Biochemistry of Blood Elements

624
Vladimíra Kvasnicová
modified and reduced by Eva Samcová

The figure is found at http://www.biosbcc.net/doohan/sample/htm/Blood%20cells.htm (March 2007)

 What to study

• Erythrocyte – structures
• Erythrocyte - metabolism
• Hemoglobin. Structure of hemoglobin
• Saturation curve
• Function of erythrocytes: Gas transport
 Blood Elements Count

erythrocytes 4 - 6 x 106 / l

thrombocytes 150 - 400 x 103 / l

leukocytes 4 - 9 x 103 / l

neutrophils 47 - 75 % hematocrit
eosinophils 1 - 4 %
muži: 42-52 %
basophils 0 - 1 %
lymphocytes 23 - 45 % ženy: 37-47 %
monocytes 2 - 11 %
 2,000,000 erythrocytes / sec into circulation

• The lifetime of erythrocytes (red blood cells - RBC)

is 120 days
New erythrocytes –reticulocytes–contain ribosomes
and components of ER - synthesis of proteins
• The life span of erythrocytes can be dramatically
reduced in the case of a series of hemolytic anemia
(in hemolytic anemia occurs increasingly hemolysis –
destruction of red blood cells)
• The production of red blood cells is regulated by
erythropoietin (EPO) - synthesized in the kidney - in
top level sport-doping is followed
 Red Blood Cells
(erythrocytes)

Structure
 large surface
(diffusion of gases)
 cytoskeletal proteins
(elasticity)
 membrane as an osmometer
(Na+/K+-ATPase)

The figure is found at http://www.biosbcc.net/doohan/sample/htm/Blood%20cells.htm (March 2007)

 Red Blood Cells
(erythrocytes)
membrane and
cytoskeletal
proteins

hereditary
spherocytosis

The figures are found at http://www.wadsworth.org/chemheme/heme/microscope/pix/spherocytes_nw.jpg and

http://www.mie.utoronto.ca/labs/lcdlab/biopic/fig/4.23b.jpg (March 2007)
 Red Blood Cells
(erythrocytes)

membrane transporters
 Na+/K+-ATPase (active transport)
 GLUT-1 (insulin independent)
 anion exchanger = band 3 protein (Cl-/HCO3-)

membrane antigens
 blood groups
Membrane antigens – example: ABO system

The figure is found at http://www.life.umd.edu/classroom/bsci422/mosser/ABO.gif (March 2007)

 Red Blood Cells
(erythrocytes)

Metabolism
Erythrocytes lack mitochondria and other organelles-
reduced metabolism. They degrade externally supplied
glucose into lactate via glycolysis
 Thus glucose is the only energy substrate
 90% anaerobic glycolysis
(2 ATP, lactate: Cori cycle; 2,3-BPG)
 10% hexose monophosphate pathway
(NADPH  antioxidative mechanisms)
 Glucose 6-Phosphate Dehydrogenase
Genetic Deficiency or Presence of Genetic Variants in
Erythrocytes
• Enzyme catalyzes the oxidation of G6P to 6-
phosphogluconate and the reduction of NADP+ in major
pathway of NADPH production – pentose cycle
• NADPH maintains glutathione in its reduced state
• GSH is necessary for the integrity of the erythrocyte
membrane – cells more susceptible to oxidative damage by
reactive oxygen species - to hemolysis.
• One of the most common enzymopathies.100 milion people
suffer from this deficiency – particularly in the area Tropical
Africa, Mediterranean region, some parts of Asia and the
Black Population in America.
• Result is usually hemolytic anemia. 300 known genetic
variants of this enzyme – wide range of symptoms.
 Cori cycle

and muscle

The figure was accepted from Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed.
Wiley-Liss, Inc., New York, 1997. ISBN 0-471-15451-2
 Red Blood Cells
Function
 erythrocyte as a bag for hemoglobin
 O2 → transport, reactive oxygen species (ROS)
 CO2 → transport, formation of HCO3-
 H+ → transport, maintaining pH
(35% of blood buffering capacity)

• superoxide dismutase
• catalase
• glutathione peroxidase antioxidative system
• glutathione reductase
• methemoglobin reductase
antioxidative enzymes
 superoxide dismutase (SOD)
O2• + O2• + 2 H+  H2O2 + O2
 catalase (CAT)
H2O2 + H2O2  2 H2O + O2
 glutathione peroxidase (GPx)
2 GSH + H2O2  GS-SG + 2 H2O
2 GSH + R-O-OH  GS-SG + H2O + ROH
 glutathione reductase
GS-SG + NADPH+H+  2 GSH + NADP+
 methemoglobin reductase - in erythrocytes
Hb-Fe3+ + e-  Hb-Fe2+ (coenzyme: NADH or NADPH)
glutathione reductase
GS-SG + NADPH+H+

2 GSH + NADP+

= „redox buffer“

Hexose Monophosphate
Pathway
The figure is found at http://www.med.unibs.it/~marchesi/ppp.html (March 2007)
 Structure of hemoglobin

• hemoprotein (complex protein: globin + prosthetic group)

• quaternary structure: 4 subunits

• prosthetic group of each of the subunit = heme

4 polypetide chains
4 molecules of heme
4 ferrous (Fe2+) ions
 Mr = 64 500
The figure is found at http://dtc.pima.edu/~biology/202alpha/lesson1/hemoglobin.jpg (March 2007)
Pyrrole

hemoglobin
The figures are found at http://www.medical-definitions.net/images/hemoglobin.jpg
and http://omlc.bme.ogi.edu/spectra/hemoglobin/hemestruct/heme-struct.gif (March 2007)
 Saturation of hemoglobin by oxygen

• quaternary structure of hemoglobin

allosteric effect the saturation curve
has sigmoidal shape

T-conformation: lower affinity to O2 (deoxy Hb)

R-conformation: higher affinity to O2 (oxyHb)

T  R
HHb + O2  HbO2- + H+
 Types of hemoglobin and its subunits
• / thalassemia
• sickle-cell anemia (HbS)
• adult hemoglobin: • congenital methemoglobinemia (HbM)
HbA1 = 22
HbA2 = 22 ( 2% from total Hb of adults)

• fetal hemoglobin
HbF = 22 ! higher affinity to O2 than HbA !

binds oxygen more firmly at lower pO2 (placenta!)

The figure is found at http://www.labcorp.com/datasets/labcorp/html/img/fethgb.jpg (March 2007)
 Synthesis of hemoglobin

• bone marrow
• in erytroblasts, not in erythrocytes
• 4 individual subunits are connected by
noncovalent bonds to form tetramer of Hb
• hemoglobin is an intracellular protein: within ery
concentration of Hb in blood:
female 120 – 162 g/l
male 135 – 172 g/l
 Synthesis of hemoglobin
Disorders:
• THALASSEMIA = group of genetically determined
disorders: absence or reduced synthesis of a globin chain
( or  thalassemia)

• ANEMIA (= decreased oxygen-carrier capacity of blood)

• Hemolytic anemia is a condition in which red blood cells

are destroyed and removed from the bloodstream before
their normal lifespan is over.
 sideropenic anemia – insufficient concentration of Fe
 sickle cell anemia – point mutation
in the -globin gene forms abnormal
HbS (Glu → Val)
 Transport of blood gases

Air composition:
78% N2 21% O2 1% water, inert gases, CO2 (0,04%)

Air pressure:
1 atm = 101 325 Pa (~ 101 kPa) = 760 Torr (= mmHg)

1 mmHg = 0,1333 kPa

1 kPa = 7,5 mmHg
 Transport of blood gases

alveols metabolizing tissue

pO2 13,33 kPa 5,33 kPa
100 mmHg 40 mmHg
In freshly inhaled air oxygen-depleted tissues
pCO2 5,33 kPa 6,13 kPa
40 mmHg 50 mmHg
 Lungs (alveolus) - Oxygenation
• Relatively high pO2 in alveolus (100 mm Hg)
• HHb + O2 → HbO2- + H +
High pO2 acts on the left side, so it helps to shift the
equilibrium to the right. The newly forming H+ equilibriate
with HCO3- in RBC and form CO2 by the help of
carbonic anhydrase
• H+ + HCO3 - → CO2 + H2O
• Coordination activity : the loss of CO2 by exhaling pulls
this and all previous equilibria to the right in the lungs
• The switch from the appearance of H+ as a product in
the first reaction to its disappearance in second reaction
as a reactant is called Isohydric shift
 Metabolizing tissue – deoxygenation of
Oxyhemoglobin
• Fully oxygenated red cell (HbO2-) arrives in a tissue. An
impetus for this diffusion is the higher pCO2 (50 mm Hg)
in active tissue versus its value in blood (40 mm Hg).
Once the CO2 arrives in the blood, it moves inside a red
cell where encounters carbonic anhydrase
• CO2 + H2O → H+ + HCO3 - the generated H+
enters in the equilibrium
• HbO2- + H + → HHb + O2 This reaction,
another isohydric shift, not only neutralizes the acid
generated by the arrival of waste CO2 , but also helps to
force oxyhemoglobin to give up its oxygen.
• Deoxygenation of HbO2- is also aided by the 2,3 BPG
anions that were pushed out when HbO2- was formed.
These anions are still inside the red cell.
 Yet to the transport of gases
• Some CO2 is carried to the lungs on Hemoglobin. Not all
the CO2 (23%) made by metabolizing cells winds up as
HCO3-. Some react with HHb
• CO2 + HHb → Hb-CO2- + H+ the product is
carbaminohemoglobin – one of the form in which some
waste CO2 travels in the blood back to the lungs.
• Chloride ion is also needed to deoxygenate hemoglobin.
Hemoglobin HHb binds chloride ion, and it binds it better
than does oxyhemoglobin :
• HHb(Cl-) + O2 → HbO2- + Cl- + H+ therefore to unload
oxygen (reverse reaction), chloride ion must be available
inside the red cell (but firstly is Cl- in blood plasma).
Mechanism called the chloride shift. Chloride ions will go
inside, bicarbonate outside the red cell. Reverse equation
to the previous. The equilibrium → now to the left.
 Transport of blood gases
- function of hemoglobin -

• it transports O2 and part of CO2 (and CO)

• it binds H+ (reacts as a buffer)

• O2 and CO: bound to Fe2+ in heme → 4 O2 / 1 Hb

„oxyhemoglobin“ HbO2 /„carbonylhemoglobin“ COHb

• CO2 is bound to globin! (-NH2 of side chains of amino acids)

„carbaminohemoglobin“ HbCO2

• H+ is bound to residues of His

„deoxyhemoglobin“ HHb
 Transport of blood gases
- transport of CO2 -

1. largely in a form of HCO3- (~ 70%)

CO2 + H2O  H2CO3  HCO3- + H+
enzyme: carbonic anhydrase spontaneous dissociation
(in erytrocytes)

2. bound to hemoglobin (~ 23%)

3. freely dissolved (~ 7%)
 Transport of blood gases
- reactions in erytrocytes -
tissues:
CO2 + H2O → H2CO3 → HCO3- + H+
H+ + HbO2- → HHb + O2 → aerobic metabolism
(HCO3- formed in the erythrocyte is then transported to plasma by an anion exchanger in
exchange with Cl-; this process is called Hamburger´s effect or „chloride shift“; in the
lungs HCO3- is transported back into the erythrocyte by the same exchange with Cl-)

lungs:
HHb + O2 → HbO2- + H+
H+ + HCO3- → H2CO3 → H2O + CO2 → excreted
 Hemoglobin saturation curve
- saturation with oxygen -

The figure is found at http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif

(March 2007)
 Saturation of hemoglobin with oxygen

Factors affecting the saturation:

 alkaline pH and  pO2 stabilize R-conformation
(IN LUNGS)

 acidic pH,  pCO2,  temperature and 2,3-BPG

stabilize T-conformation, i.e. deoxyHb
(IN PERIPHERY)

shift of the saturation curve toward right

 Bohr´s effect
= the saturation of Hb by O2 lowers because lowering pH
(shift toward right)

The figure is found at http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif

(March 2007)
 Patological forms of hemoglobin
1. methemoglobin (over 3%) metHb
 Fe3+ insted of Fe2+
 unable to transport oxygen !!!

2. glycohemoglobin (over 6%) HbA1c

 after long term increased glycemia (Glc bound to Hb)

3. carbonylhemoglobin (over 2%) COHb

 after CO poisoning

4. sulfhemoglobin, cyanhemoglobin
 poisoning by H2S, HCN or by cyanides
 Carbon monoxide poisoning

• CO has 200x higher affinity to Hb than O2

• it forms COHb = carbonyl hemoglobin
(formerly called carboxyhemoglobin)
• max. allowed concentration in the air: 0.003%
• intoxication by CO depends on pCO and a time
of its exposition (0.04%  strong headache, 2-3 hours:
unconsciousness; 1%  death after a few minutes)
 Carbon monoxide poisoning

may result due to:

• exposure to automobile exhaust

• smoke inhalation

• an improperly ventilated gas heater

• or other appliance

= incomplete burning
(incomplete oxidation of organic material)
 COHb / total Hb
Saturation of (ratio in %)

hemoglobin
with CO

physiological value:
 2%

The figure is found at

http://www.uhseast.com/134221.cfm
(March 2007)
 Carbon monoxide poisoning

TREATEMENT
• fresh air
• exposure to high concentrations of oxygen
(the 100% oxygen is administered by a face mask)
 it is recommended in patients who have a history
of loss of consciousness, carbonyl hemoglobin
saturation greater than 25%, metabolic acidosis
and cerebellar findings on neurologic exam