ACMT Board Review 2012: Molecular Mechanisms

Russ Kerns, MD, FACMT Carolinas Medical Center

Charlotte, NC

Objectives: Cover Core Content

  1.2 Molecular components/mechanisms   1.2.1 Glycolysis & oxidative phosphorylation   1.2.2 Other metabolic pathways (β-oxidation)   1.2.4 Transport proteins (hemoglobin)   1.2.5 Channels

  1.3 Cytotoxic mechanisms   Provide key example toxins

1.2.1 Glycolysis & Oxidative Phosphorylation

Oxidative Phosphorylation

  Energy is released when ATP → P + ADP   Restoration of ATP (energy stores) involves

phosphorylation of ADP via coupling of oxidation of H+ to form H20.

Oxidative Phosphorylation

carbohydrate/fatty acid

acetyl-CoA

TCA

NADH/FADH2

electron transport chain

H+ + e-

P

ADP ATP H2O ADP antiporter

H+

e- + O2- + H+

ATP synthase

H+ H+

  Main carb metab path   Cytosolic process   6C cmpd → 2 X 3 C cmpd   Net 2 ATP molecules   Make pyruvate for Kreb’s

Glycolysis glucose

glucose-6-P

fructose-6-P

glyceraldehyde-3-P + DHA-P

2 [pyruvate]

fructose-1,6-diP

2 [glyceraldehyde-3-P]

2 [1,3-diphosphoglycerate]

2 [P intermediates]

ATP

ATP

ATP

ATP

  Arsenic (V)   Substitutes for P   Fail to make 1,3-diP intermed   Fail to make ATP   Fail to make pyruvate

Glycolysis: Toxins

glyceraldehyde-3-P + DHA-P

2 [glyceraldehyde-3-P]

2 [1,3-diphosphoglycerate]

2 [P intermediates]

ATP

2NAD+ + 2 P As5+ X

Glycolysis

CH3CO-CoA acetyl-CoA

NADH

CH3COCOOH

CoA, NAD+

CH3CH2OCOOH

NAD+

lactate

pyruvate

NADH, CO2

Glycolysis: Toxins

CH3CO-CoA acetyl-CoA

CH3COCOOH

lipoamide

pyruvate

dihydrolipoamide

As3+

X

Kreb’s TCA Cycle pyruvate acetyl-CoA

NADH NAD+

citrate

Succinyl-CoA

oxalosuccinate

isocitrate

oxaloacetate

succinate

maleate cis-aconitate

α-ketoglutarate

fumarate

NAD+

NADH

NAD+ NADH

FADH FADH2

Kreb’s TCA Cycle: Toxins

Rodenticides   Sodium monofluoroacetate   Fluoroacetamide

acetyl-CoA

fluorocitrate

isocitrate

oxaloacetate

cis-aconitate monofluoroacetate

fluoroacetamide

Electron Transport Chain   Mitochondrial process   Series of oxidation-reduction reactions

  Cytochrome enzymes   FADH2 and NADH electron sources   Produce H2O and ATP

ETC: Toxins   Enzyme inhibitors   Uncouplers

X X

cytochrome oxidase aa3 NADH-CoQ reductase

X

Cytochrome Oxidase Inhibitors

acetyl CoA

lactate

glucose

pyruvate

TCA cycle

ATP H +

e- transport X X X

lactate

lactate lactate

lactate

lactate lactate

lactate

ETC: Toxins   Uncouplers

  Salicylate   Dinitrophenol (explosives & wood preservative)   Pentachlorophenol (fungicide)

  Cytochrome aa3 inhibitors   Cyanide   H2S   CO   Methanol   Phosphine gas   Sodium azide (propellant in airbags)

  NADH-CoQ reductase   Rotenone (plant derived fish poison)

1.2.2 β-Oxidation of FFA

fatty acyl-CoA

CoA carnitine

fatty acylcarnitine fatty acyl-CoA

carnitine-palmitoyltransferase

CoA

(CH)nCO-CoA (CH)n-2CO-CoA + CH3CO-CoA acetyl-CoA

NADH, FADH2

CoA

(CH)nCOOH (CH)nCO-CoA acyl-CoA synthetase

ATP

fatty acyl-CoA

ADP + P CoA

β-Oxidation of FFA: Toxins

fatty acyl-CoA

CoA carnitine

fatty acylcarnitine fatty acyl-CoA

CoA

(CH)nCO-CoA (CH)n-2CO-CoA + CH3CO-CoA acetyl-CoA

NADH, FADH2

CoA

etoh, hypoglycin

valproate

β-Oxidation of FFA: Toxins   ↑ NADH/NAD+ ratio

  ethanol   Hypoglycin(?)

  Carnitine   Valproate

  Undefined mechanism   Aflatoxin   Amiodarone   cereulide   dimethylformamide   tetracycline

www.australianprescriber.com

Mitochondrial DNA: NRTIs

  Nucleoside reverse transcriptase inhibitors   Mechanism

  Inhibit mitochondrial DNA replication   Inhibit ADP/ATP antiporter(?)

  Result   Lactic acidosis ± steatosis

  Agents   Stavudine   Didandosine   Zalcitobine   Zidovudine   Tenofovir (nucleotide)

X

1.2.4 Transport Proteins

Hemoglobin   iron-based tetrameric protein   α- and β-globin chains (2 each)   Heme complex in each chain (4 total)

  protoporhyrin ring   central iron atom

Hemoglobin: Toxins

 Site of action of toxins  Heme synthesis  Erythropoiesis  Hemorrhage  Oxidant stress  Competition for oxygen binding

Heme Synthesis: Direct Toxin

Harrison’s On-Line

Heme Synthesis: Indirect Toxins

  Acute Intermittent Porphyria   Hepatic   Autosomal dominant   Reduced HMB synthase activity   Some drugs may exacerbate AIP by

increasing ALA-synthase activity   Poorly defined mechanism

Heme Synthesis: AIP

  Barbiturates   Carisoprodol   Danazol   Ethchlorvinyl   Meprobamate   Primidone   Pyrazolones   Trimethadione

dark red urine

www.porphyriafoundation.com

Hemoglobin: Toxins

  Erythropoiesis  Nephrotoxins - ↓ erythropoietin  Pure rbc aplasia – rare

 INH  Hypoglycemics (chlorpropamide, tolbutamide)  Phenytoin  Sulfasalazine  Valproate (single case report)

Hemoglobin: Aplastic Anemia aplastic marrow normal marrow

www.hopkinsmedicine.org

Hemoglobin: Aplastic Anemia

  Immune mediated   T lymphocytes release cytokines

  Suppress hematopoietic stem cells   Apoptosis (↑ Fas receptors on stem cells)

TNF

interferon-γ

Hemoglobin: Aplastic Anemia

  DNA injury   Direct DNA injury

  Ionizing radiation   Inhibition of DNA replication

  Folate inhibitors (methotrexate)   Intermediary metabolite that binds DNA

  Benzene (quinone + free radicals)   Tubulin inhibition during cell replication

  Antimitotics (colchicine, vincristine, vinblastin)

metaphase arrest

metaphase.wordpress.com

Hemoglobin: Aplastic Anemia   Antibiotics

  Chloramphenicol   Anti-convulsants

  Carbamazepine, phenytoin   Anti-inflammatory agents

  Diclofenac, D-penicilamine, gold salts, indomethicin, phenylbutazone

  Anti-neoplastic agents   Alkylating agents (nitrogen mustards)   Antibiotics (danorubicin, adriamycin)   Antimitotics (colchicine, vinblastin, vincristine)   Antimetabolites (purine and pyrimidine analogues)

  Antipsychotics   Chlorpromazine, clozapine

Hemoglobin: Aplastic Anemia   Chemicals

  Benzene, lindane   Metals

  Arsenic   Miscellaneous

  Acetazolamide, captopril, cimetidine, chlorpromazine, dapsone, fluoxetine, meprobamate, nifedipine, PTU, ticlopidine, tocainide

  Radiation

Hemoglobin: Toxins

www.pathology.vcu.edu

Hemoglobin: Toxins

  Megaloblastic anemia   ↓ Vit B12 absorption

  Colchicine, metformin, neomycin   ↓ folate absorption

  Etoh   Impaired dihydrofolate reductase

  Methotrexate   Pyrimethamine   Pyridium   Trimethoprim

Hemoglobin: Oxidant Stress

  Heme: deoxyhgb → Methgb (Fe2+→Fe3+)   Prophyrin ring by sulfur: Sulfhgb   Globin: Heinz body hemolytic anemia

Hemoglobin: Oxidant Stress

  Protection from oxidant stress   Ascorbic acid (Vit C)   Glutathione (intact HMP, G6PD)   Enzymatic

  NADH-dependent reductase (Cytochrome b5 reduc)   NADPH-dependent reductase (intact HMP, G6PD)   Catalase   Hydrogen peroxidase

Hemoglobin: Methemoglobin

  Fe2+ state: deoxyhemoglobin carries oxygen 