LABORATORY MANUAL

BMLS- 210
Biochemical Metabolism- Practical

(For private circulation only)

Prepared By: Prof. Harpreet Singh Burmi

Name of the Student:………………………………………..

Registration Number/Roll No: ……………………………...
Section and Group No. : ………………………………….………
Department of Medical Lab Sciences, Innocent Hearts Group Institutions, Loharan, Jalandhar
Phone : +91-181-2276041,2465290, Fax : +91-181-2271809 E-mail:info@ihgi.in,
 General guidelines for the students

1. Keep the work area clear of all materials except those needed for your work. Extra books, purses, bags
etc. should be kept in the racks placed in the laboratories
2. Student is expected to be punctual in lab, should keenly perform the experiment allotted to him without
moving from one lab to another and even experimental set up should not be left until it unavoidable.
3. Mobile phones are not allowed in the labs and should be kept in the bags in silent or witch-off mode.
4. A Student is expected to maintain the decorum of the laboratory by maintaining proper discipline.
5. Clean up your work area before leaving.

Dress code:

1. Shorts and sandals should not be worn in the lab at any time. Shoes are required when working in the
laboratories.
2. Students must have lab coat, gloves and mask with them every time.

Compulsory things to be carried by the students in lab:

Lab coat, gloves, mask, calculator, butter paper, fractional weights and stationary items.

Safety Guidelines:

1. Do not use any equipment unless you are trained and approved as a user by your supervisor.
2. Wear safety glasses when working with hazardous materials or use such materials in fuming hood.
3. Wear gloves when using any hazardous or toxic agent.
4. If you have long hair or loose clothes, make sure it is tied back or confined.
 INDEX

Sr. Name of Experiment Date Remarks

1 To determine the presence of carbohydrates by Molisch’s test

2 To determine the presence of reducing sugar by Fehling solutions

3 To determine the presence of reducing sugar by Benedict’s Test

4 To determine Starch by Iodine Solution

5 To estimate level of glucose in Blood Serum/ Plasma

6 To estimate level of Blood Urea in Serum/ Plasma/ Urine

7 To estimate level of Blood Cholesterol

8. To estimate Serum Albumin

9. To estimate level of glucose in blood by Folin-Wu Method

10. To estimate level of creatinine in serum

 Experiment 1

AIM: To determine the presence of carbohydrates by Molisch’s test

Requirement: Test tubes, Beakers, Distilled water, Test tube holder, Test Tube stand, Bunsen burner,
Boiling water Bath, Pipettes/Dropper
Sample:
Principle: Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulphuric acid to
produce an aldehyde (either furfural or a derivative), which then condenses with the phenolic structure
resulting in a red or purple-coloured compound
Procedure:
a) Place 2 ml of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s reagent (10% α-
naphthol in ethanol).
b) Pour 1-2 ml of conc. H2SO4 down the side of the test tube, so that it forms a layer at the bottom of
the tube.
c) Observe the colour at the interface between two layers and compare your result with a control test.
Result:

Cautions:
a) Take clean/washed test tubes for procedure.
b) A brown colour due to charring must be ignored and the test should be repeated with a more dilute
sugar solution.

Remark filled by faculty:

 Experiment 2

AIM: To determine the presence of reducing sugar by Fehling solutions

Requirement: Test tubes, Beakers, Distilled water, Test tube holder, Test Tube stand, Bunsen burner,
Boiling water Bath, Pipettes/Dropper
Sample:
Principle: In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue
solution of copper to a red precipitate of insoluble copper oxide. The test is commonly used for reducing
sugars but is known to be NOT specific for aldehydes. A positive test is indicated by a green suspension and
a red precipitate.
Procedure:
a) To 1 ml of Fehling’s solution A (aqueous solution of CuSO4) add 1 ml of Fehling solution B
(solution of potassium tartrate).
b) Add 2 ml of the sample.
c) Mix well and boil.
d) Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.
Result:

Cautions:
a) The test is sensitive enough that even 1 mg of glucose will produce the characteristic red colour of
the compound.

Remark filled by faculty:

 Experiment 3

AIM: To determine the presence of reducing sugar by Benedict’s Test

Requirement: Test tubes, Beakers, Distilled water, Test tube holder, Test Tube stand, Bunsen burner,
Boiling water Bath, Pipettes/Dropper
Sample:
Principle: When reducing sugars are heated in basic solution, they form powerful reducing compounds
known as enedioles. Endiole further react with cupric ions which are present in Benedict’s solution to
cuprous ions. Thus we detect the presence of reducing compounds. Here is should be noted that Benedict’s
solution not only react with reducing sugars but also give positive result with other reducing compounds.
Procedure:
a) Take 5 ml of Benedict’s solution in a test tube.
b) Add 5-8 drops of sample in the test tube containing Benedict’s solution and heat it.
c) Upon boiling, it will change the colour. If it doesn’t change colour, it means the sugar in the sample
is non reducible.
Interpretation:
 If the colour upon boiling is changed into green, then there would be 0.1 to 0.5 percent sugar in
solution.
 If it changes colour to yellow, then 0.5 to 1 percent sugar is present.
 If it changes to orange, then it means that 1 to 1.5 percent sugars are present.
 If colour changes to red, then 1.5 to 2.0 percent sugar is present.
 If colour changes to brick red, it means that more than 2 percent sugar is present in solution.

Result:

Remark filled by faculty:

 Experiment 4

AIM: To determine Starch by Iodine Solution.

Requirement: Test tubes, Beakers, Distilled water, Test tube holder, Test Tube stand, Bunsen burner,
Boiling water Bath, Pipettes/Dropper
Sample:
Principle: Starch is the carbohydrate storage unit of plants. It can be detected in a solution via starch-iodine
test. Amylose in starch is responsible for the formation of a deep blue colour in the presence of iodine.
Procedure:
a) Take 1 ml of original solution and add it to a test tube.
b) Add 1 to 2 drops of drops of iodine solution in it at room temperature.
c) If the colour of the solution changes, it means that polysaccharides are present in the original
solution.
Interpretation:
Blue/ Black colour - Positive
Brown colour/ No change Negative
Result:

Remark filled by faculty:

 Experiment 5

AIM: To estimate level of glucose in Blood serum.

Sample:
Serum/Plasma (must be separated from the cells within 30 minutes. Excessive concentration of sodium
fluoride may affect result by inhibiting color development).
Reagents:
Reagent 1: Glucose Reagent
Reagent 2: Glucose diluent.
Reagent 3: Glucose standard
Working reagent preparation:
Add 50 ml of reagent 2 to reagent 1 vial and store at 2-8C in dark colour bottle because it is light sensitive.

APPARATUS USED: Test tubes, Test tube stand, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE:
Glucose estimation provides valuable information about the course, severity and therapeutic control of
Diabetes Mellitus. Fasting Glucose levels exceeding 120 mg/dL and 2 hours Post-Prandial Glucose levels
exceeding 140 mg/dL indicate a strong possibility of Diabetes Mellitus. If in an oral Glucose Tolerance
Test, the Plasma Glucose level of 2 hours Sample exceeds 200 mg/dL, the diagnosis of Diabetes Mellitus is
established. In impaired tolerance, the 2 hours Plasma Glucose lies between 140 mg/dL and 199 mg/dL.
Increased concentration: Hyperglycemia may occur in Diabetes Mellitus, in patients receiving intravenous
fluids containing Glucose and during severe stress and Cerebrovascular accidents.
Decreased concentration: Hypoglycemia may be the result of an Insulinoma, Insulin ad ministration,
inborn errors of Carbohydrate metabolism or fasting.
ASSAY PRINCIPLE: Glucose Oxidase (GOD) oxidizes glucose to gluconic Acid and hydrogen peroxide.
In presence of enzyme Peroxidase, released hydrogen peroxide is coupled with phenol and 4.-
Aminoantipyrine (4-AAP) to form coloured Quinoneimine dye. Absorbance of coloured dye is measured at
505 nm and is directly proportional to glucose concentration In the Sample.

Glucose Oxidase
Glucose + O2 + H2O Gluconic acid + H2O 2

Peroxidase
H2O 2 + Phenol + 4-AAP Quinoneimine dye + H2O
PROCEDURE:

Mark the tubes as blank, standard and test and add the content according to the table given below:
Pipettes into tube marked Blank Standard Test

Serum / Plasma 10 µL

Reagent 3 10 µL

Working Glucose Reagent 1000 µL 1000 µL 1000 µL

Mix well incubates at 370 C for 10 minutes or at room temperature (15-300 C) fir 30 minutes.
Programme the analyser as per assay parameters.
1. Blank the analyser with Reagent blank.
2. Measure absorbance of Standard followed by the test
3. Calculate results as per given calculation formula
CALCULATION:

Serum / Plasma glucose (mg/ dL) = Absorbance of Test X 100

Absorbance of Standard

OBSERVATION TABLE:

S. No. Reading for tubes Optical density (OD) at 505 nm

1. Blank

2. Standard

3. Test

Result:

Remark filled by faculty:

 Experiment 6

AIM: To estimate level of Blood Urea.

Sample: Serum or Plasma (0.01 mL is required) (Do not use anticoagulants containing Ammonium salts)
Urine: Dilute1: 20

Reagents: Reagent 1: Urea Reagent

Reagent 2: Diacetylmonoxime (DAM)
Reagent 3: Working Urea Standard, 30 mg (%)

Preparation of working solution:

Solution I: Dilute 1 mL of Reagent 1 to 5 mL with Purified water Reagent 2 and Reagent 3 (Standard) are
ready for use

APPARATUS USED: Test tubes, Pipettes, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE:
Higher level of blood urea than the normal level are usually found with retention of urine due to pre renal
and post renal factors. Dehydration, reduced rate of glomerular filtration and increase catabolism of protein
are the usual pre-renal factors. Renal factors include all sorts of kidney disease and post renal conditions are
enlargement of prostate, stones in the urinary tract, stricture urethra and tumor of the bladder.

PRINCIPLE:
For the estimation of urea we use DAM (Diacetyl Monooxime) method. Urea reacts with hot acidic
Diacetylmonoxime in presence of Thiosemicarbazide acid produces a rose-purple colored complex, the
intensity of the colour produced is directly proportional to the amount of urea present in the test sample,
which is measured colorimetrically.

PROCEDURE:
A. For Colorimeter: Mark the test tube with Blank, Test and standard and add reagents as in to tubes
according to the table given below:
 Pipettes into tube marked Blank Standard Test

Solution I
2.5 ml 2.5 ml 2.5 ml
Sample 0.01 ml

Reagent 3: Working Urea Standard, 30. mg% 0.01 ml

Reagent 2: Diacetyl monoxime (DAM) 0.25 ml 0.25 ml 0.25 ml

Mix well and keep the tubes in the boiling water exactly for 10 minutes. Cool immediately under running
water for 5 minutes, mix by inversion and measure the color intensity within 10 minutes using a green filter
against blank.

B. For Spectrophotometer: All the volumes mentioned under colorimetric procedure can be adjusted
proportionately depending on flow cell/cuvette capacity. Rest of the procedure remains unchanged. Measure
the OD at 525nm.

OBSERVATION TABLE:
S. No. Reading for tubes Optical density (OD) at 525 nm
1. Blank
2. Standard
3. Test

CALCULATIONS:
1. Serum /Plasma: Urea in mg/ 100 ml, (A) = O.D. Test X 30
O.D. Standard

Blood Urea Nitrogen in mg/100 mL = (A) x 0.467

2. Urine: Urea in g/L (B) = O.D. Test X 30X20

O.D. Standard 100

Urea Nitrogen in g/L = (B) X 0.467

PRECAUTIONS:
1. Use Chromic acid washed dry glassware.
2. Bring all the solutions to room temperature before use.
3. Prepare a standard for each series of determinations.
4. Since the method is very sensitive, sample and standard should be measured accurately.
5. Mark the test tubes properly as Blank (B), Standard (S) and Test (T) before proceeding for the
estimation, since markings may come off when the tubes are placed in a boiling water bath.

NORMAL VALUES:
Serum: 20-40 mg Urea (10-20 mg Urea Nitrogen) /100 ml
Urine: 20g Urea (10gUreaNitrogen)/ Litre

Result:

Remark filled by faculty:

 Experiment 7

AIM: To estimate level of Blood Cholesterol

Sample: Serum/Plasma
Reagents: Reagent 1: Cholesterol Reagent
Reagent 2: Working Cholesterol Standard (200 mg/dl)
Reagent 3: Precipitating Reagent
Auxiliary: Normal Saline (0.85% Saline)

APPARATUS USED: Test tubes, Test tube stand, Beaker, Pipettes, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE:
High values may be found in Diabetes Mellitus, Hypothyroidism, Obstructive Jaundice, Nephrotic
Syndrome, Biliary Cirrhosis, Atherosclerosis etc. Low values may be found in Hyperthyroidism,
Malnutrition, Gaucher’s Disease and acute Hepatitis.
Decreased levels of HDL Cholesterol lead to increased chance of coronary heart disease (CHD) while
increased levels of HDL cholesterol reduce these chances. Lower values of HDL Cholesterol and increased
ratio of Total cholesterol to HDL Cholesterol are taken as risk factor for CHD.

PRINCIPLE:
Cholesterol reacts with hot solution of Ferric Per chlorate, Ethyl acetate and sulphuric Acid (Cholesterol
Reagent) and gives a lavender colored complex, which is measured at 560nm.
High density Lipoproteins (HDL) are obtained in the supernatant after centrifugation. The cholesterol in the
HDL fraction is also estimated by this method.

PROCEDURE:
(A) For total Cholesterol: Take three tubes for blank, standard and test solution ant take the reagent as
given in the table given below:

Blank (B) Standard (S) Test (T)

Reagent 1 3.0 ml 3.0 ml 3.0ml
Reagent 2 - 0.015 ml -
Serum/Plasma - - 0.015 ml
Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds (1.5 Minutes).
0
Cool them immediately to Room Temp. (15-30 C) under running tap water. Measure the O.D. of Standard
(S) and Test (T) against Blank (B) on a colorimeter with a Yellow Green filter or on a spectrophotometer at
560 nm.
(B) For HDL - Cholesterol
Step I: Take the sample and reagent in centrifuge tube as given below:

Pipette into centrifuge tube Quantity

Sample 0.2 ml
Reagent 3 0.2 ml

Mix well and keep at Room Temperature (15 - 30 ºC) for 10 minutes and then centrifuge at 2000 rpm for 13
minutes to obtain a clear supernatant. Proceed to step II.
Step II: Mix all the content according to the table given below:
Pipette into centrifuge tube Blank (B) Standard (S) Tests (T)
Reagent 1 : 3.0 ml 3.0 ml 3.0ml
Reagent 2: - 0.015 ml -
Supernatant From Step-1 - _ 0.12 ml

Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds. Cool them
0
immediately to Room Temperature (15 to 30 C) under running tap water. Measure the OD of Standard (S)
and Test (T) against Blank (B) on a colorimeter with a Yellow Green filter or on a spectrophotometer at 560
nm.

OBSERVATION TABLE:

S. No. Reading for tubes Optical density (OD) at 560 nm

1. Blank
2. Standard
3. Test

CALCULATIONS:
1. Total Cholesterol
Serum/Plasma Cholesterol (mg/ dl) = O.D. of Test X 200
O.D. of Standard
2. HDL Cholesterol
Serum/ Plasma HDL-Cholesterol (mg /dl) = O.D. of Test X 50
O.D. of Standard
Where 50 = 200 X 2
8

PRECAUTIONS:
1. Use thoroughly clean and dry glassware.
2. This method is very sensitive; the measurement of standard and sample should be very accurate.
3. Reagent 1 is corrosive. Pipetting with mouth and contact with skin and clothing should be avoided.
4. Mark the test tubes properly as Blank (B) standard (S) and Test (T), since the marking may come off
when the tubes are placed in the boiling water bath.
Result:

Remark filled by faculty:

 Experiment 8

AIM: To estimate Serum Albumin.

Sample:
Serum/Plasma (Collected in Heparin)
Reagents: 1. BromoCresol Green
2. Albumin Standard
APPARATUS USED: Test tubes, Test tube stand, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE:
One of the most important serum proteins produced in the liver is albumin. Variation in albumin from
normal level results malnutrition, Liver diseases (for example hepatitis or cirrhosis) skin lesions such as
dermatitis and burns or dehydration. Clinical diagnosis should not be made on a single test result.

PRINCIPLE:
Colorimetric determination of Serum Albumin using bromo cresol green (BCG) at pH 4.2. Serum Albumin
in the presence of Bromo cresol Green at a slightly acid pH produces a colour change from yellow-green to
green blue.

PROCEDURE:
Mark the test tube with Blank, Test and standard and add reagents as in to tubes according to the table given
below:

Pipette into centrifuge tube Blank (B) Standard (S) Tests (T)
Bromo Cresol Green Reagent 2.0 ml 2.0 ml 2.0 ml
Albumin Standard - 0.01 ml -
Serum - _ 0.01 ml

Mix well and measure after 10 minutes. Read results against reagent blank. The colour is stable for 60
minutes.
CALCULATION:

Serum / Plasma glucose (mg/ dl) = Absorbance of Test X 5

Absorbance of Standard
NORMAL VALUE:

Adults - 3.5-5.0 g/dl

Result:

Remark filled by faculty:

 Experiment 8

AIM: To estimate Glucose level in Blood by Folin-Wu method.

Sample:
Serum/Plasma (Collected in Heparin)
REAGENTS:

1. Alkaline copper-reagent: Dissolve 40 gm of anhydrous sodium carbonate (Na2CO3) in about 400 mL of

water and transfer it into a 1-liter flask. Dissolve 7.5 gm of tartaric acid in this solution and 4.5 gms of
CuSO4, which is dissolved in 100 ml water. Mix the solution and make it up to 1 litre.
2. Phosphomolybdic acid reagent: Add 5 gm of sodium tungstate to 35 gm of phosphomolybdic acid. Add
20 ml 10% NaOH and 50 ml distilled water. Boil vigorously for 20-30 min so as to remove the NH3 present
in molybdic acid, cool and dilute to 350 ml with water. Add 125 ml of ortho phosphoric acid and make up
the volume up to 500 ml.
3. Standard glucose solution: Dissolve 100 mg of glucose in 100 ml distilled water and take 10 ml from
this stock and make it up to 100 ml.
APPARATUS USED: Test tubes, Test tube stand, Micropipettes, Spectrophotometer/ colorimeter

PRINCIPLE:
When glucose or other reducing agents are treated with alkaline copper solution they reduce the copper with
the result insoluble cuprous oxide is formed. The reaction depends on temperature, duration of heating,
degree of alkalinity. The ratio of glucose to cuprous oxide form may be varied after heating far a period.
The cuprous oxide form is allowed to react with phosphomolybdate to form molybdenum blue colored
complex which can be read colorimetrically using red filter on at 680nm.

PREPARATION OF PROTEIN FREE FILTRATE:

To 1 ml blood sample, add 8 ml distilled water, 0.5 ml of 2/3 N sulfuric acid and 0.5 ml of 10% sodium
tungstate solution in a stoppered centrifuge tube and mix the contents. Then centrifuge at 3000 rpm for 10
minutes and collect the supernatant as sample.

PROCEDURE:
Mark the Folin-Wu test tube (25ml) with Blank, Test and standard and add reagents as in to tubes according
to the table given below:

Pipette into centrifuge tube Blank (B) Standard (S) Tests (T)
Protein Free Filtrate - - 2.0 ml
Glucose Standard - 2.0 ml -
Alkaline CuSO4 2.0 ml 2.0 ml 2.0 ml

Mix well and keep in boiling water bath for exactly 8 minutes. Cool under running tap water.

Phosphomolybdic acid 2.0 ml 2.0 ml 2.0 ml

Dilute & wait for 1 minute. Fill the tubes up to mark with water & take reading at 680 nm.
CALCULATION:

Conc. Of Test= O.D of Test × 100

O.D of Std

Result:

Remark filled by faculty:

 Experiment 10

AIM: To estimate level of creatinine in serum.

Sample:
Serum/Plasma (Collected in Heparin)
Reagents: 1. Picric Acid reagent
2. Creatinine Standard
3. Buffer Reagent
APPARATUS USED: Test tubes, Test tube stand, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE:
Creatinine is the catabolic product of creatinine phosphate, which is used by the skeletal muscle. The daily
production depends on muscular mass and it is excreted out of the body entirely by the kidneys. Elevated
levels are found in renal dysfunction, reduced renal blood flow (shock, dehydration, congestive heart
failure) diabetes acromegaly. Decreased levels are found in muscular dystrophy.

PRINCIPLE:
Picric acid in an alkaline medium reacts with creatinine to form an orange coloured complex with the
alkaline picrate. Intensity of the colour formed is directly proportional to the amount of creatinine present in
the sample.
Creatinine + Alkaline Picrate Orange Coloured Complex

PROCEDURE:
1. Deproteinization of specimen: Pipette into a clean dry test tube

Picric acid reagent 2.0 ml

Sample (serum / Diluted Urine) 0.2 ml

Mix well and centrifuge at 2500-3000 rpm for 10 min. to obtain a clear supernatant.

2. Mark the test tube with Blank, Test and standard and add reagents as in to tubes according to the table
given below:

Pipette into centrifuge tube Blank (B) Standard (S) Tests (T)
PFF - - 1.1 ml
Picric Acid Reagent 1.0 ml 1.0 ml -
Distilled water - - 0.1 ml
Creatinine standard - 0.1 ml -
Buffer Reagent 0.1 ml 0.1 ml 0.1 ml

Mix well and keep at R.T & measure after 20 minutes. Read results against reagent blank.
CALCULATION:

Serum Creatinine (mg/ dl) = Absorbance of Test X 2

Absorbance of Standard
NORMAL VALUE:

Males - 0.6-1.2 mg/dl

Females - 0.5-1.1 mg/dl

Result:

Remark filled by faculty: