Decreased Paraoxonase and Arylesterase Activities in the Pathogenesis of Future Atherosclerotic Heart Disease in Women with Gestational Diabetes Mellitus

Abstract

Objective:

The aim of this study was to investigate serum paraoxonase, arlyesterase activities, and lipid hydroperoxide (LOOH) levels in patients with gestational diabetes mellitus (GDM).

Methods:

Paraoxonase and arylesterase activities, and LOOH levels were assessed for GDM cases (n = 55) and controls (n = 59). Serum basal and salt-stimulated paraoxonase and arylesterase activities were measured spectrophotometrically. LOOH levels were measured by ferrous oxidation with a xylenol orange assay.

Results:

Basal and salt-stimulated paraoxonase and arylesterase activities were significantly lower (p = 0.002, p = 0.004; and p = 0.013, respectively) in patients with GDM compared to controls, while LOOH levels were significantly higher (p < 0.001). Among gestational diabetes patients, serum paraoxonase and arylesterase activities were inversely correlated with LOOH levels (r = − 0.390, p = 0.003; and r = − 0.287, p = 0.034, respectively).

Conclusions:

Findings of the present study have shown that serum paraoxonase and arylesterase activities are significantly reduced in women with GDM. Decreased serum paraoxonase and arylesterase activities might play a role in the potential early pathogenesis for atherosclerotic heart disease in GDM beyond their antioxidant properties.

Introduction

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first diagnosis during pregnancy.¹ It is the most frequent metabolic disorder of pregnancy, occurring in 1–10% of all pregnancies.² GDM is characterized by biochemical abnormalities common to type 2 diabetes mellitus, including hyperglycemia, insulin resistance, and hyperlipidemia. GDM has been considered a “pre-diabetic” state, and the pathophysiology of the two are clearly related.³ Furthermore, up to 70% of women with a history of GDM will develop type 2 diabetes mellitus (DM) later in life.⁴ DM is an independent risk factor for the development of coronary artery disease, and is also associated with the development of an atherogenic lipid profile and increased oxidative stress. Moreover, DM plays a major role in the development of atherosclerosis.⁵

Oxidative stress plays a crucial role in the development of atherosclerosis through the oxidation of low-density lipoprotein (LDL) that subsequently leads to the formation of foam cells.^6,7 Lipid hydroperoxide (LOOH) is a well-known marker of oxidative stress formed from unsaturated phospholipids, glycolipids, and cholesterol by peroxidative reactions under oxidative stres.⁸ Oxidized LDL, in addition to membrane-bound cholesterol-derived hydroperoxide, is the main form of LOOH responsible for the development of oxidative stress-related atherosclerosis and adverse cardiovascular events.⁸ While high-density lipoprotein (HDL) is a well-known anti-oxidant molecule that prevents atherosclerosis.⁹ Paraoxonase-1 (PON1) is an HDL-associated enzyme with three mediators: paraoxonase, arylesterase, and dyazoxonase.^10,11 PON1 plays a key role in the protection of LDL and HDL from oxidation by hydrolyzing activated phospholipids¹² and lipid peroxide products,¹³ and has an important role in prevention of atherosclerosis.¹² Furthermore, several studies suggest that PON1 activity is decreased in subjects with atherosclerotic heart disease,^14

–17 hypercholesterolemia,¹⁸ type 2 diabetes,¹⁸ and renal failure.¹⁹ Additionally, streptozotocin-induced diabetes results in a progressive decrease in serum PON activity.²⁰

To the best of our knowledge, PON1 activity in women with GDM has not been evaluated. It is still unknown whether there is any relationship between PON1 activity and atherosclerosis in patients with GDM. Therefore, the goal of this study was to clarify these points by investigating paraoxonase and arylesterase activities as antioxidants, and to better define LOOH levels as an oxidative stress indicator in women with GDM.

Methods

Overall, 1475 women with singleton pregnancies between 24 and 32 weeks of gestation were registered as new patients in the Department of Obstetrics and Gynecology in Harran University Hospital between August 15, 2007, and July 15, 2008. Informed consent for study participation was obtained from all women. The study protocol conforms to the principles of the Helsinki Declaration and was approved by the Medical Ethics Committee of Harran University. The study group included 55 patients with GDM. The control group was composed of 59 healthy pregnant women who had a negative glucose challenge test (GCT). Exclusion criteria for all study participants included smoking, alcohol abuse, preeclampsia, multiple pregnancies, and pre-gestational diabetes. Controls with a family history of diabetes mellitus were also excluded. During the period mentioned above, a total of 64 (4.33%) GDM patients were diagnosed, but only 55 of them met our selection criteria. Cases and controls were compared based on gestational age, parity, maternal age, and body mass index (BMI).

In 103 of 114 (90%) of the pregnancies, gestational age was based on routine ultrasound examination performed before the end of the 19th completed gestational week. For the remaining pregnancies, gestational age was based on last menstrual period.

The 50-g GCT was carried out independently of the time of day or any previous meals,²¹ as this is our routine screening test for GDM. A 100-g, 3-h oral glucose tolerance test (OGTT) was recommended to all patients whose 1-h test result equaled or exceeded 140 mg/dl. OGTT was performed in the morning after an overnight fast of at least 8 h, and after at least 3 days of unrestricted diet (>150 g carbohydrate per day) and physical activity. Women whose 3-h oral glucose tolerance test (3h-OGTT) produced two or more abnormal values according to the Carpenter-Coustan criteria were diagnosed with GDM.²² Women whose GCT results were greater than 190 mg/dL were also identified as having GDM. The CGT and OGTT were performed during the course of the pregnancy between weeks 24 and 28. After diagnosis, 34 (61.8%) women with GDM were clinically managed by diet alone, and 21 (38.2%) women were prescribed insulin in addition to dietary management.

All blood samples were obtained in the morning from the cubital vein after an overnight fast. Samples were drawn from cubital vein into blood tubes and were immediately separated from the cells by centrifugation at 3000× g for 10 min, stored at −80°C, and then analyzed. The blood samples were collected just for this study.

Paraoxonase and arylesterase activities were measured using commercially available kits (Relassay,® Turkey). Paraoxonase activity measurements were performed both in the absence and presence of NaCl (salt-stimulated activity). The rate of paraoxon hydrolysis (diethyl-p-nitrophenylphosphate) was measured by monitoring the increase of absorption at 412 nm at 37°C. The amount of generated p-nitrophenol was calculated from the molar absorption coefficient at pH 8.5, which was 18.290 M⁻¹ cm⁻¹.²³ Paraoxonase activity was expressed as U/L serum. Phenylacetate was used as a substrate to measure the arylesterase activity. Enzymatic activity was calculated from the molar absorption coefficient of the produced phenol, 1310 M⁻¹ cm⁻¹. One unit of arylesterase activity was defined as 1 μmol phenol generated per minute under the above conditions and expressed as U/L.²⁴ Paraoxonase phenotype distribution was determined by a double substrate method, which calculates the ratio of salt-stimulated paraoxonase activity and arylesterase activity.²³

Serum LOOH levels were measured by the ferrous ion oxidation-xylenol orange (FOX-2) method previously described.²⁵ The levels of triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), and fasting glucose were determined using commercially available assay kits (Abbott, Chicago, IL) with Aeroset auto-analyzer (Abbott). HbA_1c level was measured using commercially available kits (Roche, Basel, Switzerland). The normal range was 4–6%.

Statistical analysis

All analyses were conducted using SPSS 11.5 (SPSS for Windows 11.5, Chicago, IL). Continuous variables were expressed as mean ± standard deviation (SD). Normality of distribution was evaluated with the Kolmogorov-Smirnov test. Parameter comparisons were performed using the Student's t-test and Mann-Whitney U-test. The correlation between paraoxonase activity, arylesterase activity, LOOH levels, and HDL-C and LDL-C levels were assessed by the Pearson's correlation test. Multiple linear regression analysis was performed to identify the independent predictors of the paraoxonase activity and other parameters. Standardized β-regression coefficients and their significance from multiple linear regression analysis were reported. A p-value of <0.05 was considered significant.

Results

Demographic and clinical data of women with GDM and controls are summarized in Table 1. There were no significant differences between women with GDM and controls with respect to maternal age, gestational age, and BMI.

Table 1.

Demographic and Clinical Parameters of Patients with Gestational Diabetes Mellitus (GDM) and Controls

Parameters	GDM (n = 55)	Controls (n = 59)	p value
Age (years)	29.3 ± 3.6	28.8 ± 3.7	0.474
Gestational age (weeks)	29.3 ± 2.7	29.8 ± 2.8	0.259
BMI (kg/m²)	31.1 ± 5.2	29.4 ± 4.7	0.072
Parity	2.8 ± 2.1	2.1 ± 1.9	0.040
Triglyceride (mg/dl)	277 ± 91	222 ± 78	0.001
Total cholesterol (mg/dl)	220 ± 43	231 ± 42	0.159
LDL-C (mg/dl)	147 ± 29	117 ± 35	<0.001
HDL-C (mg/dl)	57.5 ± 13.3	72.9 ± 12.4	<0.001
HbA_1c (%)	5.84 ± 0.1	4.76 ± 0.1	<0.001

BMI, body mass index; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol.

Values are mean ± SD.

While TC levels were similar in the gestational diabetes and control groups (p = 0.159), TG, LDL-C, and HbA_1c levels were significantly higher in women with gestational diabetes (p = 0.001, p < 0.001, p < 0.001, respectively) compared to the control group. In contrast, HDL-C levels were significantly lower in women with gestational diabetes (p < 0.001; Table 1).

Table 2.

Lipid Hydroperoxide, PON/HDL Ratio, Basal/Salt-Stimulated Paraoxonase and Arylesterase Activities in Women with Gestational Diabetes Mellitus (GDM) and Controls

Parameters	GDM (n = 55)	Controls (n = 59)	p value
Basal paraoxonase (U/L)	185 ± 79	234 ± 85	0.002
Salt-stimulated paraoxonase (U/L)	435 ± 307	639 ± 430	0.004a
Arylesterase (kU/L)	171 ± 34	189 ± 39	0.013
Paraoxonase/HDL-C ratio	3.19 ± 1.0	3.23 ± 1.1	0.800
LOOH (μmol H₂O₂ Eqv./L)	10.1 ± 2.7	8.2 ± 1.1	<0.001

Mann-Whitney U-test for comparison between two groups.

LOOH, lipid hydroperoxide; HDL-C, high-density lipoprotein-cholesterol.

Values are mean ± SD.

Basal and salt-stimulated paraoxonase and arylesterase activities were significantly lower in women with gestational diabetes compared to controls (p = 0.002, p = 0.004, p = 0.013, respectively), while LOOH levels were significantly higher (p < 0.001). In addition, there were no statistically significant differences between the two groups with regard to the paraoxonase/HDL-C ratio (p = 0.800; Table 2).

In patients with GDM, paraoxonase and arylesterase activities were positively correlated with HDL-C levels (r =0.677, p < 0.001; and r = 0.459, p < 0.001, respectively), while LDL-C levels were inversely correlated (r = − 0.480, p < 0.001; and r = − 0.345, p = 0.009, respectively; Table 3). Additionally, LOOH levels were inversely correlated with paraoxonase and arylesterase activities (r = − 0.390, p = 0.003; and r = − 0.287, p = 0.034, respectively), but were positively correlated with LDL-C levels (r = 0.420, p = 0.001; Table 3). With regression analysis, only HDL-C (β = 0.548, p < 0.001) and LDL-C (β = − 0.251, p = 0.028) were independent predictors of paraoxonase activity (Table 4), while arylesterase activity and LOOH levels were not associated.

Table 3.

Correlations between Paraoxonase Activity, Arylesterase Activity, LOOH, HDL-C, and LDL-C Levels in Women with Gestational Diabetes Mellitus

	Arylesterase	LOOH	HDL-C	LDL-C
Paraoxonase
r	0.397	−0.390	0.677	−0.480
p	0.003	0.003	<0.001	<0.001
Arylesterase
r		−0.287	0.459	−0.345
p		0.034	<0.001	0.009
LOOH
r			−0.360	0.420
p			0.007	0.001
HDL-C
r				−0.336
p				0.012

HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein cholesterol; LOOH, lipid hydroperoxide.

Table 4.

Relationship between Paraoxonase Activity and Other Parameters in Women with Gestational Diabetes Mellitus

	β-Regression coefficient a	p Value
Arylestarase	0.036	0.251
LOOH	−0.077	0.488
HDL-C	0.548	<0.001
LDL-C	−0.251	0.028

From multiple linear regression analysis.

HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein cholesterol; LOOH, lipid hydroperoxide.

Discussion

In the current study, we found that basal and salt-stimulated paraoxonase and arylesterase activities were significantly lower in patients with GDM than in controls, while LOOH levels were significantly higher. To our knowledge, this is the first article reporting the paraoxonase and arylesterase activities in women with GDM. A search of MEDLINE (English language; 1966 to June 2008; search terms: “paraoxonase” and “arylesterase” and “gestational diabetes mellitus”) revealed no other entries. According to the data obtained from the present study, decreased paraoxonase and arylesterase activities were correlated with increased LOOH levels and therefore may be associated with greater oxidative stress in GDM. In addition, it appears that decreased paraoxonase and arylesterase activities may play a role in the pathogenesis of subclinical atherosclerosis in women with GDM through increased susceptibility to lipid peroxidation. Besides the previous reports regarding the potential role of serum paraoxonase activity on the development of atherosclerosis,^15,16 the independent association in our study of serum paraoxonase activity with serum HDL-C and LDL-C levels, but not with oxidative parameters, suggests a role for paraoxonase on the development of atherosclerosis beyond its antioxidative properties in patients with GDM. Although conflicting results have been reported,²⁶ it is generally accepted that oxidative stress is augmented by increases in oxidant levels and/or decreases in antioxidant enzyme capacities in subjects with GDM. Similarly, Toescu et al.⁵ have shown that GDM is associated with increased oxidative stress.

LDL oxidation in arterial walls is believed to have an important role in atherogenesis,²⁷ and mechanisms that prevent the oxidation of LDL have received increased attention in recent years. One such mechanism is PON1, which reduces LDL sensitivity to lipid peroxidation. Mackness et al.²⁸ suggested that PON1 accumulates in the human artery wall as atherosclerosis progresses. Indeed, PON1 activity has been shown to decrease in subjects with risks for atherosclerosis,^{15
–17,29,30} such as those with diabetes mellitus or hypercholesterolemia.¹⁸

The relationship between PON1 and the development of atherosclerosis has been demonstrated in in vivo and in vitro trials both in human and animal models. Shih et al.³¹ reported that mice lacking PON1 activity developed significantly larger lesions than control mice. Several other studies have found similar results.^32,33 The relationship between parity and atherosclerotic heart disease risk in women has also been investigated in several studies.^34
–36 Although conflicting data have been reported,³⁷ Ness et al. showed that women with six or more pregnancies had increased risk of atherosclerotic heart disease.³⁴

Women with GDM do in fact have subclinical atherosclerosis, and therefore GDM is in and of itself a risk factor for atherosclerotic heart disease. It appears that the only current clear link between GDM and atherosclerotic heart disease involves the interval development of type 2 DM. Long-term data in this regard would be especially intriguing. Are women who go on to develop type 2 DM more likely to manifest a continued inability to manage oxidative stress, and do these women in fact go on to develop atherosclerotic heart disease? This article sought to address and better understand the early pathogenesis of atherosclerotic heart disease in women, the foremost source of mortality.

In conclusion, we found lower levels of serum paraoxonase and arylesterase activities and significantly higher LOOH levels in patients with GDM, and an independent association of serum paraoxonase activity with HDL-C and LDL-C, but not with oxidative parameters. These findings suggest that decreased serum paraoxonase and arylesterase activities (in addition to serum HDL-C and LDL-C) might play a role in the potential early pathogenesis for atherosclerotic heart disease in GDM beyond their antioxidant properties. However, long-term clinical studies are needed to clarify the pathophysiological role of serum PON1 activity in the GDM.

Footnotes

Disclosure Statement

No competing financial interests exist.

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