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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA ANNEXURE – ΙΙ PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1 NAME OF THE CANDITATE DR. SHWETHA ADDRESS 2 NAME OF THE INSTITUTE 3 COURSE OF STUDY AND SUBJECT 4 DATE OF ADMISSION TO COURSE 5 TITLE OF THE TOPIC INTRODUCTION #418, SHRINIKETAN 1ST MAIN 3RD STAGE BEML LAYOUT RAJESHWARINAGAR, BANGALORE – 560098 KARNATAKA DR. B R AMBEDKAR MEDICAL COLLEGE, K G HALLI, BANGLAORE – 560045 M.D. BIOCHEMISTRY 28/07/2010 “ TRACE ELEMENTS (MAGNESIUM, ZINC, COPPER) IN CHRONIC ALCOHOLIC LIVER DISEASE“ Alcohol is a chemical substance capable of producing pharmacological effects consistent with narcotics including central nervous system and respiratory depression and induction of euphoria, tolerance and addiction.1 Alcoholism is a repetitive pattern of drinking alcohol that has adverse effects on personal, family, occupational, social or health status. An alcoholic is a person who progressively consumes an amount of ethanol capable of producing pathological changes and who exhibit a cumulative pattern of social behaviors associated drinking including frequent intoxication, physical injuries, problem with family, job and accidents while drinking under the influence.1 Alcohol dependence is a maladaptive pattern of alcohol use leading to clinically significant impairment and distress. Dependence may be physical or psychological. Physical and psychological dependence is characterized by presence of withdrawal symptoms and tolerance. Withdrawal symptoms include sweating, hand tremors, vomiting, insomnia, anxiety, psychomotor agitation and hallucination. Tolerance is defined as need for markedly increased amounts of alcohol to achieve a desired effect.2 Percentage of alcohol in each drink Alcohol Content in Percentage 50-60% Beverage Rum, Liquor Whisky, Gin, Brandy Wine Beer 40-45% 10-15% 4-8% Alcoholic equivalents: 3 A Standard alcoholic drink is equivalent to: 30 ml of Whisky = 10 gm = 70 calories 100 ml of wine = 10 gm = 70 calories 250 ml of Beer = 10 gm = 70 calories 1 gm of alcohol yields 7 calories Drinking patterns: The average intake alcohol in a large group male alcoholic cirrhotics was 160 g / day for 8 years. Quantity, quality, duration is important. The liver injury is related to type of beverage and its alcohol content. Continued daily imbibing is more dangerous intermittent consumption when the liver is given a chance to recover. For at least two days in a week a person should not drink alcohol.3 Metabolism of alcohol: Ethanol is primarily an exogenous compound consumed in alcoholic beverages and readily absorbed from entire gastrointestinal tract. Normal absorption is complete within an hour but food intake can delay absorption by 4-6 hours. Oxidative processes, located primarily in the liver degrade most of the absorbed ethanol by following steps: 20% MEOS Acetaldehyde Ethanol Aldehyde 80% Alcohol Acetaldehyde Dehydrogenase Citric Acid cycle Acetate CO2 dehydrogenase AcetylcoA Fatty Acids The most important pathway occurs in cell cytosol where ADH produces acetaldehyde which is rapidly destroyed by ALDH in the cytosol and mitochondria. A second pathway in the microsomes of the smooth endoplasmic reticulum ( the microsomal ethanol oxidizing system) is responsible >= 10% of ethanol oxidation at high blood alcohol concentration. 4 Biochemical and metabolic alterations in alcoholism : In chronic alcoholics, there is an increase in MEOS activity that may result in an increased metabolism of upto 72%. Altered lipid, protein and carbohydrate biochemistry: Low to moderate use of ethanol (< 20 gm / day) is associated with an increase in HDL and lowers the risk of coronary artery disease. When it is > 80gm / day causes excessive serum triglycerides, VLDL and chylomicrons. +water w As seen with poor nutritional status of many alcoholics, the rate of protein degradation exceeds the rate of synthesis resulting in alcoholic myopathy, osteopathy and intestinal atrophy. Gluconeogenesis is impaired by ethanol by variety of mechanisms. Results in the fall in the amount of glucose produced from glycogen and also increase in lactate production.1 Alteration in mineral metabolism occurs in chronic alcoholics and also in alcoholic liver disease. Minerals which are markedly decreased are magnesium and zinc and which are markedly increased are copper. In this study magnesium, zinc, copper are taken into consideration. Magnesium is the fourth most abundant cation in the body. The body contains 21gm of magnesium of which 60% is in bone, 20% in the skeletal muscles, 19% in the cells and 1% in extracellular fluids. Normal level – 18 to 29 mg/ litre There are two major roles for magnesium in biological system. 1) It can compete with calcium for binding sites on membrane. 2) It can form chelates with important intracellular anionic ligands notably adenosine triphosphate Magnesium catalyses or activates more than 300 enzymes in the body. Magnesium act as an cofactor for enzymes concerned with cell respiration, glycolysis and transmembrane transport of other cation such as calcium and sodium. It affects enzyme activity by binding with active site of enzyme( pyruvate kinase, enolase) by ligand binding ( ATP requiring enzyme ) by causing changes during catalytic process ( Na – K – ATPase ) and by promoting aggregation of multienzyme complexes. Reduced extracellular magnesium concentration increases membrane excitability. Metabolism : Only 20 – 30% of ingested magnesium is absorbed. High intake of fat, phosphate, calcium, alcohol diminish absorption. Absorption is mainly at small intestine. Absorption begins with an hour of ingestion and continues at a rate of 2 – 8 hours. By this time 80% of the absorption is completed. Magnesium is excreted in faeces & the remaining 1/3 rd in urine. In alcoholic liver disease, there will be hypomagnesemia due to poor dietary intake and increased diuersis that is a direct affect of alcohol, defects in renal tubular reabsorption, starvation and vomiting. Hypomagnesemia manifests with increased muscle tone, exaggerated tendon reflexes, hyperirritability & tetany. It is associated with hypokalemia, hypocalcemia, hypoparathyroidism. Hypomagnesemia occurs in 30% of alcohol abuse. Severe depletion of magnesium affects thiamine indirectly leading to wernicks-Korsakoff’s syndrome.1 Zinc: Zinc is widely distributed in the tissues of the body. 20% is present in skin, 80% is present in prostrate, epididymis in bones, teeth in kidneys, muscles, brain & lungs Normal Level 100-1200 µg/L. Zinc is essential for normal growth & reproduction. It has a beneficial effect on tissue repair & wound healing. It is a component of a number of enzymes present in animal tissues including ADH, Alkaline Phosphatase, carbonic anhydrase, procarboxypeptidase, cytosolic superoxide dismutase. Zinc stabilizes membrane stabilizes membrane structure and gives protection at the cellular level by preventing lipid peroxidation & reducing free radical formation Metabolism:- Only a small percentage of dietary zinc is absorbed mainly from deodenum and ileum. After absorption it is taken up by liver & redistributed to other tissues primarily bone & muscle. Zinc is lost in faeces & some percentage in urine. In alcoholic liver disease, there will be hypozincemia due to poor dietary intake, increased diuresis, destruction of zinc metalloenzymes. Zinc deficiency manifest with loss of appetite, dwarfism, hypogonadism, hepatosplenomegaly , anaemia , dermatitis & delayed wound healing. Copper:- Adult human body contains 100-150 mg of copper which is distributed in muscles, bone and in liver. Normal level - 0.01to 0.2 µgm per ml Copper forms a integral part of enzymes like cytochrome oxidase, tyrosinase , MAO, lysyl oxidase, catalase, ascorbic acid oxidase, uric case and superoxide, dismutase.It plays an important role in haemoglobin synthesis, melanin formation, phospholipid synthesis, collagen synthesis, bone formation and maintaining the integrity of myelin sheath. Metabolism: - About 32% of dietary copper is absorbed mainly at the duodenum. After absorption enters the plasma where it is bound to amino acid particularly histidine and serum albumin. About 96% of serum copper is as cerruloplasmin. Under normal condition 85-99% is excreted in faeces via bile and remaining in urine. In Alcoholic liver disease copper level increases due to the failure of excretion of copper. Toxicity causes renal failure, liver failure and coma. Alcoholic liver disease Chronic and excessive alcohol ingestion is one of the major causes of liver disease. Only 15% of alcoholics develop alcoholic liver disease. Alcoholic liver disease is among the 10 most common causes of death world wide. Quantity, quality and duration of alcohol intake are the most important risk factors of alcoholic liver disease. In men, 40-80gm per day of ethanol produces fatty liver 160 gm per day for 10-20 years causes hepatitis & cirrhosis. Pathogenesis Liver is the primary target organ for alcohol induced damage. The production of acetaldehyde causes a variety of metabolic responses like increase in lipid peroxidation, inhibition of nuclear repair, interference with mitochondrial electron transport change and microtubule function, increase in collagen synthesis and superoxide formation. Stages of Alcoholic liver disease 1) Fatty Liver : It is characterized by accumulation of fat within the perivenular hepatocytes along with hepatic mitochondrial DNA deletion. Usually patients are asymptomatic. Occasionally present with upper quadrant discomfort, tender hepatomegaly, nausea and jaundice 2) Alcoholic Hepatitis: It is characterized by ballooning degeneration with sclerosing hyaline Necrosis. Mallory bodies are seen in florid case. Patient presents with fever, spider nevi, jaundice and abdominal pain. 3) Alcoholic Cirrhosis: It is also called as Laennec’s cirrhosis. Classically, alcoholic cirrhosis is micro nodular and no normal architecture can be identified. The formation of nodule is often slow because of inhibitory effect of alcohol on hepatic regeneration. With continuing Necrosis and fibrosis the cirrhosis may progress from micro nodular to macro nodular pattern. Only liver biopsy specimen will provide an accurate diagnosis of cirrhosis. Patient presents with symptoms such as vague right upper quadrant pain, Nausea, Vomiting, diarrhea, anorexia and Malaise. On examination, Hepatosplenomegaly with liver edge being firm and nodular, scleral icterus, palmar erythema, spider angiomas, Parotid enlargement, digital clubbing, Muscle wasting, Gynaecomastia and testicular atrophy due to hormonal abnormalities.4 Risk factors in alcoholic liver disease Female gender, obesity, genetic predisposition, malnutrition, co-infection with hepatitis B or C virus, Iron overload, other co-morbid condition, Hepatotoxic drug intake. Laboratory diagnosis of Alcoholic liver disease For Alcoholic fatty liver & hepatitis 4 Test Comment Increased two to seven fold, <400 U/L, greater than ALT AST ALT AST/ALT Bilirubin Increased two to seven fold, <400 U/L Usually > 1 May be markedly increased in alcoholic hepatitis despite modest elevation in alkaline phosphatase Cirrhosis:- Laboratory tests may be completely normal in patients with early compensated alcoholic cirrhosis. Alternatively in advanced cases, many abnormalities are usually present. Serum total bilirubin can be normal or mildly elevated with advanced disease. Serum amino transferases ( ALT, AST) are typical elevated with AST levels living higher than ALT levels, usually by a 2:1 ratio. 6 Brief resume of the intended work 6.1 Need for Study Alcohol is a common cause of cirrhosis all over the world including India. An alcoholic liver disease is among the ten most common cause of death worldwide. The consumption of alcohol has been steadily increasing in India during last decade due to socioeconomic status. Mineral abnormalities occur in Alcoholic liver disease. Many of the nutrients are essential for body function and a deficient state can cause alarming consequences. Hypomagnesemia is not only a laboratory symptom of fatty liver, but due to its connection with increased oxidative stress it might be a risk factor in the progression of the disease and also to know whether severity is related to the amount duration of alcohol intake and the type of alcoholic beverage. it is known that deficiency increases the susceptibility of plasma triacylglycerol- rich in lipoproteins and tissues to lipoperoxidation, but the precise mechanism by which magnesium can potentiate oxidative injury remains to be determined. Hence further studies have to be taken up.1 Alcoholic liver disease is associated with decreased zinc levels, studies using animal models have shown than Zinc supplementation prevents alcohol induced liver damaged under both acute and chronic alcohol exposure. Since few studies have been done and there is a definitive changes in the zinc levels, further study has to be taken up. Ethanol consumption/Liver damage may alter the liver content of the several trace elements like zinc and copper. Copper level is increased in alcoholic liver diseases. Liver copper and urinary zinc and copper excretion seem to be related with severity of alcoholic disease.8 Hence for the studies are required to understand the changes in the trace elements in alcoholic liver diseases. 6.2 Review of literature: it is not surprising that magnesium levels were decreased in patients with alcoholic fatty liver because the connection of hypomagnesemia with alcoholism has been known for long time.5 Hypomagnesemia is not only a laboratory symptom of fatty liver but due to its connection with increased oxidative stress it might be a risk factor in the progression of fatty liver to steatohepatitis.5 Hypomagnesemic patients more frequently had other acid base and electrolyte abnormalities, such as hypophasphatemia, hypokalemia, hypocalcemia and respiratory alkalosis, as compared with the normomagnesemic patients.6 About 25% of alcoholics are chronically hypomagnesemic because of the combination of poor nutritional intake and increased renal loss.7 Ethanol consumption and or liver damage may alter liver content of several trace elements as iron, zinc, copper and manganese. This alteration may play a role on ongoing fibrogenesis.8 Alcoholic liver disease is associated with decreased zinc and its major binding protein, metallothionein.9 Zinc deficiency affects RNA and DNA synthesis, enzymatic process ( ADH ), vitamin metabolism ( vit A ) and contributes to hypogonadism.10,11 Severe depletion of magnesium affects thiamine indirectly leading to wernicke – korsakoff syndrome.12 6.3 Objectives:To study the level of trace elements ( Magnesium, Zinc and Copper) in chronic Alcoholic liver disease. 7.Materials and Methods: 7.1 Source of data: Test Subjects: 30 patients who are diagnosed with chronic alcoholic liver disease Between the age group of 30-60 years from Dr.BRAMC Control Subjects: 30 controls with no history of chronic alcoholic liver disease Between the age group of 30-60 years from Dr.BRAMC Study period: January 2011 to January 2012 Exclusion criteria: Patients with clinical evidence of hypertension, Diabetes, Mellitus, Pancreatitis, Renal failure and malnutrition Individuals belonging to other age group . Subject on drugs affecting mineral metabolism ( gentamicin, Cisplatin) Other causes of hepatitis 7.2 Method of collection of Data ( Including Sampling procedures) Test subjects are patients with chronic alcoholic liver disease b/w age of 30-60 years ; from Dr. BRAMC Control subjects are healthy individuals with no history of alcoholic liver disease b/w the age of 30-60 years from Dr. BRAMC The Blood pressure, height, weight, BMI are recorded in both tests subjects & control subjects. The Diagnosis of chronic alcoholic liver disease is based on history of chronic significant alcohol abuse ( alcohol drinking history is assessed by interview and patient questionnaire. Data from questionnaire are used to establish the type of alcoholic beverages, frequency, pattern and duration of use), clinical signs of liver disease and supporting laboratory tests which are ALT, AST and Bilirubin levels and GGT. 5 ml of venous blood sample is collected following an overnight fasting and centrifuged. Serum is collected and analyzed by following methods: Estimation of serum magnesium by Titan Yellow Method Estimation of serum zinc by Dithizone method Estimation of serum copper by Diethyl Dithio carbamate method 7.3 Does the study require any investigation or intervention to be conducted on patient or animal? Yes, Investigations are required . No, Interventions are not required. 7.4 Has ethical clearance been obtained from your institution in case 7.3 ? yes 8. List of References: 1. Lawrence A. Kaplan textbook of Clinical Chemistry Theory, Analysis, Correlation 4th edition, Alcoholism, 641 – 642. 2 American Psychiatric Association. 1994. Diagnostic and Statistical Manual of Mental Disorders (4th edition) (DSM - IV). 3 Sheila Sherlock and James Dooley. Diseases of the liver and biliary system. 11 th edition. Alcohol and the Liver. 381-383 4 Harrison’s Principles of Internal Medicine, Volume II, 17th Edition, Alcoholic Liver Diseases, Page No.: 1969-1971 5 Turecky L, Kupcova V et al Serum Magnesium levels in patients with alcoholic and non alcoholic fatty liver. Bratisl Listy 2006; 107(3) 58-61 6 Elisaf M, Merkouropoulos M et al, pathogenetic mechanism of hypomegnesemia in alcoholic patients. J Trace elem Med Biol. 1995 Dec; 9(4): 210-4. 7 Lee Goldman, Devvis Ausiello , Cecil’s textbook of Medicine,23 rd edition Disorders of magnesium and Phosphorus. 859 8 F.Rodriguez-Morenoa, E.Gonzalez – Reimers a, et al, Zinc, Copper, Manganese and iron in chronic alcoholic liver disease. Alcohol. 1997 Jan-Feb;14(1):39-44. 9 Kang YJ, Zhou Z, Zinc prevention and treatment of alcoholic liver disease. Mol Aspects Med. 2005 Aug-Oct;26(4-5):391-404 10 Devgun MS et al; vitamin and mineral nutrition in chronic alcoholics including patients with korsakoff’s Psychosis. P Br J Nutr 45:469, 1981 11 Thomson AD, Majumdar SK: The influence of ethanol on intestinal absorption and utilization of nutrients, clin Gastroenterol 10:263, 1981 12 Smith JC, Jr, et al: Zinc: a trace element essential in vitamin A metabolism, Science 181:954, 1973 SIGNATURE OF THE CANDIDATE 9 10 REMARKS OF THE GUIDE 11 NAME AND DESIGNATION a. GUIDE DR. HEMALATHA. MD. PROFESSOR AND HOD OF BIOCHEMISTRY, DR. BRAMC, BANGALORE b. SIGNATURE c. CO-GUIDE d. SIGNATURE e. HEAD OF THE DEPARTMENT f. SIGNATURE 12 12.1 REMARKS OF THE CHAIRMAN AND PRINCIPAL 12.2 SIGNATURE DR. HEMALATHA. MD. PROFESSOR AND HOD OF BIOCHEMISTRY, DR. BRAMC, BANGALORE