LIVER FUNCTION TEST

EKTA JAJODIA

SEVERAL BIOCHEMICAL TESTS CAN BE USED –

•To detect presence of liver disease•Distinguish among different types of liver disorders•Gauge the extent of known liver disease•Follow the response to treatment

•Liver tests rarely suggest a specific diagnosis

•They suggest a general category of liver disease, such as hepatocellular or cholestatic

•This further directs the evaluation

Metabolic function

Excretory function

Synthetic function

Detoxification function

Storage function

FUNCTIONS OF LIVER

CLASSIFICATION OF LFTAccording to function of liver

Tests based on excretory function

Tests based on metabolic function

Tests based on detoxification function

Tests based in storage function

Tests based on synthetic function

1.Serum bilirubin

2.Urine bilirubin

3.Urine and feacal urobilinogen

4.Urine bile salts

5.Dye excretion tests

TESTS BASED ON EXCRETORY FUNCTION

Hippuric acid test

Determination of blood

ammonia

TESTS BASED ON DETOXIFICATION FUNCTION

Plasma proteins

Prothrombin time

TESTS BASED ON SYNTHETIC FUNCTION

Tests related toCARBOHYDRATEmetabolism

Tests related toLIPID metabolism

Tests relatedto PROTEINmetabolism

Galactose tolerance test

Serum cholesterol Serum proteinsAminoaciduria

TESTS BASED ON METABOLIC FUNCTION

ENZYMES IN DIAGNOSIS OF LIVER DISEASE

SERUM TRANSAMINASES

ASTALT

ALPGGT5’NT

1.Serum bilirubin

2.Urine bilirubin

3.Urine and feacal urobilinogen

4.Urine bile salts

5.Dye excretion tests

TESTS BASED ON EXCRETORY FUNCTION

SERUM BILIRUBINTotal bilirubin (<1mg/dl) is found in blood in 2 fractions

•Conjugated bilirubin (<0.3mg/dl)

•Soluble in water so excreted by kidney

•Unconjugated bilirubin

•Insoluble in water so bound to albumin in blood

Significance of hyperbilirubinemia

•Daily production of bilirubin <500mg

•But normal liver can conjugate upto 1500mg/day

•So plasma bilirubin concentration is an insensitive test for liver disease - since it begins to rise only after significant liver damage has occured

CAUSES OF HYPERBILIRUBINEMIA

Isolated increase in unconjugated bilirubin is due to –1. Hemolytic disease2. Genetic disorders – crigler najjar and gilbert’s syndrome3. Neonatal jaundice/physiological jaundice

Isolated increase in conjugated bilirubin is due to –4. Cholestasis5. Genetic disorders – Dubin johnson syndrome and rotor’s

syndrome

Increase in both conjugated and unconjugated bilirubin is due to –

1. Intrahepatic /liver disorders

Pigment deposition in hepatocytes in DJ syndrome

• Coarsely- granular brown pigment • Diffuse but more heavily concentrated in the

perivenular zone • grey to black colour grossly • pigment shares some of its physiochemical properties

with lipofuscin and melanin • Oil Red O-positive (in frozen sections), stains black

with the Fontana stain • autofluorescent when examined by ultraviolet

microscopy

DELTA BILIRUBIN / BILIPROTEIN•When diseases/cholestasis prevents excretion of conjugated bilirubin into bile

it enters the plasma

Filtered by the kidneys

Excreted in urine

•Some monoconjugated bilrubin can become covalently bound to albumin

•This protein bound conjugated bilirubin is known as - biliprotein or delta – bilirubin

•Normally present in very small amount

•Increases in cholestasis

•Half life is longer – 20 days (like albumin)

•Normally half life of conjugated bilirubin is 24 hrs

TESTS FOR SERUM BILIRUBIN

• Spectrophotometric method • Diazo method • Peroxidase method • Diazo- peroxidase method • HPLC • Transcutaneous bilirubinometer

DIAZO METHOD •Sample – Serum or plasma EDTA or heparin

•Note – protect from light

•Exposure to direct sunlight can decrease bilirubin in samples by 50% within one hour

Van Den Bergh reaction

Serum bilirubin Diazotized sulphanilic acid(Ehrlich diazo reagent)

Azobilirubin( red)

Direct bilirubin – reading is taken at one minute

Add activator /accelerator

2nd reading at 30 minutes – Total bilirubin

Measure absorbance at 600nm

Test Principle Comments

Evelyn Malloy method Diazo method – product is azobilirubinActivator – ethanol Readings- at one minute and 30 minutes

Sensitive to pH changes and serum protein changes Overestimates dB

Jendrassik-Groff Diazo method Product- azobilirubinActivator – caffeine sodium benzoate

Insensitive to pH changes And serum protein changes

Direct Spectrophotometric Method

Absorbance of bilirubin in serum at 455 nmAbs Hb at 455 nm is corrected by subtracting theabs at 575 nm.

Insensitive to hemolysisAffected by the presence of lipochromes and turbidityOnly used in new born infants

HPLC Alkaline methanolysis methodConjugated forms are methylated Unconjugated forms remain unchanged Extracted into chloroformDetected spectrophotometrically

URINE BILIRUBIN•Any bilirubin found in urine is conjugated bilirubin

•Presence of bilirubinuria is s/o liver disease

•Can be tested by dipstick test

•When dipstick test show presence of urine bilirubin – confirmatory test to be done

•Ictotest tablet – based on diazotization reaction – coupling of solid diazonium salt with bilirubin in an acidic medium gives a blue/purple color

•Ictotest can be used to rule out presence of any interfering substance that give false positive reaction with dipstick test

• While recovering from jaundice urine bilirubin clears before serum bilirubin

•Presence of bilirubin in urine impart it dark brown color

• Also fouchet’s test done – if bilirubin ispresent a green color develops due to formation of biliverdin

Colour Cause

Orange Conc. Urine , urobilin , drugs

Orange-reddish brown

Drugs , rhubarb ingestion

Dark brown Altered blood , myoglobin, porphyrins , phenolic drugs

Red Blood, beetroot or blackberry ingestion , food dyes

Purple-red Phenolphthalein laxative

Brown black Altered blood, melanin, homogentisic acid

URINE UROBILINOGEN

• Increase in urine is sensitive indicator of hepatocellular disease

• It is markedly increased in hemolysis• In cholestatic jaundice urobilinogen disappears

from urine • Urine strips are available • Fresh urine should be used • Ehrlich’s test – gives pink-red color

BILE SALTS

•Products of cholesterol metabolism

•Facilitate absorption of fat from intestine

•Constitute a substantial amount of bile in bilirubin excretion and can be used in diagnosing cholestasis

•Primary bile salts – cholate and chenodeoxycholate are produced in liver

Metabolised by bacteria in intestine

Produces secondary bile salts – lithocholate, deoxycholate and ursodeoxycholate

•In cirrhosis – reduced ratio of primary to secondary bile salts

•In cholestasis – as secondary bile salts are not formed – so increased ratio of primary to secondary bile salts

•In normal condition – renal excretion of bile salts is negligible

•In cholestasis – increased renal excretion of bile salts• •For measuremnet – chromatography (HPLC)

•Hay’s test – bile salts when present lower the surface tension of urine• When sulphur powder is added to the urine, sulphur particles sink to the bottom of the tube• In case of normal urine, it will float on the surface

DYE EXCRETION TEST

3 synthetic dyes are commonly employed to test liver function 1. Bromosulphthalein (BSP) 2. Indocyanine green3. Rose bengal

Bromosulphthalein excretion test – 5ml/kg weight i.v is given

BSP is taken up by hepatocytes, conjugated and excreted in bile

•A blood specimen is taken after 45 mins and 2 hrs•After 45 mins – if >50% dye is retained in blood – abnormal Liver function is present

• This test is useful in differentiating dubin johnson from rotor syndrome•In DJS – At 45 mins – normal blood levels of BSP• At 2 hrs – higher level of BSP

•In rotor syndrome – at 45 mins – higher levels• at 2 hrs – lower levels

Hippuric acid test

Determination of blood

ammonia

TESTS BASED ON DETOXIFICATION FUNCTION

BLOOD AMMONIA

•Produced in body by normal protein metabolism and by intestinal bacteria•For detoxification of ammonia

In liver

converted to urea

Excreted by kidneys

In striated muscles

Combines with glutamic acid

Forms glutamine

Plasma proteins

Prothrombin time

TESTS BASED ON SYNTHETIC FUNCTION

PROTEIN•Liver is the sole site for synthesis of most plasma proteins except immunoglobulins (gamma globulins)•Serum albumin comprises 60% of all plasma proteins

TESTS FOR PROTEINS INCLUDE-1. Total serum proteins2. Serum albumin3. Sr albumin/globulin ratio4. Serum protein electrophoresis5. Prealbumin6. Procollagen III peptide7. Ceruloplasmin8. Alpha fetoprotein9. Alpha antitrypsin

TOTAL SERUM PROTEIN

•2 methods of estimation – 1. Refractometer method 2. Biuret methodBiuret method -

• Principle: Cu2 ions present in biuret reagent complex with peptide bonds in proteins in alkaline medium and give violet colour

Absorbance is measured at 540 nm

ALBUMIN

•Synthesized exclusively by liver•Its half life is 18-20 days •Due to its slow turn over – not a good indicator of acute or mild hepatic dysfunction• In hepatitis - <3g/dl of albumin – possibility of chronic liver disease•Non hepatic causes of Hypoalbuminemia - • Protein losing enteropathy• Nephrotic syndrome

Dye binding method

Immunoassay

Chromatography

Salt -fractionation

Methods of estimation

BROMOCRESYL GREEN METHOD

•Most common method

•The complex becomes blue in color•Absorbance at 632 nm is directly proportional to the concentration of albumin

GLOBULINS

•Made up of – alpha, beta and gamma globulins•Gamma globulin is produced by plasma cells• alpha and beta globulins are synthesized in liver• In cirrhosis – gamma globulin is increased •Cirrhotic liver fails to clear bacterial antigens that normally reach the liver – Abs are formed against such Ags – so , increased gamma globulin• Polyclonal gamma globulin if increases by 100% - autoimmune hepatitis•Increased IgM – primary biliary cirrhosis•Increased IgA – alcoholic liver disease

PREALBUMIN /TRANSTHYRETIN

• Levels fall in liver disease• Half life – 2 days • Sensitive indicator of any changes affecting its

synthesis and catabolism• PAB is a negative acute phase reactant• Particularly useful in drug-induced

hepatotoxicity

• Immunoturbidimetric procedure• Sample is incubated with antibodies specific to

prealbumin • Insoluble complexes are formed

• Normal plasma levels - 0.2-0.4g/L• Acute phase protein• Decreased in multiple conditions –1. Wilson’s disease2. Menkes disease3. Aceruloplasminemia4. Copper deficiency

CERULOPLASMIN

Increased in –

1. Copper toxicity2. Pregnancy3. OCPs

PROCOLLAGEN III PEPTIDE

• Cleavage product of the type III procollagen molecule

• Radioimmunoassay• Elevated Conc. Of PIIIP- the transformation of

viable hepatic tissue into connective tissue/fibrosis

• AFP is a gp and MW – 70,000 daltons • Normally present in fetus • Liver, yolk sac and small intestine • AFP- ELISA HCC

Non seminomatous testicular cancer

Ataxia telangiectasiaHereditary tyrosinemiaNeonatal hyperbilirubinemia

Chronic active hepatitis

α- FETO PROTEIN

COAGULATION FACTORS

•With the exception of F-VIII , all other factors are synthesized in liver•Half life ranges from 6hrs for F-VII to 5 days for fibrinogen• So their measurement is the single best measure of hepatic synthetic function• Tests – Serum prothrombin time •Marked increase in PT >5secs above the control and not corrected by Vit K administration – is a poor prognostic sign in acute viral hepatitis and other acute and chronic liver disease

ENZYMES IN DIAGNOSIS OF LIVER DISEASE

SERUM TRANSAMINASES

ASTALT

ALPGGT5’NT

1.Enzymes whose elevation reflects damage to hepatocytes

2. Enzymes whose elevation reflects cholestasis3. Enzyme test that do not fit into either pattern

• ENZYMES WHOSE ELEVATION REFLECTS DAMAGE TO HEPATOCYTES

• AMINOTRANSFERASES (transaminases) –

They include AST and ALT

SERUM EMZYME TESTS ARE GROUPED IN 3 CATEGORIES

• AST(SGOT) – found in liver> cardiac muscle > skeletal muscle> kidneys >brain

• ALT(SGPT) – found primarily in liver

• Normally present in serum in low concentration (0-40 IU/L)

• When there is damage to liver cell membrane – increased permeability and so increased serum concentration

• Liver cell necrosis is not required for release of these enzymes

•Levels of >1000 IU/L occurs in –• Acute viral hepatitis• Toxin and drug induced hepatitis• Ischaemic liver injury

• In most acute hepatocellular disorders ALT is higher or equal to AST

• Normal ratio is 0.7 to 1.4

• Useful in Wilson disease, chronic liver disease and alcoholic liver disease

• AST/ALT ratio of > 2:1 is suggestive of and >3:1 is highly suggestive of ALCOHOLIC liver disease

• AST in Alcoholic live disease is rarely >300 IU/L

AST/ALT RATIO

• ALT is usually normal in alcoholic liver disease ; can be sometimes low due to an alcohol induced deficiency of pyridoxal phosphate

• AST/ALT <1 is seen in NASH and viral hepatitis

• 2 forms of AST are known- 1. Cytosolic2. Mitochondrial (mAST) – it is synthesized in

precursor form (pre-mAST)

converted to mature mAST

• Accounts for 80% of total AST activity within liver cells

• mAST/total AST ratio – marker of chronic alcohol consumption

• This distinguishes those who consume excess alcohol from normal subjects irrespective of the presence or absence of liver disease

• Isolated rise of ALT is seen in 1. Chronic HepC infection2. Fatty liver

• Isolated AST elevation

1. Alcohol -related 2. Drug -induced liver injury, 3. Hemolysis 4. Myopathic processes

•Determination of these enzymes are helpful in distinguishing hepatocellular from cholestatic jaundice

•Increase in AST and ALT is much more ( >500 IU/L)in hepatocellular jaundice than in cholestatic jaundice (>200 IU/L)

•Persistence of elevated ALT and AST beyond 6 months in a case of hepatitis indicates development of chronic hepatitis

•This AST procedure utilizes a modification of the methodology recommended by the IFCC(International Federation of Clinical Chemistry)

• In this method, aspartate aminotransferase (AST) catalyzes the transamination of aspartate and α-oxoglutarate, forming L-glutamate and oxalacetate

• The oxalacetate is then reduced to L-malate by malate dehydrogenase, while NADH is simultaneously converted to NAD+

•The decrease in absorbance due to the consumption of NADH is measured at 340 nm and is proportional to the AST activity in the sample

• L-Aspartate + α-Oxoglutarate ---------AST---------------- L-Glutamate + Oxalacetate

• Oxalacetate + NADH + H+ ------------MDH--------------- L-Malate + NAD+

• ALT procedure is based on a modification of the methodology recommended by the International Federation of Clinical Chemistry (IFCC)

• ALT transfers the amino group from alanine to α-oxoglutarate to form pyruvate and glutamate

•The pyruvate enters a lactate dehydrogenase (LD) catalyzed reaction with NADH to produce lactate and NAD+

The decrease in absorbance due to the consumption of NADH is measured at 340nm and is proportional to the ALT activity in the sample

3 enzyme activities are important – 1. Alkaline phosphatase (ALP)2. 5’nucleotidase (5’NT)3. Gamma glutamyl transferase (GGT)

ALP – found in liver , bone , placenta and small intestine

Physiological increase in ALP is seen in –4. >60 yrs5. Pregnancy

ENZYMES WHOSE ELEVATION REFLECTS CHOLESTASIS

3. Blood groups O and B – after fatty meal influx of intestinal ALP into blood

4. In children and adolescent during rapid bone growth

ALP >4 times the Normal is seen in –5. Cholestatic liver disease6. Infiltrative liver disease such as cancer and

amyloidosis7. Paget’s disease of bone

• If an isolated increase in ALP is seen , identification of the source of elevated isoenzyme is helpful-

1. Fractionation of ALP by electrophoresis2. Different isoenzymes have diiferent susceptibility

to inactivation by heat • If increased heat stable fraction is found – MC

from placenta • Most sensitive to heat inactivation is – bone ALP

3. Measure serum levels of GGT and 5’NT – they are elevated in only liver disease

• Serum GGT is increased in –1. Alcoholism- Is a helpful clue in suspected cases

of alcoholism ( even in absence of alcoholic liver disease)

2. Cholestasis

• GGT and 5’NT is especially used to assess the nature of ALP

• Normal range: 10-47 IU/L

Serum γ-Glutamyl Transferase

• Normal range: 2-17 IU/L

Serum 5’-Nucleotidase

• Normal range: 39-117 IU/L

Serum alkaline phosphatase

LFT IN ANTI TB TREATMENT

•LFT should be performed before starting anti TB treatment

•With use of rifampicin and isoniazid , onset of liver damage may be as soon as 10days after commencing therapy

•Onset of liver injury may be upto 1 year after starting therapy – so there is no point at which it is safe to stop performing routine LFT

High risk groups are –

1. Known case of liver disease – such as alcoholic liver disease or hepatitis B or C infection

2. Malnourished

3. Children and elderly

NON INVASIVE BIOMARKERS(NIBM) FOR ASSESSING LIVER FIBROSIS

•For assessing live fibrosis – liver biopsy is a preferred method•Utilization of NIBM for liver histology can reduce but not replace the requirement for liver biopsy•Classification of NIBMs

Class 1 fibrosis markers/ direct

biomarkers

Class 2 fibrosis markers/indirect

biomarkers

DIRECT BIOMARKERS –

•These are parts of liver matrix produced by stellate cells during fibrosis process•These include

Procollagen type 1 and 2

Type IV collagen

Hyaluronic acid

Laminin

MMP-1 (collagenases)

MMP-2 (gelatinase A)

MMP-9 (gelatinase B)

TIMPs (tissue inhibitor of matrix metalloproteinase)

INDIRECT MARKERS –• These are molecules released into blood due to liver inflammation• These include1. Serum ALT2. Serum AST/ALT ratio (AAR) - >1 predictive of

cirrhosisBARD score – AAR + BMI + diabetes3. AST/platelet ratio (APRI)4. Forn’s index – age + 3 lab tests (platelet count +

cholesterol level + GGT)

5. PGA index – a marker to differentiate alcoholic liver disease from cirrhosisIncludes – GTT + prothrombin index + apolipoprotein AModified – PGAA – additional alpha2 macroglobulin6. Fibrotest and fibrosure7. Fibro index8. FIB – 4 score9. Fibro Q test10. Steato test

COMBINED DIRECT AND INDIRECT MARKERS –

Fibrometer test

Fibrospect II test

SHASTA index – used in case of HIV/HCV coinfected patients

Hepascore model

European liver fibrosis panel test (ELF)

For assessing fibrosis in HIV/HBV coinfected patients –

1. Fibrometer 2. Hepascore3. Zeng’s score

REFERENCES

1.Harrison’s – text book of medicine2.Kawthalkar clinical pathology3.Henry’s textbook of clinical

pathology

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