Energy Production: Carbohydrate

Question 1

Outline how starch and glycogen are broken down in the body

Answer 1

○ Saliva contains amylase which breaks down starch and glycogen into dextrins
○ Pancreas releases amylase which breaks dextrins into monosaccharides
○ Small intestine - disaccharides attached to brush border membrane of epithelial cells
§ Lactase, sucrose, pancreatic amylase, isomaltase

Question 2

Outline how monosaccharides are absorbed into the blood

Answer 2

• Absorption of monosaccharides - active transport by sodium dependent glucose transporter 1 (SGLT1) into intestinal epithelial cells and then via GLUT2 into blood supply
○ Cotransports 2Na and 1 glucose from apical to basolateral side
○ Uptake into cells from blood via facilitated diffusion using transport proteins (GLUT1 - GLUT5)
○ GLUT2 - kidney, liver, pancreatic beta cells, small intestine
○ GLUT 4 - adipose tissue, striated muscle (insulin regulated - high insulin increase uptake of glucose by increasing number of glucose transport proteins)

Question 3

Explain the biochemical basis of lactose intolerance

Answer 3

• Lactose intolerance - unable to metabolise lactose
• Lactose moves into the colon where bacteria breaks it down
○ Presence of lactose in the lumen of colon increases the osmotic pressure - draw water into the lumen, causing diarrhoea
○ Colonic bacteria can produce hydrogen, CO2, methane gases from lactose - bloating and discomfort

Question 4

Outline the types of lactose intolerance

Answer 4

• Primary lactase deficiency - absence of lactase persistence allele
○ Only occurs in adults
• Secondary lactase deficiency - caused by injury to small intestine
○ Occurs in both infants and adults
○ Generally reversible
• Congenital lactase deficiency - extremely rare, autosomal recessive defect in lactase gene
○ Cannot digest breast milk

Question 5

Describe the glucose-dependency of some tissues

Answer 5

• Major blood sugar - glucose concentration regulated
• All tissues can metabolise glucose but some cells have an absolute requirement (can only use glucose)
○ Red blood cells (no mitochondria and nuclei)
○ Neutrophils
○ Innermost cells of kidney medulla - not much oxygen left so need glucose for energy
○ Lends of the eye - poor oxygen supply
• Uptake depends on blood glucose concentration
• CNS prefers glucose as fuel
○ Can use ketone bodies for some of energy requirements in times of starvation but needs time to adapt

Question 6

State the overall equation of glycolysis

Answer 6

Glucose + 2Pi + 2ADP + 2NAD -> 2 pyruvate + 2ATP + 2NADH + 2H + 2H2O

Question 7

Outline phase 1 of glycolysis

Answer 7

○ Phosphorylation of glucose to G-6-P
§ Makes glucose negatively charged - prevents passage back across plasma membrane
§ Increases reactivity of glucose to permit subsequent steps
§ Uses 2 moles ATP per mole glucose
§ Catalysed by hexokinase
§ Catalysed by glucokinase in liver and pancreas
○ Reactions 1 and 3 have a large -ve ∆G, so irreversible
○ Step 3 is the committing step - commits glucose to metabolism via glycolysis
§ Phosphofructokinase acts on this step

Question 8

Outline phase 2 of glycolysis

Answer 8

○ Reaction 4 - Cleavage of C6 into two C3 units
§ C3 units interconvertible
○ Reaction 6 - NAD+ converted to NADH + H
§ Reducing power captured through oxidation of G3-P
§ Total NAD+ and NADH in cell is constant, therefore glycolysis would stop when all NAD+ is converted to NADH
§ Normally, NAD+ is regenerated from NADH in stage 4 of metabolism (electron transport chain)
§ RBC have no stage 3 or 4 of metabolism
□ Stage 4 needs oxygen - supply of oxygen to muscles and gut often reduced
□ Therefore, need to regenerate NAD+ through lactate dehydrogenase
○ Reaction 7 and 10 - Transfer of phosphate onto ADP to produce ATP (substrate level phosphorylation)
Reaction 10 - large -ve ∆G, therefore irreversible

Question 9

Outline how phosphofructokinase regulates glycolysis

Answer 9

• Phosphofructokinase key regulator of glycolysis
○ Allosteric regulation (muscle)
§ Inhibited by high ATP and stimulated by high AMP
○ Hormonal regulation (liver)
§ Stimulated by insulin and inhibited by glucagon
§ Insulin aims to store glucose into glycogen
§ Glucagon aims to convert glycogen into glucose for energy

Question 10

Other than phosphofructokinase, what other glycolysis regulators are there

Answer 10

○ Hexokinase - converts glucose to glucose-6-phosphate
§ Hexokinase lower infinity for oxygen
§ Hexokinase has end product inhibition by G6-P to regulate glycolysis
§ Glucokinase in liver - no end product inhibition as glucose constantly needed
○ Metabolic regulation - high [NADH] or low [NAD+] = high energy level signal
§ Causes product inhibition of step 6 and inhibits glycolysis
○ Pyruvate kinase - increase by high insulin: glucagon ratio

Question 11

What are some important intermediates of glycolysis

Answer 11

○ 2,3-bisphosphoglycrate lowers affinity of oxygen to haemoglobin
○ Glycerol phosphate important to triglyceride and phospholipid biosynthesis
§ Produced in adipose and liver
§ Lipid synthesis in adipose requires glycolysis (liver can also phosphorylate glycerol directly)

Question 12

State the equation and enzyme of pyruvate in anaerobic conditions

Answer 12

2 pyruvate + 2NADH + 2H -> 2 lactate + 2NAD

• Lactase dehydrogenase regenerates NAD with lactate as product

Question 13

Explain how plasma lactate concentration is controlled

Answer 13

• Plasma lactate concentration determined by relative rates of production, utilisation (liver, heart, muscle) and disposal (kidney)
○ Lactate produced is transported in the circulation to the liver, heart and kidney where it is converted back to pyruvate and oxidised to CO2 or converted to glucose
○ Lactase production = rate of utilisation

Question 14

Explain consequences of high plasma lactate

Answer 14

• Normal lactate concentration - < 1mM
• Increases in lactate can be due to: strenuous exercise, hearty eating, shock, congestive heart disease, alcohol metabolism (low lactate usage)
• Hyperlactaemia - 2-5mM, below renal threshold
○ No change in blood pH (buffering capacity)
• Lactic acidosis - above 5mM, above renal threshold
○ Blood pH lowered
• In lactic acidosis, kidney can no longer excrete lactate and could lead to renal failure and death
○ Critical marker in the acutely unwell patient

Question 15

Explain the importance of the pentose phosphate pathway

Answer 15

○ Occurs when energy not needed - no need to produce pyruvate
○ Glucose-6-phosphate dehydrogenase is limiting enzyme
○ Produces C5-sugar ribose required in making DNA, RNA, nucleotides and coenzymes
○ NADPH produced which is required for reducing power of biosynthesis, maintenance of GSH levels and detoxification reactions
○ Doesn’t produce ATP and CO2 produced

Question 16

Discuss the effects of glucose 6-phosphate dehydrogenase deficiency

Answer 16

○ X-linked gene defect
○ Point mutation in gene coding for glucose 6-phosphate dehydrogenase that results in reduced activity and therefore low levels of NADPH
○ Low GSH levels - protects cell against oxidative damage
○ RBC has no sources of NADPH - risk of oxidative damage
§ Haemoglobin becomes cross-linked by disulphide bonds and form Heinz bodies
§ Destruction of RBC causes haemolysis - haemolytic anaemia

Question 17

Explain galactosaemia

Answer 17

§ Absence of galactokinase - rare, accumulation of galactose in tissues
§ Absence of uridyl transferase - accumulation of galactose and galactose 1-phosphate
§ Absence of UDP-galactose epimerase
§ Galactose accumulation - reduced to galactitol
□ Depletes some tissues of NADPH - cataracts (protein broken down) leading to blindness
§ Galactose 1-phosphate accumulation - damage to kidney, liver, brain
□ Sequestration of Pi making it unavailable for ATP synthesis

Question 18

Outline the clinical importance of fructose metabolism

Answer 18

○ Dietary sucrose broken down by sucrase into glucose and fructose
○ Metabolised in liver to glyceraldehyde 3-phosphate
○ Essential fructosuria - fructokinase missing
§ Fructose in urine - no clinical signs
○ Fructose intolerance - aldolase missing
§ Fructose 1-P accumulates in liver - liver damage
§ Treatment - remove fructose from diet

Question 19

What happens to malnourished people when they are suddenly given lots of protein

Answer 19

• If protein supplied in excess of body’s immediate requirement
○ Protein cannot be broken down into ammonia and then urea to be excreted
○ Leads to liver damage - build up of toxins

Question 20

Explain the key role of pyruvate dehydrogenase in glucose metabolism

Answer 20

• Pyruvate needs to convert to acetyl CoA
• Catalysed by pyruvate dehydrogenase
○ Oxidises pyruvate through the reduction of NAD+
○ Large multi-enzyme complex (5 enzymes)
• Bond of carboxyl group combines with CoA to create acetyl CoA of high energy state
• Occurs in the mitochondrial matrix - pyruvate transported from cytoplasm across mitochondrial membrane
• Different enzyme activities require various cofactors - B-vitamins provide these factors, so reaction is sensitive to vitamin B1 deficiency
• Reaction is irreversible, so is a key regulatory step
• High energy substrates will switch PDH off (inhibited) - phosphorylation of pyruvate dehydrogenase
○ Acetyl CoA, NADH, ATP, citrate
• Low energy substrates switch the enzyme on - dephosphorylation
○ Insulin - lots of glucose so need energy for biosynthesis
• PDH deficiency leads to lactic acidosis due to pyruvate conversion to lactate

Question 21

Discuss the role of TCA cycle in metabolism

Answer 21

• Occurs in the mitochondria
• 2 cycles for every glucose entering glycolysis
○ Produces 6 NADH, 2 FADH2, 2 GTP, 4 CO2
• 2 irreversible reactions - conversion of isocitrate (C6) and α-ketoglutarate (C5)
○ Both oxidative steps (NADH produced) with CO2 released
• From C5 to C4 - some energy comes from CoA
• Succinyl-CoA coverts GDP to GTP
• Cycle is catalytic as no net synthesis or degradation in intermediates through pathway - for acetyl CoA cycle
• Does not function in absence of oxygen
• Both a catabolic and anabolic pathway - supplies biosynthetic processes

Question 22

Explain how the TCA cycle is regulated

Answer 22

• Isocitrate dehydrogenase catalyzes isocitrate (C6) to α-ketoglutarate (C5)
○ Activated by high ADP concentration
○ Inhibited by high NADH, ATP concentration
• α-ketoglutarate dehydrogenase catalyses α-ketoglutarate to succinyl-CoA (C4)

Question 23

Describe the processes in the electron transport chain

Answer 23

• Occurs in inner membrane of mitochondria
○ Outer porous membrane and impermeable inner membrane, creating an intermembrane space and mitochondrial matrix
○ NADH passes into proton translocating complex, which takes two electrons and passes 2 protons from matrix into the intermembrane space
○ Passes through 3 translocating complexes where electrons give up their energy to move 6 proteins overall
○ ~30% of energy used to move protons across membrane (lot of energy released as heat)
○ Oxygen acts as a final electron acceptor, converting oxygen to water
• Creates both a concentration and electrical gradient
○ Proton concentration gradient (membrane potential) across inner mitochondrial membrane = proton motive force (pmf)
○ However inner membrane impermeable so for hydrogen to go across membrane, it needs to go through a carrier (ATP synthase)
• Electrons in NADH have more energy than in FADH2 - NADH uses 3 PTC, FADH2 uses 2
• The more reducing force (greater pmf), the more ATP is produced
○ 2 moles of NADH synthesises 5 moles of ATP
○ 2 moles of FADH2 synthesises 3 moles of ATP

Question 24

Outline the role ATP plays in regulating electron transport chain

Answer 24

• When ATP concentration is high and thus ADP concentration is low, no substrate for ATP synthase which stops inward flow of proton
○ Concentration of proton in intermitochondrial space increases
○ Prevents further proton pumping - stops electron transport chain
○ [H] outside increases to a level that prevents more protons being pumped

Question 25

What do electron transport inhibitors do

Answer 25

• Inhibitors - block electron transport
○ Cyanide/CO prevents acceptance of electrons by oxygen
○ No electron transport therefore no proton motive force
○ Inhibit conversion of ADP to ATP by preventing reoxydation
○ Irreversible cell damage

Question 26

What do electron transport uncouplers do

Answer 26

• Uncoupling - increase the permeability of the mitochondrial inner membrane to proton
○ Dissipate the proton gradient and thereby reducing the proton motive force
○ Proton enters matrix without driving ATP synthase
○ No inhibition of electron transport
○ Reduce ATP synthesis
Eg. Dinitrophenol, dinitrocresol, fatty acids

Question 27

Explain the significance of brown adipose tissue

Answer 27

• Brown adipose tissue - contains thermogenin (UCP1) - naturally occurring uncoupling protein
○ Degree of coupling controlled by fatty acids (uncouplers) - allows extra heat generation
○ In response to cold, noradrenaline activates
○ Lipase releases fatty acids from triglyceride
○ Fatty acid oxidation produces NADH/FAD2H, enter electron transport
○ Fatty acids activate thermogenin
○ Thermogenin transport protons back into mitochondria so electron transport uncoupled from ATP synthase
○ Energy of pmf released as extra heat
○ Brown adipose tissue found in newborn infants (maintain heat, particularly around vital organs) and hibernating animals (generate heat to maintain body temperature)

Question 28

Compare the processes of oxidative phosphorylation and substrate level phoshorylation

Answer 28

OP requires membrane associated complexes, SLP requires soluble enzymes
OP energy coupling occurs indirectly through generation and utilisation of proton gradient, SLP coupling occurs directly through formation of high energy hydrolysis bond
OP cannot occur in absence of oxygen, SLP can to limited extent
OP major process in ATP synthesis in cells requiring large amounts of energy, SLP minor process