1. (49 urban and 59 rural) to

1. Introduction
An urban rural variation in the prevalence of diabetes has been consistently reported from India. Earlier study conducted by ICMR reported that the prevalence was 2.1 per cent in urban and 1.5 per cent in rural areas. The prevalence of diabetes in India study (PODIS) was carried out in 108 centres (49 urban and 59 rural) to look at the urban-rural differences in the prevalence of T2DM and glucose intolerance showed 4.7 per cent in the urban and 2.0 percent in the rural population, according to ADA criteria while according to the WHO criteria prevalence was 5.6 and 2.7 percent among urban and rural areas respectively.(1)
Table 1:- Prevalence of diabetes in urban cities of India(2)

Prevalence (%)

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Raman et al. 1999
Hyderabad 2001 Ramachandran et al. 2001 South 16.6
Bengaluru 2001 Ramachandran et al. 2001 South 12.4
Chennai 2006 Mohan et al. 2006 South 14.3
Ernakulam 2006 Menon et al. 2006 South 19.5
Vellore 2007 Raghupathy et al. 2007 South 3.7
Tamil Nadu 2008 Ramachandran et al. 2008 South 18.6

2008 Mohan et al. 2008 (WHO- ICMR) Multi- centric

IDF estimates that as many as 175 million people worldwide or close to half of all people with diabetes are unaware of their disease. Most of these cases are T2DM. Early diagnosis of the diabetes will lead to better chances of preventing harmful and costly complications. Hence there is need to provide appropriate tools for early diagnosis.(3) .
Table 2:- Prevalence of diabetes in rural India (4)

Prevalence (%)

Delhi 1991 1.5 Ahuja 1991

Punjab 1994 4.6 Wander et al. 1994

Zargar et al. 2000

India 2001 2.7 Sadikot et al. 2004

Rajasthan 2004 1.8 Aggarwal et al. 2004

Mysore 2005 3.8 Basavanagowdappa et al. 2005

Maharashtra 2006 9.3 Deo et al. 2006

Nagpur 2007 3.7 Kokiwar et al.2007

Vellore 2007 2.1 Raghupathy et al. 2007

Ramachandran et al. 2008

Multi-centric 2008 3.1 Mohan et al. 2008

Chronic elevation of blood glucose level leads to damage of blood vessels (angiopathy). The endothelial cells lining the blood vessels take in more glucose than normal, since they do not depend on insulin. They then form more surface glycoproteins than normal and cause the basement membrane to grow thicker and weaker. In diabetes, the resulting problems are grouped under “microvascular disease” (due to damage to small blood vessels) and “macrovascular disease” (due to damage to the arteries).108
? Microvascular disease: – The damage to small blood vessels leads to a microangiopathy, which can cause one or more of the following:

• Diabetic cardiomyopathy: – damage to the heart, leading to diastolic dysfunction and eventually heart failure (5).
• Diabetic nephropathy:- damage to the kidney which can lead to chronic renal failure, eventually requiring dialysis 6
• Diabetic neuropathy:- abnormal and decreased sensation, usually in a ‘glove and stocking’ distribution starting with the feet but potentially in other nerves, later often fingers and hands. When combined with damaged blood vessels this can lead to diabetic foot. Other forms of diabetic neuropathy may present as mononeuritis or autonomic neuropathy. Diabetic amyotrophy is muscle weakness due to neuropathy.(7)

Figure 1: Complications of diabetes 8.
• Diabetic retinopathy: – abnormal growth of friable and poor-quality new blood vessels in the retina as well as macular edema (swelling of the macula), which can lead to brutal vision loss or blindness. Retinal damage (from microangiopathy) makes it the most widespread cause of blindness among non-elderly adults in the US. (9)

? Macrovascular disease: it leads to cardiovascular disease to which accelerated atherosclerosis is a contributor of:

• Coronary artery diseases (CAD) : myocardial infarctions.
• Diabetic myonecrosis: (‘muscle wasting’).
• Peripheral vascular disease: which contributes to intermittent claudication (exertion-related leg and foot pain) Stroke (mainly the ischemic type).(10)

Metformin is now the most widely used antidiabetic drug, with almost all guidelines throughout the world recommending metformin as first-line treatment for patients with type 2 diabetes mellitus (T2DM). (A-9)
Major clinical advantages of metformin include specific reduction of hepatic glucose output, with subsequent improvement of peripheral insulin sensitivity, and remarkable cardiovascular safety, but without increasing islet insulin secretion, inducing weight gain, or posing a risk of hypoglycemia (A-10). Although metformin has been widely prescribed to patients with T2D for over 50 years and has been found to be safe and efficacious both as monotherapy and in combination with other oral antidiabetes agents and insulin, the mechanism of metformin action is only partially explored and remains controversial (A-10). The effectiveness, safety, multiple metabolic and cardiovascular bene?ts of metformin, have established this oral hypoglycemic agent as ?rst line therapy for the treatment of type 2 diabetic patients (A-21)
Its effects include inhibition of insulin-dependent gluconeogenesis in the liver, promotion of incorporation of glucose into peripheral cells through improvement of insulin sensitivity, and reduction of free fatty acids in blood by inhibiting lipolysis (A-13).
The most common reported side effect of metformin is gastrointestinal discomfort along with a few non-signi?cant adverse effects of drug. Malabsorption of vitamin B12 (V B12) may occur during long-term metformin treatment (A-20).
Those patients who used metformin were found to have an increased incidence of vitamin deficiency, but not an increased incidence of neuropathy (A-11). Despite the accumulating evidence linking metformin use and B12 deficiency, assessment of B12 levels in patients treated with metformin has not been incorporated into clinical practice guidelines (A-12).

Metformin associated malabsorption of vitamin B12 is a chronic complication that may affect up to 30% of patients with Diabetes on long-term high dose metformin therapy (A-14).
Mild symptoms of vitamin B12 deficiency include anemia, fatigue, weakness, shortness of breath, and palpitations. Whereas severe vitamin B12 deficiency can cause neuropathy and peripheral nerve damage, numbness, paresthesia, ataxia, memory loss, abnormal gait, dementia, and depression (A-14).
Use of metformin is effective in lowering glycosylated haemoglobin (HbA1c) by 1 to 2 percentage points when used as monotherapy or in combination with other anti-diabetic drugs (A-19).
Vitamin B12 deficiency is common in vegans and can give rise to hematological and neurological manifestations. It can coexist with Type 2 diabetes mellitus and is an independent risk factor for cardiovascular disease (A-15/B).
Low B12 levels due to prolonged metformin use can cause or exacerbate diabetic peripheral neuropathy (DPN). Low serum B12 levels also alter cerebral functions like memory, cognition, alertness etc (A-16).
The diabetic neuropathies are heterogeneous with diverse clinical manifestations. They may be focal or diffuse. The most prevalent neuropathies are chronic sensorimotor DPN and autonomic neuropathy (A-17). Symptoms of B12 deficiency could include weakness, numbness or tingling, problems walking, and vision loss (A-20).
Low serum folate and vitamin B12 levels are thus strongly associated with increased serum homocysteine (A-23). An inverse relation has been shown between plasma homocyst(e)ine levels and plasma levels and dietary intake of folate and vitamins B6 and B12 (A-24). This may negatively impact patient’s health, as elevated homocysteine levels are associated with an increased risk of cardiovascular disease (11).

This study is a prospective open-label study where participants shall be randomized according to their nutritional status and metformin dosage as follow:
• Diet status and association with vitamin B12 level.
• Age groupand association with vitamin B12 level.
• Metformin treatment (dose and duration) and association with vitamin B12 level.
• B12 level and association with homocysteine level
• Neuropathy status association with vitamin B12 level.

Diabetic patients on long term therapy with metformin are affected by age and diet of the patient, in the same time metformin on long term therapy will affect on vitamin B12 level, neuropathy status and finally homocystine level.
This research study discusses the correlation between vitamin B12 deficiency and dose of metformin, the association between vitamin B12 deficiency and diet of the patient, the association between vitamin B12 deficiency and homocystine level and the connection between vitamin B12 deficiency and status of neuropathy.

It was observed that when there is severe vitamin B12 deficiency, the symptoms of peripheral neuropathy worsen. The National Health and Nutrition Examination Survey (NHANES) data reported that oral B12 supplementation reduced the rate of B12 deficiency by two-thirds in those without diabetes, but there was no association seen in those taking metformin (14).

Thus this study involved supplementation of vitamin B12, i.e. oral tablet and parenteral (i.m injection) and their effect on peripheral neuropathy symptoms. Peripheral neuropathy symptoms were evaluated before and after the treatment by using scores of Toronto Clinical Scoring System (TCSS) and Neuropathy Total Symptoms Score-6 questionnaire (NTSS-6) for improvement of peripheral neuropathy symptoms if any.

2. Review of literature:

2.1. Diabetes:

The Greek Apollonius of Memphi first used the term “diabetes” or “to pass through” in 230 Bc (11). Diabetes was one of the first disease described, will on Egyptian manuscript from C. 1500 BCE mentioning “too grail emptying of the urine”. The Emirs papyrus includes a recommendation for a drink to be taken in such cases. The first described cases are believed to be of type I diabetes Indian physicians around the same time identified the disease and classified it as Madhumeha or “honey urine.” nothing the urine would attract ants.

Type 1 and type 2 diabetes were identified as separate conditions for the first time by the Indian physicians Sushrula and Charaka in 400-500 CE with type 1 associated with youth and type 2 with being overweight. The terms diabetes mellitus come from two greek words; diabetes meaning ‘siphon’ and ‘mellitus’ meaning ‘honey’. The terms ‘type 1 diabetes’ has replaced several former terms including childhood onset diabetes, juvenile diabetes and insulin dependent diabetes mellitus (IDDM). Likewise, the terms ‘type 2 diabetes’ has replaced several former terms, including adult onset diabetes, obesity related diabetes and non insulin dependent diabetes mellitus (NIDDM) Diabetes mellitus is also occasionally known as ‘sugar diabetes to differentiate it from diabetes insipidus.

Diabetes – a multisystem disease due to defect in metabolism of glucose which causes multiple irregularities in the metabolism. Metabolism of glucose is well organized by multiple hormones and neurotransmitters in response to nutritional(12), emotional and environmental changes. Unger, first labelled diabetes, as a “bi-hormonal” disease(13). Conventionally the pathophysiology of T2DM was engrossed on beta cell dysfunction and insulin resistance in liver and skeletal muscle. Numerous researches in the past two decades exposed a basic understanding about mechanism and dysfunctions in gastrointestinal tract, pancreatic alpha cells, adipose tissue, brain and kidney that produced a more tough picture of Type 2DM(14). Many studies in pathology of the disease had introduced the novel drugs alike pancreatic-protein-coupled fatty-acid-receptor agonists inhibitors of the insulin-releasing glucokinase activators (15). ß-hydroxysteroid dehydrogenase, sodium-glucose cotransporter (16), glucagon like peptide-1 analogues, glucagon-receptor antagonists and quick-release bromocriptine, dipeptidyl peptidase-IV inhibitors, and metabolic inhibitors of glucose output from liver (17). Diabetes is a multifaceted process and includes managing disease complications, drug related adverse effects in addition to glycemic.

Diabetes mellitus is an endocrine-metabolic disorder in which natural flow of insulin is hampered/altered resulting into circulatory disorder primarily followed by systemic affection of organs at large. It is expressed usually by polyuria and polydypsia and polyphagia. It is classified according to its onset, as Gestational Diabetes Mellitus (GDM), Insulin Dependent Diabetes Mellitus (IDDM) or (Type I), Non Insulin Dependent Diabetes Mellitus (NIDDM) or (Type II), Maturity Onset Diabetes of the Young (MODY) etc. Its acute fatal complications include Diabetic Ketoacidosis (DKA), hyperosmolar hyperglycemia and also known as glucotoxicity may result into death if not controlled or treated timely. DM if improperly treated for long term may results into various microcircular and macrocircular complications such as Chronic Kidney Disease (CKD) due to diabetic nephropathy, loss of vision due to diabetic retinopathy, loss of foot or leg due to gross diabetic neuropathy, and macrovacular complications like Coronary Artery Disease (CAD), Cerebrovascular Disease CVD etc.

Type II DM onset is marked for several years by compensatory involvement of extra ?-cells and their more that optimum production of insulin to compensate insulin resistance but when 50 % of ? – cells die of and the remaining are exhausted for further high demand of insulin by body cells, then gradually Blood Sugar Level (BSL) starts rising above the normal excepted level and when it crosses the renal threshold of sugar, sugar starts getting detected in urine and shows morbid rise in blood sugar level.

Diabetes can be developed by several drugs like statins, steroids, diuretics antipsychotics appetizers ect. Statins are globally used for treatment and prevention of CVD in term they have adverse effect on glucose metabolism, causing raised BSL. Steroids used to treat various autoimmune diseases, like ulcerative colitis, rheumatoid arthiritis, systemic lupvs erythromatous (SLF), Idiopathic thrombocytic, purpura (ITP) etc. But at the same time it results into hyperglycaemia thus DM. Neuroleptics or antipsychotic drugs such as olinzapine etc. and some psychotrophic drugs also have potential to increase blood sugar levels giving rise to drug induced diabetes mellitus.

Similarly diuretics when used continuously for chronic heart failures and renal failures tend to decrease blood volume and increases blood viscosity and hyperglycaemia as the secondary effect. Certain steroids suppresses the natural steroidal hormone secretions thus raises shrinkage in endrocrine glands such as ?-cells and adrenal cells. Viral infections and other pathogens may contribute diabetes mellitus by infecting pancreatic ?-cells. Pancreas is also occasionally damaged by pancreatitis and various aetiology causes that such as infections, chronic alcohol consumption and obstructions due to gall stones.A

• Epidemiology of diabetes:

Diabetes mellitus is rapidly emerging as a current pandemic in this century (18).According to International Diabetic Federation 2014; nearly 183 million people are still unaware that they are living with diabetes. Therefore the identification of individuals at high risk of getting diabetes is of great importance for investigators and health care providers. (19) The target is to reduce the prevalence of the disease and its economic burden and enhance quality of life for all persons who have and are at threat of Diabetes Mellitus. It has equal priority in both developed and developing countries. It is attracting the world since the global crisis due to diabetes cripples not only the health but also the economy of every country. The glad news is that once the risk factors are accessed the development of Type 2 Diabetes can either be deferred or even prevented by healthy customs (20).

Figure 2: Number of people with diabetes by IDF Region, 2013(21)

Indian people in rural and urban areas were affected by diabetes in an equal manner in which people in urban areas have a high incidence of 5.6% while the incidence of 2.7% only was found in rural areas. This high prevalence that is observed in urban area indicts that the major role of urbanization. The most important point that should be taken seriously in consideration is that incidence of diabetes is shifting towards a younger age where Diabetes affected people ten years earlier than the age in western countries. Diabetic prevalence in Chennai was 13.5% in 2000, while in 2004 it was 14.3% and it was increased in 2006 up to 18.6% (22).
Indian population have the ability to access to screening methods and different diabetic-medications in urban cities while these advantages are not easily available to the patients living in rural areas which may in turn explain the higher predominance of type 2 DM in urban areas (23).
The boost predominance of type 2 DM in urban areas is due to that people are capable of easy access for diagnosing diabetes disease. In rural regions several factors that may contribute to the decreased predominance of the disease such as insecure food sources, the predominance of communicable diseases, ignorance or illiteracy, insufficiency or difficulty in counselling, bad sanitation and long travel to reach health care facilities which all leads to inadequate infrastructure in screening. Diverse studies concerned with Indian population all over the world were conducted expressed the great development of type 2 DM and other metabolic complication which comes in coincide with their racial group. The fundamental rises are mostly ambiguous for such problems, some unique biological variables of this race called as the “Indian phenotype”- was thought to be one of the main causes accounting to explain the increased tendency towards type 2 diabetes, in spite of low incidence of obesity (24).

Figure 3: Top 10 countries showing number of people with diabetes (age, 20-79 years), 2013(25)
• Types of Diabetes Mellitus:

Gestational Diabetes Mellitus (GDM): Gestational DM has its onset during pregnancy especially in 3rd trimester females having Polycystic Ovarian Syndrome (PCOS) or Polycystic Ovarian Disease (PCOD) are more prone for this type. Suddenly, there is rise of BSL seen during pregnancy, which is usually treated by human insulin during entire period of pregnancy. Such females are more prone to get type II DM for rest of their life. It can also be one of the stress induced DM.

Insulin Dependent Diabetes Mellitus (IDDM) or (Type I): Insulin dependent DM is one of severe form of DM, where patient suddenly starts loosing considerable weight, becomes thirsty and starts passing lot of urine especially nocturnal polyuria. The cause of this type of diabetes is either immune disorder or mostly idiopathic. All the symptoms are appearing suddenly and look like acute form but these dreadful events stay rest of life. IDDM is also termed as juvenile DM. It usually appears in childhood, but nobody is spared from infant to grown up. Because the usual onset is in early childhood where entire pancreas has not yet developed into full fledged organ, its treatment is initiated by insulin injection give subcutaneously. Urine ketone test must be done for type I type diabetic patient for confirmation of the disease. Juvenile DM having patients are very nasty, they may get worsens in their situations anytime such as severe hypoglycaemias or severe hyperglycaemias or glycosurias with Ketone bodies present. They lose weight very fast and also gain weight too quick. All this is due to very low insulin or very high insulin requirements. High level of insulin dosages is required for controlling blood sugar and too high insulin resistance gets developed as time goes on and disease starts becoming chronic. Sugars are controlled on high amount of insulin results into heavy weight gain.

Non Insulin Dependent Diabetes Mellitus (NIDDM) or (Type II): This type of diabetes forms the largest contributor of DM along the globe. It classically affects the thrifty type of genetic constitutions. Such individuals are abundantly found in Asian continents. They show heavy weight gain in the initiative phase of the disease, where pancreas are optimally active in secretion of insulin to compact insulin resistance due to presence of thrifty genes which are very good in storing the energy in the form of fat or lipids. Especially visceral is synthesized multiple times its normal need and blood tringlycerides are very high.
Such individuals develop acanthosis nigricans and hump on the posterior neck. When blood sugars measured in this 1st phase is always normal or tightly controlled, which misleads the persons as not having diabetes. As a result the person is responsible to severe hunger by eating excessively. If not eaten for long time person may get symptoms of hypoglycaemia like, sweating, lethargy tremors so in anticipation such individuals starts eating more than appetite that to sweet. There is craving for sweets. Thus all this events are resulting into massive weight gain and morbid obesity.
In second phase of the disease person gradually starts loosing flesh and gets polyuria, polydepsia and polyphagia where blood shows frank rise in glucose levels. The second stage of NIDDM is reached after several years of the onset of the metabolic or endocrinal primary disturbance. IN this stage frank diabetes is diagnosed in NIDDM, where more than 50% of ?- cells of langerhans had already been dead or lost.

Maturity Onset Diabetes of the Young (MODY): This type of diabetes may arise due to genetic problem in insulin production. Age of the patient and severity of the disease depends up on the specificity of defective gene. It is less common type of diabetes and can be controlled even without insulin.

• Oral Hypoglycemic Agents (OHA’s)
Oral Hypoglycemic Agents (OHA’s) are the most widely used drugs for treatment of diabetes disease. There are different types of these drugs as follows:

– First generation of drugs includes: Acetohexamide , Chlorpropamide, Tolbutamide, Tolazamide and Gilbenclamide
– Second generation drugs Includes : Gliclazide and Glipizide

All the above drugs are said to be secretagouge i.e. they enhance the insulin secretion by ?-cells of langerhans, present in endocrine pancreas. They actually force beta cells to secrete more and more insulin. Hence a time comes when these ?-cells gets exhausted or goes into fatigue. Still further increase in their doses actually starts killing the ?-cells functioning.

– The biguanides group of drugs include: Penformin and Metformin

These drugs causes decreasing in insulin resistance in which they do not damage ?-cells but instead they are helping ?-cells of langerhans to increase their life span for some period in the course of progression of disease. Penformin is not being in use as it has great potential effect for producing acidosis. Hence metformin remained the gold standard drug of all times to treat the individual of NIDDM

2.2. Metformin:
Metformin chemically is a l,l-dimethylbiguanide hydrochloride molecule that is affiliated to biguanide family of drugs which are essentially derivatives of guanidine. Metformin is extracted from plants known as goat’s rue (Galega officinalis) which are originally a French lilac plant that was used for treatment of diabetes mellitus (DM) in Medieval Europe (25).
Metformin is intended as prior line therapy for type 2 diabetes mellitus especially for patients suffering from obesity. It had been shown their activity by decreasing mortality rate and other complications by 30% when compared to other drugs like chlorpropamide, glibenclamide and insulin (26). In comparsion to sulfonylureas and insulin, metformin cause reduction in weight gain which leads to better controlling of glucose. This is approved in one study, where patients were treated with metformin for a period of more than 10 years and patients were get increased about one kg in their weight while other patients get increased in weight about six kg when kept on insulin therapy (27).
As one of the effective biguanide, metformin is considered as effective as sulfonylurea in their action of glycaemic control by the United Kingdom Prospective Diabetes Study. Also another suggestion was made by the American Diabetes Association to use metformin as one of the first-line medical treatment for type-2 diabetes mellitus (28).Evidence for the ongoing utilization of metformin as a front line anti-diabetic drug in both obese and non-obese patients is strongly supported (29).

• Mechanism of action of metformin:
Mechanism of action of metformin is still unclearly explained. It has different methods for the reduction of blood glucose level via nonpancreatic mechanisms in which it does not alter secretion of insulin (30). The metformin activity is mediated by insulin presence where it reduces hyperglycemia by decreasing liver production of glucose (hepatic gluconeogenesis) (31).See figure 4.
Metformin usually do not induce hypoglycemia which make it preferably a unique choice among anti-diabetic drugs (32). It have other effects like enhancement in the functions of endothelium, oxidative stress, lipid profiles, insulin resistance and fat redistribution along with their essential role in controlling glyceamic level (33).

Figure 4: Mechanism of action of metformin

An enzyme is known as an AMP-activated protein kinase (AMPK) which is must for activation of metformin. This enzyme primarily acts in signalling of insulin, energy balance of the body and the process of glucose and fats metabolism. Inhibition of liver glucose production is done by metformin which requires AMPK activation (34). Activation of AMPK is done in the brain by different metabolic stress like ischemia, hypoxia, glucose deprivation, metabolic inhibitors as well as catabolic and other ATP consuming processes which leads to prevention of ATP production (35).
Along with this effect of decreasing hepatic glucose production, metformin acts to raise the sensitivity of insulin hence, metformin is termed “insulin sensitizer (36), improve the uptake of peripheral glucose which disposed into skeletal muscle (37), boosts oxidation of fatty acids and reduces gastrointestinal tract absorption of glucose (38). Utilization of glucose may be increased because of increases in a binding of insulin to the insulin receptors (39).

In addition to this kinase interaction, metformin have the action on glucose metabolism in small intestine and small bowel absorption which may in turn affect the optimal absorption of some vitamins (40).
Metformin had been shown improvements not only in glycaemic control but also in cardiovascular complication which is the main cause of death in diabetic type 2 patients (41-42). Type 2 diabetes progressions often lead to an increased dose of metformin being required at later disease stages, however even small dosages can improve Hba1c and diabetic outcomes (43).
Further studies into alternate applications of metformin treatment and observation of additional physiological outcomes of the use of the drug have resulted in advantageous outcomes for reducing incidence and mortality in patients suffering from a variety of carcinomas (44). Studies into the effect of metformin in cancer patients has suggested that the glycaemic control of metformin, which involves the absorption of glucose by tissue such as large muscle, may aid in the reduction of tumours as it limits the amount of sugar available to the uncontrolled proliferating cells which are characteristic of carcinoma growth. Investigation has suggested that the use of metformin can directly inhibit cancer cells by initiating the LKB1/AMPK/mTOR axis which works to regulate energy metabolism as well as protein synthesis within the cells (45).

• Side effect of metformin:
In spite of its good glycemic lowering effect, metformin had been shown the effect of lowering vitamin B12 levels as such (46). In addition, Metformin has been reported for its correlation with reduction in folate concentration, even though the mechanism of such an effect has not yet been established. So the effect of lowering both folate and vitamin B-12 levels may lead to an elevation of the homocysteine concentration which is, in turn, resulting in complication of cardiovascular consequences and that can be especially observed in type 2 diabetes mellitus patients (47). Other side effects were seen in patients with heart failure and alcoholic patients where metformin can cause lactic acidosis in these patients along with vitamin B12 deficiency (48).
The popular side effects during metformin consumption are expressed in gastrointestinal complications including nausea, vomiting and diarrhoea. These usually minor side-effects are observed in about 30% of patients and treatment may be discontinued in 5% of patients (49).
The gastrointestinal side effects are commonly addressed by slow titration of metformin dosages to make patients used to the drug. Commonly, but not universally, people become more tolerant to these side effects over time. The associations of potential, serious and sometimes fatal side-effect can be seen with lactic acidosis, although there is much controversy over the real risk conferred by metformin (50).

2.3. Vitamin B12

• Overview of vitamin B12:
Vitamin B12 (cobalamin) is a vital micronutrient, important for nervous system functions, gastrointestinal tract (mucosal layer), sanguification and regulation of several processes of metabolism (51). Vitamin B12 is a water-soluble vitamin which contains cobalt atom and serves as a co-factor for metabolically significant enzymes that mainly related to DNA synthesis, amino and fatty acid metabolism and also it is also the primary feature in myelin production (52).
One of the enzymatic reactions of vitamin B12 is the production of succinyl-CoA from methylmalonyl-CoA to in which catalysis of reaction is done by methylmalonyl-CoA mutase (MCM) enzyme. In fact, succinyl-CoA enzyme is considered the significant intermediate of the Kerbs Cycle that leads to the ATP production. Vitamin B12 has another enzymatic reaction, that is the production of methionine from the conversion of homocysteine. Vitamin B12 in this reaction binds to methionine synthase enzyme which in turns leads to the production of methylcobalamin and 5-methyl tetrahydrofolate (THF) (53).
Deficiency of vitamin B12 resulting in higher serum homocysteine and higher THF levels where MS catalyzes the transfer of a methyl group from methyl-tetrahydrofolate towards homocysteine to form tetrahydrofolate and methionin in which methylcobalamin acts as a co-factor (54). Deficiency of vitamin B12 can prohibt methyl group transformation from methyl-tetrahydrofolate to get tetrahydrofolate. The THF is playing an important role for several coenzymes, where the higher levels of serum homocysteine can lead to cardiovascular complications. Methionine gets converted to S-adenosylmethionine (SAMe) in which it acts as a methyl donor. The SAMe is intended for the protein myelin, which produces the myelin sheath. The term vitamin B12 and cobalamin are interchangeable. All types of vitamin B12 (i.e hydroxocobalamin, cyanocobalamin, methylcobalamin and 5-deoxyadenosyl cobalamin (adenosyl-Cbl) are transformed intracellularly into adenosyl-Cbl and methylcobalamin which considered as the biologically active forms (55). Cyanocobalamin is taken in supplement for and it is needed to be converted first to its active form before getting absorption (56).

• Absorption and Transport of Vitamin B12:

Vitamin B12 presents in food and ready for absorption when it reaches gastric mucosa where it got cleaved by the hydrochloric acid present there. This mechanism is responsible for the absorption of at least 60% of oral coblamin.13–15 Cobalamin metabolism is complex and requires many processes, any one of which, if not present, may lead to cobalamin deficiency (57).
After releasing of cobalamin, it gets bound to R protein and reaches to the duodenum in which the bounding is destroyed and cobalamin becomes freely available for binding to another glycosylated protein i.e. Intrinsic Factor (IF). The complex of IF-cobalamin is ready then for absorption in distal ileum which requires calcium to be present (58). After Secretion of Vitamin B12 in bile, it gets again reabsorbed via ileal receptors in the presence of IF, therefore vitamin B12 deficiency will be more quickly developed in patients having pernicious anaemia because of lack of IF (59). See figure 5.
Vitamin B12 is absorbed through active and passive process, the former via ileum which is an efficient mechanism largely involved the role of intrinsic factor which is mediate secreted from the gastric parietal cells as shown in Figure (60). Active absorption for vitamin B12 in diet is mainly archived through binding to proteins and digestion by the salivary enzymes and subsequently by acids release by stomach which aids digestion and frees form of vitamin B12, combines as cobalamin-binding proteins (haptocorrin) in the salivary and gastric secretions. The IF–B12 complex unable to dissolve easily and thus passes through ileum where the IF bind to the cubilin receptors on the enterocyte, and the complex is internalized by the process of endocytosis; in the enterocyte, the IF is degraded and vitamin B12 binds with protein called as transcobalamin 1 (TC1) a transport protein (61).
The free cobalamin further bound to haptocorrin (HC) to form complex of HC-cobalamin which pushes towards duodenum where the haptocorrin is degraded by pancreatic proteases and form another complex called cobalamin-IF. The IF-cobalamin complex requires calcium and is absorbed by the distal ileum, where IF receptor is located. The complex is internalized by a receptor-mediated endocytotic process (62).
The vitamin B12 secretes in bile mainly reabsorbed through enterohepatic circulation by ileal receptors, in the intestinal cells, cobalamin separate from its complex of IF and exits as free form and form as complex of cobalamin-transcobalamin II protein (TC-II). TC-II-cobalamin complex is released into the plasma and endocytosed by membrane receptors, R-TC-cobalamin (62). The TC-II cobalamin complex soon after is hydrolysed by lysosome and the free cobalamin is transported out of the lysosome to the cytosol and finally vitamin B12 eleminated. Approximate elimination of vitamin B12 from body is in proportion to body stores with 0.1% excreted per day. Excessive vitamin B12 in the circulation exceeds the binding capacity of TC-II and thus may be excreted in the urine (63).

Figure 5: Mechanism and absorption of vitamin B12
• Physiological significance of Vitamin B12
Vitamin B12 is important in the synthesis of DNA, RNA synthesis including red blood cell formation. Along with being required for two enzymatic reactions, it also plays an important role in lipid synthesis, which produces the myelin sheath. The myelin sheath wraps around nerves and protects the axon and the nerves from being exposed (63). The conversion of homocysteine back to methionine requires the vitamin B12 enzyme. Vitamin B12 deficiency leads to excessive amounts of serum homocysteine (64). Malabsorption or deficiency of vitamin B12 can lead to megaloblastic anemia, neuropathy, folate deficiency and cardiovascular disease due to chronic high serum homocysteine levels (65).

Vitamin B12 exits as methyl cobalamine and adenosylcoblamine. The methyl cobalamin-dependent enzyme methionine synthase is largely participated in DNA synthesis which results into division of epithelial and mucosal cells. Methylmalonyl CoA mutase a adnenosylcobalamine dependent enzyme, isomerized to form succinyl CoA from methyl malonyl CoA, is a metabolic reaction of fatty acid synthesis and is involved in myelin sheath formation (66). Impairment in the functioning of this reaction results into metabolic defect subsequently manifested as neurological complications which is largely due to deficiency of vitamin B12 (67).

• Food Sources of Vitamin B12
Animal-derived products such as liver, beef, kidney, chicken, fish, shellfish, meat, eggs, milk, yoghurt and cheese are the major dietary sources of vitamin B12 (68). Plant sources do not contain larger fractions of vitamin B12. However, few herb-origins mainly mushroom and seaweeds contain some concentrations of vitamin B12 form, although these are inactive state (69). It has been recommended that Japanese seaweed (nori) considered as natural source of vitamin B12 (70). As per world health organization, daily intake of vitamin B12 as 2.4 ?g/day for adults and 2.6 and 2.8 ?g/day during pregnancy and lactation respectively (71). In India, The Indian Council of Medical Research recommends 1 ?g/day for adults and 1.2, 1.5 and 0.2?g/day for pregnancy, lactation and infants respectively (72).
2.4. Vitamin B12 Deficiency
Vitamin B12 deficiency is prevalent in populations who do not eat meat. This is of particular relevance for women of South Asian origin and has implications for the future health of their offspring. This problem has not received the public health attention that it deserves. The body of research in the Vitamin B12 study investigates the problem of B12 deficiency in South Asian women and investigates strategies to address this. Theoretical assumptions for the Vitamin B12 study include the life course model of disease aetiology and the importance of community involvement in the study (73).
• Common Symptoms of B12 Deficiency
• Paraesthesia, weakness, numbness,
• Neuropathic
• Ataxia, Abnormal gait
• Myelopathy
• cerebral causes ;
• Depression
• Memory loss
• Abnormal gait
• Anaemia (Haematological -not so common)

Deficiency for a prolonged period ends in the neuronal complications like death of the neurons, demyelination, axonal changes and apoptosis. So the interventions should be done promptly.
Vitamin B12 deficiency may results into various pathophysiological implications largely related to neurological manifestations (Table 3). Traditionally, the deficiency has been causes megaloblastic anemia and progress to a neurological condition called sub acute combined degeneration. However, this represents only the end stage of the slowly progressive process. At earlier stages clinical manifestations are subtle and highly variable, and neurological and cognitive defects may occur in the absence of hematological signs (74).
Table 3: Signs and symptoms of vitamin B12 deficiency(75).

Consequently, the diagnostic value of these unspecific symptoms and signs is low (76) Bl2 deficiency and depletion. Vitamin B12 depletion precedes deficiency and occurs at serum B 12 concentrations previously classified as within the recommended reference range of normal (77). Expected holoTC concentrations in persons with normal renal function is between 32 to 58 pmol/l (90 % confidence intervals) (78). HoloTC concentrations < 45 pmol/L are indicative of B12 depletion (79).
A serum B12 concentration of 258 pmol/L) Stage II occurs when serum B12 concentrations decrease to between 222 to 258 pmol/L. At these concentrations, serum holoHC has decreased and this indicates depletion of body B 12 stores as HoloHC maintains in equilibrium to liver and body stores of B 12. HoloHC has a half-life of 240 days so depletion takes longer to detect if using serum B 12 as the only guide to B 12 status (81). People with vegetarian dietary preferences may remain in this state of B 12 depletion for many years without progressing to overt deficiency because enterohepatic recycling of B12 is intact. In depleted states, when B 12 absorption is intact, transcobalamin receptors are up regulated, facilitating increased reabsorption of B 12 excreted via bile, keeping gastrointestinal losses of B 12 to an absolute minimum (82).
If vitamin B12 stores continue to deplete, there is deficiency of vitamin B12 for one carbon metabolism, excess accumulation of the metabolites Hcy and MMA which leads to mild cognitive impairment that remain unnoticed. An increased Hcy concentration or hyperhomocysteinaemia marks into neurological deficits associated with cardiovascular complications (83).vitamin B12 deficiency is often not diagnosed until symptoms such as macrocytic anaemia occur (84), however, the presentation of macrocytic anaemia occurs at late stage of vitamin B12 deficiency and by this stage brain glial cells are depleted of B 12 and the risk of irreversible neurological deficits is high (85). Macrocytic anaemia is changeable indictor of deficiency as not all vitamin B12 results in macrocytic changes (86).

Estimation of the B 12 levels in serum has limitations for assessing B 12 status because of the lag time in reflecting deficiency (87). Measurements of serum Hcy and MMA concentrations indicate and the index for B 12 deficiency state of patient (88), Furthermore, Hcy and MMA increase once changes in cellular biochemical functions occur in response to deficiency (89). Measurement of the holo-TC concentration is a preferable diagnostic test for B12 status as it can detect diminishing B 12 stores before irreversible cell damage from deficiency occurs, and it has a high specificity for B12 deficiency (90).MMA and Hcy are not specific for B 12 deficiency; Hcy is raised in folate and B6 deficiency and both Hcy and MMA levels were found to elevated during renal dysfunction (91).There is wide difference in the literature cut-off points used to define B 12 deficiency and insufficiency. Therefore for the Vitamin B12 study a cut-off limit of 150 pmol/L is used to define B12 deficiency (92) and a cut off of 222 pmol/L used to define insufficiency (93).

• Causes of vitamin B12 deficiency
Through various physiologic functions, the metabolism of Vitamin B12 metabolism may be check (94). As per the etiology its deficiency may be due to inadequate intake or impaired absorption (due to chronic atrophic gastritis or some other cause). An increased requirement for vitamin B12 or functional “resistance” might also cause deficiency symptoms (95). It has been postulated that gastroenterological disorder may be responsible for vitamin B12 deficiency until proven otherwise, but the Malabsorption is the common cause of vitamin B12 deficiency.
In the middle of the 19th century, patients with symptoms compatible with pernicious anemia was the first evidence identified (96). Later on it was correlated with to the stomach. Later on it was named as pernicious anemia, because it would inevitably lead to death and no cure was known. Then it was presented that pernicious anemia alleviates by ingestion of extrinsic factor present in liver, which was later purified and named vitamin B12. Later on, it was observed that lack of substance called IF in gastric juice is responsible for insufficient absorption of this extrinsic factor. The in the late 1980’s it was demonstrated that the antigen recognized by parietal cell antibodies is gastric H+/K+-ATPase. Recently, it has been suggested that pernicious anemia is developed by involvement of Helicobacter pylori (97).
The cause of vitamin B12 deficiency is pernicious anemia, but it may be the reason for vitamin B12 deficiency only in about 15-20% of subjects aged over 60 years (98). It is a consequence of end stage chronic atrophic autoimmune gastritis, characterized by atrophy of the gastric mucosa in the fundus and the corpus of the stomach (99). Parietal cell antibodies directed toward H+/K+-ATPase destroy of gastric parietal cells, impair IF production and lead to vitamin B12 malabsorption.
Another mechanism responsible for the impaired absorption of vitamin B12 in pernicious anemia is loss of parietal cells causes loss of gastric acid secretion, associated with loss of zymogenic cells and impaired pepsinogen-I production. Due to hypo-chlorhydria and loss of pepsinogen I production, protein bound vitamin B12 cannot be released and absorbed. Prevention of the formation of the vitamin B12-IF complex is found to be in approximately 50% of subjects with pernicious anemia with anti-IF antibodies.
The other autoimmune disorders viz; primary hypoparathyreoidism, chronic autoimmune thyreoiditis, Graves’ disease, Addison’s disease, primary ovarian failure, type I diabetes, vitiligo, myasthenia gravis and the Lambert-Eaton syndrome are associated with pernicious anemia (100).

• Food-vitamin B12 malabsorption:
Doscherholmen and Swaim showed that despite a normal absorption of free vitamin B12, many patients with atrophic gastritis or partial gastrectomy could not absorb food-bound vitamin B12 (101). This further confirmed the basis of the disorder that vitamin B12 could not be released from food or gastrointestinal transport proteins (HC), and consequently binding to IF was blocked (102). The chronic gastritis is the main cause for food-vitamin B12 malabsorption , which is due to Helicobacter pylori infection, and involves the antrum of the stomach and causes impaired gastric acid and pepsin secretion, and thus the absorption of unbound vitamin B12, remains unaffected. However, in autoimmune atrophic gastritis is caused due to hypochlorhydria and food-vitamin B12 malabsorption.
It was shown that food-vitamin B12 malabsorption can also be caused by other conditions than chronic gastritis, that is; hypochlorhydria caused by gastric surgery (partial gastrectomy, vagotomy, gastric bypass surgery for treatment of obesity). Long-term use of acid-suppressing drugs example, H2 receptor antagonists and proton pump inhibitors causes vitamin B12 malabsorption (103).

• The role of Helicobacter pylori:
Helicobacter pylori infection in chronic gastritis of the antrum of the stomach causes impairment in gastric acid and pepsin secretion, leads to malabsorption of food-vitamin B12.The initial assumption related to involvement of H.pylori in pernicious anemia was significantly lower in patients than in controls (11% vs. 71%) (104). Later, this was explained by an association between Helicobacter pylori and gastric autoimmunity (105), the appearance of parietal cell antibodies led to progression of corpus atrophy and disappearance of Helicobacter pylori and patients with duodenal ulcer disease found to be positive. Further, it is involved in the pathogenesis of pernicious anemia by causing atrophy of the corpus of the stomach by inducing parietal cell antibodies (106).

• Dietary insufficiency:
An extremely rare cause of vitamin B12 deficiency due to inadequate intake in industrialized countries is because of ordinary mixed food diet provides enough vitamin B12 (107). Recently it has been shown that the intake exceeds the recommended daily intake in healthy adults and aged in the United States, Canada and Europe (108). In malnourished aged persons in institutions or persons with eating disorders there is poor intake of vitamin B12 and other nutrients.
Insufficient in vitamin B12 intake in vegetarian diets contains no food of animal origin at all. Most Western people start vegetarian diet in adult life, when they have adequate hepatic stores of vitamin B12. Therefore, it may take several years before overt vitamin B12 deficiency develops, because of efficient enterohepatic circulation. The need for vitamin B12 supplementation is well recognized among vegetarians and deficiency is more likely to develop if consumed from infancy. Breast-fed infants of vegetarian mothers are also at risk, due to vitamin B12 content in breast milk may be low (109).

• Other causes for vitamin B12 deficiency:
Elimination of total and partial gastrectomy by the production of IF and gastric acid inevitably causes vitamin B12 malabsorption. Exocrine pancreatic insufficiency due to chronic pancreatitis/pancreatomy may impair vitamin B12 absorption due to the reduced production of pancreatic proteases which are needed for degradation of HC from vitamin B12 in the duodenum (110). The oral antidiabetic drug metformin impairs endocytosis of the IF-vitamin B12 complex by chelating calcium ions essential for receptor binding (111). Because absorption of vitamin B12 takes place in the terminal ileum, surgical resection of the terminal ileum and diseases like Crohn’s disease and celiac disease leads to damage in the mucosa of the terminal ileum. Further, folate deficiency may damage the ileal epithelium and impair vitamin B12 absorption.

• Biological competition:
Hypochlorhydria may also facilitate intestinal bacterial overgrowth and biological competition for vitamin B12, besides impairement of absorption by preventing its release from proteins. Long-term treatment with antibiotics may also induce intestinal bacterial overgrowth (112). Fish tapeworm (Diphyllobothrium latum) infection also causes vitamin B12 deficiency by biological competition, but it is very seldom, because of an abundant nutritional supply (113).

• Hereditary disorders
Inborn errors of cobalamin metabolism are rare and should be diagnosed in infancy. The disorders that affect vitamin B12 absorption are congenital intrinsic factor deficiency and the Imerslund-Gräsbeck syndrome, whereby the receptor for the IF vitamin B12 complex is defective due to mutations in the cubilin or amnionless gene (114). Also, IF may have an abnormal structure, and thus be unable to bind vitamin B12 or its receptor. TC deficiency causes impairment in the transport of vitamin B12 into the cells (115). But the defects in HC do not causes vitamin B12 deficiency (116). Also, the intracellular metabolism of vitamin B12 may be affected. Impairment of adenosylcobalamin synthesis (cblA and cblB), methionine synthase function (cblE and cblG) or both (cblC, cblD, cblF and cblH) are possible (117).

• B12 deficiency and diet:

In the South Asian population, the most common cause of B 12 deficiency is insufficient dietary B12 intake (118). The intergenerational inheritance of low vitamin B 12 stores combined with the accumulative effects of low B 12 diet, lead to deficiency states much more rapidly than in a person with sufficient B12 stores who commences on a vegetarian diet (119). B12 insufficiency and deficiency is also common in Asian Indian people with non-vegetarian dietary practices because even those who eat meat do so infrequently (120). Generally B12 deficiency due to inadequate intake, or absorption-inhibiting medications, takes longer to become symptomatic and is not as severe as pernicious anaemia (121).This is because existing B 12 stores undergo efficient enterohepatic recycling, so it can take between 3 to 5 years to deplete existing B 12 stores. However, for South Asian people, the long intergenerational history of inadequate B 12 intake means that sufficient B 12 stores are not passed onto subsequent generations, so B 12 deficiency can be more thoughtful.

The inter-generational actions of impaired vitamin B 12 release, pulled from one generation to the next are indicated as many contributory causes to the ballooning rate of NCD in the Indian population(122). Documented findings revealed that the phenotype for people of Indian origin differs from other populations and Indian people have a reduced muscle mass, with increased insulin resistance which are considered as higher incidence of T2DM (123).
Vegetarianism is an intrinsic part of three religious groups in South Asian culture: Janism, Hinduism and Buddhism (124). The practices are differs across numerous class with vegetarianism being practiced by only few groups. The Brahman culture peoples, vegetarianism were conventionally linked to caste, with the greater people of whose food is vegetarian while the lower castes are mainly of non-vegetarian (125). Multigenerational patriarchal families are highly valued in South Asian cultures, with several generations of one family living together (126). In India particularly and within other South Asian cultures, marriages are arranged by the family, with the bride joining her husband’s family and adopting the dominant practices and customs of that family (127). There are diverse religious practices, dietary preferences and customs that have been maintained across different cultural groups and sub groups for millenniums of generations (128).
The quality of food in South Asian people is subjective by religious beliefs and family dietary practices, region of origin and the conventional ease of use of food is as per territory wise (129). In more recent times there have been variations to the vegetarian diet. These include lactoovovegetarians who consume eggs as well as dairy, cereals, grains, fruit and vegetables; lactovegetarians who include dairy but not eggs, and vegans who only eat vegetables, fruits, cereals and grain (130).
The differences in diet of South Asian people with western cultures, is mainly with respect to meat is the main course and cereals and vegetables the accompaniment. There is high frequency of the non-vegetarian food intake per day in Western culture. In India, people prefer high vegetarian food contents as meal and frequency is little less compared to western with less frequency of non-vegetarian food (131). Socioeconomic status also influences the availability of meat. Thus, it seems that, non-vegetarian people eat less meat contain food. It was documented that minimum meat eating overall in India (132) has implications in deficiency of vitamins and proteins and thus it can be linked that non-vegetarian does not develop the deficiency and vegetarian do and hence it is proposed that Vitamin B 12 study are consistent with the participants’ cultural dietary preferences.

The interactions of vitamin B 12 deficiencies and diabetes type II were indicated as evaluation of Indian 785 pregnant women (133). Experimental findings demonstrated that maternal B12 deficiency « 150 pmol/L) were associated with increased abdominal adiposity, insulin resistance and impaired glucose tolerance. This risk was higher in those with low serum B12 and high folate concentrations. This effect existed even after adjusting for covariates such as age, socioeconomic status, parity and family history of diabetes. It is likely that no role of vitamin B 12 participation in gestational diabetes complications, since this diabetes developed B12 deficient women. More than 40% of the women in this study were B 12 deficient (134). A longitudinal follow up of offspring of these women found that at nine years of age, the children, especially females, of mothers with T2DM or gestational diabetes had a higher level of adiposity, insulin resistance and a higher systolic blood pressure than those born to non-diabetic mothers (135).
The associations between B 12 deficiency and CVD have been supported in some studies (136) but there is still debate about the direct associations of B 12 deficiency and NCD. The risks from B12 deficiency are proposed to be mediated through hyperhomocysteinaemia (137), although a study by Kumar et al. (2009) found associations between B 12 deficiency, and increased concentrations of another metabolite cysteine, rather than homocysteine. Kumar et al.’ s (2009) case control study undertaken in 816 subjects (368 participants with coronary artery disease and 448 matched controls) in India assessed the association between dietary intakes of vitamin B 12, concentrations of vitamin B 12 and rates of coronary artery disease CAD (138). The median age of subjects was fifty years. In the CAD group, 60% of subjects were found to be deficient in B 12 compared to 48% in the non CAD group. A binary logistic regression analysis of factors that significantly affected CAD risk were age (p < 0.001), male gender (p 60 years old, 41 patients were having sufficient vitamin B12 level and 15 patients were suffering from deficiency in vitamin B12 level. It was found that there is no significant correlation between vitamin B12 level and age of the patients (p-value = 0.488).

Table 1. Vitamin B12 count in correlation to age of Patient
Age group Vitamin B12 count (pg/ml) Total p-value
? 250 251 – 400 > 400
? 40 7 4 7 18 0.488
41 – 50 14 11 19 44
51 – 60 23 15 31 69
> 60 15 13 41 69
Total 59 43 98 200

The data was analyzed using Fisher’s exact test. * indicate (p 400
Male 32 18 39 89 0.199
Female 27 25 59 111
Total 59 43 98 200

The data was analyzed using Chi-square test. * indicate (p 120 months, 37 patients were having sufficient vitamin B12 level and 14 patients were suffering from deficiency in vitamin B12 level. It was found that there is no significant difference between vitamin B12 level and duration of the disease (p-value = 0.154) i.e. Diabetes itself does not cause vitamin B12 deficiency.

Table 3. Vitamin B12 count in correlation to duration of diabetes mellitus

Duration of DM Vitamin B12 count (pg/ml) Total p-value
? 250 251 – 400 > 400
? 60 month 23 12 38 73 0.154
61- 120 month 22 13 23 58
> 120 month 14 18 37 69
Total 59 43 98 200

The data was analyzed using Chi-square test. * indicate (p1500 mg/day), out of 61 patients, 19 patients only were having normal vitamin B12 level and 24 patients were suffering from deficiency in vitamin B12 level. It confirmed that there is a significant correlation between vitamin B12 level and dose of metformin (p-value = 0.014).

Table 4. Vitamin B12 count in correlation to metformin dose

Metformin dose (mg/day) Vitamin B12 count (pg/ml) Total p-value
? 250 251 – 400 > 400
? 1000 24 20 55 99 0.014*

1000 – 1500 11 5 24 40
> 1500 24 18 19 61
Total 59 43 98 200

The data was analyzed using Chi-square test. * indicate (p 36 months, 15 patients were having sufficient vitamin B12 level and 7 patients were suffering from deficiency in vitamin B12 level. It was found that there is no significant correlation between vitamin B12 level and duration of metformin treatment (p-value = 0.768).

Table 5. Vitamin B12 count in correlation to duration of metformin

Duration of Metformin Vitamin B12 count (pg/ml) Total p-value
? 250 251 – 400 > 400
? 12 month 35 32 60 127 0.768
13 – 24 month 12 5 16 33
25 – 36 month 5 2 7 14
> 36 month 7 4 15 26
Total 59 43 98 200

The data was analyzed using Chi-square test. * indicate (p 400
Vegetarian 13 4 39 56 < 0.001**
Non Vegetarian 46 39 59 144
Total 59 43 98 200

The data was analyzed using Chi-square test. ** indicate (p 400
Normal (80-95 fl) 39 29 61 129 0.823
Abnormal ( 60 42 27 69
Total 135 65 200

The data was analyzed using Chi-square test. * indicate (p 120 month 47 22 69
Total 135 65 200
The data was analyzed using Chi-square test. * indicate (p1500 mg/day), out of 61 patients 38 patients were having normal homocystine level (< 15 µmol/L) and 23 patients were having elevated homocystine level (? 15 µmol/L). It confirmed that there is no significant correlation between homocystine level and dose of metformin (p-value = 0.411).

Table 16. Homocystine level in correlation to metformin dose
Metformin dose (mg/day) Homocystine (µmol/L) Total P-value
1500 38 23 61
Total 135 65 200

The data was analyzed using Chi-square test. * indicate (p 36 months where 14 patients were having normal homocystine level (< 15 µmol/L) and 12 patients were having elevated homocystine level (? 15 µmol/L). It was found that there is no significant correlation between homocystine level and duration of metformin treatment (p-value = 0.768).

Table 17. Homocystine level in correlation to duration of metformin

Duration of metformin Homocystine (µmol/L) Total P-value
36 month 14 12 26
Total 135 65 200

The data was analyzed using Chi-square test. * indicate (p


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