Liver Function and

Lab Assessment of
Liver Function
Synthetic Function | Metabolic Function
Overview of
Lesson 15
Metabolic
01. Function

02. Synthetic
Function
 METABOLI
01. C
FUNCTION
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Metabolic Functions
• Liver: extensive metabolic capacity
• Responsiblee for metabolizing
biological compounds
• Metabolism of Carbohydrates
• When carbohydrates are absorbed and
ingested:
(1) Use glucose - cellular energy
requirements
(2) Circulate glucose
(3) Store glucose as
glycogen (liver/other tissues)
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Metabolic Functions
• Maintain Stable Glucose
Concentrations through Glycogenesis
• Degrade glycogen and break down
stored glycogen (stress or fasting
state) - Glycogenolysis
• Create glucose from nonsugar carbon
substrates - Gluconeogenesis
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Metabolic Functions
• Lipds: Metabolized in the liver
(normal circumstances
• Metabolize both lipids and the
lipoproteins
• Gather free fatty acids (diet or
liver)
Acetyl-CoA
• Greatest source of cholesterol
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Metabolic Functions
• Protein: synthesized by
liver
(except immunoglobulins
and HbA)
• Synthesized albumin
• Synthesized positive and
negative acute-phase and
coagulation proteins
Bilirubin
• Major metabolite of heme
 Iron binding tetrapyrol
ring found in hemoglobin,
myoglobin and
cytochromes
• 250 to 350 mg of bilirubin
(healthy adults)
• 85% derived from turnover
senescent red blood cells
Pathway for clearance of Bilirubin

Schematic summary of the pathway of bilirubin transport and

metabolism
Normal Bilirubin Metabolism
MACROPHAGES Methemoglobin split Free Globin
(mainly Spleen) (Red Cells) Chains and Heme

• Porphyrin ring of heme oxidized [Microsomal

Heme Oxygenase] Biliverdin and releasing Iron

• Biliverdin reduced to bilirubin by NADPH-dependent

enzyme
• Bilirubin (bound mainly to albumin)
transported to portal system to the liver
Enters hepatocytes through membrane surface
Pathway for clearance of Bilirubin
Normal Bilirubin Metabolism
• Free bilirubin enters hepatocytes
additional bilirubin
dissociates (albumin)
• Clearance of unconjugated
bilirubin: 5 mg/kg/day
• Clearance rate :with increasing
concentration of unconjugated
bilirubin
 Normal Bilirubin Metabolism
• Bilirubin enters hepatocytes by two mechanisms
 Passive Diffusion
 Receptor-mediated Endocytosis
• [Hepatocyte] - Bilirubin is “handed
off” to protein complex
• [SER] - Bilirubin becomes
substrate of glucuronyl transferase
Normal Bilirubin Metabolism
• Energy-dependent mechanism transports
conjugated bilirubin to canalicular face of
hepatocytes
• Intestinal Bacteria - further metabolize
Bilirubin in the intestinal tract
Formation of compounds
[Urobilinogen and Urobilin]

Reabsorbed from gut and

reexcreted by liver
Normal Bilirubin Metabolism
Conjugated Bilirubin covalently bound
(Serum) Albumin

• Producing Biliprotein or delta-bilirubin

• Delta-bilirubin [half-life] similar to Albumin

• Conjugated bilirubin can be filtered by Glomerulus
Conditions:
• Hyperbilirubinemia: Large quantities of
urobilinogen are found in the urine
• Impaired bilirubin metabolism: absence of
urobilinogen into stool causing clay-colored stools
• Serum Conjugated bilirubin: urinary bilirubin is
elevated
Pathway for clearance of Bilirubin

Schematic summary of the pathway of bilirubin transport and

metabolism
 Derangements of Bilirubin Metabolism
Causes of Elevated Serum Levels of Unconjugated Bilirubin
Hemolysis • [Hemolytic Anemia] unconjugated
bilirubin
rises due to abnormal high levels of Hb
• Rate of Bilirubin↑ formation exceeds
Bilirubin level in serum Rate

of
[Confirmation of Hemolytic Anemia]: In adults
Liver clearance

Elevated Indirect Bilirubin levels

[serum]
Gilbert's syndrome and Crigler - najjar syndrome
• Characterized by Mild Unconjugated
Hyperbilirubinemia Insertion of two bases
• Most common genetic lesion into UGT1A1

• High Serum levels of unconjugated bilirubin and

multiple mutations [more serious Crigler - Najjar
Syndrome] Levels can increase further with fasting [5 mg/dl]
• Total Bilirubin [unconjugated]: elevated to 2-3
mg/dl
• Frequently diagnosed in young adults
Gilbert's Syndrome
Gilbert's syndrome and Crigler - najjar syndrome
Type 1 Crigler-Najjar Syndrome
• Unconjugated Bilirubin [marked]: exceeds 5 mg/dl or
20 Causing Jaundice

mg/dl
• [Infant]: severe unconjugated hyperbilirubinemia
Basal Ganglia
Leads to Retardation
Kernicterus Severe motor dysfunction

Type 2 Crigler-Najjar Syndrome

• Enzyme activity is approx. 10% normal
Danger of Kernicterus: exceeding 20 mg/ml
Derangements of Bilirubin Metabolism

Causes of Elevated Serum Levels of Conjugated

Bilirubin • Inborn Error of Metabolism
Excretion Deficits: • Blockade of excretion of bilirubin
Dubin-Johnson
Syndrome into
Canaliculin
Associated with
increased plasma • Cause by defects in ATP [Adenosine
conjugated bilirubin
(2-5 mg/dl)
triphosphate]-binding cassette (ABC)
canalicular multi-specific organic
anion
transporter
Derangements of Bilirubin Metabolism

Causes of Elevated Serum Levels of Conjugated

Bilirubin
• Intense dark pigmentation of liver
Excretion Deficits:
Dubin-Johnson • [Conjugated Bilirubin]: Accumulates
Syndrome within hepatocytes and back-diffuses
into circulation
 Gross liver specimen from a patient with Microscopic histology of the liver in Dubin-
Dubin-Johnson Syndrome showing multiple Johnson syndrome showing multiple areas of
areas of dark pigmentation granulated pigment
Biliary Obstruction
Cholelithiasis
• Most common cause of
hyperbilirubinemia
• Results from presence of bile stones,
most commonly in common bile duct
• Elevation of total bilirubin [90% direct
bilirubin]
[Inflammatory conditions of Biliary tract]
Elevated serum level of direct bilirubin and
alkaline phosphatase

Direct bilirubin: [ 5 mg/dl ]

Gram-negative sepsis: mild elevation [ 2-3 mg/dl ]
Concominant elevation of alkaline phosphatase
[ 200 tp 300 IU/L ]
Gross photograph shows a gallbladder
filled with numerous smooth yellow Gallstones typically form in the gallbladder
cholesterol stones.
Biliary Obstruction

Hepatitis
• Blocking Conjugation of Bilirubin and
excretion of conjugated bilirubin
• Elevation of both direct and indirect
occurs
• Serum levels: variable
• Viral Hepatitis: 5-10 mg/dl [serum
Septicemia, total parental nutrition and certain
bilirubin]
drugs (androgens):Increased conjugated bilirubin
Fasting :Increased unconjugated bilirubin
SPECIMEN COLLECTION AND STORAGE FOR
BILIRUBIN
• Serum or plasma preferred.
• Fasting sample is preferred.
○ Presence of lipemia will increase in
measured bilirubin concentrations.
• Hemolyzed samples should be avoided.
○ Decreases the reaction of bilirubin
with the diazo reagent.
• Specimens should be protected from
light.
○ Bilirubin is very sensitive to and is
destroyed by light.
METHODS USED FOR BILIRUBIN
A. DIAZOTIZED SULFANILIC
•ACID
Forms a conjugated azo compound
with the porphyrin rings of bilirubin,
resulting in reaction products that
absorb strongly at 540 nm.
• Caffeine or methanol are
accelerants that are used to measure
total bilirubin.
 METHODS USED FOR BILIRUBIN
B. DIRECT BILIRUBIN ASSAY
• Direct bilirubin is equal to conjugated
bilirubin.
• Approximately 70%-80% of
conjugated bilirubin and delta-bilirubin
and a small percentage of unconjugated
bilirubin are measured.
• Direct bilirubin should measure 0 to
0.1 mg/dL in normal individuals, with
rare values of 0.2 mg/ dL in the absence
of liver or biliary tract disease.
 METHODS USED FOR BILIRUBIN
C. MALLOY-EVELYN
• Simple and widely used, but turbidity and
PROCEDURE
faintness of the color intensity may interfere.
• Diazotized sulfanilic acid reacts at the central
methylene carbon of bilirubin to split the molecule
forming two molecules of azobilirubin.
• Performed at pH 1.2 ----> red-purple in color
• Most commonly used accelerator to solubilize
unconjugated bilirubin is METHANOL.
 METHODS USED FOR BILIRUBIN
D. JENDRASSIK-GROF METHOD FOR TOTAL AND
CONJUGATED BILIRUBIN DETERMINATION
• Bilirubin pigments in serum react with a
diazo reagent which results in the
production of azobilirubin.
• Sodium acetate buffers the pH of the
diazotization reaction.
• Caffeine-benzoate accelerates the
coupling of bilirubin with the diazo
reagent.
• Ascorbic acid stops the reaction.
• Alkaline tartrate converts the purple
azobilirubin to a blue azobilirubin.
• Product azobilirubin is measured by
 METHODS USED FOR BILIRUBIN
D. JENDRASSIK-GROF METHOD FOR TOTAL
AND
CONJUGATED
● BILIRUBIN
Values for total and conjugated DETERMINATION
bilirubin are obtained by comparing
the absorbance read to that of a calibration curve prepared with
acceptable bilirubin standards.
Conjugated Direct Bilirubin - Total Bilirubin = Unconjugated Indirect
Bilirubin
CONJUGATED 0-0.2 mg/dL (0-3 µmol/L)

UNCONJUGATED 0.2-0.8 mg/dL (3-14 µmol/L)

TOTAL 0.2-1.0 mg/dL (3-17 µmol/L)

REFERENCE
RANGES POPULATION TYPE OF BILIRUBIN REFERENCE
RANGE

Adults Conjugated Bilirubin 0.0–0.2 mg/dL (0–

3 µmol/L)

Unconjugated Bilirubin 0.2–0.8 mg/dL (3–

14 µmol/L

Total Bilirubin 0.2–1.0 mg/dL (3–

17 µmol/L)
REFERENCE
RANGES POPULATION TYPE OF BILIRUBIN REFERENCE
RANGE

Premature Total Bilirubin at 24 h 1–6 mg/dL (17–

Infants 103 µmol/L)

Total Bilirubin at 48 h 6–8 mg/dL (103–

137 µmol/L)

Total Bilirubin at 3-5 10–12 mg/dL

days (171–205 µmol/L)
REFERENCE
RANGES POPULATION TYPE OF BILIRUBIN REFERENCE
RANGE

Full-term Total Bilirubin at 24 h 2–6 mg/dL (34–

Infants 103 µmol/L)

Total Bilirubin at 48 h 6–7 mg/dL (103–

120 µmol/L)

Total Bilirubin at 3-5 4–6 mg/dL (68–

days 103 µmol/L)
Other Metabolic Function
Ammonia
• is a critical and toxic compound is metabolized exclusively in the liver.
• It is derived mainly from amino acid and nucleic acid metabolism.
• Ammonia can be metabolized only in the liver because the liver uniquely
contains the critical enzymes for the Krebs-Henseleit or urea cycle.
https://www.youtube.com/watch?v=vhCF-dN6WYQ&t=648s
• To abolish liver tissue function, more than 80% of the liver must be
destroyed.
• Diseases : Cirrhosis, acute fulminant hepatic failure, including Reye’s
syndrome .
Assay for Ammonia Specimen: Arterial blood
• Enzymatic Assays • Specimens should be kept in ice water
• Dry Slide Method until separation of cells from plasma
occurs
Lipids
Cholesterol and other Lipids
• The liver is vital in lipoprotein synthesis and interconversions, hepatic disorders
often cause derangements in lipoprotein metabolism.
• None of these abnormalities is used to diagnose liver pathology, it is important
to recognize that they may result from liver disease.
• In severe liver injury, including cirrhosis.
Bile Salt
• Products of cholesterol metabolism, facilitate absorption of fat from the
intestine.
• Specimen: Serum
o Patient must be in a fasting state or taken at a specified time after meals.
• Method: High-performance liquid chromatography (are most widely used and
allow separation of different bile salts)
SYNTHETIC
FUNCTION 02.
 Protein Synthesis
• The liver is the site of synthesis for most plasma proteins.
• Synthesis of more than 90% of all protein and 100% of albumin
occurs in the liver.
• Two vital measurements of liver function, therefore, are total
protein and albumin levels in serum.
 Other major causes of low serum total protein and albumin:
o Renal disease
o Malnutrition
o Protein-losing enteropathy
o Chronic inflammatory disease
• Albumin has a half-life of about 20 days, so decreases in its serum
levels occur more slowly than those of proteins with shorter half-
lives.
Determination of Serum Protein levels
• This is based usually on the Biuret method.
○ This method reflects the ability of the peptide backbone C = O
groups of proteins to form color complexes with copper that absorb
strongly at 540 nm.
• Albumin forms a unique color complex with the dyes bromcresol green
and bromcresol purple, such that they absorb maximally at slightly
different wavelengths, thus allowing direct spectrophotometric
quantitation (Ihara et al, 1991).
○ Bromcresol purple tends to react more exclusively with albumin
than does bromcresol green (which reacts to a minor extent with
some globulins)
• The reference range: 6 to 7.8 g/dL range.
○ At least 60% of this should be albumin, the normal range for which
is about 3.5 to 5 g/dL.
Albumin
• Albumin is the major protein produced by the liver.
○ Increased by: low plasma oncotic pressure
○ Decreased by: cytokines, particularly interleukin-6.
• A decrease in albumin is one of the major prognostic features
in patients with cirrhosis.
• Albumin is a transport protein for many substances, both
endogenous (e.g., bilirubin, thyroid hormone) and exogenous
(e.g., drugs).
• Low serum albumin levels due to liver disease are almost
always caused by massive destruction of liver tissue and are
seen primarily in cirrhosis, most often secondary to
alcoholism.
• The diminution in albumin is paralleled by a fall in total
 Other Serum Proteins
α-1-Antitrypsin (AAT)
• The most abundant α-1- globulin
• The most important protease inhibitor in plasma
• AAT is coded for by the Pi gene on chromosome 14
• M = normal serum AAT levels
• S and Z variants = prevent normal protein glycation
• PiMM = “Pi” - is the protease inhibitor
other genotypes:
o PiZZ, PiSS, PiSZ, PiMZ, and PiMS
• Adults with PiZZ are most prone to develop emphysema
• AAT phenotyping can be performed using isoelectric
focusing
Ceruloplasmin
• major copper-containing protein in serum
• the enzyme present in highest circulating concentration
• Ceruloplasmin is a ferroxidase
• Low levels of ceruloplasmin are found in Wilson’s disease
• Expressed in the liver and promotes copper secretion into
plasma, coupled with ceruloplasmin synthesis, and into the
biliary tract.
• Resultant liver damage can lead to:
○ Chronic active hepatitis
○ Cirrhosis
○ fulminant hepatic failure
• Neuropsychiatric disease - copper becomes deposited in the CNS
• it can also be deposited at the edge of the iris, forming the observed
Keyser-Fleischer rings
Ceruloplasmin
• Diagnosis of Wilson’s disease:
○ including low serum ceruloplasmin
○ increased urinary copper excretion
○ increased hepatic copper content
• Acute Wilsonian hepatitis - hepatic
manifestations of Wilson’s disease
• Genetic testing is the most reliable
means to establish the diagnosis
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Clotting Factors
• Coagulation proteins are synthesized in the liver
• Inhibitors of coagulation:
○ antithrombin III
○ α-2-macroglobulin
○ α-1-antitrypsin
○ C1 esterase inhibitor
○ Protein C
• Fibrin degradation products are catabolized in the liver
• Low levels of antithrombin III:
○ decreased synthesis
○ increased consumption,
○ alteration in the transcapillary flux ratio
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Clotting Factors
• Disseminated intravascular coagulopathy (DIC) - is characterized
by increased consumption of clotting factors and platelets
○ Diagnosis of DIC be made certain by determination of elevated
blood D-dimer levels
• In some cases of liver failure, platelet counts are decreased
• Prothrombine time is the most frequently ordered laboratory test for
detecting liver associated coagulation abnormalities
• Factor VII is uniquely synthesized in the liver, its measurement can
be used to evaluate liver function status.
Some Caveats in Using PT and INR to Evaluate Liver Function
• PT and PTT measure the status of the coagulation cascades any
coagulation disorder will give rise to abnormal PT and/or PTT,
independent of liver function
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Clotting Factors
• The use of the INR in evaluating liver function can provide
misleading results

Prothrombin Times Are Used to Compute the MELD

Score
• The PT is an integral part of the model for end-stage liver disease
(MELD) score in evaluating priority for liver transplantation in liver
disease
• Although the score appears to predict accurately the 3-month
mortality for cirrhotic patients awaiting liver transplantation, it must
be used with caution
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Des-y-carboxy Prothrombin
• Also known as the protein induced by
Vitamin K absence or antagonist II
(PIVKA-II).
• It is a nonfunctional prothrombin resulting
from a lack of carboxylation of 10 glutamic
acid residues in the N-terminal portion of the
molecule.
• Prothrombin undergoes post-translational
carboxylation before release into the
peripheral blood.
• Vitamin K dependent carboxylase
responsible for the carboxylation is absent in
many HCC cells and an abnormal
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Des-y-carboxy Prothrombin
• It is considered a complementary
biomarker to alpha fetoprotein (AFP) and
AFP-L3% for assessing the risk of
developing HCC.
• Elevation of both AFP-L3 and DCP
indicate progression of HCC.
• DCP has the highest sensitivity and the
highest positive predictive value in patients
with HCC due to chronic hepatitis B and C
infections.
• Surveillance and risk assessment is used for
early detection of HCC.
• Elevated DCP Values = ≥ 7.5 ng/mL
SUMMARY
• The liver is the largest and most complex organ of the
gastrointestinal tract.
• Liver is involved in number of excretory, synthetic, and
metabolic functions.
• The liver is responsible for metabolizing many biological
compounds (Carbohydrates, lipids and proteins)
• It is compose of three systems: (1) Biochemical hepatocytic
system, (2) Hepatobiliary system and (3) Reticuloendothelial
system
• Hepatobiliary system is concerned with the metabolism of
bilirubin
• Synthesis of more than 90% of all protein and 100% of albumin
occurs in the liver.
SUMMARY
• Two vital measurements of liver function are total protein
and albumin levels in serum.
• Serum protein is based usually on the Biuret method.
• Low serum albumin levels due to liver disease are almost
always caused by massive destruction of liver tissue and
are seen primarily in cirrhosis, most often secondary to
alcoholism.
• Other Metabolic Test consist of Ammonia, Lipids, and
Drug Metabolism.
• They are used for screening for liver disease and to
determine the appropriate treatment for patients with liver
diseases.
SUMMARY
● Other serum proteins bear special importance in the
detection of congenital liver disease
● α-1-Antitrypsin (AAT) is the most important protease
inhibitor in plasma, meanwhile ceruloplasmin is the
major copper-containing protein in serum.
● Disseminated intravascular coagulopathy (DIC) is
characterized by increased consumption of clotting
factors and platelets
● Prothrombine time is the most frequently ordered
laboratory test for detecting liver associated coagulation
abnormalities
QUESTIONS
 01
Almost all proteins are synthesized by the liver
except ?

IMMUNOGLOBULIN AND
ADULT HEMOGLOBIN
 02
What are the two mechanisms involve in the
entrance of Bilirubin into the hepatocytes?

PASSIVE DIFFUSION
RECEPTOR- MEDIATED
ENDOCYTOSIS
 03
What condition is associated with increase plasma
conjugated bilirubin and intense dark pigmentation of the
liver?

DUBIN – JOHNSON
SYNDROME
 04
What are the two vital measurements of liver
function

TOTAL PROTEIN
ALBUMIN LEVELS IN
 05
A ____ in albumin is one of the major prognostic
features in patients with cirrhosis

DECREASE
 06
What is derived mainly from amino acids and
nucleic acid metabolism?

AMMONIA
 07
Bile salts are the products of ___________
metabolism, facilitate absorption of fat from the
intestine?

CHOLESTEROL
 09
What is the most common variant associated
with normal AAT levels?

M variant
 10
Low levels of ceruloplasmin are associated
with what disease?

WILSONS DISEASE
REFERENCES
Bishop, M. L., Duben-Engelkirk, J. L., & Fody, E. P. (1996). Clinical
chemistry: Principles, procedures, correlations. Philadelphia:
Lippincott.

McPherson, R. A., Pincus, M. R., & Henry, J. B. (2007). Henry's

clinical diagnosis and management by laboratory methods.
Philadelphia: Saunders Elsevier.

Asuncion, Patrisha Anne

Cuano, Ma. Joy Danielle L.
Joya, Audrey Marie
Negrosa, Pamela
Sumiog, Joyce Anne