Dr/ Ragaa Salama 1

Functions of the liver • Carbohydrate metabolism• Gluconeogenesis • Glycogen synthesis and

breakdown• Fat metabolism• Fatty acid synthesis • Cholesterol synthesis and

excretion• lipoprotein synthesis• Ketogensis • bile acid synthesis • 25-hydroxylation of vitamin D• Protein metabolism• synthesis of plasma proteins • Some coagulation factor• Urea synthesis

• Hormonal metabolism • Meabolism and excretion of

steroid hormones• metabolism of polypeptide

hormones• Drugs and foreign compounds• metabolism and excretion• Storage • Glycogen, Vitamin A , B12 , iron• Metabolism and excretion of

bilirubin

Dr/ Ragaa Salama 2

Functions of the Liver & Liver Function Tests• Classified into : • 1-Metabolic• 2-hematological function• 3-Excretory function measure bilirubin• 4-storage • 5-serum enzymes derived from liver AST, ALT, ALP.

GGT, 5’ nucleotidase.• 6- Synthetic function Serum protein , albumen,

prothrombin time• 7-detoxification and phagocytosis.

Dr/ Ragaa Salama 3

Liver Function Tests•biochemical investigation to detect the abnormalities and the extent of liver damage •Alanine aminotransferase(ALT) or SGPT•Aspatate aminotransferase (AST) or SGOT•Total protein• albumin / globulin ratio ( A / G ratio)•Alkaline phosphatase•Bilirubin•5'Nuclotidase•Gamma Glutamyl transferase ( GGT).•Alpha fetoprotein (AFP).•Lactate dehdrogenase (LDH).•Ammonia•Prothormbin time

Liver Function TestsLiver chemistry test Clinical implication of abnormality

ALT Hepatocellular damage

AST Hepatocellular damage

Bilirubin Cholestasis, impair conjugation, or biliary obstruction

ALP Cholestasis, infiltrative disease, or biliary obstruction

PT Synthetic function

Albumin Synthetic function

GGT Cholestasis or biliary obstruction

Bile acids Cholestasis or biliary obstruction

5`-nucleotidase Cholestasis or biliary obstruction

LDH Hepatocellular damage, not specific4Dr/ Ragaa Salama

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Principle: transamination reaction

Glutamate

COOH

HC

CH2

CH2

COOH

NH2

COOH

C

CH3

O +

Pyruvate

-Ketoglutarate

COOH

C

CH2

CH2

COOH

COOH

HC

CH3

+

Alanine

ONH2 GPT, ALTPLP

Glutamate

COOH

HC

CH2

CH2

COOH

NH2+

-Ketoglutarate

COOH

C

CH2

CH2

COOH

COOH

HC

CH2

+

Aspartate

ONH2 GOT, ASTPLP

COOH

COOH

C

CH2

OxaloacetateCOOH

O

Dr/ Ragaa Salama 6

Alanine aminotransferase(ALT )

Aspatate aminotransferase (AST) • Enzymes reflect hepatocellular damage.• Transfer of amino groups from aspartate or alanine to α-

Ketoglutarate leading to formation of oxaloacetate and pyruvate.

• ALT is found mainly in liver cells, more specific to liver• It is sensitive index of acute hepatocellular injury • Causes of increased level:• 1- hepatitis 2- cirrhosis 3-obstructive jaundice• AST is found mainly in cardiac, hepatic, muscle and kidney.• AST following myocardial infarction ,a peak 48-60 h after

infarction

Dr/ Ragaa Salama 7

Estimation of Alanine aminotransferase (ALT ) (SGPT)• Principle: the rate of decrease in the absorbance is proportional to ALT activity ALT• α- Ketoglutarate + alanine glutamate + pyurvate LDH• Pyurvate + NADH+H lactate + NAD+ + H2o• Procedure:

Test

Reconstituted reagent 3ml

Pre-warmed at 37 ºC then added :

Sample 0.2 ml (200 µl)

Mix and incubate at 37ºC for 1 min. Read the absorbance A1 at 340nm against d H2O . Re-incubate at 37ºC and after exactly 3 min read the absorbance (A2

) .

Calculation: ( A1 ــــ A2 ) x 857 U/L

Normal value of ALT = 5-40 U/L

Dr/ Ragaa Salama 8

Estimation of Aspartate aminotransferase (AST ) (SOPT)• Principle: the rate of decrease in the absorbance is proportional to AST activity AST• α- Ketoglutarate + aspartate glutamate + oxaloacetate MDH• oxaloacetate + NADH.H+ L-Malate + NAD+ + H2o• Procedure:

Test

Reconstituted reagent 3ml

Pre-warmed at 37 ºC then added :

Sample 0.2 ml (200 µl)

Mix and incubate at 37ºC for 1 min. Read the absorbance A1 at 340nm against d H2O . Re-incubate at 37ºC and after exactly 5 min read the absorbance (A2

) .

Calculation: ( A1 ــــ A2 ) x 514 U/L

Normal value of AST = 5-40 IU/L

Dr/ Ragaa Salama 9

BilirubinProduced by catabolism of old RBCs and other hemoproteins

( cytochrom oxidase, P-450).Normal level ( total) is ≤ 1 mg/dl (17.1 µmol /L) direct ≤ 0.2 mg/dlHyperbilirubinemia :bilirubin in the blood 1.0 mg/dL. Genral Causes of Hyperbilirubinemia :1- production of bilirubin more than liver capacity, 2-liver cell damage 3-failure of liver to excrete it in bile 4- obstruction of bile pathways. At bilirubin blood level of 2 - 2.5 mg/dL, bilirubin diffuses to

tissues (e.g., skin, mucous membranes and sclera of the eyes) and stains them yellow, a condition called jaundice or icterus.

Kernicterus: high level of unconjugated bilirubin can pass immature blood brain barrier causing a necrotic syndrome that occurs from bilirubin neurotoxicity usually in low birth weight infant.

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Bilirubin Metabolism

stercobilnogen in stool

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• Van den Bergh reaction• This is specific reaction to identify the increase in serum

bilirubin level .• Normal serum gives a negative Van den Bergh reaction .• Principle • Van den Bergh reaction is a mixture of sulfanilic acid and

sodium nitrate• sulfanilic acid + sodium nitrate → Diazotized

sulfanilic acid• Diazotized sulfanilic acid + bilirubin → Azobilirubin

( purple color).

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Blank Test

sulfanilic acid 3ml 3ml

sodium nitrate - 0.05 ml

d H2O 0.05 ml -

Mix and wait for 1min

Serum sample 0.2ml (200 µl) 0.2ml (200 µl)

After 1 min read the absorbance of test and blank at 540nm against d H2O ( direct biliurbin) then add

Methanol 3ml 3ml

Mix by inversion and wait for 5 min read the absorbance of test and blank at 540nm against d H2O ( total biliurbin)

Standard: Pour Bilirubin Equivalent Standard (2.5 mg/dl then 5 mg/dl )into a clean vial, read and record its absorbance against d H2O at 450 nm

Calculation: direct biliurbin= A (test)- A(blank) x 2.5 mg/dl A (standard)Total bilirubin= A (test)- A(blank) x 5 mg/dl Indirect bilirubin=total - direct A (standard)

Procedure : pipette in a clean dry test tubes:

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Hemolytic J aundice

Obstructive J aundice

HepaticJ aundice

Bilirubin Unconjugated Conjugated Both

VonDenBerg Indirect + Direct + Biphasic

Serum enzymes

ALT,AST,ALP normal

ALP ALT,AST

ALT,AST ALP

BilirubinIn urine

Not excreted excreted excreted

urobilinogen Excreted Normal or ↓ Normal or ↓

Comparison between 3 types of jaundice

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Types of Hyperbilirubinemias (Jaundice) I- Uncojugated hyperbilirubinemia: causes:Hemolytic diseases: Hemolytic anemia , Rh incompatability, G6PD. the unconjugated hyperbilirubinemia is mild because of the ability of adult

liver to get rid of bilirubin.Neonatal (Physiological) jaundice: ↑hemolysis due to immature hepatic

system. Treated by phenobarbital and blue light phototherapy → bilirubin excretede in

bile without conjugation. Crigler-Najjar syndrome type I and II: It is an autosomal recessive absence

or deficiency of UDP-glucuronyltransferase in the liver. Treated by phenobarbital and

blue light phototherapy may be fatal by age 15 months.Gilbert syndrome: harmless mild hyperbilirubinemia due ↓ UDP-

glucuronyltransferase.Toxic hyperbilirubinemia: liver cell damage by CCl4, cirrhosis, viral hepatitis

and chloroform.Brest feed hyperbilirubinemia: β –glucurnidase in breast milk which

deconjugate bilirubinLucy-drescall syndrom: familial ,mild,last 2-3 weeks due to inhibitor of

congjugation

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Alkaline phosphatase• ALP is present at or in cell membrane .• The response of the liver to any form of biliary tree obstruction is to induce

the synthesis of ALP. • The main site of new enzyme synthesis is the hepatocytes adjacent to the

biliary canaliculi. • Some of the newly formed enzyme enters the circulation to raise the enzme

level in serum

II - Conjugated hyperbilirubinemia: causes :Obstructive (cholestatic) jaundice: It is due to the obstruction of hepatic or common bile ducts that regurgitate conjugated bilirubin into the blood with choluria.Chronic idiopathic jaundice (Dubin-Johnson and Rotor syndromes): They are benign inherited defect in transport system of conjugated bilirubin.

Alkaline phosphatase

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test

Working reagent 1 volume of ALP substrate + 9 vol of ALP buffer →1ml

Pre-worm at 37ºC add

sample 20 µl serum

Mix and incubate at 37ºC for 1 min. Read the absorbance A1 at 405 nm against d H2O . Re-incubate at 37ºC and after exactly 3min read the

absorbance (A2 ) → ∆ A

calculation ∆ A/min = A2 -― A1 / 2 , ∆ A/min X 2720 = U/L

principleHydrolysis of p-nitrophenyl phosphate → alkaline pH→ yellow p-nitrophenoxide ions.

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Alkaline phosphatase• ALP is present at or in cell membrane, Its function by 3 ways • 1- Hydrolysis 2-Phosphotrnsferase 3- pyrophosphtase• Sources : Liver , bone ( osteoblast), intestinal epithelial cells

proximal convoluted tubules of the Kidney and placenta .• Clinical significance:• Physiological causes: growing children , healing of bone

fracture, 3rd trimester of pregnancy from placenta, lactation.• Pathological causes :• 1- hepatobiliary dis ( extrahepatic ,intra hepatic obstruction),

Infectious hepatitis • 2- bone dis. associated with increase osteoblastic activity like

Paget’s disease (10-25 fold) as osteoblast rebuild bone that resorbed by uncontrolled activity of osteoclast, osteogenic bone cancer

• Moderate rise in osteomalacia but normal in osteoprosis,• rickets (2-4 fold),Fanconi Syndrom,1ry&2ry hyperparathyrodism

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Albumin• Synthesized mainly by the liver• Osmotic effect• Transport substances• Half life is 3 weeks• It decreased in liver diseases• Non-hepatic causes of

hypoalbuminemia( Differential diagnosis) :• Decreased synthesis : malnutrition, malabsorbtion,

malignant diseases.• Increased catabolism : injury, postoperative• Acute inflammation• Chronic inflammation• Increased loss ( nephrotic syndrome, protein –losing

enteropathy, burns, exudative skin disease

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• Liver is the primary site for synthesis of plasma proteins

• In acute hepatic dysfunction , little changes • In chronic, ↓ albumen, globulin, A/G ratio is

inverted in chirrosis.• ↓ albumen, in sever liver disease, in active hepatitis

suggests a poor prognosis.• In portal hypertension, albumen leaks of liver

surface→ peritoneal cavity → osmotic pressure of peritoneal cavity → ascities.

• α 1 globulin, α 1 antitrypsin, α 2 & β globulin in cirrhosis, chronic cholestasis due to production and ↓ clearance.

• IgG autoimmune hepatitis& cirrhosis.

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Estimation of serum albumin• Principle• Albumin+ Bromocresol green Albumin bromocresol green complex

Blank Standard test

Albumen color reagent

2.5 ml 2.5 ml 2.5 ml

Standard - 0.2ml (200 µl) -

sample - - 0.2ml (200 µl)

Mix and allow to stand at room temp. For 5 min

Set wavelength at 54 0 nm and zero instrument with the blank .Read absorbance of all tubes within min.

Calculation: A (test) x 5 g/dl ( concentration of standard) A (standard)

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Estimation of serum total protein• Principle• Protein + Biurt reagent( Cu+2) Alkaline pH Cu+2-protein complex

( blue color).

Blank standard test

Biuret reagent 1 ml 1 ml 1 m l

standard - (20µl ) -

sample - - 0.02 ml (20µl)

Mix and let stand at room temp. for 10min.Read the absorbance of standard and test at 540 nm against blank

Calculation: A (test) x 5 g/dl ( concentration of standard)

A (standard)

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5'Nuclotidase• Indication: Isolated increase in alkaline

phosphatase.• Alkaline phosphatase and 5'Nuclotidase

usually increase and decrease in parallel in hepatobiliary disease.

• Alkaline phosphatase and 5’ nuleotidase: found near the bile canalicular membrane of hepatocytes REFLECT CHOLESTASIS

• Gamma Glutamyl transferase ( GGT).• in all types of liver diseases

α- fetoprotein­ hepatocelular carcinoma ,­ Acute & chronic hepatitis. ­ It is present in early fetal life and reappear

in malignant liver, used as tumor marker

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Coagulation factors:With the exception of Factor VIII, the blood clotting factors are made exclusively in hepatocytes.

Because of their rapid turnover, measurement of the clotting factors is the single best acute measure of hepatic synthetic function and helpful in both the diagnosis and assessing the prognosis of acute parenchymal liver disease

24Dr/ Ragaa Salama

• Serum prothrombin time: collectively measures factors II, V, VII, and X

Marked prolongation of prothrombin time, > 5s above control is a poor prognostic sign in acute viral hepatitis and other acute and chronic liver diseases.

25Dr/ Ragaa Salama