2. Functions of the Liver
⢠Metabolic
⢠Storage- Glycogen, vitamins (all Fat soluble
and few water soluble), iron
⢠Excretory/Secretory â bile excretion
⢠Protective (eg. kuffer cells)
⢠Coagulation â production of clotting
factors
⢠Detoxification of drugs via cytochromes.
3. Metabolic functions
⢠Carbohydrate metabolism
â Gluconeogenesis
â Glycogenolysis and glycogenesis
⢠Hormone metabolism
⢠Lipid Metabolism
â Synthesis of fatty acids, cholesterol, lipoproteins
â Ketogenesis
⢠Drug Metabolism
⢠Protein Metabolism
â Synthesis of plasma proteins
â 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 â
⢠Do not necessarily assess liver function.
⢠Lack sensitivity
⢠Lack specificity
7. Test that assess excretory function
⢠Jaundice â yellowish discoloration of skin,
sclera, and mucous membrane. Evident when
bilirubin >2 mg/dl.
⢠Classification of jaundice-
1. According to type of bilirubin increased-
â Unconjugated hyperbilirubinemia ( unconj.
Bilirubin > 85% of total)
â 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 âsignificant increase in
bilirubin values .
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:
â 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-
â Principle: BS â surface tension of urine
â Method: urine(10ml)+sulphur powder sprinkled
âparticles sink to bottom- BS present
â particles float- BS absent
15. Determination of bile pigments
⢠Harrison spot test :
⢠urine sediment + Fouchet's reagentâ
No change in colour â BP absent
change in colour to green â 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 â
â Urine + Ehrlich's reagentâ pale pink urobilinogen
normal
â cherry red- urobilinogen âââ
â 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%- â NH3 & arginine conc. Hepatic
encephalopathy
⢠Degree of hepatic encephalopathy is proportional to NH3
conc. in arterial blood .
18. Assays for Ammonia
⢠Enzyme assay
â˘Arterial blood is prefered for assay
â˘Specimen should be kept in ice water until separation of
cells from plasma
Îą ketoglutarate + NH3 glutamate
Glutamate
dehydrogenase
NADPH NADP indicator colour
â˘Dry slide method
Alkaline pH buffers convert ammonium ions to ammonia gas
â bromophenol blue - used indicator
19. Cholesterol and other lipids
⢠Lipoprotein sythesis
⢠Esterification of cholesterol
⢠Liver injury: â HDL, â LCAT, â lipoprotein lipase
⢠â TGs -â unesterified cholesterol, âphospholipids
⢠Cirrhotics with poor nutrition â â cholesterol
⢠Alcohol induced liver injury: â HDL
â apoA-1 protein
⢠Cirrhosis : â apoA-1 protein
⢠apoA-1 protein â PGA index used to differentiate ALD &
cirrhosis ( PT, GGT, and Apo- A1 protein)
20. Drug metabolism
⢠Xenobiotics are metabolised â microsomes of
liver CYP 450
⢠Detoxification â 2 phases
phase 1-oxidation/hydroxlation
phase 2-conjugation with polar compound
⢠Severe liver injury - âability to metabolise
drugs- measure extent of liver damage in
known liver disease
21. Assay : Breath tests
⢠Radiolabelled drug (usually C13
labelled) administered
â measure exhaled CO2 in pt breath
⢠Breath tests â based on rate limiting step in
metabolism- 2 groups
⢠1st
â independent of hepatic blood flow (only
microsomal enzymatic activity)
â eg: aminopyrine, caffeine, diazepam
⢠2nd
â dependent on rate of hepatic blood flow
â eg: methacetin, phenacetin, erythromycin
22. Synthetic functions:-
protein synthesis
⢠Liver â site for synthesis for most plasma
proteins( 100% albumin) exceptions-
immunoglobulins & vWF
⢠Extensive liver destruction-âserum total proteins
and albumin
⢠Cirrhosis - + â delivery of amino acids
⢠Common causes of â S.proteins : renal diseases,
liver disease, malnutrition, protein losing
enteropathy, chronic inflammatory diseases
⢠âS.proteins levels âdepend on t½ of protein
eg: albumin- 20 days , transthyretin â 1-2 days
factor VII- 4-6hrs , transferrin â 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 â Turbidity -
measured
25. Protein electrophoresis
⢠Cirrhosis :
â ââalbumin,
â âalpha-1,alpha-2 & beta band
â â polyclonal immunoglobulins: IgG & IgA
(beta-gamma bridging pattern)
⢠Autoimmune hepatitis:
-â albumin
-ââ polyclonal IgG
⢠Primary biliary cirrhosis:
-â 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- âprotein glycation â
âconc. In hepatocytes â periportal- discrete
cytoplasmic bodies- Neonatal hepatitis-
cirrhosis in 3%
âconc. In plasma - emphysema
27. Ceruloplasmin
⢠Copper containing protein in serum
⢠Enzyme present in highest circulating conc.
⢠Ferroxidase â converts Fe++
âFe+++
-allow
binding to transferrin
⢠â levels in Wilson´s disease-mutation in
Chr13
28. Coagulation factors
⢠Liver
â clotting factor synthesis
â inhibitors of coagulation synthesis
â fibrin degradation product catabolism
⢠Most common coagulopathy in liver disease -
Disseminated Intravascular Coagulopathy
⢠DIC: âconsumption of clotting factors &platelets
â PT,â PTT, â platelets,âd-Dimer levels
⢠Liver failure (some cases):
âclotting factors- âdecreased synthesis
âplatelets â sequestration in spleen
fibrin split products- 80%
⢠pt s without fibrinolysis,
â PT,â PTT, d-Dimer levels-NOT elevated
29. Prothrombin time (PT)
⢠Most frequently used â 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 â elevated in most coagulation
disorders
⢠In cholestasis â with normal hepatocyte fn
â PT - â bile salts - â absorption of vit K
- â factors II, VII, IX, X
cholestasis- precursor forms of clotting
factors increased
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 â 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. â˘Acute hepatocellular injury - < 24hrs â AST > ALT
> 24hrs - AST < ALT
since ALT has longer half life
â˘Alcoholic hepatitis (alcohol induced hepatocyte injury)- AST >ALT
- mitochondrial damage induced â ASTm released â has a
longer t½ & is the predominant AST in hepatocyte
- ââ AST:ALT â 3-4 : 1 â DeRitis Ratio
- â ASTm is s/o advanced alcoholic liver disease
â˘Chronic liver injury (mainly cirrhosis) â ALT > AST
-but as fibrosis progresses â ALTâ & â AST:ALT
â˘End stage liver cirrhosis â AST and ALT levels NOT elevated
- massive tissue destruction
â˘Acute fulminant hepatic failure - ââAST and ââ 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
â aspartate in reaction â glutamate Îą 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
â Medications and supplements
â Alcohol use
â Viral hepatitis B and C
â Hemochromatosis
â Fatty liver (hepatic steatosis and steatohepatitis )
40. Mild Chronic Elevation in Serum
Aminotransferases
⢠Step three
â Autoimmune hepatitis
â Wilson's disease
â Alpha-1-antitrypsin deficiency
41. Mild Chronic Elevation in Serum
Aminotransferases
⢠Step four
â A liver biopsy is often considered in patients in
whom all of the above testing has been
unyielding.
â However, in some settings, the best course may
be observation.
42. Lactate dehydrogenase (LDH)
⢠Cytosolic glycolytic enzyme
⢠Lactate Pyruvate
⢠5 major isomers : LD1 â LD5 LD1
& LD2 â cardiac muscle,kidney,RBCs LD4
& LD5 â liver , skeletal muscle
⢠t½ - 4-6hrs , 500X plasma conc.
⢠Normal levels upto 150 IU/dl
⢠ââ hepatitis- transient-normal âclinical presentation
⢠ââtotal LDH > 500 IU/dl
ââALP > 250 IU/dl
in absense of abnormality in AST or ALT
Indicate SOL â s/o metastatic Ca, HCC, hemangioma
oxidation
43. Enzymes â canalicular injury
Markers of Cholestasis
Alkaline Phosphatase (ALP) â
⢠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 â 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 â in ALP: biliary tract obstruction âeg: Stones
hepatitis
ascending cholangitis
44. ⢠Obstructive jaundice :
ALP > 2X UNL â rate of â in bilirubin
⢠Partial obstruction :
â ALP â â C.bilirubin :Dissociated jaundice
⢠Passive congestion & hepatic injury: mod. âALP
⢠high mol.wt ALP : malignant disease involing liver
⢠Intestinal ALP:
-â in disorders of intestinal tract &cirrhosis -
discriminate intrahepatic &extrahepatic
jaundice-absent in extrahepatic jaundice -lacks
sensitivity
⢠Cholestasis relieved â ALP levels â more slowly than
bilirubin
Alkaline phosphatase (cont..)
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 â 10 x âchronic cholestasis: PBC, SC
⢠Chronic alcohol abuse â 60-70% show â GGT
-correlation between amount of alcohol intake & GGT
activity -remains elevated 1 month after alcohol abstinence
-t½ â to 28 days
⢠Cholestasis of pregnancy : â ALP, but GGT remains normal
47. ⢠GGT is â in:-alcoholics without liver diseas
â obese pts
â high conc.of drugs: acetaminophen, phenytoin,
carbamazepine(GGT â to restore glutathione used to
metabolise drugs)
⢠Isolated increase does not require any further
evaluation, suggest watch and repeated quarterly,or
if other LFTâs become abnormal then investigate
⢠GGT Assay: substrate - ÉŁ glutamyl-p nitroanilide â
p nitroaniline liberated (chromogenic) â measured
spectrophotometrically
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 â recent or current infection
⢠Anti-HEV IgG â current or past infection
⢠HEV RNA-PCR â 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) â
follow active cirrhosis
55. ⢠PGA Index:-
âprothrombin time
âGamma Glutamyl transferase
âApolipoprotein 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
âApolipoprotein A1
âGamma Glutamyl transferase
âHaptoglobin
âTotal bilirubin
âAlpha-2 macroglobulin
âALT
Also includes pt s age and gender
57. ⢠Correlations with liver biopsy results âthen performed using
an artificial intelligence algorithm
â Fibrotest scores â computed on a scale of 0 â 1.0 â
histopathological staging system â METAVIR
â 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
â Treatment of hepatitis C with ribavirin
â Gilberts disease
â Extrahepatic cholestasis
â Acute inflammation
58. Other Indices
⢠FIBROspect II-tissue inhibitors
metalloproteinases , alpha-2 macroglobulin,
hyaluronic acid
⢠Forms index: age, platelet count, GGT,
cholesterol,-correlated âcirrhosis
⢠AST:ALT,INR,APRI