and Folic Acid in Type II Diabetes Mellitus Patients.

Transcription

1 Research Article ISSN: M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), Available online through Investigation of the Effect of on the Level of Plasma Homocysteine, and Folic Acid in Type II Diabetes Mellitus. M. Prabhu 1*, N. Srinivasan 2, M. P. Narmadha 3 and R. Radha 4 1 Department of Pharmacy Practice, Asia Metropolitan University, Cheras, Malaysia. 2 Department of Pharmacognosy, Asia Metropolitan University, Cheras, Malaysia. 3 Department of Pharmacy Practice, Nehru College of Pharmacy, Kerala, India. 4 Department of Pharmaceutical Chemistry, Asia Metropolitan University, Cheras, Malaysia. Received on: ; Revised on: ; Accepted on: ABSTRACT Background: on prolonged administration induce decreased levels of and Folic acid, and increased plasma HCY level. The aim of the present prospective study was designed to evaluate the effect of and other oral hypoglycemic agents on the level of plasma HCY, and Folic acid in Type II Diabetes Mellitus. Methods: A total number of 1 subjects were divided into five groups. were kept as control in group A, newly diagnosed Type-II Diabetes Mellitus patients in group B, Type-II Diabetes Mellitus patient treated with in group C, Type -II Diabetes Mellitus patient treated with along with folic acid in group D & finally Type II Diabetic treated with Other Oral Hypoglycemic Agents () in group E. HCY, folic acid,, HbA 1 C, lipid profiles was measured in the beginning and at the end of the study. Fasting blood sugar, Blood pressure, Kidney function test, Urinalysis was measured in every visit of the patient to the hospital. Results: In Group C, the average levels of plasma HCY at the beginning and end of the study was µmol/l and 2.35 µmol/l respectively. In Group D, the average levels of plasma HCY at the beginning and end of the study was 18.5 µmol/l and µmol/l respectively. Conclusion: The present study indicates that increases plasma HCY level in Type II Diabetes Mellitus patients, whereas administration of along with folic acid can decrease the elevated plasma HCY level, hence prevent the risk of cardiovascular diseases. KEYWORDS: Type II diabetes,, Folic acid,, Homocysteine. *Corresponding author. M.Prabhu, Asia Metropolitan University, G-8, Jalan Kemacahaya 11, Taman Kemacahaya, Batu 9, 432, Cheras, Selangor, Malaysia. INTRODUCTION A number of Cardiovascular risk factors such as Hypertension, Dyslipidemia and Obesity are more common in Type II Diabetes Mellitus. 1 Hyperhomocysteinemia is independent cardiovascular risk factor especially in Diabetic. 2,3 The prevalence estimates of hyperhomocysteinemia (> 14 µmo1/l) vary between 5% and 3% in the general population. 4,5 Homocysteine (HCY) is a thiol amino acid resulting from demethylation of methionine, an essential amino acid derived from dietary proteins. It is metabolized through two pathways: re-methylation and trans-sulfuration, which use cofactors as folate, vitamin B 6 and vitamin B 12. Normal levels of HCY are between 5 and 15 µmo1/l. There is however, and increased risk for cardiovascular disease with plasma HCY concentrations above 1 µmo1/l. Plasma HCY levels between 15 and 3 µmo1/l are described as Mild Hyperhomocysteinemia, levels between 31and 1 µmo1/l are described as Moderate and levels above 1 µmo1/l are described as Severe Hyperhomocysteinemia. 6 For prevention of cardiovascular events in patients with Type II Diabetes Mellitus in addition to control of Type II DM, identification and treatment of risk factors is very essential1. 7 is one of the most common prescribed oral anti-hyperglycemic drugs for patients with Type II DM. This drug offers both benefits and risks. suppresses Hepatic glucose production, and to a lesser extent, augmenting insulin mediated glucose uptake and enhancing insulin responses in peripheral tissues. Furthermore, it was shown that it also reduces glucose absorption in the intestine. In addition to lowering blood glucose concentrations, also has a benefit on lowering plasma lipid concentrations and promoting weight loss. 8 Among these, an elevation of HCY level in plasma has been in focus. 9 Prolonged administration of induced a decreased levels of and Folic acid, and increased plasma HCY level. 1,11,12,13 An interaction between Hyperhomocysteinemia and Type II DM with regard to cardiovascular risk may be clinically important, as it implies that HCY lowering treatment may be especially effective in Type II DM. 14 Journal of Pharmacy Research Vol.8 Issue 1.October

2 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), In view of these considerations, the present prospective study was designed to evaluate the effect of and other oral hypoglycemic agents on the level of plasma HCY, and Folic acid in Type II Diabetes Mellitus. PATIENTS: Inclusion criteria Age above 3 years old Newly diagnosed Type II diabetes mellitus patients. Patents on treatment with oral hypoglycemic agents (). Fasting plasma glucose lower than 3 mg/dl. Creatinine level lower than 1.9 mg/dl. without any associated complications. who are ready to sign in the consent form. Exclusion criteria Pediatric patients, pregnant women, patients with Thyroid diseases, Malignancy, Hypertension, or taking antihypertensive medications. Type 1 Diabetes Mellitus patients. on treatment for cerebrovascular and/or coronary heart diseases. Receiving folate antagonist medications (E.g., Methotrexate, Phenytoin and/or Carbamazepine medications). with severe uncontrolled diabetes and its complications. with mental incompetence. Study Procedure who were satisfying the inclusion criteria were enrolled in to the study after obtaining their letter of consent. Demographic details, past and present medical and medication history were obtained in suitably designed patient profile form. Glucometer For measuring the capillary blood glucose levels of the patients. One touch BASIC Plus (Company LIFESCAN, Johnson and Johnson USA) METHODS were asked to fast overnight (i.e., about 8-12 hours) prior to their appointment at the Diabetes Clinic. The following morning, 6 ml of blood was drawn from a forearm site of each candidate and placed equally into 2 separates tubes. Blood in the first tube, containing sodium fluoride, was thoroughly mixed and centrifuged. The plasma sample was taken and immediately proceeded for analysis of blood sugar. Meanwhile, 2ml of the plasma sample was kept separately in a -2 C freezer for further analysis of plasma HCY. In the second tube, serum was prepared and immediately proceeded for analysis of creatinine, cholesterol and triglyceride. Similar to the plasma sample, another 2mL of serum was saved in a -2 C freezer for further analysis of and Folic acid. Blood pressure and weight were measured on the day that blood was drawn. The demographic data (i.e. history of medications, sex age, smoking habit, occupation, family history, social history, etc. was collected from combined sources of medical records, and individual interview with patients. Laboratory Analysis The plasma HCY was measured using the particle-enhanced immunonephelometry with BN* 11 and BN prospec System. The serum vitamin B 12 and Folic acid were analyzed by the Electrochemiluminescence immunoassay (ECLIA) and Cobas e immunoassay method. Meanwhile fasting blood sugar, cholesterol, triglyceride, serum albumin, serum creatinine, uric acid, and blood urea were analyzed by using AutoAnalyzer (Herba Kemph 5 + V2) or (Bio chemical analyzer). Obtaining Patient Consent In order to conduct the demographic study, the patient was individually identified with the name from the patient record confirmed for his /her identity, given a brief personal introduction about the project researcher and explained the purpose of our project. Upon further interaction with the patients, after clarifying all the queries regarding mode, approach & utility of the project, a written & oral consent were obtained from each patient for his/her active participation. MATERIALS Health-care Screening instruments Statistical Analysis Statistical analysis was done, by using Graph Pad Insat 3 software, version 3.1. Results were expressed mean ± SD. One way ANOVA followed by student-newman-keuls multiple comparisons test was performed for analysis results from 3 to 4 groups. RESULTS AND DISCUSSION: The study of effect of on the level of Plasma HCY, Vitamin B 12 and Folic Acid in Type II Diabetic were carried out. After the preliminary assessment for anticipated patients, the initial 3 patients with Type II Diabetes mellitus were included according to Journal of Pharmacy Research Vol.8 Issue 1.October

3 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), the inclusion criteria. However, before commencing the study, 12 refused participation. In addition, for the whole period of this study, 11 patients were absent from their appointments and another 127 patients were excluded from the list. Among those excluded were 81 on Anti Hypertensive medications, 3 for having Anti Asthmatic Medications, 9 for having silicosis medications, 4 for having Thyroid disorder medications and 1 each for having congestive heart failure, AIDS, Psychotic disorder, respectively. The final total number was 15 patients throughout the study. These 15 patients were divided into two groups. Such as 8 patients were selected for the evaluation of Biochemical profiles such as HCY, Folic Acid and remaining 7 patients were given counseling regarding Drug regimen using KAP Questionnaire. Demographic Profile of Diabetic Population After the scrutiny, using Inclusion and Exclusion Criteria 8 were included into the study. They were randomized into four groups Newly diagnosed Type II Diabetic, Type II Diabetic treated with only, Type II Diabetic treated with along with Folic Acid and Type II Diabetic treated with Other Oral Hypoglycemic Agents (). Among the total patients 58 (72.5%) were males and 22 (27.5%) were females. The demographic details of the patients we shown in the below tables 1 & 2. Total Number of patients : 8 Newly diagnosed Type II Diabetic :2 Type II Diabetic treated with only :2 Type II Diabetic treated with + Folic Acid:2 Type II Diabetic treated with other except :2 Table 1: Sex distribution among study population Group Number of Number of Male Female (%) (%) Newly diagnosed Type II Diabetic 15(25.86 %) 5(22.73 %) Type II Diabetic treated with only 15(25.86 %) 5(22.73 %) Type II Diabetic treated with + Folic Acid 14(24.14 %) 6(27.27%) Type II Diabetic treated with other 14(24.14 %) 6(27.27%) Table 2: Age distribution among study population Age Group Number of Number of in Years (n) Male Female (%) (%) (%) (28.75 %) 11(13.75 %) (27.5 %) 9(11.25 %) (16.25 %) 2(2.5.%) Journal of Pharmacy Research Vol.8 Issue 1.October 214 Comparative Fasting Blood Sugar Levels at the Initiation of Study in Type II Diabetic Table 3: FBS level Fasting blood sugar in mg/dl mean ±SD (SEM) Duration Healthy Newly Type II subjects diagnosed diabetic patients type II treated with diabetic patients for 15-3 days. Initiation of Study 17.1± ± ± (2.174) (3.862) (3.862) Fasting blood sugar in mg/dl mean ±SD (SEM) Duration Type II diabetic Type II ANOVA patients treated diabetic P with patients value for 6-12 months. treated with other Initiation of Study 217.± ± <.1 (1.43) (7.597) Figure 1. FBS level of study population FBS(mg/dl) (15 to 3 days) (6 to 12months) (15 to 3 days) (6 to 12months) Healthy Subjects vs. *** P<.1,Healthy Subjects vs. (6 to 12 months) *** P<.1,Healthy Subjects vs. (15 to 3 days) *** P<.1,Healthy Subjects vs. New DM * P<.1, vs. ** P<.1, vs. (6 to 12 months) ns P>.5, vs. (15 to 3 days) ns P>.5, (15 to 3 days) vs. *** P<.1, (15 to 3 days) vs. (6 to 12 months) ns P>

4 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), Comparative Fasting Blood Sugar Levels at the End of Study in Type II Diabetic Table 4: FBS level of DM patients Fasting Blood Sugar In Mg/Dl Mean ±SD (SEM) Duration of treatment Type II Diabetic Type II Diabetic intervention for 5 Treated Treated months. With With Only Along With Folic Acid At the End of study ± ± (4.358) (4.47) Fasting Blood Sugar In Mg/Dl Mean ±SD (SEM) Duration of treatment Type II Diabetic ANOVA intervention for 5 Treated P Value months. With Other At the End of study ± <.1 (9.218) Figure 2. FBS level of DM patients FBS (mg/dl) Folicacid Folicacid vs. *** P<.1, vs. + Folic Acid** P<.1,+ Folic Acid vs. *** P<.1 Comparative HBA 1 C Levels at the Initiation of Study in Type II Diabetic Table 5: HbA 1 C level of DM patients HbA 1 C in Mean ±SD (SEM) Duration Healthy Newly Type II diabetic subjects diagnosed patients treated type II diabetic with patients for 15-3 days Initiation of Study 5.79± ± ±.3137 (.1541) (.714) (.714) HbA 1 C in Mean ±SD (SEM) Duration Type II diabetic Type II diabetic ANOVA patients treated with for 6-12 months patients treated P value with other Initiation of Study 7.85± ±.419 <.1 (.117) (.8988) Figure 3. HbA 1 C level of DM patients HbA1C(%) (15 to 3 days) 7.5 (6 to 12months) (15 to 3 days) (6 to 12months) Healthy Subjects vs. *** P<.1,Healthy Subjects vs. (6 to 12 months) *** P<.1,Healthy Subjects vs. (15 to 3 days) *** P<.1,Healthy Subjects vs. New DM*** P<.1, vs. ** P<.1, vs. (6 to 12 months) ns P>.5, vs. (15 to 3 days) ns P>.5, (15 to 3 days) vs. ** P<.1, (15 to 3 days) vs. (6 to 12 months) ns P>.5, (6 to 12 months) vs. ns P>.5 Comparative HBA1C Levels at the End of Study in Type II Diabetic Table 6: HbA1C level of DM Population HbA 1 C in Mean ± SD (SEM) Duration of Type II Diabetic Type II Diabetic treatment Treated Treated With intervention With Along With for 5 months. Only Folic Acid At the End of study 7.975± ±.5337 (.874) (.1193) HbA 1 C in Mean ± SD (SEM) Duration of Type II Diabetic ANOVA treatment Treated P intervention with other Value for 5 months. At the End of study 7.73± (.1163) Journal of Pharmacy Research Vol.8 Issue 1.October

5 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), Figure 4. HbA1C level of DM Population Type II Diabetes Mellitus patients showed statistically significant higher HCY level in comparison with. In addition 1 Diabetes Mellitus under Treatment showed further elevation in HCY level among all Diabetes HbA1C(%) Folicacid +Folicacid + Folic Acid vs. * P<.5, + Folic Acid vs. ns P>.5, vs. ns P>.5 Comparative HCY Levels at the Initiation of Study in Type II Diabetic : Table 7: Plasma HCY level of DM population Plasma HCY in µmol/l mean ±SD (SEM) Duration Healthy Newly diagnosed Type II diabetic subjects Type II diabetic patients treated patients with for 15 3 days Initiation of Study 12.7± ± ± (.584) (.5472) (.6628) Plasma HCY in µmol/l mean ±SD (SEM) Duration Type II diabetic Type II diabetic ANOVA patients treated patients treated with with other P for 6 12 months value Initiation of Study 18.5± ± <.1 (.4134) (.7483) Figure 5. Plasma HCY level of DM population Homocysteine( µmol/l) Healthy subjects (15 to 3 days) 18 (6 to 12 months 14.4 (15 to 3 days) (6 to 12 months vs months *** P<.1,Healthy subjects vs days *** P<.1, vs. ns P>.5, vs. ns P>.5, vs months*** P<.1, vs. ns P>.5, vs. ns P>.5 vs months *** P<.1, vs. ns P>.5, vs months ** P<.1 Journal of Pharmacy Research Vol.8 Issue 1.October 214 Comparative HCY Levels at the End of Study in Type II Diabetic Table 8: Plasma HCY level of DM population Plasma HCY in µmol/l Mean ±SD (SEM) Duration of treatment Type II Diabetic Type II Diabetic intervention for Treated Treated 5 months With With Along Only With Folic Acid At the end of study 2.35± ± (1.13) (.6969) Plasma HCY in µmol/l Mean ±SD (SEM) Duration of treatment Type II Diabetic ANOVA intervention for Treated P 5 months With Other value At the end of study 14.8± (.92) Figure 6. Plasma HCY level of DM population Homocystein(µmol/L ) Folic acid Folic acid vs. *** P<.1, vs. +Folic acid ns P>.5, +Folic acid vs. *** P<.1 Diabetes Mellitus patients treated with were prescribed Folic acid 5mg and those patients showed significant reduction (18.5 µmol/l to µmol/l (P <.2)) in HCY level after a period of 5 months where as patients who were not prescribed folic acid showed further increase in HCY level (18.5 µmol/l to 2.35 µmol/l)

6 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), Comparative Folic Acid Levels at the Initiation of Study in Type II Diabetic Table 9: Folic Acid level of DM population Folic acid in ng/ml mean ±SD (SEM) Duration Healthy Newly Type II diabetic subjects diagnosed patients treated type II diabetic with patients for 15-3 days Initiation of Study 14.25± ± ± (.7394) (.6125) (.8147) Folic acid in ng/ml mean ±SD (SEM) Duration Type II diabetic Type IIdiabetic ANOVA patients treated patients treated P with with other value for 6-12 months. Initiation of Study ± ± (.3362) (.4638) Figure 7. Folic Acid level of DM population Folicacid(ng/ml) (15 to 3 days) 11.7 (6 to 12months) (15 to 3 days) (6 to 12months) vs. ns P>.5, vs. ns P>.5, vs. * P<.5, vs months ns P>.5, vs. ns P>.5, vs. ns P>.5, vs months ns P>.5, 6-12 months vs. ns P>.5, vs. Healthy subjects * P<.5, vs months ns P>.5 Folic acid is significantly less in Type II Diabetes Mellitus when compared to Healthy Subjects. Folic acid levels among the different patient groups do not show significant difference. Though interferes with absorption and reduce Folic acid levels, since we have not adjusted for diet variation, Folic acid levels remain unaltered due to dietary intake of leafy vegetables having a good amount of Folic acid. Journal of Pharmacy Research Vol.8 Issue 1.October 214 Comparative Folic Acid Levels at the End of Study in Type II Diabetic Table 1: Folic acid level of DM population Folic acid in ng/ml Mean ± SD (SEM) Duration of treatment Type II Diabetic Type II Diabetic intervention for Treated Treated With 5 months. With Along With Only Folic Acid At the End of study. 11.± ± 3.42 (.8915) (.682) Folic acid in ng/ml Mean ± SD (SEM) Duration of treatment Type II Diabetic ANOVA intervention for Treated P 5 months. with Other value At the End of study ± <.1 (.7823) Figure 8. Folic acid level of DM population Folicacid(ng/ml) Folicacid Folicacid vs. + Folic acid *** P<.1, vs. ns P>.5, vs. + Folic acid *** P<.1 Comparative Levels at the Initiation of Study in Type II Diabetic Table 11: level of DM population in pg/ml mean ±SD (SEM) Duration Healthy Newly Type II diabetic subjects diagnosed type patients treated II diabetic with patients for 15-3 days Initiation of Study ± ± ± (33.495) (51.391) (51.339) in pg/ml mean ±SD (SEM) Duration Type II diabetic Type II ANOVA patients treated diabetic patients P with treated with value for 6-12 months other Initiation of Study 55.9± ± (44.56) (49.52)

7 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), Figure 9. level of DM population Figure 1. level of DM population vitamin B12(pg/ml) (15 to 3 days) (6 to 12months) (15 to 3 days) (6 to 12months) vitaminb12 (pg/ml) Folicacid Folicacid 6-12 months vs. Healthy Subjects ns P>.5, 6-12 months vs days ns P>.5, 6-12 months vs. ns P>.5, 6-12 months vs. ns P>.5, vs. Healthy Subjects ns P>.5, vs days n s P>.5, vs. ns P>.5, vs. Healthy Subjects ns P>.5, vs days ns P>.5, 15-3 days vs. Healthy Subjects ns P>.5. Analysis of status in Type II Diabetes Mellitus Clearly indicates the role of on absorption resulting in decrease of content this is clearly evident with moderate reduction in level in with treatment when compared to newly diagnosed Diabetic Mellitus. Comparative Levels at the End of Study in Type II Diabetic Table 12: level of DM population in pg/ml Mean ± SD (SEM) Duration of treatment Type II Diabetic Type II Diabetic intervention for Treated Treated 5 months. With With Only Along With Folic Acid At the End of study ± ± 21.4 (5.76) (47.47) in pg/ml Mean ± SD (SEM) Duration of treatment Type II Diabetic ANOVA intervention for Treated P 5 months. With Other value At the End of study 53.35± (51.279) vs. + Folic acid ns P>.5, vs. ns P>.5, vs. + Folic acid ns P>.5 on treatment with Folic acid supplement have helped to raise the Folate levels, but there is not remarkable increase in the Vitamin B 12 level demands dietary intake of or supplementation along with treatment. CONCLUSION: A number of cardio vascular risk factor such as Hypertension, Dyslipidemia and obesity are more common in Type II Diabetic patients. Hyperhomocysteinemia is an independent cardio vascular risk factor especially in Diabetic. For prevention of cardio vascular events in patients with Type II Diabetes in addition to control of Diabetes, identification and treatment of risk factors is very essential. We therefore prospectively investigated the level of plasma HCY in Type II Diabetic Subjects. is one of the most frequently used drugs in Type II Diabetes Therapy. Prolonged administration of induced a decrease in absorption, Folic acid level and increase in plasma HCY level (>15 µmol/l). This fact has been proved in our study. We could find an increase in plasma HCY in all Type II Diabetes Mellitus patients under in one month treatment. showed a mean HCY of 15.95µmol/ L. It increased to 18.7 µmol/l in patients with 6 month -1 year treatment. In summary our study shows that Plasma HCY levels are an independent risk factor for future Coronary Heart Disease events in patients with Type II Diabetes Mellitus. Whether lowering HCY levels, Folate Supplementation reduces cardio vascular events in patients with Type II Diabetes remains to be proven. Though the present study could Journal of Pharmacy Research Vol.8 Issue 1.October

8 M. Prabhuet al. / Journal of Pharmacy Research 214,8(1), show significant difference in the Plasma HCY level (P<.1) between patients in the and Non groups, it showed a significant difference in Serum Folic Acid (P<.1) levels between those groups. Particularly patients in the group who were apparently shown to have lower levels of the Folic Acid and Vitamin B 12. Therefore giving Vitamin therapy, particularly Folic Acid which is in expensive and has a very low risk of toxicity or any complications, would provide better health benefits to these patients. The study observed that, Clinical pharmacists can play a key role in management of Diabetes Mellitus and patient care. ACKOWLEDGEMENT: We take this opportunity to express our gratefulness to Adharsh Hospital and Anand Diagnostic Centre, Karnataka for their timely help. REFERENCES 1. Wilson PW, Kannel WB, Silbershatz H, D Agostino RB. Clustering of metabolic factors and coronary heart disease. Arch Intern Med,1999; 159(1): Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA, 1995; 274(13): Duell PB, Malinow MR. Homocyst(e)ine: an important risk factor for atherosclerotic vascular disease. Curr Opin Lipidol, 1997; 8(1): Lussier-Cacan S, Xhignesse M, Piolot A, Selhub J, Davignon J, Genest J Jr. Plasma total homocysteine in healthy subjects: sex-specific relation with biological traits. Am J Clin Nutr, 1996; 64(4): Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA, 1993; 27(22): Source of support: Nil, Conflict of interest: None Declared 6. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med, 1997; 337(4): Hoogeveen EK, Kostense PJ, Beks PJ, Mackaay AJ, Jakobs C, Bouter LM, Heine RJ, Stehouwer CD. Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, especially in non-insulin-dependent diabetes mellitus: a population-based study. Arterioscler Thromb Vasc Biol, 1998; 18(1): McEvoy GK. AHFS Drug information 2 ed. Bethesda : American Society of Health System Pharmacists Inc : 2: Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991; 324(17): Tomkin GH, Hadden DR, Weaver JA, Montgomery DA. Vitamin-B12 Status of on Long-term Therapy. Br Med J, 1971; 2 (5763): Berchtold P, Dahlqvist A, Gustafson A, Asp NG. Effects of a biguanide () on vitamin B 12 and folic acid absorption and intestinal enzyme activities. Scand J Gastroenterol, 1971; 6(8): Carlsen SM, Folling I, Grill V, Bjerve KS, Schneede J, Refsum H. increases total serum Homocysteine levels in non-diabetic male patients with coronary heart disease. Scand J Clin Lab Invest, 1997; 57(6): Gilligan MA, Joel MR. and vitamin B12 deficiency. Arch Intern Med, 22; 162: Redekop WK, Koopmanschap MA, Stolk RP, Rutten GE, Wolffenbuttel BH, Niessen LW. Health-related quality of life and treatment satisfaction in Dutch patients with type 2 diabetes. Diabetes Care, 22; 25(3): Journal of Pharmacy Research Vol.8 Issue 1.October