2. erythrocytes - hemoglobin

Erythrocytes/Red Blood Cells

Small anucleated cells, with no organelles

Erythrocytes shape

Biconcave disc shape

Large surface and thin membrane ➔ O2 diffusion

Flexible membrane ➔ passage through capillaries

Erythrocytes contain

mostly hemoglobin = O2-binding protein

Role in O2 delivery to tissues and CO2 removal

Life span of erythrocytes

120 days

erythrocyte life cycle 

1. erythrocytes form in red bone marrow

2. circulate in blood stream for 120 days

3. aged erythrocytes are phagocytized in liver + spleen

4. heme components of blood are recycled 

• heme → secreted in bile from the liver + iron stored by ferrin in the liver

5. membrane proteins + globin proteins → amino acids

• some are used to make new RBC 

Hematopoiesis

Development of all blood cell lineages from hematopoietic stem cells

primitive or definitive

Hematopoietic stem cells

self-renewing throughout the life of the host

100 billion cells produced per day!

Primitive Hematopoiesis

Transient process until embryonic w8

Cells originate from yolk sac

Definitive Hematopoiesis

During early fetal life

mostly in liver and spleen

In late fetus, the main site becomes the bone marrow

Erythropoiesis

RBC production

Erythoblasts/normoblasts → Normoblasts → Reticulocytes → mature erythrocytes

Erythoblasts/normoblasts

first recognizable red cell precursors

reticulocytes

Normoblasts decrease in size and lose nucleus

no longer need folic acid but need iron 

Released into blood stream, circulate for 1-2 days

mature erythrocytes

Reticulocytes lose mitochondria and ribosomes

Maturation in spleen

Regulation of erythropoiesis

Regulation by blood oxygenation:

1. Low blood O2 ➔ tissue hypoxia

2. In kidney, activation of hypoxia-inducible factor 1 (HIF-1) to induce synthesis of erythropoietin (EPO)

3. EPO stimulates erythropoiesis (from 2.5 million cells/second (steady state) to 17 million cells/second!!!)

4. Increased RBCs restore normoxia and EPO levels decrease

Factors that decrease oxygenation

Low blood volume (hemorrhage)

Anemia

Low hemoglobin

Poor blood flow

Pulmonary disease

Hemoglobin

1/3 of the RBC weight

Specialized protein with heme as prosthetic group

Adult Hb (HbA)

found exclusively in RBC

Heterotetrameric hemeprotein

Heterotetrameric hemeprotein

4 polypeptide (2a and 2b) chains = globins bound by noncovalent bonds

Each chain has an a-helical structure (a has 7 helices and b has
8 helices) and a hydrophobic heme-binding pocket (prosthetic group)

Heme

Flat, planar molecule

Complex of protophorphyrin IX and Fe2+

Prosthetic group for hemoglobin, myoglobin, cytochrome

Porphyrins

cyclic molecules formed by the fusion of 4 pyrrole rings through methenyl (=CH-) bridges

Side chains define the type of prophyrin

Heme synthesis + rates in what tissues

Synthesis in liver (heme for cytochromes) and bone marrow (heme for Hb)

In liver, variable rate

In bone marrow, constant rate to match RBC regeneration

Heme synthesis subcellular location

Synthesis begins in the mitochondria and continues in the cytosol

heme synthesis steps 

1. Formation of d-aminolevulinic acid (d-ALA) Catalyzed by ALA synthase (ALAS)

• rate-limiting enzyme (DOES NOT USE ATP) 

• Negative regulation by heme (its own product) 

2. 2 molecules of d-ALA condense to form porphobilinogen

• ALA dehydratase inhibited by Pb 

3. 4 porphobilinogens condense + side chains added

4. Ferrochelatase catalyzes the addition of iron ➔heme
   – Ferrochelatase inhibited by Pb

Porphyrias

inherited defects in heme synthesis, resulting in accumulation and increased excretion of porphyrins

Mostly autosomal dominant inheritance

Porphyrias symptoms

Symptoms depending on the site of the mutated enzyme

Skin eruptions and wine red urine after exposure to sunlight, alcohol, or high iron intake

Red teeth (erythrodontia)

treatment of Porphyrias

Injections with hemin (Fe3+), phlebotomies, protection from light

Iron

Iron is in ferrous state (Fe2+) and can form 6 bonds:

4 with nitrogens of the rings

1 with the nitrogen of a histidine residue in globin

1 with O2

Oxidation of Fe2+ to Fe3+ (ferric) makes the molecule incapable to bind O2

where do we get iron from?

Iron is obtained from diet (most readily from heme in meats, best form)

• Ferrous iron Fe2+ absorbed and oxidized to ferric iron Fe3+

• Free iron is toxic ➔ transported by transferrin to bone, liver, and RBC

how is iron stored?

as ferritin (plasma indicator of amount of iron stored) 

how is iron loss?

by bleeding, sweat, urine, feces, desquamation

Ideally, a balance between daily intake and output is necessary to maintain the iron stores

Globin synthesis

2 globin gene loci:
– Chr.16 – a-locus (a1, a2)
– Chr.11 – b-locus (b, d, g)

Heme is a transcriptional regulator of the globin genes

• lots of availability of heme you need more globin

States of hemoglobin

Based on the level of oxygenation, Hb can exist in 2 conformations: oxygenated and deoxygenated

States of hemoglobin: T (tense) state

deoxygenated

has low affinity for O2

Strong interactions between the a and b chains to form heterodimers

Salt bridges between ab dimers constrain the movement of the chains

States of hemoglobin: R (relaxed) state

(oxygenated

has high O2 affinity

Binding of O2 to one heme results in dimer rotation with disruption of most bonds, resulting in availability of other hemes to bind O2

Erythrocyte metabolism

No mitochondria ➔ only cytoplasmic enzymes
– no damage prevention and repair proteins 
– Generation of energy by glycolysis

Erythrocyte metabolism: Rapoport-Luebering shunt

used by erythrocytes use the to produce 2,3-bisphoshoglycerate
(2,3-BPG)

2,3-bisphoshoglycerate (2,3-BPG)

modulator of O2 binding

Erythrocyte metabolism: NADH role

necessary to regenerate hemoglobin from methemoglobin by NADH-cyt b5 methemoglobin reductase

Erythrocyte metabolism: NADPH role + where it is produced 

produced in pentose phosphate shunt

used to maintain glutathione in reduced state (defense against damage by ROS)

uses enzyme glucose-6-phosphate dehydrogenase

Lifetime of RBC correlates directly with

glucose 6-phosphate dehydrogenase activity

Erythrocyte destruction

Erythrocytes become increasingly fragile

• Proteins required to maintain plasma membrane fluidity cannot replaced

Macrophages phagocytize old RBCs (200 bil/day)

RBCs are constantly filtered through

spleen and liver

Destruction of old erythrocytes →

release of hemoglobin

Heme:
• Iron → iron cycle
• Biliverdin → bile pigments

Globin → AA metabolism

Heme degradation

Heme is oxidized and linearized to biliverdin and CO by heme oxygenases and NADPH

Fe2+ is released and returned to iron stores

Biliverdin (green) is reduced to bilirubin (red)

Bilirubin is transported to the liver bound to albumin

In the hepatocytes, bilirubin (UCB) is conjugated twice with glucuronic acid

Bilirubin diglucuronide (CB)is actively secreted in the bile

Iron cycle

Iron released from the macrophages is largely Fe2+

Multicopper ferroxidase ceruloplasmin converts Fe2+ to Fe3+

Fe3+ transported bound to transferrin and delivered to most cells via a cell surface transferrin receptor 1(TfR1)

The receptor-transferrin complex is then internalized by receptor-mediated endocytosis and bound iron is released in the cytosol and transferrin is recycled

Sickle cell disease (AR)

Common in African populations and descendants (heterozygous advantage)

Point mutation (GAG→ GTG) results in glutamate being replaced by valine in the b-chain

HbS formed by large, linear polymers (needle-like) causing the erythrocytes to become sickle-shaped under conditions of:
■ Low oxygenation
■ Low pH
■ Dehydration

Sickling

at first reversible

Cumulative damage to RBC membrane with repeated sickling episodes leads to permanently sickled RBCs even under normal physiological conditions

Fragile RBCs ➔ intravascular hemolysis

RBC aggregate in capillaries ➔ microvascular occlusions

Sickling specific to microvascular beds with slow transit times (spleen, bone marrow, inflamed vascular beds)

Sickle cells anemia (homozygotes)

85-95% HbS and rest HbF

Severity of disease increases with the increase of the proportion of HbS in deoxy (t) state

Blood smear: irreversibly sickled RBCs, reticulocytosis

Sickle cells anemia (homozygotes) manifestations

Severe hemolytic anemia, vasocclusive crises (bone, lungs, spleen, CNS), multiorgan infarctions

Sickle cells anemia (homozygotes) manifestations in spleen 

Sequestration crises ➔ massive pooling of sickle RBCs in the spleen ➔ splenic enlargement

Repeated splenic infarctions ➔ fibrosis ➔ progressive atrophy

Oral aspects of SCA

Mandibular osteomyelitis and bone marrow hyperplasia ➔ enlarged maxilla and overbite

Microvascular occlusions ➔ painful infarcts ➔ radiolucency followed by
osteosclerosis

Coarse trabecular bone pattern

Cranial X-ray: hair-on-end appearance

Smooth reddish tongue with atrophied papillae

Sickle cell trait (heterozygotes)

55-75% HbA, 25-45% HbS

Generally asymptomatic

Sickling can be triggered only by severe stress, high altitude, dehydration, or strenuous physical exercise

Normal RBC morphology

a-thallasemias

Gene deletions ➔ decreased/absent a-globin

Phenotype varies from mild to severe depending on the number of gene copies affected

autosomal recessive

types of alpha thallasemias

alpha thallasemias Symptoms due to:

Insufficient hemoglobin (all Hb types affected)

Excess unpaired globin chains polymerize ➔ forms of Hb with inadequate affinity for O2 ➔ tissue hypoxia

Excess g-chains in fetal period ➔ g4 (Hb Barts) replace HbF

• Incompatible with life

Excess b-chains in children and adults ➔ b4 (HbH) replace HbA

• HbH prone to oxidation and precipitation ➔ intracellular inclusions

• Severe anemia, enlarged spleen, bone deformities

b-thallasemias

Point mutations ➔ decreased/absent synthesis of b- globin

autosomal recessive

types of b-thallasemias

b-thallasemia major

Reduced b-globin synthesis → reduced HbA ➔ severe anemia

Aggregation of excess a-globin

• Cell damage ➔ extravascular hemolysis ➔ jaundice

• Abnormal erythroid progenitors ➔ ineffective erythropoiesis ➔anemia

Excess erythropoietin ➔ extramedullary hemopoiesis (hepatomegaly, splenomegaly)

• Severe marrow hyperplasia ➔ skeletal abnormalities

Repeated blood transfusions ➔ systemic iron overload (secondary hemochromatosis)

Bone Abnormalities in b-thallasemia major

Abnormal facial features (large cheekbones, depressed nasal bridge, protruding maxilla)

Hair-on-end appearance on cranial X-rays

Osteoporosis → fractures

Delayed puberty, endocrine disturbances (due to anemia and iron overload)

Higher incidence of chronic hepatitis B&C due to blood transfusions

Peripheral blood smear: severe hypochromia microcytosis, numerous nucleated RBCs

b-thallasemia minor trait

Usually asymptomatic

Mild anemia (1-2 g/dl below normal)

Peripheral blood smear: hypochromia, microcytosis, nucleated RBC, poikilocytosis