Carbohydrate Metabolism Notes

Introduction to Carbohydrates

• Most abundant organic molecules in nature.
• General formula: $(C<em>nH</em>2O_n)$, a "Hydrate of Carbon".
• Two main functions:
  • Energy source: Major part of the diet.
  • Storage: Stored for later use.
• Structure:
  • Bacteria: Cell walls.
  • Insects: Exoskeleton.
  • Plants: Cellulose.
• Cell membrane role: Helps in intercellular communication.

Carbohydrate Digestion

• Main sites: Mouth and intestinal lumen.
• Fast process, mostly done by the duodenum and jejunum.
• Enzymes involved:
  • Endoglycosidases: Break oligosaccharides and polysaccharides.
  • Disaccharidases: Break disaccharides.
  • Glycosidases: Hydrolyze glycosidic bonds, turning carbs into reducing sugars.
• Specificity: Enzymes recognize the structure, configuration, and type of bond.

Carbohydrate Digestion in the Mouth

• Major dietary polysaccharides:
  • Plants: Starch (amylose and amylopectin).
  • Animals: Glycogen (stored glucose).
• Enzyme activity in the mouth: Salivary α-amylase
  • During chewing, breaks α-1,4 bonds in starch and glycogen.
  • Humans lack β-1,4 endoglucosidases, so we can't digest cellulose.
• Limitations of α-amylase:
  • Can't hydrolyze α-1,6 bonds in branched amylopectin and glycogen.
  • Produces limit oligosaccharides and dextrins.
• Digestion halts in the stomach: High acidity inactivates salivary α-amylase, stopping digestion.

Carbohydrate Digestion in the Small Intestine

• Pancreatic digestion:
  • Acidic stomach contents enter the small intestine.
  • Bicarbonate from the pancreas neutralizes acidity.
  • Pancreatic α-amylase continues starch digestion.
• Final digestion in intestinal mucosal cells:
  • Occurs in the upper jejunum via disaccharidases and oligosaccharidases.
  • Key enzymes and functions:
    • Isomaltase: Breaks α-1,6 bonds in isomaltose, producing glucose.
    • Maltase: Breaks maltose, producing glucose.
    • Sucrase: Breaks sucrose, producing glucose and fructose.
    • Lactase (β-galactosidase): Breaks lactose, producing glucose and galactose.
  • Enzymes are bound to the brush border membranes of intestinal mucosal cells.

Absorption of Monosaccharides

• Where? Duodenum and upper jejunum.
• Insulin not needed for glucose uptake.
• Transporters:
  • Glucose and galactose: Active transport, needs energy, sodium-dependent (SGLT).
  • Fructose: Facilitated diffusion (GLUT5).
  • All monosaccharides: Blood portal circulation (GLUT2).

Abnormal Degradation of Disaccharides

• Normal: Carbs absorbed by the lower jejunum.
• Deficiency in disaccharidase:
  • Undigested carbs enter the large intestine.
  • Osmotic diarrhea.
  • Bacterial fermentation: \CO2, $\H</em>2$, organic acids.
  • Gas, cramps, diarrhea, flatulence.

Digestive Enzyme Deficiencies

• Causes:
  • Hereditary disaccharidase deficiency (infants, children).
  • Intestinal diseases, malnutrition, drugs (mucosa damage).
  • Severe diarrhea (temporary enzyme loss).
• Effect: Can't digest dairy, sucrose leads to diarrhea.

Lactose Intolerance

• Cause: Lactase deficiency, age-dependent, genetic.
• Common in: African, Asian adults (up to 90%).
• Symptoms: Bloating, diarrhea, dehydration.
• Treatment: Avoid milk, take yogurt, cheese, green vegetables, calcium, lactase enzyme pills.
• Diagnosis: Oral tolerance test with disaccharides.
• Breath hydrogen test: Measures $H_2$ from bacteria fermenting undigested carbs.

Glycolysis

• Purpose: Breakdown of glucose to produce ATP and intermediates for other pathways.
• Glucose: Abundant and used by all tissues.
• Central hub in carb metabolism: All sugars converted to glucose for glycolysis.
• End product: Pyruvate
  • With oxygen (mitochondria): Acetyl CoA → TCA cycle.
  • Without oxygen or mitochondria: Lactate (anaerobic).
• Key Facts:
  • 10 reactions.
  • ATP and NADH formed.
  • Intermediates link to other metabolic pathways.

Types of Glycolysis

• Aerobic glycolysis:
  • Oxygen needed, NADH produced, ATP produced.
• Anaerobic glycolysis:
  • No oxygen or mitochondria, Lactate produced, ATP produced (e.g., RBCs).
• Special intermediates:
  • 1,3-BPG: ATP production via substrate-level phosphorylation.
  • 2,3-BPG (high in RBCs): Shunt pathway, phosphoglycerate modifies glycolysis in RBCs.

Regulation of Glycolysis

• Hormonal regulation via fructose-2,6-bisphosphate:
  • Well-fed (high insulin, low glucagon): increases fructose-2,6-bisphosphate, promotes glycolysis.
  • Starvation (high glucagon, low insulin): decreases fructose-2,6-bisphosphate, inhibits glycolysis and promotes gluconeogenesis.
• Pyruvate Kinase regulation:
  • Glucagon → cAMP → PKA
    • Phosphorylation inactivates pyruvate kinase, decreasing Glycolysis.
• PFK-1 (Phosphofructokinase-1):
  • Rate-limiting, committed step.

GLUT Transporters

• Na+-independent facilitated diffusion transport:
  • Definition: Glucose transport without sodium across cell membranes.
  • Facilitated diffusion is passive, no energy required.
• GLUT Transporters: Family of 14 transporters (GLUT1 to GLUT14).
  • Found in different tissues, each with specific roles.
• Mechanism:
  • Extracellular glucose binds to GLUT transporter.
  • Transporter changes shape (2 conformational states).
  • Glucose moves into the cells.
• Key points:
  • Na+-independent: Does not need sodium or energy.
  • Facilitated diffusion: Relies on the glucose gradient.
  • Major glucose uptake system in many tissues.
• GLUT transporters by tissue:
  • GLUT 2: Liver, pancreatic β-cells (insulin-independent), helps glucose sensing.
  • GLUT3: Neurons, erythrocytes, brain (low in adult muscle).
  • GLUT4: Adipose tissue, skeletal muscle (insulin increases GLUT-4 activity).

Energy Yield from Glycolysis

• Anaerobic: 2 ATP per glucose → lactate.
• Aerobic: 4 ATP per glucose → pyruvate + 2 NADH.
  • NADH in ETC → 3 ATP per NADH.
  • NADH needs shuttle (malate-aspartate or glycerol-3-P) to enter mitochondria.

Arsenic Poisoning

• Inhibits pyruvate dehydrogenase, which needs lipoic acid.
• Arsenate replaces phosphate, bypassing 1,3-BPG → No ATP or NADH is produced, glycolysis continues without energy.

Pyruvate Kinase Deficiency

• ATP deficiency → RBC membrane damage → Hemolytic anemia.
• Compensation: increase in 2,3-BPG in RBCs.
• Second most common enzyme deficiency anemia after G6PD.

Lactate (Anaerobic End Product)

• Produced in $O_2$ shortage: exercise, shock, poor blood supply.
• Converted back to pyruvate in liver (gluconeogenesis) or heart (TCA cycle).
  • Liver: Converts lactate → pyruvate → glucose (gluconeogenesis).
  • Heart: Oxidizes lactate → \CO2 + $\H</em>2O$ (TCA cycle).
• Lactic Acidosis: Increased Lactate due to shock, MI, pulmonary embolism, hemorrhage.
  • Cells rely on anaerobic glycolysis for ATP emergency.
  • $O_2$ debt monitored by blood lactate levels.

TCA Cycle (Krebs Cycle)

• Overview:
  • Central metabolic pathway in mitochondria, fully aerobic.
  • Final oxidation of carbohydrates, amino acids, and fatty acids into \CO_2.
  • Generates NADH, FADH2, GTP/ATP, driving ATP production via oxidative phosphorylation.
• Key functions:
  • Energy production: Major source of cellular energy (ATP).
  • Biosynthesis: Provides intermediates for amino acids, nucleotides, heme, and glucose.
  • Anaplerotic reactions: Maintain cycle function by replenishing intermediates.
• Cycle steps, key intermediates, and enzymes:
  1. Acetyl CoA + Oxaloacetate → Citrate (via Citrate synthase; one-way).
  2. Citrate ↔ Isocitrate (via Aconitase; reversible).
  3. Isocitrate → α-ketoglutarate (via Isocitrate Dehydrogenase; NADH, \CO_2; one-way).
  4. α-ketoglutarate → Succinyl CoA (via α-ketoglutarate Dehydrogenase; NADH, \CO_2; one-way).
  5. Succinyl CoA ↔ Succinate (via Succinyl CoA synthetase; GTP/ATP; reversible).
  6. Succinate ↔ Fumarate (via Succinate Dehydrogenase; FADH2; reversible).
  7. Fumarate ↔ Malate (via Fumarase; reversible).
  8. Malate ↔ Oxaloacetate (via Malate Dehydrogenase; NADH; reversible).

Regulation and Medical Significance of TCA Cycle

• Energy Yield (per Cycle):
  • 3 NADH
  • 1 FADH2
  • 1 GTP
• Electron Transfer and ATP Generation:
  • 4 pairs of electrons are transferred (3 NADH, 1 FADH2).
  • NADH oxidation in the electron transport chain yields 3 ATP per NADH.
  • FADH2 oxidation yields 2 ATP per FADH2.
  • GTP is converted to ATP via substrate-level phosphorylation.
  • Total: 12 ATP produced per Acetyl CoA.
• Regulation:
  • Activated by: ADP, \Ca^{2+} (signals energy need).
  • Inhibited by: ATP, NADH, Succinyl CoA (signals sufficient energy).
• Cycle Summary:
  • Net result: Two carbons enter as Acetyl CoA and leave as 2 \CO_2.
  • Oxaloacetate is regenerated at the end of the cycle → ensures cycle continues.
  • No net loss or gain of intermediates occurs.
• Conversion of pyruvate to Acetyl CoA:
  • Pyruvate dehydrogenase complex converts pyruvate into acetyl CoA.
  • Requires CoA, produces NADH + \H^+, and releases \CO_2.
  • Acetyl CoA and NADH inhibit this reaction.
• Coenzymes involved:
  • Thiamine pyrophosphate ($E_1$).
  • Lipoic acid and CoA ($E_2$).
  • FAD and NAD ($E_3$).
• Medical significance:
  • Thiamine or niacin deficiencies can cause nervous system dysfunction.
  • Brain cells depend on ATP from the TCA cycle, making pyruvate dehydrogenase function essential.

Pyruvate Dehydrogenase Deficiency (PDH Deficiency)

• Cause: Deficiency in $E_1$ of pyruvate dehydrogenase complex.
• Congenital lactic acidosis.
• Effect: Pyruvate can't convert to acetyl CoA → turns to lactic acid via lactate dehydrogenase → acidosis.
• Brain impact: Brain relies on the TCA cycle for energy, making it sensitive to acidosis.
• Genetics: X-linked, but affects both sexes (X-linked dominant).
• Symptoms: Lactic acidosis, developmental defects, brain/nervous system, muscular spasticity/stiffness, early death.
• Treatment: No proven treatment.

Leigh Syndrome

• Definition: Progressive, neurodegenerative disorder (subacute, necrotizing encephalomyelopathy).
• Cause: Mitochondrial ATP production defects due to mutations in genes encoding:
  • PDH complex.
  • Electron transport chain.
  • ATP synthase.
• Affected DNA: Both nuclear and mtDNA.
• Consequence: Low ATP → severe brain damage → could cause death.

Gluconeogenesis

• Overview:
  • Tissues that need glucose: Brain, RBCs, kidney medulla, lens, cornea, testes, exercising muscles.
  • Liver glycogen lasts 10-18 hours without dietary carbs. After depletion, glucose is made from lactate, pyruvate, glycerol, α-ketoacids.
• Gluconeogenesis process:
  • Not glycolysis reversal; glycolysis favors pyruvate.
  • Uses mitochondrial and cytosolic enzymes.
  • Overnight fasting: 90% in liver, 10% in kidneys.
  • Prolonged fasting: Kidneys produce 40% of glucose.
• Pathway Steps, Enzymes, Energy, and Coenzymes:
  • Pyruvate → Oxaloacetate (OAA) by Pyruvate carboxylase (uses ATP, biotin-dependent; mitochondria)
  • Oxaloacetate (OAA) → Phosphoenolpyruvate (PEP) by PEP carboxykinase (PEPCK; uses GTP, releases \CO_2)
  • Phosphoenolpyruvate (PEP) → 2-phosphoglycerate by Enolase
  • 2-phosphoglycerate → 3-phosphoglycerate by phosphoglycerate mutase
  • 3-phosphoglycerate → 1,3-Bisphosphoglycerate by phosphoglycerate kinase
  • 1,3-Bisphosphoglycerate → Glyceraldehyde-3-phosphate (G3P) by G3P dehydrogenase
  • G3P ↔ Dihydroxyacetone Phosphate (DHAP) by Aldolase
  • Fructose-1,6-bisphosphate → Fructose-6-phosphate by fructose-1,6-bisphosphatase (Enzyme control step inhibited by fructose-2,6-bisphosphate; uses \H_2O hydrolysis)
  • Fructose-6-phosphate ↔ Glucose-6-phosphate by phosphoglucose isomerase
  • Glucose-6-phosphate → Glucose by Glucose-6-phosphatase (Enzyme removes phosphate only in liver and kidney; absent in muscle. Muscle glycogen cannot raise blood glucose. Uses \H_2O hydrolysis)

Cori Cycle

• Muscle and liver interaction:
  • Muscle: Converts glucose → pyruvate → lactate (anaerobic glycolysis) produces ATP quickly.
  • Lactate travels via blood to the liver.
  • Liver: Converts lactate → pyruvate (Lactate dehydrogenase, DH) → glucose via gluconeogenesis
    (ATP is energy-consuming process).
  • Glucose enters blood then goes back to muscle and repeats the cycle.

Biotin and Pyruvate Carboxylase

• Function: Converts pyruvate → oxaloacetate (OAA).
• Enzyme name: Pyruvate carboxylase.
• Coenzyme: Biotin.
• Biotin enzyme + pyruvate carboxylase + \CO_2 → biocytin complex.
• Energy source: ATP hydrolysis where mitochondria of liver and kidney.
• Purpose: Gluconeogenesis and TCA cycle replenishment if intermediates are low.
• Muscles: Use pyruvate carboxylase only for the Krebs cycle, not glucose synthesis.
• Total energy used per glucose: 4 ATP, 2 GTP, 2 NADH.

Glycogen Metabolism

• Lysosomal Degradation:
  • ~1-3% of glycogen degraded by acid maltase in lysosomes.
  • Function is unknown.
• Glucose Sources:
  • Glucose is essential: Only source for RBCs, preferred by the brain, used in muscles for energy.
  • Three sources: Diet, glycogen breakdown, gluconeogenesis.
  • Dietary glucose is unreliable, so the body stores and produces glucose.
• Role of Glycogen:
  • Glycogen is a fast, stored glucose source in liver and muscles.
  • Liver glycogen: Maintains blood glucose when fasting.
  • Muscle glycogen: Provides energy for muscle contraction.
    • \Ca^{2+} binds calmodulin → activates glycogen phosphorylase → fast glycogen for quick ATP.
    • When glycogen runs out, gluconeogenesis from amino acids takes over.
• Glycogen Breakdown Pathway:
  • Liver glycogen → glucose → blood glucose.
  • Muscle glycogen → glucose-6-phosphate (G6P) → energy (not released into blood).
• Amounts of Liver and Muscle Glycogen:
  • Muscle glycogen: ~400g (1-2% of muscle weight).
  • Liver glycogen: ~100g (2-8% of liver weight); storage limit is unknown, but in storage diseases, too much glycogen builds up.
• Structure of Glycogen:
  • Branched polysaccharide made of α-D-glucose.
  • Main chain: α-1,4 linkages.
  • Branches: α-1,6 linkages every 8-10 glucose units.
  • Stored in cytoplasmic granules with enzymes for synthesis and breakdown.
• Fluctuation of Glycogen Stores:
  • Liver glycogen: Increases when fed, depleted during fasting.
  • Muscle glycogen: Not affected by short fasting, slightly reduced in prolonged fasting, replenished/restored after exercise.
  • Synthesis and degradation happen continuously.

Regulation of Glycogen Metabolism in Liver vs. Muscle

• Liver:
  • Glycogen breakdown inhibited by ATP, glucose, G6P.
• Muscle:
  • Glycogen breakdown inhibited by ATP, G6P, but activated by AMP.
• Enzyme: Glycogen phosphorylase is key in both.

Glycogen Storage Diseases (GSDs)

• Caused by enzyme defects in synthesis or degradation.
• Leads to abnormal glycogen (structure) or excess normal glycogen buildup.
• May affect one organ (like liver) or be generalized (liver, muscle, kidney).
• Severity: From mild to fatal in infancy.

GSD Types

• McArdle (V): Muscle phosphorylase deficiency → glycogen in muscle.
• Cori (III): Debranching enzyme deficiency → abnormal glycogen with short outer branches.
• Pompe (II): Acid maltase (lysosomal glucosidase) deficiency → only lysosomal GSD, glycogen builds up in lysosomes due to faulty breakdown.
• Von Gierke(Ia, Ib): Glucose-6-phosphatase or transporter deficiency, affects liver and kidneys and causes fasting hypoglycemia

Glycogen Synthesis Pathway

• Occurs in the cytoplasm.
• It begins with glycogenin which acts as a primer.
• Steps:
  1. Glucose → Glucose-6-Phosphate (G6P) by Hexokinase (muscle) or Glucokinase (liver).
     • Uses ATP to trap glucose in the cell.
  2. G6P ↔ Glucose-1-phosphate by phosphoglucomutase.
  • Rearranges phosphate position.
  1. G1P + UTP → UDP-glucose + PPi by UDP-glucose pyrophosphorylase.
     • Activates glucose for glycogen synthesis.
  2. UDP-glucose added to glycogenin starter by Glycogen Synthase adds α-1,4 bonds
     • Glycogenin forms primer, then glycogen synthase extends the chain
  3. Branch formation catalyzed by branching enzyme (amylo α-1,4 → α-1,6 transglycosylase)
     • Creates α-1,6 branches every 8-10 glucose units.

Glycogen Degradation (Glycogenolysis)

• Occurs in the cytosol with different enzymes than synthesis.
• Steps:
  1. Glycogen → Glucose-1-Phosphate (G1P) by Glycogen phosphorylase
     • Breaks α-1,4 bonds, releases G1P.
  2. Debranching of α-1,6 branches by Debranching enzyme (4:4 transferase and α-1,6 glucosidase)
     Transfers branch and removes last glucose as free glucose.
  3. G1P ↔ Glucose-6-Phosphate (G6P) by phosphoglucomutase.
     Converts G1P to G6P for further use: glucose in liver, glycolysis in muscle.

Metabolism of Monosaccharides and Disaccharides

• Fructose Metabolism:
  • Sources: Sucrose (main), fruits, honey, corn syrup (55% fructose).
  • Entry: Not insulin-dependent, doesn't trigger insulin release.
  • Daily intake: ~50g (~10% of calories in Western diet).
  • Pathway:
    1. Fructose → Fructose-1-phosphate (Fructokinase uses ATP)
    2. Fructose-1-P → DHAP + glyceraldehyde (Aldolase B)
    3. Glyceraldehyde → Glyceraldehyde-3-P (Triose kinase uses ATP)
  • Final products: DHAP and G3P enter glycolysis, gluconeogenesis, or lipogenesis.
• Sorbitol Pathway (Glucose → Fructose):
  • Glucose → Sorbitol (Aldose reductase uses NADPH)
  • Sorbitol → Fructose (Sorbitol dehydrogenase uses NAD+)
  • Tissues:
    • Aldose reductase: widespread in Lens, retina, nerve, kidney, etc.
      • Sorbitol dehydrogenase: liver, ovaries, sperm.
  • Purpose:
    • Sperm: Fructose is the main energy source.
    • Liver: Converts sorbitol to fructose, which enters metabolism.
• Sorbitol in Hyperglycemia:
  • In diabetes: Glucose enters lens, nerve, kidney without insulin.
    • Aldose reductase makes excess sorbitol without Sorbitoldehydrogenase
      • Sorbitol is trapped(it cannot cross membranes well) → water retention → osmotic swelling → damage.
  • Diabetic Complications: Cataracts, peripheral neuropathy, nephropathy, retinopathy.
• Galactose Metabolism:
  • Sources: Lactose (milk sugar), glycoproteins, glycolipids.
  • Entry: Not insulin-dependent.
• Lactose Synthesis:
  • Lactose = galactose + β-1,4 glucose
  • Made in Golgi by “Lactose synthase”
    *Enzyme structure: protein A :β-D-galactosyltransferase usually makes glycoproteins
    *protein B: α-lactalbumin only in lactating mammary glands, stimulated by prolactin
    *A + B = Lactose synthase makes lactose instead of glycoproteins

Disorders of Monosaccharide and Disaccharide Metabolism

• Essential Fructosuria:
  • Cause: Fructokinase deficiency (AR).
  • Effect:Fructose in urine. Benign.
• Hereditary Fructose Intolerance:
  • Cause:Aldolase B deficiency (AR).
  • Effect: Fructose-1-P traps PiATPtoxic→ liver damage etc..
    Hypoglycemia, vomiting, jaundice, hepatomegaly, renal failure
  • Diagnosis: Urine enzyme & DNA test. Newborn screening
  • Treatment: Avoid fructose, sucrose, sorbitol .
• Galactosemia * Galactokinase deficiency AR
  • Galactose +P galactitol buildup
    Cataracts
    Clinical dier history
    avoid galactose
  • Glucose enters lens, nerve, kidney without insulin.
    • Aldose reductase makes excess sorbitol without Sorbitoldehydrogenase
      In lens, nerve,kidney
      Causes diabetic symptoms
      Control blood glucose
      Newborn screening
      avoid galactose, lactose
      In diabetes:Sorbiol accumulation
      Glucose enters lens, nerve,kidney without insulin
      Aldose reductaseSorbital trapped osmotic swelling
      Cataracts,heuropathy ,nephropathy,retinopathy
      Classic Galactosemia
      GALT deficiency AR
      Galactose P galactitol buildup vomiting,jaundice , liver brain damage, cataracts,ovarian failure

Pentose Phosphate Pathway (PPP) and NADPH

• * PPP Overview:
  • Location: Cytosol.
  • Two parts:
    • Oxidative (irreversible): Glucose-6-phosphate → ribulose-5-phosphate (NADPH, \CO_2; enzyme G6PD).
    • Non-oxidative (reversible): Interconverts sugars (depends on cells' need for ribose, NADPH, or glycolysis intermediates).
  • No ATP used or produced.
  • Main products and functions:
    • NADPH: Provides \H^+ for biosynthesis and ROS (reactive oxygen species) defense.
    • Ribose-5-phosphate: Provides it for nucleotides.
    • Also metabolizes 5-carbon sugars.
  • Another name: Hexose monophosphate shunt.

NADPH Functions

• Reductive biosynthesis: Fatty acid and steroid synthesis.

• ROS detoxification:

  • Glutathione peroxidase ( H2O2 → H2O) requires GSH (glutathione).
  • Glutathione reductase: Regenerates GSH using NADPH.
  • Superoxide dismutase (SOD): \O2 → $\H</em>2O_2$.
  • Catalase: $\H<em>2O</em>2$ → $\H<em>2O$ + \O2.
  • RBCs depend fully on PPP for NADPH.

• Cytochrome P450 system (RH + \O2 + NADPH → ROH + $\H</em>2O$):

  • Mitochondria: Hydroxylation for steroid, bile acid, Vitamin D synthesis.
  • Microsomes (ER): Drug/toxin detox (hydroxylation → solubility or conjugation).

• Phagocytosis respiratory burst

  • NADPH oxidase: O2- → O2
  • SOD (superoxidedismutase): O2- → H2O2
    • MPO (myeloperoxidase): H2O2 + Cl- → HOCl (kills microbes).

• Nitric Oxide (NO) synthesis (NADPH + \O_2 → NO):

  • Functions: Vasodilation, NT, anti-platelet, immunity.
  • Short half-life (3-10 sec).

Diseases and Deficiencies Related to the Pentose Phosphate Pathway

• G6PD Deficiency: *G6PD : X-linked
  • NADPH glutathione ROS
    RBC damage heinzbodies hemolysis.most common enzyme deficiency 200M :gives malaria resistance
    Effect: Haemolytic anemia (triggers from exposure to oxidants in G6PD-deficient patients).
• Chronic Granulomatous Disease (CGD):
  *Defective NADPH oxidase in phagocytes O production respiratory burst persistent infections,granulomas.
  Inherited often X linked
  Can't kill pathogens effectively
  Key triggers