J Physiol Biochem (2014) 70:535–546 DOI 10.1007/s13105-014-0333-4
ORIGINAL PAPER
Effects of warfarin and L-carnitine on hemostatic function and oxidative stress in streptozotocin-induced diabetic rats Ahmed A. ElGendy & Amr M. Abbas
Received: 20 January 2014 / Accepted: 12 March 2014 / Published online: 27 March 2014 # University of Navarra 2014
Abstract Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and L-carnitine on those parameters. Forty male Sprague–Dawley rats were divided into four groups: control, DM, and DM received warfarin or Lcarnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, L-carnitine A. A. ElGendy : A. M. Abbas (*) Department of Medical Physiology, Faculty of Medicine, Mansoura University, PO Box 35516, Mansoura, Egypt e-mail: [email protected]
decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While L-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress. Keywords Diabetes mellitus . Coagulation . L-Carnitine . Warfarin . Oxidative stress
Introduction Diabetes mellitus (DM) is a complex progressive disease, which is accompanied by multiple complications. It has been recognized as the sole independent risk factor for the development of cardiovascular disease [15]. Administration of streptozotocin (STZ) causes pancreatic beta cell destruction that leads to the development of characteristic symptoms of diabetes such as hyperglycemia, dyslipidemia, and renal dysfunction in rats [9]. Reactive oxygen species (ROS), which cause cellular damage by the oxidation ability, have been implicated in the pathogenesis of DM [18]. During diabetes, persistent hyperglycemia increases the production of ROS through glucose autoxidation [44]. In addition, oxidative stress in DM results from reduction in capacities of the antioxidant defense system including
536
scavenging enzymes such as superoxide dismutase and glutathione reductase and deficiencies of antioxidants such as vitamin C and E [1]. Besides hyperglycemia, diabetic patients also suffer from dyslipidemia, which can lead to increased atherogenesis and heart disease [38]. In addition, clinical and epidemiological observations have led to the concept of a procoagulant state in DM. Thrombophilia in diabetic patients is a wellrecognized phenomenon which contributes an additional risk of coronary heart disease. In a series of 1,980 type 2 diabetic patients, both male and female diabetic patients showed significantly shorter activated partial thromboplastin time (aPTT) [7]. Acang and Jalil [2] have reported significantly high fibrinogen and short prothrombin time (PT) and aPTT in diabetic patients, especially those who suffered from diabetes for a long time. L-Carnitine is a naturally occurring compound and an antiradical widely distributed in the body and decreases lipid peroxidation [32]. L-Carnitine prevents thiobarbituric acid-reactive substance (TBARS) formation and increases antioxidants in aged rats’ brain [32]. Although these previous studies suggest that L-carnitine possesses antioxidative activity, it remains unknown whether this compound is able to affect coagulation associated factors. It is hypothesized that L -carnitine could attenuate diabetes-associated hemostatic disorder, at least via diminishing oxidative stress. In addition, many physiologic and pathologic conditions affect blood, tissue, and urinary concentrations of carnitine in both animal and human subjects. In acute or chronic STZinduced diabetic rats, the pancreatic content of carnitine was found to be significantly lower than in non-diabetic control rats [33]. Warfarin (Coumadin) is an anticoagulant normally used in the prevention of thrombosis and thromboembolism [14]. Warfarin inhibits the vitamin K-dependent synthesis of biologically active forms of the calciumdependent clotting factors II, VII, IX, and X, as well as the regulatory factors protein C, protein S, and protein Z [3]. As there are few studies considering the anticoagulant effect of warfarin and L-carnitine in DM, the aim of the present work was to investigate the hemostatic functions and oxidative stress in STZ-induced diabetic rats, a well-characterized animal model of type 1 diabetes, and the effects of warfarin and L-carnitine on those parameters.
A.A. ElGendy, A.M. Abbas
Materials and methods Chemicals STZ, warfarin [4-hydroxy-3-(3-oxo-1phenylbutyl) coumarin], and L-carnitine were obtained from Sigma–Aldrich (St. Louis, MO, USA). Animals Forty male Sprague–Dawley rats, weighing 220–240 g, were used in the present study. They were purchased from Vaccine and Immunization Authority (Helwan, Cairo, Egypt) and housed (Animal House, Medical Physiology Department, Faculty of Medicine, Mansoura University, Egypt) under controlled conditions (temperature 23±1 °C and a 12:12 light/dark cycle). The animals were allowed free access to food and tap water. All animal procedures were performed in accordance with protocols approved by the Medical Research Ethics Committee of Mansoura University, Egypt, and in compliance with standards and regulations for the care and use of laboratory rats set by the National Institutes of Health.
Experimental design The animals were randomly divided into four groups of ten rats each. First group (GI) consisted of untreated control (normal) animals. The second group (GII) served as untreated diabetic group. The third group (GIII) included the diabetic rats treated with warfarin. The forth group (GIV) included the diabetic rats treated with L-carnitine. Diabetes was induced, in Group II, III, and IV rats, by single intraperitoneal (i.p) injection of 70 mg/kg STZ [40] (dissolved in 0.1 M sodium-citrate buffer, pH 4.5). The control rats received an equivalent amount of the sodium-citrate buffer. In order to confirm diabetes, 3 days after STZ injection, blood glucose was measured using a glucometer instrument (Accu-Chek Active, ROCHE, Germany), and animals with blood glucose over 200 mg/dL were considered as diabetics [46]. The rats with warfarin treatment (GIII) were administered 0.25 mg/L warfarin potassium [45] in drinking water from the day of STZ injection for 21 consecutive days. Because diabetic rats drink much more water, the dosage of warfarin was reduced to 0.06 mg/L [45] from 2 days after injection of STZ. Two days after administration of STZ, group IV rats were injected intraperitoneally with L -carnitine at a dose of 500 mg/kg/day for 21 consecutive days [37].
Effect of L-carnitine on hemostasis
Sampling protocol Blood samples At the end of experimental period, blood samples were collected by cardiac puncture into tubes containing EDTA, mixed well, and divided into two tubes: the first one was used for determination of hemoglobin A1c (HbA1c), and the second tube was centrifuged at 3,000 r/min (1,000g) for 10 min to obtain plasma samples which were used for measurement of glucose, insulin, malondialdehyde (MDA), and antioxidant (reduced glutathione, glutathione peroxidase, superoxide dismutase, and catalase) levels. Biochemical investigations Plasma glucose was quantitated by glucose oxidase– peroxidase method using the kit supplied by SPINREACT, Spain (ref: 1001190). Plasma insulin was determined using Ultra Sensitive Rat Insulin ELISA Kit (Cat. No. 90060, Crystal Chem INC, Spain) following manufacturer’s instructions. In addition, hemoglobin A1c (HbA1c) was measured by a commercial kit (Cat. No. 80300, Crystal Chem INC, Spain). Malondialdehyde (MDA) was analyzed by measuring the production of thiobarbituric acid-reactive substances (TBARS) using TBARS assay kit (Cat. No. 10009055, Cayman, USA) according to the manufacturer’s instructions. Reduced glutathione (GSH) (Cat. No. 703002, Cayman, USA), glutathione peroxidase (GSH-Px) (Cat. No. NWK-GPX01, Northwest Life Science Specialties [NWLSSTM] kit, Canada), superoxide dismutase (SOD) (Cat. No. 706002, Cayman, USA), and catalase (CAT) (Cat. No. NWK-CAT01, Northwest Life Science Specialties [NWLSSTM] kit, Canada) were measured according to the manufacturer’s instructions. Hemostatic functions Assessment of prothrombin time and activated partial thromboplastin time PT and APTT were measured using calcium rabbit brain thromboplastin and kaolin platelet substitute techniques (Diagen Diagnostic Reagent Ltd, Oxon, UK). Briefly, PT was assayed with 200 μL of calcium rabbit brain thromboplastin reagent placed in a clotting tube and incubated in a water bath at 37 °C for 2 min. One hundred microliters of plasma is then added, and a stop watch started. The tube is gently tilted at regular interval, and the watch was stopped when the clot formation was observed. For aPTT,
537
200 μL of kaolin platelet substitute mixture was placed in a clotting tube and incubated in a water bath at 37 °C for 2 min. One hundred microliters of plasma was then added, and the tube was gently tilted at interval for exactly 2 min. One hundred microliters of calcium chloride (preincubated at 37 °C) was then added, and a stop watch started. The tube was tilted at intervals, and the time for clot formation was recorded. Blood coagulation method Blood clotting time was measured as reported previously [20]. Briefly, the tail of the animal was warmed for 1 min in water at 40 °C. The tail was dried and cut at the tip with a razor blade. A 25-μL sample of capillary blood was collected into a microhematocrite glass capillary. The chronometer was started when the blood first made contact with the glass capillary tube. The blood was left to flow by gravity between the two marks of the tube, 45 mm apart, by tilting the capillary tube alternatively to +60° and −60° angles with respect to the horizontal plane until blood ceased to flow (reaction end point). Measurement of coagulation and anticoagulation factors Coagulation factors, FI (fibrinogen) and FVII; anticoagulation factors, antithrombin III (ATIII) and protein C; plasminogen activator inhibitor-1 (PAI-1), and fibrin degradation products (FDP) were measured. Blood samples were anticoagulated using sodium citrate according to the protocols provided by the manufacturers. Fibrinogen level was measured using TEClot FIB kit (Cat. No. 050-500, TECO, GmbH, Germany). In addition, FVII activity was determined by a commercial kit (Cat. No. 821900, Chromogenix, Lexington, USA). The activity of ATIII and protein C in plasma was measured by commercial ATIII and protein C kits (Sigma, St. Louis, MO, USA.), respectively. The activity of ATIII and protein C was expressed as a percentage related to the activity of standard plasma. PAI-1 activity (pg/mL) was assayed by a commercial kit (Cat. No. CSB-E07948r, Cusabio, China). FDP was measured by Cusabio rat FDP ELISA assay kit (Cat. No. CSB-E07942r, Cusabio, China). Assessment of platelet aggregation Platelet-rich plasma preparation To obtain platelet-rich plasma (PRP), the blood was centrifuged at about 250g at 24 °C for 10 min. Platelet count was made by coulter
538
A.A. ElGendy, A.M. Abbas
Table 1 Effect of warfarin and L-carnitine on plasma glucose, insulin, and hemoglobin A1c (HbA1c) levels in control and STZ-induced diabetic rats
Fasting plasma glucose (mg/dL) Insulin (nM) HbA1c (%)
Control rats (n=10)
Diabetic rats (STZ) (n=10)
Diabetic rats (STZ)+warfarin (n=10)
Diabetic rats (STZ)+ L-carnitine (n=10)
141±6
389±21a
403±22a
396±24a
a
0.29±0.03
0.12±0.005 a
4.8±0.1
13.1±0.5
a
0.13+0.004a
0.11+0.004 a
13.6±0.2a
13.7±0.3
The values were expressed as mean±SDM STZ streptozotocin a
Significant (p
ORIGINAL PAPER
Effects of warfarin and L-carnitine on hemostatic function and oxidative stress in streptozotocin-induced diabetic rats Ahmed A. ElGendy & Amr M. Abbas
Received: 20 January 2014 / Accepted: 12 March 2014 / Published online: 27 March 2014 # University of Navarra 2014
Abstract Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and L-carnitine on those parameters. Forty male Sprague–Dawley rats were divided into four groups: control, DM, and DM received warfarin or Lcarnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, L-carnitine A. A. ElGendy : A. M. Abbas (*) Department of Medical Physiology, Faculty of Medicine, Mansoura University, PO Box 35516, Mansoura, Egypt e-mail: [email protected]
decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While L-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress. Keywords Diabetes mellitus . Coagulation . L-Carnitine . Warfarin . Oxidative stress
Introduction Diabetes mellitus (DM) is a complex progressive disease, which is accompanied by multiple complications. It has been recognized as the sole independent risk factor for the development of cardiovascular disease [15]. Administration of streptozotocin (STZ) causes pancreatic beta cell destruction that leads to the development of characteristic symptoms of diabetes such as hyperglycemia, dyslipidemia, and renal dysfunction in rats [9]. Reactive oxygen species (ROS), which cause cellular damage by the oxidation ability, have been implicated in the pathogenesis of DM [18]. During diabetes, persistent hyperglycemia increases the production of ROS through glucose autoxidation [44]. In addition, oxidative stress in DM results from reduction in capacities of the antioxidant defense system including
536
scavenging enzymes such as superoxide dismutase and glutathione reductase and deficiencies of antioxidants such as vitamin C and E [1]. Besides hyperglycemia, diabetic patients also suffer from dyslipidemia, which can lead to increased atherogenesis and heart disease [38]. In addition, clinical and epidemiological observations have led to the concept of a procoagulant state in DM. Thrombophilia in diabetic patients is a wellrecognized phenomenon which contributes an additional risk of coronary heart disease. In a series of 1,980 type 2 diabetic patients, both male and female diabetic patients showed significantly shorter activated partial thromboplastin time (aPTT) [7]. Acang and Jalil [2] have reported significantly high fibrinogen and short prothrombin time (PT) and aPTT in diabetic patients, especially those who suffered from diabetes for a long time. L-Carnitine is a naturally occurring compound and an antiradical widely distributed in the body and decreases lipid peroxidation [32]. L-Carnitine prevents thiobarbituric acid-reactive substance (TBARS) formation and increases antioxidants in aged rats’ brain [32]. Although these previous studies suggest that L-carnitine possesses antioxidative activity, it remains unknown whether this compound is able to affect coagulation associated factors. It is hypothesized that L -carnitine could attenuate diabetes-associated hemostatic disorder, at least via diminishing oxidative stress. In addition, many physiologic and pathologic conditions affect blood, tissue, and urinary concentrations of carnitine in both animal and human subjects. In acute or chronic STZinduced diabetic rats, the pancreatic content of carnitine was found to be significantly lower than in non-diabetic control rats [33]. Warfarin (Coumadin) is an anticoagulant normally used in the prevention of thrombosis and thromboembolism [14]. Warfarin inhibits the vitamin K-dependent synthesis of biologically active forms of the calciumdependent clotting factors II, VII, IX, and X, as well as the regulatory factors protein C, protein S, and protein Z [3]. As there are few studies considering the anticoagulant effect of warfarin and L-carnitine in DM, the aim of the present work was to investigate the hemostatic functions and oxidative stress in STZ-induced diabetic rats, a well-characterized animal model of type 1 diabetes, and the effects of warfarin and L-carnitine on those parameters.
A.A. ElGendy, A.M. Abbas
Materials and methods Chemicals STZ, warfarin [4-hydroxy-3-(3-oxo-1phenylbutyl) coumarin], and L-carnitine were obtained from Sigma–Aldrich (St. Louis, MO, USA). Animals Forty male Sprague–Dawley rats, weighing 220–240 g, were used in the present study. They were purchased from Vaccine and Immunization Authority (Helwan, Cairo, Egypt) and housed (Animal House, Medical Physiology Department, Faculty of Medicine, Mansoura University, Egypt) under controlled conditions (temperature 23±1 °C and a 12:12 light/dark cycle). The animals were allowed free access to food and tap water. All animal procedures were performed in accordance with protocols approved by the Medical Research Ethics Committee of Mansoura University, Egypt, and in compliance with standards and regulations for the care and use of laboratory rats set by the National Institutes of Health.
Experimental design The animals were randomly divided into four groups of ten rats each. First group (GI) consisted of untreated control (normal) animals. The second group (GII) served as untreated diabetic group. The third group (GIII) included the diabetic rats treated with warfarin. The forth group (GIV) included the diabetic rats treated with L-carnitine. Diabetes was induced, in Group II, III, and IV rats, by single intraperitoneal (i.p) injection of 70 mg/kg STZ [40] (dissolved in 0.1 M sodium-citrate buffer, pH 4.5). The control rats received an equivalent amount of the sodium-citrate buffer. In order to confirm diabetes, 3 days after STZ injection, blood glucose was measured using a glucometer instrument (Accu-Chek Active, ROCHE, Germany), and animals with blood glucose over 200 mg/dL were considered as diabetics [46]. The rats with warfarin treatment (GIII) were administered 0.25 mg/L warfarin potassium [45] in drinking water from the day of STZ injection for 21 consecutive days. Because diabetic rats drink much more water, the dosage of warfarin was reduced to 0.06 mg/L [45] from 2 days after injection of STZ. Two days after administration of STZ, group IV rats were injected intraperitoneally with L -carnitine at a dose of 500 mg/kg/day for 21 consecutive days [37].
Effect of L-carnitine on hemostasis
Sampling protocol Blood samples At the end of experimental period, blood samples were collected by cardiac puncture into tubes containing EDTA, mixed well, and divided into two tubes: the first one was used for determination of hemoglobin A1c (HbA1c), and the second tube was centrifuged at 3,000 r/min (1,000g) for 10 min to obtain plasma samples which were used for measurement of glucose, insulin, malondialdehyde (MDA), and antioxidant (reduced glutathione, glutathione peroxidase, superoxide dismutase, and catalase) levels. Biochemical investigations Plasma glucose was quantitated by glucose oxidase– peroxidase method using the kit supplied by SPINREACT, Spain (ref: 1001190). Plasma insulin was determined using Ultra Sensitive Rat Insulin ELISA Kit (Cat. No. 90060, Crystal Chem INC, Spain) following manufacturer’s instructions. In addition, hemoglobin A1c (HbA1c) was measured by a commercial kit (Cat. No. 80300, Crystal Chem INC, Spain). Malondialdehyde (MDA) was analyzed by measuring the production of thiobarbituric acid-reactive substances (TBARS) using TBARS assay kit (Cat. No. 10009055, Cayman, USA) according to the manufacturer’s instructions. Reduced glutathione (GSH) (Cat. No. 703002, Cayman, USA), glutathione peroxidase (GSH-Px) (Cat. No. NWK-GPX01, Northwest Life Science Specialties [NWLSSTM] kit, Canada), superoxide dismutase (SOD) (Cat. No. 706002, Cayman, USA), and catalase (CAT) (Cat. No. NWK-CAT01, Northwest Life Science Specialties [NWLSSTM] kit, Canada) were measured according to the manufacturer’s instructions. Hemostatic functions Assessment of prothrombin time and activated partial thromboplastin time PT and APTT were measured using calcium rabbit brain thromboplastin and kaolin platelet substitute techniques (Diagen Diagnostic Reagent Ltd, Oxon, UK). Briefly, PT was assayed with 200 μL of calcium rabbit brain thromboplastin reagent placed in a clotting tube and incubated in a water bath at 37 °C for 2 min. One hundred microliters of plasma is then added, and a stop watch started. The tube is gently tilted at regular interval, and the watch was stopped when the clot formation was observed. For aPTT,
537
200 μL of kaolin platelet substitute mixture was placed in a clotting tube and incubated in a water bath at 37 °C for 2 min. One hundred microliters of plasma was then added, and the tube was gently tilted at interval for exactly 2 min. One hundred microliters of calcium chloride (preincubated at 37 °C) was then added, and a stop watch started. The tube was tilted at intervals, and the time for clot formation was recorded. Blood coagulation method Blood clotting time was measured as reported previously [20]. Briefly, the tail of the animal was warmed for 1 min in water at 40 °C. The tail was dried and cut at the tip with a razor blade. A 25-μL sample of capillary blood was collected into a microhematocrite glass capillary. The chronometer was started when the blood first made contact with the glass capillary tube. The blood was left to flow by gravity between the two marks of the tube, 45 mm apart, by tilting the capillary tube alternatively to +60° and −60° angles with respect to the horizontal plane until blood ceased to flow (reaction end point). Measurement of coagulation and anticoagulation factors Coagulation factors, FI (fibrinogen) and FVII; anticoagulation factors, antithrombin III (ATIII) and protein C; plasminogen activator inhibitor-1 (PAI-1), and fibrin degradation products (FDP) were measured. Blood samples were anticoagulated using sodium citrate according to the protocols provided by the manufacturers. Fibrinogen level was measured using TEClot FIB kit (Cat. No. 050-500, TECO, GmbH, Germany). In addition, FVII activity was determined by a commercial kit (Cat. No. 821900, Chromogenix, Lexington, USA). The activity of ATIII and protein C in plasma was measured by commercial ATIII and protein C kits (Sigma, St. Louis, MO, USA.), respectively. The activity of ATIII and protein C was expressed as a percentage related to the activity of standard plasma. PAI-1 activity (pg/mL) was assayed by a commercial kit (Cat. No. CSB-E07948r, Cusabio, China). FDP was measured by Cusabio rat FDP ELISA assay kit (Cat. No. CSB-E07942r, Cusabio, China). Assessment of platelet aggregation Platelet-rich plasma preparation To obtain platelet-rich plasma (PRP), the blood was centrifuged at about 250g at 24 °C for 10 min. Platelet count was made by coulter
538
A.A. ElGendy, A.M. Abbas
Table 1 Effect of warfarin and L-carnitine on plasma glucose, insulin, and hemoglobin A1c (HbA1c) levels in control and STZ-induced diabetic rats
Fasting plasma glucose (mg/dL) Insulin (nM) HbA1c (%)
Control rats (n=10)
Diabetic rats (STZ) (n=10)
Diabetic rats (STZ)+warfarin (n=10)
Diabetic rats (STZ)+ L-carnitine (n=10)
141±6
389±21a
403±22a
396±24a
a
0.29±0.03
0.12±0.005 a
4.8±0.1
13.1±0.5
a
0.13+0.004a
0.11+0.004 a
13.6±0.2a
13.7±0.3
The values were expressed as mean±SDM STZ streptozotocin a
Significant (p
