21-Liver function tests 240

21-Functional Tests
Q 1. Classify jaundice. Write the principle, test procedure and interpretation of Van den
Bergh test.
Jaundice is classified into three groups
i. Prehepatic or hemolytic jaundice
ii. Hepatic or hepatocellular jaundice
iii. Post hepatic or obstructive jaundice
Different types of jaundice can be differentiated with the help of van den Bergh test.
Principle of van den Bergh test
Diazotized sulfanilic acid (sulfanilic acid in HCl and sodium nitrite) couples with bilirubin to
form a purple colored complex, azobilirubin.
Test Procedure
Direct bilirubin: Pipette 0.2 ml of serum and 4.3 ml of water each into two test tubes. Add 0.5 ml
of diazo reagent to the first test tube (unknown, direct), and 0.5 ml of 1.5% HCl to the second
test tube (blank, direct).
Total bilirubin: Pipette 0.2 ml of serum, 1.8 ml of water and 2.5 ml of methanol each, into two
other test tubes. To the first tube (unknown total) add 0.5 ml of diazo reagent to the second tube
(blank, total) add 0.5 ml of 1.5% HCl.
To the fifth test tube (standard), add 0.2ml of standard bilirubin solution (0.02 mg). To the
sixth test tube (blank) add 0.2 ml of water. Add 1.8ml of water, 2.5ml of methanol and 0.5 ml of
diazo reagent to fifth and sixth tubes.
After 30 minutes, read the optical density (O.D) values of all the tubes at 540 nm (green
filter).
Indirect bilirubin = Total bilirubin – Direct bilirubin
Interpretation of van den Bergh test:
Normal serum does not give a positive test. Conjugated bilirubin, being water soluble,
develops purple color directly on addition of diazo reagent within 30 seconds. This is referred to
as direct positive van den Bergh reaction and the conjugated bilirubin is known as the direct
reacting bilirubin. Unconjugated bilirubin, being water insoluble, develops color with diazo
reagent only in the presence of methanol (alcohol), which dissolves it. This response is referred
to as indirect positive Van den Bergh reaction and the unconjugated bilirubin is known as
indirect reacting bilirubin. When both conjugated and unconjugated bilirubin are present in
increased amounts in serum, purple color is produced immediately and the color increases further
on adding methanol. This response is known as biphasic.
Increase of unconjugated bilirubin in serum (indirect positive van den Bergh reaction)
indicates hemolytic jaundice, whereas increase of conjugated bilirubin in serum (direct postive
van den Bergh reaction) indicates obstructive jaundice. Increase of both conjugated and
unconjugated bilirubin in serum (biphasic van den Berg reaction) indicates hepatocellular
jaundice.
Q 2. An 18 year-old boy with jaundice has the following LFT report: ALT 1100 IU/L, ALP
350 IU/L, GGT 150 IU/L, total bilirubin 18 mg/dl, and direct bilirubin 16 mg/dl. Write the
physiological range for each of the parameters, and your comments.
21-Liver function tests 241

Normal range of values

Serum alanine transaminase (ALT): 0-40 IU/L at 370 C
Serum alkaline phosphate (ALP): 40-125 IU/L
Serum γ-glutamyl transferase (GGT): 10-30 IU/L
Serum total bilirubin: 0.2-1 mg/dl
Serum direct bilirubin: 0.1-0.4 mg/dl
The LFT report indicates elevated serum ALT, ALP, GGT, total bilirubin and direct
bilirubin. Direct (conjugated) bilirubin more than the indirect (unconjugated) bilirubin, along
with elevated ALP and GGT indicates obstructive jaundice. Marked increase of serum ALT
indicates acute hepatitis. The condition appears to be intrahepatic cholestasis due to infective
(viral) hepatitis.
Q 3. Classify liver function tests (LFTs) based on liver functions. How will you assess the
secretory function of liver?
Classification of liver function tests
Functions of liver Liver function tests
a. Synthetic function i. Estimation of serum albumin
ii. Prothrombin time
b. Secretory function i. Estimation of serum conjugated bilirubin
ii. Estimation of urobilinogen in urine
c. Excretory function i. Estimation of bromsulfthalein (BSP)
ii. Estimation of indocyanine green
d. Detoxification function i. Estimation of hippuric acid in urine
ii. Estimation of blood ammonia
e. Metabolic function i. Galactose tolerance test
ii. Antipyrine breath test
f. Storage function i. Estimation of serum iron
g. Serum enzymes i. Estimation of serum alanine transaminase
ii. Estimation of serum alkaline phosphatase (ALP)
iii. Estimation of serum γ-glutamyl transferase.
Secretory function of liver:
Liver secretes bile and very low-density lipoprotein. Bile contains bile pigments, bile salts,
alkaline phosphatase etc. In the intestine conjugated bilirubin is converted to urobilinogen, a part
of which is absorbed into the circulation and excreted in urine.
Secretory function of liver can be assessed by 1) estimation of serum conjugated bilirubin by
van den Bergh test, 2) estimation of urobilinogen in urine by Ehrlich’s test, and 3) detection of
bilirubin in urine by fouchet’s test. When the liver is functioning normally, serum conjugated
bilirubin level is normal with normal amount of urobilinogen excreted in urine, and bilirubin is
absent in urine. When secretory function of liver is defective, serum conjugated bilirubin level is
increased due to regurgitation and urinary excretion of urobilinogen is absent, and bilirubin is
excreted in urine.
Secretory function of liver can also be assessed by bromsulfthalein (BSP) test, after
administering 250 mg of BSP intravenously. In normal persons blood contains not more than 5%
of original value at 45 minutes, and not more than 2% at 2 hours. When the secretory function of
liver is defective, BSP level in serum at two hours is more than 5%. This is because the secretory
21-Liver function tests 242

defect of liver causes the regurgitation of conjugated BSP into the blood. Regurgitation leads to
increased value at two hours.
Q 4. Name the commonly employed LFTs in clinical practice. Write the test to assess
detoxification function of liver.
Liver function tests that are commonly employed in clinical practice are estimation of serum
bilirubin, albumin, alkaline phosphatase, alanine transaminase, aspartate transaminase, and
serum γ-glutamyl transpeptidase.
Hippuric acid test is carried out to assess the detoxification function of the liver. Oral intake
of 6 g of sodium benzoate will result in urinary excretion of 50% of benzoate as hippuric acid in
normal individual within 4 hours. Lesser quantities are excreted in acute and chronic liver
diseases.
Q 5. Define prothrombin time (PT), and write its normal value and significance.
Prothrombin time (PT) is defined as the time required for clotting of plasma to which
calcium and thromboplastin are added. Normally it is 11 to 15 seconds. Increase of PT indicates
a defect in synthetic function of liver. In vitamin K deficiency also PT is prolonged.
Q 6. Define A/G ratio, and write its normal value and significance.
Ratio of albumin to globulin or albumin/globulin is known as A/G ratio. The normal A/G
ratio is about 1.5:1. A/G ratio is reversed in cirrhosis of liver due to hypoalbuminemia and
associated hypergammaglobulinemia
Q 7. Name the marker enzymes of obstructive liver disease and hepatocellular damage.
Marker enzymes of obstructive liver disease
i. Highly increased levels of serum alkaline phosphatase
ii. Elevated levels of serum γ-glutamyl transferase
iii. Increased levels of serum 5`-nucleotidase
iv. Elevated levels of serum leucine amino peptidase
In obstructive liver disease, there may be slight increase of serum alanine transaminase, but
the increased levels return to normal when the obstruction persists.
Enzymes indicating hepatocellular damage
i. Highly increased levels of serum alanine transaminase
ii. Moderately elevated levels of serum aspartate transaminase
Both alanine transaminase and aspartate transaminase levels are increased in hepatitis, but
the elevation of serum alanine transaminase is much more than that of serum aspartate
transaminase. The degree of elevation of transminases may reflect the extent of hepatocellular
damage.
Mild increase (2-3 times) of serum alkaline phosphatase (α-2 heat labile isoenzyme) is seen
in hepatic parenchymal disease.
Q 8. What are renal (kidney) function tests and why are they performed? Enumerate the
common biochemical tests performed to assess renal functions.
Renal function tests is a collective term for a variety of individual tests and procedures that
can be done to evaluate how well the kidneys are functioning.
They are performed to:
 identify renal dysfunction.
21-Liver function tests 243

 diagnose renal disease.

 monitor disease progress.
 monitor response to treatment.
 assess changes in renal function that may have impact on other therapy (e.g. digoxin,
chemotherapy).
The common tests performed to assess renal function are as follows:
a) Routine urinalysis
b) Estimation of plasma creatinine and urea. These tests are relatively insensitive assays of
renal function since more than 60% of the kidney can be destroyed before their values are
significantly altered.
c) Measurement of glomerular filtration rate (GFR)/clearance tests. Urea and creatinine
clearance tests are commonly used to measure glomerular filtration and to assess renal
function. Neither gives an absolutely accurate value. Urea gives a slightly low value since
it diffuses back into the bloodstream from the proximal tubule, while creatinine gives a
slightly high value because it is secreted from the proximal tubule. Inulin would give a
more accurate estimate of glomerular function but is rarely used since it has to be
injected.
d) Cystatin (cysteine proteinase inhibitor) clearance test appears better than any of the other
available tests. Because it is an endogenous protein that is produced at a constant rate by
all nucleated cells, no known extra- renal excretion routes, and not influenced by muscle
mass, diet or subjects sex
e) Tubular function tests. Proximal tubular function is assessed by phosphate reabsorption
test and fractional bicarbonate excretion test. Distal tubular function is assessed by
acidification and concentration tests.
Q 9. What is glomerular filtration rate (GFR), and how is the test performed? Write the
various other clearance values.
The GFR is the most frequently performed test to assess renal functions. The measurement is
based on concept of clearance. It is the determination of the volume of plasma from which a
substance (e.g. creatinine/urea/inulin) is removed by glomerular filtration, during its passage
through the kidney over a period of 1 minute.
The clearance equals GFR, when the substance used for clearance test is freely filtered by
glomerulus, its sole route of excretion from the body is through glomerular filtration, and it is
neither secreted nor reabsorbed by renal tubules.
The substance used for assessing clearance should be non-toxic and easily measurable.
Determination of Clearance
Clearance = (U x V)/P
Where U is the urinary concentration of substance x
V is the rate of urine formation (ml/min)
P is the plasma concentration of substance x
Units = volume/unit time (ml/min)
The glomerular filtration rate is the volume of fluid filtered from the glomeruli into
Bowman's space per unit time. It is approximately 125ml/min in the average-sized 70kg healthy
male. Estimation of GFR is an extremely important tool in the assessment of renal function since
its value is proportional to the number of intact nephrons. The value obtained must be corrected
21-Liver function tests 244

for height and weight (usually standardized to a surface area of 1.73 m squared), sex and age of
the individual before it can be used to identify patients with impaired renal function.
Abnormal GFR indicates among other things glomerulo-tubular imbalance and hence
impaired function.
Creatinine clearance test is performed on a urine specimen collected over a two to twenty-
four hour period. Determination of the blood creatinine level is also required to calculate the
creatinine clearance value.
Ucr (mg/dl) x V (ml/min)
Creatinine clearance (Ccr) = =GFR
Pcr (mg/dL)
Example: Patient A has
urine volume (V) = 1.2 L/24 hour (must be complete)
urine creatinine concentration (Ucr) = 100 mg/dL
plasma creatinine concentration (Pcr) = 1.2 mg/dL
UxV 100 mg/dl x 1.2 L/24 hr
Ccr = =
P 1.2 mg/dL
= 100 x (L/24 hr) = 100 X (1000 ml/1440 min) = 100 X 0.7 ml/min = 70 ml/min
Clearance values:
Inulin clearance value: 125 ml/min
Creatinine clearance value (corrected value): 100 ml/min
Maximum clearance value: 75 ml/min
Standard clearance value: 54 ml/min
p-Aminohippurate (PAH) clearance value: 700 ml/min.

Q 10. What is renal plasma flow (RPF) and filtration fraction?

RPF can be measured with a substance that is 100% extracted from the plasma in one
passage. It must be freely filtered at the glomerulus and secreted into the tubular lumen.
Clearance of such a hypothetical substance would equal RPF. Para-aminohippuric acid (PAH)
can be used in practice although its extraction ratio is only 90%. It measures the effective renal
plasma flow (ERPF), which underestimates true RPF by 10%. Alternatively RPF can be
measured directly using an electromagnetic or ultrasonic flow meter or by collecting blood from
renal vein. RPF values outside the range 550-600ml/min, after correction for age, sex and surface
area- may be linked to impaired renal function.
The filtration fraction (GFR/RPF) is another indicator of renal function and can be calculated
if GFR and RPF are known. Its normal value is 0.16-0.20 (no units).
Q 11. Briefly discuss the tests for renal tubular functions
Urinary concentrating ability
The formation of maximally concentrated urine requires the delivery of an adequate amount
of fluid to the loop of Henle and normal urea production. Therefore, concentration of urine gives
an indication of tubular function. It may be measured by a variety of methods including the
impregnated stick, refractive index, and specific gravity. The most usual test in clinical practice
involves water deprivation and ADH administration for measuring urine osmolality first thing in
the morning. If an osmometer is used, this is a highly accurate method. A patient with an
osmolality of less than 800 mOsmol/kg should alert the doctor.
21-Liver function tests 245

Urinary acidification
A major function of the kidneys is to maintain acid-base balance mainly by the secretion of
hydrogen ions, the reabsorption of bicarbonate, and the production and excretion of ammonia. A
test can be employed to identify a possible impairment of this homeostatic mechanism.
Ammonium chloride (100 mg/kg body weight) can be given orally to the patient and urine
samples are collected over the next 8 hours. Plasma bicarbonate levels must be tested to ensure
sufficient ammonium chloride absorption. Absence of at least one urine sample of pH <5.3
indicates renal dysfunction.
Q 12. Enumerate the commonly used tests for assessment of thyroid function. Discuss
briefly each one of the tests.
Routinely three hormones are often measured to assess thyroid function. They are thyroxin
(T4), tri-iodothyronine (T3) and thyroid stimulating hormone (TSH).
Thyroxine (T4)
Thyroxine is found in the blood in two forms; one is bound to proteins, and the other is free.
The free component is the active form of the hormone and comprises only 0.03% of the
circulating total T4. Some laboratories still measure total thyroxine, which comprises both the
bound and free forms. The trouble with this is that the level of total thyroxine very much depends
on the amount bound to proteins and therefore the level of binding proteins in the blood. The
major thyroxin binding protein is called thyroid-binding globulin (TBG). The TBG can be low in
some patients due to an inherited defect but seemingly harmless deficiency. In these patients
total thyroxine is low but the free and active component is normal. Medicines such as the
contraceptive pill and life events such as pregnancy can also alter TBG giving spuriously high
levels of total thyroxine. For these reasons most laboratories have now introduced the
measurement of free thyroxine (FT4) and this is what we measure.
Triiodothyronine (T3)
There are two assays available, one measures total T3 and the other free T3 (FT3). The total
T3 comprises of both protein bound and FT3. The free component is the active form and
comprises 0.3% of total circulating T3. Gradually laboratories are moving over to FT3
measurements as more reliable FT3 assays become available. T3 is the biologically active thyroid
hormone, possessing 5 times the metabolic power of T4. In man some 80% of T3 is produced
from T4 by conversion in liver and kidney. Therefore very little is produced in the thyroid itself.
The conversion of T4 to T3 can depend on a number of situations such as chronic illness or
surgical stress, which cause a fall in T4 to T3 conversion (called low T3 syndrome). Starvation
also alters T4 to T3 conversion with a fall in T3 as the body tries to reduce its metabolism to
conserve energy.
The T3 alters the metabolism of the body. It alters protein synthesis, cellular activity, and is
essential for growth and well being. Without T3 the patient develops hypothyroidism. This
condition can go unnoticed by the patient and their relatives for many years. It is however easily
diagnosed with a simple blood test. The level of FT3 in the blood is low. TSH levels are usually
high because the pituitary gland is trying to make the failing thyroid gland work harder. Very
rarely it is the pituitary that is not working and in such cases thyroid hormones (T3 + T4) and
TSH levels are all low.
When T3 levels are too much and the patient develops an overactive thyroid it is called
thyrotoxicosis. This is easily diagnosed with a blood test. The levels of thyroid hormones in the
blood are high. The thyroid gland is usually under control of another gland called the pituitary
situated at the base of the brain. It does this by releasing the hormone TSH. Since the thyroid
21-Liver function tests 246

gland has gone out of control, the pituitary tries to stimulate the thyroid as little as possible, and
hence TSH levels are very low or undetectable.
Thyroid stimulating hormone (TSH)
TSH is released by the pituitary gland and circulates in the blood stream to the thyroid, where
it controls release of the thyroid hormones T4 and T3. TSH release is very sensitive to alterations
in the blood thyroid hormones, with small decreases augmenting TSH secretion and small
increases reducing release. Therefore in hypothyroidism, TSH is raised above normal reference
range, whereas in thyrotoxicosis TSH is suppressed to an undetectable range. In thyrotoxicosis
the thyroid automatically manufactures too much T4 and T3 without the need for TSH to switch
on. As TSH is so sensitive to changes in thyroid hormone levels it is used as the number one test
for screening for thyroid disease. If the laboratories notice a raised TSH then automatically T4 is
measured. If the laboratories notice a suppressed TSH then both T4 and T3 are measured. In some
patients thyroid over secretes only T3, this condition is called T3 toxicosis. Then both hormones
need to be measured to detect this form of thyrotoxicosis. It is usually seen in those who have
undergone thyroidectomy or radioactive iodine therapy for thyrotoxicosis in the past. In primary
hyperthyroidism, both T3 and T4 are elevated, but TSH is decreased. In primary hypothyroidism
the reverse situation prevails.
Q 13. Discuss briefly TRH test and thyroid antibodies. Write the reference range for TSH,
TRH, T3 and T4.
TRH test
Thyrotropin releasing hormone (TRH) is a hormone released by the hypothalamus, which
controls the pituitary release of TSH. The TRH test is done as a screening after the age of 35-
years, or when the level of TSH is borderline low, or high. By using this simple test one can tell
whether a patient has in reality an overactive, normal or hypoactive thyroid. After TRH
administration, in normal individuals the serum TSH value double, in hypothyroidism TSH rises
markedly, whereas in hyperthyroidism TSH does not rise.
Thyroid antibodies
These are circulating antibodies to various thyroid gland components and indicate something
may be faulty when the body is directing the immune response against itself, a so-called
"autoimmune" phenomenon. The major antibody measured is thyroid microsomal
antibody; another antibody now less often measured is thyroglobulin antibody. These tests
indicate a possible disease of the thyroid and the need for thyroid hormone surveillance, because
a certain low number of patients with thyroid antibodies develop hypothyroidism over years if
not decades. A third antibody commonly measured is TRAB (Thyroid Receptor Activating
Antibody), which is raised in some forms of thyrotoxicosis and is the cause of the disease.
Obviously, if TRAB remains elevated then the disease process is still active.
Reference range of T4, T3, TSH, and TRH
Test Reference Range
T4 Total 4.5 - 12.0 μg/dl (60 – 150 nmol/L)
Free T4 8 – 24 ng/L
T3 Total 120 – 190 ng/dl (1.8 – 3 nmol/L)
TSH 0.34 - 5.6 μU/ml (0.34 – 5.6 mU/L)
TRH 5 – 60 ng/L