Download HEMOGLOBIN

HEMOGLOBIN
Hemoglobin is a conjugated protein.The non-protein part(prosthetic
group) is heme which is iron protoporphyrin.The protein part is globin.
Hemoglobin is a chromoprotein and gives blood its red colour. The most
important property of hemoglobin is its ability to form stable complex
with oxygen (oxyhemoglobin) which serves in O2 transport in the body.
Iron is found in the ferrous form (Fe++) in both hemoglobin and
oxyhemoglobin. Iron is in the oxidised form (Fe+++) in
cyanmethemoglobin.
Normal concentration of Hb in blood___14-18 gms/dl in males
12-16 gms/dl in females
DETERMINATION OF HEMOGLOBIN
BY CYANMETHEMOGLOBIN TECHNIQUE
PRINCIPLE: Blood is treated with the Drabkin’s reagent which contains
potassium ferricyanide, potassium cyanide,and NaHCO3.
Hb + ferricyanide
met-Hb
Met-Hb +cyanide
cyan met-Hb
PROCEDURE:
1) Take 3ml of Drabkin’s reagent(POISON) into a test tube
2) Puncture the finger so that blood comes out freely.
3) Place the end of the Sahli’s pipette against the drop of blood. The tube
will fill by capillary action.(Work quickly so no clotting takes place)
4) Wipe the pipette carefully and bring the level of blood upto the mark
0.02ml. Blow it into the Drabkin reagent and mix well. Incubate
5mins at room temperature.
5) Read at 546nm against distilled water.
WARNING: DRABKIN’S REAGENT CONTAINS POISON.
CALCULATION:
Conc of hemoglobin (gms/dl)= 36.77 x A
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CLINICAL INTERPRETATION:
A decrease in blood hemoglobin level is called ANEMIA. It could be due
to: 1) Decreased production of hemoglobin as in:
a) Protein deficiency (b) iron deficiency
c) Vitamin deficiency:vit B12, Folic acid, Vit C e.t.c.
d) Leukemia
(2)Increased destruction of hemoglobin as in:
a) Hemolytic anemia
b) Heavy metal poisoning:Lead
c) Infectious diseases:malaria,
An increase in blood hemoglobin level is called POLYCYTHEMIA. It
could be due to: (a) High altitudes
(b) Congenital heart diseases
(c) Emphysema
BILIRUBIN
Bilirubin is the major pigment present in human bile. It is the end product
of destruction of hemoglobin.
Hemoglobin
Globin
Amino acid pool
For reuse
Iron
iron pool
for reuse
iron free porphyrin
dedraded in reticulo-endothelial cells
of liver,spleen, bone marrow
TYPES OF BILIRUBIN:
1) Conjugated or soluble or direct bilirubin:In the liver bilirubin is
conjugated with glucouronic acid which makes it water soluble.
2) Unconjugated or insoluble or indirect bilirubin: before conjugation in
the liver ,bilirubin is lipid soluble.
NORMAL LEVELS:
Total bilirubin: 0-1mg/dl
Direct bilirubin: up to 0.25mg/dl
Indirect bilirubin: up to 0.75mg/dl
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CLINICAL SIGNIFICANCE:
Hyper bilirubinemia is called JAUNDICE
CAUSES OF JAUNDICE:
1) PRE-HEPATIC JAUNDICE: there is an increase of indirect bilirubin
due to: (a) incompatible blood transfusion (b) hemolytic anemia
2) HEPATIC JAUNDICE: can be due to:
A) Decreased glucouronyl transferase as in physiologic jaundice of
newborn. There is increase in unconjugated lipid soluble bilirubin which
can cross the blood brain barrier and produce encephalopathy.
B) Hepato-cellular damage as in viral hepatitis, toxic hepatitis, cirrhosis.
There is increase in both conjugated and unconjugated bilirubin
3) POST-HEPATIC JAUNDICE:can be due to obstruction of the common
bile duct by stone tumor, inflammation. There is increase mostly of
conjugated bilirubin.
IRON
The total amount of iron in an adult is 4-5gms/dl.
70-75% is in active form and 25% is stored in reticulo-endothelial cells of
liver and spleen.
Physiologically active iron:
a) Oxygen carrying chromoprotein e.g: hemoglobin, myoglobin.
b) Enzymes: cytochromes, catalase, peroxidase.
Storage iron: ferritin and hemosidrin.
Main site of absorption of iron is the duodenum and jejunum.In order to
cross cell membranes iron must be in Fe+2 form. Storage iron is Fe+3 form
DETERMINATION OF SERUM IRON
Principle:
1) Acidification: transferrin-Fe+3
HCl
2) Reduction: Fe+3 +thioglycolic acid
3) Colouring: Fe+2 +bathophenanthrolene
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transferrin + Fe+3
Fe+2
pink colour
Procedure:
Normal level: 60-160 mcg/ dl
a)
b)
c)
d)
CLINICAL SIGNIFICANCE:
Increase in serum iron may be due to:
Increased destruction of RBC as in hemolytic anemia
Increase iron absorption
Iron overload(blood transfusion)
Increased release from body stores e.g:viral hepatitis.
Decrease in serum iron may be due to:
a)
lack of sufficient intake
b)
Increase loss of iron in hemorrhage, menstruation.
c)
Pregnancy-increases demand on body stores.
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Enzymes
Definition: Enzymes are highly specific biologic catalysts that greatly
speed up the rate of a chemical reaction occuring in living cells. Enzymes
are found in low concentration in body fluids.
The enzyme activity is expressed in the international unit (I.U).
Definition of (IU): The amount of an enzyme that will convert one
micro-mole of substrate per minute in an assay system.
Techniques for determination of enzyme activity:
1. TWO- POINT ASSAY
a- A sample is incubated with substrate for a fixed
time.
b- Stop the reaction
c-Measure the amount of product formed or
substrate used
N.B. Must follows zero order reaction
2. KINETIC OR RATE REACTION ASSAY
Changes are measured at short time intervals or
continuously monitored.
- Co-enzyme NAD or NADP is used in this
type of reaction.
- Using chromogenic substrate
Example: In determination of lactate dehydrogenase
(LDH) activity:
Pyruvate + NADH + H+
Lactate + NAD+
NAD
1.5
NADH
1.0
0.5
256
340
Wave length  (nm)
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The extinction at 256 nm will increase due to the formation of NAD and
it will decrease at 340 nm due to the consumption of NADH
3. Radio- Immuno- Assay (RIA): it measures the concentration of
enzyme but not the catalytic activity.
Classification of Enzymes
1. Oxidoreductases: there is a hydrogen donor and a hydrogen
acceptor. a. Aerobic oxidases: use O2 as H-acceptor forming H2O
e.g.. Tyrosinase
b. Aerobic dehydrogenases: use O2 as H-acceptor
forming H2O2 e.g.Glucose oxidase
c. Anaerobic dehydrogenases: use co-enzyme as H-acceptor
e.g. LDH
2. Transferases: Transfer a group from one organic compound to
another (CH3 , NH2 , Phosphate etc…).
ex. Aminotransferases, Kinases, Transketolases…
3. Hydrolases: hydrolyse the substrate ex. Enzymes acting on
Ester bond (lipase, cholinestrase)
Peptide bond (pepsin, trypsin)
C-N bond (urease).
4. Lyases: Remove groups without hydrolysis leaving a double bond.
Ex. Decarboxylases
5. Isomerases: convert one pair of isomers into another.
Ex. Racemases.
6. Ligases = Synthetases: linking two molecules together
coupled with the breakdown of phosphate bond.
FACTORS AFFECTING ENZYMATIC REACTION:
a.
b.
c.
d.
e.
f.
g.
h.
Substrate concentration
Enzyme concentration
Product concentration
PH
Temperature
Activators and Co-enzymes
Inhibitors
Specificity of enzymes
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Determination of lactate dehydrogenase activity (LDH)
Type: anaerobic dehydrogenase enzyme
Occurrence: Heart > liver > skeletal muscle> erythrocytes > pancreas
Principle: kinetic determination of LDH according to the following
reaction:
LDH
+
Pyruvate + NADH + H
L-Lactate + NAD+
Normal serum level: 200 - 480 U/l at 37 C
Wave length: 340 nm
Procedure:
Calculation:  A / min = ( (Ao – A1) + ( A1 – A2) + (A2 – A3) ) / 3
LDH activity (U/l) =  A / min X
Clinical significance: The total LDH is non –specific index of cell
damage LDH activity indicates:
a. Myocardial infarction.
b.High value 20 X normal is seen in pernicious anaemia.
c.Moderate increase in viral hepatitis and skeletal muscle
disease.
Determination of Aminotransferases
1. Glutamate oxaloacetate transaminase (GOT) OR Aspartateamino transferase (ASAT)
Occurrence: Heart > liver > skeletal muscle > kidney > panceas
Principle: kinetic determination of GOT activity according to the
following reaction:
L-aspartate + - ketoglutarate
oxaloacetate + L-glutamate
Oxaloacetate + NADH + H+
MDH : malate dehydrogenase
L-malate + NAD+
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Normal serum range: up to 37 U/l at 37 C.
Wave length: 340 nm
Procedure:
Calculation:  A / min =
(Ao – A1) + (A1 – A2) + (A2 – A3)
GOT activity (U/l) =  A/min X
3
Clinical significance: GOT activity increase in
1.
Myocardial infarction.
2.
Hepatobiliary disease.
2.Glutamate pyruvate transaminase (GPT) OR Alanine amino
transferase (ALAT)
Occurrence: Liver > Heart > Kidney > skeletal muscle > spleen
Principle: Kinetic determination of GPT according to the following
reaction:
L- alanine + - ketoglutarate
pyruvate + L-glutamate
Pyruvate + NADH + H+
Normal serum level: up to 40 U/l
Wave length: 340 nm
Procedure:
L- lactate + NAD+
at 37 C.
Calculation:
GPT activity (U/l) =  A / min
Clinical significance:
a. Liver damage and toxic hepatitis (high level)
b. Myocardial infarction.
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PHOSPHATASES
Phosphatases are enzymes which catalyze the splitting of phosphoric acid
from mono-phosphate esters.
They are hydrolases.
Organic phosphate esters + water
alcohol +phosphate ion
Two types are commonly estimated in the serum
1) Alkaline phosphatase with maximum activity at pH10
2) Acid phosphatase with maximum activity at pH5
ALKALINE PHOSPHATASE
OCCURANCE:in most tissues of the body, mainly in
Osteoblasts in bone
Bile canaliculi in liver
Small intestinal epithelium
Proximal tubules of kidney
Breasts during lactation.
In all these sites ALP seems to be involved in transport of phosphates
across cell membranes.
ALP is activated by Mg+2, Mn+2,Co+2.
It is inactivated by Zn+2,Cu+2,Hg+2,EDTA.
DETERMINATION OF ALKALINE PHOSPHATASE:
Principle:
ALP
Nitrophenyl phosphate
nitrophenol +phosphate
The reaction is carried out at pH9.8 in diethanolamine buffer
Procedure:
Calculation:
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Normal value:
Adults---100-290 IU/l
Children---180-1200 IU/l
Clinical significance:
Increase in ALP occurs mainly in:
1) Bone diseases like Paget’s disease (highest level), rickets,
hyperparathyroidism, bone cancer.
2) Liver diseases like obstructive jaundice, biliary cirrhosis, carcinoma
liver abscess
3) Drugs producing cholestasis like androgens, sulfonamides.
Or hepatotoxic drugs like aspirin,gentamycin,cyclophosphamide,
halothane
Decrease in ALP occurs in anemia, scurvy, and cretinism.
ACID PHOSPHATASE
Occurance:
Highest concentration is found in prostate (prostatic ACP), also inRBCs,
leucocytes and platelets (non prostatic ACP). Maximum activity at pH5.6
DETERMINATION OF SERUM ACID PHOSPHATASE:
A variety of substrates have been used for determination of serum ACP
activity .These include:
Nitrophenylphosphate-attacked by phosphatases of non-prostatic origin.
Β-Glycerophosphate, α naphthylphosphate, phenolphthalein
monophosphate are all non specific substrates for both.
Principle:
ACP
1-naphthylphosphate +H2O
phosphate +1-naphthol
pH6.7
1-naphthol +4-chloromethylphenyl diazonium
Procedure;
Calculation:
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azo dye.
Normal level:
Total ACP: up to 5.4 U/L
Prostatic ACP: up to 1.7 U/L
Clinical significance:
ACP is used mostly to detect prostatic carcinoma when it may reach very
high level. In benign hypertrophy of prostate ACP is normal.
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CREATINE KINASE
Creatine kinase is a phosphotransferase enzyme which catalyses reactions
responsible for formation of ATP in tissues.
Ph8.9
ATP + creatine
ADP + creatine phosphate
pH6.8
The enzyme is activated by Mg+2
When muscle contraction occurs, ATP is hydrolysed to ADP to produce
energy for contraction process. An important step in regenaration of ATP
is the reaction with creatine phosphate.
SAMPLE COLLECTION:
1) Serum sample is used for CK assay.
2) Anti-coagulants inhibit ennyme activity.
3) CK is light sensitive and loses significant amounts of activity when
exposed to light for long periods.
TISSUE DISTRIBUTION:
CK is widely distributed in skeletal muscle, brain and cardiac muscle.
DETERMINATION OF CK ACTIVITY IN SERUM.
Principle
Creatine phosphate +ADP CK
ATP + glucose
HK
creatine +ATP
glucose-6-P+ADP
Glucose-6-P+ NADP+ G-6PDH
CK :creatine kinase.
HK: hexokinase.
G-6PDH: Glucose6-P dehydrogenase.
Procedure
Calculation
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Gluconate-6-P + NADP + H+
Normal value:
Males-25 to 195 U/l
Females-25 to175U/l
The difference is related to muscle mass.
Clinical Significance:
CK levels increase in a variety of disorders in which cardiac or skeletal
muscle tissue is affected.
Myocardial infarction
Muscular dystrophy
Polymyositis
Malignant hyperthermia
In strenuous physical exertion and IM injection there is a transient
elevation of CK.
Malignant neoplasms of brain
Brain infarction.
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α-AMYLASE
Amylase is an enzyme secreted by the salivary glands of the oral cavity
and is known as salivary amylase or ptylin. It is also secreted by the
pancreas and is known as pancreatic amylase or amylopsin.
FUNCTION:
Amylase catalyses the hydrolysis of starch, splitting the 1-4glycosidic
linkages.This hydrolysis occurs randomly along the polysaccharide chain
with the production of maltriose and dextrins.The carbohydrate digestion
by amylase begins in the mouth and continues breifly in the stomach until
the pH drops too low .It is completed in the small intestine by the action
of amylase secreted by the pancreas.
DETERMINATION OF α AMYLASE BY ENZYMATIC
COLOURIMETRIC METHOD
Principle:
5-ethylidene-G7PNP
α amylase
2G2PNP +2G3PNP+ 10 H2O
2ethylidene-G5+ 2G2PNP
+ 2ethyledene-G4+ 2G3PNP
+ ethyledene-G3 +G4PNP
α glucosidase
4PNP + 10G
PNP= paranitrophenol G=glucose
Procedure:
Calculation:
Normal Value:
At 370C---220U/l
Clinical Significance:
Very high serum amylase is seen in acute pancreatitis and obstruction of
pancreatic duct by stone tumor e.t.c.
Moderate increase is seen in : acute peritonitis
Perforated peptic ulcer
Mesentric thrombosis
Small intestinal obstruction
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