PHYSIOLOGY OF THE METABOLIC GASESClaude A. Piantadosi, M.D.Professor of MedicineDirector, Center for Hyperbaric Medicine And Environmental Physiology

THE METABOLIC GASES

• Questions for today• What are the physiological gases?• What is physiological O2 sensing?

• What is hypoxic vasodilation? • What is ROS signaling?• When does ROS production become pathological?

THE METABOLIC GASES

• Metabolic gases• CO2

• O2

• NO• CO• H2S

• ROS

• Inert gases• N2

• Ar• He• H2

THE METABOLIC GASES

• Metabolic Gas• CO2

• O2

• NO• CO• H2S

Physiology ToxicitypH/Vasodilation NarcosisRespiration/

OxidationVasoconstrictionVasodilation NitrationVasodilation AsphyxiaVasodilation Asphyxia

Metabolic gas concentrations vary not just with solubility and partial pressure, but with quantity and number of binding targets in cells and tissues— this defines their reactivity

THE METABOLIC GASES

• Physiological, adaptive, and toxic effects of all metabolic gases depend on dose and time

Conce

ntr

ati

on

Time

Toxic

AdaptivePhysiological

CARRIAGE OF CO2 IN BLOOD

CO2 is produced by mitochondrial TCA cycle and transported in the blood from tissue to lungs in three ways: Dissolved in solution Buffered with water as carbonic acid Bound to proteins, particularly hemoglobin

About 75% of CO2 is transported in RBCs and 25% in plasma

CARRIAGE OF CO2 IN BLOOD*

*Approximate values (CO2 content of blood is influenced by hemoglobin concentration and saturation, 2, 3-DPG, and pH). Estimates include bicarbonate and CO2 inside the RBC

100%

0%

60%90%

30%

10%5%

5%100%

0%

Carbamino

Arterial Venous

CO2 distribution in arterial & venous blood

HCO3-

Dissolved

THE METABOLIC GASES

• CO2 transport: RBC plays a critical role

Bohr effect

PaO2

SaO2

CO2 production by tissues favors O2 unloading

CO2 +H2O

H+ + HCO3-

Lungs

HCO3-

CA

CA

H2CO3

+ H2OCO2

Tissue

RBC

—CO2CO2—

Cl-Band 3

Plasma

THE METABOLIC GASES: CO2

• CO2 dissociation curve of blood

PO2 or PCO2 (mmHg)150100500

O2 or

CO

2 con

tent

(m

L/1

00 m

L)

40

20

60

0

Haldane effect

Oxyhemoglobin

Deoxyhemoglobin

Dissolved CO2

Dissolved O2

HbO2Bohr Effect

• O2 dissociation curve of blood

OXYGEN TRANSPORT TO TISSUES

• The O2 cascade

150

100

50

0

PO2

(mmHg)

Air Alveolus Capillary MitochondrionArtery

THE METABOLIC GASES

• Mitochondrial sink for cellular O2 diffusion

10-8 10-5 10-4 10-310-7 10-6

100

0

50 Cyt a,a3NADH

MbHb

State 3State 4

Oxygen concentration (M)

Hb or Mb(% Oxygenation)

NADHor

Cyt a,a3

(% Oxidation)

100

0

50

Increasing oxygen affinity

THE MAIN CONSUMER OF O2

6O2+ C6H12O6+ 30Pi2-+ 30ADP3-+ 30H+ 6CO2+ 30ATP4-+ 36H2O

Mitochondria ~95%

Respiration:

THE METABOLIC GASES

• Rate of O2 consumption depends on the Michaelis-Menton constant:

• O2 is rarely, if ever, rate-limiting under hyperbaric conditions

VO2

[O2]

Vmax

50%

KM

Diving and hyperbaric range3-4 ATA

PHYSICAL PROCESSES OF O2 TRANSPORT

• Diffusion— alveolus to blood• Chemical combination— hemoglobin• Convective transport— tissues• Chemical release— hemoglobin• Diffusion— blood plasma to cells• Chemical reduction to water— mitochondria

THE METABOLIC GASES

• HBO2 and the HbO2 dissociation curve

PO2 (mmHg)

CaO2

(ml/dl)

0

10

20

1000 1000 1500500

25

5

15Normal AVO2 difference

Dissolved oxygen

THE METABOLIC GASES

• Arterial O2 content— Sea level Air O2 and HBO2 at 2.5 ATA

CaO2 = 1.34 ml/g [Hb](SaO2) + 0.003 ml O2/dl/mmHg

= 1.34ml/g [15.0g/dl](1.0) + 0.003 ml/dl/mmHg O2x100 mmHg

= 1.34ml/g [15.0g/dl](1.0) + 0.3 ml O2

= 20 ml O2/dl + 0. 3 ml O2/dl= 20.3 ml O2/dl (Air)= 20 ml O2/dl + 2. 1 ml O2/dl= 22.1 ml O2/dl (O2)= 20 ml O2/dl + 5.4 ml O2/dl= 25.4 ml O2/dl

Dissolved Oxygen

THE METABOLIC GASES

• Determinants of PO2 in tissue• Capillary hematocrit• Position of hemoglobin O2 dissociation curve

• Adequacy and uniformity of perfusion• O2 shunting

• Capillary transit time• Rate of cell respiration

THE METABOLIC GASES

HBO2

THE METABOLIC GASES

• O2 diffusion into tissue—Krogh cylinder model

rVenous

r

PO2

Arterial

A

V

r

}r

PO2

A

V

}

Dead corner VO2 max

THE METABOLIC GASES

• O2 diffusion into tissues

rr

VenousAir r = 12mHBO2 r = 60m

ArterialAir r = 60mHBO2 r = 300m

PaO2

r

HBO2Air

Nitric oxide synthase: O2 + L-arginine NO.. + L-citrulline

Heme oxygenases: O2+ heme CO+ Fe + biliverdin

ROS generation: O2 .O2- H2O2 .OH 2H2O

e- e- e- e-

+2H+

NADPH

NADPH

+2H+

~5%

Cytochrome P450: O2 + RH + 2H+ + 2e– ROH + H2O NADPH

NADPH oxidases:: 2O2 + NADPH NADP+ + 2.O2- + H+

OTHER O2 CONSUMERS

THE METABOLIC GASES

• High PO2 promotes ROS generation• Protein oxidation• Thiol (SH) oxidation• Lipid peroxidation• DNA oxidation

O2 .O2- H2O2 .OH H2O

e- e- e- e-

+2H+

REDOX SIGNALING BY ROSPhysiological

StatesPathological

States

High ROS

Levels

LowROS

Levels

Cell proliferationAdaptation to stressPromote injury repairChange cell phenotype

Kills pathogensInterferes with cell functionBlocks cell repairCauses apoptosis/necrosisPromotes tissue injury

Chronic anti-oxidant therapyineffective or harmful

Chronic anti-oxidant therapymore likely to be effective

Localized De-localized

THE METABOLIC GASES: VASCULAR CONTROL BY NO

• Many vascular control events require NO production• Examples:

• CO2-induced vasodilation• NO plays a permissive role

• O2-induced vasoconstriction• Profound vasoconstriction at PO2 >500 mmHg• Arterial and venous vessels• Reduces cerebral, retinal, and renal blood flow• Limits inert gas clearance from tissues

THE METABOLIC GASES

• O2-induced vasoconstriction

• HBO2 decreases vasodilator activity of NO by generating superoxide (.O2

-)

• .O2- inactivates NO forming the strong oxidant

peroxynitrite (ONOO-)• Hyperoxia prevents allosteric unloading of NO from RBCs

by SNO-hemoglobin

nNOS

iNOSVascular NOS:

eNOS

METABOLIC GASES: NO

• NOS isoforms• nNOS (type I constitutive)• iNOS (type II inducible) • eNOS (type III constitutive and inducible)• mtNOS (nNOS variant)

METABOLIC GASES: NO

• O2-induced vasoconstriction

NO. + .O2 -

(superoxide) ONOO-

(peroxynitrite) ONOOH(peroxynitrous acid)

NO2 + OH.

H+

O2e-

Dilation ToxicityConstriction

(6.7 X 109 M-1 s-1)

NOS

Reactive nitrogen species (RNS)

Effector cell(endothelial)

Target cell(smooth muscle)

L-citrulline + NO

NOS

L-arginine + O2

NO-heme-sGC

GTP cGMP

NO

L-arginine

Arginosuccinate NG-OH-L-arginine

ArginaseL-ornithine

(-)

R-SNO + H+

R-SH

METABOLIC GASES: NO

• The L-arginine-nitric oxide pathway

Fleming I. Molecular mechanisms underlying the activation of eNOS. Pflugers Arch. May;459(6):793-806, 2010

METABOLIC GASES: NO

• Multiple levels of eNOS regulation• Transcriptional control• Translational control

• Cytokine-driven mRNA degradation • Post-translational modification

• Phosphorylation/ Myristoylation/ Palmitoylation• Protein-protein interactions (enzyme

localization) • Calmodulin/ Hsp90/ Caveolin

• Uncoupling • BH4/ L-arginine deficiency

H2O2 IS A PLEIOTROPIC VASODILATOR• H2O2 mediates endothelium-

dependent or independent vasorelaxation

• NO-dependent • NO- independent

• H2O2 activates eNOS in large vessels, leading to eNOS-dependent relaxation

• In small vessels, e.g. coronary arterioles, mitochondrial-derived H2O2 is responsible for flow-mediated vasodilation (NO-independent)

• In disease, e.g. atherosclerosis and hypertension, H2O2 produced by large vessels mediates compensatory, endothelial-dependent, but NO.-independent relaxation

• H2O2 may cause endothelium-independent relaxation via catalase compound I activation of smooth muscle cGMP

METABOLIC GASES: CO

• Carboxyhemoglobin (COHb) derived from endogenous and exogenous sources

Condition COHb

Normal 1-2%

Pregnancy 2-4%

Hemolytic anemia 2-6%

Cigarette smoking 4-5% /pack/day

CO poisoning 20-50%

METABOLIC GASES: CO

• CO decreases blood O2 content and tissue PO2

20

15

5

10

01000 50 7525

PaO2 (mm Hg)

CaO2 or CvO2

(ml/dl)

100% HbO2

50% COHb

AVDO2

AVDO2

METABOLIC GASES

• CO and CO body stores• OSHA 8-hour exposure limit is 50 ppm• Endogenous CO production by HO reflects ~ 1-5

ppm

Endogenous CO production

Metabolism to CO2

Hemoproteinenzymes

Myoglobin

CO

ExtravascularIntravascular

COHbAlveolar gas

METABOLIC GASES: CO

Dual mechanism of CO poisoning Chemical asphyxia (CO hypoxia)

COHb has increased O2 affinityCOHb does not carry O2

Haldane’s First Law:[COHb]/[HbO2]= M (PCO/PO2), M=220

Cellular poisoning—heme protein binding Warburg constant: K= (n/1-n)(CO/ O2) Where n, the fraction bound to CO, is equal to 0.5 K is the ratio of CO:O2 to half-saturate the binding site

Tissue hypoxia

PGC-1

REDOX-REGULATION OF MITOCHONDRIAL BIOGENESIS

PI3K/PTEN

GSK3

Sepsis-induced inflammation

Akt1

NRF-1

Nrf2

Nrf2Keap1

P

Nucleus

-SH oxidation

PP

ARE

HO-1/CO

NRF-1

NRF-1

P

Proteasome

Nrf2

AREHmox1

Nrf2

Ub

Nrf2

Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1. Piantadosi CA, Carraway MS, Babiker A, Suliman HB. Circ Res. 2008 Nov 21;103(11):1232-40.

Mitochondrial biogenesisAnti-oxidant enzyme inductionAnti-apoptosis (Bcl2)Counter-inflammation (IL-10)Mitophagy (p62)

TLRs

MyD88

NFkB

NO

THE METABOLIC GASES

• CO binds iron and other transition metals allowing it to interact with ROS and NO

Fe IIFe IIIe-

NO CO

.O2-

O2

Pro-oxidant

Anti-oxidant

ONOO-.OH

RSH

H2O2

.O2-

METABOLIC GASES: H2S

• Hydrogen sulfide• Sewer gas (rotten eggs)• Poisons mitochondrial ETC at high levels• Generated enzymatically by cells and plays several

physiological roles• Relationship to O2 mainly involve sulfide oxidation

Hydrogen sulfide chemosynthesis: 6CO2 + 6H2O + 3H2S = C6H12O6 + 3H2SO4

HYDROGEN SULFIDE CHEMOSYNTHESIS

• Chemosynthesis • Biological conversion of one or more carbons (usually CO2

or CH4) into organic matter by oxidation of inorganic molecules (H2 or H2S) or CH4 as a source of energy, rather than by sunlight (photosynthesis)

• Some bacteria do this, e.g. purple sulfur bacteria, instead of photosynthetic release of O2

• Yellow sulfur globules produced that are visible in the cell• Proposed that chemosynthesis may support life below the

surfaces of Mars, and Jupiter's moon Europa

METABOLIC GASES: H2S

Kabil O, Motl N, Banerjee R. H2S and its role in redox signaling. Biochim Biophys Acta 2014 Jan 11

METABOLIC GASES: H2S

• Enzymatic H2S Production

• 3-mercaptopyruvate sulfurtransferase (MST)• Cystathione gamma lyase (CSE)• Cystathionine beta-synthase (CBS)

• CBS normally condenses serine and homocysteine to cystathionine:

L-serine + L-homocysteine = L-cystathionine + H2O

•Only pyridoxal phosphate-dependent enzyme that contains a heme co-factor that functions as a redox sensor; modulates activity in response to redox potential. •Resting form of CBS has ferrous heme (Fe II) that is activated under oxidizing conditions by conversion to ferric state

•Fe (II) form is inhibited by CO or NO binding; activity doubles when Fe (II) Fe (III)

METABOLIC GASES: H2S

Controversies surround the sometimes conflicting effects of H2S (e.g. both pro- and anti-inflammatory) Highlights problems associated with interpreting

studies Very wide concentration range of H2S Technical challenges of handling a redox-active gas

Multiple mechanisms of H2S-based signaling Protein persulfidation Sulfhydration of electrophiles Interaction with S-nitrosothiols Interaction with metal centers

H2S SYNTHESIS AND DEGRADATION Tissue H2S concentration is

low 10–30 nM except in aorta

Sulfur flux into H2S in murine liver is comparable to GSH (6–10 mM at steady-state)

Thus, sulfide clearance rate must be high to account for low steady-state H2S concentrations

Sulfide biosynthesis

Sulfide clearance

Kabil O, Motl N, Banerjee R. H2S and its role in redox signaling. Biochim Biophys Acta 2014 Jan 11

METABOLIC GASES: H2S

Cysteine H2S

BiosynthesisCBSCSEMST

DegradationOxidation

Methylation

InteractionsNeuromodulation Muscle relaxation

Hibernation-like state

StorageIron sulfide (Fe-S)

Sulfane sulfurPolysulfides

KATP channel

NMDA receptorSulfhemoglobin

Sulfmyoglobin

Cytochrome oxidase

O2

O2

O2

O2

Olson KR, Whitfield NL (2010) Hydrogen sulfide and oxygen sensing in the cardiovascular system. Antioxid Redox Signal 12:1219–1234.

H2S is degraded mainly in mitochondria through a series of oxidations that convert the gas to sulfite (SO3

-2), thiosulfate (S2O3

-2), and sulfate (SO4-2 )

THE METABOLIC GASES

Summary O2’s role is not limited to aerobic metabolism,

but is involved in the production of and interactions with other metabolic gases Of the O2 used in the body, ~95% is reduced to H2O

by respiration Non-respiratory processes use ~5% (ROS, NO, and CO) An increase in tissue PO2 above that needed to

support respiration does not increase VO2, but does increase O2 utilization by the other processes (depending on Km) This may interfere with O2 regulation of these

processes Excessive ROS production leads to delocalization of

redox signaling, and macromolecular damage (oxidative stress), disordered repair and cell death