LIVER FUNCTION TEST

1. By
Dr KHALED ALGARIRI
CAMS- QASSIM UNIVERSITY
March 2020

2. INTRODUCTION
The liver is a reddish brown wedge shaped organ with
four lobes of unequal size and shape.
A human liver normally weighs 1.44–1.66 kg (3.2–
3.7 lb).
Liver function tests are blood tests used to help diagnose and monitor
liver disease or damage.
The tests measure the levels of certain enzymes and proteins in our
blood.
 Some of these tests measure how well the liver is performing its normal
functions of producing protein and clearing bilirubin, a blood waste
product.

3. FUNCTIONS OF THE LIVER
1-Synthetic functions : synthesis of plasma proteins,
cholesterol, triacyl glycerol, lipoprotein
2-Metabolic function: protein metabolism, ketogenesis,
TCA cycle, production of ATP
3-Detoxification & excretion: ammonia to urea, bilirubin,
cholesterol, drug metabolites.
4- Homeostasis : blood glucose regulation
5-Storage function: Vitamin A,D,K,B12
6-Production of bile salts

4. Why it's done
 Screening: They are non invasive yet sensitive modality for liver
dysfunction.
 Pattern of disease: They are helpful to recongnize pattern of
various diseases. Like being helpful in differentiating between
acute viral hapatitis and various cholestatic disorders and chronic
liver disease.(CLD)
 Assess severity : They are heplful to assess the severity and predict
the outcome of certain diseases like primary biliary cirrhosis.
 Follow up: They are helpful in the follow up of certain liver
diseases and also helpful in evaluating response to therapy like
autoimmune hepatitis

5. CLASSIFICATION OF LIVER FUNCTIONS
TESTS

6. 1.Serum bilirubin
2.Urine bilirubin
3.Urine and feacal urobilinogen
4.Urine bile salts
5.Dye excretion tests
TESTS BASED ON
EXCRETORY
FUNCTION

7. TESTS BASED ON
DETOXIFICATION FUNCTION
Hippuric acid
test
Determination
of blood
ammonia

8. TESTS BASED ON SYNTHETIC
FUNCTION
Prothrombin
time
Protein
time

9. TESTS BASED ON METABOLIC
FUNCTION

10. ENZYMES IN DIAGNOSIS OF
LIVER DISEASE
SERUM
TRANSAMINASES
AST
ALT
ALP
GGT
5’NT

11. 1.Serum bilirubin
2.Urine bilirubin
3.Urine and feacal urobilinogen
4.Urine bile salts
5.Dye excretion tests
TESTS BASED ON
EXCRETORY
FUNCTION

12. Serum bilirubin
Bilirubin: is the end product of heme degradation derived from
breakdown senescent (aging) erythrocytes by mononuclear
phagocytes system specially in the spleen, liver and bone marrow.
The major pigment present in bile is the orange compound
bilirubin.
It is highly soluble in all cell membranes (hydrophobic) and is also
very toxic. Therefore, its excretion in the bile is one of the very
important functions of the liver.
Classification of bilirubin into direct & indirect bilirubin is based
on original van den bergh method of measuring bilirubin.

13. Extravascular Pathway for RBC Destruction
(Liver, Bone marrow,
& Spleen)
Hemoglobin
Globin
Amino acids
Amino acid pool
Heme Bilirubin
Fe2+
Excreted
Phagocytosis & Lysis
Recycled
The globin is
recycled or
converted into
amino acids, which
in turn are recycled
or catabolized as
required

14. BILIRUBIN METABOLISM
 .Bilirubin is the excretory product formed by the catabolism of heme part of
haemoglobin.
 .Porphyrin part of heme are converted to bilirubin in reticuloendothelial cells
of liver, spleen, bone marrow.
 Unconjugated bilirubin is bound to serum albumin &transferred to liver
where it is conjugated to glucuronate by UDP GLUCURONYL TRANSFERASE.
 Conjugated bilirubin is excreted into bile. A fraction of bilirubin from stool is
reabsorbed into blood via portal circulation (enterohepatic circulation)

15. Bilirubin Is the Major Component of Bile
Pigments,Steps of Execretion
1. Hemoglobin is first dissociated into heme and globin.
2. In the presence of NADPH and O2, the Heme oxygenase
enzyme hydroxylates Heme and converts it into Biliverdin.
3. Biliverdin is then reduced or converted into bilirubin by
biliverdin reductase enzyme. Bilirubin is transported in
blood bound to albumin forming a water soluble compound
called hemobilirubin (unconjugated bilirubin, free bilirubin)
which is rapidly transported to hepatocytes for further
metabolism .

16. RBCs Breakdown
Hemoglobin Produces & Breakdown
Heme
Biliverdin
Bilirubin
Heme
Oxygenase
Biliverdin
Reductase

17. Role of Blood Proteins in the Metabolism of
Bilirubin
1. Albumin
Dissolved in Blood

18. Blood
Liver
Ligandin
(-) charge
Ligandin
(-) charge
Ligandin Prevents bilirubin from
going back to plasma

19. 4-The liver removes bilirubin from the circulation
rapidly, mediated by a carrier protein (receptor),
and conjugates it with glucuronic acid. This
reaction is catalyzed by the enzyme glucuronyl
transferase in the smooth endoplasmic reticulum
to have conjugated bilirubin, which is more water
soluble than bilirubin.
5-The bilirubin-glucuronide (conjugated bilirubin)
is secreted into the bile canaliculi.
Note: the unconjugated bilirubin is normally not secreted.
6-In the small intestine, bilirubin glucuronide is
poorly absorbed. In the gut, however, bacteria
deconjugate it back to bilirubin, and convert it to
the highly soluble colorless compound called
Urobilinogen

20. 7- Only 20% of Urobilinogen can be absorbed by
the small intestine (this represents the
enterohepatic circulation of bile pigments). 70%
of the Urobilinogen can be oxidized in the large
intestine to Stercobilin and stercobilinogen (by
bacteria).
10 % of Urobilinogen is
excreted in either urine
(where it is converted to
yellow urobilin in the
kidney) or feces (after it
is converted to
Stercobilin which is
responsible for the
brown color of feces).
(entero-hepatic circulation)

22. Insoluble in water
Present normally in plasma
Tightly complex to albumin
Not filtered through renal
glomeruli, is not excreted in urine
Toxic substance
The chief form of bilirubin in the
blood
Unconjugated
Water soluble and
Present normally in bile
Loosely bound to albumin
Filtered through renal glomeruli
and excreted in urine
Non-toxic
Present in low concentration in the
blood
Conjugated
Differences between the conjugated and
unconjugated bilirubin

23. Diagnostic Importance of Bilirubin
• Disruption of bilirubin metabolism and excretion can
cause hyperbilirubinaemia and subsequent
jaundice
• Hyperbilirubinaemia maybe unconjugated (indirect)
or conjugated (direct) depending on the type of
bilirubin present in plasma.

24. A-Unconjugated Hyperbilirubinemia
• It is due to overproduction of bilirubin by
reticuloendothelial system over the capacity of the liver to
remove and clear from blood. It is characterized by high
level of indirect or unconjugated bilirubin. This type of
bilirubin can cross the blood-brain barrier into the
central nervous system and cause kernicterus

25. Unconjugated hyperbilirubinemia occurs in the
following conditions
I-Neonatal or Physiologic Jaundice: This is the most
common cause of jaundice in neonatal age. It results from
accelerated hemolysis and immature hepatic system for
uptake, conjugation and secretion of bilirubin.
II-Hemolytic jaundice (Anemia)

26. III-Congenital Syndromes related to uptake and
conjugation of bilirubin as follow :
1- Crigler-Najjar Syndrome
 Type 1: Due to severe decrease in the activity of UDP- glucuronyl
transferase.
 Type II: Due to decreased activity of UDP-glucuronyl transferase that
adds the second glucuronide group.
2- Gilbert Disease
 It is mainly due to hepatic defect in the uptake of bilirubin by
liver cells.
3-Toxic Hyperbilirubinemia
 This is due to toxin-induced liver dysfunctions e.g.
chloroform, carbon tetrachloride and mushroom poisoning.

27. B-Conjugated Hyperbilirubinemia
• Conjugated hyperbilirubinemia is due to reflux
of direct or conjugated bilirubin into blood due to
biliary obstruction, conjugated bilirubin is water
soluble, so it is excreted in urine and darken its
color.

28. Conjugated hyperbilirubinemia occurs in the following
conditions
1-Obstructive Jaundice (Cholestatic Jaundice)
Conjugated hyperbilirubinemia results from blockage of hepatic or common bile
duct (stones and tumors).
2- Micro-obstruction of intrahepatic biliary ductules by swollen
damaged hepatocytes e.g. viral hepatitis and liver cirrhosis. Both cases are
associated with marked increase of conjugated bilirubin and slight to moderate
increase of unconjugated bilirubin (mixed hyperbilirubinemia)

29. 3- Rotor’s/Dubin-Johnson syndrome – defective excretion of
conjugated bilirubin into the biliary cannaliculi therefore elevated conjugated
bilirubin

30. • Jaundice becomes clinically evident when the serum
bilirubin level exceeds 2.5mg/dL.
• Several types of Jaundice:
– Hemolytic
– Hepatocellular
– Obstructive
• Symptoms:
– Yellow discoloration of the skin, sclera and mucous membranes
– Itching (pruritus) due to deposits of bile salts on the skin
– Stool becomes light in color
– Urine becomes deep orange and foamy

31. Normal range
Bilirubin type Bilirubin level
Total bilirubin 0.0-1.4 mg/dL or 1.7-20.5 mcmol/L
Direct bilirubin 0.0-0.3 mg/dL or 1.7-5.1 mcmol/L
Indirect bilirubin 0.2-1.2 mg/dL or 3.4-20.5 mcmol/L

34.  The presence of urine bilirubin indicates hepatobiliary disease.
Unconjugated bilirubin is tightly bound to albumin and not
filtered by the glomerulus and thus not present in urine.
Measurable amount of conjugated bilirubin in serum are found
only in hepatobiliary disease.
 Because the renal threshold for conjugated bilirubin is low and
the laboratory methods can detect low levels of bilirubin in
urine so conjugated bilirubin may be found in urine when the
serum bilirubin levels are normal. This is the case in early acute
viral hepatitis.
URINE BILIRUBIN

35. 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

36. URINE UROBILINOGEN
 An increase in the urobilinogen in urine is a sensitive indicator
of hepatocellular dysfunction. Itis a good indicator of alcoholic
liver damage, well compensated cirrhosis or malignant disease
of liver.
 In viral hepatitis it appears early in urine. It is markedly
increased in hemolysis.
 In cholestatic jaundice urobilinogen disappears from urine. It
is intermittently present in case of gallstones.
 Urobilinogen gives a purple reaction to Ehrlich’s aldehyde
reagent

37. 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

38.  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

39. ENZYMES IN DIAGNOSIS OF LIVER DISEASE

40. SERUM ENZYMES REFLECT THE DAMAGE OF
HEPATOCYTES
A large number of enzyme estimations are available which are
used to ascertain liver function.
They are be divided into two groups:
 A). Enzymes indicating hepatocellular damage.
 B). Enzymes indicating cholestasis (obstruction).
In liver cells injury , damage to the membrane of cells &
organelles allows intracellular enzymes to leak into the blood

41. A- Enzymes indicating hepatocellular damage
 AST (Aspartate transaminase)/SGOT (serum glutamate oxaloacetate
transaminase)
Normal range: 10-45 U/L.
 AST is found in both cytoplasm & mitochondria
 AST/GOT also reflects damage to the hepatic cells & is less specific for
liver disease.
 > skeletal
 muscle> kidneys >brain
 • AST help diagnose various heart, muscle or brain disorders, such as a
myocardial infarct (heart attack).

42. Alanine transaminase ALT sGPT
Normal Range: 5-40 U/L.
 ALT(SGPT) – found primarily in liver .
ALT is specific for liver disease.
Its elevations favor liver cell necrosis as a cholestasis.

43. Serum aminotransferases (AST-ALT) are the sensitive
markers of acute hepatocellular injury.
Serum alanine aminotransferase or ALT (formerly called
serum glutamic-pyruvic transaminase or SGPT) is a cytosolic
enzyme.
When necrosis or death of cells containing these enzymes
occurs, aminotransferases are released into the blood and
their concentration in blood increases whose level
correlates with extent of tissue damage.

44. Most marked elevations of ALT and AST (>15 times normal)
are seen in acute viral hepatitis, toxin-induced
hepatocellular damage (e.g. carbon tetrachloride), and
centrilobular necrosis due to ischemia (congestive cardiac
failure).
Moderate elevations (5-15 times) occur in chronic hepatitis,
autoimmune hepatitis, alcoholic hepatitis, acute biliary tract
obstruction, and drug-induced hepatitis.
Mild elevations (1-3 times) are seen in cirrhosis,
nonalcoholic steatosis, and cholestasis.
Clinical Significant of Transaminase
Enzymes

45. Clinical Significant of Transaminase Enzymes
Increase of AST and ALT is much more in hepatocellular jaundice
(>500 units/ml) than inn cholestatic jaundice (<200 units/ml).
ALT and AST are elevated in acute viral hepatitis even before the
appearance of jaundice.
Persistence of elevated ALT and AST beyond 6 months in a case
of hepatitis indicates development of chronic hepatitis
Normal ALT:AST ratio is 0.7 to 1.4.

46. 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.
Clinical Significant of Transaminase
Enzymes

47. ELEVATION OF SERUM AST MAY INDICATE
Acute hemolytic anemia
Cirrhosis of the liver
Hepatitis
Acute pancreatitis or inflammation of pancreas
Acute renal failure or loss of kidney function.
Heart attack
Primary muscle disease
Recent surgery

48. ELEVATION OF SERUM ALT MAY INDICATE
Alcoholic liver disease
Cancer of liver
Hepatitis or inflammation of the liver
Noncancerous tumor of the liver
Use of medicines or drugs toxic to the liver
Cirrhosis or scarring of the liver
Death of liver tissue.

49. AST:ALT ratio
Normal ratio is 0.7 to 1.4
Useful in Wilson disease, chronic liver disease and alcoholic
liver disease
 AST/ALT ratio > 3:1 is highly suggestive of Alcoholic liver disease
AST in Alcoholic live disease is rarely >300 IU/L.
 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 viral hepatitis.

50. Clinical Significant of serum AST/ALT ratio

51. B- ENZYMES THAT DETECT CHOLESTASIS
1. ALKALINE PHOSPHATASE
2. Gamma-GLUTAMYL TRANSPEPTIDASE
3. 5-NUCLEOTIDASE

52. 1-ALKALINE PHOSPHOTASE
ALP occurs in in all tissues, especially liver, bone, bile duct, kidney & the
placenta.
The ALP used to help diagnose certain liver diseases and bone disorders.
Normal range: 30 - 95 IU/L (3-13 kings unit)
ALP is a hydrolase enzyme responsible for removing phosphate groups from
many types of molecules, including nucleotides & proteins.
Levels are significantly higher in growing children.
A rise in serum ALP usually associated with elevated serum bilirubin is an
indicator of biliary obstruction (obstructive/post hepatic jaundice).
ALP is also elevated in cirrhosis of liver & hepatic tumors.

53. Physiological increase in ALP is seen in –
1. >60 yrs
2. Pregnancy
3. Blood groups O and B – after fatty meal influx of intestinal ALP into blood
4. In children and adolescent during rapid bone growth
If an isolated increase in ALP is seen , identification of the source of
elevated isoenzyme is helpful- by fractionation of ALP by electrophoresis.
Measure serum levels of GGT and 5’NT – they are elevated in only liver
disease

54. Isoenzymes of Alkaline Phosphatase
Hepatic Isoenzyme – Travels fastest towards the anode .Its level
rises in extra hepatic biliary obstruction.
2. Bone Isoenzyme-Increases due to osteoblastic activity and is
normally elevated in children during periods of
active growth .
3. Placental Isoenzyme - Rises during last 6 weeks of pregnancy.
4. Intestinal Isoenzyme- Rise occurs after a fatty meal. May
increase during various GI disorders.

55. Clinical Significant of ALP
 Normal range Adult: 42.0-128.0 IU/L. Children: 82.0-390 IU/L
 In acute viral hepatitis, alkaline phosphatase is usually either normal or moderately
increased. Hepatitis A may present a cholestatic picture with marked and prolonged
itching and elevation of alkaline phosphatase. Tumor may secrete alkaline
phosphatase into plasma and there are tumor specific isoenzymes such as Regan,
Nagao and Kasahara isoenzymes.
 Hepatic and bony metastasis can also cause elevated levels of alkaline phosphatase.
Other diseases like infiltrative liver disease, abscesses, granulomatous liver disease
and amyloidosis may also cause a rise in alkaline phosphatase. Mildly elevated levels
of alkaline phosphatase may be seen in cirrhosis and hepatitis ofncongestive cardiac
failure.
 Low levels of alkaline phosphatase occur in hypothyroidism, pernicious anemia, zinc
deficiency and congenital hypophosphatasia.

56. Enzymes Prehepatic
Jaundice
Hepatic
Jaundice
Obstructive
Jaundice
ALS or ALT Normal Marked
Elevation
Elevation
ALP Normal Slightly
increase
Marked
Elevation
Enzyme assays in differential
diagnosis of Jaundice

57. Abbreviations: GGT, γ-glutamyltranspeptidase; L-AP, liver-specific alkaline
phosphatase; B-AP, bone-specific alkaline phosphatase; 5′nT, 5′- nucleotidase;
HFP, hepatic function panel; THP, transient hyper-alkaline phosphatemia; Ca/P,
calcium/phosphorus; PTH, parathyroid hormone

58. Gamma Glutamyl transpeptidase(GGT) is a membrane bound glycoprotein
which catalyzes the transfer of gamma glutamyl group to other peptides,
amino acids and water.
Large amounts are found in the kidneys, pancreas, liver, intestine and
prostate.
 The levels of gamma glutamyl transpeptidase are high in neonates and
infants up to 1 year and also increase after 60 years of life. Men have higher
values. Children more than 4 yr old have serum values of normal adults.
The normal range is 0-30IU/L.
In acute viral hepatitis the levels of g glutamyl transpeptidase may reach its
peak in the second or third week of illness and in some patients they
remain elevated for 6 weeks.
2- Gamma GLUTAMYL TRANSPEPTIDASE GGT

59. Clinical Significant of GGT
Often clinicians are faced with a dilemma when they see
elevated alkaline phosphatase levels and are unable to
differentiate between liver diseases and bony disorders and in
such situations measurement of gamma glutamyl transferase
helps as it is raised only in cholestatic disorders and not in bone
diseases.
Other conditions causing elevated levels of Gamma glutamyl
transpeptidase include uncomplicated diabetes mellitus, acute
pancreatitis and myocardial infarction. Drugs may increase the
levels of Gamma glutamyl transpeptidase..

60. Clinical Significant of GGT
The enzyme present in serum appears to originate primarily from
the Hepatobiliary system.
GGT activity is elevated in all forms of liver disease like
1. Obstructive jaundice
2. Cholangitis
3. Cholecystitis
4. Biliary atresia
5. Infectious hepatitis

61. Clinical Significant of GGT
Relatively high sensitivity and specificity because of their
measurement is easy and in expensive.
 Elevated earlier in liver diseases.
 Early detection of chronic alcohol misuse.
Enzyme level found correlate with the duration of the drug
action.

62. 3- 5'-Nucleotidase
Normal range: 2-15 U/L
 The serum activity of 5'-nucleotidase is elevated
 in hepatobiliary disease & this parallels ALP.
 It is highest in post-hepatic obstructive jaundice.
The 5'-nucleotidase is normal in patients with bone
disease where as serum ALP increased.
 GGT and 5’NT is especially used to assess the nature of
ALP.

63. TESTS BASED ON SYNTHETIC
FUNCTION
Prothrombin
time
Protein
time

64. SYNTHETIC FUNCTION OF LIVER
Liver is involved in synthesis of many plasma proteins like BLOOD CLOTTING
FACTORS, LIPO PROTEINS, ETC..
• SYNTHESIS of these compounds may be affected in pathological conditions of
liver..
• i..e their concentration in plasma may decrease, however of their long HALF
LIIFE and REGENARATION capacity of liver decrease may be apparent only on
long standing liver dieases
• THESE INCLUDE
1. Serum Albumin
2. Prothrombin
3. Serum Glubins

65. 1-Serum Albumin
In severe liver diseases, hypoalbunemia occurs.
• Since half life of albumin is 20 days, decrease in albumin
occurs in chronic liver diseases.
• Albumin
• it is most abundant protein in serum [120 mg/kg/day]
• Reduction of albumin occurs in
• Impaired synthesis (malnutrition, malabsorption, hepatic
dysfunction,cirrhosis)
1. Loss (ascites, protein losing-nephropathy, enteropathy)
2. May result in peripheral oedema.

66.  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 -
1. Protein losing enteropathy
2. Nephrotic syndrome

67. 2-Serum Globulins
 In chronic liver dieases globulins increase due to
decrease clearance by hepatocytes
IgA level increased in all types of cirrhosis
IgG level increases in Auto-immune hepatitis and
cirrhosis
IgM elevates in biliary cirrhosis

68. 3-Prothrombin Time PT
Since prothrombin is one coagulation factor synthesized by Liver,
any damage to liver can causes decrease in synthesis of Prothrombin
i.e Hypoprothrombinaria indicates liver dysfunction
i.e Increased in prothrombin time
Since PT is also prolonged in Vitamin-K Deficiency it is carefully
ruled out by estimating PT BEFORE and AFTER Vitamin-K
administration.
PT also be prolonged in chronic obstructive Jaundice due to
resistant in Vitamin-K reabsorption (malabsorption of Vitamin-K)
THUS PT IS USEFUL IN DIAGNOSIS OF JAUNDICE AND LIVER
FUNCTION

69. Normal value : 10 to 15 sec.

70. TESTS BASED ON
DETOXIFICATION FUNCTION
Hippuric acid
test
Determination
of blood
ammonia

72. Hippuric acid test
Hippuric acid test
• 6gm of sodium
benzoate dissolved in 250ml water
• Collect urine for next 4 hour
Hippuric acid is a normal component of urine and is typically
increased with increased consumption of phenolic compounds
(tea, wine, fruit juices). These phenols are converted into
benzoic acid which is then converted into hippuric acid and
excreted in the urine.

73. THANK YOU