Biochimica II – Lezione 9 – Slides

NADPH2 ° Scenario

La cellula ha bisogno solo di NADPH, non di zuccheri con 5 atomi di carbonio. Una delle sei molecole di glucosio-6P viene trasformata in CO (ossidata).

23 ° Scenario

La cellula ha bisogno di NADPH e di energia. Il globulo rosso necessita di una fonte di energia per:

1. mantenere il ferro dell'Hb in forma ridotta (Fe²⁺)
2. mantenere alti livelli intracitoplasmatici di K+ e bassi livelli di Na+ e di Ca+
3. mantenere in forma ridotta i gruppi -SH di Hb, enzimi e membrana
4. mantenere la sua forma biconcava

Fonte di energia = glucosio

Il globulo rosso maturo non ha mitocondri, quindi usa le vie:

• glicolisi ATP, 2-3DPG, NADH
• shunt ossidativo via dei pentoso fosfati NADPH
• gluconeogenesi
• glicolisi Via pentoso fosfati NADPH
• NADH riduzione glutatione
• metemoglobina attività 2-3DPG
• anti-ossidante modulazione affinità Hb ossigeno ATP metabolismo
• Glutatione perossidasi
• Perossido Glutatione ossidato
• Glutatione ridotto Proteina
• Glutatione ossidato Proteina ossidata

ridotto ridotta

Glutatione reduttasi+ +GS - SG + NADPH + H 2GSH + NADP

La glutatione riduttasi contiene FAD e, nella cellula, agisce essenzialmente in modo unidirezionale.

Infatti nelle cellule il rapporto [GSH] / [GS - SG] = 500 / 1

Negli eritrociti il glutatione ridotto contribuisce al mantenimento del ferro dell'eme dell'emoglobina nello stato di ossidazione Fe2+

NADPH is needed to transform GS--GSSG into GSH, which reduces selenocysteine in glutathione peroxidase

Proposed mechanism of glutathione peroxidase

Glutathione peroxidase has selenocysteine, a rare amino acid that contains selenium. The story of selenocysteine incorporation into proteins is unusual.

The predominant pathways of carbohydrate metabolism in the red blood cell (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate (2,3-BPG) metabolism

Glycolysis provides ATP for membrane ion pumps and NADH for reduction of methemoglobin. The PPP supplies the RBC with NADPH to maintain the reduced state of glutathione.

The inability

To maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H₂O₂, that in turn results in a weakening of the cell wall and concomitant hemolysis. Accumulation of H₂O₂ also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell membrane.

Glutathione removes peroxides via the action of glutathione peroxidase.

The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.

Deficiency in the level of activity of glucose-6-phosphate dehydrogenase (G6PDH) is the basis of favism, primaquine (an anti-malarial drug) sensitivity, and some other drug-sensitive hemolytic anemias, anemia and jaundice in the newborn, and chronic nonspherocytic hemolytic anemia.

In addition, G6PDH deficiencies are associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of

Mediterranean and African descent. The basis for this resistance is the weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.

G6PD Patologie da deficienze della:

• Glucose-6-Phosphate Dehydrogenase
• One of more common enzyme deficiencies.
• Occurs worldwide.
• Isolated more than 400 variants of G6PD enzyme. Almost all of variants resulted from point mutations that produced structurally abnormal or functionally defective enzymes.

< 1% Class 1: Enzyme deficiency with chronic nonspherocytic hemolytic anemia

Class 2: Severe enzyme deficiency (less than 10%) ~ 79%

Class 3: Moderate to mild enzyme deficiency (10-60%) ~ 21%

Class 4: Very mild or no enzyme deficiency (60%)

Class 5: Increased enzyme activity

G6PD Mode of Inheritance:

• Transmitted by mutant gene located on X chromosome.
• Disease is fully expressed in males; only

expressed inhomozygous females.

Heterozygous females have• enzymatically dimorphic red cell(have both normalpopulationG6PD activity and decreasedG6PD activity in red cells).

Expression of G6PD activity• varies widely in heterozygotes•Gene Xq28, 18,5 Kb, 13 esoni

The Heinz body test for primaquine sensitivity (G6PD deficiency). The cells(right) are from a primaquine-sensitive (G6PD-deficient) donor; the cells in the leftpanel, from a normal control•

Under normal conditions, individual with G6PD deficiencycompensates for shortened erythrocyte life span byreticulocytosis.

Oxidative stress, however, can lead to mild to severehemolytic episode.

More than 50 chemical agents known to induce• hemolysis in G6PD deficient erythrocytes. favism

Certain individuals exhibit sensitivity to fava beans ( );After eating fava beans or after inhaling fava bean pollen,may have a severe hemolytic episode.

Favism• found in some individuals with G6PD deficiency•

Highest incidence in darkly pigmented racial and ethnic groups - African Americans, Mediterranean ancestry and Asians.

• Whites tend to have Mediterranean form of G6PD deficiency (Gd Med), which is associated with favism.

Plasmodium falciparum111,

From: Ernest Beutler, BLOOD 16-24 (2008)

An indirect histochemical stain of erythrocytes from a heterozygote for G6PD deficiency who was suffering from naturally acquired malaria. The more pigmented cells are those that contain normal G6PD activity. They contain the highest percentage of malaria parasites, as shown by the arrows. (Courtesy Professor Lucio Luzzatto.)

transketolase requires thiamine pyrophosphate (vitamin B1)