Glycocalyx and Lactate SOTAs

Where is the endothelial glycocalyx located under normal conditions?

Covers the luminal aspect of all blood vessels

What is the endothelial glycocalyx composed of?

Scaffolding of proteoglycans (PGs), glycoproteins (GPs), and glycosaminoglycans (GAGs) associated with the underlying endothelial cells

What do the endothelial glycocalyx and its associated molecules and fluid comprise?

The endothelial surface layer (ESL)

What is the function of proteoglycans in the glycocalyx?

Form the main scaffolding

What are the 2 main proteoglycans in the glycocalyx?

Syndecans

Glypicans

What is the function of syndecans in the glycocalyx?

• Transmembrane proteins comprised of extracellular, transmembrane, and cytosolic domains

  • Extracellular domain binds GAGs and detects extracellular signals, such as shear stress, which are transduced to the intracellular environment via the transmembrane portion and the cytoplasmic tail

What is the function of glypicans in the glycocalyx?

• Its ectodomain binds only the GAG heparan sulfate (HS)

• Its anchor molecule is thought to localize it around lipid rafts and caveolae

  • Caveolae - membrane structures rich in signaling molecules that serve as communication hubs in the cell membrane

  • Location allows glypicans to partake in various signaling cascades

What is the most abundant component of the glycocalyx?

Glycosaminoglycans

Form up to 95% of the proteoglycan composition

What are the five main GAGs in the glycocalyx?

Heparan sulfate

Chondroitin sulfate

Dermatan sulfate

Keratin sulfate

Hyaluronan

What is the most abundant GAG in the glycocalyx?

Heparan sulfate

What gives the endothelial glycocalyx a net negative charge?

Sulfate groups attached to GAGs

Synthesis and Attachment of Syndecan and Glypican

• Synthesis of core proteins syndecan and glypican occur on membrane bound ribosomes

  • They are transferred to the lumen of the endoplasmic reticulum followed by the Golgi apparatus where attachment, polymerization, and sulfation of GAG side chains occur

  • Core protein + GAG transferred to the cell surface where it is incorporated into the cell membrane (syndecans) or attached to the cell surface with an anchor molecule (glypican)

  • HA is not attached to a core protein and is synthesized on the cell membrane rather than the Golgi apparatus

Glycoproteins of the Glycocalyx

• Located on the EC surface

• Covered by the EG in health

• Endothelial GPs are membrane-bound cell adhesion molecules that are separated into 3 different families based on structural and functional characteristics

What are the families of glycoproteins

Selectins

Immunoglobulins

Integrins

What are selectins involved in?

Initial contact and adhesion of leukocytes and platelets to the activated endothelium

P-Selectin

• Found on endothelium and platelets

• Constitutively expressed

• Stored within granules of Weibel-Palade bodies of EC and platelets

• Translocated to the cell surface following stimulation by complement components, thrombin, histamine, or fibrin

  • Occurs within minutes

E-Selectin

• Found exclusively on the endothelium

• Inducible

• Requires transcription, translation, and translocation to the cell surface

• Upregulated following cytokine or antigenic stimulation

  • Reaches maximal levels within 4-6 hours

  • Returns to baseline within 24-48 hours

What is the function of immunoglobulins in the glycocalyx?

Support the adhesion and transmigration of leukocytes between ECs

What is the function of integrins in the glycocalyx?

Important for firm attachment of leukocytes and platelets to the endothelial cells as well as transduction of mechanical or chemical signals from the extracellular to the intracellular microenvironment

Endothelial Glycoproteins and the Leukocyte Recruitment Cascade

• Presence of endothelial GPs required for functioning of leukocyte recruitment cascade

  • Rolling

  • Adhesion

  • Transmigration

  • Diapedesis of neutrophils, monocytes, eosinophils, and some lymphocytes is achieved when these adhesion molecules bind to their respective leukocyte integrins

Soluble Components of the Glycocalyx

• Soluble plasma components are incorporated into the EG and form the ESL

• Enhance the EG by altering its physical properties such as thickness and permeability

• Albumin is one of the key soluble components within the EG

  • Appears to be required to impart normal barrier function to the ESL

  • Evidence suggests that albumin alone is not sufficient to maintain the barrier function of the ESL

• One study found that in addition to charge and size affecting the inclusion of molecules into the ESL, the shape of the molecule also appears to be important

• Different GAGs also bind to other molecules, which facilitate their integration into the ESL

Perfused Boundary Region

• In health, the negatively charged EG repels RBCs from its surface to facilitate laminar flow

  • An increase in the perfused boundary region (PBR, area accessible to RBCs within the vasculature) has been shown to be associated with a loss of EG thickness

  • PBR shown to significantly increase in critically ill people, with septic patients having the largest PBR

What are the crucial roles of the endothelial glycocalyx?

Maintaining normal vascular permeability and transvascular fluid flux

Cell-to-cell interactions (inflammation and coagulation)

Vascular mechanotransduction

Starling Hypothesis

• Movement of fluid between the intracapillary and interstitial spaces depends on the osmotic pressure generated by plasma proteins and the hydrostatic pressure (HP) differences between these two compartments

  • Intravascular fluid is filtered out of the vessel at the arterial end of the capillary and reabsorbed at the venous end

Starling Hypothesis Modified with the Discovery of the Endothelial Glycocalyx

• Colloid osmotic pressure (COP) within the interstitium contributes much less to the transendothelial fluid flux than previously thought

• Revised hypothesis suggests that the main COP gradient is not the difference of forces between the plasma and the interstitium, but rather between the plasma and the space immediately below the EG, the subglyceal space (SGS)

• The very low COP within the SGS causes the net COP difference between the SGS and the capillary lumen to be much larger than the COP difference between the interstitium and the lumen

  • Possible that the most physiologically relevant COP gradient exists between the ESL and the SGS, which is an even greater gradient than between the capillary lumen and the SGS

• Under steady state conditions, the rate of fluid extravasastion is low, but occurs along the entire length of the capillary and without net absorption of fluid at the venous end of the capillary

• The COP gradient, which is entirely intravascular, opposes but does not reverse fluid filtration

Why is the colloid osmotic pressure so low in the subglyceal space?

• Within the SGS, the COP is very low for 2 reasons

  • Albumin incorporation into the ESL increases its filter function by effectively excluding entry of larger macromolecules

  • Any back diffusion of proteins from the interstitium into the SGS is prevented by the high velocity of filtered fluid funneled through the interendothelial clefts directionally toward the interstitium

    • These clefts are the main sites of fluid movement from the vascular lumen to the interstitium

What controls solute exchange? What is passage of solutes restricted based on?

Negatively charged GAGs + the EG's ultrastructure control solute exchange, restricting passage of solutes based on size, shape, and charge

Fiber-Matrix Theory of the Endothelial Glycocalyx Microstructure

• a fibrous mesh (the EG) covers the entire endothelial surface and confers the molecular sieving properties to the vessel

  • Has been experimentally confirmed - enzymatic degradation of the EG resulted in a 60% increase in permeability

What size molecules is the endothelial glycocalyx permeable to?

• EG excludes large molecules while maintaining a relative permeability to smaller molecules, specifically those with radii less than 4-5 nm

  • Gaps of <10 nm significantly restrict permeability of the matrix for larger molecules

Effect of Albumin on the Endothelial Glycocalyx

• Albumin alters the EG structure to a regular, lattice-like structure which, in conjunction with its net negative charge, enhances the EGs selectivity to macromolecules

  • Albumin's effect of reducing vascular permeability is likely not solely due to an increase in COP but also its ability to become incorporated into the EG

    • Other plasma macromolecules, such as fibrinogen and orsomucoid, are also important to maintain permeability characteristics due to their incorporation into the ESL

Effect of the Glycocalyx on RBC Movement

• Thickness of the EG helps maintain normal RBC movement while simultaneously modulating the amount of fluid sheer stress on the EC

• EG aids in maintaining the laminar flow pattern (inner region of RBC's surrounded by outer layer of plasma and platelets) by preventing RBC from attaching to the EC

  • Negatively charged RBC glycocalyx repelled by the negatively charged EG

• An intact EG may improve microcirculatory flow by causing RBCs to develop a more elongated conformation which improves the efficiency and speed of RBC transit

  • Leads to an increase in oxygen exchange capacity and reduction in friction as blood moves through the microcirculation

• Changes in EG leads to changes in RBC glycocalyx and vice versa

Endothelial Glycocalyx and WBCs in Health

• In health, the GPs responsible for WBC recruitment and initiation of coagulation are hidden beneath the EG so the ESL is anti-inflammatory and anticoagulant

  • Physical thickness of the EG and its charge prevent circulating WBCs, which have their own glycocalyces, from accessing ECs

  • EG must be partially shed under inflammatory conditions to allow exposure of EC adhesion molecules and subsequent WBC diapedesis

Shedding of the Glycocalyx and WBCs

Shedding of the EG exposes WBC activators which upregulates WBC integrin expression and potentiates binding to their respective EC adhesion molecules

The Endothelial Glycocalyx and Regulation of Coagulation

• In health, the EG regulates coagulation by acting as a physical barrier to prevent EC and platelet adhesion molecule interaction as well as concentrating anticoagulant molecules within its structure

  • vWF is constitutively expressed on the surface of ECs and hidden beneath the EG, preventing unwanted platelet adhesion and activation

  • EG also binds many anticoagulant molecules including antithrombin (AT), thrombomodulin [TM], protein C, and TFPI

Endothelial Glycocalyx and Antithrombin

Within the EG, AT binds to regions of heparan sulfate, which enhances its anticoagulant activity on the EC surface

Endothelial Glycocalyx and Thrombomodulin

• TM, an integral membrane protein containing chondroitin sulfate, is also constitutively expressed on ECs beneath the EG

  • Association with chondroitin sulfate is essential to TM's anticoagulant ability and including into the EG

• Binding of TM to thrombin and further complex formation with protein C receptor expressed on the EC surface potentiates activation of the protein C anticoagulant pathway

Endothelial Glycocalyx and TFPI

TFPI is bound to heparan sulfate within the EG, inhibits the formation of TF-FVII complex

Mechanotransduction

The transformation of a mechanical force into a biochemical response

Endothelial Glycocalyx and Mechanotransduction

• EG core proteins sense shear stress and transmit this signal to the actin cytoskeleton via their transmembrane domain

  • Under shear stress, ECs produce NO

    • Production of NO occurs via activation of the enzyme endothelial NO synthase (eNOS), which leads to relaxation of subendothelial smooth muscle cells and thus vasodilation

• Degradation of the EG is associated with the loss of shear-induced NO release

• Studies suggest that heparan sulfate and/or HA is responsible for sensing vascular shear stress

• Without an intact EG, the vasculature cannot appropriately respond to hemodynamic forces, which could lead to direct mechanical damage to the EC and the inability to regulate vascular tone

• EG can reorganize its structure under conditions of high flow

Alteration of Vascular Permeability and Transvascular Fluid Flux with Damaged Endothelial Glycocalyx

• Damage to the EG can lead to alterations in transvascular fluid movement, capillary leak, and development of edema

• The large COP gradient across the EG supports retention of fluid within the vascular lumen

  • When the EG is damaged, the COP gradient is reduced or lost and fluid movement becomes more dependent on intravascular HP and the transendothelial HP gradient

• Destruction of the EG also results in the loss of its molecular sieving properties

  • Leads to extravasation of large macromolecules into the interstitium, which decreases intravascular COP and increases interstitial COP

  • In severe vasculitis, the relevant COP gradient is then between the plasma and the interstitial fluid rather than between the plasma and the SGS

• Vascular inflammation is associated with the loss of the structure of the EG

  • Inflamed vessels develop larger gaps in the EG leading to increased permeability

Damaged Endothelial Glycocalyx and WBCs

• In health, the EG shields numerous WBC adhesion molecules from circulating cells and creates an anti-inflammatory phenotype

• During inflammation, loss or thinning of the EG is physiologically advantageous by exposing the adhesion molecules required for WBC transmigration and ultimately the resolution of infection or tissue injury

  • With the loss of the EG, the endothelium changes from an anti-inflammatory phenotype to a proinflammatory phenotype

  • With widespread loss of the EG or with persistent activation of the endothelium, this adaptive response can become detrimental

• Excessive WBC adhesion can become detrimental as it physically obstructs the vascular lumen, particularly within microvessels

  • Causes increased resistance to blood flow and if widespread, microvascular dysfunction can occur

Damaged Glycocalyx and Freely Circulating GAGs

• Freely circulating GAGs shed from the EG may also directly affect inflammation in critical illness

  • Circulating GAGs can bind and impede the action of locally released antimicrobial peptides and activated complement fragments, impairing the body's innate defense mechanisms

  • Freely circulating GAGs could be associated with a reduced ability to clear infection in sepsis

  • Circulating GAG fragments may act as local and distant inflammatory stimulators and activate both the innate and adaptive immune systems

How does loss of the endothelial glycocalyx help transition the endothelium from an antithrombotic to a prothrombotic state?

• Loss of the EG is a key step in the transition of the endothelium from an antithrombotic to a prothrombotic state that is common in critical illness

  • Loss of the EG results in an increase in platelet adhesion and markers of hypercoagulability

  • Increased platelet-endothelial binding triggers EC activation resulting in activation of WBCs and complement

  • With EG loss the anticoagulant molecules are shed into the systemic circulation

    • May act at distant sites and contribute to a generalized hypocoagulable state

Damaged Glycocalyx and Mechanotransduction

• Loss of the EG leads to a reduction in normal vascular reactivity preventing required alterations in vessel tone

  • May increase requirement for vasopressor use

Biomarkers of Endothelial Glycocalyx Degradation

• Circulating EG degradation products as biomarkers to identify EG alterations or dysfunction are of interest

• Matrix metalloproteinase (MMPs) are a family of cell surface proteases that are responsible for degrading extracellular matrices

  • Thought to be some of the key enzymes responsible for the shedding of the EG in many diseases

• Syndecan-1, heparan sulfate, chondroitin sulfate, and HA are all considered valid markers of EG integrity

• EG shedding is exaggerated under pathological conditions such as SIRS and sepsis, trauma, ischemia and reperfusion (I-R) injury, hyperglycemia, hypervolemia, and major surgery

SIRS and Sepsis Leading to Glycocalyx Dysfunction

• An increase in circulating biomarkers of EG degradation such as SDC1, HS, and HA occurs in animals and people with SIRS or sepsis

• Multiple molecules have been identified as possible instigators of EG degradation during sepsis: TNF-a, ROS, MMPs, C-reactive protein (CRP), endogenous catecholamines, and heparanases

TNF-a and Glylcocalyx Dysfunction

• Linked to the degradation of the EG in sepsis

• Plays at least a partial role in EG degradation during inflammation

• During inflammation TNF-a release may activate MMPs and lead to degradation of the EG

C Reactive Protein and Glycocalyx Dysfunction

• Commonly used marker of inflammation but also implicated in directly contributing to EG degradation in sepsis

• CRP may not only be a marker of inflammation but an active contributor to the associated evolution of vascular dysfunction

Endogenous Catecholamines and Glycocalyx Dysfunction

• A large catecholamine surge may be partly responsible for EG degradation in sepsis

• In people with naturally occurring sepsis, the level of increase in both catecholamine and SDC1 concentrations was correlated with disease severity

Heparanase and Glycocalyx Dysfunction

• An enzyme specific for the cleavage of heparan sulfate

• Implicated in the development of ALI in septic patients

• Speculated that heparanase expression is induced by ECs after their stimulation by circulating molecules termed "danger signals"

  • "Danger signals" - endogenous molecules or molecular structures produced or released from cells that are damaged or undergoing cell death or exogenous molecules from pathogens, both of which activate the immune system

  • Heparanases then partially degrade the EG allowing the exposure of EC leukocyte adhesion molecules to facilitate WBC diapedesis, which ultimately clears the insult

• During sepsis, where there is a large concentration of circulating danger signals, there may be diffuse and persistent stimulation of pulmonary heparanase expression, which may lead to widespread pulmonary EG shedding

Systemic Effects of Loss of the Glycocalyx in Sepsis

• Development of tissue edema

  • Identification of high EG biomarkers at presentation to the ICU may be useful to help identify patients at risk of harm from large volume fluid resuscitation and who may benefit from early vasopressor support

• Excessive inflammation

• Alterations in coagulation

  • Initial EC activation has been associated with an initial period of hypercoagulability which can later progress to hypocoagulability

  • Studies have identified an association but not causality between increased EG degradation products and the development of hypocoagulability in sepsis

• Reduced vasomotor tone

Using Biomarkers of the Endothelial Glycocalyx to Aid Prognostication in Patients with Sepsis

• Multiple studies in people with sepsis have demonstrated an association between increased levels of plasma EG biomarkers and an increase in mortality

• Studies have also looked at GAG biomarkers in the urine rather than plasma which found that increased urinary GAG concentrations were associated with higher mortality and morbidity

Ischemia Reperfusion Injury Leading to Glycocalyx Dysfunction

• In companion animals, I-R is clinically relevant in the setting of postcardiac arrest (PCA) care and cases of thromboembolic disease

  • Postcardiac arrest syndrome is associated with the exposure of the body to widespread I-R injury and an increase in EG degradation biomarkers has been demonstrated in this condition

  • Studies suggest that global I-R may be the cause for EG degradation following cardiac arrest

  • Increased circulating catecholamine concentrations have also been associated with EG degradation in PCA human patients

  • The loss of the EG likely contributes to the common sequelae see in these cases: increased vascular permeability, hyperinflammation, coagulopathy, and reduced vascular responsiveness

Trauma and Hemorrhage Leading to Glycocalyx Dysfunction

• In people, clinical studies have demonstrated an increase in EG biomarkers, including HS, HA, SDC1, and CS after trauma or hemorrhagic shock

• There are no clinical veterinary studies investigating the link between EG degradation and trauma, but animal hemorrhagic shock models have demonstrated EG loss after serious hemorrhage

• Suggested mechanism for EG degradation in trauma is related to the large catecholamine release and tissue hypoperfusion that occurs with shock

  • Hypothesized that this leads to the direct loss of the EG

• Studies have found that catecholamine release activates the inflammatory response in trauma and induction of TNF-a may be the mechanism for EG loss

• Massive EG loss in trauma is associated with increased vascular permeability, increased systemic inflammation, hypocoagulability, and reduced vascular responsiveness

Glycocalyx Biomarkers and Hypocoagulability in Trauma

A correlation between increased EG biomarkers and hypocoagulability in human trauma patients has been demonstrated, suggesting that EG damage may play a role in acute traumatic coagulopathy

Endogenous Heparinization

• GAGs, particularly HS and CS, which are shed after a traumatic insult, act systemically as anticoagulants

• Traumatic coagulopathy may represent an adaptive evolutionary response that in some cases becomes maladaptive when severe, unregulated, and widespread or exacerbated by medical interventions such as fluid therapy

• Hypothesized that trauma creating an increasingly hypocoagulable state through endogenous heparanization counterbalances the proinflammatory and procoagulant state of an activated endothelium, reducing clot formation and maintaining perfusion through the microcirculation

  • Shedding of the EG may also be advantageous as it allows for increased vascular permeability and shifting of fluid from the intravascular to extravascular space

    • Reduces blood pressure, reducing ongoing hemorrhage and shifting of fluid to the extravascular space allows for a pool of fluid for later mobilization

Hypervolemia Leading to Glycocalyx Dysfunction

• Hypervolemia leads to atrial distension and release of ANP

  • ANP reduces intravascular volume by vasodilating vascular beds, increasing renal excretion of fluid, and increasing vascular permeability

• ANP release leads to EG shedding resulting in extravasation of fluid and colloids from the vasculature

  • May lead to detrimental edema formation and reduced oxygenation

• Multiple studies in human medicine and veterinary medicine have shown that fluid overload is associated with increased morbidity and mortality

  • Emphasize that resuscitative fluid therapy is not a benign intervention and call for a judicious and rational approach

Hyperglycemia Leading to Glycocalyx Dysfunction

• Transient and chronic hyperglycemia also lead to EG degradation

• Findings suggest a link between hyperglycemia-induced EG degradation and an increased risk of cardiovascular disease in patients with diabetes mellitus

Antioxidants to Reduce Glycocalyx Shedding

Protective effects of N-acetylcysteine only present when administered prior to hyperglycemia and before EG loss

Activated Protein C for Reduction in Glycocalyx Loss

• Also associated with reduced markers of endothelial oxidative stress, improved microcirculatory function, and improved response to vasopressor therapy in animal endotoxemia models but no mortality benefit found in human studies of severe sepsis and septic shock

• Septic people treated with APC had increased risk of developing bleeding complications

• Drug is no longer commercially available

Exogenous GAGs to Repair the Glycocalyx

• Exogenous GAGs may help reconstitute the EG after shedding but unknown how this may translate to a clinical population

• Treatment of people with diabetes mellitus with soludexide for 8 weeks resulted in an increased thickness of the EG compared to controls

  • Soludexide or administration of other exogenous GAGs would need to thicken the EG and improve its function more quickly than 8 weeks to be clinically relevant to the critical care population

Heparin to Reduce Glycocalyx Shedding

• Speculated that unfractionated heparin mobilizes intracellular pools of SDC1, leading to reformation of the EG

  • Also may inhibit heparanase, which cleaves HS in sepsis

• Hypothesized that LMWH binds to components of the EG, such as HS, and inhibits or reduces the release of heparanase from the EC

Colloids to Reconstitute the Glycocalyx

• The ESL is the in vivo structure responsible for normal vascular integrity

  • Treatment with plasma proteins may therefore aid in EG reconstitution

    • Infusion of 5% human albumin led to a reduction in the extravasation of fluid after I-R injury

    • Albumin appears to be able to penetrate and bind within the EG, reforming the ESL and restoring vascular integrity

  • Evidence suggests that synthetic colloids are not superior to crystalloids for the preservation of the EG

  • Possible that providing albumin to reconstitute the EG in critical illness could help restore microvascular barrier function, but appropriate albumin dose to achieve a beneficial effect remains unknown

    • Also not possible to know prior to administration whether any EG scaffolding remains to absorb albumin

  • Risk that administration of endogenous or synthetic colloids could lead to extravasation of the macromolecules into the interstitium, worsening edema

Fresh Frozen Plasma (FFP) for Repairing the Glycocalyx

• Animal hemorrhagic shock models have demonstrated that fluid resuscitation with FFP compared to crystalloids successfully restores the EG

• Use of FFP leads to improved microhemodynamics, vascular hemostasis, and reduced leukocyte-endothelium interaction compared to crystalloids or synthetic colloids

• A possible mechanism is that FFP may restore the structural scaffolding of the EG by replacing SDC1 and preventing its further loss

• FFP also contains albumin and other plasma proteins that are important in maintaining the ESL

• FFP may be beneficial as a method to restore the ESL

Protease Inhibitors such as AT to Reduce Glycocalyx Shedding

• n experimental I-R and sepsis models, treatment with AT reduced EG shedding and attenuated vascular permeability and tissue edema

• Multiple possible mechanisms for protective effects of AT

  • Inhibition of cleaving enzymes such as heparanase at the site of inflammation

  • Thrombin reduction

  • Reduction in the amount of heparanase released from mast cells during inflammation

• AT found in FFP

• Use of AT in critical illness is controversial

• Given lack of conclusive evidence for benefit of AT and documented risk for clinically significant hemorrhage, the 2017 Surviving Sepsis Campaign Guidelines recommend against the use of AT in sepsis and septic shock

Doxycycline to Preserve the Glycocalyx

At subantimicrobial doses, doxycycline reduces EG shedding through inhibition of MMPs

Glycocorticoid Administration to Reduce Glycocalyx Shedding

• Suggested mechanisms of protective effects

  • Stabilization of mast cells because their degranulation releases proteases that can degrade the EG

  • Suppression of MMPs

• Currently the Surviving Sepsis Campaign Guidelines do not advocate the use of corticosteroids in sepsis

Lactic Acid at Physiologic pH

At physiologic pH, lactic acid is essentially fully dissociated into lactate anions and protons (H+)

What are the two stereoisomeric forms of lactate?

L-lactate

D-lactate

L-Lactate

• In health, accounts for >99% of total body lactate

• Isomer of major physiological significance

D-Lactate

Formed either by the glyoxalase pathway or produced by commensal bacterial in the mammalian GI tract and absorbed into circulation

Hyperlactatemia

Serum, plasma, or blood lactate concentration is above the relevant reference interval

Lactic acidosis

Moderate to severe hyperlactatemia with concurrent metabolic acidosis

What does glycolysis produce?

3 molecules of pyruvate, 2 molecules of ATP, 2 molecules of NADH

What does glycolysis require to produce ATP?

Constant supply of glucose and NAD+ but does not require oxygen

Glycolysis Steps

Pyruvate after Glycolysis

• Pyruvate transported into the mitchondrion

  • Undergoes decarboxylation to produce acetyl-CoA

  • Reduction is irreversible, requires NAD+, and is catalyzed by pyruvate dehydrosenase complex

• Acetyl-CoA proceeds through the tricarboxylic acid (TCA) cycle to produce CO2, NADH, and FADH2

• Protons from NADH and FADH2 create the proton gradient required for the production of ATP by the electron transport chain (ETC)

What does glycolysis + TCA cycle + ETC produce?

36 molecules of ATP from oxidation of one molecule of glucose

Under healthy, resting conditions, what % of pyruvate is converted into lactate by LDH?

~10%

Conversion of Pyruvate into Lactate by LDH

• Reversible, cytosolic reaction

• NADH is oxidized to NAD+

What occurs to continue to supply ATP when oxygen demand exceeds supply?

• When oxygen demand exceeds supply, NAD+ stores become depleted, pyruvate and NADH accumulate in the cytosol and LDH activity is upregulated

  • Lactate formation reduces cytosolic pyruvate and H+ while replenishing NAD+, enabling glycolysis to continue supplying ATP

  • Once oxygen supply is restored, LDH transforms lactate back into pyruvate which can enter the TCA cycle or be used for gluconeogenesis

What is metabolic acidosis that occurs with hyperlactatemia the result of?

Hydrolysis without concurrent proton consumption by the ETC and subsequent proton accumulation

Lactate as a Strong Anion

• Lactate is a strong anion and has an acidifying effects similar to chloride according to Stewart approach

  • Increase in lactate causes a decrease in the SID

  • Decreasing the SID results in an increase in [H+] -> acidosis

In the quantitative approach to acid base how does lactate affect SBE?

For each 1 mmol/L increase in lactate, SBE will decrease by 1 unit

Lactate Transport

• Transport across cell membranes occurs predominantly via facilitate passive transport by proton-linked monocarboxylate transporters (MCT) and sodium-coupled MCTs

  • MCT1 and MCT4 most important in mammalian tissues

• Lactate transporters also play an essential role in "lactate shuttles," a form of energy currency exchange

  • Shown to exist in the brain, striated muscle, liver, kidneys, and myocardium

Where does the majority of lactate produced at rest come from?

Skeletal muscle (40-50%), the brain (13%), and adipose tissue (variable)

What % of lactate production in the blood are RBCs responsible for?

80%

What % of lactate production in the blood are leukocytes (predominately neutrophils) responsible for?

13%

What % of lactate production in the blood are platelets responsible for?

7%

What are the most important lactate consuming tissues?

Liver (20-30%), the renal cortex (20%), and the myocardium (5-15%)

• Hepatic lactate uptake is a saturable process

Where in the kidney is lactate reabsorbed?

Proximal convoluted tubule

What causes Type A hyperlactatemia?

Due to insufficient oxygen supply

What causes relative Type A Hyperlactatemia?

Due to insufficient oxygen supply from increased oxygen demand

What causes Absolute Type A Hyperlactatemia?

Due to insufficient oxygen supply from inadequate oxygen delivery

What causes Type B Hyperlactatemia?

In the face of apparently adequate oxygen availability

What causes Type B1 Hyperlactatemia?

Associated with underlying disease

What causes Type B2 Hyperlactatemia?

Associated with drugs or toxins

What causes Type B3 Hyperlactatemia?

Resulting from congenital errors in metabolis

Relative Type A Hyperlactatemia

• Can occur due to exercise, seizure activity, shivering, trembling, and struggling

  • Exercise induced hyperlactatemia is highly variable

    • In healthy animals, lactate concentrations fall rapidly following cessation of muscle activity, with an estimated half life of 20-60 minutes

  • Seizure-induced hyperlactatemia results primarily from vigorous muscle activity and is associated with a similar half-life

    • A persistent increase in lactate after cessation of seizure activity is concerning

Absolute Type A Hyperlactatemia Due to Shock

• Likely the most common cause of pathologic hyperlactatemia in veterinary ECC

• Shock is associated with inadequate oxygen delivery to the tissues, leading to impaired mitochondrial respiration and increased anaerobic metabolism

• Onset of hyperlactatemia relative to oxygen delivery is similar in hypovolemic, cardiogenic, and obstructive shock, but occurs earlier in maldistributive shock due to impaired oxygen extraction from mitochondrial and microcirculatory dysfunction

• Hyperlactatemia of shock is unlikely to be solely due to impaired oxygen delivery leading to increased anaerobic metabolism

• Several causes of Type B hyperlactatemia are believed to be important contributors to shock-associated hyperlactatemia