Pharmacist. Drugs. body physiology. ( molecular constituents)

Transcription

1 Why? Pharmacist Drugs body physiology ( molecular constituents)

2 Mechanistic levels of response: Altered patient response physiologic systems Vascular system blood, muscle, liver tissues / organs cellular processes erythrocytes (proteins) hemoglobin myoglobin

3 UNIT OVERVIEW: ERYTHROCYTES AND OXYGEN DELIVERY 1. Biology of erythrocytes / vasculature 2. Hemoglobin and Myoglobin function 3. Energy metabolism in erythrocytes 4. 2,3 Diphosphoglycerate 5. Drugs / toxins which affect erythrocyte function Recommended reading: (Devlin) pp , (Stryer) pp

4 Primary RBC function: transport of O 2 / CO mm Hg mm Hg (5 mm Hg) *Blood is a colloid, in addition to RBC s, blood also contains: Additional cell types: leucocytes (WBC s), platelets (clotting) Free proteins: albumin, globulins (Ig), ferritins (transport), enzymes (clotting), hormones Other non-cellular components: electrolytes

5 Erythrocytes: Key erythrocyte features: From Tina Carvalho (MicroAngela) 95% of cellular protein is hemoglobin (35% by weight) humans: 300x 10 6 Hb molecules/rbc, x 10 6 RBC s/cc blood hematocrit 35-50%, produce / destroy 2.5 x 10 6 RBC s/sec RBC s harbor variety of membrane transporters (glucose) on cell surface

6 Erythrocyte development: (spleen)

7 BIOLOGY OF ERYTHROCYTES/VASCULATURE * Fetal development RBC s produced in liver. Bone marrow production commences at 4 mo. in humans. From the 7 mo. on RBC production occurs ONLY in the bone marrow. Adult erythrocyte production occurs only in bone marrow. (bone marrow) Phys. Mech. of Disease: 4 th Ed. 1 * Mitochondria, nuclei and endoplasmic reticulum lost as reticulocytes mature into adult erythrocytes. Thus NO mito. respiration * Therefore NO gene transcription or protein translation occurs in RBC s all proteins within the erythrocyte must be produced at the time of genesis. * No mitochondrial respiration, thus low ATP formation. Energy requirements of the cell must be met largely through GLYCOLYSIS.

8 HEMOGLOBIN and MYOGLOBIN - Physical structure of hemoglobin - Developmental expression of globin genes - Mechanisms of O 2 regulation - Allosterism and important conformational changes - Regulation by external agents

9 Structure of Myoglobin:

10 Hemoglobin / Myoglobin: Heme-containing proteins Heme - a cyclic tetrapyrrole (Fe(II)-protoporphyrin IX) N H pyrrole ring The iron atom in heme can form 6 bonds. Catabolism: Fe Globin Heme reused (Tf) peptidase (AA) Bilirubin O 2 binding to heme of Mb or Hb is reversible Devlin Fig. 9.31, 9.32, pyrrole - Harpers Fig. 7-1

11 Myoglobin (Mb) O 2 binding curve: Myoglobin: single chain protein, 1 heme/protein, first x-ray solution structure solved. Because it contains only a single subunit, it does NOT display cooperativity or allosterism (hyperbolic O2 curve). P 50 Mb: 2.8 torr low P 50 = high O 2 affinity modified from Stryer Fig 10.17, see also Devlin 9.35 P O2 lungs: 100 mm Hg tissues: 20 mm Hg working muscle: 5 mm Hg ~1,100 m. years ago

12 Hemoglobin Structure: HbA (alpha2, beta2) alpha beta (146 aa) beta alpha (141 aa) Heme group

13 Hemoglobin (Hb) O 2 binding curve: Hb: tetrameric protein (~2-500 m years) 4 subunits and heme groups/protein alpha beta The tetrameric structure of Hb imparts it with several important properties: Allosterism: The binding of a ligand (O 2 ) at one site affects the binding of other ligands at distal sites. Thus Hb exhibits sigmoidal O 2 kinetics. Devlin 9.35 Positive cooperativity: The affinity of Hb for the 4 th O 2 is 100x greater than for the first, due to conformation changes in Hb.

14 Measures of cooperativity, Hill plot: Y = number of binding sites occupied total number of binding sites Y/1-Y = po 2 / po2 (50) log Y/1-Y = log po2 - log po2 (50) Hill plot log (y/1-y) 1.0 Mb 2.8 Hb Hill coefficient (slope) log po 2

15 Perutz mechanism: Histidine I Fe Porphyrin Plane On the basis of the X-ray structure of oxyand deoxyhemoglobin, Perutz formulated a mechanism for hemoglobin oxygenation. Perutz postulated that hemoglobin has 2 stable conformational states; the dexoy "T -state, and the fully oxygenated "R -state. The conformation of subunits in T-state hemoglobin differ from those in the R-state. O2 binding initiates a series of coordinated movements that result in a shift from the T to the R state in a few microseconds. Fe R = relaxed = oxy state, T = tense = deoxy state

16 Oxygen Binding Site of Hemoglobin:

17 Oxygen Binding Site of Hemoglobin:

18 Oxygen Binding Site of Hemoglobin: Histidine F8 Histidine E7 Heme Plane

19 Structural states of Hb: Deoxy Hb (T state) Oxy Hb (R state)

20 Structural states of Hb: Deoxy Hb (T state) Oxy Hb (R state)

21 The Bohr effect: Blood ph: T-Hb 2CO 2 + 2H 2 O 2H 2 CO 3 carbonic anhydrase Carbonic acid 2H + + 2HCO 3 - Bicarbonic ion CO 2 and H + produced during metabolism causes ph in RBCs, resulting in protonation of some amino acid groups in Hb. These effects decrease the affinity of Hb for O 2 in RBCs (protons bind to the T form of hemoglobin thus stabilizing it). See Devlin 9.42, and Stryer Fig

22 Globin synthesis during development: yolk sac liver spleen bone marrow % total globin synth zeta (a) epsilon (b) gamma alpha (1,2) Gestational age (weeks) beta HbA (alpha2, beta2) HbF (alpha2, gamma2) delta

23 Isohydric transport: isohydric Transport (70-80%)

24 CO2 induces chloride shift: tissues 2CO 2 The chloride content of red cells in venous blood greater than 20 fold higher than that in arterial blood. 0.5 million channels/rbc 10 billion anions/rbc Anion channel releases HCO 3- in lung. antiport Tissue 2CO 2 + 2H 2 O 2H 2 CO 3 2H + + 2HCO 3- [Cl - ] 2CO 2 + 2H 2 O Lung 2H 2 CO 3 2CO 2 + 2H 2 O Lung

25 Systemic O2 delivery: CO2 Hb Hb O2 O2 HbO2 O2 HbO2 Lungs: low CO2 Hb picks up O2 Tissues: high CO2, low ph Hb releases O2 See Devlin 9.43

26 One more trick: 2,3 bisphosphoglycerate (BPG): -2,3 DPG is synthesized as a side reaction from glycolysis (Raport-Luenberg) - 2,3 DPG decreases the O2 affinity of Hb by stabilizing the deoxygenated form of hemoglobin through ionic cross-linking of beta chains (salt bridges). It therefore acts to enhance O 2 release. Glyceraldehyde-3-phosphate Fetal hemoglobin shows decreased 2,3 DPG binding activity, therefore it exhibits higher O2 affinity 1,3-Bisphosphoglycerate Pi 2,3- Bisphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate defects in glycolysis will alter oxygen transfer kinetics. Drugs which alter the activity of in hexokinase, pyruvate kinase, etc. can thus alter tissue oxygenation levels.

27 Role of 2,3 bisphosphoglycerate in O2 transport: - O C O H C OPO 3 2- H C OPO 2-3 H 2,3-BPG 5 negative charges and binds electrostatically 2,3-BPG binds tightly to deoxyhb, weakly to oxyhb (i.e. stabilizes the T form of Hb through B-B interations) O 2 affinity of Hb by keeping Hb in deoxy. conformation allows unloading of O 2 in tissues (increases P 50 of Hb)

28 Interaction of 2,3 BPG with Hemoglobin: (from Devlin 9.47)

29 Interaction of 2,3 BPG with Hemoglobin: Lys 82 BPG His 143 His 2 The five negative charges on DPG coordinate with positive charge on the globin chain. Coordination stoichimetry is 1:1. NH 3 + NH 3 + Lys 82 His 2 His 143 Stryer Fig. 7-26

30 Regulatory features of 2,3 BPG: High altitudes adaptation: 2,3-BPG levels double after 2 days P 50 so more O 2 unloaded in tissues Fetal RBCs: In the fetus, BPG binding to Hb is weaker than mother s Hb, Therefore: P 50 so O 2 transfer to fetus from mothers Hb.

31 Effect of CO2, 2,3 BPG on Hemoglobin O2 dissociation curve: P 50 Hb: 26 torr (blood) Voet fig 7-14

32 ENERGY METABOLISM (ERYTHROCYTES) Adequate dietary intake (North America): Carbohydrates and Fats used as primary fuel, or stored (as glycogen or in adipose tissue). Common monosaccharides: glucose(6), galactose(6), fructose(6); Disaccharides: sucrose (g+f), lactose (g+ga) and maltose (g+g) Starch, glycogen and cellulose are all polysaccarides (carbohydrates). Proteins (amino acids) used for cellular protein and nucleotide metabolism. Starvation conditions (24 hours): Blood glucose and glycogen used as primary fuel Glycerol from fat, amino acid from protein begin to be converted to glucose through gluconeogenesis (liver). Glucose remains dominant fuel supply for brain, erythrocytes, bone marrow, WBC s and renal medulla. Prolonged starvation (weeks): Fat and protein degradation can no longer maintain bodily needs, ketone body formation begins. Brain begins to utilize ketone bodies (max. starv. 100 days).