Metabolism Cases I

Question 1

Glutathione is synthesized from what amino acids? What is it’s significance? Does it need ATP for it’s synthesis? Reduction of glutathione (SS -> 2-SH) requires what? What two enzyme deficiences can contribute to decreased function of glutathione?

Answer 1

Glutathione is an antioxidant (ie. it is reduced and can reduce an ROS) and neutralizes reactive oxygen species (ROS). Synthesized from glutamate, cysteine and glycine with ATP as it’s power source. Enzymes: G6PD and pryuvate Kinase deficies contribute to decreased function of glutathione. Reduction of glutathion requeires NADPH!

Question 2

Ribose-5-P is produced by the oxidative branch of the HMP shunt. Why is nucleotide biosynthesis not compromised in individuals with G6PD deficiency?

Answer 2

Ribose 5-P can also be produced via HMP shunt-nonoxidative branch-reverse direction. The nonoxidative branch-reverse direction allows the nonoxidative pathway to generate ribose-5-P without the need for 6PD. If it were not for this, a deficiency in G6PD might prove to be more lethal.

Question 3

Common events that result from oxidative damage to RBCs

Answer 3

• Heinz bodies (found in association with G6PD deficieny; consis of Hb covalently bound to plasma membrane lipids)
• Rigidity of cytoskeleton (signal for premature destruction in the spleen)
• Increased RBC removal results in hemolytic anemia and/or hemolytic jaundice)

Question 4

Events that may increase oxidative stress in erythrocytes

Answer 4

• infections: macrophages produce H2O2 as a killing mechanism. This is the most common cause of hemolysis associated with G6PD deficiency.
• oxidant drugs (such as antibiotic Bactrim, generate free oxygen radicals (ROS) that are destroyed in a reaciton catalyzed by the endogenous enzyme superoxide disumuatse (SOD). The product of this rxn is H2O2
• Fava beans: contain compounds that indirectly stimulate the production of H2O2, much like that of oxidant drugs (favism is a form of G6PD deficiency, but not all G6PD=deficiencies manifest after fava bean ingestion)

Question 5

The PK deficiency that produces hemolytic anemia is RBC-specific. Individuals with G6PD deficiency have a defect of this enzyme in all cells, but only the RBCs are affected. Explain why RBCs are the only cell type to show pathology.

Answer 5

In RBCs, and other cells without mitochondria, the sole source of NADPH is the HMP shunt oxidative branch. Glutathione reduction is impaired because of decreased NADPH availability.
In liver and muscle, which do have mitochondria, malic enzyme can also be used to produce NADPH. This NADPH can then be used in the reduction of glutathione.

Question 6

G6PD deficiency
Impaired production of:
Impact on glutathione production:
Impact on GS-SG/G-SH ratio:
Impact on H2O2 levels:
Types of hemolysis (acute or chronic)

Answer 6

Impaired production of: NADPH
Impact on glutathione production: Unchanged
Impact on GS-SG/G-SH ratio: Increased
Impact on H2O2 levels: increased
Types of hemolysis (acute or chronic): acute (induced by cellular oxidative stress)

Question 7

PK deficiency: 
Impaired production of:
Impact on glutathione production:
Impact on GS-SG/G-SH ratio:
Impact on H2O2 levels:
Types of hemolysis (acute or chronic)

Answer 7

Impaired production of: ATP
Impact on glutathione production: decreased
Impact on GS-SG/G-SH ratio: unchanged
Impact on H2O2 levels: increased
Types of hemolysis (acute or chronic): chronic (caused by ATP debt and reduced glutathione levels) as well as Acute (by induced cellular oxidation stress)

Question 8

PK deficiency: what results in the reduction of ATP synthesis? In PK deficiency is hemolysis a chronic or acute condition?

Answer 8

The chronic reduction of ATP synthesis impairs many proteins and cellular functions, including:
-Na+/K+-ATPase: Ion imbalance and osmotic stress
-Biosynthesis of glutathione: Increased oxidative stress
Like G6PD deficiency, the resulting RBC rigidity results in premature erythrocyte destruction
(i.e., hemolysis) by the spleen. In PK deficiency, however, this hemolysis is a chronic condition
that can occur even in the absence of external stresses (infection, drugs, etc.)

Question 9

Do individuals with RBC-specific PK deficiency experience acute or chronic hemolytic
anemia? Why?

Answer 9

Chronic . PK deficiency -> chronic ATP deficit in RBCs -> reduced activity of Na+-K+-
ATPase and decreased production of glutathione -> increase in H2O2 levels -> membrane
damage -> hemolysis.

Question 10

Would you expect a PK-deficient individual to suffer increased hemolysis in response to
infection? Why or why not?

Answer 10

Yes. In addition to chronic hemolysis, decreased levels of glutathione will impair the ability of PK deficient RBCs to adequately detoxify H2O2, thus inducing more oxidative stress.

Question 11

Explain how bactrim treatment may result in hemolytic anemia in a G6PD-deficient patient.

Answer 11

Bactrim treatment may indirectly result in the increased production of H2O2. Individuals with G6PD deficiency cannot produce adequate amounts of NADPH. Under stress, glutathione disulfide (G-S-S-G) cannot be reduced back to G-SH. A decrease in G-SH will result in increased H2O2 accumulation. (this would also happen in a patient with PK deficiency)

Question 12

Why didn’t the patient experience chronic hemolytic anemia in the absence of Bactrim
treatment?

Answer 12

Individuals with G6PD deficiency typically retain 40-50% of normal G6PD activity. This level of activity is sufficient to prevent oxidative stress under non-aggravating conditions.

Question 13

Why does a person with cytochrome b5 reductase deficiency need to avoid exposure to compounds/foods that contain nitrites (NO2-) or nitrates (NO3 -)?

Answer 13

Nitrites and nitrates are oxidants that lead to production of methemoglobin. Exposure (topical or oral ingestion) to these compounds can lead to increased methemoglobinemia. In individuals with congenital cause of methemoglobinemia such as a cytochrome b5 reductase deficiency should be extra cautious with these agents that can exacerbate the existing methemoglobinemia

Question 14

If someone has a cytochrome b5 reductase deficiency, what is the treatment of choice? Will this treatment work as well with someone who also has a G6PD deficiency?

Answer 14

No. The methylene blue treatment will not work effectively. An inadequate supply of NADPH due to G6PD deficiency will result in a decrease in reduction of methylene blue (less leukomethylene blue to reduce MetHb). As a result, MetHb concentration will be high. Methylene blue therapy is contraindicated in G6PD deficient patients, since the MB will consume NADPH.

Question 15

Cytochrome b5 reductase uses NADH or NADPH?

Answer 15

NADH!

Question 16

Functional Hb has Ferrous (Fe2+) or Ferric (Fe3+)?

Answer 16

Functional hemoglobin contains ferrous (Fe2+) iron. Methemoglobin (metHb) is hemoglobin which carries within its heme group an oxidized form of iron, ferric iron (Fe3+). This form of hemoglobin cannot bind oxygen. Methemoglobin is continuously being formed via auto-oxidation in the erythrocyte, but is continuously being reduced back to functional hemoglobin. At any given time under normal physiological conditions, methemoglobin makes up less than 1% of total hemoglobin.

Question 17

What are some results and causes of methemoglobinemia?

Answer 17

Tissue hypoxia can occur if methemoglobin is not properly reduced, and its levels exceed approximately10% (1.5 g/dL) of total hemoglobin. Cyanosis, or a bluish discoloration of the skin, is commonly observed in patients with methemoglobinemia. ”Chocolate cyanosis” refers to the brownish color of methemoglobin. Multiple pathologies can give rise to abnormal levels of methemoglobin, including mutations in the genes encoding cytochrome b5 (rare) and cytochrome b5 reductase

Question 18

What is the role of thiamine in glucose oxidation? Which metabolic processes will slow as a result of thiamine deficiency?

Answer 18

Thiamine is a precursor of TPP, which is required by enzymes that participate in complete glucose oxidation such as PDHC and a-ketoglutarate dehydrogenase. PDHC is necessary for converting pyruvate -> acetyl CoA; a-ketoglutarate dehydrogenase functions in the TCA cycle.

Question 19

Explain why comatose alcoholics may develop localized cerebral lactic acidosis after glucose infusion if thiamine is not present in the IV-dextrose solution.

Answer 19

Due to malnutrition commonly seen in severe alcoholics, they tend to be thiamine-deficient, resulting in decreased activity of TPP-dependent enzymes. As a result of inadequate PDHC activity, pyruvate will accumulate in the cytosol after the glucose infusion. Further favored by the high [NADH] produced by ethanol catabolism, LDH will convert pyruvate to lactate. Even though many tissues will exhibit lactic acidosis, it is most damaging in the brain.

Question 20

What two enzymes in the cytosol break down ethanol? Where else and what enzyme? What increases in the cytosol as a result of alcohol catabolism?

Answer 20

cytosol: alcohol dehydrogenase and aldehyde dehydrogenase
SER: CYP2E1
NADH increases! this messes with gluconeogenesis

Question 21

What effect will increased alcohol catabolism have on malate dehydrogenase and lactate dehydrogenase?

Answer 21

The malate-aspartate shuttle is utilized to shuttle e- of cytosolic NADH into the mito. This shuttle contains reversible reactions. The direction of the reaction is dictated by the substrate concentrations, such as NADH.
-Lactate dehydrogenase is able to regenerate NAD+ in the pyruvate to lactate direction. LDH catalyzes both directions, depending on the substrate concentrations. Too much NADH in the cytosol and your fucked.

Question 22

How will alcohol catabolism affect gluconeogenesis?

Answer 22

When cytosolic NADH levels are very high due to alcohol catabolism, the cells need to regenerate NAD+ in the cytosol. The rate of gluconeogen is greatly reduced due to the reactions favoring NAD+ regeneration. LDH and malate dehydrogenase are both taking NADH and making NAD+ while making gluconeogenic intermediates. When there is too much NADH around this makes those reactions run a lot slower.

Question 23

Fructose-induced hypoglycemia caused by FBPase-1 deficiency.
What is the cause for the hypophosphatemia in a fasting patient with FBPase-1 defieciency, and is given some fructorse?

Answer 23

During fasting gluconeogensis is on and FBPase-1 is on. When given frutose Due to the metabolic block at the reaction catalyzed by FBPase-1 in the fasting state, inorganic phosphate is sequestered in compounds such as fructose-1-P, DHAP, glyceraldehyde 3-P and fructose-1,6-bisP.

Question 24

Fructose-induced hypoglycemia caused by FBPase-1 deficiency. Why the severe hypoglycemia after fructose intake during fasting?

Answer 24

1) a decrease in hepatic glycogenolysis, because the phosphates were sequestered in compounds like fructose 1-P, DHAP, glyceraldehyde 3-P and F-1,6-BP. Inorganic phosphates are needed for liver glycogenolysis.
2) A decrease in gluconeogenesis: decreased Pi leads to decrease in ATP synthesis. Gluconeogen requires ATP, this its activity is decreased!