1. Oxygen: What’s it good for anyways?

2.  Outline Basic Concepts Basic Concepts DiffusionDiffusion Hemoglobin bindingHemoglobin binding Oxygen equationsOxygen equations Mitochondrial functionMitochondrial function Type IV Respiratory Failure Type IV Respiratory Failure Critical DO 2Critical DO 2 Cytopathic hypoxiaCytopathic hypoxia Microcirculation shuntingMicrocirculation shunting

3. Oxygen Diffusion  Partial pressure of O 2 at standard pressure and temperature is 21.3 kPa but falls to 14.7 kPa at the alveoli.  Diffusion of O 2 into the blood and then into the tissue is determined by Fick’s law. K=permeability of O 2 within the diffusion medium K=permeability of O 2 within the diffusion medium S=surface area S=surface area P=pressure gradient P=pressure gradient =diffusion distance =diffusion distance

4. Oxygen Diffusion  In the lung, the diffusion barrier is the alveolar-capillary membrane.  The PO 2 is 100 mmHg on the alveolar side and 90 mmHg on the capillary side.  At the tissue level, the capillary wall is the primary barrier.  The diffusion distance can vary but the pressure gradient is much higher as the PO 2 at the mitochondria is about 1 mmHg.

5. Hemoglobin Binding  Once oxygen has crossed the capillary membrane, it enters the red blood cells and binds to hemoglobin.  Why is the oxyhemoglobin dissociation curve sigmoid? Cooperativity Cooperativity  When the curve shifts to the left or right, it alters the P 50 (oxygen tension at which hemoglobin is 50% saturated).  Shift to the left – P 50 decreases (i.e. lower PO 2 needed to saturate 50% of the hemoglobin)  Shift to the right – P 50 increases (i.e. higher PO 2 needed to saturate 50% of the hemoglobin).

7. Hemoglobin Binding  Name four conditions that shift the oxyhemoglobin curve to the left. Hypothermia Hypothermia Alkalosis Alkalosis CO CO Decreased 2,3-diglycerophosphate Decreased 2,3-diglycerophosphate  Name four conditions that shift the oxyhemoglobin curve to the right. Hyperthermia Hyperthermia Acidosis Acidosis Hypercarbia Hypercarbia Increased 2,3-DPG Increased 2,3-DPG  What happens to red cells from the blood bank?  What is the purpose of 2,3 DPG? Binds deoxyhemoglobin to stabilize the T-state and forces release of oxygen. A lack of 2,3 DPG mimics fetal hemoglobin. Binds deoxyhemoglobin to stabilize the T-state and forces release of oxygen. A lack of 2,3 DPG mimics fetal hemoglobin.  Trivia – What is the normal shifting of the oxyhemoglobin curve in the lungs and the tissue called?

8. Oxygen Equations  1 gm of hemoglobin binds 1.34 mL of O 2.  The solubility of oxygen in serum is 0.03 mL of O 2 / (L)(mmHg).  Since there is no other way to transport oxygen, the total oxygen content of blood is the sum of: That bound to hemoglobin: (1.34 mL/g)(Hgb g/L)(Saturation) That bound to hemoglobin: (1.34 mL/g)(Hgb g/L)(Saturation) That dissolved in serum: (0.03 mL/(L)(mmHg))(PO 2 mmHg) That dissolved in serum: (0.03 mL/(L)(mmHg))(PO 2 mmHg)

9. Oxygen Equations  In order to calculate the total amount of oxygen delivery (global), multiply the cardiac output by the oxygen content.  Normal oxygen delivery is 1000 ml O 2 /min (assuming a cardiac output of 5 L/min and hemoglobin of 150 g/L)

10. Oxygen Equations  The amount of oxygen consumed in any tissue can be calculated by measuring the oxygen content in both the arterial and venous limb of the tissue.  The normal global oxygen consumption is 250 mL/min.  What would be the required cardiac output in the absence of hemoglobin to support a VO 2 of 250 mL/min?

11. Oxygen Equations  The ratio of VO 2 /DO 2 is the oxygen extraction ratio (ER).  How can you calculate the ER without knowing the Hgb?  The ER increases in conditions such as exercise, CHF, and anemia as a result of a lower CVO 2.  The converse occurs in sepsis.  Each organ has its own metabolic needs so individual organ ER vary.  The brain and the heart extract much more oxygen and thus are more susceptible to decreased delivery.

12. Mitochondrial Function  All reversible reactions proceed in the direction that results in a net decrease in the Gibbs energy for the system.  In order for living systems to carry out reactions that require a positive Gibbs energy, they must be coupled to reactions that is energically favorable.  If the total Gibbs energy for the two reactions is negative then the reactions can proceed.

13. Mitochondrial Function  The reaction of oxygen to NADH or FADH has a very negative Gibbs energy whereas the phosphorylation of ADP to ATP has a low positive Gibbs energy.  To capture the released energy efficiently, mitochondria step down the reaction.  The electrons are transferred through a series of intermediate compounds that have progressively lower reducing potentials.

14. Mitochondrial Function  Aerobic generation of ATP occurs as a result of series of stepwise reactions that couple the oxidation of substrates to oxygen with the phosphorylation of ATP.  To review: Reducing agents donate electrons. Reducing agents donate electrons. Oxidizing agents accept electrons. Oxidizing agents accept electrons. Oxygen is a very strong oxidizer while NADH and FADH are very strong reducers. Oxygen is a very strong oxidizer while NADH and FADH are very strong reducers.

16. Mitochondrial Function  This respiratory chain is located on the inner membrane of the mitochondria.  The energy thus released is used to pump protons from the mitochondrial matrix to the intermembrane space.  The protons then follow their gradient through the F 0 F 1 ATPase that catalyzes the formation of ATP.  Oxygen’s job is to act as the final electron acceptor in the respiratory transport chain.

18. Type IV Respiratory Failure

19. Critical DO 2  With moderate reductions in DO 2, the ER increases to satisfy VO 2.  What is the ER when DO 2 is 1000 mL/min? (assume VO 2 = 250 mL/min)  What is the ER when DO 2 is 500 mL/min?  What is the ER when DO 2 is 150 mL/min?  The level at which VO 2 begins to decline with declining DO 2 is the critical DO 2.  At this point, VO 2 becomes supply dependant and the tissues turn to anaerobic metabolism.  The average critical DO 2 is 4.2 mL/min/kg.

20. Cytopathic Hypoxia  There are four different but mutually compatible mechanisms to explain decreased oxygen consumption in sepsis: Inhibition of pyruvate dehydrogenase Inhibition of pyruvate dehydrogenase NO mediated inhibition of cytochrome a,a3 NO mediated inhibition of cytochrome a,a3 Peroxynitrite inhibition of mitochondrial enzymes Peroxynitrite inhibition of mitochondrial enzymes Poly(ADP-ribose) polymerase Poly(ADP-ribose) polymerase

21. Inhibition of Pyruvate Dehydrogenase (PDH)  End product of glycolysis is pyruvic acid.  It can be reduced to either lactate or enter TCA.  PDH converts pyruvate to acetyl-coenzyme A in the presence of NAD+ and coenzyme A.  PDH kinase phosphorylates PDH to inactive form.

22. Inhibition of Pyruvate Dehydrogenase (PDH)  In sepsis, the activity of PDH kinase is increased.  The inactivation of PDH limits the flux of pyruvate through TCA cycle.  Excess pyruvate accumulates and leads to increased production of lactate.  Hyperlactatemia is not just evidence of low DO 2.

23. NO-mediated inhibition of Cytochrome a,a3  Sepsis induces iNOS to produce NO.  When NO binds to cytochrome a,a3 (last step in the ETC) out competing O 2 for the same binding site.  This causes a rapid but reversible inhibition of the enzyme.  NO reacts with a limited range of intracellular targets and should not be regarded as toxic BUT…

24. Peroxynitrite Inhibition of Mitochondrial Enzymes  NO also can react with O 2 - to form peroxynitrite (ONOO-) with is a powerful oxidizing and nitrosating agent.  ONOO- inhibits F 0 F 1 ATPase and Complex I and II.  ONOO- also inhibits aconitase (TCA enzyme).  Unlike NO, these inhibitions are irreversible.

25. Poly(ADP-ribose) Polymerase (PARP-1)  PARP-1 is a nuclear enzyme involved in the repair of single strand breaks of DNA.  It catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide and then polymerizes the ADP-ribose into homopolymers.  ROS and ONOO- can induce single strand breaks in DNA which activates PARP-1.  The PARP-1 causes the NAD+/NADH content to fall which impairs the cells ability to use O 2 in ATP production.

26. Microcirculation shunting  The endothelium is an important regulator of oxygen delivery.  In response to local blood flow and other stimuli, it signals upstream to dilate feeding arterioles.  RBC can sense hypoxia and release vasodilators such as NO and ATP.  The goal is to control heterogeneous flow patterns but ensure homogeneous oxygenation.

27. Microcirculation shunting  In sepsis, endothelial cells: Are less responsive to vasoactive agents. Are less responsive to vasoactive agents. Lose their anionic charge and glycocalyx. Lose their anionic charge and glycocalyx. Become leaky Become leaky Massively over express NO. Massively over express NO.  RBC and WBC cell deformability reduces, causing plugging.  The WBC and endothelium interact in ways to induce inflammation and coagulation pathways.

28. Microcirculation shunting  Inflammatory activation of NO is one of the key mechanism responsible for shunting.  Inhomogeneous expression of iNOS interferes with regional blood flow and promotes shunting from vulnerable microcirculatory units.  Inhomogeneous expression of endothelial adhesion molecules also contribute through their effects on WBC kinetics.

29.  Outline Basic Concepts Basic Concepts DiffusionDiffusion Hemoglobin bindingHemoglobin binding Oxygen equationsOxygen equations Mitochondrial functionMitochondrial function Type IV Respiratory Failure Type IV Respiratory Failure Critical DO 2Critical DO 2 Cytopathic hypoxiaCytopathic hypoxia Microcirculation shuntingMicrocirculation shunting

30. Questions???