CLINICAL SIGNIFICANCE

of
Proteins in Blood and urine

Lac.3

By
Dr. Muna M. Yaseen
 Objective

1. Type of proteins in blood

2. Clinical Diagnostic & Utility
of Proteins Measurements in blood
3. Causes of Proteinuria
• Proteins are Polypeptide group of nutrients in human body. All enzymes, receptors,
membrane channels such as those of Na-K, Ca channels, coagulation factors and peptide
hormones
(GH, prolactin,... ),..., etc. are proteins in nature.
• All proteins are synthesized in the liver, with exception of complement systems
( C1-C9 these are components of immune system synthesized by liver and macrophages),
and Immunoglobulin's (Igs) (by plasma cells of immune system).
• Proteins may be linear structural (such as collagen component of connective tissue) or
globular functional such as enzymes & peptide hormones.
Amounts of proteins in blood depend on balance:

rate of synthesis ↔ (rate of catabolism + rate of clearance).

However, protein distribution between the Intravascular (IV) and Extra vascular
compartments is also important and therefore blood protein concentrations
are affected by dehydration & over hydration.
Proteins in blood involved two types:
Albumin & total Globulin.
Albumin is the major single protein accounts to 60 % of total serum protein,
while globulin is consisted of 4-5 fractions; α 1, α 2, β 1, β 2, and γ globulins.
These Proteins components are separated by electrophoresis technique in which
serum is introduced to filter paper in a media of PH 8.6 to make protein which are polar
substances negatively charged. Then electrical current is passed into media and the
serum proteins are separated according to their MW and charge intensity into five–six fractions
or bands: albumin, α1- globulin, α2-globulin, β-globulin (may be β1 & β2), and γ globulin.

Total Serum Protein=S. albumin + total serum globulin.

Hyperproteinemia
are rare and are of no clinical significance value and may obtained from
prolonged vein stasis during blood collection, posture (due to fluid redistribution)
and from excessive dehydration.
Hypoalbuminemia:
It is clinically an important condition because albumin is one of the major
components of osmotic colloid pressure of blood vessels and involved in normal
fluid distribution between the Intravascular and Extra vascular compartments and
in maintenance of normal blood pressure.
Albumin is also the major transporter substance in the blood; transporting bilirubin, fatty
acids, steroid drugs, steroid & thyroid hormones,

Hypoalbuminemia
1. Chronic liver disease ; liver cirrhosis
2. Advanced kidney disease; Nephroteic syndrome &Chronic renal failure
3. Malnutrition (Kwashiorkor & Marasmus diseases) and Malabsorption like in Tropical
intestinal diseases; Celiac disease
4. Loss through Enteropathy
5. skin lesions; extensive burns.
Clinical consequences Hypoalbuminemia :

1. edema due to migration of fluid from IV to interstitum compartment

2. transporting and binding capacity defects; such as for fatty acids, bilirubin,
steroid Hs and drugs which may leads to toxicity with appropriate dose.

Analbuminemia is a rare disorder characterized by low blood albumin

(s. albumin 10 gram/l; but of no edema or other symptoms and signs).
 Globulin
This include 4-5 fractions (alpha 1, alpha 2, beta, and gamma fractions).

Increased in globulin may be due to increased in one or more of its fractions;α,β, and γ.

The α-1 and -2 include :

α1 -Antitrypsin,
haptoglobin,
ceruloplasmin,
C- reactive protein(CRP),
α2- macroglobulin.... etc.
α1-Antitrypsin(AAT)
•Protease inhibitor that binds to, and inactivates macrophage enzymes like
trypsin, limit their actions during infection, and protects the body.
•Deficiency is associated with
– Pulmonary emphysema.
– Liver Cirrhosis (direct hyperbilirubinemia; Jaundice is one of tests used in investigation of
prolonged neonatal )
•α1 -Fetoprotein(AFP)

– Principal fetal protein, used in screening for fetal abnormalities (neural tube
defects) and in adult for liver
carcinoma investigation.

α2 -Macroglobulin

• Largest non-immunoglobulin in blood ~750 KD

•Protease inhibitor
• Increased in Nephrotic syndrome (largest in size)
(α -globulin) Ceruloplasmin (Cp)

•Copper transporting protein

•Participates in plasma redox reactions like Fe+2 Fe+3.

•serum CP measurement is used in investigation of Wilson’s disease (Liver cirrhosis-Copper

storage disease) in

which serum Cp level is decreased due to genetic defect in incorporation of Cu with

apoceruloplasmin in the liver,

leading to precipitation of toxic Cu ion and damage of liver .

(α2) Haptoglobin

•Binds to, and preserves hemoglobin and its content of iron during hemolysis.

•Hemolytic diseases can deplete haptoglobin levels (α2) .

(β) Transferrin

•Iron transporting protein

•Transferrin is increased in iron deficiency anemia.

Apotransferrin + Fe+3=Transferrin
Β2 -Microglobulin BMG

•Smallest blood protein (MW=11.8K)

•BMG is filtered through the glomerulus, but is reabsorbed by

renal tubules.

- Urinary BMG levels are a sensitive measure of renal tubular function

γ -Region
•Includes Immunoglobulin's (IgG, IgM, IgA, IgD & IgE).
They are involved in specific immune system.
•CRP is the most sensitive indicator of Acute Phase Reaction (non
specific early immune defense system)
– Serum CRP (high sensitive -CRP) increased in Inflammation, trauma,
infection, etc.
Protein in urine

normally less than 100 mg/day of proteins appears in urine,

in kidney disease this value increased according to degree of kidney damage which

reflect mainly the glomerular damage.

Normally glomerulus is permeable to

proteins of MW ˂ 60 KD (D Dalton unit of

MW.
In kidney damage(mainly of glomerulus) excess amounts of proteins of large

MW˃60 KD will pass in the urine and may reach 5-50 gr/day.

Presence of low MW of proteins, like BMG in the urine

indicates the renal tubules damage as these tubules normally catabolize and

reabsorbed the low MW proteins. In tubules damage these proteins will

escape from the damaged tubules and appear in the urine(Low MW).
Amino Acids Metabolism

Lac.1
By
Dr. Muna M. Yaseen
CHOLESTEROL METABOLISM
By
Dr. Muna M. Yaseen
Oxidation of Fatty acids

by
Dr. Muna M. Yaseen
Enzymes
Luc. 1
By
Dr. Muna M. Yaseen
 Objective

• Definition
• Nomenclature
• Classification of enzymes
• Factors affecting enzyme activity.
• Application of enzyme inhibition.
• Isoenzymes.
• Enzyme in the Diagnosis of Pathology
• Definition
Enzyme : It is a protein, catalyst, synthesized in all living cells that regulate a biochemical
reaction without being changed.

• Characteristics
They are high catalytic rate.
They catalyze reaction without being changed.
They are very specific .
Enzyme distribution
Cofactor
Definition: A non-protein unit ,its presence is important in many enzymes.
Types:
1-Inorganic metals: Mn ,Zn ,Fe ,Cu.
2-Organic Complex ( Coenzyme ) .
Cofactors
Metal-activated enzymes:
- active in the presence of metal ions as K+, Mg+ or Ca++.
- Example: Kinase uses Mg++ , ATP.
Metalloenzyme:
- Firmly bound metal ion in the active site as Iron , copper , Zn & Co.
Examples:
1-Carbonic Anhydrase Zn.
2- Cytochrome oxidaseFe2+.
 COENZYMES

Many enzymes require for their action on substrate, specific ,heat stable ,low M. wt.

and organic substance called coenzymes

Enzyme which requires a coenzyme for its catalytic action is called apoenzyme and complete catalytic unit which

contain enzyme and its coenzyme is called holoenzyme.

Catalytic unit (Apoenzyme + Coenzyme ==== Holoenzyme)

Apoenzyme: inactive protein part.

Cofactor: Non protein part.

Holoenzyme: Active enzyme .

Coenzyme itself may covalently or non covalently bound to enzyme and when coenzyme is
covalent linked to its enzyme it will be then called PROSTHETIC GROUP.
Majority of enzyme in the body required coenzyme in their action

(Nomenclature)
Unsystematic nomenclature:
1- Enzyme is named by adding (ase) to the name of the substrate e.g. (Urease ).
2-Some other enzymes as (Trypsin ,pepsin ) are known by their historic names.
one enzyme has one name or many enzymes have the same name.
 Systematic Nomenclature

Adopted by (IUB) ; According to the type of reaction which is catalyzed.

It divided the enzymes into 6 classes.
Classification of enzymes
Class no I Oxidoredoctase
Class no II Transferase
Class no III Hydrolases
Class no IV Lyases
Class no V Isomerases cis and Trans
Class no VI Ligases
Class 1: Oxido-Reductase:
Catalyses Oxidation ,reduction reactions as : Dehydrogenase ,Oxidase ,Hydroxylase
,Peroxidase.
Usually they require coenzymes as : (NAD+,NADP+,FAD,FMN ).
Class 2: Transferase
Catalyze transfer of functional group between donor & acceptor molecule as methyl , formyl ,carboxyl
,nitrogenous, phosphorus & sulfur containing groups

Class 3: Hydrolases
Catalyze hydrolytic reaction by adding H2Ocleavage of bond between C & others as : C-O , C-N & C-S.
Class 4 : Lyases
Catalyze non-hydrolytic reaction
Examples: Decarboxylase .

Class 5 : Isomerase
Catalyze transfer of groups within a molecule (rearrange).
Class 6:Ligase

Catalyze bond formation coupled to ATP-hydrolysis joining 2 molecules.

Substrate
The molecule being utilized and/or modified by a particular enzyme at its active site

Enzyme Specificity
The most significant properties in the enzyme catalytic reaction is the ability of the enzyme in
catalyze one specific reaction and no other that is a characteristic of enzyme and when these
enzyme is absent the respective reaction will not occur and this behavior is called specificity of
enzyme and this behavior is usually appear in the following TWO properties:

I-optical specificity
II- Selective group
I-optical specificity

The enzyme has an absolute specificity in particular optical region of the substrate. Almost all human enzyme are

being specific for an optical part of substrate . ex: enzyme acting on CHO. Metabolism (sugar breakdown )are

usually specific for D-sugar not act on L-sugar or other enzyme acting on amino acid metabolism are usually

acting on L- amino acid (not D-amino acid ) with exception of D- amino acid oxidase in the kidney .

II- Selective group:

In this properties enzyme is usually affective on specific chemical group that is present in the structure of

substrate. ex: glycosidase, glycosidase catalyze hydrolysis of Glycosidic bond between sugar and alcohol are highly

specific for sugar portion not specific for alcohol.

Trypsin and pepsin act on peptide bond.
Some enzymes have a higher degree of specificity ex: amino peptidase act on amino group , carboxypeptidase act on carboxy
end of peptide bond .
Chymotrypsin will act on peptide bond on which carboxy terminal end of peptide bond is being contributed to an aromatic a.a.
Which may be phenyl alanine , tyrosine and tryptophan split of a.a one at a time from the carboxy or amino terminal end of
polypeptide chain respectively.
Tyrosine
Tyrosine
CH NH2COOH
CH NH2COOH
CH2
CH2
-HO
-HO
Enzyme velocity (V)
It is moles of product (P) appearing or substrate (S) disappearing per unit of time. (Mole / liter /sec.)
Enzyme units
International unit (IU): a mount of enzyme that converts one micromole (μmol) of substrate per minute at 25°C
under the optimal conditions of the measurement.
Katal: amount of enzyme that converts one mole of substrate to product/sec

(Active site)
Active site: is an important structural feature to recognize and to bind substrates.
It is very specific.
Catalytic Site:
The large size of the enzyme molecule in comparison with substrate size that a small part or limited number of
amino acids in the enzyme molecule is being responsible for the catalytic reaction these size is called
CATALYTIC SITE or ACTIVE SITE or ACTIVE CENTER of the enzyme.
There are two theory or mode or type to explain the interaction between the substrate and enzyme.

Type I
The lock & key model:
- Enzyme fits substrate as a lock & key .
- Its rigid type.
Type II Induced fit (Koshland model ):
the substrate induces conformational changes in the active site rearrangement of the A.A Enzyme fits substrate
exactly.
This type discovered by Koshland in which there is a source of flexibility in substrate – enzyme binding in which
certain physical changes take place in the enzyme that include arrangement of certain (a.a )s both to the substrate
binding site and at catalytic site.
These changes are called (conformational changes) and the site in which these changes take place are called
Allosteric site being important for the enzyme catalytic reaction. This type is more flexible than the lock and key
type and it has wide application in explaining the mechanism of the majority of enzyme – catalytic reaction.
Catalytic efficiency
Most enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed
reactions.

Factors affecting Enz. Activity

[Link] concentration.
[Link].
[Link]
[Link] concentration.
5. Inhibiters
6. Activators
Enzyme concentration:
The rate of the reaction is directly proportional to [ enzyme ]
Temperature
The rate of the reaction increases with the temperature increasing until reaching the ( Maximal velocity ) at the
(Optimal temperature) . Increasing of the temperature after the optimal temperature decreasing in the reaction
velocity.
The velocity decreases due to (enzyme denaturation )
Effect of PH
Each enzyme has its own (Optimal PH ).
Any change in the PH decreasing in the reaction velocity due to change in the ionization of the active site A.A.
This ionization inactivation of the active site decrease in enzyme activity.
Substrate concentration
Rate of the catalytic enzyme increases rapidly constant.
1-low [S] active sites are not saturated rapid reaction .
2-High [S] Saturated active sites slow reaction.

Substrate concentration
The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time and
is usually expressed as μmoles product formed per minute.
The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax)
is reached.
A. Low [S] B. 50% [S] or Km C. High, saturating [S]
The Michaela's- menten constant (Km).
The quantitative relationship between substrate concentration and Vmax. For different enzymes, it is defined as
that substrate conc. at which a given enzyme give one – half it maximum velocity . In many cases the Km is an
inverse measure of the affinity of the enzyme for its substrate : the lower the Km the higher the affinity .
Characteristics of Km
The Michaelis constant is characteristic of an enzyme and a particular substrate, and reflects the affinity of the enzyme for that
substrate.
Km does not vary with the concentration of enzyme. A numerically small (low) Km reflects a high affinity of the enzyme for
substrate because a low concentration of substrate is needed to half-saturate the enzyme.
Large Km:
A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of, substrate is needed
to half-saturate the enzyme.
The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations.
When [S] is much less than Km, the velocity of the reaction is proportional to the substrate concentration.
Uses of Km
Experimentally, Km is a useful parameter for characterizing the number and/or types of substrates that a particular enzyme will
utilize. It is also useful for comparing similar enzymes from different tissues or different organisms. Also, it is the Km of the rate-
limiting enzyme in many of the biochemical metabolic pathways that determines the amount of product and overall regulation of
a given pathway. Clinically, Km comparisons are useful for evaluating the effects mutations have on protein function for some
inherited genetic diseases
Introduction to Biochemistry