Chapter23

Practicals in Biochemistry
CARBOHYDRATES
1.

WHAT IS A SUGAR?
Ans. Sugar is a white crystalline substance, soluble in water and sweet in taste. Sugars are classified
into two types:
(a) Reducing sugars.
(b) Non-reducing sugars.

2.

WHAT IS A REDUCING SUGAR? GIVE EXAMPLES.

Ans. Reducing sugar reduces cupric ions in the Benedicts and Fehlings solutions to cuprous
ions and it answers these tests.

Examples
Reducing monosaccharide.
Aldohexose
:
Keto hexose
:
Reducing disaccharides
:

Glucose
Fructose
Lactose (milk sugar)
Maltose (malt sugar, obtained by the hydrolysis of starch)
Reducing sugars contain sugar groups in their structure.

Example
R1
(i) Aldelyde group H C = O in aldoses

(ii)

3.

WHAT IS A NON-REDUCING SUGAR?

Ans. Non reducing sugars do not have free sugar group in their structure and they fail to answer
Benedicts and Fehlings tests.

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Example
Sucrose (table sugar)

4. WHAT ARE OSAZONES?

Ans. Reducing sugars react with pheynylhydrazine hydrochloride at 100C for 30 minutes and
form osazones. Reducing sugars can be identified by seeing the shape of osazones under
microscope.

Example
Glucosazone
Lactosazone
Maltosazone

5.

Needle shape crystals.

Hedgehog shape crystals.
Petals of flower (sun flower).

WHY GLUCOSE, FRUCTOSE AND MANNOSE FORM SAME SHAPE OF

OSAZONES (NEEDLE SHAPE)?
Ans. The spatial configuration of atoms and groups in lower (four) carbons is same in them.

PROTEINS
6.

WHAT ARE PROTEINS?

Ans. Proteins are polymers of amino acids. There are 20 different amino acids which are present
in proteins. These amino acids are joined by peptide linkages.

7.

WHAT ARE COLOUR REACTIONS OF A PROTEIN? GIVE EXAMPLES.

Ans. Certain specific reactions give particular colour due to presence of amino acids in their
structures. These are called colour reactions.

Example
(a) Biuret Test
Gives violet colour. This is due to the presence of peptide linkages in
the protein structure. It is a general test for the identification of a protein.

This test is called biuret because it was first demonstrated in the product called Biuret,
which is formed by the decomposition of urea on heating at 180C.

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(b) Ninhydrin reaction

Blue colour due to presence of alpha-amino acid radical.
It is also a general test for the identification of a protein.
(c) Xantho proteic reaction
Yellow colour due to presence of Benzoid radical
containing amino acids (Phenylalanine, tyrosine, tryptophan)

(d) Millons reaction Red colour due to presence of hydroxy benzene radical (tyrosine).

(e) Aldelyde test: Purple colour due to presence of indole ring containing amino acid tryptophan.

(f) Sakaguchi test: Bright red colour due to presence of guanidine group containing amino acid
(Arginine)

(g) Test for cysteine and cystine black colour due to SH or SS radicals.

8. CLASSIFY THE PROTEINS AND GIVE EXAMPLES.

Ans. Proteins are classified into :
1. Simple proteins Example: Albumin (Egg white).
2. Conjugated proteins Example: Casein (Phosphoprotein present in milk).
3. Derived proteins Example: Peptones formed by the hydrolysis of a native protein.

9. WHAT ARE PROTEINS OF HIGH BIOLOGICAL VALUE?

Ans. Proteins of high biological value contain all the essential amino acids in optimum concentration.

Example
Albumin, Casein

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10. WHAT ARE PROTEINS OF LOW BIOLOGICAL VALUE?

Ans. Proteins of low biological value lack one or two essential amino acids.

Example
Zein of corn lacks amino acids tryptophan and lysine.

11. WHAT ARE THE PROPERTIES OF PROTEINS AT ITS ISOELECTRIC pH

(I.E.P.)?
Ans. (a) The proteins carry equal number of positive and negative charges, so that it is electrically
neutral.
(b) The conductivity and osmotic pressure of a protein solution is at minimum.
(c) The solubility is at minimum.
(d) The viscosity is at minimum.

ABNORMAL URINE
12. WHAT ARE THE ABNORMAL SUBSTANCES WHICH ARE LIKELY TO BE
EXCRETED IN VARIOUS DISEASES?
Ans. (a)
(b)
(c)
(d)
(e)
(f)

Glucose
Ketone bodies (acetone and acetoacetate)
RBC (blood)
Protein (albumin)
Bile pigments (bilirubin)
Bile salts

13. WHAT IS GLYCOSURIA (GLUCOSURIA)? WHAT ARE THE CAUSES OF THIS

CONDITION?
Ans. Excretion of glucose in urine is called glycosuria. The causes are:
(a) Diabetes mellitus.
renal threshold. Normal 180 mg/dl).
(b) Renal glycosuria (

14. WHAT ARE THE CAUSES OF KETONURIA?

Ans. (a) Severe untreated diabetes mellitus.
(b) Starvation (prolonged fasting).

15. WHAT ARE THE CAUSES OF HAEMATURIA?

Ans. (a) Renal calculus (renal colic).
(b) Acute glomerulonephritis.

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16. WHAT ARE THE CAUSES OF ALBUMINURIA?

Ans. (a) Nephrotic syndrome.
(b) Acute glomerulonephritis.

17. WHAT ARE THE CAUSES OF EXCRETION OF BILIRUBIN AND BILE SALTS?

Ans. (a)

Intra hepatic
Extra hepatic

Viral hepatitis
Gallstones, carcinoma of head of pancreas.

N.P.N. SUBSTANCES
18. WHAT ARE THE N.P.N. SUBSTANCES?
Ans. The non-protein nitrogen (NPN) containing substances are :
(a) Urea
(b) Uric Acid
(c) Creatinine

19. WHAT IS NORMAL BLOOD UREA LEVEL AND NAME THE CLINICAL
DISORDERS IN WHICH IT IS RAISED?
Ans. Normal value : 10 40 mg/dl
Blood urea level.
Pre-renal Dehydration (diarrhoea and vomiting).

Post-Renal Renal calculus and enlarged prostrate.

20. WHAT IS NORMAL URIC ACID LEVEL AND NAME THE CLINICAL DISORDERS
IN WHICH IT IS RAISED?
Ans. Normal value : 2.0 6.0 mg/dl. Gout and renal failure.

21. WHAT IS NORMAL CREATININE LEVEL AND NAME THE CLINICAL DISORDERS
IN WHICH IT IS RAISED?
Ans. Normal value : 0.6 1.2 mg/dl. Renal insufficiency and renal failure.

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QUANTITATIVE ANALYSIS
PHOTOELECTRIC COLORIMETER
22. WHAT ARE THE PARTS OF PHOTOELECTRIC COLORIMETER?
Ans. The following are the parts of photoelectric colorimeter:

The Parts of Photoelectric Colorimeter

1. A Source of Light
Tungsten lamp is the source of visible light in the colorimeters (Spectrophotometers)
(a) Tungsten lamp (400700 nm).
(b) Deuterium lamp U.V. (200400 nm).
2. Monochromator
(Specific wave length of light) obtained by
(i) Selective filters.
(ii) Prisms.
(iii) Diffraction by a grating.
3. Slit
This is to allow a narrow beam of selected monochromatic light to pass through the sample
solution.
4. Cuvette
The glass container to keep the test solution.
5. Photocells
Which convert quanta of radiation (light) to electrical energy, which may be amplified,
detected and recorded.

Fig. 1

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23. WHAT IS BEERS LAW AND LAMBERT LAW?

Ans. Beers law states that when monochromatic light passes through a coloured solution the
amount of light transmitted decreases with the increase in concentration of the coloured

substance i.e. the absorbance or optical density increases (

) with the concentration of the
substance.
Lamberts law states that when monochromatic light passes through a coloured solution the

amount of light transmitted decreases with increase (

) in optical path.

24. WHAT DO YOU RECORD WITH THE COLORIMETER?

Ans. The absorbance or optical density of the coloured solution.

25. WHAT ARE ANTICOAGULANTS?

Ans. Anticoagulants prevent the clotting of blood and these are used for the separation of plasma.

Example
Potassium Oxalate, Heparin, Sodium Citrate and EDTA.

26. WHAT IS THE DIFFERENCE BETWEEN SERUM AND PLASMA?

Ans. Fibrinogen is absent in serum and fibrinogen is present in plasma.

27. WHAT IS THE FORMULA FOR THE CALCULATION OF CONCENTRATION OF

SUBSTANCE IN COLORIMETRY?
Ans. Formula for the calculation of concentration of substance in colorimetry.
O.D. of test O.D. of Blank
Concentrat ion of standard

100
O.D. of standard O.D. of blank Volume of fluid taken into test
(ii) When the volume of sample equals to the volume of standard, so that same conditions are
fulfilled for both standard and test the following formula is applied.

(i)

T
Concentration of standard
S

28. WHAT ARE THE FUNCTIONS OF THE FOLLOWING EQUIPMENTS?

Ans. Centrifuge : Used for the separation of serum or plasma.
Hot Air Oven : For drying the glassware after washing.
Incubator/37C water bath : For the incubation of the sample with the reagents for allowing
the reaction to occur.

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29. HOW DO YOU WASH THE TEST TUBES, BEAKERS, VOLUMETRIC FLASKS,
AND GLASS CYLINDERS ETC.?
Ans. Soak the glassware in the detergent solution for few hours and then wash them with the tap
water. Afterwards rinse them with the distilled water and keep them in Hot Air Oven for one
hour.

Pipettes
Keep them in the chromic acid over night and wash them with tap water, rinse them with the
distilled water and keep them in Hot Air Oven for one hour.

30. WHAT IS END POINT ASSAY?

Ans. In End point assay the samples are treated with the reagent and incubated at 37C for a fixed
time. The colour developed after incubation time at the end of reaction is compared in
colorimeter and the concentration of the substance is calculated by applying the following
formula.
T
Concentration of substance
S

31. WHAT IS KINETIC ASSAY? AND HOW AN ENZYME ESTIMATION IS DONE

BY KINETIC ASSAY?
Ans. In kinetic assay optimum pH is provided by the suitable buffer. In kinetic assay the sample is
treated with buffer and other reagents which are required for reaction to occur and then it is
incubated at 37C water bath for one minute. The change in absorbance after one minute
will be recorded in spectrophotometer and enzyme activity will be calculated by applying
formula.
Formula : Change in absorbance Factor per minute = Units per litre.

32. WHAT IS NORMAL FASTING PLASMA OR SERUM GLUCOSE LEVEL?

Ans. 70110 mg/dl.

33. WHAT IS THE LATEST CRITERIA APPLIED FOR THE DIAGNOSIS OF

DIABETES MELLITUS?
Ans. When fasting serum glucose is greater than 125 mg/dl and when 2 hours post prandial
serum glucose is 200 mg/dl or more than 200 mg/dl, the patient is diagnosed as diabetes
mellitus.

34. WHAT IS IMPAIRED GLUCOSE TOLERANCE?

Ans. When the fasting serum glucose is between 111 mg/dl to 125 mg/dl, it is called impaired
glucose tolerance.

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35. WHAT ARE THE DIFFERENT METHODS OF BLOOD SUGAR

ESTIMATION?
Ans.

1. GODPOD (glucose oxidase-peroxidase method).

2. Orthotoluidine method.
3. Folin and Wu method.

36. WHAT IS THE PRINCIPLE OF GOD-POD METHOD?

Ans. Test principle:

4-(p-benzoquinone-mono-imino) phenazone + 4H2O

The phenazone gives pink colour and the absorbance of this colour is read at 470560 nm
wave length in the colorimeter.

37. WHAT ARE THE DIFFERENT METHODS OF BLOOD UREA ESTIMATION?

Ans.

1. Berthelot reaction.
2. DAM (diacetylmonoxime) method.

38. WHAT IS THE PRINCIPLE OF BERTHELOT REACTION?

Ans. Urease splits urea into ammonia and carbondioxide. Ammonia released in this reaction reacts
with hypochlorite and phenolic chromogen to produce green colour. The absorbance of this
green colour is read at 585 nm (570620) which is directly proportional to the concentration
of urea in specimen.

39. NAME THE METHOD OF ESTIMATION OF URINE CREATININE AND WHAT IS

THE PRINCIPLE OF IT?
Ans. Creatinine in urine is determined by its reaction with picric acid in an alkaline medium to
form the orange coloured tautomer of creatinine picrate (Jaffes reaction). Since creatinine
content of urine is high, it is suitably diluted. The intensity of orange colour is read using a
green filter (520 nm).

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40. WHAT IS THE NORMAL VALUE OF ENDOGENOUS CREATININE CLEARANCE

TEST? GIVE ITS INTERPRETATION.
Ans. Normal value 91130 ml/mt. Creatinine clearance indicates the renal insufficiency. In
renal failure it is grossly decreased.

41. WHAT IS THE NORMAL EXCRETION OF CREATININE PER DAY?

Ans. 12 G/day.

42. WHAT IS THE PRINCIPLE OF SERUM TOTAL PROTEIN ESTIMATION?

Ans. The peptide bonds of protein react with cupric ions in alkaline solution to form blue-violet
colour complex. The colour formed is proportional to the protein concentration and is measured
at 546 nm (520560 nm).

43. WHAT IS THE NORMAL VALUE OF SERUM TOTAL PROTEINS? GIVE ITS
LINICAL INTERPRETATION.
Ans. Normal value of total protein

6.0 8.0 g/dl

Normal value of total albumin

3.5 5.0 g/dl

Normal value of total globulin

2.0 3.5 g/dl

The concentration of total proteins and albumin are decreased in:
1. Nephrotic syndrome 2. Protein loosing enteropathy
3. Liver disease
4. Malnutrition.

44. WHAT ARE THE DIFFERENT FRACTIONS OBTAINED BY THE SEPARATION

OF SERUM PROTEINS BY ELECTROPHORESIS IN ORDER OF RATE OF
MIGRATION?
Ans. In order of rate of migration
(a) Albumin
Fastest moving fraction.
Movement is less than albumin
(b) 1-globulin
(c) 2-globulin
Movement is less than 1-globulin
(d) -globulin
Movement is less than 2-globulin
(e) -globulin
Least mobile fraction.

45. WHAT IS THE ABNORMAL PATTERN IN THE SEPARATION OF SERUM

PROTEINS BY ELECTROPHORESIS?
Ans. There are three possible abnormal patterns:
(i) In polyclonal gammopathy
The gamma band is most prominent. It is observed in:
(a) Chronic infections.
(b) Collagen diseases.
(c) Sarcoidosis.

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(ii) In monoclonal gammopathy (Multiple myeloma)

The M band is seen in regions or between and regions.
(iii) In nephrotic syndrome
(a) 2 band is prominent
(b) Albumin band is less prominent.

46. WHAT IS NORMAL A.G. RATIO? GIVE ITS INTERPRETATION.

Ans. 1.5:1
A.G. ratio occurs in liver diseases.

CEREBRO SPINAL FLUID (C.S.F)

47. WHAT ARE THE NORMAL VALUES OF BIOCHEMICAL SUBSTANCES
PRESENT IN C.S.F. AND GIVE THEIR CLINICAL INTERPRETATION?
Ans. (a)

Reducing sugar 40 70 mg/dl

in Meningitis.
(b) Proteins 10 40 mg/dl
in Meningitis and obstruction to flow of C.S.F.
(c) Chlorides 116 to 122 m.mol/dl
in Meningitis.

LIVER FUNCTION TESTS (L.F.T.)

48. WHAT ARE THE IMPORTANT TESTS INCLUDED IN L.F.T.? GIVE THEIR
NORMAL VALUES.
Ans. (a) Van den Bergh reaction (VBR): Immediate direct negative (I.D. ve)
(b) Serum bilirubin (SB) 0.2 0.8 mg/dl.
(c) (i) AST 820 U/L
(ii) ALT 1040 U/L
(iii) ALP 40125 U/L

49. CLASSIFY THE JAUNDICE AND GIVE INTERPRETATION OF L.F.T. VALUES

IN THEM.
Ans. Classification

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LIPID PROFILE
50. WHAT ARE THE SUBSTANCES INCLUDED IN LIPID PROFILE AND GIVE
THEIR NORMAL VALUES?
Ans.
1.
2.
3.
4.

Serum cholesterol
HDL-Cholesterol
LDL-Cholesterol
Triglycerides (TAG)

150 180 mg/dl desirable

40 60 mg/dl desirable
Below 100 mg/dl desirable
60150 mg/dl

Borderline upto 200 mg/dl

Lower side of borderline 3539 mg/dl
Borderline upto 130 mg/dl

51. GIVE CLINICAL INTERPRETATION OF SERUM CHOLESTEROL LEVEL.

Ans.

cholesterol level observed in:

1. Hypothyroidism (Myxoedema).
2. Nephrotic syndrome.
3. Primary biliary cirrhosis and biliary obstruction.
4. Familial hypercholesterolemia.