Biochemistry of Blood Elements 624 Vladimíra Kvasnicová modified and reduced by Eva Samcová The figure is found at (March 2007)

Blood Elements Count erythrocytes4 - 6 x 10 6 /  l thrombocytes x 10 3 /  l leukocytes4 - 9 x 10 3 /  l neutrophils % eosinophils % basophils % lymphocytes % monocytes % hematocrit muži: % ženy: %

2,000,000 erythrocytes / sec into circulation The lifetime of erythrocytes (red blood cells - RBC) is 120 days New erythrocytes – reticulocytes - contain more ribosomes and components of ER 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

What to study Erythrocyte – structures Erythrocyte - metabolism Hemoglobin. Structure of hemoglobin Saturation curve Function of erythrocytes: Gas transport

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 (March 2007)

The figures are found at and (March 2007) Red Blood Cells (erythrocytes) membrane and cytoskeletal proteins hereditary spherocytosis

Red Blood Cells (erythrocytes) membrane transporters  Na + /K + -ATPase (active transport)  GLUT-1 (insulin independent)  anion exchanger = band 3 protein (Cl - /HCO 3 - ) membrane antigens  blood groups

The figure is found at (March 2007) Membrane antigens – example: ABO system

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, ISBN 0 ‑ 471 ‑ ‑ 2

Red Blood Cells Function  erythrocyte as a bag for hemoglobin  O 2 → transport, reactive oxygen species (ROS)  CO 2 → transport, formation of HCO 3 -  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) O 2 + O H +  H 2 O 2 + O 2  catalase (CAT) H 2 O 2 + H 2 O 2  2 H 2 O + O 2  glutathione peroxidase (GPx) 2 GSH + H 2 O 2  GS-SG + 2 H 2 O 2 GSH + R-O-OH  GS-SG + H 2 O + ROH  glutathione reductase GS-SG + NADPH+H +  2 GSH + NADP +  methemoglobin reductase- in erythrocytes Hb-Fe 3+ + e -  Hb-Fe 2+ (coenzyme: NADH or NADPH)

The figure is found at (March 2007) Hexose Monophosphate Pathway glutathione reductase GS-SG + NADPH+H + 2 GSH + NADP + = „redox buffer“

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 (Fe 2+ ) ions

The figure is found at (March 2007) M r =

The figures are found at and (March 2007) Pyrrole hemoglobin

Saturation of hemoglobin by oxygen quaternary structure of hemoglobin allosteric effect T-conformation: lower affinity to O 2 (deoxy Hb) R-conformation: higher affinity to O 2 (oxyHb) T  R HHb + O 2  HbO H + the saturation curve has sigmoidal shape

Types of hemoglobin and its subunits adult hemoglobin: HbA 1 =  2  2 HbA 2 =  2  2 (  2% from total Hb of adults) fetal hemoglobin HbF =  2  2 ! higher affinity to O 2 than HbA ! binds oxygen more firmly at lower pO 2 (placenta!)  /  thalassemia sickle-cell anemia (HbS) congenital methemoglobinemia (HbM)

The figure is found at (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 male135 – 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% N 2 21% O 2 1% water, inert gases, CO 2 (0,04%) Air pressure: 1 atm = Pa (~ 101 kPa) = 760 Torr (= mmHg) 1 mmHg = 0,1333 kPa 1 kPa = 7,5 mmHg

Transport of blood gases arterial bloodvenose blood pO 2 13,33 kPa5,33 kPa 100 mmHg40 mmHg pCO 2 5,33 kPa6,13 kPa 40 mmHg46 mmHg (alveols)

Transport of blood gases - function of hemoglobin - it transports O 2 and part of CO 2 (and CO) it binds H + (reacts as a buffer) O 2 and CO: bound to Fe 2+ in heme → 4 O 2 / 1 Hb  „oxyhemoglobin“ HbO 2 /„carbonylhemoglobin“ COHb CO 2 is bound to globin! (-NH 2 of side chains of amino acids)  „carbaminohemoglobin“ HbCO 2 H + is bound to residues of His  „deoxyhemoglobin“ HHb

Transport of blood gases - transport of CO largely in a form of HCO 3 - (~ 70%) CO 2 + H 2 O  H 2 CO 3  HCO H + enzyme: carbonic anhydrase spontaneous dissociation (in erytrocytes) 2.bound to hemoglobin (~ 23%) 3.freely dissolved (~ 7%)

The figure is found at (March 2007)

Transport of blood gases - reactions in erytrocytes - tissues: CO 2 + H 2 O → H 2 CO 3 → HCO H + H + + HbO 2 - → HHb + O 2 → aerobic metabolism (HCO 3 - 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 HCO 3 - is transported back into the erythrocyte by the same exchange with Cl - ) lungs: HHb + O 2 → HbO H + H + + HCO 3 - → H 2 CO 3 → H 2 O + CO 2 → excreted

The figure is from (March 07) O2O2 O2O2

Hemoglobin saturation curve - saturation with oxygen - The figure is found at (March 2007)

The figure is found at (March 2007) Right shifted = oxygen is more easily released from Hb but worse bound to it

Saturation of hemoglobin with oxygen Factors affecting the saturation:  alkaline pH and  pO 2 stabilize R-conformation (IN LUNGS)  acidic pH,  pCO 2,  temperature and 2,3-BPG stabilize T-conformation, i.e. deoxyHb (IN PERIPHERY) shift of the saturation curve toward right

The figure is found at (March 2007) Bohr´s effect = the saturation of Hb by O 2 lowers because lowering pH (shift toward right)

Patological forms of hemoglobin 1.methemoglobin (over 3%)metHb  Fe 3+ instad of Fe 2+  unable to transport oxygen !!! 2.glycohemoglobin (over 6%)HbA 1c  after long term increased glycemia (Glc bound to Hb) 3.carbonylhemoglobin (over 2%)COHb  after CO poisoning 4.sulfhemoglobin, cyanhemoglobin  poisoning by H 2 S, HCN or by cyanides

Carbon monoxide poisoning CO has 200x higher affinity to Hb than O 2 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)

The figure is found at (March 2007)

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)

Saturation of hemoglobin with CO The figure is found at (March 2007) COHb / total Hb ( ratio in %) physiological value:  2%

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 Carbon monoxide poisoning