CHAPTER I INTRODUCTION

1.1. General Background:

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the -cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The basis of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues. Deficient insulin action results from inadequate insulin secretion and/or diminished tissue responses to insulin at one or more points in the complex pathways of hormone action. Impairment of insulin secretion and defects in insulin action frequently coexist in the same patient, and it is often unclear which abnormality, if either alone, is the primary cause of the hyperglycemia.(ADA, 2009) Diabetes mellitus is the commonest endocrine disorder encountered in clinical practice. It may be defined as a syndrome characterized by hyperglycemia due to an absolute or relative lack of insulin and/ or insulin resistance.(clinical biochemistry, second edition, Churchill livingstone)

Diabetes is not one disease, but rather a heterogenous group of syndrome characterized by an elevation of fasting blood glucose caused by relative or avsolute deficiency in insulin. (Lippincotts biochemistry 3rd edition) Diabetes mellitus (type1, or insulin-dependent diabetes mellitus; IDDM) is characterized by decreased glucose tolerance as a result of decreased secretion of insulin as a result of progressive destruction of pancreatic -islet cells.(Harpers illustrated biochemistry, 27th edition) 1.2. Statement of problem: Diabetes is of two types, type 1 accounting for 5% prevalence and type 2 for 95% prevalence among diabetics. This calls for improved treatment of hyperglycaemia and other risk factors associated with metabolic syndrome. Since it is possible to dramatically lower the risk of both micro and macrovascular complications. Persistent elevations in blood sugar increase the risk for the long-term vascular complications of diabetes such as coronary disease, heart attack, stroke, heart failure, kidney failure, blindness, erectile dysfunction, neuropathy (loss of sensation, especially in the feet), gangrene and gastro paresis (slowed emptying of the stomach). Poor blood glucose control also increases the risk of short-term complications of surgery such as poor wound healing (Sultanpur C M, Deepa K, et al. 2010). Assigning a type of diabetes to an individual often depends on the circumstances present at the time of diagnosis, and many diabetic individuals do not easily fit into a single class. For example, a person with gestational diabetes mellitus (GDM) may continue to be hyperglycemic after delivery and may be determined to have, in fact, type 2 diabetes. Alternatively, a person who acquires diabetes because of large doses of exogenous steroids may become

normoglycemic once the glucocorticoids are discontinued, but then may develop diabetes many years later after recurrent episodes of pancreatitis. Another example would be a person treated with thiazides who develops diabetes years later. Because thiazides in themselves seldom cause severe hyperglycemia, such individuals probably have type 2 diabetes that is exacerbated by the drug. Thus, for the clinician and patient, it is less important to label the particular type of diabetes than it is to understand the pathogenesis of the hyperglycemia and to treat it effectively, (ADA, Diabetes Care; vol.29, 2006). 1.3. Rationale of study: About 2 million people in our country are today affected with diabetes mellitus. It is generally due to genetic or by metabolic defect. Diabetes mellitus is the leading cause of mortality of people across the globe causing various secondary disorders such as CHD, renal dysfunction, neuropathy, and various ocular disorders such as retinitis pigmentosa. Diabetes mellitus is associated with several metabolic alternations. Most important

among

them are hyperglycemia,

ketoacidosis,

hypertrygyceridemia and dyslipidemia. Hyperglycemia: elevation of blood glucose concentration is the hallmark of uncontrolled diabetes. Hyperglycemia is primarily due to the reduced glucose uptake by tissues and its increased production via gluconeogenesis and glcogenolysis. When the blood glucose level goes beyond renal threshold, glucose is excreted into urine (glycosuria).

Ketoacidosis: Increased Mobilisation of overproduction ketoacidosis. of ketone bodies

fatty acids results in often leads to

which

Hypertrygyceridemia:

Conversion

of

fatty

acids

to

triacylglycerols and the secretion of VLDL and chylomicrons are comparatively higher in diabetics. Further, the activity of the enzyme lipoprotein lipase is low in diabetic patients. Consequently, the plasma level of VLDL, chylomicrons and triacylglycerols are increased.

Hypercholesterolemia: It is also frequently seen in diabetics as the Acetyl CoA pool is increased and more molecules are channeled to cholesterol.

Hyperlipidemia: Hyperlipidemia is the elevation of on or more of the lipoprotein fractions constitute hyperlipidemia is also called as dyslipidemia.

Dyslipidemia in Diabetes mellitus is seen as the result of inherited disorders of lipoproteins metabolism and transport. It is genetic defect where there is defect in LDL-receptors, enzyme lipoprotein lipase deficiency or overproduction of endogenous triacylglycerols with rise in VLDL. According to Fredericksons classification of dyslipidemia it is of following types:

Type I: This is due to familial lipoprotein lipase deficiency. The enzyme defect causes increase in plasma chylomicrons and TG levels.

Type II a: This is also known as hyperbetalipoproteinemia and is caused by a defect in LDL receptors. It is observed in

Diabetes mellitus, hypothyroidism, nephrotic syndrome etc. it is characterized by hypercholesterolemia.

Type II b: Both LDL and VLDL increase along increase with elevation in plasma cholesterol and triacylglycerol. It is due to overproduction of apo B.

Type III: This is commonly known as broad beta disease and is characterized by the appearance of broad beta band corresponding to IDL on electrophoresis.

Type IV: This is due to overproduction of endogenous triacylglycerols with rise in VLDL. It is usually associated with obesity, alcoholism, diabetes mellitus etc.

Type V: Both chylomicrons and VLDL are increased. It is mostly due to secondary condition such as obesity, diabetes mellitus and excessive alcohol intake.

Thus Hyperlipidemia is categorized as a risk factor for diabetes mellitus which leads to other neuropathy, ocular dystrophy etc. secondary disorders like CHD,

1.4. OBJECTIVE OF THE STUDY 1.4.1. General objective: To study lipid profile in diabetes mellitus type 2 patients. To estimate serum cholesterol in type 2 diabetic patients. To estimate serum LDL-C in type 2 diabetic patients. To estimate serum HDL-C in type 2 diabetic patients. To estimate serum Triglyceride in type 2 diabetic patients. 1.4.2. Specific objective:

CHAPTER II LITERATURE REVIEW

2.1. Definition: Diabetes Mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the -cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The basis of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues. Deficient insulin action results from inadequate insulin secretion and/or diminished tissue responses to

insulin at one or more points in the complex pathways of hormone action. (American Diabetes Association, 2010)

Classification of diabetes mellitus:

Type 1 diabetes (Beta-cell destruction, usually leading to absolute insulin deficiency): Type 1 diabetes is a chronic (lifelong) disease that occurs when the pancreas does not produce enough insulin to properly control blood sugar levels. Type 1 diabetes can occur at any age, but it is most often diagnosed in children, adolescents, or young adults. Insulin is a hormone produced by special cells, called beta cells, in the pancreas, an organ located in the area behind your stomach. Insulin is needed to move blood sugar (glucose) into cells, where it is stored and later used for energy. In type 1 diabetes, these cells produce little or no insulin. (Medline plus, 2010). Although the cause of type 1 diabetes is still not fully understood it is believed to be of immunological origin. There is a growing body of evidence that diet may play a role in the development of type 1 diabetes, through influencing gut flora, intestinal permeability, and immune function in the gut; wheat in particular has been shown to have a connection to the development of type 1 diabetes, although the relationship is poorly understood. Type 1 can be distinguished from type 2 diabetes via a C-peptide assay, which measures endogenous insulin production. (Wikipedia, 2010) Normally, the body's immune system fights off foreign invaders like viruses or bacteria. But for unknown reasons, in people with type 1 diabetes, the immune system attacks various cells in the body. This

results in a complete deficiency of the insulin hormone. (WebMD, 2010) Immune-mediated diabetes: This form of diabetes, which accounts for only 510% of those with diabetes, previously, encompassed by the terms insulin dependent diabetes, type I diabetes, or juvenile-onset diabetes, results from a cellular-mediated autoimmune destruction of the cells of the pancreas. Markers of the immune destruction of the cells include islet cell autoantibodies, Autoantibodies decarboxylase to insulin, and autoantibodies autoantibodies to to glutamic the acid (GAD65), tyrosine

phosphatases IA-2 and IA-2. One and usually more of these autoantibodies are present in 8590% of individuals when fasting hyperglycemia is initially detected. Also, the disease has strong HLA associations, with linkage to the DQA and DQB genes, and it is influenced by the DRB genes. These HLA-DR/DQ alleles can be either predisposing or protective. In this form of diabetes, the rate of betacell destruction is quite variable, being rapid in some individuals (mainly infants and children) and slow in others (mainly adults). Some patients, particularly children and adolescents, may present with ketoacidosis as the first manifestation of the disease. Others have modest fasting hyperglycemia that can rapidly change to severe hyperglycemia and/or ketoacidosis in the presence of infection or other stress. Still others, particularly adults, may retain residual _-cell function sufficient to prevent ketoacidosis for many years; such individuals eventually become dependent on insulin for survival and are at risk for ketoacidosis. At this latter stage of the disease, there is little or no

insulin secretion, as manifested by low or undetectable levels of plasma C-peptide. Immunemediated diabetes commonly occurs in childhood and adolescence, but it can occur in any age, even in the 8th and 9th decades of life. Autoimmune destruction of _-cells has multiple genetic predispositions and is also related to environmental factors that are still poorly defined. Although patients are rarely obese when they present with this type of diabetes, the presence of obesity is not incompatible with the diagnosis. These patients are also prone toother autoimmune disorders such as Graves disease, Hashimotos thyroiditis, Addisons disease, vitiligo, celiac sprue, autoimmune hepatitis, myasthenia gravis, and pernicious anemia. (ADA, 2005)

Idiopathic diabetes: Some forms of type 1 diabetes have no known etiologies. Some of these patients have permanent insulinopenia and are prone to ketoacidosis, but have no evidence of autoimmunity. Although only a minority of patients with type 1 diabetes fall into this category,of those who do, most are of African or Asian ancestry. Individuals with this form of diabetes suffer from episodic ketoacidosis and exhibit varying degrees of insulin deficiency between episodes. This form of diabetes is strongly inherited, lacks immunological evidence for Betacell autoimmunity, and is not HLA associated. An absolute requirement for insulin replacement therapy in affected patients may come and go. (ADA, 2005) Type 2 diabetes (ranging from predominantly insulin

resistance with relative insulin deficiency to predominantly an insulin secretory defect with insulin resistance):

10

Diabetes mellitus type 2 formerly non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Diabetes is often initially managed by increasing exercise and dietary modification. Unlike type 1 diabetes, there is very little tendency toward ketoacidosis though it is not unheard of. One effect that can occur is nonketonic hyperglycemia. Long-term complications from high blood sugar can include increased risk of heart attacks, strokes, amputation, and kidney failure. (Wikipedia, 2010). People who are overweight are more likely to have insulin resistance, because fat interferes with the body's ability to use insulin. Type 2 diabetes usually occurs gradually. Most people with the disease are overweight at the time of diagnosis. However, type 2 diabetes can also develop in those who are thin, especially the elderly. Family history and genetics play a large role in type 2 diabetes. Low activity level, poor diet, and excess body weight (especially around the waist) significantly increase your risk for type 2 diabetes. (Medlineplus, 2010). In type 2 diabetes, either the body does not produce enough insulin or the cells ignore the insulin. Insulin is necessary for the body to be able to use glucose for energy. When you eat food, the body breaks down all of the sugars and starches into glucose, which is the basic fuel for the cells in the body. Insulin takes the sugar from the blood into the cells. When glucose builds up in the blood instead of going into cells, it can lead to diabetes complications. (ADA, 2010) This form of diabetes, which accounts for 9095% of those with diabetes, previously referred to as non-insulin-dependent diabetes, type II diabetes, or adult-onset diabetes, encompasses individuals who have insulin resistance and usually have relative (rather than

11

absolute) insulin deficiency At least initially, and often throughout their lifetime, these individuals do not need insulin treatment to survive. There are probably many different causes of this form of diabetes. Although the specific etiologies are not known, autoimmune destruction of -cells does not occur, and patients do not have any of the other causes of diabetes listed above or below. Most patients with this form of diabetes are obese, and obesity itself causes some degree of insulin resistance. Patients who are not obese by traditional weight criteria may have an increased percentage of body fat distributed predominantly in the abdominal region. Ketoacidosis seldom occurs spontaneously in this type of diabetes; when seen, it usually arises in association with the stress of another illness such as infection. This form of diabetes frequently goes undiagnosed for many years because the hyperglycemia develops gradually and at earlier stages is often not severe enough for the patient to notice any of the classic symptoms of diabetes. Nevertheless, such patients are at increased risk of developing macrovascular and microvascular complications. Whereas patients with this form of diabetes may have insulin levels that appear normal or elevated, the higher blood glucose levels in these diabetic patients would be expected to result in even higher insulin values had their cell function been normal. Thus, insulin secretion is defective in these patients and insufficient to compensate for insulin resistance. Insulin resistance may improve with weight reduction and/or pharmacological treatment of hyperglycemia but is seldom restored to normal The risk of developing this form of diabetes increases with age, obesity, and lack of physical activity. It occurs more frequently in women with prior GDM and in individuals with hypertension or dyslipidemia, and its frequency varies in different racial/ethnic subgroups. It is often

12

associated with a strong genetic predisposition, more so than is the autoimmune form of type 1 diabetes. However, the genetics of this form of diabetes are complex and not clearly defined.(ADA, 2005)

Other specific types of Diabetes Genetic defects of the -cell: Several forms of diabetes are associated with monogenetic defects in -cell function. These forms of diabetes are frequently characterized by onset of hyperglycemia at an early age (generally before age 25 years). They are referred to as maturity-onset diabetes of the young (MODY) and are characterized by impaired insulin secretion with minimal or no defects in insulin action. They are inherited in an autosomal dominant pattern. Abnormalities at six genetic loci on different chromosomes have been identified to date. The most common form is associated with mutations on chromosome 12 in a hepatic transcription factor referred to as hepatocyte nuclear factor (HNF)-1. A second form is associated with mutations in the glucokinase gene on chromosome 7p and results in a defective glucokinase molecule. Glucokinase converts glucose to glucose-6phosphate, the metabolism of which, in turn, stimulates insulin secretion by the -cell. Thus, glucokinase serves as the glucose

13

sensor for the -cell. Because of defects in the glucokinase gene, increased plasma levels of glucose are necessary to elicit normal levels of insulin secretion. The less common forms result from mutations in other transcription factors, including HNF-4, HNF-1, insulin promoter factor (IPF)-1, and NeuroD1. Point mutations in mitochondrial DNA have been found to be associated with diabetes mellitus and deafness the most common mutation occurs at position 3243 in the tRNA leucine gene, leading to an A-to-G transition. An identical lesion occurs in the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike syndrome); however, diabetes is not part of this syndrome, suggesting different phenotypic expressions of this genetic lesion. Genetic abnormalities that result in the inability to convert proinsulin to insulin have been identified in a few families, and such traits are inherited in an autosomal dominant pattern. The resultant glucose intolerance is mild. Similarly, the production of mutant insulin molecules with resultant impaired receptor binding has also been identified in a few families and is associated with an autosomal inheritance and only mildly impaired or even normal glucose metabolism.(ADA, 2005)

Genetic defects in insulin action: There are unusual causes of diabetes that result from genetically determined abnormalities of insulin action. The metabolic abnormalities associated with mutations of the insulin receptor may range from hyperinsulinemia and modest hyperglycemia to severe diabetes. Some individuals with these mutations may have acanthosis

14

nigricans. Women may be virilized and have enlarged, cystic ovaries. In the past, this syndrome was termed type A insulin resistance. Leprechaunism and the Rabson-Mendenhall syndrome are two pediatric syndromes that have mutations in the insulin receptor gene with subsequent alterations in insulin receptor function and extreme insulin resistance. The former has characteristic facial features and is usually fatal in infancy, while the latter is associated with abnormalities of teeth and nails and pineal gland hyperplasia. Alterations in the structure and function of the insulin receptor cannot be demonstrated in patients with insulin-resistant lipoatrophic diabetes. Therefore, it is assumed that the lesion(s) must reside in the postreceptor signal transduction pathways. (ADA, 2005)

Diseases of the exocrine pancreas: Any process that diffusely injures the pancreas can cause diabetes. Acquired processes include pancreatitis, trauma, infection, pancreatectomy, and pancreatic carcinoma. With the exception of that caused by cancer, damage to the pancreas must be extensive for diabetes to occur; adrenocarcinomas that involve only a small portion of the pancreas have been associated with diabetes. This implies a mechanism other than simple reduction in -cell mass. If extensive enough, cystic fibrosis and hemochromatosis will also damage -cells and impair insulin secretion. Fibrocalculous pancreatopathy may be accompanied by abdominal pain radiating to the back and pancreatic calcifications identified on X-ray examination. Pancreatic fibrosis and calcium stones in the exocrine ducts have been found at autopsy. (ADA, 2005)

15

Endocrinopathies: Several hormones (e.g., growth hormone, cortisol, glucagon,

epinephrine) antagonize insulin action. Excess amounts of these hormones (e.g., acromegaly, Cushings syndrome, glucagonoma, pheochromocytoma, respectively) can cause diabetes. This generally occurs in individuals with preexisting defects in insulin secretion, and hyperglycemia typically resolves when the hormone excess is resolved. Somatostatinomaand aldosteronoma-induced hypokalemia can

cause diabetes, at least in part, by inhibiting insulin secretion. Hyperglycemia generally resolves after successful removal of the tumor. (ADA, 2009) Drug- or chemical-induced diabetes: Many drugs can impair insulin secretion. These drugs may not cause diabetes by themselves, but they may precipitate diabetes in individuals with insulin resistance. In such cases, the classification is unclear because the sequence or relative importance of -cell dysfunction and insulin resistance is unknown. Certain toxins such as Vacor (a rat poison) and intravenous pentamidine can permanently destroy pancreatic -cells. Such drug reactions fortunately are rare. There are also many drugs and hormones that can impair insulin action. Examples include nicotinic acid and glucocorticoids. Patients receiving -interferon have been reported to develop diabetes associated with islet cell antibodies and, in certain instances, severe insulin deficiency. (ADA, 2005)

16

Infections: Certain viruses have been associated with -cell destruction. Diabetes occurs in patients with congenital rubella, although most of these patients have HLA and immune markers characteristic of type 1 diabetes. In addition, coxsackievirus B, cytomegalovirus, adenovirus, and mumps have been implicated in inducing certain cases of the disease. (ADA, 2005)

Uncommon forms of immune-mediated diabetes: In this category, there are two known conditions, and others are likely to occur. The stiff-man syndrome is an autoimmune disorder of the central nervous system characterized by stiffness of the axial muscles with painful spasms. Patients usually have high titers of the GAD autoantibodies, and approximately one-third will develop diabetes. Anti-insulin receptor antibodies can cause diabetes by binding to the insulin receptor, thereby blocking the binding of insulin to its receptor in target tissues. However, in some cases, these antibodies can act as an insulin agonist after binding to the receptor and can thereby cause hypoglycemia. Anti-insulin receptor antibodies are occasionally found in patients with systemic lupus erythematosus and other autoimmune diseases. As in other states of extreme insulin resistance, patients with anti-insulin receptor antibodies often have acanthosis nigricans. In the past, this syndrome was termed type B insulin resistance. (ADA, 2005)

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Other

genetic

syndromes

sometimes

associated

with

diabetes: Many genetic syndromes are accompanied by an increased incidence of diabetes mellitus. These include the chromosomal abnormalities of Downs syndrome, Klinefelters syndrome, and Turners syndrome. Wolframs syndrome is an autosomal recessive disorder characterized by insulin-deficient diabetes and the absence of -cells at autopsy. Additional manifestations include diabetes insipidus, hypogonadism, optic atrophy, and neural deafness.(ADA, 2009)

Gestational diabetes mellitus (GDM): GDM is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. The definition applies regardless of whether insulin or only diet modification is used for treatment or whether the condition persists after pregnancy. It does not exclude the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy. GDM complicates 4% of all pregnancies in the U.S., resulting in 135,000 cases annually. The prevalence may range from 1 to 14% of pregnancies, depending on the population studied. GDM represents nearly 90% of all pregnancies complicated by diabetes.(ADA, 2005) Gestational diabetes (or gestational diabetes mellitus, GDM) is a condition in which women without previously diagnosed diabetes exhibit high blood glucose levels during pregnancy. Gestational diabetes generally has few symptoms and it is most commonly diagnosed by screening during pregnancy. Diagnostic tests detect inappropriately high levels of glucose in blood samples. Gestational

18

diabetes affects 3-10% of pregnancies, depending on the population studied. The cause is believed to be due to deficiency in Vitamin D. (Wikipedia, 2010) In some cases, though, gestational diabetes can affect the pregnancy and baby. Some potential risks include (National institute of health, 2008)

The babys body is larger than normalcalled macrosomia. A large baby may need to be delivered by a surgical procedure called cesarean section, instead of naturally through the vagina. The babys blood sugar is too lowcalled hypoglycemia. Starting to breastfeed right away can help get more glucose to the baby. The baby may also need to get glucose through a tube into his or her blood. The babys skin turns yellowish and the whites of the eyes may change colorcalled jaundice. This condition is easily treated and is not serious if treated. The baby may have trouble breathing and need oxygen or other helpcalled Respiratory Distress Syndrome. The baby may have low mineral levels in the blood. treated by giving the baby extra minerals. This

problem can causes muscle twitching or cramping, but can be

Table 2. Criteria for the diagnosis of Diabetes mellitus: (ADA, 2009) Symptoms of diabetes plus casual plasma glucose concentration 200 mg/dl (11.1 mmol/l). Casual is defined as any time of day

19

1.

without regard to time since last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss.

2.

FPG 126 mg/dl (7.0 mmol/l). Fasting is defined as no caloric intake for at least 8 h.

3.

2-h post load glucose 200 mg/dl (11.1 mmol/l) during an OGTT. The test should be performed as described by WHO, using a glucose load containing the equivalent of 75 gm anhydrous glucose dissolved in water.

Dyslipidemia: Dyslipidemia is one of the major risk factors for cardiovascular disease in diabetes mellitus. The characteristic features of diabetic dyslipidemia are high plasma triglyceride concentration, low HDL cholesterol concentration and increased concentration of small dense LDL-cholesterol particles. The lipid changes associated with diabetes mellitus are attributed to increased free fatty acid flux secondary to insulin resistance. The availability of multiple lipid-lowering drugs and supplements provide new opportunities for patients to achieve target lipid levels. However, the variety of therapeutic options poses a challenge in the prioritization of drug therapy. The prevalence of hypercholesterolemia is not increased in patients with diabetes mellitus, but mortality from coronary heart disease increases exponentially as a function of serum cholesterol levels, and lowering of cholesterol with statins reduces diabetic patients relative cardiovascular risk. Although drug therapy for dyslipidemia must be individualized, most people with diabetes mellitus are candidates for statin therapy, and often need treatment with multiple agents to achieve therapeutic goals.

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(The above information was reviewed from the article published by Arshag D Mooradian which was published in English manuscript that was identified through the search done on google. The term used to make the search was dyslipidemia in diabetes) Dyslipidemias are disorders of lipoprotein metabolism, including lipoprotein overproduction and deficiency. They may manifest as one or more of thefollowing: elevated total cholesterol, low-density lipoprotein cholesterol (LDL), and triglyceride levels or as decreased high-density lipoprotein cholesterol (HDL) level. Dyslipidemia is closely associated with atherosclerosis and is a major causal factor in the development of ischemic diseases. Ischemic cardiovascular and cerebrovascular events are leading causes of morbidity and mortality. Knowledge of pathophysiology of dyslipidemia has grown dramatically in the last few years, leading to effective treatment strategies. (The above two paragraph were taken from the Essence Series, essential information in brief, a cipla initiative, which was identified through the search made in google.) Patients with type 2 diabetes have an increased frequency of dyslipidemia, which is invariably linked to the presence of insulin resistance and central obesity.(Taskinen MR 1990, Syvanne M et al 1997, Garg A 1998, Belteridge DJ 1999 ) Elevated concentrations of triglyceride-rich lipoproteins, especially very-low-density lipoprotein (VLDL), and decreased levels of high-density lipoprotein (HDL), measured as HDL cholesterol, are the most characteristic lipoprotein abnormalities in type 2 diabetes.(Syvanne M et al 1997, Belteridge DJ 1999) Most patients with type 2 diabetes have concentrations of LDL

21

cholesterol which are similar to those of nondiabetic subjects; (Belteridge DJ 1999) however, in type 2 diabetes there are important qualitative changes in LDL, including the preponderance of smaller and denser particles, and modifications through glycation and oxidation which increase their atherogenicity.( Taskinen MR 1990, Syvanne M et al 1997, Garg A 1998, Belteridge DJ 1999) Recently, studies investigating derangements of postprandial lipid metabolism in type 2 diabetes and their atherogenic potential, (Ebenbichler CF et al 1995, De Man FH et al 1996, Mero N et al 1996 ) have revived interest in the concept that atherosclerosis may be a postprandial phenomenon, which was formulated by Zilversmit more than 20 years ago.(Zilversit DB 1979) Indeed, evidence from clinical studies suggests that postprandial lipemia, characterized by a long residence time of triglyceride-rich remnants in the circulation and subsequent decrease in HDL levels, is an independent risk factor for CHD.(Ebenbichler CF et al 1995, Patsh Jr. et al 1992) In view of their lipid profile, patients with type 2 diabetes may be regarded as permanently-postprandial. This review elaborates upon the lipoprotein abnormalities in type 2 diabetes and the underlying mechanisms, all of which are strongly associated with insulin resistance. In view of the interrelationship of the lipoprotein abnormalities and their atherogenic potential, it is suggested that both fasting and postprandial levels of triglyceride-rich lipoproteins (collectively termed non-HDL cholesterol), rather than LDL cholesterol, may be a more appropriate therapeutic target in type 2 diabetes.

Lipids and lipoproteins:

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Lipids are ubiquitous in the body tissue and have important role in virtually ll aspects of life-serving as hormones or hormone precursors, aiding in digestion, providing energy storage and metabolic fuels, acting as functional and structural components in cell membranes, and forming insulation to allow nerve conduction or to prevent heat loss. The term lipid applies to compound that are soluble in organic solvent and nearly insoluble in water. Chemically, lipids are either compound that yields fatty acids on hydrolysis or complex alcohols that combine with fatty acids to form esters. (TIETZ Textbook of clinical biochemistry and molecular diagnostics, fourth edition) Lipids are a heterogeneous group of organic bimolecules soluble in non-polar solvents such as ether and benzene. (Biochemistry for clinical medicine, 2001) Cholesterol is widely distributed in all cell of the body but particularly in nervous tissue. It is a major constituent of the plasma membrane and of plasma lipoproteins. It can be obtained from the diet or synthesized de novo. The absorption of triglyceride is essentially complete whereas that of cholesterol varies between 30-50%. Endogenous synthesis of cholesterol in the liver is controlled by the rate limiting step involving the microsomal enzyme 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase. (Harpers illustrated biochemistry, 27th edition.) Lipids are transported in plasma as components of lipoprotein complexes. Lipoproteins are spherical complex particles made up of

23

hundreds

of

lipids

and

protein

molecules.

Proteins

called

apolipoproteins occupy the surface of lipoproteins. These serve as an additional interface between lipid and aqueous environments and play an important role in the regulation of lipid transport and lipoprotein metabolism. Lipids are transported in the plasma as lipoproteins. Plasma lipids consist of triglycerols (16%), phospholipids (30%), cholesterol (14%), and cholesteryl ester (36%) and a much smaller fraction of unesterified long chain fatty acids (4%). Four major groups of plasma lipoprotein have been identified that are important physiologically and in clinical diagnoisis. They are (1) chylomicrons, (2) very low density lipoproteins (VLDL), (3) low density lipoprotein (LDL) and (4) high density lipoprotein (HDL). The lipoprotein consist of the protein part known as apolipoprotien or apoprotein, constituiting nearly 70%os some HDL and as little as 1% of chylomicrons.

24

Table 2. Composition of the lipoprotein in plasma of humans.

Lipopro tein chylomi cron Source Diam eter 901000 Density (g/ml) Less than 0.95 Less than 1.006 compositio n Prot Lipi ein d 1-2 9899 Main apolipopr lipid otein compone nts Triacylgl A-I, A-II, ycerol A-IV, B48, C-I, C-II, C-III, E Triacylgl B-48, E ycerol, phosphol ipids, cholester ol Triacylgl B-100, Cycerol I, C-II, CIII Triacylgl B-100, E ycerol, cholester ol cholester B-100 ol Phosphol A-I. A-II, ipids, A-IV, C-I, cholester C-II, C-III, ol D, E

Intesti ne

chylomi cron remnan ts VLDL IDL

chylom 45icron 150

6-8

9294

Liver (intesti ne) VLDL

3090 2535 2025 4-10

0.951.006 1.0061.019 1.0191.063 1.0631.210

7-10 11

9093 89

LDL HDL

VLDL Liver, intesti ne, VLDL, chylom icrons

21 50

79 50

25

Lipoprotein metabolism:
The pathways of lipoprotein metabolism are complex. They include exogenous and endogenous pathways based on whether they carry lipids of dietary or hepatic origin and other pathways such as the intracellular LDL receptor pathway and HDL reverse cholesterol transport pathway.

Exogenous pathway:
Nascent chylomicrons are assembled from dietary TG and cholesterol in the enterocytes and packaged in secretory vesicles in the golgi apparatus. These particles are then transported by exocytosis into the extracellular space and introduced into circulation through the intestinal villi. The lipid content of nascent chylomicrons consists mainly of TG (90% by mass), whereas the protein components include apo B-48 and the A lipoprotein (2% by mass). Shortly after entering the circulation , these particles acquire the C apolipoprotein and apo E from circulating surface of chylomicrons, activities the LPL attached to the luminal surface of endothelial cells, which rapidly hydrolyzes the TG to free fatty acids. The fatty acids taken up by muscle cells as an energy source or into adipose cell for storage. Simultaneously, some of the phospholipids and the apo A apolipoproteins and transferred from the chylomicron particle onto HDL. The newly formed particle,

26

the chylomicron remnant, contains 80% to 90% of the TG content of the original chylomicron. Because of the presence of apo B-48 and apo E on its surface, the chylomicron remnant can be recognized by specific hepatic remnant receptors and internalized by endocytosis, the components of particle are then hydrolyzed in the lysosomes. The cholesterol released can form bile acids, be incorpoted into newly synthesized HMG-CoA lipoprotein, the or be stored limiting as cholesteryl of ester. Furthermore, the cholesterol from these remnants can down regulate reductase, rate enzyme cholesterol biosynthesis.

Figure1: Exogenous and endogenous pathway

27

Endogenous pathway:
Hepatocytes have the ability to synthesize TG from carbohydrates

and fatty acids. In addition, when dietary cholesterol acquired from the receptor mediated uptake of chylomicron remnantsis insufficient, hepato cytes also synthesize their own cholesterol by increasing the activity of HMG-CoA reductase. The endogenously made TG and cholesterol are packaged in secretory vesicles in the golgi apparatus, transported by exocytosis into the extracellular space, and introduced into circulation through the fenestrae of the hepatic sinusoidal endothelium in the form of nascent VLDL. The TG rich particle (55% by mass) contains apo B-100, apo E, and small amount of C apolipoproteins on its surface. Additional C lipoproteins are transferred after secretion from circulating HDL. As in the case of chylomicron metabolism, apo C-II present on the surface of VLDL activates LPL on endothelial cells, which leads to the hydrolysis of VLDL TG and the release of free fatty acids. It is important to note, however, that the rate of hydrolysis of VLDL TG is significantly lower than that of chylomicron triglyceride, the average residence time of VLDL triglyceride is 15-60 minutes, compared with the 5 to 10 minutes of chylomicron triglyceride. This difference may be attributed to the fact that VLDL particles are smaller and bind to fewer LPL molecules than chylomicron.

28

During the hydrolysis of VLDL triglyceride, the C apolipoproteins are transferred back to HDL. VLDL particles are thus converted to VLDL remnants, some of which are taken up by the liver and rest converted to smaller, denser particles called IDL. Large IDL particles, which also have several molecules os apo E, bind the hepatic remnant receptors and are removed from circulation. In humans, about 50% of IDL is removed by hepatocytes. Surface materials from IDL, including some phospholipids, free cholesterol, and apolipoproteins, are transferred to HDL, or from HDL de novo in the circulation. Cholesteryl esters are transferred from HDL to LDL. The net result of the coupled lipolysis and the cholesteryl esters exchange reaction is the replacement of much of the triglyseride core of the original VLDL with cholesteryl esters. IDL undergoes a further hydrolysis in which most of the remaining triglyceride are removed and all apolipoproteins except B-100 are transferred to other lipoproteins. This process ends with ultimate formation of LDL.

Low density lipoprotein receptor pathway:

The mechanism by which LDL is removed from circulation is well understood. Specific receptors present in coated pits on plasma membrane recognize and bind apo B-100 of LDL. The LDL particles are internalized in coated vesicles, which then fuse to form an endosome, because of the acidic medium of the endosome, LDL dissociates from the receptor, which returns to the cell surface for reuse, whereas LDL migrate to the lysosome. Once the LDL is delivered to the lysosome, apo B-100 is degrade to small peptides and

29

amino acids. Cholesterol esters are also hydrolysed, with the cholesterol ten available for the synthesis of cell membranes, steroid hormones in tissue that make them, and bile acids in hepatocytes. Cells have the ability to regulate their cholesterol content. Compared with VLDL and chylomicrons, LDL has a relatively long residence in circulation, about 3 days. LDL is also taken up by extrahepatic tissue through scavenger receptor or non-receptor mediated pinocytosis. The non-receptor mediated uptake becomes important as plasma LDL comncentration increase, as in familial hypercholesterolemia. Non-receptor mediated uptake is not saturable amd not regulated. Scavenger receptor are unregulated as well and recognize LDL that has been modified in various way. They are found in macrophages and other cells. Macrophages that become engorged with cholesteryl esters are called foam cells and are considered the earliest components of the atherosclerotic lesion. Two thirds of LDL is normally removed by LDL receptors and remainder by the scavenger cell system.

Reverse Cholesterol Transport:

Reverse cholesterol transport refers to the process by which cholesterol is removed from the tissues and returned to the liver. HDL is the key lipoprotein involved in reverse cholesterol transport. and the transfer of cholesteryl esters between lipoproteins. (TIETZ Textbook of clinical biochemistry and molecular biology, fourth edition)

Physiology of lipid metabolism:

In order to enable understanding of the lipoprotein abnormalities in type 2 diabetes,the physiology of the lipoprotein metabolic cascades

30

is summarized

below.

Lipoprotein metabolism can be divided into two major pathways, the apolipoprotein (apo) B-lipoprotein and HDL pathways.(Brewer HB Jr. 1995, 1999) The apo B-lipoprotein pathway consists of a cascade of lipoproteins containing either the apo B-48 or the apo B-100 isoform of the B apolipoprotein secreted from the intestine or liver, respectively. The triglyceride-rich apo B-48-containing chylomicrons transport dietary lipids from the intestine to the liver and peripheral tissues. The catabolism of the triglyceride-rich chylomicrons is initiated by the endothelial enzyme, lipoprotein lipase (LPL), which hydrolyzes the triglyceride core of the chylomicrons and releases fatty acids for energy production in muscle and for storage in adipose tissue (Figure 1).

31

Figure density

2.

Schematic

overview

of

lipoprotein elevated

metabolic postprandial

pathways in type 2 diabetes. Increased endogenous very-lowlipoprotein (VLDL) production, triglyceride (TG) concentrations, and impaired endothelial lipoprotein lipase (LPL) activity lead to raised plasma concentrations of TG-rich lipoproteins (LP). High TG-rich lipoprotein concentrations increase the transfer of TG to low-density lipoprotein (LDL) and high-density lipoprotein (HDL), and simultaneous transfer of cholesteryl esters (CE) from LDL and HDL to TG-rich lipoproteins, mediated by cholesteryl ester transfer protein (CETP). Hydrolysis of core TG by hepatic lipase (HL) produces small, dense (lipid-poor and protein-enriched) LDL that are modified (eg. by oxidation or glycation). Similarly, small, dense HDL particles are produced that have a higher catabolic rate and may be dysfunctional with regard to reverse cholesterol transport. IDL, intermediate-density lipoprotein; FA, fatty acids. (Syvanne M et al 1997) The chylomicron remnants are taken up by the liver by the LDLreceptor-related protein and LDL receptors, which have a high affinity for apoE. (Kreiger M, 1994) In a parallel cascade, the liver assembles triglyceride-rich VLDLcontaining apo B-100, which, similarly to chylomicrons, undergoes hydrolysis by LPL and is remodeled to intermediate-density lipoprotein (IDL) and partly further to LDL (Figure 1). The latter step involves another enzyme, hepatic lipase (HL). VLDL remnants and IDLcontaining apo E are removed by hepatic LDL receptors and LDLreceptor-related protein receptors.(Krieger M 1994, Goldstein JL 1985) LDL contains only apo B-100 and is metabolized by two pathways: it may be taken up into the liver by an LDL-receptor-mediated mechanism, but it also may become modified or oxidized and

32

removed by the scavenger receptor-A or CD36 scavenger receptors on macrophages. In the HDL pathway, HDL is synthesized as nascent HDL particles from both the liver and intestine as well as by transfer of lipids and apolipoproteins during the metabolism of triglyceride-rich chylomicrons and VLDL. A major function of HDL is the transport of excess cellular cholesterol from peripheral cells to the liver, a process called reverse cholesterol transport (RCT).(Glosmet JA 1968, Fielding CJ 1995, Bruce C 1995) RCT is initiated by the removal of nonesterified or free cholesterol from cells mediated by the ATPbinding cassette transporter ABCA1 (formerly ABC1).(Oram JF 2000) In plasma, free cholesterol on nascent HDL is converted to cholesteryl ester (CE) by lecithin cholesterol acyltransferase.(Glosmet JA 1968, Bruce C 1995) The CE synthesized on HDL can be transferred to apo B-containing lipoproteins by the cholesteryl ester transfer protein (CETP) in exchange for triglycerides (Figure 1).(Bruce C 1995) Hepatocytes remove CE from HDL and apo B-containing lipoproteins either by direct uptake of the whole lipoprotein particle (mediated by LDL- and LDL-receptor-related protein receptors) or by selective removal of CE from the particle (mediated by the scavenger receptor BI).(Action S, Rigotti A, Landschulz KT, Xu s Hobbs HH, Krieger M 1996)

Lipoprotein metabolism in type 2 diabetes: the impact

of

insulin resistance:

In type 2 diabetes, insulin resistance and visceral obesity have a major impact on the regulatory processes of both the apo Blipoprotein and the HDL pathways. Many steps in the lipoprotein metabolic cascades are insulin-sensitive. (Taskinen MR 1990, Syvanne

33

et

al

1997)

Apo B-lipoprotein pathway in diabetes:

The insulin-resistant state impairs the normal suppression of free fatty acid (FFA) release from the abundantly present adipose tissue. This increased flux of FFA to the liver is a major stimulus for overproduction of VLDL. Postprandially, independent of the FFA flux, hepatic VLDL production is not normally suppressed, resulting in competition for LPL with exogenous triglycerides carried on chylomicrons.(Malmstrom R et al 1997) LPL activity is lower in type 2 diabetics than in nondiabetic subjects and increases with improved glycemic control.[8] Thus, the raised concentration of triglyceride-rich lipoproteins in type 2 diabetes is due to hepatic overproduction of VLDL and impaired clearance of apo B-containing remnant particles (Figure-1). The prolonged circulating time of high triglyceride-rich lipoprotein particles allows longer exposure to CETP, which facilitates transfer of cholesterol from LDL and HDL to VLDL and chylomicrons in exchange for triglycerides. (Bagdade JD et al 1990) The enhanced transfer of triglycerides to LDL renders them better substrates for HL. HL hydrolyzes triglycerides from the core of LDL and turns them into smaller and denser LDL particles. The same holds true for modification of HDL. Small, dense LDL particles and triglyceride-rich lipoproteins readily enter the artery wall and show substantial intimal retention, an important step in the development of atherosclerosis (Mamo JC et al 1998, Tabas I 1999).

34

Retained lipoprotein particles may then undergo enzymatic or oxidative modifications.(Tabas I 1999)These qualitative changes may increase the atherogenicity of LDL particles in type 2 diabetes. (Syvanne M et al 1997, Belteridge DJ 1999) Epidemiological studies have shown a relation between small-sized LDL particles and the risk of myocardial infarction in nondiabetic populations.(Stampfer MJ et al 1996, Lamarche B et al 1998) In type 2 diabetes, small LDL particles from patients were associated with reduced endothelium-dependent arterial dilation, which is a surrogate marker for cardiovascular risk. (Tan KC et al 1999) It should be noted, however, that prospective studies evaluating the role of small, dense LDL particles in atherogenesis in type 2 diabetes are still awaited. An inverse relationship exists between hypertriglyceridemia and the number of small, dense LDL particles.( Scheffer PG et al 1998, Steiner G 1998) Indeed, lowering triglyceride levels in type 2 diabetes using fibrates was shown to increase LDL particle size.(Lahdenpera S 1993)

HDL pathway

in type 2

diabetes:

The above-described changes in apo B-lipoprotein metabolism in type 2 diabetes strongly affect the HDL pathway and consequently its antiatherogenic potential. In type 2 diabetes, analogous to LDL, triglyceride-enriched HDL is readily modified into small, dense HDL, which has an increased metabolic rate and helps to explain the low HDL concentration. Also, modified HDL in type 2 diabetes has been associated with CHD because its compositional changes may lead to impaired RCT.(Syvanne M et al 1995, 1996) It has been suggested that other abnormalities in the RCT cascade may contribute to the development of CHD in type 2 diabetes. Both increased and decreased CETP activity have been described in type 2 diabetes.

35

(Jones RJ 1996, Quntao EC 2000) Although as yet this controversy has not been conclusively settled, population studies indicate that with regard to the development of atherosclerosis, plasma HDL rather than CETP levels maybe of more importance. Postprandial lipid abnormalities in type 2 diabetes In healthy subjects, the duration of the postprandial state depends on the composition of the meal: after a meal consisting mainly of carbohydrates, a return to the basal state occurs within 23 h, after a mixed meal it takes 35 h, and after a fat-rich meal the return to baseline may take as long as 810 hours.(Schrezenmeir J 1993) Since most individuals eat intermittently throughout daylight hours, they are postprandial for about 18 h of each 24-h day.(Kreiberg KA 1998) In view of their abnormal lipoprotein profile, type 2 diabetic patients may be regarded as postprandial throughout 24 h. Following a fatenriched meal, the rise in triglyceride-rich lipoprotein concentrations is greater in type 2 diabetic patients than in nondiabetic subjects, and the fasting level of triglyceride-rich lipoproteins is positively correlated with the area under the curve for plasma triglyceride levels after the meal (Figure 2).(Levis GF et al 1991)

36

Figure 3. Postprandial changes in triglyceride levels in patients with type 2 diabetes mellitus: relation to fasting triglyceride level. (Levis GF et al 1991) This postprandial lipemia is due to an impaired suppression of hepatic VLDL synthesis after meals and the competition of chylomicrons and their remnants with endogenous VLDL particles for common removal pathways through LPL (Figure 1). These mechanisms, which are linked to insulin resistance, collectively result in prolonged exposure of arteries to potentially atherogenic triglyceride-enriched particles. Also, postprandial lipid disturbances have been associated with alterations in coagulation mechanisms that predispose them to arterial thrombosis.(Selvieria A 1996, Georgopoulus A et al 1998) Recent studies have demonstrated impaired endothelium-dependent vasodilation following a fatty meal both in healthy subjects and in type 2 diabetic patients.(Vogel RA 1997, Shige H 1999) In addition, an association was found between postprandial levels of triglyceride-rich remnants and the severity of coronary artery disease in type 2 diabetic patients.(Mero N et al 2000) The true atherogenic potential of postprandial dyslipidemia in type 2 diabetes, however, still has to be demonstrated in outcome studies.

Atherogenecity of diabetic dyslipidemia: the case of non-HDL particles:

There have been frequent attempts to determine which of the dyslipidemic alterations in type 2 diabetes is the most atherogenic, and to assign an independent risk status to changes in the individual lipoproteins.

37

Based on a large body of evidence, current clinical guidelines have identified LDL as the major atherogenic lipoprotein and the primary target of lipid-lowering therapy.(JAMA 1993) As stated before, patients with type 2 diabetes do not have marked elevations of LDL cholesterol; however, the importance of LDL as a risk factor for CHD in diabetic patients has been demonstrated in subgroup analyses of the major secondary prevention trials, such as the Scandinavian Simvastatin Survival Study, the Cholesterol and Recurrent Events trial, and the Long-term Intervention with Pravastatin in Ischemic Disease trial.(Sacks FM et al 1996, N Engl J Med 1999) In all these trials intensive LDL-lowering therapy reduced recurrent CHD events in type 2 diabetic patients. In view of the close association between the lipoprotein abnormalities in type 2 diabetes, mere estimates of LDL cholesterol levels may underestimate the total atherogenic risk potential associated with the total apo B-containing lipoprotein fractions and low (modified) HDL. And, although no prospective trials have been conducted on the effects of lipid-lowering agents on subsequent CHD specifically in diabetic populations, several prospective studies in type 2 diabetics have convincingly shown a strong association between the different lipoprotein abnormalities and CHD in these patients. Thus, triglyceride levels were positively associated with increased risk for CHD in populations with type 2 diabetes.(Foutbonne A et al 1989, Leato S et al 1993, Laakso M et al 1993 ) In a 7-year prospective study in type 2 diabetics, low HDL, HDL2 cholesterol, and high levels of total and VLDL triglycerides and VLDL cholesterol were all found to be powerful risk indicators for CHD events.(Laakso M et al 1993) In the UKPDS, beside LDL cholesterol, decreased HDL cholesterol was identified as

38

an indicator of CHD.(Turner RC, Millus H, Neil HA, Stratton IM, Manly SE, Matthews DR, 1998) Thus, it appears that in type 2 diabetes all major lipoprotein abnormalities, including elevated triglyceride-rich lipoproteins, low (modified) HDL cholesterol, and small, dense (modified) LDL particles, constitute the atherogenic phenotype independent of LDL cholesterol levels. (Garg A 1998, Vogel RA 1997) In patients with type 2 diabetes, all these derangements are present in the fasting and, even more so, in the postprandial state. In view of their atherogenic properties, therapeutic interventions in patients with type 2 diabetes should be aimed at lowering fasting and postprandial levels of triglyceride-rich lipoproteins, also termed non-HDL particles. (Garg A 1998) Table I lists the lipoprotein abnormalities in type 2 diabetes and their proposed atherogenic potential, which needs to be confirmed in prospective studies. The study done by Regmi P, Gyawali P, Shrestha R, Sigdel M, Mehta D K, Majhi S, department of biochemistry, BP Koirala Institute of Health Sciences, Dharan, and department of biochemistry, IOM, TUTH, Kathmandu, among the 144 type 2 diabetic individual and 56 healthy controls showed that hypercholesttrolemia was found in 51(35.4%) individuals, similarly hypertriglyceridemia was found in 90(62.5%) individuals, decreased HDL C was found in 54(37.5%) individuals and increased LDL C was found in 69(47.9%)individuals. Study by Curtis et al in african-american type 2 diabetic individuals found higher LDL in 58% individuals, low HDL-C in 41% individuals an higher TG in about 50% individuals. The study of Sehran et al in pakistan, 54% diabetic individuals had LDL-C, greater than 50% individuals had increased TG. They also found decreased HDL-C in 73% individuals. Similarly 16%, 48% and

39

34% individuals respectively had one, two and more than two abnormal parameters. Studies have generally suggested a positive association between dyslipidemia and Inflammation and end-stage renal disease or advanced chronic kidney failure, but the relation between these biomarkers and mild or moderate renal dysfunction has Not been well characterized, especially in type 2 diabetes mellitus (DM). Type 2 DM is now Recognized as an inflammatory condition associated with insulin resistance and abnormal endothelial vascular reactivity. As the leading cause of kidney disease and an important cause of cardiovascular disease (CVD) in the Western world, type 2 DM is an increasingly prevalent etiology of micro vascular and macro vascular disease. A chronic, systemic inflammatory state has been proposed to underline this increased risk for atherosclerotic disease, including renal dysfunction and CVD; however, investigations have not focused on the relation between glomerular filtration rate and dyslipidemia or inflammatory biomarkers in individuals with type 2 diabetes. Identification of such serologic markers may reveal new approaches to the prevention of progressive renal insufficiency and CVD in this population. (Julie Lin et al, 2006). Defining the relationship between body weight and metabolic diseases is critical toward better understanding of the underlying pathophysiological processes leading to these diseases. Data from the two national surveys reported here support the common clinical observation that patients with higher BMI are at higher risk for having diabetes mellitus, hypertension and dyslipidemia. They also confirm the converse the majority of patients with these metabolic diseases are either overweight or obese (H. E. Bays et al, 2007).

40

Hypertension and Hyperlipidemia management in type 2 diabetes patients has improved in the past decade but further improvement is possible. Greater effort is needed to stimulate medication adjustments in patients with insufficiently controlled hypertension and combined risk factors, (Jacoba P Greving et al, 2007). Adolescents with type 1 DM had the lowest tHcy values which were reflective of the limited extant research with this population. Lipid profiles and dietary energy did not differ significantly among the 3 groups. Hemoglobin A1C was related to total cholesterol and triglycerides in those with type 1 DM, confirming the importance of promoting better control in lipid management for these youth. Future research should continue to explore the validity of tHcy and lipids as predictors of CV risks for youth with type 1 and type 2 DM. (Wei-Hsun Chao et al, 2006). Among patients with type 2 diabetes, the association between LDL cholesterol and cancer was V Shaped, whereby both low and high levels of LDL cholesterol were associated with elevated risk of cancer. (Xilin Yang et al, 2008) Two measurements of fasting triglyceride levels obtained 5 years apart can assist in identifying apparently healthy young men at increased risk for diabetes, independent of traditional risk factors and of associated changes in BMI and lifestyle parameters. ( Rafael Bitzur et al, 2008) As an additional measure of the quality of care, measuring therapy modifications in response to poor control in a large population is feasible. Many patients with poorly controlled hypertension, dyslipidemia, or diabetes had their therapy modified and, thus,

41

seemed to receive clinically appropriate care with this new quality measure. (Nicolas Rodondi et al, 2006) Women have a significantly higher prevalence of hypertension than men (76.6% vs. 63.0%) and a significantly lower rate of control when treated pharmacologically (42.9% vs. 57.9%). Dyslipidemia prevalence is 60.3% overall, and women are significantly more likely to be aware of their condition than men (71.1% vs. 59.1%). Diabetes affects 21.2% of older adults for and all 50.9% of prevalent among cases those are treated is (48.8%), pharmacologically. problematic Goal attainment conditions treated

three

hypertension

dyslipidemia (64.9%), and diabetes (50.4%). Having two or more doctor visits annually is associated with goal attainment for dyslipidemia. (Margaret McDonald et al, 2009) Dyslipidemia commonly associated with type II diabetes includes elevated triglycerides, low Levels of HDL-C, and preponderance of small dense LDL particles, with LDL cholesterol generally being similar to that of no diabetic patients. The cornerstone of treatment for diabetic dyslipidemia is therapeutic lifestyle change. In addition to these measures, recent clinical trials have demonstrated the benefits of statin therapy. Therapy with 3-hydroxy-3-methylglutaryl (HMG) Coreductase inhibitor (statins) in both the Heart Protection Study (HPS) and the Collaborative Atorvastatin Diabetes Study (CARDS) has shown that there is clear reduction in cardiovascular events in type 2 diabetes. (Ishwarlal Jialala et al, 2009)

42

CHAPTER III RESEARCH METHODOLOGY

Study design: Cross sectional, descriptive Study site: Study was conducted in Manmohan Memorial Community Hospital, Lekhnath Marg, Thamel, Kathmandu. Study duration: Study was conducted for the duration of 3 month From July 2010 to September 2010. Study method: Quantitative Study population: People with diabetes mellitus type 2 Study subjects:

43

Patient attending Out Patient Department (OPD) and wards of Hospital Sample size: 40 type 2 diabetic patients and 40 non-diabetic patients.
Instrumentation:

Laboratory investigation Inclusion Criteria: Patient with Diabetes mellitus . Patient not suffering from any mental illness and malignant diseases. Patient not suffering from coronary heart disease.

Exclusion Criteria :

Patient who were unconscious. Patients who refused to give consent for blood examination.

Ethical consideration: Approval from institution was taken before carrying out study. Before drawing blood, verbal consent of patients was taken. The patient who refused to give consent was not included in the study.

Reliability and Validity: Appropriate sample size was selected. Standard study design was applied.

44

 Study variables: Biochemical indicators Serum TG Serum LDL-C Serum HDL-C Serum total cholesterol.

CHAPTER IV MATERIALS AND METHOD This study was conducted in the department of pathology, Clinical laboratory services, Manmohan Memorial Hospital, ThamelKathmandu, Nepal. Total 40 type 2 diabetic individuals and 40 nondiabetic individuals were included in the study. Blood sample was collected from 10-12 hr fasting subjects. Serum was separated within 30 min of blood collection. Serum glucose was measured by enzymatic glucose oxidase-peroxidase method. Serum total cholesterol (TC) level was measured by enzymatic methods. Highdensity lipoprotein cholesterol (HDL-C) was measured by precipitation followed by enzymatic methods. Low-density lipoprotein cholesterol (LDL-C) and very-low density lipoprotein cholesterol (VLDL-C) were calculated by Fridewalds formula. For serum lipids, a National Cholesterol Education Programme-Adult Treatment Panel III (NCEP-ATPIII) guideline was referred. According to NCEP-ATPIII guideline, hypercholesterolemia is defined as TC>200mg/dl, hypertriglyceridemia as >150mg/dl, LDL-C as 150mg/dl

45

and LDL-C as >150mg/dl. Dyslipidemia is defined by presence of one or more than one abnormal serum lipid concentration. Statistical analysis was done using SPSS (version 16.0).

Estimation of serum HDL-C and LDL-C:

(Peg/Chod-PAP method) PRINCIPLE: Lipoproteins are the proteins which mainly transport fats in the blood stream. They can be grouped into chylomicrons, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Chylomicrons and VLDL transport mainly triglyceride(TG), though VLDL also transport cholesterol. LDL carries cholesterol to peripheral tissues where it can be deposited and increase the risk of coronary heart disease (CHD). Hence, low LDL level is atherogenic. HDL transport cholesterol from peripheral tissue to liver for excretion, of total thus and LDL HDL has protective effect. The measurement cholesterol provide valuable

information for the risk of CHD.

PROCEDURE:

46

Wavelength/filter: 505 nm/green Temperature: 37*C Light path: 1 cm Precipitation of VLDL and LDL: Precipitating reagent Sample 0.1 ml 0.1 ml

This was mixed well and was incubated at room temperature for 5 minutes, and then it was centrifuged at 2500-3000 rpm to obtain clear supernatant.

Cholesterol assay: Three clean dry test tubes were taken and labeled and then pipetted with reagent as follows: Addition sequence Working reagent D/W HDL standard Supernatant 0.05 0.05 0.05 1.0 1.0 1.0 Blank (ml) Standard (ml) Test (ml)

47

This was mixed well and was incubated at 37*C and OD was measured. Calculations: HDL (mg/dl) = Absorbance of test/absorbance of std, x 25 x 2. Where 2 is the dilution factor due to depolarizing step. LDL = total cholesterol-TG/5-HDL (Freidwalds formula)

Estimation of serum triglyceride:

PRINCIPLE:

48

Lipoprotein lipase hydrolyses TG to glycerol and fatty acid. The glycerol formed with ATP in the presence of glycerol kinase forms glycerol-3-phosphate oxidase to form H2O2. The H2O2 further reacts with phenolic compound and 4-amino-antipyrine by the catalytic action of peroxidase to produce a red colored quinine imino complex. The presence of TG in serum is directly proportional to the concentrations of substances in serum.

PROCEDURE: Three test tubes were taken and labeled as blank (B), standard (S) and test (T) respectively. Addition sequence Working reagent D/W TG Serum 1.0 0.01 1.0 0.01 1.0 0.01 Blank (ml) Standard (ml) Test (ml)

These was mixed well and incubated at 37*C and absorbance was measured. Calculation: Concentration of test = OD of test/OD of std. x 200 Normal range: serum/plasma (suspicious) = 150mg/dl (Elevated) = 300 mg/dl

49

CHAPTER V RESULT
The study include 40 type 2 diabetic individuals and 40 healthy controls who were the patients attending Manmohan Memorial Community Hospital. Baseline values of glucose and lipid is presented with their SD. Table 3: Baseline character of diabetic subjects and controls. Biochemical parameters Diabetic subjects Mean(SD) mg/dl Healthy controls Mean(SD) mg/dl

Total cholesterol(TC)

182.20(49.840)

170.08(19.248)

Triglyceride(TG)

214.32(119.942)

154.40(45.760)

HDL-C

46.98(8.328)

52.15(5.816)

LDL-C

92.35(44.763)

88.68(22.085)

According to my study the following result was found:

50

1. Hypercholesterolemia was found in 13 diabetic patient among 40 i.e. 32.5%. 2. Hypertriglyceridemia was found in 25 diabetic patient i.e. 62.5%. 3. Increased LDL-C was found in 19 (47.5%) individuals. 4. Decreased HDL-C was found in 7 (17.5%) individuals. 5. Among 40 diabetic patients 30 individuals had any two abnormalities, 27 individuals had three abnormalities and 7 individuals had all four abnormalities.

Figure

4:

Relation

between

diabetic

and

non-diabetic

individuals in regard to lipid profile. The above bar diagram represents the relation between the type 2 diabetic patient and healthy control on basis of lipid profile. It was

51

found that there was no any significant corelation between diabetic and healthy control on basis of Cholesterol, HDL-C and LDL-C. but it was found a marked significant on triglyceride, the diabetic patient had much more higher TG level compared to the healthy control.

CHAPTER VI

52

DISCUSSION
The objective of my study was to evaluate dyslipidemia in type 2 diabetic individuals. The study reveals the prevalence of hypercholesterolemia, hypertriglycerademia, abnormally high LDL-C, and low HDL-C levels which are well-known risk factors for cardiovascular disease. There are several factors that are responsible for diabetic dylipidemia: 1. Insulin effects on liver apoprotein production. 2. Regulation of lipoprotein lipase (LpL). 3. Action of cholesterol ester transfer protein(CETP). 4. Peripheral actions of insulin on muscle and adipose tissue. Hepatic lipase is an enzyme synthesized by hepatocytes that hydrolyses phospholipids and triglycerides on HDL remnant lipoproteins. Some studies suggest that this enzyme is reduced byinsulin deficiency. One effect of hepatic lipase deficiency is to decrease the clearance of remnant lipoproteins. LpL is the major enzyme responsible for convertion of lipoprotein triglyceride into free fatty acids. The reason for decrease in HDL found in patient with diabetes maybe due to increased concentration of plasma VLDL drive the exchange of triglyceride from VLDL for the cholesteryl esters found in HDL. In the study of Regmi et. al. in Kathmandu, 62.5% diabetic individual had hypertriglycedemia, 35.4% had hypercholesterolemia and 47.9% had increased LDL-C. These findings are similar to my study. But compared to our study they found decreased HDL-C in 37.5%.

53

In another study carried by Sehran et al in pakistan, >50% individuals had increased TG, 54% individuals had elevated LDL-C. these findings are also similar to my findings but they found decreased HDL-C in73% which is higher compared to my study. In my study dyslipidemia was found in almost all the diabetic patient with one or more than one abnormal lipid concentration. The main factor responsible for hypertriglyceridemia in our population could be diets that are rich in carbohydrates and the standard life style among the rich people. Diet with high fat and calorie intake and lack of physical activity could be the major factor contributing dyslipidemia among our population. Besides it also involves the over-cooking of food, frying and refrying in the same oil which causes trans fatty acid formation which contributes to increase of dyslipidemia in our population.

CHAPTER VII

54

CONCLUSION
Dyslipidemia in diabetes is a major problem and the individuals who are diabetic with dyslipidemia are at higher risk for cardiovascular disease. Diabetic individuals must frequently monitor their lipid profile as they are higher risks associated with dyslipidemia. As per my study I came to the conclusion that in the most cases of type 2 diabetic patients they come across the risk for cardiovascular disease which is reflected by the increased TG, Cholesterol or LDL and decreased HDL. My study concludes me to a point that most of the type 2 diabetic patients are more likely to have hypertriglyceridemia which was found in most of my cases taken.

CHAPTER VIII

55

RECOMMENDATION
The present study would provide information that lipid profile seems destined to continue to be the most valuable parameter for assessing the complication of diabetes. However the present study due to various constraints and time limitation is preliminary so some recommendations are listed below. This study was conducted with low sample size, it show the necessities of large sample size so that more accurate result would be produce. This study do not categorize the patient with duration of diabetes mellitus, so for further best result, the categorization of the patient would be more beneficial.

56

CHAPTER IX BIBLIOGRAPHY
1. ADA, International Expert Committee Report on the Role of the

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2. American Diabetes Association: Diagnosis and Classification of

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3. American

Optometric

Association,

OPTOMETRIC

CLINICAL

PRACTICE GUIDELINE CARE OF THE PATIENT WITH DIABETES MELLITUS, 2009. 4. American Diabetes Association, Standards of Medical Care in Diabetes, 2010. 5. American Diabetes Association, Standards of Medical Care in Diabetes, 2009.
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7. Stamler J, Vaccaro O, Neaton JD, Wentworth D, Department of

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DR, Holman RR, BMJ 1998 Mar 14;316(7134):823-8

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MR, Hyperlipidaemia in diabetes, Baillieres Clin

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risk factors in non-insulin-dependent diabetes mellitus, Lancet 1997 Jul;350 Suppl 1:SI20-3
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Bruin TW, Triglyceride-rich lipoproteins in non-insulin-dependent diabetes mellitus: post-prandial metabolism and relation to premature atherosclerosis, Eur J Clin Invest 1996 Feb;26(2):89108
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WORK PLAN

66

Activities

July week) 1-2

(in August 3-4 1-2 3-4

September October 1-2 3-4 1-2 3-4

Selection of study area, objectives and drafting of proposal and defense Visit to Manmohan Memorial Community HospitalMMCH and data collection Data entry and analysis

Finalizing the report

Thesis defense and dissemination

67