Chapter 1

Liver Function
 Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-
Ethiopia
 Chapter Objectives
Upon completion of this chapter the student will be
able to:
• Describe the anatomy and physiological role of the liver,
including formation of bilirubin.
• Discuss the metabolic processes and organs involved in
the formation & excretion of forms of bilirubin
• Explain the clinical significance of bilirubin
• Describe methods of analysis of serum bilirubin (Direct &
total), sources of errors and interpretation of bilirubin
results
 Learning Objectives
Upon completion of this lecture the student will
be able to:
1. Describe the physiological functions of the
liver including:
a. Carbohydrate, protein and lipid metabolism
b. Conjugation, detoxification, excretion
c. Vitamin storage
d. Formation of bile
2. Describe bilirubin metabolism, including
formation, conjugation and excretion.
 Learning Objectives
3. Define the terms jaundice and icterus, and
how the two are related
4. List 3 types of jaundice
5. List the specimen collection and handling
requirements, and pre-analytical
interferences for the Evelyn-Malloy,
Jendrassik-Grof and Walters-Gerarde
methods
 Learning Objectives
6. Describe the Evelyn-Malloy, Jendrassik-Grof,
Walters-Gerarde and the Icterus Index
methods for measuring bilirubin in serum
7. Correlate increased or normal levels of total
and conjugated bilirubin and urobilinogen
with hepatic disorders
 Outline
• Introduction
– Anatomy of the liver
• Physiological role of the liver
• Tests for liver function
– Bilirubin
• Formation & excretion of bilirubin
• Clinical significance of bilirubin
• Determination of serum Bilirubin (Direct & total)
– Interpretation of bilirubin results
Introduction: Anatomy of the Liver
 Microscopic Cross-Section
of a Hepatic Lobe
K = Kupffer cells K

CV = a central vein CV

B = a bile duct – connected to

the biliary tree
B
V = branch to portal vein
V
A = branch to hepatic artery
A
P = parenchymal cells
P
(hepatocytes)
Physiological Functions of the Liver
Right lobe Left lobe
• Carbohydrate metabolism
• Protein metabolism
• Lipid metabolism
• Conjugation, detoxification
and excretion
• Vitamin storage
• Digestion and formation of
bile
• Enzymes
• Bilirubin metabolism

Gall bladder Bile duct Pancreas

 Excretory Functions

• Bile
• Bilirubin esters
• Bile Acid (salt or conjugates)
• Cholesterol
• Other
• Excreted waste products
• Bile emulsifies ingested fats for digestion
 Tests for Liver Function

• Total and Direct bilirubin *

• Total protein *
• Albumin*
• Cholesterol
• Triglycerides
• Urea
• Ammonia
• Liver Inflammatory Enzymes: AST*, ALT*, ALP*, GGT
* Standard liver Panel
Metabolism of Bile Acid

Cholesterol  Bile Acid

Metabolism of Bilirubin
 Bilirubin Metabolism
1. Hemoglobin released from 5. Conjugated bilirubin
red cells excreted in bile (via the
2. Iron and globin are gallbladder and common
conserved bile duct)
3. Insoluble albumin- bound 6. Enters sm. intestine as
unconjugated bilirubin component of bile
4. Albumin removed and 7. Bacteria convert to
conjugated to glucuronic urobilinogen (UBG)
acid by hepatocytes 8. ~15% UBG is reabsorbed
by intestinal mucosa
 Metabolism of Bilirubin
From circulation & RES –
hemolysis of senescent RBCs Globin to protein reserves &
releases Hgb Iron to Iron stores
Blood
Unconjugated Bilirubin + Albumin

•Albumin removed in hepatic sinusoids Kidney

Liver •Unconj. Bili conjugated to glucoronic acid

by hepatocytes → Conjugated Bilirubin
(soluble) enters biliary tree
Portal Vein

Intestines
Bile contains Conjugated
bilirubin.
 Bilirubin Becomes Urobilinogen in
the Intestines
2%-5% reabsorbed
Blood Urobilinogen
urobilinogen re-enters
circulation and is
excreted in urine.

Urobilinogen Kidney
Liver Most re-
excreted in
bile Portal Vein

Intestines 15% reabsorbed

Bilirubin + bacterial flora + alk. pH =
Urobilinogen 85%
Feces
 Urobilinogen
1. Conjugated bili. (bili- 4. 15% UBG is reabsorbed
rubin gluconoride) and returns to the liver
enters the high pH of 5. All re-excreted into bile
small intestine. except 2-5% via
2. ß-glucoronidase kidneys
converts bilirubin back 6. Most (85%) UBG is
to unconjugated form. oxidized into urobilin,
3. Bacteria reduce biliribin the brown pigment of
to three compounds feces
known as urobilinogen
(UBG) UBG is colorless
 Definitions Related to Bilirubin

1. Jaundice: A condition characterized by a

brownish-yellow pigmentation of skin, sclera
& mucous membranes
2. Icterus: A condition characterized by an
increase of bilirubin in blood
3. Conjugated bilirubin (a.k.a.- direct): Bilirubin
coupled with glucuronic acid to enhance
solubility
 Definitions Related to Bilirubin
• Unconjugated bilirubin (a.k.a.- indirect):
Bilirubin that has not been coupled with
gluconic acid and is not water soluble until
bound to albumin

• Total Bilirubin: Conjugated + unconjugated

bilirubin in the bloodstream

• Delta Bilirubin: Conjugated bilirubin

covalently bound to albumin, usually a very
small fraction of total
 Jaundice
• A symptom, not a disease or
disorder. The yellow staining of
connective tissue from excess
bilirubin.
• Prominently - sclera of the eyes
and skin. Above photo is courtesy of the Centers for
Disease Control and Prevention
• Icterus describes the dark
yellow-brown color of serum
with increased bilirubin.
• Normal serum
• Icteric serum
 Pre-Hepatic Jaundice
• Due to hemolytic anemia - acquired and inherited
• Caused by destruction of RBCs by either:
• Extravascular Hemolysis - RBCs sensitized w/ IgG or
complement, removed by phagocytes in tissues
(spleen and Kupffer cells of the liver).
• Intravascular hemolysis – less freq. than
extravascular.
• Laboratory Results: ↑ serum bilirubin (mostly
unconjugated), ↑ urine urobilinogen, ↓ Hgb ↓ Hct
MCV ↓ MCHC ↑ RDW & ↑ reticulocytes
 Neonatal Jaundice
Other Causes of Prehepatic Jaundice
• Neonatal physiologic jaundice
• Hemolytic disease of newborn

• Phototherapy
 Hepatic Jaundice
• Liver inflammation and/ or cellular damage due to
various causes - viral hepatitis, parasites, malignancy,
drug-induced hepatitis. Clinically HAV & HBV most
common.
• Also, metabolic liver diseases, alcoholic cirrhosis,
autoimmune hepatitis.
• Degree of bilirubin uptake varies (normal to
decreased).
• Laboratory results: ↑ serum bili (unconjugated &
conjugated), positive urine bilirubin, liver enzymes
elevated (esp. ALT and AST)
 Other Causes of Hepatic Jaundice
• Transport Failure
– Dubin-Johnson syndrome
• Conjugation Failure
– Crigler-Najjar syndrome
• Intrahepatic obstruction
– Drug induced [e.g., chlorpromazine]
 Post-Hepatic Jaundice
• An obstruction of the bile ducts, which serve as the
conduit of bile from the liver to the duodenum
• The most common cause is gallstones, but tumors in
or near to the bile ducts can also impede bile flow
into the small intestine
• Since conjugated bilirubin is normally excreted as a
component of bile, bilirubin accumulates in
circulation, leading to jaundice
• Post-hepatic jaundice includes increased conjugated
bilirubin, detected in both serum and urine
Post-Hepatic Jaundice
The absence of bilirubin in bile
negatively effects digestion
1. The brown pigment urobilin is
not produced -feces become
pale and clay-colored
2. Causes poor absorption of
fats and fat-soluble vitamins
(A, D, E, K). Deficiencies of
these nutrients are possible
 Specimens for Bilirubin /
UBG Analysis
• Non-hemolyzed serum or
heparinized plasma
• Fresh urine
• Protect from light (e.g., wrap
collection tube in aluminum
foil)
• Light exposure will reduce
bilirubin and UBG detected
 Semi-quantitative Analytical
methodology
Icterus Index Test
• Measures the degree of icterus in plasma or
serum and correlates with a rough estimation
for bilirubin concentration.
• Take absorbance at 420nm, result is expressed
in icterus index units obtained in comparison
with standard potassium dichromate solution
of assigned icterus index value.
• Low specificity because of interference due to
presence of hemoglobin, carotene, and
different yellow pigments found in sample.
 Measuring Bilirubin in Serum or
Plasma
• Bilirubin in serum or plasma is commonly
measured by photometric methods based upon
the diazo reaction
• Conjugated bilirubin + diazotized sulfanilic acid →
azobilirubin + alkaline tartrate (green to blue-
green color)
• Measured with photometer at 555 - 600 nm
depending on specific reagent used
• Unsoluble uncojugated-bilirubin requires an
accelerating agent to react with the diazo reagent
 Two Classic Diazo Reagent
Methods
• Malloy and Evelyn uses • Jendrassik-Grof uses a
methanol as an accelerator caffeine benzoate
• Limitations: interference accelerator
from hemoglobin and • After 10-min. ascorbic acid,
turbidity due to protein dilute HCl or benzoate, plus
precipitation by methanol alkaline tartrate solutions
are added
• A blue-green azobilirubin
mixture is read at 600 nm
• Caffeine is omitted for
conjugated bilirubin only
 Modern Adaptations of Diazo
Methods
• Some systems use a dimethyl sulfoxide (DMSO)
accelerator, known as the Walters and Gerarade
modification
• Sodium nitrate is used to diazotize sulfanilic acid
• Enzymatic and metal binding reagents are used
in some automated analyzers
• Dry-slide system - a modification of Jendrassik-
Grof
 Quality Control
• A normal & abnormal quality control sample
should be analyzed along with patient samples,
using Westgard or other quality control rules for
acceptance or rejection of the analytical run.
– Assayed known samples
– Commercially manufactured (Humastar)

• Validate patient results

• Detects analytical errors.
Sources of Interference in Bilirubin
Testing
• Samples over-exposed to light
• Hemolysis interferes with diazo reaction
• Manual procedure requires carefully timing and
pipetting to avoid analytic error
• Outdated or poor calibration concentration curve
• Improperly maintained or operated instrument
Reference Ranges for Bilirubin for
Interpretation of Results
Patient conjugated total bilirubin Urine Urine
(direct) unconj. + bilirubin, urobilino-gen
bilirubin conjugated conjugated
Newborn Not < 12.0 mg/dL
(1-2 d old) applicable
Adult 0-0.2 mg/dL 0.3-1.2 negative <0.8 EU*
mg/dL

• Compare patient result with reference range

• * EU = “Ehrlich unit”, is equivalent to 1 mg/dL
• Newborn range for full term,1-2 days old
 Clinical Correlations
**Urobilinogen = UBG; *Total Bili = (conjugated + unconjugated)
Jaundice Clinical Serum Serum Urine Urine
Type Condition Conjugated Total Bilirubin (UBG)**
Bilirubin Bilirubin *
None Normal Normal level Normal Neg Normal
level <1 mg/dL
Pre-hepatic Hemolytic Normal or Increase Neg Increase
anemia Sl Increase
Hepatic Hepatitis Increase Increase Positive Normal or
increase

Post-hepatic Obstruct-ion of Increase Increase Positive Normal or

bile duct none.
Documentation of Bilirubin Results
• Record patient results in result logbook
• Record QC results in QC logbook
• Retain records for recommended time
 Practice Problems
• The tube containing patient’s serum was left
in the test rack for 7 hours on the laboratory
bench before it was analyzed for total and
direct bilirubin. Evaluate this situation for any
possible source of errors.
Answer: This sample was likely exposed to light for 7
hours which would falsely decrease total bilirubin levels. If
serum was not separated from clotted red blood cells,
hemolysis may have occurred over this time which could
interfere with bilirubin results.
 Review Questions
• Differentiate unconjugated bilirubin,
conjugated bilirubin and urobilinogen in terms
of:
– A. where they are produced
– B. general chemical characteristics
– C. how they react in the Jendrassik-Groff method
– D. normal amounts in serum and urine
 Summary
• This chapter reviewed anatomy and
physiology of the liver to relate to liver
function testing, the biochemistry, clinical
significance and methods of bilirubin and
correlation with liver function and liver
inflammatory tests
 References
• Burtis, Carl A., and Ashwood, Edward R. Tietz: Fundamentals
of Clinical Chemistry. WB Saunders, Co., Philadelphia, 2001
• Arneson, W and J Brickell: Clinical Chemistry: A Laboratory
Perspective 1st ed. FA Davis Co., Philadelphia. 2007