The Effect of Metformin on Mean Platelet Volume

 

Sherin Dib¹, Rana Makhous²

¹Master Student  in Pharmacology and Toxicology Department, Faculty of Pharmacy, Tishreen University. Lattakia, Syria

²Doctor in Pharmacology and Toxicology Department, Faculty of Pharmacy, Tishreen University.

Lattakia, Syria

 

ABSTRACT:

Background and objective: Diabetes mellitus (DM) is a global pandemic. The increased Mean platelet volume (MPV) and its activity may play a role in the development of vascular complications of this metabolic disorder. Metformin, the first-line therapy for T2DM, the only drug demonstrated to reduce cardiovascular complications in diabetic patients. However, whether metformin can effectively prevent thrombosis and its potential mechanism of action is not fully understood. In this study, the first aim is to determine whether there is a difference in MPV between diabetics with and without macro- and microvascular complications,compared to nondiabetics. The second aim is to examine the effects of metformine on MPV values in newly diagnosed type 2 DM patients on metformin monotherapy, and to investigate whether a correlation exists between MPV and fasting blood glucose changes after and before treatment. Methods: MPV values were measured MPV in 87 newly diagnosed Type 2 diabetic patients, 25 insulin-dependent diabetic patients and 40 nondiabetic control subjects, who had complete blood count on venous blood sample taken into tripotassium EDTA, using automatic blood counter (Diagon D-Cell 60 CBC Eurup). The blood glucose level was measured by glucose oxidase method. Statistical evaluation was performed by SPSS for Windows statistics programme using linear regression analysis, Student's t, one way Anova, and Pearson correlation tests. Results: MPV values were significantly higher in Insulin- dependent T2DM group compared to the newly diagnosed T2DM and control [9.7 ± 0.78 FL vs 8.52 ± 0.8 FL and 8.48 ± 0.9 FL ( P=0)], respectively. Among the newly diagnosed patients MPV values showed a low positive correlation with patient age (R = 0.37, P= 0 ) but no correlation was with BMI ( R=-0.001 ، P=0.99 ) and initial fasting plasma glucose (P= 0.111, R=0.172 ). MPV values were significantly reduced after 6-month metformin therapy [8.56±0.78 vs 8.18±0.70 (P<0.05)]. There were no statistically significant associations of ΔMPV with ΔFBG levels (beta coefficient=0.41,P=0.51) after metformin treatment Conclusions:  Our results showed that MPV values significantly higher in insulin- dependent T2DM patients than in the newly diagnosed T2DM patients and nondiabetic controls.The increased MPV may be due to the diabetic complication which increase with disease progression. Our results suggest that metformin had decreased MPV in diabetes mellitus Regardless of glucose lowering effect.These findings may provide a further explanation for the anti-atherogenic effect of metformin.

 

 

INTRODUCTION:

All over the world, 451 million people, or 1 in 11 adults, are estimated to have diabetes mellitus (DM). Approximately 5.0 million people died from DM in 2017, which is equal to one death every six seconds [1, 2]. Thrombosis is the leading cause of morbidity and mortality in diabetic patients, with a reported 65% of diabetic patients eventually dying from thrombotic diseases [3,4].

 

Metformin is the most commonly used drugs in the world, due to its antihyperglycemia effect. It has also some effects, namely pleiotropic, which are not directly related to glucose lowering effect. Recent clinical observations have demonstrated that one of the beneficial effects of metformin exerts its action via directly interfering with platelet formation and aggregation. Especially, it is known that MPV is increased in type 2 diabetes mellitus (T2DM) and associated with an increased risk of cardiovascular complications (coronary artery disease, transient ischaemic attack, stroke and peripheral arterial disease) that are the leading cause of morbidity and mortality in diabetic patients [5-7]. Given that platelets play a key role in hemostasis, their dysfunction in diabetes is related to increased thrombus formation therefore, macrovascular disease [8].

 

Furthermore, all these alterations of platelet function that are observed in T2DM may result in resistance to anti-platelet agents, as compared with non-diabetic patients [9,10].

 

Mean platelet volume (MPV) which is a simple measure of platelet activation, has recently become an interesting matter in cardiovascular research. When platelets become activated, MPV increases and change from quiescent disks to swollen spheres. Largeplatelets are more adhesive and likely to aggregate than small ones [11]. Accordingly, we aimed to investigate the effects of metformin on MPV in newly diagnosed diabetic patients.

 

MATERIALS AND METHODS:

The study group included 87 newly diagnosed Type 2 diabetic patients according to American Diabetes Association criteria [13] (44 females and 43 males), mean age was 52.36±9.48 years (range 25–77 years), without any diabetic complications, who had applied to the outpatient clinic of Al Basel hospital.

 

The age, body mass index, and sex-matched control group consisted of 40 healthy controls (22 females, 18 males; age 48.55±.67 years; range 23–73 years).

 

We also added a new group to this study included 25 Insulin- dependent T2DM (11 females and 14 males), mean age was 63.16±9.7 years, with micro- and macro vascular disease complications.

 

Subjects having renal failure, acute or chronic infection, cancer, hepatic and hematological disorders, pregnancy and a history of drug use that affect the MPV (warfarin, heparin, statin, anticoagulant medications) and who were younger than 18 years were excluded from the study. also male patients with hemoglobin below 13g/dl and female patients below 12g/dl were excluded from the study.

 

An automatic blood counter (Diagon D-Cell 60 CBC Eurup) was used to obtain mean Platelet volume of the patients and controls within 15 min of collection, on venous blood samples taken into tubes containing ethylenem diamine tetraacetic acid (EDTA) after a follow-up 8-hour fasting. The refferance MPV values in our laboratory range from 7.0–11 fl. The blood glucose level was determined by glucose oxidase method.

 

Newly diagnosed diabetic Participants were followed for 6 months from the start of 500 mg of metformin twice daily. At the end of 6 months, only 42 patients were completed the required treatment period, The drop outs (n=45) were due to infectious disease, need for surgery, non-compliance and sampling difficultie. Blood glucose levels and MPV values were measured again.

 

STATISTICAL ANALYSIS:

Statistical evaluation was performed by statistical package for the social sciences (SPSS) version 20 (Chicago, IL) for Windows statistics program using Student’s independent sample two-tailed t-test, paired Student's t test to measure changes in MPV and FBG after 6 months of treatment, Pearson's correlation coefficients were calculated to evaluate the relationships between MPV and several clinical variables (age, sex, BMI), Differences in baseline patient characteristics measurements were analyzed by one-way analysis of variance (ANOVA). Delta differences (Δ) of MPV, FBG were calculated by subtracting the values of before-metformin treatment from that of after-treatment. Linear regression analysis was made to find a possible correlation of ΔMPV with ΔFBG levels. Data were expressed as mean ± standard deviation. A P-value <0.05 was considered statistically significant.

 

RESULTS:

The demographic and clinical characteristics of patients and controls are shown in Table I.

 

In our study There were 43 male diabetics and 44 female diabetics (87 Newly diagnosed T2DM in total). There were 18 nondiabetic males and 22 nondiabetic females (40 control in total). The mean age of the diabetic population was 52.36±9.48 years, whereas that of nondiabetic population was 48.55±11.67 years (P >0.05). The mean BMI in the diabetic group was 30.73±6.42 kg/m² whereas it was 29.27±5.43 kg/m² in the nondiabetic group ( P >0.05 ).The mean MPV was similar in the diabetic group (8.52 ± 0.8 fl) as compared to the non-diabetic group (8.48 ± 0.9 fl; P=0.837). But when we compared with Insulin- dependent T2DM (14 male and 11 female), the mean MPV ​​was significantly higher in Insulin-dependent T2DM group than control and Newly diagnosed T2DM ( P=0). In another word the mean MPV in subjects with complications was higher than that of subjects without complications.

 

Table I : Comparison of Newly diagnosed  T2DM, Insulin- dependent T2DM and control group in Clinical and demographic characteristics

	Newly diagnosed T2DM (n=87)	Control (n=40)	Insulin- dependent T2DM (n=25)
Age (year)	52.36 ± 9.48	48.55± 11.67	63.16±9.7
Sex (F/M)	44/43	22/18	11/14
BMI (kg/m2)	30.73 ± 6.42	5.43 ± 29.27	27.86 ± 4.39
MPV (FL)	8.52 ± 0.8	8.48 ± 0.9	9.7 ± 0.78
FBG (mg/dl)	201.95 ± 61.96	-	139.4 ± 31.7

BMI, body mass index; MPV, mean platelet volume; FBG, Fasting blood glucose

 

Among the newly diagnosed patients the MPV values did not show any significant correlation with BMI (( R=-0.001 ، P=0.99 )), initial fasting plasma glucose ((P= 0.111, R=0.172 )), also there was no difference in the mean MPV between male and female. But a low positive correlation was seen between MPV and patient age (R = 0.37, P= 0 ). So we also divided the newly diagnosed diabetic category based on the age into groups, we found statistically significant differences in MPV values among these groups ( p=0.008). We observe an increase in the mean platelets volume with age. Table II, Figure 1

 

Table II. Comparison of The Mean MPV Among Age Groups of Newly Diagnosed Patients

Fingure 1. Correlation between age and MPV of Newly Diagnosed before Metformin Treatment

 

MPV values ​​were significantly reduced after 6-month metformin therapy (P<0.05), FBG levels were significantly decreased too (P<0.05) Table III. Linear regression analysis revealed that there were no statistically significant associations of ΔMPV with ΔFBG levels (beta coefficient=0.41, P = 0.51 ) after metformin treatment. this means that is no correlation between MPV values reduction and blood glucose levels reduction.

 

Table III. MPV and FBG levels before and after treatment  with metformin period (mean ± SD)

	Pretreatment	After 6 months	P - Value
MPV (FL)	8.56±0.78	8.18±0.70	P<0.05
FBG (mg/dl)	201.95 ± 61.96	147.98 ± 38.9	P<0.05

MPV, mean platelet volume; FBG, Fasting blood glucose

 

We also divided the treatment group based on sex, age, BMI, into groups as shown in Table IV. we found that mean platelet volume decreases by treatment with metformin more as age and body mass increase, and the decrease by treatment with metformin was more statistically significant in older age than younger one.

 

Table IV. Changes In MPV Values Before and After  Metformin Period  Among Sex, Age, BMI Groups of Treated Patients (Mean ± SD)

		Before 6 months	After 6 months	ΔMPV	P- value
Sex	Male (17)	8.83 ± 1.01	8.44 ± 0.81	0.38 ± 0.42	P* = 0.002
	Female (25)	8.37 ± 0.53	8.01 ± 0.57	0.36 ± 0.47	P* = 0.001
BMI	(1) weight Low	7.8	7.9	-	-
	Normal weight (4)	8.45 ± 0.31	8.50 ± 0.52	-0.05 ± 0.45	P = 0.83
	Overweight (12)	8.98 ± 0.86	8.36 ± 0.66	0.61 ±0.53	P* = 0.002
	Obesity (25)	8.40 ± 0.75	8.06 ± 0.75	0.34 ± 0.34	P* = 0
Age (year)	30-39 (1)	8.5	8.2	-	-
	40-49 (16)	8.16 ± 0.55	7.86 ± 0.61	0.30 ± 0.61	0.69P =
	(13) 50-59	8.49 ± 0.83	8.12 ± 0.69	0.36 ± 0.37	0.004P* =
	(11) 60-69	9.20 ± 0.72	8.72 ± 0.64	0.48 ± 0.26	0P* =
	(1) > 70	8.7	8.3	-	-

*Paired sample t-test, weight  Low (BMI < 18.5 Kg/m²), Normal weight (BMI = 18.5 – 24.9 Kg/m²), Overweight (BMI =25 – 29.9 Kg/m²), Obesity (BMI > 30  Kg/m²)

 

DISCUSSION:

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia responsible for complications affecting the kidneys, eyes, peripheral nerves, and micro and macrovascular systems [14]. People with type-2 diabetes mellitus (DM) have a two to four times higher risk of coronary heart disease (CHD) morbidity and mortality, a four-to eight-fold higher risk of congestive heart failure, a two- to six-fold higher risk of stroke, and a poorer prognosis of cardiovascular events than people without diabetes [15,16]

 

Platelets are small discoid blood cells that circulate and participate in hemostasis. Primary plug formation due to platelets seals the vascular defects and provides the required phospholipid surface for the recruited and activated coagulation factors. In response to stimuli generated by the endothelium of blood vessels, platelets change shape, adhere to subendothelial surfaces, secrete the contents of intracellular organelles, and aggregate to form a thrombus. [17] Thus, platelets may suppose an important role in signaling of the development of advanced atherosclerosis in diabetes.[17-19]

 

Changed platelet morphology and function have been reported in diabetic patients. they have increased platelet activation compared to nondiabetic subjects. Platelet hyperactivity was accompanied by an increased synthesis of thromboxane and a decreased prostacycline production. MPV is a marker of platelet function and activation. Whereas Larger platelets are more reactive and aggregable. They contain denser granules, secrete more serotonin and b-thromboglobulin, and produce more thromboxane A2 than smaller platelets. [20-23]

 

All these can produce a pro-coagulant effect and cause thrombotic vascular complications. This suggests a relationship between the platelet function particularly MPV and diabetic vascular complications thus indicating changes in MPV reflect the state of thrombogenesis. [19, 20] There might be small bleeds due to the rupture of atherothrombotic plaques leading to increased platelet recruitment, hyper reactivity, and bone marrow stimulation. High MPV is emerging as a new risk factor for the vascular complications of DM of which atherothrombosis plays a major role.[24]

 

In this study, Insulin-dependent T2DM patients had significantly larger MPV than nondiabetic controls and newly diagnosed T2DM patients who didn’t have complications. In another word the mean MPV in diabetic subjects with complications were higher than that of diabetic subjects without complications. Our finding in agreement with the result of Kodiatte et al (2012) [25] But it was in contrast to the study done by Hekimsoy (2012) which showed a significantly higher MPV in diabetic patients than in the nondiabetic controls, and no significantly different in the patients with diabetic retinopathy and coronary heart disease from that in diabetic patients without these complications [20]

 

Diabetes mellitus is considered to be a prothrombotic state with chronic platelet activation, activation of the coagulation system and decreased fibrinolytic potential [26], three main explanations for abnormal platelet function in DM have been proposed: (1) Immature, larger and more reactive platelets are synthesized in the bone marrow. (2) Platelets are activated when exposed to the metabolic milieu in DM. (3) Platelets are activated due vascular damage

 

There is possible explanation for the increased platelet size in diabetics, vascular lesions which occure because of biochemical mechanisms of hyperglycaemia involve non-enzymatic glycation (irreversible formation of advanced glycation end products, AGE, during chronic hyperglycaemia), increased sorbitol pathway, protein kinase C (PKC) activation, decreased production of NO, oxidative stress and haemodynamic changes that may cause endothelial dysfunction and inflammation [27-33] This leads to accelerated atherosclerosis and earlier advanced in diabetes [34], causing increase platelet activation, effectiveness and size [35]. Increased size of platelets make them more effective and more likely to form thrombosis, which has made to this indicator essential role of the formation of clots and complications of vascular, especially cardiovascular disorders, which constitute 75% of deaths in diabetes [36].

 

In our study there was no correlation between MPV and fasting plasma glucose,sex and BMI. But a positive correlation was seen between MPV and patient age. Therefore, it may be concluded that the increase in MPV due to the diabetic complication which increase with disease progression. However, Sharpe ( 1993 ), Papanas (2004), and Hekimsoy (2004) found no correlation between MPV and fasting plasma glucose, sex and age in diabetic patients [7, 20, 37]. But Kodiatte have found that there are positive correlation between MPV and fasting plasma glucose but no correlation between MPV and BMI in diabetic patients. [25]

 

The main finding of our study is that metformin treatment had significantly decreased MPV, and this effect had no relation to glucose lowering effect. Decreased MPV value correlates with decreased platelet enzymatic and metabolic effectiveness, the number of platelet glycoproteins (GPIb and GPIIb/IIIa) on the platelet membrane, the thromboxane synthesizing capacity, and the platelet granule contents of various platelet specific proteins. This agreed with the findings seen in studies done by Dolasık et al [38].

 

Data from both in vivo and in vitro studies show a favorable action for Metformin therapy on platelet function. Few studies have assessed the effect of metformine therapy on platelet function. As a study in 26 newly diagnosed T2DM patients showed that metformin improved oxidative stress, preserved anti-oxidant function and limited platelet activation [39]. While Guang Xin et al showed that metformin prevents both venous and arterial thrombosis with no significant prolonged bleeding time by inhibiting platelet activation and extracellular mitochondrial DNA (mtDNA) release. [4] On the other hand, Roussel et al had observed that metformin use may decrease mortality among patients with diabetes when used as a means of secondary prevention [40]

 

CONCLUSIONS:

The increased MPV may be due to the diabetic complication which increase with disease progression and this increase may be a factor in the increased risk of atherosclerosis and another vascular complications which associated with diabetes mellitus and associated. However, the increased MPV as the cause or the end result of vascular complications needs to be further explored.

 

Our study showed that metformin had decreased MPV in diabetes mellitus regardless of glucose lowering effect.These findings may provide a further explanation for the anti-atherogenic effect of metformin. However, the exact pathogenic mechanism underlying the favorable effect of metformin in not fully understood.

 

DISADVANTAGES:

We know that the lack of information on glycated hemoglobin level may be detrimental to the analysis. Principally this variable is the criterion standard for estimation of glucose control. We also acknowledge Our sample was small so, we need to be further confirmed in larger studies.

 

REFERENCES:

1.      Cho, N., et al., IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Research and Clinical Practice, 2018. 138: p. 271-281.

2.      Tang, W.H., et al., Aldose Reductase–Mediated Phosphorylation of p53 Leads to Mitochondrial Dysfunction and Damage in Diabetic Platelets. Circulation, 2014. 129(15): p. 1598-1609.

3.      Vazzana, N., et al., Diabetes mellitus and thrombosis. Thrombosis Research, 2012. 129(3): p. 371-377.

4.      Xin, G., et al., Metformin uniquely prevents thrombosis by inhibiting platelet activation and mtDNA release. Scientific Reports, 2016. 6: p. 36222.

5.      Viles-Gonzalez, J.F., et al., Update in atherothrombotic disease. The Mount Sinai Journal of Medicine, New York, 2004. 71(3): p. 197-208.

6.      Yuri Gasparyan, A., et al., Mean platelet volume: a link between thrombosis and inflammation? Current Pharmaceutical Design, 2011. 17(1): p. 47-58.

7.      Papanas, N., et al., Mean platelet volume in patients with type 2 diabetes mellitus. Platelets, 2004. 15(8): p. 475-478.

8.      Yngen, M., et al., Effects of improved metabolic control on platelet reactivity in patients with type 2 diabetes mellitus following coronary angioplasty. Diabetes and Vascular Disease Research, 2006. 3(1): p. 52-56.

9.      Natarajan, A., A.G. Zaman, and S.M. Marshall, Platelet hyperactivity in type 2 diabetes: role of antiplatelet agents. Diabetes and Vascular Disease Research, 2008. 5(2): p. 138-144.

10.   Angiolillo, D.J., Antiplatelet therapy in diabetes: efficacy and limitations of current treatment strategies and future directions. Diabetes Care, 2009. 32(4): p. 531-540.

11.   Martin, J., et al., The biological significance of platelet volume: its relationship to bleeding time, platelet thromboxane B2 production and megakaryocyte nuclear DNA concentration. Thrombosis research, 1983. 32(5): p. 443-460.

12.   Gin, H., et al., Study of the effect of metformin on platelet aggregation in insulin-dependent diabetics. Diabetes Research and Clinical Practice, 1989. 6(1): p. 61-67.

13.   Association, A.D., 2. Classification and diagnosis of diabetes. Diabetes care, 2017. 40 (Supplement 1): p. S11-S24.

14.   Demirtunc, R., et al., The relationship between glycemic control and platelet activity in type 2 diabetes mellitus. Journal of Diabetes and its Complications, 2009. 23(2): p. 89-94.

15.   Gerich, J.E., Type 2 diabetes mellitus is associated with multiple cardiometabolic risk factors. Clinical cornerstone, 2007. 8(3): p. 53-68.

16.   Fox, C.S., et al., Increasing cardiovascular disease burden due to diabetes mellitus. Circulation, 2007. 115(12): p. 1544-50.

17.   Mitchell, R.N., Hemodynamic disorders, thromboembolic disease, and shock. Pathologic Basis of Diseases, 8th ed.; Kumar, V., Abbas, AK, Fausto, N., Aster, JC, Eds, 2005: p. 111-134.

18.   Angiolillo, D.J., et al., Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes, 2005. 54(8): p. 2430-2435.

19.   Bae, S.H., et al., Platelet activation in patients with diabetic retinopathy. Korean Journal of ophthalmology, 2003. 17(2): p. 140-144.

20.   Hekimsoy, Z., et al., Mean platelet volume in Type 2 diabetic patients. Journal of Diabetes and its Complications, 2004. 18(3): p. 173-176.

21.   Colwell, J.A. and R.W. Nesto, The platelet in diabetes: focus on prevention of ischemic events. Diabetes care, 2003. 26(7): p. 2181-2188.

22.   Chang, H.A., et al., The Role of Mean Platelet Volume as a Predicting Factor of Asymptomatic Coronary Artery Disease. Korean Journal of Family Medicine, 2010. 31(8): p. 600-606.

23.   Ateş, O., et al., Association of mean platelet volume with the degree of retinopathy in patients with diabetes mellitus. Eur J Gen Med, 2009. 6(2): p. 99-102.

24.   Zuberi, B., N. Akhtar, and S. Afsar, Comparison of mean platelet volume in patients with diabetes mellitus, impaired fasting glucose and non-diabetic subjects. Singapore Medical Journal, 2008. 49(2): p. 114.

25.   Kodiatte, T.A., et al., Mean platelet volume in type 2 diabetes mellitus. Journal of laboratory physicians, 2012. 4(1): p. 5.

26.   Carr, M.E., Diabetes mellitus: a hypercoagulable state. Journal of Diabetes and its Complications, 2001. 15(1): p. 44-54.

27.   Queen, L., et al., Nitric oxide generation mediated by β-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus. Diabetologia, 2003. 46(11): p. 1474-1482.

28.   Tripathi, B.K. and A.K. Srivastava, Diabetes mellitus: Complications and therapeutics. Medical Science Monitor, 2006. 12 (7): p. RA130-RA147.

29.   Thomas, N. and N. Kapoor, A Practical Guide to Diabetes Mellitus- 7th Edition. 2016.

30.   Morohoshi, M., et al., Glucose-dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes, 1996. 45(7): p. 954-959.

31.   Guha, M., et al., Molecular mechanisms of tumor necrosis factor α gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and-independent pathways. Journal of Biological Chemistry, 2000. 275(23): p. 17728-17739.

32.   Cosentino, F., et al., High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation, 2003. 107(7): p. 1017-1023.

33.   Ceriello, A. and E. Motz, Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arteriosclerosis, Thrombosis, and Vascular Biology, 2004. 24(5): p. 816-823.

34.   Stratmann, B. and D. Tschoepe, Atherogenesis and atherothrombosis–focus on diabetes mellitus. Best Practice and Research Clinical Endocrinology and Metabolism, 2009. 23(3): p. 291-303.

35.   Wang, R.-T., et al., Increased mean platelet volume is associated with arterial stiffness. Platelets, 2011. 22(6): p. 447-451.

36.   Walker, B., et al., Davidson’s Principle and Practice of Medicine. 22nd. Edinburgh, Elsevier.

37.   Sharpe, P. and T. Trinick, Mean platelet volume in diabetes mellitus. QJM: An International Journal of Medicine, 1993. 86(11): p. 739-742.

38.   Dolasık, I., et al., The effect of metformin on mean platelet volume in dıabetıc patients. Platelets, 2013. 24(2): p. 118-121.

39.   Formoso, G., et al., Decreased in vivo oxidative stress and decreased platelet activation following metformin treatment in newly diagnosed type 2 diabetic subjects. Diabetes Metabolism Research and Reviews, 2008. 24(3): p. 231-237.

40.   Roussel, R., et al., Metformin use and mortality among patients with diabetes and atherothrombosis. Archives of Internal Medicine, 2010. 170(21): p. 1892-1899.

 

Accepted on 09.10.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(5): 2329-2334.