Many mechanisms, including oxidative stress, contribute to hypertension. This study investigated the possible associations between oxidative stress, blood pressure and arterial stiffness in black South Africans. Ambulatory blood pressure measurements were taken for 101 black South African men and 99 women. The stiffness indices included ambulatory arterial stiffness index (AASI) and pulse pressure (PP). Reactive oxygen species (ROS) levels (P<0.0001) were higher in the African women compared with men. ROS levels were also higher in hypertensive compared with normotensive men. The 24 h systolic blood pressure (SBP; P<0.01), 24 h diastolic blood pressure (DBP; P<0.0001) and pulse wave velocity (PWV; P<0.01) were significantly higher in African men compared with women. There were unadjusted positive associations of 24 h SBP (r=0.33; P=0.001), 24 h DBP (r=0.26; P=0.008) and 24 h PP (r=0.29; P=0.003) with ROS in African men only. A positive association between AASI and ROS existed only in hypertensive men (r=0.27; P=0.035), but became nonsignificant (B=0.0014; P=0.14) after adjustments. Adjusted, positive associations of 24 h SBP (B=0.181; P=0.018) and 24 h PP (B=0.086; P=0.050) with ROS were again only evident in African men. ROS is positively associated with SBP and PP in African men, suggesting that increased ROS levels may contribute to hypertension in this population group.
It is well established that hypertension is one of the most common cardiovascular risk factors in the black South African population, especially in men.1, 2, 3 An increase in blood pressure could be the result of many factors, including age, environmental factors (such as stress and socioeconomic status), genetic factors, diet, unhealthy lifestyle and functional and structural alterations of the arterial wall because of haemodynamic and/or humoral factors, including oxidative stress.4, 5
An increase in reactive oxygen species (ROS) causes oxidative stress, and contributes significantly to the functional and structural alterations present in hypertension.4 Large amounts of ROS produced by vascular cells, including superoxide (•O2−) and hydrogen peroxide (H2O2), act as important intracellular signals.4 Physical and environmental stressors contribute to an overproduction of these molecules, and may cause injury to the cells of the vasculature. These stressors may therefore contribute to oxidative stress and/or decreased antioxidant reserve, which in turn contribute to arterial dysfunction and stiffness.6 The structural and cellular changes of the aforementioned conditions portray the link existing between oxidative stress, arterial stiffness and hypertension.7 This link is not one of cause and effect, but rather the contribution of various factors or conditions (that is, sun exposure, smoking, chronic inflammation, the use of alcohol, stress and so on) that can lead to a nonphysiological production of ROS.3 Animal studies reveal that the increase in O2− generation in hypertension impacts on the production and activity of endogenous vascular nitric oxide. Also, endothelium-dependent relaxation is strongly impaired following experimental elevation of blood pressure because of O2− generation8 and was confirmed in Caucasians with essential hypertension.9
No studies regarding the associations of ROS with blood pressure and arterial stiffness have been done in black South Africans. The aims of this study were, therefore, to compare ROS levels between black South African men and women, as well as the associations of ROS with measures of cardiovascular function.
This study forms part of the SABPA (Sympathetic Activity and Ambulatory Blood Pressure in Africans) study, which included 200 urbanised black South African educators working in the Dr Kenneth Kaunda district in the North West Province of South Africa. The reason for this target selection was to obtain a homogenous sample from a similar socioeconomic class. The total group consisted of 101 men (mean age 43.2±8.1 years) and 99 women (mean age 45.4±7.7 years). Participants between the ages of 25 and 65 years were included in this study. The exclusion criteria were an oral temperature above normal (37.8 °C), usage of α- and β-blockers, psychotropic substance dependence or abuse, regular blood donors and individuals vaccinated in the past 3 months. Three participants were excluded from all analyses because of missing data from basic variables. Sessions informing the participants about the study were done before their recruitment. Assistance was available to participants who preferred elucidation of information in their home language. All the participants signed an informed consent form. This study was approved by the ethics committee of the North-West University (Number 00036-07-S6) and the study protocol conformed to the ethical guidelines of the Declaration of Helsinki (2004) for investigation of human participants.
Ambulatory blood pressure measurements were conducted during the week from Monday to Thursday. At ∼0800 hours every morning, trained fieldworkers connected an ambulatory blood pressure measurement (CE120 Cardiotens; Meditech, Budapest, Hungary) apparatus and two-lead electrocardiogram to the participants at their workplace. The ambulatory blood pressure measurement apparatus was programmed to measure blood pressure at 30-min intervals during the day (0800–2200 hours) and every hour during night time (2200–0600 hours). Blood pressure cuffs were attached to the non-dominant arm. Participants were also asked to continue with their daily activities and note any abnormalities such as headache, nausea and stress on their diary cards.
At 1630 hours, the participants arrived at the Metabolic Unit Research Facility of the North-West University. This facility consists of 10 bedrooms, 2 bathrooms, a living room and a kitchen. All participants received a standardised dinner and had their last beverages (tea/coffee and two biscuits) at 2030 hours. They were then allowed to relax by reading, watching television or social interaction, and were encouraged to go to bed at 2200 hours. At 0600 hours, the ambulatory blood pressure measurement apparatus was removed. Participants with a daytime systolic blood pressure (SBP) ⩾140 mm Hg and/or diastolic blood pressure (DBP) ⩾90 mm Hg were considered hypertensives.10 The ambulatory arterial stiffness index (AASI) was calculated by initially computing the regression slope of diastolic over systolic blood pressures from unedited 24-h recordings for each participant and then subtracting the slope from one.11 The stiffer the arterial tree, the closer the regression slope and AASI are to zero and one, respectively. The carotid dorsalis-pedis pulse wave velocity (PWV) was measured using the Complior SP Acquisition system (Artech-Medical, Pantin, France) on the left side of each participant in the supine position. The carotid intima-media thickness was determined using a SonoSite Micromaxx ultrasound system (SonoSite, Bothell, WA, USA) and a 6–13 MHz linear array transducer. The images were obtained from at least two optimal angles of the left and right common carotid artery. The images were analysed using the Artery Measurement Systems automated software.12, 13 The far walls of the carotid arteries were used for measurements in this study. The carotid intima-media thickness of <0.9 mm was regarded as subclinical atherosclerosis.14
Body height (stature), weight and waist circumference were measured with calibrated instruments (Invicta Stadiometer, IP 1465, London, UK; Precision Health Scale, A&D Company, Tokyo, Japan; Holtain unstretchable flexible 7 mm wide metal tape, Crosswell, Wales) while participants were in their underwear. Subsequently, the body mass index was calculated for each participant. All measurements were done in triplicate by registered anthropometrists according to standard procedures.12
After the cardiovascular and anthropometric measurements were done, a registered nurse obtained a fasting blood sample with a sterile winged infusion set from the vena cephalica or medial ante-cubital vein. EDTA whole blood and serum were stored at −80 °C. Venous samples for fasting blood glucose were collected in sodium fluoride tubes. Serum glucose was determined using a timed-end-point method (Unicel DxC 800; Beckman and Coulter, Krefeld, Germany). Fasting serum samples for total cholesterol, C-reactive protein and creatinine were analysed using the sequential multiple analyser computer (Konelab 20i; Thermo Scientific, Vantaa, Finland). The major metabolite of nicotine, cotinine, was measured in the serum for each participant using a DRI (Diagnostic Reagents Inc., Sunnyvale, CA, USA) enzyme immunoassay kit.13 For alcohol intake, γ-glutamyltransferase levels were measured with the UniCel DxC 800 analyser on an enzyme rate method.14 Oestrogen and progesterone levels, for quantitative determination of the specific menstrual phase of the women, involved a radioimmunometric assay done with the Auto-Gamma Cobra Counting System (Packard Instrument Company, Meriden, CT, USA). We calculated the estimated creatinine clearance using the Cockcroft–Gault formula,15 that is, estimated creatinine clearance (ml min–1)=(140–age) × mass (kg) × constant/serum creatinine (μmol l–1), where constant is 1.23 for men and 1.04 for women.
Serum ROS were determined by an improved assay system based on the principle of the derivatives of reactive oxygen metabolites test, which is recognized as an efficient method for evaluating oxidative stress in the body. The Bio-Tek FL600 Microplate Fluorescence Reader (Bio-Tek, Instruments, Inc., Highland Park, Winooski, VT, USA) was used to measure ROS levels, where 1.0 mg l–1 H2O2 represents one unit of ROS.16
Statistica software v8.0 was used for database management and statistical analyses (Statsoft, Inc., 2008, Tulsa, OK, USA). The distribution of serum glucose, high sensitivity C-reactive protein, physical activity, cotinine and γ-glutamyltransferase were normalised by logarithmic transformation. The central tendency and spread of these variables were represented by the geometric mean and the 5th and 95th percentile intervals. The t-tests were used to determine differences in mean values between groups. The χ2 tests were used to compare proportions between the two groups. Mean values of ROS were plotted by quartiles of the cardiovascular variables to ensure that linear correlation techniques were appropriate. Pearson's correlations were done to determine unadjusted associations between cardiovascular variables and ROS. Independent associations between cardiovascular variables and oxidative stress were done using multiple linear regression analyses after adjusting for significant covariates, which included age, body mass index, serum glucose, total cholesterol, C-reactive protein, physical activity, cotinine, γ-glutamyltransferase, mean arterial pressure and dipping status.
Characteristics of participants
Table 1 lists the characteristics of the African men and women. The 24 h SBP, 24 h DBP and PWV were significantly higher in the African men compared with the women, although ROS levels were significantly higher in the women compared with the men. Out of the 97 women, 9 (9.3%) used oral contraceptive medication. When the total group of men was divided into normotensives and hypertensives (data not shown), the ROS levels were significantly higher (P=0.031) in the hypertensive compared with the normotensive men (mean±s.d: 87.5±18.9 vs 79.0±18.2 units). No differences existed between normotensive and hypertensive women (P=0.49).
In single regression analyses (Table 2), significant positive correlations of 24 h SBP, 24 h DBP and 24 h pulse pressure (PP) with ROS existed in the African men. However, no significant correlation between measures of arterial stiffness and ROS was found in the total group of African men (AASI: r=0.17, P=0.11; PWV: r=0.03, P=0.79). No significant associations were observed in the African women. By separately dividing the total group of African men and women into normotensives and hypertensives, the significant correlations observed previously disappeared in the normotensive men and remained nonsignificant in normotensive and hypertensive women. However, the associations between SBP (r=0.30; P=0.017) and PP (r=0.30; P=0.017) remained significant in the hypertensive men, and a positive correlation (r=0.27; P=0.035) between AASI and ROS was established in the hypertensive men only.
In exploratory analyses (Figure 1), 24 h SBP, 24 h PP and AASI were plotted by quartiles of ROS, with adjustments applied for dipping status. By doing so, the significant positive associations between 24 h SBP (P for trend, 0.007) and 24 h PP (P for trend, 0.015) with ROS were still evident in the African men and absent in the women. Again, no correlation existed between AASI and ROS in the total group of men and women. However, a tendency existed in the men only (P for difference between the lowest and highest quartile, 0.045). The independent associations between the cardiovascular variables and ROS are shown in Table 3. With adjustments applied for significant covariates (age, body mass index, serum glucose, total cholesterol, C-reactive protein, mean arterial pressure, physical activity, cotinine, γ-glutamyltransferase and dipping status), the above associations were confirmed. In the men, 24 h SBP (P=0.041) and 24 h PP (P=0.050) correlated positively with ROS, whereas these associations were absent in the women (24 h SBP: P=0.74; 24 h PP: P=0.72). By repeating the analyses in the normotensive and hypertensive men and adjusting for the above-mentioned covariates, the positive association between 24 h SBP and ROS became borderline significant in the hypertensive (B=0.153±0.086; P=0.080) as well as the normotensive (B=0.144±0.074; P=0.060) men. However, the positive association between 24 h PP was still evident in the hypertensive men only (B=0.124±0.059; P=0.039). In the women, no associations between 24 h SBP (total group: B=−0.039±0.066, P=0.78; normotensive: B=−0.0088±0.050, P=0.86 and hypertensive: B=0.073±0.11, P=0.52) and 24 h PP (total group: B=−0.013±0.039, P=0.77; normotensive: B=−0.0042±0.036, P=0.91 and hypertensive: B=0.064±0.087, P=0.47) with ROS were evident. This was also the case after additionally adjusting for progesterone and oestrogen.
Previous studies suggested that the use of contraception increases the risk of oxidative stress.17 Therefore, because of the use of oral contraception in the African female group, the multiple regression analyses were repeated by additionally adjusting for oral contraception. By doing so, the results remained unchanged.
This study compared ROS levels between African men and women and investigated gender-specific associations with measures of arterial stiffness. African men had lower ROS levels compared with the women, but hypertensive men had higher ROS levels compared with normotensive men, which was not seen in the women. In addition, men had higher blood pressure, PP and PWV compared with the women. In the men only, associations suggested that SBP and PP increase with increasing ROS levels, indicating a possible contributing role of oxidative stress in blood pressure elevation, especially in hypertensive men. Also, a positive correlation existed between AASI and ROS in the men. However, when adjusting for dipping status, this correlation between AASI and ROS became nonsignificant, confirming the suggestion by Schillaci et al.18 that AASI is strongly confounded by dipping status.
Previous studies have shown that ROS levels are higher in men compared with women.18 However, in this study the women had higher ROS levels compared with the men, but significantly lower blood pressure and PWV. A possible reason is that women tend to have lower blood pressure and PWV because of the protective effect of oestrogen.19 Ongoing research shows that part of the high ROS levels in women may be because of the use of contraceptive medication.19 No significant associations were found between any cardiovascular variables and ROS in the women. After excluding the women using contraceptive medication, the above associations were confirmed. As black South African women become more urbanised, they tend to use oral contraceptives more than DMPA (depot medroxyprogesterone acetate) injections, which has a long-acting effect, and it is evident that the use of oral contraception creates a greater risk for venous thrombosis and other health problems.20
The cardiovascular health of the black South African population is a major concern with regard to the high prevalence of stroke and hypertension.1 The pathogenesis of hypertension appears to be different in the African population compared with Caucasians. This is influenced via multiple factors including renal disparities, demographic differences regarding socioeconomic status, unhealthy lifestyle (smoking, alcohol intake and diet) as well as environmental influences due to urbanisation.21 Previous studies on urbanisation in the same population group did show increases in blood pressure levels with increasing westernisation.3 The participating roles of factors associated with urbanisation such as smoking, alcohol abuse, socioeconomic stress, diet and physiological processes increase oxidative stress and, therefore, is the possible link between urbanisation and blood pressure. Therefore, oxidative stress may contribute partially to the onset and/or maintenance of hypertension.19
Studies have shown altered endothelial function by changes in nitric oxide biosynthesis when exposed to smoking and confirmed that oxidative stress has a central role in smoking-mediated dysfunction of nitric oxide biosynthesis in endothelial cells.22 Alcohol intake, on the other hand, reduces glutathione production, causing increased levels of H2O2, which in turn reacts with transition metals in the mitochondria of the cells and contributes to oxidative stress. Glutathione in the mitochondria is the only protection available to metabolize H2O2. Therefore, excessive alcohol intake depletes cells of glutathione in the mitochondria because of a faulty operation of the carrier responsible for transport of glutathione from the cytosol into the mitochondrial matrix.23, 24 In this study, the prevalence of smoking and alcohol intake were the highest in the black South African men compared with the women. This could possibly explain why the associations with ROS were more prominent in the men who had higher blood pressure levels.
Increased levels of ROS contribute to a cascade of reactions in cells and cause vascular injury via lipid peroxidation, protein oxidation, DNA damage and altered gene expression, to name a few.25 It is clear that one could not pinpoint a specific factor contributing to the onset and/or maintenance of hypertension, but rather explore the vast collection of contributing factors. It is evident from this study that ROS seems to have a role in cardiovascular dysfunction, as reflected by the association with SBP and PP in the men. This study has to be interpreted within the context of its limitations and strengths. This was a cross-sectional study and therefore one cannot infer causality. Although the results were consistent after multiple adjustments, one cannot exclude that the results were because of residual confounding or because of unknown factors that are associated with both cardiovascular function and oxidative stress. Additionally, the lack of association with AASI could be reasoned on the suggestion of Westerhof et al.26 who proposed that AASI is not a direct measure or predictor of arterial stiffness, but rather a measure of ventriculo-arterial coupling, and increased arterial stiffness results in an increase in both AASI and pulse pressure, and AASI correlates with indicators of arterial stiffness. Overall, this was a well-designed study implemented under strict and controlled conditions, and was the first study to investigate associations between arterial stiffness and ROS in Africans from South Africa.
In conclusion, ROS levels were higher in African women compared with the men, but higher in hypertensive compared with normotensive men only. ROS was positively associated with SBP and PP in African men, especially hypertensive men, suggesting that increased ROS levels may contribute to increased arterial stiffness and hypertension in this population group.
Opie LH, Seedat YK . Hypertension in sub-Saharan African populations. Circulation 2005; 112: 3562–3568.
Seedat Y, Seedat M, Hackland D . Prevalence of hypertension in the urban and rural Zulu. Br Med J 1982; 36: 256–261.
Van Rooyen JM, Huisman HW, Eloff FC, Laubscher PJ, Malan L, Steyn HS et al. Cardiovascular reactivity in Black South-African males of different age groups: the influence of urbanization. Ethn Dis 2002; 12: 69–75.
Zalba G, Jose GS, Moreno MU, Fortuno MA, Fortuno A, Beaumont FJ et al. Oxidative stress in arterial hypertension: role of NAD(P)H oxidase. Hypertension 2001; 38: 1395–1399.
Huisman HW, van Rooyen JM, Malan NT, Eloff FC, Malan L, Laubscher PJ et al. Prolactin, testosterone and cortisol as possible markers of changes in cardiovascular function associated with urbanization. J Hum Hypertens 2002; 16: 829–835.
Cai H, Harrison DG . Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87: 840–844.
Zieman SJ, Melenovsky V, Kass DA . Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol 2005; 25: 932–943.
Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG . Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation 1997; 95: 588–593.
Zeiher AM, Drexler H, Saurbier B, Just H . Endothelium-mediated coronary blood flow modulation in humans. J Clin Invest 1993; 92: 652–662.
O’Brien E, Asmar R, Beilin L, Imai Y, Mancia G, Mengden T et al. Practice guidelines of the European Society of Hypertension for clinic, ambulatory and self blood pressure measurement. J Hypertens 2005; 23: 697.
Dolan E, Li Y, Thijs L, McCormack P, Staessen JA, O’Brien E et al. Ambulatory arterial stiffness index: rationale and methodology. Blood Press Monit 2006; 11: 103.
Norton K, Olds T . Anthropometrica: A textbook of body measurements for sports and health courses. Sydney: UNSW Press, 1996.
Yao JK, Reddy R, van Kammen DP . Reduced level of plasma antioxidant uric acid in schizophrenia. Psychiatry Res 1998; 80: 29–39.
Herzum I, Rieger T, Funke J . Evaluation of the consolidated Beckman Coulter UniCel (R) DxC 880i analyser: 1714_A. Clin Chem Lab Med 2008; 46: A72.
Cockcroft D, Gault M . Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41.
Hayashi I, Morishita Y, Imai K, Nakamura M, Nakachi K, Hayashi T . High-throughput spectrophotometric assay of reactive oxygen species in serum. Mut Res 2007; 631: 55–61.
Mitrunen K, Sillanpaa P, Kataja V, Eskelinen M, Kosma VM, Benhamou S et al. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk. Carcinogenesis 2001; 22: 827.
Schillaci G, Parati G, Pirro M, Pucci G, Mannarino MR, Sperandini L et al. Ambulatory arterial stiffness index is not a specific marker of reduced arterial compliance. Hypertension 2007; 49: 986.
Touyz RM . Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: what is the clinical significance? Hypertension 2004; 44: 248–252.
Vandenbroucke JP, Rosing J, Bloemenkamp KWM, Middeldorp S, Helmerhorst FM, Bouma BN et al. Oral contraceptives and the risk of venous thrombosis. N Engl J Med 2001; 344: 1527–1535.
Sutton-Tyrrell K, Najjar SS, Boudreau RM, Venkitachalam L, Kupelian V, Simonsick EM et al. Elevated aortic pulse wave velocity, a marker of arterial stiffness, predicts cardiovascular events in well-functioning older adults. Circulation 2005; 111: 3384–3390.
Barua RS, Ambrose JA, Srivastava S, DeVoe MC, Eales-Reynolds LJ . Reactive oxygen species are involved in smoking-induced dysfunction of nitric oxide biosynthesis and upregulation of endothelial nitric oxide synthase an in vitro demonstration in human coronary artery endothelial cells. Circulation 2003; 107: 2342–2347.
Fernandez-Checa J, Kaplowitz N, Garcia-Ruiz C, Colell A, Miranda M, Mari M et al. GSH transport in mitochondria: defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol Gastrointest Liver Physiol 1997; 273: 7–17.
Schutte R, Schutte AE, Huisman HW, van Rooyen JM, Malan NT, Péter S et al. Blood glutathione and subclinical atherosclerosis in African men: the SABPA study. Am J Hypertens 2009; 22: 1154–1159.
Diep QN, Amiri F, Touyz RM, Cohn JS, Endemann D, Neves MF et al. PPARá activator effects on ang II-induced vascular oxidative stress and inflammation. Hypertension 2002; 40: 866–871.
Westerhof N, Lankhaar JW, Westerhof BE . Letter to the editor: Ambulatory arterial stiffness index is not a stiffness parameter but a ventriculo-arterial coupling factor. Hypertension 2007; 49: e7.
The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study would not have been possible without the voluntary collaboration of the participants and the Department of Education, North West Province, South Africa. We gratefully acknowledge the technical assistance of Mrs Tina Scholtz, Mrs C van Deventer and Sr Chrissie Lessing. Research included in the present study was partially funded by the National Research Foundation, South Africa; the North-West University, Potchefstroom, South Africa; and the Metabolic Syndrome Institute, France.
The authors declare no conflict of interest.
About this article
Cite this article
Kruger, R., Schutte, R., Huisman, H. et al. Associations between reactive oxygen species, blood pressure and arterial stiffness in black South Africans: the SABPA study. J Hum Hypertens 26, 91–97 (2012). https://doi.org/10.1038/jhh.2010.134
- arterial stiffness
- blood pressure
- oxidative stress
- reactive oxygen species
Untargeted Metabolomics of Fermented Rice Using UHPLC Q-TOF MS/MS Reveals an Abundance of Potential Antihypertensive Compounds
Nitric oxide-related markers link inversely to blood pressure in black boys and men: the ASOS and African-PREDICT studies
Amino Acids (2020)
Left ventricular mass and urinary metabolomics in young black and white adults: The African-PREDICT study
Nutrition, Metabolism and Cardiovascular Diseases (2020)
Implementing a new variant load model to investigate the role of mtDNA in oxidative stress and inflammation in a bi-ethnic cohort: the SABPA study
Mitochondrial DNA Part A (2019)
Three-year change in oxidative stress markers is linked to target organ damage in black and white men: the SABPA study
Hypertension Research (2019)