Pentose Phosphate Pathway

Question 1

Where is the pentose phosphate pathway active?

Answer 1

Liver, lactating mammary gland, adipose tissue (NADPH for fatty acid biosynthesis)

Adrenal cortex, testis, ovaries (NADPH for synthesis of steroid hormones)

Erythrocytes (NADPH for reduction of glutathione )

White blood cells and macrophages (NADPH for phagocytosis )

Question 2

What is the oxidative pathway of the pentose phosphate pathway?

Answer 2

• Irreversible reactions
• forms NADPH and pentose phosphate
• Enzymes: dehydrogenase (G6PD)

Question 3

Describe the non- oxidative pathway of the pentose phosphate pathway

Answer 3

• reversible reactions
• Takes place under conditions where pentoses are not required by cells

Involves interconversion of sugars resulting in formation of glycolyticontermedkates

Enzymes: transketolase (TPP) and transaldolase

Question 4

What is the regulated enzyme (and disorder associated of) the oxidative pathway?

Answer 4

Glucose-6 phosphate dehydrogenase (G6PD)

Question 5

What are the reactions of the oxidative phase if the pathway?

Answer 5

Two reactions: catalyzed by G6PD 1 and 6-phosphogluconate dehydrogenase

Both reactions form NADPH+ H+ (reducing equivalents)

Oxidative decarboxylation by 6-phosphogluconate dehydrogenase enzyme forms Ribulose 5-phosphate (pentose phosphate)

Regulatory enzyme if pathway: G6PD

Question 6

Where and when does the non-oxidative phase of the pentose pathway occur?

Answer 6

• Occurs in cells/ tissues that do not require pentose phosphate
• Convert excess Ribulose 5-phosphate into glycolytic intermediates (excess pentose s are metabolized by major pathways)- to recycle carbon atoms

To understand the non-oxidative phase, we Imagine that 3 molecules of glucose 6-phosphate are converted to 3 molecules of Ribulose 5-phosphate by oxidative reactions

Question 7

Which pentose does epimerase interconvert?

Answer 7

Ribulose 5-P(formed from G6P)

And xylulose 5-P

Question 8

What pentoses does does isomerase interconvert in the oxidative phase?

Answer 8

Ribulose 5-P (from G6P)

And ribose 5-P

Question 8

What pentoses does does isomerase interconvert in the oxidative phase?

Answer 8

Ribulose 5-P (from G6P)

And ribose 5-P

Question 9

What is the significance of thiamine (vitamin B1) to transketolase?

Answer 9

Transketolase uses TPP (thiamine pyrophosphate) as coenzyme

Transketolase activity in RBCs used an index of nutritional thiamine (vitamin B1) status

Laboratory test to identify nutritional thiamine deficiency

Patients with beriberi (thiamine deficiency) have low erythrocyte transketolase activity

Question 10

What cells require both NADPH and pentoses?

Answer 10

• Only oxidative phase operates, to form NADPH and peptide phosphate
• Ribise 5-phosphate required for nucleotide biosynthesis is formed from Ribulose 5-P by isomerization

Question 11

What cells only require NADPH?

Answer 11

RBCs

Oxidative phase operates to produce NADPH- Used in various reactions

Pentoses formed in oxidative phase are converted to glycolytic intermediates (recycling of unwanted/excess pentoses)

Question 12

What cells require only pentoses ?

Answer 12

• Non-oxidative reversible reactions (transketolase, transaldolase, epimerase abd isomerase) convert glycolytic intermediates into pentose phosphates
• Only the non-oxidative phase operates to form pentose phosphates

Question 13

What are the uses of NADPH?

Answer 13

• Reductive biosynthesis freactions in fatty acid synthesis and synthesis of steroid hormones
• RBCs: detoxification of hydrogen peroxide and reactive oxygen species
• Cytochrome P450 system (drug metabolizing enzymes)
• Phagocytosis in WBCs (forms reactive oxygen species)
• Synthesis of nitric oxide(NO)

Question 14

Explain phagocytosis by WBCs

Answer 14

• On phagocytosis, neutrophils and macrophages increase their oxygen consumption and increase generation of reactive oxygen species- ‘respiratory or oxidative burst’
• Superoxide free radicals (O2-) formed using NADPH oxidase (requires NADPH)
• Superoxide converted to hydrogen peroxide by superoxide dismutase (SOD)
• H2O2 forms hydroxyl radicals (toxic to bacteria)
• H2O2 forms HOCl (hydrochloride acid) which kills bacteria and fungi
• Infections result in increased production of oxidants

Question 15

What is NADPH activity responsible for?

Answer 15

Responsible for respiratory burst (increased oxygen consumption) following phagocytosis

Question 16

Explain the results of NADPH oxidase deficiency

Answer 16

Chronic granulomatous disease (CGD):
Severe persistent bacterial and fungal infections
-Neutrophils show lack of increased oxygen consumption on phagocytosis (defective respiratory burst/ negative respiratory burst test)

Question 17

What are the results from Myeloperoxidase deficiency ?

Answer 17

Increased risk of fungal infections
-Normal respiratory burst (positive respiratory burst) but decreased HOCl formation

-G6PD deficiency usually does NOT affect WBCs

Question 18

Explain detoxification of H2O2 (Reactive oxygen species)

Answer 18

1. Oxidant drugs (sulfa drugs/primaquine) or infection or Fava bean increase production of free radicals (reactive oxygen species)
2. In RBCs, H2O2 detoxified to H2O by glutathione peroxidase
   
   • Hydrogen donated by reduced glutathione (GSH)
   • Selenium containing enzyme
3. Oxidized glutathione (GS-SG) converted back to reduced glutathione (GSH) by glutathione reductase, using NADPH formed by pentose phosphate pathway
   - GSH functions as antioxidant to detoxify H2O2 (ROS) in RBCs
   - Defect in pentose pathway results in H2O2 accumulation (reactive oxygen species) that damage RBC membrane resulting in hemolysis

Question 19

Explain the case report of G6PD deficiency

Answer 19

A 16 year old boy is diagnosed with pneumonia. He is treated with sulfamethoxazole (sulfa drugs/ sulfonamides)

On the third day of treatment, he is slightly jaundiced (increased serum bilirubin) and he feels fatigued.

His hemoglobin has fallen by 3.5 g/dL

Urine is reddish brown and is positive fir hemoglobin

Evidence of acute hemolysis

Question 20

What is the most common enzyme abnormality?

Answer 20

G6PD deficiency

Question 21

What is G6PD deficiency?

Answer 21

X-linked recessive disorder resulting in hemolysis

Highest prevekance in Middke East, Africa, Asia and Mediterranean

Reduced activity of G6PD (pentose phosphate pathway)—> impairs NADPH formation

Decreases cellular reduced glutathione pool (remember: GSH is major antioxidant in RBCs)

Also, mature RBCs are unable to synthesize new enzyme (do not have nucleus and cellular organelles)
Older RBCs more susceptible to hemolysis

Question 22

What are the effects of G6PD deficiency in RBC ?

Answer 22

Impaired detoxification of free radicals and peroxide’s
-H2O2 accumulates and damages RBC membrane (unsaturated fatty acids in membrane phospholipids), resulting in hemolysis

• GSH maintains thiol (-SH) groups in proteins
  
  • G6PD deficiency results in formation of denatured insoluble proteins (Heinz bodies)
• G6PD deficiency most severe in RBCs
  
  • (PPP is ONLY pathway generating (NADPH in RBCs)

Question 23

Summarize the mechanism of hemolysis in G6PD deficiency

Answer 23

Oxidative stress: infections/fava beans/ antimalarials/sulfa drugs—> GSH converted to GS-SG fir detoxifying oxidants (GSH used up for detoxification of oxidants)

Thus leads to increased requirement of NADPH to convert oxidized glutathione to reduced form (GSH)-(remember NADPH production in G6PD deficiency is low)

On one hand this leads to oxidants to accumulate, causing damage of fatty acids containing double bonds (cell membrane phospholipids), leading to RBC lysis

On the other hand this leads to denaturation of proteins due to GSH depletion, this causes formation of Heinz bodies

Question 24

Explain the precipitating factors of G6PD deficiency

Answer 24

Old (senile) RBCs are vulnerable to G6PD deficiency, as mature RBC have no nucleus or ribosomes, and cannot synthesize new enzyme.

Precipitating factors (oxidants)
Oxidant drugs
  -antibiotics (sulfa drugs)
  -antimalarials (promaquine )
  -antipyreptics (aspirin)

Fava (broad ) beans- ‘favism’ (Mediterranean)

Infections

Question 25

What are the G6PD variants?

Answer 25

About 400 pathogenic variants of G6PD known

G6PD A-: moderate hemolysis

• Young RBCs with G6PD A- have sufficient levels of the enzyme
• lower enzyme stability
• Hemolysis in presence of precipitating factors

-G6PD Mediterranean variant: severe hemolysis