LIVER FUNCTION TESTS (LFTs)

Introduction
The liver is in the upper right part of the abdomen. The functions of the liver include:

1. Storing glycogen (fuel for the body) which is made from sugars.
2. Helping to process fats and proteins from digested food.
3. Making proteins that are essential for blood to clot (clotting factors).
4. Processing many medicines which you may take.
5. Helping to remove poisons and toxins from the body.
Liver Anatomy
The liver, is a large, bilobed, complex organ receiving a large amount of blood and nutrients from
the gastrointestinal system in order to convert nutrients from the diet into usable or storage forms.
The organ is richly vascularized with two main supply vessels: the hepatic artery and the portal
vein. The liver also contains Kupffer cells as well as tissue lymphocytes and fibroblasts, key
contributors in immune defenses. Hepatic structure can be viewed both macroscopically and
microscopically.
Microscopically, the structural and functional unit of the liver is the lobule, which consists of:
 Cords, or hepatocytes, that surround a central vein
 Sinusoids consisting of blood spaces lined with endothelial cells and Kupffer’s cells that
surround the cords, which drain into a central vein.
 Bile canaliculi, or small channels between hepatocytes that carry bile formed by the
hepatocytes to the bile ducts
Liver Physiology
The liver performs four major functions: excretion/secretion, synthesis, detoxification, and
storage. The liver is so important that if the liver becomes nonfunctional, death will occur with 24
hours due to hypoglycemia.
 Excretion and Secretory-One of the most important functions of the liver is the processing
and excretion of endogenous and exogenous substances into the bile or urine such as the
major heme waste product, bilirubin. Bilirubin is the principal pigment in bile, and it is
derived from the breakdown of red blood cells. Approximately 120 days after the
emergence from the reticuloendothelial tissue, red blood cells are phagocytized and
haemoglobin is released. Haemoglobin is broken down into haem, globin, and iron. The
iron is bound by transferrin and is returned to iron stores in the liver or bone marrow for
reuse. The globin is degraded to its constituent amino acids, which are reused by the body.
The haem portion of haemoglobin is converted to bilirubin in 2–3 hours. Bilirubin is bound
by albumin and transported to the liver. This form of bilirubin is referred to as unconjugated
or indirect bilirubin. This form of bilirubin is insoluble in water and cannot be removed
from the body until it has been conjugated by the liver.
Once at the liver cell, unconjugated bilirubin flows into the sinusoidal spaces and is
released from albumin so it can be picked up by a carrier protein called ligandin. Ligandin,
which is located in the hepatocyte, is responsible for transporting unconjugated bilirubin
to the endoplasmic reticulum, where it may be rapidly conjugated. The conjugation
(esterification) of bilirubin occurs in the presence of the enzyme uridyldiphosphate
glucuronyl transferase (UDPGT), which transfers a glucuronic acid molecule to each of the
two proprionic acid side chains of bilirubin to form bilirubin diglucuronide, also known as
conjugated bilirubin. This form of bilirubin, is water soluble and is able to be secreted from
 the hepatocyte into the bile caniliculi. Once in the hepatic duct, it combines with secretions
from the gallbladder through the cystic duct and is expelled through the common bile duct
in to the intestines. Intestinal bacteria (especially the bacteria in the lower portion of the
intestinal tract) work on conjugated bilirubin to produce mesobilirubin, which is reduced
to form mesobilirubinogen and then urobilinogen (a colorless product). Most of the
urobilinogen formed (roughly 80%) is oxidized to an orange-colored product called
urobilin (stercobilin) and is excreted in the feces. The urobilin or stercobilin is what gives
stool its brown color.
 Synthetic-The liver has extensive synthetic capacity. Albumin, α- and β-globins, blood-
clotting factors, glycogen, carbohydrates, fat, some lipids, ketones, and some enzymes are
synthesized in the hepatocytes. The metabolism of carbohydrates is one of the most
important functions of the liver. When carbohydrates are ingested and absorbed, the liver
can do three things: (1) use the glucose for its own cellular energy requirements, (2)
circulate the glucose for use at the peripheral tissues, or (3) store glucose as glycogen
(principal storage form of glucose) within the liver itself or within other tissues. The liver
is the major player in maintaining stable glucose concentrations due to its ability to store
glucose as glycogen through glycogenesis and degrade glycogen through glycogenolysis
depending on the body’s needs.
 Detoxification and Drug Metabolism-The liver serves as a gatekeeper between
substances absorbed by the gastrointestinal tract and those released into systemic
circulation. Every substance that is absorbed in the gastrointestinal tract must first pass
through the liver; this is referred to as first pass. Hepatocytes have the capability to
conjugate (and thus inactivate) a substance or to modify it chemically. This system is
responsible for the detoxification of many drugs through oxidation, reduction, hydrolysis,
hydroxylation, carboxylation, and demethylation.
 Storage Function- Iron, glycogen, amino acids, and some lipids are stored in hepatocytes.
Liver Function Alterations During Disease
The target cell determines the pattem of injury, with hepatocyte injury leading to hepatocellular
disease and biliary cell injury leading to cholestasis. All cellular injury may induce fibrosis as an
adaptive or healing response, with the duration of injury and genetic factors determining whether
cirrhosis and ultimately carcinoma occur.
Cell death occurs by necrosis or apoptosis or both. Cellular necrosis occurs as the result of an
injurious environment and has been referred to as "murder." Toxic injury from compounds such
as carbon tetrachloride, aspirin, and acetaminophen occurs for the most part by necrosis. Apoptosis
occurs as the result of accelerated programmed death in which the cell participates in its own
demise and thus commits "suicide." Regardless of the cause, cell death typically leads to leakage
of cytoplasmic enzymes.
Jaundice- Jaundice is used to describe the yellowish discoloration of skin, is caused by abnormal
bilirubin metabolism or by retention of bilirubin. Although the upper limit of normal for total
bilirubin is 1.0–1.5 mg/dL, jaundice is usually not noticeable to the naked eye (known as overt
jaundice) until bilirubin levels reach 3.0 mg/dL. Although the terms jaundice and icterus are used
interchangeably, the term icterus is most commonly used in the clinical laboratory to refer to a
serum or plasma sample with a yellow discoloration due to an elevated bilirubin level.
Jaundice is most commonly classified based on the site of the disorder: prehepatic, hepatic, and
posthepatic jaundice.
 1. Prehepatic jaundice is the result of excessive bilirubin presented to the liver. It can occur
in newborns and in people with haemolytic anaemia or ineffective erythropoiesis. This
condition produces increased serum unconjugated bilirubin.
2. Hepatic jaundice is present in people with hepatobiliary disease. This disorder exhibits
increases in both unconjugated and conjugated bilirubin levels.
3. Posthepatic jaundice is produced by obstruction of the flow of bile into the gut either by
gallstones or a tumor, which causes increased conjugated bilirubin levels in serum and
urine, but low urobilinogen levels in urine and colorless stool.
Cirrhosis-Cirrhosis is a clinical condition in which scar tissue replaces normal, healthy liver tissue
or better still it can be defined as the destruction of the liver’s architecture. As the scar tissue
replaces the normal liver tissue, it blocks the flow of blood through the organ and prevents the
liver from functioning properly. The most common cause of cirrhosis is chronic alcoholism and
chronic hepatitis C virus infection. Other causes of cirrhosis include chronic hepatitis B and D
virus infection, autoimmune hepatitis, inherited disorders.
Tumor-Cancers of the liver are classified as primary or metastatic. Primary liver cancer is cancer
that begins in the liver cells. Metastatic cancer occurs when tumors from other parts of the body
spread (metastasize) to the liver. Metastatic liver cancer is much more common than primary liver
cancer; 90%–95% of all hepatic malignancies are classified as metastatic. Cancers that commonly
spread to the liver include colon, lung, and breast cancer. Cancers of the liver may also be classified
as benign or malignant. The common benign cancers of the liver include hepatocellular adenoma
and hemangiomas. Malignant tumors of the liver include hepatocellular carcinoma (HCC),
hepatocarcinoma, and hepatoma. Of those, HCC is the most common malignant tumor of the liver.
Hepatitis-Hepatitis is defined as inflammation of the liver and subsequent hepatocellular damage
caused by bacterial infection, drugs, toxins, or viral infections. Types of viral hepatitis include:
Hepatitis A (“infectious” hepatitis), Hepatitis B (“serum” hepatitis), or hepatitis B virus (HBV),
Hepatitis C (HCV) and Delta hepatitis.
Liver function tests

LFTs are group of clinical biochemistry laboratory blood assays designed to give information
about the state of a patient’s liver. LFTs are helpful to detect the abnormalities and extent of liver
damage can contribute to making an accurate diagnosis of the specific liver disorder ( in addition
to careful history and physical exam). As the liver performs it's various functions it makes a
number of chemicals that pass into the bloodstream and bile. Various liver disorders alter the blood
level of these chemicals. Some of these chemicals can be measured in a blood sample. Some tests
that are commonly done on a blood sample are called 'LFTs' (liver function tests).

Classification of LFTs

a) Tests of excretion by the liver (bilirubin).

b) Evaluation of synthesis and excretion in liver (albumin, total proteins, blood
clotting tests, prothrombin time-PT and ammonia).
c) Evaluation of enzyme activity (ALT, AST, ALP, GGT).
d) Tests of infective agents (Hepatitis A, B, C, D and E)
Liver function tests are most often employed to determine:
  The presence of liver disease.
 The type of liver disease.
 The extent and progression of liver disease.
The diagnosis of liver disease depends upon a complete history, complete physical examination,
and evaluation of liver function tests and further invasive and noninvasive tests.

Laboratory tests are helpful in distinguishing the (1) pattern of injury (hepatocellular versus
cholestatic), (2) chronicity of injury (acute versus chronic), and (3) severity of injury (mild versus
severe). In general, (1) the aminotransferase enzymes and ALP are used to distinguish the pattern,
(2) plasma albumin to determine the chronicity, and (3) the PT or factor V concentration to
determine the severity. At the present time, the only way to accurately detect fibrosis is by a liver
biopsy.
Liver Enzymes

(See AST, ALT, ALP, GGT in the previous lectures)

Measurement of Serum Bilirubin (Total, direct & indirect)

Bilirubin is a yellow breakdown product of normal haem catabolism. Its levels are elevated in
certain diseases and it is responsible for the yellow color of bruises and the brown color of faeces.
Bilirubin reduction in the gut leads to a product called urobilinogen, which is excreted in urine.
Like other pigments, bilirubin changes its conformation when exposed to light. This is used in the
phototherapy of jaundiced newborns: the illuminated version of bilirubin is more soluble than the
unilluminated version.

Serum bilirubin concentration depends on the rate of removal of bilirubin from destruction of
haemoglobin. A bilirubin test measures the amount of bilirubin in a blood sample. Total and direct
bilirubin levels can be measured from the blood, but indirect bilirubin is calculated from the total
and direct bilirubin.

Types of Bilirubin: Bilirubin is present in plasma as: indirect bilirubin (unconjugated bilirubin)
and direct bilirubin (conjugated bilirubin).

Principle

Van der Bergh diazo reaction has been used for many years to determine bilirubin in serum. This
involves treating the serum with diazotized sulphanilic acid to form the azobilirubin complex. The
conjugated bilrubin reacts directly with the diazo reagent while the unconjugated bilirubin
(indirect) bilirubin reacts with diazo reagent only in the presence of an accelerator such as caffeine
benzoate reagent and takes about 10minutes for the colour development. The azobilirubin is purple
in the acid medium and is converted to blue colour by addition of alkaline tartarate solution. The
diazo reagent is terminated at the end by the addition of ascorbic acids. This method is otherwise
referred to as the Jendrassik and Grof.
Samples:

For use with serum or plasma samples. Haemolysis interferes with the test. Do not expose sample
to sunlight or other light. Samples can be stored for up to 3 months at –20 C, 4 days at 2-8 C and
1 day at 15-25 C

Calculation:

Total Bilirubin (mg/dl)=Absorbance tube Total x 17.5

Direct Bilirubin(mg/dl)=Absorbance tube Direct x 17.5

(mg/dl) x 17.1 = μmol/L

Linearity:

The method is linear up to 20mg/dl (342μmol/l).

In case of higher results, dilute sample 1:2 with NaCl 0.9% solution and repeat test. Multiply result
by 2.

Normal Values: Serum:

Total Bilirubin up to 1.1mg/dl (18.8μmol/l)

Direct Bilirubin up to 0.25mg/dl (4.3μmol/l)

Causes of hyperbilirubinaemia

 Unconjugated (indirect) hyberbilirubinaemia

1. Overproduction of bilirubin
a) Congenital (e.g. haemoglobinpathies)
b) Haemolytic anaemia
c) Liver disease (Hepatitis and cirrhosis)

2. Decreased excretion
a) Hereditary-Gilbert’s syndrome
b) Acquired e.g. post viral hepatitis
c) Drugs (e.g flavispidic acid, novobiocin)
d) Crigler-Najjar syndrome

 Conjugated (direct) hyberbilirubinaemia

 1. Intrahepatic cholestasis (obstruction) which may be due to:
a) Cirrhosis (occasional)
b) Hepatitis (often)
c) Alcoholic liver disease
d) Drugs (e.g. chlorpromazine)
e) Primary biliary cirrhosis
2. Extrahepatic obstruction which may be due to:
a) Gall stones
b) Carcinoma of the bile duct or pancreas
c) Bile duct stricture
d) Biliary atresia

Measurement of Urobilinogen in Urine and Faeces

Urobilinogen is a colorless end product of bilirubin metabolism that is oxidized by intestinal
bacteria to the brown pigment urobilin. In the normal individual, part of the urobilinogen is
excreted in feces, and the remainder is reabsorbed into the portal blood and returned to the liver.
A small portion that is not taken up by the hepatocytes is excreted by the kidney as urobilinogen.
Urine urobilinogen is measured qualitatively using a dipstick. Increased levels of urinary
urobilinogen are found in haemolytic disease and in defective liver-cell function as in decreased
uptake into liver due to hepatocellular dysfunction. Absence of urobilinogen from the urine and
stool is most often seen with complete biliary obstruction. Fecal urobilinogen is also decreased in
biliary obstruction, as well as in HCC.
Most quantitative methods for urobilinogen are based on a reaction first described by Ehrlich in
1901: the reaction of urobilinogen with p-dimethyl amino-benzaldehyde (Ehrlich’s reagent) to
form a red color. Many modifications of this procedure have been made over the years to improve
specificity. However, because the modifications did not completely recover urobilinogen from the
urine, most laboratories use the less laborious, more rapid, semi-quantitative method.

Measurement of Plasma Proteins

The liver synthesizes all proteins except immunoglobulin. Albumin is decreased in chronic liver
disease, but is an insensitive index of liver function. Prothrombin time and thromboplastin time
maybe prolonged in liver disease due to secondary vitamin K deficiency resulting from fat
malabsorption.
Methods of estimating total serum proteins-There are several methods of estimating total serum
proteins. Some of these methods are:
 1. Kjeldahl method-By boiling in the presence of sulphuric acid and a catalyst, proteins are
digested. The calculation is based on estimating the amount of nitrogen released either by
the use of Nessler’s reagent or by titration with standard acid. Sixteen percent of proteins
is nitrogen so that the results of nitrogen is multiplied by the factor 6.5. The validity of this
factor has been in dispute. The Kjeldahl methods which are time consuming and of very
little value in routine use are employed extensively in reference laboratory.
2. Biuret methods-Proteins molecules are made up of amino acids which are arranged in
long chains called peptides chains; and the links joining the amino acids together are called
peptide bonds. The Biuret methods are based on the reaction which occurs between cupric
ions in the reagent and peptide bonds of the protein molecules in an alkaline solution to
form blue-violet or purple coloured complexes. The absorbance of the colour is measured
in a colorimeter (yellow-green filter) or in a spectrophotometer at 540nm.

Prothrombin time
Prothrombin is synthesized by the liver and a marker of liver function. Half-life: 6 hrs. (indicates
the present function of the liver). PT is prolonged only when liver loses more than 80% of its
reserve capacity. Vitamin K deficiency also causes prolonged PT. Intake of vitamin K does not
affect PT in liver disease