Oxygen: What’s it good for anyways?

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Oxygen Whats it good for anyways?
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• Outline
• Basic Concepts
• Diffusion
• Hemoglobin binding
• Oxygen equations
• Mitochondrial function
• Type IV Respiratory Failure
• Critical DO2
• Cytopathic hypoxia
• Microcirculation shunting

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Oxygen Diffusion

• Partial pressure of O2 at standard pressure and
  temperature is 21.3 kPa but falls to 14.7 kPa at
  the alveoli.
• Diffusion of O2 into the blood and then into the
  tissue is determined by Ficks law.
• Kpermeability of O2 within the diffusion medium
• Ssurface area
• Ppressure gradient
• ?diffusion distance

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Oxygen Diffusion

• In the lung, the diffusion barrier is the
  alveolar-capillary membrane.
• The PO2 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 PO2 at the
  mitochondria is about 1 mmHg.

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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
• When the curve shifts to the left or right, it
  alters the P50 (oxygen tension at which
  hemoglobin is 50 saturated).
• Shift to the left P50 decreases (i.e. lower PO2
  needed to saturate 50 of the hemoglobin)
• Shift to the right P50 increases (i.e. higher
  PO2 needed to saturate 50 of the hemoglobin).

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Hemoglobin Binding

• Name four conditions that shift the oxyhemoglobin
  curve to the left.
• Hypothermia
• Alkalosis
• CO
• Decreased 2,3-diglycerophosphate
• Name four conditions that shift the oxyhemoglobin
  curve to the right.
• Hyperthermia
• Acidosis
• Hypercarbia
• 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.
• Trivia What is the normal shifting of the
  oxyhemoglobin curve in the lungs and the tissue
  called?

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Oxygen Equations

• 1 gm of hemoglobin binds 1.34 mL of O2.
• The solubility of oxygen in serum is 0.03 mL of
  O2/ (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 dissolved in serum (0.03 mL/(L)(mmHg))(PO2
  mmHg)

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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 O2/min
  (assuming a cardiac output of 5 L/min and
  hemoglobin of 150 g/L)

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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 VO2 of 250
  mL/min?

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Oxygen Equations

• The ratio of VO2/DO2 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 CvO2.
• 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.

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Mitochondrial Function

• All reversible reactions proceed in the direction
  that results in a net decrease in the Gibbs
  energy for the system. (GH-TS)
• In order for living systems to carry out
  reactions that require a positive Gibbs energy,
  they must be coupled to a reaction that is
  energically favorable.
• If the total Gibbs energy for the two reactions
  is negative then the reactions can proceed.

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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.
• Oxidizing agents accept electrons.
• Oxygen is a very strong oxidizer while NADH and
  FADH are very strong reducers.

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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.
• First it has to generate NADH and FADH via the
  citric acid cycle.

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Mitochondrial Function

• The electrons are transferred through a series of
  intermediate compounds that have progressively
  lower reducing potentials.
• 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 F0F1ATPase that catalyzes the formation of
  ATP.
• Oxygens only job is to act as the final electron
  acceptor in the respiratory transport chain.

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Type IV Respiratory Failure
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Critical DO2

• With moderate reductions in DO2, the ER increases
  to satisfy VO2.
• What is the ER when DO2 is 1000 mL/min? (assume
  VO2 250 mL/min)
• What is the ER when DO2 is 500 mL/min?
• What is the ER when DO2 is 150 mL/min?
• The level at which VO2 begins to decline with
  declining DO2 is the critical DO2.
• At this point, VO2 becomes supply dependant and
  the tissues turn to anaerobic metabolism.
• The average critical DO2 is 4.2 mL/min/kg.

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Cytopathic Hypoxia

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

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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.

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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.
• Therefore, hyperlactatemia is not just evidence
  of low DO2.

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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) it out competes O2 for the same binding
  site.
• This causes a rapid but reversible inhibition of
  the enzyme.
• Since the reaction is reversible, this should not
  pose a major problem BUT

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Peroxynitrite Inhibition of Mitochondrial Enzymes

• NO also can react with O2- to form peroxynitrite
  (ONOO-) with is a powerful oxidizing and
  nitrosating agent.
• ONOO- inhibits F0F1 ATPase and Complex I and II.
• ONOO- also inhibits aconitase (TCA enzyme).
• Unlike NO, these inhibitions are irreversible.

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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 O2 in ATP
  production.

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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 local flow patterns to
  ensure global oxygenation.

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Microcirculation shunting

• In sepsis, endothelial cells
• Are less responsive to vasoactive agents.
• Lose their anionic charge and glycocalyx.
• Become leaky
• 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.

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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.

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Lactate

• Sympathy for the Devil

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Background

• For years lactate was considered a waste product
  of metabolism.
• Recent bench work points to its important role in
  intracellular signaling and energy transport in
  the muscle, brain and sperm.
• In sepsis, the classic explanation for
  hyperlactatemia has been anaerobic glycolysis due
  to insufficient oxygen delivery.

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The Case

• Recent it has been suggested that the
  hyperlactatemia in sepsis may be from an
  adrenaline surge that stimulates Na/K/ATPase
  activity and coupled aerobic glycolysis.
• To review, adrenaline binds B2-adrenoreceptors,
  activating adenylate cyclase, catalyzing ATP to
  cAMP.
• cAMP activates PKA which activates Na/K/ATPase.
• The ATPase pump derives its energy from
  glycolysis.
• Therefore, hyperlactatemia in the face of
  hemodynamic stability may be adrenaline
  stimulated aerobic glycolysis rather than tissue
  hypoxia.

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The Evidence

• Aerobically incubated muscle from septic rats had
  an increased rate of lactate production that was
  partially inhibited by ouabain (Na/K/ATPase
  inhibitor).
• Endotoxemia in heathy humans causes a rise in
  adrenaline and lactate levels.
• The lactate rise in the leg was matched by a fall
  in regional potassium levels in the blood and
  increase in uptake.
• No evidence of hypoxia or hypoperfusion.

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More Evidence

• A similar mechanism is in play with hemorrhage.
• Blocking adrenergic receptors in bleeding rats
  caused a significant fall in lactate levels with
  a increased Na/K ratio (implying decreased
  Na/K/ATPase activity).
• Administering ouabain to muscles that were either
  bled or infused with adrenaline reduced the
  lactate level equally compared to controls.

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Conclusion

• Tissue hypoperfusion, hypoxia and anaerobic
  glycolysis are probably not the only cause of
  increased lactate production in shock.

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• Outline
• Basic Concepts
• Diffusion
• Hemoglobin binding
• Oxygen equations
• Mitochondrial function
• Type IV Respiratory Failure
• Critical DO2
• Cytopathic hypoxia
• Microcirculation shunting
• Lactate Maybe not the boogie man after all.

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Questions???