We evaluated the association of indices of paraoxonase (PON1) and oxidative status with subclinical cardiovascular disease (CVD) in mixed-ancestry South Africans. Participants were 491 adults (126 men) who were stratified by diabetes status and body mass index (BMI). Carotid intima-media thickness (CIMT) was used as a measure of subclinical CVD. Indices of PON1 and oxidative status were determined by measuring levels and activities (paraoxonase and arylesterase) of PON1, antioxidant activity (ferric reducing antioxidant power and trolox equivalent antioxidant capacity), and lipid peroxidation markers (malondialdehyde and oxidized LDL). Diabetic subjects (28.9%) displayed a significant decrease in PON1 status and antioxidant activity as well as increase in oxidized LDL and malondialdehyde. A similar profile was apparent across increasing BMI categories. CIMT was higher in diabetic than nondiabetic subjects
Early identification of people at high risk of atherosclerotic cardiovascular diseases (CVDs), followed by the implementation of lifestyle and drug interventions with proven beneficial effects, has been largely emphasized in strategies to reduce the mortality and morbidity from cardiovascular disease [
The study setting has been described in detail elsewhere [
All consenting participants completed a questionnaire designed to obtain information on lifestyle factors such as smoking and alcohol consumption, physical activity, diet, family history of CVD, diabetes mellitus, and demographics. Blood pressure measurements were performed according to the World Health Organization (WHO) guidelines [
Two qualified sonographers measured CIMT in longitudinal section at the far wall of the distal common carotid arteries, 2 cm from the bifurcation, at 3 consecutive end-points, 5–10 mm apart. The mean of six readings (3 from each side) was calculated for each participant using a portable B-mode and spectral Doppler ultrasound scanner equipped with cardiovascular imaging software. The GE LOGIQ e (General Electric Healthcare, Germany) high performance multipurpose colour compact ultrasound system included new imaging CrossXBeam technologies with multifrequency virtual apex on phased array cardiac transducer (3S-RS wide band phased probe 1.7–4.0 MHz) for echocardiography and a linear wideband vascular transducer (8L-RS 4.0–12 MHz linear probe) used for improved diagnostic confidence and imaging clarity for the carotids.
Blood samples were collected after an overnight fast and processed for further biochemical analyses. The Cobas 6000 immunometric analyzer (Roche Diagnostics) was used to measure levels of fasting plasma glucose (FBG), glycated haemoglobin (HbA1c), total cholesterol, high density lipoprotein cholesterol (HDL-c), triglycerides (TG) and
The total antioxidant capacity in plasma samples was assessed using the ferric reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. FRAP was done according to the method of Benzie and Strain [
Paraoxonase (PONase) and arylesterase (AREase) activities were measured using paraoxon and phenylacetate (Sigma Aldrich, SA) as substrates, respectively. PONase activity was measured using Richter and Furlong’s method [
Plasma MDA and ox-LDL were used as markers of lipid peroxidation (LPO). The method of Jentzsch et al. [
Data are presented as mean standard deviation, SD, or median of 25th–75th percentiles for continuous variables and as count and percentage for categorical variables. For group (sex, diabetes status, and BMI, quarters of CIMT) comparisons, chi square test, student’s
Of the 651 participants (men 170, 26%) who took part in the study, 160 (25%) were excluded from this analysis as they had missing values for CIMT and/or other relevant variables. Baseline characteristics of participants included and excluded from analyses were very similar. The final analytic sample comprised 491 participants (men 126, 25.7%) with a mean age of 54.6 (13.2) years. Among them, 142 (29%) had diabetes, 137 (28%) were overweight, and 261 (53%) were obese. The average BMI was 31.4 (8.1) kg/m2 (Table
General characteristics of the participants.
Variables | Overall | Sex | Diabetes | BMI | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Men | Women |
|
No | Yes |
|
Normal | Overweight | Obese |
| ||
|
491 | 126 | 365 | 349 | 142 | 93 | 137 | 261 | |||
Female, |
365 (74.3) | 0 | 365 (100) | 257 (73.6) | 108 (76.1) | 0.578 | 47 (50.5) | 93 (67.9) | 235 (90.0) | <0.0001 | |
Age (years) | 54.6 (13.2) | 55.4 (13.3) | 54.3 (13.2) | 0.401 | 52.5 (13.4) | 59.6 (11.5) | <0.0001 | 55.3 (15.2) | 53.6 (13.3) | 54.8 (12.6) | 0.568 |
BMI (kg/m2) | 31.4 (8.1) | 27.5 (7.0) | 32.8 (8.0) | <0.0001 | 30.6 (7.3) | 33.4 (9.5) | 0.002 | 21.5 (3.1) | 27.6 (1.4) | 37.0 (6.7) | <0.0001 |
Waist circumference (cm) | 96.4 (15.4) | 94.9 (14.5) | 96.9 (15.7) | 0.208 | 94.6 (16.1) | 100.8 (12.9) | <0.0001 | 80.5 (17.1) | 90.7 (8.4) | 105.1 (11.2) | <0.0001 |
Waist/hip ratio | 0.89 (0.12) | 0.93 (0.12) | 0.87 (0.12) | <0.0001 | 0.88 (0.12) | 0.92 (0.12) | 0.001 | 0.85 (0.19) | 0.89 (0.09) | 0.90 (0.10) | 0.010 |
Systolic BP (mmHg) | 136 (26) | 138 (24) | 136 (27) | 0.309 | 133 (23) | 144 (31) | 0.0001 | 133 (31) | 135 (25) | 136 (25) | 0.138 |
Diastolic BP (mmHg) | 82 (14) | 84 (15) | 81 (13) | 0.030 | 81 (14) | 84 (14) | 0.055 | 78 (12) | 81 (12) | 83 (15) | 0.002 |
FPG (mmol/L) | 6.4 (2.9) | 6.2 (2.5) | 6.5 (3.1) | 0.292 | 5.1 (0.7) | 9.5 (3.9) | <0.0001 | 5.3 (1.5) | 6.6 (3.4) | 6.7 (3.0) | 0.0003 |
HbA1c (%) | 6.6 (1.6) | 6.3 (1.4) | 6.7 (1.7) | 0.025 | 5.9 (0.4) | 8.3 (2.2) | <0.0001 | 5.9 (1.0) | 6.8 (2.0) | 6.7 (1.6) | <0.0001 |
Creatinine (µM) | 74.8 (25.5) | 89.5 (35.5) | 69.6 (18.5) | <0.0001 | 75.3 (20.0) | 81.1 (42.8) | 0.21 | 79.4 (19.1) | 75.0 (19.3) | 73.7 (30.0) | 0.480 |
C-reactive protein (mg/L) | 5.1 [2.0–8.9] | 3.7 [1.5–7.6] | 5.6 [2.2–9.2] | 0.025 | 5.0 [1.9–8.4] | 5.5 [2.5–10.2] | 0.126 | 3.0 [1.0–5.6] | 2.9 [1.4–6.6] | 6.8 [3.5–11.1] | 0.375 |
GGT (IU/L) | 27 [19–43] | 31 [24–46] | 25 [17–41] | <0.0001 | 25 [18–41] | 29 [23–50] | 0.003 | 26 [18–42] | 25 [18–43] | 28 [20–43] | 0.451 |
Total cholesterol (mmol/L) | 5.5 (1.1) | 5.4 (1.0) | 5.5 (1.1) | 0.494 | 5.5 (1.1) | 5.5 (1.2) | 0.360 | 5.4 (1.1) | 5.7 (1.1) | 5.4 (1.0) | 0.072 |
HDL cholesterol (mmol/L) | 1.4 (0.4) | 1.3 (0.5) | 1.4 (0.4) | 0.058 | 1.4 (0.4) | 1.3 (0.4) | 0.009 | 1.6 (0.5) | 1.3 (0.4) | 1.3 (0.3) | <0.0001 |
LDL cholesterol (mmol/L) | 3.4 (1.0) | 3.4 (0.9) | 3.4 (1.0) | 0.525 | 3.4 (0.9) | 3.4 (1.0) | 0.990 | 3.3 (1.1) | 3.6 (1.0) | 3.4 0.9) | 0.026 |
Triglycerides (mmol/L) | 1.5 (0.9) | 1.7 (1.1) | 1.5 (0.8) | 0.047 | 1.4 (0.8) | 1.8 (1.0) | <0.0001 | 1.2 (0.8) | 1.6 (0.9) | 1.6 (0.9) | 0.0001 |
CIMT (mm) | 0.82 [0.67–1.02] | 0.95 [0.72–1.22] | 0.80 [0.65–0.95] | <0.0001 | 0.77 [0.65–0.93] | 0.98 [0.77–1.15] | <0.0001 | 0.85 [0.65–1.08] | 0.80 [0.70–0.92] | 0.82 [0.68–1.00] | 0.227 |
PON1 (µg/mL) | 96 [74–111] | 88 [69–105] | 98 [78–112] | 0.002 | 99 [77–112] | 91 [66–107] | 0.0005 | 107 [92–115] | 99 [77–111] | 91 [71–108] | 0.0006 |
PONase (U/L) | 184 [131–220] | 194 [158–218] | 183 [124–221] | 0.091 | 204 [167–225] | 156 [96–182] | <0.0001 | 221 [186–244] | 198 [156–226] | 172 [102–206] | <0.0001 |
AREase (kU/L) | 107 [82–132] | 91 [65–104] | 117 [90–137] | <0.0001 | 113 [91–137] | 90 [63–115] | <0.0001 | 118 [96–147] | 107 [80–132] | 98 [77–123] | <0.0001 |
FRAP (µM) | 677 [545–827] | 732 [592–861] | 655 [537–811] | 0.006 | 722 [588–852] | 614 [471–732] | <0.0001 | 754 [642–911] | 725 [583–852] | 638 [523–749] | <0.0001 |
TEAC (nM) | 1279 [944–1654] | 1366 [1018–1742] | 1271 [936–1631] | 0.086 | 1423 [1077–1746] | 1051 [681–1320] | <0.0001 | 1570 [1238–1882] | 1366 [1153–1846] | 1206 [879–1505] | <0.0001 |
Ox-LDL (ng/mL) | 4408 [3143–5911] | 5141 [3979–6333] | 4110 [2834–5484] | <0.0001 | 3916 [2755–5138] | 5885 [4602–6944] | <0.0001 | 3535 [2511–4747] | 4337 [2859–5736] | 4780 [3683–6216] | <0.0001 |
TBARS (nM) | 2277 [1559–3132] | 2042 [1527–2772] | 2304 [1625–3214] | 0.203 | 1982 [1406–2672] | 3152 [2380–3655] | <0.0001 | 1546 [1046–2369] | 2238 [1546–3115] | 2533 [1924–3281] | <0.0001 |
FPG: fasting plasma glucose; BMI: body mass index; BP blood pressure; GGT: gamma glutamyl transferase; HDL: high density lipoprotein; LDL: low density lipoprotein; CIMT: carotid intima-media thickness; PON1: paraoxonase 1; PONase: paraoxonase; AREase: arylesterase; FRAP: ferric reducing ability of plasma; TEAC: trolox equivalent antioxidant capacity; Ox-LDL: oxidized LDL; TBARS: thiobarbituric acid reactive substances.
Men had significantly higher FRAP (732 versus 655
The median CIMT was 0.82 mm. It was higher in men than in women (0.95 versus 0.80 mm,
Robust correlation of CIMT with PON and antioxidant status.
Correlates | Overall | No diabetes | Diabetes |
|
---|---|---|---|---|
PON1 | −0.09 (−0.17 to 0.00) | −0.04 (−0.14 to 0.07) | −0.01 (−0.17 to 0.16) | 0.74 |
PONase | −0.17 (−0.25 to −0.08) | −0.11 (−0.21 to 0) | 0.04 (−0.13 to 0.20) | 0.15 |
AREase | −0.24 (−0.32 to −0.15) | −0.20 (−0.30 to −0.09) | −0.05 (−0.21 to 0.12) | 0.13 |
FRAP | −0.17 (−0.26 to −0.09) | −0.12 (−0.22 to −0.02) | 0.03 (−0.13 to 0.20) | 0.12 |
TEAC | −0.22 (−0.30 to −0.13) | −0.16 (−0.26 to −0.06) | 0.04 (−0.12 to 0.21) | 0.04 |
Ox-LDL | 0.25 (0.17 to 0.34) | 0.21 (0.10 to 0.30) | −0.03 (−0.20 to 0.13) | 0.02 |
TBARS | 0.26 (0.17 to 0.34) | 0.19 (0.09 to 0.29) | −0.01 (−0.17 to 0.16) | 0.04 |
Paraoxonase 1; PONase: paraoxonase; AREase: arylesterase; FRAP: ferric reducing ability of plasma; TEAC: trolox equivalent antioxidant capacity; Ox-LDL: oxidized LDL; TBARS: thiobarbituric acid reactive substances.
Characteristics of the participants by quarters of CIMT.
Variables | Quartiles of CIMT |
|
|||
---|---|---|---|---|---|
Q1 | Q2 | Q3 | Q4 | ||
|
123 | 131 | 118 | 119 | NA |
CIMT [min–max] | 0.30–0.67 | 0.68–0.82 | 0.83–1.02 | 1.03–2.18 | NA |
Female, |
101 (82.1) | 104 (79.4) | 97 (82.2) | 63 (52.9) | <0.0001 |
Age (years) | 44.9 (12.9) | 53.3 (11.8) | 57.6 (11.3) | 63.0 (9.8) | <0.0001 |
BMI (kg/m2) | 30.5 (6.7) | 31.5 (8.0) | 32.3 (8.0) | 31.5 (9.5) | 0.234 |
Waist circumference (cm) | 94.0 (18.7) | 97.1 (13.5) | 96.3 (14.0) | 98.2 (14.9) | 0.060 |
Waist/hip ratio | 0.86 (0.15) | 0.87 (0.12) | 0.89 (0.10) | 0.93 (0.10) | <0.0001 |
Systolic BP (mmHg) | 125 (19) | 135 (26) | 138 (21) | 149 (31) | <0.0001 |
Diastolic BP (mmHg) | 78 (12) | 83 (16) | 82 (11) | 84 (15) | 0.007 |
FPG (mmol/L) | 5.7 (2.2) | 6.0 (2.3) | 6.4 (2.8) | 7.6 (3.9) | <0.0001 |
HbA1c (%) | 6.2 (1.1) | 6.2 (1.0) | 6.7 (1.6) | 7.4 (2.3) | <0.0001 |
Creatinine (µM) | 68.8 (14.3) | 72.8 (19.0) | 72.2 (18.6) | 85.8 (39.8) | <0.0001 |
C-reactive protein (mg/L) | 4.4 [1.4–7.6] | 5.1 [2.2–8.7] | 5.4 [2.5–9.2] | 5.3 [2.0–9.2] | 0.222 |
GGT (IU/L) | 24 [17–38] | 29 [19–49] | 28 [19–41] | 29 [21–43] | 0.145 |
Total cholesterol (mmol/L) | 5.2 (0.9) | 5.4 (1.1) | 5.7 (1.2) | 5.7 (1.1 ) | <0.0001 |
HDL cholesterol (mmol/L) | 1.4 (0.4) | 1.3 (0.4) | 1.4 (0.4) | 1.4 (0.4) | 0.708 |
LDL cholesterol (mmol/L) | 3.2 (0.8) | 3.4 (1.0) | 3.6 (1.0) | 3.6 (1.0) | 0.003 |
Triglycerides (mmol/L) | 1.3 (0.7) | 1.5 (0.9) | 1.7 (1.0) | 1.6 (0.9) | 0.0008 |
PON1 (µg/mL) | 102 [78–112] | 94 [77–110] | 98 [77–112] | 94 [70–110] | 0.290 |
PONase (U/L) | 204 [170–233] | 184 [130–215] | 182 [136–218] | 177 [119–217] | 0.005 |
AREase (kU/L) | 118 [97–141] | 103 [84–126] | 94 [75–132] | 95 [76–120] | <0.0001 |
FRAP (µM) | 734 [602–887] | 672 [573–809] | 636 [533–795] | 667 [534–785] | 0.014 |
TEAC (nM) | 1484 [1068–1858] | 1316 [947–1628] | 1206 [929–1595] | 1235 [904–1568] | 0.001 |
Ox-LDL (ng/mL) | 3618 [2641–5102] | 4576 [3230–5844] | 4747 [3638–6176] | 4975 [3545–6333] | <0.0001 |
TBARS (nM) | 1857 [1251–2504] | 2280 [1544–3042] | 2447 [1919–3256] | 2571 [1905–3666] | <0.0001 |
FPG: fasting plasma glucose; BMI: body mass index; BP blood pressure; GGT: gamma glutamyl transferase; HDL: high density lipoprotein; LDL: low density lipoprotein; CIMT: carotid intima-media thickness; PON1: paraoxonase 1; PONase: paraoxonase; AREase: arylesterase; FRAP: ferric reducing ability of plasma; TEAC: trolox equivalent antioxidant capacity; Ox-LDL: oxidized LDL; TBARS: thiobarbituric acid reactive substances.
Correlations of paraoxonase, paraoxonase activity, arylesterase ferric reducing antioxidant power, TEAC, oxidized LDL, and thiobarbituric acid reactive substances activity with carotid intima-media thickness. Participants are grouped by diabetes status with the triangles representing those with diabetes and the solid circles representing those without diabetes. The superimposed regression lines are for the overall sample (solid line), and participants with (broken line) and without diabetes (dotted lines). Accompanying correlations coefficients are shown in Table
In a model comprising sex, age, and BMI, each of the three variables was significantly associated with CIMT. This basic model explained 26.4% of the variation in CIMT levels. When this model was expanded with the inclusion of diabetes status, all variables in the model remained significantly associated with CIMT and further explained 29.2% of the variation. However, as shown in Table
Regression coefficients from multiple robust linear models for the prediction of CIMT by indices of PON1 and antioxidant status accounting for the potential effect of sex, age, diabetes, and adiposity.
Age—sex—BMI | Diabetes | PON1 | PONase | AREase | FRAP | TEAC | Ox-LDL | TBARS | ||||||||||
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— | — | — | — | −0.0002 | 0.723 | −0.00006 | 0.730 | −0.0003 | 0.390 | −0.00002 | 0.743 | −0.00002 | 0.571 | 0.000004 | 0.539 | 0.00001 | 0.255 |
Age | 0.009 | <0.0001 | 0.008 | <0.0001 | 0.008 | <0.0001 | 0.008 | <0.0001 | 0.008 | <0. 0001 | 0.008 | <0.0001 | 0.008 | <0.0001 | 0.008 | <0.0001 | 0.008 | <0.0001 |
Sex (men) | 0.151 | <0.0001 | 0.150 | <0.0001 | 0.149 | <0.0001 | 0.150 | <0.0001 | 0.141 | <0.0001 | 0.151 | <0.0001 | 0.150 | <0.0001 | 0.145 | <0.0001 | 0.145 | <0.0001 |
BMI | 0.004 | 0.002 | 0.003 | 0.017 | 0.003 | 0.029 | 0.003 | 0.030 | 0.003 | 0.041 | 0.003 | 0.030 | 0.003 | 0.035 | 0.003 | 0.027 | 0.003 | 0.043 |
Diabetes | — | — | 0.124 | <0.0001 | 0.122 | <0.0001 | 0.121 | <0.001 | 0.118 | <0.0001 | 0.121 | <0.0001 | 0.120 | <0.0001 | 0.119 | <0.0001 | 0.114 | <0.0001 |
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0.264 | 0.292 | 0.292 | 0.293 | 0.291 | 0.295 | 0.293 | 0.292 | 0.294 |
BMI: body mass index; paraoxonase 1; PONase: paraoxonase; AREase: arylesterase; FRAP: ferric reducing ability of plasma; TEAC: trolox equivalent antioxidant capacity; Ox-LDL: oxidized LDL; TBARS: thiobarbituric acid reactive substances.
The prominent role of oxidative stress in the occurrence of atherosclerosis and related complications is well recognized, with suggestions that measurement of oxidative stress may aid risk stratification and effective prevention of atherothrombotic diseases. In this study, we evaluated the profile of several indices of PON1 and oxidative status and their relationship with CIMT, which is a marker of subclinical cardiovascular disease. Our findings demonstrate that measures of oxidative stress together with PON1 are only superficially associated with CIMT. Instead, conventional risk factors such as age, gender, adiposity, and chronic hyperglycemia and not measures of oxidative stress may be more important in estimating CVD risk in our population.
The physiological role of PON1 in reducing atherosclerosis stems from its ability to inhibit the oxidation of low density lipoprotein (LDL) [
Proinflammatory markers and oxidative stress have been shown to modulate and inactivate PON1 activity [
Measurements of oxidative stress have previously been proposed as a predictor of atherosclerosis in end stage renal disease patients [
Some limitations should be accounted for when interpreting our findings. First, the cross-sectional design of our study precludes drawing inferences on the direction of the associations. Second, we did not establish the intraobserver variability between the sonographers who performed CIMT measurements; however we used multiple measurements at different points. Third, because our study population was ethnically exclusive, our results can only be generalized to mixed-ancestry South African subjects. Fourth, we used BMI as the marker of obesity although it cannot distinguish between visceral and subcutaneous fat. Visceral fat is particularly strongly associated with atherosclerotic CVD risk [
In conclusion, although atherosclerosis is considered an inflammatory/oxidative condition, our results argue against a major role of PON1 and oxidative status in prediction of atherosclerotic risk as none of these indices impacted on the model’s value in explaining the variability of CIMT. Rather, the findings reaffirm the importance of conventional risk factors such as age, gender, adiposity, and chronic hyperglycemia in estimating CVD risk in this mixed-ancestry population.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank the Bellville South Community of Cape Town, South Africa. This study was funded by the University Research Fund of the Cape Peninsula University of Technology, South Africa, South African Medical Research Council, Harry Crossley Foundation, and University of Stellenbosch.