2. Introduction

Hepatitits B virus (HBV) infection is a global health problem

and it is estimated by the World Health Organization (WHO), that

approximately one-third of the world’s population has been infected with HBV,

with serological evidence of past or present infection with HBV. Of the 2

billion people who have been infected world wide, more than 350 million (5 -

7% of the world’s population) suffer from chronic HBV infection.

Approximately 15 - 40% of patients infected with HBV will develop life –

threatening liver consequences (including cirrhosis, liver failure and

hepatocellular carcinoma) resulting in 600,000 to 1.2 million deaths per year

due to HBV.1-4

Based on the prevalence of Hepatitis B surface antigen (HBsAg),

countries are classified as having high (where ≥ 8% of population of the

population is HBsAg positive), intermediate (2-7%) or low (< 2%) HBV

endemicity. Areas of high endemicity include South-East Asia, China, most of

Africa, most of Pacific islands, the Amazon basin and parts of the Middle-

East. The areas of intermediate endemicity include South Asia, Eastern and

Southern Europe, Russia, Central and South America. On the other hand,the

areas with low endemicity include the United states, Western Europe and

Australia.5

Every year over the world, there are 4 million acute clinical cases of

HBV, and about 1 million die from chronic active hepatitis, cirrhosis or primary

liver cancer.

South Asia including India has been grouped in countries with

intermediate endemicity (3.7%).The sheer enormity of the population of the

1

region accounts for a large chunk of the entire pool of HBV carriers of the

world.6

India has over 40 million HBV carriers and accounts for 10-15% of the

entire pool of HBV carriers of the world. Of the 25 million infants born every

year in India, it is estimated that over 1 million run the life time risk of

developing chronic HBV infection. Every year 100,000 Indians die due to

illnesses related to HBV infection7,8. There are varying reports of overall rate

of HBsAg positivity ranging from 2 - 4.7%9,10.

Very high levels of HBsAg positivity has been reported in the tribes of

Andaman and Nicobar islands.11. There are hyperendemic foci of HBV

infection in Arunachal Pradesh. While it is generally accepted that the

modality of transmission of HBV in India is horizontal, the recent report by

Dwivedi,et .al.12 showing a high prevalence of replicative markers in India

suggest that there may be a significant role of vertical transmission as well.

The present study involves study of biochemical variables (Serum

Total bilirubin, Direct bilirubin, Alanine transaminase, Aspartate transaminase,

Albumin,Globulin, Copper, Zinc) in liver disorders (Chronic active hepatitis B

and Cirrhosis). This topic of study was choosen, as it has been least explored

in this region and to draw conclusions, to progress further in diagnosis and

management of patients.

2

GROSS ANATOMY OF THE LIVER13

Liver,the largest gland of the body, is wedge shaped. It occupies whole of the

right hypochondrium, upper part of the epigastrium and part of the left

hypochondrium upto the left lateral plane.It weighs about1.4 -1.8 kgs.

The liver presents 5 surfaces(Superior,inferior, anterior,posterior and

right lateral surfaces),3 borders(the postero-superior border, postero-inferior

and inferior border), 5 lobes( anatomical right and left lobes, caudate,

quadrate lobes and sometimes riedel’s lobe). The H fissure is located in the

inferior and posterior surface – the right limb includes groove for vena cava

and fossa for gall bladder, the left limb includes fissures for ligamentum

venosum and ligamentum teres,and the horizontal limb of the fissure is

formed by the porta hepatis.

The microscopic organization of liver cells is grouped into three cell

clusters:

1. Classical hepatic lobules: centre around the central vein (a tributary of

hepatic vein).

2. The Portal lobules: centres around the portal triads.

3. The liver acinus: centres on the pre terminal transverse vessels and bile

ductules derived from adjacent portal triad.

Blood supply – hepatic artery and portal vein.

3

Lymphatic drainage - Superficial lymphatics drain into supra diaphragmatic,

hepatic, coeliac and paracardiac lymph nodes. Deep lymphatics drain into

supradiaphragmatic and hepatic lymph nodes.

Nervous supply- by hepatic nerves, which arise from the hepatic plexus.

FUNCTIONS OF THE LIVER14,15

Liver performs certain endocrine functions,

has a role in Immune and inflammatory response .

synthesizes blood clotting factors.

Liver is an important homeostatic regulator of blood glucose

The liver plays a key role in the metabolism of liver and lipoproteins(fatty acid

synthesis ,beta oxidation of fatty acids,ketogenesis,etc)

Liver is responsible for the formation and secretion of bile in the intestine. The

bilirubin formed from heme catabolism is conjugated in liver cells and is

secreted in the bile.

Liver synthesizes most of the plasma proteins.

liver is involved in detoxification and protective function-Kupffer cells of the

liver remove foreign bodies from blood by phagocytosis. Liver cells detoxify

drugs, hormones and convert them into less toxic substances for excretion.

Liver stores glucose in the form of glycogen. It stores vitamin A, vitamin B 12,

iron, aminoacids and lipids.

4

CHRONIC HEPATITIS B16

Chronic hepatitis represents a series of liver disorders of varying

causes and severity in which hepatic inflammation and necrosis continue for

at least 6 months.

Clinical and serological features allow the establishment of a diagnosis

of chronic viral hepatitis caused by Hepatitis B.

Laboratory diagnostic tests include those of HBsAg, IgG anti HBc,

HBeAg and HBV DNA.

CHB can be divided into 2 phases based on relative level of HBV

replication. The relative replicative phase is characterized by the presence in

serum of markers of HBV replication (hepatitis B antigen HBeAg, HBV DNA),

by presence in liver of detectable intrahepatocyte nucleocapsid antigens

(primarily hepatitis B core antigen HBcAg),

The relatively non replicative phase is characterized by association

with anti-HBe, absence of intra hepatocytic HBcAg.

CHRONIC ACTIVE HEPATITIS16

Chronic active hepatitis occurs 2 -3 times less often than chronic

persistent hepatitis.HBV is the causative agent in from 10 % to 50 % of cases.

Clinically silent, slow progression to cirrhosis is common.

Intermittent deepening of jaundice and recurrence of malaise and anorexia,

worsening fatigue, are reminiscent of acute hepatitis.

5

Extrahepatic complications of CHB are associated with deposition of

circulating hepatitis B antigen – antibody complexes. These include

arthralgias, arthritis, purpuric cutaneous lesions (leukocytic vasculitis),

immune complex glomerulonephritis, and generalized vasculitis (polyateritis

nodosa).

The principal diagnostic histologic features of chronic active hepatitis

are portal inflammation and necrosis involving the periportal parenchyma.

There is a progressive deposition of new collagen in these areas, with

formation of irregular fibrous septa at the periphery. The degenerating

hepatocytes, often individually or in small groups, are entrapped and

encompassed by the fibrous tissue of expanding portal areas. Destruction of

periportal liver cell plate results in liver cell regeneration.

Serum Aspartate tansaminase (AST), Alanine transaminase (ALT) are

commonly elevated, Serum globulins tend to be increased, and the bilirubin

levels is often high. Aminotransferases elevations tend to be modest for CHB

but may fluctuate in the range of 100 – 1000 units. ALT tends to be more

elevated than AST. Levels of alkaline phosphatase activity tend to be normal

or only marginally elevated.In severe cases, moderate elevations in serum

bilirubin (3-10 mg/dl) occur. Hypoalbuminemia and prolongation of

prothrombin time occur in severe or end stage cases.The most consistent

globulin change in chronic liver disorders is an increase in gamma globulin

concentration

6

CIRRHOSIS19

Cirrhosis as the end stage of chronic liver disease is defined by three

characteristics:

1. Bridging fibrous septae in the form of delicate bands or broad scars

linking portal tracts with one another and portal tracts with terminal

hepatic veins.

2. Parenchymal nodules containing proliferating hepatocytes encircled by

fibrosis, with diameters varying from very small (< 3mm, micronodules ) to

large (several cm, macronodules).

3. Disruption of architecture of the entire liver.

The cardinal pathologic features reflect irreversible chronic injury of the

hepatic parenchyma and include extensive fibrosis in association with the

formation of regenerative nodules. These features result from hepatocyte

necrosis, collapse of the supporting reticulin network with subsequent

connective tissue depositon, distortion of the vascular bed, and nodular

regeneration of remaining liver parenchyma.

The central event leading to hepatic fibrosis is activation of the hepatic

stellate cell. Upon activation by factors released by hepatocytes and kupffer

cells, the stellate cells assumes a myofibroblast- like conformation and , under

the influence of cytokines such as transforming growth factor beta (TGF –

beta), produces fibril forming type I collagen.

CLINICAL FEATURES - Loss of functioning hepatocellular mass may

lead to jaundice, edema, coagulopathy, and a variety of metabolic

7

abnormalities; fibrosis and distorted vasculature lead to portal hypertension

and its sequelae, including gastroesophageal varices and splenolmegaly.

Ascites and hepatic encephalopathy result from both hepatocellular

insufficiency and portal hypertension.

ALCOHOLIC CIRRHOSIS -Historically referred as Laennec’s cirrhosis,

is characterized by diffuse fine scarring, fairly uniform loss of liver cells, and

small regenerative nodules, thus referred as micronodular cirrhosis.

Quantity and duration of alcohol intake are the most important risk

factors involved in the development of alcoholic liver disease. The threshold

for developing severe disease in men is an intake of > 60-80 g/d of alcohol for

10 years.

With continued alcohol intake and destruction of hepatocytes,

fibroblasts (including activated hepatic stellate cells that have transformed into

myofibroblasts with contractile properties) appear at the site of injury and

deposit collagen. Web like septa of connective tissue appear in periportal and

pericentral zones and eventually connect portal triads and central veins. This

fine connective tissue network surrounds small hepatocyte destruction and

collagen deposition, the liver shrinks in size, acquires a nodular appearance,

and becomes hard as end stage cirrhosis develops.

SIGNS AND SYMPTOMS- Anorexia and malnutrition lead to weight loss and

reduction in skeletal muscle mass. Easy bruising is experienced. Eventually

clinical manifestations of hepatocellular dysfunction and portal hypertension

ensue, including progressive jaundice, bleeding from gastoesophageal

varices, ascites and encephalopathy.

8

A firm, nodular liver may be an early sign of disease.Jaundice, palmar

erythema, spider angiomas, parotid and lacrimal gland enlargement, clubbing

of fingers, splenomegaly, muscle wasting and ascites with or without

peripheral edema.Men may have decreased body hair, gynaecomastia and

testicular atrophy. In women, signs of virilization or menstrual irregularities

may be encountered. Dupuytren’s contractures resulting from fibrosis of

palmar fascia are associated with alcoholism. Most patients with advanced

cirrhosis die in hepatic coma, commonly precipitated by hemorrhage from

esophageal varices or intercurrent infection.

LABORATORY FINDINGS

Anemia may result from gastrointestinal blood loss, hypersplenism,

suppressive effect of alcohol on bone marrow and nutritional deficiencies. Mild

or pronounced hyperbilirubunemia may be found, usually in association with

varying elevations of serum alkaline phosphatase levels. Levels of serum AST

are frequently elevated but levels more than 300 units are unusual. The

serum AST is usually disproportionately elevated relative to ALT, i.e. AST /

ALT ratio > 2.

The serum prothrombin time is prolonged, reflecting reduced synthesis

of coagulation proteins. The serum albumin level is usually depressed, while

serum globulins are increased.Elevated blood ammonia levels in hepatic

encephalopathy are seen.Central hyperventilation may lead to respiratory

alkalosis. Dietary deficiency and increased urinary losses lead to

hypomagnesemia and hypophosphatemia. In ascites and dilutional

9

hyponatremia, hypokalemia may occur from increased potassium losses due

in part to hyperaldosteronism.

DIAGNOSIS

Alcoholic cirrhosis is suspected in patients with a history of prolonged

or excessive alcohol intake and physical signs of liver disease. The clinical

features and lab findings are usually sufficient to provide presence and extent

of injury. Biopsy and ultrasonography are helpful diagnostic tools to evaluate

patients.

POSTHEPATITIC CIRRHOSIS

Coarsely nodular cirrhosis and multilobular cirrhosis are synonymous

with posthepatitic cirrhosis.1/4 -3/4 of these cases are due to hepatitis B or

hepatitis C.

Cirrhosis following viral hepatitis is macronodular, although a mixed

micro and macro nodular cirrhosis can develop. Cirrhosis following viral

hepatitis is called post necrotic, implying that it follows a single episode of

acute hepatitis with massive necrosis. Most cases of cirrhosis develop from

progression of Chronic active hepatitis.

The posthepatitic liver is shrunken in size, distorted in shape,

composed of regenerating nodules of varying shape and size, usually 1- 5 cm.

The fibrous septa are also irregular and often broad, following collapse of

several lobules. The newly formed regenerated nodules and vascular

distortion produced by central- portal bridging leads to the development of

portosystemic shunts and complete reconstruction of the original architecture

10

of the liver. The end result of this continuous process is a small, scarred

coarsely nodular liver.

Dysplasia of liver cells may appear in the liver with macronodular

cirrhosis, especially in Hepatitis B carriers. In patients with cirrhosis of known

etiology, the manifestations are an extension of those resulting from the initial

disease process. Clinical symptoms are related to portal hypertension and its

sequelae. The hematological and liver function abnormalities resemble those

seen with other types of cirrhosis.

DIAGNOSIS

Suspected in patients with signs and symptoms of cirrhosis or portal

hypertension. Biopsy confirms the diagnosis. About 75% of patients have

progressive disease despite supportive therapy and die within 1- 5 years from

complications.

MAJOR COMPLICATIONS OF CIRRHOSIS19

These include portal hypertension and its consequences (eg.

Gastroesophageal varices and splenomegaly), ascites, hepatic

encephalopathy,spontaneousbacterialperitonitis,coagulopathy,hepatopulmona

ry syndrome, hepatorenal syndrome, and hepatocellular carcinoma.

ASPARTATE AMINO TRANSFERASE ALANINE AMINO TRANSFERASE -

Elevations in the levels of the serum amino transferases suggest hepatocyte

injury.Chronic active hepatitis exhibit elevated AST and ALT.In cirrhotic liver

disease, serum transaminases activities are generally not elevated above 300

U/L21.

11

.

12

AIMS AND OBJECTIVES

1) To estimate biochemical variables (Total bilirubin, Direct bilirubin, liver

enzymes Alanine transaminase, Aspartate transaminase, Albumin,

A/G ratio, Copper, Zinc, Cu/Zn ratio) among patients with liver

disorders like Chronic active hepatitis B and Cirrhosis.

2) To estimate biochemical variables in age and sex matched healthy

controls.

3) To compare the biochemical variables between patients with liver

disorders and controls.

4) To evaluate correlation of AST, ALT, Total bilirubin with serum zinc and

copper in liver disorders like chronic active hepatitis B and Cirrhosis.

13

ZINC

(atomic number: 30, atomic weight :65.39) lies at the end of the

transition series of the periodic table15.

DAILY REQUIREMENT

Recommended is about 0.3 mg zinc / kg body weight, Adult men and

women require 15 -20mg.Food sources of zinc include meat products,

oysters, and legumes.22

Normal range (serum): 70–130 mcg/dL or 10.7–19.9 μmol/L

FUNCTIONS15

ROLE IN GROWTH AND DEVELOPMENT

The key role of zinc in protein and nucleic acid synthesis explains the

failure of growth and impaired would healing observed in individuals with zinc

deficiency. Proteins can form domains, able to bind tetrahedral zinc atoms by

coordination with histidine and cysteine to form folded structures that have

become known as “zinc fingers”23. These biologically active molecules have

important roles in gene expression by acting as DNA – binding transcription

factors and play key role in development biology and also in regulation of

steroid, thyroid and other hormone synthesis24.

ROLE IN WOUND HEALING-Zinc has been found to accumulate in

granulation tissues in around the healing wounds.

STORAGE AND SECRETION OF INSULIN-In secretory vesicles of

pancreatic β cells, insulin is stored as crystalloid like hexamers, each

14

stabilized by binding of Zinc to either thiol or imidazole side chain aminoacid

of insulin.

REMOVAL OF FREE RADICALS-Because of its presence in cytoplasmic

superoxide dismutase (SOD), zinc plays a role in the removal of superoxide

free radicals by that enzyme.

ROLE IN VITAMIN A METABOLISM - Zinc has been claimed to stimulate the

release of vitamin A from liver into the blood, and thus increases its plasma

level and its utilization in rhodopsin synthesis. zinc containing mettaloenzyme,

retinene reductase participates in the regeneration of rhodopsin in the eye,

during dark adaptation after illumination with light.

ROLE IN BIOSYNTHESIS OF MONONUCLEOTIDES - The biosynthesis of

mononucleotides and their incorporation into nucleic acids has been found to

be impaired in zinc deficiency. Zinc has a role in immune response.

GUSTATOTY SENSATION: Zinc protein, Gusten is involved in taste

sensation25.

ABSORPTION AND TRANSPORTATION

Food zinc is largely bound to proteins and released below the common duct

for absorption by the ileum.

Foods rich in calcium, dietary fiber, or phytate may interfere with zinc

absorption, as also can folic acid supplements. 20-30% of the dietary zinc is

absorbed mainly by small intestine. Zinc is transported from the small

intestine to the portal circulation where it binds to proteins such as albumin,

transferrin, and other globulins.

15

Circulating zinc is bound mostly to serum proteins; 2/3rds are loosely

bound to albumin and transthyretin while 1/3rd is bound tightly to β2-

macroglobulin. Only 2% to 3% (3 mg) of zinc is either in free ionic form or

bound to amino acids.

DISTRIBUTION -Normal adult body contains 1.5 – 2.5 g of Zinc. Tissues high

in zinc include liver, pancreas, spleen, lungs, eyes (retina, iris, cornea, and

lens), prostate, skeletal muscle, and bone.

EXCRETION -Zinc undergoes enteropancreatic recirculation and is excreted

primarily in pancreatic and intestinal secretion. It is also lost dermally through

sweat, hair and nail growth, and skin shedding. Zinc is mainly excreted

through gastrointestinal tract in the stool and to a lesser extent in urine. Urine

output is about 0.5 mg/day.

HYPOZINCEMIA15

Individuals with serum zinc concentrations below 70 mcg/dL (<10.7

μmol/L) are at an increased risk for developing symptomatic zinc deficiency.

ETIOLOGIES OF ZINC DEFICIENCY

1. LOW INTAKE: Anorexia, Nutritional deficiencies, Alcoholism, Chronic

kidney disease, Premature infants, Certain vegetarian diets, Use of

hyperalimentation solutions.

2. DECREASED ABSORPTION: Acrodermatitis enteropathica,

Malabsorption syndromes

3. INCREASED UTILIZATION: Adolescence, Lactation, Menstruation,

Pregnancy16

4. INCREASED LOSS: Alcoholism, β-thalassemia, Cirrhosis, Diabetes

mellitus, Diarrhea, Diuretic therapy, Enterocutaneous fistula drainage,

Exercise (long term, strenuous), Glucagon, Loss of enteropancreatic

recycling, Nephrotic syndrome, Protein-losing enteropathies, Sickle cell

anemia, therapy with hyperalimentation solutions.

5. UNKNOWN CAUSES: Arthritis and other inflammatory diseases,

Down’s syndrome.

SIGNS - Anemia, Anergy to skin test antigens, Complicated pregnancy,

Excessive bleeding, Maternal infection, Premature or stillborn birth,

Spontaneous abortion, Toxemia, Decreased basal metabolic rate, Decreased

circulating thyroxine (T4) concentration ,Decreased lymphocyte count and

function, Effect on fetus, infant, or child, Congenital defects of skeleton, lungs,

and CNS, Fetal disturbances, Growth retardation, Hypogonadism, Impaired

neutrophil function, Impairment and delaying of platelet aggregation,

Increased susceptibility to dental caries, Increased susceptibility to infections,

Mental disturbance, Pica, Poor wound healing, Short stature in children,

Skeletal deformities.

In zn deficiency, there is impairment of cell- mediated immunity26.

Neurological effects: Severe zn deficiency is known to affect mental

well being, with varying degree of confusion and depression being consistent

with zn enzymes having important activity in brain development and

function.27

SYMPTOMS:

17

Acne and recurrent furunculosis, Ataxia, Decreased appetite, Defective

night vision, Hypogeusia, Hyposmia, Erectile dysfunction, Mouth ulcers

The patients with end-stage liver disease frequently have depleted zinc

storage due to decreased functional hepatic cell mass.

HYPERZINCEMIA

Clinical manifestations include drowsiness, lethargy, and increased serum

lipase and amylase concentrations. Nausea, vomiting, and diarrhea may also

oocur. More than 60 mg zn/day can result in copper depletion by causing

intestinal blockade in intestinal absorption.28

ZINC and chronic liver disease

Supplementation of zinc may have a high potential to be developed as

an effective agent in the prevention and treatment of chronic liver

diseases29.Supplementation with zn has shown to improve glucose disposal

in cirrhotic patients30.

Metallothionine is a important zn binding protein ( formed by liver) and is

involved in zn metabolism, homeostasis and its release in number of oxidants,

the released zinc will inhibit the activity of enzymes involved in fibrogenesis

(fibrosis) in the liver31.

COPPER (Cu)

Normal range: 65–155 mcg/dL (10–24.6 μ mol/L) for serum.

18

Chemistry : Copper(atomic number :29,atomic weight : 63.54) has cu1+ and

cu2+ oxidation status in biological systems; the exchange between these ions

gives the element important redox properties.

Adult human contains 100-150 mg of copper, out of which 65 mg is

found in muscles, 23 mg in bones, and 18 mg in liver. It occurs as

Erythrocuprein (in RBC), Hepatocuprein (in liver) and cerbrocuprein32

(in brain)

DIETARY SORCES22

Found in high concentration in organ meats, such as liver and kidney,

with high amounts also found in shellfish, nuts, whole grain cereals, bran and

all cocoa containing products. Lower amounts of copper are found in white

meats and in dairy products.

ABSORPTION, TRANSPORT, METABOLISM and EXCRETION15

Copper absorption occurs in small intestine although gastric uptake

has been shown to occur in a small extent. The extent of intestinal copper

absorption varies with dietaty copper content and is around 50% at low

copper intake (< 1 mg cu per day) but only 20% at higher intakes (> 5 mg

cu /day)33.

Absorption is reduced by zinc, molybdate, iron and increased by

aminoacids and by dietary sodium32.Absorbed copper is transported to the

liver in portal blood bound to albumin where it is incorporated by hepatocytes

into cuproenzymes and other proteins and then exported in peripheral blood

to tissues and organs. Liver is the key organ in copper homeostasis33.

ROLE OF LIVER IN COPPER METABOLISM32

19

Liver processes absorbed copper through two routes: Copper is

excreted in the bile into gastrointestinal tract from which it is not reabsorbed.

Second route: Incorporation as an integral part of cerulopasmin, a

glycoprotein synthesized exclusively in liver.

More than 90% of the copper exported from the liver into peripheral

blood is in the form of the glycoprotein ceruloplasmin.A small amount is bound

to plasma by specific peptide sequences. 0.5-2.0 mg/day is excreted via bile

into feces. Copper urine and sweat are <3% of dietary intake. Urine copper

output is normally less than 60µg/day.

REQUIREMENTS

Infants and children : 0.05 mg/kg body weight per day. Adults – 2.5 mg/day.

FUNCTIONS

Copper is a catalytic component of numerous enzymes and is also

structural component of important proteins in humans.

ENERGY PRODUCTION - Cytochrome c oxidase is a multisubunit complex

containing copper and iron. The enzymes catalyzes a four electron reduction

of molecular oxygen, establishing a high energy proton gradient across the

inner mitochondrial membrane necessary for ATP production.

CONNECTIVE TISSUE FORMATION - Protein lysine – 6 oxidase is a

cuproenzyme essential for stabilization of extracellular matrices specifically

the enzymatic cross linking of collagen and elastin.

IRON METABOLISM - Copper containing enzymes ferroxidase 1

(ceruloplasmin) and ferroxidase 2 and the recently described haphestin in the

20

RBC – oxidizes ferrous to ferric iron. This allows incorporation of Fe3+ into

hemoglobin.

CENTRAL NERVOUS SYSTEM- Dopamine monoxygenase is an enzyme

that requires copper as a cofactor and uses ascorbateas an electron donor.

Monoamine oxidase, is a copper containing enzyme The formation of the

phospholipids necessary for myelin sheath formation is affected by

cytochrome c oxidase depletion.

MELANIN SYNTHESIS - Tyrosinase present in melanocytes catalyzes the

synthesis of melanin.

ANTI OXIDANT FUNCTIONS - Both intracellular and extracellular Superoxide

dismutases are copper and zinc containing enzymes, able to convert

superoxide radicals to hydrogen peroxide, which is removed by catalase and

other anti oxidant defenses.

REGULATION OF GENE EXPRESSION AND INTRACELLULAR COPPER

HANDLING - Copper dependent proteins acts as transcription factors for

specific genes such as those regulating transcription factors and this protein is

important in regulating intracellular distribution of copper35.

DEFICIENCY

MALNOURISHED INFANTS- develop iron resistant anemia, neutropenia,

other hematological disorder and bone lesions36.

PREMATURE INFANTS - premature infants fed with formula lacking

21

NUTRITIONAL SUPPORT- Adults and children fed intravenously develop

symptomatic copper deficiency.

MENKE’S SYNDROME - mutation is x- linked, occurs in male infants at 2-3

months.There is low copper in plasma, liver and brain; occurs due to impaired

intestinal absorption.

MALABSORPTION SYNDROME - Patients at risk include those with celiac

disease, tropical sprue, cystic fibrosis, and short bowel syndrome.

CARDIOVASCULAR DISEASE - Coronary artery pressure is decreased.

TOXICITY

Wilson’s disease (hepatolenticular degeneration) is a genetic disorder

of cu metabolism that causes an increase in copper to toxic level. Metabolic

defects - defect in incorporation of cu into newly synthesized

apoceruloplasmin to form ceruloplasmin. Plasma cu and ceruloplasmin which

will usually be low, there are abnormalities in liver function tests and

increased urine cu output (> 500µg cu/ L). Liver biopsy for cu analysis is

useful in suspected cases and result above 250µg /g.

COPPER AND LIVER DISEASE

Cu is an essential trace element which participates in many enzymatic

reactions. It is important in redox processes. Reactive copper can participate

in liver damage directly or indirectly through kupffer cell stimulation37.Redox

cycling between cu2+ and cu1+ can catalyze the production of toxic hydroxyl

radicals, which play important role in pathogenesis of liver cirrhosis38.

22

The interaction between zinc and copper in their intestinal absorption

and their competing for binding sites on their carrier proteins and cellular

uptake may be the regulator of homeostasis. May be this can explain inverse

concentrations of zinc and copper39.

CLINICAL RELATIONSHIP BETWEEN CHRONIC HEPATITIS B AND

SERUM TRACE ELEMENT LEVELS, MUHANNAD M. QASSIME; HIND M.

HAMMAD.; Department of Chemical and Pharmacological Analysis (M.O.S.T)

IRAQ.

In the present study serum trace elements Cupper (Cu), and Zinc (Zn), were

determined in sera of patients with viral hepatitis (B)cases (n=40) and

statistically compared with controls (n=20). The results showed that, in viral

hepatitis Cu level was significantly higher than in control group(p<0.05), Zn

level found to be significantly low in viral hepatitis patients, when compared to

healthy individuals(p<0.05) . It is suggested that the decrease in Zn level and

elevation in Cu levels are probably resulted from defense strategies of

organism and induced by the hormone-like substances.

Journal of Natural Sciences Research ISSN 2224-3186, Vol.3, No.6, 2013.

Correlation of Trace Elements and Chronic Hepatitis B Infections in

Babylon. Ali Abdul Hamid Salih Al-Jubbawy.

Hepatitis B virus (HBV) infection is a serious global public health problem.

Various trace elements play an important role in the course of HBV infection.

23

To determine the quantitative estimation of some trace element zinc in the

serum of patients with chronic HBV infection and compare with serum of

healthy group, and also to evaluate trace elements concentrations in chronic

HBV infected patients in regard to their serum transaminase levels. serum

concentration of Zn was determined in 40 patients with chronic HBV

infections, while the control group comprised 30 healthy volunteers.The study

showed that Zn concentrations in serum of hepatitis B patients with high liver

function test was 554 ± 292 ppb(parts per billion). While in patients with

hepatitis B virus infection with normal liver function test, these concentrations

was 735 ± 138ppb. In healthy individuals the concentration was 786 ± 333

ppb. All of these results were statistically significant (p < 0.05). This study

confirms the variation of trace elements concentration in Hepatitis B Virus

affected patients compared with healthy volunteers.

Copper and Iron determination in Biological Samples of Viral Hepatitis

(A–E) Female Patients. Hassan Imran Afridi & Tasneem Gul Kazi,Published

online: 23 December 2008# Humana Press Inc.

There is accumulative evidence that the metabolism of copper is altered in

viral hepatic diseases, and it might have specific role in their pathogenesis

and progress. The aim of study was to compare the level of copper in

biological samples (serum, urine, and scalp hair) of female patients suffering

from different viral hepatitis (A, B, C, D, and E; n=253) of age range 31–45

years. For comparative study, 95 healthy females of the same age group were

selected. The elements in the biological samples were analyzed by flame

atomic absorption spectrophotometry. The results of this study showed that

24

the mean value of Cu was higher in sera and scalp hair samples of hepatitis

patients than age-matched control subjects, while the difference was

significant (p<0.001), in the cases of viral hepatitis B and viral hepatitis C as

compared to viral hepatitis A, D, and E. These results are consistent with

literature-reported data, confirming that hepatic copper overload can directly

cause lipid peroxidation and eventually hepatic damage.

J Viral Hepat. 2006 Oct;13(10):671-7.

Predictive value of serum globulin levels for the extent of hepatic

fibrosis in patients with chronic hepatitis B infection. Schmilovitz-Weiss

H, Tovar A, Halpern M, Sulkes J, Braun M, Rotman Y, Tur-Kaspa R.

Immunoglobulins stimulate the proliferative activity of hepatic stellate

cells in vitro. A strong association was found between serum immunoglobulin

levels and hepatic fibrosis in patients with hepatitis C virus infection. Our

objective was to determine if the same index could also be used in patients

with chronic HBV infection. The records of 100 patients with evidence of

chronic HBV infection were reviewed for background factors and serum

globulin and immunoglobulin levels. Of the factors found to be significant on

univariate analysis, the only significant predictors of severe hepatic fibrosis

(stage > or = 2) on multivariate analysis were serum globulin level [ P =

0.0004], immunoglobulin G (IgG) level ( P < 0.042) For each increase of 0.33

mg/dL in serum globulin, there was a 0.5 point increase in the stage of hepatic

fibrosis. There appears to be a strong association between levels of serum

globulin and IgG and extent of hepatic fibrosis in patients with chronic HBV

infection. They can serve as noninvasive markers of hepatic fibrosis and, if

25

http://www.ncbi.nlm.nih.gov/pubmed?term=Tur-Kaspa%20R%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Rotman%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Braun%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Sulkes%20J%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Halpern%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Tovar%20A%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Schmilovitz-Weiss%20H%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed?term=Schmilovitz-Weiss%20H%5BAuthor%5D&cauthor=true&cauthor_uid=16970598

http://www.ncbi.nlm.nih.gov/pubmed/16970598

confirmed, have important implications for the management of patients with

chronic HBV infection.

Research Journal of Biological Sciences 4(5): 638-642, 2012.

The Effect of Chronic Liver Diseases on Some Biochemical Parameters

in Patients Serum, Essam F. Al-Jumaily and Faiha'a M. Khaleel

Genetic Engineering and Biotechnology Institute, Department of Chemistry,

Baghdad.

62 patients with chronic liver disease and 26 healthy individuals were

included as normal controls. Blood analysis was carried which include serum

bilirubin, total protein, and liver enzyme tests (GOT, GPT) levels. Results

showed that: (total bilirubin and direct bilirubin (μ mol/L)) have a higher level in

Chronic viral hepatitis than in the cirrhosis, but all groups showed increased

bilirubin levels more than in the control group (p<0.001).The concentration of

serum ALT, AST levels are increased highly significant in cirrhotic patients

compared to other groups of control patients (p<0.001). There was a

decrease in total protein concentrations observed in cirrhosis and liver cancer

patients compared to the control group.

A STUDY OF TRACE ELEMENTS ( ZINC, COPPER) IN LIVER CIRRHOSIS

PATIENTS, Gupta, Sunil; Meena, Shravn Kumar; Ahuja, Jitendra; Bohra,

Vishnu Dutt, International Journal of Current Research & Review;Aug2012,

Vol. 4 Issue 16, p69

The aim of present study is to examine serum level of Copper, Zinc in

Liver cirrhosis patients and Compare these in Patients with age, sex matched

normal control subjects. study was designed to evaluate serum zn, and cu

26

levels in blood and liver function test (Bilirubin, Serum Glutamate Oxalate

Transaminase, Serum Glutamate Pyruvate Transaminase, Akaline

Phosphatse, Total Protein, Albumin, and Globulin). Results -- the values of

serum Bilirubin, copper levels in blood were increased statistically significant

(p<0.00l) in liver cirrhosis when compared with the control subjects.

Statistically significant decrease (p<0.00l) in zn and Albumin were also found

in subjects of liver cirrhosis.

Copper, and zinc levels and copper to zinc ratios in serum of patients at

different stages of viral hepatic diseases. ching chang lin, jee fuhuang,

Department of laboratory medicine, Taiwan.

To examine the status of trace elements during the development of hepatic

carcinoma, we determined the cu and zn levels and cu to zn ratios in the

serum of patients at different stages of viral hepatic disease. The mean serum

cu level in patients with HCC was significantly higher than that of the control

group. In contrast, the mean zn levels in patients having HCC were

significantly low than those of the control group. The mean zinc level in the

serum of patients with hepatic cirrhosis was significantly lower than that of the

control group ( p < 0.05).Moreover, we found markedly elevated cu: zn ratios

(p < 0.05) in patients having hepatic cirrhosis or HCC. Our findings imply that

the levels of some trace elements ,such as cu, zn, and cu: zn ratios might

serve as biomarkers for the increased severity of viral hepatic damage.

Serum concentration of zinc, copper in patients with liver cirrhosis.

Rahelić D, Kujundzić M, Romić Z, Brkić K.

27

Serum concentrations of zinc, copper were determined in 105 patients with

alcoholic liver cirrhosis and 50 healthy subjects by means of plasma

sequential spectrophotometer. Serum concentrations of zinc were significantly

lower (median 0.82 vs. 11.22 micromol/L, p < 0.001) in patients with liver

cirrhosis in comparison to controls. Serum concentrations of copper were

significantly higher in patients with liver cirrhosis (median 21.56 vs. 13.09

micromol/L, p < 0.001).There were no differences in the concentrations of

zinc, copper between male and female patients with liver cirrhosis. The

correction of trace elements concentrations might have a beneficial effect on

complications and may be progression of liver cirrhosis.

Relationship of Copper and Zinc as Pathogenic Factors in Liver

Diseases. A.sawa, k.okita

In order to clarify the roles of copper and zinc in a progress of chronic liver

diseases, levels of copper and zinc in both sera and liver tissues were

measured. As a result, serum concentration of zinc decreased significantly, in

accordance with an aggrevation of liver disease, while serum and hepatic

levels of copper increased. The ratio of serum copper to zinc elevated

significantly in parallel with a progress of liver disease. Patients with the ratio

exceeding 2.0 generally suffered from cirrhosis or chronic active hepatitis.

This ratio coincided with changes of liver function test which reflected liver

fibrosis and residual liver function.

Serum immunoglobulin G levels in patients with chronic liver disease in

comparison to patients with autoimmune hepatitis Hind I. Fallatah* and

Hisham Akbar, Libyan J Med 2010, 5: 4857 - DOI: 10.3402.

28

Hypergammaglobulinemia is frequently observed in patients with chronic liver

disease (CLD) of different causes. Elevated levels of serum immunoglobulin

G (IgG) are the best diagnostic marker for autoimmune hepatitis (AIH). Thus,

the ability to distinguish AIH patients from patients with other liver disease,

especially patients with advanced liver cirrhosis, is important since most AIH

patients will a have favorable treatment response if diagnosed properly. We

conducted this study to evaluate the significance of elevated IgG levels in

patients with non-autoimmune CLD and to compare these IgG levels with

those in patients with AIH upon diagnosis. The serum IgG levels in 27 patients

with AIH were compared to the serum IgG levels in 27 patients with other

CLDs of variable severity. AIH patients had significantly higher serum IgG

levels than the non-autoimmune hepatitis CLD patients and the cirrhosis

patients in the CLD group (p<0.001 and p<0.044, respectively). Most patients

with elevated serum IgG of the AIH group (67%) and the CLD group (75%)

had significant hypergammaglobulinemia, not just isolated elevated IgG

levels. Elevated serum IgG levels with hypergammaglobulinemia are

commonly found in patients with advanced CLD.

Predictive markers of hepatocellular carcinoma in chronic hepatitis B.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is a major

global health problem. This study aimed to assess the predictability of HCC

risk in chronic hepatitis B patients, using a combination of liver-related

seromarkers combined with or without HBV seromarkers. A prospective

cohort of 1,822 anti-HCV-seronegative chronic HBV carriers was included in

this study. Liver-related seromarkers including AST, alanine aminotransferase

(ALT), total bilirubin, total protein, albumin, serum globulins were examined.

29

During a median follow-up of 5.9 years, 48 newly-developed HCC cases were

ascertained. Elevated serum levels of ALT (≥28 U/L), increased AST/ALT

ratio (AAR, ≥1), and lowered serum levels of albumin (≤4.1 g/dL) were

significantly associated with an increased HCC risk (P<0.05).Liver-related

seromarkers may be combined into useful risk models for predicting HBV-

related HCC risk. World Applied Sciences Journal 16 (8): 1053-1059,, 2012

30

MATERIALS AND METHODS

The samples from the following groups were analysed in the clinical

laboratory of Department of Biochemistry, Government general Hospital,

Kakinada:

1. 50 Chronic active hepatitis B patients.

2. 80 Cirrhosis patients.

3. 40 age and sex matched normal controls.

Selection of patients in this case control clinical study :

INCLUSION CRITERIA

1) Chronic active hepatitis B patients, diagnosed to be chronic hepatitis B

positive since 2 ± 0.2 years, serologically HBsAg and anti-HBc positive by

immunological assays, with age of 46.76 ± 4.02 years, with presence of

risky behaviour and risk factors as cigarette smoking and alcohol

consumption.

2) Cirrhosis patients diagnosed based on history, clinical signs and abnormal

ultrasonograms of abdomen since 3 ± 0.5 years ,with age of 57.03 ± 2.12

years, with presence of risk factors as alcohol consumption and cigarette

smoking. (Alcoholic and post necrotic cirrhosis)

EXCLUSION CRITERIA

31

Patients with positive serological tests for Hepatitis C virus / Human

immunodeficiency virus (HIV)

Delta virus co-infection.

Other causes of liver diseases due to drug toxicity.

Impaired renal function ( creatinine clearance <60 ml/min)

Multiorgan failure.

Wilson’s disease.

Any autoimmune disorders/ immunodeficiency disorder.

Acute or chronic diarrhea.

If on any mineral supplements.

Malnutrition.

Other causes of cirrhosis.

On drugs affecting mineral metabolism.

On cancer chemotherapy

On hormonal therapy

Diabetics and hypertensives.

Prior permission was taken from the Institutional Ethics Committee of

Rangraya Medical College, Kakinada to conduct the study. All of the subjects

provided their informed consent as approved by the ethics committee.

5 ml of random venous blood sample was collected from subjects in

sterile bottles and serum was separated taking precautions to avoid

hemolysis. All samples were analyzed immediately by kit methods using UV

1800 spectrophotometer.

Statistical analysis

32

Results are shown as Mean ± S.D. (standard deviation). For univariate

analysis, Pearson correlation coefficient (r) and its significance (p) were

calculated between the variables. To analyse statistically significant

differences in means of continuous variables between 2 groups of patients,

student t – test was used. P ≤ 0.05 was considered statistically significant.

ESTIMATION OF SERUM TOTAL AND DIRECT BILIRUBIN

Modified Jendrassik Grof Method40.41

PRINCIPLE

Bilirubin (Total and direct) in the sample reacts with diazotized

sulphanilic acid to form a coloured compound (azobilirubin), that can be

measured by spectrophotometer at 546nm.

REAGENTS

1. Bilirubin total reagent 1 : Sulphanilic acid(10 mmol/L), Conc HCL(40

mmol/L),Caffeine(25 mmol/L).

2. Bilirubin total reagent 2 : Sodium nitrite (1.5 mmol/L).

3. Bilirubin direct reagent 1 : Sulphanilic acid(10 mmol/L), Conc HCL (40

mmol/L).

4. Bilirubin direct bilirubin 2 : Sodium nitrite ( 0.26mmol/L) .

SAMPLE: Serum, EDTA sample.Bilirubin in serum is stable for 2 days at 2 –

8oC serum is protected from light.

33

PROCEDURE

Light path – 1 cm, Temperature – 37oC, Prewarm the sample, reagent

cuvettes to reaction temperature.

Sample Blank (ml) Sample (ml)

Bilirubin total reagent - 1 1.000 1.000

Bilirubin total reagent – 2 --- 0.025

Sample 0.050 0.050

Mix and incubate for 10 min at room temperature(RT) (or) 5 min at

37oC and read the absorbance at 546 nm against a serum blank immediately.

CALCULATIONS

Normal value -> Total bilirubin upto 1.0 mg/dl.

(Abs of sample- Abs of sample blank ) (26.31) = Total bilirubin (mg/dL)

DIRECT BILIRUBIN

Procedure is similar to the total bilirubin, for direct bilirubin use

reagents –bilirubin direct reagent 1 and 2 replacing the total bilirubin reagents.

CALCULATIONS

34

Normal value direct bilirubin upto 0.3 mg/dl.

(Abs of sample – Abs of sample blank) (26.31) = Direct bilirubin (mg/dl)

The assay is linear upto 25 mg/dl of bilirubin total / direct. For higher

concentrations dilute the sample ½ with saline. Multiply the result with 2.

ESTIMATION OF SERUM ALANINE TRANSAMINASE (ALT)

IFCC (International Federation Of Clinical Chemistry) Method42,43.

PRINCIPLE:

Alpha ketoglutarate + LAlanine L-Glutamic acid + Pyruvate

(catalysed by ALT)

Pyruvic acid + NADH + H+ L-Lactic acid+ NAD+ (catalysed by lactate

dehydrogenase)

REAGENTS:

The concentration in the reagent solution are: Tris –HCL buffer (90

mmol/L), L-Alanine (500 mmol/L), alpha-ketoglutaric acid (15 mmol/L), NADH

(0.18 mmol/L), LDH (800 U/L), available as enzyme reagent (R1) and

Substrate reagent (R2).

Working reagent is prepared in the ratio of 4 parts R1 : 1 part R2.

Monoreagent is stable for 30 days at 2-8oC, for 4 days at RT.

35

SAMPLE: Serum / plasma with EDTA / heparin. Samples free from

hemolysis should be used. Sera at 2-8oC loses 10% of its activity after 3 days.

PROCEDURE

Blank (ml) Sample(ml)

Deionised water 1.000 ---

Worki`ng reagent --- 1.000

Sample --- 1.000

Prewarm the sample, working reagent and cuvettes to reaction

temperature. Wavelength : 340 nm, light path : 1 cm.

Blank the photometer with deionised water. Mix, read the absorbance

after 1 min, and start the stop watch. Read again the absorbance after 1, 2

and 3 min.

CALCULATIONS

X = Initial absorbance – Absorbance after 1st, 2nd &or 3rd min.

Determine X / min for every reading and find the mean value.

Calculate U/L from: (Average X / min) (3490)

Normal values: At 37degree C – Men : 40 U/L, Women : 31 U/L.

Linearity: Upto 500 U/L For higher values the sample is diluted 1/10 with

normal saline and the result is multiplied with 10.

36

ESTIMATION OF SERUM ASPARTATE TRANSAMINASE (AST)

IFCC (International federation of clinical chemistry ) Method44,45,46

PRINCIPLE

Alpha-ketoglutaric acid + L-Aspartic acid L-Glutamic acid+ oxaloacetic

acid (catalysed by AST).

Oxaloacetic acid + NADH+ H+ L- Malic acid + NAD+ (catalysed by malate

dehyrogenase)

REAGENT: The concentrations in the reagent solution are : Tris-HCL buffer

pH 7.8 (80 mmol/L), L-Aspartic acid (240 mmol/L), Alpha-ketoglutaric acid (12

mmol/L), NADH (0.18 mmol/L), MDH (600U/L), LDH (800U/L).

Working Reagent preparation : The reagent solution is divided into

reagent R1 and reagent R2 .Working reagent is prepared by mixing 4 parts

R1(Enzyme reagent) and 1 part of R2 (Substrate reagent),

SAMPLE: Serum with EDTA / heparin. Samples free from hemolysis should

be used. Sera at 2-8oC loses 10% of its activity after 3 days.

37

PROCEDURE

Blank (ml) Sample (ml)

Deionised water 1.000 ----

Working Reagent ---- 1.000

Sample ---- 0.050

Prewarm the sample, working reagent and cuvettes to reaction

temperature. Blank the photometer with deionised water, mix, read the

absorbance after 1 min, and start the stopwatch. Read again the absorbance

after 1, 2 and 3 min. Wavelength :340 nm, light path : 1 cm.

CALCULATIONS

X = Initial absorbance-absorbance after 1st, 2nd and 3rd min.

Determine X / min, for every reading and find the mean value. Calculate the

U/L from (X / min) 3490.

Normal values: At 37oC – Men : 37 U/L, Women : 31 U/L.

Linearity: Upto 500 U/L of AST. For higher values, dilute the sample 1/10

with normal saline and multiply the result with 10.

38

ESTIMATION OF PLASMA TOTAL PROTEINS

(Biuret Method)47,48

PRINCIPLE

Proteins in an alkaline medium, bind with the cupric ions present in the

biuret reagent to form a blue- violet coloured complex. The intensity of the

colour formed is directly proportional to the amount of proteins present in the

sample.

REAGENTS: 1 Biuret reagent .2.Standard protein solution (Conc. = 6 gm/dl).

SAMPLE: Heparinised / EDTA Plasma, serum .Proteins are reported to be

stable in sample for 6 days at 2-8oC.

PROCEDURE

Blank (ml) Standard (ml) Test (ml)

Biuret reagent 1.00 1.00 1.00

Distilled water 0.01 -- --

Protein standard -- 0.01 --

Sample -- -- 0.01

Mix well and incubate at 37oC or at R.T. for 5 min. Measure the

absorbance of the standard (Abs S), of Test (Abs T) against the blank at 550

nm, with 1cm light path.

39

CALCULATIONS

Concentration of total proteins in the plasma = (Abs T /Abs S) 6

Normal reference value : 6 – 8 gm/dl

Linearity: The procedure is linear upto 15 gm/dl. If the values exceed the

limit, dilute the sample with distilled water, repeat assay, calculate value using

dilution factor.

ESTIMATION OF SERUM ALBUMIN - BROMOCRESOL GREEN

(BCG) METHOD49

PRINCIPLE

Determination of albumin in serum is based on the binding behavior of

albumin with dye 33’55’ tetra bromo M cresol sulfonapthalein (BCG) in the

acidic medium at pH 4.2. The blue green coloured complex is formed, the

intensity of which is proportional to the concentration of the albumin present in

the sample and is measured at 600 nm (600 – 650 nm ) or red filter.

REAGENTS: BCG reagent and Albumin standard, 4 gm/dl

SAMPLE: Serum.

PROCEDURE

Reagent Blank (ml) Standard (ml) Test (ml)

Working reagent 1.00 1.00 1.00

Standard -- 0.01 --

Sample -- -- 0.01

40

Mix well and incubate for 1 minute at room temperature. Measure the

absorbance of the standard (Abs S) and test (Abs T) against the reagent

blank at 600 nm (600 – 650 nm).

CALCULATIONS: Serum albumin concentration (gm/dl) = (Abs T /Abs S) 4

Normal reference value : 3.4 – 5.5 gm/dl.

CALCULATION of serum GLOBULINS : Plasma Total proteins –

Serum Albumin (g/dl). Normal range : 1.8-3.6 g/dl.

ESTIMATION OF SERUM COPPER

Colorimetric Method50.

PRINCIPLE

Copper, released from ceruloplasmin, in an acidic medium, reacts with

Di- Br- PAESA (di bromo pyridylazo –N- ethyl- N- sulfopropyl aniline) to form a

coloured complex. Intensity of the complex formed is directly proportional to

the amount of copper present in the sample.

REAGENTS: R1.Buffer reagent .R2.Colour reagent.

Working reagent is prepared by mixing equal volumes of R1 and R2.

The reagent is stable at 2-8oC for atleast 3 weeks.

SAMPLE: Serum, free from hemolysis. Copper is reported to be stable in the

sample for 6 days when stored at 2-8oC.

PROCEDURE



41


Copper standard -- 0.05 --

Sample -- -- 0.05

Wavelength: 580 nm, light path : 1cm. Mix well and incubate at R.T.(25oC) for

10 min. Measure the absorbance of the standard (Abs S) and test (Abs T)

against the blank, within 30 min.

CALCULATIONS:

Serum Copper = (Abs T /Abs S) (200).

Normal reference values: Serum (males) = 80 – 140 µg/dl, (females) = 80 –

155 µg/dl, (newborns) = 12 – 67 µg/dl, children upto 10 years = 30 – 150

µg/dl.

Linearity: Upto 500 µg/dl. For higher values dilute the sample with normal

saline and repeat the assay. Calculate the value using proper dilution factor.

Chelating agents as EDTA, oxalate and citrate present even in traces,

prevent the formation of colour complex. Highly lipemic samples could

interfere and should be cleared by centrifugation or filtration.

SERUM COPPER STANDARD CURVE

Standard: The concentration of Copper in standard solution is 2 µg /ml.

The working standards (S) are prepared by dilution of the standard.

S 1 S 2 S 3 S 4 S 5

Standard (ml) 0.2 0.4 0.6 0.8 1.0

Distilled water (ml) 0.8 0.6 0.4 0.2 0.0

Concentration(µg/dl) 40 80 120 160 200

42

These standards are reacted with the reagent and incubated at room

temperature for 10 min, and absorbance of the standards and duplicates of

the standards are taken at 580 nm and a standard curve is plotted with

absorbance on the x – axis and concentration of Copper on Y – axis.

Concentration of Copper (µg/dl)

Absorbance of standards

Absorbance of duplicates

Blank 0 0.00 0.00

S 1 40 0.03 0.03

S 2 80 0.06 0.06

S 3 120 0.09 0.09

S 4 160 0.12 0.12

S 5 200 0.15 0.15

ESTIMATION OF SERUM ZINC

Colorimetric Method51

PRINCIPLE

Zinc in alkaline medium reacts with Nitro – PAPS to form a purple

coloured complex, the intensity of the complex is directly proportional to the

concentration of the amount of zinc present in the sample.

REAGENTS

R1 : Buffer reagent.R2 : Colour reagent.

Working reagent is prepared by mixing together 4 parts of R1 and I part

of R2. The working reagent is stable for at least 2 weeks when stored at

2-8oC.

SAMPLE: Serum free from hemolysis. Zinc is reported to be stable for

2-8oC.

PROCEDURE 43




Standard -- 0.05 --

Sample -- -- 0.05

Mix well and incubate at R.T. (25 degree C ) for 5 min. Measure the

absorbance of the standard (Abs S) and Test (Abs T) against the blank within

20 min. wavelength : 570 nm, light path : 1 cm.

CALCULATIONS

Serum Zinc = (Abs T / Abs S) (200) µg/dl.

Normal reference value : Serum : 60 -120 µg/dl.

Linearity : Upto 700 µg/dl. For higher values, dilute the sample with distilled

water and calculate the results using dilution factor.

Chelating agents such as EDTA, oxalate, citrate even in trace

amounts, prevent the formation of coloured complex. Highly lipemic samples

could interfere and should be cleared by centrifugation or filtration.

SERUM ZINC STANDARD CURVE

Zinc standard: The concentration of zinc in standard solution is 2 µg /ml.

The working standards (S) are prepared by dilution of the standard:

S 1 S 2 S 3 S 4 S 5

Standard (ml) 0.2 0.4 0.6 0.8 1.0

Distilled water (ml) 0.8 0.6 0.4 0.2 0.0

Concentration (µg/dl) 40 80 120 160 200

44

These standards are reacted with the reagent and incubated at room

temperature for 5 min, and absorbance of the standards and duplicates of the

standards are taken at 570 nm and a standard curve is plotted with

absorbance on the x – axis and concentration of zinc on Y – axis.

Concentration of Zinc (µg/dl)

Absorbance of standards

Absorbance of duplicates

Blank 0 0 0

S 1 40 0.06 0.06

S 2 80 0.12 0.12

S 3 120 0.18 0.18

S 4 160 0.24 0.24

S 5 200 0.30 0.30

TABLE - 1

NUMBER OF CONTROLS AND CASES

(Chronic active hepatitis B and Cirrhosis)

GROUP NUMBER

1. CONTROLS 40

2. CHRONIC ACTIVE HEPATITIS B 50

3. CIRRHOSIS 80

TABLE - 2

AGE DISTRIBUTION OF PATIENTS STUDIED

Age (years) ControlsChronic active

hepatitis B Cirrhosis

35 – 40 15 (37.5%) 06 (12.0 %) 00 ( 0.0 %)

40 – 45 12 (30.0 %) 10 (20.0 %) 04 ( 5.0 %)

45 – 50 06 (15.0 %) 14 (28.0 %) 11 (13.75 %)

50 – 55 04 (10 .0%) 12 (24.0 %) 30 (37.5 %)

45

55 – 60 03 (7.5%) 08 (16.0%) 35 (43.75 %)

Total 40 (100 %) 50 (100 %) 80 (100 %)

35 - 40 40 -45 45 - 50 50 - 55 55 - 600%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CIRRHOSISCHRONIC ACTIVE HEPATITIS BCONTROLS

TABLE - 3

MEAN ± S.D. OF BIOCHEMICAL VARIABLES IN CONTROLS

PARAMETER MEAN ± S.D. Reference Range

Total Bilirubin (mg/dl) 0.40 ± 0.20 0.1 - 1.0

Direct Bilirubin (mg/dl) 0.12 ± 0.03 Upto 0.3

ALT (U/L) 28 ± 10.0 Upto 40

AST (U/L) 21 ± 8.5 Upto 37

AST / ALT 0.75 ±0.05 0.8

Albumin (g/dl) 3.95 ± 0.25 3.4 - 5.5

Globulins (g/dl) 2.50 ± 0.45 1.8 - 3.6

A / G 1.35 ± 0.04 1.2:1 - 2.5:1

46

Copper (µg/dl) 110.24 ± 8.9 80 – 140

Zinc (µg/dl) 88.17 ± 7.04 60 – 120

Cu / Zn 1.2 ± 0.23

The Mean ± S.D. of Serum Total bilirubin, Direct bilirubin, ALT, AST,

AST / ALT, Albumin, globulins, A/ G ratio, copper, Zinc, Cu / Zn ratio of the

control group are represented in the above table. They are within the

established normal values.

TABLE - 4

MEAN ± S.D. AND P VALUES BETWEEN CHRONIC ACTIVE

HEPATITIS B and CONTROLS

++ PARAMETERChronic active

Hepatittis BControls P value

Total Bilirubin (mg/dl) 5.16 ± 1.30 0.40 ± 0.20 < 0.01

Direct Bilirubin (mg/dl) 2.20 ± 0.31 0.12 ± 0.03 < 0.01

ALT (U/L) 266 ± 10.0 28 ± 10.0 < 0.01

AST (U/L) 223 ± 12.3 21 ± 8.5 < 0.01

47

AST /ALT 0.84 ± 0.12 0.75 ± 0.05 < 0.01

Albumin (g/dl) 3.0 ± 0.02 3.95 ± 0.25 <0.01

Globulins (g/dl) 4.1 ± 0.21 2.50 ± 0.45 < 0.01

A / G 0.73 ± 0.23 1.35 ± 0.04 < 0.01

Copper(µg/dl) 148.21 ± 4.5 110.24 ± 8.9 < 0.01

Zinc (µg/dl) 55.9 ± 7.2 88.17 ± 7.04 < 0.01

Cu / Zn 2.4 ± 0.04 1.2 ± 0.23 < 0.01

The above table shows the Mean ± S.D. of both Chronic active

hepatitis B and controls, which shows statistically significant increase in

serum total bilirubin, direct bilirubin, ALT, AST, AST /ALT ratio, Globulins,

copper and Cu /Zn ratio and statistically significant decrease in serum

Albumin, A / G ratio and Zinc.

TABLE - 5

MEAN ± S.D. AND P VALUES OF BIOCHEMICAL VARIABLES

BETWEEN CIRRHOSIS AND CONTROLS.

PARAMETER Cirrhosis Controls P value



ALT (U/L) 58 ± 8.0 28 ± 10.0 < 0.01

AST (U/L) 79 ±10.2 21 ± 8.5 < 0.01

48

AST /ALT 1.36 ± 0.20 0.75 ± 0.05 < 0.01

Albumin (g/dl) 2.71 ± 0.12 3.95 ± 0.25 <0.01

Globulins (g/dl) 4.38 ± 0.13 2.50 ± 0.45 < 0.01

A / G 0.63 ± 0.03 1.35 ± 0.04 < 0.01

Copper(µg/dl) 156.23 ± 7.2 110.24 ± 8.9 < 0.01

Zinc (µg/dl) 50.2 ± 13.88 88.17 ± 7.04 < 0.01

Cu/ Zn 2. 7± 0.12 1.2 ± 0.23 < 0.01

The above table shows the Mean ± S.D. of both Cirrhosis and controls,

which shows statistically significant increase in serum total bilirubin, direct

bilirubin, ALT, AST, AST/ALT ratio, Globulins, copper and Cu /Zn ratio and

statistically significant decrease in serum Albumin, A / G ratio and Zinc.

TABLE - 6

CORRELATION BETWEEN SERUM ZINC AND COPPER, AST,

ALT, TOTAL BILIRUBIN IN CHRONIC ACTIVE HEPATITIS B

PARAMETER r VALUE P VALUE

Copper -0.385 < 0.05

AST -0.649 < 0.01

ALT -0.724 < 0.01

Total Bilirubin -0.631 < 0.01

49

This table shows that there was a negative correlation between serum

zinc and the following parameters: serum Copper, AST, ALT and Total

bilirubin (P < 0.01) in chronic active hepatitis B.

TABLE - 7

CORRELATION BETWEEN SERUM COPPER AND ALT, AST,

TOTAL BILIRUBIN IN CHRONIC ACTIVE HEPATITIS B

PARAMETER r VALUE P VALUE

AST 0.705 < 0.01

ALT 0.772 < 0.01

Total bilirubin 0.673 < 0.01

The above table shows that there was a positive correlation between

serum Copper and the following parameters : serum AST, ALT, Total bilirubin

( p < 0.01)

TABLE - 8

CORRELATION BETWEEN SERUM ZINC AND COPPER, ALT,

AST, TOTAL BILIRUBIN IN CIRRHOSIS

Parameter r value P value

AST - 0.429 < 0.05

ALT - 0.402 < 0.05

Total bilirubin - 0.361 < 0.05

Copper - 0.585 < 0.01

50

The above table shows a significant negative correlation of serum zinc

with AST, ALT, Total bilirubin and Copper in Cirrhosis.

TABLE – 9

CORRELATION OF SERUM COPPER WITH AST, ALT, TOTAL

BILIRUBIN IN CIRRHOSIS

Parameter r value P value

AST 0.581 <0.01

ALT 0.533 <0.01

Total bilirubin 0.405 <0.05

The above table shows positive significant correlation between serum

copper and serum AST, ALT and total bilirubin in cirrhosis.

CHART - 1

REPRESENTATION OF MEAN VALUES OF SERUM TOTAL

BILIRUBIN, DIRECT BILIRUBIN IN CONTROLS, CHRONIC

ACTIVE HEPATITIS B AND CIRRHOSIS

51

Scale : Y axis -> 1 cm = 1 mg/dl.

TOTAL BILIRUBIN

DIRECT BILIRUBIN

0

1

2

3

4

5

6

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

CHART - 2

REPRESENTATION OF MEAN VALUES OF SERUM AST AND ALT IN CONTROLS, CHRONIC ACTIVE

HEPATITIS B AND CIRRHOSIS

Scale : Y axis -> 1 cm = 50 U/L.52

ALT AST0

50

100

150

200

250

300

CONTROLS


CIRRHOSIS

CHART - 3

Representation of MEAN VALUES OF AST / ALT in

CONTROLS, CHRONIC ACTIVE HEPATITIS B and CIRRHOSIS

Scale – Y axis : 1 cm = 0.2 .53

AST / ALT0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

CONTROLS


CIRRHOSIS

CHART - 4

REPRESENTATION OF MEAN VALUES OF SERUM ALBUMIN,

GLOBULIN IN CONTROLS, CHRONIC ACTIVE HEPATITIS B

AND CIRRHOSIS.

Scale – Y axis : 1 cm = 0.5 gm/dl.

54

ALBUMIN GLOBULIN0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

CONTROLS


CIRRHOSIS

CHART - 5

REPRENTATION OF MEAN VALUES OF A / G RATIO IN

CONTROLS, CHRONIC ACTIVE HEPATITIS B AND

CIRRHOSIS

Scale – Y axis : 1 cm = 0.2

55

A / G0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

CONTROLS


CIRRHOSIS

CHART - 6

REPRESENTATION OF MEAN VALUES OF SERUM COPPER

AND ZINC IN CONTROLS, CHRONIC ACTIVE HEPATITIS B

AND CIRRHOSIS.

Scale – Y axis : 1 cm = 20 µg/dl.

56

COPPER ZINC0

20

40

60

80

100

120

140

160

180

CONTROLS


CIRRHOSIS

CHART - 7

REPRESENTATION OF MEAN VALUES OF CU / ZN IN

CONTROLS, CHRONIC ACTIVE HEPATITIS B AND

CIRRHOSIS

Scale – Y axis : 1 cm = 0.5

57

Cu / Zn0

0.5

1

1.5

2

2.5

3

CONTROLS


CIRRHOSIS

TABLE - 10

MEAN ± S.D. AND P VALUES OF BIOCHEMICAL VARIABLES

BETWEEN CHRONIC ACTIVE HEPATITIS B AND CIRRHOSIS

PARAMETERChronic active

Hepatittis B Cirrhosis P value



58

ALT (U/L) 266 ± 10.0 58 ± 8.0 < 0.001

AST (U/L) 223 ± 12.3 79 ±10.2 < 0.001

AST /ALT 0.84 ± 0.12 1.36 ± 0.20 < 0.001

Albumin (g/dl) 3.0 ±0.02 2.71 ±0.12 < 0.001

Globulins (g/dl) 4.1 ± 0.21 4.38 ±0.13 < 0.001

A / G 0.73 ±0.23 0.63 ±0.03 0.007

Copper(µg/dl) 148.21±4.5 156.23 ± 7.2 < 0.001

Zinc (µg/dl) 55.9 ± 7.2 50.2 ± 13.88 < 0.001

Cu / Zn 2.4 ± 0.04 2.7 ± 0.12 < 0.001

The above table shows the Mean ± S.D. of both Chronic active

hepatitis B and Cirrhosis, which shows statistically significant increase in

serum total bilirubin, direct bilirubin, ALT, AST, AST /ALT ratio, Globulins,

copper and Cu /Zn ratio and statistically significant decrease in serum

Albumin, A / G ratio and Zinc.

DISCUSSION

The present study consists of analysis of Serum Total bilirubin, Direct

bilirubin, ALT, AST, AST / ALT ratio, Albumin, Globulins, A / G ratio, Copper,

Zinc, Cu / Zn ratio in 50 Chronic active hepatitis B patients, 80 Cirrhosis

patients and 40 healthy controls.

The Serum Total bilirubin & direct bilirubin in controls was 0.40 ± 0.02

and 0.12 ± 0.03 mg/dl respectively. Serum total bilirubin and direct bilirubin in

59

Chronic active hepatitis B and Cirrhosis was 5.6 ± 1.3 and 2.2 ± 0.31 mg/dl &

3.61 ± 1.16 and 1.08 mg/dl respectively.

The study shows statistically significant increase in Serum Total

bilirubin and direct bilirubin in both Chronic active hepatitis B and Cirrhosis

when compared to controls, & statistically significant increase in Chronic

active hepatitis B when compared to Cirrhosis(p value< 0.001).This

observation in the present study is in accordance with the study of Essam F.

Al-Jumaily and Faiha'a M. Khaleel52.

Jaundice in Chronic active hepatitis and Cirrhosis is of hepatocellular

type. The hypothesis of increased levels of total bilirubin and direct bilirubin is

(a) Defective conjugation- there may be a reduction in the number of

functioning liver cells so that conjugation is impaired. (b) Infective cause-

there is extensive damage to liver cells effecting bilirubin excretion. (c) there is

considerable degree of intrahepatic obstruction with occlusion of bile

canaliculi lumen by desquamated and disintegrated cells and bile thrombi

resulting in appreciable absorption of conjugated bilirubin53.

In active hepatitis, there is hepatocyte derangement and because of

edema (due to inflammation) there may be obstructive impairment of bile

excretion. The result is amount of unconjugated bilirubin is increased because

of hepatic failure and conjugated bilirubin may increase because of

obstructive pathology. In advanced cirrhosis, there is both hepatocyte failure

and some degree of obstructive pathology as result of diffuse fibrotic changes

within liver53.

60

Serum ALT and AST levels in controls were 28 ± 10 and 21 ± 8.5 IU/L

respectively. Serum ALT & AST values in Chronic active hepatitis B were

266 ± 10 and 223 ± 12.3 IU/L, and in Cirrhosis were 58 ± 8.0 and 79 ±10.2

IU/L respectively.

There is statistically significant increase in ALT and AST in Chronic

active hepatitis B and Cirrhosis when compared to controls & significant

increase in Chronic active hepatitis B when compared to Cirrhosis (p < 0.001).

These observations are in correlation with the study done by Essam F.,

Al-Jumaily and Fiaha’a M.khaleel52 .

Serum ALT and AST are sensitive indicators for liver injury, released

into blood due to defective membrane permeability, degeneration, necrosis,

and inflammation of hepatocytes in chronic active hepatitis and cirrhosis54.

AST /ALT ratio in controls was 0.75 ± 0.05 and in chronic active

hepatitis B and Cirrhosis was 0.84 ± 0.12 & 1.36 ± 0.2 respectively. There is

statistically significant increase in AST / ALT ratio (De Ritis ratio) in Chronic

active hepatitis B and Cirrhosis when compared to controls, and significant

increase in cirrhosis than chronic active hepatitis B(p < 0.01)This observation

in the study is in accordance with the study of Paul L Wolff.etal55.

De ritis ratio implies the degree of parenchymal damage caused to

hepatocytes in the liver. As more cells become completely destroyed as in

cirrhosis, AST rises to above that of ALT, this explain the rise in the ratio in

cirrhosis.

61

AST / ALT ratio is one of the eldest markers of liver fibrosis that is

easily available and applicable. It has been validated in different forms of liver

disease and a ratio of > 1 is predictive of cirrhosis56.

Serum albumin in controls was 3.95 ± 0.25g/dl, in Chronic active

hepatitis B and cirrhosis was 3.0 ± 0.02 and 2.71 ± 012g/dl respectively.

There is statistically significant decrease in serum albumin levels in Chronic

active hepatitis B and Cirrhosis when compared to controls, and statistically

significant decrease in serum albumin level in Cirrhosis than Chronic active

hepatitis B( p < 0.001).These observations in the study correlates with the

study done by Ching chiang, Jee etal57.

Serum Albumin indicates the residual functional mass of liver with

synthetic capacity. As its half life is of 21 days, its concentration is reduced in

chronic active hepatitis B and Cirrhosis, with further reduction in cirrhosis as

functioning liver mass is much reduced in cirrhosis than chronic active

hepatitis B54.

Serum globulin was 2.50 ± 0.45 g/dl in controls, in chronic active

hepatitis B and cirrhosis was 4.1 ± 0.21 & 4.38 ± 0.13g/dl respectively. There

is statistically significant increase in serum levels of globulins in chronic active

hepatitis B and cirrhosis when compared to controls and statistically

significant increase in cirrhosis than chronic active hepatitis B

(p < 0.001).These observations in the study correlate with the study done by

Schmilovitz, Weiss H, Tovar etal58.

62

In chronic active hepatitis B, the gamma globulin increase may be due

to production of antibodies to altered liver cell proteins induced by Hepatitis B

virus. In cirrhosis, diffuse hyper gamma globulinemia is due to Ig

(Immunoglobulin) resonse involving Ig G and A, is probably the result of

distorted hepatic architecture that allows antigens absorbed from intestine to

bypass normal filtering in hepatic sinusoids38.Another possible factor is

chronic inflammatory and necrotizing lesions in liver that results in the reactive

proliferation of mesenchymal cells, which are concerned with production of

gamma globulins.

A / G ratio in controls was 1.35 ± 0.04, in chronic active hepatitis B and

cirrhosis was 0.73 ±0.03 and 0.63 ± 0.02 respectively. There is statistically

significant decreased A / G ratio in Chronic hepatitis B and Cirrhosis when

compared to controls, statistically significant decrease was more in cirrhosis

than chronic active hepatitis B cases ( p = 0.007).These observations in the

study are in correlation with the study done by Gupta, Sunil; Meena, Shravn

Kumar; Ahuja etal59. Further reduced serum albumin and increased globulins

in cirrhosis compared to the chronic active hepatitis B is the cause of

significant reduction in A/G ratio in cirrhosis. Serum Albumin and Globulin

levels can be used as predictive markers of extent of liver fibrosis according to

study of Schmilovitz, Towar et. al.

Serum copper in controls was 110.24 ± 8.9 µg/dl, in chronic active

hepatitis B & cirrhosis was 148.21 ± 4.4 and 156.23 ± 7.2µg/dl respectively.

There is statistically significant increase in serum copper in Chronic active

hepatitis B and cirrhosis when compared to controls, significant increase was

63

more in cirrhosis than chronic active hepatitis B (p< 0.001). This observation

in the study is in correlation to the study of Muhanad, Ammar etal,

Pramoolsinsap etal60 and Rahelic et al38.

Serum zinc in controls was 88.17± 7.04 and in chronic active hepatitis

B and cirrhosis was 55.9± 7.2 and 50.2 ± 13.88 µg/dl respectively. The study

shows statistically significant decrease in serum zinc in Cirrhosis and Chronic

active hepatitis B when compared to controls, with significance in cirrhosis

when compared to chronic active hepatitis B ( p < 0.001).This study is in

correlation with the study of N.R.P. Reddy et al61 and Rachelic et al38. The

present study shows that serum Cu/Zn ratio was significantly higher in the

Chronic active hepatitis B and cirrhosis groups than the control (P<0.001) and

mores significant increase in cirrhosis when compared to chronic active

hepatitis B.This finding is in accordance with the study of A.Sawa, K.Okita et

al62.

The elevated levels of serum copper is due to cholestasis as a result of

either a functional defect in bile formation at the level of the hepatocytes, or

from impairment in bile secretion and flow at the bile ducts level, which

causes impaired biliary excretion of Cu and excess Cu absorption, as bile

ducts are the main way to excrete Cu from the body.

Copper being oxidative, and hepatotoxic in nature causes

progression of chronic liver diseases38.Cu binds to sulfhydryl groups of

enzymes, as glutathione reductase, thus interfering with their protection of

cells from free radical damage. Redox cycling between cu2+and cu1+ can

catalyze the production of toxic hydroxyl radicals.

64

The mechanisms contributing to zn deficiency are poor dietary intake,

reduced intestinal absorption, reduced hepato-intestinal extraction, portal

systemic shunting, altered protein and aminoacid metabolism, protein

restriction and increased clearance of Zn in pancreatic or intestinal fluids,

which leads to loss of Zn in the stool which is the main route of Zn excretion63.

The interaction between zinc and copper in their intestinal absorption

and their competition for binding sites on the carrier proteins and cellular

uptake may be the regulators of their homeostasis. May be this can explain

the inverse concentration of zinc and copper 38.

There was a positive correlation between serum Cu level and AST,

ALT and total bilirubin in chronic hepatitis B and cirrhosis. These findings are

in accordance with those of shwetha et al64.This may indicate a positive

correlation between serum Cu level and biochemical parameters of liver

damage.

There was a negative correlation between serum Zn level and serum

Cu, AST, ALT and total bilirubin (P<0.001) in chronic active hepatitis B and

cirrhosis. This finding is in accordance with study of shwetha et al64. The

significant negative correlation between Zn and the biochemical parameters of

liver damage (AST and ALT) can reflect the presumed protective role of Zn

against progression of liver diseases.

Zn administration has been shown to inhibit accumulation of hepatic

collagen in experimentally produced hepatic necrosis and to significantly

improve neurological signs in hepatic encephalopathy in humans. Also dietary

65

zinc supplementation could improve liver regeneration by increasing the

expression of genes involved in hepatic cellular proliferation65.

SUMMARY AND CONCLUSION

In present study Serum total bilirubin, direct bilirubin, ALT, AST, AST /

ALT ratio, Globulins, copper and Cu / Zn ratio were significantly high in both

Chronic active hepatitis B and Cirrhosis, when compared to controls, but total

bilirubin, direct bilirubin, ALT, AST, globulins, copper, Cu/Zn ratio were more

highly significant in Chronic active hepatitis B than Cirrhosis (p < 0.001), and

66

Serum Albumin, A/ G ratio and zinc were statistically significantly low in all

liver disorders.

Serum Copper was positively correlated with ALT, AST levels and total

bilirubin in Chronic active hepatitis B patients and cirrhosis significantly.

Serum Zinc was negatively correlated significantly with copper, AST,

ALT, and total bilirubin in chronic active hepatitis B and cirrhosis Decrease in

serum zinc in various chronic liver diseases is due to decreased serum

albumin, poor dietary intake and increased clearance of zinc. As the severity

of the disease worsened, Zinc levels decreased., so; Zinc may be used as a

prognostic indicator of chronic liver diseases.

So this study concludes

1. Serum Zinc, Copper could be included in the routine assessment of

patients with Chronic liver diseases as chronic active hepatitis and

cirrhosis.

2. Zinc supplementation may be encouraged in patients with Chronic

active hepatitis and cirrhosis as it is an antioxidant and it is negatively

correlated with liver damage parameters.

3. Caution regarding Cu intake either dietary or medicinal; should be

taken in patients with Chronic hepatitis and cirrhosis.

4. The level of certain trace elements such as Cu, Zn and Cu/Zn ratio

may serve as biomarkers for monitoring the increased severity of liver

damage in chronic hepatitis and cirrhosis.

67

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ANNEXURE - 1

PROFORMA

S.No.:

74

Name of the patient: Age: Sex:

Address: Occupation: Chief complaint:

HISTORY OF PRESENT ILLNESS:

Gastro – intestinal symptoms

Yellow discolouration of sclera

Edema

Sleep disturbances

Tremors

Fatigue

Weight loss

PAST HISTORY: H/O Hypertension, Diabetes, Asthma, Tuberculosis,

other chronic infections, any surgeries.

FAMILY HISTORY: H/O Hypertension, Diabetes, liver disease, renal

disease.

PERSONAL HISTORY: H/O Smoking, tobacco chewing, Alcoholism,

risky behaviour regarding unprotected sexual contact.

H/O diet, appetite. sleep, bowel and bladder.

H/O drug intake.

GENERAL PHYSICAL EXAMINATION:

Built and nourishment

Height : weight : BMI :

Pallor, icterus, cyanosis, clubbing, oedema, lymphadenopathy :

VITAL EXAMINATION :

Pulse : Blood pressure : Respiratory rate :

Temperature:

75

SYSTEMIC EXAMINATION :

Gastrointestinal system and genitals -

Inspection: Abdomen - umbilical hernia - caput medusae -

Palpation: liver - spleen –

Percussion : Shifting dullness - Fluid thrill -

Auscultation: any bruits

Respiratory system

Cardiovascular system

Central nervous system

PROVISIONAL DIAGNOSIS: chronic active hepatitis B / Cirrhosis

INVESTIGATIONS :

Serum total bilirubin :

Serum direct bilirubin :

Serum Alanine transaminase:

Serum Aspartate transaminase :

Serum AST / ALT :

Serum Albumin :

Serum Globulins: Serum A / G ratio :

Serum copper:

Serum zinc : Serum Cu/Zn ratio :

ANNEXURE - 2

RANGARAYA MEDICAL COLLEGE, KAKINADA

INSTITUTIONAL ETHICS COMMITTEE APPROVAL FORM –

HUMAN RESEARCH PROJECTS

76

PROJECT TITLE : STUDY OF BIOCHEMICAL VARIABLES

IN LIVER DISORDERS.

NAME OF THE APPLICANT : Dr. V. Saumya.

INSTITUTION : Rangaraya Medical College, Kakinada.

POSITION IN THE INSTITUTE: Post Graduate. (M.D., Biochemistry)

GUIDE : Dr. G. Rajeswari, M.D.

Professor & Head,

Department of Biochemistry.

This is to certify that the project, “Study of biochemical variables in liver

disorders” conducted by Dr. V.Saumya under the guidance of

Dr. G.Rajeswari, M.D., Professor and Head, Department of Biochemistry has

been approved by the Institutional Ethics Committee, Rangaraya Medical

College.

Signature of the Applicant : Signature of Member Secretary:

On behalf of the Institutional Ethics

Committee.

77

ANNEXURE - 3

INFORMED CONSENT FORM

Title of the study : Study of biochemical variables in liver

disorders.

Name of the Participant :

Name of the Principal Investigator : Dr. V.Saumya.

Name of the institution : Government General Hospital,

Kakinada.

I ,……… S/O, D/O, W/O ……… am admitted in the medical ward of the

Government General Hospital, Kakinada. I have been explained in my own

language regarding the need for the study and the investigations to de done.

I am hereby, giving my consent for taking the blood sample and to include me

as a participant in the study.

Date :……………….. Signature of the Patient.

Time :………………..

Signature of the witness.

78

79

2. Introduction

Documents

hbv endemicity

chronic hbv infection

hbv infection7

entire pool of hbv carriers

acute clinical cases

areas of high endemicity

present infection

low endemicity