Dr. Bahoran Singh

Moderator- Dr. Swati

LIVER FUNCTION TESTS

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.

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

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

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

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

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%)

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

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 .

Bilirubin Metabolism

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

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

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

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

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

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: • 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 .

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

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)

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

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

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

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

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

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

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

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)

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

Aminotransferases (Transaminases)

• Aspartate Aminotransferase(AST) =Serum Glutamate Oxaloacetate Transaminase (SGOT)

• Alanine Aminotransferase(ALT) = Serum Glutamate Pyruvate Transaminase(SGPT)

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.

•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..)

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

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 )

Mild Chronic Elevation in Serum Aminotransferases

• Step two– Muscle disorders– Thyroid disease– Celiac disease (less)– Adrenal insufficiency (less)– Anorexia nervosa (less)

Mild Chronic Elevation in Serum Aminotransferases

• Step three – Autoimmune hepatitis – Wilson's disease – Alpha-1-antitrypsin deficiency

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.

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

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

• 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..)

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 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

Other canalicular enzymes

• 5‘ Nucleotidase • Leucine aminopeptidase

↑ in cholestatic disorders

Autoimmune markers

• Primary Biliary cirrhosis : AMA • Primary Sclerosing Cholangitis:- p ANCA

- ANA

- ASMA• Autoimmune hepatitis: - ANA

- ASMA - anti-LKM1

Markers of Hepatitis Viral Infections

Hepatitis A :• Anti-HAV - Measures

IgG and IgM Indicates exposure and immunity

• Anti-HAV, IgM -Indicates acute infection

Hepatitis B

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

Hepatitis E:• Anti-HEV IgM – recent or current infection

• Anti-HEV IgG – current or past infection

• HEV RNA-PCR – definite indicator of acute infection

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

• 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

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

• 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

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