LIVER FUNCTION TEST

Members:

Bangquiao, Nina Marie Darullo, Christine

Cuenca Elaine Sangilan, Kate

Carreon, Doughtie Wilkom, Fritz

Contents:

1. Anatomy and Physiology of the Liver

● The liver is the largest organ in the body, weighing approximately 1500g or 3

pounds.

● It is shaped like a cone and dark reddish- brown in color.

● The liver is located in the upper right-hand portion of the abdominal cavity,

beneath the diaphragm, and on top of the stomach, right kidney, and intestines.

The liver holds about one pint (13%) of the body's blood supply at any given moment.

> Functions of the Liver

Liver is a versatile organ which is involved in metabolism and independently involved in

many other biochemical functions. Regenerating power of liver cells in tremendous.

● Metabolic functions: Liver is the key organ and the principal site where the

metabolism of carbohydrates, lipids and proteins take place.

> ammonia is converted to urea

> cholesterol is synthesized, and catabolized to form bile acids and bile salts

> esterification of cholesterol takes place

● Secretory functions:

- Liver: responsible for the formation and secretion of bile in the intestine

> Bilirubin is conjugated in liver cells and secreted in the bile

● Excretory functions:

- BSP (bromsulphthalein) and Rose Bengal dye are exclusively excreted through liver

cells

● Synthesis of Certain Blood Coagulation Factors:

- Liver cells are responsible for conversion of preprothrombin (inactive) to active

prothrombin in the presence of Vitamin K

- produces other clotting factors, factor V, VII and X

- fibrinogen in blood coagulation is also synthesized in liver

● Synthesis of Other Proteins:

- Albumin is solely synthesized and also α and β globulins

 ● Detoxification Function and Protective Function:

- Kupffer cells remove foreign bodies from blood by phagocytosis

- it can detoxicate drugs, hormones and convert them into less toxic substances for

excretion

● Storage function:

- stores glucose in the form of glycogen

- also stores vitamin B12 and A, etc.

● Miscellaneous Function:

- Liver: involved in blood formation in embryo and in some abnormal states

- also forms blood in adult

2. Bilirubin

a. Metabolism

Bilirubin is the catabolic product of heme metabolism. Within physiologic range, bilirubin has

cytoprotective and beneficial metabolic effects, but in high levels it is potentially toxic.

Normal bilirubin metabolism can be summarized as a series of steps, including (1) production,

(2) uptake by the hepatocyte, (3) conjugation, (4) excretion into bile ducts, and (5) delivery to

the intestine. Jaundice can result from defects in any of these steps of bilirubin metabolism.

A late phase of heme breakdown, accounting for 80% of bilirubin, is from senescent red blood

cells. This occurs approximately 110 days after administration of radioactive labeled heme and

is consistent with the life span of red blood cells. Heme is initially broken down into a green

biliverdin by heme oxygenase, which is then broken down into the orange bilirubin by biliverdin

reductase.

1 – Creation of Bilirubin

Reticuloendothelial cells are macrophages which are responsible for the maintenance of the

blood, through the destruction of old or abnormal cells. They take up red blood cells and

metabolise the haemoglobin present into its individual components; haem and globin. Globin is

further broken down into amino acids which are subsequently recycled.

Meanwhile, haem is broken down into iron and biliverdin, a process which is catalysed by haem

oxygenase. The iron gets recycled, while biliverdin is reduced to create unconjugated

bilirubin.
2 – Bilirubin Conjugation

In the bloodstream, unconjugated bilirubin binds to albumin to facilitate its transport to the liver.

Once in the liver, glucuronic acid is added to unconjugated bilirubin by the enzyme glucuronyl

transferase. This forms conjugated bilirubin, which is soluble. This allows conjugated bilirubin

to be excreted into the duodenum in bile.

3 – Bilirubin Excretion

Once in the colon, colonic bacteria deconjugate bilirubin and convert it into urobilinogen. Around

80% of this urobilinogen is further oxidised by intestinal bacteria and converted to stercobilin

and then excreted through faeces. It is stercobilin which gives faeces their colour.

Around 20% of the urobilinogen is reabsorbed into the bloodstream as part of the enterohepatic

circulation. It is carried to the liver where some is recycled for bile production, while a small

percentage reaches the kidneys. Here, it is oxidised further into urobilin and then excreted into

the urine

Heme to Bilirubin

Heme is a ring of four pyrroles joined by carbon bridges and a central iron atom. Bilirubin is generated

by a two-stage sequential catalytic degradation reaction that primarily takes place in the cells of the

reticuloendothelial system, notably the spleen. Other cells include phagocytes and the Kupffer cells of the

liver. Heme is taken up into These cells take up the heme, and enzyme heme oxygenase acts on them. The

enzyme liberates the chelated iron by catalyzing the oxidation of the alpha carbon bridge. This reaction

produces an equimolar amount of carbon monoxide which is excreted by the lungs and leads to the

formation of the green pigment, biliverdin. This green pigment is acted upon further by the nicotinamide

adenine dinucleotide phosphate (NADPH) dependent enzyme, biliverdin reductase. This process releases

an orange-yellow pigment known as bilirubin.

Types

Unconjugated bilirubin is otherwise also known as indirect bilirubin. Unconjugated bilirubin is

formed during the breakdown process of heme.

Unconjugated bilirubin is water-insoluble and is extremely toxic to the body.

The normal value for unconjugated bilirubin in adults should be in 0.2 to 0.8 mg/dL. If

unconjugated bilirubin is present in the body in larger quantities, then it may cause damage to

the brain. Unconjugated bilirubin is absent in the bile.

It needs to be converted into conjugated bilirubin so it can be excreted from the body.

Indirect bilirubin levels can be maintained by ;

● Staying hydrated

● Including lots of fruits and vegetables in the diet

● Avoiding alcohol intake

High Levels of Unconjugated Bilirubin

High unconjugated bilirubin levels in the plasma refer to unconjugated hyperbilirubinemia.

Unconjugated hyperbilirubinemia can also cause jaundice in neonates. Various factors might be

responsible for unconjugated hyperbilirubinemia. These include:

● An increase in the production of bilirubin or increased haemoglobin degradation.

● Impaired hepatic bilirubin uptake.

● Reduced or impaired conjugation of bilirubin.

● Having inherited disorders like hemolytic anaemia and Gilbert's syndrome.

What is Conjugated Bilirubin?

Conjugated bilirubin is otherwise also known as direct bilirubin. Conjugated bilirubin is water

soluble and is not toxic to the body.

An adult's normal value of conjugated bilirubin should be between 0.0 to 0.3 mg/dL.

Since conjugated bilirubin is water soluble, it can be filtered by the kidney.

Conjugate bilirubin is formed when unconjugated bilirubin is conjugated by adding glucuronic

acid. Normally, conjugated bilirubin is not present in the urine. However, in certain conditions or

diseases, it may be seen in the urine. The presence of conjugated bilirubin in the urine will give

it a dark cola or tea-like colour (brown).

High Levels Conjugated Bilirubin Causes

Elevating the conjugated bilirubin level leads to conjugated hyperbilirubinemia. Various causes

lead to an increase in the level of conjugated bilirubin. Some of these causes of conjugated

hyperbilirubinemia include:

● Cholelithiasis

● Viral hepatitis

● Liver cirrhosis

● Certain medications and toxins

● Sepsis

● Alcohol

● Autoimmune disorders

● Certain metabolic diseases like Gaucher disease, Niemann-Pick disease, etc.

Jaundice

PRE-HEPATIC JAUNDICE is caused by increased haemolysis. This results in the increased

presence of unconjugated bilirubin in the blood as the liver is unable to conjugate it all at the
same rate.

HEPATIC JAUNDICE is caused by liver impairment. This causes the decreased ability of the
liver to conjugate bilirubin, resulting in the presence of conjugated and unconjugated bilirubin in
the blood.

POST-HEPATIC JAUNDICE is caused by the blockage of bile ducts. This results in backflow of
conjugated bilirubin into the blood as it cannot move past the obstruction.

b. Methodologies

Bilirubin Assays

Principle: Van den Berg Reaction (diazotization of bilirubin to produce

azobilirubin)

Malloy-Evelyn Procedure

● Bilirubin pigments in serum or plasma are reacted with a diazo reagent.

● This method is commonly used at pH 1.2, where the azobilirubin generated is
red-purple in color and has a maximum absorption wavelength of 560 nm.
● The diazotized sulfanilic acid interacts at the central methylene carbon of bilirubin
to divide the molecule generating two molecules of azobilirubin.
● Uses methanol as a coupling accelerator.

Jendrassik-Grof Method

● The most commonly used method and popular technique for discrete analyzers.
● Principle: Bilirubin pigments in serum or plasma are reacted with a diazo reagent
resulting in the production of the purple product azobilirubin.
● Measures total and conjugated bilirubin.
● Individual bilirubin fractions are measured by collecting two aliquots of material
and treating one with only the diazo reagent and the other with the diazo reagent
plus an accelerator—Caffeine–benzoate.
The Jendrassik-Grof method has the following advantages over the Malloy-Evelyn
method:

● Not affected by pH changes.

● Insensitive to a 50-fold variation in protein concentration of the sample.
● Maintains optical sensitivity even at low bilirubin concentrations.
● Has minimal turbidity and a relatively constant serum blank.
● Is not affected by hemoglobin up to 750 mg/dL
c. Reference Range

Reference: Bishop, Fody, & Schoeff. (2013). Clinical Chemistry: Principles, Techniques and
Correlations (7th ed.). ‎Jones & Bartlett Learning.

d. Clinical Significance

High amounts of both forms of bilirubin will accumulate in your blood if your liver's
capacity to handle bilirubin is impaired. It might potentially be an indication of either
acute or chronic liver disease. Among the causes are:

● Gilbert’s syndrome, Wilson disease and Crigler-Najjar syndrome

● Cirrhosis and/or chronic liver failure

3. Methods for Evaluation of Liver Function

3.1 Excretory Test

3.1.1. BSP- RETENTION TEST (Bromsulphalein Test)

a. PRINCIPLE:
i. The ability of the liver to excrete certain dyes, e.g., BSP is utilized in this
test.
ii. In normal healthy individual, a constant proportion (10%-15% of the dye)
is removed per minute. In hepatic damage and insufficiency, BSP removal
is impaired by cellular failure, as damaged liver cells fail to conjugate
the dye or dude to decrease blood flow.
iii. Removal of BSP by the liver involves conjugation of the dye as
mercaptide with the cysteine component of glutathione. The reaction of
conjugation of BSP with glutathione is RATE-LIMITING, and thus it exerts
a controlling influence on the rate of removal of the dye.

B. INTERPRETATIONS

● In normal healthy individuals, not more than 5% of the dye should remain in the
blood at the end of 45 minutes. The bulk of the dye is removed in 25 minutes and less
than 15% is left at the end of 25 minutes
● In parenchymatous liver disease, removal proceeds more slowly. In advanced
cirrhosis removal is very slow and 40-50% of the dye is retained in 45 minutes sample.

C. Contraindications
 ● Since the dye is removed in the bile after conjugation, this test can only be used in cases
in which there is no obstruction to the flow of bile. Hence, the test is no value if
obstruction of biliary tree exists (OBSTRUCTIVE JAUNDICE)

D. CLINICAL SIGNIFICANCE

● BSP-excretion test is USEFUL INDEX OF LIVER DAMAGE, particularly when the

damage is diffuse and extensive.
● The test is most useful in:
○ Liver cell damage without jaundice
○ Cirrhosis liver; and
○ Chronic hepatitis

a. Rosenthal White (Double Collection Method)

Dose- 2mg/kg body weight of the patient

Specimen collection - after 5 minutes and after 30 minutes

Normal value:

-After 5 minutes - 50% dye retention

-After 30 minutes - 0% dye retention

b. Mac Donald Method(Single Collection Method)

Dose- 5mg/kg body weight of the patient

Specimen Collection - after 45 minutes

Normal Value (After 45 mins.)- +/-5% dye retention

3.1.2. ROSE BENGAL DYE TEST

● Is another dye which can be used to ASSES EXCRETORY FUNCTION. 10 mL of a 1%

solution of the dye is injected IV slowly.
INTERPRETATION

● Normally, 50% or more of the dye DISAPPEARS WITHIN 8 MINUTES.

3.1.2.1. I-labelled Rose Bengal

- I-Rose Bengal has been used where ISOTOPE LABORATORY IS PRESENT.

- IT IS ADMINISTERED THROUGH IV
- The count is taken over the neck and abdomen.
- As the dye is EXCRETED through the liver, NECK COUNT goes DOWN and COUNT
OVER ABDOMEN INCREASES
- In parenchymal liver disease, HIGH COUNT in the neck persists and there is HARDLY
RISE in count over abdomen, as the dye is retained

Bilirubin Tolerance Test

● One mg/kg body weight of bilirubin is injected IV

● If more than 5% of the injected bilirubin is retained after 4 hours, the excretory and
conjugating function of the liver is considered ABNORMAL.
● The bilirubin excretion test has been recommended by some authorities as a
BETTER TEST OF EXCRETORY FUNCTION OF THE LIVER.
● The test is not used routinely and extensively DUE TO ITS HIGH COST.

3.2 Metabolic Test

A, Glucose Tolerance Test

● NOT OF MUCH VALUE in liver disease

● Although glucose tolerance is sometimes diminished, it is often difficult to separate the
part played by the liver from other factors influencing glucose metabolism

B. Galactose Tolerance Test

Basis: The normal liver is able to convert galactose into glucose, but this function is
IMPAIRED in INTRAHEPATIC DISEASE and the amount of blood galactose and
galactose in urine is excessive.

ADVANTAGES:

● It is used primarily TO DETECT LIVER CELL INJURY.

● It can be performed in presence of jaundice.
● As it measures an intrinsic hepatic function, it may be used TO DISTINGUISH
 OBSTRUCTIVE AND NON-OBSTRUCTIVE JAUNDICE

Two Types of Galactose Tolerance Test:

a. Oral galactose tolerance test (Maclagan)

b. IV galactose tolerance test (King)

1. ORAL GALACTOSE TOLERANCE TEST (MACLAGAN)

● The test is performed IN THE MORNING AFTER AN OVERNIGHT FAST.
● A FASTING BLOOD SAMPLE is collected which serves as “CONTROL”
● 40 gm of galactose dissolved in a cup-full of water is given ORALLY.
● Further, FOUR BLOOD SAMPLES are collected at ½ HOURLY INTERVALS
FOR TWO HOURS (similar to GTT)

INTERPRETATIONS:

● Normally or in OBSTRUCTIVE JAUNDICE, 3 GM OR LESS OF GALACTOSE are

EXCRETED IN THE URINE within 3-5 hours and the blood galactose returns to
NORMAL WITHIN ONE HOUR.
● In INTRAHEPATIC (parenchymatous) jaundice, the excretion amounts to 4-5 gm or
more during the FIRST FIVE HOURS.

GALACTOSE INDEX (MACLAGAN)

- It is obtained by ADDING THE FOUR BLOOD GALACTOSE LEVELS.

Interpretations:

● Upper normal limit of normal was taken as 160.

● IN HEALTHY MEDICAL STUDENTS range varied from 0 to 110 and in hospital patients
not suffering from liver disease the value range 0-160.
● IN LIVER DISEASE very high values are obtained.
● IN INFECTIVE AND TOXIC HEPATITIS VALUES UP TO 500 ARE SEEN, decreasing
slowly as the clinical condition improves.
● IN CIRRHOSIS LIVER, INCREASED VALUES MAY BE OBTAINED UP TO 500,
depending on the severity of the disease.

2. INTRAVENOUS GALACTOSE TOLERANCE TEST (KING)

● The test is performed in the MORNING AFTER A NIGHT’S FAST
● A fasting blood sample is collected which serves as “CONTROL”
● An IV injection of galactose, equivalent to 0.5 gm/kg body weight is given as a sterile
50% solution.
● Blood samples are collected after 5 minutes, ½,1,½,2 and 2 ½ hours after IV injection
and blood galactose level is estimated.

INTERPRETATIONS:

● A normal response should have a curve beginning on the average at about 200 mg
GALACTOSE/100 dL, falling steeply during the one hour and reaching a figure between
0 and 10 mg% by end of 2 hours.
● In most cases of obstructive jaundice, similar results are obtained, unless there is
parenchymal damage.
● In PARENCHYMATOUS DISEASES with liver cell damage, the fall in blood galactose
 takes place MORE SLOWLY.
● Normally, no galactose is detected in 2 1⁄2 hours sample, but in parenchymatous
disease, value is GREATER THAN 20 MG/DL.

C. Fructose Tolerance Test

Method

● Fasting blood sugar is estimated.

● 50 gm of fructose is given to the fasting patient.

● Samples are taken at 1⁄2 hourly intervals for 2 1⁄2 hours after giving the oral fructose.
Blood sugar is estimated in all the samples.

Interpretations

• Normal response shows little or no rise in the blood sugar level. The highest blood sugar
value reached during the test should not exceed the fasting level by more than 30 mg%.

● Similar result is obtained in most cases of OBSTRUCTIVE JAUNDICE CASES

(provided no parenchymal damage).

• In INFECTIOUS HEPATITIS and PARENCHYMATOUS LIVER CELLS DAMAGE, rise in

blood sugar is greater than above, but the increases obtained are never very great.

D. Epinephrine Tolerance Test (Storage Function)

Principle: The response to epinephrine as evidenced by elevation of blood sugar is a

manifestation of glycogenolysis and is directly influenced by glycogen stores of liver.

METHOD

• The patient is kept on a high carbohydrate diet for three days before the test.

• After an overnight fast, the fasting blood sugar is determined.

METHOD

• 0.01 ml of a 1 in 1000 solution of epinephrine per kg body weight is injected.

• The blood sugar is then determined in samples collected at 15 minutes intervals up to one
hour.

3.3 Conjugation Test

Delta bilirubin

● It is conjugated bilirubin tightly bound to albumin.

● It has a longer half-life than other forms of bilirubin.

● It is formed due to prolonged elevation of conjugated bilirubin in biliary obstruction.

● It helps in monitoring the decline of serum bilirubin following surgical removal of

gallstones.

● It reacts with diazo reagent in the direct bilirubin assay.

 ● It is computed by using this formula: TB - DB + IB = Delta bilirubin

● It is not calculated on neonatal patients (<14 days ).

● Reference value: < 0.2 mg/dL ( < 3 umol/L)

Bilirubin Assay

● Principle: Van den Berg Reactions diazotization of bilirubin to produce azobilirubin.

a. Evelyn and Malloy Method

b. Jendrassik and Grof

A. Evelyn and Malloy Method

● Coupling Accelerator: Methanol

● Diazo Reagents:

- Diazo A = 0.1% Sulfanilic Acid + HCI

- Diazo B = 0.5% Sodium Nitrite

- Diazo Blank = 1.5% HCI

● Final reaction: pink to purple azobilirubin

B. Jendrassik and Grof

● It is the most commonly used method.

● It is a popular technique for discrete analyzers.

● It is more sensitive than Evelyn and Malloy method

● is not affected by hemoglobin up to 750 mg/dL and pH changes.

● Main reagent: Diazo reagent

● Coupling Accelerator: Caffeine Sodium Benzoate

● Buffer: Sodium Acetate,

● Ascorbic acid: Terminates the initial reaction and destroys the excess diazo reagent

● Alkaline tartrate solution: Provides an alkaline pH

● Final reaction: pink to blue abilirutin

Notes to Remember:

- Conjugated bilirubin produced a color in aqueous solution.

- Unconjugated bilirubin is the fraction that produces a color only after the addition of

alcohol.

- Delta bilirubin is the conjugated bilirubin bound to albumin; elevated in obstructive

jaundice.
 - The purpose of adding a methanol or caffeine solution is to allow indirect bilirubin to

react (solubilize) with the color reagent.

- Total bilirubin is measured 15 minutes after adding methanol or caffeine solution.

- Caffeine-benzoate is preferred over methanol because the latter promotes protein

precipitation and increases turbidity.

- Measurement of total bilirubin involves solubilization of the unconjugated form before

chemical quantitation.

- Bilirubin absorbs light maximally at 450nm and imparts a yellow color to amniotic fluid.

3.4 Detoxification Test

● ENZYME TESTS- It is used to asses tge extent of liver damage and to differentiate
hepatocellular (functional) form obstructive (mechanical) disease

- INDICATION OF CELLULAR INJURY IN EARLY OR LOCALIZED LIVER DISEASE

● AMMONIA- it arises from deamination of amino acids, which occurs mainly through the
action of digestive and bacterial enzymes (bacterial proteases, ureases, and amino
oxidases) on proteins in the intestinal tract

-REFERENCE VALUE: 19-60 ug/dL (11-35 mmol/L)

4. Differential Diagnosis of Jaundice

CASE STUDY

An 84-year-old woman presented with a 2-day history of jaundice, fever and abdominal pain.
Physical examination showed scleral icterus and right upper quadrant tenderness without
inspiratory arrest at palpation (absent Murphy's sign.)

The patient noted that the color of her stool on the other day is clay colored.

Analyte Results Reference Range

WBC Count (cells/cumm) 12,400 cells/cumm 4,500-11,500 cells/cumm

C-reactive protein 8.3 mg/L 2.5 mg/L

Aspartate 231 U/L 8-33 U/L

Aminotransferase (AST)

Alanine Aminotransferase 178 U/L 4-36 U/L

(ALT)

Alkaline Phosphatase 893 U/L 44-147 U/L

Gamma-glutamyl 1143 UL 5-40 U/L

Transferase

Bilirubin 5.4 mg/dL 0.1 to 1.2 mg/dL

The physician requested for an endoscopic retrograde cholangiopancreatography (ERCP) that
clearly showed common hepatic duct compression by a large gallstone (20mm) impacted in the
cystic duct (Fig. 1), compatible with the diagnosis of Mirizzi syndrome.

Questions:

1. What is most likely the diagnosis?

-Obstructive Jaundice due to Cholelithiasis or gallstone.

2. Which of the findings led the physician to diagnose Obstructive Jaundice?

-The patient presented the clinical triad of jaundice, fever, right upper quadrant pain, showing
in the laboratory elevations in the serum concentrations of alkaline phosphatase and bilirubin.
The patient presented a clay colored stool which is present in the Obstructive Jaundice.

3. What is most likely to be the treatment for Obstructive Jaundice due to cholelithiasis?

=If gallstones are diagnosed, laparoscopic cholecystectomy or keyhole surgery is

recommended for the removal of gallstones. Endoscopic removal of stones obstructing the bile
duct is sometimes necessary prior to laparoscopic surgery to fully clear all the stones.