07 - Oxidation of NADH

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TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Metabolic Biochemistry::07 - Oxidation of NADH

Reoxidation of Glycolytic NADH

Glycolysis is an oxidative pathway that reduces $NA{D}^{+}$ to $NADH$ at the step of glyceraldehyde-P dehydrogenase.
Two $NADH$/glucose are produced.

How is $NADH$ reoxidized in order to allow glycolysis to proceed?

Remember that glycolysis takes place in the cytosol, whereas $NADH$-linked oxidative phosphorylation occurs in mitochondria.

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Reoxidation of Glycolytic NADH

Condition	Mechanism
Anaerobiosis	Reduction of pyruvate to lactate in the cytosol
Aerobiosis	Reducing equivalents from NADH are transported to mitochondria via shuttles

Aerobic shuttles:

• Glycerol-3-P shuttle
• Malate/Aspartate shuttle

Under conditions favouring biosynthesis (such as insulin), glycolytic NADH serves as a source of reducing power in the cytosol, supporting lipid synthesis.

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Reoxidation of Glycolytic NADH – Glycolysis Overview

${\text{Glucose (C}}_6\text{)}\stackrel{\text{-2 ATP (preparatory)}}{\to }2\times {\text{C}}_3\stackrel{+2,NA{D}^{+}\to 2,NADH}{\to }\stackrel{+4,ATP}{\to }2\times \text{Pyruvate}$

Net Gain from Glycolysis

• Net gain: 2 ATP and 2 NADH per glucose
• +1 NADH and +2 ATP per C3 triose (×2 = 2 NADH, 4 ATP gross; 2 ATP net after preparatory cost)

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Reoxidation of Glycolytic NADH – Fates of Pyruvate

Under anaerobic conditions, glycolysis stops until $NA{D}^{+}$ can be replenished by fermentation.

Under aerobic conditions, $NA{D}^{+}$ is regenerated by mitochondrial electron transfer.

Condition	Pathway	Products
Hypoxic/Anaerobic	Lactate fermentation	2 Lactate
Anaerobic (yeast)	Ethanol fermentation	2 Ethanol + $2,C{O}_2$
Aerobic	Mitochondrial electron transfer	$2,Acetyl\text{-}CoA\to C{O}_2+H_{2}O$

Nelson & Cox, Lehninger Principles of Biochemistry, 8e, © 2021 W. H. Freeman and Company

What happens to glycolysis under anaerobic conditions if NAD⁺ is not replenished?

Glycolysis stops. NAD⁺ must be replenished by fermentation to allow glycolysis to continue.

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Reoxidation of Glycolytic NADH – Fermentations

Definition Fermentations:

ways to anaerobically regenerate $NA{D}^{+}$ from $NADH$ to maintain a high rate of glycolysis.

1) Pyruvate to Lactate

(Athletes; bacteria making yoghurt; tumor cells)

$\text{Pyruvate}\stackrel{\text{lactate dehydrogenase}}{\to }\text{Lactate}\Delta G^{{\circ }^{\prime }}=-25.1,\text{kJ/mol}$

$\text{Glucose}+2,NA{D}^{+}\to 2,\text{Pyruvate}+2,NADH$

$2,\text{Pyruvate}\stackrel{+2,NADH}{\to }2,\text{Lactate}+2,NA{D}^{+}$

CC(=O)C(=O)O

(Pyruvate)

CC(O)C(=O)O

(Lactate)

Stereochemistry

The lactate produced is specifically L-lactate (L stereoconfiguration).

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The conversion of pyruvate to lactate has a $\Delta G^{{\circ }^{\prime }}=$ {1:−25.1 kJ/mol} and is catalysed by {1:lactate dehydrogenase}.

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2) Pyruvate to Ethanol

(In yeast)

$\text{Glucose}+2,ADP+2,P_i⟶2,\text{Ethanol}+2,C{O}_2+2,ATP+2,H_{2}O$

Two-step reaction:

$\text{Pyruvate}\stackrel{\text{pyruvate decarboxylase (TPP)}}{\to }\text{Acetaldehyde}\underset{\text{NADH}\to NA{D}^{+}}{\overset{\text{alcohol dehydrogenase}}{\to }}\text{Ethanol}$

CC(=O)C(=O)O

(Pyruvate)

CC=O

(Acetaldehyde)

CCO

(Ethanol)

Thiamine Pyrophosphate (TPP)

Cofactor of pyruvate decarboxylase. Derived from Vitamin B1(Thiamine).

What vitamin is the precursor of Thiamine Pyrophosphate (TPP)?

Vitamin B1 (Thiamine).

What are the two enzymes involved in the conversion of pyruvate to ethanol in yeast?

1. Pyruvate decarboxylase (requires TPP)
2. Alcohol dehydrogenase (oxidises NADH → NAD⁺)

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Reoxidation of Glycolytic NADH – Pasteur Effect

Pasteur Effect

The rate and total amount of glucose consumption under anaerobic conditions is many times greater than under aerobic conditions.

This occurs because the ATP yield from glycolysis alone is much smaller (2 ATP per glucose) than complete oxidation to $C{O}_2$ (30 or 32 ATP per glucose).

What is the Pasteur Effect?

The rate and total amount of glucose consumption under anaerobic conditions is many times greater than under aerobic conditions, because the ATP yield from glycolysis alone (2 ATP/glucose) is much smaller than complete oxidation to CO₂ (30 or 32 ATP/glucose).

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The Pasteur Effect explains why anaerobic glucose consumption is {1:greater} than aerobic, because glycolysis only yields {1:2} ATP/glucose vs. {1:30 or 32} ATP/glucose from complete oxidation.

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Lactate Oxidation

$\text{Lactate}+NA{D}^{+}\to \text{Pyruvate}+NADH+H^{+}$

Two fates of pyruvate from lactate oxidation:

1. For energetic purposes — further oxidation of pyruvate to acetyl-CoA and TCA cycle (e.g., in heart muscle, red fibres of the skeletal muscle, etc.): acidification resulting from ionisation of lactic acid in muscle and blood limits the period of vigorous activity.
2. For gluconeogenesis in the liver (Cori cycle).

Unless lactate formed by glycolysis is transported out of the cell, the intracellular pH will decrease due to the accumulation of intracellular lactic acid. The low pH decreases PFK-1 activity, thereby inhibiting further lactic acid production by glycolysis.

Red Blood Cells and Lactate

RBCs have no mitochondria → the only way they can produce ATP is glycolysis. Excess lactate in RBCs lowers pH → decreases PFK-1 activity → must be efficiently expelled to maintain a constant pH and a good rate of PFK-1 activity.

Cori Cycle

The Cori Cycle

1. Muscle produces lactate under hypoxic conditions.
2. Blood transports lactate to the liver.
3. Liver converts lactate back to glucose (gluconeogenesis — the opposite pathway of glycolysis, starting from pyruvate).
4. Glucose exits the liver, enters the bloodstream, and is delivered back to muscles.

The Cori cycle transfers the energetic cost from muscle to liver.

What is the effect of lactate accumulation within a cell?

Intracellular pH decreases → decreased PFK-1 activity → inhibition of further lactic acid production by glycolysis.

What are the two main fates of lactate oxidation products?

1. Energetic purposes: further oxidation of pyruvate → acetyl-CoA → TCA cycle (e.g., heart muscle, red skeletal muscle fibres).
2. Gluconeogenesis in the liver (Cori cycle).

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Lactate Oxidation – Tissue Profiles

Tissue	Key Features
Red Blood Cells	Glucose → Glucose-6-P; produces 2 Lactate
Muscle and Heart Tissue	Glucose → Pentose phosphates / Glycogen / Pyruvate → Lactate; Pyruvate → Acetyl-CoA → 4 CO₂

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Lactate Dehydrogenase (LDH)

LDH is a tetramer...

with two types of subunits: M (muscle) and H (heart). There are 5 combinations(isozymes).

Isozyme	Subunit Composition	Predominant Location
LDH1	H4 (HHHH)	Myocardium and RBC
LDH2	H3M (HHHM)	Myocardium and RBC
LDH3	H2M2 (HHMM)	Brain and kidney
LDH4	HM3 (HMMM)	Liver and skeletal muscle
LDH5	M4 (MMMM)	Liver and skeletal muscle

Kinetic Properties of LDH Isozymes

• M forms are kinetically favorable to pyruvate reduction (anaerobic muscle).
• H forms are kinetically favorable to lactate oxidation (heart).

Clinical Pearl

LDH isozymes are used as diagnostic tools (e.g., heart attack): elevated LDH1/LDH2 ratio is characteristic of myocardial infarction.

Mnemonic – LDH Isozyme Distribution

“Hearts Have More Hope, Muscles Make More”

• Hearts → Heavy in H subunits (LDH1, LDH2)
• Muscles/liver → More M subunits (LDH4, LDH5)

How many LDH isozymes exist and what subunits compose them?

5 isozymes composed of combinations of M (muscle) and H (heart) subunits in a tetramer: LDH1 (H4), LDH2 (H3M), LDH3 (H2M2), LDH4 (HM3), LDH5 (M4).

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M forms of LDH are kinetically favourable to {1:pyruvate reduction} (anaerobic muscle), while H forms favour {1:lactate oxidation} (heart).

Why are LDH isozymes used as diagnostic tools in heart attack?

Because different tissues express different LDH isozymes (LDH1/LDH2 predominate in the myocardium), and their elevation in blood indicates tissue-specific damage.

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Lactate Threshold

Lactate Threshold:

The point at which blood lactic acid suddenly rises during incremental exercise. Also called: Anaerobic threshold.

Mechanisms for lactate threshold:

• Low muscle oxygen
• Accelerated glycolysis — role for epinephrine
• Recruitment of fast-twitch muscle fibres with different isozymes
• Reduced rate of lactate removal from the blood

Clinical/Practical Uses

• Prediction of performance
• Marker of exercise intensity
• Onset of Blood Lactate Accumulation (OBLA)

Trained subjects show a right-shifted lactate threshold curve compared to untrained subjects — i.e., they can sustain higher relative exercise intensity (% $\dot{V}{O}_2$ max) before blood lactate accumulates.

OBLA Detail

The Onset of Blood Lactate Accumulation (OBLA) is defined at a threshold concentration of ~4 mmol/L blood lactate, used by athletes to monitor performance. Blood lactate concentration is related to % VO₂ max: lactate accumulation increases with exercise intensity and reaches high levels when production exceeds clearance.

What are the four mechanisms proposed for the lactate threshold?

1. Low muscle oxygen
2. Accelerated glycolysis (role for epinephrine)
3. Recruitment of fast-twitch muscle fibres with different isozymes
4. Reduced rate of lactate removal from the blood

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The point at which blood lactic acid suddenly rises during incremental exercise is called the {1:lactate threshold} or {1:anaerobic threshold}.

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Shuttles

General Principles of Shuttles

• The inner mitochondrial membrane (IMM) is impermeable to $NA{D}^{+}$/$NADH$.
• There are two pools of NAD: cytosolic and mitochondrial.
• A redox-active metabolite permeable through IMM carries the reducing equivalents.
• Two sets of isoenzymes: cytosolic and mitochondrial.

Feature	Detail
IMM permeability	Impermeable to NAD⁺/NADH
NAD pools	Cytosolic and mitochondrial (separate)
Transfer agent	Redox-active metabolite permeable through IMM
Enzymes	Two isoenzyme sets: cytosolic and mitochondrial

General shuttle mechanism:

$NADH+H^{+}+X\to NA{D}^{+}+X{H}_2\text{(cytosol)}$

$X{H}_2\stackrel{\text{crosses IMM}}{\to }\text{matrix}$

$X{H}_2+NA{D}^{+}\to X+NADH+H^{+}\text{(matrix)}$

$NADH\stackrel{\text{respiratory chain}}{\to }\text{oxidised}$

$X\stackrel{\text{crosses IMM}}{\to }\text{cytosol}$

Why are shuttles necessary for reoxidation of cytosolic NADH under aerobic conditions?

Because the inner mitochondrial membrane is impermeable to NAD⁺/NADH, so reducing equivalents must be transferred via redox-active metabolites that can cross the IMM.

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Malate/Aspartate Shuttle

$L\text{-Malate}\underset{\Delta G^{{\circ }^{\prime }}=+29.7,\text{kJ/mol}}{\overset{\text{malate dehydrogenase}}{\to }}\text{Oxaloacetate}+NADH+H^{+}$

$NA{D}^{+}+NADH+H^{+}\rightleftharpoons$

Components:

• Malate–$\alpha$-ketoglutarate transporter (IMM)
• Glutamate–aspartate transporter (IMM)
• Malate dehydrogenase (cytosolic and mitochondrial isoforms)
• Aspartate aminotransferase (cytosolic and mitochondrial isoforms)

Transamination (requires PLP, Vitamin B6):

$\alpha \text{-Ketoglutarate}+\text{L-Amino acid}\underset{\text{PLP}}{\overset{\text{aminotransferase}}{\to }}\text{L-Glutamate}+\alpha \text{-Keto acid}$

The malate/aspartate shuttle...

transfers reducing equivalents as malate into the mitochondrial matrix, where oxaloacetate is regenerated and NADH is produced for the respiratory chain. Aspartate and glutamate are used to regenerate oxaloacetate in the cytosol via transamination.

Detailed Mechanism

1. In the cytosol, NADH reduces oxaloacetate (OAA) → malate (via cytosolic malate dehydrogenase).
2. Malate crosses the IMM via the malate–α-ketoglutarate antiporter.
3. In the matrix, malate is oxidised back to OAA (via mitochondrial malate dehydrogenase), regenerating NADH for the respiratory chain.
4. OAA cannot cross the IMM directly → it is transaminated to aspartate (by aspartate aminotransferase, cofactor PLP/Vitamin B6): OAA + glutamate → aspartate + α-ketoglutarate.
5. Aspartate exits the matrix via the aspartate–glutamate antiporter.
6. In the cytosol, aspartate is reconverted to OAA: aspartate + α-ketoglutarate → OAA + glutamate.
7. α-Ketoglutarate re-enters the matrix to complete the cycle.

Why α-ketoglutarate is essential: one transporter moves α-keto acids (malate ↔ α-KG) and one moves amino acids (aspartate ↔ glutamate). Without α-ketoglutarate, the shuttle cannot operate.

Thermodynamics

The OAA → malate reaction has $\Delta G^{{\circ }^{\prime }}=+29.7\text{kJ/mol}$ (unfavourable in standard conditions), but under physiological conditions it operates in equilibrium and is fully reversible, allowing reducing equivalents to travel in both directions between cytosol and mitochondria.

Tissue Distribution

The malate/aspartate shuttle is the main shuttle in the liver and heart.

What vitamin is the cofactor for transamination reactions in the malate/aspartate shuttle?

Vitamin B6 (Pyridoxal phosphate, PLP).

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The malate/aspartate shuttle uses two IMM transporters: the {1:malate–α-ketoglutarate} transporter and the {1:glutamate–aspartate} transporter.

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Glycerol-3-P Shuttle

During the glycerol-P shuttle, dihydroxyacetone-P (DHAP) is not removed from glycolysis, because it is continuously regenerated.

$\text{DHAP}+NADH+H^{+}\stackrel{\text{cytosolic glycerol-3-P dehydrogenase}}{\to }\text{Glycerol-3-P}+NA{D}^{+}$

$\text{Glycerol-3-P}\stackrel{\text{mitochondrial glycerol-3-P dehydrogenase (FAD)}}{\to }\text{DHAP}+FAD{H}_2$

Detailed Mechanism & Entry Point into Respiratory Chain

1. In the cytosol, DHAP + NADH → glycerol-3-phosphate + NAD⁺ (cytosolic glycerol-3-phosphate dehydrogenase).
2. Glycerol-3-phosphate is reoxidised at the outer face of the IMM by the mitochondrial glycerol-3-phosphate dehydrogenase (cofactor: FAD) → DHAP + FADH₂.
3. FADH₂ transfers electrons to ubiquinone (CoQ) → electrons enter the respiratory chain at Complex III, bypassing Complex I.

Key consequence: cytosolic NADH is effectively converted to mitochondrial FADH₂, yielding only ~1.5 ATP instead of ~2.5 ATP. This is why the glycerol-3-P shuttle is less efficient than the malate/aspartate shuttle.

Tissue Distribution

The glycerol-3-P shuttle is the main shuttle in muscle and brain.

Why is DHAP not depleted during the glycerol-3-P shuttle?

Because DHAP is continuously regenerated by the mitochondrial glycerol-3-phosphate dehydrogenase reaction.

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After Glycolysis

Glycolysis provides the carbon atoms for lipid biosynthesis. It also provides the required reducing power ($NADH$; $NADPH$). After a carbohydrate meal, insulin promotes glycolysis and lipid biosynthesis.

Metabolic fates in the liver:

• Glucose → Glucose-6-phosphate
• → Glycogen
• → Pentose phosphate pathway → Nucleotides, Ribose-5-phosphate
• → Glycolysis → Pyruvate → Fatty acids, Cholesterol (via Acetyl-CoA)
• → Oxidative phosphorylation → $C{O}_2$, $H_{2}O$

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After Glycolysis – ATP Yield Summary

ATP Yield Comparison

Condition	Pathway	ATP from substrate level	ATP from OXPHOS	Total ATP
Anaerobiosis	Glucose → 2 Pyruvate; 2 NADH → 2 Lactate	2 ATP	0	2 ATP
Aerobiosis (malate/aspartate shuttle)	2 NADH → respiratory chain ($2.5\times 2$)	2 ATP	5 ATP	7 ATP
Aerobiosis (glycerol-P shuttle)	2 glycerol-P → respiratory chain ($1.5\times 2$)	2 ATP	3 ATP	5 ATP

N.B: More ATP in further oxidation of pyruvate!

What is the total ATP yield from glycolysis under anaerobic conditions?

2 ATP per glucose (substrate level only).

What is the total ATP yield from glycolysis when NADH is shuttled via the malate/aspartate shuttle?

7 ATP: 2 ATP (substrate level) + 5 ATP from OXPHOS (2.5 × 2 NADH).

What is the total ATP yield from glycolysis when NADH is shuttled via the glycerol-P shuttle?

5 ATP: 2 ATP (substrate level) + 3 ATP from OXPHOS (1.5 × 2).

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The malate/aspartate shuttle feeds NADH worth {1:2.5} ATP each into the respiratory chain, while the glycerol-P shuttle feeds equivalents worth {1:1.5} ATP each.

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After Glycolysis – Summary of Fates

Starting Point	Pathway	End Products
Glucose	Glycolysis	Pyruvate
Pyruvate	Alcoholic fermentation	$C{O}_2$, $H_{2}O$ (ethanol)
Pyruvate	Homolactic fermentation	Lactate, $C{O}_2$
Pyruvate	Citric acid cycle + oxidative phosphorylation	$C{O}_2$, $H_{2}O$

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TLDR - 07 - Oxidation of NADH

Complete Study Reference: Reoxidation of Glycolytic NADH

Core Problem

• Glycolysis produces 2 NADH/glucose at the glyceraldehyde-3-phosphate dehydrogenase step (cytosol).
• The IMM is impermeable to NAD⁺/NADH → cytosolic NADH must be reoxidised separately.

Anaerobic Solutions (Fermentations)

• Lactate fermentation: Pyruvate → Lactate (lactate dehydrogenase); $\Delta G^{{\circ }^{\prime }}=-25.1,\text{kJ/mol}$; regenerates NAD⁺. Occurs in athletes, bacteria (yoghurt), tumour cells.
• Ethanol fermentation (yeast): Pyruvate → Acetaldehyde (pyruvate decarboxylase, TPP/Vitamin B1) → Ethanol (alcohol dehydrogenase, NADH consumed). Net: $\text{Glucose}\to 2,\text{Ethanol}+2,C{O}_2+2,ATP$.
• Pasteur Effect: Anaerobic glucose consumption >> aerobic because glycolysis yields only 2 ATP vs. 30–32 ATP from full oxidation.

Lactate Oxidation

• Reaction: $\text{Lactate}+NA{D}^{+}\to \text{Pyruvate}+NADH+H^{+}$
• Fates: (1) energetic oxidation via TCA (heart, red skeletal muscle); (2) gluconeogenesis (liver, Cori cycle).
• Lactate accumulation → ↓ intracellular pH → ↓ PFK-1 activity → inhibits glycolysis.

Lactate Dehydrogenase (LDH) Isozymes

Isozyme	Composition	Location
LDH1	H4	Myocardium, RBC
LDH2	H3M	Myocardium, RBC
LDH3	H2M2	Brain, kidney
LDH4	HM3	Liver, skeletal muscle
LDH5	M4	Liver, skeletal muscle

• M forms → favour pyruvate reduction (anaerobic muscle).
• H forms → favour lactate oxidation (heart).
• Clinical use: LDH isozymes as markers of myocardial infarction.

Lactate Threshold

• Point where blood lactate suddenly rises during incremental exercise (= anaerobic threshold / OBLA).
• Mechanisms: low muscle O₂, accelerated glycolysis (epinephrine), fast-twitch fibre recruitment, reduced lactate clearance.
• Trained subjects: right-shifted threshold.

Aerobic Shuttles (to transfer cytosolic NADH reducing equivalents into mitochondria)

Shuttle	Carrier	ATP yield per NADH	Cofactors	Main Tissues
Malate/Aspartate	Malate / OAA / Aspartate / Glutamate	2.5 ATP	PLP (Vitamin B6)	Liver, Heart
Glycerol-3-P	Glycerol-3-phosphate / DHAP	1.5 ATP	FAD (mitochondrial enzyme)	Muscle, Brain

• Malate/aspartate shuttle delivers NADH to the matrix → 2.5 ATP/NADH.
• Glycerol-P shuttle delivers FADH₂ equivalent to the matrix → 1.5 ATP.
• DHAP is not depleted in glycerol-P shuttle (continuously regenerated).
• Transamination in malate/aspartate shuttle requires PLP (Vitamin B6).

ATP Yield Summary (Glycolysis only)

Condition	Total ATP
Anaerobic	2 ATP
Aerobic + malate/aspartate shuttle	7 ATP
Aerobic + glycerol-P shuttle	5 ATP

After Glycolysis (Biosynthetic Role)

• Glycolysis provides carbon atoms for lipid biosynthesis and reducing power (NADH, NADPH).
• Insulin (post-carbohydrate meal) promotes glycolysis and lipid biosynthesis.
• Pyruvate can enter: TCA cycle, alcoholic fermentation, homolactic fermentation, or oxidative phosphorylation.

Cori Cycle

• Muscle (hypoxic) → lactate → bloodstream → liver → gluconeogenesis → glucose → bloodstream → muscle.
• Transfers the energetic cost from muscle to liver.
• RBCs also produce lactate (no mitochondria; glycolysis is their only ATP source) → must expel lactate to maintain intracellular pH and PFK-1 activity.

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Anki

What is the stereochemistry of lactate produced by lactate dehydrogenase?

L-lactate (L stereoconfiguration).

Why do red blood cells produce lactate?

RBCs have no mitochondria, so glycolysis is their only source of ATP. Pyruvate must be reduced to lactate to regenerate NAD⁺ and maintain glycolysis. Lactate must be expelled to prevent pH drop and PFK-1 inhibition.

Describe the Cori cycle.

1. Muscle produces lactate under hypoxic conditions.
2. Blood transports lactate to the liver.
3. Liver converts lactate → pyruvate → glucose (gluconeogenesis).
4. Glucose re-enters bloodstream and is delivered to muscle.
   The Cori cycle transfers the energetic cost from muscle to liver.

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The Cori cycle transfers the energetic cost from {1:muscle} to {1:liver}, where lactate is converted back to {1:glucose} via {1:gluconeogenesis}.

What is the OBLA threshold concentration and why is it used?

~4 mmol/L blood lactate. It marks the Onset of Blood Lactate Accumulation and is used by athletes as a marker of exercise intensity and performance.

Why does the glycerol-3-P shuttle yield less ATP than the malate/aspartate shuttle?

Because cytosolic NADH is effectively converted to FADH₂ (not NADH) in the mitochondria, entering the respiratory chain at Complex III (bypassing Complex I), yielding ~1.5 ATP instead of ~2.5 ATP per NADH.

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The glycerol-3-P shuttle enters the respiratory chain at {1:Complex III} via {1:ubiquinone (CoQ)}, bypassing {1:Complex I}, because the mitochondrial enzyme uses {1:FAD} as cofactor rather than NAD⁺.

Why is α-ketoglutarate essential for the malate/aspartate shuttle?

Because one IMM transporter moves α-keto acids (malate ↔ α-KG) and another moves amino acids (aspartate ↔ glutamate). α-Ketoglutarate is required to link these two transport steps; without it, the shuttle cannot operate.

Which shuttles predominate in which tissues?

Malate/aspartate shuttle: liver and heart. Glycerol-3-P shuttle: muscle and brain.