13.1 Carbohydrate Chemistry, Digestion, Glycolysis, Pyruvate Dehydrogenase & the Pentose Phosphate Pathway

Why this area matters

The 2025 Biochemistry TOS lists for carbohydrates: "carbohydrate biochemistry (structure and functions, classifications, properties and reactions), digestion and metabolism, ketogenesis, lipidogenesis and disorders, diseases of hormonal metabolism". This section covers chemistry and the glucose-oxidation pathways. The next section covers storage, gluconeogenesis, lipogenesis, ketogenesis and hormonal control. G6PD deficiency and galactosemia are on the Philippine expanded newborn screening panel, so they appear in both Biochemistry and Pediatrics items.

Structure, classification and reactions

Key reactions:

• Free aldehyde or ketone groups reduce Benedict's and Fehling's reagents. Urine reducing-substance tests detect glucose, galactose and fructose; dipstick glucose oxidase strips detect only glucose.
• Glucose reacts non-enzymatically with proteins (glycation), forming HbA1c and advanced glycation end-products.
• Aldose reductase reduces glucose to sorbitol, which causes osmotic damage in the lens, nerves and retina in diabetes.

Digestion and absorption

1. Salivary and pancreatic alpha-amylase split starch into maltose, maltotriose and alpha-limit dextrins.
2. Brush-border disaccharidases (lactase, sucrase-isomaltase, maltase) release monosaccharides.
3. SGLT1 absorbs glucose and galactose with sodium; GLUT5 absorbs fructose; GLUT2 releases all three into the portal blood.

Lactase non-persistence, common in Asians, causes bloating and osmotic diarrhea after milk. Undigested lactose is fermented to gas and acids (low stool pH, positive hydrogen breath test).

Glucose transporters:

• GLUT1: red cells and the blood-brain barrier.
• GLUT2: liver, beta cells, kidney and intestine (bidirectional, high Km).
• GLUT3: neurons.
• GLUT4: muscle and fat, insulin-dependent.

The Glycolytic Pathway: Key Regulatory Steps and Erythrocyte Shunt

Glycolysis is the ubiquitous cytosolic pathway that converts one molecule of glucose ($6\text{C}$) into two molecules of pyruvate ($3\text{C}$), generating a net yield of $2\text{ ATP}$ and $2\text{ NADH}$. Glycolysis operates in two phases: the preparatory (energy investment) phase, consuming $2\text{ ATP}$, and the payoff phase, generating $4\text{ ATP}$ and $2\text{ NADH}$.

Hexokinase versus Glucokinase: Kinetic and Tissue Specialization

The initial irreversible step traps intracellular glucose by phosphorylation to glucose-6-phosphate (G6P), catalyzed by two distinct isozymes:

Phosphofructokinase-1 (PFK-1): The Primary Rate-Limiting Pacemaker

The phosphorylation of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F-1,6-BP) by Phosphofructokinase-1 (PFK-1) represents the definitive commitment and rate-limiting step of glycolysis:

$\text{Fructose-6-Phosphate} + \text{ATP} \xrightarrow{\text{PFK-1}} \text{Fructose-1,6-Bisphosphate} + \text{ADP}$

PFK-1 is subject to complex allosteric regulation:

• Allosteric Inhibitors: High intracellular ATP (reflecting abundant energy charge) and Citrate (signaling active downstream Krebs cycle activity).
• Allosteric Activators: High AMP and Fructose-2,6-bisphosphate (F-2,6-BP), the most potent physiological activator.

F-2,6-BP is synthesized and degraded by a single bifunctional tandem enzyme containing two distinct catalytic domains: PFK-2 (kinase domain) and Fructose-2,6-bisphosphatase-2 (FBPase-2) (phosphatase domain):

• Fed State (Insulin Dominance): Insulin signaling stimulates protein phosphatase-1, which dephosphorylates the bifunctional enzyme. Dephosphorylation activates the PFK-2 domain and inactivates FBPase-2, driving the synthesis of F-2,6-BP. Elevated F-2,6-BP allosterically activates PFK-1, accelerating glycolysis.
• Fasting State (Glucagon Dominance): Glucagon binds G-protein-coupled receptors, elevating cAMP and activating Protein Kinase A (PKA). PKA phosphorylates the bifunctional enzyme. Phosphorylation inactivates PFK-2 and activates FBPase-2, degrading F-2,6-BP. Reduced F-2,6-BP relieves PFK-1 activation and derepresses gluconeogenesis.

Pyruvate Kinase and the Rapoport-Luebering Shunt

The final irreversible glycolytic reaction is catalyzed by Pyruvate Kinase, which transfers a high-energy phosphate from phosphoenolpyruvate to ADP, producing pyruvate and ATP. Pyruvate kinase is stimulated feedforward by upstream fructose-1,6-bisphosphate and allosterically inhibited by ATP and alanine. In the liver, glucagon-stimulated PKA phosphorylates and inactivates pyruvate kinase during fasting.

In mature erythrocytes, which lack mitochondria, glycolysis is the sole source of ATP. Erythrocytes feature a specialized collateral pathway: the Rapoport-Luebering shunt. In this bypass, bisphosphoglycerate mutase converts 1,3-BPG into 2,3-bisphosphoglycerate (2,3-BPG). 2,3-BPG binds selectively to the central cavity of deoxygenated hemoglobin tetramers, stabilizing the T (taut) conformation and shifting the oxygen-hemoglobin dissociation curve to the right, facilitating oxygen unloading to hypoxic peripheral tissues. Because this bypass circumvents phosphoglycerate kinase, no ATP is generated from that triose branch.

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Pyruvate Fates and the Pyruvate Dehydrogenase Complex

Under anaerobic conditions, pyruvate is reduced to lactate by cytosolic lactate dehydrogenase (LDH), oxidatively regenerating $NAD^+$ to allow glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to sustain glycolysis. In aerobic tissues, pyruvate enters the mitochondrial matrix via the mitochondrial pyruvate carrier (MPC) to undergo irreversible oxidative decarboxylation to acetyl-CoA, catalyzed by the multi-enzyme Pyruvate Dehydrogenase (PDH) Complex:

$\text{Pyruvate} + \text{NAD}^+ + \text{CoA-SH} \xrightarrow{\text{PDH Complex}} \text{Acetyl-CoA} + \text{NADH} + \text{CO}_2 + \text{H}^+$

The Five Essential Coenzymes of the PDH Complex

The PDH complex comprises three catalytic subunits requiring five specific coenzymes derived from dietary vitamins:

1. $E_1$ (Pyruvate Dehydrogenase): Requires Thiamine Pyrophosphate (TPP), derived from Vitamin B1 (Thiamine).
2. $E_2$ (Dihydrolipoyl Transacetylase): Requires Lipoic Acid and Coenzyme A (CoA), derived from Vitamin B5 (Pantothenic acid).
3. $E_3$ (Dihydrolipoyl Dehydrogenase): Requires FAD, derived from Vitamin B2 (Riboflavin), and $NAD^+$, derived from Vitamin B3 (Niacin).

Mnemonic: "The Tender Loving Care For Nancy" (Thiamine, Lipoic acid, CoA, FAD, $NAD^+$).

Toxicological and Clinical Correlations

• Arsenic Poisoning: Arsenite binds covalently to the vicinal sulfhydryl groups of dihydrolipoic acid, arresting both the PDH complex and $\alpha$-ketoglutarate dehydrogenase. Patients present with acute vomiting, profuse "rice-water" watery diarrhea, garlic odor on breath, and QT prolongation, progressing to encephalopathy and death.
• PDH Complex Deficiency: Most commonly an X-linked mutation in the $E_1$-$\alpha$ subunit gene (PDHA1). Pyruvate accumulates and is shunted to lactate (causing severe lactic acidosis) and alanine. Affected infants exhibit psychomotor retardation, microcephaly, and hypotonia. Management involves a strict ketogenic diet high in fats and low in carbohydrates, supplemented with purely ketogenic amino acids (lysine and leucine), which generate acetyl-CoA directly without requiring the PDH complex.

The Pentose Phosphate Pathway and G6PD Deficiency

The Pentose Phosphate Pathway (Hexose Monophosphate Shunt) is a cytosolic collateral pathway of glucose oxidation that produces NADPH and Ribose-5-phosphate (for nucleotide synthesis) without consuming or generating ATP. The irreversible, rate-limiting oxidative step is catalyzed by Glucose-6-Phosphate Dehydrogenase (G6PD):

$\text{Glucose-6-Phosphate} + 2\text{ NADP}^+ + \text{H}_2\text{O} \xrightarrow{\text{G6PD}} \text{Ribulose-5-P} + 2\text{ NADPH} + 2\text{ H}^+ + \text{CO}_2$

Physiological Roles of NADPH

1. Reductive Biosynthesis: Fatty acid, cholesterol, and steroid hormone synthesis.
2. Respiratory Burst: Substrate for phagocyte NADPH oxidase to generate superoxide ($O_2^{\bullet-}$).
3. Maintenance of Reduced Glutathione: Erythrocytes depend on NADPH to reduce oxidized glutathione (GSSG) back to reduced glutathione (GSH) via glutathione reductase. GSH detoxifies hydrogen peroxide ($H_2O_2$) and reactive oxygen species via glutathione peroxidase.

G6PD Deficiency (Favism / Drug-Induced Hemolysis)

• Genetics: X-linked recessive disorder; most prevalent red cell enzymopathy worldwide (common in Mediterranean, African, and Southeast Asian/Filipino populations).
• Pathophysiology: In erythrocytes (which lack mitochondria and alternative NADPH sources), G6PD deficiency impairs GSH regeneration. Oxidative stress leads to cross-linking of hemoglobin sulfhydryl groups, precipitating insoluble aggregates known as Heinz bodies.
• Morphology: Splenic macrophages remove Heinz bodies, generating characteristic bite cells (degmacytes).
• Triggers: Ingestion of fava beans (Vicia faba); infections; oxidizing medications including the antimalarials primaquine and tafenoquine, sulfonamides (sulfamethoxazole), dapsone, nitrofurantoin, and rasburicase.

Fructose and galactose disorders

Lactic acidosis in a child may come from PDH deficiency, pyruvate carboxylase deficiency, mitochondrial disease or glycogen storage disease type I. A high lactate-to-pyruvate ratio points to a respiratory chain defect; a normal ratio points to a PDH defect.