ESTIMATION OF

VARIOUS
BIOCHEMISTRY
PARAMETRES IN
HUMAN BLOOD
 INTRODUCTION
Clinical Chemistry is a science, a service, and an industry. As science, Clinical Chemistry links the
knowledge of general chemistry, organic chemistry, and biochemistry with an understanding of
human physiology. The primary purpose of a clinical chemistry laboratory is to perform analytic
procedures that yield accurate and precise information, adding in patient diagnosis and treatment.
The achievement of reliable results requires that the clinical laboratory scientist be able to correctly
use basic supplies and possess an understanding of fundamental concepts.

There are various biochemistry elements like glucose, calcium, HDL, Alkaline phosphate, AST, ALT,
Urea, creatine etc. glucose is a carbohydrate which chemically named dextrose-Glucose (C6H12O6)
and it is the main source of energy to our body. Calcium helps in blood clotting and helping
muscles to contract. Alkaline phosphate is an enzyme responsible for hydrolysing esters and
liberating in organic phosphate. HDL is high density lipoprotein can lower high risk for heart
diseases and stroke. ALT and AST stand for Alanine Transaminase and Aspartate
Transaminase respectively and found in the liver that helps covert proteins into energy for
the liver cells. Creatine (C4H9N3O2)is an amino acid help as to maintain a continuous supply of
energy to work muscles by keep production up in working muscles.

Urea (CH₄N₂O) helps to maintain metabolism of nitrogen compounds in our body.

For detection of various biochemistry parameters in blood biochemical reactions are done.
Reactions are the chemical process in which substances act mutually on each other and are changed
into different substances, or one substance changes into other substances called products.

A biochemical reaction is the transformation of one molecule to a different molecule inside a cell.

Biochemical reactions are usually mediated by enzymes, which are biological catalysts that
can alter the rate and specificity of chemical reactions inside the cells.

Reactant 1 + Reactant 2+ ………...+Reactant n⟶products.

When proper quantity of reagent is mixed with the sample and incubated for specific time @ 37-
degreeCelsius temperature the end point can be detected at reactions like colour formed, turbidity
formed, also colourless reaction may happen. The reactions are read by semi-automated analyzer to
get the result

TYPES OF BIOCHEMICAL REACTIONS

1.END POINT REACTION

In End point reaction result is measured at the end after specified incubation. Based on the no of
reagents adopted in the reaction the end point chemistry can be represented by two ways endpoint
1 chemistry and end point 2 chemistry.
 a. END POINT 1
In endpoint 1 reaction is normally sample and a single reagent are added to a cuvette and
incubated for a certainamount of time. after the time of absorbance is read the signal is
mostly stable for a period. this kind of reaction is usually associated with mono reagent
substrate and electrolyte e.g.; -glucose

b. ENDPOINT 2
In a 2-point endpoint reaction normally sample and a reagent 1is added to a
cuvette, incubated for a certain amount of time and then the absorbance is read.
This sample is blank reading. Reagent2 is added and the mixture is again
incubated for a certain time. After this absorbance is read again e.g.;-HDL
2.KINETIC REACTIONS
Kinetic reactions basically deal with rate of change in absorbance while the reaction is
progressing
In kinetic method the Sample absorbance is measured several times in pre-established time
intervals. At the beginning and at the end of the delay time the absorbance values ABS1 and
ABS 2 measured respectively. The difference between ABS1 and ABS2 allows the
differentiation between normal and abnormal activities e.g., - Alkaline phosphate, AST, ALT

3. FIXED TIME
Enzyme activity is determined after a fixed period of time at the end of which the amount of
product formed or substrate consumed is measured.
A Fixed time substrate is used for test which shows a first order kinetic reaction where
substrate depletion occurs. This kind of measurement can also be used in latex enhanced
protein tests where the high absorbance of the latex in reagent requires to have the first
measuring point after its addition.eg;- Urea,Creatine
 REVIEW OF LITERATURE
1. GLUCOSE

Tu6urtrjyttrsi
 CHAPTER 1
EXPEIMENTAL PROCEDURES

OBJECTIVE
Quantitative determination of Glucose in serum using Endpoint 1 Reaction

INTRODUTION
Blood glucose test is a blood test that screens for diabetes by measuring the level of glucose
in a person’s blood. Normal blood glucose level range between 70 to 99 LL

PRINCIPLE
Enzymatic colorimetric determination of glucose according to following end point reaction
Glucose + H2O→(glucose oxidase) Gluconoic acid +H2O2

2H2O+phenol +4-Aminoantipyrine----→(peroxidase) Quinonimine +4H2O

MATERIALS REQUIRED

.Pipettes and Tips

.Test tube and Racks
.Timer
.Incubator

.Semi automated Analyzer

.Glucose reagent R1
PROCEDURE
Glucose is a mono reagent. 1000 micro litre of glucose is taken as blank to nullify colour
interference of the reagent and it is incubated for 10 minutes at 370 C and it is aspirate in a
semi automated analyzer which works under the principle of beer lamberts law and
spectroscopy. For standardization 1000 micro litre mixed with 10 micro litres is incubated
for 10 minutes at 370 C and it is also aspirate in the analyzer to get a standard value. Then
we have to analyse the sample.1000 microlitere glucose reagent and 10 micro litre sample
the analyser shows the result .The slope of this reaction is increasing so it is a positive
reaction. The analyser absorbs wave length of 505 m and it also calculates the absorbance
of sample and standard. Hence the glucose in the serum can be calculated. The experiments
are repeated for several samples and can be aware of glucose content in our blood.

EXP NO:-2
AIM
Quantitative determination of Calcium using Endpoint 1 Reaction in a Semi Automated
Analyzer

INTRODUCTION
Heart and skeletal muscle contractility are affected by calcium ions. Hypocalcaemia result of
overactive parathyroid glands.

MATERIALS REQUIRED

.Calcium Dye Reagent R1 contains Diethyl amine

.Calcium Base Reagent contains o-cresophthalein complex
. Pipettes and Tips
. Timer
. Incubater
.Semi Automated Analyzer
PROCEDURE
Calcium is a monoreagent. 1000 microletre calcium reagent is incubated for 1 minutes at
room temprature taken as blank to nullify the colour interference and it is introduced in to
semiautomated analyser to aspirate it. For standerdisation 1000 microliter reagent and 10
microleter standred of concentration 100 mg/dl is incubated for 1 minute at room
temperature and aspirate in semi automated analyzer.And then 1000 microlitere of calcium
reagent and 10 microlitere is incubated for 1 minutes at room temperature. And aspirate
in a semi automated analyzer which works under the principle of spectroscopy and Beer
lamberts law .The experiments are repeated for several values.
EXP NO:-3

AIM
Quntative determination of HDL in serum

INTRODUCTION
HDL can lower the heart diseases and stroke .The HDL concentration in serum in 36.16 -
86.0 mg/dl.

PRINCIPLE
(𝑝𝑜𝑙𝑦𝑎𝑛𝑖𝑜𝑛𝑠)
Lipoprotein than HDL − − − − − − − − −→Suppress reaction with enzyme
(𝑎𝑡𝑜𝑚𝑖𝑐 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒)
(𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑟𝑜𝑙 𝑒𝑠𝑡𝑒𝑟𝑠𝑒)
HDL (Cholestrol esters) + H2O → HDL (Free cholesterol) + Free fatty acids
(𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑟𝑜𝑙𝑜𝑥𝑖𝑑𝑒)
HDL (free cholesterol) + O2 + H2 → Cholestenone + H2O2
(𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒)
2H2O2 + 4AA + EMSE+ H3+ O→ Violet quinone+ 5H2O

MATERIALS REQUIRED
❖ HDL−D Reagent 1
❖ HDL−𝐷 Reagemt2
❖ HDL Caliberator
❖ Pipettes & Tips
❖ Test tube & Racks
❖ Timer
❖ Incubater
❖ Semiauotomated analyser

PROCEDURE
HDL is estimated by two reagents.
ALAKALINE PHOSPHATASE

EXP NO:-4

AIM
Quatitative determination of alkaline phosphate in serum using kinetic reaction

INTRODUCTION
Alkaline phosphate is widely distributed throught the body,but clinically importance one for
diagnostic reasons are in bone,liver,placenta,and intestine.Growing bone is associated with
the release of ALP and so in childhood the level of ALP is around 3times of that adult.
Elevated levels are seen in bone dieases
Eg:-Paget dieases, osteoblastic,metastatic and in obstructive dieases of billarytract.

PRINCIPLE
Kinetic determination of ALP according to following reaction.
𝐴𝐿𝑃
P−nitrophenyl phosphate + H2O → P−nitrophenol + Inorganic Phosphate

MATERIALS REQUIRED
❖ Alkaline phosphate Reagent 1 contains diethanolamine buffer of 1mmolL and
magnesium chloride of 0.5 mmolL
❖ Alkaline phosphate Reagent 2 contains P−nitrophenyl phosphate 10mmolL
❖ Pippettes and Tips
❖ Test tubes & Racks
❖ Timer
❖ Incubator
❖ Semi Automaited analyser

PROCEDURE
Alkaline phosphate is estimated by two reagent 800ml R1 and 200ml R2 20ml sample and
aspirate in the semi automated analyzer which works in the principle of spectroscopy and
Beer lamberts law. The slop of the reaction is increases there for if is a positive reaction the
wavelength absorbed by the analyzer is 505m.
 𝐴𝑆𝑇
EXP NO: 5
AIM
Quantitave determination of AST in human serum

INTRODUCTION
ASPARTATE AMINOTRANSFERASE is an enzyme that is found mostly in the liver .The
deficiency of liver diseases

PRINCIPLE
Kinetic determination of as Aspirate Aminotransferase based upon the following reaction
𝐴𝑆𝑇
L-Asparate +alpha-ketoglurate − − − − − − − − −−→ + L−Glutamate
𝑂𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒
Oxaloacete + NaDH + H+ − − − − − − −→ L−Malate + NAD

MATERIALS REQUIRED

❖ AST reagent R1
❖ AST reagent R2
❖ Pipettes and tips
❖ Test tube and racks
❖ Timer
❖ Incubater
❖ Semi automated analyzer

PROCEDURE
AST is determined by kinetic reaction.400 microlitere reagent R 1 is taken as Blank to nullify
the colour intereference.400 microlitere R1 AST reagent and 100 microlitere R1 reagent are
mixed and 50 micro litre sample is added to the mixture the it will introduced into semi
automated analyzer . The experimental time as 60sec . the experiments are repeated for
several times.
 UREA

EXPERIMENT NO:6

AIM
Quantitative determination of Urea using Fixed time reaction
INTRODUCTION
The nitrogen combines with other elements such as carbon, hydrogen and oxygen
to form urea which is a chemical waste product.

PRINCIPLE
Urea + H2O → 2NH3 +CO2
𝑈𝑟𝑒𝑎𝑠𝑒
𝐺𝐿𝐷𝐻
2NH3 + 2𝑎 −Ketoglutarate + 2NaDH→ 2L−𝐺𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 2NAD+2H2O

MATERIALS REQUIRED
❖ Urea Reagent R1
❖ Uea Reagent R2
❖ Urea standard
❖ Pipettes and tips
❖ Test tube and racks
❖ Timer
❖ Incubater
❖ Semi auto analyzer
 CREATINE
EXPERIMENT:7

AIM
Quantitave determination of creatinine in serum plasma and urine

INTRODUCTION
Creatine react with picric acid to produce a colored compound, creatine alkaline picrate
the change in absorbance is propotional to the creatine concentration.

MATERIALS REQUIRED
❖ Creatine base Reagent R1
❖ Creatine Dye Reagent R2
❖ Creatine Standred
❖ Pipettes & Tips
❖ Test tubes & Tips
❖ Analyser
RESULTS AND DISCUSION

Sample
1.GLUCOSE
Blank Resp
MC Resp
Factor
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
Sample 13
Sample 14
Sample 15
Sample 16
Sample 17
Sample 18
Sample 19
Sample 20
Sample 21
Sample 22
Sample 23
Sample 24
Sample 25
Sample 26
Sample 27
Sample 28
Sample 29
Sample 30
Sample 31
Sample 32
Sample 33
Sample 34
Sample 35
Sample 36
Sample 37
Sample 38
Sample 39
Sample 40
Sample 41
Sample 42
Sample 43
Sample 44
Sample 45