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INTRODUCTION
Chapter -1
INTRODUCTION
I ndigenous plants have been the traditional source of raw

materials for the manufacture of medicines. India is sitting on a
gold mine of well recorded and traditionally well-practiced
knowledge of herbal medicine, and rightly called the botanical
garden of the world. About 6000 plants are estimated to be used
in traditional, Folk and herbal medicine in India representing
about 75% of the medicinal needs of the third world countries.
In order to promote Indian herbal drugs, there is an urgent

need to evaluate the therapeutic potentials of the drugs as per
WHO guidelines.1
1.1 THE LIVER

In the upper right quadrant of the abdominal cavity is a
large red mass that may look like a sleeping giant. It doesn't
pulsate, it doesn't move much, only passively, and you don't
ordinarily see it secreting anything. This soft, almost jelly-like
mass is the liver. Fortunately, a body can survive on about one-
third the amount of liver that the normal person has!
Location of the Liver2
The liver, the largest internal organ in the body, is situated

under the diaphragm. It is further protected by the costal
cartilage of the ribs. The undersurface of the liver, also known as
the visceral surface, is more irregular in appearance than is the
domed convex upper surface. This irregularity is caused by the
fact that the inferior surface is in contact with:
The lower esophagus
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Hepatoprotective studies of Carissa carandas Linn. fruit Extract on
carbontetrachloride intoxicated rats.
INTRODUCTION
The stomach, and

The right kidney and adrenal gland
Liver lobes
The liver has two main lobes due to the falciform ligament
which divides it. The left lobe is smaller than the right which is
further subdivided into the caudate and quadrate lobes.
Surrounded by a fibrous capsule, the liver is made up of

liver lobules (the functional units of the liver). Each lobule is
constructed around a central vein that empties into the right and
left hepatic veins which then drain into the vena cava. The lobule
is composed of cellular plates that radiate from the central vein.
Each cellular plate is two cells thick and between the two cells
are small bile canaliculi that empty into terminal ducts.
Blood supply 3
The average rate of blood flow through the liver is 1400

ml/min. Measured pressure in the hepatic vein, however, is
normally 0 mm Hg, while in the portal vein the pressure is 8 mm
Hg. The elevated portal venous and capillary pressures make the
liver more susceptible than other organ systems to increases in
resistance to circulation; e.g. cirrhosis of the liver. The liver
receives a dual blood supply:
1. From the hepatic artery
2. From the portal vein
The portal vein carries blood to the sinusoids of the liver

from the alimentary canal. One of the three branches of the
celiac trunk (off the aorta) is the hepatic artery. The hepatic
artery enters the substance of the liver in front of (anterior to)
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INTRODUCTION
the portal vein and to the left of the bile duct. Once the artery
enters the liver it divides into the left and right hepatic arteries.
The portal vein, arising from the gut, enters the substance of the
liver behind not only the bile duct but also the hepatic artery. At
the hilum of the liver (portal hepatis) the vein divides into left and
right branches. The inferior vena cava receives blood from the
liver via a series of hepatic veins which drain the central vein.
The hepatic vein series can be enumerated as follows:
1. Left hepatic v. - drains left lobe
2. Middle hepatic v. - drains central portion and may join the

left branch to form a common trunk
3. Right hepatic v. - drains most of the right lobe
The hepatic portal vein is the venous drainage for the large
and small intestine, the stomach and terminal esophagus and the
spleen. Since the function of the spleen is to filter out worn out
and broken down red blood cells, the splenic vein of the hepatic
portal system carries the products of red cell breakdown to the
liver. The mesenteric veins (they drain the large and small
intestine) carry deoxygenated blood and the products of
intestinal digestion and absorption (amino acids, mono and
disaccharides and short chain fatty acids; long chain fatty acids
are absorbed via the lymphatic system). Outside the liver these
veins come together to form the hepatic portal vein. About 60%
of the blood perfusing the liver is from the hepatic portal vein.
Entering the liver next to the hepatic portal vein is the hepatic
artery.. These two vessels remain separate in their passage
through the liver until they reach the lobule. At each corner of the
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INTRODUCTION
hexagonal liver lobule is a group of three structures: a branch of

the hepatic portal vein, a branch of the hepatic artery, and a bile
duct. These three structures comprise the portal triad.
As the two blood vessels leave the portal triad, they empty
into the sinusoids. This is a large endothelial lined space and in it
the blood from the two sources begins to mix. It percolates
through the sinusoids toward the center of the lobule where the
central vein is located. It passes through a series of veins that
collect from many lobules to enter the right and left hepatic veins
which empty into the inferior vena cava.
The venous sinusoids are lined with two different cell types:
1. Endothelial cells- have large pores, allows H2O and plasma

proteins to pass freely.
2. Kupffer cells - reticuloendothelial cells capable of phagocytizing bacteria

and other foreign matter in the blood.
Portal triad
A portal triad is an arrangement of three structures within

the liver lobule. At the corner of the hexagonally arranged lobule
the following structures are seen together:
1. Branch of hepatic portal vein
2. Branch of hepatic artery
3. Bile duct.
1.1.1 Functions of the Liver4
These functions include:
1. Carbohydrate storage
2. Amino acid metabolism

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INTRODUCTION
3. Metabolism of steroidal hormones
4. Metabolism of fat
Bile is a complex solution secreted by the cells of the liver

into the bile duct. Approximately 250-1000 ml/day are secreted.
It is golden yellow in color due to the presence of bile pigments
(bilirubin and biliverdin). These pigments, it should be noted, are
the breakdown products of hemoglobin. Also found in bile are the
bile salts which are sodium and potassium salts of bile acids.
Bile, containing the substances just mentioned as well as

cholesterol, phospholipids, water, Na, K, C1, Ca, and HCO, is
secreted into the bile duct which eventually drains into the
duodenum. D3uring periods where the digestive processes are
somewhat slowed, as in between meals, the duodenal orifice of
the duct is closed, causing the bile to back ups and eventually
enter the gallbladder where it is stored.
Bile is formed by the liver cells (the liver cells are epithelial
cells), and excreted into tiny bile canaliculi located between the
cells. Bile does not enter the sinusoids. Instead, the canaliculi
come together at the portal triad where the portal ductule is
formed. These bile ductules coalesce as they approach the
surface of the liver (near where the hepatic portal vein and the
hepatic artery enter) to form the hepatic duct which emerges
from the inferior surface of the liver.
When food enters the duodenum, cholecystokinin is

released from the intestinal mucosa which will cause gallbladder
contraction, leading to the secretion of bile into the small
intestine. Bile is an active emulsifying (suspension of fats) agent
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INTRODUCTION
and thus plays a part in the digestion and absorption of fat from
the intestine.
The second item to be considered is the production of

plasma proteins. The liver plays an intricate role in the synthesis
of these plasma proteins and is able to provide for an
interconversion (i.e. converting one type of amino acid to another
by the process of transamination) of are the” building blocks “of
protein structures. The source of the amino acids necessary for
this plasma protein production is:
1. Metabolic turnover of proteins
2. Dietary proteins
3. Glucose (glucogenic amino acids)
Because proteins are stored for only limited periods of time,

any imbalance between the amino acids required and those
which are available is handled quite readily by the liver’s amino
acid interconversion capability.
Two major categories of proteins produced by the liver are

the albumins and the globulins. The albumins are large colloidal
protein molecules which have an influence on osmotic pressure,
plasma volume and tissue fluid balance. The globulins are
involved in many functions such as: the transport of several key
substances (iron, copper, and lipids); serving as a precursor to
fibrin (fibrinogen); serving as antibodies or immunoglobins
(Gamma globulin, IgG, IgE, IgA, IgD, and IgM).
Furthermore, several proteins concerned with blood

coagulation are produced by the liver, such as:
1. Fibrinogen
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INTRODUCTION
2. Prothrombin
3. Factor VII
4. Factor IX
5. Factor X
Another function of the liver is detoxification. You will recall

from an earlier pharmacology mini- course that in this process,
which can manifest itself as either conjugation, oxidation, or
reduction, the liver metabolizes the by-products of cellular
metabolism and exogenous materials such as drugs. Of particular
importance is the removal of ammonia which is toxic to the
human organism. This ammonia is removed from amino acids via
deamination and converted to a normally nontoxic material
called urea. Urea is formed during a series of reactions called the
urea cycle (Krebs-Henseleit cycle) and is excreted in the urine.
Because of the vast enzyme system of the liver, this organ plays
an important role in drug metabolism. Knowledge of how a drug
is handled by the liver is very important in therapeutics.
As one can imagine, liver damage must be taken into

account when drugs that are metabolized in the liver are given to
a patient. Not all types of liver disease affect drug metabolism
equally, since drug metabolism is not uniform throughout the
liver. However, those disease states which damage areas which
actively metabolize drugs can have serious consequences. In
order for a patient’s ability to metabolize drugs to be
compromised, the liver parenchymal cells surrounding the central
vein must be damaged. In some cases, such as liver damage
associated with alcohol- ism, this area is not damaged and there
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INTRODUCTION
is little change in the drug metabolizing capabilities. On the other

hand, toxic substances like carbon tetrachloride (CC14), a
cleaning fluid, specifically damages the parenchymal cells
surrounding the central vein and, therefore, severely compromise
an individual’s drug metabolizing capacity.
1.1.2 Hepatic Responses to Injury 6
The liver is a major target organ of chemically induced toxicity. Serious

drug-induced liver injury is the leading single cause for withdrawal of approved
drugs from the U.S. market. It also accounts for more than 50% of the cases of
liver failure in the United States today. The liver is centrally located between the
gastrointestinal tract where absorption of ingested drugs occurs and the organs that
are targets of these drugs and is central to the metabolism of nearly all xenobiotics.
Most drugs are lipophilic, which enables them to be absorbed by the mucosal
surfaces of the intestinal mucosa. Biochemical processes in the hepatocyte
metabolize many drugs, so they are more hydrophilic, resulting in metabolites that
are water-soluble and can be excreted in the bile or urine.
The morphologic assessment of the gross and microscopic

appearance of the liver can provide a broad base of knowledge
concerning the potential toxicity of a drug or chemical. This
information may lead to an understanding of the underlying
mechanism of toxicity or guide further study that will assist in
determining the mode of action of the hepatotoxicity. In standard
regulatory bioassays, toxicity studies are conducted during phase
1 and phase 2 of the development process to define the acute,
subchronic and chronic toxicity of the test compound. In acute
and subchronic bioassays, a range of doses is commonly used to
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INTRODUCTION
establish a "no-observed-effect level" (NOEL), to establish

maximum tolerated doses (MTD) for chronic toxicity studies, and
to aid in the prediction of potential effects of long-term exposure
to the test article.
1.1.3 Diseases of Liver
Liver problems include a wide range of diseases and conditions

that can affect your liver. Your liver is an organ about the size of
a football that sits just under your rib cage on the right side of
your abdomen. Without your liver, you couldn't digest food and
absorb nutrients, get rid of toxic substances from your body or
stay alive. Liver problems can be inherited, or liver problems can
occur in response to viruses and chemicals. Some liver problems
are temporary and go away on their own, while other liver
problems can last for a long time and lead to serious
complications.
Symptoms
Signs and symptoms of liver problems include:
 Discolored skin and eyes that appear yellowish
 Abdominal pain and swelling
 Itchy skin that doesn't seem to go away
 Dark urine color
 Pale stool color
 Bloody or tar-colored stool
 Chronic fatigue
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INTRODUCTION
 Nausea
 Loss of appetite
Problems of Liver
Acute liver failure, Alcoholic hepatitis, Alpha-1-antitrypsin

deficiency, Autoimmune hepatitis, Bile duct obstruction, Chronic
liver failure, Cirrhosis, Enlarged liver, Gilbert syndrome,
Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis
D, Hepatitis E, Liver adenoma, Liver cancer, Liver cyst, Liver
hemangioma, Liver nodule (focal nodular hyperplasia),
Nonalcoholic fatty liver disease, Parasitic infection, Portal vein
thrombosis, Primary biliary cirrhosis, Toxic hepatitis, Wilson's
disease.
Risk factors
Factors that may increase your risk of liver problems include:
A job that exposes to other people's blood and body fluids,

Blood transfusion, Body piercing, Certain herbs and supplements,
Certain prescription medications, Diabetes, Heavy alcohol use,
High levels of triglycerides in blood, Injecting drugs using shared
needles, Obesity, Tattoos, Unprotected sex, Working with
chemicals or toxins without following safety precautions .
1.1.4 Tests and procedures used to diagnose liver

problems
 Blood tests. A group of blood tests called liver function

tests can be used to diagnose liver problems. Other blood
tests can be done to look for specific liver problems or
inherited conditions that affect the liver.
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INTRODUCTION
 Imaging tests. Procedures that create pictures of your

liver, such as computerized tomography (CT) scan,
magnetic resonance imaging (MRI) and ultrasound, can
reveal liver problems.
 Tests of liver tissue. A procedure to remove tissue from

your liver (liver biopsy) may help in diagnosing liver
problems. Liver biopsy is most often done using a long
needle inserted through the skin to extract a tissue sample
(needle biopsy). The tissue sample is sent to a laboratory
where it can be examined under a microscope.
7
1.1.5 Pharmacotherapy of liver diseases
Liver diseases are a major problem of worldwide

proportions. However, the number of drugs actually used
successfully in humans is very small. In this review some of the
most promising/studied drugs utilized for liver diseases were
chosen and analysed critically from the basic to the clinical point
of view. Antiviral agents are not discussed because excellent
reviews have appeared on this topic. The
compounds/preparations described herein are, alphabetically:
colchicine, corticosteroids, curcumin, glycyrrhizin, interferons (for
their antifibrotic properties), Liv 52, nitric oxide, resveratrol,
silymarin, sulfoadenosylmethionine, and thalidomide. Colchicine
and corticosteroids have been studied extensively in animals and
humans; most clinical studies suggest that these compounds are
not useful in the treatment of liver diseases. Glycyrrhizin is an
herbal medicine with several components that has interesting
hepatoprotective properties in patients with subacute liver failure
but deserves more prospective controlled trials. Interferon has
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INTRODUCTION
shown interesting antifibrotic properties in animals and humans;

prospective studies on their antifibrotic/fibrolytic activity are
required. Curcumin, resveratrol and thalidomide are very
attractive newly discovered protective and curative compounds
on experimental hepatic diseases. Their mechanism of action is
associated with the ability to down-regulate NF-kappaB and to
decrease pronecrotic and profibrotic cytokines. Unfortunately,
clinical studies are lacking. Sulfoadenosylmethionine and
silymarin are also promising drugs utilized mainly in cholestasis
but the benefits can be expanded if more controlled trials are
performed. The future is to carry out controlled prospective
double-blind multicenter studies with the newly discovered drugs
with proven beneficial effects on animals. Fundamental
hepatobiology should also be encouraged.
The enhanced effect of drugs in patients with liver disease

is primarily due to decreased drug metabolism. Fortunately,
glucuronidation, a common method by which lipid soluble drugs
are metabolised in dogs, appears to be relatively unaffected by
hepatic disease. Hepatic elimination of drugs is influenced by
hepatic clearance and hepatic extraction that are in turn
dependant on hepatic blood flow, protein binding, and intrinsic
hepatic clearance. For some drugs such as propranolol, the rate
of hepatic elimination is influenced by hepatic blood flow but they
are insensitive to changes in hepatic metabolism. Clearance of
lignocaine is also prolonged by poor hepatic perfusion (e.g., in
heart failure, in shock, and with propranolol administration).
For other drugs, such as diazepam, phenylbutazone,

prednisolone, theophylline and cimetidine, changes in hepatic
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INTRODUCTION
metabolism but not changes in hepatic blood flow will affect

hepatic elimination. However, for many drugs, the effects of
hepatic disease on drug disposition are complex and difficult to
predict.
Unfortunately, in veterinary laboratory medicine, there are

no satisfactory indices of liver dysfunction that can be used to
predict the magnitude of changes in hepatic clearance of drugs.
In general, when administering drugs that are extensively
metabolised by the liver such as benzodiazepines, NSAIDs and
opioids to patients with liver disease the dosage interval should
be prolonged. Use of barbiturates and several cytotoxic drugs
(which have a narrow therapeutic index) such as
cyclophosphamide, dacarbazine, thiotepa and L-asparaginase
should be avoided in patients with liver disease.
For drugs that have high plasma protein binding and are
predominantly cleared by the liver, liver disease would be
expected to increase the volume of distribution of the drug and
decrease drug clearance. However, reduced protein binding due
to reduced albumin levels associated with advanced hepatic
disease may actually increase hepatic clearance and therefore
compensate for reduced hepatic metabolism (if this is occurring).
Increased serum globulin levels may occur in inflammatory
hepatic disease or when the hepatic reticuloendothelial system is
compromised. In these circumstances, increased protein binding
can occur for some basic drugs such as lignocaine due to
increased production of acute-phase proteins.
The dose of drugs that are primarily eliminated in bile

should be reduced in patients with significant cholestasis
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INTRODUCTION
particularly if the drug has a narrow therapeutic window (e.g.,

digitoxin). Although only about 15% of digoxin is metabolized in
the liver, bile duct ligation increases its half-life from an average
of 26 hours to 35 hours in experimental dogs.
Antimicrobial drugs that are believed to potentially

accumulate in hepatic disease and may cause toxicity include
chloramphenicol, lincosamides, macrolides, metronidazole,
sulphonamides and tetracyclines.
Drugs that have been reported to directly cause hepatic

toxicity in dogs include primidone, phenobarbitone (rarely),
rifampin, and triazole antifungals such as ketoconazole.
Lignocaine is administered parenterally because it is extensively
metabolized by the liver to toxic metabolites if given orally.
Whether the use of certain drugs should be avoided in

animals experiencing liver dysfunction is controversial.
Ultimately, it depends on whether that drug leads to toxicity at
concentrations close to therapeutic concentrations (i.e., those
drugs with a low therapeutic index) and whether other available
drugs are suitable alternatives.
Specific drugs used in the management of liver disease 7
Ursodeoxycholic acid (Ursodiol)
 Clinical applications. Ursodeoxycholic acid is a naturally

occurring bile acid found in the bile of the Chinese black bear.
Black bear bile has been used for many years by practitioners
of Eastern medicine and has been commercially synthesised
and available for use as a hepatoprotective agent in Japan
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INTRODUCTION
since the 1930s. Since the 1970s, ursodeoxycholic acid has

been used in Western human medicine for dissolution of
gallstones. More recently, ursodeoxycholic acid has been used
in the management of chronic hepatic diseases in humans
such as primary biliary cirrhosis, biliary disease secondary to
cystic fibrosis, nonalcoholic steatohepatitis, idiopathic chronic
hepatitis, autoimmune hepatitis, primary sclerosing
cholangitis, and alcoholic hepatitis. However, its therapeutic
efficacy in some of these disorders has not been firmly
established.
In veterinary medicine, ursodeoxycholic acid has been

used in the management of dogs with chronic hepatitis and
cats with lymphocytic plasmacytic cholangitis. It is believed to
be most beneficial in disorders where bile toxicity plays an
important role in the ongoing pathology. The efficacy of
ursodeoxycholic acid in veterinary patients has not been
definitely established although anecdotal reports suggest it
may have some benefit in patients with chronic inflammatory
hepatobiliary disease. It may be of some benefit in slowing
disease progression especially if used at an early stage of the
disease. Some authors recommend ursodeoxycholic acid
treatment for all cats with cholangiohepatitis where
extrahepatic biliary obstruction has been eliminated.
 Mechanism of action. Ursodeoxycholic acid decreases

intestinal absorption and suppresses hepatic synthesis and
storage of cholesterol. This is believed to reduce cholesterol
saturation of bile and thereby allowing solubilization of
cholesterol-containing gall stones. It has little effect on
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INTRODUCTION
calcified gallstones or on radiolucent bile pigment stones and

therapy is only successful in patients with a functional gall
bladder. Ursodeoxycholic acid, a relatively hydrophilic bile
acid, is also believed to protect the liver from the damaging
effects of hydrophobic bile acids, which are retained in
cholestatic disorders. The hepatoprotective effect may
however, be less in cats and dogs than in humans as the major
circulating bile acid in dogs and cats is taurocholate. This is
more hydrophilic and less hepatotoxic than the major
circulating bile acids in humans. The immunomodulatory
effects of ursodeoxycholic acid are believed to involve
decreased immunoglobulin production by B lymphocytes,
decreased interleukin-1 and interleukin-2 production by T
lymphocytes, decreased expression of hepatocyte cell surface
membrane HLA class I molecules and possibly stimulation of
the hepatocyte glucocorticoid receptor.
 Adverse effects. Ursodeoxycholic acid appears to be well

tolerated by dogs and cats; vomiting and diarrhoea are
reported rarely. There is some concern in human patients that
taurine depletion may be potentiated by chronic treatment
with ursodeoxycholic acid. This may be important in cats that
are obligate taurine conjugators. This potential for taurine
depletion may be exacerbated in some cats with hepatobiliary
disease that have increased urinary excretion of taurine-
conjugated bile acids. Dogs are less likely to become taurine
depleted by this mechanism as they can shift to glycine
conjugation. Ursodeoxycholic acid should not be used in
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INTRODUCTION
patients with extra-hepatic biliary obstruction, biliary fistulas,

cholecystitis or pancreatitis.
Colchicine
 Clinical applications. Colchicine is used in the

management of gout in humans providing acute relief of
symptoms as well as prophylaxis. In veterinary medicine, it
has been used in the management of amyloidosis and chronic
hepatic fibrosis. Controlled clinical trials are lacking but there
has been anecdotal evidence from a few case studies that
colchicine may improve liver function and slow the progression
of hepatic fibrosis.
 Mechanism of action. Collagen secretion from lipocytes

requires microtubles, the assembly of which is inhibited by
colchicines, thereby interfering with the transcellular
movement of collagen. The drug increases collegenase activity
and therefore may promote degradation of existing collagen. It
has anti-inflammatory effects by inhibiting leukocyte migration
—this may suppress fibrogenesis. It may also have a direct
hepatoprotective effect by stabilising hepatocyte membranes.
 Colchicine is marketed in combination with probenicid in

some countries—this combination should be avoided as
probenecid can cause nausea, vomiting, and lethargy. Dose:
0.025–0.03 mg/kg/day
 Adverse effects. Because of the limited veterinary

experience with colchicine, little is known about its potential
toxicity in dogs and cats.
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INTRODUCTION
o In humans, the therapeutic window for colchicine is quite

narrow with toxic effects occurring after only small
overdoses.
o Nausea, vomiting and diarrhoea have been reported in

dogs.
o Bone marrow suppression has occurred in humans after

prolonged use.
o Myopathy and peripheral neuropathy has been reported

rarely in humans
o Severe local irritation occurs if the drug is inadvertently

administered perivascularly. Thrombophlebitis has also
been reported.
o Colchicine is contraindicated in patients with serious renal,

GI or cardiac disease and should be used with caution in
patients with less severe disease of these organs.
o Colchicine is teratogenic in mice and hamsters; therefore, it

should not be used in pregnant patients unless the
benefits outweigh the risks.
o Colchicine may decrease spermatogenesis.
o Safety to nursing neonates is unknown, as it is not known

whether it is excreted in milk.
o NSAIDs, especially phenylbutazone increase the risk of

thrombocytopenia, leukopenia or bone marrow
suppression when used concurrently with colchicine
o Many antineoplastic and other potentially marrow-

suppressing drugs may cause additive myelosuppression
when used concurrently with colchicine.
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INTRODUCTION
Penicillamine
Penicillamine is a degradation product of penicillin but has

no antimicrobial activity. It was first isolated in 1953 from the
urine of a patient with liver disease who was receiving penicillin
 Clinical applications. Penicillamine is a monothiol

chelating agent which is used in veterinary medicine in the
treatment of copper-storage hepatopathy (e.g., Bedlington
Terriers), lead toxicity, and cystine urolithiasis. It has also
been used in the management of rheumatoid arthritis in
humans.
 Mechanism of action. Penicillamine chelates several

metals including copper, lead, iron, and mercury, forming
stable water soluble complexes that are renally excreted. It
also combines chemically with cystine to form a stable,
soluble, readily excreted complex. Although it usually takes
months to years for hepatic copper levels to decrease, clinical
improvement is often seen in Bedlington Terriers after only a
few weeks suggesting the drug has other beneficial effects
other than copper depletion. Penicillamine induces hepatic
metallothionein, which may bind and sequester copper in a
nontoxic form. It may also have antifibrotic effects as it inhibits
lysyl oxidase, an enzyme necessary for collagen synthesis and
directly binds to collagen fibrils, preventing cross-linking into
stable collagen fibres. However, its efficacy as an antifibrotic
agent in humans is doubtful and it has not been evaluated in
veterinary medicine. Penicillamine may have
immunomodulatory effects and has been demonstrated to
reduce IgM rheumatoid factor in humans with rheumatoid
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INTRODUCTION
arthritis. However, its mechanism of action in this disease

remains uncertain.
 Dosage and formulations. For management of copper-

associated hepatopathy, a dose of 10–15 mg/kg q12h PO is
given on an empty stomach. However, if GIT adverse effects
are experienced, these may be reduced if it is given with food,
although absorption may be reduced. Alternatively, reduce
dose and gradually build up to full dose.
 Adverse reactions: GIT adverse effects are common

resulting in nausea and vomiting. Smaller doses on a more
frequent basis may alleviate adverse effects. Alternatively, the
drug can be given with food although this will reduce
absorption.
Other adverse effects observed infrequently or rarely include:

o Fever
o Lymphadenopathy
o Skin hypersensitivity reactions
o Immune-complex glomerulonephropathy
o Leucopenia, aplastic anemia and agranulocytosis have
been reported in humans
The Progression of Liver Disease
Liver helps fight infections and cleans blood. It also helps

digest food and stores energy. A healthy liver has the amazing
ability to grow back, or regenerate, when it is damaged. Anything
that keeps the liver from doing its job – or from growing back
after injury – may put the life in danger!
Inflammation
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INTRODUCTION
In the early stage of any liver disease, liver may become

inflamed. It may become tender and enlarged. Inflammation
shows that the body is trying to fight an infection or heal an
injury. But if the inflammation continues over time, it can hurt the
liver permanently.
When most other parts of the body become inflamed, the

area becomes hot and painful. But an inflamed liver may cause
no discomfort at all to a person. This is the stage which has to
treated to avoid further liver complications.
Fibrosis
If left untreated, the inflamed liver will start to scar. As

excess scar tissue grows, it replaces healthy liver tissue. This
process is called fibrosis. (Scar tissue is a kind of fibrous
tissue.).Scar tissue cannot do the work that healthy liver tissue
can. Moreover, scar tissue can keep blood from flowing through
your liver. As more scar tissue builds up, your liver may not work
as well as it once did. Or, the healthy part of your liver has to
work harder to make up for the scarred part.
If the liver disease is diagnosed and treated successfully at

this stage, there’s still a chance that the liver can heal itself over
time.
Cirrhosis
Cirrhosis of the liver refers to a disease in which normal

liver cells are replaced by scar tissue caused by alcohol and viral
hepatitis B and C. Cirrhosis can lead to a number of
complications, including liver cancer. In some people, the
symptoms of cirrhosis may be the first signs of liver disease. This
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INTRODUCTION
disease leads to abnormalities in the liver's ability to handle

toxins and blood flow, causing internal bleeding, kidney failure,
mental confusion, coma, body fluid accumulation, and frequent
infections. Symptoms include yellowing of the skin, itching, and
fatigue.
Drug combinations used to handle cirrhosis
 Bumetanide, Bumex
 Cholestyramine, Questran
 Furosemide, Lasix
 Hydrochlorothiazide, Hydrodiuril, Ezide, Hydro-Par,

Microzide, and many combinations
 Lactulose-oral, Chronulac, Constilac, Constulose, Duphalac,

Evalose
 Spironolactone, Aldactone
 Vitamin k-injection, Aqua-Mephyton, Vitamin K
 Vitamin k-oral, Mephyton, Vitamin K
Liver Cancer
Cancer that starts in the liver is called primary liver cancer.

Cirrhosis and hepatitis B are leading risk factors for primary liver
cancer.
Liver failure
Liver failure means that the liver is losing or has lost all of
its function. It is a life-threatening condition that demands urgent
medical care.
 22 
INTRODUCTION
The first symptoms of liver failure are often nausea, loss of

appetite, fatigue, and diarrhea. Because these symptoms can
have any number of causes, it may be hard to tell that the liver is
failing.
But as liver failure progresses, the symptoms become more

serious. The patient may become confused and disoriented, and
extremely sleepy. There is a risk of coma and death. Immediate
treatment is needed. The medical team will try to save whatever
part of the liver that still works. If this is not possible, the only
option may be a liver transplant.
When liver failure occurs as a result of cirrhosis, it usually

means that the liver has been failing gradually for some time,
possibly for years. This is called chronic liver failure.
Chronic liver failure can also be caused by malnutrition.

More rarely, liver failure can occur suddenly, in as little as 48
hours. This is called acute liver failure and is usually a reaction to
poisoning or a medication overdose.
Cirrhosis, liver cancer, and liver failure are serious

conditions that can threaten the life treatment options may be
very limited at these stages.
That’s why it’s important to catch liver disease early, in the

inflammation and fibrosis stages. If you are treated successfully
at these stages, your liver may have a chance to heal itself and
recover test is sufficient to provide complete estimate of function
of liver. Often clinicians are faced with reports that do not tally
with the clinical condition of the patient and they face difficulty in
interpreting the LFT.
 23 
INTRODUCTION
Liver has to perform different kinds of biochemical,

synthetic and excretory functions, so no single biochemical test
can detect the global functions of liver.
1.1.6 Liver function tests8
All laboratories usually employ a battery of tests for initial

detection and management of liver diseases and these tests are
frequently termed “Liver function tests”,
USES
The various uses of Liver function tests include:
 Screening: They are a non-invasive yet sensitive screening

modality for liver dysfunction.
 Pattern of disease: They are helpful to recognize the

pattern of liver disease. Like being helpful in differentiating
between acute viral hepatitis and various cholestatic
disorders and chronic liver disease. (CLD).
 Assess severity: They are helpful to assess the severity

and predict the outcome of certain diseases like primary
biliary cirrhosis.
 Follow up: They are helpful in the follow up of certain liver

diseases and also helpful in evaluating response to therapy
like autoimmune hepatitis.
 Limitations
 Lack sensitivity: The LFT may be normal in certain liver

diseases like cirrhosis, non cirrhotic portal fibrosis,
congenital hepatic fibrosis, etc.
 24 
INTRODUCTION
 Lack specificity: They lack specificity and are not specific

for any particular disease. Serum albumin may be
decreased in chronic disease and also in nephritic
syndrome. Aminotransferases may be raised in cardiac
diseases and hepatic diseases. Except for serum bile acids
the LFT are not specific for liver diseases and all the
parameters may be elevated for pathological processes
outside the liver. Thus, we see that LFT have certain
advantages as well as limitations at the same time. Thus, it
is important to view them keeping the clinical profile of the
patient in mind.
Classification of Liver Function Tests
A. Tests of the liver’s capacity to transport organic anions

and to metabolize drugs- Serum bilirubin, urine bilirubin,
urobilinogen etc.
B. Tests that detect injury to hepatocytes (serum enzyme

tests) – Aminotransferases, alkaline phosphatase,
C. Tests of the Liver’s biosynthetic capacity- Serum

proteins, albumin, prealbumin, serum ceruloplasmin, procollagen
III peptide, α-1 antitrypsin, α-feto protein, and prothrombin time
etc.
A. Tests of the liver’s capacity to transport organic anions

and to metabolize drugs
1. SERUM BILIRUBIN
Bilirubin is an endogenous anion derived from hemoglobin

degradation from the RBC. The classification of bilirubin into
direct and indirect bilirubin is based on the original van der Bergh
 25 
INTRODUCTION
method of measuring bilirubin. Bilirubin is altered by exposure to

light so serum and plasma samples must be kept in dark before
measurements are made. When the liver function tests are
abnormal and the serum bilirubin levels more than 17μmol/L
suggest underlying liver disease.4 Types of bilirubin:-
I. Total bilirubin: This is measured as the amount, which reacts

in 30 minutes after addition of alcohol. Normal range is 0.2-0.9
mg/dl (2-15μmol/L). It is slightly higher by 3-4 μmol/L in males as
compared to females. It is this factor, which helps to diagnose
Gilbert syndrome in males easily.
II. Indirect Bilirubin: This is the water-soluble fraction. This is

measured by the reaction with diazotized sulfanilic acid in 1
minute and this gives estimation of conjugated bilirubin. Normal
range 0.3mg/dl (5.1μmol/L).
III. Indirect bilirubin: This fraction is calculated by the

difference of the total and direct bilirubin and is a measure of
unconjugated fraction of bilirubin.The diazo method of bilirubin
estimation is not very accurate especially in detecting low levels
of bilirubin.
Direct bilirubin over estimates bilirubin esters at low

bilirubin levels and under estimates them at high concentration.
Thus slight elevation of unconjugated bilirubin not detected,
which is of value in detecting conditions like Gilbert syndrome.A
newer highly accurate method of estimation involves alkaline
methanolysis of bilirubin followed by chloroform extraction of
bilirubin methyl esters and later separation of these esters by
 26 
INTRODUCTION
chromatography and spectrophotometric determination at 430

nm.
Hyperbilirubinemia: It results from overproduction /impaired

uptake, conjugation or excretion / regurgitation of unconjugated
or conjugated bilirubin from hepatocytes to bile ducts. Other
causes of extreme hyperbilirubinemia include severe
parenchymal disease, septicemia and renal failure.
2. URINE BILIRUBIN
The presence of urine bilirubin indicates hepatobiliary

disease. Unconjugated bilirubin is tightly bound to albumin and
not filtered by the glomerulus and thus not present in urine.
Measurable amounts of conjugated bilirubin in serum are found
only in hepatobiliary disease. Because the renal threshold for
conjugated bilirubin is low and the laboratory methods can detect
low levels of bilirubin in urine so conjugated bilirubin may be
found in urine when the serum bilirubin levels are normal. This is
the case in early acute viral hepatitis. Tests strips impregnated
with diazo reagent are easy to use and detect as little as 1-2m
mol bilirubin/L.
3. UROBILINOGEN
An increase in the urobilinogen in urine is a sensitive

indicator of hepatocellular dysfunction. It is a good indication of
alcoholic liver damage, well compensated cirrhosis or malignant
disease of the liver. In viral hepatitis it appears early in urine. It is
markedly increased in hemolysis.In cholestatic jaundice
urobilinogen disappears from urine. It may be intermittently
present in case of gallstones.3 Urobilinogen gives a purple
 27 
INTRODUCTION
reaction to Ehrlich’s aldehyde reagent. A dipstick containing this

reagent allows rough and ready quantification. Freshly voided
urine should be used.
B. Tests that detect injury to hepatocytes (serum enzyme

tests):
The liver contains thousands of enzymes and these enzymes

have no function and behave as serum proteins.
1. Enzymes That Detect Hepatocellular Necrosis –
α- AMINOTRANSFERASES
The aminotransferases (formerly transaminases) are the

most frequently utilized and specific indicators of hepatocellular
necrosis. These enzymes- aspartate aminotransferase (AST,
formerly serum glutamate oxaloacetic transaminase-SGOT) and
alanine amino transferase (ALT, formerly serum glutamic
pyruvate transaminase-SGPT) catalyze the transfer of the á
amino acids of aspartate and alanine respectively to the á keto
group of ketoglutaric acid. ALT is primarily localized to the liver
but the AST is present in a wide variety of tissues.
Enzyme reaction
AST: alanine + a ketoglutarate = oxaloacetate +glutamate
ALT: alanine + a ketoglutarate = pyruvate +glutamate
Whereas the AST is present in both the mitochondria and

cytosol of hepatocytes, ALT is localized to the cytosol.The
cytosolic and mitochondrial forms of AST are true isoenzymes
and immunologically distinct.About 80% of AST activity in human
liver is contributed by the mitochondrial isoenzyme, whereas
 28 
INTRODUCTION
most of the circulating AST activity in normal people is derived

from the cytosolic isoenzyme.
Large increases in mitochondrial AST occur in serum after

extensive tissue necrosis. Because of this, assay of mitochondrial
AST have been advocated in myocardial Infarction. Mitochondrial
AST is also increased in chronic liver disease. Their activity in
serum at any moment reflects the relative rate at which they
enter and leave circulation. Of the numerous methods used for
measuring their levels, the most specific method couples the
formation of pyruvate and oxaloacetate.
MILD, MODERATE AND SEVERE ELEVATIONS OF

AMINOTRANSFERASES
1. Severe (> 20 times, 1000 U/L): The AST and ALT levels
are increased to some extent in almost all liver diseases.
The highest elevations occur in severe viral Hepatitis, drug
or toxin induced hepatic necrosis and circulatory shock.
Although enzyme levels may reflect the extent of
hepatocellular necrosis they do not correlate with eventual
outcome. In fact declining AST and ALT may indicate either
recovery of poor prognosis in fulminant hepatic failure.
2. Moderate (3-20 times): The AST and ALT are moderately

elevated in acute hepatitis, neonatal hepatitis, chronic
hepatitis, autoimmune hepatitis, drug induced hepatitis,
alcoholic hepatitis and acute biliary tract obstructions. The
ALT is usually more frequently increased as compared to
AST except in chronic liver disease. In uncomplicated acute
viral hepatitis, the very high initial levels approach normal
levels within 5 weeks of onset of illness and normal levels
 29 
INTRODUCTION
are obtained in 8 weeks in 75% of cases. For reasons, which

are not, understood AST levels appear disproportionately
low in patients with Wilson disease.
3. Mild (1-3 times): These elevations are usually seen in

sepsis induced neonatal hepatitis, extrahepatic biliary
atresia (EHBA), fatty liver, cirrhosis, non alcoholic
steatohepatitis (NASH), drug toxicity, myositis, duchenne
muscular dystrophy and even after vigorous exercise. One
third to one half of healthy individuals with an isolated
elevation of ALT on repeated testing has been found to be
normal.
AST: ALT ratio:
The ratio of AST to ALT is of use in Wilson disease, CLD and

alcoholic liver disease and a ratio of more than 2 is usually
observed. The lack of ALT rise is probably due to pyridoxine
deficiency. In NASH the ratio is less than one in the absence of
fibrosis on liver biopsy. In viral hepatitis the ratio is usually less
than one. The ratio invariably rises to more than one as cirrhosis
develops possibly because of reduced plasma clearance of AST
secondary to impaired function of sinusoidal cells.ALT exceeds
AST in toxic hepatitis, viral hepatitis, chronic active hepatitis and
cholestatic hepatitis
Mitochondrial AST: Total AST ratio:
This ratio is characteristically elevated in alcoholic liver

disease. Abstinence from alcohol improves this ratio. It is also
seen to be high in Wilson’s disease.
Falsely low aminotransferase levels:
 30 
INTRODUCTION
They have been seen in patients on long term hemodialysis

probably secondary to either dialysate or pyridoxine deficiency.
Low levels have also been seen in uremia.
Other enzymes tests of hepatocellular necrosis
None of these tests have proved to be useful in practice

than the aminotransferases.These include glutamate
dehydrogenase, isocitrate dehydrogenase, lactate
dehydrogenase and sorbitol dehydrogenase
b. Enzymes that detect cholestasis
1. ALKALINE PHOSPHATASE
Alkaline phosphatases are a family of zinc metaloenzymes,

with a serine at the active center; they release inorganic
phosphate from various organic orthophosphates and are present
in nearly all tissues. In liver, alkaline phosphatase is found
histochemically in the microvilli of bile canaliculi and on the
sinusoidal surface of hepatocytes. Alkaline phosphatase from the
liver, bone and kidney are thought to be from the same gene but
that from intestine and placenta are derived from different
genes.In liver two distinct forms of alkaline phosphatase are also
found but their precise roles are unknown. In healthy people most
circulating alkaline phosphatase originates from liver or
bone.Internationally recommended reference method uses p-
nitrophenol phosphate as substrate, in alkaline buffer. Fresh
unhemolysed serum is the specimen of choice for the estimation.
Heparinized plasma may also be used. The test should not be
done on plasma if citrate,oxalate or EDTA were used as
anticoagulants, they form a complex with zinc and the alkaline
 31 
INTRODUCTION
phosphatase,causing irreversible enzyme inactivation.Average

values of alkaline phosphatase vary with age and are relatively
high in childhood and puberty and lower in middle age and higher
again in old age. Males usually have higher values as compared
to females. The levels correlate with person’s weight and
inversely with the height of person.Not uncommonly isolated
elevated levels of alkaline phosphatase in otherwise healthy
persons return to normal on follow up.Highest levels of alkaline
phosphatase occur in cholestatic disorders. Elevations occur as a
result of both intrahepatic and extrahepatic obstruction to bile
flow and the degree of elevation does not help to distinguish
between the two. The mechanism by which alkaline phosphatase
reaches the circulation is uncertain; leakage from the bile
canaliculi into hepatic sinusoids may result from leaky tight
junctions.and the other hypothesis is that the damaged liver fails
to excrete alkaline phosphatase made in bone, intestine and
liver.In acute viral hepatitis, alkaline phosphatase is usually
either normal or moderately increased. Hepatitis A may present a
cholestatic picture with marked and prolonged itching and
elevation of alkaline phosphatase. Tumours may secrete alkaline
phosphatase into plasma and there are tumour specific
isoenzymes such as Regan, Nagao and Kasahara isoenzymes.
Elevated serum levels of intestinal alkaline phosphatase

have been found in patients with cirrhosis, particularly those with
blood group type O, and may be associated specifically with
intrahepatic disease as opposed to extrahepatic obstruction.
Hepatic and bony metastasis can also cause elevated levels of
alkaline phosphatase. Other diseases like infiltrative liver
 32 
INTRODUCTION
diseases, abscesses, granulomatous liver disease and

amyloidosis may also cause a rise in alkaline phosphatase. Mildly
elevated levels of alkaline phosphatase may be seen in cirrhosis
and hepatitis of congestive cardiac failure.Low levels of alkaline
phosphatase occur in hypothyroidism, pernicious anemia, zinc
deficiency and congenital hypophosphatasia.Wilson’s disease
complicated by hemolysis and FHF may also have very low levels
of alkaline phosphatase. Ratio of alkaline phosphatase and
bilirubin is low in fulminant Wilson disease. This might be the
result of replacement of cofactor zinc by copper and subsequent
inactivation of alkaline phosphtase.Regardless of the cause of
acute hepatic failure a low ratio of alkaline phosphatase to
bilirubin is associated with a poor prognosis. Drugs like
cimetidine, frusemide, phenobarbitone and phenytoin may
increase levels of alkaline phosphtase.
2. ‫ץ‬-GLUTAMYL TRANSPEPTIDASE
‫ץ‬-Glutamyl transpeptidase(GGT) is a membrane bound

glycoprotein which catalyses the transfer of g glutamyl group to
other peptides, amino acids and water.Large amounts are found
in the kidneys, pancreas,liver, intestine and prostate. The gene
for ‫ץ‬-glutamyl transpeptidase is on chromosome. The levels of ‫ץ‬-
glutamyl transpeptidase are high in neonates and infants up to 1
yr and also increase after 60 yr of life. Men have higher values.
Children more than 4 yr old have serum values of normal adults.
The normal range is 0-30 IU/L In acute viral hepatitis the levels of
‫ץ‬-glutamyl transpeptidase may reach its peak in the second or
third wk of illness and in some patients they remain elevated for
6 weeks. Often clinicians are faced with a dilemma when they see
 33 
INTRODUCTION
elevated alkaline phosphatase levels and are unable to

differentiate between liver diseases and bony disorders and in
such situations measurement of ‫ץ‬-glutamyl transferase helps as it
is raised only in cholestatic disorders and not in bone diseases. In
liver disease g glutamyl transpeptidase activity correlates well
with alkaline phosphatase levels but rarely the ‫ץ‬- glutamyl
transpeptidase levels may be normal in intra hepatic cholestasis
like in some familial intrahepatic cholestasis.Other conditions
causing elevated levels of g glutamyl transpeptidase include
uncomplicated diabetes mellitus, acute pancreatitis and
myocardial infarction. Drugs like phenobarbitone, phenytoin,
paracetamol, tricyclic antidepressants may increase the levels of
‫ץ‬-glutamyl transpeptidase. Non-hepatic causes of increased
levels of the enzyme include anorexia nervosa, Gullian barre
syndrome,hyperthyroidism, obesity and dystrophica
myotonica.As a diagnostic test the primary usefulness of
gglutamyl transpeptidase is limited to the exclusion of bone
disease, as ‫ץ‬-glutamyl transpeptidase is not found in bone.
OTHER ENZYMES THAT DETECT CHOLESTASIS
These are the other enzymes that are not routinely

estimated to detect cholestasis.
5- Nucleotidase
Leucine aminopeptidase
C. Tests of the Liver’s biosynthetic capacity.
1. SERUM PROTEINS
The liver is the major source of most the serum

proteins.The parenchymal cells are responsible for synthesis of
 34 
INTRODUCTION
albumin, fibrinogen and other coagulation factors and most of the

and b globulins.
1. ALBUMIN
Albumin is quantitatively the most important protein in

plasma synthesized by the liver and is a useful indicator of
hepatic function. Because the half life of albumin in serum is as
long as 20 days, the serum albumin level is not a reliable
indicator of hepatic protein synthesis in acute liver disease.
Albumin synthesis is affected not only in liver disease but also by
nutritional status, hormonal balance and osmotic pressure. Liver
is the only site of synthesis of albumin.The serum levels are
typically depressed in patients with cirrhosis and ascites. In
patients with or without ascites, the serum albumin level
correlates with prognosis. In addition the rate of albumin
synthesis has been shown to correlate with the Child- Turcotte or
Child- Pugh score. Normal serum values range from 3.5g/dl to 4.5
g/dl. The average adult has approximately 300 to 500 g of
albumin. The serum levels at any time reflect its rate of
synthesis, degradation and volume of distribution. Corticosteroids
and thyroid hormone stimulate albumin synthesis by increasing
the concentration of albumin mRNA and tRNA in hepatocytes.The
serum albumin levels tend to be normal in diseases like acute
viral hepatitis, drug related hepatotoxicity and obstructive
jaundice. Albumin levels below 3g/dl in hepatitis should raise the
suspicion of chronic liver disease like cirrhosis which usually
reflects decreased albumin synthesis. In ascites there may be
normal synthesis but the levels may appear reduced because of
increased volume of distribution. Hypoalbuminemia is not specific
 35 
INTRODUCTION
for liver disease and may occur in protein malnutrition, nephrotic

syndrome and chronic protein losing enteropathies.
2. PREALBUMIN
The serum prealbumin level is 0.2- 0.3 g/L. these levels fall
in liver disease presumably due to reduced synthesis. Because of
its short half life, changes may precede alteration in serum
albumin. Determination of prealbumin has been considered
particularly useful in drug-induced hepatotoxicity.
3. SERUM CERULOPLASMIN
Normal plasma levels are 0.2-0.4g/L. It is synthesized in the

liver and is an acute phase protein. The concentration rise in
infections, rheumatoid arthiritis, pregnancy, non Wilson liver
disease and obstructive jaundice.
This is an important diagnostic marker in Wilson disease, in

which the plasma level is usually low. Low levels may also be
seen in neonates, Menke’s disease, kwashiorkor, and marasmus,
protein losing enteropathy, copper deficiency and
aceruloplasminemia.
4. PROCOLLAGEN III PEPTIDE
The serum concentration of this peptide appears to

increase not only with hepatic fibrosis but also with inflammation
and necrosis. Serial measurement of procollagen III may be
helpful in the follow up of chronic liver disease.
5. α-1 ANTITRYPSIN
α-1 antitrypsin is a glycoprotein synthesized by the liver

and is an inhibitor of serine proteinases, especially elastase. Its
 36 
INTRODUCTION
normal concentration is 1- 1.6g/L. it is an acute phase protein,

serum levels increase with inflammatory disorders, pregnancy
and after oral contraceptive pills (OCP). The various alleles coded
are M, F, S, Z and null forms. PiZZ homozygotes are associated
with neonatal hepatitis. Cirrhosis in adults has been found with
ZZ, MZ, SZ and FZ phenotypes. Liver disease is usually seen with
deficiency of α-1 antitrypsin, an inherited disorder. Deficiency
should be confirmed by quantitative measurement.
6 α-.FETO PROTEIN
This protein, the principal one in fetal plasma in early

gestation is subsequently present at very low levels. (<25mg/L) It
is increased in hepatocellular carcinoma (HCC) and more than
90% of such patients have raised levels. Raised values are also
found in other liver diseases like chronic hepatitis, in
regeneration phase of acute hepatitis and in hepatic metastasis.
This is also raised in adenomas associated with tyrosinemia. A
feto protein elevation is less frequent when HCC arises in non
cirrhotic liver. Serial determination is of value in cirrhotic patients
and rise in the values should raise the suspicion of HCC.
7. PROTHROMBIN TIME (PT)
Clotting is the end result of a complex series of enzymatic

reactions that involve at least 13 factors. The liver is the major
site of synthesis of 11 blood coagulation proteins: fibrinogen,
prothrombin, labile factor, stable factor, Christmas factor, Stuart
prowe factor, prekallikrein and high molecular wt kininogen.Most
of these are present in excess and abnormalities of coagulation
only result when there is substantial impairment in the ability of
 37 
INTRODUCTION
the liver to synthesize these factors. The standard method to

assess is the one stage prothrombin time of quick, which
evaluate the extrinsic coagulation pathway.
The results of this test may be expressed in sec or as a

ratio of the plasma prothrombin time to control plasma time.
Normal control usually is in the range of 9-11 seconds. A
prolongation of more than 2 seconds is considered abnormal.The
prolonged PT is not specific for liver diseases and is seen in
various deficiencies of coagulation factors, DIC, and ingestion of
certain drugs. In acute and chronic hepatocellular disease the PT
may serve as a prognostic indicator. In acute hepatocellular
disease worsening of PT suggests an increased likelihood of acute
hepatic failure. The PT is a predictor of outcome in cases of
acetoaminophen over dosage and acute alcoholic hepatitis.
Prolongation of PT is also suggestive of poor long-term outcome
in chronic liver disease. If the PT returns to normal or improves
by at least 30% within 24 hr of a single parenteral injection of
vitamin K1 (5-10 mg), it may be surmised that parenchymal
function is good and that hypovitaminosis K was responsible for
the original prolongation of PT. Patients with parenchymal
disease by contrast will show only minimal improvement. Most
patients with extra hepatic obstruction like EHBA would respond
promptly to a single injection of vitamin K1.The PT is particularly
important in the management of patients with liver disease. It is
important to perform before procedures like liver biopsy and
kidney biopsy and it permits an assessment of the tendency to
 38 
INTRODUCTION
bleed. In many centers the International normalized ratio (INR) is

done in place of PT.
A single liver function test is of little value in screening for

liver disease as many serious liver diseases may be associated
with normal levels and abnormal levels might be found in
asymptomatic healthy individuals. The use of battery of liver
function tests however constitutes a highly sensitive procedure.
The number of false negatives must be reduced by this
technique. The use of battery of liver tests is also associated with
high specificity especially when more than one test is abnormal.
The pattern of enzyme abnormality, interpreted in the context of
the patient’s characteristics, can aid in directing the subsequent
diagnostic work-up. Awareness of the prevalence of determined
liver disease in specific populations and of possible hepatic
involvement during systemic illnesses or drug therapies may help
the clinician identify the cause of alterations efficiently.
1.2 PLANT PROFILE
Name- Carissa carandas,
Synonym-Carissa congests
Common Name-Christ’s thorn (Eng.), Karaunda (Hindi)
Family- Apocynacea
1.2.1 Distribution And Cultivation Of The Plant9
Description
 39 
INTRODUCTION
This species is a rank-growing, straggly, woody, climbing

shrub, usually growing to 10 or 15 ft (3-5 m) high, sometimes
ascending to the tops of tall trees; and rich in white, gummy
latex. The branches, numerous and spreading, forming dense
masses, are set with sharp thorns, simple or forked, up to 2 in (5
cm) long, in pairs in the axils of the leaves. The leaves are
evergreen, opposite, oval or elliptic, 1 to 3 in (2.5-7.5 cm) long;
dark-green, leathery, glossy on the upper surface, lighter green
and dull on the underside. The fragrant flowers are tubular with 5
hairy lobes which are twisted to the left in the bud instead of to
the right as in other species. They are white, often tinged with
pink, and borne in terminal clusters of 2 to 12. The fruit, in
clusters of 3 to 10, is oblong, broad-ovoid or round, 1/2 to 1 in
(1.25-2.5 cm) long; has fairly thin but tough, purplish-red skin
turning dark-purple or nearly black when ripe; smooth, glossy;
enclosing very acid to fairly sweet, often bitter, juicy, red or pink,
juicy pulp, exuding flecks of latex. There may be 2 to 8 small, flat,
brown seeds
Varieties
Formerly there were believed to be 2 distinct varieties: C.

carandas var. amara–with oval, dark-purple, red-fleshed fruits, of
acid flavor; and var. dulcis–round, maroon, with pink flesh and
sweet-subacid flavor. However, David Sturrock, a Florida
horticulturist who took a special interest in the karanda, observed
these and other variations throughout seedling populations.
Origin and Distribution
 40 
INTRODUCTION
The karanda is native and common throughout much of

India, Burma and Malacca and dry areas of Ceylon; is rather
commonly cultivated in these areas as a hedge and for its fruit
and the fruit is marketed in villages. It is rare in Malaya except as
a potted plant in the north; often grown in Thailand, Cambodia,
and South Vietnam and in East Africa. It was introduced into Java
long ago as a hedge and has run wild around Djakarta. The
karanda first fruited in the Philippines in 1915 and P.J. Wester
described it in 1918 as "one of the best small fruits introduced
into the Philippines within recent years."
Season
The karanda may bloom and fruit off and on throughout the
year. For use unripe, the fruits are harvested from mid-May to
mid-July. The main ripening season is August and September. The
5-pointed calyx remains attached to the plant when the fruit is
picked, leaving a gummy aperture at the base.
Food Value
Analyses made in India and the Philippines show the

following values for the ripe karanda: calories, 338 to 342/lb
(745-753/kg); moisture, 83.17-83.24%; protein, 0.39-0.66%; fat,
2.57-4.63%; carbohydrate, 0.51-0.94%; sugar, 7.35-11.58%;
fiber, 0.62-1.81%; ash, 0.66-0.78 %. Ascorbic acid content has
been reported as 9 to 11 mg per 100 g.
Other Uses 10
 Fruit: The fruits have been employed as agents in tanning

and dyeing.
 41 
INTRODUCTION
 Leaves: Karanda leaves have furnished fodder for the

tussar silkworm.
 Root: A paste of the pounded roots serves as a fly

repellent.
 Wood: The white or yellow wood is hard, smooth and

useful for fashioning spoons, combs, household utensils and
miscellaneous products of turnery. It is sometimes burned
as fuel.
1.2.2 Medicinal Uses: 11
The unripe fruit is used medicinally as an astringent.

The ripe fruit is taken as an antiscorbutic and remedy for
biliousness. The leaf decoction is valued in cases of
intermittent fever, diarrhea, oral inflammation and earache.
The root is employed as a bitter stomachic and vermifuge
and it is an ingredient in a remedy for itches. The roots
contain salycylic acid and cardiac glycosides causing a
slight decrease in blood pressure. Also reported are
carissone; the D-glycoside of B-sitosterol; glucosides of
odoroside H; carindone, a terpenoid; lupeol; ursolic acid and
its methyl ester; also carinol, a phenolic lignan. Bark, leaves
and fruit contain an unnamed alkaloid.
1.2.3 Taxonomy 14
Domain : Eukaryota Whittaker & Margulis,

1978
Kingdom : Plantae Haeckel, 1866
Subkingdom : Viridaeplantae Cavalier-Smith, 1981
 42 
INTRODUCTION
Phylum : Tracheophyta Sinnott, 1935 ExCavalier-

Smith, 1998
Subphylum : Euphyllophytina
Infraphylum : Radiatopses Kenrick & Crane, 1997
Class : Magnoliopsida Brongniart, 1843 -

Dicotyledons
Subclass : Lamiidae Takhtajan Ex Reveal,

1992
Superorder : Gentiananae Thorne Ex Reveal, 1992
Order : Apocynales Bromhead, 1838
Family : Apocynaceae A.l De Jussieu, 1789-Dogbane

Family
Subfamily : Rauvolfioideae
Tribe : Carisseae
Genus : Carissa Linnaeus, Mant.

Pl.1:52.1767.-Bellflower
1.3 DISEASE PROFILE
Hepatic injury progresses in the following fashion:-
1. Degeneration and intracellular accumulation of toxins and

immunologic insults which leads to ballooning, foamy
degeneration, steatosis (accumulation of fat droplets).
2. Necrosis and apoptosis, which is the mode of damage by

CCl4.
3. Inflammation, caused by scavenger macrophages release

after necrosis and apoptosis.
4. Regeneration
 43 
INTRODUCTION
5. Fibrosis, which is a irreversible process due to deposition of collagen..
1.3.1 Free Radicals and Antioxidants in Health and Diseases25, 27
It is ironic that oxygen, an element indispensable for life

can, under certain situations, have severely deleterious effects
on the human body. Most of the potentially harmful effects of
oxygen are due to the formation and activity of a number of
chemical compounds, known as reactive oxygen species, which
have a tendency to donate oxygen to other substances. Many
such reactive species are free radicals and have a surplus of one
or more free-floating electrons rather than having matched pairs
and are, therefore, unstable and highly reactive. Types of free
radicals include the hydroxyl radical (OH.), the superoxide radical
(O.2), the nitric oxide radical (NO.) and the lipid peroxyl radical
(LOO.).
Production of free radicals in the human body
Free radicals and other reactive oxygen species are derived

either from normal essential metabolic processes in the human
body or from external sources such as exposure to X-rays, ozone,
cigarette smoking, air pollutants and industrial chemicals.
Free radical formation occurs continuously in the cells as a

consequence of both enzymatic and non-enzymatic reactions.
Enzymatic reactions which
serve as sources of free radicals include those involved in the
respiratory chain, in phagocytosis, in prostaglandin synthesis and
in the cytochrome P450 system. Free radicals also arise in non-
enzymatic reactions of oxygen with organic compounds as well
 44 
INTRODUCTION
as those initiated by ionizing radiations. Some internally

generated sources of free radicals are:
• Mitochondria
• Phagocytes
• Xanthine oxidase
• Reactions involving iron and other transition metals
• Arachidonate pathways
• Peroxisomes
• Exercise
• Inflammation
• Ischemia/reperfusion.
Some externally generated sources of free radicals are :
• Cigarette smoke
• Environmental pollutants
• Radiation
• Ultraviolet light
• Certain drugs, pesticides, anesthetics and industrial

solvents
• Ozone.
If free radicals are not inactivated, their chemical reactivity

can damage all cellular macromolecules including proteins,
carbohydrates, lipids and nucleic acids.Their destructive effects
on proteins may play a role in the causation of cataracts. Free
radical damage to DNA is also implicated in the causation of
cancer and its effect on LDL cholesterol is very likely responsible
 45 
INTRODUCTION
for heart disease. In fact, the theory associating free radicals with
the aging process has also gained widespread acceptance.
The free radical diseases
A well accepted fact is the increasing incidence of disease

with advancing age. A plausible explanation for the association of
age and disease is based on the implication of free radical
reactions in the pathogenesis of several disorders.
Free radical reactions are expected to produce progressive

adverse changes that accumulate with age throughout the body.
Such "normal" changes with age are relatively common to all.
However, superimposed on this common pattern are patterns
influenced by genetics and environmental differences that
modulate free radical damage. These are manifested as diseases
at certain ages determined by genetic and environmental factors.
Cancer and atherosclerosis, two major causes of death, are
salient "free radical" diseases. Cancer initiation and promotion is
associated with chromosomal defects and oncogene activation. It
is possible that endogenous free radical reactions, like those
initiated by ionizing radiation, may result in tumor formation. The
highly significant correlation between consumption of fats and
oils and death rates from leukemia and malignant neoplasia of
the breast, ovaries and rectum among persons over 55 years
may be a reflection of greater lipid peroxidation. Studies on
atherosclerosis reveal the probability that the disease may be
due to free radical reactions involving diet-derived lipids in the
arterial wall and serum to yield peroxides and other substances.
 46 
INTRODUCTION
These compounds induce endothelial cell injury and produce

changes in the arterial walls.
Free radicals, however, are not always harmful. They also

serve useful purposes in the human body. Several observations
indicate that the oxygen radicals in living systems are probably
necessary compounds in the maturation processes of cellular
structures. Furthermore, white blood cells release free radicals to
destroy invading pathogenic microbes as part of the body's
defense mechanism against disease. Hence, the complete
elimination of these radicals would not only be impossible, but
also harmful.
Counteracting free radical damage
The human body has several mechanisms to counteract

damage by free radicals and other reactive oxygen species.
These act on different oxidants as well as in different cellular
compartments.
One important line of defense is a system of enzymes,

including glutathione peroxidases, superoxide dismutases and
catalase, which decrease concentrations of the most harmful
oxidants in the tissues. Several essential minerals including
selenium, copper, manganese and zinc are necessary for the
formation or activity of these enzymes. Hence, if the nutritional
supply of these minerals is inadequate, enzymatic defenses
against free radicals may be impaired.
The second line of defense against free radical damage is

the presence of antioxidants. An antioxidant is a molecule stable
 47 
INTRODUCTION
enough to donate an electron to a rampaging free radical and

neutralize it, thus reducing its capacity to damage. Some such
antioxidants, including glutathione, ubiquinol and uric acid, are
produced during normal metabolism in the body. Other lighter
antioxidants are found in the diet. Although about 4000
antioxidants have been identified, the best known are vitamin E,
vitamin C and the carotenoids. Many other non-nutrient food
substances, generally phenolic or polyphenolic compounds,
display antioxidant properties and, thus, may be important for
health.
Although a wide variety of antioxidants in foods contribute

to disease prevention, the bulk of research has focused on three
antioxidants which are essential nutrients or precursors of
nutrients. These are vitamin E, vitamin C and the carotenoids.
Each of these antioxidant nutrients has specific activities and
they often work synergistically to enhance the overall antioxidant
capability of the body.
The balance between the production of free radicals and

the antioxidant defenses in the body has important health
implications. If there are too many free radicals produced and too
few antioxidants, a condition of "oxidative stress" develops which
may cause chronic damage. As mentioned above, free radicals
have been implicated in several health problems. Cancer,
atherosclerosis, cerebrovascular accidents, myocardial infarction,
senile cataracts, acute respiratory distress syndrome and
rheumatoid arthritis are just a few examples. Numerous studies
have shown the protective effects of antioxidant nutrients on
these health problems.
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INTRODUCTION
Vitamin E is the collective name for eight compounds, four

tocopherols and four tocotrienols, found in nature. It is a fat-
soluble substance present in all cellular membranes and is mainly
stored in adipose tissue, the liver and muscle. Vitamin E is a
principal antioxidant in the body and protects polyunsaturated
fatty acids in cell membranes from peroxidation. It is a singlet
oxygen quencher, neutralizing these highly reactive and unstable
singlet oxygen molecules. In fact, singlet oxygen can damage
DNA and be mutagenic. Vitamin E also protects the double bonds
of b-carotene from oxidation and thus exhibits a sparing effect.
Due to the ability of vitamin E to work at higher oxygen
pressures, free radicals are scavenged and tissue injury is
minimized. Besides its anti-aging properties, vitamin E is known
to afford protection against cancer, ischemia and reperfusion
injury, cataract, arthritis and certain neurological disorders.
1.3.2 Mechanism of CCl4 damage 26, 28, 29
The use of many halogenated alkanes such as carbon

tetrachloride (CCl4), chloroform (CHCl3) or iodoform (CHI3), has
been banned or severely restricted because of their distinct
toxicity. Yet CCl4 continues to provide an important service today
as a model substance to elucidate the mechanisms of action of
hepatotoxic effects such as fatty degeneration, fibrosis,
hepatocellular death, and carcinogenicity. In a matter of dose,
exposure time, presence of potentiating agents, or age of the
affected organism, regeneration can take place and lead to full
recovery from liver damage. CCl4 is activated by cytochrome
(CYP) 2E1, CYP2B1 or CYP2B2, and possibly CYP3A, to form the
 49 
INTRODUCTION
trichloromethyl radical, CCl3*. This radical can bind to cellular

molecules (nucleic acid, protein, lipid), impairing crucial cellular
processes such as lipid metabolism, with the potential outcome of
fatty degeneration (steatosis). Adduct formation between CCl3*
and DNA is thought to function as initiator of hepatic cancer. This
radical can also react with oxygen to form the
trichloromethylperoxy radical CCl3OO*, a highly reactive species.
CCl3OO* initiates the chain reaction of lipid peroxidation, which
attacks and destroys polyunsaturated fatty acids, in particular
those associated with phospholipids. This affects the
permeabilities of mitochondrial, endoplasmic reticulum, and
plasma membranes, resulting in the loss of cellular calcium
sequestration and homeostasis, which can contribute heavily to
subsequent cell damage. Among the degradation products of
fatty acids are reactive aldehydes, especially 4-hydroxynonenal,
which bind easily to functional groups of proteins and inhibit
important enzyme activities. CCl4 intoxication also leads to
hypomethylation of cellular components; in the case of RNA the
outcome is thought to be inhibition of protein synthesis, in the
case of phospholipids it plays a role in the inhibition of lipoprotein
secretion. None of these processes per se is considered the
ultimate cause of CCl4-induced cell death; it is by cooperation
that they achieve a fatal outcome, provided the toxicant acts in a
high single dose, or over longer periods of time at low doses. At
the molecular level CCl4 activates tumor necrosis factor
(TNF)alpha, nitric oxide (NO), and transforming growth factors
(TGF)-alpha and -beta in the cell, processes that appear to direct
the cell primarily toward (self-)destruction or fibrosis. TNFalpha
 50 
INTRODUCTION
pushes toward apoptosis, whereas the TGFs appear to direct

toward fibrosis. Interleukin (IL)-6, although induced by TNFalpha,
has a clearly antiapoptotic effect, and IL-10 also counteracts
TNFalpha action. Thus, both interleukins have the potential to
initiate recovery of the CCl4-damaged hepatocyte. Several of the
above-mentioned toxication processes can be specifically
interrupted with the use of antioxidants and mitogens,
respectively, by restoring cellular methylation, or by preserving
calcium sequestration. Chemicals that induce cytochromes that
metabolize CCl4, or delay tissue regeneration when co-
administered with CCl4 will potentiate its toxicity thoroughly,
while appropriate CYP450 inhibitors will alleviate much of the
toxicity. Oxygen partial pressure can also direct the course of
CCl4 hepatotoxicity. Pressures between 5 and 35 mmHg favor
lipid peroxidation, whereas absence of oxygen, as well as a
partial pressure above 100 mmHg, both prevent lipid
peroxidation entirely. Consequently, the location of CCl4-induced
damage mirrors the oxygen gradient across the liver lobule.
Mixed halogenated methanes and ethanes, found as so-called
disinfection byproducts at low concentration in drinking water,
elicit symptoms of toxicity very similar to carbon tetrachloride,
including carcinogenicity.
 51 

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INTRODUCTION

I ndigenous plants have been the traditional source of raw

In order to promote Indian herbal drugs, there is an urgent

1.1 THE LIVER

Location of the Liver2

The liver, the largest internal organ in the body, is situated

The lower esophagus

The stomach, and

Surrounded by a fibrous capsule, the liver is made up of

The average rate of blood flow through the liver is 1400

1. From the hepatic artery

2. From the portal vein

The portal vein carries blood to the sinusoids of the liver

1. Left hepatic v. - drains left lobe

2. Middle hepatic v. - drains central portion and may join the

3. Right hepatic v. - drains most of the right lobe

hexagonal liver lobule is a group of three structures: a branch of

1. Endothelial cells- have large pores, allows H2O and plasma

2. Kupffer cells - reticuloendothelial cells capable of phagocytizing bacteria

A portal triad is an arrangement of three structures within

1. Branch of hepatic portal vein

2. Branch of hepatic artery

1.1.1 Functions of the Liver4

These functions include:

2. Amino acid metabolism

3. Metabolism of steroidal hormones

Bile is a complex solution secreted by the cells of the liver

Bile, containing the substances just mentioned as well as

When food enters the duodenum, cholecystokinin is

The second item to be considered is the production of

1. Metabolic turnover of proteins

3. Glucose (glucogenic amino acids)

Because proteins are stored for only limited periods of time,

Two major categories of proteins produced by the liver are

Furthermore, several proteins concerned with blood

Another function of the liver is detoxification. You will recall

As one can imagine, liver damage must be taken into

is little change in the drug metabolizing capabilities. On the other

1.1.2 Hepatic Responses to Injury 6

The liver is a major target organ of chemically induced toxicity. Serious

The morphologic assessment of the gross and microscopic

establish a "no-observed-effect level" (NOEL), to establish

1.1.3 Diseases of Liver

Liver problems include a wide range of diseases and conditions

Signs and symptoms of liver problems include:

 Discolored skin and eyes that appear yellowish

 Abdominal pain and swelling

 Itchy skin that doesn't seem to go away

 Dark urine color

 Pale stool color

 Bloody or tar-colored stool

Acute liver failure, Alcoholic hepatitis, Alpha-1-antitrypsin

Factors that may increase your risk of liver problems include:

A job that exposes to other people's blood and body fluids,

1.1.4 Tests and procedures used to diagnose liver

 Blood tests. A group of blood tests called liver function

 Imaging tests. Procedures that create pictures of your

 Tests of liver tissue. A procedure to remove tissue from

Liver diseases are a major problem of worldwide

shown interesting antifibrotic properties in animals and humans;

The enhanced effect of drugs in patients with liver disease

For other drugs, such as diazepam, phenylbutazone,

metabolism but not changes in hepatic blood flow will affect