This document provides information on various biochemistry practical procedures and principles. It contains sections on colorimetry, abnormal constituents found in urine like proteins, carbohydrates, ketone bodies, bile salts, and blood. It also outlines the principles, normal ranges and formulas for estimating various serum components like total proteins, albumin, alkaline phosphatase, transaminases, bilirubin, urea, creatinine, uric acid, glucose, electrolytes, lipids, and others. Qualitative tests are described for detecting sugars, proteins, bile in urine.

1. BIOCHEMISTRY
PRACTICALS
FOR FIRST YEAR MBBS
By—
Aheed Khan

2. Contents---
1-Colorimetry
2-Abnormal Urine

3. that
interpretation
and the
procedures are
hereby not

4. Principle of Colorimetry

5. Total serum Proteins
PRINCIPLE Normal range
Proteins contain CONH2 6—8 mg%
group, which in the presence of
alkali and copper ions form
complexes called Biurets. These
are PURPLE coloured. Reagant
contains Na ions as antioxidant, Na
tartarate , NaOH and CuSO4 Formula/Calculations
solution.
CODt x Amt of subs x 100
CODs x volume of Serum

6. Total Albumin
PRINCIPLE Normal range
Below pH of 4.5—5.5 mg%
4.7, albumin exists as a
cation. At such a pH it
binds with anionic dyes
such as Bomocresol Formula/Calculations
green which turns blue CODt x Conc of Standardx 100
in bound form and CODs
change the Absorption
maxima.

7. Serum ALP
PRINCIPLE Normal range
ALP is secreated by growing 35-94 IU/L
bones, placenta, bile
canaliculus, intestinal epethelium. 10-40 KAU
Disodium Phenyl PO4 Phenyl
Formula/Calculations
Test-control x 10 x 1000 x 1
Red coloured complex
on addition of Amino antipyrene and Std-blank 94 0.1 15
potassiom ferrocyanide.
IU/L

8. ALP Km Estimation.
PRINCIPLE Double reciprocal graph
The principle is same as was for
ALP. Only that different standards 1/[COD]
are first formed which are then
subjected for OD calculation. The
procedure involves reciprocating
the OD and the standards
concentrations for the double
reciprocal curve. When the valued -1/Km
are plotted, the curve is produced 1/[S]
backwards till it intersects the
negative x-axis, i.e it gives us -
1/Km. The graph is also known a Formula/Calculations
Lineweaver-Burk plot.
-1/Km= -a
Km= 1/a

9. Effect of pH and temp on ALP
PRINCIPLE Normal graph
The principle is same as that of
ALP estimation. For pH, instead of
the usually used neutral
bicarbonate buffer, different COD
buffers are used. For
temperature, incubation
temperatures are set at different
levels.
Graphs are then plotted between
the modality concerned and the
COD. The graph hence obtained is
as shown.
pH / Temp

10. Serum transaminase(SGPT)
Normal range
PRINCIPLE
On addition of alpha keto
5-45 IU/L
glutarate and alanine as the Formula/Calculations
substrate, Serum glutamate
Test-control x 1 1000 x 0.4 IU/L
pyruvate transaminase forms
Std-blank 30 0.1
pyruvate and glumate.
T-C x 134 IU/L
Pyruvate combines with S-B
Dinitro phenyl hydrazine
(DNPH) to form a brown
colored hydrazone complex

11. Serum Bilirubin
PRINCIPLE Normal range
Van der Berg reaction from the basis 0.2-1 mg%
of the bilirubin test. Bilirubin exist as Direct—0.2-0.4 mg &
conjugated and non-conjugated form
in blood. Bilirubin reacts with Indirect---0.2-0.7 mg &
Diazotized Sulphanilic acid to form
Azobilirubin, which is reddish purple.
This is called Malloy and Evelyn
method. The reagant used is Ehrlich’s
reagant. Diazo blank is also used
Formula/Calculations
which is only HCl. CODtest x amount of subs x 100
COD control x volume of Serum

12. Urea Estimation
PRINCIPLE Normal range
Urea combine with Serum-15-40 mg%
Diacetyl Monooxime to
form Diacetyl and
Hydroxylamine. Diacetyl
combines with Urea to Formula/Calculations
give a pink colour in CODt x amount of standardx 100 x DF
boiling water. CODs x Volume of serum

13. Urea Clearance
PRINCIPLE Normal range
PRINCIPLE
Clearance– 75
Urea combine with
Diacetyl Monooxime to
mL/min
form Diacetyl and
Hydroxylamine. Diacetyl
combines with Urea to Formula/Calculations
give a pink colour in Clearance= U.V
boiling water.
P

14. Creatinine Clearance
PRINCIPLE Normal ranges
Creatinine combines with Picric Male-0.7-1.5 mg %
acid to form creatinine picrate Female-0.6-1.2mg%
which turns into its red tautomer. Clearance– 110-130 mL/min
This is called Jaffe’s Alkaline Picrate
method. <90 mL/min=diminished
Formula/Calculations
CODt x amt of subs x 100 x DF
CODs volume of serum

15. Uric Acid Estimation
PRINCIPLE Normal range
In ECF uric acid exists as Male-3-7 mg%
urate ions. These urate ions
combines with phospho
Female-2.5-6.5mg%
tungstic acid to form
tungsten blue in the
presence of Sodium Formula/Calculations
carbonate. Water turns into CODt x amount of subs x 100
Allantoin and CO2 is
CODs volume of Serum
released. Lithium is added to
create turbidity.

16. Glucose Oxidase test
PRINCIPLE
Glucose turns into gluconic acid Normal ranges
and Hydrogen peroxide in the Post prandial fasting
presence of glucose oxidase 1.Normal < 140 <100
enzyme. Second reaction is--
Gluconic Acid + 4- 2.Impaired glucose Impaired fasting
aminoantipyrene + 4-hydroxy Tolerance 140-199 glucose = 100-125
benzoic Acid
3.DM >200mg% >126 mg%
Quinone- imine dye
+ water
The reaction is catalyzed by
Peroxidase. The quinoneimine dye
is pink.
Formula/Calculations
CODt x conc of standard
CODS

17. Glucose tolerance Test
Normal graph
PRINCIPLE
The principle is same as Glucose
oxidase method, but three 180
different valued are taken---one Glu conc
before meal, one immediately
after a meal and one later after
some time having a meal. A graph
is hence obtained which should
peak just after the meal but not
time
above 180mg%. If the values
plotted exceed the glucose
threshold then its called impaired
glucose tolerance graph. Formula/Calculations
CODt x conc of standard
CODS

18. Gastric Function test
PRINCIPLE Normal range
BAO- 1-5 meq/hr
It’s the principle of titration which MAO-18-28 meq/hr
works here. Gastric acid consisting
of predominantly HCl is titrated
against a known normality of Formula/Calculations
NaOH. The gastric juice taken after 5mL of gastric juice ‘a’ mL of 0.05N NaOH
basal metabolism is called Basal 1mL “ “  a/5 x 0.05
Acid output or BAO. The juice = 0.01a
taken after giving stimulants such
as histamine is called maximal acid 50 mL  0.01a x 50 = a/2 = BAO
output or MAO. 100 mL  0.01a x 100 = a = MAO
‘a’ is the amount of alkali found by
titrations.

19. Acsorbic Acid Estimation
PRINCIPLE Formula/Calculations
Vitamin C is a reducing agent There are 2 samples—Ascorbic acid
which reduces the dye sample and the lemon extract.
DCPIP(dichlorophenol-indo- Conc of Ascorbic Acid sample-0.1mg/mL
phenol) to its stotiometrically
coloured form which is PINK. The X ml of dye 0.1 x 5 = 0.5mg Acid
stablilizing medium used is
Y mL of dye 5 mL extract
Metaphosphoric Acid and glacial
Acetic acid. so 5 mL extract (0.5). Y Acid
X
Expressed in mg/100 mL

20. Serum Bicarbonate
PRINCIPLE Normal range
1. the normality of the NaOH 20-30 meq/L
solution is determined by
titrating the HCl with NaOH. Formula/Calculations
2. The normality of NaOH is Meq HCl = meq HCO3- + meq NaOH
checked by titrating HCl and Meq HCO3- = meq HCl – meq NaOH
HCO3- solution. = 1 x 0.1 - 0.01a
3. 1mL of 0.1 N HCl is pipetted
into a beaker and 1 mL of
plasma is added and swirled
for 30 seconds. Titrations is
then done with NaOH.

21. Cholesterol Estimation
PRINCIPLE Normal range
<200 mg% is desirable
Cholesterol esters are first
converted to free cholesterol by 200-239mg% is borderline high.
Cholesterol ester hydrolase. After > 240 mg% is high.
that Cholesterol is acted upon by
Cholesterol oxidase enzyme which
is obtained from Nocardia
erythrolpolis. It converts it into Formula/Calculations
Cholest-4-en-3-one and H2O2 is
released. CODt x Conc of Std
H2O2 reacts with aminoantipyrene
to form pink colored CODs
Quinoneimine dye and water.

22. Triacylglycerol estimation
PRINCIPLE Normal range
Acid Lipases <100mg% Optimal
1.TAG Glycerol + fa 130-159mg% borderline high
Glycerol Kinase > 190 mg% Very high
2.Glycerol + ATP Glycerol 3 PO4
GP Oxigenase
3.Gycerol 3-PO4 DHAP + H2O2
Formula/Calculations
4.H2O2 + amino antipyrene
CODt x Conc of Std
Peroxidase CODs
Quinoneimine + H2O

23. Serum Calcium
PRINCIPLE Normal range
Calcium ions in the presence of 8.5-10.5 mg/dL
alkaline medium binds with dye to
form coloured chromophore. The
dye used is Cresoplthalein
complexone. Arsenazo III is also a
dde which specifically binds with
Calcium and has a strong affinity
Formula/Calculations
with Calcium ions. CODt x Conc of Std
CODs

24. Serum Phosphate
PRINCIPLE Normal range
The method is called as Fiske and 2.5-5 mg/dL
Subbarow method. Protein is first
precipitated by Trichloroacetic
acid. Phosphate then reacts with
Molybdate to form Ammonium
phospho Molybdate.
Formula/Calculations
Ammonium phospho molybdate
reacts with AMINAPTHOL CODt x amt of std x 100
SULPHONIC ACID (ANSA) to form
molybdenum blue. CODs volume of serum

25. Abnormal
Constituents of Urine

26. Carbohydrates
• Benedicts’ Test: 8 drops of urine are added to
5 mL of Benedicts reagant.
• This is a semi quantitative test which shows
different colors for different concentrations of
sugars.
• Presence of sugars, especially glucose idicates
glycosuria and Diabetes mellitus.

27. Proteins
• Coagulation test: A serum test tube is taken and its upper part is heated
and the lower part is left for as control. The heated urine sample is then
acidized with acetic acid so that turbidity cause d by phosphates is
eliminated.
• Esbach’s test: some urine is added to Esbach’s reagant. A yellow ppt
indicates protein.
• Heller’s test: take 3-5 mL of conc HNO3 in a test tube. Incline the tube and
add 2-3 mL of urine. White zone of precipitated protein formed confirms
the test.

28. Bile Salts
• Hay’s surface tension test: the urine is first put
in a serum test tube and sulphur powder is
then added to it. If the sulphur particles sink
then bile salts are present as they reduce the
surface tension. Otherwise the S particles will
float on the surface.

29. Bile pigments
• Gmelin’s test : to 5mL of conc HNO3 add an equal amount of urine. Add
urine carefully so that the two fluids don’t mix. At the junction there is
formation of coloured rings. This confirms bile pigments.
• Fouchet’s test: to 5mL of urine add an equal amount of 10% BaCl2. Filter
the contents which are collected over a filter paper. Few drops of
Fouchet’s reagant are then added. A green colour is obtained which
confirms presence of bile salts.
• fouchet’s reagant
• Bilirubin Biliverdin

30. Ketone bodies
• Nitroprusside test: 2 mL of urine is added to 5% aq solution of sodium
nitroprusside. The solution is made alkaline with the addition of NaOH. A
red color indicates presence of acetone.
• Modified Nitroprusside test: to few drops of urine add few drops of
nitroprusside. Then add few drops of NH4OH. A purpe ring will be formed.

31. Blood
• Benzidine test: to 3 mL of urine add drops of
saturated solution of benzidine in glacial
acetic acid and 1mL of 3% H2O2. immediate
blue or green colour indicates presence of
blood in urine.

32. THANK YOU