|CLINICAL CHEMISTRY (lecture)

LIVER FUNCTION TEST MICROSCOPIC ANATOMY

Overview:
● Anatomy
○ Gross Anatomy ● Liver is divided into lobules
○ Microscopic Anatomy ● Functional units responsible for the metabolic and
● Biochemical Functions excretory functions performed by the liver
○ Excretory and Secretory Functions ● All lobules is six sided structure with centrally located
○ Metabolism vein which is central vein with portal triads and
○ Detoxification and Drug Metabolism corners
● The portal triad comprises:
● Liver Function Alterations During Jaundice
○ Haptic artery
○ Jaundice ○ Portal vein
○ Cirrhosis ○ Bile duct
○ Tumors ■ Surrounded by protective/connective
○ Reye's Syndrome tissues
○ Drug and Alcohol-Related Disorders
● Assessment of Liver Function Liver / Function 2 MAJOR CELL TYPES:
Test
○ Bilirubin HEPATOCYTES KUPFFER CELLS
● Methods
○ Urobilinogen in Urine and Feces 80% of the organ Macrophages that line the
○ Serum Bile Acids sinusoids of the liver
○ Test Measuring Synthetic Ability
Large cells that mediate Act a phagocyte capable of
○ Test Measuring Nitrogen Metabolism upward from the central engulfing bacteria, toxin,
vein and periphery of the other substances
lobules

Performed the major

GROSS ANATOMY
functions associated with Responsible for the
the liver and responsible for immune response
the liver regenerative
process

● Location: Right upper quadrant (RUQ)

● Number of Lobes: 2 (right and left lobe)
○ Functionally insignificant
○ Right lobe is 6x larger than the left lobe BIOCHEMICAL FUNCTIONS
● Divided by falciform Ligament
● Organs: stomach, gallbladder ● 4 MAJOR FUNCTIONS:
○ Excretion/Secretion
A. LIVER: BLOOD SUPPLY ○ Metabolism
○ Detoxification
LIVER: BLOOD SUPPLY ○ Storage

HEPATIC ARTERY PORTAL VEIN

Branch of abdominal aorta

● Processing and excretion of endogenous and
exogenous substances into bile or urine
Supply oxygen from the Supply nutrients collected
● Composition of bile:
heart from the digestive tract as
○ Bile acids / salts
the food is digested
○ Bile pigments
○ Cholesterol
25% of total blood supply to 75% of total blood supply ○ Other substances extracted from the blood
the liver ● The body produces approximately 3 liters of bile
per day and excretes 1 liter of what is produced
● Bilirubin:
B. LIVER: EXCRETORY SYSTEM ○ Principal pigment of bile derived from the
● Begins at the Bile Canaliculi - small breakdown of red blood cells
spaces between the hepatocytes that ○ Unconjugated and Conjugated
formed Intrahepatic ducts
● Intrahepatic ducts - excretory product of
cell can drained
● Right and left hepatic duct will merged
forming Common Hepatic Duct
● Common Hepatic Duct - eventually joined
cystic duct (gallbladder) to form Common
Bile Duct
● Common Bile Duct - drain the duodenum

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 |CLINICAL CHEMISTRY (lecture)

A. Carbohydrate metabolism
● The liver performs majority of the pathways:
○ Glycolysis
■ It uses glucose to form
pyruvate and lactate
○ Glycogenesis
■ Synthesis of glycogen
from glucose
○ Glycogenolysis
■ Breakdown of glycogen to
form glucose
○ Gluconeogenesis
■ Formation of glucose
from non-carbohydrate
sources
B. Lipid metabolism
○ Fatty Acid Oxidation
■ Especially when glucose is not
enough so fatty acid will be the
source of energy
○ Lipogenesis
■ Secretion and synthesis of fatty
acid
○ Cholesterol synthesis
■ It happens in the liver
C. Protein metabolism
○ Liver is the major site of protein synthesis
○ Transamination of Amino Acids
EXPLANATION: ○ Deamination of Amino Acids

1. In RBC destruction, there is hemoglobin that will

liberate
○ Hemoglobin is a protein that has heme
and globin component ● First pass
■ Heme - iron ○ Any substance or drugs that intake it will
■ Globin - protein first to the liver before to the target site of
2. Heme will degrade into bilirubin that will result action
Unconjugated Bilirubin ● 2 mechanisms for detoxification of foreign materials
○ Insoluble to plasma so that is why (drugs and poisons) and metabolic products
Bilirubin is bind to Albumin (bilirubin and ammonia):
■ Albumin will acts as carrier ○ Binds the material reversibly to inactovate
protein of Bilirubin to the compound
Unconjugated Bilirubin ○ Chemically modifies the compound

3. Albumin will carry the Unconjugated Bilirubin to

liver that needs to be Conjugated Bilirubin
○ Once at the liver, Unconjugated Bilirubin ● It takes in the liver microsomes via the cytochrome
flows into sinus spaces and is released P450 isoenzymes:
from albumin ○ Oxidation
○ Ligandin is a carrier protein inside the ○ Reduction
liver (hepatocytes) ○ Hydrolysis
4. Ligandin will carry the Unconjugated Bilirubin to go ○ Hydroxylation
to Endoplasmic Reticulum ○ Carboxylation
○ In the ER, the conjugation of bilirubin will ○ Demethylation
happen and occurs on enzyme UDPGT
■ Uridine diphosphate
-glucuronosyltransferases LIVER FUNCTION ALTERATION DURING DISEASE
5. UDPGT - it transfers the glucuronic acid molecule
to each two propionic acid side chains of bilirubin
to form bilirubin diglucuronide / conjugated
bilirubin
6. Conjugated Bilirubin is water soluble and it can ● Yellow discoloration of the skin, eyes and mucous
be secreted from the hepatocytes into bile membranes
canaliculi, then once in the hepatic duct, it ○ Due to the abnormal levels of bilirubin
combines with secretions from gall bladder to ● Bilirubin Level: reached 3.0 to 5.0 mg/dL
cystic to the common bile duct to the intestines ● Icterus
7. Conjugated Bilirubin will be converted into ○ Term used in clinical laboratory refers to the
Urobilinogen by the GI Bacteria in the intestine serum or plasma sample with a yellow
8. It will be secreted into feces 50-250 mg/day discoloration due to an elevated bilirubin
○ If it will go pass by systemic circulation, level
the small portion will secreted into urine ● Classified based on the site of the disorder
1-4 mg/day

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 |CLINICAL CHEMISTRY (lecture)
CLASSIFICATION OF JAUNDICE
in the absence of
1. PRE-HEPATIC hemolysis
○ Caused by abnormalities BEFORE the liver
○ Acute and Chronic Hemolytic Anemia - Manifest during
○ Commonly caused by increased amount of adolescence or early
bilirubin being presented to the liver adulthood
■ Increased Unconjugated
Bilirubin - Total Serum
■ The liver response by functioning Bilirubin: usually
at the maximum capacity and fluctuates between
individuals with pre-hepatic 1.5 & 3.0 merely
jaundice barely have bilirubin exceeds 4.5
levels seen in 5.0 mg/dL, since
liver still function and working - rare , more severe
efficiently to handle its increase & dangerous from
workload (+) 2 types
○ Also called as Unconjugated
Hyperbilirubinemia Crigler - Najjar Genetic - Type 1: Complete
Syndrome mutation in the Absence of
2. HEPATIC UGT1A1 gene Enzymatic Bilirubin
○ Caused by the problems with the liver itself conjugation
○ Intrinsic liver defect or disease
○ - Type 2: With
3. POST-HEPATIC Mutation causing
○ Caused by abnormalities AFTER the liver Severe Deficiency of
○ Biliary Obstructive Disease Enzyme
■ Caused by stones (Gallstones,
Tumors)
- Possible
complication:
NOTE: Kernicterus (high
● Pre-hepatic and Post-hepatic jaundice that the levels of bilirubin
liver function is NORMAL or it is functioning at its deposits in the brain
maximum to compensate the for abnormalities leads to brain
occurs elsewhere Neonatal Deficiency in damage or death)
● Manifestation of Yellow Discoloration, since Physiologic UDPGT
unconjugated bilirubin is bound to albumin in the Jaundice - In newborns &
bloodstream and is not water soluble premature babies,
● Unconjugated Bilirubin, since bind to albumin it conjugation system
does not pass thru glomerular basement is not readily
membrane and therefore not detected in urine available, that's why
they manifest
jaundice

- Babies undergo
Phototherapy
○ Disorders of Bilirubin Metabolism and
Transport Defects
■ Crigler - Najjar Syndrome NOTE:
■ Gilbert’s Disease ● UGT1A1 gene - produces enzyme for UDPGT
■ Neonatal Physiologic Jaundice

○ Disorders Resulting in Hepatocellular CONJUGATED HYPERBILIRUBINEMIA

Injury/Destruction (Increase Conjugated Bilirubin)
■ Cirrhosis
■ Hepatitis DISEASE ETIOLOGY OTHER INFO

UNCONJUGATED HYPERBILIRUBINEMIA Dubin- Deficiency of the - (+) Delta Bilirubin

(Increase Unconjugated Bilirubin) Jonson canalicular
multidrug ○ Means
DISEASE ETIOLOGY OTHER INFO resistance/multi- conjugated
specific organic bind to albumin
Genetic - NO morbidity/ anionic ○ Increase Delta
mutation in the mortality, no clinical transporter Bilirubin poses
UGT1A1 gene consequences protein a problem in
Gilbert’s (MDR2/cMOAT) the lab, since
Disease - Characterized by delta bilirubin
intermittent fraction reacts
unconjugated as conjugated
Hyperbilirubinemia bilirubin

- Defective - (+) Dark stained

conjugation system granules on a liver

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 |CLINICAL CHEMISTRY (lecture)
● No color of stool, it will be clay colored
biopsy
MECHANISMS OF HYPERBILIRUBINEMIA
- Mild in nature with
excellent prognosis,
excellent survival rate,
normal life
expectancy that’s why
no treatment is
necessary

- The problem is the

liver’s ability to uptake
and conjugate
bilirubin is functional.
However, removal of
conjugated from the
liver and excretion
into the bile are
defective ● Uptake defect - OATP1B1/3?
● Rotor - OATP1B1/3
- accumulation of ● Gilbert Crigler-Najjar - UGT1A1
conjugated bilirubin in ● Dubin-Johnson - MRP2/ABCC2
the blood leading to
hyperbilirubinemia
CHANGES IN CONCENTRATION OF BILIRUBIN IN
and will cause
THOSE WITH JAUNDICE
bilirubinuria

- Rare Syndrome

- Total Bilirubin
Concentration:
remains between 2 &
5 mg with more than
50% due to
conjugated fraction

Rotor Mutations in both - (+) Abnormally short

Syndrome the SLCO1B1 & proteins
SLCO1B3 genes
on ch.12 ○ Bilirubin is less
efficiently taken
up by the liver
and remove ● Scar tissue replaces normal, healthy liver tissue
● S/sx: fatigue, nausea, unintended weight loss,
- Rare, benign jaundice, bleeding from the gastrointestinal tract,
intense itching, and swelling in the legs and
- (+) Chronic jaundice abdomen
● CHRONIC ALCOHOLISM: most common cause
- Not require ● Liver damage cannot easily be reversed
treatment ● Other infections:
○ Chronic infection with Hepatitis B, C & D
○ Autoimmune Hepatitis
○ Inherited disorders of Alpha 1
NOTE: Antitrypsin deficiency
● SLCO1B1 & SLCO1B3 genes - The genes ○ Wilson’s disease
function is to provide instructions where making ○ Hemochromatosis
proteins found on liver cells that mediate the ○ Galactosemia
cellular uptake of compounds such as bilirubin,
bile, acid and steroids.

● Primary Liver Cancer: Hepatocellular carcinoma

○ the cancer begins in the liver cells
● Metastatic Liver Cancer: Colon, lung & breast
○ happened in the other tissues, it
● Gallstones metastasized in the liver
● Tumors ● Benign: Hepatocellular adenoma & hemangioma
● Gallstones and Tumors forms physical obstruction, it ● Malignant: Hepatocellular carcinoma & Bile duct
will prevent the flow of conjugated bilirubin to the bile carcinoma
canaliculi
● The Liver normally functioning, but can’t excrete
Bilirubin properly (wala sa liver ang problem, nasa
biliary tract since hindi maka-excrete)
● Since bile is not being brought into the intestine, no ● Cause: infectious, metabolic or drug-induced
conversion of bilirubin into urobilinogen disease

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 |CLINICAL CHEMISTRY (lecture)
● Almost exclusively in children
● Preceded by a viral syndrome or upper respiratory Hemobilirubin Cholebilirubin
tract infection
○ viral such as (varicella, gastroenteritis and Slow reacting Fast Reacting
influenza)
● (+) Acute illness: non inflammatory encephalopathy Pre-Hepatic Bilirubin Post-Hepatic Bilirubin/
& fatty degeneration of the liver Hepatic Bilirubin/
● Patient would present with: profuse vomiting Obstructive & Regurgitative
accompanied by varying degrees of neurologic Bilirubin
impairment such as (fluctuating, personality changes,
and deterioration in consciousness)
○ Encephalopathy: is characterized by
progression from mild, confusion then ● Conjugated bilirubin that is covalently bound to
progressive loss of neurologic function to albumin
the loss of brain stem reflexes ● Seen only when there is significant hepatic
obstruction
● React in most laboratory method as conjugated
bilirubin
● TB = C + UC + DB
● Immune-mediated injury to hepatocytes ○ TB = Total Bilirubin
● Alcoholic fatty liver —> Alcoholic hepatitis —> ○ C = Conjugated
hepatic cirrhosis ○ UC = Unconjugated
● Fatty liver disease: ○ DB = Delta Bilirubin
○ (+) slightly elevated AST, ALT & GGT
○ (+) fatty infiltrates in the vacuoles of the liver
● Alcoholic hepatitis:
○ (+) moderately elevated AST, ALT, GGT,
● Serum or Plasma
ALP
○ Serum - preferred for Evelyn Malloy method
○ increase TB >5 mg/dL
since the addition of alcohol in the analysis
○ The elevation in AST is more than twice the
can precipitate proteins and can cause
upper reference normal but rarely exceeds
interference with the method
300 international units per ml.
● Fasting sample: preferred
○ The elevation in ALT is comparatively lower
○ Presence of lipemia will increase measured
than AST, resulting in an AST ALT ratio or
bilirubin concentration
de ritis ratio greater than 2.
● Avoid hemolyzed samples
● Alcoholic cirrhosis:
○ Hemolyzed samples = decreased reaction
○ increased AST, ALT, GGT, ALP & TB
of bilirubin in diazo reagent
○ decreased albumin & prolonged
● Protect specimen from light
prothrombin time
○ Bilirubin can be easily destroyed by light
○ The risk for development of cirrhosis
○ Bilirubin sample can be covered by dark
increases proportionally with consumption
cloth or aluminum foil
of more than 30 grams (3-4 drinks of
○ When exposed to light, bilirubin will be
alcohol per day) so kapag inaraw- araw mo
converted to biliverdin
ang pag inom ng alak mabilis mamatay ang
● Stability of serum/plasma separated from the cells
liver cells mag li-lead agad to liver cirrhosis)
and stored in the dark
DON'T DRINK GUYS.

TEMPERATURE STABILITY
ANALYSIS OF BILIRUBIN
Room temp 2 days
● Determination of different fractions of Bilirubin
4C 1 week

-20 C Indefinitely

● NO preferred method/standardization of bilirubin

● Accelerator is added to futherly detect indirect analysis
bilirubin ● Modified Jendrassik-Grod procedure:
○ Candidate reference method for total
bilirubin
TYPES OF BILIRUBIN ○ Caffeine benzoate is used as solubilizer to
also detect B1
B1 B2 ● Most widely used methods
1. Malloy Evelyn Procedure
Unconjugated bilirubin Conjugated bilirubin 2. Jendrassik-Grof Method

Water Insoluble Water soluble MALLOY-EVELYN PROCEDURE

Non-polar Bilirubin Polar Bilirubin Principle: Bilirubin pigments in serum or plasma
are reacted with diazo reagent. The
Indirect Reacting Direct Reacting diazotized sulfanilic acid reacts at the

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 |CLINICAL CHEMISTRY (lecture)
central methylene carbon of bilirubin to
split the molecule, forming 2 molecules
or azobilirubin

pH: 1.2

Color Reaction: Red-purple (azobilirubin)

Maximal 560 nm
absorption:

Accelerator: Methanol
○ Colorless end product of bilirubin
JENDRASSIK-GROF METHOD metabolism oxidized by intestinal bacteria to
the brown pigment urobilin
Principle: Bilirubin pigments in serum or plasma ○ Most quantitative methods: based in
are reacted with diazo reagent Ehrlich’s reaction
(sulfanilic acid in hydrochloric acid and ■ The reaction of urobilinogen with
sodium nitrate), resulting in the p-dimethylaminobenzaldehyde
production of the purple product, (Ehrlich’s reagent) to from a red
azobilirubin color

ADDITIONAL PROCEDURES:
➢ The individual fractions of bilirubin are determined by
● Principle:
taking two aliquots of sample and reacting one aliquot
○ Urobilinogen reacts with
with the diazo reagent only and the other aliquot with
p-dimethylaminobenzaldehyde (ehrlich’s
the diazo reagent and an accelerator (caffeine
reaction) to form a red color, which is then
benzoate)
measured spectrophotometrically
○ Caffeine benzoate is used to make the
● Ascorbic acid is added as a reducing agent to
unconjugated bilirubin MORE soluble
maintain urobilinogen in the reduced state
and be measured as well
● Specimen:
➢ After a short period of time, the reaction of the
○ Fresh 2 hour specimen (keep cool and
aliquots with the diazo reagent is terminated by the
protected from light)
addition of ascorbic acid
● Comments & sources of error:
○ Ascorbic acid = destroys the excess
○ Results - reported in Ehrlich’s units
diazo reagent
○ Compound other than urobilinogen may be
➢ The solution is then alkalized using an alkaline
present in the urine and react with Ehrlich’s
tartrate solution, which shifts the absorbance
reagent
spectrum of the azobilirubin to a more intense blue
○ Fresh urine: important; test must be
color
performed WITHOUT delay to prevent
➢ The final blue product is measured at 600 nm, with
oxidation of urobilinogen to urobilin
intensity of color produced directly proportional to
○ Spectrophotometric readings: should be
bilirubin concentration
made within 5 minutes after color
➢ Indirect (unconjugated) bilirubin may ba calculated by
production
subtracting the conjugated bilirubin concentration
● Reference range:
from the total bilirubin concentration
○ 0.1 to 1.0 = Ehrlich units every 2 hours
○ 0.5 to 4.0 = Ehrlich units per day
Color Reaction: Blue

Maximal 600 nm
absorption:
● Serum bile acids
Accelerator: Caffeine benzoate ○ Methods: involve extraction with organic
solvents, partition chromatography, gas
chromatography mass spectrometry,
spectrophotometry, ultraviolet, light
absorption, fluorescence,
radioimmunoassay, and enzyme
● Hemolysis immunoassay (EIA) methods
● Lipemia ○ Total concentration: extremely variable
● Exposure to fluorescent and indirect and direct and adds no diagnostic value to other tests
sunlight of liver function

LIVER ENZYMES

● Aminotransferases: AST & ALT

● Phosphatases: ALP and 5’- nucleotidase
UROBILINOGEN ● Gamma-Glutamyl Transferase
● Lactate dehydrogenase

TEST MEASURING HEPATIC SYNTHETIC ABILITY

● Urobilinogen

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 |CLINICAL CHEMISTRY (lecture)
● 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 gamma-globulin levels
○ Transiently increased in acute liver disease
and remain elevated in chronic liver disease
● Marked prolongation of the prothrombin time
○ Sever, diffuse liver disease and poor
prognosis

TEST MEASURING NITROGEN METABOLISM

● Plasma ammonia level: reflection of liver’s ability to

convert ammonia to urea

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