- Email: [email protected]
Blunted hemodynamic and hormonal responses to tilt-test in patients with sickle-cell disease
International Congress Series 1237 (2002) 169 – 177
Blunted hemodynamic and hormonal responses to tilt-test in patients with sickle-cell disease Eduardo Romero-Vecchione*, Jose´ Va´squez, Olimpia Pe´rez, Rafael Antequera Faculty of Medicine, Vargas Medical School, Central University of Venezuela, Plaza San Lorenzo, San Jose, Caracas 1010, Venezuela
Abstract Sickle-cell disease patients often show microvascular obstructions responsible for tissue hypoxia; a high proportion of them have a reduced left ventricle contractility and diastolic dysfunction. Also, these patients have a sympathetic dysfunction as evidenced by a reduced response to Valsalva’s maneuver and Cold pressor test, together with a lower urinary catecholamines output. We evaluated the hemodynamic response to passive orthostatic body change by leaning the patients, without feet support, at 60j for 15 min to avoid presyncopal symptoms; 18 sickle-cell disease patients (Hb SS, N = 8, HbS + h thalassemia, N = 4, HbF SS, N = 2, HbSC, N = 2, HbS, N = 1) and 20 healthy subjects were used as controls. During passive orthostatism, sickle-cell disease patients showed lower systolic blood pressure (peak 6.0 F 2.4 vs. 12.0 F 3.0 mm Hg, p < 0.05) and diastolic blood pressor response (peak 10.0 F 2.8 vs. 17.0 F 2.2 mm Hg, p < 0.05) than healthy subjects. Heart rate increase was lower in sickle-cell disease patients (7.0 F 2.0 vs. 13.0 F 3.0 beats/min, p < 0.05). Resting plasma cortisol and aldosterone concentration did not differ between sickle-cell disease patients and healthy volunteers. During tilt-test, cortisol increased up to 44.0% of basal values in healthy subjects and 13% in sickle-cell disease patients ( p < 0.05). Aldosterone also showed a reduced increase during tilt-test in sickle-cell anemia patients (13.3% vs. 30.2%, p < 0.05) when compared to healthy volunteers. These findings further confirm the presence of a sympathetic dysfunction in sickle-cell disease patients. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Sickle-cell disease; Sympathetic reflexes; Tilt-test; Aldosterone; Cortisol
1. Introduction Sickle-cell anemia has 3 – 5% incidence in afro-american Venezuelan [1] and Brazilian population [2]. The most frequent abnormal hemoglobins found in *
Corresponding author.
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 5 8 4 - 2
170
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
Venezuela are HbS (a2h2Glu –Val) and HbC. Although HbS or HbC heterozygotes are asymptomatic, homozygotes or combined hetherozygotes have a disease of variable severity. Patients with sickle-cell anemia often have microvascular obstructions which reduce blood and oxygen supply to peripheral tissues [3] and a significant proportion of them suffer systolic and diastolic heart contractility abnormalities, not fully explained by the severe chronic anemia they carry [4,5]. In a previous work [6], we demonstrated a blunted hypertensive response to the cold pressor test, a lower tachycardic response to Valsava’s maneuver and a reduced urinary noradrenaline output in patients whit sickle-cell anemia when compared to patients having iron deficiency anemia. The objective of this work was to explore further the cardiovascular abnormalities of the sympathetic reflex responses observed in sickle-cell anemia patients and the acute hormonal changes to tilt-test as an acute stressing stimulus, in sickle-cell disease patients; the sympathetic discharge induced by this test is dependent on barorreflex activity.
2. Patients and methods This study included 18 (8 female, 10 male gender) sickle-cell disease patients and 20 healthy volunteers (10 female, 10 male gender), age range: 16 –30 years old (Table 1). The sickle-cell disease patients regularly attended control visits at the hemathology department of the Municipal Blood Bank (Caracas, Venezuela). All patients and healthy subjects were clinically assessed by a routine clinical examination and the following laboratory evaluations: red and white blood cells count, hematocrit, total hemoglobin concentration, cellulose acetate separation of abnormal hemoglobins, plasma glucose, BUN, uric acid, creatinine, total colesterol, triglycerides, plasma sodium and potassium concentration; antibodies anti Hbs, hepatitis-B, HIV,
Table 1 Characteristcs of sickle-cell disease patients and healthy volunteers Parameter
Sickle-cell anemia
Healthy volunteers
N Male/female Age range (years) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) HR (beats/min) Hemoglobin (g/dl) Hematocrit
18 8/10 16 – 29 19.4 F 0.08 100.0 F 5.0 60.0 F 4.0 * 72.0 F 3.0 8.6 F 1.2 * 27.08 F 1.4 *
20 10/10 16 – 30 23.2 F 0.07 115.0 F 4.5 75.0 F 3.0 74.0 F 5.0 14.5 F 0.6 42.0 F 3.1
BMI = Body mass index, BDP = diastolic blood pressure, SBP = systolic blood pressure, HR = heart rate. * p < 0.05 vs. healthy volunteers.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
171
Chagas, disease and VDRL were assayed. A chest-X ray and a 12 leads rest ECG were obtained from all patients and healthy subjects. 2.1. Inclusion and exclusion criteria Patients carrying sickle-cell disease without pain crisis and receiving no blood transfusions during the 6 months previous to the hemodynamic tests were included. Patients receiving any medication 15 days before the tests were excluded; only 5 mg/day folic acid was allowed in all patients. All the experiments were conducted between 9 and 11 AM to avoid circadian variations of cortisol plasma levels. The objective and details of the procedures were explained to all subjects and patients who did not give their informed consent were excluded from the study. 2.2. Tilt-test Patients and healthy subjects were allowed to rest in supine position for 30 min and a Jelco catheter was introduced in the antecubital vein of the right arm at the beginning of the resting period. Blood pressure was recorded every 5 min in the left arm and D-II lead of the electrocardiogram (ECG) was continuously monitored with disposable electrodes placed on thorax and abdomen. Subjects were tilted in upright posture at a 60j angle leaning, during 15 s time, supported only by a pelvic harness, without feet support. Maximal tilt-time was 15 min to avoid presyncopal symptoms in sickle-cell anemia patients. Blood samples, systolic (SBP) and diastolic (DBP) blood pressure, and heart rate were obtained every 5 min in the left arm; D-II lead of the electrocardiogram (ECG) was continuously monitored. The patients returned to resting supine position for 15 additional minutes and the same parameters were taken again. Blood serum was immediately obtained after centrifugation at 1000 g for 10 min in a refrigerated (4 jC) centrifuge serum samples were stored frozen at 70 jC for no longer than 1 month until cortisol and aldosterone were assayed. 2.3. Cortisol and aldosterone assay Cortisol and aldosterone plasma levels were assayed by a commercial radioinmmunoassay (Biochemical Diagnostic Products, USA). Radioactivity from duplicate samples was measured in a liquid scintillation counter (Packard Ins, USA). Plasma Na + and K + ions were assayed by ion selective electrodes (Easylyte Lithium Medical, USA) and Chloride-ion by spectrophotometric assay (Stat-Fax, USA). 2.4. Statistical analysis Differences between samples were calculated by Student’s t-test for unpaired data, at a level of significance of p < 0.05. When repeated measures were used for comparison, the Bonferroni correction for t-test was applied.
172
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
3. Results All patients were diagnosed for sickle-cell disease during childhood or adolescence. The groups were comparable and matched for age, sex and body mass index (Table 1). Resting supine diastolic blood pressure was significantly lower in the anemic patients (60.0 F 4.0 mm Hg, p < 0.05) compared to healthy volunteers (75.0 F 3.0 mm Hg). Resting supine systolic blood pressure and heart rate were not different between groups. Hemoglobin concentration and hematocrit were lower in the sickle-cell disease group (Table 1). Abnormal hemoglobins found in the sickle-cell disease group were the following: HbSS (N = 8), Hbs + h thalassemia (N = 4), HbSS + HbF (N = 2), HbSC (N = 2) and HbS (N = 1). The resting ECG in both groups showed no abnormalities. All other plasma biochemical analyses were within normal range. Chest X-ray showed no alterations. 3.1. Tilt-test During the tilt-test, sickle-cell diseased patients showed a lower 6 F 2 mm Hg systolic blood pressure increase when compared to 12 F 3 mm Hg increase observed in healthy volunteers ( p < 0.05) (Fig. 1); at 15 min of tilt-test; SBP started to fall in sickle-cell disease patients, but not in healthy volunteers. Diastolic blood pressure showed a lower (9 F 2 mm Hg, p < 0.05) increase in sickle-cell disease patients when compared to healthy volunteers (17 F 3 mm Hg) (Fig. 2). Heart rate also showed a lower progressive increase (7 beats/min) than in healthy volunteers (13 beats/min) at 15 min of tilt-test ( p < 0.05) (Fig. 3).
Fig. 1. Systolic blood pressure (SBP) increase observed during head up tilt-test at 60j angle in sickle-cell disease (SCD) patients and control healthy volunteers (C); N = 18/20, respectively. * p < 0.05. Supine SBP is showed after 15-min post tilt-test.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
173
Fig. 2. Diastolic blood pressure (DBP) increase observed during head up tilt-test at 60j angle in sickle-cell disease (SCD) patients and control healthy volunteers (C); N = 18/20, respectively. * p < 0.05. Supine DBP is showed after 15-min post tilt-test.
All hemodynamic variables returned to normal resting values after 15 min recovery in supine position in both groups. 3.2. Cortisol and aldosterone Resting plasma cortisol concentration in supine position, between 9 and 11 AM, did not differ between control and sickle-cell anemia groups (Fig. 4); however, sickle-cell anemia patients exhibited a little lower cortisol concentration.
Fig. 3. Heart rate (HR) changes observed during head up tilt-test at 60j angle in sickle-cell disease (SDC) patients and control healthy volunteers (C); N = 18/20, respectively.
174
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
Fig. 4. Cortisol plasma levels during 15-min head up tilt-test at 60j angle in healthy volunteers (filled circles) and sickle-cell disease patients (open circles). * p < 0.05.
In healthy volunteers, cortisol progressively increased during tilt-test, up to 44.4%, of basal values, at the end of the tilting procedure; compared to 13.0% increase observed in sickle-cell anemia patients ( p < 0.05). In sickle-cell diseased patients, plasma aldosterone showed a 13.3% slight increase (Fig. 5), which was statistically different when compared to the greater increase this hormone showed in healthy volunteers 30.2% ( p < 0.05).
Fig. 5. Aldosterone plasma levels during 15-min head up tilt-test at 60j angle in healthy volunteers (filled circles) and sickle-cell disease patients (open circles). * p < 0.05.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
175
4. Discussion 4.1. Hemodynamic changes Tilt-test is a strong sympathetic stimulus produced by blood volume pooling in the lower extremities and lower abdomen, after leaning the body without feet support [8]. This maneuver decreases blood pressure at carotid and thoracic barorreceptors, which reflexly stimulates a sympathetic discharge from vasopressor neurons of the brain medulla. Blunting of this response in patients with sickle-cell disease is the expression of a sympathetic dysfunction these patients show when submitted to different stimuli like the cold pressor test and Valsalva’s maneuver [6,7]. These patients also showed a reduced 24 h urine noradrenaline excretion [6], meaning the overall release of this neurotransmitter is also decreased. The pathophysiology of this sympathetic response attenuation in sickle-cell diseased patients is not fully understood; we put forward several hypothesis to explain the fact; chronic hypoxia and vasooclussive events during this chronic anemia may be conducted to: (1) postganglionc sympathetic nerve fibers damage and (2) reduced amounts of noradrenaline released from sympathetic fibers, when submitted to stressing conditions. Damage to postganglionic sympathetic nerve fibers may be caused by long-term tissue iron deposits, as a consequence of the continuous sickle-cell hemolisis. Homozygous sickle-cell anemia patients suffer continuous hemolisis and the released hemoglobin is a constant source of iron ions that can be intra- and extracellularly trapped, producing direct tissue damage including the sympathetic postganglionic nerve fibers, or indirect membrane cell deterioration through lipid peroxidation [9]. In agreement with this argument, it is interesting that iron-limited erythropoiesis, achieved by periodic red blood-cell apheresis, improved the signs and symptoms of thalassemia and sickle red blood cells anemia [10]. Thalassemic patients, which also suffer hemolysis, show increased iron deposit levels in heart myocardium that are probably related to atrial dilatation; however, atrial function of these patients remain unaltered while they are asymptomatic [11]. For unknown reasons, endothelial cell of sickle-cell patients detach to plasma in increased amounts, in comparison to other hemolytic anemias regardless of patient’s clinical status, both during acute painful crisis or in between them. Endothelial activation promotes platelets and white cell adhesion so in contributing to the vasooclussive phenomenon, these patients suffer. The sympathetic dysfunction we observed could prevent excessive vosoconstriction that could aggravate the vasooclussive episodes [12]. Vasooclusive phenomenon observed in sickle-cell disease patients could also be stimulated by the shear stress endothelial dysfunction recently described by Belhassen et al. [13]. 4.2. Hormones plasma levels Resting supine plasma concentration of cortisol and aldosterone was not different between groups; however, the physiological increase of both hormones observed in healthy volunteers upon tilt-test was decreased in patients carrying sickle-cell anemia, this fact indicates that the adrenal cortex of these patients is not properly functioning.
176
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
The lower aldosterone secretion in sickle-cell anemia patients also indicates that adrenal cortex is failing to release adequate amounts of this mineralocorticoid hormone under a stress condition like the upright posture during tilt-test. As we did not measure adrenocorticotrophic plasma levels, we cannot rule out that this hypophysial hormone has been adequately released during tilt-test. The reason for this stimulated reduction of adrenocortical hormone secretion could be a local tissue gland dysfunction imposed by the chronic hypoxia exerted upon the gland, which conducts to a lower aldosterone release induced by the hemodynamic stimulus. In conclusion, the hemodynamic and some hormonal responses to sympathetic stress are decreased in patients with sickle-cell anemia and the reduced release of the adrenocortical hormones cortisol and aldosterone, observed during tilt-test, indicates a slower adrenocortical gland function in these anemic patients when submitted to stressing stimuli.
Acknowledgements We acknowledge the technical assistance of Lic. Lennis Lara for her skill with biochemical assays, and Mrs. Ingrid Gonza´lez for typing the manuscript. This work was partially sponsored by Consejo de Desarrollo Cientı´fico y Humanı´stico de la Universidad Central de Venezuela, Grant No. 09-11-0031-96.
References [1] T. Arends, Epidemiologı´a de las variants hemoglobı´nicas en Venezuela, Gac. Med. Caracas 2 (1984) 189 – 192. [2] M.A. Zago, W.A. Silva, J.R. Franco, Hemoglobinopathies and other hereditary hematological diseases in the Brazilian population, Cien. Cult. J. Braz. Assoc. Adv. Sci. 51 (1999) 226 – 234. [3] M.E. Fabry, D.K. Kaul, Sickle cell vaso-occulsion, Hematol. Oncol. 5 (1991) 375 – 385. [4] I. Balfour, W. Govitz, F.W. Arensman, C. Eubig, M. Garrido, C. Jones, Left ventricular filling in sickle cell anemia, Am. J. Cardiol. 61 (1998) 395 – 399. [5] Cardiovascular function during rest and exercise in patients with sickle-cell anemia and coexisting alpha thalassemia—2, Am. J. Hematol. 52 (1996) 96 – 102. [6] E. Romero-Vecchione, O. Perez, M. Wessolosky, S. Liberatore, J. Vasquez, Respuestas cardiovasculares auto´nomas anormales en pacientes con anemia drepanocı´tica, Sangre 40 (1995) 393 – 399. [7] W. Covitz, C. Eubig, J.C. Balfour, R. Jerath, B.S. Alpert, W.B. Strong, R. Dv Rant, B. Hadden, Exerciseinduced cardiac dysfunction in sickle anemia: a radio nuclide study, Am. J. Cardiol. 51 (1983) 570 – 575. [8] T. Vubrial, R.J. Bryg, M. Maddens, W.E. Boden, Effect of passive tilt on sympathetic and parasympathetic components of heart rate variability in normal subjects, Am. J. Cardiol. 63 (1989) 1117 – 1120. [9] O. Shalev, T. Repka, M. Maddens, W.E. Boden, Defepirone (Li) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo, Blood 86 (1995) 2008 – 2013. [10] O. Castro, W.N. Poillon, H. Finke, E. Massac, B.C. Kim, Improvement of sickle cell anemia by iron-limited erythropoiesis, Am. J. Hematol. 47 (1994) 74 – 81. [11] L. Antoniades, K. Simamonian, G. Daniel, D. Petrondas, C. Zambartas, Left atrial mechanical function in a symptomatic patients with thalassemia major, Eur. Heart J. 21 (2000) 412 (P2203)(Abstract Suppl. XXII Cong. Eur. Soc. Cardiol.).
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
177
[12] A. Solovey, Y. Lin, P. Browne, S. Choong, E. Wayner, R. Hebbel, Circulating activated endothelial cells in sickle cell anemia, N. Eng. J. Med. 337 (1977) 1584 – 1590. [13] L. Belhassen, G. Pelle, S. Sediane, C. Carville, C. Bucherer, F. Lacombe, Endothelial dysfunction in patients with sickle cell disease is related to selective impairment of shear stress-mediated vasodilatation, Blood 97 (2001) 1584 – 1589.
Blunted hemodynamic and hormonal responses to tilt-test in patients with sickle-cell disease Eduardo Romero-Vecchione*, Jose´ Va´squez, Olimpia Pe´rez, Rafael Antequera Faculty of Medicine, Vargas Medical School, Central University of Venezuela, Plaza San Lorenzo, San Jose, Caracas 1010, Venezuela
Abstract Sickle-cell disease patients often show microvascular obstructions responsible for tissue hypoxia; a high proportion of them have a reduced left ventricle contractility and diastolic dysfunction. Also, these patients have a sympathetic dysfunction as evidenced by a reduced response to Valsalva’s maneuver and Cold pressor test, together with a lower urinary catecholamines output. We evaluated the hemodynamic response to passive orthostatic body change by leaning the patients, without feet support, at 60j for 15 min to avoid presyncopal symptoms; 18 sickle-cell disease patients (Hb SS, N = 8, HbS + h thalassemia, N = 4, HbF SS, N = 2, HbSC, N = 2, HbS, N = 1) and 20 healthy subjects were used as controls. During passive orthostatism, sickle-cell disease patients showed lower systolic blood pressure (peak 6.0 F 2.4 vs. 12.0 F 3.0 mm Hg, p < 0.05) and diastolic blood pressor response (peak 10.0 F 2.8 vs. 17.0 F 2.2 mm Hg, p < 0.05) than healthy subjects. Heart rate increase was lower in sickle-cell disease patients (7.0 F 2.0 vs. 13.0 F 3.0 beats/min, p < 0.05). Resting plasma cortisol and aldosterone concentration did not differ between sickle-cell disease patients and healthy volunteers. During tilt-test, cortisol increased up to 44.0% of basal values in healthy subjects and 13% in sickle-cell disease patients ( p < 0.05). Aldosterone also showed a reduced increase during tilt-test in sickle-cell anemia patients (13.3% vs. 30.2%, p < 0.05) when compared to healthy volunteers. These findings further confirm the presence of a sympathetic dysfunction in sickle-cell disease patients. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Sickle-cell disease; Sympathetic reflexes; Tilt-test; Aldosterone; Cortisol
1. Introduction Sickle-cell anemia has 3 – 5% incidence in afro-american Venezuelan [1] and Brazilian population [2]. The most frequent abnormal hemoglobins found in *
Corresponding author.
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 5 8 4 - 2
170
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
Venezuela are HbS (a2h2Glu –Val) and HbC. Although HbS or HbC heterozygotes are asymptomatic, homozygotes or combined hetherozygotes have a disease of variable severity. Patients with sickle-cell anemia often have microvascular obstructions which reduce blood and oxygen supply to peripheral tissues [3] and a significant proportion of them suffer systolic and diastolic heart contractility abnormalities, not fully explained by the severe chronic anemia they carry [4,5]. In a previous work [6], we demonstrated a blunted hypertensive response to the cold pressor test, a lower tachycardic response to Valsava’s maneuver and a reduced urinary noradrenaline output in patients whit sickle-cell anemia when compared to patients having iron deficiency anemia. The objective of this work was to explore further the cardiovascular abnormalities of the sympathetic reflex responses observed in sickle-cell anemia patients and the acute hormonal changes to tilt-test as an acute stressing stimulus, in sickle-cell disease patients; the sympathetic discharge induced by this test is dependent on barorreflex activity.
2. Patients and methods This study included 18 (8 female, 10 male gender) sickle-cell disease patients and 20 healthy volunteers (10 female, 10 male gender), age range: 16 –30 years old (Table 1). The sickle-cell disease patients regularly attended control visits at the hemathology department of the Municipal Blood Bank (Caracas, Venezuela). All patients and healthy subjects were clinically assessed by a routine clinical examination and the following laboratory evaluations: red and white blood cells count, hematocrit, total hemoglobin concentration, cellulose acetate separation of abnormal hemoglobins, plasma glucose, BUN, uric acid, creatinine, total colesterol, triglycerides, plasma sodium and potassium concentration; antibodies anti Hbs, hepatitis-B, HIV,
Table 1 Characteristcs of sickle-cell disease patients and healthy volunteers Parameter
Sickle-cell anemia
Healthy volunteers
N Male/female Age range (years) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) HR (beats/min) Hemoglobin (g/dl) Hematocrit
18 8/10 16 – 29 19.4 F 0.08 100.0 F 5.0 60.0 F 4.0 * 72.0 F 3.0 8.6 F 1.2 * 27.08 F 1.4 *
20 10/10 16 – 30 23.2 F 0.07 115.0 F 4.5 75.0 F 3.0 74.0 F 5.0 14.5 F 0.6 42.0 F 3.1
BMI = Body mass index, BDP = diastolic blood pressure, SBP = systolic blood pressure, HR = heart rate. * p < 0.05 vs. healthy volunteers.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
171
Chagas, disease and VDRL were assayed. A chest-X ray and a 12 leads rest ECG were obtained from all patients and healthy subjects. 2.1. Inclusion and exclusion criteria Patients carrying sickle-cell disease without pain crisis and receiving no blood transfusions during the 6 months previous to the hemodynamic tests were included. Patients receiving any medication 15 days before the tests were excluded; only 5 mg/day folic acid was allowed in all patients. All the experiments were conducted between 9 and 11 AM to avoid circadian variations of cortisol plasma levels. The objective and details of the procedures were explained to all subjects and patients who did not give their informed consent were excluded from the study. 2.2. Tilt-test Patients and healthy subjects were allowed to rest in supine position for 30 min and a Jelco catheter was introduced in the antecubital vein of the right arm at the beginning of the resting period. Blood pressure was recorded every 5 min in the left arm and D-II lead of the electrocardiogram (ECG) was continuously monitored with disposable electrodes placed on thorax and abdomen. Subjects were tilted in upright posture at a 60j angle leaning, during 15 s time, supported only by a pelvic harness, without feet support. Maximal tilt-time was 15 min to avoid presyncopal symptoms in sickle-cell anemia patients. Blood samples, systolic (SBP) and diastolic (DBP) blood pressure, and heart rate were obtained every 5 min in the left arm; D-II lead of the electrocardiogram (ECG) was continuously monitored. The patients returned to resting supine position for 15 additional minutes and the same parameters were taken again. Blood serum was immediately obtained after centrifugation at 1000 g for 10 min in a refrigerated (4 jC) centrifuge serum samples were stored frozen at 70 jC for no longer than 1 month until cortisol and aldosterone were assayed. 2.3. Cortisol and aldosterone assay Cortisol and aldosterone plasma levels were assayed by a commercial radioinmmunoassay (Biochemical Diagnostic Products, USA). Radioactivity from duplicate samples was measured in a liquid scintillation counter (Packard Ins, USA). Plasma Na + and K + ions were assayed by ion selective electrodes (Easylyte Lithium Medical, USA) and Chloride-ion by spectrophotometric assay (Stat-Fax, USA). 2.4. Statistical analysis Differences between samples were calculated by Student’s t-test for unpaired data, at a level of significance of p < 0.05. When repeated measures were used for comparison, the Bonferroni correction for t-test was applied.
172
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
3. Results All patients were diagnosed for sickle-cell disease during childhood or adolescence. The groups were comparable and matched for age, sex and body mass index (Table 1). Resting supine diastolic blood pressure was significantly lower in the anemic patients (60.0 F 4.0 mm Hg, p < 0.05) compared to healthy volunteers (75.0 F 3.0 mm Hg). Resting supine systolic blood pressure and heart rate were not different between groups. Hemoglobin concentration and hematocrit were lower in the sickle-cell disease group (Table 1). Abnormal hemoglobins found in the sickle-cell disease group were the following: HbSS (N = 8), Hbs + h thalassemia (N = 4), HbSS + HbF (N = 2), HbSC (N = 2) and HbS (N = 1). The resting ECG in both groups showed no abnormalities. All other plasma biochemical analyses were within normal range. Chest X-ray showed no alterations. 3.1. Tilt-test During the tilt-test, sickle-cell diseased patients showed a lower 6 F 2 mm Hg systolic blood pressure increase when compared to 12 F 3 mm Hg increase observed in healthy volunteers ( p < 0.05) (Fig. 1); at 15 min of tilt-test; SBP started to fall in sickle-cell disease patients, but not in healthy volunteers. Diastolic blood pressure showed a lower (9 F 2 mm Hg, p < 0.05) increase in sickle-cell disease patients when compared to healthy volunteers (17 F 3 mm Hg) (Fig. 2). Heart rate also showed a lower progressive increase (7 beats/min) than in healthy volunteers (13 beats/min) at 15 min of tilt-test ( p < 0.05) (Fig. 3).
Fig. 1. Systolic blood pressure (SBP) increase observed during head up tilt-test at 60j angle in sickle-cell disease (SCD) patients and control healthy volunteers (C); N = 18/20, respectively. * p < 0.05. Supine SBP is showed after 15-min post tilt-test.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
173
Fig. 2. Diastolic blood pressure (DBP) increase observed during head up tilt-test at 60j angle in sickle-cell disease (SCD) patients and control healthy volunteers (C); N = 18/20, respectively. * p < 0.05. Supine DBP is showed after 15-min post tilt-test.
All hemodynamic variables returned to normal resting values after 15 min recovery in supine position in both groups. 3.2. Cortisol and aldosterone Resting plasma cortisol concentration in supine position, between 9 and 11 AM, did not differ between control and sickle-cell anemia groups (Fig. 4); however, sickle-cell anemia patients exhibited a little lower cortisol concentration.
Fig. 3. Heart rate (HR) changes observed during head up tilt-test at 60j angle in sickle-cell disease (SDC) patients and control healthy volunteers (C); N = 18/20, respectively.
174
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
Fig. 4. Cortisol plasma levels during 15-min head up tilt-test at 60j angle in healthy volunteers (filled circles) and sickle-cell disease patients (open circles). * p < 0.05.
In healthy volunteers, cortisol progressively increased during tilt-test, up to 44.4%, of basal values, at the end of the tilting procedure; compared to 13.0% increase observed in sickle-cell anemia patients ( p < 0.05). In sickle-cell diseased patients, plasma aldosterone showed a 13.3% slight increase (Fig. 5), which was statistically different when compared to the greater increase this hormone showed in healthy volunteers 30.2% ( p < 0.05).
Fig. 5. Aldosterone plasma levels during 15-min head up tilt-test at 60j angle in healthy volunteers (filled circles) and sickle-cell disease patients (open circles). * p < 0.05.
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
175
4. Discussion 4.1. Hemodynamic changes Tilt-test is a strong sympathetic stimulus produced by blood volume pooling in the lower extremities and lower abdomen, after leaning the body without feet support [8]. This maneuver decreases blood pressure at carotid and thoracic barorreceptors, which reflexly stimulates a sympathetic discharge from vasopressor neurons of the brain medulla. Blunting of this response in patients with sickle-cell disease is the expression of a sympathetic dysfunction these patients show when submitted to different stimuli like the cold pressor test and Valsalva’s maneuver [6,7]. These patients also showed a reduced 24 h urine noradrenaline excretion [6], meaning the overall release of this neurotransmitter is also decreased. The pathophysiology of this sympathetic response attenuation in sickle-cell diseased patients is not fully understood; we put forward several hypothesis to explain the fact; chronic hypoxia and vasooclussive events during this chronic anemia may be conducted to: (1) postganglionc sympathetic nerve fibers damage and (2) reduced amounts of noradrenaline released from sympathetic fibers, when submitted to stressing conditions. Damage to postganglionic sympathetic nerve fibers may be caused by long-term tissue iron deposits, as a consequence of the continuous sickle-cell hemolisis. Homozygous sickle-cell anemia patients suffer continuous hemolisis and the released hemoglobin is a constant source of iron ions that can be intra- and extracellularly trapped, producing direct tissue damage including the sympathetic postganglionic nerve fibers, or indirect membrane cell deterioration through lipid peroxidation [9]. In agreement with this argument, it is interesting that iron-limited erythropoiesis, achieved by periodic red blood-cell apheresis, improved the signs and symptoms of thalassemia and sickle red blood cells anemia [10]. Thalassemic patients, which also suffer hemolysis, show increased iron deposit levels in heart myocardium that are probably related to atrial dilatation; however, atrial function of these patients remain unaltered while they are asymptomatic [11]. For unknown reasons, endothelial cell of sickle-cell patients detach to plasma in increased amounts, in comparison to other hemolytic anemias regardless of patient’s clinical status, both during acute painful crisis or in between them. Endothelial activation promotes platelets and white cell adhesion so in contributing to the vasooclussive phenomenon, these patients suffer. The sympathetic dysfunction we observed could prevent excessive vosoconstriction that could aggravate the vasooclussive episodes [12]. Vasooclusive phenomenon observed in sickle-cell disease patients could also be stimulated by the shear stress endothelial dysfunction recently described by Belhassen et al. [13]. 4.2. Hormones plasma levels Resting supine plasma concentration of cortisol and aldosterone was not different between groups; however, the physiological increase of both hormones observed in healthy volunteers upon tilt-test was decreased in patients carrying sickle-cell anemia, this fact indicates that the adrenal cortex of these patients is not properly functioning.
176
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
The lower aldosterone secretion in sickle-cell anemia patients also indicates that adrenal cortex is failing to release adequate amounts of this mineralocorticoid hormone under a stress condition like the upright posture during tilt-test. As we did not measure adrenocorticotrophic plasma levels, we cannot rule out that this hypophysial hormone has been adequately released during tilt-test. The reason for this stimulated reduction of adrenocortical hormone secretion could be a local tissue gland dysfunction imposed by the chronic hypoxia exerted upon the gland, which conducts to a lower aldosterone release induced by the hemodynamic stimulus. In conclusion, the hemodynamic and some hormonal responses to sympathetic stress are decreased in patients with sickle-cell anemia and the reduced release of the adrenocortical hormones cortisol and aldosterone, observed during tilt-test, indicates a slower adrenocortical gland function in these anemic patients when submitted to stressing stimuli.
Acknowledgements We acknowledge the technical assistance of Lic. Lennis Lara for her skill with biochemical assays, and Mrs. Ingrid Gonza´lez for typing the manuscript. This work was partially sponsored by Consejo de Desarrollo Cientı´fico y Humanı´stico de la Universidad Central de Venezuela, Grant No. 09-11-0031-96.
References [1] T. Arends, Epidemiologı´a de las variants hemoglobı´nicas en Venezuela, Gac. Med. Caracas 2 (1984) 189 – 192. [2] M.A. Zago, W.A. Silva, J.R. Franco, Hemoglobinopathies and other hereditary hematological diseases in the Brazilian population, Cien. Cult. J. Braz. Assoc. Adv. Sci. 51 (1999) 226 – 234. [3] M.E. Fabry, D.K. Kaul, Sickle cell vaso-occulsion, Hematol. Oncol. 5 (1991) 375 – 385. [4] I. Balfour, W. Govitz, F.W. Arensman, C. Eubig, M. Garrido, C. Jones, Left ventricular filling in sickle cell anemia, Am. J. Cardiol. 61 (1998) 395 – 399. [5] Cardiovascular function during rest and exercise in patients with sickle-cell anemia and coexisting alpha thalassemia—2, Am. J. Hematol. 52 (1996) 96 – 102. [6] E. Romero-Vecchione, O. Perez, M. Wessolosky, S. Liberatore, J. Vasquez, Respuestas cardiovasculares auto´nomas anormales en pacientes con anemia drepanocı´tica, Sangre 40 (1995) 393 – 399. [7] W. Covitz, C. Eubig, J.C. Balfour, R. Jerath, B.S. Alpert, W.B. Strong, R. Dv Rant, B. Hadden, Exerciseinduced cardiac dysfunction in sickle anemia: a radio nuclide study, Am. J. Cardiol. 51 (1983) 570 – 575. [8] T. Vubrial, R.J. Bryg, M. Maddens, W.E. Boden, Effect of passive tilt on sympathetic and parasympathetic components of heart rate variability in normal subjects, Am. J. Cardiol. 63 (1989) 1117 – 1120. [9] O. Shalev, T. Repka, M. Maddens, W.E. Boden, Defepirone (Li) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo, Blood 86 (1995) 2008 – 2013. [10] O. Castro, W.N. Poillon, H. Finke, E. Massac, B.C. Kim, Improvement of sickle cell anemia by iron-limited erythropoiesis, Am. J. Hematol. 47 (1994) 74 – 81. [11] L. Antoniades, K. Simamonian, G. Daniel, D. Petrondas, C. Zambartas, Left atrial mechanical function in a symptomatic patients with thalassemia major, Eur. Heart J. 21 (2000) 412 (P2203)(Abstract Suppl. XXII Cong. Eur. Soc. Cardiol.).
E. Romero-Vecchione et al. / International Congress Series 1237 (2002) 169–177
177
[12] A. Solovey, Y. Lin, P. Browne, S. Choong, E. Wayner, R. Hebbel, Circulating activated endothelial cells in sickle cell anemia, N. Eng. J. Med. 337 (1977) 1584 – 1590. [13] L. Belhassen, G. Pelle, S. Sediane, C. Carville, C. Bucherer, F. Lacombe, Endothelial dysfunction in patients with sickle cell disease is related to selective impairment of shear stress-mediated vasodilatation, Blood 97 (2001) 1584 – 1589.