Red blood cell - production and survival

Question 1

RBC production

Answer 1

2x10power11 a day

Erythropoiesis starts of in the bone marrow with a stem cell (hemocytoblast) and it goes through multiple stages under the influence of cytokines.

We start off with the stem cell, which becomes the proerythroblast. This differentiates into an early (basophilic) erythroblast.

At this stage, Phase 1 is occurring in the cell which is ribosome synthesis. The early erythroblast then differentiates into the late (polychromatophil) erythroblast and haemoglobin starts to accumulate in the cell as it is synthesised (phase 2).

The erythroblast then differentiates into a normoblast. Here, the nucleus gets extruded to make the reticulocyte, which has no nucleus (Hb is still being formed from left over mRNA).

This eventually forms the erythrocyte.

Question 2

Regulation of RBC production

Answer 2

Number of rbc is inversely related to ambient O2 pressure -> hence if o2 levels low in blood means there is hypoxia. the blood will pas through kidneys and sense the low o2 and begin to produce epo which will be released to circulation and stimulate rbc production in bone marrow

Key regulator of feedback loop is EPO

Question 3

what is epo?

Answer 3

Epo is a glycosylated polypeptide hormone.
90% produced by the kidneys as a result of low oxygen tension in the kidneys
It stimulates marrow production of rbc

Question 4

EPO and HIF

Answer 4

Studies of EPO regulation led to the discovery of hypoxia-inducible transcription factors (HIFs) which is produced either in liver or kidneys

HIF determines Epo gene expression in kidneys;
it also enhances expression of iron-absorbing genes

Iron absorbing proteins is ferroportin
While EPO is a growth factor for erythroid precursors, iron is their most important substrate, being essential for the synthesis of haemoglobin, the molecular oxygen carrier.

Question 5

What regulates Iron?

Answer 5

Hepcidin, which is predominately produced by the liver, serves as a master regulator of iron homeostasis.

Hepcidin inhibits intestinal iron reabsorption and iron release from macrophages, thereby reducing iron availability by binding to ferroportin and induces it internalisation and degradation.

The hormoneerythroferrone, produced by erythroblasts, acts on hepatocytes to suppress hepcidin production.

Question 6

HIF-α regulation

Answer 6

In the presence of sufficient oxygen, HIF-α is hydroxylated by PHD (prolyl hydroxylase domain). Hydroxylated HIF-α is recognized by vHL, which results in proteasomal degradation

In hypoxic conditions or PHD inhibition, HIF-α accumulates in the cytosol and forms a heterodimer with HIF-β, the hypoxia-insensitive unit. The heterodimer translocates to the nucleus and acts as a transcriptional factor that binds to HRE (hypoxia related element) and acts as a transciption factor to stimulate gene transcription for production of the agent needed for RBC production

Question 7

oxygen-sensing mechanism

Answer 7

The oxygen-dependency of prolyl-hydroxylase domain enzymes (PHDs), which target HIFα for proteasomal degradation, provides the basis of a widespread oxygen-sensing mechanism

Question 8

Iron soruces and absoprtion

Answer 8

Iron
Sources:
meat, eggs, vegetables, dairy foods

Absorption:
Normal Western diet provides 15mg daily.
5-10% absorbed (1mg) principally in duodenum and jejunum.
gastric secretion (HCl) and ascorbic acid help absorption.

Question 9

iron absorption regulation

Answer 9

Iron absorption is regulated by DMT-1 and ferroportin;

Controlled by: total body iron status; intracellular iron levels; Erythropoiesis

DMT-1 at the brush border of the enterocyte transporting iron into cells and ferroportin at the basal membrane then transport iron from enterocytes into circulation.

Question 10

Causes of Iron deficiency

Answer 10

Causes of Iron deficiency
• Decreased uptake due to inadequate intake in diet or malabsorption in GI tract
• Increased demand by our body e.g. in pregnancy or if having a growth spurt
• Increased loss of iron due to GI bleed or during menstruation

Iron deficiency leads to microcytic anaemia, where RBCs are smaller and have low levels of haemoglobin

Question 11

Vitamin B12 and Folic acid

Answer 11

Both essential for RBC maturation & DNA synthesis

Both needed for formation of thymidine triphosphate.
B12 is coenzyme for methionine synthase in methylation of homocysteine to methionine.

Deficiency leads to failure of nuclear maturation hence RBC may not be the only cell affected

Question 12

Causes of vitamin B12 deficiency

Answer 12

Causes of vitamin B12 deficiency include:
• Inadequate uptake e.g. vegans
• An absorption defect like blind loop syndrome or tropical sprue
• IF deficiency (B12 usually binds to intrinsic factor (IF) so if you have a deficiency you cannot transport B12 to the bone marrow) may be caused by pernicious anaemia, gastrectomy (as IF found in small intestine).

Question 13

where is folate abd b12 absorbed?

Answer 13

Folate is absorbed in proximal SI

B12 is absorbed in distal SI by the help of IF

Question 14

Causes of folate deficiency

Answer 14

Causes of folate deficiency include:
• Inadequate intake, a folate free diet causes deficiency within a few weeks
• Malabsorption e.g. coeliac disease
• Excess utilisation of folate e.g. pregnancy, haemolysis, cancer
• Drugs e.g. anticonvulsants may induce malabsorption of folate

Question 15

Effects of folate & B12 deficiencies

Answer 15

If we have folate and or B12 deficiency we end up with macrocytic (large RBC) anaemia.
We may also have reduced WBC and platelet count, megaloblastic (larger nuclei) change in bone marrow, megaloblastic anaemia, sore tongue, abnormal gut mucosa.

Question 16

only b12 defiency

Answer 16

In B12 deficiency you also may get demyelination of CNS.

Question 17

What else can affect RBC prod?

Answer 17

Renal dx - ineffective erythropoiesis (low epo)

Reduced BM erythroid cells
Aplastic anaemia (serious condition affecting the blood, where the bone marrow and stem cells do not produce enough blood cells)
Marrow infiltration by leukaemia or other malignancies

Question 18

Classification of Haemolytic Anaemia

Answer 18

Haemolytic Anaemia can be classified as:
Hereditary (congenital) or Acquired)
Intrinsic or extrinsic
Intravascular or extravascular

Question 19

RBC survival

Answer 19

RBC circulate for approx. 120 days without nuclei or cytoplasmic organelles
300 miles travelled through microcirculation
7.8 microns diameter
capillaries as small as 3.5 microns

Question 20

RBC metabolism and survival

Answer 20

Components needed for function and survival are present in matured rbc.

Function does not require the consumption of much energy.
rbc therefore are capable of limited metabolic activity.

Metabolic processes include :
Embden-Meyerhof pathway 
Hexose monophosphate shunt (or PPP)
Luebering Rapaport Shunt
Methaemoglobin reductase pathway

Question 21

RBC metabolic pathways

Answer 21

Two main enzymes are involved in red cell metabolism that allow the red cell to stay alive during its 120-day life span.

These are:

1. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
2. Pyruvate Kinase (PK)

These two enzymes support two main metabolic pathways; the pentose phosphate pathway and glycolytic pathway.

• G6PD helps to generate NADPH, NADPH is used for glutathione (GSSG) reduction.
• Reduced glutathione (GSH) maintains Hb solubility and membrane, it also protects red cell proteins from oxidative stress (i.e. it is an antioxidant).

Question 22

RBC metabolic pathways in and out overview

Answer 22

1.Glycolysis – energy- ATP
Na/K pump
3 Na+ out 2 K+ in
ATP -> ADP+Pi

2.HMS – reducing power - NADPH/GSH

3.Rapoport Luebering shunt
2,3 Bi-PhosphoGlycerate (2,3 BPG) – modulates O2 binding to Hb

Question 23

Glycolytic pathway

Answer 23

Generates energy in ATP;
to maintain red cell shape and deformability

regulates intracellular cation conc. via cation pumps (Na/K pump),

ADP is catalysed by pyruvate kinase to produce ATP and pyruvate

Question 24

Pyruvate Kinase (PK) deficiency

Answer 24

PK deficiency will lead to ATP depletion, ATP depleted cells lose a large amount of potassium and water, this makes them dehydrated and rigid
As a result
• The cation pumps fail to function
o This causes chronic non-spherocytic haemolytic anaemia -> shape of RBC changes
o Leads to excess haemolysis leads to jaundice and gallstones.

Question 25

Pentose Phosphate pathway

Answer 25

Oxidants cause RBC damage and cause Hb to precipitate out (producing Heinz bodies).
GSH acts as an antioxidant (prevents oxidative stress) by removing these oxidants to convert to GSSG.

1. Glucose is converted to glucose-6-phosphate (G6P) which in turn is converted to 6 phosphoglycerate (6PG), by the enzyme G6PD (glucose-6-phospate dehydrogenase).
2. This produces NADPH, which is used to reduce glutathione (GSSH) to reduced glutathione (GSH) which acts as the antioxidant as mentioned.

The pentose phosphate “shunt” pathway provides NADPH to maintain GSH

Question 26

G6PD deficiency

Answer 26

NADPH and GSH generation impaired

Acute haemolysis on exposure to oxidant stress: oxidative drugs, fava (broad) beans or infections

Hb precipitation – Heinz bodies

G6PD deficiency most common known enzymopathy, estimated to affect 400 million people worldwide.

Question 27

Luebering Rapoport Shunt

Answer 27

2,3 DPG binds to deoxyHb to stabilise at lower O2 affinity state
this makes it harder for O2 to bind to Hb; favours O2 release

Question 28

Methaemoglobin reductase pathway

Answer 28

it reduces cytochrome b5(cb5), which in turn reduces oxidized ferric ion of haemoglobin.

without this reaction, haem iron would be oxidized to methaemoglobin, which is not a functional oxygen transporter

Question 29

Red Cell membrane disorders

Answer 29

Hereditary spherocytosis
Loss of membrane integrity, the RBCs become spherical
Common hereditary haemolytic anaemia in N. Europ.
deficiency in proteins with vertical interactions between the membrane skeleton and the lipid bilayer: e.g. ankyrin def

Hereditary elliptocytosis
mutations in horizontal interactions e.g. spectrin, ankyrin; actin, protein 4.1 deficiency.

Question 30

Haemoglobinopathies

Globin disorders

Answer 30

The genes for globin chains occur in clusters on chromosomes:
•On chromosome 11 (epsilon, gamma, delta, beta globins)
•On chromosome 16 (zeta, alpha 1, alpha 2 globins)

Normal adult blood type has three types of Hb –
•HbA (made from α2β2 chains) – Forms about 90% of Hb
•HbF (α2γ2)
•HbA2 (α2δ2)

Mutations or deletions may lead to abnormal synthesis of globin chain as in the Sickle Cell Diseases.
•Or it could lead to a reduced rate of synthesis of normal alpha or beta globin chains as in the Thalassaemia.

Question 31

Sickle cell disease

Answer 31

Sickle Cell Disease is a group of Hb disorders with an inherited sickle β-globin gene. The most common is homozygous Sickle Cell Anaemia (Hb SS). But there are also heterozygote conditions (HbS/ βthal, HbSC, HbSD)

Point mutation in the β globin gene: e.g. glutamic acid → valine (HbS)
Insoluble Hb tetramer when deoxygenated → polymerisation
“Sickle” shaped cells

Question 32

Features of SCD

Answer 32

clinical 
Painful crises
Aplastic crises
Infections
Acute sickling:
Chest syndrome
Splenic sequestration
Stroke
Chronic sickling effects:
Renal failure
Avascular necrosis bone

lab
Anaemia
Hb often 65-85
Reticulocytosis
Increased NRBC
Raised bilirubin
Low creatinine

Question 33

globin disorders

Thalassaemia

Answer 33

Beta-thalassaemia

Loss of 1 B-chain causes mild microcytic anaemia (thalassaemia trait)
Loss of both (B0) causes thalassaemia major
Excess α-chains precipitate in erythroblasts causing haemolysis and ineffective erythropoiesis.

Alpha-thalassaemia
There can be loss of 1, 2, 3 or 4 alpha chains.

Question 34

diagnosis of thalassaemia

Answer 34

Asymptomatic
Microcytic hypochromic anaemia
Low Hb, MCV, MCH
Increased RBC

Question 35

Alpha-thalassaemia

Answer 35

Alpha Thal:
Loss of 1 or 2 causes mild microcytic anaemia
Loss of 3 causes moderate anaemia - Hb H disease
Loss of 4 causes death in utero (hydrops fetalis)

Question 36

Beta Thalassaemia Major

Answer 36

Transfusion dependent in 1st year of life

If not transfused:
Failure to thrive
Progressive hepatosplenomegaly
Bone marrow expansion – skeletal abnormalities
Death in 1st 5 years of life from anaemia

Side effects of transfusion:
Iron overload
Endocrinopathies
Heart failure
Liver cirrhosis

Question 37

Haemolysis/ RBC breakdown

Answer 37

Normal RBC breakdown is extravascular, in macrophages in the spleen which break it down into amino acids (recycled), Iron (recycled, binds to transferrin) and unconjugated bilirubin which travels to the liver, gets conjugated and then is then passed out in urine or faeces. (if too much breakdown, liver overwhelmed so can’t conjugate bilirubin hence increase conjugated bilirubin )

Abnormal breakdown of RBC is intravascular and leads to blood being found in urine.

Question 38

Low Hb level
LOw red count
Low MCV
High RDW

Answer 38

Microcytic, hypochromic anaemia