2. Chapter objective
At the end of this chapter the learners should
able to :
The main functions of liver
Abnormal or /diseases condition of liver
Laboratory diagnostic method of liver
abnormality
3. Introduction
The liver is the largest internal organ of the human body.
It is functionally complex organ that plays a critical biochemical
role in the metabolism, digestion, detoxification, and elimination
of substances from the body.
The liver is involved in a number of excretory , synthetic, and
metabolic functions, all of which are essential to life.
The liver is unique organ that can regenerate in short term injury
or diseases.
However, if the liver is damaged repeatedly over a long period of
time, it may undergo irreversible changes that permanently
interfere with its essential functions.
5. Gross anatomy of liver
• Weight ≈ 1.2–1.5 kg in the
healthy adult.
• Location= beneath and is
attached to the diaphragm
• Held in place by ligamentous
attachments
• Divided unequally into two
lobes- right & left by the
falciform ligament
6. Hepatic blood supply
• Extremely vascular
organ.
• Receives its blood supply
from two sources unlike
other organs :
1. Hepatic artery
2. Portal vein
7. Hepatic blood…………..
The hepatic artery:
Supplies oxygen-rich
blood from the heart to
the liver.
Responsible for providing
≈ 25% of the total blood
supply to the liver.
The portal vein:
Supplies nutrient-rich
blood (collected as food
is digested) from the
digestive tract.
Responsible for
providing ≈ 75% of the
total blood supply to
the liver.
8. Microscopic anatomy
Lobules are :
The microscopic units of the liver
The functional units of the liver
Responsible for all metabolic and excretory
functions performed by the liver.
9. Major cell types in the liver
The major cell types of liver are:
1. Hepatocytes
2. Kupffer cells
3. Stellate cells or Ito cells
Other type of cells are
including cholangiocytes,
portal fibroblasts, endothelial
cells , macrophages, pit cells
and oval cells.
10. The hepatocytes
make up ≈80% of the volume of the liver .
responsible for:
o most of the metabolic and synthetic functions
o the regenerative properties of the liver
11. Kupffer cells
Derived from monocytes and contain lysosomes that
break down phagocytized bacteria.
Responsible for innate immune response including
phagocytosis, pinocytosis , mediator
formation(cytokines ,interlukin ).
Are the main site for clearance of Ag-Ab complexes
from blood.
capable of engulfing bacteria, debris, toxins, and
other substances flowing through the sinusoids.
12. Stellate cells or Ito cells
Located between the endothelial lining of sinusoids
and the hepatocytes
Store vitamin A, and synthesize nitric oxide(NO).
o NO= main biological effect is relaxation of vascular
smooth muscle cells.
When stimulated, stellate cells are transformed to
collagen producing cells, and are responsible for
fibrosis and eventually, cirrhosis.
o Fibrosis is the thickening and scarring of
connective tissue.
13. Hepatic function
Synthetic function
E.g. protein synthesis= albumin , globulin , acute phase reactant
protein(Haptoglobin, C-reactive protein , Ceruplasmin , α-Fetoprotein,etc
),coagulation proteins.
Metabolic function :
• lipid metabolism = lipogenesis , β-oxidation,
ketogenesis, cholesterol biosynthesis and lipoprotein
metabolism.
• Carbohydrate metabolism =gluconeogenesis ,
glycolysis
14. Function……..
• Amino acid and nitrogen metabolism
o In nitrogen metabolism , carbamoylphosphate synthetase
=synthesis of urea, and glutamine synthetase=synthesis of
glutamine
• Bile formation and secretion
o Metabolism of key components of bile, including bile acids,
bilirubin and glutathione
• Metabolism of xenobiotics or detoxification
• Excretory function = bilirubin
15. Function……..
The liver is composed of three systems:
1. The biochemical hepatocytic system = responsible
for vast majority of metabolic activities
2. The hepato-biliary system= concerned with
metabolism of bilirubin .
3. The reticulo-endothelial system = concerned with :
a. The immune system= a major site of defense against intestinal
bacteria and removal of Ag–Ab complexes from the circulation
b. The breakdown of hemoglobin from dead erythrocytes, giving rise
to bilirubin, which, together with bilirubin from the spleen, enters
the hepatocyte
16. Hepatic protein synthesis
• Most plasma proteins are synthesized in the liver
,except immunoglobulins (Igs) and von willebrand
factor.
• Synthesis of >90% of all protein and 100% of albumin
occurs in the liver.
• Thus extensive destruction of liver tissue will result in
low serum levels of total protein and albumin.
• In cirrhosis, besides hepatocyte destruction ,another
cause of diminished protein production is portal
hypertension , which decreases delivery of amino acids
to the liver.
17. Ammonia metabolism
• Most ammonia is metabolized to urea in hepatocytes in
the krebs-Henseleit urea cycle.
• The major source of circulating ammonia is the action
of bacterial proteases, ureases, and amine oxidases
acting on GI tract contents.
• An elevated concentration of ammonia
(hyperammonemia) exerts toxic effects on the CNS.
Causes of hyperammonia :
1. Inherited : inherited deficiencies of urea cycle
enzymes . E.g., Ornithine carbamoyl transferase
(OCT), an enzyme that is unique to the liver.
2. Acquired: advanced liver disease and renal failure
18. Metabolism of xenobiotics
Xenobiotics:
• Foreign substances that are cleared and metabolized by
the liver. e.g., bromsulfophthalein (BSP), indocyanine
green(ICG), aminopyrine, caffeine, lidocaine.
• Some have been used as tests of liver function.
• The liver is a major site for xenobiotic metabolism
• Are lipophilic and their elimination from the body is
facilitated by enzymatic modifications to render them
more hydrophilic.
• Besides foreign substances , a number of endogenous
substances, are also modified to facilitate their
excretion, e.g.,
• The presence of xenobiotics can cause oxidative stress
19. Metabolism….
• While molecular oxygen is essential for a wide array of
organisms, O2 and reactive oxygen species (ROS) can
be toxic.
• Oxidative stress is an imbalance b/w antioxidant defense
mechanisms and ROS , which can irreversibly modify
cellular components.
• Therefore, very effective antioxidant systems are
essential for humans and other organisms using oxygen
to control ROS.
• The antioxidant capabilities of higher organisms are due
to the sum of enzymatic and non-enzymatic
mechanisms.
20. Metabolism….
The concentration of ROS is controlled by:
Internal defense mechanism of antioxidants including
hydrophilic (uric acid, vitamin C, bilirubin, glutathione)
and hydrophobic (vitamin E).
Enzymes directly metabolize ROS (Superoxide
dismutase and catalase), or replenish the supply of
reduced ROS scavengers (Glutathione reductase).
• If not controlled ,oxidative damage occurs to proteins,
lipids, and DNA, leading to cytotoxicity, genotoxicity,
and carcinogenesis.
21. Metabolic function: Bilirubin Metabolism
• Bilirubin is produced from
metabolism of heme,
primarily in the spleen,
• It is transported to the liver
bound to albumin.
• It enters the hepatocyte by
binding to a transporter
protein and crosses the cell
membrane ,thus entering the
cell.
• It binds to Y and Z proteins
and then to ligandin for
transport to the smooth
endoplasmic reticulum (SER).
Fig. Schematic summary of the pathway
of bilirubin transport and metabolism
22. Bilirubin ……..
In the SER, bilirubin is
conjugated to glucuronic
acid by UDP-glucuronyl
transferase , producing
monoglucuronides and
diglucuronides of bilirubin .
Conjugated bilirubin is then
secreted into the canaliculi
by the adenosine
triphosphate(ATP)-binding
cassette transporter protein
Ultimately, intestinal urobilinogen is converted to stool
pigments (stercobilin); their absence leads to clay-
colored stools, often an early sign of impaired bilirubin
metabolism
23. Derangements of Bilirubin Metabolism
• Elevated Serum Levels of Unconjugated Bilirubin
• Elevated Serum Levels of conjugated Bilirubin
24. Unconjugated hyperbilirubinemia : causes
Hemolysis, e.g. hemolytic anemia
o unconjugated bilirubin is produced at rates that exceed the
ability of the liver to clear it.
o ≈ 250–350 mg of bilirubin is produced daily in healthy
adults.
Gilbert’s Syndrome and the Crigler-Najjar Syndrome
o Are Caused by Gene Mutations and Deletions.
o mutations in the gene 1 encodingUDG-
transferase1(UDPGT1A1)
In Gilbert’s syndrome, there may also be a defect in the
bilirubin transporter protein
25. Conjugated hyperbilirubinemia : causes
Excretion Deficits:
Dubin-Johnson syndrome: inborn error of metabolism
there is a blockage of the excretion of bilirubin into the
canaliculi, caused by :
o defects in the ATP-binding cassette (ABC) canalicular
multispecific organic anion transporter.
Biliary Obstruction:
Cholelithiasis is the most common cause of
hyperbilirubinemia
o results from bile stones most commonly in the common
bile duct (choledocholithiasis).
26. Conjugated…….
• Inflammatory conditions of the biliary tract (ascending
cholangitis), also give rise to direct bilirubin and ALP.
• The most common cause of conjugated
hyperbilirubinemia in adults :
o blockage of any of the major bile ducts, especially the
common bile duct, by stones or space-occupying
lesions such as tumors
• Viral induced Rotor syndrome can cause conjugated
hyperbilirubinemia
27. Conjugated…….
Aside from liver disease, elevations of conjugated
bilirubin may occur with a few other disorders.
o Septicemia ,
o Total parenteral nutrition
o certain drugs (androgens), but the mechanism is not
understood
• Fasting causes increases in unconjugated bilirubin in
normal individuals.
28. Physiologic jaundice of neonate
Is a result of glucuronyl transferase deficiency :
w/c is one of the last liver functions to be activated in
prenatal life since bilirubin processing is handled by the
mother of the fetus.
In premature births, infants may be born without
glucuronyl transferase.
This deficiency results in the rapid buildup of
unconjugated bilirubin, which can be life threatening.
29. Physiologic……..
• When this type of bilirubin builds up in the neonate, it
cannot be processed and it is deposited in the nuclei
of brain and nerve cells, causing kernicterus.
o Results In cell damage and death in the newborn
o typically occurs at bilirubin levels >20 mg/dL in infants
due to their immature blood-brain barrier.
o less likely to cause brain damage in adults due to the
natural barrier in the brain, called the blood-brain
barrier.
30. Physiologic…………
Phototherapy is a method of treating
neonatal hyperbilirubinemia in which the
baby is placed periodically under a light
source emitting 450-nm wavelength light.
Light diffuses through layers of skin and
converts unconjugated bilirubin to stable
water soluble forms that can be excreted.
The baby’s eyes are protected during this
process from harmful ultraviolet (UV) and
near-UV rays.
32. Cholestasis
o mechanical or functional stoppage of the bile flow in
intrahepatic or extrahepatic bile ducts — with bile
components passing into the blood.
o Cholestasis can occur both with and without jaundice
Cerulo-plasmin=
o A copper-binding protein which contains most of the
copper in plasma
Wilson’s disease
o Disorder of copper metabolism.
o Hepatic excretion of copper into the bile is impaired,
leading to toxic deposition of copper in tissues.
33. Hepatic injury
The liver has a limited number of ways of responding to
injury.
Acute injury to the liver may be asymptomatic, but
often presents as jaundice.
The two major acute liver diseases are :
1. Acute hepatitis
2. Cholestasis
Chronic liver injury generally takes the clinical form
of chronic hepatitis; its long-term complications include
cirrhosis and HCC.
34. Hepatic ……..
Hepatitis = inflammation in the liver tissue
Cirrhosis = scar tissue replaces normal, healthy liver
tissue.
It blocks the flow of blood through the organ and
prevents the liver from functioning properly.
The most common cause of cirrhosis is:
Chronic alcoholism and
Chronic HCV infection
Liver damage from cirrhosis cannot easily be reversed,
but treatment can stop or delay further progression of
the disorder
35. Hepatic viral infection
Five viruses are identified as causes of infection that
primarily targets the liver.
HAV,HBV,HCV,HDV,HEV
Megalo-virus (CMV), epstein-barr virus (EBV), and
herpes simplex virus (HSV) may also infect the liver
36. HAV
Most common cause of acute
viral hepatitis
Its epidemic is associated
with water borne and food-
borne contamination.
Most adults with acute HAV
infection become jaundiced,
Most children remain
asymptomatic.
No chronic form of hepatitis
A, but cholestasis may occur
in some adults.
Test methods for HAV
Two tests are commonly
used
Total antibody to HAV
Develops after natural
exposure or
Following immunization
IgM anti-HAV:
Develops rapidly with
acute exposure
37. HBV
The most common chronic
viral infection.
Transmitted from mother to
child, usually at or after
delivery (termed vertical
transmission).
Much of the transmission is
vertical
Diagnostic test for
HBV
HBs Ag :
Produced in excess by the virus
Is used to detect current HBV
infection.
typically present with both acute
and chronic infection.
Antibody to the hepatitis B core
antigen (anti-HBc):
The most commonly detected
antibody against HBV.
Hepatitis B viral DNA
A direct measure of circulating
virus.
38. HCV infection
Most common cause of chronic hepatitis
Its infection is primarily occurs through plasma
Injection drug use and transfusion before testing the
blood supply are the major risk factors.
Has a high rate of spontaneous mutation.
Six major genotypes (<70% nucleotide
homology),along with 67 subtypes (77% to 80%
homology).
39. Diagnostic test for HCV
Antibody to HCV (anti-HCV)
The principal screening test for HCV exposure
HCV RNA:
Is used to detect active infection.
Rapid separation of serum from clot is critical for
accurate measurement of HCV RNA.
HCV genotype
Genotype is determined by direct sequencing or line
probe assay.
40. Toxic hepatitis
Refers to direct damage of hepatocytes by a
toxin or toxic metabolite .
The most common cause is acetaminophen, a
widely used non-prescription pain reliever.
The first laboratory abnormality to appear is
an increase in PT.
41. Tumors
Cancers of the liver are classified as :
Primary liver cancer =begins in the liver cells.
Metastatic cancer occurs when tumors from other parts
of the body spread (metastasize) to the liver.
Metastatic liver cancer is much more common than
primary liver cancer;90%–95% of all hepatic
malignancies are classified as metastatic.
Cancers that commonly spread to the liver include colon,
lung, and breast cancer.
42. Cancers of the liver may also be classified as benign or
malignant.
Malignant tumors of the liver include:
Hepatocellular carcinoma (HCC),
Hepatocarcinoma
Hepatoma.
HCC is the most common malignant tumor of the liver
The most common benign cancers of the liver include:
hepatocellular adenoma and
hemangiomas
43. Malignant tumors are :
Cancerous
Are made up of cells that grow out of control.
Cells in these tumors can invade nearby
tissues and spread to other parts of the body
Benign tumors are:
not malignant,
one that does not invade surrounding tissue
or spread to other parts of the body; it is not
a cancer
44.
45. Jaundice
• It is a disorder in the metabolism of bilirubin
• it is thus neither directly related to the bile acid
metabolism nor to cholestasis.
• Jaundice is a symptom and not a disease.
• Observed by naked eye (overt jaundice) when
bilirubin concentration is reaching 3.0 mg/dl.
• It can occur with and without cholestasis.
46. Jaundice is most commonly classified based
on the site of the disorder as :
Pre-hepatic
hepatic, and
Post hepatic jaundices
47. Pre-hepatic jaundice
• Increased bilirubin production
• Decreased liver uptake
• Decreased conjugation
• Some causes are
o Physiological neonatal jaundice
o Haemolysis
o Glucuronyl transferase deficiency
• Its typical serum bilirubin pattern is increased
unconjugated bilirubin(usually 1.5-3.0 mg/dl) and
normal conjugated bilirubin
48. Hepatic jaundice
• Result of hepatocyte damage
• Usually some cholestasis (therefore mixed
picture of unconjugated and conjugated
bilirubin but as disease progresses more
cholestatic – obstructive jaundice occurs)
• Two main causes are viral hepatitis and alcohol
• Other causes = drugs, cirrhosis, autoimmune
hepatitis
49. Obstructive jaundice
• Obstruction (e.g common bile duct) causes increased
conjugated bilirubin in blood
• Is water-soluble so urine = dark
• Less conjugated bilirubin enters the bowel – faeces =
pale
• Causes =
o Gallstones
o Pancreatic cancer
o PSC (primary sclerosing choleangitis)
o PBC (primary biliary cirrhosis
50. Biliary obstruction
Extrahepatic obstruction
Intrahepatic obstruction
o mechanical
o toxic
Special forms
Recurrent intrahepatic cholestasis
Recurrent cholestasis in pregnancy
Postoperative jaundice (on occasions
52. Skin stigmata in liver diseases
Palmar erythema
in liver cirrhosis
White nails and paper
money skin in liver
cirrhosis
53. Smooth red tongue with
angular cheilosis in liver
cirrhosis
Gynaecomastia in
liver cirrhosis
54. Xanthoma on the elbow in
biliary cirrhosis as a result of
primary biliary cholangitis
Multiple xanthomas in
chronic cholestasis due to
congenital biliary tract
atresia
55. Scleral icterus in acute
viral hepatitis A
Kayser-Fleischer corneal ring occurs
in Wilson’s disease. It has also been
observed in alcoholic liver disease.
(47) Deposits of copper compounds
form a brownish green corneal ring
56. Drumstick fingers and hour-glass nails
in liver cirrhosis (and moderate paper
money skin:
Cutaneous haemorrhages
(confluent and streak-like)
57. • Vein dilatation and
tortuosity in the
abdominal wall of a
cirrhotic patient
suffering from ascites
and jaundice
58. Liver function tests (LFT)
• LFT profile is used to evaluate liver function by
determining serum analytes , many of whose
components are not unique to liver ; however,
when evaluated together ,allow for accurate
diagnosis of abnormality of liver function.
May include :
• Enzymatic assay =hepatocellular(AST,ALT,LDH) +
canalicular membrane enzymes(ALP,GGT,5`-N)
• Total bilirubin measurement :-direct + indirect
• Total Protein (Biuret) and albumin(Dye binding)
measurement
59. Bilirubin diagnostic method
Jendrassik-Grof method
Azo-bilirubin produced
is purple
Performed at a PH of 13
Azo-bilirubin is
measured at 600nm.
Accelerator used is
caffeine-benzoate.
Malloy and Evelyn method
Azo-bilirubin produced
is red-purple
Performed at PH of 1.3
Azo-bilirubin is
measured at 560 nm
Accelerator used is 50%
methanol solution
60. Bilirubin
o Direct bilirubin + Indirect bilirubin= Total
bilirubin
o HCL + sodium nitrite = diazotized sulfanilic
acid(DSA)= (Diazo reagent)
o Bilirubin + DSA= azobilrubin direct
o Bilirubin + DSA + accelerator= azobilrubin total
o Total bilirubin – direct bilirubin=indirect
bilirubin
61. Aminotransferases (Transaminases)
• Two diagnostically very useful enzymes are:
o AST (serum glutamate oxaloacetate transaminase)
o ALT( formerly called serum glutamate pyruvate
transaminase).
They catalyze reversibly the transfer of an amino
group of AST or ALT to α-ketoglutarate to yield:
o glutamate plus the corresponding ketoacid of the
starting amino acid (i.e., oxaloacetate or pyruvate,
respectively).
Both enzymes require pyridoxal phosphate
(vitamin B6) as a cofactor.
63. Alkaline Phosphatase(ALP)
It catalyzes the hydrolysis
of various
phosphomonoesters at
an alkaline PH.
It is a non-specific
enzyme capable of
reacting with many
different substrates.
The optimal pH for the
reaction is 9.0 to 10.0
Requires Mg2+ as an
activator.
64. Bowers –McComb method
The IFCC recommended
method for ALP.
P-nitro-
phenylphosphate
(colorless) is hydrolyzed
to p-nitro-phenol
(yellow).
The increase in
absorbance at 405 nm
is directly proportional
to ALP activity.
65. Electrophoresis
Is considered the most useful
single technique for ALP iso-
enzyme analysis.
However , some degree of
overlap between the
fractions occurs in this
technique
The liver fraction migrates
the fastest, followed by
bone, placental, and
intestinal fractions
66. Gamma glutamyl transferase(GGT)
It transfers γ-glutamyl residue
from γ-glutamyl peptides to
amino, H2O, and other small
peptides acids.
Its specific physiologic
function is not clearly
established.
However, it is suggested that
GGT is involved in:
o peptide and protein synthesis
o regulation of tissue
glutathione levels
o the transport of amino acids
across cell membranes
67. Assay for Enzyme Activity
Most widely accepted
substrate for use in GGT
analysis is :
γ -glutamyl-p-
nitroanilide.
The γ–glutamyl residue is
transferred to
glycylglycine, releasing p-
nitroaniline
p-nitroaniline is a
chromogenic product with
a strong absorbance at 405
to 420 nm.
68. 5′-nucleotidase(5′-NT)
It is a phosphoric monoester hydrolase.
It is also known as 5′-ribonucleotide
phosphohydrolase
It is a cytoplasmic membrane–bound phosphatase
with a wide specificity for 5′-ribonucleotides .
It acts only on nucleotides.
It is a metalloprotein, and zinc is believed to be an
integral part of the enzyme.
It is widely distributed in the body,
predominantly attached to cell membranes
(similar to ALP and GGT).
69. 5′-nucle………….
Plasma 5′-NT is derived predominantly from the
liver.
Its activity is increased in cholestatic disorders
with virtually no increase in activity in patients
with bone disease
It catalyses :
o 5-Ribonucleotide + H2O Ribonucleoside +
Phosphate
Similar to ALP and GGT, antiepileptic drugs can
increase 5′-NT activity.
70. Lactate dehydrogenas (LDH)
Has very wide distribution throughout the body.
Released into circulation when cells of the body
are damaged or destroyed, serving as a general
Non-specific marker of cellular injury.
It is a zinc-containing enzyme that is part of the
glycolytic pathway
Found in the cytoplasm of all cells and tissues in
the body
71. LD is a tetramer of two different polypeptide
chains
H( heart)
M(muscle )
Combinations of the subunits produce five iso-
enzymes:
LD1 =H4
LD2=HHHM(H3M)
LD3= HHMM(H2M2)
LD4=HMMM(HM4)
LD5=MMMM(M4)
72. Assay for Enzyme Activity
• LD catalyzes the inter-
conversion of lactic and
pyruvic acids using the
coenzyme NAD+.
73. LD iso-enzyme analysis
Can be accomplished by:
Electrophoresis
Immuno-inhibition or
Chemical inhibition
methods
The electrophoretic
procedure has been
widely used historically.
74. Assignment-3
1. Discuss the difference between hepatocelular
biomarkers and cholestatic biomarkers ; their
clinical significance ; laboratory diagnosis .
Submission date : the same with assignment-1
and 2.
75. References
Michael L. Bishop; Clinical Chemistry
techniques, principles, correlations ……6th
edition
Wendy Arson Clinical Chemistry A Laboratory
Perspective……2007
Henary`s clinical diagnosis and management
by laboratory methods…..22nd edition
Tietz fundamentals of clinical chemistry
……..6th editio
