1ST SEMESTER

A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)

○ Portal Triads
■ Composed of the hepatic artery,
LIVER FUNCTION
portal vein, and bile duct.
○ Central Vein
GROSS ANATOMY
● 2 Major Cell Types
1. Hepatocytes
● Liver • Make up approx, 80% of the organ.
○ Location: RUQ • Perform the major functions of the
○ Number of Lobes: 2 liver
○ Falciform Ligament • Responsible for the liver
■ Divides the right, and left lobe of regenerative prooperties
the liver 2. Kupffer cells
○ Right lobe is 6x larger than the left lobe; • Macrophages that line the sinusoids
of the liver
● Liver: Blood Supply • Acts as phagocytes that’s capable
○ Hepatic Artery of engulfing bacteria, debris, toxin
■ Is a branch of the abdominal and other substances flowing from
aorta the sinusoids.
■ Supply oxygen-rich blood from
the heart
■ Provides approx. 25% of the total
blood supply to the liver
○ Portal Vein
■ Supplies nutrients from the
digestive tract as the food is
digested
■ Provides 75% of the total blood
supply

● Liver Excretory System

○ Bile Canaliculi
■ Excretory system starts at the bile
canaliculi
■ Small spaces between the
hepatocytes that form
intrahepatic ducts
○ Intrahepatic ducts
■ Where the excretory products of
the cells can drain
BIOCHEMICAL FUNCTIONS
■ Right and left ducts can join to
form;
○ Common Hepatic Duct 4 MAJOR FUNCTIONS
■ Eventaully joined by the cystic • Excretion/Secretion
duct of the bladder, to form; • Metabolism
○ Common Bile Duct • Detoxification
■ Combined digestive sequations • Storage
are then expelled into the
duodenum
EXCRETION & SECRETION

• Procession and excretion of endogenous and

exogenous substances into bile/urine.
• Bile Composition
o Composed of bile acids or salts, bile
pigments, cholesterol and other
substances extracted from the blood
o Body produces 3 liters of bile every
day, and 1 liter of produced is excreted.
• Bilirubin: Unconjugated and Unconjugated
o Principal pigment of bile
o Derived from the breakdown of RBC’s

● Microscopic Anatomy
○ Lobules
■ Functional units, and is
responsible for the metabolic and
the excretory functions performed
by the liver
■ Each lobule is a six sided
structure, with a centrally located
vein with portal triads

1
 1ST SEMESTER
A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)
METABOLISM LIVER FUNCTION ALTERATION DUTING
DISEASE

JAUNDICE

● Yellow discoloration of the skin, eyes, and

mucous membranes
● Bilirubin levels: reaches 3.0 to 5.0 mg/dL

PRE-HEPATIC HEPATIC POST-

HEPATIC
• Caused by • Caused • Caused
abnormaliti by the by
es problem abnorm
BEFORE s with alites
the liver the liver AFTER
• Acute and itself the liver
Chronic • Intrinsic • Biliary
Hemolytic liver Obstruct
Anemia defect ive
• Unconjugat or Disease
ed bilirubin disease • Can be
is increased caused
• Rarely have by
bilirubin gallston
levels es and
CARBOHYDRATE METABOLISM exceeding tumors
● Glycolysis 5.0 mg/dL
● Glycogenesis • Aka.
● Glycogenolysis Unconjugat
● Gluconeogenesis ed
Hyperbilirub
inemia
LIPID METABOLISM ● Icterus
○ Serum or plasma sample with a yellow
● Fatty Acid Oxidation dicsoloration
● Lipogenesis ● Classified based on the site of the disorder
● Cholesterol Synthesis ● In pre, and post-hepatic jaundice, liver function
is normal
● Unconjugated Bilirubing binded with albumin, it
PROTEIN METABOLISM
does not pass through the Glomerular Filtration
Membrane, therefore cannot be detected in
● Protein Synthesis urine
● Transamination of Amino Acids
● Deamination of Amino Acids
HEPATIC JAUNDICE

DETOXIFICATION OF WASTE PRODUCTS

● Disorder of Bilirubin Metabolism and
Transport Defects
● “First Pass” ○ Crigler-Najjar syndrome
● 2 Mechanisms for detoxification of foreign ○ Dublin-Johnson syndrome
materials and metabolic products ○ Gilbert’s Disease
1. Binds the material reversibly to inactivate the ○ Neonatal Physiologic Jaundice
compound ● Disorders Resulting in Hepatocellular
2. Chemically modifies the compound Injury/Destruction
○ Cirrhosis
DRUG DETOXIFICATION ○ Hepatitis
● Unconjugated Hyperbilirubinemia
○ Crigler-Najjar syndrome
● Oxidation ○ Gilberts disease
● Reduction ○ Neonatla physiologic jaundice
● Hydrolysis ● Conjugated Hyperbilirubinemia
● Hydroxylation ○ Dubin-Johnson
● Carboxylation ○ Rotor Syndrome
● Demethylation
UNCONJUGATED HYPERBILIRUBINEMIA
DISEAS ETIOLOFY OTHER INFO
E
Gilbert - Genetic NO
Syndro mutation in the morbidity/mortal
me UGT1A1 gene ity, no clinical
- Characterized consequences
by intermittent
2
 1ST SEMESTER
A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)
unconjugated found in liver
hyperbilirubine cells that
mia mediate the
- Manifest in cellular uptake
adolescence of compound
/early such as
adulthood bilirubin, bile
- TSB fluctuates acids, and
between 1.5- steroids.
3.0, rarely - The proteins
exceeds 3.5 are abnormally
short therefore
bilirubin is less
Crigler- Genetic mutation in Rare, more efficiently taken
Najjar the UGT1A1 gene severe and up by the liver,
Syndro dangerous form and removed.
me (+) 2 types
- Type 1 POST- HEPATIC JAUNDICE
Complete
absence of
enzymatic ● Problems are in the biliary tract due to
bilirubin obstructions
conjugation ● Biliary Obstructive Disease
- Type 2 ○ Gallstones
Severe ○ Tumors
deficiency of the
enzyme MECHANISMS OF HYPERBILIRUBINEMIA
Neonata Deficiency in UDPGT Possible
l complication:
hysiologi Kernicterus
c - high
Jaundic levels of
e bilirubin
deposit
ed in
the
brain

CONJUGATE HYPERBILIRUBINEMIA
Dubin-Johnson - Deficiency of (+) Delta
Syndrom the canalicular Bilirubin
multi drug - can be counted
resistance/multi as conjugated
specific organic bilirubin CHANGES IN CONCENTRATION OF BILIRUBIN IN
anionic (+) Dark Stained THOSE WITH JAUNDICE
transporter granules on a
protein live biopsy
(MDR2/cMOAT)
- Mild in nature
- The liver’s
ability to uptake
and conjugate
bilirubin is
functional,
however
removal of
conjugated
from the liver,
and excretion
into the bile
ducts is CIRRHOSIS
defective.
Rotor - Mutations in (+) abnormally
Syndrome both the short proteins ● Scar tissue replaces normal, healthy liver tissue
SLCO1B1 and Rare, benign ● S/sx: fatigue, nasue, unintended weight loss,
SLCO1B3 (+) chronic jaundice, bleeding from the gastrointestinal tract,
genes on ch.12 jaundice; not intense itching, and swelling in the legs and
required abdomen
SLCO1B1 and treatment ● Chronic Alcoholism – most common cause
SLCO1B3 ● Other Infections
genes ○ Chronic infections with Hepatitis B, C,
- These genes and D
provide ○ Autoimmune Hepatits
instructions in ○ Inherited disorders of Alpha 1
making proteins Antitrypsin deiciencies

3
 1ST SEMESTER
A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)
■ Wilson’s Disease
■ Hemochromatosis
■ Galactosemia
● Liver damage CANNOT be easily reversed

TUMORS
TYPES OF BILIRUBIN
● Primary Liver Cancer – hepatocellular carinoma
○ The cancer begins in the liver cells ○ Disadvantage: Flushing or even
● Metastatic Liver Cancer: colon, lung, and breast hepatotoxicity but newer, timed-
● Benign: Hepatocellular adenoma and release preparations may ameliorate
hemangioma these effects.
● Malignant: Hepatocellular carcinoma and bile ○ Lifestyle modifications and treatment of
duct carcinoma any coexisting disorders that increase
CHD risk are also important in these
patients.

REYE’S SYNDROME
LIPID AND LIPOPROTEIN ANALYSIS

● Cause: infectious, metabolic or drug induced

disease ● B1
● Almost exclusive in children o Unconjugated Bilirubin
● Proceeded by a viral syndrome (varicella) or o Water Insoluble
UPPER respiratory tract infection (influenza) o Non-polar Bilirubin
● (+) acute illness: noninflammatory o Indirect Reacting
encephalopathy and fatty degeneration of the liver o Hemobilirubin
○ Profuse vomiting accompanied with o Slow Reacting
varying degrees of neurologic impairment o Pre-Hepatic Bilirubin
such as fluctuating personality changes, ● B2
and deterioration of consciousness o Conjugated Bilirubin
○ Encelopathy: Progression from mild o Water soluble
(confusion) to progressive loss of o Polar Bilirubin
neurological function to the loss of brain o Direct Reacting
stem reflexes o Cholebilirubin
o Fast Reacting
o Post-hepatic Bilirubin/Hepatic
Bilirubin/Obstructive Bilirubin and
DRUG AND ALCOHOL RELATED DISORDERS Regurgitative Bilirubin

● Immune-mediated injury to hepatocytes DELTA BILIRUBIN

● Alcoholic fatty liver -> Alcoholic Hepatitis ->
hepatic cirrhosis
● Conjugated Bilirubin that is covalently bound
● Fatty Liver Disease: (+): sl elevated AST, ALT to albumin
and GGT and (+) fatty infiltrates in the vacuoles of ● Seen only when there is significant hepatic
the liver obstruction
● React in most laboratory methods as conjugated
● Alcoholic Hepatitis (+): mod. Elevated AST, bilirubin
ALT, GGT, ALP and increased TB >5 mg/dL
○ Elavations of AST: more than twice the
upper reference normal but rarely exceed
300 international units per ml
○ Elavations in ALT: comparatively lower
compared to AST resulting in a de ritis
ratio of >2 SPECIMEN COLLECTION AND HANDLING
● Alcoholic Cirrhosis: increased AST, ALT, GGT,
ALP and TB, decreased albumin and prolonged
prothrombin time ● Serum or Plasma
○ Risk of development of cirrhosis ● Fasting sample: preferred
increases proportionally with ○ Presence of lipemia will increase
consumption of more than 30 grams or 3- measured bilirubin conc.
4 drinks ● Avoid HEMOLYZED samples
○ With the highest degree of consumption of ○ May decrease the reaction of bilirubin
> 120 grams = 12-16 drinks per day from diazo reaction
● ● Protect specimen from light
○ May convert Bilirubin -> Biliverdin
ASSESSMENT OF LIVER/ LFT ● Stability of serum/plasma separated from the
cells and stored in the dark

4
 1ST SEMESTER
A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)
METHODS
● NO preferred methodtandardization of bilirubin
analysis
● Modified Jendrassik-Grof procedure: candidate
reference method for total bilirubin
o `Uses caffeine benzoate as a solubilizer.
● Most widely used method
o Malloy Evelyn Procedure
o Jendrassik-Grof Method

MALLOY-EVELYN PROCEDURE

● Principle: Bilirubin pigments in serum or plasma

are reacted with a diazo reagent. The diazotized
sulfanilic acid reacts at the central methylene
carbon of bilirubin to split the molecule, forming
two molecules of azobilirubin
o pH: 1.2
o Color reaction: Red-purple (azobilirubin)
o Maximal absorption: 560 nm
o Accelerator: Methanol
- to solubilize unconjugated bilirubin
UROBILINOGEN IN URINE AND FECES
JENDRASSIK-GROF METHOD

• Urobilinogen: colorless end product of bilirubin

• Principle: Bilirubin pigments in serum or plasma metabolism oxidized by intestinal bacteria to the
are reacted with a diazo reagent (sulfanilic acid in brown pigment urobilin
hydrochloric acid and sodium nitrite), resulting in • MOST QUANTITATIVE METHODS: based on
the production of the purple product, azobilirubin. Ehrlich's reaction - the reaction of urobilinogen
with p-dimethylaminobenzaldehyde (Ehrlich's
• The individual fractions of bilirubin are determined reagent) to form a red
by taking two aliquots of sample and reacting one
aliquot with the diazo reagent only and the other
aliquot with the diazo reagent and an accelerator DETERMINATION OF URINE UROBILINOGEN
(caffeine benzoate) (SEMIQUATITATIVE)

• \After a short period of time, the reaction of the • PRINCIPLE: Urobilinogen reacts with p-
aliquots with the diazo reagent is terminated by dimethylamino-benzaldehyde (Ehrlich's reagent)
the addition of ascorbic acid. to form a red color, which is then measured
o Ascorbic acid – destroys the excess spectrophotometrically.
diazo reagent • Ascorbic acid is added as a reducing agent to
maintain urobilinogen in the reduced state.
• The solution is then alkalinized using an alkaline • Specimen: fresh 2-hour specimen (keep cool
tartrate solution, which shifts the absorbance and protected from light)
spectrum of the azobilirubin to a more intense blue
color
SOURCES OF ERROR
• The final blue product is measured at 600 nm, with
the intensity of color produced directly proportional • Comments & Sources of Error
to bilirubin concentration. o Results: reported in Ehrlich's units
o Compounds other than urobilinogen
• Indirect (unconjugated) bilirubin may be calculated may be present in the urine and react
by subtracting the conjugated bilirubin with Ehrlich's reagent
concentration from the total bilirubin o Fresh urine: important; test must be
concentration. performed WITHOUT delay to prevent
oxidation of urobilinogen to urobilin
o Spectrophotometric readings: should
be made within 5 minutes after color
SOURCES OF ERROR production
• REFERENCE RANGE:
• Hemolysis and Lipemia o 0.1 to 1.0 Ehrlich units every 2 hours
o Will alter the concentration of bilirubin o 0.5 to 4.0 Ehrlich units per da
• Exposure to fluorescent and indirect and direct
sunlight DETERMINATION OF FECAL UROBILINOGEN
o Serious loss of bilirubin will occur

• FECAL UROBILINOGEN
REFERENCE RANGES o Same principle for urine
o carried out in an aqueous extract of
fresh feces
o any urobilin present is reduced to
urobilinogen by treatment with alkaline

5
 1ST SEMESTER
A.Y. 2024– 2025 |CLINICAL CHEMISTRY (lecture)
ferrous hydroxide before Ehrlich's reagent
is added.
• NORMAL REF RANGE: 75 to 275 Ehrlich units
per 100 g of fresh feces or 75 to 400 Ehrlich units
per 24-hour specimen

DETERMINATION OF FECAL
UROBILINOGEN

• SERUM BILE ACIDS

• Methods: involve extraction with organic
solvents, partition chromatography, gas
chromatography mass spectrometry,
spectrophotometry, ultraviolet light
absorption, fluorescence,
radioimmunoassay, and enzyme
immunoassay (EIA) methods
• total concentration: extremely variable
and adds no diagnostic value to other
tests of liver function

Abbreviations & symbols for commonly occurring aaS

LIVER ENZYMES

• Aminotransferases: AST & ALT

• Phosphatases: ALP and 5'-nucleotidase
• Gamma-Glutamyl Transferase
• Lactate

TEST MEASURING HEPATIC SYNTHETIC ABILITY

• Decreased serum albumin: decreased liver

protein synthesis
• Serum a-globulins: tend to decrease with chronic
liver disease
• low or absent a-globulin suggests a-antitrypsin
deficiency as the cause of the chronic liver
disease
• Serum y-globulin levels: transiently increased
in acute liver disease and remain elevated in
chronic liver disease
• Marked prolongation of the prothrombin time:
severe, diffuse liver disease and poor prognosis

TEST MEASURING HEPATIC NITROGEN

METABOLISM

• Plasma ammonia level: reflection of liver’s ability

to convert ammonia to urea