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Physiology of the Blood II. Red Blood Cells (Erythrocytes)

Prof. Szabolcs Kéri

University of Szeged, Faculty of Medicine, Department of Physiology

2019

Ferenc Domoki

1. Number, structure, physical

propertiesLO9: The general features of red blood cells

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- number: 4-5 million/µl, life: 120 days

- diameter: 7-8 µm

- genesis: red bone marrow, facilitated by ERYTHROPOIETIN

(produced by kidney, trigger: hypoxia)

- mature form: in humans no nucleus, mitochondria, endoplasmatic

reticulum; metabolism: glycolysis

- RETICULOCYTE: young form (~1 day old, ~1%) with endoplasmatic

reticulum (protein synthesis)

reticulocyte

Erythrocytes

Parameters of red blood cells

MCH – mean corpuscular hemoglobin (29 pg)MCHC – mean corpuscular hemoglobin concentration (330 g/L)MCV – mean corpuscular volume (94 fL)

HEMATOCRIT44%

HEMO-GLOBIN160 g/L

NUMBER4-5 mill/µl

MCHC

MCV

MCH

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Distribution of the size of red blood cells: PRICE-JONES curve

Normal

Small size,

narrow variance:

MICROCYTOSIS

Normal

Large size,

wide variance:

MACROCYTOSIS,

ANISOCYTOSIS

RDW-CV = (Standard deviation of MCV ÷ MCV) × 100. (11.5-14.5%)

Erythrocyte sedimentation rate (ESR)

- Venous blood sample diluted with

anticoagulant citrate solution

- Filled into Westergren tube- red blood cells aggregate and sink

toward the bottom of tube

- Aggregation is promoted by globulins,

inhibited by albumin (A/G ratio!)

- The height of plasma on top is read

after 1hour

- <20 mm/hour, higher in women

- Very sensitive, but non-specific test!

Albumin/Globulin ≈ 2

DECREASES, if

Albumin cc decreases: -decreased synthesis (liver

damage, malnutrition)

-increased loss (renal diseases)

Globulins increase: - acute phase proteins (inflammation)

- immunglobulins (infections, tumours)

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The osmotic resistance of the red blood cells

Isotonicsolution

Hypertonic solutin

Hypotonic solution

Hypotonic solution → H2O in →

spheroid shape → membrane

rupture → hemolysis

Minimal resistance: hemolysis

not yet (0.44%)

Maximal resistance: full

hemolysis (0.3%)

Anemia, old red blood cells, membrane diseases

(e.g., spherocytosis) – decrease of osmotic

resistance

Glucose transporter (GLUT1)Carbohydrates

Ankyrin

Spectrin

Actin/Tropomyosin

Band 3 aniontransporter

Proteins: aquaporin-1, Na+/K+ and Ca2+ ATP-ase, Cl- /HCO3-, Na+/H+, Na+/K+/2Cl-

transporter, integrin/laminin binding adhesion molecules, nitrogen monoxide (NO) andhydrogen sulfide (H2S) synthesis (vasodilatation), antigens (AB0 and Rh)

Membrane proteins of the erythrocytes

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2. Genesis: iron and vitaminsLO10: Erythropoesis

Constitutive erythropoesis

• To replace normal loss of

erythrocytes

• ~1% of all RBCs/day ~ 250

000 000 000 cells/ day!

• Needs: normal genome, red

bone marrow, nutrients

(proteins, iron, vitamins), and

hormonal stimulation

(erythropoetin)

• In bone marrow: erythroblastic

islands on a central

macrophage

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The genesis of erythrocytes

IRON

- uptake: 1-2 mg/day (total need: 10-15 mg/day)

- better absorption: Fe2+ (vitamin C and gastric acid: Fe3+→ Fe2+) and heme-

bound iron (from meat)

- duodenum - proximal jejunum (inhibited by cereals, oxalic acid [sorrel,

spinach], tannic acid [tea])

- intestine: binding to ferritin; circulation: to transferrin

- store: liver, spleen, bone marrow’s macrophages in the form of ferritin,

insoluble denatured form: hemosiderin

- ferroportin: release of iron from storage cells, inhibited by hepcidin

produced in liver (e.g. infection, tumors)

- iron deficiency: microcyter hypochrom anaemia

Accessory minerals- copper, nickel, cobalt (facilitates iron absorption)

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Hepcidin: a liver hormone

• Produced by the liver (peptide, 25 AA)

• Induced by both elevated plasma and store iron levels

(homeostasis!), also stimulated by inflammatory cytokines

(acute phase response).

• Its receptor is the ferroportin iron tranporter itself – that it

inhibits, thus inhibiting both iron absorption and release of stores

VITAMIN B12/FOLIC ACID

- DNA-synthesis

- B12 bound to R-protein (saliva) and then to intrinsic factor

(apoeritein, produced by stomach) in intestine

- absorption: ileum

- in blood bound to transcobalamin

- deficiency: macrocyter hyperchrom anaemia (anaemia perniciosa)

HORMONES

Stimulation: growth hormone, testosterone, thyroxin

Inhibition: estrogens

ERYTHROPOIETIN

- produced by kidney due to hypoxia

- stimulation of the erythroid line in bone marrow

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Folic acid and B12 vitamin biochemistry

(in part)

• In B12 deficiency the regeneration of THF is being impaired, there is relative folate deficiency and impaired DNA synthesis

• THF: tetrahydrofolate

• Required dietary allowance: B12: 1-2 µg/day, folic acid: 200 µgFolic acid

dTMP

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What can be the „use” of

reticulocyte count?

• The ratio of 1-day old RBCs is determined (relative reticulocyte count)

• Depends on the ACTUAL erythropoesis (TODAY how much the marrow produces)

• In anemia diagnosis: too few is produced (low), too many die early (high)?

• To check therapy: did the marrow respond??

• Absolute reticulocyte count: more informative, can be calculated, but apps exist too ☺

3. Degradation of hemoglobin:

the question of bilirubin LO11: Hemoglobin degradation, bilirubin metabolism

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Degradation of hemoglobin 1.

1. Old erythrocytes: extraction from blood by macrophages

(liver, spleen)

2. Haptoglobin transiently binds hemoglobin in circulation

(hemopexin: heme-binding protein in blood)

3. Fe2+ dissociation (used again or stored) & proteolysis of β-globin

4. Porphyrin degradation: CO + biliverdin (green), then bilirubin

(yellow)

Circulation: bilirubin binds to albumin – indirect bilirubin

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Haptoglobin

-physiology

• Alpha-2 globulin

• Can bind myoglobin or

hemoglobin

• Enters macrophage

with receptor-mediated

endocytosis (CD163

scavenger-receptor)

• Vitally important

mechanism to protect

the kidney

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6. Liver takes up bilirubin and conjugates that with glucuronide –

direct bilirubin

7. From liver to gut with bile where further transformation occurs

(urobilinogen - urobilin, stercobilinogen - stercobilin;

oxidoreductive process mediated by bacteria)

8. Some of them are reabsorbed to liver with bile acids via the

portal vein: Enterohepatic circulation

9. Secretion with faces (gives its color) and urine

Degradation of hemoglobin 2.

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Urobilinogen in urine:- ↑degradation of redblood cells- hepatic diseasewith icterus

- Disappears after

bile obstruction

Indirect and direct bilirubin

• Indirect bilirubin aka unconjugated or free bilirubin –

albumin bound (water insoluble), can be measured only

INDIRECTLY from the plasma, after giving an enhancer

• Direct bilirubin aka conjugated bilirubin (water soluble)

can be measured DIRECTLY from the plasma

• The lab determines TOTAL bilirubin from the sample

(with enhancer) and DIRECT bilirubin (no enhancer).

• Indirect bilirubin = total – direct, physiologically indirect =

total bilirubin = 5-17 µM.

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The definition of icterus

(jaundice) >20 µM bilirubin

Icterus galbula

Baltimore Oriole

Causes of jaundice:

1. Prehepatic (hemolysis, indirect ↑)

2. Hepatic (liver disease, indirect/direct ↑)

3. Posthepatic (bile obstruction, direct ↑

urine: bilirubin + UBG - )

4. Blood groupsLO13: The ABO and Rh blood groups

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The AB0 blood group, LANDSTEINER rulesIn the membrane of erythrocytes: glycolipid antigens0 blood group: basic H-antigenA and B groups: additional sugars are bound to the H-antigen

(e.g. N-acethyl galactosamine (A) or galactose (B)

the GENES code for the ENZYMES building the chain

A (44%) – A1: many antigens, A2: few antigens

Circulating antibody (IgM): anti-B

Genotype: AA/A0

B (10%)

Circulating antibody : anti-A

Genotype: BB/BO

0 (42%) – „universal graft”

Circulating antibody: anti-A+B

Genotype: 00

AB (4%)

Circulating antibody: none

Genotype: AB

Bombay: H-antigen misses terminal sugar (fucose) → no more sugars can bind to it (0 group with othergenotype) anti-A+B+H!

The antibodies are NOT inherited, but the ABO antigens are found in bacteria so everybody is immunized with them.

Cross reactions:

Major test: donor cell,

host serum

Minor test: donor serum,

host cell

Determination of blood groups

Transfusion of non-compatible blood

group: HAEMOLYSIS

(degradation of erythrocytes caused by the

antigen-antibody reaction, release of

hemoglobin, kidney dysfunction and

multiple organ failure)

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Rh (Rhesus) blood group

- Protein antigens, 3 alleles: c/C, d/D, e/E

- D is the only strong antigen – Rh+ blood group

- there is no spontaneous immunization (antibodies) only if:

a. Rh- receives Rh+ blood

b. Rh- mother has a Rh+ baby

(Rh prophylaxis by giving incomplete Rh-antibodies

that suppress production, otherwise danger of erythroblastosis fetalis)

5. Hemoglobin structure and

function: gas transport(more details in respiration physiology)

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Hemoglobin (Hb) – Gas transport

- β-globin + heme (= [Fe2+] porphyrin)

- Fe3+: methemoglobin (loss of function)

- 4 subunits, 4 oxygen binding

- mainly HbA, α2β2 subunit-composition

- artery: 97% saturation

- Hüffner-number: 1.3 ml oxygen/1 g Hb

Oxygen-affinity decreased:

1. Temperature

2. H+ (CO2↑, pH↓) - Bohr-effect

3. 2,3-bis-phosphoglycerate (2,3-BPG, byproduct of glycolysis)

Oxygen-affinity increased:

1. Fetal hemoglobin (HbF, α2γ2 – no 2,3-BPG binding)

2. Carboxy-hemoglobin (CO-Hb, unable to let oxygen to tissue)

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Changes of globin-chains with age

%

Gamma-chain (fetal)

Epsilon-chain(embrional)

Beta-chain(adult)

Delta-chain

Alpha-chain

Pregnancy (months) Age (months)

BIRTH

CO2 in tissue:

1. Carbonic acid is

produced by carbonic

anhydrase enzyme

2. Hb lets oxygen and

takes up proton (H+)

dissociated from the acid

3. Bicarbonate is

exchanged for chloride

(Hamburger shift)

4. Non-enzymatic solution

and binding to proteins