EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

occurs when the experiment is carried out using either concentrated alcohol or diluted alcohol used for a
longer time.

Take a clean tube and add the solution of the proteins. Then add couple of drops of concentrated alcohol
(Figure 21, I1). White precipitate is obtained which does not dissolve in the surplus of water (I2).

Precipitation of proteins by heating: high temperature causes denaturation of proteins by disruption of

hydrogen and hydrophobic bonds within protein molecules. That is also how proteins coagulate, forming
coagulum. By heating, proteins molecules vibrate due to increase of their kinetic energy causing the
hydrophobic and hydrogen bonds to break.

Precipitation of proteins by heating is carried out in two clean tubes. In the both tubes add 1-2 ml of egg
whites solution, but only to the second tube add couple of drops of diluted CH3COOH, and heat both
tubes on the heater (Figure 21 J1 and I2). Obtained precipitate in both tubes is not soluble in surplus of
distilled water. Measure the time needed for the precipitate to form. Note how the coagulum is being
formed in the tube with acid much faster. The acid will move pH value to the acidic area, accelerating
thermal denaturation and coagulation.

Figure 21: Reversible and irreversible precipitation of proteins

2. Electrophoresis
Electrophoresis is a technique used for separation of charged molecules in a sample by means of an electric
current. Depending on their net charge, charged particles that are placed in an electric field migrate either
to the anode (positive pole) or the cathode (negative pole). The rate of migration directly depends on
the net charge of a molecule, the strength of the electric field and the weight or size of each particle. In
electrophoretic analysis, the apparatus (Figure 22) is designed so that the electrical current passes between
the two electrodes, through buffer and the sample that is put on supporting medium and immersed into
both chambers to make an electrical circuit. An electrophoresis chamber consists of two compartments
that are separated by a dividing wall. One side contains the anode, a positively charged electrode that
will attract anions – negatively charged ions. The second chamber contains cathode, a negatively charged
electrode that attracts cations as a positivelly charged ions. Each compartment is filled to the same height
with a buffer. Biological sample (e.g. serum, urine, cerebrospinal fluid) where the proteins are to be
separated using electric current.

54
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Figure 22: Electrophoresis system

Legend: A-cover, B-electrophoresis chamber, C-dividing wall, D-buffer compartments, E-electrical

bridge, F-power socket, G-command table of the apparatus, H-power supply.

The supporting medium is a physical support through which the charged molecules get separated; it
is composed of an agarose gel, acrylamide gel, or a moist cellulose acetate membrane. The supporting
medium provides the adsorption and sieving of the molecules that undergo the electrophoretic separation
and analysis. Then electrophoretic bridge is placed above the dividing wall in a way that the end portions
of the bridge are placed inside the buffer and touching the bottom of the chamber.

The contact between the medium and buffer in each compartment should be provided. The only electrical
connection between the two compartments is supporting material of the bridge. Finally, before the electric
power is applied, the electrophoretic chamber should be covered.

The power supplies used for electrophoresis can be adjusted to provide either constant current or constant
voltage. The support medium, buffer and electrical field affect the final separation of components during
electrophoresis.

The actual distance of migration and the resolution of the separated bands are affected by many variables
in the system like isoelectric point (IEP) of proteins and the pH of the buffer and the size of the molecules.

Isoelectric point is the environmental pH value at which the proteins are in form of zwitter ions (net charge
of amino acids in the protein molecule is zero). In this case, proteins carry no charge and will not migrate. If
the environmental pH is greater or lesser than pH of the isoelectric point, protein will be charged negatively
or positively, respectively.Therefore, according to the charge they have, molecule will migrate towards the
anode or cathode.

On the large-pore support mediums, the separation is primarily determined by the charge of the molecule.
On the fine-pore, highly cross-linked gel, the separation is affected additionally by the size of the molecules.

55
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Electrophoresis of serum proteins and its application in clinical practice

Electrophoresis is used for separation of serum proteins, types of haemoglobin (fetal hemoglobin,
carboxyhemoglobin), lipoproteins, and isoenzymes (lactate dehydrogenase, creatine kinase etc.), as well
as also for separation and analysis of DNA and RNA.

Plasma contains more than hundred different types of proteins where they exert their specific functions.
Their structures and concentrations are subject to different variations in various diseases or in physiological
conditions (pregnancy). The electrophoresis of serum proteins is helpful in diagnosing various medical
conditions such as renal failure, hypogammaglobulinemia, liver cirrhosis etc. In conditions such as stress
or inflammation caused by infection, injury or trauma the immediate response of plasma proteins results
in decrease of albumin and increase of alpha 1 and alpha 2 globulins. These proteins are called acute phase
proteins. Therefore albumin is classified as negative acute phase protein, but alpha globulins are positive
acute phase reactants. Using electrophoresis, serum protein will be separated into 5 bands on cellulose
acetate, 6 bands on agarose gel and more than 25 bands in polyacrylamide gel. Usage of polyacrilamide gel
(a polimer of acrylamide gel) for serum protein separation is not recommended in routine laboratory work
except for specialized purposes because of its high separating power. Many protein bands obtained by this
type of separation are hard to interpret and contribute very little to diagnosis.

Buffer used in serum proteins electrophoresis (commonly used Tris-barbital) provides environmental pH
value of 8.6 and acetoacetate is used as a supporting medium. In this case serum proteins carry a negative
net charge and migrate towards the anode. Albumins are relatively small and carry the largest charge so
they will move the fastest. The gamma globulins have the smallest net charge so they exceed the minimum
distance.

After the separation of serum proteins by electrophoresis, the proteins need to be fixed by immersion of
supporting medium in fixative dye, like Ponceau - S, to allow visualisation and proper reading of the sample.
After removal of excess dye, membrane is cleared by carefully timed immersion in a dehydrating agent and
a mixture of organic solvents. Finally, the membrane is heated, in order to provide the evaporation of
the excess of solvent forming a thin, transparent film with the protein bands stained red. The transparent
membrane is scanned and evaluated in a densitometer in order to get quantity of proteins that are separated.

A densitometer is a colorimeter that measures an intensity of a light beam passing through a filter to a
photodetector specially designed to scan and quantitate electrophoresis patterns. As the light beam
passes over the transparent portion of the membrane, the instrument records a baseline level at the
electrophoretogram (densitometer graph). On the contrary, as the light beam passes over electrophoresis
protein bands, less light will reach the detector and the instrument will record the absorbance of light
of the protein bands as a peak on the graph. Serum protein separation on cellulose acetate membranes
generally have five bands that appear as five peaks on the densitometer graph. The densitometer also
integrates the area under each peak so the relative concentration (Table 3) of each fraction (% of total
protein) is recorded; if the concentration of the total protein is known, the actual concentration of each
protein fraction is calculated. Five serum fractions are: albumin, alpha one (α1) globulin, alpha two (α2)
globulins, beta (β) globulins, and gamma (γ) globulins, ranged with the greatest proximity to the anode
(Figure 23). Each fraction is actually a group of many individual proteins. Two beta fractions (b 1 and b 2)
are visible if the electrophoresis is preformed on agarose gel.

56
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Serum albumins represent a single species of proteins and they are the most prominent band in
electrophoretogram. Serum albumins take up to 60% out of the total serum proteins. They are the fastest
fraction and take the position closest to the anode. Albumin is a protein synthesized in the liver and its
concentration in plasma increases in body dehydration. A decrease of albumin plasma concentration is
seen in liver diseases, chronic infection, kidney disease, and malnutrition.

Alpha one globulin is the second fastest band. They are the mixture of many proteins. Some of the
prominent individual proteins in the α1 band are α1- antitrypsin, α1-acid glycoprotein and thyroxin-
binding globulin. This band increases in a nonspecific response to inflammation (acute-phase reactant)
arising from various causes, such as infection, trauma and neoplasms.

Alpha two globulins are the third visible band on cellulose acetate electrophoresis. Some of the well-
known proteins in this fraction include haptoglobin, α2-macroglobulin, and ceruloplasmin. Haptoglobin
binds free hemoglobin and form complex metabolized in the reticuloendothelial system. That complex
preserves the body’s stores of iron. Haptoglobin is elevated in plasma under condition of acute or chronic
inflammation and reduced in liver diseases and hemolytic anemia.

The fourth band is β globulin fraction which contains the iron-transporting protein (transferrin), fibrinogen
etc. Transferrin is a major iron transporting protein in plasma. Concentration of transferrin in plasma is
increased in iron-deficiency anemia as body compensatory response. As a negative acute phase protein, it
is reduced in plasma in liver disease, malnutrition, malignant and inflammatory conditions.

Gamma globulin fraction is the slowest fraction. It contains the immunoglobulins or circulating antibodies,
essential for body defense against foreign proteins of all sorts. They represent immunoglobulins synthesized
in plasma cells. This fraction is mainly composed of immunoglobulins G class. Gamma globulins are
elevated in polyclonal or monoclonal gammaglobulinemia, liver diseases, chronic infections and decreased
in hypoimmune syndrome.

Figure 23: Protein fractions separated on cellulose acetate and electrophoretogram

The protein profiles (electrophoretic tracings) usually present distinctive patterns in several diseases
like: nephrotic syndrome, hepatocellular disease, hypogammaglobulinemia, multiple myeloma,
macroglobulinemia, 1-antitrypsin deficiency, and occasionally in nutritional assessment, etc.

57
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Table 3: Concentration of each protein fraction

Fraction % Protein fraction

Albumins 55.8 – 67

α1 – globulins 2.2 – 4.8 α1-antitrypsin, apolipo A, α-fetoprotein

α2 – globulins 8.2 – 12.5 haptoglobin,ceruloplasmin, α2-macroglobulin

β 1 – transferrin, apolipoprotein B, C3 complement

β – globulins 7.2 – 14.2
β2 – fibrinogen, C3 complement, β2 – microglobulin

γ – globulins 11.5 – 18.8 IgG, IgA, IgM, CRP

Figure 24: Examples of protein electrophoresis of serum (A-F) and urine (G-I).

Legend: A-Typical normal pattern of serum protein electrophoresis; B-Hypoalbuminemia; C-Nephrotic

syndrome; D-Multiple myeloma; E-alpha1-antitrypsin deficiency; F-Polyclonal gammopathy;G-Normal
urine electrophoretic pattern; H-Nephrotic syndrome; I-Multiple myloma

58
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Hypoalbuminemia (Figure 24B) is found in malnutrition, liver disease, in excess urinary excretion and
reduced reabsorption due to kidney failure (nephrotic syndrome), burns etc. The electrophoretic pattern
of nephrotic syndrome (Figure 24C) represents a decrease of albumin and gamma globulin fractions and
normal alpha 2 fractions in serum. Albumins and gamma globulins are lost in the urine due to their low
molecular weight, but alpha 2 fraction proteins are retained, because they are composed of high molecular
weight proteins. The basic characteristic of nephrotic syndrome is the loss of glomerular selectivity for
proteins. Multiple myeloma electrophoretic pattern (Figure 24D) shows a sharp gammaglobulin band due
to abnormal synthesis of monoclonal immunoglobulins as a product of a single abnormal clone of plasma
cells or B-lymphocytes. If monoclonal gammopathy or a low globulin fraction is detected, quantitative
immunoglobulin tests should be ordered. Alpha1-antitrypsin deficiency is a common genetic disease that
can be found in obstructive pulmonary and liver diseases. Deficiency is manifested as a decrease of alpha1-
protein band (Figure 24E). Polyclonal gammopathy (Figure 24F) is identified as a broad diffuse band in
electrophoretogram in the area of immunoglobulins as a result of multiple line cells activation by infection,
neoplastic and inflammatory diseases.

Under normal physiological conditions in the body, urine contains less than 150 mg of protein/ 24
hours. Predominant proteins in urine are albumins originated from serum and Tamm-Horsfall proteins
produced by the urinary tract itself. Due to low protein content, a urine sample is concentrated before an
electrophoretic separation. Urine protein electrophoresis can be used to evaluate proteinuria in nephrotic
syndrome (Figure 24H) and to detect Bence Jones protein (light chain of immunoglobulins, Figure 24I),
which is associated with myeloma, Waldenströmmacroglobulinemia, and Fanconi syndrome.

3. Lipids
Lipids are a diverse group of compounds of biochemical importance. They are hydrophobic molecules
insoluble in H2O, but dissolving well in non-polar organic solvents like benzene, chloroform, ether, alcohol,
acetone, etc. Water is the most abundant inorganic compound in the body, so in order to be dissolved
in aqueous environment lipids form complexes with proteins giving stabilized emulsion. Together with
carbohydrates, proteins and nucleic acids, lipids are one of the four major classes of biologically essential
organic molecules found in all living organisms. Their amounts and quality in diet are able to influence cell,
tissue and body physiology. Lipids are classified into three major groups:

1. Simple lipids. They are esters of fatty acids with glycerol or other higher alcohols (triacylglycerol or
triglycerides or neutral fat)
2. Complex lipids are fatty acids esterified with alcohol that, in addition, contain other groups.
Depending on the type of these groups the following complex lipids are known: Phospholipids that
contain phosphoric acid and glycolipids that contain carbohydrate component.
3. Isoprenoid lipids contain either isoprene as a part of their structure (carotene) or it is one of
intermediary products in biosynthesis pathways (cholesterol).

The roles of lipids in human organism are multiple:

• Storage form of energy (triglycerides). They release more energy per gram than any other types of
organic compounds in the body (9 kcal /g or 37 kJ/g).
• Structural components of biomembranes (phospholipids and cholesterol).

59
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

• Synthesis of bile acids that act as surfactants, detergents and emulsifying agents, enhancing
emulsification, digestion, absorption of lipids and other hydrophobic compounds such as liposoluble
vitamins (A, D, E and K) .
• Lipids are involved in neurotransmission by participating in myelin sheath synthesis that acts as
electric insulators in neurons.
• As a poor heat conductor, lipids provide insulation against changes in external temperature
(subcutaneous triacylglycerols).
• Give shape and contour to the body.
• Protect internal organs by providing a cushioning effect (the kidney is protected from trauma and
damage by perinephritic adipose tissue).
• Help in absorption of fat soluble vitamins (A, D, E and K).
• Improve taste and palatability of food.
• Lipids act as metabolic regulators by synthesis of steroid hormons and eicosanoids. Steroid homones
such as corticosteroids of adrenal glands and sex hormones of gonads or placenta are synthesized
from lipids. Lipids are precursor molecules of eicosanoids (autocoids) synthesis. Autocoids are
biological factors acting as local homones (leukotrienes, prostaglandins and tromboxanes) that
control many physiological and pathophysiological functions in the body such as blood pressure,
uterine contraction, inflammation, blood clotting etc.
• Lipids contribute to the amount of vitamin D in the human body. A cholesterol precursor called 7
hydroxycholesterol is converted into vitamin D3 when the skin is exposed to sunlight.

Cholesterol
Cholesterol is cyclic, monovalent, secondary non saturated alcohol (sterol). It falls into isoprenoid category
of lipids named according to isoprene as intermediate molecule in the metabolic pathway of cholesterol
synthesis. Cholesterol is synthesized in animal cells as the essential structural component of all animal cell
membranes.

Chemical structure of cholesterol is complex, as shown in (Figure 25). It is composed of hydrocarbon

or cyclopentaphenanthrene ring with 17 carbon atoms. There are methyl groups at positions 10 and
13, hydroxyl group (-OH) at position 3 and branched side chain containing eight carbon atoms
(trimethylpentane or isooctane) at position 17. Also, there is unsaturated bond between fifth and sixth
carbon atoms of the ring.

Figure 25: Cholesterol structure

Cholesterol is referred as an amphipathic molecule regarding the water-soluble (hydroxyl group) and fat-
soluble (non-polar tail containing the ring and the side chain of the molecule) regions of the molecule.

60
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

The body contains endogenous and exogenous cholesterol. Exogenous cholesterol is ingested through
food of animal origin, and endogenous cholesterol is synthesized in human body from acetyl CoA that
can be produced from the metabolism of glucose, fatty acids and amino acids. Cholesterol shows poor
solubility along with other lipids in the blood, so it has to be transported in blood in the form of complexes
known as lipoproteins (complex compounds of lipids and proteins) like low density lipoproteins (LDL)
and high density lipoproteins (HDL). Low density lipoproteins deliver cholesterol from the liver (where
the synthesis mainly takes place) to the peripheral tissues. On the contrary, high density lipoproteins are
responsible for the reverse transport of the cholesterol from peripheral tissues to the liver. It means that HDL
is a major molecule responsible for transport the cholesterol back into the liver where it is catabolized.
This way, HDL prevents oxidation of LDL cholesterol into oxidized LDL (oxLDL) that is considered to
be highly atherogenic molecule. Oxidation of LDL molecules and its phagocytosis by macrophages is a
hallmark of the atherosclerosis process (hardening and narrowing of arteries due to building up of lipid
plaque inside the artery wall).

All tissues in the body are capable of cholesterol synthesis, but it is predominantly synthesized in the liver,
than other tissues like intestines, gonads, skin, and immature brain. Cholesterol with other complex lipids,
is the main component of all cell membranes and myelin sheath. It is also needed for the synthesis of the bile
acids in the liver and vitamin D in the skin under the influence of ultraviolet light. Vitamin D should also be
considered as steroid hormone because it is activated in the liver and kidneys after being synthesized in the
skin from 7-dehydrocholesterol or absorbed from the food. It acts as a hormone affecting intestinal cells,
osteoblasts and kidney tubular cells functions. The 4-ring structure of cholesterol is the basic of all steroid
hormones produced in the gonads and adrenal glands (sex hormones, mineralocorticoids, glucocorticoids
and androgens).

The major route of cholesterol excretion from the body is its metabolic transformation into bile acids
which are then excreted into the bile. Bile enters the gallbladder where it is concentrated or is excreted
directly to the intestinal lumen. The amount of the cholesterol in the body can vary considerably among
healthy people with the mean value of about 100 grams. Approximately 60 grams are moving dynamically
in the organism.

Around 70% of cholesterol in the blood appears in the esterified form with fatty acids located in a core
of lipoprotein molecules and the rest, 30% as non-esterified in the surface monolayer. The value of the
total cholesterol in serum/plasma below 5 mmol/l, (for children, less than 4.7 mmol/l) is considered
healthy, whereas LDL should not exceed 3 mmol/l contrary to HDL levels that should be more than 20%
of the total cholesterol. Atherosclerotic plaque formation within the arterial wall is consequence of the
elevated blood cholesterol, especially LDL cholesterol considered as atherogenic contrary to HDL that
is considered as protective molecule. The measurement of two major lipoproteins in blood such as LDL
and HDL are important in cardiovascular disease risk assessment by atherogenic risk calculation (LDLc/
HDLc=1.2-4.0).

Elevated levels of cholesterol in blood (hypercholesterolemia) can be primary (genetic) and secondary when
hypercholesterolemia is a consequence of another disease that disturbs the cholesterol metabolism. A number
of secondary causes exist, including diabetes mellitus type 2, obesity, alcohol abuse, nephrotic syndrome,
hypothyroidism, anorexia nervosa and medications such as corticosteroids. One of the consequences of
the elevated cholesterol blood level is xanthelasma that is reffered as condition caused by a collection of
yellowish cholesterol deposits underneath the skin around the eyes. Moreover, the formation of cholesterol
gallstones and atherosclerosis are also consequences of disturbed cholesterol metabolism in the body.

61
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Practical work:

SALKOWSKI’S REACTION (H2SO4 test)

Add 1-2 ml chloroform solution of cholesterol into a clean tube and carefully add a couple of drops of
concentrated sulphuric acid along the side of the test tube. Always use sulphuric acid previously poured
into another clean tube; never pour it directly from the reagent bottle! The acid is heavier and goes under
the solution of cholesterol. Do not mix the content of the tube! Note the appearance of a red coloured
ring on the contact surface of two reagents because cholesterol molecule is dehydrated by concentrated
sulphuric acid at position C3 (Figure 26, A).

LIEBERMANN-BURCHARD REACTION (ACETIC ANHYDRIDE SULPHURIC ACID TEST)

Use 1-2 ml of the same reagent prepared for the first reaction (cholesterol dissolved in chloroform). Add
a few drops of acetic anhydride into the tube and, with great caution, add a few drops of sulphuric acid,
down the wall of the test tube. Do not mix the content of the tube! Note the solution becoming red, then
blue and finally, bluish green (Figure 26, B1). The same reaction can be performed on the watch glass
(Figure 26, B2).

Same reaction can be used as a Liebermann-Burchard’s method for spectrophotometric determination

of cholesterol concentration in a biological sample. The intensity of color that is formed is directly
proportional to the concentration of the cholesterol in the sample.

Figure 26: The sample color changes in Salkowski’s and Liebermann-Burchard reactions

PROBLEM 1
•• Explain the significance of the cholesterol for human organism

62
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

4. Bile acids
Bile acids belong to steroid class of compounds and are the final products of metabolism of endogenous
cholesterol in the liver. They are secreted into bile and via bile canaliculi reach the gallbladder where the
bile is stored and concentrated during the fasting state. The bile is released from the gallbladder under
action of cholecystokinin. In the form of conjugated bile salts with sodium or potassium, they represent
the constant pool in the gallbladder (3-5 grams). Cholecystokinin, peptide hormone released by the
enteroendocrine cells of the duodenum, stimulates the gallbladder contraction and secretion of the bile
and pancreatic juice into duodenal lumen providing a proper digestion in the digestive tract upon the
arrival of the digestion products of stomach. Synthesis and secretion of the bile acids into digestive tract is
the significant way of cholesterol excretion from the human body but not sufficient if cholesterol is taken
in excess.

Directly synthesized from cholesterol in hepatocytes, bile acids have the chemical structure similar to
cholesterol with the differences in the side chain at the position 17 (isovaleric acid or gamma-methylbutanoic
acid instead of the trimethylpentan) and cyclopentaphenanthren ring which is saturated contrary to
cholesterols’ unsaturated one. There are four different kinds of bile acids (cholic, chenodeoxycholic,
deoxycholicand and lithocholic acids); the only difference between them is the number of hydroxyl (–
OH) groups in a molecule. So, lithocholic acid has one –OH group at position 3 of the ring (3-alpha-
monohydroxy acid). Chenodeoxycholic (3 -alpha, 7- alpha-dihydroxy acid) and deoxycholic acid (and
3-alpha,12 -alpha dihydroxy acid) have two hydroxyl groups in the molecules. Finally, cholic acid with
three OH groups (3-alpha, 7-alpha, 12-alpha-threehydroxy acid). The cholic and chenodeoxycholic acids
make up to 80% of all bile acids in the bile.

The bile acids secreted into the bile are amphipathic molecules (Figure 27) that contain both the hydrophilic
side (this side faces water molecules) and hydrophobic sides (facing to a lipid molecule). Having both sides
provides the bile acids with function of a biological detergent (surfactant) which enhances the solubility of
cholesterol and other lipids presented in the bile and also dietary lipid digestion and absorption.

Figure 27: The amphipathic nature of bile acids

The synthesis and secretion of the bile acids

The synthesis of bile acids represents the route of cholesterol excretion from the human body. There are
primary and secondary bile acids (Figure 28) according to the place where the synthesis of bile acids

63
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

takes. The primary bile acids such as cholic and chenodeoxycholic are synthesized in the liver cells. Before
being secreted into the bile canaliculi, they are conjugated forming an amide bond with either glycin or
taurin giving the glycocholic (glycochenodeoxycholic) or taurocholic (taurochenodeoxycholic) acid,
respectively. The conjugation improves the ionization of bile acids giving them the better detergents
quality followed by enhanced emulsifying capacity of the bile acids. As a consequence, the bile acids are
ionized in a broader range of pH values forming bile salts with sodium or potassium (sodium glycocholate
or sodium taurocholate). Upon acting in the intestinal tract, bile salts are either excreted in the feces (small
portion) or reabsorbed (95%) in the terminal ileum; the process termed as enterohepatic circulation of bile
salts. The reabsorption of bile salts represents the way how the body preserves the cholesterol used for the
bile salts synthesis. If the bile salts escape the reabsorption they undergo modification in the colon such
as deconjugation and dehydroxylation forming secondary bile acids (litocholic and deoxycholic acids).
Due to their poorer solubility than the primary bile acids, some of secondary bile acids are reabsorbed.
Through circulation, they will reach liver, but will not be rehydroxylated, and as a result they will re –
excreted. Therefore, the bile is the liver secretion product containing primary bile acids synthesized de
novo (starting from the beginning) and secondary ones synthesized in the colon.

Figure 28: The structure and place of the primary and secondary bile acids synthesis

The importance of bile acid in the human body

The main role of bile acids in human body is the preparation of fats for digestion by acting as detergents and
enabling the emulsification of hydrophobic fat molecules. In this way, breaking the lipid molecules down into
small particles, their surface area is greatly increased and the surface tension is reduced. This makes the lipid
droplets available for digestion by lipases that are not able to access the inside of lipid droplets otherwise.

64
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

Besides facilitating the digestion of fat molecules by emulsification, other important roles of bile acids are:

• They activate the pancreatic lipase and act together with colipase (small peptide cofactor) that
aids the function of lipase in the digestion of triacylglycerols by binding lipase to the surface of fat
droplets.
• Bile acids and phospholipids (both soluble in water) help solubilize cholesterol in the bile; thereby
prevent the precipitation of cholesterol in the gallbladder by forming mixed micelles.
• They enhance the intestinal absorption of fat-soluble vitamins such as A, D, E and K.
• Regarding their surfactant activity (due to amphipathic nature), bile acids promote the delivery of
dietary lipids to the liver by enhancing the absorption of lipids with formation of mixed micelles. The
final products of lipids digestion like fatty acids, monoacylgycerols, phospholipids and cholesterol
are poorly solubilised and tend to aggregate in an aqueous environment, so bile acids prevent it by
forming the micelles followed by absorption into the intestinal endothelial cells.

Clinical significance of bile acids

Total amount of bile acids measured in the blood serve as marker for the assessment of the liver function.
Increased values in serum are seen in patients with acute hepatitis, chronic hepatitis, liver sclerosis,
liver cancer, cholestasis, portal-vein thrombosis, Budd-Chiari syndrome, cholangitis, Wilson disease
and hemochromatosis. Cholestasis, retention of bile acids, is manifested with bile salts elevation in
systemic circulation leading to accumulation of bile salts in the skin what is manifested by the skin itching
(cholestatic pruritis). Additional symptom in those patients is steatorrhea (an excessive amount of lipids
in feces) due to malabsorption of lipids in the digestive system. Another clinical condition associated with
malabsorption of bile acids is diarrhea (three or more loose or watery stools per day) that can be induced
by overproduction or failed reabsorption of bile acids in the intestinal tract.

Normally, bile salts are not found in the urine but in the case of biliary obstruction, they might be detected.
Total bile acids concentration is increased after meals so biological samples intended for analysis should be
collected under fasting conditions.

Practical work:

Pettenkoffer’s test

Take a clean test tube and pour couple of milliliters of urine and, add a couple of milliliters of 10% sucrose
solution. Then, carefully sublayer the mixture with concentrated H2SO4, along the side of the test tube.
Note the appearance of the red ring at the touching surfaces of the two layers (bile and sulphuric acid).
Sucrose has been hydrolyzed, glucose/fructose form methylfurfural and oxymethylfurfural and react with
bile salts, forming hydroxymethylfurfural which is red coloured solution (Figure 29, A).

Hay’s sulphur test

Take 2-3 ml of urine and pour it in the clean test tube. Then, gently sprinkle a pinch of sulphur powder on
the top of it (avoid shaking).

65
 EXPERIMENTAL PROCEDURES AND CLINICAL CORRELATIONS IN MEDICAL BIOCHEMISTRY 1

In urine that contains bile, sulphur powder sinks on the bottom of the tube, forming characteristic flower-
like formation (Reaction of sulphur flower). Bile salts reduce the surface tension of the solution by
weakening the forces between water molecules to allow the sulphur powder to sink. If the bile acids are
absent from urine, the powder will stay at the surface of the urine (Figure 29, B1 and B2).

Figure 29: Urine bile acid testing

PROBLEM 2
•• Explain what molecule is used for the bile acids synthesis? List the roles of bile acid in the
human body

SIGNATURE________________ DATE________________

66