Serum Zinc Level in Cirrhosis of Liver

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Serum Zinc Level in Cirrhosis of Liver Introduction Cirrhosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrous scar leading to progressive loss of liver function. It is most commonly caused by alcoholism and hepatitis B or C but has many other possible causes (Heidelbaugh & Bruderly 2006, pp. 756-7). Epidemiology of liver cirrhosis varies between gender, ethnic groups and geographical distribution. Prevalence Rate of Cirrhosis of the liver was approximately 1 in 679 people in USA. Average life years lost for Cirrhosis of the liver was 23.3 in North Carolina in 1994. In 2000 liver cirrhosis was the fifth leading cause of death in Mexico where as it was 12th most common cause of death in the United States (Kochanek et al. 2011, pp. 1-30).

A diagnosis of cirrhosis of the liver may be overlooked or delayed because in its early stages there generally are symptoms, such as poor appetite, fatigue, weight loss, and weakness, are vague and easily attributed to less serious conditions, such as aging and stress. Majority of patients remain symptom free until the advance stage called decompensate cirrhosis, characterized by ascites, spontaneous bacterial peritonitis, hepatic coma or Variceal bleeding from portal hypertension. The physical examination of patients with cirrhosis may reveal variety of findings that necessitate a hepatic or a gastrointestinal based work-up to determine the etiology. There is correlation observed between persistently disturbed liver function tests and biopsy-proven underlying hepatic disease, so to diagnose the cirrhosis liver biopsy should be considered when all initial and specific measures have failed to confirm (Heidelbaugh & Bruderly 2006, pp. 756-62).

The most effective treatment plan for cirrhosis of the liver uses a multifaceted approach and varies depending on the cause of the disease. Treatment plans are individualized to best fit the patient's age, medical history, and stage of the disease. The goal of treatment is to stop or slow the progression of damage to the liver and minimize and quickly treat any complications, such as portal hypertension, esophageal varices, ascites, liver failure, hemorrhage and kidney failure. Bleeding esophageal varices are managed by endoscopic sclerotherapy or rubber band ligation. Ascites and edema are often responsive to low sodium, diuretic therapy and peritoneal paracentesis. A low protein diet and agents such as lactulose are used to manage hepatic encephalopathy. Infections such as spontaneous bacterial peritonitis must be aggressively rapidly treated with appropriate antibiotics. Coagulation disorders will sometimes respond to vitamin K and fresh frozen plasma. However liver transplantation is highly effective for the treatment of end-stage cirrhosis (Mazeaferro et al.

1996, pp.693-700).

The Zinc (Zn) is a metallic element; symbol Zn, atomic number 30 and it is a transition metal in group 12 of the periodic table (Lehto 1968, pp. 822-4). The Recommended Dietary Allowance (RDA) is 8 mg/day for women and 11 mg/day for men. Red meats, especially beef, lamb and liver have some of the highest concentrations of zinc in food (United States National Research Council 2001, pp. 442-445). Nearly two billion people in the developing world are deficient in zinc (Prasad 2003, pp. 409–10). Zinc affects growth and plays a role in the synthesis of proteins and nucleic acids, as well as in the synthesis of insulin like growth factor 1 (IGF-1) and in its effects on target tissues (MacDonald 2000, pp. 1507-8). Zinc

deficiency predisposes to growth retardation, neurosensory deficit, disorders in the metabolism of several hormones and enzymes that participate in growth and bone development, wound healing retardation, skin lesions, delayed sexual maturation, and immunodeficiency (Hambridge & Krebs 2007, pp. 1101-1105). This trace element also acts as an antioxidant by means of two different mechanisms: protection against oxidation and inhibition of the production of reactive types of oxygen by transition metals, such as iron and copper, both metals linked to liver injury (Prasad 2008, pp. 354).

The low serum zinc level is common in patient with liver cirrhosis due to anorexia and reduced intake of animal proteins, increase in cytokines or hormones that may affect zinc metabolism, increase of renal loss of zinc and also portal hypertension, which are responsible for poor absorption of nutrients (Stamoulis et al. 2007, p. 1598). There is reduced liver protein synthesis in patients with cirrhosis of liver, the metallothionein (MT) is an important zinc-binding protein (formed by liver) and is involved in zinc metabolism, homeostasis and its release in number of oxidants, the released zinc will inhibit the activity of the enzymes involved in fibrogenesis (fibrosis) in the liver, all these are yet known pathophysiological mechanisms (Maret 2003, pp. 1460s-62s). Zinc is also essential for some of the neutrophil functions and it appears that zinc has a role in the maintenance of human immunity. Recent evidence suggests that thymic-dependent lymphocytes (T cells) are zinc dependent. T-helper and suppressor cells, T-effector cells and T-natural killer cells appear to be zinc dependent (McClain et al. 1986, pp. 582-9). In a study of Stamoulis et al. published by Digestive Diseases and Sciences in 2007 the prevalence of low serum zinc level in cirrhotic patients was 65.3%.

Some alterations of patients with cirrhosis may be associated with zinc deficiency: alopecia, wound healing disorders, lack of appetite, hypogeusia, gonadal growth retardation, and it is also associated with hepatic encephalopathy (Chetry & Choudhuri 2003, pp. 28–30). A study with adult patients with decompensate cirrhosis found that these patients have plasma zinc concentrations significantly lower than patients with compensated disease, and that zinc concentrations are negatively associated with fasting ammonia concentrations (Yoshida et al.

2001, pp. 349-55).

It has long been speculated that Zn has a protective effect against liver fibrosis and Zn intake in cirrhosis are based mostly on observations of reduced Zn levels in cirrhotic patients and on the beneficial effects of Zn supplementation on liver metabolism (Capocaccia et al. 1991, pp.

386–91).

Zinc serum examination is not an examination that can be routinely done in every laboratory.

The zinc examination requires instrument preparation and unpractical specimen container because the validity of zinc measurement depends mainly to the successful analysis in avoiding contamination of ambient zinc (Reinhold 1975, pp. 476-500).

Therefore keeping all such important points and views in mind, the focus and aim of this study is to evaluate and assess the serum zinc level in patients with cirrhosis of liver. So far no study has been conducted on this topic in Bangladesh. As such this study will fill the gap, open new forum of discussion and will provide knowledge and information regarding the medical workup of patients with cirrhosis of liver.

Hypothesis

Serum zinc level is low in patients with cirrhosis of liver and the level is inversely related with the severity of the disease.

AIMS AND OBJECTIVES General: To observe the association of serum zinc level in cirrhosis of liver and to find out its relationship with the severity of the disease.

Specific: • • • • • • •

To estimate serum zinc level in patients with cirrhosis of liver (cases).

To estimate serum zinc level in healthy control.

To estimate serum bilirubin, serum total protein, serum albumin, prothrombin time, AST, ALT, serum glucose and serum creatinine in study subjects to diagnose cirrhosis of liver and to establish selection criteria.

To evaluate the findings of ultrasonography of hepatobiliary system and endoscopy of upper GIT to diagnose cirrhosis of liver.

To assess ascites and encephalopathy of the cases for scoring the severity of the disease by Child-Pugh score.

To compare statistically the serum zinc level between cases and controls.

To find out the relationship statistically between serum zinc level and the severity of cirrhosis of liver.

Review of Literature Cirrhosis of Liver Cirrhosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function. It can occur at any age, has significant morbidity and is an important cause of premature death. Worldwide, the most common causes of cirrhosis are chronic viral hepatitis, prolonged excessive alcohol consumption and fatty liver disease. Cirrhosis is the most common cause of portal hypertension and its associated complications (Heidelbaugh & Bruderly 2006, pp. 756-7).

The incidence of cirrhosis of liver in Bangladesh has been recorded as 2.6%. Nutritional deficiency was considered to be the important etiological factor whereas alcoholism did not appear to have any significant role. Almost all the cases (94.2%) were non-alcoholic (Islam & Khan 1975, pp. 39-40). Studies in recent past revealed Hepatitis B virus is responsible for 41 per cent of cirrhosis of liver in Bangladesh (Alam 2010, p. 1).

Causes of cirrhosis Cirrhosis has many possible causes; sometimes more than one cause is present in the same patient. In the Western World, chronic alcoholism and hepatitis C are the most common causes.

1. Chronic viral hepatitis (B or C) 2. Alcohol 3. Non-alcoholic fatty liver disease

4. Immune 5. Primary sclerosing cholangitis 6. Autoimmune liver disease 7. Biliary 8. Primary biliary cirrhosis 9. Secondary biliary cirrhosis 10. Cystic fibrosis 11. Genetic 12. Haemochromatosis 13. Wilson’s disease 14. α1-antitrypsin deficiency 15. Cryptogenic (unknown-15%) 16. Chronic venous outflow obstruction (Collier & Webster 2010, p. 942) Pathophysiology The cardinal feature of cirrhosis is an increase in fibrous tissue, progressive and widespread death of liver cells, and inflammation leading to loss of normal liver architecture. Following liver injury, stellate cells in the space of Disse are activated by cytokines produced by Kupffer cells and hepatocytes. This transforms the stellate cells into myofibroblast-like cell, capable of producing collagen, pro-inflammatory cytokines and other mediators which promote hepatocyte damage and cause tissue fibrosis.

Destruction of liver architecture causes distortion and loss of the normal hepatic vasculature with the development of portosystemic vascular shunts and the formation of nodules.

Cirrhosis evolves slowly over years to decades and normally continues to progress even after removal of the etiological agent. These changes usually affect the whole liver, but can be patchy (Collier & Webster 2010, p. 943).

Classification • • •

Cirrhosis can be classified histologically into two types: Micronodular cirrhosis, characterized by small nodules about 1 mm in diameter and seen in alcoholic cirrhosis.

Macronodular cirrhosis, characterized by larger nodules of various sizes. Areas of previous collapse of the liver architecture are evidenced by large fibrous scars (Sharlock & Dooley 2000, p.375).

Clinical features of cirrhosis of liver Some of the following signs and symptoms may occur in the presence of cirrhosis or as a result of the complications of cirrhosis. Many are nonspecific and may occur in other diseases and do not necessarily point to cirrhosis. Likewise, the absence of any does not rule out the possibility of cirrhosis.

1.

2.

3.

4.

5.

6.

Hepatomegaly (although liver may be small) Jaundice Ascites Circulatory changes Spider talengiectasia Palmar erythema

7. Cyanosis 8. Endocrine changes 9. Loss of libido 10. Hair loss 11. Men: 12. Gynaecomstia 13. Testicular atrophy 14. Impotence 15. Women: 16. Breast atrophy 17. Irregular menses 18. Amenorrhea 19. Hemorrhagic tendency 20. Bruises 21. Parpura 22. Epistaxis 23. Portal hypertension 24. Splenomegaly 25. Collateral vessels 26. Variceal bleeding 27. Hepatic (portosystemic) encephalopathy 28. Other features 29. Pigmentation 30. Digital clubbing 31. Dupytren’s contracture (Collier & Webster 2010, p. 944) Complications of cirrhosis of liver The clinical course of patients with advanced cirrhosis is often complicated by a number of important sequelae that are independent of the etiology of the underlying liver disease. In some people, these may be the first signs of the disease. These include— 1.

2.

3.

4.

5.

6.

7.

8.

Portal hypertension Gastro esophageal varices Splenomegaly Ascites Hepatic encephalopathy Spontaneous bacterial peritonitis Hepatorenal syndrome Hepatocellular carcinoma (Chung & Podolsky 2005, p.1860)

Diagnosis of cirrhosis of liver The gold standard for diagnosis of cirrhosis is a liver biopsy, through a percutaneous, transjugular, laparoscopic, or fine-needle approach. A biopsy is not necessary if the clinical, laboratory, and radiologic data suggests cirrhosis. Furthermore, there is a small but significant risk to liver biopsy, and cirrhosis itself predisposes for complications due to liver biopsy (Grant & Neuberger 1999).

Biochemical and Hematological findings The following findings are typical in cirrhosis: 1. Aminotransferases - AST and ALT are moderately elevated, with AST > ALT. However, normal aminotransferases do not preclude cirrhosis.

2. Alkaline phosphatase (ALP) - usually slightly elevated.

3. Gamma-glutamyl transferase – correlates with ALP levels. Typically much higher in chronic liver disease from alcohol.

4. Bilirubin - may elevate as cirrhosis progresses.

5. Albumin - levels fall as the synthetic function of the liver declines with worsening cirrhosis since albumin is exclusively synthesized in the liver 6. Prothrombin time - increases since the liver synthesizes clotting factors.

7. Globulins - increased.

8. Serum sodium – decreased.

9. Platelet count - Rarely results in < 50,000/mL. 10. Total count of WBC- decreased with neutropenia.

Coagulation defects- the liver produces most of the coagulation factors and thus coagulopathy correlates with worsening liver disease (Heidelbaugh & Bruderly 2006, pp. 760-1).

There is now a validated and patented combination of 6 of these markers as non-invasive biomarker of fibrosis (and so of cirrhosis): FibroTest (Halfon et al. 2008, pp. 22-29).

Ultrasound of liver It is routinely used in the evaluation of cirrhosis, where it may show a small and nodular liver in advanced cirrhosis along with increased echogenicity with irregular appearing areas.

Ultrasound may also screen for Hepatocellular carcinoma, portal hypertension and BuddChiari syndrome (by assessing flow in the hepatic vein).

Other tests performed in particular circumstances include abdominal CT and liver/bile duct MRI (MRCP).

Endoscopic examination Gastroscopy (endoscopic examination of the esophagus, stomach and duodenum) is performed in patients with established cirrhosis to exclude the possibility of esophageal varices.

Rarely diseases of the bile ducts, such as primary sclerosing cholangitis, can be causes of cirrhosis. Imaging of the bile ducts, such as ERCP or MRCP (MRI of biliary tract and pancreas) can show abnormalities in these patients, and may aid in the diagnosis (Heidelbaugh & Bruderly 2006, pp. 760-2).

Grading of cirrhosis of liver The severity of cirrhosis is commonly classified with the Child-Pugh score. This score uses bilirubin, albumin, INR, presence and severity of ascites and encephalopathy to classify patients in class A, B or C; class A has a favorable prognosis, while class C is at high risk of death. It was devised in 1964 by Child and Turcotte and modified in 1973 by Pugh et al.

More modern scores, used in the allocation of liver transplants but also in other contexts, are

the Model for End-Stage Liver Disease (MELD) score and its pediatric counterpart, the Pediatric End-Stage Liver Disease (PELD) score.

The hepatic venous pressure gradient, i.e., the difference in venous pressure between afferent and efferent blood to the liver, also determines severity of cirrhosis, although hard to measure. A value of 16 mm or more means a greatly increased risk of dying (Patch et al.

1999).

The Child- Pugh classification is a means of assessing the severity of liver cirrhosis.

Child-Pugh Score for Cirrhosis Mortality Score bilirubin (μmol/L) albumin (g/L) PT (seconds prolonged) encephalopathy ascites

1 <34 >35 <4 none none

2 34-50 28-35 4-6 mild mild

3 >50 <28 >6 marked marked

If there is primary biliary cirrhosis or sclerosing cholangitis then bilirubin is classified as <68=1; 68-170=2; >170=3.

(To convert bilirubin in μmol/L to mg/dL, divide by 17.)

The individual scores are summed and then grouped as: <7 = Child’s A 7-9 = Child’s B >9 = Child’s C A Child’s C classification forecasts a survival of less than 12 months. (Pugh et al. 1973, p.

649) Survival of cirrhosis Child-Pugh Survival (%) grade 1 year A 82 B 62 C 42

5 years 45 20 20

10 years 25 7 0

Hepatic (%)

deaths

43 72 85

(Collier & Webster 2007, p. 945) Epidemiology Cirrhosis and chronic liver disease were the 10th leading cause of death for men and the 12th for women in the United States in 2001, killing about 27,000 people each year. Also, the cost of cirrhosis in terms of human suffering, hospital costs, and lost productivity is high (Anderson & Smith 2003).

Established cirrhosis has a 10-year mortality of 34-66%, largely dependent on the cause of the cirrhosis; alcoholic cirrhosis has a worse prognosis than primary biliary cirrhosis and cirrhosis due to hepatitis. The risk of death due to all causes is increased twelvefold; if one excludes the direct consequences of the liver disease, there is still a fivefold increased risk of

death in all disease categories (Sørensen et al. 2003, pp. 88-93).

Little is known on modulators of cirrhosis risk, apart from other diseases that cause liver injury (such as the combination of alcoholic liver disease and chronic viral hepatitis, which may act synergistically in leading to cirrhosis). Studies have recently suggested that coffee consumption may protect against cirrhosis, especially alcoholic cirrhosis (Klatsky et al.

2006).

Zinc Metabolism Zinc (from German: Zink), also known as spelter, is a metallic chemical element; it has the symbol Zn and atomic number 30. It is the first element in group 12 of the periodic table (Lehto 1968, pp. 822-24). It is called an ‘essential trace element’ because very small amounts of zinc are necessary for human health.

Biological role of zinc Zinc is an essential trace element, necessary for plants, animals and microorganisms. Zinc is found in nearly 100 specific enzymes (other sources say 300), serves as structural ions in transcription factors and is stored and transferred in metallothionein. It is the second most abundant transition metal in organisms after iron and it is the only metal which appears in all enzyme classes (United States National Research Council 2001, pp. 442-454).

In proteins, Zn ions are often coordinated to the amino acid side chains of aspartic acid, glutamic acid, cysteine and histidine. In humans, zinc interacts with a wide range of organic ligands, and has roles in the metabolism of RNA and DNA, signal transduction, and gene expression. It also regulates apoptosis.

In the brain, zinc is stored in specific synaptic vesicles and can modulate brain excitability. It plays a key role in learning. However it has been called "the brain's dark horse" since it also can be a neurotoxin, suggesting zinc homeostasis plays a critical role in normal functioning of the brain and central nervous system (Hambidge & Krebs 2007, pp. 1101-5).

Zinc is a good Lewis acid, making it a useful catalytic agent in hydroxylation and other enzymatic reactions. In blood plasma, zinc is bound to and transported by albumin (60%, low-affinity) and transferrin (10%). Since transferrin also transports iron, excessive iron reduces zinc absorption, and vice-versa (Rink & Gabriel 2000, p. 541).

Zinc may be held in metallothionein reserves within microorganisms or in the intestines or liver of animals. Metallothionein in intestinal cells is capable of adjusting absorption of zinc by 15–40%. However, inadequate or excessive zinc intake can be harmful; excess zinc particularly impairs copper absorption because metallothionein absorbs both metals (Hambidge & Krebs 2007, pp. 1101-5).

Dietary sources and daily requirement of zinc Foods high in protein are high in zinc. Red meats, especially beef, lamb and liver have some of the highest concentrations of zinc in food. Carbohydrate, cereal, fruit and fat contain low zinc. The Recommended Dietary Allowance (RDA) is 8 mg/day for women and 11 mg/day for men (United States National Research Council 2001’ p. 442).

Absorption of zinc Dietary zinc usually absorbed throughout the entire small intestine. Negligible absorption occurred from the caecum and colon. Zinc absorption seems to be a carrier mediated transport process. The rate and extent of zinc absorption is under the homeostatic control programmed by the zinc status of the individual. Zinc absorption is influenced by a variety of factors including dietary zinc content, metabolic demand and presence or absence of potential inhibitors of in the diets (Sandström et al. 1992).

Intestinal Lumen

Intestinal Cell

Plasma

Zinc Metalloproteins

Zinc

A

Zinc Pool

DNA

B

Zinc Carrier

m-RNA Thionein

Polysome

Zinc–metallothionein

C

Zinc

Zinc–Carrier

Figure: Proposed mechanism for intestinal zinc absorption (Cousin 1979, p. 342) Storage distribution and excretion of zinc Zinc is present in all tissues and fluids of the body. The total body content has been calculated as 2-3 gram (30-45 mmol) in adult, of which skeletal muscle accounts for approximately 60% and bone for about 30%. Plasma zinc represents only about 0.1% of total body content; it has a rapid turnover and the level appear to be under close homeostatic control. The majority of zinc in plasma is bound to albumins which act as the transport vehicle.

Various vascular components such as erythrocytes, leucocytes and platelets may provide immediate information about zinc. Of these erythrocytes expected to give a long term indication of zinc status since their half life is 30-40 days. Platelets with a shorter half life would be more indicative of acute changes, as would be in some of the leucocytes subset (Hallberg et al. 2000, pp. 192-208).

Underwood (1977) stated that zinc absorbed from the intestine is carried to the liver and released into the peripheral circulation. In venous plasma, zinc is mostly bound to albumin to some extent transferrin and α2 macroglobulin.

Dietary Intake 8-15 mg/day

Pancreas

Liver 130mg

Intestine Plasma 3mg

Bone 770mg

Skin 160mg

Loses: 7â&#x20AC;&#x201C;14 mg/day

0.5 mg/day

Muscle 1500mg

Kidney 20mg

Urine

0.5 mg/day

Figure: Zinc Metabolism in adult (Hallberg et al. 2000, p. 198).

The most rapid accumulation and turnover of zinc occur in pancreas, liver, kidney, spleen and prostate. The liver is the major organ involved in zinc metabolism, where the metal remains bound to metallothionein (MT). Metallothionein is unique in that, it may be synthesized de novo, depending on increased zinc status, which then serve as a storage protein for zinc, prior to its utilization in essential functions (Gordon et al. 1981, pp. 341-349).

Zinc is excreted from the body via the kidneys, skin and intestine. The intestine is the major route of zinc excretion and zinc is lost via the digestive juices & the shaded intestinal cells.

Endogenous intestinal looses can range from 0.5-3.0 mg/day depending on zinc intake.

Looses of zinc can be substantially increased in many diseases (Hallberg et al. 2000, pp. 192208).

Deficiency Zinc deficiency is usually due to insufficient dietary intake, but can be associated with malabsorption, acrodermatitis enteropathica, chronic liver disease, chronic renal disease, sickle cell disease, diabetes, malignancy and other chronic illnesses. Symptoms of mild zinc deficiency are diverse. Clinical outcomes include depressed growth, diarrhea, impotence and delayed sexual maturation, alopecia, eye and skin lesions, impaired appetite, altered cognition, impaired host defense properties, defects in carbohydrate utilization and reproductive teratogenesis. Mild zinc deficiency depresses immunity, although excessive zinc

does also (United States National Research Council 2001, pp. 442-455). The normal range of serum zinc is 700 – 1500 µg/L. The value less than 700µg/L are considered as low (Trumbo et al. 2001, pp.297-8).

Nearly two billion people in the developing world are deficient in zinc. In children it causes an increase in infection and diarrhea, contributing to the death of about 800,000 children worldwide per year (Prasad 2003, pp. 409–10).

Assessment of zinc status Circulating levels of zinc in plasma or serum are the most widely used indices of zinc status.

Although plasma zinc is decreased in severe zinc deficiency, the level can be affected by a number of conditions that are unrelated to zinc status. Other indices such as zinc level in WBC, RBC & functional indices such as taste, visual acuity and dark adaption have so far not proven useful in identifying marginal zinc deficiency in human (Hallberg et al. 2000, pp. 199208).

Plasma zinc concentration Zinc is an intracellular element and so only 0.01-0.02% of the body content circulates in plasma where it is highly bound to plasma proteins, particularly albumin. It is not surprising that plasma level in an individual gives limited information of body zinc status. Zinc rises after meal, interestingly falls below the baseline two hours after meal, rises on short term starvation and undergoes a diurnal rhythm. Plasma levels are also correlated with albumin levels, to which zinc is chiefly bound in plasma (Lifschitz & Henkin 1971, pp. 88-92).

Zinc in cirrhosis of liver As liver plays an important role in zinc homeostasis and different zinc compartments have been recognized to explain Zn kinetics in humans; the liver represents a fast-exchangeable Zn pool with an important role in the metabolism of Zn and other trace elements (Echejoh et al.

2008). Whereas Kalkan et al. also identified zinc deficiency in patients with liver disease in his study published in 2002. It has long been speculated that Zn has a protective effect against liver fibrosis and Zn intake in cirrhosis are based mostly on observations of reduced Zn levels in cirrhotic patients and on the beneficial effects of Zn supplementation on liver metabolism (Capocaccia et al. 1991, pp. 386–91).

Majority of zinc deficient cirrhotic patients one belonged to rural population in the study of Ma et al. on ‘Assessment of intake inadequacy and food sources of zinc of people in China’ published in 2007. In fulminant hepatic failure and hepatic encephalopathy, biochemical parameters suggesting liver dysfunction presenting inverse correlation with serum Zn levels (Chetri et al. 2003, pp. S28-30).

As Gur et al. (1998) states that serum and hepatic Zn levels were reduced in hepatitis B virus infected patients with cirrhosis. The Zn depletion in cirrhosis has been attributed to decreased intestinal Zn absorption, increased urinary loss, malnutrition, hypoalbuminemia, portosystemic shunts, and diminished hepatic Zn extraction. Majority of the published studies have suggested that Zn absorption was reduced in cirrhotic patients. Two mechanisms were proposed for Zn malabsorption in liver cirrhosis- (1) damage of the small bowel mucosa, (2) impairment of pancreatic exocrine function accompanied by reduced synthesis of ligands

such as picolinic acid in the liver (Ijuin 1998, pp. 1-5).

It has been suggested that some of the clinical features of liver cirrhosis, such as testicular atrophy, loss of body hair, night blindness, poor wound healing, poor appetite, decreased taste and smell acuity, susceptibility to infections, enhanced sensitivity to drugs, and decreased neurocognitive performances, may be related to conditioned Zn deficiency. In some cases Zn supplementation was beneficial to these patients (Grungreiff 2002, pp. 67-68). The zinc supplementation also reduces the inflammation and contributes to faster inflammation resolution, therefore further advance, modified and related studies are needed to update the data, knowledge and information regarding medical workup of patients with liver cirrhosis.

Materials and Methods Study Design: Case-control observational study.

Period of Study: The study was conducted from July 2010 – June 2011.

Place of Study: The study was carried out in the Department of Biochemistry, Dhaka Medical College in collaboration with the Department of Medicine, Dhaka Medical College Hospital and the Department of Biochemistry, Bangabandhu Seikh Mujib Medical University (BSMMU), Shahbag, Dhaka.

Study population: Study population was the diagnosed cases of liver cirrhosis attending in department of Medicine, Dhaka Medical College Hospital, Dhaka aged 18 years and above of both male and female.

Sample: Hundred adult subjects were selected as per selection criteria of which fifty diagnosed cases of liver cirrhosis and fifty healthy controls.

Sample Size: Calculation:

n=

{Zα 2P2 (1 − P2 ) + Zβ P1 (1 − P1 ) + P2 (1 − P2 )}2 (P1 − P2 ) 2

Here, n = Sample Size P1= Anticipated probability of exposure among cases P2= Anticipated Probability of exposure among controls =0.5(50%) Zα= Z value (2 tail) at a definite level of significance = 1.96 at 95% confidence level (assumed) Zβ= Z value (1 tail) at a definite power = 1.64 at 95% power

P1 =

OR × P2 1 + P2 (OR − 1)

Using above formula, P1 = 0.6 (assuming OR = 2)

Now, n

{1.96 × 2 × 0.5 × 0.5 + 1.64 × 0.6 × 0.4 + 0.5 × 0.5}2 = (0.6 − 0.5) 2 {1.96 × 0.5 + 1.64 × 0.49}2 = (0.1) 2 6.4208 = ≈ 642 0.01

So, sample size should be 642. But for the convenience 100 were taken.

Sampling techniques: Purposive consecutive sample was selected.

Selection criteria: All study subjects were selected under the following inclusion and exclusion criteria.

Inclusion Criteria: For cases: Age group: 18 years and above.

Sex: Both male and female.

Diagnosed cases of cirrhosis of liver.

Diagnosis was done on the basis of Compatible clinical history Biochemical evidence of liver dysfunction:Serum bilirubin – increased Abnormal liver enzymes (ALT, AST) Serum total protein – reduced Serum albumin– reduced Prolonged prothrombin time Radiological evidence (by ultrasound of hepatobiliary system):– Altered liver size Increased liver echo pattern Portal vein diameter – increased Evidence of ascites.

Endoscopic evidence of:– Esophageal varices Congestive gastropathy Liver Biopsy – in selective cases.

For control: Age group: 18 years and above.

Sex: Both male and female.

Subjects are apparently healthy, no acute or chronic systemic diseases.

Subjects without history of any type of hepatitis.

Subjects with normal liver function test.

Exclusion Criteria: For both cases and control: Patients on zinc therapy.

Patients with acute or chronic diarrhea.

Patients suffering from chronic debilitating diseases (renal failure, diabetes mellitus, malignancy).

Patients on hormonal therapy.

Pregnant woman.

Methods This observational case control study was conducted from July 2010 to June 2011. All study subjects were selected purposively according to selection criteria. Purpose of the study was explained in detail in each subject. Written informed consent was taken. Data were collected in a pre-designed data collection sheet including particulars of the patients, history and relevant investigations. Complete physical and relevant clinical examinations were performed. Diagnosis of cirrhosis was determined on the basis of clinical, biochemical and radiological findings. Severity of liver cirrhosis was assessed by Child-Pugh score.

Collection & Preservation of Samples: After full aseptic precaution 10ml of blood sample was collected from antecubital vein in the morning following 8 hours fasting. Before collection of blood specimen special effort was made to make the plastic wares free from metallic contamination particularly from zinc. All plastic wares used were kept immersed in detergent water for at least half an hour. Then all were washed thoroughly with tap water and were allowed to dry in air. Then all were kept immersed for 24 hours in 20% Nitric acid. After 24 hours the equipments were washed for 3 times in de-ionized water and were air dried. The air dried container were stored in a capped plastic container and used for sample collection.

Five ml of blood was transferred to de-ionized plastic test tube, centrifuged at 3500 rpm for 5 minutes. Serum was transferred to a clean polypropylene tube and preserved at -35째C until the test was carried out.

Parameters estimated were: 1. Serum zinc: Concentration was determined Spectrophotometry (AAS).

2. Serum bilirubin 3. Serum total protein 4. Serum albumin 5. Serum aspartate aminotransferase (AST) 6. Serum alanine aminotransferase (ALT) 7. Prothrombin time 8. Serum creatinine 9. Serum glucose

using

Atomic

Absorption

Ethical issues Ethical clearance for the study was taken from the Ethical Committee and the Department of Biochemistry, Dhaka Medical College.

Permission for the study was taken from the Department of Medicine, Dhaka Medical College Hospital and the Department of Biochemistry, Bangabandhu Seikh Mujib Medical University, Shahbag, Dhaka.

All the study subjects were thoroughly appraised about the nature purpose and implications of the study as well as entire spectrum and benefits and risk of the study.

Interests of study subjects were not compromised to safeguard their rights and health.

All the study subjects were assured of adequate treatment of any risk developed to study purpose.

Finally, written consents of all study subjects were taken free of duress and without exploiting of the subjects.

Data Analysis After meticulous checking & rechecking all data were tabulated and processed using Statistical Package for Social Science (SPSS) version 17.0. Qualitative data were expressed as frequency and percentage and were analyzed by Chi-square test (x2 test). Quantitative variables were expressed by mean ± SD. Values of different parameters were compared to see the difference between two groups using Student’s t-test. Different Child-Pugh classes were compared by ANOVA test (F-test). Spearman’s correlation was used to correlate the serum zinc level with different CTP classes. The P-value < 0.05 was considered as statistically significant. 95% confidence limit was taken as the level of significance.

Flow Chart Study population

Potential Participant (By inclusion criteria)

Excluded from the Study (By exclusion criteria)

Invitation of Patients for consent

Participants

History Physical Examination Investigations

Patients with Cirrhosis of Liver

Healthy Control

Outcome (Serum Zinc)

Comparison

Data Analysis

Result

Discussion, Summary & Conclusion

WORKING DEFINITIONS: Cirrhosis of Liver: Cirrhosis of Liver is a consequence of chronic liver disease, diagnosed on the basis of compatible clinical history, abnormal liver function tests (serum bilirubin, serum total protein, serum albumin, AST, ALT, prothrombin time), ultrasound studies (altered liver size & echo pattern, signs of portal hypertension), endoscopy results (esophageal varices) and by histological examination (in selective cases) (Heidelbaugh & Bruderly 2006, pp. 756-758).

Serum Zinc Level: The normal range of serum zinc is 700 – 1500 µg/L. The value less than 700µg/L are considered as low (Trumbo et al. 2001).

The Child- Pugh Score: The Child- Pugh classification is a means of assessing the severity of liver cirrhosis.

Child-Pugh Score for Cirrhosis Mortality Score Bilirubin (μmol/L) Albumin (g/L) PT (seconds prolonged) Encephalopathy Ascites

1 <34 >35 <4 none none

2 34-50 28-35 4-6 mild mild

3 >50 <28 >6 marked marked

If there is primary biliary cirrhosis or sclerosing cholangitis then bilirubin is classified as <68=1; 68-170=2; >170=3.

(To convert bilirubin in μmol/L to mg/dL, divide by 17.)

The individual scores are summed and then grouped as: <7 = Child’s A 7-9 = Child’s B >9 = Child’s C A Child’s C classification forecasts a survival of less than 12 months. (Pugh et al. 1973, p.

649) Results And Observations An observational case-control study of 50 diagnosed adult patients of cirrhosis of liver and 50 healthy control was conducted in the Department of Biochemistry, Dhaka Medical College, from July 2010 to June 2011. The fasting serum zinc level of both cases and controls were assessed. Then serum zinc level was statistically compared to observe the association with cirrhosis of liver and its relationship with the severity of the disease. The results obtained are presented bellow.

Table I Distribution of study subjects by age Age Case Group Frequency Percentage < 40 21 42.0

Control Frequency 19

Percentage 38.0

40 – 60 > 60 Total

11 18 50

22.0 36.0 100.0

14 17 50

28.0 34.0 100.0

Table I shows the age distribution of the study subjects. Most of the subjects belonged to age group less than 40 years. In this group, 21 (42%) were case & 18 (36%) were control. Belong to the group 40 – 60 years, the frequency of case and control was 11 (22%) and 17 (34%) respectively, whereas 18 (36%) case and 15 (30%) control subjects were included in the age group of more than 60 years.

Figure I: Age distribution of case and control group Table II Mean age of the study subjects Group Mean ± SD (Years) Control 47.96 ± 13.29 (n- 50) Case 48.72 ± 16.28 (n- 50)

Range (Years) 21-68

t

P

-0.256

0.799ns

18-72

Values are expressed as Mean± SD n- Total number of subjects t = Value obtained by Unpaired student’s t test.

P = Level of significance. ns = Not significant (P>0.05) Table II shows the mean age of the study subjects. In case group, the mean age was 48.72 ± 16.28 years (Range 18 – 72) and in control group, the mean age was 47.96 ± 13.29 years

(Range 21 â&#x20AC;&#x201C; 65). No statistically significant difference was found among the study subjects by age.

Figure II: Mean age of Case and Control Table III Distribution of study subjects by sex Sex Male Female Total

Case (n- 50) 31 (62.0) 19 (38.0) 50 (100.0)

Control (n- 50) 28 (56.0) 22 (44.0) 50(100.0)

Total (n- 100) 59 (59.0) 41 (41.0) 100(100.0)

x2

P

3.240

0.07ns

Figure in parenthesis indicate percentage.

X2 = Value obtained by Chi-Square test.

P = Level of significance. ns- Not significant (P>0.05).

Table III shows the sex distribution of the study subjects. There were 31 (62%) of male and

19 (38%) of female cirrhotic patients enrolled in this study. In control group 28 (56%) were male and 22 (44%) were female. Among the total of 100 subjects, male & female were 59 (59%) and 41 (41%) accordingly. No statistically significant difference was found among the study subjects by sex.

Figure III: Distribution of study subjects by sex Table IV Distribution of cirrhotic patients in relation to serum zinc level Serum zinc level (Îźg/L) Low

Normal

Frequency

Percentage

Frequency

Percentage

36

72.0%

14

28.0%

Table IV shows the distribution of cirrhotic patients in relation to serum zinc status. Among the total of 50 patients with cirrhosis of liver the serum zinc level was low in 36 (72%) patients while remaining 14 (28%) patients had normal serum zinc level.

Figure IV: Distribution of cases in relation to serum zinc level Table V Mean serum zinc level in case and control Serum zinc level (μg/L)

Case (n- 50)

Control (n- 50)

t

P

Mean ± SD Range

610.32 ± 169.60 183.0-938.0

827.66 ± 267.32 429.0-1830.0

4.854

0.0001***

Values are expressed as Mean± SD. n- Total number of subjects. t = Value obtained by Unpaired student’s t test.

P = Level of significance. *** Highly significant at the 0.01 level.

Table VIII shows Mean serum zinc level in case and control. The mean serum zinc level was 610.32 ± 169.60 μg/L in case group and it was 827.66 ± 267.32 μg/L in control group. A statistically significant mean difference was found; indicating case group had lower serum zinc concentration than control group.

(Îźg/L)

Figure V: Mean serum zinc level in case and control Table VI Distribution of cases in relation to CTP score CTP Score Group A Group B Group C Total

Male 2 (4.0) 16 (32.0) 13 (26.0) 31 (62.0)

Female 2 (4.0) 11 (22.0) 6 (12.0) 19 (38.0)

Total 4 (8.0) 27 (54.0) 19 (38.0) 50 (100.0)

Figure in parenthesis indicate percentage.

Table VII shows Distribution of case in relation to CTP score. According to Childâ&#x20AC;&#x201C;Pugh score system, 4 (8%) patient were in group A and 19 (38%) were in group C. Majority of the patients i.e. 27 (54%) were remained in group B.

Figure VI: Distribution of case in relation to CTP score Table VII Mean serum zinc level of different CTP class Serum zinc Group A (μg/dL) Mean ± SD 748.0 ± 128.49 Range

654.5 – 938.0

Group B

Group C

F

644.61 ± 532.60 ± 156.02 4.401 164.03 201.5 – 928.5 183.0 – 845.5

P 0.01**

Values are expressed as Mean± SD. F = Value obtained by one way ANOVA (F-test).

P = Level of significance. ** Significant (P<0.05) Table VII shows the mean serum zinc level of cirrhotic patients according to CTP class. Here mean serum zinc level of Group A was 748.0 ± 128.49 μg/dL (range 654.5 – 938.0) Group B was 644.61 ± 164.03 μg/dL (range 201.5 – 928.5) and Group C was 532.60 ± 156.02 μg/dL (range 183.0 – 845.5). A significant mean difference was found by one way ANOVA test among the groups indicating stepwise decline of serum zinc with worsening child class.

Figure VII: Mean serum zinc level of different CTP class

Figure VIII: Negative correlation of serum zinc with different CTP class Table VIII Correlation between serum zinc and severity of cirrhosis of liver Serum zinc Correlation-Coefficient (r) P Variables CTP Group A CTP Group B -0.761 < 0.01** CTP Group C r = Value obtained by Spearman rank correlation test.

P = Level of significance. **Correlation is significant at the 0.01 level (2 tailed).

Table VIII shows correlation between serum zinc and severity of cirrhosis of liver according to CTP classification. Here Correlation-Coefficient or â&#x20AC;&#x2DC;râ&#x20AC;&#x2122;-value is -0.761 which means there is strong negative correlation between serum zinc level and severity of the disease. And the relationship is statistically significant.

Discussion The magnitude of liver disease in Bangladesh is progressively increasing. It is one of the major issues causing mortality and morbidity both in urban and rural population. It affects all

age group from children to elderly people. Hepatitis B and C viruses are the commonest culprit causing cirrhosis of liver in our country. It has been observed that, even in the rural population of Bangladesh, about one-third of common people are affected by Hepatitis B virus infection sometimes in their life. About 0.5% of the population was found to posses Hepatitis C virus infection. Furthermore, due to consumption of adulterated foods, fruits, edible oils etc. may be causative factor for long term inflammation of the liver leading to chronic liver disease, cirrhosis and Hepatocellular carcinoma in a vicious cycle. Excess and fatty food intake leads to fatty liver which may progress to cirrhosis of liver in the long run.

The current trend of first food culture in young generation is one of the reasons of fatty liver, which is prevailing in our country (Alam 2010).

The liver is important for the regulation of zinc homeostasis, while zinc is necessary for proper liver function. Decreased zinc levels have been implicated in both acute and chronic liver disease states, but its hepatoprotective property has not been fully elucidated (Stamoulis et al. 2007). It should be noted that zinc level is usually related to the nutritional pattern of each population. Zinc deficiency is widespread in people living in developing countries like Bangladeshi populations who consume rice-based diets (Hambridge & Krebs 2007).

Therefore keeping all such important points and views in mind, the present study was carried out to evaluate and assess the serum zinc level in patients with cirrhosis of liver. With this objective serum zinc level of adult patients with cirrhosis of liver was studied. For this reason a data collection sheet was prepared and laboratory investigations were done in all subjects for serum zinc level. All the data were filled up carefully and after plotting the results of laboratory investigation statistical analysis was done by using appropriate tests.

In the present study 59 were male and 41 were female among the total of 100 subjects. In case group 31 (62%) were male and 19 (38%) were female; in control group, 28(56%) were male and 22 (44%) were female. The mean age (Mean ± SD) of the case group was 48.72 ± 16.28 years, ranging from 18 – 72 years. In control group, it was 47.96 ± 13.29 years, ranging from 21 – 68 years. No statistically significant difference was found among the study subjects by age and sex. It is similar to the study of Ma et al. published in 2007.

This study was found a significant plasma zinc deficiency (72%) in patients with cirrhosis of liver when compared with normal control subjects in the same age group. The results of this study are compatible with Soomro et al. (2009, p. 988) who evaluated the cirrhotic patients of a tertiary care hospital in Pakistan. They found low level of zinc in 69% of cirrhotic patients.

Triwikatmani et al. (2009, p. 5) found 66.7% & stamoulis et al. (2007) found 65.3% of cirrhotic patients with hypozincemia. A study in Brazil conducted by Ana et al. (2009, p. 361) showed that 43% had concentration below the normal level. That was not consistent with this study.

Mean serum zinc concentration of cirrhotic patients was 610.32±169.60 μg/L and the mean of healthy controls was 827.66±267.32 μg/L. A statistically significant mean difference was found; indicating case group had lower serum zinc level than control group.

In a study of Triwikatmani et al. (2009, p. 5) the mean value of zinc serum level was 637.0±248.5 μg/L. However Yoshida et al. (2001, p. 354) demonstrated the mean serum zinc level in decompensate liver cirrhosis patients 563.0±137.0 μg/L, while in compensated hepatic cirrhosis patients 750.0±150.0 μg/L. Mean serum zinc level was 694.0±229.5 μg/L when studied by Ana et al. (2009, p. 362). These results were consistent with the present

study. Poo et al. (1995, p. 141) found mean serum zinc level 589.0±161.0 μg/L in cirrhotic patients from Mexico City. They showed that the levels were unexpectedly lower compared to those found in other countries. Whereas the level was 751.5±114.0 μg/L in the study conducted by Nurdjanah et al. (2006) in Indonesia that was quite different compared to the present result.

The exact percentage of zinc deficiency in this study may be higher. Some researcher assumed that serum zinc measurement in zinc deficiency is relatively less sensitive because mild zinc deficiency can occur with normal zinc serum concentration. The examination of zinc concentration in granulocyte and lymphocyte give more sensitive diagnostic criteria for marginal zinc deficiency compared to plasma zinc concentration (Grahn et al. 2001, p. 112), but cannot be done in our laboratory.

According to Child-Pugh classification 54% of cirrhotic patient were in class B, 38% in class C and 8% in class A. Triwikatmani et al. (2009, p. 4) also found a nearly similar result when studied in Serdjito hospital, Indonesia. The mean serum zinc level of child class A, B and C was 748.0 ± 128.49 μg/L, 644.61 ± 164.03 μg/L and 532.60 ± 156.02 μg/L. A significant mean difference was found by ANOVA test among the groups indicating stepwise decline of serum zinc with worsening child class. The similar results were obtained by Poo et al. (1995, p. 141) and stamoulis et al. (2007). But Triwikatmani et al. (2009, p. 5) did not find statistically significant difference in the mean serum zinc concentration of CTP score B (676.8 ± 215.5 μg/L) and CTP score C (540.4 ± 322.5 μg/L). Hambridge et al. (1987, p. 875) and Loguercio et al. (2001, p. 253) did not find any association between zinc deficiency and the increase in the severity of cirrhosis.

Ana et al. (2009, p. 362) stated the mean serum zinc level of cirrhotic patients as follows: 912.6 ± 228.8 μg/L for Child-Pugh A; 596.2 ± 148.5 μg/L for Child-Pugh B+C. There was no statistical difference between Child-Pugh A patients and control subjects, but there was a statistically significant difference between Child-Pugh B+C patients and Child-Pugh A patients.

To find out the relationship between serum zinc level and severity of cirrhosis of liver (classified according to CTP score) Spearman’s rank correlation test was done in this study.

The value of ‘r’ was -0.761.The negative value indicates indirect or negative relation. The degree of relationship was strong and statistically significant. So the present study showed that serum zinc level was inversely related with the severity of cirrhosis of liver. Similar conclusion was drawn by Ramzy et al. (2008, p. 104). Ana et al. (2009, p. 362) found a negative correlation between zinc level and disease severity with r = -0.59 and the association was significant.

From above discussion it may be concluded that serum zinc level remains low in patients in cirrhosis of liver compared to healthy subjects. The level was deteriorating while the severity of the cirrhosis of liver increases according to the Child Pugh score. Therefore, zinc status should be investigated in all patients regardless of cirrhosis severity. Zinc should be included among the micronutrients that are given particular consideration in the management of cirrhosis in order to prevent zinc deficiency.

Summary And Conclusion The present study titled ‘Serum Zinc Level in Cirrhosis of Liver’ was carried out in the

Department of Biochemistry, Dhaka Medical College in collaboration with the Department of Medicine, Dhaka Medical College Hospital and the Department of Biochemistry, Bangabandhu Seikh Mujib Medical University, Shahbag, Dhaka. This observational case control study was conducted from July 2010 to June 2011.

The aim of the study is to observe the association of serum zinc level in patient with cirrhosis of liver. A total of 100 subjects were included in this study, having the age 18 years and above. Among them 50 subjects were cases of cirrhosis of liver and 50 subjects were age and sex matched healthy controls. All study subjects were selected purposively according to selection criteria. Purpose of the study was explained in detail to each subject. Written informed consent was taken. Data were collected in a pre-designed data collection sheet including particulars of the patients, history and relevant investigations. Complete physical and relevant clinical examinations were performed. Diagnosis of cirrhosis was determined on the basis of clinical, biochemical and radiological findings. Severity of liver cirrhosis was assessed by Child-Pugh score. The fasting serum zinc level of both cases and control were assessed. The value less than 700Âľg/L were considered as low. Then the serum zinc level was statistically compared to observe the association with cirrhosis of liver and its relationship with the severity of the disease.

The present study had identified the lower level of serum zinc in patients with cirrhosis of liver compared to healthy subjects. The 72% prevalence of low serum zinc concentrations with cirrhosis of liver was observed in the study. This study found an inverse relationship between zinc concentration and the cirrhosis severity.

Conclusion: A stepwise decline of serum zinc with worsening Child-Pugh class was observed in this study. It is important to note that some patients classified as Child-Pugh A also had hypozincemia. Therefore a routine biochemical assessment of zinc status in patients with liver cirrhosis is an important step in the management protocol and to reduce progression of the disease. We conclude that zinc should be included among the micronutrients that are given particular consideration in the management of cirrhosis in order to prevent zinc deficiency.

Recommendations To improve better management of cirrhosis of liver we recommend that, Further prospective studies with large sample should be carried out to evaluate the association of zinc status with cirrhosis of liver.

Clinician should perform the biochemical assessment of zinc status and supplement the zinc as micronutrient in management of cirrhosis of liver.

Further studies should be carried out in larger population including other parameters like zinc level in erythrocytes, granulocyte, lymphocyte and also urinary zinc excretion in patients with cirrhosis of liver.

List Of Abbreviations ALT AST CT CTP dL DNA

: Alanine Transaminase : Aspertate Transaminase : Computed Tomography : Child-Turcott-Pugh : Deciliter : Deoxyribonucleic acid

ERCP : Endoscopic Retrograde Cholangio-Pancreatography g : Gram GIT : Gastro Intestinal Tract L : Liter mg : Milligram MRCP : Magnetic Resonance Imaging Cholangio-Pancreatography MRI : Magnetic Resonance Imaging PT : Prothrombin Time RBC : Red Blood Cell RNA : Ribonucleic acid SD : Standard Deviation SPSS : Statistical Package for Social Science WBC : White Blood Cell WHO : World Health Organization Zn : Zinc μg : Micro gram μmol : Micro mol Bibliography Alam, M 2010, ‘Prevalence of Liver Disease in Bangladesh’, The Stethoscope, 24 October, pp 1-2.

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Appendices

APPENDIX II CONSENT FORM (English Version) Thesis Title: Study of Serum Zinc in Cirrhosis of Liver I, …………............................................ do hereby give my well informed and coercion free consent to participate in the study conducted by Dr. Farhana Atia. I have been explained the purpose of the study and fully understand that my participation in the study may facilitate generation of new medical information for many others including myself in future.

I am convinced that during participation in the study, I shall not be exposed to any physical, physiological, social and legal risk. My confidentiality will be safeguarded and any anonymity will be protected. I would not like to be monetarily compensated because of the loss of my work time. I also give my consent to use my blood and urine for the study.

Signature of Principal Investigator

Signature of Subject/ Thumb impression

Date: .............................

Date:.........................

APPENDIX III Data Collection Form Department of Biochemistry Dhaka Medical College, Dhaka.

Thesis Title: Study of Serum Zinc level in Cirrhosis of Liver ID No: Reg. No: Name of the Patient: Address with Contact No.: Particulars of the Subject: Age (Years): Sex: Male Lived in: Rural Area History: History of hepatitis: Yes Duration of Liver Cirrhosis: < 5yrs Hepatic Diet: Yes Pregnancy: Yes Treatment history: Zinc Supplement: Yes Diuretics: Yes Hormonal Therapy: Yes Associated Diseases: Diarrhea: Yes i

Date:

Female Urban Area No > 5 yrs No No No No No No

Renal Failure: Diabetes Mellitus: Malignancy: On Examination: Encephalopathy: No Lab Investigations: Serum Zinc: Serum Bilirubin: Serum total protein: Serum Albumin: AST: ALT: Prothrombin Time: Serum Creatinine: Serum Glucose: Ultrasound of HBS: Evidence of cirrhosis of liver: Ascites: No 8. Endoscopic examination: Esophageal varices: Congestive gastropathy: Liver biopsy: Evidence of cirrhosis of liver:

Yes Yes Yes

No No No Mild

Marked

Yes Mild

No

Yes Yes

No No

Yes

No

Marked

Signature of data collector:

ii

APPENDIX V Statistical Formulae Sample size,

n=

{Zα 2P2 (1 − P2 ) + Zβ P1 (1 − P1 ) + P2 (1 − P2 )}2 ( P1 − P2 ) 2

Here, n = Sample Size P1= Anticipated probability of exposure among cases P2= Anticipated Probability of exposure among controls Zα= Z value (2 tail) at a definite level of significance Zβ= Z value (1 tail) at a definite power

P1 =

OR × P2 1 + P2 (OR − 1)

∑x Mean, (x) = n Here, ∑x = Summation of individual observation n = Number of observations (x − x)2 Standard Deviation (SD) = ∑ n −1 2 ( x − x ) Here, ∑ = Summation of square deviation from mean n = Number of observations Unpaired students ‘t’ test m1 − m 2 t= (SE1 ) 2 + (SE 2 ) 2 df = (n1 − 1) + (n 2 − 2) Here, m1 = mean of first group m2 = mean of second group SE1 = Standard error of first group SE2 = Standard error of second group n1 = Sample size of first group n2 = Sample size of second group Chi-square test (x2–test) (O − E ) 2 x2 = ∑ E Here, O = Observed value E = Expected value =

Row Total × Column Total Grand Total

df = (No. of Row – 1) × (No. of Column – 1) ANOVA (F-test)

F=

S2bet S2wit Here, F = F-ratio or F-test statistic S2bet = between group variance iii

S2wit = within group variance

Spearman’s Rank Correlation Test Correlation Coefficient, rs =

∑(R x − R x )(R y − R y ) ∑(R x − R x ) 2 ∑(R y − R y ) 2

Here, df = n-2 Rx = Rank of independent (x) variable Ry = Rank of dependent (y) variable APPENDIX VI Estimation of serum zinc Method: Determination of serum zinc concentration by Atomic Absorption Spectrophotometry, model Perkin Elmer – 3110.

Principle: Elements are dissociated from its chemical bonds by spraying over a flame and brought to unexcited ground state. These dissociated elements are capable of absorbing light of definite wavelength. The intensity of light absorption is proportional to the concentration of the element in the sample.

Reagents: Glycerol solution Nitric acid Zinc Standard De-ionized water Heparin sodium Preparation of glycerol / water solution: 5 ml of glycerol is diluted to 100 ml with de-ionized water (5/90 v/v) as blank.

Preparation of standard: Stock standard: Primary standard, 1000 mg of zinc per Liter. Dilute 10 ml of nitric acid to 50 ml; dissolve 1gm of Zinc metal in this and further diluted to 100 ml. Our zinc metal was zinc powder.

Working standard: 100, 200, 300 and 400 mg of Zinc/Liter from the stock standard 1ml were taken into a 100 ml volumetric flask. Then it was diluted up to 100 ml with glycerol/water solution (5/95 by volume). It was then mixed by inverting at least 16 times.

Aliquots of 1, 2, 4, & 8 ml from these intermediate stock solutions were taken into four 100 ml volumetric flasks. They were diluted to the volumes with the glycerol / water mixture.

Thus concentration of working standard became 100, 200, 400 & 800 mg of Zinc/Liter.

Procedure: Plasma samples, after removal from the refrigerator were allowed at first to come to room temperature. Then each sample was mixed by gently inverting the tube six times.

0.5 ml of plasma sample was delivered with a micropipette into a plastic test tube and 2.0 ml of de-ionized water was added to it, then immediately mixed the solution for 30 seconds.

Instrument and gas flow settings and aspiration rate were precisely established to minimize background noise and optimize signal. Once the aspiration rate was optimized with 10 ml of aliquots of H2O, the nebulizer flow adjustment was locked in place.

Glycerol / water solution (5/95 by volume), as blank, was aspirated into the luminescent flame and the baseline was set to read 0.000 ± 0.001 extinction. A baseline reading was taken before and after each sample application and the baseline was reset frequently.

The zinc working standards were sampled sequentially from most dilute to most iv

concentrated, aspirating until the reading become stable (Âą 0.001 extinction).

Then reading for plasma samples were taken at wavelength 214 nm.

Three readings were taken for all blank standards or plasma samples (1second integration reading for each sample.)

Calculation: The concentration of Zinc in plasma are calculated using the following formula a, which are also compared with the working (standard) curve. The working curve is prepared by plotting the concentration of Zinc in different working standards along the abscissa against their extinction along the ordinate.

Concentration of Zinc in serum (ppm) =

Extinction of test Ă&#x2014; Concentration of standard Ă&#x2014; dilution factor Extinction of standard

Precaution A baseline is taken before and after each sample application and the baseline is resetted frequently and after each sample application Instrumental and gas flow setting and aspiration rate are precisely established to minimize background noise and optimize signal.

Preparation of standard curve for Zinc Strength of stock standard solution was 1000 mg of Zinc per liter. From these stock solutions, the different standard solutions were prepared by diluting the stock solutions. For estimation of plasma zinc, dilution was done by glycerol / water solution. Thus the strength of working standard was 100, 200, 400 and 800 mg / L of Zinc.

Test Tube No. Concentration of Zinc Standard (mg / L) Extinctions 1 100 0.030 2 200 0.060 3 400 0.121 4 800 0.246 The curve was constructed by taking the extinctions as the ordinate and concentrations as abscissa.

v

Reference Range 700-1500 μg/L Appendix VII Estimation of serum Bilirubin (Total) Method: Determination of serum Total Bilirubin was done by DMSO (dimethylsulphoxide) method.

Principle: The azobilirubin produced by the reaction between bilirubin and the diazonium salt of sulfanilic acid shows maximum absorption at 555 nm in acid medium. The intensity of the color produced is proportional to the quantity of bilirubin which has reacted. In the absence of an accelerator only conjugated bilirubin reacts. In the presence of an accelerator, the dimethylsulphoxide (DMSO), the non-conjugated bilirubin also participates to the reaction, thus to determine the level of total bilirubin.

Reagent Concentration R1: Sulfanilic acid 32.2 mmol/L HCl 0.12 N Sodium chloride 0.145 mmol/L Ethylene glycol 1.8 mmol/L DMSO 6 mmol/L R2: Sodium nitrite 1 mmol/L Preparation of Reagents: All reagents are ready for use.

Procedure: Test tubes are arranged in racks labeled as Blank and Sample. Then reagents are added as follows– Sample Blank Sample R1 1.0 ml 1.0 ml Distilled H2O 0.2 ml --R2 --0.2 ml Serum 0.1 ml 0.1 ml Then tubes were shaken & allowed to stand 5 minutes at room temperature. Then the absorbance of the sample was measured against the sample blank at 530 nm in a colorimeter.

Calculation Total bilirubin concentration mg/dL = Absorbance x 13.50 Total bilirubin concentration μmol/L = Absorbance x 195 Reference range Total bilirubin: Up to 1 mg/dL (17.1 μmol/L)

vi

APPENDIX VIII Estimation of serum Total Protein Method: Estimation of serum Total Protein was done by Biuret method described by Weichselbaum (1946).

Principle: Cupric ion in alkaline medium, interact with protein peptide bond resulting in the formation of a violet colored complex. Color produced is proportional to amount of protein in solution Reagent Composition Contents Initial Concentration of Solutions Biuret reagent Sodium Hydroxide 100 mmol/L Na-K-tartrate 16 mmol/L Potassium iodide 15 mmol/L Cupric sulphate 6 mmol/L Blank reagent

Sodium hydroxide Na-K-tartrate

100mmol/L 16 mmol/L

Standard Protein 6.0 g/dL Preparation of Reagents: Biuret reagent: Dilute the contents of bottle I with 400 ml of double distilled water, rinsing the bottle thoroughly.

Blank Reagent: Dilute the contents of bottle 2 with 400 ml of double distilled water, rinsing the bottle thoroughly.

Standard: Ready for use.

Procedure: Test tubes are arranged in racks labeled as Blank, Standard and Sample. Then reagents are added as followsâ&#x20AC;&#x201C; Reagent Blank Standard Sample Distilled H2O 0.02 ml --Standard --0.02 ml Serum or Plasma ----0.02 ml Solution 1 1.0 ml 1.0 ml 1.0 ml Then tubes were shaken & allowed to stand 10 minutes at room temperature. Then the extinction of the sample and standard were measured against the reagent blank at 530 nm in a colorimeter.

Calculation Total protein concentration g/dL =

Extinction of sample Ă&#x2014; Concentration of Standard Extinction of Standard

Preparation of standard curve for standard total protein The strength of the standard solution supplied was 6 g/dL. From this stock solution, different standard solutions were prepared by dilution with de-ionized water.

Standard Concentration Concentration of Protein (g/dL) 6 5 4 3 2 1 Standard Protein Solution (ml) 100 0.83 0.67 0.50 0.33 0.17

De-ionized Water (ml) 0.00 0.17 0.33 0.50 0.67 0.83 For each standard, 3 tests were done separately. The average of the 3 readings of each standard was taken to construct the curve.

Test Tube Actual Protein Standard Calculated Protein Standard Extinctions No. (g/dL) (g/dL) 1 6 0.268 6.00 2 5 0.222 4.97 3 4 0.180 4.03 4 3 0.135 3.03 5 2 0.09 2.01 6 1 0.046 1.03 The curve was constructed by taking the extinctions as the ordinate and concentrations as abscissa.

Reference Range in serum 6.4 – 8.3 g/dL (64 – 83 g/L) Appendix IX Estimation of serum Albumin Principle The measurement of serum albumin is based on its quantitative binding to the indicator 3, 3’, 5, 5’-tetrabromo-m cresol sulphonephthalein (bromocresol green, BCG). The albumin-BCGcomplex absorbs maximally at 578 nm, the absorbance being directly proportional to the concentration of albumin in the sample.

Sample Serum of subjects.

Reagent Composition Contents Initial Concentration of Solutions R1.

BCG concentrate Succinate buffer 75 mmol/L; pH 4.2 Bromocresol green 0.15 mmol/L Brij 35

CAL.

Preservative Standard Human serum albumin Tris Buffer

45 g/L 100mmol/L; pH 7.3

Procedure Test tubes were arranged in a rack and labeled as Blank, Standard & Sample. Then reagents are added as followsReagent Standard Sample Distilled H2O 0.01 ml ------Standard (CAL) ---0.01 ml ---Serum of Plasma ------0.01 ml BCG reagent (R1) 3.00 ml 3.00 ml 3.00ml Tubes were shaken and allowed to stand for 10 minutes at room temperature. Then the absorbance of the sample and standard were measured against the reagent Blank at 530 nm in a colorimeter.

Calculation The albumin concentration in the sample may be calculated from the following formula: Albumin Concentration (g/L) =

Absorbance of Sample × Concentration of Standard Absorbance of Standard

Normal values in serum Adults 38 – 44 g/L (3.8 – 4.4 g/dL) APPENDIX X Estimation of ALT (Alanine aminotransferase) by Humalyzer – 2000 (Semi Auto Analyzer) Method Optimized Triss Buffer, kinetic method is used for estimation of ALT. Principle ALT L-alanine + α-ketoglutarate Pyruvate + L-glumate

LDH Pyruvate + NADH + H+ L-Lactate + NAD + H2O ALT catalyses the transfer of amino group from L-alanine to α-ketoglutarate resulting in formation of Pyruvate + L-glutamate. Lactate dehydrogenase (LDH) catalyses the reduction of pyruvate and simultaneous oxidation of NADH (Nicotinamide adnine dinucleotide dehydrogenase) and NAD (Nicotinamide adnine dinucleotide).

Contents of reagents Reagent 1 (buffer / substrate) Triss buffer, pH 7.3 100 mmol/L L-alanine 500 mmol/L Reagent 2 (Enzyme / Co-enzyme / α-oxaloglutarate) α-oxaloglutarate 15 mmol/L LDH ≥ 1200 U/L NADH 0.18 mmol/L Reagent preparation and stability Contents of the Reagent 2 were dissolved with corresponding volume of Reagent 1 (buffer / substrate). Working reagent was stable for 30 days at +2 to +8°C. Specimen Non hemolyzed serum.

Test procedure

Assay: Method Wavelength Temperature Pre-incubation Zero Read Pipetting Scheme

Kinetic 340 nm 37°C 1 min H2O Every 60 second, during 3 minutes

Pipette into test tube Sample 0.1 ml Working Reagent 1.0 ml Mixed well and initial absorbance was read after 1 minute. Again after every 60 second during 3 minutes.

Calculate ∆A/min Calculation of Result ΔA / min ×106 × TV = ΔA / min × F = U/L 6.3 ×103 ×1 × V Here, ∆A = Change in absorbance min = Minute 3 = Molar absorptivity of NADH at 340 nm 6.3 ×10 6 10 = Conversion of mmol to μmol 1 = Light path in cm TV = Total reaction volume in ml V = Sample volume in ml ΔA 340nm / min ×1746 = U/L ALT Normal values in serum Up to 40 U/L. APPENDIX XI Estimation of serum AST (Aspartate amino transferase) Method Colorimetric method is used for estimation of AST Principle The enzyme AST catalyzes the following reaction: L-aspartate +2-oxoglutarate ASToxalacetate + L-glutamate The oxalacetate in reaction 2, 4-dinitro-phenylhydrazine forms oxalacetate hydrazones which are brown in alkaline medium. The product is determined photometrically at 505 nm.

Sample Nonhemolyzed serum of subjects Reagents Buffer-substrate (1×100 ml) Triethanolamine-EDTA buffer, pH 7.5 50 mmol/L L-aspartate 200 mmol/L 2-oxoglutarate 2 mmol/L Color reagent (1×100 ml) 2, 4-dinitrophenylhydrazine (DNPH) 1 mmol/L

Standard (1×10 ml) Sodium pyruvate 2 mmol/L Additional reagent NaOH 0.4 mmol/L Procedure Wavelength: 505 nm (490-520) Cuvette 1 cm light path Temperature 37 °C Color stability 60 min.

Zero Reagent blank Test tubes were arranged in a rack and labeled as Blank & Sample. Then reagents are added as followsPipette into test tubes Sample Reagent blank Buffer-substrate 0.5 ml 0.5 ml Sample 0.1 ml --Distilled water --0.1 ml Mix and incubate for exactly 30 minutes at 37 °C DNPH 0.5 ml 0.5 ml Mix well and let stand for exactly 20 minutes at 20 to 25 °C NaOH 5.0 ml 5.0 ml Mix and after 5 minutes read the absorbance of sample against the reagent blank.

Calculation Using the absorbance values of the samples, read off the enzyme activity in U/L from calibration curve.

Preparation of calibration curve Test tube no.

Pipette into tubes Blank 1 2 3 4 5 6 Redistilled water 0.20 0.20 0.20 0.20 0.20 0.20 0.20 Buffer-substrate 1.00 0.95 0.90 0.85 0.80 0.75 0.70 Standard --0.05 0.10 0.15 0.20 0.25 0.30 DNPH 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.4 mol/L NaOH 10 10 10 10 10 10 10 AST U/L 0 10 20 32 47 67 93 Plot (millimeter paper) the absorbance values determined on the ordinate against the U/L values (from the table) on the abscissa and use these points to draw a calibration curve.

Reference Range of AST: Up to 19 U/L.

APPENDIX XII Estimation of serum creatinine Method Serum creatinnine was estimated by alkaline picrate method.

Principle Creatinine reacts with picrate ions in an alkaline picrate solution to form an orange red colored complex (Janovsky complex). The intensity of orange red color is measured by colorimeter.

Creatinine + Alkaline picrate Creatinine picrate Reagent Reagent – 1 Picric acid 17.5 mmol/L Reagent – 2 Sodium hydroxide 0.29 mmol/L Standard Creatinine solution 2 mg/dL Picric acid (reagent – 1) was mixed with sodium hydroxide (reagent – 2) in proportionately 1:1 (0.5 ml each) to make working solution.

Sample Serum of study subjects Procedure Pipette into the tubes: Standard Sample Serum --100 μL Standard 100 μL --Reagent mixture 1.0 ml 1.0 ml Immediately after proper mixing the colored solution were given into the colorimeter and absorbance were taken at 492 nm wavelengths against distilled water blank at the end of 1 minute.

Calculation Creatinine concentration in the sample was calculated by using the following formula Creatinine concentration (mg/dL) =

Absorbance of sample × Concentration of standard (2 mg/dL) Absorbance of standard

Reference Range in serum 0.6 – 1.4 mg/dL APPENDIX XIII Estimation of Serum Glucose Method Serum glucose was estimated by glucose oxidase method.

Principle The glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The formed hydrogen peroxide reacts under catalysis of peroxidase with phenol and 4aminophenazone to a red-violet quinoneimine dye as indicator.

Contents RGT 100 ml or 1000 ml Enzyme reagent Phosphate buffer (pH 7.5) 0.1 mol/L 4-aminophenazone 0.25 mmol/L Phenol 0.75 mmol/L Glucose oxidase > 15 KU/L Peroxidase > 1.5 KU/L

Mutarotase > 2.0 KU/L Stabilizers STD 3 ml standard Glucose 100 mg/dL or 5.55 mmol/L Procedure: Test tubes are arranged in racks labeled as Blank, Standard and Sample. Then reagents are added as follows– Reagent Blank Standard Sample Standard --0.01 ml Serum ----0.01 ml Reagent 1.0 ml 1.0 ml 1.0 ml Then tubes were shaken & allowed to stand 10 minutes at room temperature. Then the extinction of the sample and standard were measured against the reagent blank at 530 nm in a colorimeter.

Calculation Serum Glucose concentration (mg/dL) =

Extinction of sample × Concentration of Standard Extinction of Standard

Normal values Serum (fasting):

75 – 115 mg/dL or 4.2 – 6.4 mmol/L