Citric Acid Cycle Overview

ITRIC ACID CYCLE (aka Krebs cycle and tricarboxylic acid cycle)

  • Occurs under aerobic conditions (has many exits and entry points)
  • Occurs in the mitochondrial matrix (pyruvate transported from cytosol)
  • First intermediate is Acetyl CoA (two carbon molecule)
  • 8 more intermediates to complete the cycle: Oh, Can I Keep Studying Science For Med-school?

ACETYL CoA

  • 2-carbon molecule
  • Can come from carbohydrates (via pyruvate), fatty acids and amino acids
  • Pyruvate dehydrogenase complex: Pyruvate + NAD + –> Acetyl CoA + CO2 + NADH

CITRATE AND ISOCITRATE

  • 6-carbon molecules
  • Oxaloacetate (4 carbons) + Acetyl CoA (2 carbons) = Citrate
  • Isomerized to form isocitrate

ALPHA-KETOGLUTARATE

  • 5-carbon molecule
  • 1 carbon dioxide and 1NADH released in its production

SUCCINYL CoA

  • 4-carbon molecule
  • 1 carbon dioxide and 1 NADH released in its production

SUCCINATE

  • 4-carbon molecule
  • Lost CoA and 1 ATP produced via substrate level phosphorylation

FUMARATE

  • 4-carbon molecule
  • FADH2 released in its production (F for FADH2 and Fumarate)

MALATE TO OXALOACETATE

  • Last reaction in the cycle
  • Both are 4-carbon molecules
  • Last NADH released in the cycle

TOTAL OUTPUT:

  • Pyruvate decarboxylation: 1 NADH and 1 carbon dioxide
    (x2 per glucose)
  • Per turn: 2 carbon dioxide molecules, 3 NADH and 2 FADH2
    (2 turns per glucose)
    *NADH and FADH2 deliver electrons to electron transport chain on inner mitochondrial membrane

Pyruvate Dehydrogenase Complex Part I

PYRUVATE DEHYDROGENASE COMPLEX (PDC)

  • Pyruvate + CoA + NAD+ –> Acetyl CoA + CO2 + NADH
  • Located in mitochondrial matrix
  • Irreversible reaction

PDC ENZYMES

E1, pyruvate dehydrogenase/pyruvate decarboxylase

  • Catalyzes pyruvate to acetyl (releases CO2)
  • Cofactor: thiamine pyrophosphate (Vitamin B1)

E2, dihydrolipoyl transacetylase

  • Attaches CoA to acetyl
  • Cofactor: lipoic acid (not vitamin-derived) & coenzyme A (pantothenic acid/vitamin B5)

E3, dihydrolipoyl dehydrogenase

  • Reduces NAD+ to NADH
  • Cofactor: NAD+ (niacin/vitamin B3) & FAD (riboflavin/vitamin B2)

Lipoic acid is only cofactor for PDC that is not vitamin-derived

CLINICAL CORRELATION

PDC-based pathology

  • Deficiencies in vitamins or PDC cofactors produce initial neurological/muscular symptoms

Fates of Pyruvate

KEY FATES OF PYRUVATE

  1. Acetyl CoA: substrate for citric acid cycle and fatty acid synthesis
  2. Oxaloacetate: intermediate in CAC and substrate for gluconeogenesis
  3. Lactate: produced by eukaryotes in absence of oxygen
  4. Ethanol: produced by yeast and some bacteria (including intestinal flora) in absence of oxygen.

AEROBIC CONDITIONS

  1. Cellular respiration: Pyruvate converts to acetyl CoA
  • Fed conditions (glucose abundant)
  • Occurs in mitochondrial matrix
  • Pyruvate dehydrogenase complex
  • Irreversible reaction: produces 1 CO2 and 1NADH
  • Acetyl CoA enters the citric acid cycle and oxidative phosphorylation
  • Final product is ATP
  1. Gluconeogenesis: Pyruvate converts to oxaloacetate
  • Fasting conditions (glucose in demand)
  • Occurs in liver (minor process in kidneys): mitochondrial matrix
  • Pyruvate carboxylase
  • Irreversible reaction
  • Oxaloacetate is substrate for gluconeogenesis and CAC intermediate

ANAEROBIC CONDITIONS

  1. Lactic acid fermentation (humans)
  • Occurs in exercising muscle and red blood cells: cytosol
  • Glycolysis: 1 glucose = 2 pyruvates + 2 ATP + 2 NADH
  • Lactate dehydrogenase: 2 pyruvate + 2NADH = 2 lactate + 2 NAD+
  • Reversible reaction
  • Lactate can enter bloodstream and travel to liver: lactate dehydrogenase catalyzes reverse reaction (lactate to pyruvate)

Clinical correlation: intense exercise can produce lactic acidosis; lactate accumulates in muscle cells and causes intracellular drop in pH

  1. Ethanol production (yeast and select bacteria)
  • Can occur in inteestinal flora
  • Glycolysis: 1 glucose = 2 pyruvates + 2 ATP + 2 NADH
  • 2 step rxn: pyruvate to acetaldehyde to ethanol
  • Ethanol formation consumes 2 NADH in second step and produces 2 NAD+ for reuse
  • Irreversible reaction
  • Fermentation in yeast used to make beer and wine

Pyruvate Kinase

  • Last enzyme in glycolysis
  • Irreversibly dephosphorylates phosphoenolpyruvate (PEP) to form pyruvate
  • 1 ATP produced by substrate level phosphorylation
  • Several isozymes: M-type (muscle) and L-type (liver)
  • All isozymes allosterically regulated (L-type also hormonally regulated)

M-TYPE ISOZYMES

Allosteric regulation

Activation

  • AMP: marker of ATP depletion or low energy
  • Fructose 1,6-bisphosphate: product of rate-limiting reaction in glycolysis
    (feed-forward activation: stimulates downstream glycolytic enzymes)

Inhibition

  • ATP: sufficient energy
  • Acetyl CoA: first intermediate of citric acid cycle
  • Alanine: can be produced from pyruvate; sufficient pyruvate in the cell

L-TYPE ISOZYME

  • Allosteric and hormonal regulation (similar to PFK-2)

Hormonal regulation

Activation

  • Insulin activates phosphatases, which remove phosphate from PK
  • Makes PK susceptible to positive allosteric regulators

Inhibition

  • Glucagon promotes phosphorylation of PK via cAMP-dependent pathway
  • Makes PK susceptible to negative allosteric regulators

CLINICAL CORRELATION

Pyruvate kinase deficiency

  • Produce hemolytic anemia (spiculated RBC’s)
  • RBC biconcave shape maintained by sodium-potassium pumps (require ATP)
  • RBC’s do not have mitochondria: rely on glycolysis for ATP

Phosphofructokinase

PHOSPHOFRUCTOKINASE-1 (PFK-1)

  • Catalyzes rate-limiting step in glycolysis
  • Catalyzes irreversible phosphorylation of F6P to F1,6P.
  • Allosterically regulated (hormonally regulated in liver)

PFK-1 INHIBITION

  • Citrate, intermediate of citric acid cycle
  • ATP, final product of glycolysis and cellular respiration
  • H+, symptom of lactic acid buildup in exercising muscle

PFK-1 ACTIVATION

  • AMP, marker of ATP depletion.
  • Fructose-2,6-bisphosphate (special case)

PFK-2/FBP-2 (BIFUNCTIONAL ENZYME)

  • PFK-2: F6P –> F2,6P (activates PFK-1)
  • FBP-2 (fructose-2,6-bisphosphatase): F2,6P –> F6P (deactivates PFK-1)
  • PFK-2/FBP-2 regulated differently in different tissues

Skeletal muscle

  • Feed-forward activation
  • Substrate-level regulation: F6P
  • When F6P HIGH: FBP-2 inactive and PFK-2 active (activate PFK-1)
  • When F6P LOW: FBP-2 active and PFK-2 inactive (inhibits PFK-1 activity)

Liver

  • Hormonal regulation: PFK-2 has phosphorylation site (unlike muscle)
  • PFK-2 is INACTIVE when phosphorylated
  • Glucagon activates protein kinase A (PKA), which phosphorylates PFK-2
  • Insulin activates phosphoprotein phosphatase (PPP), which dephosphorylates PFK-2

FED STATE

  • HIGH blood glucose
  • INSULIN secreted –> PPP activated –> PFK-2 dephosphorylated (ACTIVE)
  • HIGH F2,6P activates PFK-1
  • Promotes glycolysis

FAST

  • LOW blood glucose
  • GLUCAGON secreted –> PKA activated –> PFK-2 phosphorylated (INACTIVE)
  • LOW F2,6P deactivates PFK-1
  • NO glycolysis

Liver responds to entire body’s glucose needs

  • Site of gluconeogenesis: glucose synthesized from non-carbohydrate precursors and released into the bloodstream

Hexokinase

  • Catalyzes phosphorylation of glucose to form glucose-6P
  • Traps glucose inside the cell
  • 1st regulated enzymes in glycolysis – catalyze an irreversible reaction
  • 4 isozymes – I, II, III, and IV (glucokinase)

HEXOKINASE (I, II & III) vs. GLUCOKINASE (IV)

  • Tissue distribution
  • Kinetics (Km and Vmax)
  • Regulation (allosteric vs hormonal)

Hexokinase

  • Ubiquitous in mammals
  • Low Km & low Vmax
  • Allosteric regulation – inhibited by glucose-6P

Glucokinase

  • Liver & pancreatic beta cells
  • High Km & high Vmax: spares glucose for brain, muscle & other tissues (glucose sensor)
  • Hormonal regulation: inhibited by glucagon, activated by insulin
  • Glucokinase regulatory protein (GKRP): nuclear protein that reversibly binds/inactivates glucokinase
  • High [glucose] inhibits GKRP & promotes glucokinase release
  • Fructose-6P (glycolytic intermediate in equilibrium with glucose-6P): promotes GKRP-GK binding
  • Liver glucose-6P shunts into one of three pathways: glycolysis, glycogen or fatty acid synthesis

what you should know about conscious sedation

  1. Introduction:

                     Minimally depressed level of consciousness that retains the patient’s ability to independently and continuously maintain an airway and respond appropriately to physical stimulation or verbal command and that is produced by a pharmacological or non-pharmacological method or a combination.

  • 2. Features:
  • Retains the patient’s ability to maintain a patent airway independently and continuously
  • Permits appropriate response by the patient to physical stimulation or verbal command
  • Maintains protective reflexes.
  • Different routes of administration:
  • IV route:     deep conscious sedation

       IV sedation are more effective than the same drugs taken orally  

Advantage: surgeon or anaesthesiologist has complete control of the entire procedure

       Disadvantage: profound amnesia

Entral/oral route: sedation dentistry

  • All body functions remain normal and the person is able to breathe on their own.
  • The patient will often fall asleep. Some degree of amnesia is common.
  • The disadvantage with this method of sedation is that the level of sedation for each person is not predictable
  • Inhalation conscious sedation :  nitrous oxide/ oxygen sedation also known as ‘laughing gas’
  • This is the most frequently used sedation method in dentistry.
  •  All bodily functions remain normal.

  •  Clinical effects:
  • For 20–30 min acute detachment and later a state of relaxation.
  • Anterograde amnesia (loss of memory following administration of the drug) for the same period.
  • Cardiovascular depression is minimal (the relative hypotension and bradycardia due to the relief of hypertension and tachycardia caused by anxiety).
  • In most cases, minimal respiratory depression is seen. The exception is in patients with impaired respiratory function or in those who have taken other depressants such as opiates, alcohol, where the effect may be more marked. However, excessively rapid intravenous injections have the potential to cause respiratory depression leading to apnoea and respiratory arrest, which is life-threatening if not diagnosed and treated promptly.
  • Additional properties of benzodiazepines:
  • Muscle relaxant
  • Anticonvulsant (used to treat status epilepticus).
  • Objectives:
  • To allay apprehension, anxiety or fear
  • To decrease stress associated with traumatic or prolonged procedures
  • To control gagging
  • To stabilise the blood pressure for patients with hypertension or history of cardiovascular disease.
  • Commonly used pharmacological agents:

Benzodiazepines:

  1. Antianxiety
  2.  anticonvulsant,
  3.  sedative,
  4.  muscle relaxant
  5. amnesic properties.

 Midazolam and diazepam are the medications used in the dental operating set-up.

        Midazolam:

                           Midazolam is a short acting benzodiazepine CNS depressant.    

         Indication: short diagnostic and surgical procedure

 Properties:

  • Water soluble
  • Nonirritating to veins
  • Faster and shorter acting
  • Three times more potent than diazepam
  • It may be administered IV, IM, PO, rectally or nasally. The most common route of Midazolam is IV.
  • Sedative agent that should be administered slowly over 2 min for a single large bolus dose. Rapid or excessive IV doses may result in respiratory depression or arrest. If not recognised and treated promptly, death or hypoxic encephalopathy may result.
  • The initial IV dose may be as little as 0.5–1.0 mg.
  • Onset of sedation after IV injection is achieved within 3–5 min.
  • The duration of effect ranges from 1 to 6.
  • The half-life ranges from 1.2 to 12.3 h.

Adverse effect:

  • Hiccups
  •  Nausea
  •  Vomiting
  •  Headache
  • coughing and pain at the injection site

  Diazepam:

  • Indicated for conscious sedation prior to short diagnostic or surgical procedures, either alone or with a narcotic.
  • It may be administered IV, IM or PO, although IM administration is very painful and hence not recommended.
  • It cannot be mixed with other medications or diluted as it carries the risk of precipitation.

Adverse effect:

  • Extremely irritating to the tissues
  •  Venous thrombosis
  • Phlebitis
  • Apnoea
  • Hypotension
  • Diazepam can be given orally as a premedication prior to many nonpainful surgical procedures.
  • Effective anxiolytic.

Fentanyl:

  • Fentanyl is a synthetic opioid. It is indicated for analgesic action short duration procedures.
  • If given alone, dosage should begin at 1–2 μg/kg, which is about 75–150 μg for an average size adult. 
  • Fentanyl has an immediate response and effective excellent analgesia.
  • It has a half-life of 2–4 h .
  • Rapid IV administration can lead to a rigid chest wall and difficulty in

breathing. This effect may be reversed with naloxone (Narcan) 

Dose: 2-4mg/kg

  • Fentanyl is used effectively as sedation, adjunct to regional/local

anaesthetics.

  • Combined with benzodiazepines, it can obviate the need for inhaled anaesthetics for diagnostic, endoscopic, angiographic and other minor procedures in poor risk patients, as well as for burn dressing.

Ketamine: Dissociative anasthesia

  • Ketamine is a nonbarbiturate hypnotic with very high margin of safety. It has good tissue compatibility (no irritation to veins).
  • Profound analgesia, immobility, amnesia with light sleep and feelings of dissociation from one’s own body.

             DOSE: A dose of 1–3 mg/kg IV (average 1.5 mg/kg) or 6.5–13 mg/kg IM (average 10 mg/kg).

  • Ketamine produces the above effects within a minute and recovery start after 10–15 min, but patient remains amnesic for 1–2 h.
  • Ketamine is effective for short procedures as operations on head and neck especially in asthmatics.
  • It is also useful for repeated use particularly for burn dressings
  • Combined with diazepam, it is useful in angiography, cardiac catheterisation and trauma surgery.

Disadvantage:

  • Increased intracranial and intraocular pressure with increased occurrence of nausea and vomiting.

Propofol:

  • It is the recent IV anaesthetic agent, used for induction/maintenance of anaesthesia.
  • Major advantage of propofol is rapid recovery irrespective of the duration of infusion.

Dose

1–2 mg/kg/min (for sedation)

• Possess significant antiemetic property at low doses

• Rapid recovery without hangover effect

• Full orientation returns within 5–10 min