Effect of Glimepiride on Paraoxonase Activity

Abstract

In this study, the effect of glimepiride on serum paraoxonase activity (EC 3.1.8.1.) has been investigated in vitro.. The inhibition and activation effects of this drug on serum paraoxonase activity was measured spectrophotometrically using paraoxon as the substrate. The phenotypic distribution of paraoxonase was determined using the dual substrate paraoxon and phenyl acetate as substrates. It was observed that glimepiride inhibited serum paraoxonase activity. In addition, an in vivo. study was performed for glimepiride in Sprague-Dawley rats. It was demonstrated that paraoxonase in serum, liver, and heart of Sprague-Dawley rat was significantly inhibited.

Keywords:

• Glimepiride
• heart
• liver
• paraoxonase
• serum

Introduction

The existence of organophosphatase in mammalian plasma was first reported nearly 50 years ago (Mazur, 1946). Subsequently, studies have shown this enzyme to be paraoxonase/arylesterase (PON; aryldialkylphosphatase, EC 3.1.8.1), an organophospahatase with broad substrate specificity, including aromatic carboxylic acid esters such as phenyl acetate (La Du, 1992).

The PON gene family includes at least three members termed PON1, PON2, and PON3, and it is mapped on human chromosome 7q21-q22. PON1 and PON3 gene products are constituents of high-density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low-density lipoprotein (LDL) oxidation (Campo et al., 2004). Serum paraoxonase is a glycoprotein that binds to HDLs and may prevent oxidation of LDL by hydrolyzing lipid peroxides. Two polymorphisms identified in the paraoxonase gene have been associated with cardiovascular disease. Oxidative LDL is also toxic to retinal capillary endothelial cells and pericytes, so that mildly modified LDL may contribute to the development of diabetic retinopathy (Walker & Mackness, 1987).

Mammals tend to exhibit high A-esterase activity in the blood and the liver, and this is apparently an important factor in determining the resistance of these organisms to organophosphate toxicity (Graham et al., 1997). Initial interest in PONs was, therefore, toxicological. In addition, PON is involved in drug metabolism and is being used for drug inactivation. There have been a few verbal reports suggesting that some drugs require PON1 for either their activation from a prodrug form or their inactivation. (Davies et al., 1996; Tougou et al., 1998; La Du et al., 1999; Biggadike et al., 2000; Jakubowski, 2000). In recent years, it has become apparent that PONs play an important role in atherosclerosis (Klimov et al., 1993; Watson et al., 1995).

Glimepiride is an oral blood sugar–lowering drug used for controlling diabetes, and is a sulfonylurea. It is used in type II diabetes, the most common type of diabetes found in 90% of patients with diabetes. In type II diabetes, insulin usually is not necessary to control blood sugar. Instead, diet and oral medications often are sufficient. Intolerance to sugar that results in elevated blood sugar is caused by reduced insulin secretion by the pancreas and resistance to insulin's effects by the body's cells. Glimepiride lowers the sugar level in the blood by stimulating insulin to be secreted (Kabadi & Kabadi, 2004; Korytkowski, 2004).

In the current study, the effects of glimepiride were investigated on human serum paraoxonase and arylesterase activities in in vitro. and in vivo. rat erythrocyte, liver, and heart.

Materials and Methods

Preparation of human serum

Erythrocytes were obtained from healty human blood at the Center of Research Hospital at Atatürk University. The blood samples were centrifuged at 2500 × g. for 20 min and the serum was obtained.

In vitro studies

To determine percent activity values of the drug, five different volumes (0.1, 0.2, 0.3, 0.4, and 0.5 ml) of glimepiride at a constant concentration were added to the enzyme activity determination medium (total volume: 3 ml). Paraoxonase activities with medical drugs were assayed by following using paroxon (O,O.-diethyl-p.-nitrophenylphosphate; Sigma Chemical Co.) and phenyl acetate as the substrates (Reiner & Radic, 1985; Kuo & La Du, 1995). Percent activity values of human serum paraoxonase for five different concentrations of medical drug were drawn by using regression analysis graphs on a computer. Paraoxonase activity without a medical drug was accepted as 100% activity.

In vivo studies

Fifteen adult Sprague-Dawley rats with individual weights of 200–250 g were used for the experiment. Before glimepiride administration, an 0.5-ml blood sample was taken from a tail vein for control, and then rats were fed with a food tablet that included 3 mg kg⁻¹ glimepiride. At 1, 3, and 6 h after glimepiride administration, 0.5-ml blood samples were taken again.

All blood samples were centrifuged at 2500 × g. for 15 min at 5°C (HERMLE Z383K) to separate the erythrocytes from the serum. Studies were carried out at 4°C. Paraoxonase activity was assayed by using paroxon (O,O.-diethyl-p.-nitrophenylphosphate; Sigma Chemical Co.) and phenyl acetate as the substrates.

Preparation of liver and heart homogenates

At 1, 3, and 6 h after glimepiride administration, rats were sacrificed and livers and hearts were excised. Rats livers (20 g) were removed, placed in beakers on ice, minced with 5 mM Tris/HCl (pH 7.4), 0.25 M sucrose, 0.15% Triton, minced with scissors, and then placed in 4 vol of ice-cold 5 mM Tris/HCl (pH 7.4), 0.25 M sucrose. The obtained final solution was homogenized by Sonic Dismembrator for 4 h, then it was centrifuged (15,000 × g., 30 min). The supernatant was dialyzed against distilled water to remove Triton X-100 and then against 5 mM Tris/HCl (pH 7.4), 0.25 M sucrose (Gil et al., 1983). The heart homogenate was prepared by using the same procedure.

Determination of paraoxonase (PON) activity

Paraoxonase activity was determined, using paroxon (O,O.-diethyl-p.-nitrophenylphosphate; Sigma Chemical Co.) as the substrate, by measuring the change in the absorbance at 412 nm due to formation of 4-nitrophenol. Activity was masured by adding 50 µl serum to 1 ml Tris/HCl buffer (100 mm, pH = 8) containing 2 mm CaCl₂ and 5.5 mm paraoxon. The rate of the generation of 4-nitrophenol was determined at 412 nm, 25°C, by use of a UV-Visible spectrophotometer. A molar extinction coefficient of 17,100 M⁻¹ cm⁻¹ was obtained and used for calculations of activity, and units were expressed as nmol of 4-nitrophenol formed per minute (Benedek et al., 1995).

Determination of arylesterase activity

Arylesterase activity was also measured spectrophotometrically. The assay contained 1 mM phenyl acetate in 20 mM Tris/HCl (pH = 8.0). The reaction was started by addition of serum, and the increase in absorbance was read at 270 nm. Blanks were included to correct the spontaneous hydrolysis of phenyl acetate. Enzyme activity was calculated using the molar extinction coefficient 1310 M⁻¹ cm⁻¹. Arylesterase activity was expressed in units per liter. One unit is defined as 1 μ.mol phenyl acetate hydrolyzed per minute (Hsyu et al., 1989).

Statistical methods

The SAS TM for Windows TM 6.11 software was used to perform statistical analyses. Data are presented by descriptive analysis (case number, mean, standard deviation). The comparisons between groups were performed by t.-test and ANOVA. The p < 0.05 probability was accepted as the significance level.

Results and Discussion

PON1 is widely distributed in tissues such as kidney, liver, intestine, and serum (La Du et al., 1993; La Du, 1996; Mackness et al., 1996; Nevin et al., 1996). Serum PON1 activity is decreased in patients with myocardial infarction, hypercholesterolemia, and diabetes mellitus (Mackness et al., 1991; Abbot et al., 1995; Letellier et al., 2002; Ikeda et al., 2003).

Glimepiride is a sulfonylurea used to treat type II, or non-insulin dependent diabetes, when diet and exercise alone have been ineffective, usually in those patients with adult maturity onset or non-insulin dependent diabetes mellitus (NIDDM). It works by lowering blood sugar levels by stimulating the production and release of insulin from the pancreas. It also promotes the movement of sugar from the blood into the cells in the body that need it. These two mechanisms, in conjunction with a diet low in sugar and fat, allows diabetics to control their blood sugar levels.

The effects of increasing concentrations of glimepiride on human serum paraoxonase activity were undertaken in this study. The range of glimepiride concentrations used was considered adequate to show enzyme inhibition or activation effects. In this regard, it was evident from in vitro. studies that human serum paraoxonase activity was inhibited by glimepiride at 0.40–2.04 mM concentrations (Fig. 1).

Figure 1 Percent activity versus [Glimepiride] regression analysis graph for paraoxonase at five different concentrations.

With in vivo. studies, maximal inhibition of rat erythrocyte, heart, and liver paraoxonase and arylesterase activities by 3 mg kg⁻¹ glimepiride occurred within 1 h (p < 0.001; Tables 1, 2, and 3) after glimepiride administration, and inhibition significantly continued even after 3 h (p < 0.001; Tables 1, 2, and 3). In our study, maximal inhibition of paraoxonase enzyme activity was seen within 1 h after drug administration, and significant inhibition continued even after 3 h (p < 0.001; Tables 1, 2, and 3). Our data showed that there is concordance between in vivo. and in vitro. effects of glimepiride on paraoxonase activity.

Table 1. Effect of glimepiride (3 mg kg⁻¹) on erythrocyte paraoxonase activity.

		Hydrolysis (mmol/min per mg of protein)
Medical drug	Hours	PAF	Phenyl acetate
	Control	31.1 ± 2.8	168.5 ± 20.2
Glimepiride	1 h	15.2 ± 1.1	75.2 + 11.1
	3 h	23.2 ± 2.3	165.8 ± 18.1
	6 h	33.5 ± 4.3	213.3 ± 20.3

Table 2. Effect of glimepiride (3 mg kg⁻¹) on rat liver paraoxonase activity.

		Hydrolysis (mmol/min per mg of protein)
Medical drug	Hours	PAF	Phenyl acetate
	Control	35.2 ± 3.2	356.8 ± 32.2
Glimepiride	1 h	20.2 ± 1.4	155.2 ± 18.1
	3 h	26.3 ± 3.2	265.7 ± 26.2
	6 h	33.5 ± 4.3	359.3 ± 32.3

Table 3. Effect of glimepiride (3 mg kg⁻¹) on rat heart paraoxonase activity.

		Hydrolysis (mmol/min per mg of protein)
Medical drug	Hours	PAF	Phenyl acetate
	Control	27.2 ± 2.2	138.4 ± 28.8
Glimepiride	1 h	11.2 ± 1.1	98.2 ± 19.1
	3 h	18.3 ± 2.3	125.7 ± 21.2
	6 h	28.5 ± 4.3	140.3 ± 33.5

In this Report, in vitro. studies of human serum paraoxonase and arylesterase activities and in vivo. studies after 1 h in Sprague-Dawley rats have demonstrated that serum, liver, and heart paraoxonase and arylesterase activities were significantly inhibited by glimepiride.