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Diabetes mellitus is a metabolic disease in which carbohydrate utilization is reduced whilst that of lipid and protein is enhanced1. It is caused by an absolute or relative deficiency of insulin and is characterized, in more severe cases, by chronic hyperglycaemia, glycosuria, water and electrolyte loss, ketoacidosis, and coma1,2. There are two classifications of diabetes mellitus which are namely primary and secondary classes13. The primary class is divided into diabetes type 1 and diabetes type 22,13. Diabetes type 1 is an autoimmune disorder characterised by insulin deficiency1,2. In diabetes type 2 the cells are unable to respond to normal levels of insulin because of decreased insulin receptors and relative insulin deficiency2.3. The secondary class of diabetes mellitus includes maturity onset type diabetes of the young (MODY), pancreatic disease, endocrine disease and gestational diabetes. MODY is characterised by onset prior to 25 years of age due to impairment of β cell function because of mutations of the various genes which includes hepatic nuclear factor -4α ,pancreatic glucokinase and homeodomain transcription factor insulin promoter factor(IPF-1)13.
1.1.1 Epidemiology of diabetes mellitus
Diabetes mellitus affects 6% of the world's population14. WHO estimates that more than 180 million people worldwide have diabetes mellitus and this number will most likely double by 203014,15. In 2005 an estimated 1.1 million people died from diabetes and 80 % of these deaths occurred in low and middle income countries such as Zimbabwe, half of these people being under the age of 70 and 55 % being women16. Before the 1990s, diabetes was considered a rare medical condition in Africa16. Almost all the reports published between 1959 and 1985 showed a prevalence of diabetes below 1.4 %, except those from South Africa, where higher prevalence was reported15,16.
The prevalence of diabetes in Africa was approximately 3 million in 1994; but the region has experienced a two-to threefold increase by the year 201017. Among the population of Indian origin in South Africa and Tanzania, the prevalence is between 12 and 13 %18. The prevalence in blacks follows a Westernization gradient, with that of rural Africa generally below 1 % and that of urban Africa between 1 and 6 %. In general the prevalence of type 2 diabetes is low in both rural and urban communities of West Africa except in urban Ghana, where a high rate of 6.3 % was recently reported19. Moderate rates have been reported from South Africa: 4.8 % in a semi-urban community in the Orange Free State, 6.0 % in an urban community of the Orange Free State, 5.5 % in Durban and 8 % in Cape Town17. The incidence of type 1 diabetes ranges from 1, 9 to 7, 0/100 000/year in Africa14,15. The prevalence of type 2 diabetes is 0, 3 -1, 17 in Africa14,15. Type 2 diabetes accounts 90% of diabetics worldwide14. Type 1 diabetes affects 3 in 1 000 children14,15.
The prevalence of diabetes mellitus in Zimbabwe from 1991-97 increased from 150 to 550 per 100 000 people. According to the Zimbabwe National Health profiles (1996-98) the number of new cases recorded in the ages 15years and above rose from 2734 cases in1996 to 5114 in 1998 which is an increase of 87% of recorded cases. In Zimbabwe diabetes mellitus is among the top five chronic conditions seen in the OPD Clinics. The 2005 survey noted that the prevalence among the adult population is 10% with a large number unaware of their increased glucose levels.
1.1.2 Pathogenesis of diabetes mellitus
Type 1 diabetes mellitus is an auto-immune disorder whereby tolerance is broken down resulting in destruction of insulin producing β cells20. Risk factors of developing type 1 diabetes mellitus include disease of the pancreas that inhibits insulin production, exposure to cow's milk proteins, environmental factors, genetics and family history20,21.
The main gene associated with the development of type 1 diabetes is major histocompatibility complex (MHC). The MHC region is associated with the genes for immune system recognition known as HLA-DQ, HLA-DR and HLA-DP22. The pancreas is a sequestered organ so its antigens are not presented to the T-lymphocytes and B-lymphocytes during their development23. T and B-lymphocytes are reactive against self-antigens of the pancreas mounting an immune response and destroying the pancreatic cells20,23. Damage to the membrane sequestering the pancreas exposes the pancreatic antigens on islet cells resulting in destruction of the β cells by the production of the following antibodies islet cell cytoplasmic antibodies that react with antigens located in the cytoplasm of the pancreatic islet cell, islet cell antibody that targets islet antigens on β cells ,anti-glutamic acid decarboxylase which destroys glutamic acid decarboxylase expressed on pancreatic cells therefore destroying β cells resulting in insulin deficiency20.
Environmental factors such as viruses also lead to the destruction of β cells. Infection of the β cells with a virus such as the coxasckievirus, adenovirus, cytomegalovirus (CMV) causes destruction of β cells due to molecular mimicry of the viral protein to the β cell so when the body mount an immune response against the viral proteins it also destroys the β cells leading to type 1 diabetes22. The infected β cell releases cytokines which activates anti-viral cytotoxic lymphocytes, cytokines and radicals produced by macrophages augment the cytotoxic response to β cells22. Other environmental factors such as vaccines ,toxins and diet for example if cow's milk is given to a baby its leads to β cell destruction because cow's milk have the same sequence with β cell so an immune response to the cow's milk also destroys the β cells causing type 1 diabetes25. Strain of foot and mouth virus damage the β cells directly resulting in type 1 diabetes25.
Inflammatory response to pancreatitis especially the reactive oxygen species (ROS) damages the β cells because β cells weakly express the enzyme that reduces the superoxide24. Continued release of cytokines at the inflammatory site leading to overexpression of HLA-1 on β cells potentiating their destruction by production of antibodies, antibody dependent cell cytotoxicity (ADCC) and cytotoxic cytokines ( IL-7 ,IL-1)24.
Under normal circumstances plasma glucose concentration is maintained within a narrow range and is tightly regulated with a dynamic interaction between tissue sensitivity to insulin and insulin secretion28. In type 2 diabetes this mechanism is broken-down due to defects in impaired insulin secretion and action due to resistance. Risk factors of developing type 2 diabetes mellitus include obesity, sedentary life style, family history, above 45 years of age, insulin resistance, history of gestational diabetes, increased triglycerides levels and hypertension26.
The aetiology of type 2 diabetes includes genetic defects in insulin action due to mutation of the insulin gene and receptor, disease of the pancreas e.g. hemochromatosis, pancreatic carcinoma and chronic necrotizing pancreatitis which decrease the β cell size and drugs that impair insulin action e.g. cyclosporin A .
The pathogenesis of type 2 diabetes mellitus is insulin resistance and insulin insufficiency26. Insulin resistance involves normal levels of insulin being produced but the receptors are insensitive26. The receptors are insensitive due to mutations in the receptor gene therefore it codes for a defective receptor which is not sensitive to insulin26,28. The receptor can be normal but the insulin produced is abnormal or there is incomplete conversion of the proinsulin molecule due to mutation in the proinsulin to insulin cleavage sites, the insulin will have a defective biological function26, 28. The insulin can be normal but are circulating antagonists such as anti-insulin receptor antibody which destroys the insulin receptors26,28. Insulin and its receptors can be normal but circulating anti-insulin antibodies trap the insulin in plasma compartment and changes the time course of insulin therefore cause insulin resistance29. The presence of non-hormonal antagonist such as free fatty acids (FFA) in elevated amounts aids to insulin resistance because when oxidised they are associated with reduction in peripheral glucose utilisation and insulin resistance29. Low serum bicarbonate levels and high anionic gap is associated with insulin resistance because metabolic acidosis decreases the binding of insulin to its receptors30.
In relative insulin deficiency the size of the β cell is reduced so that the amount of insulin produced is reduced31. The cell size can be reduced after suffering from chronic relapsing pancreatitis so the pancreatic tissue is replaced with fibrous tissue31. Aging is characterised by progressive alterations in insulin secretion and action because they take multiple drugs which interfere with insulin action27. Islet cell amyloidosis is a diabetogenic factor because increase in islet amyloid polypeptide (IAPP) fibirilar aggregates inhibits insulin secretion and reduces β cell size28.
1.1.3 Complications of diabetes mellitus
Acute complications of diabetes mellitus due to severe hyperglycaemia are polyuria, polydipsia, polyphagia, weight loss and increased susceptibility to infections1,2. Chronic complications include development of neuropathy, retinopathy, and nephropathy and generalized degenerative changes in large and small blood vessels1,2.
1.1.4 Alanine transaminase
Liver function tests (LFTs) are used to screen for liver disease, monitor the progression of known disease, and monitor the effects of potentially hepatotoxic drugs10. Elevations of aminotransferases greater than eight times the upper limit of normal reflect acute viral hepatitis, ischemic hepatitis, or drug- or toxin-induced liver injury10. ALT is an enzyme involved in cellular nitrogen metabolism, amino acid metabolism and liver gluconeogenesis4.ALT is found mainly in the cytoplasm of hepatocytes and in cardiac cells ,damage to hepatocytes due to infection or fat accumulation leads to leakage of ALT in serum and damage to hepatocytes due to ischemia leading to myocardial infraction elevates serum ALT4,10. ALT serves as a marker of hepatocyte and cardiac cells injury 4,10.
1.1.5 Pathophysiology of diabetes mellitus
Insulin facilitates intake of glucose in cells, glycogen synthesis and inhibits gluconeogenesis7. Diabetic mellitus patients have insulin impairment and loss of insulin leads to reduced entry of glucose into cells8. There is a decrease in intracellular glucose which stimulates gluconeogenesis7,8. Proteins are catabolised to amino acids and the amino acids are used in the generation of glucose. ALT is the major enzyme involved in amino acid transamination leading to its increase in circulation8. Loss of insulin also leads to lipolysis of the stored lipids to triglycerides and free fatty acids (FFA)8. Adipose tissue is unable to re-use glycerol formed as a result of hydrolysis of triglycerides; instead it requires newly synthesized glycerol phosphate2. Glucose is the source of the substrate for the formation of glycerol phosphate, which is needed for the re-esterification of FFA in the formation of triglycerides1. Lack of the circulating insulin leads to failure of entry, of glucose into adipose tissue hence shortage of glycerol phosphate and the amount of FFA is increased1. The excess FFA are toxic to the hepatocytes leading to hepatic injury therefore leakage of ALT in circulation9,10.
In poorly controlled diabetes mellitus there is an insulin independent inflow of glucose in hepatocytes31,34. The chronic hyperglycaemic state inactivates glycogen phosphorlylase therefore inhibiting glycogenolysis and activates glycogen synthase resulting in excessive glycogen accumulation in the hepatocytes leading to glycogenic hepatopathy31,32. Glycogenic hepatopathy causes liver injury and there is a marked elevation of ALT in serum33,34,35.
Non-alcoholic fatty liver disease (NAFLD) is a chronic liver condition characterised by insulin resistance and hepatic fat accumulation in the absence of alcohol abuse, viral hepatitis and autoimmune hepatitis35. People with diabetes mellitus have a higher risk of developing NAFLD and liver related deaths35. NAFLD is the common cause liver test abnormalities and it accounts for 70% of all cases of asymptomatic elevated ALT in US adults35. The prevalence of NAFLD in diabetes mellitus is 70% and is associated with fatty liver and chronically elevated ALT35, 36. NAFLD is a marker of CVD risk and mortality in diabetic patients 35. Diabetes with NAFLD have a 7.2 fold increased risk of mortality from cirrhosis and CVD compared with those without diabetes35. The relative risk of a liver related death is directly related to the severity of DM with those requiring insulin having a 6.8 fold increased risk, those requiring hypoglycaemic medication having 4.9 increased risk 36. Hypertension is the leading cause NAFLD and 50-62 patients with diabetes have hypertension making them prone to NAFLD.
Hyperinsulinemia and hyperglycaemia in type 2 diabetes promotes lipogenesis by up regulating sterol regulatory element binding protien1c (SREBP1c) and carbohydrate regulatory element binding protein (ChREP) activity5. The up-regulation of SREBP-1c and subsequent simulation of de novo lipogenesis in the liver leads to increased intracellular availability of triglycerides, promoting fatty liver5. Excess FFA are directly toxic to hepatocytes and the putative mechanisms include cell membrane disruption at high concentration, mitochondrial dysfunction, toxin formation, and activation and inhibition of key steps in the regulation of metabolism5. Increase in mitochondrial oxidative stress due to increased intracellular FFA produces free radicals which induces inflammation and cellular necrosis35. Cellular necrosis elevates ALT due to the rupturing of the hepatocytes releasing the ALT36. Insulin resistant state is also characterized by an increase in proinflammatory cytokines such as tumour necrosis factor-α (TNF-α), which may also contribute to hepatocellular injury37.
NAFLD is associated with the presence of vascular disease which is the most common cause of death in DM. Cross-sectional studies of population s with DM have demonstrated that NAFLD is associated with increased carotid intima media thickness, carotid athermanous plaques, cardiovascular disease and myocardial infraction. Diabetes mellitus affects ≈6% of the US population but is present in as many as 30% of patients hospitalized with acute coronary syndromes. It has been recognized for some time that diabetics experience a greater mortality during the acute phase of myocardial infarction. Before the advent of coronary care as we know it today, mortality among diabetic patients in MI was reported to be as high as 40% .
Diabetes alone or in combination with a variety of risk factors (hypertension, hypercholesterolemia) can impair endothelial function. Increased oxidative stress brought about by hyperglycaemia may be an important link between diabetes and vascular events. Advanced glycosylated end products may quench nitric oxide through the generation of oxygen free radicals, leading to impaired endothelial vasodilatation. Angiotensin II augments oxidative stress by increasing the vascular production of superoxide radicals, which in turn interfere with the bioavailability of nitric oxide. By increasing free radical production, angiotensin II increases leukocyte adhesion to the endothelium, platelet aggregation, and cytokine expression, resulting in macrophage infiltration at the site of atherosclerotic plaques, leading to increased plaque vulnerability. The plaque becomes unstable, ruptures and form a thrombus which occludes the artery blocking the flow of blood leading to ischemia and the cardiac cells die releasing ALT in circulation.
Greater 40% of diabetic patients studied had previously had angina, ischemic preconditioning might have mitigated the extent of left ventricular dysfunction. Myocardial infarction in diabetic patients usually is more extensive and more severe than in non- diabetic patients. The long-term survival rate after acute myocardial infarction among diabetic patients is also lower than that among non-diabetic patients. In fact, the 5-year survival rates for diabetic patients after the first major coronary event have been found to be 38% and only 25% for those with subsequent events, compared with the corresponding figures in non-diabetic patients of 75% and 50%, respectively.
Individuals with diabetes mellitus have a higher incidence of liver function test abnormalities as compared with non-diabetics10. Mild chronic elevations of alanine transaminase often reflect underlying insulin resistance or no insulin production10,11. Alanine transaminase is elevated three times the upper limits of normal7.In a study done in Jordan the prevalence of elevated serum ALT in diabetic patients is 9.4% , males had a higher prevalence of 10.6% and females had a prevalence of 8.2%11. The normal reference range of serum ALT in males is ≤45U/L and in females is ≤34U/L11. The prevalence is higher in males and poor metabolic control12. The prevalence of ALT is decreased in elderly people due to decrease in the release of muscle proteins for gluconeogenesis and reduced hepatic function12.
1.1.6 Diagnosis and management of diabetes mellitus
Different tests can be used to diagnose and monitor blood glucose levels in diabetes mellitus. For diagnosis the OGTT, fasting and random blood glucose tests are ideal and according to the WHO guidelines, if the plasma glucose is <5.5 mmol/L then diabetes is unlikely. A fasting plasma glucose of 7.0 mmol/L or more, or a random glucose of 11.1 mmol/L or more makes diabetes more likely.
In the management and control of diabetes , blood glucose determined at the time of the clinic attendance can only give limited information and may not represent the overall closeness of control at other times therefore it has limitations in monitoring glucose control. The HbA1c test provides a better index of diabetic control than plasma glucose since it is not greatly affected by short-term fluctuations in plasma glucose. Other tests such as blood urea (BUN), blood insulin, blood fructosamine, macroalbumin, glycosylated haemoglobin (HbA1c), blood gases, C-peptide and electrolytes are used in the monitoring of diabetes.
1.2 Statement of the problem
Diabetic patients have elevated serum ALT levels due to impairment of insulin7 .Diabetic patients have liver disease due to excess FFA which are toxic to the hepatocytes and therefore leads to hepatic injury leading to the release of ALT in the serum3.Fats also accumulates in hepatocytes causing NAFLD which chronically elevates ALT. The abnormal lipid profile promotes atherogenesis leading to atherosclerosis which causing silent ischemic myocardial infarction. The infarcted myocardial cells release ALT in circulation. The mortality of NAFLD is 70% in diabetics and of silent ischemic myocardial infarction is greater than 40%. Unfortunately clinicians are unaware of these conditions in diabetic patients. The proposed research is to determine the serum ALT levels in diabetic patients , to estimate the prevalence of elevated ALT in diabetic mellitus patients and the prevalence of people who are at risk of liver related deaths and CVD.
Null hypothesis - The prevalence of elevated serum ALT levels in diabetes mellitus patients at Parirenyatwa Group of Hospitals is less than 9.4%11
Alternative hypothesis - The prevalence of elevated serum ALT levels in diabetes mellitus patients at Parirenyatwa Group of Hospitals is greater than 9.4%11
This study aims to:
Measure the serum levels of ALT in diabetes mellitus patients attending Parirenyatwa Group of Hospitals Diabetic Clinic.
Estimate the prevalence of elevated ALT in diabetes mellitus patients attending Parirenyatwa Group of Hospitals Diabetic Clinic.
CHAPTER TWO: MATERIALS AND METHODS
Mindray BS120 chemistry analyse
2.2 Study design
• A cross sectional analytical study will be done on blood samples of diabetes mellitus patients attending Parirenyatwa Group of Hospitals Diabetic Clinic.
2.3 Sample size determination
S = Z2pq/E2
Where Z = Test statistic
E = Standard Error
P = Population Proportion with desired characteristic
S = 1,962 x 0,094 x 0, 86 (q = 1 -p)
S = 124, 2
A minimum of 124 samples will be used in this study.
2.4 Study population and setting
The study population will consist of diabetic patients aged between 18 and 65 years attending Parirenyatwa Group of Hospitals Diabetic Clinic.
2.5 Sampling technique
2.5.1 Sampling procedure
Sequential Convenience sampling will be used for selection of diabetic patients at Parirenyatwa Diabetic Clinic who would have come for routine check-up or monitoring. Participants who will meet the eligibility criteria will be enrolled into the study.
2.5.2 Inclusion criteria
Diabetic patients of an age range between 18 and 65 years attending Parirenyatwa Group of Hospitals Diabetic Clinic between 1st of November 2012 and 30th of April 2013.
• Non-diabetic patients
• Diabetic patients below 18 years and above 65 years of age
2.6 Ethical considerations
Permission will be sought from the Parirenyatwa Group of Hospitals Clinical Director, Head of Department of the Chemistry Laboratory, the Consultants at the Diabetic Clinic, Matrons and Sister-in-charge working at PGH Diabetic Clinic to collect demographic data and use residual routine samples handed over for analysis in the laboratory. Ethical clearance will be sought from the Joint Research Ethics Committee of the Parirenyatwa Group of Hospitals and University of Zimbabwe College of Health Sciences. Confidentiality will be maintained and no names will be used for identification to ensure privacy. Subjects on any kind of treatment or assessment of patients will not be altered in any way because of the project. Results will not be accessible to any unauthorised persons. Safe disposal of residual samples and raw data will be done after the project has been successfully rated.
2.7 Laboratory methods
2.7.1 Collection of samples
Left over diabetic patient samples handed in for routine assays to the laboratory in plain tubes will be used.
2.7.2 Principle of ALT assay
α-oxoglutarate + L-alanine ALT L-glutamate + pyruvate
Pyruvate + NADH + H+ LDH L-lactate + NAD-
Alanine aminotransferase catalyzes the reversible transamination of L-alanine and α - oxoglutarate to pyruvate and L- glutamate. The pyruvate is then reduced to lactate in the presence of lactate dehydrogenase(LDH) with the concurrent oxidation of reduced β-nicotinamide adenine dinucleotide (NADH) to β-nicotinamide adenine dinucleotide (NAD).This change in absorbance is directly proportional to the activity of ALT in the sample.
Samples will be centrifuged at 3 000 revolutions per minute (rpm). The serum will be aliquoted into plastic made serum pots using a micropipette and then stored in a refrigerator at a temperature maintained between 2 - 8oC and then processed in batches. Prior to processing of the samples they are brought to room temperature and the analyser will be calibrated. After calibration the control samples will be analysed. The samples will be loaded in the sectors of the machine (Mindray BS 120) and assayed for ALT.