Biochemistry of Erythrocytes

Hemolytic anemia

a condition in which red blood cells are destroyed and removed from the bloodstream before their normal lifespan is over

normal RBC structure and function

Red blood cells are disc-shaped and look like doughnuts without holes in the center

• Carry O2 to the body

• Remove CO2 from the body

rbc made in and lifespan

Are made in the bone marrow, live for about 120 days in the bloodstream and then die

Causes of hemolytic anemia

Intrinsic

extrinsic

intrinsic

(result from deficit within RBC itself)

1. Hemoglobinopathies

-Sickle cell anemia-

Thalassemia

2. Membranopathies

-Hereditary Spherocytosis

-Hereditary Elliptocytosis

3. Enzymopathies

G6PD deficiency

pyruvate kinase deficiency

Extrinsic

1. Immune (lupus)

2. Non-immune (burns, toxins, drugs)

3. Malaria (parasites enter rbc and rupture it)

4. Blood transfusion (agglutination, not compatible)

Membranopathies

Mutations in genes expressing membrane proteins cause RBCs to change shape

Dysfunctional membrane proteins leading to different shape:

-interfere with the cell's ability to be flexible to travel from the arteries to the smaller capillaries

-make the red blood cells more prone to rupture

-this can cause hereditary spherocyctosis and elliptocytosis

Spectrin

a cytoskeletal protein that lines the intracellular side of the plasma membrane in eukaryotic cells

mutations in spectrin

can cause hereditary defects of the erythrocyte, including hereditary spherocytosis and hereditary elliptocytosis

Enzymopathies

Defective enzymes inside the RBC cause its premature breakdown

defective enzyme example

glucose-6-phosphate dehydrogenase or G6PD deficiency

G6PD deficiency

RBCs do not have mitochondria: this is the only source for NADPH

NADPH is used in the RBC to make reduced glutathione

Reduction of NADPH → Reduction of Reduced Glutathione → Increased Reactive Oxygen Species (ROS) oxidizes excess hemoglobin to make HEINZ bodies (aggregates of denatured, precipitated hemoglobin within erythrocytes) leading to premature destruction of RBCs

pyruvate kinase deficiency

less glycolysis, less ATP

Hemoglobin structure

Hemoglobin is a tetramer composed of 4 subunits (2 of each type)

Each subunit has a porphyrin ring which holds an iron molecule

-This is the binding site of oxygen

why Hb inside the RBC

iron atoms could cause oxidative damage if they were not inside the cell membrane of the RBC

HbA chains

a2b2

HbA affinity and proportion

(A) stands for Adult Dominates in adult blood Range 95-98%

HbA2 chains

a2da

HbA2 proportion

Range 2-3%

HbF chains

a2g2

HbF proportion

F stands for Fetus Range 0.8-2% Higher affinity for O2

Qualitative Hemoglobinopathies

The amino acid sequence is altered due to DNA mutation (Sickle Cell Anemia)

Quantitative Hemoglobinopathies

Production of one or more globin chains is reduced or absent (Thalassemia)

-Hereditary persistence of Fetal Hemoglobin (HPFH)

Complete or partial failure of γ globin to switch to β globin

Sickle Cell Anemia mutation

point (substitution) mutation in B gene

Defective B subunit : Glutamic Acid switched to Valine position

glutamic acid is hydrophillic and negatively charged while valine is hydrophobic.

Under low O2 conditions they stick together hydrophbic patches

Clinical Findings of Sickle Cell Anemia

 Clinical signs appear at 6 months of age

 Have all physical symptoms of anemia

 Growth and sexual maturation slower

 Crisis - very painful. Anything that deoxygenates blood acts as trigger (exercise, illness and airplane flights) - Sickle cells get stuck in capillaries

 Strokes

what drug is used in sickle-cell disease to decrease the number of attacks

Hydroxyurea taken orally

Hydroxyurea MOA

stimulates the body to produce more HbF in place of HbS that causes the pathology

- High levels of more HbF prevent more HbS to be produced

sickle cell and malaria

• Heterozygosity – 1 normal allele – 1 mutated allele

- Confers some resistance to malaria parasitization of red blood cells as RBCs are removed from circulation quickly not allowing parasite to replicate

Thalassemia

Production of a or B subunit is reduced leading to excess of the other subunit

a-subunit is encoded by

2 genes

2 a genes → 4 alleles

b-thalassemia

Production of B subunit is reduced

In β-Thalassemia minor:

Instead of B subunit the body produces delta Subunits forming HbA2- Good Marker for B-Thalassemia

β-Thalassemia major

increased HbF level, which is associated with stress erythropoiesis, which may result from the recruitment of erythroid progenitor cells that prematurely undergo terminal differentiation and are committed to producing γ-globin chains

Hemoglobin C

Hemoglobin C (Hb C) is one of the most common structural hemoglobin variants in the human population

• Individuals with hemoglobin C trait (Hb AC) are phenotypically normal, with no clinically evident limitations or symptoms

• Those with hemoglobin C disease (Hb CC) may have a mild degree of hemolytic anemia, splenomegaly and borderline anemia

-mutation in same position as B thalassemia where glutamic acid becomes lysine (hyrdophillic) in position 6

Hemoglobin Electrophoresis

Hemoglobin electrophoresis is a test that measures the different types of hemoglobin in the blood

• Gel electrophoresis is a method for separation and analysis of macromolecules, such as proteins based on their size and/or charge

It uses the principles of gel electrophoresis to separate the various types of hemoglobin

how is it done

• The sample (lysed red blood cells from the patient) is loaded into a well in the gel material

• The gel is placed in an electrophoresis tank filled with buffer solution

• The tank is then connected to a power supply in order for the molecules to start migrate from the cathode to the anode

Anelectric field is applied through a support medium (a gel) in a buffer at a fixed pH and the macromolecules migrate in that field, according to their size and/or net charge

• The electric field consists of a negative charge (cathode) at one end which pushes the molecules through the gel, and a positive charge (anode) at the other end that pulls the molecules through the gel

what buffer is used in thalassemia or hemoglobinopathy

When a thalassemia or hemoglobinopathy is suspected, a hemoglobin electrophoresis (alkaline or acid) is performed in order to identify different variants of hemoglobin • At the alkaline hemoglobin electrophoresis, the sample is loaded into a well of a cellulose acetate gel and is electrophoresed in a buffer at pH for the hemoglobin molecules to migrate from cathode to anode 8.4-8.6, in order • Separation of hemoglobins is based on the variable rates of migration of charged hemoglobin molecules in an electrical field

how is relative percent of each band is determined

The hemoglobin bands are visualized by the application of a dye, which also makes them measurable by densitometry. The patterns are scanned on a scanning densitometer, and the relative percent of each band is determined

Alkaline hemoglobin electrophoresis problem

s can be used to separate common variants such as HbA, HbF, HbS and HbC, but HbC and HbA2 are unresolved from each other

acid electrophoresis

Hemoglobins with identical mobility can be differentiated by acid haemoglobin electrophoresis. The acid hemoglobin electrophoresis, is similar to the alkaline hemoglobin electrophoresis with the exception that the sample (lysed red blood cells from the patient) is dispensed in a citrate gel and is electrophoresed in a buffer at pH 6.0-6.2

B-major can be normal newborn

HPLC

• It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material • Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column

what is separation based on

in column, if has higher affinity to whats in the column it will be released last and you record the volume and the time of what was released