about the liver function test

1. Dr. Bahoran Singh
Moderator- Dr. Swati
LIVER FUNCTION TESTS

2. Functions of the Liver
• Metabolic
• Storage- Glycogen, vitamins (all Fat soluble
and few water soluble), iron
• Excretory/Secretory – bile excretion
• Protective (eg. kuffer cells)
• Coagulation – production of clotting
factors
• Detoxification of drugs via cytochromes.

3. 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
– Urea synthesis

4. LFTs are classified as:
• Excretory function tests:
Bile pigments,salts,acids,bilirubin and BSP
• Metabolic functions tests :
Carbohydrates, Protiens, Fats
• Synthetic capabilities :
Protiens(albumin), coagulation factors
• Detoxification :
Ammonia, drugs
• Tests of liver injury :
Enzyme assays,autoimmune markers,markers of
hepatitis virus infections

5. Uses of LFTs
• Diagnosis of type of jaundice- etiology
• Assess severity & follow trend of liver disease
• Detect latent liver disease
• Screening of infective hepatitis
• Screen drug hepatotoxicity

6. 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.
• Lack sensitivity
• Lack specificity

7. 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)
– Conjugate hyperbilirubinemia ( conj. Bilirubin >
20%)

8. • According to site of disease
• Prehepatic-
• Hemolytic
• Ineffective erythropoesis
• Resorption of large hematoma
• Hepatic
• Predominantly unconjugated

9. Bilirubin
• Bilirubin is the major metabolites of heme
• Sources:-hemoglobin, myoglobin & cytochromes.
• 250-350mg of bilirubin is produced daily
• Normal levels upto 1mg%
• Strenuous exercise –significant increase in
bilirubin values .

10. Bilirubin Metabolism

11. Lab tests for bilirubin
• Bilirubin 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

12. Bilirubin - interpretation
• Normal bilirubin level- upto 1mg%
• Direct bilirubin upto 0.3mg%
• direct bilirubin
<20% of total-hemolytic jaundice
=20-40%of total- hepatocellular jaundice
>50%of total- post hepatic
jaundice

13. Bile Salt Assay
• Analysis done in fasting state
• Assay done using chromatographic metods,
HPLC
• Sulphur Test-
– Principle: BS ↓ surface tension of urine
– Method: urine(10ml)+sulphur powder sprinkled
→particles sink to bottom- BS present
→ particles float- BS absent

14. Clinical
condition
Normal
Bile
Salts
absent
Bile
Pigments
absent
Urobilinog
en
trace
Prehepatic Absent Absent Very high +
++ to++++
Hepatic Present
trace to+++
+
Present Increased +
+
Post
hepatic
Present ++
to ++++
Present Absent

15. Determination of bile pigments
• Harrison spot test :
• urine sediment + Fouchet's reagent→
No change in colour – BP absent
change in colour to green – BP present
Positive result graded as trace - ++++ as per
intensity of colour of sediment

16. 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

17. Metabolic functions:-
protein and ammonia metabolism
• Ammonia derived from amino acid and nucleic acid
metabolism.
• Metabolised only in the liver:
• Urea cycle or Krebs Henseleit cycle
• Ammonia Urea .
• Liver damage >80%- ↑ NH3 & arginine conc. Hepatic
encephalopathy
• Degree of hepatic encephalopathy is proportional to NH3
conc. in arterial blood .

18. Assays for Ammonia
• Enzyme assay
•Arterial blood is prefered for assay
•Specimen should be kept in ice water until separation of
cells from plasma
α ketoglutarate + NH3 glutamate
Glutamate
dehydrogenase
NADPH NADP indicator colour
•Dry slide method
Alkaline pH buffers convert ammonium ions to ammonia gas
– bromophenol blue - used indicator

19. Cholesterol and other lipids
• Lipoprotein sythesis
• Esterification of cholesterol
• Liver injury: ↓ HDL, ↓ LCAT, ↓ lipoprotein lipase
• ↑ TGs -↑ unesterified cholesterol, ↑phospholipids
• Cirrhotics with poor nutrition – ↓ cholesterol
• Alcohol induced liver injury: ↑ HDL
↑ apoA-1 protein
• Cirrhosis : ↓ apoA-1 protein
• apoA-1 protein – PGA index used to differentiate ALD &
cirrhosis ( PT, GGT, and Apo- A1 protein)

20. 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

21. Assay : Breath tests
• Radiolabelled drug (usually C13
labelled) administered
– measure exhaled CO2 in pt breath
• Breath tests – based on rate limiting step in
metabolism- 2 groups
• 1st
– independent of hepatic blood flow (only
microsomal enzymatic activity)
– eg: aminopyrine, caffeine, diazepam
• 2nd
– dependent on rate of hepatic blood flow
– eg: methacetin, phenacetin, erythromycin

22. 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
• Cirrhosis - + ↓ delivery of amino acids
• 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

23. Protein Assays
• Biuret method :
• peptide backbone C=O +copper
• Dye binding method :
protein + Coomassie blue dye
• Albumin + bromocresol green/
purple
COLOURED
COMPLEX
Spectrophotometric
quantitation
Normal total protein levels : 6-7.8 g/dl
Albumin levels: 3.5- 5 g/dl

24. 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

25. 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

26. 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

27. Ceruloplasmin
• Copper containing protein in serum
• Enzyme present in highest circulating conc.
• Ferroxidase – converts Fe++
→Fe+++
-allow
binding to transferrin
• ↓ levels in Wilson´s disease-mutation in
Chr13

28. Coagulation factors
• Liver
– clotting factor synthesis
– inhibitors of coagulation synthesis
– fibrin degradation product catabolism
• Most common coagulopathy in liver disease -
Disseminated Intravascular Coagulopathy
• DIC: ↑consumption of clotting factors &platelets
↑ PT,↑ PTT, ↓ platelets,↑d-Dimer levels
• Liver failure (some cases):
↓clotting factors- ↓decreased synthesis
↓platelets – sequestration in spleen
fibrin split products- 80%
• pt s without fibrinolysis,
↑ PT,↑ PTT, d-Dimer levels-NOT elevated

29. Prothrombin time (PT)
• Most 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
-computed no.- based on values of bilirubin,
creatinine and PT (INR)

30. 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

31. 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

32. Cellular location of enzymess

33. Algorithm for Diagnosis of Liver Diseases

34. Aminotransferases
(Transaminases)
• Aspartate Aminotransferase(AST) =Serum
Glutamate Oxaloacetate Transaminase (SGOT)
• Alanine Aminotransferase(ALT) = Serum
Glutamate Pyruvate Transaminase(SGPT)

35. AST- liver, heart skeletal muscle, kidneys, brain, RBCs
• In liver 20% activity is cytosolic and 80% mitochondrial
• Clearance performed by sinusoidal cells,
• AST cytosolic t½ -17hrs,7000 X plasma conc.
• AST mitochondrial t½ - 87hrs
• Used for monitoring therapy with hepatotoxic drugs if levels
>3 X normal stop therapy
ALT – more specific to liver, very low concentrations in kidney
and skeletal muscles.
• In liver totally cytosolic.
• t½ - 47hrs , 3000 X plasma conc.
• Used in non alcoholic, asymptomatic pts.

36. •Acute hepatocellular injury - < 24hrs – AST > ALT
> 24hrs - AST < ALT
since ALT has longer half life
•Alcoholic hepatitis (alcohol induced hepatocyte injury)- AST >ALT
- mitochondrial damage induced – ASTm released – has a
longer t½ & is the predominant AST in hepatocyte
- ↑↑ AST:ALT → 3-4 : 1 – DeRitis Ratio
- ↑ ASTm is s/o advanced alcoholic liver disease
•Chronic liver injury (mainly cirrhosis) – ALT > AST
-but as fibrosis progresses – ALT↓ & ↑ AST:ALT
•End stage liver cirrhosis – AST and ALT levels NOT elevated
- massive tissue destruction
•Acute fulminant hepatic failure - ↑↑AST and ↑↑ ALT ,
AST:ALT > 1
Aminotransferases (cont..)

37. Assay for AST & ALT
• Vit B6 important requirement for AST and ALT assays
• Normal serum levels upto 40 IU/dl for both
↑ aspartate in reaction → glutamate α ketoglutararte
Glutamate
dehydrogenase
NAD NADH(Colour indicator)
Oxaloacetate malate
malate
dehydrogenase
NAD NADH (Colour indicator)
Measured
spectrophotometrically

38. Mild Chronic Elevation in Serum
Aminotransferases
• Step one
– Medications and supplements
– Alcohol use
– Viral hepatitis B and C
– Hemochromatosis
– Fatty liver (hepatic steatosis and steatohepatitis )

39. Mild Chronic Elevation in Serum
Aminotransferases
• Step two
– Muscle disorders
– Thyroid disease
– Celiac disease (less)
– Adrenal insufficiency (less)
– Anorexia nervosa (less)

40. Mild Chronic Elevation in Serum
Aminotransferases
• Step three
– Autoimmune hepatitis
– Wilson's disease
– Alpha-1-antitrypsin deficiency

41. Mild Chronic Elevation in Serum
Aminotransferases
• Step four
– 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.

42. Lactate dehydrogenase (LDH)
• Cytosolic glycolytic enzyme
• Lactate Pyruvate
• 5 major isomers : LD1 – LD5 LD1
& LD2 – cardiac muscle,kidney,RBCs LD4
& LD5 – liver , skeletal muscle
• t½ - 4-6hrs , 500X plasma conc.
• Normal levels upto 150 IU/dl
• ↑↑ hepatitis- transient-normal –clinical presentation
• ↑↑total LDH > 500 IU/dl
↑↑ALP > 250 IU/dl
in absense of abnormality in AST or ALT
Indicate SOL – s/o metastatic Ca, HCC, hemangioma
oxidation

43. 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

44. • Obstructive jaundice :
ALP > 2X UNL ≈ rate of ↑ in bilirubin
• Partial obstruction :
↑ ALP ≈ ↑ C.bilirubin :Dissociated jaundice
• Passive congestion & hepatic injury: mod. ↑ALP
• high mol.wt ALP : malignant disease involing liver
• Intestinal ALP:
-↑ in disorders of intestinal tract &cirrhosis -
discriminate intrahepatic &extrahepatic
jaundice-absent in extrahepatic jaundice -lacks
sensitivity
• Cholestasis relieved – ALP levels ↓ more slowly than
bilirubin
Alkaline phosphatase (cont..)

45. Clinical significance of AST: ALT ratio

46. 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

47. • GGT is ↑ in:-alcoholics without liver diseas
– obese pts
– 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

48. Other canalicular enzymes
• 5‘ Nucleotidase
• Leucine aminopeptidase
↑ in cholestatic
disorders

49. Autoimmune markers
• Primary Biliary cirrhosis : AMA
• Primary Sclerosing Cholangitis:- p ANCA
- ANA
- ASMA
• Autoimmune hepatitis: - ANA
- ASMA
- anti-LKM1

50. Markers of Hepatitis Viral Infections
Hepatitis A :
• Anti-HAV - Measures
IgG and IgM Indicates
exposure and
immunity
• Anti-HAV, IgM
-Indicates acute
infection

51. Hepatitis B

52. Hepatitis C
• Anti-HCV - Indication infection with HCV. Does
not indicate immunity
• HCV RNA - Active Virus. Used to detect HCV
when anti-HCV is negative

53. Hepatitis E:
• Anti-HEV IgM – recent or current infection
• Anti-HEV IgG – current or past infection
• HEV RNA-PCR – definite indicator of
acute infection

54. Diagnosing & following cirrhosis,
fibrosis & necroinflammation of Liver
• Definitive diagnosis of fibrosis, necrosis,&
inflammation of the liver is Liver Biopsy-
invasive
• Indices -Levels of serum analytes measuring
liver function- used to follow these disease
process Non-invasively
• Pro-collagen type III pro-peptide (PIIIP) –
follow active cirrhosis

55. • PGA Index:-
–prothrombin time
–Gamma Glutamyl transferase
–Apolipoprotein A1
Higher PGA scores correlate with degree of
hepatic fibrosis & severity of cirrhosis
-judged by clinical grading & liver biopsy
-good correlation with levels of PIIIP

56. • Fibrotest & Actitest :
measurement of 6 analytes
–Apolipoprotein A1
–Gamma Glutamyl transferase
–Haptoglobin
–Total bilirubin
–Alpha-2 macroglobulin
–ALT
Also includes pt s age and gender

57. • Correlations with liver biopsy results –then performed using
an artificial intelligence algorithm
– Fibrotest scores – computed on a scale of 0 – 1.0 –
histopathological staging system – METAVIR
– Actitest scores- computed on scale 0-1.0 correlated with
necroinflammatory activity aslo using METAVIR grading
system
• More effective and of value in detecting fibrosis False +ve
results with
– Treatment of hepatitis C with ribavirin
– Gilberts disease
– Extrahepatic cholestasis
– Acute inflammation

58. Other Indices
• FIBROspect II-tissue inhibitors
metalloproteinases , alpha-2 macroglobulin,
hyaluronic acid
• Forms index: age, platelet count, GGT,
cholesterol,-correlated –cirrhosis
• AST:ALT,INR,APRI