Liver FunctionTest

Dr. Aastha Gupta

 Functions of the
Liver
• Excretory function : bilirubin
• Synthetic function : albumin, globulins, transport
proteins and many coagulation proteins
• General metabolic functions : carbohydrate, lipid and
protein metabolism
• Storage site for iron, glycogen, and vitamins
• During fetal life, hematopoiesis occurs in the liver
• catabolism of steroid hormones
 Metabolic
functions
• Carbohydrate metabolism
– Gluconeogenesis
– Glycogenolysis and glycogenesis
• Hormone metabolism

• Lipid Metabolism
– Synthesis of fatty acids, cholesterol,
lipoproteins
– Ketogenesis

• Drug Metabolism

• Protein Metabolism
– Synthesis of plasma proteins
 LFTs are classified
• Excretory function tests:
as:
Bile pigments, salts, acids, bilirubin and
BSP
• Metabolic functions tests :
Carbohydrates, Protiens,
Fats
• Synthetic functions :
Protiens(albumin), coagulation factors

• Detoxification :
Ammonia,
drugs
• Tests of liver injury :
Enzyme assays, autoimmune markers, markers
of hepatitis virus infections
 Uses of
•
LFT s
Diagnosis of type of jaundice- etiology
• Assess severity & follow trend of liver
disease
• Detect latent liver disease
• Screening of infective hepatitis
• Screening of drug hepatotoxicity
 Indication and limitation of
• LFT
Indication-
• Screen for liver diseases
• Identifying the nature of
liver
• diseases( hepatocellular,
• cholestatic, or infiltrative)
• Assess severity and prognosis of liver
• disease Follow up the course of liver
• disease.
• Limitations –
Do not necessarily assess liver function
 Test that assess excretory
•function
Jaundice – yellowish discoloration of skin,
sclera, and mucous membrane. Evident when
bilirubin >2 mg/dl.
• Classification of jaundice-
1. According to type of bilirubin increased-
• Unconjugated hyperbilirubinemia ( unconj.
Bilirubin > 85% of total)
• causes are hemolysis, resorption of a large
hematoma, ineffective erythropoiesis, Gilbert’s
syndrome, physiologic jaundice of newborn, and
Crigler-Najjar syndrome
• Conjugated hyperbilirubinemia ( conj. Bilirubin > 50% of total)

• causes are hepatitis, cirrhosis, cholestasis, drugs

(anabolic steroids, oral contraceptives), toxins, Dubin-
Johnson syndrome, and Rotor syndrome

• Mixed (conjugated + unconjugated) hyper-bilirubinemia:

Conjugated bilirubin is 20-50% of total; it results from
viral or alcoholic hepatitis
• According to etiology:
• Hemolytic: increased rate of red cell destruc-
tion leads to increased haemoglobin breakdown to
bilirubin saturates the capacity of conjugation in liver

• Hepato-cellular: Inability of hepatocytes to

conjugate and/or excrete bilirubin

• Obstructive: Failure of excretion of conjugated

bilirubin into the intestine, causing its regurgitation in
circulation
 According to site of disease
• Pre-hepatic
• Hepatic
• Post-hepatic
 Pre-hepatic jaundice
•Hemolytic anemia
•Ineffective erythropoiesis (megaloblastic anemia, thalassemias)
•Resorption of a large hematoma
Hepatic jaundice
•Predominantly unconjugated hyperbilirubinemia
•  Gilbert’s syndrome
•  Crigler-Najjar syndrome
• Physiologic jaundice of newborn
•Predominantly conjugated hyperbilirubinemia
•  Hepatocellular diseases: viral hepatitis, toxic hepatitis, alcoholic hepatitis, active
cirrhosis
•  Intrahepatic cholestasis: Dubin-Johnson syndrome, drugs, primary biliary
cirrhosis, primary sclerosing cholangitis, biliary atresia
Post-hepatic jaundice
•Carcinoma of head of pancreas
•Carcinoma of ampulla of Vater
•Secondaries in porta hepatis
•Gallstones in or stricture of common bile duct
 Bilirubin
• Bilirubin is the major metabolite of heme
• Sources:- hemoglobin, myoglobin & cytochromes. 250-350mg
• of bilirubin is produced daily
• Normal levels upto 1mg%

• Strenuous exercise –significant increase

in bilirubin values
Bilirubin Metabolism
 Lab tests for
• Bilirubinbilirubin
is measured using diazotised
sulfanilic acid -form a reddish purple
coloured complex- spectrophotometrically

• total bilirubin=unconjugated +conjugated

+delta bilirubin

• direct bilirubin assays measure:

– C bilirubin +delta bilirubin +small% of UC
bilirubin
Fig.: Estimation of serum bilirubin by diazo method
Fig.: Investigation of jaundice. ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase
 Bile Salt
Assay
• Analysis done in fasting state
• Assay done using chromatographic
methods, HPLC
• Hay Sulphur Test-
– Principle: BS ↓ surface tension of urine
– Method: urine(10ml)+sulphur powder
sprinkled
→particles sink to bottom- BS present
→ particles float- BS absent
 Urobilinogen
• determination
Freshly collected normal fasting urine
sample-
+ve reaction for urobilinogen
• On air exposure oxidized to urobilin
(pinkish brown)
• Test –
– Urine + Ehrlich's reagent→ pale pink
urobilinogen normal
– cherry red- urobilinogen ↑ ↑ ↑
– graded as per colour intensity
• Metabolic functions:- Protein and
ammonia metabolism

Ammonia derived from amino acid and nucleic acid

metabolism

Metabolised only in the liver:

Degree of hepatic encephalopathy is proportional to NH3conc.
in arterial blood .
 Assays for
Ammonia
•Arterial blood is prefered for assay
•Specimen should be kept in ice water until separation of
cells from plasma
• Enzyme assay
Glutamate
dehydrogenase
α ketoglutarate + NH3 glutamate

NADPH
NADP indicator colour

•Dry slide method

Alkaline pH buffers convert ammonium ions to ammonia
gas
– bromophenol blue - used indicator
 Drug
metabolism
• Xenobiotics are metabolised – microsomes
of liver CYP 450
• Detoxification – 2 phases
phase
1-oxidation/hydroxlation
phase 2-conjugation with
polar compound
• Severe liver injury - ↓ability to
metabolise drugs- measure extent of
liver damage in known liver disease
 Synthetic functions:- protein
synthesis
• Liver – site for synthesis for most
plasma proteins( 100% albumin)
exceptions- immunoglobulins & vWF
• Extensive liver destruction-↓serum total
proteins and albumin
• Common causes of ↓ S.proteins : renal
diseases, liver disease, malnutrition, protein
losing enteropathy, chronic inflammatory
diseases
• ↓S.proteins levels –depend on t½ of protein
eg: albumin- 20 days , transthyretin – 1-2
days
factor VII- 4-6hrs , transferrin – 6 days
 Protein
• Biuret method : Assays
• peptide backbone C=O
+copper
• Dye binding method : COLOURED
protein + Coomassie blue COMPLEX
Spectrophotom
dye etric
quantitation
• Albumin + bromocresol green/
purple
Normal total protein levels : 6-7.8 g/dl
Albumin levels: 3.5- 5 g/dl
 Protein
Assay
• Thymol turbidity test :
determination of serum
proteins
-Principle: reaction ɣ & β glogulin
with phenolic group of thymol
-Serum + buffered soln Thymol → Turbidity
- measured
 Protein
•
electrophoresis
Cirrhosis :
– ↓↓albumin,
– ↓alpha-1,alpha-2 & beta band
– ↑ polyclonal immunoglobulins: IgG &
IgA (beta-gamma bridging pattern)
• Autoimmune hepatitis:
- ↓ albumin
- ↑ ↑ polyclonal IgG
• Primary biliary cirrhosis:
- ↑ polyclonal IgM
 Other proteins alpha-1-
antitrypsin
• Most abundant alpha-1 globulin
• Most imp protease inhibitor in plasma
• Inhibits trypsin & other serine proteases
• Coded by Pi gene on Chromosome14
• Mutation- ↓protein glycation →
↑conc. In hepatocytes – periportal-
discrete cytoplasmic bodies- Neonatal
hepatitis- cirrhosis in 3%
↓conc. In plasma - emphysema
 Prothrombin time
• Most (PT)
frequently used – liver associated coagulation
abnormalities- best index of severity
• Efficacy of extrinsic clotting system- factor VII
• Factor VII- synthesized in liver- evaluate liver
function
• PT part of MELD score- Model for End stage
Liver Disease
- evaluating priority
- Liver Transplantation
- predicts 3 month mortality for cirrhotic pts
- based on values of bilirubin, creatinine and
PT INR
 Problems with using
PT
• Non-specific – elevated in most
coagulation disorders
• In cholestasis – with normal hepatocyte fn
↑ PT - ↓ bile salts - ↓ absorption of
vit K
- ↓ factors II, VII, IX, X
cholestasis- precursor forms of
clotting
factors increased
 Tests of Liver
Injury
Plasma Enzyme Levels
• Aspartate Aminotransferase (AST)
• Alanine Aminotransferase (ALT)
• Lactate Dehydrogenase (LDH)
• Alkaline Phosphatase (ALP)
• Gamma Glutamyl Transferase
(GGT)
• 5‘- Nucleotidase
Cellular location of
enzymess
Algorithm for Diagnosis of Liver
Diseases
 Mild Chronic Elevation in
Serum
• Step one Aminotransferases
– Medications and supplements
– Alcohol use
– Viral hepatitis B and C
– Hemochromatosis
– Fatty liver (hepatic steatosis and
steatohepatitis
)
 Mild Chronic Elevation in
Serum
• Step two Aminotransferases
– Muscle disorders
– Thyroid disease
– Celiac disease (less)
– Adrenal insufficiency
(less)
– Anorexia nervosa (less)
 Mild Chronic Elevation in
Serum
• Step threeAminotransferases
– Autoimmune hepatitis
– Wilson's disease
– Alpha-1-antitrypsin
deficiency
 Mild Chronic Elevation in
Serum
• Step four Aminotransferases
– A liver biopsy is often considered in patients
in whom all of the above testing has been
unyielding.
– However, in some settings, the best course
may be observation.
 Enzymes – canalicular injury
Markers of
Cholestasis
Alkaline Phosphatase (ALP)
• liver and bone (placenta, kidneys, intestines or WCC)
–
• Hepatic ALP present on surface of bile duct epithelia-
canalicular surface and accumulating bile salts increase
its release from cell surface – in biliary dysfunction (not
cell injury).
• Takes time for induction of enzyme levels so may not be
first enzyme to rise and half-life is 3 days
• ALP isoenzymes, 5-NT or gamma GT may be necessary
to evaluate the origin of ALP
• Normal = 30-120 IU/L
• Causes of ↑ in ALP: biliary tract obstruction –eg:
Stones hepatitis
ascending cholangitis
Clinical significance of AST: ALT
ratio
 Gamma Glutamyl transferase
(GGT)
• Regulates transport of AA across cell membrane
• hepatocytes and biliary epithelial cells, pancreas, renal
tubules and intestine
• Confirm hepatic source for a raised
• ALP Half life -10 days
• Very sensitive but Non-specific
• Raised in ANY liver disease hepatocellular or
• cholestatic Highest values – 10 x –chronic cholestasis:
• PBC, SC Chronic alcohol abuse – 60-70% show ↑ GGT
-correlation between amount of alcohol intake & GGT
activity -remains elevated 1 month after alcohol
abstinence
• -t½ ↑ to 28 days
Cholestasis of pregnancy : ↑ ALP, but GGT remains normal
• GGT is ↑ in:-alcoholics without liver
– obese pts
diseas
– high conc.of drugs: acetaminophen, phenytoin,
carbamazepine(GGT ↑ to restore glutathione used
to metabolise drugs)
• Isolated increase does not require any further
evaluation, suggest watch and repeated
quarterly,or if other LFT’s become abnormal then
• investigate
GGT Assay: substrate - ɣ glutamyl-p
nitroanilide
→ p nitroaniline liberated (chromogenic) –
measured spectrophotometrically
 Other canalicular
enzymes
• 5‘ Nucleotidase ↑ in cholestatic
• Leucine disorders
aminopeptidase
 Autoimmune
markers
• Primary Biliary cirrhosis : AMA
• Primary Sclerosing Cholangitis:- p
ANCA
- ANA
• Autoimmune - ANA - ASMA
hepatitis: - ASMA
- anti-LKM1
Interpretation of LFTs:
A stepwise approach consists of laboratory tests for:
Presence of liver disease: History, physical findings, and liver function tests
Nature of liver disease: whether hepatocellular (cell injury) or cholestatic
Specific cause of liver disease
Assessment of severity of liver disease