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Liver Function Test

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LIVER FUNCTION TESTLIVER FUNCTION TESTliverliver AnatomyAnatomy

Liver is the largest gland of the body , and Liver is the largest gland of the body , and is the soft pinkish brown organ ,weighing is the soft pinkish brown organ ,weighing 1000 – 1600 gram in adult. Its anatomical 1000 – 1600 gram in adult. Its anatomical position is in theposition is in the Rt upper abdominal area Rt upper abdominal area under the diaphragm ,it makes a bed for the under the diaphragm ,it makes a bed for the gall bladder .gall bladder .The liver is supplied by two main blood The liver is supplied by two main blood vessels : The hepatic artery and the portal vessels : The hepatic artery and the portal vein.vein.

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• dual blood supply

• portal vein from GI tract, pancreas, spleen

• artery from aorta

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• LIVER PHYSIOLOGY • NORMAL LIVER FUNCTION :- 1. Carbohydrate metabolism

2. Lipid metabolism

3. Synthetic function

4. Storage function

5. Excretory function

6. Detoxification

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BLOOD CELLS

LIVER

Bilirubin diglucuronide(water-soluble)

2 UDP-glucuronic acid

via bile duct to intestines

Stercobilin excreted in feces

Urobilinogen formed by bacteria

KIDNEY

Urobilin excreted in urine

CO

Biliverdin IX

Heme oxygenase

O2

Bilirubin (water-insoluble)

NADP+

NADPH

Biliverdinreductase

HemeGlobin

Hemoglobin

reabsorbed into blood

Bilirubin (water-insoluble)via blood

to the liver

INTESTINE

Catabolism of hemoglobin 08/04/2308/04/23 Mutaz - Yassir - MullahMutaz - Yassir - Mullah

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• 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

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Genetic Disorders of Bilirubin Metabolism

Condition Defect BilirubinClinical

Findings

Crigler-Najjar syndrome

severely defective UDP-glucuronyltransferase

Un conjugated bilirubin

Profound jaundice

Gilberts syndrome

reduced activity of UDP-glucuronyltransferase

Un conjugated bilirubin

Very mild jaundice during illnesses

Dubin -Johnson syndrome

abnormal transport of conjugated bilirubin into the biliary system

Conjugated bilirubin

Moderate jaundice

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• 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 .

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

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

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• 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

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• Liver failure :-Advanced liver cirrhosis characterized by :

1. Hypotension

2. Hepatic-Renal syndrom

3. Impaired de amination of amino acid

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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

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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

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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

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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 :-

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• (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)

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• (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)

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• © 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

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• (A).1: Plasma total protein Method of estimation :-

1. Biuret method • Principle • Reagent preparation • Procedure

2.Kjeldahl method • Principle • Disadvantage

3.Turbidimetry method

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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

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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

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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 .

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Protein Fractions: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.

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Methods For Albumin Estimation: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.

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It is not most used because:

• Direct methods of albumin are available.

• Labor intensive.

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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.

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• 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.

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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.

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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.

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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.

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Rocket ImmunoelectrophoresisRocket Immunoelectrophoresis

+

-

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Total GlobulinsTotal 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.

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Cellulose acetate Cellulose acetate electrophoresis of serum electrophoresis of serum

proteinprotein

albumin

-regionSerum no.

123456

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Separated protein bands can be Separated protein bands can be quantified using Densitometerquantified using Densitometer

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Serum Protein ElectrophoresisSerum Protein Electrophoresis

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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)

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• Liver disease

Normal

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Alpha-1-anti-trypsin deficiency

Normal

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• 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

Clinical Significance of AlbuminClinical Significance of Albumin

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Analysis of bilirubinAnalysis 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.

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Sources of ErrorSources of Error

• Lipochrome pigments.

• Hemolyzed specimen → ↓serum bilirubin.

• Lipemic causes interference, a fasting specimens are preferable.

• Exposure to fluorescent and indirect and direct sunlight.

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Serum bilirubin methodsSerum 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.

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Jendrassik and Grof 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.

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• 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.

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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.

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Reference Ranges for Bilirubin Reference Ranges for Bilirubin ConcentrationsConcentrations

AGE Total Bilirubin Conjugated Bilirubin

Infants <1 month of age

4.0 – 8.0 mg/dL

68 – 137 mol/L

0 – 2.0 mg/dL

0 – 34 mol/L

Adults0.2 – 1.0 mg/dL

3.4 – 17 mol/L

0 – 0.2 mg/dL

0 – 3.4 mol/L

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Urine Bile PigmentUrine 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.

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• 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.

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Lab. Finding in JaundicesLab. Finding in JaundicesHemolytic Hepatic Obstructive

Serum bilirubin ↑ (Mainly unconjugated)

↑ Later (Mainly conjugated)

↑ (Mainly conjugated)

Urine bilirubin Normal Normal & ↑ Later

↑ ↑

Urine urobilinogen

↑ ↑ Normal & absent later

Absent

AST & ALT Mildly ↑ ↑ ↑ ↑ Mildly ↑

ALP Normal Slightly ↑ ↑ ↑ ↑

LDH ↑ Slightly ↑ Slightly ↑

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liver enzymes are:liver enzymes are:

• ALKALINE PHOSPHATASE

• GAMMA GLUTAMYLTRANSPEPTIDASE

• ASPARTATE TRANSAMINASE

• ALANINE TRANSAMINASE

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GAMMA GAMMA GLUTAMYLTRANSFERASEGLUTAMYLTRANSFERASE

• LOCATION:

• GGT PRESENT IN THE CELLS OF LIVER,KIDNEYS,PANCREASE AND PROSTATE.

• PLASMA GTT ACTIVITY IS HIGHER IN MALE THAN FEMALE.

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CAUSES OF RAISED PLASMA CAUSES OF RAISED PLASMA GTT ACTIVITYGTT 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)

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• 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.

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GAMMA GGTGAMMA 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

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• 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

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ALANINE AMINO ALANINE AMINO TRANSFERASETRANSFERASE

• 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.

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CAUSES OF RAISED PLASMA CAUSES OF RAISED PLASMA ALT ACTIVITIESALT 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).

- DRUGS.08/04/2308/04/23 Mutaz - Yassir - MullahMutaz - Yassir - Mullah

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DETERMINATION OF SERUM DETERMINATION OF SERUM ALTALT

• 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.

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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.

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REITMAN-FRANKELREITMAN-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.

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ASPARTATE ASPARTATE AMINOTRANSFERASEAMINOTRANSFERASE

• 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.

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Causes of raised plasma AST Causes of raised plasma AST activitiesactivities

• 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.

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• 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.

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DETERMINATION OF SERUM DETERMINATION OF SERUM ASTAST

• 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.

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• 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

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ALKALINE PHOSPHATASEALKALINE 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.

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CAUSES OF RAISED PLASMA CAUSES OF RAISED PLASMA ALP ACTIVITYALP 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

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• 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

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• LIVER DISEASE:

- INTRA/EXTRA HEPATIC CHOLESTASIS

- TUMORS,GRANULOMAS

- INTESTINAL OBSTRUCTION.

- MALIGNANCY.

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DETERMINATION OF ALKALINE DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITYPHOSPHATASE 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.

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• 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.

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• 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

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SEPARATION OF ALP SEPARATION OF ALP ISOENZYMESISOENZYMES

• 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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