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.
1.
2.
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.
(3-10
mg/dl)
occur.
Hypoalbuminemia
and
prolongation
of
CIRRHOSIS19
Cirrhosis as the end stage of chronic liver disease is defined by three
characteristics:
1.
2.
3.
10
alkalosis.
Dietary
hypomagnesemia
deficiency
and
and
increased
hypophosphatemia.
In
urinary
ascites
losses
and
lead
to
dilutional
11
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
2)
3)
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
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,
19
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
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
24
25
26
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
Serum
Glutamate
Pyruvate
Transaminase,
Akaline
29
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
33
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
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
Multiorgan failure.
Wilsons disease.
34
Malnutrition.
On cancer chemotherapy
On hormonal therapy
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.
PRINCIPLE
35
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 (ml)
1.000
1.000
---
0.025
0.050
0.050
Sample
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
37
PRINCIPLE:
Alpha ketoglutarate + LAlanine
L-Glutamic
acid
Pyruvate
(catalysed by ALT)
Pyruvic acid + NADH + H+
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.
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.
39
PRINCIPLE
Alpha-ketoglutaric acid + L-Aspartic acid
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
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.
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
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.
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.
S2
S3
S4
S5
Standard (ml)
0.2
0.4
0.6
0.8
1.0
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
PRINCIPLE
45
Absorbance of
duplicates
0.00
0.03
0.06
0.09
0.12
0.15
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
S2
S3
S4
S5
Standard (ml)
0.2
0.4
0.6
0.8
1.0
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
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
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
5.16 1.30
0.40 0.20
< 0.01
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
3.61 1.16
0.40 0.20
< 0.01
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
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
CHART - 1
REPRESENTATION OF MEAN VALUES OF SERUM TOTAL
BILIRUBIN, DIRECT BILIRUBIN IN CONTROLS, CHRONIC
ACTIVE HEPATITIS B AND CIRRHOSIS
CONTROLS
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
CONTROLS
CIRRHOSIS
100
50
0
ALT
AST
54
CHART - 3
Representation of MEAN VALUES OF AST / ALT in
CONTROLS, CHRONIC ACTIVE HEPATITIS B and CIRRHOSIS
55
1.6
1.4
1.2
1
0.8
CONTROLS
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.
56
5
4.5
4
3.5
3
2.5
CONTROLS
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
1.6
1.4
1.2
1
0.8
CONTROLS
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
180
160
140
120
100
80
CONTROLS
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
CONTROLS
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
5.16 1.30
3.61 1.16
< 0.001
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
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
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
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
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
67
68
69
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Goldstein ST, Zhou F, Hadler SC, et al. Model to estimate global hepatitis
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Falchuk KH. The molecular basis for the role of zinc in developmental
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King JC, Shames DM, Lowe NM et al. Effects of acute zinc depletion on
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Kang YJ, Zhou Z. Zinc prevention and treatment of liver disease. Mol
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Turuland JR, Keyes WR. Peiffer GL, Scott KC. Copper absorption,
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Soomro AA, Devrajini BR, et al, serum zinc in patients with liver cirrhosis.
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Kang YJ, Zhou Z. Zinc in prevention and treatment of liver disease. Mol
Aspects Med, 2005;26(4-5) :391-404.
ANNEXURE - 1
PROFORMA
S.No.:
Name of the patient:
Age:
Sex:
Address:
Occupation:
Chief complaint:
76
Edema
Sleep disturbances
Tremors
Fatigue
Weight loss
weight :
BMI :
VITAL EXAMINATION :
Pulse :
Blood pressure :
Respiratory rate :
Temperature:
SYSTEMIC EXAMINATION :
Gastrointestinal system and genitals Inspection: Abdomen - umbilical hernia -
77
caput medusae -
Respiratory system
Cardiovascular system
Central nervous system
Serum A / G ratio :
Serum copper:
Serum zinc :
ANNEXURE - 2
RANGARAYA MEDICAL COLLEGE, KAKINADA
INSTITUTIONAL ETHICS COMMITTEE APPROVAL FORM
HUMAN RESEARCH PROJECTS
PROJECT TITLE
Dr. V. Saumya.
78
INSTITUTION
GUIDE
conducted
by
Dr.
V.Saumya
under
the
guidance
of
79
ANNEXURE - 3
INFORMED CONSENT FORM
Dr. V.Saumya.
Kakinada.
Date :..
Time :..
80
81