Liver Function Test

LIVER FUNCTION TEST
liver Anatomy
Liver is the largest gland of the body , and
is the soft pinkish brown organ ,weighing
1000 – 1600 gram in adult. Its anatomical
position is in the Rt upper abdominal area
under the diaphragm ,it makes a bed for the
gall bladder .
The liver is supplied by two main blood
vessels : The hepatic artery and the portal
vein.
02/11/21 Mutaz - Yassir - Mullah 3
• dual blood
supply
• portal vein
from GI
tract,
pancreas,
spleen
• artery from
aorta

• LIVER PHYSIOLOGY
• NORMAL LIVER FUNCTION :-
1. Carbohydrate metabolism
2. Lipid metabolism
3. Synthetic function
4. Storage function
5. Excretory function
6. Detoxification

BLOOD
Stercobilin
CELLS Urobilin
excreted in feces
Hemoglobin excreted in urine
Globin
Urobilinogen
Heme
O2 formed by bacteria KIDNEY
reabsorbed
Heme oxygenase INTESTINE into blood
CO
Biliverdin IX via bile duct to intestines

NADPH
Biliverdin Bilirubin diglucuronide
reductase (water-soluble)
NADP+ 2 UDP-glucuronic acid

Bilirubin Bilirubin
(water-insoluble) LIVER
(water-insoluble) via blood
to the liver
Catabolism of hemoglobin
• LIVER DISORDER :-
• 1: Jaundice :-
refer to the yellowish discoloration of the skin and
sclera ,resulting from hyper bilirubinemia . it is
not clinically apparent until the bilirubin level
exceed 2 mg/dl
Type of Jaundice :-
1. Pre hepatic Jaundice
2. Hepatic Jaundice
3. Post hepatic Jaundice

Genetic Disorders of Bilirubin Metabolism
Clinical
Condition Defect Bilirubin
Findings
Crigler-Najjar severely defective Un conjugated Profound
syndrome UDP- bilirubin  jaundice
glucuronyltransferase
Gilberts reduced activity of Un conjugated Very mild
syndrome UDP- bilirubin  jaundice during
glucuronyltransferase illnesses
Dubin abnormal transport of Conjugated Moderate
-Johnson conjugated bilirubin into bilirubin  jaundice
syndrome the biliary system

• LIVER CIRRHOSIS :
Refer to the irreversible scarring

process by which normal liver
architecture is transformed into
abnormal nodular architecture .
Cirrhosis can classified to macro
nodular and micro nodular
cirrhosis .

Portal hypertension occur when
blood flow through the portal vein
is obstructed by cirrhosis ,this
may be result in splenomegaly ,
esophageal varices

• The synthetic ability is reduced
causing hypo albuminemia )
ascites) and clotting factor
deficiency (bleeding )

• Causes of liver cirrhosis :-
1. Auto immune hepatitis
2. Chronic alcohol intake
3. Persistent of hepatitis B and C
4. Inherited metabolic disorder such as :
• Wilson,s disease
• Haemochromatosis
• Alpha-1- anti trypsin deficiency
• Galactosaemia

• Liver failure :-
Advanced liver cirrhosis characterized by :
1. Hypotension
2. Hepatic-Renal syndrom
3. Impaired de amination of amino acid

Liver tumor :
Most cases of hepato cellular carcinoma
can be related to previous infection
with a hepatitis virus. Also there is
metastatic tumor to the liver from
primary site ( lung , ovary ,etc ) .
Benign tumor of the liver is not
common .
Any malignancy of the liver is a serious
finding with a poor prognosis

Acute hepatitis :
Caused by :-
1. A,B,C,D and E hepatitis virus
2. Epstein Barr virus (EBV)
3. Cytomegalovirus (CMV)
4. Alcohol, toxin, paracitamol and fungal toxin
Outcome of acute hepatitis :-
1. Complete resolution in most cases
2. Chronic hepatic damage
3. Progress to acute hepatic failure

Reyes syndrome
It is a form of hepatic destruction that usually
occur following recovery from a viral
infection such as chickenpox and influenza
It has been related to aspirin therapy
Patient develops neurological abnormalities
Liver function are always abnormal

LIVER FUNCTION TEST (LFT)
Definition : are group of clinical biochemistry
laboratory blood assays designed to give
information about the state of the liver ,
these tests are performed by a medical
technologist on a patient serum or plasma
obtained by phlebotomy .
LFTs fall into three main categories :-

• (A): STANDARED PANEL TEST :-
Used as indicator of liver disease ,
include :
1. Plasma total protein
2. Plasma albumin
3. Plasma total bilirubin
4. Liver enzymes (AST,ALT,ALP)

• (B) TRUE LIVER TEST -:
Reflect the ability of the liver to perform it is
vital function , include :-
1. Bromo sulphathaline test (BST)
2. Bile acid
3. Blood ammonia
4. Blood clotting factor (INR)

• © SPECIFIC LIVER TEST :-
To diagnose underlying cause of liver disease ,
include :-
1. Serum iron (haemochromatosis)
2. Blood ceruloplasmin (Wilson,s disease)
3. Alpha 1-anti trypsin (A-1-anti trypsin dif )
4. Anti nuclear, anti smooth muscle Abs
(autoimmune CH )
5. Anti mitochondrial Abs (p.biliary cirrhosis)
6. Alpha 1-feto protein (Hepato.C.carcinoma)
7.Hepatitis A,B,C and D Abs
• (A).1: Plasma total protein
Method of estimation :-
1. Biuret method
• Principle
• Reagent preparation
• Procedure
2.Kjeldahl method
• Principle
• Disadvantage
3.Turbidimetry method

4. Specific gravity method :
• Principle :
The serum when dropped in cuppric
sulfate solution (159 g/L ) it is either
float if the protein is low or sinks if the
protein concentration is high
• Disadvantage :
- Less sensitive
- Not specific

5. Ultra violet method :
• Principle :
Most of the protein absorbs UV light at
280nm ,so it can be measured at that
wave length using spectrophotometer
• Disadvantage :
- Turbid sample cause interference

6. Refractometer method:
Principle :
Refract meter is a machine that used to read the
refraction of different solution . The protein in the
serum changes the refractive index of the
diluted sample , this change is proportional to
the protein concentration in the sample .
Advantage : - 1: small drop of sample need .
2:No reagent, STD are need .
3: Get the result directly from the scale .

Protein Fractions:
In the assay of total proteins, useful diagnostic
information can be obtained by determined
the albumin fraction & total globulins. A
several or significant change in the ratio of
albumin and total globulin was first noticed
in diseases of the kidney and liver.

Methods For Albumin Estimation:
1. Salt fractionation (precipitation):
Globulins can be separated from albumin
by salting-out ( Na2SO4, Na2SO3), by
decreasing the water available for
hydration of hydrophilic groups, will cause
precipitation of the globulins. The albumin
that remains in solution in the supernatant
can be measured by any of the routine total
protein methods.
It is not most used because:
• Direct methods of albumin are available.
• Labor intensive.

2. Dye binding:
• The most widely used methods.
• The pH of the solution is adjusted to that
albumin is positively charged. Then, by
electrostatic forces, the albumin is attracted
to and binds to an anionic dye, the dye
binding causes shift in absorption maximum.
The amount of albumin can be quantitated
by measurement of absorbance of the
albumin-dye complex.

• Dye binding include:
a)Methyl orange: Non specific for albumin, -
lipoprotein & some 1- and 2-globulins also
will bind to this dye.
b)HABA (2-4-hydroxy-azobenzene-benzoic
acid): More specific for albumin, has a low
sensitivity. Several compounds, such as
salicylates, pencillin, conjugated bilirubin
and sulfonamides, interfere with the binding
of albumin to the dye.
c) Bromocresol purple (BCP):
• Specific, sensitive, & precise.
• BCP binding to albumin is impaired in the
presence of covalently bound bilirubin,
BCG binding is unaffecting in these
situations.

d) Bromocresol green (BCG):
o Is not affected by interfering substances
such as bilirubin & salicylates however, Hb
can bind to the dye (100 mg/dl of Hb, the
albumin is increased by 0.1 g/dl).
o It is sensitive: overestimates low albumin
levels.
o Most commonly used.
o Ceruloplasmin & 1-acid glycoprotein react
with BCG after incubation times exceed 5
minutes.
3. Electrophoresis.
4. Rocket immunoelectrophoresis:
Reagent Ab is mixed with agarose Ag is
placed in well and electrophoresed. As the
antigens moves through the agarose, it
reacts with the reagent Ab and forms a
rocket with stronger precipitation along the
edges. The light of the rocket is proportional
to the concentration of Ag present, the
concentration is determined based on a
calibration curve.
Rocket Immunoelectrophoresis

Total Globulins
• Globulins are calculated by subtracting the
albumin from the total protein.
Total protein – Albumin = Globulin
• Direct colorimetric method ( Glyoxylic acid):
Principle: Glyoxylic acid, in the presence of
Cu2+ & acid medium ( acetic acid & H2SO4),
condenses with tryptophan found in
globulins to produce a purple color.
Cellulose acetate electrophoresis
of serum protein
Serum no.
albumin
 -region
1
2
3
4
5
6
 

Separated protein bands can be
quantified using Densitometer

Serum Protein Electrophoresis

Normal SPE
Albumin 47-71% (3.63-4.91 g/dL)

Alpha-1 globulin 2.7-5.8% (0.11-0.35 g/dL)
Alpha-2 globulin 5.1-12.0% (0.65-1.17 g/dL)
Beta- globulin 4.5-15.7% (0.74-1.26 g/dL)
Gamma globulin 11.3-24.0% (0.58-1.74 g/dL)

Normal
• Liver disease

Normal
Alpha-1-anti-trypsin deficiency

Clinical Significance of Albumin
• Reference range 3.5-5.5 g/dL

• Hyperalbuminemia dehydration
• Hypoalbuminemia
– Low intake, synthesis
– Increase loss:
• kidney; nephrotic syndrome, wound, burn
• GI tract; protein-losing enteropathy
– Increase catabolism

Analysis of bilirubin
Specimen Collection and Storage:
• Fasting serum specimen.
• Neither hemolyzed nor lipemic.
• Before testing, serum should be stored in dark &
measured as soon as possible (within 2-3 hours)
after collection.
• Serum may be stored in the dark in a refrigerator
for up to 1 week and in freezer for 3 months.

Sources of Error
• Lipochrome pigments.
• Hemolyzed specimen → ↓serum bilirubin.
• Lipemic causes interference, a fasting
specimens are preferable.
• Exposure to fluorescent and indirect and
direct sunlight.

Serum bilirubin methods
• Van den Bergh method: used alcohol as an
accelerator for coupling of bilirubin to diazotized
sulfanilic acid.
• Malloy and Evelyn method: using 50%
methanol as an accelerator for coupling of
bilirubin to diazotized sulfanilic acid, a
technique that avoided the precipitation of
proteins that was a source of error in the Van
den Bergh method.
Jendrassik and Grof method:
• Principle: Serum or plasma is added to a

solution of sodium acetate and caffeine-
sodium benzoate, which is then added to
diazotized sulfanilic acid (sulfanilic acid,
sodium nitrite,& HCl) to form purple
azobilirubin.

• The sodium acetate buffers the pH.
• The caffeine-sodium benzoate
accelerates the coupling of bilirubin with
diazotized sulfanilic acid.
• Ascorbic acid is terminated reaction, which
destroys the excess diazo reagent.
• A strongly alkaline tartrate solution is then
added to convert the purple azobilirubin to
blue azobilirubin.
• And the intensity of the color is read at
600 nm.

Direct Spectrophotometric method:
• Principle: The absorbance of bilirubin in
serum at 455 nm is proportional to its
concentration. The absorbance of
hemoglobin at 455 nm is corrected by
subtracting the absorbance at 575 nm.

Reference Ranges for Bilirubin
Concentrations
AGE Total Bilirubin Conjugated Bilirubin
Infants <1 month of 4.0 – 8.0 mg/dL 0 – 2.0 mg/dL

age 68 – 137 mol/L 0 – 34 mol/L
0.2 – 1.0 mg/dL 0 – 0.2 mg/dL

Adults
3.4 – 17 mol/L 0 – 3.4 mol/L

Urine Bile Pigment
• Tincture iodine test: Layer some tincture iodine
carefully on to some of the urine in a test tube. A
green ring at the junction of the two fluids indicates
the presence of bilirubin.
• Fouchet's test: Barium chloride reacts with the
sulfate radicals in the urine to form a precipitate of
barium sulfate. Any bile pigment present adheres to
the precipitate and is detected by the oxidation of
bilirubin (yellow) to biliverdin (green) on treatment
with ferric chloride in the presence of TCA, a blue
color is given by bilicyanin.

• Strip test: The bilirubin reaction is base on
the coupling of bilirubin with 2,6-
dichlorobenzene-diazonium fluoroborate in
acid medium to give a reddish-violet azo
dye.
• Ehrlich's reagent for urobilinogen:
Urobilinogen reacts with p-dimethyl amino-
benzaldehyde to form a red color.

Lab. Finding in Jaundices
Hemolytic Hepatic Obstructive
Serum bilirubin ↑ (Mainly ↑ Later (Mainly ↑ (Mainly

unconjugated) conjugated) conjugated)
Urine bilirubin Normal Normal & ↑ ↑↑
Later
Urine ↑↑ Normal & Absent
urobilinogen absent later
AST & ALT Mildly ↑ ↑↑↑ Mildly ↑
ALP Normal Slightly ↑ ↑↑↑
LDH ↑ Slightly ↑ Slightly ↑

liver enzymes are:
• ALKALINE PHOSPHATASE
• GAMMA GLUTAMYLTRANSPEPTIDASE
• ASPARTATE TRANSAMINASE
• ALANINE TRANSAMINASE

GAMMA
GLUTAMYLTRANSFERASE
• LOCATION:
• GGT PRESENT IN THE CELLS OF
LIVER,KIDNEYS,PANCREASE AND
PROSTATE.
• PLASMA GTT ACTIVITY IS HIGHER IN
MALE THAN FEMALE.

CAUSES OF RAISED PLASMA
GTT ACTIVITY
• INDUCTION OF ENZYME SYNTHESIS
WITHOUT CELL DAMAGE BY DRUGS OR
ALCOHOL.
• CHOLESTATIC LIVER DISEASE (CHANGE IN
GTT USUALLY PARALLEL TO THOSE OF
ALP).
• HEPATOCELLULAR DAMAGE AS IN
INFECTIOUS HEPATITIES
(AMINOTRANSFERASE IS MORE SENSITIVE)

• VERY HIGH PLASMA GTT ACTIVITIES
NOT ASSOCIATED WITH INCREASE OF
THOSE OF TRANSEAMINASES ARE
SEEN IN:
• ALCOHOLIC HEPATITIES.
• ALCOHOL,DRUG INTAKE.
• CHOLESTATIC LIVER DISEASE.
• FATTY LIVER.
GAMMA GGT
• THIS ENZYME CATALYSE THE TRANSFER OF GAMMA-
GLUTAMYL GROUP FROM GLUTAMYL PEPTIDE TO
ANTHOR PEPTIDE OR AMINO ACID.
• KINETIC METHOD:
- GG-P-ANILIDE+GLYCYL GLYCINE GGT GG GLYCYL
GLYCINE+P-NITROANILINE.
- GGT CATALYSES THE TRANSFER OF THE G-GLUTAMYL
GROUP FROM THE SUBSTRATE G-GLUTAMYLE
PEPTIDES TO ANTHOR PEPTIDE LIKE GLYCYL GLYCINE
FORMING G-GLUTAMYL GLYCYL GLYCINE AND P-
NITROANILINE.
- P-NITOANILINE HAS MAXIMUM ABSORBANCE AT 450nm

• REAGENT:
- TRIS BUFFER PH 8.25
- GLYCYLGLYCINE L-GG-3-CARBOXYL-P-
NITROANILIDE.
• SAMPLE: SERUM&DON’T USED PLASMA.
• STABILITY: 8H AT 15-25C,5 DAYS AT -20C
• WL 405nm,BLANK AIR OR D.W
• CALCULATION: EXTENTION INCREASED
EVERY MIN FOR 10MINXFACTOR=U/L
• LINEARITY UP TO 250U/L
• RV:WOMEN 5-25U/L,MEN 8-38U/L

ALANINE AMINO
TRANSFERASE
• LOCATION: IS PRESENT IN HIGH CONENTERATION
IN THE LIVER AND TO LESS EXTEND IN SKELETAL
MUSCLE,KIDNEY AND HEART.
• DAMAGE TO ANY OF THESE TISSUE MAY CAUSE
ELEVATION IN PLASMA ALT LEVEL.
• ALT CATALYSE THE TRANSFER OF AMINO GROUP
FROM ALANINE TO ALPHA KETO-GLUTARATE,AS
ARESULT ALANINE IS CONVERTED TO PYRUVATE
AND ALPA-KETOGLUTARATE IS CONVERTED TO
GLUTAMATE.

ALT ACTIVITIES
• MARKED INCREASE(5 TO 10 TIMES URL):
- CIRCULATORY FAILURE WITH SHOCK AND HYPOXIA.
- ACUTE VIRAL OR TOXIC HEPATITIES.
• MODERATE INCREASE(LESS THAN 5 TIMES URL):
- CIRRHOSIS(MAY BE NORMAL).
- INFECTIOUS MONONUCLEOSIS.
- LIVER CONGESTION SECONARY TO CONGESTIVE
HEART FAILURE.
- CHOLESTATIC JAUNDICE.
- SURGERY OR EXTENSIVE TRAUMA AND SKELETAL
MUSCLE DISEASE(AST MOR AFFECTED).
-02/11/21
DRUGS. Mutaz - Yassir - Mullah 67
DETERMINATION OF SERUM
ALT
• AMINOTRANSFERASE.
• KINETIC METHOD:
- L-ALANINE+ALPHP-KG ALT PYRUVAT+
L-GLUTAMATE
- PYRUVATE+NADH LDH LACTATE +NAD
- NADH IS CONVERTED TO NAD AND THE
DECREASE IN ABSORBANCE IS MEASURED AT
340nm,THE CHANGE IN ABSORBANCE/MIN IS
CALCULATED AND EXPRESSED AS U/L.
• NORMAL VALUE IN SERUM OR PLASMA 5-40U/L.
• AVOID THE USE OF HAEMOLYSED SAMPLE.

REAGENT:
- NADH /LDH/ALPHAOXOGLUTRATE.
- TRIS BUFFER PH 7.8+L-ALANINE.
SAMPLE: SERUM OR HEPARNIZED PLASMA.
WL: 340nm BLANK:AIR OR DW.
RV:MEN UP TO 30U/L FEMALE UPTO 22U/L.
AVOID HAEMOLYSED SAMPLE.

REITMAN-FRANKEL
• SERUM GOT/GPT CATALYSE THE TRANSFER OF
THE AMINO GROP FROM ASPARTATE/ALAINE TO
2,OXOGLUTARATE DURING REVERSIBLE REACTION
WITH FORMATION OF GLUTAMATE AND
OXALOACETATE/PYRUVATE TO FORM HIGHLY
COLOURED HYDRAZONE WITH ADDITION OF 2,4-
DNPH IN ALKALINE MEDIA (0.4N NAOH)AS ACOLOUR
DEVELOPER ,ABSORBANCE OF HYRAZONE IS
MEASURED COLORIMETRICALLY AT 520nm WHICH
IS DIRECTLY PROPORTIONAL TO ACTIVITY OF GPT
OVER DEFINED PERIOD OF TIME.
• NORMAL VALUE 2.4-14.5 U/L.

ASPARTATE
AMINOTRANSFERASE
• LOCATION:- IS PRESENT IN HIGH CONCENTRATION
IN CELLS OF CARDIAC & SKELETAL MUSCLES,
LIVER, KIDNEY, & ERYTHROCYTE.
• DAMAGE TO ANY OF THESE TISSUES MAY
INCREASE PLASMA AST LEVEL.
• AST TRANSFER THE AMINO GROUP FROM
ASPARTATE TO ALPHA-KETOGLUTRATE,AS
ARESULT ASPARTATE IS CONVERTED TO
OXALOACETATE AND ALPHA-KETOGLUTARATE IS
CONVERTED TO GLUTAMATE AND THIS
REVERSIBLE REACTION REGUIRES PYRIDOXAL
PHOSPHATE AS ACO-ENZYME.
Causes of raised plasma AST
activities
• ARTEFACTUAL: DUE TO INVITRO RELEASE FROM
ERYTHROCYTE IF THERE IS HAEMOLYSIS OR
DELAYED PLASMA SEPARATION FROM THE CELLS.
• PHYSIOLOGICAL: DURING NEONATAL PERIOD AST IS
1.5 TIMES THE UPPER REFERANCE LIMIT.
• MARKET INCREASE(5 TO 10 TIMES THE URL):
-CICULATORY FAILURE WITH SHOCK AND HYPOXIA.
-MYOCARDIAL INFARCTION
- ACUTE VIRAL OR TOXIC HEPATITIES.

• MODERATE INCREASE(LESS THAN 5 TIMES URL:
- CIRRHOSIS(MAY BE NORMAL)
- INFECTIOUS MONONUCLEOSIS.
- MALIGNANT INFILTERATION OF THE LIVER(MAY
BE NORMAL).
- SKELETAL MUSCLE DISEASE.
- CHOLESTATIC JAUNDICE.
- AFTER TRAUMA OR SERGERY(ESPECIALLY
CARDIAC SERGERY).
- SEVER HAEMOLYTIC DISEASE(FROM
ERYTHROCYTE ORIGN).
- DRUGS.

DETERMINATION OF SERUM
AST
• 1- KINETIC METHOD:
• L-ASPARTATE+ALPHA-KG AST
OXALOACETATE+GLUTAMATE
• OXALOACETATE RODUCED IS CONVERTED TO MALATE WITH
THE HELP OF MALATE DEHYDROGENASE AND NADH
PRODUCED IS CONVERTED TO NAD
• NADH HAS THE MAXIMUM ABSORBANCEAT 340nm AND NAD
HAS LESS ABSPORBANCE AT 340nm,SO CONTINEOUSE OF
THE REACTION CAUSING DECREASE IN THE ABSORBANCE.
• CHANGE IN THE ABSORBANCE PER MIN IS CALCULATED AND
THE ACTIVITY OF THE ENZYME IS EXPRESSED AS U/L.
• NORMAL VALUE IN SERUM OR PLASMA ABOUT 5-40 U/L.
• AVOID HAEMOLYSED SAMPLE.

• REAGENT:
- TRIS BUFFER PH7.8 +L-ASPARTATE
- NADH LDH MDH
- ALPA OXOGLUTRATE
• STABILITY 6MONTHES AT 2-8C 3WEEK AT
15-25C
• SAMPLE: SERUM OR HEPARNIZED PLASMA
• BLANK AIR/DW
• WL 340nm
• AVOID HAEMOLYSED SAMPLE

ALKALINE PHOSPHATASE
• ENZYME HYDROLYSE ORGANIC PHOSPHATE AT
HIGH PH.
• LOCATION: ARE PRESENT IN MOST TISSUES AND
PARTICULARY AT HIGH CONCENTRATION IN THE
OSTEOCLASTS OF BONE AND CELLS OF THE
BILIARY TRACT,INTESTINAL WALL,RENAL TUBULES
AND PLACENTA.
• A DULT PLASMA ALP IS DRIVED MAINLY FROM
BONE AND LIVER.

ALP ACTIVITY
• PHYSIOLOGICAL:
- DURING LAST TRIMESTER OF PREGNANCY,THE
PLASMA TOTAL ALP ACTIVITY RAISED DUE TO
PLASENTAL ISOENZYME(ALP INCREASE UP TO
FIVE TIMES AND RETURN TO NORMAL LEVEL BY 1
MONTH POST-PARTUM).
- IN PRETERM INFANTS TOTAL ALP ACTIVITY IS UP
TO 5 TIMES THE URL IN ADULT DUE TO BONE
ISOENZYME.
- IN CHELDREN THE TOTAL ACTIVITY ABOUT 2.5
TIMES URL DUE TO BONE GROWTH

• BONE DISEASE:
- RICKETS AND OSTEOMALACIA.
- PAGET”S DISEASE OF BONE.
- SECONDARY MALIGNANCY IN BONE.
- OSTEOGENIC SARCOMA
- PRIMARY HYPERPARATHYROIDISM
WITH EXTENSIVE BONE DISEASE.
- SECONDAEY HYBER
PARATHYROIDISM

• LIVER DISEASE:
- INTRA/EXTRA HEPATIC CHOLESTASIS
- TUMORS,GRANULOMAS
- INTESTINAL OBSTRUCTION.
- MALIGNANCY.

DETERMINATION OF ALKALINE
PHOSPHATASE ACTIVITY
• KING AND KIND METHOD:
- DISODIUM PHENYL PHOSPHATEPHENOL +PHOSPHATE
- PHENOL+4-AMINOANTIPYRINE ALP ORANGE-RED
COLORED PRODUCT
- WHEN SERUM INCUBATED WITH PHENYLPHOSPHAT BUFFER
AT PH 10 FOR 15 MIN AT 37C THE HYDROLYTIC PRODUCT IS
CONDENSED WITH 4-AMINOANTIPYRINE AND OXIDISED IN
THE PRESNCE OF ALKALINE OXIDIZING AGENT(K-
FERRICYANIDE),4-AMINOANTIPYRIN GIVES RED-PURPLE
COLOUR WITH COMPOUND CONTAINING APHENOLIC GROUP
- THE COLOUR DEVELOPED IS READ AT 510nm.
- THE ACTIVITY OF ALP EXPRESS IN KAU(PRODUCTION OF 1MG
OF PHENOL IN 15 MIN UNDER DEFIND CONDITION.

• COLORIMETRIC METHOD:
- P-NITROPHENYL PHOSPHATE ALP P-NI
TROPHENOL+PHOSPHATE
- P-NITRO PHENYL PHOSPHATE USED AS
SUBSTRATE
- ALP ACT ON PNPP AND LIBERATE P-
NITROPHENOL(YELLOW)WHICH MEASURED
COLORIMETRICALLY AT 405nm .
- RESULT ARE EXPRESSED IN U/L.
- NR:3-13KAU/DL(35-140U/L).
- SPECIMEN:SERUM OR HEPARNIZED PLASMA.

• KINETIC METHOD:
- P-NITROPHENYL-PHOSPHATE ALP P-
NITROPHENOL+PHOSPHATE
• REAGENT:
- DIETHANOLAMINE BUFFER PH
10.4+MgCL
- P-NITROPHENYL-PHOSPHATE
• SAMPLE:SERUM-PLASMA
• WL:405nm BLANK:AIR-DW
• CALCULATION:A/MIN X 3300= U/L
• RV:CHILDREN LESS THAN 480U/L
-ADULT LESS
THAN 207 U/L
SEPARATION OF ALP
ISOENZYMES
• THE ISO ENZYMES OF ALP ARE SEPARATED BY THE
FOLLOWING TECHNIGUE:
• ELECTROPHORESIS
• CHEMICAL INHIBITOR:
• EDTA IN ACTIVATES ALL ISOENZYMES OF ALP EXCEPT
PLACENTAL ALP
• HEAT STABILITY:
• PLACNTAL ALP IS STABLE TO HEAT WHERE OTHER ENZYMES
ARE INACTIVATED BY HEAT AT 56C FOR 10MIN.
• UREA:
• LIVER ENZYME LABILE,PLACENTAL ENZYME ARE STABLE.


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LIVER FUNCTION TEST

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Biliverdin IX via bile duct to intestines

NADP+ 2 UDP-glucuronic acid

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Refer to the irreversible scarring

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Albumin 47-71% (3.63-4.91 g/dL)

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• Reference range 3.5-5.5 g/dL

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• Principle: Serum or plasma is added to a

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Infants <1 month of 4.0 – 8.0 mg/dL 0 – 2.0 mg/dL

0.2 – 1.0 mg/dL 0 – 0.2 mg/dL

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Serum bilirubin ↑ (Mainly ↑ Later (Mainly ↑ (Mainly

ALP Normal Slightly ↑ ↑↑↑

LDH ↑ Slightly ↑ Slightly ↑

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