CHEMICAL PATHOLOGY I

(MLS 425)
LECTURE NOTE
BY
MR F.O. OYAKHIRE
DEPARTMENT OF MEDICAL
LABORATORY SCIENCE
JOSEPH AYO BABALOLA
UNIVERSITY

(JABU)
OSUN STATE
EXTRACTING PURE PROTEINS FROM CELLS

Many different proteins exist in a single cell. A detailed study of the properties of any one
protein requires a homogeneous sample consisting of only one kind of molecule. The separation
and isolation, or purification, of proteins constitutes an essential first step to further
experimentation. In general, separation techniques focus on size, charge, and polarity-the sources
of differences between molecules. Many techniques are performed to eliminate contaminants and
to arrive at a pure sample of the protein of interest. As the purification steps are followed, we
make a table of the recovery and purity of the protein to gauge our success. Table 5.1 shows a
typical purification for an enzyme. The percent recovery column tracks how much of the
protein of interest has been retained at each step. This number usually drops steadily during the
purification, and we hope that by the time the protein is pure, sufficient product will be left for
study and characterization. The specific activity column compares the purity of the protein at
each step, and this value should go up if the purification is successful.
HOW DO WE GET THE PROTEINS OUT OF THE CELLS?

Before the real purification steps can begin, the protein must be released from the cells and
subcellular organelles. The first step, called homogenization, involves breaking open the cells.
This can be done with a wide variety of techniques. The simplest approach is grinding the tissue
in a blender with a suitable buffer. The cells are broken open, releasing soluble proteins. This
process also breaks many of the subcellular organelles, such as mitochondria, peroxisomes, and
endoplasmic reticulum.

A gentler technique is to use a Potter–Elvejhem homogenizer, a thick-walled test tube through

which a tight-fitting plunger is passed. The squeezing of the homogenate around the plunger
breaks open cells, but it leaves many of the organelles intact. Another technique, called
sonication, involves using sound waves to break open the cells. Cells can also be ruptured by
cycles of freezing and thawing. If the protein of interest is solidly attached to a membrane,
detergents may have to be added to detach the proteins. After the cells are homogenized, they are
subjected to differential centrifugation.

Spinning the sample at 600 times the force of gravity (600 x3g) results in a pellet of unbroken
cells and nuclei. If the protein of interest is not found in the nuclei, this precipitate is discarded.
The supernatant can then be centrifuged at higher speed, such as 15,000 x 3g, to bring down the
mitochondria. Further centrifugation at 100,000x3g brings down the microsomal fraction,
consisting of ribosomes and membrane fragments. If the protein of interest is soluble, the
supernatant from this spin will be collected and will already be partially purified because the
nuclei and mitochondria will have been removed. Figure 5.1 shows a typical separation via
differential centrifugation.
After the proteins are solubilized, they are often subjected to a crude purification based on
solubility. Ammonium sulfate is the most common reagent to use at this step, and this procedure
is referred to as salting out. Proteins have varying solubilities in polar and ionic compounds.
Proteins remain soluble because of their interactions with water. When ammonium sulfate is
added to a protein solution, some of the water is taken away from the protein to make ion–
dipole bonds with the salts. With less water available to hydrate the proteins, they begin to
interact with each other through hydrophobic bonds. At a defined amount of ammonium sulfate,
a precipitate that contains contaminating proteins forms. These proteins are centrifuged down
and discarded. Then more salt is added, and a different set of proteins, which usually contains the
protein of interest, precipitates. This precipitate is collected by centrifugation and saved. The
quantity of ammonium sulfate is usually measured in comparison with a 100% saturated
solution. A common procedure involves bringing the solution to around 40% saturation and then
spinning down the precipitate that forms. Next, more ammonium sulfate is added to the
supernatant, often to a level of 60%–70% saturation. The precipitate that forms often contains the
protein of interest. These preliminary techniques do not generally give a sample that is very pure,
but they serve the important task of preparing the crude homogenate for the more effective
procedures that follow.

Summary

To begin the process of purification, proteins are released from cells with homogenization using
a variety of physical techniques.
Initial purification steps are accomplished using differential centrifugation and salting out with
ammonium sulfate.

Chromatography

The word chromatography comes from the Greek chroma, “color,” and graphein, “to write”; the
technique was first used around the beginning of the 20th century to separate plant pigments with
easily visible colors. The term chromatorgraphy was originally applied by Micheal Tswett, a
Russian Botanist, in 1906 to a procedure where a mixture of different coloured pigments
(chlorophylls and xanthophylls) was separated from each other.
It has long since been possible to separate colorless compounds, as long as methods exist for
detecting them. Chromatography is based on the fact that different compounds can distribute
themselves to varying extents between different phases, or separable portions of matter. One
phase is the stationary phase, and the other is the mobile phase.

The mobile phase flows over the stationary material and carries the sample to be separated along
with it. The components of the sample interact with the stationary phase to different extents.
Some components interact relatively strongly with the stationary phase and are therefore carried
along more slowly by the mobile phase than are those that interact less strongly. The differing
mobilities of the components are the basis of the separation.

Many chromatographic techniques used for research on proteins are forms of column
chromatography, in which the material that makes up the stationary phase is packed in a
column. The sample is a small volume of concentrated solution that is applied to the top of the
column; the mobile phase, called the eluent, is passed through the column. The sample is diluted
by the eluent, and the separation process also increases the volume occupied by the sample. In a
successful experiment, the entire sample eventually comes off the column. Figure 5.2 diagrams
an example of column chromatography. One of the tasks of biochemists is to

s identify, separate and purify one or more biological components in a mixture of such
compounds in a biological sample. One of the most important convenient methods for achieving
such separation is the use of chromatographic techniques.

Principle : The basis of all forms of chromatography is the partition or distribution coefficient
which describes the way in which a compound distributes itself between two immiscible phases.
For a compound distributing itself between equal volumes of two immiscible solvents A and B
(Fig. 10.1), the value of distribution co-efficient is a constant at a given temperature and is given
by the expression. Basically all chromatographic systems consists of two phases. One is the
stationary phase which may be a solid, liquid or a solid liquid mixture which is immobilized. The
mobile phase may be a liquid or a gas and flows over or through the stationary phase.

Separation starts to occur when a compound to be separated is held more firmly by the stationary
phase than the other which tends to move on slower in the mobile phase. Thus, the underlying
principle of chromatorgraphy is to adsorb the components of the mixture on an insoluble material
and then to differentially remove or elute these components one by one with suitable solvents.

The term effective distribution co-efficient is defined as the total amount as distinct from the
concentration of substance present in one phase divided by the total amount present in the other
phase. Thus, a distribution co-efficient of a substance between alumina (stationary phase) and
butanol (mobile phase) might be 0.25 which means that the concentration of the substance in
butanol is four times that in the alumina.The choice of stationary or mobile phases is made so
that the compounds to be separated have different distribution co-efficient.
In practice separations may be achieved by using different types of chromatographic techniques

Types of chromatography

Column chromatography: In this type, the stationary phase is packed into a glass or metal
columns (wide tubes or cylinders). The mixture to be separated is layered on the top of the
column in the form of a solution at particular concentration. After equilibration the components
are eluted out of the column one by one using specific mobile phases (Fig.10.2).The solvent used
to elute the separated components is known as eluant.
Thin layer chromatography: In this type, the stationary phase is thinly coated on to a glass,
plastic or foil plates. The mixture to be separated is applied on the stationary phase at one end
and kept vertical in the petridish containing the mobile phase. When the mobile phase reaches
the other end of the plate the plate is removed from the petri dish and the compounds separated
are identified by using specific staining reagents

Paper chromatography: In this type, the stationary column chromatography phase is

supported by the cellulose fibres of a paper sheet. The mobile phase flows through the stationary
phase and effects separation.

Each of these three types of chromatography have their specific advantages, applications and
method of operation.

Column chromatography:

The word chromatography comes from the Greek chroma, “color,” and graphein, “to write”; the
technique was first used around the beginning of the 20th century to separate plant pigments with
easily visible colors. It has long since been possible to separate colorless compounds, as long as
methods exist for detecting them. Chromatography is based on the fact that different compounds
can distribute themselves to varying extents between different phases, or separable portions of
matter. One phase is the stationary phase, and the other is the mobile phase.

The mobile phase flows over the stationary material and carries the sample to be separated along
with it. The components of the sample interact with the stationary phase to different extents.
Some components interact relatively strongly with the stationary phase and are therefore carried
along more slowly by the mobile phase than are those that interact less strongly. The differing
mobilities of the components are the basis of the separation.

Many chromatographic techniques used for research on proteins are forms of column
chromatography, in which the material that makes up the stationary phase is packed in a
column. The sample is a small volume of concentrated solution that is applied to the top of the
column; the mobile phase, called the eluent, is passed through the column. The sample is diluted
by the eluent, andthe separation process also increases the volume occupied by the sample. In a
successful experiment, the entire sample eventually comes off the column. Figure 5.2 diagrams
an example of column chromatography.

All the major types of chromatography are routinely carried out using column type (Fig. 10.3).
The different types of column chromatography are

i. adsorption chromatography

ii. partition chromatography

iii. ion-exchange chromatography

iv. exclusion chromatography

v. affinity chromatography
i. Adsorption chromatography

Principle:

An adsorbent may be described as a solid which has the property of adsorbing molecules at its
surface, particularly when it is porous and finely divided. Adsorption can be specific so that one
solute may be adsorbed selectively from a mixture. Separation of components by the method
depends upon differences both in their degree of adsorption by the adsorbent and solubility in the
solvent used for separation. Adsorption chromatography can be carried out in both the column
and thin layer modes.

ii.Partition chromatography Principle:

This technique is based on the partitioning of compounds between a liquid stationary phase and a
liquid mobile phase. The liquid stationary phase can be held on any solid support like paper. This
technique is otherwise known as liquid- liquid chromatography.

iii. Gas liquid chromatography

Principle: This technique is based upon the partitioning of compounds between a

liquid stationary phase and a gas mobile phase . It is a widely used method for the qualitative and
quantitative analysis of a large number of compounds (eg. fatty acids) because it has high
sensitivity, reproducibility and speed of resolution. A stationary phase of liquid material such as
a silicone grease is supported on an inert granular solid. This material is packed into a narrow
coiled glass or steel column 1 to 3 meter long and 2 to 4mm internal diameter. Through this
column an inert carrier gas (the mobile phase) such as nitrogen, helium or argon is passed. The
column is maintained in an oven at an elevated temperature which volatilizes the compounds to
be separated.

The basis for the separation is the difference in the partition coefficients of the volatilized
compounds between the liquid and gas phases as the compounds are carried through the column
by the carrier gas. As the compounds flow, they leave the column and pass through a detector
which is connected to a recorder and record a peak. The area of the peak corresponds to the
concentration of the compound separated.

iv. Ion exchange chromatography is logistically similar to affinity chroma-tography. Both use
a column resin that binds the protein of interest. With ion-exchange chromatography, however,
the interaction is less specific and is based on net charge. An ion-exchange resin has a ligand
with a positive charge or a negative charge. A negatively charged resin is a cation
exchanger, and a positively charged one is an anion exchanger. Figure 5.7 shows some typical
ion-exchange ligands. Figure 5.8 illustrates their principle of operation with three amino acids of
different charge. Figure 5.9 shows how cation exchange chromatography would separate
proteins. The column is initially equilibrated with a buffer of suitable pH and ionic strength. The
exchange resin is bound to counterions. A cation-exchange resin is usually bound to Na+ or
K+ ions, and an anion exchanger is usually bound to Cl– ions. A mixture of proteins is loaded on
the column and allowed to flow through it. Proteins that have a net charge opposite to that of the
exchanger stick to the column, exchanging places with the bound counterions. Proteins that have
no net charge or have the same charge as the exchanger elute. After all the nonbinding proteins
are eluted, the eluent is changed either to a buffer that has a pH that removes the charge on the
bound proteins or to one with a higher salt concentration. The latter outcompetes the bound
proteins for the limited binding space on the column. The once-bound molecules then elute,
having been separated from many of the contaminating ones.

Principle: The principle of this form of chromatography is the attraction between

oppositely charged particles . Many biological materials, such as amino acids and proteins, have
ionisable groups and the fact that may carry a net positive or negative charge can be utilized in
separating mixtures of such compounds. The net charge carried by such compounds depend on
their pKa and on the pH of the solution.

Ion exchange separations are mainly carried out in columns packed with an ion exchanger, which
contain the core matrix molecule with exchangeable ionic groups on its surface. There are two
types of ion exchangers, namely cation and anion exchangers. Cation exchangers posses
negatively charged groups and they will attract positively charged molecules. Anion exchangers
have positively charged groups which will attract negatively charged molecules. The actual ion
exchange mechanism composed of four steps;

❖ Selective adsorption of the molecules to be separated by the ion exchange resins. B

release of the exchangeable group from the matrix.
❖ Elution of the absorbed molecule by specific eluants.
❖ Regeneration of the matrix by recharging with the original exchangeable groups.
Cation exchanger
Some of the ion exchange materials used in this technique are Amberlite IRC 50, Bio- Rex,
Dowex 50, Sephadex etc.

v. Molecular exclusion chromatography/Size-exclusion chromatography, also called gel-

filtration chromatography, separates molecules on the basis of size, making it a useful way to
sort proteins of varied molecular weights. It is a form of column chromatography in which the
stationary phase consists of cross-linked gel particles. The gel particles are usually in bead form
and consist of one of two kinds of polymers.

The first is a carbohydrate polymer, such as dextran or agarose; these two polymers are often
referred to by the trade names Sephadex and Sepharose, respectively (Figure 5.3). The second is
based on polyacrylamide (Figure 5.4), which is sold under the trade name Bio-Gel. The cross-
linked structure of these polymers produces pores in the material. The extent of cross-linking can
be controlled to select a desired pore size. When a sample is applied to the column, smaller
molecules, which are able to enter the pores, tend to be delayed in their progress down the
column, unlike the larger molecules. As a result, the larger molecules are eluted first, followed
later by the smaller ones, after escaping from the pores. Molecular-sieve chromatography is
represented schematically in Figure 5.5. The advantages of this type of chromatography are (1)
its convenience as a way to separate molecules on the basis of size and (2) the fact that it can be
used to estimate molecular weight by comparing the sample with a set of standards. Each type of
gel used has a specific range of sizes that separate linearly with the log of the molecular weight.
Each gel also has an exclusion limit, a size of protein that is too large to fit inside the pores. All
proteins that size or larger elute first and simultaneously.
This chromatography is otherwise known as gel permeation chromatography.

Princple: This technique is based on the separation of molecules on the basis of their molecular
size and shape and the molecular sieve properties of a variety of porous materials which serve as
the solid stationary phase.

A column of gel particles or porous glass granules is in equilibrium with a suitable solvent for
the molecules to be separated. Large molecules which are completely excluded from the pores
will pass through the interstitial spaces and smaller molecules will be distributed between the
solvent inside and outside the molecular sieve and will then pass through the column at a lower
rate . So the larger particles will come out of the column first followed by smaller particles (fig.
10.4).
The gel materials generally used for this technique are cross-linked dextrans, agarose,
polyacrylamide, poly styrene etc.
vi. Affinity chromatography: uses the specific binding properties of many proteins. It is
another form of column chromatography with a polymeric material used as the stationary phase.
The distinguishing feature of affinity chromatography is that the polymer is covalently linked to
some compound, called a ligand, that binds specifically to the desired protein (Figure 5.6). The
other proteins in the sample do not bind to the column and can easily be eluted with buffer, while
the bound protein remains on the column. The bound protein can then be eluted from the column
by adding high concentrations of the ligand in soluble form, thus competing for the binding of
the protein with the stationary phase. The protein binds to the ligand in the mobile phase and is
recovered from the column. This protein–ligand interaction can also be disrupted with a change
in pH or ionic strength. Affinity chromatography is a convenient separation method and has the
advantage of producing very pure proteins.

Principle: This technique is based on the specific biological interaction of the compounds to be
separated with the special molecules attached on the stationary phase called as ligands. This
technique requires that the material to be isolated is capable of reversibly binding to a specific
ligand which is attached to an insoluble matrix (stationary phase)

Under suitable experimental conditions when a complex mixture containing the specific
compound to be purified is added to the insolubilised ligand generally contained in a
chromatography column, only that compound will bind to the ligand. All the other compounds
can be washed away and the compound subsequently recovered by displacement from the ligand.
The purification of an enzyme by this technique is shown diagrammatically in Fig. 10.5.
In practice, particles which are uniform, spherical and rigid are used as matrix materials such as
polystyrene, cellulose, porous glass and silica etc.
Thin Layer chromatography

Principle: Partition, adsorption, exclusion chromatography can be carried out in a thin

layer mode. In this technique the stationary phase is made in the form of a slurry and applied as a
thin coating on the surface of a glass plate.After activating the plate, the sample to be separated
is applied at one end of the plate . The plate is kept vertically in a chamber specially designed for
this purpose( TLC chamber) and allowed the sample and the mobile phase to raise through the
stationary phase by capillary action. The whole procedure consists of.
a. Thin layer preparation : A slurry of the stationary phase, generally applied to a glass, plastic
or foil plate as a uniform thin layer by means of a plate spreader starting from one end of the
plate and moving progressively to the other. Calcium phosphate is incorporated into the slurry in
order to facilitate the adhesion of the adsorbent to the plate. The plate is heated in an oven at
1000 C to activate the adsorbent.

b. Sample application : The sample is applied to the plate by means of a micropipette or syringe
as spot or as a band on the stationary phase.

c. Plate development : Separation takes place in a glass tank which contains mobile phase to a
depth of about 1.5 cm. This is allowed to stand for at least an hour with a lid over the top of the
tank to ensure that the atmosphere within the tank becomes saturated with solvent vapour.

d. Component detection : The components separated are detected by (i) spraying the plate with
50% sulphuric acid or 25% sulphuric acid in ethanol and heating; (ii) examining the plate under
ultraviolet light; (iii) spraying of plates with specific colour reagents, for example ninhydrin for
amino acids.

Paper chromatography

Principle: The cellulose fibres of chromatography paper act as the supporting matrix for
the stationary phase. The stationary phase may be water or a non-polar material such as liquid
paraffin. The components get separated between the liquid stationary phase and the liquid mobile
phase. The procedure consists of

a. Paper development: There are two techniques which may be employed for the development
of paper, ascending and descending methods. In both cases, the solvent is placed in the base of a
sealed tank or glass jar to allow the chamber to become saturated with the solvent paper. The
sample spots should be in a position just above the surface of the solvent so that as the solvent
moves vertically up the paper by capillary action, separation of the sample is achieved.

b. Component detection: The separated components can be detected by (i) examining the paper
under ultraviolet light; (ii) spraying of papers with specific colour reagents, for example
ninhydrin for amino acids and sulphuric acid for simple sugars.
The identification of a given compound may be made on the basis of its R f value (retardation
factor) which is the distance moved by the component during development divided by the
distance moved by the solvent from the point of origin (Fig. 10.6).

The value of Rf is constant for a particular compound under standard conditions and closely
reflects the distribution coefficient for that compound.

Applications of chromatography

❖ Thin layer chromatography is used for the separation of alkaloids, phospholipids and
other lipids.
❖ Gas liquid chromatography is applied for the separation of fatty acids in a lipid mixture.
❖ Ion exchange chromatography can be used for the separation and identification of amino
acids in a mixture of protein hydrolysate. This principle is used in auto-analyzer.
❖ Exclusion chromatography can be applied for the determination of the molecular weight
of the components separated.
 ❖ Affinity chromatography is applied for the purification of a wide range of enzymes and
other proteins like immunoglobulins

Summary
Column chromatography refers to several common techniques for purification of proteins.
In gel-filtration chromatography, proteins are separated by size.
In ion-exchange chromatography, molecules with a specific charge are selectively bound to a
column, separated from proteins that don’t bind, and then eluted.
In affinity chromatography, molecules are bound to the column via specific interactions for a
bound ligand. Once nonbinding proteins are removed, the protein of interest can be eluted.

Electrophoresis

Electrophoresis is based on the motion of charged particles in an electric field toward an

electrode of opposite charge. Macromolecules have differing mobilities based on their charge,
shape, and size. Although many supporting media have been used for electrophoresis, including
paper and liquid, the most common support is a polymer of agarose or acrylamide that is similar
to those used for column chromatography. A sample is applied to wells that are formed in the
supporting medium. An electric current is passed through the medium at a controlled voltage to
achieve the desired separation (Figure 5.10). After the proteins are separated on the gel, the gel is
stained to reveal the protein locations, as shown in Figure 5.11.
ELECTROPHORESIS

1. General principle: Many important biological molecules such as amino acids, peptides,
proteins, nucleotides and nucleic acids posses ionisable groups and can therefore be made to
exists in solution as electrically charged species either as cations (+) or anions (-). When a
mixture of these components are subjected to electric field, they migrate differentially and thus
can be separated.

2. Types of electrophoresis

(i) low voltage thin sheet electrophoresis; (ii) high voltage electrophoresis, (iii) gel
electrophoresis – native poly acrylamide gel electrophoresis and sodium dodecyl sulphate (SDS)
poly acrylamide gel electrophoresis, (iv) isoelectric focussing and (v) isotachophoresis.

Gel electrophoresis
The most commonly used electrophoresis is gel electrophoresis. In this technique either agarose
or poly acrylamide is used as supporting media. Electrophoresis units are available for running
either vertical or horizontal gel systems. Vertical slab gel units are commercially available and
routinely used to separate proteins in acrylamide gels. The gel is formed between two glass
plates that are clamped together but held apart by plastic spacers. Gel dimensions are mostly 12
cm x 14 cm with a thickness of 0.5 mm to 1.0 mm. A plastic comb is placed in the gel solution
and removed after polymersization to provide loading wells for the samples. When the apparatus
is assembled, the lower electrophoresis tank buffer surrounds the gel plates and effect cooling of
the gel plates.

In horizontal gel system, the gel is cast on a glass or a plastic sheet and placed on a cooling plate.
Connection between the gel and electrode buffer is made by using a thick wetted filter paper
(wick). The power pack supplies direct current between the electrodes in the electrophoresis unit.
All electrophoresis is carried out in appropriate buffer to maintain constant state of ionization of
the components being separated. Any variation in pH may alter the over all charge and so the
mobility of the molecules being separated

WHAT IS THE DIFFERENCE BETWEEN AGAROSE GELS AND

POLYACRYLAMIDE GELS?

Agarose-based gels are most often used to separate nucleic acids and will be discussed. For
proteins, the most common electrophoretic support is polyacrylamide (Figure 5.4), although
sometimes agarose is used. A polyacrylamide gel is prepared and cast as a continuous cross-
linked matrix, rather than being produced in the bead form employed in column chromatography.
In one variation of polyacrylamide-gel electrophoresis, the protein sample is treated with the
detergent sodium dodecyl sulfate (SDS) before it is applied to the gel. The structure of SDS is
CH3(CH2)10CH2OSO3Na+. The anion binds strongly to proteins via nonspecific adsorption. The
larger the protein, the more of the anion it adsorbs. SDS completely denatures proteins, breaking
all the noncovalent interactions that determine tertiary and quaternary structure. This means that
multisubunit proteins can be analyzed as the component polypeptide chains. All the proteins in a
sample have a negative charge as a result of adsorption of the anionic SO 3–. The proteins also
have roughly the same shape, which is a random coil. In SDS–polyacrylamide-gel
electrophoresis(SDS–PAGE), the acrylamide offers more resistance to large molecules than
tosmall molecules.

Because the shape and charge are approximately the same for all the proteins in the sample, the
size of the protein becomes the determining factor in the separation: small proteins move faster
than large ones. Like molecular-sieve chromatography, SDS–PAGE can be used to estimate the
molecular weights of proteins by comparing the sample with standard samples. For most
proteins, the log of the molecular weight is linearly related to its mobility on SDS–PAGE, as
shown in Figure 5.12. Proteins can also be separated on acrylamide without the SDS, in which
case the gel is called a native gel. This is useful for times when the study calls for a protein in its
native conformation. In this case, however, the mobility is not correlated with size specifically,
as three variables control the movement down the gel-size, shape, and charge. Agarose Gels:
Agarose is a linear polysaccharide made up of repeating units of agarobiose which contains
galactose and 3, 6 anhydro galactose. This is isolated from seaweeds. Agaroses gel is usually
prepared at the concentration of 1-3% solutions. The gels can be prepared by suspending dry
agarose in suitable aqueous buffer then boiling the mixture until a clear solution forms. This is
poured and allowed to cool to room temperature to form a rigid gel. Agarose gels are used for the
electrophoresis of both proteins and nucleic acids.

Polyacrylamide Gels: Electrophoresis in acrylamide gels is referred as PAGE being an

abbreviation for PolyAcrylamide Gel Electrophoresis. Polyacrylamide gels are prepared by
dissolving required quantity of acrylamide with a small amount of N, N’-methylene
bisacrylamide in suitable buffer. The polymerization is initiated by ammonium persulphate and
N, N, N’, N’-tetramethylene diamine (TEMED).The polymerization is free radical mediated
reaction. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis is the most widely
used method for analyzing protein mixture qualitatively. It is particularly useful for monitoring
protein purification. SDS is an anionic detergent. Samples to be run on SDS-PAGE are first
boiled for 5 minutes in sample buffer containing beta mercapitoethanol and SDS. The mercapto
ethanol reduces any disulphide bridges and cleave the protein into different sub-units. So, by this
electrophoresis different units of proteins can be identified.
Isoelectric focusing is another variation of gel electrophoresis. Because different proteins have
different titratable groups, they also have different isoelectric points. Recall that the isoelectric
pH (pI) is the pH at which a protein (or amino acid or peptide) has no net charge. At the pI, the
number of positive charges exactly balances the number of negative charges. In an isoelectric
focusing experiment, the gel is prepared with a pH gradient that parallels the electric-field
gradient. As proteins migrate through the gel under the influence of the electric field, they
encounter regions of different pH, so the charge on the protein changes. Eventually each protein
reaches the point at which it has no net charge-its isoelectric point-and no longer migrates. Each
protein remains at the position on the gel corresponding to its pI, allow- ing for an effective
method of separation. An ingenious combination, known as two-dimensional gel electrophoresis
(2-D gels), allows for enhanced separation by using isoelectric focusing in one dimension and
SDS–PAGE run at 90° to the first (Figure 5.13).
Summary

Electrophoresis separates molecules on a gel medium by passing electri-cal current through the
gel.
Proteins are separated on the gel based on their size, shape, and charge.
With SDS–polyacrylamide-gel electrophoresis, proteins separate based on molecular weight.

UNIT DETECTION OF SEPARATED COMPONENTS

❖ Proteins can be detected by using the dye-solution Coomassie Brilliant Blue R-250
(CBB). Staining is usually carried out using 0.1% CBB in methanol : acetic acid : water
in the ratio 5:1:5. The protein bands will look blue in colour.
❖ Glycoproteins are detected by using periodic acid – schiff (PAS) stain. The bands will
appear in red colour.
❖ Nucleic acids can be detected by using the fluorescent dye ethidium bromide. The
nucleic acids bands will appear colourless in black background.
PROTEINS PRECIPITATION

The basic goal of protein precipitation is to remove interferences or purify the protein by
isolating it from the solvent.

The efficiency of different precipitation techniques varies, often depending on the protein's
solubility and molecular structure.

The hydration shell of a protein is the primary focus of all protein precipitation techniques
because of its crucial role in protein folding and function. Protein hydration refers to the water
that forms around proteins and facilitates their uniform distribution in solution and their
interactions via hydrogen bonds. This translates to a high degree of solvability in water.

Adding an organic solvent (methanol, ethanol, or acetonitrile) to a biological sample is a typical

method for removing proteins. To remove the mass of precipitated protein, the sample matrix is
typically diluted with the precipitating agent (2-4 parts), mixed with a vortex, and then
centrifuged or filtered. An aliquot of the filtrate or supernatant is injected for further study. Keep
in mind that trichloroacetic acid (10%, w/v), perchloric acid(6%, w/v), and metaphosphoric acid
(5% w/v) are all examples of acids, salts, and metal ions that can be employed to precipitate
proteins. Acetonitrile, trichloroacetic acid, and zinc sulfate have all been reported to be
particularly effective at precipitating out proteins (with efficiencies of >96, 92, and 91%,
respectively, when used at a 2: 1 ratio of precipitant to plasma). As a universal approach
(needing no method development), protein precipitation is frequently used for sample
preparation. This is because it just requires adding the solvent to the sample once. Analysts are
drawn to the speed and low cost of this method. In most cases, the nonselective cleansing
approach is outclassed by the great resolving power of LC-MS/MS analytical methods. Plasma,
serum, whole blood, tissue homogenates, and in vitro incubation mixtures are common sample
matrices utilized with protein precipitation procedures. The protein precipitation method has a
few drawbacks that should be taken into account. Precipitation is a viable approach only when
analyte concentrations are quite high and the detection limits allow appropriate quantification
because samples are diluted by a factor of 3 or more from the addition of organic solvent.
Evaporating the supernatant with nitrogen and heat allows for concentration of the analyte,
although this process adds a transfer step (with potential transfer loss of analyte) and time to the
dry-down and reconstitution steps. An evaporation step may decrease recovery of an analyte if it
is volatile and sensitive to heat.

Acid Precipitation

The pH of the solution is adjusted in this technique. Since the structure of a protein is sensitive to
small shifts in medium pH, each protein is assigned an isoelectronic point (pI) value. When acid
is added to a solution, the pH drops and the amino groups on the protein become more
electronegative, giving the protein a positive charge. When a protein is dissolved in water, its
surrounding hydration sphere is damaged, causing a structural imbalance that ultimately results
in precipitation.

TCA (Trichloroacetic Acid) is the acid of choice for this process since it is extremely reactive
and may be utilized in very low concentrations. It's crucial to understand that following this
procedure, the protein will continue to be inactive.

Salting out precipitation

The hydrogen bonds in proteins are broken when salt ions are present in the solution because the
amount of water available to the proteins is reduced. Proteins aggregate and precipitate because
their association with one another is stronger than their contact with the available water
molecules.
Zinc sulfate and ammonium sulfate are two such compounds. This is the gold standard for first
protein fractionation based on solubility. Proteins can be re-dissolved after a salting precipitation
experiment without losing their biological properties.

Precipitation of Alcohol

It is also possible to employ organic solvents. Proteins in a solution lose some of their hydration
when alcohol is added; this causes them to aggregate and precipitate.

Experiments should be conducted at low temperatures to protect their biological functions.

Verify the protein concentration and solution pH while you're at it.

Ethanol, methanol, and acetone are the three most common solvents.

SOLID/DRY PHASE CHEMISTRY

Wet chemistry analysis has its roots in dry chemistry. In dry chemistry, a liquid test sample is
combined with a dry reagent designed for another purpose. In order to conduct chemical
analysis, the moisture present in the test sample is used as a solvent to set off a targeted chemical
reaction. The analytical technique known as "dry chemistry" is based on the use of enzymes.
Sometimes referred to as "solid phase chemistry," dry chemistry is a subfield of analytical
chemistry. In dry chemistry, measurements are taken with reflectance photometry or the
differential electrode technique.

Dry chemistry has many benefits, including: 1) quick turnaround time for results (as little as
three to four minutes), 2) simple operation (no need for daily calibration), 3) no additional
reagents or solution preparation required, 4) no pretreatment of specimen is required due to the
multilayer membrane's selective filtration function, and 5) low specimen and water consumption.
The outcomes of routine tests, etc., can be evaluated methodologically using this tool.

Dry chemistry has applications beyond just qualitative analysis. It has also advanced as a
quantitative and semi-quantitative technique for analysis. Clinical testing has seen a rise in the
use of dry chemistry techniques. The quality of urine testing has come a long way because to the
development of dry chemical techniques. These days, many reagents can measure several
different things at once. Protein, sugar, bilirubin, urobilinogen, ketone bodies, specific gravity,
nitrite, bacteriuria, and occult blood can all be detected in a urine analysis. Dry chemistry
methods are not limited to analyzing urinalysis specimens; they can also be applied to testing
whole blood, serum, and plasma.

Paulingmann, a business based out of Germany, developed the Reflomat device for measuring
blood glucose in 1974. In the 1980s, medical testing often involved the use of dry automated
biochemical analyzers.

Dry chemistry analyzers have a number of benefits.

1) Timely Patients spend less time waiting when reports are processed quickly. This enhances
the quality of care by helping doctors identify and treat patients faster.

Two) Precision

Wet chemistry is commonly used as the standard of comparison for dry chemistry analyzers.
Results from the methodological comparison and recovery tests indicate that the dry chemistry
approach is accurate enough for most applications.

3. Accuracy

The dry chemistry method is more accurate than the wet chemistry approach since it requires
fewer steps to complete an experiment.

Whole blood, plasma, or serum can be analyzed with just 100ul on the Seamaty fully automated
dry chemistry analyzer. Because of its user-friendliness, it can be run by virtually anyone with
minimal training.

DETERMINATION OF ENZYMES

Measuring the activity or mass in serum or plasma of enzymes that are primarily intracellular
and that are physiologically present in low quantities only is of interest to the clinical laboratory
scientist. Disease-related shifts in the levels of these enzymes can provide insight into the sites
and causes of tissue damage.
MEASUREMENT OF REACTION RATE

When an enzyme is present, the reaction it catalyses proceeds at a rate proportional to the
enzyme's activity. Therefore, the concentration of enzyme in a sample like serum can be
calculated by measuring the rate of reaction under standard laboratory conditions. The amount of
change produced in a given time interval is the kinetic measurement used to determine the
response rate. Reaction times can be measured in either a discrete period of time or continuously.
The fixed-time method involves stopping the reaction after a predetermined amount of time and
then measuring the amount of change caused by the enzyme. In the continuous-monitoring
technique, the reaction's development is constantly tracked. There are benefits and drawbacks to
both of these approaches. They can be understood by thinking about the time-dependent rate of
an enzyme reaction. By measuring the declining concentration of the substrate or the rising
concentration of the products, one can track the pace at which the substrate is being converted
into products in the presence of an enzyme. Because measuring a decrease in concentration from
an initial high concentration is less reliable analytically than measuring the formation of a
product, measuring the formation of a product is preferred. There is no rate of reaction when the
enzyme and substrate are together. After afterwards, the rate usually increases sharply to a
maximum, where it stays stable for some time. During the steady-state phase of a reaction, the
rate is independent of the substrate concentration and depends only on the enzyme concentration.
Since the rate of the reaction is independent of the substrate concentration, it is considered to
have zero-order kinetics. First-order dependence on substrate concentration sets in as the reaction
rate decreases with increasing substrate consumption. The accumulation of potentially inhibitory
products, the increasing significance of the reverse reaction, and even enzyme denaturation all
contribute to the slowing of the reaction rate. Under first-order conditions, it is conceivable to
compare the reaction rates produced by different amounts of an enzyme, but it is obviously easier
to standardise such comparisons when the concentration of the enzyme is the sole variable
influencing the reaction rate. Therefore, enzyme tests are typically performed at substrate
concentrations that initially saturate the enzyme. The rate of reaction in the zero-order phase
can be calculated by monitoring the amount of product generated over a predetermined
incubation time.
Isoenzymes and isoforms have been measured using a variety of analytical approaches.
Electrophoresis, chromatography, chemical inactivation, and differences in catalytic
characteristics are among them. However, immunochemical principles underpin the
approaches that are currently in widespread use.

DETREMINATION OF TOTAL PROTEIN

Protein concentrations in plasma and serum are typically 6.5 to 8.5 g/dL and 4% lower,
respectively. Because the protein content of biologic fluids varies, the carbohydrate composition,
charge, and physical features of proteins in the combination also vary, making total protein
determination in biologic fluids more difficult than analysis of a specific protein. Many protein
analysis techniques have issues when used on samples with a wide range of protein
concentrations or types. Because of the buildup of peptides and tiny proteins in renal failure, the
interaction of most methods other than the biuret method with these substances has not been
adequately investigated. The total protein concentration of biological fluids can be determined in
a number of ways. There are several discussed here.

The Kjeldahl Methods

Despite its historical significance and occasional use as a reference method, this procedure is
rarely employed in clinical laboratories. When heated with sulfuric acid in the presence of a
catalyst, nitrogen in proteins is transformed to ammonium ion. It is possible to determine the
concentration of ammonium ion using alkalinization, distillation, acid titration, or Nessler's
reagent. The nitrogen content of protein is predicted to be 16%. If the amino acid composition is
out of the ordinary, or if the nitrogen level is lower than 16%, the estimated protein content will
be off. Protein precipitation may be necessary when measuring nonprotein nitrogen sources like
urea and amino acids. Even though it was one of the first methods employed, the Kjeldahl
method for measuring total protein is too time-consuming and complex to be utilised on a regular
basis. The biuret method's reference materials have been valued using this technique.

Biuret Method

Multivalent complexes of Cu2+ ions with peptide bonds are formed under highly basic
circumstances. The biuret reaction describes the transition from a blue to a violet color caused by
the binding of molecules that modify the absorption spectrum of Cu2+ ions to shorter
wavelengths. The spectrophotometric measurement of absorbance at 540 nm that can be
attributed to protein. Many chemicals that bind Cu2+ ions alter the 540 nm absorbance spectrum
because they form chelates with five- or six-member rings that contain amino, amide, or
hydroxyl groups. Serine, asparagine, ethanolamine, and the TRIS buffer are all examples of such
chemicals. Proteins cause a larger change in the spectrum than tiny molecules. Absorbance
changes occur with some amino acids, with amino acid amides, and with dipeptides, despite the
fact that they were not previously thought to react with the biuret method. At first, it was
believed that the biuret action would react similarly to all proteins and peptides longer than two
amino acids; however, it was later shown that peptides containing proline reacted less strongly.
Assuming the biuret reaction is carried out in the conventional endpoint fashion, it is likely that
proteins of varying compositions have similar reactivities. Although the Biuret rate assays are
available, they should be treated independently from the endpoint assays. The rate at which a
protein unfolds and exposes more binding sites for Cu2+ ions under very alkaline conditions
affects the rate at which Cu2+ ions complex with small molecules and accessible sites in
proteins.

DIRECT OPTIC METHOD

It is usual practice to use UV absorbance between 200 and 225 nm and 270 and 290 nm to track
peptide and protein chromatographic separations and calculate protein concentrations. The
concentrations of tryptophan and tyrosine in a protein are the primary determinants of its
absorbance at 280 nm. For proteins that have been purified and whose absorptivity is known, this
method is ideal. Absorbance of low molecular weight substances such free amino acids, uric
acid, and bilirubin can affect the accuracy and specificity of a test for complex mixtures.
Absorption of UV light between 200 and 225 nm is primarily due to peptide bonds. Proteins
have a 10- to 30-fold higher absorption at these wavelengths than they do at 280 nm. Absorbance
at wavelengths below 220 nm is also present in many low molecular weight molecules like urea.
Removal of low molecular weight molecules before performing absorbance measurements may
be necessary for accurate protein measurement with this approach. Other optical techniques, such
as infrared or Raman examination of samples, provide total protein determination strategies
predicated on intricate spectral analysis.
Dye-Binding Techniques.

Dye-binding techniques rely on the changes in dye absorbance spectra caused by protein binding.
Dye binding to proteins can be highly variable. Protein mixtures present unique challenges for
defining reliable calibration standards. Albumin is a pure protein that can be used for calibration,
but it may not accurately represent the complex protein mixture found in serum and plasma.
Coomassie blue gives twice the reaction to albumin and transferrin at the same concentration, but
only 60% of the response to immunoglobulins. The absorbance of this dye is diminished at 465
nm and enhanced at 595 nm when it attaches to polypeptide chains in an acidic environment.
There are dyes that can detect even minute amounts of protein. For example, pyrogallol red is
one of the most popular dyes for analyzing low-protein fluids like urine and cerebrospinal fluid
(CSF).

Method of Lowry (Folin-Ciocalteu).

The Lowry method has a detection limit roughly one hundred times lower than the biuret
approach. The Folin-Ciocalteu reagent is added to a mixture of specimens and an alkaline copper
solution. Copper complexed with peptide, as well as tyrosine and tryptophan residues, can
catalyse the reduction of phosphotungstic acid and phosphomolybdic acid to tungsten blue and
molybdenum blue, respectively. Products are tested for their ability to absorb light between 650
and 750 nm. Proteins' reactivity changes according to how much tyrosine and tryptophan they
contain. Tryptophan, tyrosine, salicylates, chlorpromazine, tetracyclines, and certain
sulfonylureas are all low molecular weight substances that might cause interference. Removal of
low molecular weight components is necessary prior to measuring protein concentration in a
fluid with high quantities of phenolic compounds, such as urine.

Refractometry.

Refractometry is a fast method for determining protein concentrations in solution. Since salts,
glucose, and other low molecular weight substances have a greater proportionate effect on
refractive index at protein concentrations below 3.5 g/dL, accuracy falls at those levels. Rather
than calculating total protein, refractometry is typically employed in clinical laboratories to
evaluate the concentration of solutes in urine samples.
Light Scattering Method.

Proteins have been aggregated using reagents such as trichloroacetic acid, sulfosalicylic acid,
benzethonium chloride, and benzalkonium salts in alkaline conditions for turbidimetric and
nephelometric tests. The formation of a suspension of homogeneous, insoluble protein particles
that scatter incident light is central to precipitation methods for total protein measurement. In
precipitation techniques, albumin and globulins typically respond differently.

CREATININE DETERMINATION

The kidney function test serum creatinine is used in almost all clinical laboratories.
Measurements in serum and urine are often performed using modified versions of the same assay
in most labs. Creatinine levels in the blood and other bodily fluids can be measured using either
chemical or enzymatic methods.

CREATININE: Jaffe Reaction

The reaction with alkaline picrate is the cornerstone of most chemical techniques for detecting
creatinine. An orange-red complex is produced when creatinine interacts with picrate ion in an
alkaline media, a reaction first described by Jaffe in 1886. There is a lot written about this topic,
but the details of the reaction and the structure of the result are still murky. Creatinine is not a
requirement for the Jaffe reaction. Protein, glucose, ascorbic acid, ketone bodies, pyruvate,
guanidine, haemoglobin F, blood-substitute products, streptomycin, and cephalosporins are only
some of the many chemicals that have been documented to produce a Jaffe-like chromogen. The
level of disruption caused by these chemicals is sensitive to the specifics of the reaction
conditions. Therefore, it is essential to be aware that there are various Jaffe assays with varying
performance characteristics and that the versions of the Jaffe reaction employed by different
manufacturers will respond to interferences in different ways. Despite being technique
dependant, the effects of ketones and ketoacids are likely the most clinically significant
interferences.

ENZYMATIC METHOD
The enzymatic determination of creatinine has been studied with enzymes from a variety of
metabolic pathways. All of the procedures follow a series of stages that culminate in a
photometric reading. The following sections detail the three main methods that are employed.

Creatininase.

The conversion of creatinine to creatine is catalysed by the enzyme creatininase (EC 3.5.2.10;
creatinine amidohydrolase). The presence of creatine is subsequently determined using a
sequence of enzyme-mediated reactions (creatine kinase, pyruvate kinase, and lactate
dehydrogenase; the drop in absorbance at 340 nm is measured). Removing endogenous creatine
and pyruvate requires a preincubation process, which can be triggered by adding creatininase. It
takes 30 minutes of incubation for the reaction to achieve equilibrium due to the slow kinetics.
This drawback can be remedied by adopting a kinetic strategy, albeit at the expense of even
lower sensitivity.

Dry Chemistry Systems: Have not gained much traction because of the high price of reagents
and the low sensitivity and precision they provide. Methods for measuring creatinine via
enzyme-mediated reactions using multilayer dry reagents have been described. The ammonia
produced by the reaction of creatinine deaminase with bromophenol blue resulted in a rise in
absorbance at 600 nm in an early "two-slide" method. To correct for blanks, we measured
endogenous ammonia using a second multilayer film that lacked the enzyme. The creatininase-
creatinase reaction sequence was used in a later, more accurate single-slide approach. Lidocaine
metabolites may cause problems with this technique. A point-of-care testing device based on the
previously published creatinine deaminase system has been developed and deployed.

Reflectance is used universally as a measurement of the film's output colour. The reaction with
3,5-dinitrobenzoic acid has been characterised as the basis for a dry chemistry system that
employs a nonenzymatic method.

Other Methods
Isotope dilution (ID) GCMS and liquid chromatography-mass spectrometry (LCMS) are both
listed as reference techniques for measuring creatinine in serum and urine by the JCTLM. Due to
its high specificity and low imprecision, GCMS quickly gained acceptance as the gold standard
for determining the actual quantity of creatinine in serum in the 1980s. Due to its polarity,
creatinine must be derivatized in these processes before GC analysis. Because creatine is
derivatized into the same chemical species as creatinine, a cation-exchange purification step is
required prior to GC analysis. Direct creatinine quantification using HPLC and IDMS has been
reported. Because only a simple protein precipitation without derivatization is required, and
equal performance may be obtained from GC and LC procedures, this technique is convenient
and expedient for analysis.

UREA MEASUREMENT

Urea in body fluids has been quantified using two main approaches: chemical methods based on
direct interactions with urea, and enzymatic methods using urea as the enzyme substrate.
Chemical procedures were originally commonplace, but have since been mostly replaced by
enzymatic ones. Taylor and Vadgama provide a more in-depth explanation of direct chemical
urea measurement.

ENZYMATIC METHOD

Enzymatic methods relying on the generation of ammonium ion by the preparatory hydrolysis of
urea with urease (urea amidohydrolase,EC 3.5.1.5; primary source jack bean meal) are used for
the vast majority of urea tests in clinical laboratories. End-point, kinetic, conductimetric, and dry
chemistry systems are all examples of applications for this method. Berthelot reaction and
glutamate dehydrogenase (l-glutamate:NAD(P) oxido-reductase (deaminating), EC 1.4.1.3)
enzymatic test are two spectrophotometric methods for ammonium quantification. This latter
strategy was proposed as a reference method in 1980 and has since been ported to several
analytical frameworks. In most serum tests, urease is included in the reaction system formulation
to facilitate the initiation of the reaction upon the addition of a sample containing urea. The
glutamate dehydrogenase process is followed by measuring the decrease in absorbance at 340
nm. Ammonia formed from urea by urease combines with glutamate and adenosine triphosphate
in the presence of glutamine synthetase (EC 6.3.1.2) to form glutamine, another example of a
coupled-enzyme test method for urea. In the third and fourth phases, pyruvate kinase (EC
2.7.1.40) and pyruvate oxidase (EC 1.2.3.3) quantify the adenosine diphosphate created in the
second enzymatic reaction, resulting in the production of peroxide. Peroxidase (EC1.11.1.7)
catalyses the last step, in which peroxide combines with phenol and 4-aminophenazone to
produce a quinone-monoamine dye that can be measured spectrophotometrically.

DRY CHEMISTRY

Dry chemistry systems employing the urease method and several detection techniques have been
described for the determination of urea. Sample and a urease-containing reagent are incubated in
a conductivity cell, and the rate of change in the conductivity is monitored as the urea is
transformed to an ionic species, allowing for a conductimetric measurement of urea.
Potentiometric methods use an ammonium ion-selective electrode and a membrane-bound form
of urease. A similar method has been implemented to track how well dialysis is working in real
time. Morishita and coworkers describe a technique that includes leucine dehydrogenase (EC
1.4.1.9) in addition to urease for measuring urea, which prevents interference from endogenous
ammonium. Because blood is rarely analyzed for nitrogen (rather, the proper measurand is urea
in serum), the term blood urea nitrogen (BUN) continues to be used in some countries when
ordering serum urea tests with findings reported as mg/dL of BUN. Although the SI system
suggests using urea, expressed in mmol/L, the custom of reporting and presenting urea assay
findings in units of urea nitrogen appears to be deeply rooted in the United States. The mass
concentration of urea in a sample is 60/28 (2.14 times the urea nitrogen concentration) due to the
fact that urea comprises two nitrogen atoms (each Mr 14) out of a total Mr of 60. Due to the
change in the denominator from dL to L, the molar unit conversion is as follows: 1 mg/dL
BUN=10x2.14/60=0.357mmol/Lurea.
MEASUREMENT OF HORMONE

Due to their low concentration in the blood, hormones are notoriously difficult to measure
without the use of a highly sensitive assay, such as a competitive immunoassay. The first
technique, radioimmunoassay, used an antibody specific to the hormone along with a
radiolabeled version of the hormone to detect its presence. There is competition between labelled
and unlabeled hormones for binding sites on antibodies. The concentration of hormone in patient
samples is determined by comparison to a standard curve containing known levels of hormone.
The introduction of radioactive tags enables the detection of hormone concentrations in the pico-
(10-12) or nanomolar (10-9) range, which are commonly found in circulation. Recent years have
seen the development and widespread adoption of nonradioactive tags (e.g. chemiluminescence),
"sandwich-type" assays, and ELISA methods for the detection of hormones. Hormone
measurement devices used at the point-of-care or in the clinic are also widely used in veterinary
medicine. Normal amounts of a given hormone might vary greatly between species, creating
special hurdles for accurate assessment in veterinary animals. For instance, canine and feline
normal total T4 concentrations are around one-fourth that of human normal values. Cross-
reactivity is a major issue since different species produce hormones that differ in amino acid
content and other structural features (such as glycosylation patterns). Because of this, it's
possible that anti-hormone antibodies developed in one species won't work on another. Despite
the structural similarity between steroid hormones of different species (canine and human
cortisol, for example), chemicals present in the serum of a given species can occasionally
interfere in a test, leading to erroneous results. Overall, it's crucial for a laboratory offering
hormone measurement to show that the assay is valid in the species in question and to have
established normal ranges for that hormone.

LIPIDS MEASUREMENTS

Physical procedures, such as ultracentrifugation, electrophoresis, column chromatography, and

others, have also been used to separate and quantify plasma lipids and lipoproteins and
lipoprotein subfractions. Enzymatic, immunochemical, and chemical precipitation reagents have
also been utilised. In addition, there are systematic biases amongst methods that profess to
quantify the same component since, while different methods of lipoprotein separation may yield
similar lipoprotein fractions, they usually do not produce identical fractions. Here, we'll talk
mostly about the standard reference methods and protocols for measuring lipids and lipoproteins
in a clinical setting.

Reference Methods

For the most prevalent analytes, like cholesterol, triglycerides, LDL, and HDL cholesterol,
reference methods have been created as the "gold standards" or accuracy targets. Cholesterol's
gold standard method has been thoroughly verified and accredited by the Joint Committee for
Traceability in Laboratory Medicine. Although the other approaches lack official credentials,
they have been widely accepted nonetheless.

Routine Methods

Complex, time-consuming, and usually manually-intensive, reference procedures necessitate a

high level of skill for reliable operation. This has led to the development of approaches that are
both easier to implement and more suitable for regular clinical use.

Cholesterol Determination

Cholesterol assessment with enzymes is a straightforward process that yields precise results after
proper calibration. The enzymes and other components needed to measure cholesterol levels are
typically combined into a single photometric reagent that is sold commercially. Colour
development is induced by incubating the reagent with a small volume of serum or plasma under
strict laboratory conditions, and absorbance is read at a wavelength of roughly 500 nm.
Cholesteryl ester hydrolase, which is normally produced by bacteria, is used to produce the
reagents. Other coloured chemicals or those that compete with the oxidation reaction or react
with peroxide, such as bilirubin, ascorbic acid, and haemoglobin, may cause interference with
these procedures. Up to roughly 1000 mg/dL (25.9 mmol/L), most assays are linear. The effects
of hemolysis can now be reduced with the help of enhanced reagents that include chemicals like
bilirubin oxidase and dual wavelength measurements; interference from bilirubin is typically not
an issue at concentrations below 5 mg/dL (85.5 umol/L).

Because -hydroxy sterols and plant sterols (such as sitosterol) might react with enzyme reagents,
they are not completely selective for cholesterol. However, because these interfering sterols are
typically present in relatively low concentrations in human blood or plasma.

Triglycerides,

This is not a big problem. Enzyme reagents can be used to analyze triglycerides in blood plasma
or serum. There are commercially available reagents that include all of the necessary enzymes,
cofactors, and buffers. Like cholesterol, these reagents are tailored to the specific calibrator
systems used by individual instruments. Multiple enzyme-based chemical processes have been
implemented. First, triglycerides are hydrolyzed into glycerol and fatty acids by lipase-catalyzed
hydrolysis in all approaches.Then, glycerokinase catalyses a phosphorylation of glycerol that
uses ATP.

High Density Lipoprotein (HDL).

Current recommendations for characterising the cardiovascular disease risk in patients emphasize
the need of measuring HDL and LDL, the two primary cholesterol-carrying lipoproteins. The
density range (1.063-1.21g/mL) obtained using ultracentrifugation has traditionally been utilized
as the gold standard against which the efficacy of alternative HDL techniques can be evaluated.
Since the density range of Lp(a) (1.04-1.08 g/mL) overlaps that of HDL, ultracentrifugation at
1.063 g/mL would result in an overestimation of the real HDL content in individuals with high
Lp(a) concentrations. So, like many other research methods, the CDC reference method (already
stated) uses precipitation to separate HDL. The newer direct or homogeneous tests became
accessible in the early 1990s, and they are currently widely used in ordinary laboratories.
Because they may be fully automated, the homogeneous tests offer a number of advantages.
However, specimens from patients with atypical lipoprotein distributions have revealed that
homogenous assays lack specificity (see below). Pretreatment precipitation methods will be
discussed first because they were the norm for many years before the advent of the more modern
homogenous tests.

Precipitation Methods.

Prior to the development of agents like polyanions in the presence of divalent cations, HDL C
was typically evaluated in supernatant solutions following the direct precipitation of apo B 100-
containing lipoproteins (VLDL, IDL, Lp[a], LDL, and, when present, chylomicrons) from
plasma or serum. Total cholesterol in healthy people is mostly made up of LDL and HDL, with
LDL making up about two thirds of total cholesterol and HDL making up about one third.
Low Density Lipoprotein

Typical LDLC methods quantify a "broad cut fraction," which includes not only the principal
LDL species (density range: 1.019–1.063 kg/L) but also IDL (density range: 1.006–1.019 kg/L)
and Lp(a). Therefore, the following formula is generally accepted as the standard for calculating
total cholesterol from a lipid-free fasting sample.

TC = VLDL +LDLC+HDL

Major studies that showed the association between LDL C concentration and risk for CHD used
both indirect and direct methods to assess LDL C.

Indirect Method.

To determine the LDL C concentration in a sample, indirect techniques first measure a panel of
lipid-related analytes. Methods such as the quantification and the Friedewald equation are
utilised. Equation of Friedewald. Cholesterol, triglyceride, and HDL C are measured in the most
common indirect approach, and LDL C is estimated from these using an empirical equation
published by Friedewald and colleagues.

DETERMINATION OF TRACE ELEMENT

Many elements occur in many matrices at concentrations too low to be detected by traditional
analytical methods. The evolution of analytical technology revealed their presence, leading to the
coinage of the phrase "trace elements." The International Union of Pure and Applied Chemistry
(IUPAC) defines a trace element as one with a concentration below 100 mg/kg on average. The
term "ultratrace elements" first originated in the second half of the twentieth century, coinciding
with the fast expansion of analytical techniques' detection capabilities. Despite the existence and
widespread use of the term, it has no precise definition. Elements with a mass fraction of 1 ppm
or less are considered ultratrace.

There are many areas of science, industry, and technology that would benefit greatly from an
understanding of trace and ultratrace elements. Some trace elements can drastically alter the
functionality of developed devices, and ultralow concentrations of elements may be just as
important as harmful quantities for organisms. For this reason, precise measurements of even
minute quantities of substances are crucial.

The proper management of contamination and verification of the accuracy of determination is

essential for the widespread use of extraordinarily sensitive instruments. Increases in analytical
sensitivity have led to a proliferation of new challenges, including widespread contamination.
Because of this, proper care must be taken when analysing trace elements with concentrations in
the parts per billion range and lower. Improper sampling, storage, sample preparation, and
analysis can all introduce error into trace and ultratrace elemental analysis. As a result, it's
crucial to always verify the precision of an analysis.

The ability to analyze trace and ultratrace elements has become more possible thanks to the wide
variety of analytical methods now in use. Atomic absorption spectrometry using flame
atomization is perhaps the most common method for detecting trace elements present in
the parts per million concentration range. As the concentration of ultratrace elements
declines to the parts-per-billion range or lower, the number of viable analytical methods
decreases. Potentiometry, voltammetry, atomic spectroscopy, X-ray, and nuclear techniques are
frequently used for the determination of trace elements. Some elements' oxidation states can be
determined using electrochemical analysis, and the measurement of free ions in solution
(potentiometry) or free ions along with ions bound in labile complexes (voltammetry) is also
possible. A sample's matrix can have an impact on the accuracy of atomic spectrometric
techniques, which are otherwise exceedingly sensitive and can be used to estimate the total
element content within a sample. Since X-ray and nuclear techniques operate on fundamentally
different principles than the rest of the analytical methods, they are utilised for comparison of
data and have a very low limit of detections and matrix insensitivity. That's why it's unlikely
you'll encounter the same kind of systematic prejudices while dealing with them. The advantages
and disadvantages of each method ought to be evaluated in terms of the number of analytes that
may be measured with the method, the frequency of interferences and difficulties, the detection
limits, the sample throughput, and the costs involved.

Extensive sample preparation and/or extraction is sometimes necessary for the determination of
trace elements and contaminants in complex matrices prior to instrumental analysis. Food,
environmental, clinical, and biological samples all need to have their concentration of important
and harmful components assessed. Inductively coupled plasma atomic emission spectrometry
(ICPAES), inductively coupled plasma mass spectrometry (ICPMS), electrothermal atomic
absorption spectrometry (ETAAS), and flame atomic absorption spectrometry (FAAS)
have all been used regularly for the identification of trace metals. Many samples (biological,
clinical, environmental, etc.) have a complex matrix that includes both substantial amounts of
inorganic chemicals (such as salts of Ca, K, Na, Mg, chlorides, phosphates, and sulphates) and a
large number of soluble solid substances. Sample introduction, nonspectral and spectral
interferences in measurements by atomic absorption spectroscopic methods, inductively coupled
plasma atomic emission, or mass spectrometry all provide challenges for the direct study of
various types of samples. In order to eliminate the presence of organic material, samples must be
mineralized prior to examination, or at the very least diluted.

ATOMIC ASORPTION SPECTROMETRY(AAS)

Due to the ease of use and low cost of the necessary equipment, FAAS has become one of the
most common methods for analysing trace metal ions. This method involves putting a sample
into a flame, at which point the atoms in the sample are broken apart. When electromagnetic
radiation in the ultraviolet and visible ranges is shone through a flame, some of it is absorbed by
the atoms. It is possible to apply the FAAS method for direct determination of trace elements in a
wide range of sample materials because the methodology for most elements is well established
However, the existing analytical instrumentation often lacks the sensitivity required for the
detection of natural materials and is plagued by matrix interferences. In order to reduce the
detection limits, improve the precision and accuracy of analytical results, and bring the analyte
concentration within the dynamic range of the detector, several methods have been developed for
preconcentration and separation of trace metals prior to instrumental determination. Trace
analysis of lead, cadmium, copper, cobalt, chromium, nickel, tin, gold, palladium, iron, and zinc
in a variety of research materials was commonly performed using preconcentration techniques
such as solvent extraction, ion exchange, adsorption, and coprecipitation .One of the most
effective strategies for both the preconcentration and separation of trace elements from the
sample matrix is the coprecipitation technique, which is excellent for the preconcentration of
trace metal ions. The use of Ni(II)--benzoin oxime as a coprecipitation agent can be successfully
utilised without overextending the technique for the determination of Cr(III), Cu(II), Fe(III), Pb
(II), Pd(II), and Zn(II) in food samples .To prepare samples, liquid-liquid extraction is commonly
employed to move analyte from the aqueous sample to a solvent that is immiscible with water.
Cloud point extraction (CPE), which is similar to liquid-liquid extraction in that it involves
moving analyte from an aqueous sample to a water-immiscible solvent, is commonly employed
for sample preparation in conjunction with the AAS method. CPE relies on the fact that
surfactants can produce micelles, which, under the right conditions (temperature and
concentration), will split into a small, surfactant-rich phase and a much larger, water-based
phase. Hydrophobic complexes of metallic elements present in such media are captured in the
hydrophobic micellar core and removed in the surfactant-rich phase, which is channelled to an
AAS detector. Applications of CPE linked with FAAS exist, but the tiny volume of the
surfactant-rich phase formed following the CPE approach appears to be perfect for coupling with
electrothermal AAS.

However, the aforementioned procedures are labor-intensive, necessitating the use of a variety of
chemicals and sophisticated apparatus. Single-drop microextraction, hollow fibre liquid-phase
microextration, and dispersive liquid-liquid microextraction (DLLM) are three miniature liquid
extraction techniques that drastically reduce the extractant phase volume to separate and
preconcentrate organic and inorganic contaminants in a single operation. Due to the need for a
sample volume between 2 and 4 mL for FAAS analysis, a microsample injection system (MIS)
may be used in the event of small quantities acquired after preconcentrations procedures .

A wide variety of sorbents, chelating agents, and eluents can be used in an online separation and
preconcentration based on the adsorption of an analyte on an appropriate material representing
the solid phase (SP), and then the solution is directed to the FAAS detector via an elution step.
Sorbents can be used to keep the analytes in their complexed or ionic forms, or they can be
functionalized with certain ligands. Minicolumns containing stable material are positioned
immediately behind the injection valve or its sampling loop in an online separation or
preconcentration system. An SP extraction (SPE) has often been linked with FAAS because of
its high enrichment factor, high recovery, low cost, minimal consumption of organic solvents,
and the possibility to combine with diverse detection techniques. When doing SPE, the analytical
parameters selectivity, affinity, and capacity are all affected by the sorbent used. Sorbents for
online preconcentration include modified silica gel, modified chitosan resins, chelating resins,
magnetic nanoparticles, carbon materials, cellulose, egg shell membrane, and a number of other
materials.

Carbon nanotubes (CNTs) have garnered a lot of interest because of their unusual thermal and
mechanical stability, high surface-to-volume ratio, and ease of derivatization methods. Carbon
nanotubes (CNTs) range in size from a few nanometers in diameter to tens of nanometers in
length. They can be used as sorbents because their surface areas are between 150 and 1,500 m 2/g.
CNTs can have their selectivity enhanced by being functionalized with a variety of organic
compounds. CNTs can be treated with a targeted ligand to boost sorbent performance by
enhancing sorbent capacity and selectivity. Because of their ability to perform three distinct yet
interdependent tasks—ion exchange, chelate formation, and physical adsorption—chelating
resins are far more selective than solvent extraction and ion exchange. Oxygen, nitrogen, and
sulphur are examples of functional group atoms that can form chelate rings. Different factors,
such as the chemical activity of the complexing group, the nature and sort of the metal ions, the
pH of the solution, the ionic strength, or the polymetric matrix, might affect the selectivity and
sorption capabilities of these resins .

Carbon dots (CDs) have proven to be a selective and sensitive method for separating and
determining Cr in a wide variety of samples, among other applications for carbon materials. CDs
with added functional groups can be used in SPE as a separation and preconcentration material in
either online or offline modes by attracting analytes through electrostatic interaction, anion
exchange, chelate interaction, or their physical structure. Cr(VI) was analysed using a novel
water-soluble CDs encapsulated with branching polyethyleneimine polymer, extracted using a
dispersed particle extraction coupled with a slurry sampling approach, and then detected by
flame atomic absorption spectrometry. CDs treated with cationic surfactant promoted small
droplets production during the aspiration and nebulization processes, acting as a selective sorbent
for separation and preconcentration of Cr(VI) and increasing the sensitivity of its determination .

Improving the sample introduction efficiency of the FAAS approach for elemental analysis by
modifying the instruments and using thermospray flame-furnace (TS-FF) AAS is an intriguing
solution. If the TS-FF is a nickel tube, then the sample solution is nebulized using a ceramic
capillary and delivered to the standard burner head of an FAAS instrument. The TS-FF allows
for significantly more sample time in the flame than conventional FAAS systems and introduces
a complete sample to the atomizer. Thus, it's possible that measurement sensitivity could
improve by an order of magnitude. Samples are analysed using a chemical vapour generation
(CVG) system for the identification of elements generating hydrides or volatiles species (As, Bi,
Ge, Pb, Se, Sb, Sn, Te, Hg, Cd, Co, Cu, Ag, Au). Directly transferring volatile substances to an
atomizer reduces the number of stages beyond atomization, increasing sensitivity. In contrast to
ETAAS, the online generation systems of volatile species are not incompatible with the FAAS
method. Preconcentrating analytes is simple when you catch them right on the flame atomizer.
Even though [26] there are methods of linking CVG for both FAAS and ETAAS, it is not yet
widely used.

ETAAS is distinct from FAAS since it employs atomization temperatures of up to 3,000 K. It is

common practise to utilise FAAS for determining trace amounts of elements (e.g., Al, Ca, Co,
Cr, As, Cd, Cu, Fe, Mn, Ni, Pb, Zn) without first preconcentrating the analytes. Typically, just
one or two factors are considered while taking a measurement. Although only little amounts of
solid and liquid samples can be analysed, there are ways to do so. Background absorbance, the
matrix influence on atomic absorbance values, and changes in chemical form are just some of the
interferences that might arise while using this method, just as they can when using FAAS.
Preliminary mineralization of a sample, separation of elements to be determined from interfering
components, chemical modifiers, Zeeman effect background correction, and devices with
separated zone of evaporation and atomization, such as a graphite "filter furnace" (FF)
atomizer and an L'vov platform, are all methods used to get rid of or significantly reduce
these interferences.