06_BIOCHEM_LIVER_EN_2025

Page 1

BIOCHEMISTRY OF THE LIVER – 2025
• Lecturer: Endre Kristóf (based on materials of Prof. Dr. László Fésüs)
• Contact: kristof.endre@med.unideb.hu

Page 2 — Outline

1. Basic architecture of the liver
2. Liver in the service of other organs
3. Acute-phase proteins (APPs)
4. Biotransformation of xenobiotics
5. Cytochrome P450 (CYP) enzymes
6. Conjugation reactions (Phase II)
7. Excretion of metabolites by ABC multidrug transporters (Phase III)
8. Oxidation of alcohol
9. Pathobiochemistry of liver diseases

Page 3 — Theme Block

Faculty of Medicine, Dept. of Biochemistry & Molecular Biology
Topic block: BASIC ARCHITECTURE OF THE LIVER

Page 4 — Principles Governing Liver Structure & Function

1. Double blood supply
   • Splanchnic venous blood via portal vein → second cellular interface (hepatocytes).
2. Architectural/zonal arrangement for efficient exchange between blood & liver parenchyma.
3. Space of Disse (perihepatocellular) with no basement membrane ⇒ maximal metabolite exchange.
4. Dual roles of hepatocyte: simultaneous excretion & metabolic tasks for whole body.
5. Separate biliary vs. blood lumens – clear excretory compartment.
6. Unique gene expression defines hepatocyte biochemistry.
7. Kupffer macrophages & Ito (stellate) cells integrated into liver function network.

Page 5 — Microscopic Organizational Units

• Classic (hepatic) lobule: hexagon; central vein center, portal triads (hepatic artery, portal vein, bile duct) at vertices. Blood: portal vein/hepatic artery → sinusoids → central vein → IVC.
• Portal lobule: triangle; portal triad in center, central veins at corners; organized along bile flow.
• Hepatic acinus (see next page): perfusion-based diamond.

Page 6 — Functional Significance of the Acinus

• Liver ≈ 2.5\% body weight; ≈ 300\,\text{ml} blood.
• Acinus diamond: short-axis corners = portal triads, long-axis corners = central veins.
• Sinusoidal endothelium: fenestrated, no tight junctions or basement membrane.
• Bile canaliculi: formed between adjacent hepatocytes (canalicular membranes).

Page 7 — Liver Cell Types & Regeneration

• Hepatocytes: polygonal, 20\text{–}30\,\mu\text{m}; abundant cytoplasm, mitochondria. Membrane domains: sinusoidal (microvilli), canalicular, apicolateral.
• Kupffer cells (macrophages) in sinusoidal lumen.
• Stellate (Ito) cells in Space of Disse: vitamin A storage, fibrogenesis.
• Progenitor (oval) cells present.
• Liver contains ≈ 2.5\times 10^{11} hepatocytes; can regrow after \le 90\% resection.

Page 8 — Theme Block

THE LIVER IN THE SERVICE OF OTHER ORGANS

Page 9 — Integrating Role in Metabolic Homeostasis

Blood-glucose homeostasis

• First-pass effect on portal blood.
• Insulin-independent uptake via GLUT2, phosphorylation by glucokinase.
• Glycogen synthesis/storage & glucose release via GLUT2.
• Gluconeogenesis substrates: Cori-lactate cycle, alanine cycle, glycerol, amino acids.
• Fructose metabolism: fructokinase → \text{fructose-1-P} → aldolase B ⇒ dihydroxyacetone-P + glyceraldehyde.
• Galactose metabolism: galactokinase → \text{gal-1-P} → UDP-Gal ↔ glucose-1-P → glucose-6-P.

Energy production & export

• Fatty-acid & triacylglycerol (TAG) synthesis in fed state.
• Ketone-body formation during starvation (large mitochondrial mass).
• Pentose phosphate pathway → NADPH for FA synthesis, mevalonate, detox, antioxidants.
• Carnitine final hydroxylation; Creatine: guanidino-acetate + SAM → creatine.

Page 10 — Hormonal Control of Blood Glucose (schematic)

• High glucose → pancreatic β-cells secrete insulin → liver, muscle, adipose: glycogen & lipid synthesis, glucose uptake.
• Low glucose → α-cells secrete glucagon → liver: glycogenolysis + gluconeogenesis.

Page 11 — Metabolic Fates of Glucose-6-P in Hepatocyte

Branches:

1. Glycogen synthesis/breakdown.
2. Glycolysis → pyruvate → TCA.
3. Pentose phosphate pathway → \text{NADPH} + ribose-5-P.
4. Lipogenesis: acetyl-CoA → FA, TAG, cholesterol.
5. Free glucose export via Glc-6-phosphatase.

Page 12 — Fed-State Lipid Traffic

• Chylomicron remnants deliver dietary TAG/Chol to liver.
• Liver synthesizes VLDL, de novo FA & cholesterol; secretes bile acids.
• HDL trafficking of cholesterol.

Page 13 — “Lipogenic Liver” in Fed State

Pathway: intestinal glucose → portal vein → liver (first pass) → glycogen & acetyl-CoA → FA & TAG → VLDL → adipose.
Amino acids partially oxidized (TCA), nitrogen → urea.

Page 14 — Starvation Metabolism

• Mobilized adipose FA → liver β-oxidation → acetyl-CoA accumulates (OAA depleted) → ketogenesis (\text{acetoacetate},\, \beta\text{-OH-butyrate}).
• Glycerol, amino acids → gluconeogenesis → glucose export (brain).

Page 15 — Fasting State “Glucogenic Liver”

Glucagon stimulates glycogenolysis (early), then gluconeogenesis (pyruvate, lactate, AA, glycerol).
Ketone bodies produced for peripheral use.

Page 16 — Prolonged Fasting / Diabetes

• After glycogen depletion, gluconeogenesis dominant.
• Glucogenic AA from muscle proteolysis.
• Adipose FA → liver oxidation → ketone bodies for brain.
• Excess ketones lost in urine.

Page 17 — Cholesterol & Lipoprotein Homeostasis

• Liver orchestrates: dietary uptake (unlimited CM-remnants), de novo synthesis, VLDL secretion, LDL-R mediated reuptake, HDL reverse transport, bile acid & cholesterol excretion.

Page 18 — Central Cholesterol Pool Changes

Decreases: VLDL/bile acid synthesis, biliary cholesterol efflux (\approx 800\,\text{mg day}^{-1}).
Increases: unlimited CM-remnant uptake, LDL-R recycling, HDL-R (SR-B1) uptake, de novo production.

Page 19 — Nitrogen, Vitamin & Metal Handling

• Urea cycle + glutamine synthesis remove ammonia, regulate \text{pH}.
• Plasma protein turnover (half-life \approx 10\,\text{days}) supplies amino acids.
• Vitamin A storage, D 25-hydroxylation, vitamin K storage/“regeneration”.
• Iron stored in ferritin (\approx 10\% body ferritin in liver).
• Copper stored/secreted as ceruloplasmin.
• Major plasma proteins synthesized: albumin, transferrin, ceruloplasmin, apo-lipoproteins, clotting factors (vit-K-dependent \gamma-carboxylation), APPs, etc.

Page 20 — Amino-Acid Metabolism Schema

Hepatic roles summarized: urea formation, glutamine, plasma protein synthesis, lysosomal catabolism of aged plasma proteins.

Page 21 — Hepatokines

• Liver-secreted signaling factors (proteins, metabolites, ncRNA) act in autocrine/paracrine/endocrine manner regulating systemic metabolism (e.g., FGF21, fetuin-A).

Page 22 — Zonal Heterogeneity of Hepatocytes

Zone 1 (periportal): high \text{O}2, gluconeogenesis, β-oxidation, urea cycle, glutaminase. Zone 2 (mid-lobular): proliferative reserve. Zone 3 (perivenous): low \text{O}2, glycolysis, lipogenesis, ketogenesis, Mevalonate, CYP450, glutathione S-transferase (GST), bilirubin conjugation.

Page 23 — Inter-cellular Glutamine Cycle & Acid–Base

• Periportal cells: glutaminase + GDH + CPS-I + urea cycle → ammonia → urea.
• Perivenous cells: glutamine synthetase “scavenges” residual NH4^+. • Kidney: glutaminase again liberates NH4^+ for proton excretion (acidosis increases Gln synthesis in liver).

Page 24 — Theme Block: ACUTE PHASE PROTEINS

Page 25 — Acute Phase Reaction

Inflammation → cytokines (IL-6, IL-1β, TNF-α) re-program hepatocyte transcription → dramatic change in serum protein profile (“positive” vs “negative” APPs).

Page 26 — Classes of APPs

Positive APPs:
• Complement (C2,3,4,5,9) – opsonization/lysis.
• Coagulation (fibrinogen, vWF).
• Protease inhibitors (α1-antitrypsin, α2-antiplasmin, C1-inh).
• Metal handling: hepcidin (↓Fe), haptoglobin (binds Hb), hemopexin (heme).
• “Major APPs”: serum amyloid A (SAA), C-reactive protein (CRP), manganese SOD, etc.
Negative APPs (decreased synthesis): albumin, transferrin, Apo A-I/A-II.

Page 27 — Dynamics & CRP

• CRP rises within 6\text{–}8\,\text{h}, peaks \sim48\,\text{h}; normal <8\,\text{mg L}^{-1}.
• Pentraxin structure (five identical sub-units).
• Functions: binds pneumococcal C-polysaccharide, opsonization, debris clearance.

Page 28 — Secondary Amyloidosis

Chronic inflammation → sustained SAA → proteolysis → Amyloid A fibrils (β-sheet) deposit in organs → dysfunction.

Page 29 — Theme Block: BIOTRANSFORMATION OF XENOBIOTICS

Page 30 — First-Pass Effect & Phase Concept

• Xenobiotic: non-nutritive foreign compound. Mostly hydrophobic.
• Phases:
– Phase 0: uptake/efflux transporters (e.g., intestine ABCG5/8).
– Phase I: functionalization (mostly oxidation by CYP, FMO).
– Phase II: conjugation (↑size, ↑water solubility).
– Phase III: export via ABC transporters to bile/urine.

Page 31 — Phase 0 Example — Sitosterol vs Cholesterol

• Enterocyte ABCG5/8 pump plant sterols (sitosterol) back to lumen; cholesterol partly effluxed (maintains balance).

Page 32 — Phase I vs II Overview

Phase I
• Oxidation, reduction, hydrolysis.
• Enzymes: Flavin-containing monooxygenases (FMO), CYPs, oxidases.
Phase II
• Conjugations: glucuronidation, sulfation, acetylation, amino-acid, glutathione (mercapturate).
• Induction of CYP transcription via AhR/XenoRs; species & genetic variability.

Page 33 — Detox vs Bioactivation

• Biotransformation usually lowers toxicity, but can generate reactive intermediates (bioactivation).
• Hydrophilic drugs may skip Phase I/II and be excreted directly.

Page 34 — Zonal Distribution of Biotransformation Enzymes

• CYP450 high in zone 3.
• Phase II enzymes (GST, UDP-GT) also higher perivenously but glutathione highest in zone 1 → gradient affects susceptibility to toxicants.

Page 35 — Flavin-Containing Monooxygenases (FMO)

• ER membrane.
• NADPH donates 2 e^-; forms stable peroxyflavin; substrate binds after O_2.
• Broad specificity; important for soft-nucleophile oxidation (S, N).

Page 36 — Theme Block: CYTOCHROME P450 ENZYMES

Page 37 — CYP Family Overview

• Heme monooxygenases in ER (microsomal) or mitochondrial inner membrane.
• Inducible transcriptionally.
• Xenobiotic: CYP1-4 (drug metabolism; \sim75\% clinical drugs by CYP1-3).
• Endogenous: CYP7-51 (cholesterol, eicosanoids, steroids, vit D).
• Origin: gene duplications; polymorphic.

Page 38 — Microsomal CYP System Components

• NADPH-CYP reductase (FAD/FMN) shuttles electrons one-by-one.
• Cytochrome b5 may assist.
• Reaction consumes \text{O}2 (one O → product, one → \text{H}2\text{O}).

Page 39 — CYP Catalytic Cycle Steps

1. Substrate binding (RH) to ferric CYP.
2. e^- from NADPH → Fe^{2+}.
3. O_2 binding.
4. Second e^- → peroxo; protonations → “Compound I” (Fe^{4+}-oxo).
5. Oxygen insertion: \text{RH}+O \rightarrow ROH+H_2O.

Page 40 — Representative CYP Reactions

• Aliphatic / aromatic hydroxylation, epoxidation, N/O/S-dealkylations, N- & S-oxidation, dehalogenation.

Page 41 — CYPs in Biosynthesis

• Vitamin D activation/degradation.
• Mevalonate → cholesterol.
• Steroidogenesis (adrenal, gonad).
• Bile-acid synthesis (CYP7A1, etc.).

Page 42 — Lanosterol → Cholesterol (≈20 steps)

Multiple CYPs perform C-demethylations, double-bond migrations, etc.; mevalonate pathway most active in liver.

Page 43 — Bile-Acid Synthesis Regulation

• CYP7A1 (cholesterol 7α-hydroxylase) = rate-limiting.
• Bile acids activate FXR → SHP → ↓CYP7A1 transcription (feedback).

Page 44 — Vitamin D Metabolism

• Cholecalciferol (skin/diet) → 25-hydroxylase (liver CYP2R1) → calcidiol.
• Kidney: 1α-hydroxylase (CYP27B1) → calcitriol (active) or 24-hydroxylase → inactive.
• Liver/kidney failure disrupts calcitriol synthesis.

Page 45 — Mitochondrial CYPs & Steroidogenesis

• e^- flow: NADPH → adrenodoxin reductase (FAD) → adrenodoxin (2Fe-2S) → CYP (inner membrane).

Page 46 — CYP-Generated Epoxides (EETs)

• Eicosanoid epoxides (EETs) – vasodilatory, anti-inflammatory; degraded by soluble epoxide hydrolase.

Page 47 — CYP Induction via AhR

• Ligands (dioxin, PAHs) bind cytosolic AhR → nucleus with ARNT → bind XRE → transcription of CYP1A1, 1A2, others, plus Phase II enzymes.

Page 48 — Induction via Nuclear XenoRs

• RXR heterodimers: PXR, CAR, PPARγ, etc. Drug/metabolite binds → dimer → XRE in CYP 2/3 promoters.

Page 49 — Species Specificity & Pharmacogenomics

• Rodent vs human CYP profiles differ – translational caveats.
• CYP2D6 polymorphic – >70 alleles; \approx 10\% Caucasians are poor metabolizers → adverse drug effects.

Page 50 — Liver Hyperplasia & Bioactivation

• Some xenobiotics cause hepatocyte proliferation (“hepatostat”).
• Bioactivation mechanisms:
– Stable toxic metabolites (e.g., CH2Cl2 → CO).
– Electrophilic intermediates (acetaminophen).
– Free-radical formation (CCl_4).
– Redox cycling compounds (paraquat).

Page 51 — PAH Bioactivation (Benzo[a]pyrene)

CYP1 → epoxide → epoxide hydrolase → diol → further CYP → BPDE (mutagenic) → DNA adducts; detox via glucuronide, sulfate, GSH conjugates.

Page 52 — Theme Block: CONJUGATION REACTIONS

Page 53 — Glucuronidation

• Enzyme: UDP-glucuronosyl-transferase (UGT) (ER lumen).
• Cofactor: UDP-glucuronic acid (synth. from UDP-glucose + 2 NAD^+).
• Substrates: OH, COOH, NH_x, SH groups (bilirubin, drugs).

Page 54 — UDP-Glucuronic Acid Synthesis Reaction

\text{UDP-glucose}+2\,\text{NAD}^+ \xrightarrow{\text{dehydrogenase}} \text{UDP-glucuronic acid}+2\,\text{NADH}+2H^+

Page 55 — Sulfation & Acetylation

• Sulfotransferases (cytosolic) use \text{PAPS} (3′-phosphoadenosine-5′-phosphosulfate).
• Acetyltransferases use acetyl-CoA (N-, O-, S-acetylations).

Page 56 — Bile-Acid Conjugation

• Cholyl-CoA + glycine/taurine in peroxisome → glyco- or tauro-cholic acid (↓pK, ↑solubility).

Page 57 — Detox Reactions Alternative to Urea Cycle

• When ammonia excess, glutamine formation & amino-acid conjugations (benzoate → hippurate).

Page 58 — Mercapturate Pathway

1. GST catalyzes GSH conjugation (thioether).
2. γ-glutamyl-transferase removes γ-Glu.
3. Dipeptidase removes Gly.
4. N-acetyl-transferase → N-acetylcysteine conjugate (mercapturate) → urine.

Page 59 — GSTs

• ≈5 human genes; up to 10\% cytosolic protein in liver.
• Bind hydrophobic ligands (bilirubin, heme, drugs) – “intracellular albumin”.
• GST expression induced by electrophilic xenobiotics via Keap1-Nrf2 pathway.

Page 60 — Glutathione Metabolism

• Hepatic [GSH] =4\text{–}10\,\text{mM} (~99 % reduced).
• Synthesis: γ-glutamyl-cysteine synthetase (rate-limiting) + GSH synthetase.
• Recycling: γ-glutamyl cycle uses NADPH (PPP) to regenerate GSH.

Page 61 — Acetaminophen Overdose

• CYP2E1 forms NAPQI (reactive).
• Detox via GSH; depletion → hepatotoxicity.
• Antidote: N-acetylcysteine replenishes GSH pool.

Page 62 — Theme Block: EXCRETION BY ABC TRANSPORTERS

Page 63 — Canalicular (Apical) ABC Transporters

• MDR1 (ABCB1/P-gp) – hydrophobic drugs.
• MDR3 (ABCB4) – phosphatidylcholine.
• MRP2 (ABCC2) – glucuronide/sulfate/GSH conjugates.
• MRP4 (ABCC4) – cyclic nucleotides, anionic drugs.
• ABCG2 (BCRP) – drugs, porphyrins.
• ABCG5/8 – sterol efflux.
• ABCA1 – HDL formation, cholesterol export.

Page 64 — Basolateral (Sinusoidal) Export to Blood

• MRP1 (ABCC1), MRP3, MRP5, MRP6 pump conjugates to blood → kidney → urine.

Page 65 — Theme Block: OXIDATION OF ALCOHOL

Page 66 — Methanol Toxicity

• ADH converts methanol → formaldehyde → formic acid → metabolic acidosis, ocular toxicity.
• Therapy: ethanol or fomepizole (ADH inhibitor) – competitive blockade.

Page 67 — Ethanol Oxidation Pathways

1. Alcohol dehydrogenase (ADH) (cytosolic; K_m=0.2\text{–}2 \text{mM}). Not inducible; dominates at \text{BAC}<0.1\%.
2. CYP2E1 (MEOS) (ER; K_m=5\text{–}15 \text{mM}). Inducible 5\text{–}10\times; active at high BAC; consumes NADPH (energy cost).
3. Catalase (peroxisome) minor (requires H2O2).
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