liver function test
TRANSCRIPT
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Dr. Bahoran Singh
Moderator- Dr. Swati
LIVER FUNCTION TESTS
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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.
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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
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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
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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
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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
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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%)
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• According to site of disease• Prehepatic-• Hemolytic• Ineffective erythropoesis• Resorption of large hematoma• Hepatic• Predominantly unconjugated
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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 .
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Bilirubin Metabolism
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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
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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
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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
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Clinical condition Normal
Bile Salts absent
Bile Pigments absent
Urobilinogen trace
Prehepatic Absent Absent Very high +++ to++++
Hepatic Present trace to++++
Present Increased ++
Post hepatic
Present ++ to ++++
Present Absent
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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
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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
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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 .
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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
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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)
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Drug metabolism
• Xenobiotics are metabolised – microsomes of liver CYP 450
• Detoxification – 2 phasesphase 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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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Cellular location of enzymess
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Algorithm for Diagnosis of Liver Diseases
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Aminotransferases (Transaminases)
• Aspartate Aminotransferase(AST) =Serum Glutamate Oxaloacetate Transaminase (SGOT)
• Alanine Aminotransferase(ALT) = Serum Glutamate Pyruvate Transaminase(SGPT)
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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.
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•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..)
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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
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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 )
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Mild Chronic Elevation in Serum Aminotransferases
• Step two– Muscle disorders– Thyroid disease– Celiac disease (less)– Adrenal insufficiency (less)– Anorexia nervosa (less)
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Mild Chronic Elevation in Serum Aminotransferases
• Step three – Autoimmune hepatitis – Wilson's disease – Alpha-1-antitrypsin deficiency
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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.
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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 ALTIndicate SOL – s/o metastatic Ca, HCC, hemangioma
oxidation
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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
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• 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..)
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Clinical significance of AST: ALT ratio
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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
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• 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
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Other canalicular enzymes
• 5‘ Nucleotidase • Leucine aminopeptidase
↑ in cholestatic disorders
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Autoimmune markers
• Primary Biliary cirrhosis : AMA • Primary Sclerosing Cholangitis:- p ANCA
- ANA
- ASMA• Autoimmune hepatitis: - ANA
- ASMA - anti-LKM1
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Markers of Hepatitis Viral Infections
Hepatitis A :• Anti-HAV - Measures
IgG and IgM Indicates exposure and immunity
• Anti-HAV, IgM -Indicates acute infection
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Hepatitis B
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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
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Hepatitis E:• Anti-HEV IgM – recent or current infection
• Anti-HEV IgG – current or past infection
• HEV RNA-PCR – definite indicator of acute infection
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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
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• 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
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• Fibrotest & Actitest : measurement of 6 analytes– Apolipoprotein A1– Gamma Glutamyl transferase– Haptoglobin– Total bilirubin– Alpha-2 macroglobulin– ALTAlso includes pt s age and gender
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• 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
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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