Vilnius University

Faculty of Medicine

Arvydas Kaminskas, Asta Maeikien

BIOCHEMISTRY
LABORATORY MANUAL

Vilnius, 2012

Discussed and recommended to publish at the staff hearing of Physiology, biochemistry,

microbiology and laboratory medicine department of Medicine Faculty on August 29, 2012;
Protocol No. 7
Reviewer: PhD Dovil Kariauskait

ISBN 978-609-459-125-9

Arvydas Kaminskas, 2012

Asta Maeikien, 2012
Vilnius University, 2012

3
Content
RULES FOR WORKING IN A BIOCHEMISTRY LABORATORY......................................... 4
1. COLOUR REACTIONS OF PROTEIN (PEPTIDES) AND AMINO
ACIDS........................................................................................................................................ 7
2. DETERMINATION OF PROTEIN CONCENTRATION IN URINE.................................. 9
3. DETERMINATION OF PROTEIN CONCENTRATION IN SERUM...............................10
4. ELECTROPHORESIS OF SERUM PROTEINS.................................................................. 11
5. ISOLATION OF PROTEINS FROM TISSUE. PAPER CHROMATOGRAPHY
OF AMINO ACIDS................................................................................................................ 11
6. QUALITATIVE REACTIONS OF OXIDOREDUCTASES.
DETERMINATION OF CATALASE ACTIVITY IN BLOOD PLASMA......................... 12
7. DETERMINATION OF ASPARTATE AMINOTRANSFERASE (AST) AND
ALANIN AMINOTRANSFERASE (ALT) ACTIVITY IN BLOOD SERUM................. 14
8. DETERMINATION OF LACTATE DEHYDROGENASE (LDG) ACTIVITY
IN BLOOD SERUM............................................................................................................... 15
9. ANALYSIS OF TRYPSIN ACTION....................................................................................... 15
10. DETERMINATION OF -AMYLASE ACTIVITY IN BLOOD SERUM.......................... 16
11. QUALITATIVE ANALYSIS OF VITAMINS.
DETERMINATION OF VITAMIN C IN URINE.............................................................. 17
12. DETERMINATION OF SEROMUCOIDS CONCENTRATION IN SERUM.................. 18
13. THE DETERMINATION OF GLUCOSE CONCENTRATION IN SERUM.................... 19
14. QUALITATIVE REACTIONS FOR URINE CARBOHYDRATES... 20
15. GLUCOSE CONCENTRATION IN URINE..... 22
16. QUALITATIVE REACTIONS FOR KETONE BODIES. ACETONE
CONCENTRATION IN URINE.. 22
17. DETERMINATION OF TRIACYLGLYCEROLS CONCENTRATION IN
SERUM........................................................................................................................... 26
18. QUALITATIVE REACTIONS FOR BILE PIGMENTS AND ACIDS.... 28
19. DETERMINATION OF TOTAL CHOLESTEROL CONCENTRATION IN
SERUM 28
20. DETERMINATION OF LOW DENSITY LIPOPROTEIN (LDL)
CONCENTRATION IN SERUM......................................... 30

21. DETERMINATION OF UREA CONCENTRATION IN

SERUM.................................................................................................................................... 30
22. DETERMINATION OF CREATININE CONCENTRATION IN URINE............ ......... 32
23. DETERMINATION OF URIC ACID CONCENTRATION IN SERUM........................ 33
24. DETERMINATION OF TOTAL BILIRUBIN CONCENTRATION IN SERUM......... 34
25. QUALITATIVE REACTIONS FOR THE DETECTION
OF BLOOD AND ITS PIGMENTS IN URINE.. 34

5
RULES FOR WORKING IN A BIOCHEMISTRY
LABORATORY

There are two major concerns to consider when working in a biochemistry laboratory. The first is
safety: this can never be overemphasized. General guidelines for safety are discussed below. The
second is efficiency in the laboratory work. Although the latter very much depends on the
individuals doing the experiments, there are general rules students are advised to follow:
1. Keep the benches and shelves clean and well-organized.
2. Avoid contaminating the chemicals; use only clean glassware and spatulas; label glassware in
use.
3. Plan your experiments before starting to carry them out.
4. Pay attention to others in the laboratory.

SAFETY IN THE LABORATORY

Students working in a biochemistry laboratory must always be aware that the chemicals used are
potentially toxic, irritating and flammable. Such chemicals are hazardous, however, only when they
are mishandled. Students who come to the laboratory session must have a complete understanding
of the laboratory procedures to carry out and be familiar with both the physical and chemical
properties of chemicals and reagents to be used. Since the carelessness on the part of one student
can often cause injury to other students, one must have a special concern for the safety of
classmates. Students must be familiar with general safety pratices, facilities and emergency actions.

General safety rules

1. Do not work alone in the laboratory.
2. Unauthorized experiments are not allowed.
3. Eating, drinking and smoking in the laboratory are strictly prohibited.
4. Become familiar with the location and use of standard safety features in the laboratory. The
laboratory is equipped with fire extinguishers, eye washes, safety showers, fume hoods and first-aid
kits. Any questions regarding the use of these facilities should be addressed to your instructor.

6
5. Special care for eye protection is required. Safety glasses must be used when certain procedures
are being carried out. The instructor will call the students' attention to those procedures. The use of
contact lenses is not recommended, since they reduce the rate of self-cleansing of the eye.

Special safety rules

1. While heating a solution, one should make sure not to overheat it; therefore, a vigorous mixing of
the solution by shaking or stirring is required. The mouth of the glassware containing the solution to
be heated should never be pointed toward anyone.
2. Handling of strong acids and bases requires special attention. When diluting concentrated acids,
the acid should be poured into water and never the opposite.
3. The pipettes should never be filled with solutions of toxic substances, biological fluids, strong
acids and bases by mouth suction. Use either automatic pipettes or pipette pumps.
4. Volatile liquids and solids that are toxic or irritating should be handled under fume hoods.
5. While handling flammable liquids such as ether, alcohols, benzene, naked flame (burners,
matches) must not be in use. The above liquids must not be stored near radiating heat sources, such
as laboratory ovens.
6. Before using electrical appliances, make sure they are grounded.
7. Flasks with flat bottoms or thin walls should not be desiccated.
8. Before leaving the laboratory, electrical equiment should be turned off and gas burners
extinguished. No tap water should be left running.

Rules to follow in the case of accidents and injuries

Chemical splatters into the eye. First the eyelid should be opened by using the thumb and the
pointing finger. Then, by using the eye wash kit, the eye should be rinsed with large amounts of
water. When an acid or alkaline solution gets into the eye, the eye should be rinsed with 1%
NaHCO3 or 1% boric acid, respectively. The victim should be taken to the doctor as soon as
possible.
Burning. The burned spot on the skin should not be treated with water; rather, a special bandage
should be used. See a doctor if necessary.
Poisoning. Prompt medical treatement should be obtained.
All injuries and accidents must be reported to the instructor.

7
1. COLOUR REACTIONS OF PROTEIN (PEPTIDES) AND AMINO ACIDS
Procedure. One test-tube fill with a diluted serum and another with gelatin (which is collagen
hydrolyzate, a mixture of polypeptides) solution. Into both test-tubes add 10 drops of NaOH and 1
drop of 1% CuSO4. The solution in both tubes turns purple.
The Ninhydrin reaction. This reaction is the most important method of detecting proteins, peptides
and - amino acids and indentifying their content of a free -NH2 group. For example, when amino
acids with a free -NH2 group are treated with ninhydrin solution, performing the oxidation of amino acids and decomposition into aldehyde, CO2 and NH3, ninhydrin is becoming reduced; then
the two ninhydrin molecules are bound by N derived from the -NH2 group.

Ninhydrin

Bluish purple coloured compound

Aldehyde

Procedure. To one tube add 5 drops of diluted egg protein or serum and to another 5 drops of 1%
gelatin. Into each tube add 3 drops of 0.5% ninhydrin and keep all the tubes in a boiling water bath.
After 2 3 minutes they yield a red and later a bluish purple colour.
The xanthoproteic (yellow protein) reaction. The xanthoproteic test is a method that can be used
to determine the amount of protein soluble in a solution, using the concentrated nitric acid. Upon
heat the protein with concentrated nitric acid, the solution turns yellow. The solution alkalinization
makes it orange. During the reaction, tyrosine, tryptophan and phenylalanine aromatic rings are
nitrifying to form yellow-coloured compounds.

Tyrosine

Dinitrotyrozine

8
Nitrated benzene derivatives in an alkaline medium transfer to a chinoidic structure. Their solutions
turn orange.
Procedure. To the test-tube add a diluted serum or egg protein, 3 drops of concentrated nitric acid
and heat (carefully!); the solution turns yellow. After cooling, into the content of the tube add 10
drops of concentrated ammonia and 30% NaOH, which yield an orange colour.
The Sakaguchi reaction. Proteins treated with hypobromite and -naphthol develop an intensive
red colour in an alkaline medium. In this case, hypobromite oxidizes the guanidine group of
arginine, and the resulting compound condenses with -naphthol a coloured solution:

Arginine

Red coloured compound

Procedure. To one test-tube add 5 drops of diluted blood serum or egg protein and to another 5
drops of 1% gelatin (partly hydrolyzed collagen). To each tube add 5 drops of 10% NaOH solution,
3 drops of 0.1% -naphthol in ethanol solution and 1 5 drops of 2% sodium hypobromite.
Solutions in the tubes get a red colour.
The Milon reaction. The heated protein solution with the Milon reagent (mercury in nitric acid
solution) produce a reddish-brown precipitate. Protein containing tyrosine (the phenol ring) with
HgNO3 result in a coloured nitrocompound. The Milon reaction does not occur with gelatin,
because it does not contain tyrosine.

Tyrosine

Coloured nitrocompound

Procedure. To one test-tube add 10 drops of diluted egg protein or blood serum and to the other
1% gelatin solution. Into both tubes add 1 2 drops of the Milon reagent and heat (carefully!). The
solution in the first test-tube gets a red colour.

9
Lead acetate test. This is a specific test for sulfur-containing amino acids such as methionine and
cysteine. The protein solution upon adding a few drops of lead acetate and heating in an alkaline
medium becomes dark. The reaction takes place also with free amino acids. By heating in an
alkaline medium, they produce sulfur and sodium sulphide:

Cysteine
Lead acetate with sodium hydroxide produce Pb(ONa)2:

Sodium sulfide reacts with Pb(ONa)2 to form dark lead sulphide sediments:

Procedure. To one test-tube add 5 drops of diluted egg protein or blood serum and to the other
test-tube 5 drops of 1% gelatin. To both tubes add 5 drops of 30% sodium hydroxide and one drop
of 5% lead acetate. The solution of the first tube after intensive boiling darkens, and the second
does not change because there are no gelatine amino acids containing sulfur.

REVIEW QUESTIONS
1.
2.
1.
2.
3.
4.
5.
6.

Characterization of protein biological function.

Principles of the classification of proteins.
Amino acid classification principles: their properties and formulas.
Peptide link, its properties.
Protein structures.
Biologically important peptides.
The chemical composition of proteins. The nitrogen content of proteins.
Protein physical and chemical properties: solubility, osmotic properties, amphoteric properties,
isoelectric point, optical properties of amino acids. Protein denaturation and renaturation.
7. Scleroproteins, their importance.
8. Colour reaction of proteins (peptides) and amino acid.

2. DETERMINATION OF PROTEIN CONCENTRATION IN URINE

Procedure. To the test-tube add 1.25 ml of filtered urine and 3.75 ml of 3% sulfosalicylic acid
solution and mix the contents. After 5 min, measure the extinction with a colorimeter-nephelometer,
use a red filter and a 5 mm cell. To the control sample add 1.25 ml of urine and 3.75 ml of 0.9%
NaCl solution. Estimate the calibration curve (will be presented at the laboratory). It has drawn on
graph paper: abscissa axis plotted on a different standard (albumin) levels, and the axis of ordinates
- their reactions with sulfosalicylic acid, the extinction values.
There is the standard sample preparation table needed to draw a calibration curve (standard albumin
concentration - 10 mg/ml).

10
Serial
No.

Standard
solution (ml)

0.9% NaCl Diluted standard

(ml)
solution (ml)

Sulfosalicylic acid
(ml)

1
2
3
4
5

0.05
0.1
0.2
0.5
1.0

9.95
9.9
9.8
9.5
9.0

3.75
3.75
3.75
3.75
3.75

1.25
1.25
1.25
1.25
1.25

Protein
concentration
g/l
0.05
0.1
0.2
0.5
1.0

REVIEW QUESTIONS
1. Proteinuria and its classification.
2. Proteinuria mechanism. Why plasma albumin in urine appears more easily than many of
globulins? How many and what proteins are there in normal human urine?
3. Uromucoproteins (glycoproteins), the clinical significance of determination.
4. The Bence-Jones protein in urine and its cause.
5. Protein detection method in urine. Is it possible to change sulfosalicylic acid by sulfuric
acid?
6. Importance of electrophoresis for detection of Bence-Jones protein and diagnostics of renal
proteinuria (organic).
3. DETERMINATION OF PROTEIN CONCENTRATION IN SERUM
REAGENT

PROCEDURE

CALCULATION

REACTION

R1: biuret reagent

Potassium iodide
30 mmol/l
Potassium sodium tartrate
100 mmol/l
CuSO4
30 mmol/l
NaOH
3.8 mmol/l
R2: standard
Solution of albumin
60 g/l
1. To the first test-tube add 1 ml of R1 reagent; this tube is called
"blank".
2. To the second test-tube add 1 ml of R1 reagent, 10 l of
standard R2 (known concentration of protein solution); this
tube is called "standard".
3. To the third test-tube add 1 ml of R1 reagent and 10 l of an
unknown protein sample (blood serum); this tube is called
"sample".
"Blank"
"Standard"
"Sample"
R1 reagent
1 ml
1 ml
1 ml
R2 standard
-10 l
-Blood serum
--10 l
4. Mix and incubate 5 min. at +37 C.
5. The analyzer measures light absorption rates and shows the
protein concentration in g / l.
ABS sample
----------------- * standard concentration = protein concentration
ABS standard
(g/l)
(ABS absorbance)
Wavelength
546 nm (520570 nm)

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CONDITIONS

Temperature
Cell

+37 oC
1 cm

RECOMMENDED
VALUES

Serum: Adult subjects

6585 g/l

REVIEW QUESTIONS
1.
2.
3.
4.
5.
6.
7.

Blood plasma proteins and their concentration.

What is the difference between serum and blood plasma? Conversion of fibrinogen to fibrin.
Serum albumin, 1, 2, , -globulin place of synthesis, their importance, molecular weight.
Immunoglobulins.
Hypo-, hyper- and disproteinemias. Their cause. Relative and absolute hyperproteinemia.
Paraproteins, their causes. Diagnostic possibilities of paraproteinemias.
The principle of the biuret method. What is a biuret?

4. ELECTROPHORESIS OF SERUM PROTEINS

Procedure
1. Preparation for electrophoresis. To chambers add 300 400 ml of buffer (the level must be
equal in both chambers) when the power is switched off.
2. Preparation of the plate(s). Before electrophoresis on cellulose acetate plate(s), a plate is
placed for 2 3 minutes into the buffer, then dried (to remove the excess buffer) and mounted
on a flexible part of the stretched bridge. Place the ends immersed in the buffer solution.
3. Apply the sample to the plate. Take 10 13 l of blood serum with a micropipette and move
the sample into a cellulose acetate plate by a dry applicator.
4. Electrophoresis. The plate with a specimen is placed into the electrophoresis chamber so
that the test samples will be near the cathode (-) field. The buffer is used with a pH higher
than the protein isoelectric point (pH 8.6). Proteins contain the negative charge and move
toward the positive electrode. The electrophoresis chamber is closed with a cover, electric
power must be switched on. After the migration, electrophoresis is switched off; carefully
remove the cover and remove the plate.
5. Visualization of the protein bands. The plate is placed in the dye solution and kept for about
10 minutes.
6. Fade. The plate is transferred to a destaining solution, then placed on a glass plate. If a
solution forms the excess air burbles, they are removed.
7. Drying. The plate is placed into a drying oven at 120 180 C for 5 10 minutes until it
becomes dry and transparent.
8. Evaluation of the protein bands. Scan the plates in a densitometer using a 525 nm filter. The
integrated lens of the densitometer provides a light flux which passes through the slot, and
the protein fractions. The photodetector light energy is converted into electric signals
resulting in a curve which shows 5 6 protein fractions: albumin, 1, 2, (1, 2), and
globulins. Also, the densitometer calculates the relative proportions of the fractions. When
albumin makes about 60% of the total serum protein, globulin represents about 40% of it,
1-globulin fraction about 4%, 2 8%, 12%, 16%.

REVIEW QUESTIONS
1.
2.
3.
4.

Protein isoelectric point. Dissociation of protein, depending on pH.

The essence of the electrophoresis method. Free and zone electrophoresis. Disc electrophoresis.
Procedure of zone electrophoresis.
Blood serum protein fractions, their relative quantity.

12
5. Disproteinemias.
5. ISOLATION OF PROTEINS FROM TISSUE. PAPER CHROMATOGRAPHY OF
AMINO ACIDS
Procedure. From the chromatography paper, there is cut a circle a petri dish in size. In the centre of
this circle, draw a small circle with the radius of 1 cm and from the big circular edge up to the small
one cut a 1 cm wide tape, which will be the wick. In the centre of the chromatography paper, draw a
small circle the starting line on which to drip amino acids. The paper disc is placed on a petri dish
and the wick is immersed into the petri dish filled up with the solvent mixture (ethanol and 1M
ammonium acetate 7:3). The chromatogram develops from 1 to 1.5 hours until the solvent reaches
the edge of the paper. Then the chromatography paper is put into a drying oven at a temperature of
50 C. The detection of amino acids on the chromatography paper is performed by dyeing
(sprinkling) with 0.1% ninhydrin solution, and then the sample is put into a drying oven for 5 10
minutes at 50 C to reveal purple spots. You have to measure the distance of moved amino acids in
mm (a), the distance of the moved solvent in mm (b) and to calculate the partition coefficient Rf for
each amino acid by the formula:
The partition coefficient is a constant characteristic of each amino acid and depending on test
conditions.

REVIEW QUESTIONS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Characterization of chromatographic methods.

The phases of protein extraction from its mixture.
Ion-exchange chromatography.
Determination of protein concentration in eluates.
Molecular filtration using sephadex.
Affinity chromatography.
The criteria of protein homogeneity.
The protein hydrolysis.
Determination of amino acid composition in hydrolysates of proteins (peptides).
Paper chromatography.
Amino acids, their classification, physico-chemical properties.

6. QUALITATIVE REACTIONS OF OXIDOREDUCTASES. DETERMINATION OF

CATALASE ACTIVITY IN BLOOD PLASMA
The first class of enzymes oxidoreductases catalyze oxidation-reduction reactions in the body.
There are four groups of them (i.e. no subclasses):
a) anaerobic dehydrogenases, which transmit protons (H+) and electrons (e-) from one
substrate to another, but not to oxygen. They have coenzymes: nicotinamide adenine dinucleotide
and its phosphate (NAD+ and NADP+), flavin mononucleotide (FMN), flavin adenine dinucleotide
(FAD);
b) aerobic dehydrogenases (oxidases); they oxidize substrates, are electron transferring
enzymes and catalyze the removal of one or two hydrogen atoms from substrate by directly using
O2 as a hydrogen acceptor. They have coenzymes FAD and FMN;
c) microsomal oxygenases which incorporate O2 into their substrates, but are not related to the
energy production. They have two subclasses: dioxygenases, which catalyze the incorporation of
both the atoms of O2 into the substrate, e.g., tryptophan-2,3-dioxygenases and monoxygenases or

13
hydroxylases, which incorporate one oxygen atom into the substrate to form a hydroxyl group on it,
e.g., mitochondrial cytP450 monooxygenase. These enzymes transfer reducing equivalents from
NADPH or NADH;
d) oxidoreductases containing the heme or similar compounds. These enzymes belong to a
group of cytochromes, catalase (mainly in red blood cells) and peroxidase (found in plants).
Hydrogen peroxide formation and detoxification. Hydrogen peroxide is formed in cells: 1) the
oxidation reduction reactions catalyzed by oxidase, where their flavin coenzymes take from the
substrate hydrogen and transfer it directly to oxygen, 2) by superoxide dismutase (SOD) catalysis:

Catalase acts in two ways: if the intracellular H2O2 is limited, it acquires the properties of
peroxidase and uses H2O2 for the oxidation of the substrate:

if H2O2 is abundant, then catalase cleaves it for protecting cells from the toxic action:

Peroxidase is catalyzing substrate oxidation reactions by hydrogen peroxide (H2O2).

Glutationperoxidase is very important for human red blood cells (it requires selenium). This enzyme
catalysing reaction is important for hydrogen peroxide inactivation.
Determination of tyrosinase and oxidases (of the plant) activity. Tyrosinase is oxidase
containing 0.2 0.23% of copper. It is found in mushrooms, potatoes and animal melanocytes. This
enzyme catalyzes the oxidation of tyrosine and some phenols to the corresponding quinones
precursor of melanin.

Tyrosine

DOPA

Galachrom
(red coloured compound)

Procedure. Peel a raw potato and cut the upper layers into the blender. Add 10 ml of distilled
water, crush potato chips in it, filtrate the mashed potato. To a clean test tube add 2 3 drops of the
tyrosine solution, 1 2 ml of the filtrate of mashed potato and put it into the thermostat at 37 C.
The solution turns pink and then brown.
Plant oxidase oxidizes Guaiacum resin acid to ozonide it turns blue in colour.
Procedure. Add a few drops of the alcoholic solution of Guaiacum resin onto upper layers of
peeled potato pellet. The edges of potatoes turn blue. Repeat the same reaction with boiled potato;
in this case, there remains the same colour as when cooking a inactivate the enzyme.

14
Determination of catalase activity in blood plasma:
a) a qualitative study.
Procedure. To a test tube add 1 2 ml of 1% H2O2 solution and a drop of blood. There is an intense
release of oxygen;
b) a quantitative study. The enzyme activity is determined from the decrease of hydrogen
peroxide content in the test sample. The rest of H2O2 in it, as well as all infused H2O2 in the control
sample are titrated with KMnO4.
Procedure. Into a 100 ml flask, add a small amount of distilled water and 0.1 ml of fresh blood
taken from a finger. The solution is diluted to 100 ml (blood diluted 1000 times). To two
Erlenmeyer (flat) flasks, add 7 ml of distilled water and 0.1 ml of the diluted blood solution. One
flask contents gently boil for 2 minutes (for catalase inactivation); it is the control sample. After
cooling, to each flask add 2 ml of 1% H2O2 and then leave for 30 minutes at room temperature.
Then to both flasks add 3 ml of 10% sulfuric acid solution. It stops catalase functioning in the acidic
medium. This is required for H2O2 titration. Titrate with 0.02 M KMnO4 solution to develop a pale
pink colour. The difference of potassium permanganate solution in ml consumed in the test (b) and
the control (a) sample, shows the amount of H2O2 fragmented by catalase; 1 ml of 0.02 M KMnO4
is equivalent to 1 ml of 0.05 M (1.7 g / l) H2O2. The value of catalase activity, i.e. the content of
hydrogen peroxide in mg, cleaved in 1 ml of blood within 30 minutes is calculated by the formula:
K=1,7(a-b)1000 mg
REVIEW QUESTIONS
1. Enzymes as biological catalysts.
2. Classification of enzymes, their nomenclature and code.
3. Environmental conditions acting enzymes.
4. The active zone (centre) of enzymes.
5. Inhibition of enzymes. Inhibitors and activators.
6. A zymogen (or proenzyme) as an inactive enzyme precursor. Coenzymes.
7. The presence of enzymes in cells.
8. Describe the four groups of oxidoreductases.
9. NAD+, NADP+, FMN, FAD structure, importance.
10. Generation of hydrogen peroxide and other reactive oxygen compounds, detoxification.
Antioxidants.
11. Method of detecting catalase activity in blood.
7. DETERMINATION OF ASPARTATE AMINOTRANSFERASE (AST) AND ALANIN
AMINOTRANSFERASE (ALT) ACTIVITY IN BLOOD SERUM
Procedure. 1 ml of sample solution is mixed with 100 ml of blood serum and incubated for 3 min
at 37 C. Extinction is measured at a wavelength of 340 nm. Repeat measuring after 2 or 3
minutes. Calculate the change in extinction per minute (A1 and A2). The activity of AST and
ALT is calculated in international units by the formula:
AST U/L = 1746 x A1
ALT U/L = 1746 x A2
Normal AST and ALT activity rates at 37 C: up to 35 U/L for women and up to 40 U/L for men.
REVIEW QUESTIONS
1. Classification of enzymes.
2. Units of enzyme activity.

15
3. Transferases, their subclasses.
4. Write the reaction of aminotransferase. Indicate the importance of the formed product.
The mechanism of aminotransferase reaction, importance of the pyridoxal phosphate
coenzyme.
5. Relation between enzymes and vitamins.
6. The diagnostic value of AST and ALT activity determination.
7. Method of detecting AST and ALT activity.

8. DETERMINATION OF LACTATE DEHYDROGENASE (LDG) ACTIVITY IN BLOOD

SERUM
Procedure. 1 ml of sample solution is mixed with 20 l of non hemolyzed serum. Incubate for 30
seconds at 37 C and measure the extinction at the wavelength of 340 nm. The measurement is
repeated after 1, 2 and 3 minutes. Calculate the change in extinction A / min. LDG activity in
international units (U) is calculated by the formula: LDG U / l = A x 8095 (+37 C).
Normal LDG activity range is 230 460 U / l (+37 C).
REVIEW QUESTIONS
1.
2.
3.
4.

Anaerobic dehydrogenases, their coenzymes.

Isoenzymes. Creatine kinase, AST, and LDG isoenzymes .
Location of LDG isoenzymes, their diagnostic importance.
Method of determining LDG activity. The Warburg test and its applications.

9. ANALYSIS OF TRYPSIN ACTION

The principle of work. Trypsin hydrolyzes the internal peptide bonds of protein by forming shorter
peptides with free amino and carboxyl groups:
trypsin

Amino groups react with formaldehyde in a slightly acidic medium (pH 6.8) (the methylene group
is bound to the N atom, and the amino group loses its alkaline properties); free carboxyl groups
must have titrate with alkali.

Procedure. To the Erlenmeyer flask add 50 ml of casein solution and 10 ml of trypsin. Mix.
Transfer 10 ml of this mixture into another flask and place the rest of it in a thermostat at 37 40
C. To the flask containing 10 ml of the mixture, add 2 ml of 0.4% HCl, 3 ml of formalin and a few
drops of phenolphthalein. Titrate with NaOH up to a light pink colour. Every 20 minutes to a 10 ml
mixture add specified quantities of HCl, formalin and phenolphthalein, then titrate with NaOH
again. Repeat it four times. According to the obtained results draw a graph. Mark the time to on the
abscissa axis and the amount of NaOH titration ml on the ordinate. Plot the curve. An increase in
the content of carboxyl groups shows trypsin activity.

16
REVIEW QUESTIONS
1. Proteolytic enzymes. The importance of proteolysis processes in the body.
2. Cathepsins.
3. Proenzymes (zymogens).
4. Enzymatical degradation of dietary protein in the human gastrointestinal tract.
5. Proteolytic hydrolases synthesized and secreted by the exocrine cells of the pancreas.
6. Convertion of trypsinogen (zymogen) to trypsin.
7. The specificities of trypsin and other proteases in the gastrointestinal tract.
8. The optimal pH of trypsin.
9. Casein. Phosphoproteins.
10. Procedure for the determination of trypsin activity.

10. DETERMINATION OF -AMYLASE ACTIVITY IN BLOOD SERUM

The principle of the assay is that the substrate of the kit, p-nitrophenol maltoheptaoside (glucose
polymer called pNP-G7) is hydrolyzed by -amylase in the sample. Complete the breakdown of
pNP-G7 to shorter oligosaccharide chains with p-nitrophenol (p-NP-G). This product is acted by
two helper enzymes, glucoamylase and -glucosidase; both of which are included in the reagent to
form glucose and p-nitrophenol (yellow compound), whereupon the absorbance is read at 405 nm.
REACTIONS

-amylase
pNP-G7

pNP-G
glucoamylase
-glucosidase

pNP-G
REAGENTS

PROCEDURE

p- nitrophenol (yellow) + glucose

R1: buffer
GOODS buffer (pH 7.2)
Sodium chloride
Calcium chloride
R2: substrate
pNP-G7
-glucosidase + - glucoamylase

50 mmol/l
50 mmol/l
5 mmol/l
>0.5 mmol/l
(8 + 2) U/l

CALCULATION

Prepare a working solution: gently mix (not shake) reagents R1 and

R2.
1 ml of working solution mix with 25 l of the sample and incubate
for 1 minute at +37 C.
Measure the change in extinction during 3 minutes (A/min).
Wavelength 450 nm.
A / min x 10480 = U/l

RECOMMENDED
VALUE

Serum, plasma
Urine

Up to 180 U/l
Up to 900 U/l

REVIEW QUESTIONS
1. Hydrolases, the subclasses of hydrolases. Glycoside hydrolases.

17
2.
3.
4.
5.
6.

Dietary carbohydrates and their daily intake. Digestion of carbohydrates.

How -amylase is released into the blood and urine?
Enzyme activity in the blood serum and urine of healthy persons.
Diagnostic value of -amylase activity determination.
Procedure of -amylase activity detection.

11. QUALITATIVE ANALYSIS OF VITAMINS. DETERMINATION OF VITAMIN C IN

URINE
Measurement of thiamine (vitamin B1). Vitamin B1 is composed of two heterocyclic rings:
pyrimidine and thiazole. The vitamin contains an amino group and sulfur, and therefore its chemical
name is thiamine. Thiamin interacts with K3[FeCN)6] (potassium hexacyanoferrate(III) red blood
salt) in an alkaline environment, oxidizes and forms a yellow pigment thiochrome which, if exposed
to ultraviolet light, fluoresces blue.

Procedure. To 1 2 drops of thiamine solution add 5 10 drops of 10% NaOH or KOH and 1..2
drops of K3[FeCN)6], mix. The heated solution turns yellow.
Measurement of pyridoxol (vitamin B6). Vitamin B6 is a pyridine derivate. It is soluble in water
and alcohol and destroyed by acids. It is degraded rapidly by oxidizing agents such as potassium
permanganate (KMnO4), hydrogen peroxide (H2O2) and others.
Procedure. Mix 5 drops of pyridoxol with 1 drop of 5% FeCl3. The yield is a red complex of iron
phenolate.
Measurement of cholecalciferol (vitamin D3). Vitamin D3 is fat-soluble, steroid-dependent.
Procedure. Mix 1 drop of fish oil with 5 drops of chloroform (the tube must be dry), add 1 drop of
aniline. After heating, the solution turns pink.
Measurement of phylloquinone (vitamin K1). Phylloquinone is a pale yellow viscous liquid
(oil), water-insoluble but soluble in organic solvents ether, ethanol, benzene. Thermostable, easily
inactivated by ultraviolet rays. Chemists have successfully synthesized water-soluble compounds:
vicasol, menadione, and others.
Procedure. Mix 5 drops of vicasol, 5 drops of cysteine solution, and 1 drop of 10% NaOH.
Initially, the solution turns yellowish in colour, but it quickly changes into orange.
REVIEW QUESTIONS
1. Water and fat soluble vitamins.
2. Enzymes and vitamins: connection.
3. B1, B6, D3, and K vitamins, their structure and properties.

18
4. B1 and B6 vitamin conversion to coenzymes. Significance of its for specific metabolic
pathway.
5. Conversion of vitamin D3 to 1,25-dihydroxycholecalciferol, its mechanism of action and the
importance of Ca and P metabolism; the importance of other hormones in the metabolism
of Ca and P (parathormone and calcitonin).
6. The importance of vitamin K for the postsynthetic modification of blood coagulation
factors.
7. The deficiency of vitamins B1, B6, D3 and K.
8. Symptoms of D3 hypervitaminosis.
9. Dietary sources of vitamins B1, B6, D3 and K, their dietary reference intakes.

VITAMIN C DETERMINATION IN URINE

The method of determining vitamin C is based on its reduction features. Ascorbic acid reduces to
the blue 2,6-dichlorphenolindophenol (Tillmans reagent), making it colourless and in an acidic
environment pink. During the reaction, ascorbic acid is oxidized to dehydroascorbic acid.
Procedure. To a 200 ml flask containing 10 ml of urine, add 100 ml of distilled water and 1 ml of
concentrated acetic acid. In order to monitor changes in colour, prepare a control flask containing
the same amount of urine, water, and acetic acid. The mixture is titrated with Tillmans reagent
until appear a pink colour. One ml of the Tillmans reagent reduces 0.0877 mg of vitamin C, and its
content in mg is calculated as:

here C the amount of vitamin C (mg),

a Tillmans reagent used for titration (ml),
1500 the total amount of urine per day (ml).
REVIEW QUESTIONS
1. Vitamin C, its structure, properties.
2. The importance of vitamin C for hydroxylation (prolyl oxidase, lysyl oxidase and other
enzyme-catalyzed reactions).
3. Vitamin C as an antioxidant.
4. Daily intake of vitamin C, sources in the food, deficiency of vitamin C.
5. Method of detecting vitamin C in urine.

12. DETERMINATION OF SEROMUCOIDS CONCENTRATION IN SERUM

Procedure. Add 0.5 ml of serum and 4.5 ml of saline solution into a test tube. After mixing
(thoroughly), add 2.5 ml of 6% perchloric acid (HClO4), mix again, incubate at room temperature
for 15 minutes and let the tube stand for 15 minutes at room temperature (16 25 C). During this
time, other serum proteins precipitate, and seromucoids dissolve in perchloric acid. Use filtration
paper to filter the precipitated proteins. Add 0.5 ml of 5% phosphowolframic acid to 2.5 ml of the
filtrate (the remainder of the filtrate will be used as a control solution for nefelometry). After 10
minutes perform the nefelometry (use red light filter). The results are expressed in conditional units,
which are obtained by multiplying the numeric value of the extinction coefficient by 1000. The
reference value of seromucoides is about 100 conditional units.

19
REVIEW QUESTIONS
1. Characteristics of glycoproteins.
2. Proteoglycans: their structure and functions.
3. Major glycosaminoglycans (GAGs) (hyaluronic acid, heparin, chondroitin sulphate,
keratan sulphate, dermatan sulphate): structure, location, physiological role.
4. Mucopolysaccharidoses diagnosis, causes, and consequences.
5. The structure of plasma glycoproteins.
6. The structure of fucosis and sialic acid.
7. The physiological role of ceruloplasmin, transferrin, haptoglobin, orosomucoid (alpha1 acid
glycoprotein), glycophorin, fibronectin.
8. The method for the determination of seromucoid concentration in serum; Changes of
seromucoid concentration in case of a pathology.

13. THE DETERMINATION OF GLUCOSE CONCENTRATION IN SERUM

METHOD

Enzymatic (glucose oxidation) colourimetric

PRINCIPLE

In the presence of oxygen, glucose is oxidized by glucose oxidase

(GOD) to gluconic acid and hydrogen peroxide (H2O2). The H2O2
reacts whit 4-chlorophenol and 4-aminoantipyrine in the presence of
peroxydase (POD) to form quinoneimine dyes. The intensity of
colour formed is proportional to the glucose concentration and can
be measured photometrically.
1. Glucose + O2

GPO

gluconic acid + H2O2

2. 2H2O2 + 4 AA + Phenol
REAGENTS

R: reagent
Phospahate buffer (pH 7.4)
Phenol
4 aminoantipyrine (4 AA)
Glucose oxidase (GOD)
Peroxidase (POD)
ST: standard
Glucose

POD

Quinoneimine (red) + 4H2O

13.8 mmol/l
10 mmol/l
0.3 mmol/l
10000 U/l
700 U/l
5.55 mmol/l
100 mg/dl

SAMPLE

Blood serum free of hemolysis; plasma collected on heparin or any

glycolysis inhibitor.

REACTION
CONDITIONS

Wavelength
500 nm
Temperature
+37 C
Cuvette
1 cm light path
1. Pipette 1 ml of the reagent (R) into the first cuvette (the cuvette
is called a "blank").
2. Pipette 1 ml of the reagent (R) and 10 l of the standard (ST)
into the second cuvette (the cuvette is called a "standard").
3. Pipette 1 ml of the reagent (R) and 10 l of the sample into the
third cuvette (the cuvette is called "sample").

OPERATING
PROCEDURE

20

"Blank"
1 ml

Reagent (R)
Standard (ST)
Sample

Cuvettes:
"Standard"
1 ml
10 l

"Sample"
1 ml

10 l

4. Mix and incubate the cuvettes at +37 C for 15 mitutes.

5. The analyzer reads light absorption rates (ABS) of the standard
and the sample against the reagent blank and calculates
glucose concentration.
CALCULATION
ABS sample
x standard concentration = glucose
concentration
ABS standard
(mmol/l, mg/dl)
(mmol/l, mg/dl)
Convertion factor:
REFERENCE VALUE

mg/dl * 0.0555 = mmol/l

mg/dl * 0.01 = g/l
Blood serum: 4.1 6.4 mmol/l
Capillary blood: 3.33 5.55 mmol/l
Cerebrospinal fluid: 2.78 3.89 mmol/l

REVIEW QUESTIONS
1. Glucose concentrations in the capillary blood, plasma (serum), cerebrospinal fluid. Hyper
and hypoglycemia. The importance of glucose for the brain.
2. Glucose-6-phosphate metabolism.
3. Glycolysis and its regulation.
4. Glucogenic compounds. Gluconeogenesis and its regulation.
5. The mechanism of the hormones action (insulin, glucagon, adrenaline, glucocorticoids and
STH), their importance in carbohydrate metabolism.
6. The formation cAMP and its biological role.
7. Renal threshold for glucose. Glucosuria process.
8. Alimentary and renal glucosuria.
9. Diabetes. Glucose tolerance test.
10. The method for determining serum glucose concentration.

14. QUALITATIVE REACTIONS FOR URINE CARBOHYDRATES

Qualitative reaction for glucose in a urine sample
Glucose is a reducing carbohydrate because of a free aldehyde group. On this property are based
Tromers, Fehling's and Nilenders reactions for a qualitative detection of glucose in the urine.
Tromers and Fehlings reaction:
CuSO4 + 2NaOH

Cu(OH)2 + Na2SO4
Blue colour

2 Cu(OH)2 + glucose

2CuOH + H2O + gluconic acid

Yellow colour

2CuOH

Cu2O + H2O
Red colour

21
During the reduction process of Cu(OH)2 to CuOH by the glucose aldehyde group, following group
is converted into a carboxyl group. CuOH and its degradation product, Cu 2O, are coloured
compounds. In excess of CuSO4, the high Cu(OH)2 content may lead to the formation of black
CuO particles. They dont form in the Fehlings reaction. Fehlings reaction is a modified Tromers
reaction, in which the reagent containing Segnets salt reacts with the formed excess of Cu(OH)2.
Procedure.
Tromers reaction. Add to the tube 1 2 ml of urine containing glucose, and an equal volume of
10% NaOH solution. Mix gently and add a few drops of 5% CuSO4. The content of the tube is
heated by holding the tube over a heater. In the beginning the colour of the solution is yellow, and
more heated it becomes red. If there is no glucose in the urine, the colour of the mixture does not
change.
Nilenders reaction. Add into the test tube 1 ml of pathological urine, 1 ml of Nilenders reagent
(which contains bismuth salt), and place the tube in a boiling water bath. After a few minutes, the
liquid begins to get darker glucose reduces Bi(OH)3 until free bismuth and black precipitate are
formed. This reaction is sensitive and allows detecting glucose even when its concentration is low
(about 0.5 g/l).
Qualitative reactions for the detecting lactose and other monosaccharides in urine
Fischer reaction. Glucose, lactose, and other carbohydrates with a free aldehyde group (reducing
carbohydrates), heated with an excess of phenyl hydrazine, form ozazones, nearly insoluble crystals
of various shape. During the reaction of phenyl hydrazine with glucose, crystals in the shape of a
whisk, and with lactose in the shape of sea urchins are formed. Usually, this reaction is used to
distinguish between glucose and lactose. However, it can be used to identify other monosaccharides
on the basis of the different melting temperatures of their ozazones: glucozazons, fructozazons melt
at +210 C, galactozazon melts at +180 C, and pentozazons melt at +160 C.
Procedure. Add into the tube 0.3 g of phenyl hydrazine, 0.1 g of sodium acetate (CH3COONa), a
drop of glacial acetic acid and 1 ml of 1% glucose solution. Place the tube for 30 minutes in a
boiling water bath and then rapidly cool its content to fall out yellow needle shaped crystals of
glucose ozazones (the crystals are examined under a microscope).
The Selivanov reaction. It is used to separate ketoses (fructose) from glucose and other aldoses.
During the reaction of fructose with hydrochloric acid, oxymethyfurfural is forming, which reacts
with resorcinol to form a red coloured compound. With aldoses this reaction is very slow.
Procedure. Add into the tube 1 2 ml of the Selivanov reagent (0.05 g resorcinol in 100 ml of 20%
hydrochloric acid) and 1 ml of 1% fructose solution. The tube is placed in a boiling water bath. The
boiling content of the tube turns red.
The reaction to separate pentoses from hexoses. Pentoses react with acids to form furfural.
Furfural, interacting with aniline, gives a red compound and with orcinol green (BIAL reaction).
Procedure. Add into the tube 1 2 ml of orcinol reagent. Bring the content of the tube to the boil
and add quickly a few drops of pentose solution. After the heating for 2 3 minutes, the content of
the tube turns blue.
REVIEW QUESTIONS
1. The biological role of carbohydrates, dietary norms. Food carbohydrates.
2. Classification of carbohydrates.
3. Monosaccharides and their derivatives: acids, alcohols (sorbitol, xylitol), hexosamines,
deoxyhexoses (methylpentoses), phosphoric acid esters.
4. Disaccharides: maltose, lactose, sucrose. Their structure, properties, significance.
5. Concentration of lactose in milk.
6. Lactosuria and its causes.

22
7. Fructose and galactose metabolism, the potential disoders.
8. Methods of the determination of glucose and other sugars in urine.

15. GLUCOSE CONCENTRATION IN URINE

The principle of Fehlings reaction is described in the previous laboratory work. Fehlings reagent
consists of CuSO4 (Fehling I), Segnet salt and a mixture of NaOH solutions (Fehling II). In one ml
of Fehlings reagent there is the amount CuSO4; for its reduction to the Cu 2O 0.005 g of glucose
should be used. After measuring the volume of urine, used for titration, the glucose concentration
could be calculated.
Procedure. Add into the flask 2.5 ml of Fehling-I, 2.5 ml of Fehlings solution-II and 20 ml of
distilled water. Bring the content of the flask to the boiling point and by heating titrate it with urine
containing glucose (pipette a few drops, wait a few seconds). The blue colour of the solution turns
green, then yellow. When the reddish colour appears, the titration is completed.
Glucose concentration ( g/l) can be calculated using the formula:

here X the glucose concentration (g/l),

a the volume of urine, used for titration,
5 the volume of the Fehling reagent.
REVIEW QUESTIONS
1. Glycogen structure, biological role and content in tissues.
2. Glycogenesis and glycogenolysis in the liver and muscles. Regulation of these processes.
Activation of glycogen phosphorylase and glycogen synthase.
3. Glycogen hydrolysis in the digestive tract and tissues (-amylase).
4. Glycogenosis.
5. Glucosuria.
6. Diabetes.

16. QUALITATIVE REACTIONS FOR KETONE BODIES. ACETONE

CONCENTRATION IN URINE
In the human liver three endogenous ketone bodies are formed: acetone, acetoacetic acid, and
beta-hydroxybutyric acid.

Acetoacetic acid

Acetone

-hydroxybutiric acid

23
Ketone bodies can be used a source of energy. They are transported from the liver to peripheral
tissues, where acetoacetate and beta-hydroxybutyrate can be reconverted to acetyl-CoA to produce
energy, via the citric acid cycle. Acetone is produced by spontaneous (without enzyme)
decarboxylation of acetoacetate in the liver in cases of overproduction of acetoacetate, when it cant
be used by peripheral tissues. Healthy human body is producing only little amount of acetone.
Acetone is harmful, because it melts body lipids, i.e. may be vulnerable to the membrane. Acetone
cannot be converted back to acetyl-CoA, so it is excreted in the urine and skin, or exhaled by the
lungs.
Under physiological conditions (when is enough of carbohydrates, especially oxalacetate) the
production of ketone bodies is low, because acetyl-CoA is oxidased in Krebs cycle. Also the excess
acetyl-CoA of can be moved from the cytoplasm to the mitochondria, in form of citrate, and can be
used to synthesized fatty acids and cholesterol (Figure 2).

Figure 2. The formation and use of Acetyl CoA.

Synthesized in the liver, acetoacetic and beta-hydroxybutyric acids are transported with the
blood to the mitochondria of peripheral tissue (brain, heart, kidney, skeletal muscle) cells, where
following acids can be reconverted to acetyl-CoA to produce energy via the citric acid cycle
(ketolysis, Figure 3) . Oxidation of hydroxybutyric acid can produce 26 mol of ATP and of
acetoacetic acid 23 mol of ATP.
When fasting, only the brain can used these compounds for energy, because only they
contain oxaloacetate (formed from blood glucose) required for acetyl-CoA oxidation. In the event

24
of low blood glucose, most other tissues have additional energy sources besides ketone bodies (such
as fatty acids), but the brain has not, because fatty acid -oxidation doesnt happens in the brain.
In normal individuals, there is a constant production of ketone bodies by the liver and their
utilization by extrahepatic tissues. The concentration of ketone bodies in blood is maintained around
0.01 0.02 mmol /L (0.15 2 mg%). When the rate of the synthesis of ketone bodies exceeds the
rate of utilization, their concentration in blood increases; this is known as ketonemia. It is followed
by ketonuria excretion of ketone bodies in urine. The overall picture of ketonemia and ketouria is
commonly referred to as ketosis. The concentration of ketone bodies in blood in case of ketosis can
reach 20 mmol/l; it lowers the pH of the blood and leads to metabolic acidosis.

Figure 3. Ketogenesis and ketolysis

25
In healty individuals, the excretion of ketone bodies in urine is very low (1020 mg) and is
undetectable by routine urine tests. In case of ketosis, the excretion of ketone bodies reaches 150 g,
and it will cause the further removal of water and electrolytes from the blood. This can lead to
exsiccosis water deficiency in the body.
Qualitative reactions of ketone bodies
Libens reaction. The reaction of acetone with iodine in an alkaline medium gives iodoform:

Procedure. Add into the test tube containing 3 ml of study sample 5 drops of iodine solution and 5
drops of 10% NaOH. Mix. If the sample contains acetone, the characteristic odor iodoform
precipitate comes out.
Legals reaction. The reaction of acetone and acetoacetic acid with sodium nitroprusside in an
alkaline medium gives a red orange coloured complex compound.

The addition of acetic acid changes the structure of the complex, and its red-orange colour turns
cherry.
Procedure. Add to the test tube containing 3 ml of urine (study sample) 5 drops of 10%
Na2Fe(CN)5NO and 1 ml of 10% NaOH; they give an orange colour; upon adding 2 ml of
concentrated acetic acid, it turns cherry.
Determination of acetone concentration in urine
The method is based on the detection of iodine, required to convert acetone into iodoform. Acetone
is blown into alkaline iodine solution. Non-reacted iodine is titrated with sodium thiosulfate
solution.
Procedure. Add 20 ml of the study sample, 0.2 g of oxalic acid, and 10 g NaCl to the flask A. Add
into the flask B 20 ml of 0.1 N iodine solution, 15 ml of 25% KOH and 25 ml of water. Stoppered
flasks are connected with each other to the air stream for 30 minutes. Acetone with a stream of air
passes to the flask B, where acetone reacts with iodine to form iodoform. Then add concentrated
HCl to the flask B, until a bright yellow colour appears. Add a couple of drops of starch solution
into the flask. Titrate iodine non-reacted with acetone with 0.1 N Na2S2O3 solution until the blue
colour disappears.

26
Calculation. The quantity of acetone (mg) per 100 ml of study sample (mg/dl) can be calculated
using the formula:

here a = the volume (ml) of iodine solution, added to flask B;

b = the volume (ml) of 0.1 N Na2S2O3, used to titrate iodine non-reacted with acetone;
c = the volume of study sample (20 ml).

REVIEW QUESTIONS
1. Write the formulas of ketone bodies; indicate the relationship between these compounds.
2. Synthesis of ketone bodies (ketogenesis). Ketogenic amino acids.
3. Oxidation of hydroxybutyric and acetoacetic acids in peripheral tissues (ketolysis). Write
the reaction, note the energy value.
4. Why hyperketonemia occurs in cases of diabetes, after a long fasting or receiving inadequate
amounts of dietary carbohydrates? What is the concentration of ketone bodies in urine?
5. Why in the above mentioned cases acetone is formed in the liver?
6. Ketosis and its consequences.
7. Qualitative reactions of ketone bodies.
8. The method for the quantitative determination of ketone bodies in the urine.

17. DETERMINATION OF TRIACYLGLYCEROLS CONCENTRATION IN SERUM

METHOD

Enzymatic colorimetric
Endpoint

PRINCIPLE

The method is based on the enzymatic hydrolysis of serum or plasma

triglyceride to glycerol and free fatty acids (FFA) by lipoprotein
lipase (LPL). The glycerol is phosphorylated by adenosine
triphosphate (ATP) in the presence of glycerolkinase (GK) to form
glycerol 3 phosphate (G 3 P) and adenosine diphosphate (ADP).
G3 P is then oxidized by glycerophosphate oxidase (GPO) to form
dihydroxyacetone phosphate (DHAP) and hydrogen peroxide
(H2O2). The H2O2 reacts whit 4 aminoantipyrine (4 AA) and
phenol in the presence of peroxydase (PO) to produce red
chromogen. The intensity of colour formed is proportional to the
concentration of triglycerides in the sample and can be measured
photometrically.
1. Triglycerides +3H2O
2. Glycerol + ATP
3. Glycerol 3 P + O2

LPL

GK

GPO

4. H2O2 + 4 AA + 4Phenol
REAGENTS

R: reagent
Buffer (pH 6.8)
Phenol

Glycerol + 3FFA
Glycerol 3 P + ADP
DHAP + H2O2
POD

Quinoneimine (red) + H2O

50 mmol/l
3 mmol/l

27
4 aminoantipyrine (4 AA)
ATP
Mg2+
Lipoprotein lipase (LPL)
Glycerolkinase (GK)
Peroxidase (POD)
ST: standard
Glycerol

0.5 mmol/l
2 mmol/l
40 mmol/l
1200 U/l
1000 U/l
10000 U/l
2.26 mmol/l

SAMPLE

Blood serum obtained by the patient after an overnight fast.

REACTION
CONDITIONS

Wavelength
500 nm (20 nm)
Temperature
+37 C
Cuvette
1 cm light path
1. Pipette 1 ml of the reagent (R) into the first cuvette (the cuvette
is called a "blank");
2. Pipette 1 ml of the reagent (R) and 10 l of the standard (ST)
into the second cuvette (the cuvette is called a "standard");
3. Pipette 1 ml of the reagent (R) and 10 l of the sample into the
third cuvette (the cuvette is called a "sample").
Cuvettes:
"Blank"
"Standard"
"Sample"
Reagent (R)
1 ml
1 ml
1 ml
Standard (ST)
10 l

Sample
10 l

OPERATING
PROCEDURE

4. Mix and incubate the cuvettes at +37 C for 5 minutes.

5. The analyzer reads light absorption rates (ABS) of the standard
and the sample against the reagent blank and calculates
triglyceride concentration (mmol/l).
CALCULATION
ABS sample
* standard concentration = triglycerides
ABS standard
(mmol/l)
concentration (mmol/l)
REFERENCE VALUE

1.8 mmol/l

REVIEW QUESTIONS
1. Characterization of lipids, their biological importance.
2. Lipid classification.
3. TAG classification and their properties.
4. Fatty acids and their properties. The human body's fatty acids. Essential fatty acids.
5. The synthesis of 6 and 3 polyunsaturated fatty acids.
6. The synthesis of eicosanoids and their biological role.
7. TAG hydrolysis in the body: in the digestive tract, adipose tissue, and blood.
8. Emulsifying of lipids. Emulsifiers.
9. Soaps. What kind of soaps can be produced in the human digestive tract?
10. Fat hydrogenation. Margarine. The advantages and disadvantages.
11. The diagnostic significance of the determination of serum TAG concentration.

28

18. QUALITATIVE REACTIONS FOR BILE PIGMENTS AND ACIDS

Reactions of bile pigments
The reaction to recognize the bile pigments are based on their oxidation, which leads to the
composition of coloured oxidation products: greenish biliverdin and other blue, pink, yellow
derivatives.
Procedure. Gmelins reaction. Add 1 ml of bile to the tube. On the wall of the tube, pour
carefully 1 ml of HNO3. In the liquid junction, residues of bile acids and protein and coloured rings
of bile pigments are formed.
Rozenbachs reaction. Filter the bile through the filter paper several times. On the filter paper,
there remain the bile pigments. Then spread out the filter paper and add a drop of concentrated
HNO3 in the middle. This results in coloured bile rings.
Razins reaction. Add a few drops of acetic acid to the tube with the bile. On the wall of the tube,
carefully pour 1 ml of iodine solution in the spirit. In the liquid junction, there appears a greenish
ring.
Reactions of bile acids
Procedure. Add 2 ml of distilled water into two test tubes. Pour a few drops of bile to one of them
and mix. Then add a sulfur powder to both tubes. In the tube containing bile, sulfur sinks. The
reaction is based on the ability of bile acids to reduce the surface tension.
Pettenkofers reaction. Mix 1 ml of H2SO4 and 1 ml of sucrose. Pour the bile into another test tube
and add a mixture of sulfuric acid and sucrose. In the liquid junction, there are formed residues of
bile acids and a purple ring. After cooling the tube and gently shaking its content, the liquid
acquires a cherry colour. It depends on the formation of a coloured compound during the reaction of
cholic acid and oxymethylfurfural (derived from the reaction of sucrose by sulfuric acid).
REVIEW QUESTIONS
1. The qualitative and quantitative composition of the bile.
2. What is the difference between hepatic bile and gall bladder bile? The factors that stimulate
the secretion of bile.
3. What amount of bile acids is produced per day?
4. The synthesis of bile acids and their importance.
5. Fat digestion. Cholein acids.
6. The formation and removal of bile pigments.
7. Gallstones.
8. Mechanical jaundice and cholemia. Other jaundices.
9. Reactions of bile pigments and acids.
19. DETERMINATION OF TOTAL CHOLESTEROL CONCENTRATION IN SERUM
METHOD

Enzymatic-colorimetric

PRINCIPLE

All cholesterol esters present in a specimen are hydrolyzed

quantitatively into free cholesterol and free fatty acids (FFA) by
cholesterol esterase (CHE). In the presence of oxygen, free
cholesterol is then oxidized by cholesterol oxidase (CHO) to
cholesten 4 ene 3 one and hydrogen peroxide (H2O2). The H2O2
reacts whit 4 chlorophenol and 4 aminoantipyrine in the presence
of peroxydase (POD) to form quinoneimine dyes. The intensity of
colour formed is proportional to the cholesterol concentration and
can be measured photometrically between 480 and 520 nm.

29
1. Cholesterol ester + H2O
2. Cholesterol + O2

cholesterol esterase

cholesterol oxidase

Cholesterol + FFA

Cholesten 4 ene 3 one + H2O2

3. 2 H2O2 + 4 chlorophenol + 4 aminoantipyrine

Quinoneimine (red) + 4H2O
REAGENTS

R: reagent
Buffer (pH 7.2)
p chlorophenol
Sodium chlorate
4 aminoantipyrine
Cholesterol esterase (CHE)
Cholesterol oxidase (CHO)
Peroxidase (POD)
ST: standard
Cholesterol

peroxidase

50 mmol/l
2 mmol/l
8 mmol/l
0.6 mmol/l
400 U/l
200 U/l
500 U/l
5.17 mmol/l

SAMPLE

Blood serum obtained from the patient after an overnight fast

REACTION
CONDITIONS

Wavelength
510 nm (480520 nm)
Temperature
+37 C
Cuvette
1 cm light path
1. Pipette 1 ml of the reagent (R) into the first cuvette (the cuvette
is called a "blank").
2. Pipette 1 ml of the reagent (R) and 10 l of the standard (ST)
into the second cuvette (the cuvette is called a "standard").
3. Pipette 1 ml of the reagent (R) and 10 l of the sample into the
third cuvette (the cuvette is called a "sample").

OPERATING
PROCEDURE

Reagent (R)
Standard (ST)
Sample

"Blank"
1 ml

Cuvettes
"Standard"
1 ml
10 l

"Sample"
1 ml

10 l

4. Mix and incubate the cuvettes at +37 C for 5 minutes.

5. The analyzer reads light absorption rates (ABS) of the standard
and the sample against the reagent blank and calculates
cholesterol concentration (mmol/l).
CALCULATION
ABS sample
* standard concentration = cholesterol
ABS standard
(mmol/l)
concentration (mmol/l)
REFERENCE VALUE

<5.2 mmol/l

REVIEW QUESTIONS
1. The importance of cholesterol.
2. The molecular formula of cholesterol. Formation of cholesteryl esters in tissues.
3. Exogenous and endogenous cholesterol.

30
4.
5.
6.
7.

Synthesis of endogenous cholesterol.

Cholesterol metabolism in the gut and liver.
Cholesterol and lipoprotein interface. The role of lecithin cholesterol acyl transpherase.
Total, LDL, and HDL cholesterol concentrations in plasma. Antiatherogenity of HDL
cholesterol.

20. DETERMINATION OF LOW DENSITY LIPOPROTEIN (LDL) CONCENTRATION IN

SERUM
Procedure. LDLs form a complex with heparin which, in the presence of CaCl2, forms residues.
Add into two test tubes 2 ml of 0.025 M CaCl2 and 0.2 ml of blood serum in each. Into one of them,
pour 0.04 ml of 1% heparin solution. Mix and after 4 minutes perform a colorimetric measurement,
using a red light filter.
LDL concentration can be calculated using the formula:

here Ek light absorption of the control solution,

Ex light absorption of the test solution.

REVIEW QUESTIONS
1.
2.
3.
4.
5.
6.
7.
8.
9.

Characterization of lipoproteins.
Lipoprotein fractions in plasma, concentrations, methods of their fractionation.
Blood plasma lipoprotein structure their lipids and proteins.
Metabolism of chylomicrons. Lipoprotein lipase.
Metabolism of very low density lipoproteins (VLDL).
Metabolism of low density lipoproteins (LDL). Atherogenity of cholesterol.
Metabolism of high density lipoproteins (HDL). Antiatherogenity of cholesterol.
Dyslipoproteinemia.
The principle of detecting LDL concentration in blood plasma and its diagnostic value.

21. DETERMINATION OF UREA CONCENTRATION IN SERUM

METHOD

Enzymatic colorimetric

PRINCIPLE

Urea (H2N CO NH2) is hydrolysed by urease into amonia (NH3) and

carbon dioxide (CO2). The amonia generated reacts with alkaline
hypochlorite (NaClO) and sodium salicylate in presence of sodium
nitroprusside as a coupling agent to yeld a blue cromophore called
indophenol. The intensity of the colour formed is proportional to the
concentration of urea in the sample.
1. H2N CO NH2 + H2O

urease

2NH3 + CO2

2. NH4+ + salycilate + NaClO + nitroprusside OH indophenol + NaCl

(blue)
REAGENTS

R1a: enzyme reagent

31
Urease
Stabilizers
R1b: buffered chromogen
Phosphate buffer (pH 6.9)
EDTA
Sodium salycilate
Sodium nitroprusside
R2 : Alkaline hypochlorite
Sodium hypochlorite
NaOH
ST: standard
Urea
SAMPLE
REACTION
CONDITIONS
OPERATING
PROCEDURE

> 500 U/ml

20 mmol/l
2 mmol/l
60 mmol/l
3.4 mmol/l
10 mmol/l
3.4 mmol/l

8.3 mmol/l
(50 mg/dl)
Serum or heparinized plasma free of hemolysis and urine
Wavelength
600 nm (10 nm)
Temperature
+37 C
Cuvette
1 cm light path
1. Preparation of the working reagent No.I: mix the enzyme
reagent (R1a) with the buffered chromogen (R1b).
2. Preparation of the working reagent No.II: dilute alkaline
hypochlorite (R2) in 100 ml of distilled H2O.
3. Pipette 1 ml of working reagent No.I into the first cuvette (the
cuvette is called "blank").
4. Pipette 1 ml of working reagent No.I and 10 l of the standard
(ST) into the second cuvette (the cuvette is called a
"standard");
5. Pipette 1 ml of working reagent No.I and 10 l of the sample
into the third cuvette (the cuvette is called "sample").
"Blank"
Working reagent 1 ml
No.I (R1a+R1b)
Standard (ST)

Sample

"Standard"
1 ml

"Sample"
1 ml

10 l

10 l

6. Mix and incubate the cuvettes at +37 C for 5 minutes.

7. Pipette 1 ml of working reagent No.II into all cuvettes:
Working
reagent No. II

CALCULATION

REFERENCE
VALUES

1 ml

1 ml

1 ml

8. Mix and incubate the cuvettes at +37 C for 5 minutes.

9. The analyzer reads light absorption rates (ABS) of the standard
and the sample against the reagent blank and calculates urea
concentration (mmol/l).
ABS sample
* standard concentration = urea
ABS standard
(mmol/l)
concentration
(mmol/l)
Serum or plasma:
1,78,3 mmol/l
Urine:
1530 g/24h

32
REVIEW QUESTIONS
1.
2.
3.
4.
5.
6.
7.
8.

Ammonia formation in the body. Its concentration in the blood.

Ammonia uptake and detoxification ways.
Ammonia detoxification in the brain. Glutamine synthetase.
Ammonia detoxification in the liver urea synthesis. The origin of urea nitrogen atoms.
Urea concentrations in plasma and amount in the daily urine. Hyperuremia.
Renal glutaminase. Formation of ammonium salts. Hyperammoniurea and acidosis.
Nitrogen-containing compounds in the urine.
The method for the determination of urea concentration in serum.

22. DETERMINATION OF CREATININE CONCENTRATION IN URINE

Qualitative reactions
Procedure. Weill reaction. Add 3 4 ml of urine and 3 5 drops of 10% NaOH solution in a test
tube. Add a few drops of sodium nitroprusside. The colour of the solution rapidly turns from pink
to yellow.
Procedure. Jaffe reaction. Add 3 4 ml of urine, 3 5 drops of 10% NaOH and several drops of
picric acid into a test tube. Creatinine picrate is forming. The colour of the solution turns orange.
Quantitative determination of creatitine concentration
Creatinine is a colourless compound, so it shall be determined by using the colour Joffe reaction.
Procedure. Take two 50 ml volumetric flasks. Add 1 ml of standard creatinine solution into one of
them, and 1 ml of urine into the other. Then add 1 ml of 10% NaOH solution and 1.5 ml of picric
acid into both flasks. Gently mix the content of the flasks and leave for 5 minutes at room
temperature. Then fill up both flasks with distilled water until the line and perform a colorimetric
measurement, using a green light filter. The control cuvette is filled whit distilled water.
Calculation:
Ex * C2
C1 = (g /dl);
Est
C1 * 1500
X = g;
100

X * 8.8 = mmol/24h;
here C1 concentration of study solution (g/dl);
C2 concentration of the standard solution (0.1 g/dl);
Ex extinction coefficient of the study solution;
Est extinction coefficient of the standard solution;
X creatinine quantity (g) per day in urine;
8.8 convertion factor to mmol.

33
REVIEW QUESTIONS
1. Synthesis of creatine phosphate. Its importance.
2. S-adenosyl (SAM) formation and the importance of the methylation process.
3. Creatinine generation. How much creatinine is excreted in the urine per day? Creatinine
ratio.
4. Creatine and creatinine levels in blood serum. Hypercreatininemia.
5. Hypercreatinemia, creatinuria.
6. The method for the determination of creatinine concentration in urine.

23. DETERMINATION OF URIC ACID CONCENTRATION IN SERUM

Principle. Reduction of uric acid with phospho wolframic acid (the part of Folins reagent) forms
coloured compounds, which can be measured using photoelectrocolorimetry.
Procedure. Add 1.5 ml of serum, 1.5 ml of distilled water and 1.5 ml of 20% trichloroacetic acid
(CCl3COOH) into the centrifuge tube. Mix the contents of the tube thoroughly. After 30 minutes
centrifuge the tube at 3000 rpm. Then add into two empty tubes the following reagents:
Sample

Standard

Supernatant CCl3COOH

I (ST)
II(S)

0.5 ml

1.5 ml

0.5 ml

Distilled
H2O
0.5 ml

Na2CO3
0.7 ml
0.7 ml

Folins
reagent
1 drop
1 drop

After 10 minutes perform a calorimetric measurement, using a green light filter. Uric acid
concentration is calculated using the formula:

Cs (mg/100ml) *59 = Cs (mol/l),

here Cs concentration of uric acid in the serum (mg/dl);
CST concentration of uric acid in the standard (I) solution (0.02 mg/ml);
Es extinction of the sample (II);
EST extinction of the standard solution;
a volume of the supernatant.
59 the convertion coefficient from mg/100ml to mol/l.

REVIEW QUESTIONS
1.
2.
3.
4.
5.
6.
7.

The molecular formula of purine and pyrimidine bases. Properties of these bases.
The structure, importance and properties of mononucleotides.
Polynucleotides: DNA and RNA. Polynucleotide backbone.
Double-helical stucture of DNA. Nucleotide complementarity.
Hydrolysis of nucleic acids. Endo- and exonucleases. Restrictase.
Degradation of purine nucleotide. Uric acid and its salts (urate).
Contentration of uric acid in the blood serum and the amount in the daily urine.
Hyperuricemia. Xanthinuria.
8. Gout and its causes.

34
9. Synthesis of purine nucleotides. Sources of carbon and nitrogene atoms in the purine ring.
10. Functional role of DNA. Nucleosome.
11. RNA types, functions. Ribosome.
12. The method for the determination of uric acid concentration in the blood serum.

24. DETERMINATION OF TOTAL BILIRUBIN CONCENTRATION IN SERUM

Procedure. Add into three tubes the following reagents in the sequence shown below (mix after
each addition):
Reagents

Serum
Caffeine reagent
NaCl 0.9%
Ehrlich's
diazo reagent

First tube
(total bilirubin)
0.5 ml
1.75 ml

0.25 ml

Test tubes
Second tube
(direct bilirubin)
0.5 ml

1.75 ml
0.25 ml

Third tube
(control)
0.5 ml
1.75 ml
0.25 ml

After 10 minutes, transfer the content of the second tube (direct bilirubin) to the cuvet and perform
a calorimetric measurement. After 20 minutes, transfer the solutions from the first (total bilirubin)
and the third tubes (control) to cuvets and perform a calorimetric measurement.
For these measurements, use a green light filter (compared with water) and a cuvette with a 5 mm
ply.
Calculation. Take the extinction value of the control sample from the extinction values of the first
and second samples. Use the calibration curve to find the total and direct bilirubin concentrations.
The indirect bilirubin is the difference between the total and the direct bilirubin. If the concentration
value, expressed in mg/dl, is multiplied by a conversion factor equal to 17.104, the concentration of
bilirubin, expressed in mol/l can be obtained.

REVIEW QUESTIONS
1.
2.
3.
4.

The formation of bilirubin in the body.

The concentration of total bilirubin and bilirubin fractions in blood serum.
The formation of bilirubin glucuronide in the liver. Bilirubin transformation in the gut.
Stercobilinogen excretion. Stercobilinogen content in stool and urine. Causes of the
increased or decreased stercobilinogen levels in stool and urine.
5. Changes of total bilirubin and its fractions concentrations in blood serum in cases of
mechanical, parenchymal (hepatic) and haemolytic jaundice.
6. Bilirubinuria.
7. Urobilinogen; the causes of its appearance in urine.

25. QUALITATIVE REACTIONS FOR THE DETECTION OF BLOOD AND ITS

PIGMENTS IN URINE
Principle. In the presence of hemoglobin, organic compounds are oxidized with hydrogen peroxide
(H2O2) and converted into coloured compounds.
Procedure. Benzidine reaction. This is a very sensitive reaction and can be performed with a highly
diluted blood up to 200,000 times. Dissolve some benzidine crystals in 0.5 ml of concentrated

35
acetic acid; add 1 ml of urine containing blood, and a few drops of H2O2. The solution turns green
or blue.
Gaiac resin reaction. Add into the test tube some urine containing blood; add 2 ml of Gaiac resin
and a few drops of H2O2. The solution turns blue.
The formation of hemochromogen crystals. During the forensic medical examination of blood
stains, hemoglobin is converted to hemochromogen, which can be identified by its characteristic
absorption spectrum or by getting its crystals.
Add on the slide a drop of blood and a few drops of the reagent (containing pyridine, sodium
hydroxide, and glucose) beside. Gently warm the slide at 37 C. The heme interacts with pyridine;
the formed compound composes crystals. They can be observed through a microscope.
The formation of hemin chloride crystals (Teichmann's sample). By the examination of blood
stains, Teichmann's reaction can be also carried out. The reaction is based on the formation of
hemin chloride crystals (hemin is a heme product).
Add a drop of blood on the slide and dry it out (the temperature cannot be higher than 30
C). Then place several crystals of NaCl on top of dry blood and put a cover glass; drip 1 2 drops of
glacial acetic acid on the top of the cover glass. Heat up the slide. A small needle shape hemin
chloride crystals can be observed through the microscope.

REVIEW QUESTIONS
1.
2.
3.
4.
5.

The physiological role of hemoglobin and its concentration in blood serum.

Hemoglobin structure. Heme structure.
Compounds containing heme-like substances. The physiological role of these compounds.
Fetal and adult forms of hemoglobin.
Pathological forms of hemoglobin. Hemoglobinopathies: thalassemia, hemoglobin variants
and their causes.
6. The structural properties and incidence of the hemoglobin variant HbS.
7. Iron metabolism. Transferrin and ferritin and their physiological role. Hemosiderin.
Hemosiderosis (haemochromatosis).
8. Heme synthesis.
9. Porphyrias, their classification, clinical and biochemical features. Anemia.
10. Hematuria, hemoglobinuria, myoglobinuria.
11. Qualitative reactions for the detection of blood and its pigments in urine.