oxygen: what’s it good for anyways?. outline basic concepts basic concepts diffusiondiffusion...

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

OutlineOutline Basic ConceptsBasic Concepts

• DiffusionDiffusion• Hemoglobin bindingHemoglobin binding• Oxygen equationsOxygen equations• Mitochondrial functionMitochondrial function

Type IV Respiratory FailureType IV Respiratory Failure• Critical DOCritical DO22

• Cytopathic hypoxiaCytopathic hypoxia• Microcirculation shuntingMicrocirculation shunting

Oxygen DiffusionOxygen Diffusion

Partial pressure of OPartial pressure of O22 at standard pressure and at standard pressure and temperature is 21.3 kPa but falls to 14.7 kPa at the temperature is 21.3 kPa but falls to 14.7 kPa at the alveoli.alveoli.

Diffusion of ODiffusion of O22 into the blood and then into the tissue is into the blood and then into the tissue is determined by Fick’s law.determined by Fick’s law.

K=permeability of OK=permeability of O22 within the diffusion medium within the diffusion medium S=surface areaS=surface area P=pressure gradientP=pressure gradient =diffusion distance=diffusion distance

PS

KDiffusion

Oxygen DiffusionOxygen Diffusion

In the lung, the diffusion barrier is the In the lung, the diffusion barrier is the alveolar-capillary membrane.alveolar-capillary membrane.

The POThe PO22 is 100 mmHg on the alveolar side is 100 mmHg on the alveolar side and 90 mmHg on the capillary side.and 90 mmHg on the capillary side.

At the tissue level, the capillary wall is the At the tissue level, the capillary wall is the primary barrier.primary barrier.

The diffusion distance can vary but the The diffusion distance can vary but the pressure gradient is much higher as the pressure gradient is much higher as the POPO22 at the mitochondria is about 1 mmHg. at the mitochondria is about 1 mmHg.

Hemoglobin BindingHemoglobin Binding Once oxygen has crossed the capillary Once oxygen has crossed the capillary

membrane, it enters the red blood cells and membrane, it enters the red blood cells and binds to hemoglobin.binds to hemoglobin.

Why is the oxyhemoglobin dissociation curve Why is the oxyhemoglobin dissociation curve sigmoid?sigmoid? CooperativityCooperativity

When the curve shifts to the left or right, it alters When the curve shifts to the left or right, it alters the Pthe P5050 (oxygen tension at which hemoglobin is (oxygen tension at which hemoglobin is 50% saturated).50% saturated).

Shift to the left – PShift to the left – P5050 decreases (i.e. lower PO decreases (i.e. lower PO22 needed to saturate 50% of the hemoglobin)needed to saturate 50% of the hemoglobin)

Shift to the right – PShift to the right – P5050 increases (i.e. higher PO increases (i.e. higher PO2 2 needed to saturate 50% of the hemoglobin).needed to saturate 50% of the hemoglobin).

Hemoglobin BindingHemoglobin Binding Name four conditions that shift the oxyhemoglobin curve to the left.Name four conditions that shift the oxyhemoglobin curve to the left.

HypothermiaHypothermia AlkalosisAlkalosis COCO Decreased 2,3-diglycerophosphateDecreased 2,3-diglycerophosphate

Name four conditions that shift the oxyhemoglobin curve to the right.Name four conditions that shift the oxyhemoglobin curve to the right. HyperthermiaHyperthermia AcidosisAcidosis HypercarbiaHypercarbia Increased 2,3-DPGIncreased 2,3-DPG

What happens to red cells from the blood bank?What happens to red cells from the blood bank? What is the purpose of 2,3 DPG?What is the purpose of 2,3 DPG?

Binds deoxyhemoglobin to stabilize the T-state and forces release of Binds deoxyhemoglobin to stabilize the T-state and forces release of oxygen. A lack of 2,3 DPG mimics fetal hemoglobin.oxygen. A lack of 2,3 DPG mimics fetal hemoglobin.

Trivia – What is the normal shifting of the oxyhemoglobin curve in Trivia – What is the normal shifting of the oxyhemoglobin curve in the lungs and the tissue called?the lungs and the tissue called?

Oxygen EquationsOxygen Equations

1 gm of hemoglobin binds 1.34 mL of O1 gm of hemoglobin binds 1.34 mL of O22..

The solubility of oxygen in serum is 0.03 mL of The solubility of oxygen in serum is 0.03 mL of OO22/ (L)(mmHg)./ (L)(mmHg).

Since there is no other way to transport oxygen, Since there is no other way to transport oxygen, the total oxygen content of blood is the sum of:the total oxygen content of blood is the sum of: That bound to hemoglobin: (1.34 mL/g)(Hgb g/L)That bound to hemoglobin: (1.34 mL/g)(Hgb g/L)

(Saturation)(Saturation) That dissolved in serum: (0.03 mL/(L)(mmHg))(POThat dissolved in serum: (0.03 mL/(L)(mmHg))(PO22

mmHg)mmHg)

222 03.034.1 OPOSHgbOC aaa


In order to calculate the total amount of oxygen In order to calculate the total amount of oxygen delivery (global), multiply the cardiac output by delivery (global), multiply the cardiac output by the oxygen content.the oxygen content.

Normal oxygen delivery is 1000 ml ONormal oxygen delivery is 1000 ml O22/min /min (assuming a cardiac output of 5 L/min and (assuming a cardiac output of 5 L/min and hemoglobin of 150 g/L)hemoglobin of 150 g/L)

)/(min)/( 22 LmLOCLCODO a


The amount of oxygen consumed in any tissue can be The amount of oxygen consumed in any tissue can be calculated by measuring the oxygen content in both the calculated by measuring the oxygen content in both the arterial and venous limb of the tissue.arterial and venous limb of the tissue.

The normal global oxygen consumption is 250 mL/min.The normal global oxygen consumption is 250 mL/min. What would be the required cardiac output in the What would be the required cardiac output in the

absence of hemoglobin to support a VOabsence of hemoglobin to support a VO22 of 250 mL/min? of 250 mL/min?

222 OCOCCOOV va

Oxygen EquationsOxygen Equations The ratio of VOThe ratio of VO22/DO/DO22 is the oxygen extraction ratio (ER). is the oxygen extraction ratio (ER). How can you calculate the ER without knowing the Hgb?How can you calculate the ER without knowing the Hgb? The ER increases in conditions such as exercise, CHF, and anemia The ER increases in conditions such as exercise, CHF, and anemia

as a result of a lower CvOas a result of a lower CvO22.. The converse occurs in sepsis.The converse occurs in sepsis. Each organ has its own metabolic needs so individual organ ER Each organ has its own metabolic needs so individual organ ER

vary.vary. The brain and the heart extract much more oxygen and thus are The brain and the heart extract much more oxygen and thus are

more susceptible to decreased delivery.more susceptible to decreased delivery.

Mitochondrial FunctionMitochondrial Function

All reversible reactions proceed in the direction All reversible reactions proceed in the direction that results in a net decrease in the Gibbs that results in a net decrease in the Gibbs energy for the system. (G=H-TS)energy for the system. (G=H-TS)

In order for living systems to carry out reactions In order for living systems to carry out reactions that require a positive Gibbs energy, they must that require a positive Gibbs energy, they must be coupled to a reaction that is energically be coupled to a reaction that is energically favorable.favorable.

If the total Gibbs energy for the two reactions is If the total Gibbs energy for the two reactions is negative then the reactions can proceed.negative then the reactions can proceed.


Aerobic generation of ATP occurs as a Aerobic generation of ATP occurs as a result of series of stepwise reactions that result of series of stepwise reactions that couple the oxidation of substrates to couple the oxidation of substrates to oxygen with the phosphorylation of ATP.oxygen with the phosphorylation of ATP.

To review: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 Oxygen is a very strong oxidizer while NADH

and FADH are very strong reducers.and FADH are very strong reducers.


The reaction of oxygen to NADH or FADH The reaction of oxygen to NADH or FADH has a very negative Gibbs energy whereas has a very negative Gibbs energy whereas the phosphorylation of ADP to ATP has a the phosphorylation of ADP to ATP has a low positive Gibbs energy.low positive Gibbs energy.

To capture the released energy efficiently, To capture the released energy efficiently, mitochondria step down the reaction.mitochondria step down the reaction.

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


The electrons are transferred through a series of The electrons are transferred through a series of intermediate compounds that have progressively lower intermediate compounds that have progressively lower reducing potentials.reducing potentials.

This respiratory chain is located on the inner membrane This respiratory chain is located on the inner membrane of the mitochondria.of the mitochondria.

The energy thus released is used to pump protons from The energy thus released is used to pump protons from the mitochondrial matrix to the intermembrane space.the mitochondrial matrix to the intermembrane space.

The protons then follow their gradient through the The protons then follow their gradient through the FF00FF11ATPase that catalyzes the formation of ATP.ATPase that catalyzes the formation of ATP.

Oxygen’s only job is to act as the final electron acceptor Oxygen’s only job is to act as the final electron acceptor in the respiratory transport chain. in the respiratory transport chain.

Type IV Respiratory Type IV Respiratory FailureFailure

Critical DOCritical DO22

With moderate reductions in DOWith moderate reductions in DO22, the ER , the ER increases to satisfy VOincreases to satisfy VO22..

What is the ER when DOWhat is the ER when DO22 is 1000 mL/min? is 1000 mL/min? (assume VO(assume VO22 = 250 mL/min) = 250 mL/min)

What is the ER when DOWhat is the ER when DO22 is 500 mL/min? is 500 mL/min? What is the ER when DOWhat is the ER when DO22 is 150 mL/min? is 150 mL/min? The level at which VOThe level at which VO22 begins to decline with begins to decline with

declining DOdeclining DO22 is the critical DO is the critical DO22.. At this point, VOAt this point, VO22 becomes supply dependant and becomes supply dependant and

the tissues turn to anaerobic metabolism.the tissues turn to anaerobic metabolism. The average critical DOThe average critical DO22 is 4.2 mL/min/kg. is 4.2 mL/min/kg.

Cytopathic HypoxiaCytopathic Hypoxia

There are four different but mutually There are four different but mutually compatible mechanisms to explain compatible mechanisms to explain decreased oxygen consumption in sepsis:decreased oxygen consumption in sepsis: Inhibition of pyruvate dehydrogenaseInhibition of pyruvate dehydrogenase NO mediated inhibition of cytochrome a,a3NO mediated inhibition of cytochrome a,a3 Peroxynitrite inhibition of mitochondrial Peroxynitrite inhibition of mitochondrial

enzymesenzymes Poly(ADP-ribose) polymerasePoly(ADP-ribose) polymerase

Inhibition of Pyruvate Inhibition of Pyruvate Dehydrogenase (PDH)Dehydrogenase (PDH)

End product of glycolysis End product of glycolysis is pyruvic acid.is pyruvic acid.

It can be reduced to It can be reduced to either lactate or enter either lactate or enter TCA.TCA.

PDH converts pyruvate to PDH converts pyruvate to acetyl-coenzyme A in the acetyl-coenzyme A in the presence of NAD+ and presence of NAD+ and coenzyme A.coenzyme A.

PDH kinase PDH kinase phosphorylates PDH to phosphorylates PDH to inactive form.inactive form.

Inhibition of Pyruvate Inhibition of Pyruvate Dehydrogenase (PDH)Dehydrogenase (PDH)

In sepsis, the activity of PDH kinase is In sepsis, the activity of PDH kinase is increased.increased.

The inactivation of PDH limits the flux of The inactivation of PDH limits the flux of pyruvate through TCA cycle.pyruvate through TCA cycle.

Excess pyruvate accumulates and leads to Excess pyruvate accumulates and leads to increased production of lactate.increased production of lactate.

Therefore, hyperlactatemia is not just Therefore, hyperlactatemia is not just evidence of low DOevidence of low DO22..

NO-mediated inhibition of NO-mediated inhibition of Cytochrome a,a3Cytochrome a,a3

Sepsis induces iNOS to produce NO.Sepsis induces iNOS to produce NO. When NO binds to cytochrome a,a3 (last When NO binds to cytochrome a,a3 (last

step in the ETC) it out competes Ostep in the ETC) it out competes O22 for the for the

same binding site.same binding site. This causes a rapid but reversible This causes a rapid but reversible

inhibition of the enzyme.inhibition of the enzyme. Since the reaction is reversible, this should Since the reaction is reversible, this should

not pose a major problem BUT…not pose a major problem BUT…

Peroxynitrite Inhibition of Peroxynitrite Inhibition of Mitochondrial EnzymesMitochondrial Enzymes

NO also can react with ONO also can react with O22- to form - to form peroxynitrite (ONOO-) with is a powerful peroxynitrite (ONOO-) with is a powerful oxidizing and nitrosating agent.oxidizing and nitrosating agent.

ONOO- inhibits FONOO- inhibits F00FF11 ATPase and Complex ATPase and Complex I and II.I and II.

ONOO- also inhibits aconitase (TCA ONOO- also inhibits aconitase (TCA enzyme).enzyme).

Unlike NO, these inhibitions are Unlike NO, these inhibitions are irreversible.irreversible.

Poly(ADP-ribose) Polymerase Poly(ADP-ribose) Polymerase (PARP-1)(PARP-1)

PARP-1 is a nuclear enzyme involved PARP-1 is a nuclear enzyme involved in the repair of single strand breaks of in the repair of single strand breaks of DNA.DNA.

It catalyzes the cleavage of NAD+ into It catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide and then ADP-ribose and nicotinamide and then polymerizes the ADP-ribose into polymerizes the ADP-ribose into homopolymers.homopolymers.

ROS and ONOO- can induce single ROS and ONOO- can induce single strand breaks in DNA which activates strand breaks in DNA which activates PARP-1.PARP-1.

The PARP-1 causes the NAD+/NADH The PARP-1 causes the NAD+/NADH content to fall which impairs the cells content to fall which impairs the cells ability to use Oability to use O22 in ATP production. in ATP production.

Microcirculation shuntingMicrocirculation shunting

The endothelium is an important regulator The endothelium is an important regulator of oxygen delivery.of oxygen delivery.

In response to local blood flow and other In response to local blood flow and other stimuli, it signals upstream to dilate stimuli, it signals upstream to dilate feeding arterioles.feeding arterioles.

RBC can sense hypoxia and release RBC can sense hypoxia and release vasodilators such as NO and ATP.vasodilators such as NO and ATP.

The goal is to control local flow patterns to The goal is to control local flow patterns to ensure global oxygenation.ensure global oxygenation.


In sepsis, endothelial cells: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 leakyBecome leaky Massively over express NO.Massively over express NO.

RBC and WBC cell deformability reduces, RBC and WBC cell deformability reduces, causing plugging.causing plugging.

The WBC and endothelium interact in ways to The WBC and endothelium interact in ways to induce inflammation and coagulation pathways.induce inflammation and coagulation pathways.


Inflammatory activation of NO is one of the Inflammatory activation of NO is one of the key mechanism responsible for shunting.key mechanism responsible for shunting.

Inhomogeneous expression of iNOS Inhomogeneous expression of iNOS interferes with regional blood flow and interferes with regional blood flow and promotes shunting from vulnerable promotes shunting from vulnerable microcirculatory units.microcirculatory units.

Inhomogeneous expression of endothelial Inhomogeneous expression of endothelial adhesion molecules also contribute adhesion molecules also contribute through their effects on WBC kinetics.through their effects on WBC kinetics.

LactateLactate

Sympathy for the DevilSympathy for the Devil

BackgroundBackground

For years lactate was considered a waste For years lactate was considered a waste product of metabolism.product of metabolism.

Recent bench work points to its important Recent bench work points to its important role in intracellular signaling and energy role in intracellular signaling and energy transport in the muscle, brain and sperm.transport in the muscle, brain and sperm.

In sepsis, the classic explanation for In sepsis, the classic explanation for hyperlactatemia has been anaerobic hyperlactatemia has been anaerobic glycolysis due to insufficient oxygen glycolysis due to insufficient oxygen delivery.delivery.

The CaseThe Case Recent it has been suggested that the Recent it has been suggested that the

hyperlactatemia in sepsis may be from an hyperlactatemia in sepsis may be from an adrenaline surge that stimulates Na/K/ATPase adrenaline surge that stimulates Na/K/ATPase activity and coupled activity and coupled aerobicaerobic glycolysis. glycolysis. To review, adrenaline binds B2-adrenoreceptors, To review, adrenaline binds B2-adrenoreceptors,

activating adenylate cyclase, catalyzing ATP to activating adenylate cyclase, catalyzing ATP to cAMP.cAMP.

cAMP activates PKA which activates Na/K/ATPase.cAMP activates PKA which activates Na/K/ATPase. The ATPase pump derives its energy from glycolysis.The ATPase pump derives its energy from glycolysis.

Therefore, hyperlactatemia in the face of Therefore, hyperlactatemia in the face of hemodynamic stability may be adrenaline hemodynamic stability may be adrenaline stimulated aerobic glycolysis rather than tissue stimulated aerobic glycolysis rather than tissue hypoxia.hypoxia.

The EvidenceThe Evidence

Aerobically incubated muscle from septic Aerobically incubated muscle from septic rats had an increased rate of lactate rats had an increased rate of lactate production that was partially inhibited by production that was partially inhibited by ouabain (Na/K/ATPase inhibitor).ouabain (Na/K/ATPase inhibitor).

Endotoxemia in heathy humans causes a Endotoxemia in heathy humans causes a rise in adrenaline and lactate levels.rise in adrenaline and lactate levels. The lactate rise in the leg was matched by a The lactate rise in the leg was matched by a

fall in regional potassium levels in the blood fall in regional potassium levels in the blood and increase in uptake.and increase in uptake.

No evidence of hypoxia or hypoperfusion.No evidence of hypoxia or hypoperfusion.

More EvidenceMore Evidence

A similar mechanism is in play with A similar mechanism is in play with hemorrhage.hemorrhage. Blocking adrenergic receptors in bleeding rats Blocking adrenergic receptors in bleeding rats

caused a significant fall in lactate levels with a caused a significant fall in lactate levels with a increased Na/K ratio (implying decreased increased Na/K ratio (implying decreased Na/K/ATPase activity).Na/K/ATPase activity).

Administering ouabain to muscles that were Administering ouabain to muscles that were either bled or infused with adrenaline reduced either bled or infused with adrenaline reduced the lactate level equally compared to controls.the lactate level equally compared to controls.

ConclusionConclusion

Tissue hypoperfusion, hypoxia and Tissue hypoperfusion, hypoxia and anaerobic glycolysis are probably not anaerobic glycolysis are probably not the only cause of increased lactate the only cause of increased lactate

production in shock.production in shock.

OutlineOutline Basic ConceptsBasic Concepts

• DiffusionDiffusion• Hemoglobin bindingHemoglobin binding• Oxygen equationsOxygen equations• Mitochondrial functionMitochondrial function

Type IV Respiratory FailureType IV Respiratory Failure• Critical DOCritical DO22

• Cytopathic hypoxiaCytopathic hypoxia• Microcirculation shuntingMicrocirculation shunting

Lactate – Maybe not the boogie man after all.Lactate – Maybe not the boogie man after all.

Questions???Questions???

oxygen: what’s it good for anyways?. outline basic concepts basic concepts diffusiondiffusion...

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