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 worlds 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 worlds 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 MiddleEast. 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

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.

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 riedels 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.

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.

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.

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

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
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 Laennecs 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.
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. Dupuytrens 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

10

alkalosis.

Dietary

hypomagnesemia

deficiency
and

and

increased

hypophosphatemia.

In

urinary
ascites

losses
and

lead

to

dilutional

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

11

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

portosystemic shunts and complete reconstruction of the original architecture
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

12

disease, serum transaminases activities are generally not elevated above 300
U/L21.

13

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.

14

15

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): 70130 mcg/dL or 10.719.9 mol/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 fingers23. 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 cells, insulin is stored as crystalloid like hexamers, each

16

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.

17

Circulating zinc is bound mostly to serum proteins; 2/3 rds are loosely
bound to albumin and transthyretin while 1/3 rd is bound tightly to 2macroglobulin. 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
mol/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:

Malabsorption syndromes

18

Acrodermatitis

enteropathica,

3. INCREASED UTILIZATION: Adolescence, Lactation, Menstruation,

Pregnancy
4. INCREASED LOSS: Alcoholism, -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,
Downs 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 immunity 26.
Neurological effects: Severe zn deficiency is known to affect mental
well being, with varying degree of confusion and depression being consistent

19

with zn enzymes having important activity in brain development and

function.27
SYMPTOMS:
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.

20

COPPER (Cu)
Normal range: 65155 mcg/dL (1024.6 mol/L) for serum.
Chemistry : Copper(atomic number :29,atomic weight : 63.54) has cu 1+ 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 cerbrocuprein 32
(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 sodium 32.Absorbed copper is transported to the
liver in portal blood bound to albumin where it is incorporated by hepatocytes

21

into cuproenzymes and other proteins and then exported in peripheral blood
to tissues and organs. Liver is the key organ in copper homeostasis 33.
ROLE OF LIVER IN COPPER METABOLISM32
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 60g/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.

22

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
RBC oxidizes ferrous to ferric iron. This allows incorporation of Fe 3+ 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 copper 35.

23

DEFICIENCY
MALNOURISHED INFANTS- develop iron resistant anemia, neutropenia,
other hematological disorder and bone lesions 36.
PREMATURE INFANTS - premature infants fed with formula lacking
NUTRITIONAL SUPPORT- Adults and children fed intravenously develop
symptomatic copper deficiency.
MENKES 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
Wilsons 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 (> 500g cu/ L). Liver biopsy for cu analysis is useful in
suspected cases and result above 250g /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

24

in liver damage directly or indirectly through kupffer cell stimulation 37.Redox

cycling between cu2+ and cu1+ can catalyze the production of toxic hydroxyl
radicals, which play important role in pathogenesis of liver cirrhosis 38.
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.

25

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.
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
(AE) 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 3145
years. For comparative study, 95 healthy females of the same age group were

26

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

atomic absorption spectrophotometry. The results of this study showed that
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

27

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
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 ( 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

28

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
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.

29

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

Raheli D, Kujundzi M, Romi Z, Brki K.
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

30

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.
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

31

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.
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

32

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.

33

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

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.

Wilsons disease.

Any autoimmune disorders/ immunodeficiency disorder.

Acute or chronic diarrhea.

If on any mineral supplements.

34

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
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

35

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.

PROCEDURE
Light path 1 cm, Temperature 37 oC, Prewarm the sample, reagent
cuvettes to reaction temperature.

36

Sample Blank (ml)

Sample (ml)

Bilirubin total reagent - 1

1.000

1.000

Bilirubin total reagent 2

---

0.025

0.050

0.050

Sample

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
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 with saline. Multiply the result with 2.

ESTIMATION OF SERUM ALANINE TRANSAMINASE (ALT)

IFCC (International Federation Of Clinical Chemistry) Method 42,43.

37

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 2-8 oC, for 4 days at RT.

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.

38

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.

ESTIMATION OF SERUM ASPARTATE TRANSAMINASE (AST)

39

IFCC (International federation of clinical chemistry ) Method 44,45,46

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.

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

40

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.

ESTIMATION OF PLASMA TOTAL PROTEINS

(Biuret Method)47,48

41

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
Biuret reagent
Distilled water
Protein standard
Sample

Blank (ml)
1.00
0.01
---

Standard (ml)
1.00
-0.01
--

Test (ml)
1.00
--0.01

Mix well and incubate at 37 oC 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.
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

42

PRINCIPLE
Determination of albumin in serum is based on the binding behavior of
albumin with dye 3355 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)

1.00

1.00

1.00

Standard

--

0.01

--

Sample

--

--

0.01

Working reagent

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.

43

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
Blank (ml)

Standard (ml)

Test (ml)

Working reagent

1.00

1.00

1.00

Distilled water

0.05

--

--

Copper standard

--

0.05

--

Sample

--

--

0.05

Wavelength: 580 nm, light path : 1cm. Mix well and incubate at R.T.(25 oC) 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 g/dl, (females) = 80
155 g/dl, (newborns) = 12 67 g/dl, children upto 10 years = 30 150
g/dl.

44

Linearity: Upto 500 g/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 g /ml.
The working standards (S) are prepared by dilution of the standard.
S1

S2

S3

S4

S5

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(g/dl)

40

80

120

160

200

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.

Blank
S1
S2
S3
S4
S5

Concentration of
Copper (g/dl)
0
40
80
120
160
200

Absorbance of
standards
0.00
0.03
0.06
0.09
0.12
0.15

ESTIMATION OF SERUM ZINC

Colorimetric Method51

PRINCIPLE

45

Absorbance of
duplicates
0.00
0.03
0.06
0.09
0.12
0.15

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
Working reagent
Distilled water
Standard
Sample

Blank (ml)
1.0
0.05
---

Standard (ml)
1.0
-0.05
--

Test (ml)
1.0
--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) g/dl.
Normal reference value : Serum : 60 -120 g/dl.
Linearity : Upto 700 g/dl. For higher values, dilute the sample with distilled
water and calculate the results using dilution factor.

46

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 g /ml.
The working standards (S) are prepared by dilution of the standard:
S1

S2

S3

S4

S5

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 (g/dl)

40

80

120

160

200

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 (g/dl)

Absorbance of
standards

Absorbance of
duplicates

Blank

S1

40

0.06

0.06

S2

80

0.12

0.12

S3

120

0.18

0.18

S4

160

0.24

0.24

S5

200

0.30

0.30

TABLE - 1
NUMBER OF CONTROLS AND CASES
(Chronic active hepatitis B and Cirrhosis)
GROUP

NUMBER

1. CONTROLS

40

47

2. CHRONIC ACTIVE HEPATITIS B

50

3. CIRRHOSIS

80

TABLE - 2
AGE DISTRIBUTION OF PATIENTS STUDIED
Chronic active

Age (years)

Controls

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 %)

55 60

03 (7.5%)

08 (16.0%)

35 (43.75 %)

Total

40 (100 %)

50 (100 %)

80 (100 %)

hepatitis B

Cirrhosis

100%
90%
80%
70%
60%

CIRRHOSIS

50%

CHRONIC ACTIVE
HEPATITIS B

40%

CONTROLS

30%
20%
10%
0%
35 - 40

40 -45

45 - 50

50 - 55

55 - 60

TABLE - 3
MEAN S.D. OF BIOCHEMICAL VARIABLES IN CONTROLS

PARAMETER
Total Bilirubin (mg/dl)

MEAN S.D.

Reference Range

0.40 0.20

0.1 - 1.0

48

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

Copper (g/dl)

110.24 8.9

80 140

Zinc (g/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

++ PARAMETER

Chronic active
Hepatittis B

Controls

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

49

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(g/dl)

148.21 4.5

110.24 8.9

< 0.01

Zinc (g/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

Total Bilirubin (mg/dl)

3.61 1.16

0.40 0.20

< 0.01

Direct Bilirubin (mg/dl)

1.08 0.06

0.12 0.03

< 0.01

ALT (U/L)

58 8.0

28 10.0

< 0.01

AST (U/L)

79 10.2

21 8.5

< 0.01

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(g/dl)

156.23 7.2

110.24 8.9

< 0.01

Zinc (g/dl)

50.2 13.88

88.17 7.04

< 0.01

2. 7 0.12

1.2 0.23

< 0.01

Cu/ Zn

50

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

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

51

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

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

52

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

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

6
5
4
3

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

2
1
0
TOTAL BILIRUBIN

DIRECT BILIRUBIN

53

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.

300
250
200
150

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

100
50
0
ALT

AST

54

CHART - 3
Representation of MEAN VALUES OF AST / ALT in
CONTROLS, CHRONIC ACTIVE HEPATITIS B and CIRRHOSIS

Scale Y axis : 1 cm = 0.2 .

55

1.6
1.4
1.2
1
0.8

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

0.6
0.4
0.2
0
AST / ALT

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.

56

5
4.5
4
3.5
3
2.5

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

2
1.5
1
0.5
0
ALBUMIN

GLOBULIN

CHART - 5
REPRENTATION OF MEAN VALUES OF A / G RATIO IN
CONTROLS, CHRONIC ACTIVE HEPATITIS B AND CIRRHOSIS

57

Scale Y axis : 1 cm = 0.2

1.6
1.4
1.2
1
0.8

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

0.6
0.4
0.2
0
A/G

CHART - 6
REPRESENTATION OF MEAN VALUES OF SERUM COPPER
AND ZINC IN CONTROLS, CHRONIC ACTIVE HEPATITIS B
AND CIRRHOSIS.

58

Scale Y axis : 1 cm = 20 g/dl.

180
160
140
120
100
80

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

60
40
20
0
COPPER

ZINC

CHART - 7
REPRESENTATION OF MEAN VALUES OF CU / ZN IN
CONTROLS, CHRONIC ACTIVE HEPATITIS B AND CIRRHOSIS

59

Scale Y axis : 1 cm = 0.5

3
2.5
2
1.5

CONTROLS

CHRONIC ACTIVE HEPATITIS B

CIRRHOSIS

1
0.5
0
Cu / Zn

TABLE - 10
MEAN S.D. AND P VALUES OF BIOCHEMICAL VARIABLES
BETWEEN CHRONIC ACTIVE HEPATITIS B AND CIRRHOSIS

PARAMETER

Chronic active
Hepatittis B

Cirrhosis

P value

Total Bilirubin (mg/dl)

5.16 1.30

3.61 1.16

< 0.001

Direct Bilirubin (mg/dl)

2.20 0.31

1.08 0.06

< 0.001

60

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(g/dl)

148.214.5

156.23 7.2

< 0.001

Zinc (g/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
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.

61

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 causethere 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 bilirubin 53.
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.
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

62

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 Fiahaa 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 cirrhosis 54.
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.etal 55.
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.
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 cirrhosis 56.

63

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

(p < 0.001).These observations in the study correlate with the study done by
Schmilovitz, Weiss H, Tovar etal58.
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

64

(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 sinusoids 38.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 g/dl, in chronic active
hepatitis B & cirrhosis was 148.21 4.4 and 156.23 7.2g/dl respectively.
There is statistically significant increase in serum copper in Chronic active
hepatitis B and cirrhosis when compared to controls, significant increase was
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.

65

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 g/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 al 61 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 cu 2+and cu1+ can
catalyze the production of toxic hydroxyl radicals.
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

66

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 excretion 63.
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 al 64.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 al 64. 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
zinc supplementation could improve liver regeneration by increasing the
expression of genes involved in hepatic cellular proliferation 65.

67

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
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.

68

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.

69

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ANNEXURE - 1
PROFORMA
S.No.:
Name of the patient:

Age:

Sex:

Address:

Occupation:

Chief complaint:

HISTORY OF PRESENT ILLNESS:

Gastro intestinal symptoms

76

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:

SYSTEMIC EXAMINATION :
Gastrointestinal system and genitals Inspection: Abdomen - umbilical hernia -

77

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

PROJECT TITLE

STUDY OF BIOCHEMICAL VARIABLES IN

LIVER DISORDERS.

NAME OF THE APPLICANT

Dr. V. Saumya.

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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.

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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.

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