Racial Differences in Plasma Endothelin-1 Concentrations in Individuals With Essential Hypertension
Abstract
Hypertension is more prevalent in blacks than whites, and the reasons for this difference remain unclear. To test whether endothelin may play a role in these racial variations, we analyzed plasma samples from black and white women and men with high blood pressure by an enzyme-linked immunoassay specific for endothelin-1 (ET-1), a potent vasoconstrictor, and compared them with those obtained from similar subjects with normal blood pressure. Both female and male hypertensive blacks had elevated levels of immunoreactive ET-1 (11.3±1.0 and 12.3±1.3 nmol/L, respectively) compared with values in normotensive control blacks (1.5±0.2 and 1.4±0.2 nmol/L). Corresponding values in female and male hypertensive whites were 3.8±0.6 and 3.8±0.6 nmol/L, respectively, compared with respective values of 1.4±0.1 and 2.8±0.4 nmol/L in normotensive control whites. These results indicate that plasma concentrations of immunoreactive ET-1 levels differ significantly between black and white individuals with high blood pressure. This finding may be an important factor in the etiology of racial differences in the prevalence and severity of hypertension and deserves further study.
Endothelial cells are important in the regulation of vascular tonus and homeostasis, in part by secreting vasoactive substances.1 One of these factors, ET-1, is a 21–amino acid residue peptide with potent vasopressor actions.2 In addition to its contractile effects, it has also been shown to stimulate mitogenesis in a number of cell types, including vascular and airway smooth muscle cells in vitro.34 Moreover, ET-1 displays modulatory effects on the endocrine system, including stimulation of Ang II and aldosterone production.56 Interestingly, it inhibits renin release in vitro but causes a significant increase in plasma renin activity when administered to anesthetized animals.7
On the basis of the broad spectrum of biological effects associated with ET-1, it has been suggested to be involved in a variety of diseases, such as hypertension, atherosclerosis, and asthma. Plasma ET-1 levels have been shown to be elevated in individuals with pulmonary hypertension, atherosclerosis, and ischemic heart disease.8910 The role of ET-1 in the pathogenesis of hypertension is still unknown. Although a direct correlation between the concentration of circulating ET-1 and high BP has not been demonstrated, it has been shown that plasma ET-1 levels tend to increase with age and are higher in men than women.1112 No studies have been reported on the relationship of race and circulating ET-1 concentrations.
It is well documented that a substantial difference exists in hypertension among people of African and European heritage.131415 Hypertension among blacks is more prevalent, earlier in onset, and associated with more end-organ damage, including left ventricular hypertrophy, renal failure, stroke, and death.131415 There are, however, few explanations for this difference despite substantial clinical and laboratory investigation. Further work is needed to identify factors that contribute to the problem.
This study reports the first investigation of racial variations in plasma ET-1 concentrations among hypertensive and normotensive individuals. Concentrations of irET-1 in the samples obtained from both male and female black hypertensive subjects were found to be significantly higher than those in normotensive black control subjects and white hypertensive subjects.
Methods
Subject Selection
This study was conducted under the guidelines of the Medical Center of Central Georgia Institutional Review Committee. Fifty hypertensive and 50 normotensive individuals participated; each group was divided into four categories by sex and race. In each of these groups, 12 or 13 subjects were included. The mean ages (years) and arterial BP (millimeters of mercury) values (mean±SE) for each group were as follows: black normotensive women, 51±7 and 91±3; black normotensive men, 48±5 and 90±4; black hypertensive women, 61±4 and 130±2; black hypertensive men, 59±5 and 132±4; white normotensive women, 52±5 and 88±4; white normotensive men, 55±4 and 87±3; white hypertensive women, 62±5 and 125±1; and white hypertensive men, 61±4 and 126±2. After consent was obtained from each participant, BP was measured with a mercury sphygmomanometer after subjects had rested 15 minutes in the supine position. Normal BP was defined as systolic pressure equal to or less than 140 mm Hg and diastolic pressure equal to or less than 80 mm Hg. Hypertension was defined as systolic pressure equal to or greater than 160 mm Hg and diastolic pressure equal to or greater than 95 mm Hg. Borderline hypertensive subjects (systolic pressure between 140 and 160 mm Hg and diastolic pressure between 85 and 90 mm Hg) were not included in this study. Individuals with renal failure, pulmonary edema, cardiogenic shock, or a myocardial infarction within the last 6 months were excluded. Individuals with known hypertension on medical therapy were accepted if their hypertension was uncontrolled at the time of examination and blood collection. The BP medication used by hypertensive subjects included Ca2+ channel blockers, angiotensin-converting enzyme inhibitors, β-blockers, diuretics, and vasodilators.
Preparation and Analysis of Blood Samples
Blood samples (3 mL) were collected into EDTA-containing tubes, and plasma was aliquoted into plastic Eppendorf tubes (0.5 mL each) and stored at −80°C until the day of the assays. The amount of irET-1 in the plasma was determined with an enzyme-linked immunoassay (ELISA) kit specifically designed for direct measurement of irET-1 in plasma (American Research Products) that eliminates the extraction step of plasma with Sep-Pak C18 cartridges. To provide maximum sensitivity, an immunoaffinity purified polyclonal capture antibody and monoclonal detection antibody, both highly specific for ET-1, were used in the assay. The sensitivity of the assay was 0.1 to 15.6 nmol/L. The cross-reactivities of the antibodies used were reported to be less than 1% with big ET-1-(1-38) and big ET-1-(22-38), less than 5% with ET-3, and 100% with ET-2. The manufacturer reported the intra-assay and interassay variabilities of the kit to be 3.3% and 3.5%, respectively.
Each plasma sample was analyzed three times in separate ELISA runs. The correlation coefficient between each run ranged between .87 and .91. The samples that contained irET-1 above the maximum standard concentration were diluted with assay buffer provided in the kit, and diluted samples were also assayed in three individual experiments.
Statistical Analysis
Data were analyzed for multiple comparisons by one-way ANOVA written with the Statistical Analysis Program (SAS); a value of P<.05 was considered significant. The results are given as mean±SE of three independent measurements.
Results
The distribution of plasma irET-1 concentration in each group is shown in Fig 1. Levels of irET-1 in black hypertensive women and men (11.3±1.0 and 12.3±1.3 nmol/L, respectively) were significantly higher (P<.001) than in black normotensive women and men (1.5±0.2 and 1.4±0.2 nmol/L, respectively). Interestingly, as shown in Fig 2, irET-1 concentrations were found to be much lower in hypertensive whites (women and men both 3.8±0.6 nmol/L). Compared with irET-1 levels in white normotensive subjects (1.4±0.1 and 2.8±0.4 nmol/L, women and men, respectively), the difference was significant (P<.05). Correlation coefficients of mean arterial BP with ET-1 levels, calculated separately for hypertensive and normotensive subjects, were .155 (P=.28) and .07 (P=.62), indicating that elevated ET-1 levels are independent of the degree of hypertension. In addition, the majority of the hypertensive subjects were on medical treatment, as reported above. However, the number of subjects in each group taking different medications was not sufficient for statistical analysis to conclude any association between any of these drugs and ET-1 levels.
Among normotensive subjects, there were no significant differences between sex (P=.147) and race (P=.129). The difference between hypertensive blacks and whites was found to be extremely significant (P<.001). The means of irET-1 levels in black hypertensive women and men were fourfold and threefold higher than in white hypertensive women and men, respectively.
The Table summarizes the statistical analysis of irET-1 concentrations in hypertensive subjects (n=50) versus normotensive subjects (n=50), including white and black women and men and effects of race and sex on ET-1 levels.
Discussion
In the present study, we investigated plasma irET-1 concentration in black and white subjects with high and normal BP and also assessed the effect of sex factors. We found irET-1 levels in hypertensive blacks to be sevenfold to eightfold higher than in normotensive blacks and threefold to fourfold higher than in hypertensive whites. To our knowledge, this is the first report on racial differences in plasma irET-1 concentrations.
Hypertension is characterized by an increase in peripheral vascular resistance that is associated with an elevation in vascular tone and structural changes in the blood vessel wall. Since ET-1 has potent contractile and proliferative properties, it has been suggested to be involved in the pathogenesis of hypertension.2 ET-1 has been proposed to modulate BP through several pathways. ET-1 exerts its contractile effects via endothelin-A receptors on vascular smooth muscle cells, and it also causes vasodilation via nitric oxide release mediated by endothelin-B receptors on endothelial cells. In addition, ET-1 interacts with nonendothelial pathways, eg, the sympathetic system, renin-angiotensin system, and central nervous system.16 Although in vitro studies have demonstrated the interaction of ET-1 with these systems, there is no direct evidence that ET-1 is involved in the pathogenesis of hypertension or in the in vivo regulation of vascular tonus. Several groups have reported that plasma irET-1 levels were elevated in moderate hypertension171819 ; however, other studies found no significant increase.1220 In an age-matched study, Miyauchi et al12 reported no difference in irET-1 levels between Japanese hypertensive and normotensive control individuals, but they found irET-1 concentrations to be higher in men than women and to increase with age. A sex-related difference in irET-1 levels in healthy people was found to be mediated by sex hormones.11 It was also suggested that this difference might contribute to the higher incidence of atherosclerotic cardiovascular disease in men. In our study, normotensive white men were found to have slightly higher levels of irET-1 than normotensive white women (twofold). However, we did not observe any significant sex-associated differences in irET-1 levels. We also analyzed the effect of age factors on plasma irET-1 concentrations and found them to have no significant effect. Thus, our results suggest that there might be a differential regulation of irET-1 levels in blacks and/or in hypertensive individuals.
The prevalence of essential hypertension in blacks is much higher compared with that in whites. Racial differences in renal physiology and socioeconomic factors have been suggested as possible causes of this difference.131415 Recently, it was reported that the T235 allele of the angiotensinogen gene is linked to human hypertension and that T235 is the predominant angiotensinogen gene allele in blacks.21 It was also demonstrated that angiotensinogen concentrations are higher in hypertensive blacks than hypertensive whites.22 Interestingly, ET-1 has been shown to enhance the production of Ang II from Ang I in cultured endothelial cells by an angiotensin-converting enzyme–sensitive mechanism.5 Two groups have reported that BP elevation in ET-1–infused rats can be prevented by angiotensin-converting enzyme inhibitors.2324 Furthermore, Ang II stimulates ET-1 synthesis.25 On the basis of these observations, ET-1 and Ang II have been proposed to act synergistically to induce vasoconstriction and aldosterone secretion from the adrenal cortex.67 Our finding that irET-1 levels are elevated in hypertensive blacks compared with normotensive blacks and hypertensive whites suggests that interaction of Ang II and ET-1 might be an important factor in BP regulation in blacks. In addition, since ET-1 has diverse biological effects on cardiovascular and renal systems,7 elevated levels of ET-1 in hypertensive blacks may contribute to the higher incidence of complications in this population.14
In conclusion, we have demonstrated racial differences in plasma irET-1 concentrations in individuals with high BP. Further studies are required for determination of whether the elevated concentrations of plasma ET-1 in hypertensive blacks are involved in the development of complications of hypertension and to elucidate the possible interaction of ET-1 with Ang II.
Selected Abbreviations and Acronyms
| Ang I, II | = | angiotensin I, II |
| BP | = | blood pressure |
| ET-1 | = | endothelin-1 |
| irET-1 | = | immunoreactive endothelin-1 |
Figure 1. Distribution of irET-1 in hypertensive (H) and normotensive (N) subjects of different sex (M and F) and race (B and W). In each group, 12 or 13 subjects were analyzed.
Figure 2. Bar graph representation of irET-1 concentrations in hypertensive and normotensive subjects of different sex and race. Results are given as mean+SE. Abbreviations as in Fig 1 legend.
| Contrast | F | P>F |
|---|---|---|
| Hypertension vs normotension* | 38.36 | .0001 |
| Sex effects in normotension | 2.18 | .147 |
| Racial effects in normotension | 2.39 | .129 |
| Sex effects in hypertension | 0.08 | .777 |
| Racial effects in hypertension | 27.81 | .0001 |
| Age effects† | 1.06 | .420 |
This research was supported by the American Heart Association, Georgia Affiliate (Grant-in-Aid to D. Puett); University of Georgia Research Foundation Biotechnology Award; and the Clinical Research Center of the Medical Center of Central Georgia, The Mercer University School of Medicine, Macon. We would like to thank Dr Randall L. Tackett for bringing the race issue in hypertension to our attention. We also thank Dr Stephen L. Rathbun, Zhuobei Zhou, and Marcus D. Durham for their assistance with the statistical analysis of data and Bruce Burns, JMS, for his assistance with the subject selection.
Footnotes
References
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