G6PD deficiency

Question 1

RBC metabolism

Answer 1

• Enucleate, cannot reactive mRNA or make proteins
• Energy generated from glycolysis
• Deficiencies in glycolytic nzs produce chronic hemolysis (except LDH)
• Retics have high levels of nzs that decline over time (retics have 5x level of G6PD than old RBCs)
• Normal T1/2 for G6PD is 60 days
• In nz deficiencies, the nz activity becomes deficient at earlier times than normal RBC nzs (in G6PD deficiency, G6PD A- t1/2 is 13 days)
• G6PD is part of hexosemonophosphate shunt (HMP, or PPP), the first nz in the pathway, and it generates NADPH

Question 2

Glutathione

Answer 2

• High levels in RBCs, used to detoxify superoxide, hydroxyl radicals, low levels of H2O2
• Maintains thiol (-SH) groups on Hb, RBC membrane, and other nzs
• Depends on NADPH for reduction (GSSG-> GSH)
• Depletion of GSH allows sulfide bonds btwn Hb molecules, and globin binding to membrane band 3
• Insufficient GSH leads to oxidant damage, Hb degradation, free globin chains, and heinz body formation (bound to band 3)
• Low levels of GSH due to lack of NADPH (which comes from G6PD)

Question 3

Rapid onset anemia

Answer 3

• Either due to bleeding or hemolysis
• Deficiencies take time to develop anemia
• Heinz bodies seen in thalassemia, HbE, G6PD

Question 4

Genetics of G6PD deficiency

Answer 4

• G6PD is located on X chromosome, and deficiency can manifest in 3 ways
• Sub-normal/absent nz production, nzs w/ reduced activity, unstable nzs (lose protein and activity over time)
• Most defects in gene are mis-sense mutations
• Provides partial protection against falciparum and vivax malaria (due to changes in band 3 that inhibit parasite invasion)
• If a parasite does enter the cell, it rapidly undergoes oxidative stress and is destroyed by macrophages

Question 5

Hemolytic isoforms of G6PD

Answer 5

• Only 2 isoforms cause episodic hemolysis
• G6PD A- (2 mutations on the nz) cause some hemolysis, has a t1/2 of 13 days, and confers partial protection against malaria (found in Africa)
• G6PD has 10-60% activity of normal (class 3)
• G6PD Med (1 mutation) causes severe hemolysis and is found in the mediterranean
• G6PD has 0-10% activity of normal (class 2)
• Chronic hemolysis is observed in class 1 (hereditary nonspherocytic hemolytic anemia, or HNSHA)
• Both types are exacerbated when confronted w/ excess ROS generation

Question 6

Things that generate ROS

Answer 6

• In G6PD patients, things that generate ROS can lead to hemolytic events
• These include ketoacidosis, hyperglycemia, drugs, infections, fauvism (divicine and isouramil)
• Infections are most common cause of ROS, b/c of ROS bursts from macrophages and PMNs
• ROS lead to thiol group cross-linking, oxidative damage to Hb and other molecules, membrane rigidity and loss (leading to dehydration, fragmentation, and lysis)

Question 7

Acute hemolysis and RBC morphology

Answer 7

• Acute hemolysis severity determined by: potency of stimulus, and proportion of cells w/ deficient G6PD vs those w/ normal nz
• RBC morphology: initially “bite” cells are seen (very short lifespan), later normocytic and normochromic anemia develops
• Can see heinz bodies (also appear in thalassemias and unstable Hbopathies
• Hemolysis is primarily intravascular (Hburia, followed by hemosiderin in urine)

Question 8

Diseases that cause acute intermittent hemolysis (differential Dx)

Answer 8

• G6PD deficiency
• Unstable Hbopathies
• PNH (complement binding to RBC due to lack of membrane proteins, happens at night)
• Malaria

Question 9

Lyon (beutler) hypothesis

Answer 9

• Males have 1 X chrom, females express only 1 X and inactive the other (barr body)
• Therefore both males and females have similar nz activity
• In females, an organ’s cell pop is a mixture of active paternal and active maternal X chrom
• In females, a single tissue may exhibit all levels of nz activity (homozygous for normal, for deficient, or heterozygous)

Question 10

Dx of G6PD deficiency

Answer 10

• Heinz body prep, not definitive
• Spot test determines if sufficient G6PD activity exists in RBC to generate NADPH
• Nz quantification (definitive test): correlated w/ another age-dependent RBC nz to show one nz is inappropriately low compared to the other (necessary in females and immediately after hemolytic event in both sexes)
• Hexokinase level is often elevated b/c more glc is fluxing through glycolysis due to lack of PPP shunt