f.

BACTICAL CLINICAL BIOCll£MisTJlr

. Death is usuallv in the first or second year with ker .

phenobarb 1ta1.
10 • n1cte
While mild unconjugated hyperbilirubinemia respond to enzyme-induci

agents as phenobarbital.
J. Post.hepatic hyperbilirubinaemia: Mainly occurs due to obstruction of the
bile ducts. leading to a deficiency in bilirubin excretion (conjugated
hyperbilirubinaemia). Obstruction can be located either within the liver or in the
bile duct (cholestatic hyperbilirubinaemia).
Pathology of bile secretion and defect in the transportation into bile canaliculi as
in DUBIN-JOHNSON SYNDROME: The syndrome consists of chronic benign
jaundice due to conjugated hyperbilirubinemia. The hepatocytes contain an
abundance of coarse dark-brown pigment similar to melanin. The liver is black
but nonnal. Serum bilirubin ranges between 2 and 20mg/dl, 60% conjugated.
Jaundice appears in the first 3 decades of life and is intermittent. Sometimes the
onset is acute, simulating hepatitis. The prognosis is excellent.
ROTOR SYNDROME: This is a condition similar to Dubin-Johnson. There is
intermittent jaundice with conjugated hyperbilirubinemia, similar clinical
course, excellent prognosis but no pigment in the liver tissue.
LA ~c) ;... e cl; pk~Jp kwf,e_ :- 4 0 f
Higll Bilirubi11 in neo11ates
Neonates are especially vulnerable to high bilirubin levels due 10 an
immature blood-brain barrier that predisposed them to kemicterus. bilirubin
encephalopathy which can result in pennanent neurological damage. Neonates
also have a low amount of functional UDP-glucuronyl-transferase enzyrneaoa
can have elevated unconjugated bilirubin, since conjugation is limited.
For 1h'15 reason, newborns are often treated with UV ltght · to turn th ~
h) drophob·
alb umm-bmdmg
· · • unconjugated bilirubm
. into
. a "',orm that is. niorc
1
IC
hd hT ' brain-
y rop I tc and able lo be secreted out via urine, sparing the neonate 5 '"
H 1 ~mni~ -
ea thy newborns especially those who are premature are ess
susceptible to jaundice because their immature livers are slow to proc
 PRACTICAL aJNICAL BIOCHEMJSTRY

. . b'n Jaundice is also fairly common among breast feeding babies who are
b11tru 1 ·
0 .,tting enough milk.
not=-"
Most mild cases resolve spontaneously. but because of high bilirubin levels
use hearing loss and hrain damage in babies, therefore, all infants should
ma) Ca
be examined for jaundice soon after birth. If the doctor feels that the baby is
more jaundiced than would be expected, a blood test is done to detennine the
exact level of bilirubin in the blood.
Babies with high levels may need phototherapy (treatment with a special
light that makes bilirubin easier for the liver to be processed), or in rare cases, an
exchange transfusion in which the infant's bilirubin level is brought down by
removing bilirubin-rich blood from the baby and replacing it with blood that
contains normal levels of bilirubin.
High bilirubin levels in infants may also be the result of hemolytic disease,
a condition that occurs when there is an incompatibility between the blood types
of the mother and baby leading to more rapid breakdown of the infant's red
blood cells.

Measurement of total serum bilirubin

Principle

Based on the reaction of bilirubin with diazotized sulphanilic acid, after

releasing albumin bound bilirubin by caffeine. Conjugated bilirubin reacts in
aqueous solution directly with diazo reagent and give colored complex that is
measured spectrophotometrically and compared with standard bilirubin; whereas
unconjugated bilirubin requires an accelerator or solubilizer to react with diazo
reagent, such as methanol (because it is water insoluble).
In the presence of accelerator both direct and indirect react and total
bilirubin is measured. In the presence of water only direct bi]irubin reacts.
subtraction of direct from total will give the indirect bilirubin concentration.
Procedure

Water
Serum 0.2 0.2 0.2
Standard
Diazo R 0.5 0.5
Diazo B
Methanol
Water
2.5
2.5
0.5

2.5
-- 2.5
==-------
0.5

Stir all tubes and allow standing in the dark for 30 min read the absorb
----
' ance ai
540 nm.

Calcu/atio11

Ar,-Ac,
Total bilirubin = - - - X 171 Tt: Total test ; ct: control ; st standard
{mmol/L) As, - Aste
A0t-Ac1
Direct bilirubin - - - - X 171; Dt: Direct test; stc: standard control
(mrnol/L) As, - Aste

Indirect bilirubin (mmol/L) = total bilirubin - direct bilirubin

NOTE: 1 mg/di ofbilirubin =I 7 µmol/L .

Reagents .
. • . 0 3 ml soluuon
Diazo R consists of the following solutions ( 10 ml solution A + ·
8): 'd
Jfanilic acr.
Solution A: 15 ml concentrated HCL in 985 ml water with Jgm su
Solution B: 0.5 gm Na-Nitrite in 100 ml water.
Diazo B (blank) consists of 15 ml HCL in 1 liter water.
 PRACTICAL CLINICAL BIOCHEM[STRf

principle . . ..
Sulfanilic acid reacts with sodmm nitrite to form diazotized sulfanilic acid.
Total bilirubin reacts with diazotized sulfanilic acid in the presence of dimethyl
suJfoxide to form azobilirubin. In the absence of dimethyl sulfoxide, only direct
• . bi·n reacts. with diazotized sulfanilic· acid to form azobilirubin .
1
~IN

Total bilirubin

50 µI 50 µI
Sample 50 µI 50 µI

1 ml I ml
Working solution 1 ml I ml

•Working solution mix 20 volume R 1 with I volume R3

• Wave length 555 nm
•Measurement against blank
•Mix well and incubate exactly 5 min at 37 C 0

•Read the absorbance (A) of standard and test against their blanks

Calculation

Ar
Total hilirubin = - - - X Standard concentration
As,
 l'.RACT/Cdl, CL/N/Q1L B ! q ~

Direct /Ji/iru/Ji11

.
Sample
'. '
-
50 µI

R2(D) J ml
-
Working
solution (D)
I ml
-
• Working solution mix 20 volume R2 with l volume R3
• Wave length 555 nm
• Measurement against blank
• Mix well and incubate exactly 5 min at 37 C 0

• Read the absorbance (A) of standard and test against their blanks

Calculation
Ar
Direct bilirubin = - - X Standard concentration
As,
Reagents:
R1: sulfanilic acid 30 mmol/L

Hydrochloric acid 150 mmol/L

Dimethyl sulfoxide 7 mmol/L
R2: sulfanilic acid 30 mmol/L
Hydrochloric acid J so mmol/L
R3: sodium nitrite 20 mmol!L
Rt: standard
 PRACTICAL CLINICAL BIOCHEMISTRY

tRANSAMINASES
3.
r,ansamination
ls the process in which an amino group is transferred from a-amino acid to
a-keto acid. The two enzymes that can take part in such reaction are ALT and
AST. These enzymes are intracellular and widely distributed in human tissues.
Both enzymes are normally present at low level in the blood; they leak into the
blood and increase their levels when injury or damage occurs to the tissues
containing these enzymes.
Both aminotransferases are good markers of liver cells damage that occurs
in disorders such as viral hepatitis. Both enzymes are normally present at low
levels in the blood, but if liver cells are damaged, some of the enzymes leak into
the blood and increase their levels. Virtually any injury to liver cells can raise
aminotransferase levels. However, the level of enzymes does not necessarily
reflect how severely the liver is damaged. Reference values for ALT are less
than 36IU/L and for AST are less than 42IU/L.

Aminotransferases

a. SERUM ALANINE TRANSAMINASE (ALT)

Is a liver specific enzyme, also called glutamate pyruvate transaminase
(GPT). It plays important role in the metabolism, released into the blood stream
in case of liver injury. Its level is especially high with acute viral hepatitis.
Alanine+ a-ketoglutaric acid < ➔ pyruvic acid + glutamic acid

Causes of increased ALT

a. markedly raised levels in:
1· Viral hepatitis: in acute hepatitis both AST and ALT are increased, while in
chronic hepatitis serum ALT is much more sensitive than AST and persist for a
long period than AST this is .because AL~ has -long half-life than AST in
add'·
ition the latter may be normal.
 fllACTICAL CLINICAL BIQCH~

2. Toxic liver necrosis: with mild cellular damage ALT levels are h'
1gher
AST, wherease AST is predominates in more sever cellular d
arnage
necrosis.
3. Circulatory failure.
b. moderately raised levels as in cirrhosis; cholestatic,· 1·1ver c·on
gesr;
secondary to cardiac failure; extensive trauma; muscular disease and 1. ,r,
11.Jecti
mononucleosis (liver involvement).
Measurement of serum ALT activity

Principle
The reagent 2,4 dinitrophenylhydrazine (DNPH) reacts with
ketocarboxylic acid to give brown colored complex (pyruvate hydrazine) whi
is measured spectrophotometrically at 510 nm.

Procedure
Wann the substrate by putting in 37 C" water bath for 3 min.

ALT or AST
substrate

Serum 0.1

Incubate for 30 min in 37 C' in water bath

Standard 0.1
D.W 0.1

After incubation of the test, add to all tubes:

DNPH 0.5 0.5 0.5

Serum 0.1
Wait for 20 min at room temperature

0.4 N (NaOH) 5 5 5
5

Wait for 10 min at room temperature then read the absorbance at 5 IO nm

 PBACTK4£ a!NIC4£ BIOCHEMISIRX. .

vate fonned by the serum is responsible for the difference between

'fhe pyrU
d control (T - C). The pyruvate in 0.1 ml of the working standard o4
the test an . .
duces the difference between the standard and blank (Std - B).
µtnol pro

Calculation
Obtain the activity of ALT in the serum from the fo\lowing equation:
Ar Ac 1 1000
ALT activity = - - - - X 0.4 X - - X---
30 0.1

Ar Ac
Pyruvate /min/L = X 133
Ast -As

Or can obtain the activity of ALT in the serum from the table below:

0.275 48

0.050 8 0.300 52

0.075 12 0.325 57

O.lOO 17 0.350 62

0.125 21 0.375 67

0.150 25 0.400 72

0.175 29 0.425 77

0.200 34 0.450 83

0.225 39 0.475 88
0.250 43 0.500 94
 PRACTICAL CLINICAL BIQCHfMJS7:.Bx b
b. SERUM ASPARTATE TRANSAMINASE (AST)
q
ls called glutamate oxaloacetate transaminase (GOT). It is simila
r to ALT .
that it is another enzyme associated with liver parenchymal ce1ls so . . . in
. . . is raised
it
in acute liver damage, but 1s also present cardiac, skeletal muscle k"
111
s, idnevs
brain pancreas lungs, leukocytes and erythrocytes, while ALT . e: • ·
, ' is iounct
primarily in the liver. Elevated AST levels are not specific for liver dama
ge, and
have also been used as a cardiac marker ; therefore, it is important to
measure ALT as liver specific enzyme at the same time.
Aspartic acid + a-ketoglutaric acid < > oxaloacetic acid+ glutamic acid

Causes of increased AST

1. Physiological: in newborn approximately 1 ½ titnes more than normal adult
value; during and immediately after labor a moderate raises are noted.
2. Pathological:
a. markedly increased activity in myocardial infarction (Ml) starts rapidly after
6-8 hrs from the onset of Ml, it may be 2-20 times the upper normal limit are
reached within 24-48 hrs and normal levels are regained typically within 3-6
days.
b. viral hepatitis: in the prodromal phase transaminase levels are raised, reach
their peak at about the time jaundice is noticed and remain elevated for 2 weeks.
c. toxic liver necrosis: due to hepatotoxic drugs or chemicals

d. circulatory failure: in shock and hypoxia

e. moderately raised levels as in cirrhosis (up to twice nonnal); cho/eSWtic
jaundice (up to 10 times nonnal); malignant infiltration of the liver; skeletal
,y. serer
muscle disease as in muscular dystrophy; after trauma or surge '
hemolytic anaemia and infectious mononucleosis (liver involvement)
Artefactully: occur in hemolysed specimen
 PRACTICAL UINICAL BIOCHEMISTRY

ent of serum AST activity

Measurem
same steps for ALT measurement, except:
follow the
trate is different (Aspartic and a-ketoglutarate).
1, The subs,
I, The incubation time is 60 min instead of 30 min.

Calculation
Ar-Ac 1000
AST activity = - - - - X 0.4 X - - X - - -
60 0.1

Ar Ac
= --- X67
As,-AB

Reagents

1. Phosphate buffer (pH 7.4)

2. ALT substrate
3. Stock pyruvate standard (20 mM)
4. Working pyruvate standard
5. 2,4-DNPH (1 mM)
6. 0.4 N sodium hydroxide.

4. ENZYMES OF BILIARY TRACT

4.a. ALKALINE PHOSPHATASE (ALP)

ls an enzyme present in the cells lining the biliary ducts of the liver. ALP
levels in plasma will rise with large bile duct obstruction, intrahepatic
cholestasis or infiltrative diseases of the liver. ALP is also present in the bone
so it is higher in growing children and placental tissues in the 3rd trimester of
pregnancy at which ALP is ~2-3 times higher than the normal also in elderly
patients with Paget's disease.