1. 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)http://www.biosbcc.net/doohan/sample/htm/Blood%20cells.htm

2. Blood Elements Count erythrocytes4 - 6 x 10 6 /  l thrombocytes150 - 400 x 10 3 /  l leukocytes4 - 9 x 10 3 /  l neutrophils47 - 75 % eosinophils 1 - 4 % basophils 0 - 1 % lymphocytes23 - 45 % monocytes 2 - 11 % hematocrit muži: 42-52 % ženy: 37-47 %

3. 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

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

5. 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)http://www.biosbcc.net/doohan/sample/htm/Blood%20cells.htm

6. 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)http://www.wadsworth.org/chemheme/heme/microscope/pix/spherocytes_nw.jpg http://www.mie.utoronto.ca/labs/lcdlab/biopic/fig/4.23b.jpg Red Blood Cells (erythrocytes) membrane and cytoskeletal proteins hereditary spherocytosis

7. 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

8. The figure is found at http://www.life.umd.edu/classroom/bsci422/mosser/ABO.gif (March 2007)http://www.life.umd.edu/classroom/bsci422/mosser/ABO.gif Membrane antigens – example: ABO system

9. 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 )

10. 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.

11. 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

12. 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

13. antioxidative enzymes  superoxide dismutase (SOD) O 2 + O 2 + 2 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)

14. The figure is found at http://www.med.unibs.it/~marchesi/ppp.html (March 2007)http://www.med.unibs.it/~marchesi/ppp.html Hexose Monophosphate Pathway glutathione reductase GS-SG + NADPH+H + 2 GSH + NADP + = „redox buffer“

15. 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

16. The figure is found at http://dtc.pima.edu/~biology/202alpha/lesson1/hemoglobin.jpg (March 2007)http://dtc.pima.edu/~biology/202alpha/lesson1/hemoglobin.jpg M r = 64 500

17. 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)http://www.medical-definitions.net/images/hemoglobin.jpghttp://omlc.bme.ogi.edu/spectra/hemoglobin/hemestruct/heme-struct.gif Pyrrole hemoglobin

18. 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 2 - + H + the saturation curve has sigmoidal shape

19. 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)

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

21. 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

22. 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)

23. Transport of blood gases Air composition: 78% N 2 21% O 2 1% water, inert gases, CO 2 (0,04%) Air pressure: 1 atm = 101 325 Pa (~ 101 kPa) = 760 Torr (= mmHg) 1 mmHg = 0,1333 kPa 1 kPa = 7,5 mmHg

24. 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)

25. 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

26. Transport of blood gases - transport of CO 2 - 1.largely in a form of HCO 3 - (~ 70%) CO 2 + H 2 O  H 2 CO 3  HCO 3 - + H + enzyme: carbonic anhydrase spontaneous dissociation (in erytrocytes) 2.bound to hemoglobin (~ 23%) 3.freely dissolved (~ 7%)

27. The figure is found at http://fig.cox.miami.edu/~cmallery/150/physiol/sf41x11.jpg (March 2007)http://fig.cox.miami.edu/~cmallery/150/physiol/sf41x11.jpg

28. Transport of blood gases - reactions in erytrocytes - tissues: CO 2 + H 2 O → H 2 CO 3 → HCO 3 - + 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 2 - + H + H + + HCO 3 - → H 2 CO 3 → H 2 O + CO 2 → excreted

29. The figure is from http://science.kennesaw.edu/~jdirnber/Bio2108/Lecture/LecPhysio/42-29-BloodCO2Transport-AL.gif (March 07)http://science.kennesaw.edu/~jdirnber/Bio2108/Lecture/LecPhysio/42-29-BloodCO2Transport-AL.gif O2O2 O2O2

30. Hemoglobin saturation curve - saturation with oxygen - The figure is found at http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif (March 2007)http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif

31. The figure is found at http://dr-amy.com/rich/oxygen/fig1.gif (March 2007)http://dr-amy.com/rich/oxygen/fig1.gif Right shifted = oxygen is more easily released from Hb but worse bound to it

32. 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

33. The figure is found at http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif (March 2007)http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/MbHbbindcurve.gif Bohr´s effect = the saturation of Hb by O 2 lowers because lowering pH (shift toward right)

34. 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

35. 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)

36. The figure is found at http://dr-amy.com/rich/oxygen/fig1.gif (March 2007)http://dr-amy.com/rich/oxygen/fig1.gif

37. 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)

38. Saturation of hemoglobin with CO The figure is found at http://www.uhseast.com/134221.cfm (March 2007) http://www.uhseast.com/134221.cfm COHb / total Hb ( ratio in %) physiological value:  2%

39. 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