Abstract
Aim
The sodium-sparing effect of insulin leads to increase in total sodium pool of the body which is a chronic stimulus for atrial natriuretic peptide (ANP). In our study we aimed to determine the relationship between ANP and microvascular complications of diabetes.
Methods
60 patients, 30–70 years old, with the diagnosis of type 2 diabetes mellitus (DM) are enrolled into the study. Patients with a chronic disease other than DM are excluded. Blood samples for routine biochemical tests are taken after at least 12 h fasting at 8–9 am. Blood samples for glucose and insulin levels are taken 2 h after a standard meal. Blood tubes with EDTA are used for ANP levels. The microvascular complications of the patients are evaluated.
Results
32 of the patients had microvascular complications. Age, BMI, waist and hip circumferences, and ANP levels were significantly higher in the group with microvascular complications. There were no significant differences in waist-to-hip ratio, blood glucose, HbA1c, fasting insulin, postprandial insulin, fasting HOMA, postprandial HOMA as well as sodium, potassium, magnesium, calcium and lipid levels between the two groups. When the relationship between ANP and obesity, retinopathy, neuropathy, nephropathy, diabetes time, HbA1c, or sex are evaluated separately, the only significant parameters related to ANP were obesity and retinopathy.
Conclusion
In our study we have found that there was a significant relationship between ANP levels and microvascular complications of diabetes. Future studies are needed to show if ANP is the stimulus of microvascular complication development/progression or only an epiphenomenon.
Keywords: BMI, Cardiometabolic risk, Diabetes mellitus, Obesity, Paraoxonase 1 activity, Brachial artery intima-media thickness
Introduction
Oxidative stress is one of the first steps in the atherosclerotic process and the common mechanism underlying the increased risk for the development of cardiovascular diseases that are associated with many predisposing factors such as age, gender, diabetes mellitus (DM), renal failure, cigarette smoking, and hypertension [1]. Among these factors, DM, which is characterized by increased oxidative stress [2], ranks as the major risk factor for the development of coronary artery disease (CAD) despite current treatments for DM [3]. Moreover, methods to assess CAD risk adequately in asymptomatic individuals are not yet available [4].
Human paraoxonase 1 (PON1), which has PON, arylesterase (ARE), and diazoxonase activities, is a calcium-dependent ester hydrolase exclusively bound to the serum high-density lipoprotein (HDL) [5]. By virtue of its antioxidant and anti-atherogenic properties that enable protection of both low-density lipoproteins (LDLs) and HDLs against lipid peroxidation [5, 6], PON1 activity has been demonstrated to be inversely related to the risk of cardiovascular disease [7], and a reduction in PON1 activity has been reported in states of high oxidative stress such as metabolic syndrome, obesity, uncontrolled diabetes, and dyslipidemia [8].
Reduced PON1 activity is considered as a major cause of dysfunctional HDL in patients with type 2 DM, which has been suggested to lead accelerated atherosclerosis and thereby increased mortality due to CAD [9]. Additionally, given the role of hyperglycemia in inducing oxidative stress directly by depleting natural anti-oxidants and facilitating the production of reactive oxygen species, the protective effects of PON1 activity against peroxidation of LDL particles has been speculated to be more important in type 2 diabetic patients than in non-diabetic subjects [3].
In relation to dramatic increases in its prevalence that reached epidemic proportions, obesity has become one of the most serious public health problems worldwide. Obesity together with metabolic syndrome has been associated with oxidative damage of lipoproteins and thereby increased risk for the development of DM, hypertension, atherosclerosis, dyslipidemia, cancer, and CAD [8]. Notably, lower PON1 activity has also been reported among obese subjects when compared with control subjects and is considered to expose them to an increased risk of oxidative damage [8, 10].
Early stages of atherosclerosis consist of a functional impairment of endothelial surface with a consequent impairment of arterial vasodilation capacity and the thickening of intima-media space [11]. Carotid intima-media thickness (IMT) is considered a validated parameter for detecting subclinical atherosclerosis as it is significantly related to the presence and severity of coronary atherosclerosis [11]. Additionally, brachial artery IMT (BA-IMT) has also been shown to be correlated with carotid IMT and thereby might serve as a marker of cardiovascular risk and the initial steps of the atherosclerotic process [12]. An increase in IMT has been reported in type 2 diabetic patients with increased LDL peroxidability and also in individuals with enhanced pro-oxidant status [13]. Additionally, direct involvement of PON has also been noted in atherosclerotic lesions based on its identification in the arterial wall in amounts ranging from fatty streak to characterized plaque [14].
Despite the documented association of low levels of serum PON1 with susceptibility to CAD [15] and the evidences showing low PON1 activity in both diabetic [6] and obese patients [10], to the best of our knowledge no data are available on the relationships of the PON and ARE activities with atherosclerosis in patients with diabetes with or without obesity. Therefore, the present study aimed to evaluate the relationships of serum PON and ARE activities with biochemical variables, brachial artery diameter (BAd), and BA-IMT in patients with diabetes with or without obesity.
Patients and Methods
Patients
Outpatients aged between 18 and 75 years who had type 2 DM were included in the present study. Patients who had a fasting blood glucose level of >126 mg/dl or a blood glucose level of >200 mg/dl at random measurements or a hemoglobin A1c (HbA1c) level of >6.5% were diagnosed with type 2 DM. Patients who were diagnosed with type 1 DM from our polyclinic were not included in the study. Patients who had psychiatric disorders, cancer history, chronic renal failure, chronic liver disease or hepatic failure, CAD, or a history of acute myocardial infarction were excluded from the study. The present study was conducted in accordance with the ethical principles stated in the ‘Declaration of Helsinki’ and approved by the Institutional Ethics Committee. Written informed consent was obtained from each patient following a detailed explanation of the objectives and protocol of the study. The patients included in the present study were grouped according to their BMI and evaluated in two groups as obese group (patients with a BMI of ≥30 kg/m2) and non-obese group (patients with a BMI of ≤29.99 kg/m2).
Assessments
Patients' data regarding demographics (age, gender) and lifestyle characteristics (smoking status, alcohol consumption, and regular physical exercise) were recorded. Moreover, anthropometric measurements (height, weight, BMI), complete blood count, blood biochemistry (measurements of glucose, HbA1c, serum lipids, aspartate and alanine aminotransferases, urea, creatinine, uric acid, parathormone (PTH), vitamin B12, C-peptide, and homocysteine levels), homeostatic model of assessment-insulin resistance (HOMA-IR) measurements as well as measurements of BAd and BA-IMT were performed in each group. All measurements were evaluated with respect to serum PON and ARE activities.
Measurements of PON and ARE Activities
For the measurement of PON1 activity, venous blood samples of the patients were collected from the antecubital vein after an overnight fasting. The blood samples were centrifuged at 2,000 × g for 10 min to separate the plasma and serum. The plasma and serum samples were kept at −80 °C until analysis of PON1 activity.
The PON and ARE activities were determined using a novel automated measurement method developed by Erel (Rel Assay Diagnostics®; Erel, Gaziantep, Turkey). In brief, an increased absorbance at 412 nm at 25 °C was measured as the rate of paraoxon hydrolysis. Spectrophotometric measurement of ARE activity was performed using phenyl acetate as substrate. The reaction was initiated by the addition of the serum, and the increase in absorbance was measured at 270 nm. Enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol. One unit of ARE activity was defined as 1 μmol phenol generated per minute under the defined assay conditions. Both PON and ARE activities were expressed as U/l serum. The coefficient of variation was 1.8% for PON activity and 3.3% for ARE activity for individual samples.
Measurements of Other Biochemical Variables
Serum levels of LDL cholesterol, HDL cholesterol, triglyceride, high-sensitivity C-reactive protein, thyroid stimulating hormone, free triiodothyronine, free thyroxine, and other biochemical parameters were determined using the Abbott Architect c16200 clinical chemistry analyzer and commercial kits (Abbott Laboratories, Abbott Park, IL, USA). Serum insulin, homocysteine, vitamin B12, and folate levels were determined using the IMMULITE 2000 automated chemiluminescent analyzer and commercial kits (Siemens, Washington, D.C., USA). Reference ranges were as follows: 5–12 µg/l for homocysteine, 126.5–505 pg/ml for vitamin B12, and 2.34–17.56 ng/ml for folate. Complete blood count was performed with a Coulter LH 750 autoanalyzer (Beckman Coulter, Brea, CA, USA).
Measurement of BA-IMT
All ultrasonographic examinations, namely the IMT and BAd measurements, were performed by the same radiologist. After a 5-min rest in the supine position, the brachial artery was examined in a longitudinal plane between the antecubital fossa and the axilla by continuous grayscale imaging with a linear, high-resolution Dynamic Micro Slice (7–18 MHz) transducer. Sonographic examinations were performed using a Toshiba Aplio 500 (Toshiba Medical Systems Corporation, Nasu, Japan). Measurement of IMT in the brachial artery was performed at a proper site where the IMT was thought to be the thickest and where the clearest B mode image of the anterior and posterior intimal interfaces between the lumen and vessel wall was obtained above the antecubital fossa. IMT measurement was performed three times, and the mean of these three measurements was defined as BA-IMT. At the same obtained image where the IMT was measured, the distance between the two intimal interfaces was estimated and defined as the BAd.
Statistical Analysis
Data analyses were performed using the MedCalc Statistical Software version 12.7.7 (MedCalc Software bvba, Oostende, Belgium). Mann-Whitney U test was used to analyze independent non-parametric variables. The correlations of PON and ARE activities with laboratory parameters, BAd, and BA-IMT were performed using Pearson's and Spearman's correlation analyses for parametric and non-parametric variables, respectively. Data were expressed as mean ± standard deviation (SD), minimum-maximum, and percentage, where appropriate. A p value of <0.05 was considered statistically significant.
Results
The present study included 201 type 2 diabetic patients (mean age 52.4 ± 13.4 years; 73.6% female). The general characteristics of the whole study group are summarized in table 1. Of 201 type 2 diabetic patients, 89 were in the obese group (mean age 52.8 ± 11.7 years; 83.1% female) and 112 were in the non-obese group (mean age 52.2 ± 14.6 years; 65.5% female). The obese and non-obese groups were homogenous in terms of age.
Table 1.
Patient characteristics (n = 201)
| Age, years, mean ± SD | 52.4 ± 13.4 |
| ≤50 years, n (%) | 85 (42.5) |
| >50 years, n (%) | 115 (57.5) |
| Gender, n (%) | |
| Female | 148 (73.6) |
| Male | 53 (26.4) |
| Anthropometrics, mean ± SD | |
| Height, cm | 164.1 ± 7.5 |
| Weight, kg | 79.9 ± 14.9 |
| BMI, kg/m2 | 29.8 ± 6.1 |
| Obese (BMI ≥ 30kg/m2), n (%) | 89 (44.3) |
| Non-obese (BMI ≤ 29.99 kg/m2), n (%) | 112 (55.7) |
| Smoking status, n (%) | |
| Active smoker | 28 (13.9) |
| None-smoker | 169 (84.1) |
| Ex-smoker | 4 (2.0) |
| Alcohol consumption, n (%) | |
| Regular | 10 (10.0) |
| None | 179 (89.1) |
| Seldom | 2 (1.0) |
| Physical exercise, n (%) | |
| Regular | 57 (28.4) |
| None | 143 (71.1) |
| Seldom | 1 (0.5) |
| Family history for diabetes mellitus | 96 (47.8) |
| Hypoglycemia | 15 (7.5) |
In the whole study population, the mean PON activity level was 119.8 ± 37.5 U/l, and the mean ARE activity level was 149.0 ± 39.9 U/l. Evaluations of the PON and ARE activity levels in the whole study population revealed that there were no significant differences between patients ≤50 years and those >50 years as well as between female and male patients in terms of PON and ARE activities. In the obese group, the mean PON and ARE activities were 119.5 ± 35.6 U/l and 150.4 ± 39.0 U/l, respectively. In the non-obese group, the mean PON and ARE activities were 120.0 ± 39.1 U/l and 147.9 ± 40.7 U/l, respectively. There were no significant differences between obese and non-obese patients in terms of PON and ARE activities. The PON and ARE activities in the whole study population according to age, gender, and presence/absence of obesity are shown in table 2.
Table 2.
PON and ARE values with respect to patient demographics and obesity*
| PON, U/l | p valuea | ARE, U/l | p valuea | |
|---|---|---|---|---|
| Overall | 119.8 ± 37.5 | 149.0 ± 39.9 | ||
| Age | ||||
| <50 (n = 84) | 119.6 ± 43.8 | 0.505 | 149.4 ± 42.7 | 0.892 |
| >50 (n = 115) | 120.3 ± 32.4 | 148.4 ± 37.9 | ||
| Gender | ||||
| Female | 120.8 ± 38.5 | 0.363 | 152.5 ± 41.8 | 0.068 |
| Male | 115 ± 33.0 | 139.8 ± 32.9 | ||
| Obesity | ||||
| Obese | 119.5 ± 35.6 | 0.879 | 150.4 ± 39.0 | 0.464 |
| Non-obese | 120.0 ± 39.1 | 147.9 ± 40.7 | ||
Data are shown as mean ± SD.
Mann-Whitney U test.
Laboratory findings (blood biochemistry and complete blood count) in the whole study population as well as in the obese and non-obese groups are presented in table 3.
Table 3.
Blood biochemistry, complete blood count and urinalysis findings
| Total (n = 201) |
Obese (n = 89) |
Non-obese (n = 112) |
||||
|---|---|---|---|---|---|---|
| mean | SD | mean | SD | mean | SD | |
| HbA1c, % | 7.4 | 1.9 | 7.2 | 1.7 | 7.5 | 2.0 |
| Glucose, mg/dl | 148.2 | 61.6 | 143.9 | 54.1 | 151.6 | 67.1 |
| Urea, mg/dl | 32.9 | 13.1 | 32.6 | 12.4 | 33.1 | 13.7 |
| Creatinine, mg/dl | 1.2 | 6.5 | 1.7 | 9.7 | 0.8 | 0.6 |
| Uric acid, ng/dl | 4.8 | 1.4 | 4.9 | 1.5 | 4.6 | 1.4 |
| AST, U/l | 23.6 | 13.3 | 24.1 | 17.6 | 23.2 | 8.6 |
| ALT, IU/l | 25.5 | 24.5 | 28.0 | 33.4 | 23.6 | 13.7 |
| PTH, pg/ml | 51.0 | 32.7 | 47.8 | 17.0 | 53.6 | 41.0 |
| Vit B12, pg/ml | 310.2 | 267.5 | 292.2 | 279.2 | 324.5 | 258.2 |
| C peptide, ng/ml | 2.7 | 1.4 | 3.1 | 1.6 | 2.4 | 1.3 |
| Homocysteine, nmol/l | 12.3 | 4.3 | 12.4 | 4.9 | 12.1 | 3.7 |
| WBC, 103/mm3 | 235.2 | 3075.1 | 539.0 | 4697.4 | 7.4 | 2.3 |
| RBC, 106/mm3 | 4.6 | 0.6 | 4.6 | 0.6 | 4.6 | 0.5 |
| Hemoglobin, g/dl | 12.8 | 1.6 | 12.7 | 1.5 | 12.8 | 1.6 |
| Hematocrit, % | 40.4 | 22.7 | 38.8 | 4.1 | 38.9 | 4.7 |
| MCV, µm3 | 86.3 | 5.7 | 85.6 | 5.4 | 87.0 | 5.8 |
| MPV, µm3 | 9.1 | 0.9 | 9.0 | 0.9 | 9.1 | 0.8 |
| Platelet, 103/mm3 | 278.9 | 67.5 | 293.5 | 67.2 | 267.3 | 65.7 |
| Total cholesterol, mg/dl | 201.7 | 42.3 | 203.1 | 35.5 | 200.7 | 47.1 |
| HDL-C, mg/dl | 48.4 | 11.8 | 47.2 | 10.5 | 49.4 | 12.7 |
| LDL-C, mg/dl | 124.1 | 36.5 | 125.4 | 31.1 | 123 | 40.3 |
| Triglyceride, mg/dl | 163.2 | 187.0 | 181.7 | 264.7 | 148.5 | 83.4 |
AST = Aspartate aminotrasferase; ALT = alanine aminotransferase; PTH = parathormone; WBC = white blood cell, RBC = red blood cell; MCV = mean corpuscular volume; MPV = mean platelet volume; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol.
Correlation analysis revealed that the PON activity was negatively correlated with HbA1c (r = −0.533; p < 0.001), plasma glucose (r = −0.457; p < 0.001), and HOMA-IR (r = −0.265; p < 0.001) in the whole study population, as were in the obese and non-obese groups (table 4). Moreover, the PON activity was found to be positively correlated with uric acid (r = 0.299; p = 0.005) and PTH (r = 0.213; p = 0.046) in the obese group (table 4).
Table 4.
Correlation of PON and ARE values to laboratory findings
| HbA1c | Glu | HOMA | Urea | Cre | UA | AST | ALT | PTH | VitB12 | Cpep | Homocys | WBC | RBC | Hb | MPV | TChol | HDL | LDL | TG | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PON | ||||||||||||||||||||
| Non-obese | ||||||||||||||||||||
| r | −0.684 | −0.527 | −0.281 | −0.037 | 0.021 | 0.105 | −0.015 | −0.088 | 0.048 | 0.012 | −0.018 | 0.028 | −0.223 | 0.006 | −0.043 | −0.033 | −0.107 | −0.047 | −0.011 | −0.167 |
| p | <0.001 | <0.001 | 0.007 | 0.699 | 0.830 | 0.271 | 0.879 | 0.356 | 0.612 | 0.898 | 0.848 | 0.770 | 0.023 | 0.948 | 0.655 | 0.747 | 0.263 | 0.621 | 0.907 | 0.078 |
| N | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 104 | 112 | 112 | 112 | 112 | 112 | 112 | 112 |
| Obese | ||||||||||||||||||||
| r | −0.331 | −0.340 | −0.263 | 0.053 | 0.019 | 0.299 | 0.031 | 0.024 | 0.213 | −0.135 | 0.090 | 0.072 | −0.159 | −0.090 | −0.030 | 0.071 | 0.032 | 0.100 | −0.075 | −0.117 |
| p | 0.002 | 0.001 | 0.039 | 0.621 | 0.863 | 0.005 | 0.774 | 0.821 | 0.046 | 0.209 | 0.407 | 0.507 | 0.168 | 0.406 | 0.779 | 0.531 | 0.764 | 0.355 | 0.487 | 0.276 |
| N | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 77 | 88 | 88 | 88 | 88 | 88 | 88 | 88 |
| Total | ||||||||||||||||||||
| r | −0.533 | −0.457 | −0.265 | 0.017 | 0.017 | 0.185 | 0.005 | −0.049 | 0.120 | −0.047 | 0.032 | 0.047 | −0.185 | −0.020 | −0.030 | −0.002 | −0.057 | 0.015 | −0.018 | −0.144 |
| p | <0.001 | <0.001 | <0.001 | 0.810 | 0.816 | 0.009 | 0.940 | 0.495 | 0.091 | 0.504 | 0.656 | 0.510 | 0.013 | 0.783 | 0.676 | 0.983 | 0.426 | 0.832 | 0.801 | 0.041 |
| N | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 181 | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
| ARE | ||||||||||||||||||||
| Non-obese | ||||||||||||||||||||
| r | −0.596 | −0.577 | −0.334 | 0.053 | −0.150 | −0.024 | −0.016 | −0.100 | −0.027 | −0.059 | 0.047 | −0.026 | −0.296 | 0.028 | −0.131 | −0.067 | −0.072 | −0.069 | −0.014 | −0.055 |
| p | <0.001 | <0.001 | 0.001 | 0.580 | 0.114 | 0.798 | 0.870 | 0.296 | 0.779 | 0.534 | 0.622 | 0.784 | 0.002 | 0.773 | 0.169 | 0.515 | 0.450 | 0.467 | 0.883 | 0.566 |
| N | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 112 | 104 | 112 | 112 | 112 | 112 | 112 | 112 | 112 |
| Obese | ||||||||||||||||||||
| r | −0.456 | −0.586 | −0.269 | −0.154 | −0.151 | 0.160 | −0.008 | −0.068 | 0.217 | −0.086 | 0.061 | −0.072 | −0.214 | −0.019 | 0.033 | 0.049 | 0.156 | 0.177 | 0.151 | −0.094 |
| p | <0.001 | <0.001 | 0.035 | 0.152 | 0.159 | 0.137 | 0.944 | 0.530 | 0.042 | 0.425 | 0.574 | 0.507 | 0.061 | 0.861 | 0.763 | 0.665 | 0.146 | 0.098 | 0.159 | 0.383 |
| N | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 88 | 77 | 88 | 88 | 88 | 88 | 88 | 88 | 88 |
| Total | ||||||||||||||||||||
| r | −0.544 | −0.584 | −0.298 | −0.031 | −0.153 | 0.067 | −0.013 | −0.088 | 0.084 | −0.073 | 0.062 | −0.041 | −0.265 | 0.011 | −0.060 | −0.040 | 0.028 | 0.040 | 0.068 | −0.063 |
| p | <0.001 | <0.001 | <0.001 | 0.660 | 0.031 | 0.344 | 0.851 | 0.218 | 0.234 | 0.304 | 0.382 | 0.561 | <0.001 | 0.878 | 0.398 | 0.577 | 0.693 | 0.571 | 0.338 | 0.374 |
| N | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 181 | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
r = Spearman correlation coefficient. Glu = glucose; HOMA = HOMA-IR; cre = creatinine; UA = uric acid; AST = aspartate aminotrasferase; ALT = alanine aminotransferase; PTH = parathormone; Cpep = C-peptide; Homocys = homocysteine; WBC = white blood cell, RBC = red blood cell; Hb = hemoglobin; Tchol = total cholesterol; HDL = high density lipoprotein; LDL = low density lipoprotein; TG = triglyceride.
Correlation analysis revealed that ARE activity was negatively correlated with HbA1c (r = −0.544; p < 0.001), plasma glucose (r = −0.584; p < 0.001), and HOMA-IR (r = −0.298; p < 0.001) in the whole study population, as were in the obese and non-obese groups (table 5). In addition, ARE activity was negatively correlated with creatinine (r = −0.153: p = 0.031) in the whole study population and positively correlated with PTH (r = 0.217:p = 0.042) in the obese group (table 5). In obese and non-obese patients, the HOMA-IR was negatively correlated with PON (r = −0.263; p = 0.039 and r = −0.281; p = 0.007, respectively) and ARE (r = −0.269; p = 0.035 and r = −0.334; p = 0.001, respectively) activities.
Table 5.
Correlation of PON and ARE values to brachial artery diameter and IMT in study groups
| PON |
ARE |
|||
|---|---|---|---|---|
| obese | non-obese | obese | non-obese | |
|
Brachial artery IMT Right |
||||
| r | −0.084 | 0.203 | −0.010 | 0.094 |
| p | 0.592 | 0.161 | 0.950 | 0.522 |
| Left | ||||
| r | −0.340 | 0.098 | −0.303 | −0.004 |
| p | 0.026 | 0.502 | 0.048 | 0.978 |
|
Brachial artery diameter Right |
||||
| r | 0.073 | 0.069 | −0.052 | 0.042 |
| p | 0.641 | 0.636 | 0.741 | 0.777 |
| Left | ||||
| r | 0.063 | 0.027 | −0.017 | 0.016 |
| p | 0.690 | 0.854 | 0.914 | 0.911 |
r = Spearman correlation coefficient.
Correlation analysis revealed that PON (r = −0.340; p = 0.026) and ARE (r = −0.303; p = 0.048) activities were negatively correlated with the left BA-IMT in the obese group.
Discussion
The findings of the present study which was conducted in a cohort of type 2 diabetic patients revealed no differences in PON and ARE activities with respect to age, gender, and obesity. The PON and ARE activities were correlated negatively with blood glucose, HbA1c, and HOMA-IR in both obese and non-obese groups. Additionally, the PON and ARE activities were positively correlated with PTH in the obese group and negatively correlated with WBC in the non-obese group. Moreover, the PON and ARE activities were negatively correlated with the left BA-IMT in the obese group.
Serum PON1 activity has been consistently reported to be decreased in conditions such as type 2 DM, which are associated with low HDL cholesterol [15, 16, 17, 18]. On the other hand, studies on measurement of serum PON enzyme activity as a marker of CAD have revealed controversial findings with varying degrees of association in different communities; these findings have suggested that expression of these factors is under genetic control [19, 20].
There is a paucity of data on alterations in PON1 status in obesity due to inconclusive data resulting from measurements of different activities of PON1 (such as esterase, PON, and ARE activities) in different studies and not considering the possible influence of genetic polymorphisms on the PON1 activity in most studies [21]. In this regard, following the first data on the decreased PON activity and increased lipid peroxidation levels in isolated HDL from adult obese [10], decreased serum ARE activity has been consistently reported in obese adults [22, 23]. On the other hand, studies have reported inconsistent findings on PON activity in obesity; some reported decreases in PON activity [24], while some others reported no significant changes [21, 25]. Within this context, measurement of PON1 activity through serum 5-thiobutyl butyrolactone (TBBLase) or ARE activities rather than PON activity has been suggested for investigating the associations among PON1, obesity, and metabolic syndrome [21].
Studies on PON1 activity in patients with obesity, metabolic syndrome or insulin resistance but without DM revealed inconsistent findings, indicating a negative correlation of PON1 activity with morbid obesity [23] and metabolic syndrome [26], similar PON1 activity in obese non-diabetic subjects with or without metabolic syndrome when compared to control levels [25], and a higher PON1 protein concentration but lower specific enzyme activity in obese non-diabetic males with metabolic syndrome [27]. Moreover, PON1 activity was also reported to be similar in diabetic patients, in subjects with impaired fasting glucose, and in normoglycemic controls [28, 29], which suggested the loss of PON1 activity to occur later in the course of DM and hyperglycemia rather than in the stage of insulin resistance [8].
The lack of a control group limited interpretation of our findings with respect to the findings of above-mentioned studies. The similarity in terms of serum PON and ARE activities as well as serum lipids between patients with diabetes with or without obesity in the present study seemed in line with the results from another study investigating a Turkish population, which indicated that PON1 activities were not different between non-diabetic subjects with and without metabolic syndrome [25].
Although there are previous studies demonstrating the relationship between PON1 activity and the lipid profile (particularly HDL cholesterol level) [30], in the present study, the PON and ARE activities were not correlated with lipid fraction concentrations in our diabetic patients, which was consistent with the data from past research on atherosclerosis [31, 32] and type 1 DM and familial hypercholesterolemia [6, 31]. Notably, the concentration of the HDL cholesterol fraction has been suggested to influence PON1 activity only in cases with dramatic decreases of HDL cholesterol such as in Tangier disease or fish-eye disease, but not in those with moderate decrease, e.g. during a prolonged state of ischemia in patients with atherosclerosis [32]. Besides, it was reported that PON1 activity was more closely related to the HDL particle concentration or large HDL particles than to HDL cholesterol in type 2 diabetic and non-diabetic patients [33].
In the present study, PON and ARE activities were negatively correlated with glycemic parameters in our diabetic patients regardless of obesity. This finding was in agreement with the data from a previous study indicating inverse correlations of PON1 with blood glucose, insulin, and HOMA-IR in patients with type 2 DM [4] and the data from another study reporting a correlation between HbA1c and PON1 activity in both types of DM [34]. Our findings were also in line with those of an experimental study in which administration of PON1 in vivo to mice or in vitro to β-cells attenuated development of DM and increased secretion of insulin from pancreatic β-cells as a result of PON1's unique antioxidant properties and β-cell insulin biosynthesis stimulated by PON1 [2]. PON1 activity plays a critical role in the relationship between insulin resistance, metabolic syndrome, and subsequent progression to type 2 DM [4], which was also emphasized in our findings. Additionally, PON1 activity is likely to provide protection against oxidative damage in pancreatic β-cells via increasing β-cell survival and increasing insulin secretion [2].
It has been suggested that measurement of PON1 activity may provide an early indicator of metabolic disturbances before the onset of measurable arterial changes [4], which was supported by the findings of our study. The negative correlations of PON and ARE activities with left BA-IMT in obese but not in non-obese diabetics in our study population supported the statement that the contribution of PON1 activity to endothelial function and atherogenesis may differ in patients with respect to cardiovascular risk status [35]. Considering obese diabetic patients, our findings were consistent with the previously reported importance of PON1 activity as a predictor of coronary atherosclerosis in patients with ischemic heart disease [7, 31] and emphasized the likelihood of PON1 to modulate early atherogenesis steps in obese diabetic patients. However, similar to our findings, in non-obese patients, no evidence was shown for a relationship between PON1 genotype and subclinical atherosclerosis in an unselected group of type 2 DM patients based on either the measurement of carotid IMT as a surrogate continuous variable or non-invasive vascular investigations [36].
The genetic polymorphisms of PON1 have been suggested to be responsible for a wide variation in serum PON1 activity among individuals and populations and its relation to CAD [31, 37]. Taking into consideration that there was no correlation between PON1 activity and BA-IMT in our non-obese diabetics and no association of BAd with PON1 activity, regardless of obesity in our study population, it cannot be ruled out that the PON1 polymorphisms are likely to differentially affect the atherosclerosis process or that the PON1 gene has a potential role in modulating later steps of atherosclerosis [38]. Regardless of its genetic or environmental determinants, a low PON1 activity was reported to be associated with endothelial dysfunction expressed as brachial artery flow-mediated vasodilation (FMV) in patients with symptomatic peripheral artery disease [35], while BAd and male gender were shown as independent predictors of brachial FMV in a multivariate analysis [35]. Moreover, interethnic variability in PON1 polymorphism along with unimodal distribution of PON1 activity in non-Europid populations was suggested to cause the lack of a significant difference in PON1 activity between diabetic CAD patients and non-CAD controls in a previous study [20]. .
In most studies using popular commercial kits it was assumed that PON activity reflected PON1 activity [27]. In the present study, we were able to demonstrate that PON and ARE activities in patients with diabetes with or without obesity were similar, supporting the hypothesis that the results and changing trends among the three types of PON1 enzyme activities are all compatible with measurements in DM and obese patients [27, 39, 40].
The present study has some limitations. Firstly, the cross-sectional design of the study, the lack of a non-diabetic control group, and the relatively small sample size did not allow to draw extensive causal conclusions and to generalize our findings to the diabetic population in general. Secondly, we did not evaluate the surrogate serum biomarkers of oxidative stress that could have impacts on PON1 protein expression or activity. Thirdly, data regarding treatments with classical inducers of PON1 activity such as statins, fibrates, acetylsalicylic acid, glucocorticoids, and phenobarbital [8] were not evaluated. Nevertheless, as it is known that data on this topic are scant, our findings represent a valuable contribution to the literature.
In conclusion, we were able to show that there are no differences between patients with diabetes with or without obesity in terms of serum PON and ARE activities, that there are negative correlations of serum PON and ARE activities with glycemic parameters regardless of obesity, and that negative correlations of PON and ARE activities with the left BA-IMT could only be measured in obese diabetic patients. In this regard, our findings emphasized the possible role of low serum PON and ARE activities in predicting poor glycemic control and identified PON1 as a potential therapeutic target to improve glycemic control among diabetics regardless of obesity. Although the current findings suggests that low PON1 activity is a potential marker of early atherosclerosis in diabetes, at least in obese patients, further verification and deeper understanding of PON1 functions and modulators via larger-scale prospective case control studies are needed to unambiguously clarify whether or not the determination of PON1 activities might be useful in the identification and prevention of atherosclerosis and other oxidative stress-related diseases.
Conclusion
Obesity and type 2 DM are associated with atherosclerosis. The importance of PON1 activity and its decrease in atherosclerosis has been shown in several studies. Accordingly, in the present study, including a large number of patients, we demonstrated the association of endothelial dysfunction with PON1 activity and obesity.
Disclosure Statement
The authors declare no conflict of interest.
References
- 1.Gbandjaba NY, Ghalim N, Hassar M, et al. Paraoxonase activity in healthy, diabetic, and hemodialysis patients. Clin Biochem. 2012;45:470–474. doi: 10.1016/j.clinbiochem.2012.01.005. [DOI] [PubMed] [Google Scholar]
- 2.Koren-Gluzer M, Aviram M, Meilin E, Hayek T. The antioxidant HDL-associated paraoxonase-1 (PON1) attenuates diabetes development and stimulates β-cell insulin release. Atherosclerosis. 2011;219:510–518. doi: 10.1016/j.atherosclerosis.2011.07.119. [DOI] [PubMed] [Google Scholar]
- 3.Elnoamany MF, Dawood AA, Azmy RM, Elnajjar MM. Paraoxonase 1 gene (Gln192-Arg) polymorphism and the risk of coronary artery disease in type 2 diabetes mellitus. Egypt Heart J. 2012;64:55–62. [Google Scholar]
- 4.Mackness B, Marsillach J, Elkeles RS, et al. Paraoxonase-1 is not associated with coronary artery calcification in type 2 diabetes: results from the PREDICT study. Dis Markers. 2012;33:101–112. doi: 10.3233/DMA-2012-0910. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mackness MI, Mackness B, Durrington PN, Connelly PW, Hegele RA. Paraoxonase: biochemistry, genetics and relationship to plasma lipoproteins. Curr Opin Lipidol. 1996;7:69–76. doi: 10.1097/00041433-199604000-00004. [DOI] [PubMed] [Google Scholar]
- 6.Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett. 1991;286:152–154. doi: 10.1016/0014-5793(91)80962-3. [DOI] [PubMed] [Google Scholar]
- 7.Mackness B, Davies GK, Turkie W, et al. Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype? Arterioscler Thromb Vasc Biol. 2001;21:1451–1457. doi: 10.1161/hq0901.094247. [DOI] [PubMed] [Google Scholar]
- 8.Kota SK, Meher LK, Kota SK, Jammula S, Krishna SV, Modi KD. Implications of serum paraoxonase activity in obesity, diabetes mellitus, and dyslipidemia. Indian J Endocrinol Metab. 2013;17:402–412. doi: 10.4103/2230-8210.111618. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Haffner SM, Lehto S, Rönnemaa T, Pyörälä K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998;339:229–234. doi: 10.1056/NEJM199807233390404. [DOI] [PubMed] [Google Scholar]
- 10.Ferretti G, Bacchetti T, Moroni C, et al. Paraoxonase activity in high-density lipoproteins: A comparison between healthy and obese females. J Clin Endocrinol Metab. 2005;90:1728–1733. doi: 10.1210/jc.2004-0486. [DOI] [PubMed] [Google Scholar]
- 11.Stein JH, Fraizer MC, Aeschlimann SE, Nelson-Worel J, McBride PE, Douglas PS. Vascular age: integrating carotid intima-media thickness measurements with global coronary risk assessment. Clin Cardiol. 2004;27:388–392. doi: 10.1002/clc.4960270704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Iwamoto Y, Maruhashi T, Fujii Y, et al. Intima-media thickness of brachial artery, vascular function, and cardiovascular risk factors. Arterioscler Thromb Vasc Biol. 2012;32:2295–2303. doi: 10.1161/ATVBAHA.112.249680. [DOI] [PubMed] [Google Scholar]
- 13.Picard S, Talussot C, Serusclat A, Ambrosio N, Berthezène F. Minimally oxidised LDL as estimated by a new method increase in plasma of type 2 diabetic patients with atherosclerosis or nephropathy. Diabetes Metab. 1996;22:25–30. [PubMed] [Google Scholar]
- 14.Mackness B, Hunt R, Durrington PN, Mackness MI. Increased immunolocalization of paraoxonase, clusterin, and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 1997;17:1233–1238. doi: 10.1161/01.atv.17.7.1233. [DOI] [PubMed] [Google Scholar]
- 15.Mackness B, Mackness MI, Arrol S, et al. Serum paraoxonase (PON1) 55 and 192 polymorphism and paraoxonase activity and concentration in non-insulin dependent diabetes mellitus. Atherosclerosis. 1998;139:341–349. doi: 10.1016/s0021-9150(98)00095-1. [DOI] [PubMed] [Google Scholar]
- 16.Dullaart RPF, de Vries R, Sluiter WJ, Voorbij HA. High plasma C-reactive protein (CRP) is related to low paraoxonase-I (PON-I) activity independently of high leptin and low adiponectin in type 2 diabetes mellitus. Clin Endocrinol (Oxf) 2009;70:221–226. doi: 10.1111/j.1365-2265.2008.03306.x. [DOI] [PubMed] [Google Scholar]
- 17.Jornayvaz FR, Brulhart-Meynet MC, James RW. Myeloperoxidase and paraoxonase-1 in type 2 diabetic patients. Nutr Metab Cardiovasc Dis. 2009;19:613–619. doi: 10.1016/j.numecd.2008.12.005. [DOI] [PubMed] [Google Scholar]
- 18.Stefanović A, Kotur-Stevuljević J, Spasić S, et al. HDL 2 particles are associated with hyperglycaemia, lower PON1 activity and oxidative stress in type 2 diabetes mellitus patients. Clin Biochem. 2010;43:1230–1235. doi: 10.1016/j.clinbiochem.2010.08.005. [DOI] [PubMed] [Google Scholar]
- 19.Karakaya A, Ibiş S, Kural T, Köse SK, Karakaya AE. Serum paraoxonase activity and phenotype distribution in Turkish subjects with coronary heart disease and its relationship to serum lipids and lipoproteins. Chem Biol Interact. 1999;118:193–200. doi: 10.1016/s0009-2797(99)00085-x. [DOI] [PubMed] [Google Scholar]
- 20.Rahmani M, Raiszadeh F, Allahverdian S, Kiaii S, Navab M, Azizi F. Coronary artery disease is associated with the ratio of apolipoprotein A-I/B and serum concentration of apolipoprotein B, but not with paraoxonase enzyme activity in Iranian subjects. Atherosclerosis. 2002;162:381–389. doi: 10.1016/s0021-9150(01)00715-8. [DOI] [PubMed] [Google Scholar]
- 21.Ferré N, Feliu A, García-Heredia A, et al. Impaired paraoxonase-1 status in obese children. Relationships with insulin resistance and metabolic syndrome. Clin Biochem. 2013;46:1830–1836. doi: 10.1016/j.clinbiochem.2013.08.020. [DOI] [PubMed] [Google Scholar]
- 22.Bajnok L, Seres I, Varga Z, et al. Relationship of endogenous hyperleptinemia to serum paraoxonase 1, cholesteryl ester transfer protein, and lecithin cholesterol acyltransferase in obese individuals. Metabolism. 2007;56:1542–1549. doi: 10.1016/j.metabol.2007.06.022. [DOI] [PubMed] [Google Scholar]
- 23.Bajnok L, Csongradi E, Seres I, et al. Relationship of adiponectin to serum paraoxonase 1. Atherosclerosis. 2008;197:363–367. doi: 10.1016/j.atherosclerosis.2007.06.001. [DOI] [PubMed] [Google Scholar]
- 24.Aslan M, Horoz M, Sabuncu T, Celik H, Selek S. Serum paraoxonase enzyme activity and oxidative stress in obese subjects. Pol Arch Med Wewn. 2011;121:181–186. [PubMed] [Google Scholar]
- 25.Tabur S, Torun AN, Sabuncu T, et al. Non-diabetic metabolic syndrome and obesity do not affect serum paraoxonase and arylesterase activities but do affect oxidative stress and inflammation. Eur J Endocrinol. 2010;162:535–541. doi: 10.1530/EJE-09-0732. [DOI] [PubMed] [Google Scholar]
- 26.Garin MC, Kalix B, Morabia A, James RW. Small, dense lipoprotein particles and reduced paraoxonase-1 in patients with the metabolic syndrome. J Clin Endocrinol Metab. 2005;90:2264–2269. doi: 10.1210/jc.2004-1295. [DOI] [PubMed] [Google Scholar]
- 27.Liang KW, Lee WJ, Lee IT, et al. Persistent elevation of paraoxonase-1 specific enzyme activity after weight reduction in obese non-diabetic men with metabolic syndrome. Clin Chim Acta. 2011;412:1835–1841. doi: 10.1016/j.cca.2011.06.018. [DOI] [PubMed] [Google Scholar]
- 28.Beer S, Moren X, Ruiz J, James RW. Postprandial modulation of serum paraoxonase activity and concentration in diabetic and non-diabetic subjects. Nutr Metab Cardiovasc Dis. 2006;16:457–465. doi: 10.1016/j.numecd.2005.09.005. [DOI] [PubMed] [Google Scholar]
- 29.Kopprasch S, Pietzsch J, Kuhlisch E, Graessler J. Lack of association between paraoxonase 1 activities and increased oxidized low-density lipoprotein levels in impaired glucose tolerance and newly diagnosed diabetes mellitus. J Clin Endocrinol Metab. 2003;88:1711–1716. doi: 10.1210/jc.2002-021561. [DOI] [PubMed] [Google Scholar]
- 30.Blatter Garin MC, Moren X, James RW. Paraoxonase-1 and serum concentration of HDL-cholesterol and apoA-1. J Lipid Res. 2005;47:515–520. doi: 10.1194/jlr.M500281-JLR200. [DOI] [PubMed] [Google Scholar]
- 31.Ayub A, Mackness MI, Arrol S, Mackness B, Patel J, Durrington PN. Serum paraoxonase after myocardial infarction. Arterioscler Thromb Vasc Biol. 1999;19:330–335. doi: 10.1161/01.atv.19.2.330. [DOI] [PubMed] [Google Scholar]
- 32.Kasprzak M, Iskra M, Majewski W, Wielkoszyński T. Arylesterase and paraoxonase activity of paraoxonase (PON1) affected by ischemia in the plasma of patients with arterial occlusion of the lower limbs. Clin Biochem. 2009;42:50–56. doi: 10.1016/j.clinbiochem.2008.09.117. [DOI] [PubMed] [Google Scholar]
- 33.Dullaart RP, Otvos JD, James RW. Serum paraoxonase-1 activity is more closely related to HDL particle concentration and large HDL particles than to HDL cholesterol in Type 2 diabetic and non-diabetic subjects. Clin Biochem. 2014;47:1022–1027. doi: 10.1016/j.clinbiochem.2014.04.013. [DOI] [PubMed] [Google Scholar]
- 34.Flekac M, Skrha J, Zídková K, Lacinová Z, Hilgertová J. Paraoxonase 1 gene polymorphisms and enzyme activities in diabetes mellitus. Physiol Res. 2008;57:717–726. doi: 10.33549/physiolres.931285. [DOI] [PubMed] [Google Scholar]
- 35.Pasqualini L, Cortese C, Marchesi S, et al. Paraoxonase-1 activity modulates endothelial function in patients with peripheral arterial disease. Atherosclerosis. 2005;183:349–354. doi: 10.1016/j.atherosclerosis.2005.03.030. [DOI] [PubMed] [Google Scholar]
- 36.Cao H, Girard-Globa A, Serusclat A, et al. Lack of association between carotid intima-media thickness and paraoxonase gene polymorphism in non-insulin dependent diabetes mellitus. Atherosclerosis. 1998;138:361–366. doi: 10.1016/s0021-9150(98)00031-8. [DOI] [PubMed] [Google Scholar]
- 37.La Du BN, Piko JI, Eckerson HW, Vincent-Viry M, Siest G. An improved method for phenotyping individuals for the human serum paraoxonase arylesterase polymorphism. Ann Biol Clin (Paris) 1986;44:369–372. [PubMed] [Google Scholar]
- 38.Campo S, Sardo MA, Trimarchi G, et al. The paraoxonase promoter polymorphism (−107)T>C is not associated with carotid intima-media thickness in Sicilian hypercholesterolemic patients. Clin Biochem. 2004;37:388–394. doi: 10.1016/j.clinbiochem.2003.12.012. [DOI] [PubMed] [Google Scholar]
- 39.Rosenblat M, Karry R, Aviram M. Paraoxonase 1 (PON1) is a more potent antioxidant and stimulant of macrophage cholesterol efflux, when present in HDL than in lipoprotein-deficient serum: relevance to diabetes. Atherosclerosis. 2006;187:74–81. doi: 10.1016/j.atherosclerosis.2005.08.026. [DOI] [PubMed] [Google Scholar]
- 40.Kotani K, Sakane N, Sano Y, et al. Changes on the physiological lactonase activity of serum paraoxonase 1 by a diet intervention for weight loss in healthy overweight and obese women. J Clin Biochem Nutr. 2009;45:329–334. doi: 10.3164/jcbn.09-26. [DOI] [PMC free article] [PubMed] [Google Scholar]