ml of distilled water. The catecholamines were eluted with 3 ml of 1.5 M perchloric acid collecting the effluent in conical tubes with caps. Two millilitres of 1.5 M Tris buffer (pH 9.3) containing 0.06 M EDTA and 20 mg of acid-washed alumina (1) were added to the solution. The tube was vortexed for 2 min on a whirl-mixer, the supernatant removed by vacuum aspiration and the alumina washed 3 times with 1 ml of water. The catecholamines were eluted with 100 Ixl of 0.1 M acetic acid, vortexed for 2 min, allowed to settle and centrifuged at 3000 x g for 2 min. The supernatant was removed and 25 txl were injected into the chromatograph. Retention times and intraassay coefficients of variation were 4 min and 3% for NE, 6 min and 7% for E, and 13 min and 3% for DA, respectively. Reproducibility of the method was established by analyzing a plasma pool (n = 8); the relative coefficients of variation for NE, E, DA were 3.1%, 6.5% and 3.2%, respectively. Recoveries of the prepurification step, obtained by analyzing plasma spiked with known amounts of substances (n = 6), ranged from 90% (DA) to 94% (E). PA and PRA were determined by RIA method using commercial kits obtained from Sorin Co. (Saluggia, Italy). Statistical Analysis The statistical analysis of data was performed with the BMDP statistical package (10). The statistical tests used were analysis of variance (ANOVA), t-test and the Mann-Whitney U-test for two samples (30). RESULTS

HPLC Determination of Plasma Catecholamines The chromatographic procedure includes original aspects and was obtained by improving several parameters of a previous method (22).

Blood Pressure and Heart Rate During the bicycle ergometer test in the N group, systolic blood pressure (BP), which in supine position displayed mean values of

PLASMA CATECHOLAMINES AND PHYSICAL STRESS

687

TABLE 1 BLOOD PRESSURE (BP) IN NORMAL SUBJECTS (N) AND HYPERTENSIVE PATIENTS (EH) DURING THE 6 PHASES OF THE TEST

Phases Cases

1

2

3

4

5

6

124.9 - 2.98* 78.1 -4- 1.89

124.1 --+ 4.80 82.0 --- 2.49

128.7 --- 4.52 86.2 -+ 3.14

148.3 -+ 5.05 90.0 +__2.38

193.7 ~ 8.68 99.5 --- 4.01

121.2 - 2.69 81.2 - 2.05

165.0 _ 4.62 103.1 -4- 4.00

159.0 - 5.12 105.4 - 5.49

148.1 - 5.48 107.7 - 4.63

177.0 --- 7.78 119.5 - 4.91

220.0 --- 4.71 121.3 _ 4.91

141.8 - 6.26 99.1 - 4.41

N Systolic BP Diastolic BP EH Systolic BP Diastolic BP

*Mean ___ SE (mmHg).

pg/ml, A% of 721.7; p