Biochemistry And Genetics Of RBCs

Question 1

Structure of RBC overview

Answer 1

Lack of nucleus and membrane bound granules

Shaped in a biconcave disc to maximize the cell surface for oxygen exchange

Short life span (120 days on average) and very fast production (10^12 on average daily)

Question 2

Hereditary elliptocytosis

Answer 2

RBC assumes an elliptic shape, usually caused by abnormalities in spectrin protein (also possible in band 4.1 and glycoproteins C proteins, but this is less common)

Question 3

Hereditary spherocytosis

Answer 3

Caused by deficiency and/or abnormalities in spectrin protein (can also becaused by ankyrin, band 4.1, 4.2 proteins but is less common)

Symptoms:

• presence of spherocytes w/ really short lifespan
• hemolytic anemia and splenomegaly

Affects people of Northern European descent usually.

Question 4

RBC metabolism

Answer 4

Glycolysis:

• to generate ATP
• production of 2,3 BPG
• Reduces Iron

PPP/HMP:

• generates NADPH which is used to fuel G3P and F6P production
• NADPH is used to reduce glutathione which is used to protect RBCs from oxidizing damage

Question 5

RBC energy metabolism

Answer 5

Only uses anaerobic glycolysis
(does not have mitochondria)

NAD+ is restored via lactate production

Question 6

Pyruvate kinase deficiency

Answer 6

Results in hemolytic anemia
- 2nd most common deficiency after G6PD

RBCs cant produce lactate so it cant regenerate its NAD+ which is required to generate ATP
- specifically cant generate G3P -> 1,3 BPG

Symptoms:
- fatigue, pale skin, shortness of breath, jaundice, increased gall stone appearance

• NO HEINZ BODIES*

Question 7

Difference between Pyruvate kinase and G6PD deficiencies

Answer 7

G6PD deficiency produces Heinz bodies (are precipitated hemoglobin)

Pyruvate kinase deficiency does not produce these

Question 8

What is the most abundant organophosphate in RBCs?

Answer 8

2,3 BPG

Is an allosteric regulator of oxygen binding to hemoglobin and is generated via mutase and 1,3 BPG
- presence increases efficiency of blood cells

Binds to the Beta chains of hemoglobin taut structures.

Is rapidly degraded in blood stored for transfusions and is increased in people that live in high altitudes

Question 9

How is 2,3 BPG generated?

Answer 9

From 1,3 BPG via mutate enzyme

can enter the Rapoport-Luebering shunt to generate 3 phosphoglycerate via phosphatase if levels are low

Question 10

PPP (or HMP shunt) steps

Answer 10

Rate limiting step= G6PD

Oxidative reactions are 3 irreversible steps
-purpose is to generate NADPH to reduce glutathione

• nonoxidative reactions are reversible and use two enzymes
• transaldolase
• transketolase
• purpose is to generate nucleotides for precursors

Question 11

Transketolase activity

Answer 11

Requires TPP (thiamine / Vit. B1) and its activity in RBCs can be directly measured

• low activity of Transketolase = thiamine deficiency

Question 12

G6PD deficiency

Answer 12

Causes episodic hemolytic anemia induced via oxidative stress within the cell since glutathione cant be reduced
- contains Heinz bodies in affect people

• very common single gene disorder that is x-linked (mainly affects males)

Question 13

Role of NADPH

Answer 13

Electron donor for biosynthesis of

• Fatty acids
• cholesterol
• steroid

Electron donor for the neutralization of ROS

• hydrogen peroxide
• superoxide
• hydroxyl radicals

Helps reduce Cytochrome P450

Aids in destructions of pathogens by macrophages and neutrophils

Substrate for nitric oxide synthesis

Question 14

What is the primary role of NADPH in RBCs?

Answer 14

Reducing glutathione (GSH)

• only source of NADPH in RBCs is via PPP and through the oxidation of GSSG from Glutathione reductase
• this is done to make GSH able to fight/ capture free radicals and prevent damage

Question 15

Heme structure

Answer 15

A Porphyrin cyclic molecule containing 4 pyrrole rings joined via methenyl bridges and a metal ion in the middle
- iron group is ferrous (Fe2) to allow reversible binding to oxygen

Question 16

Heme biosynthesis and degradation

Answer 16

Most of the heme (85%) is synthesized in bone marrow and requires the presence of iron to do so.

Heme is degraded via mononuclear phagocyte system (MPS) in the spleen, liver and bone marrow

Question 17

Hemoglobin structure

Answer 17

Two diners composed each of identical heme chains

• strong hydrophobic bonds form between heme chains of each dimer to form stable diners
• each dimer weakly binds to the other via ionic in the deoxygenated state (Taut structure)
• the bonds between each dimer are broken when bound to oxygen (relaxed structure)
• increasing relaxed structure of the hemeglobin as it binds oxygen molecules causes increased affinity for oxygen until all 4 heme groups (positive cooperatively)

Question 18

Hemoglobin Oxygen dissociation curve

Answer 18

Follows a positive cooperatively model. (The more oxygen bound to heme groups, the high the affinity to oxygen for the non bound heme groups)
- this is caused because hemoglobin becomes relaxed when it binds to oxygen, allowing more open sites for oxygen binding

Oxygen dissociative curve is steepest at the Po2 inside the tissues and at its highest value in the lungs
- sigmoidal curve

Question 19

Hepcidin

Answer 19

Presence down-regulates transports of iron (tranferrin protein and its receptors on cells) in the GI system and prevents its absorption in the diet.

Hepcidin is turned off when erythropoiesis is needed

primary molecule used in iron metabolism

Question 20

Hemoglobin vs myoglobin in oxygen dissociation curve

Answer 20

Myoglobin saturates much quicker than hemoglobin due to less total heme groups

• myoglobin P50 = 1mmHg
• hemoglobin P50 = 26 mmHg

Question 21

P50

Answer 21

The amount of Po2 required to saturate 50% of the hemoglobin/myoglobin

• increases in P50 results in decreased affinity (shift to the right of the curve)
• decreases in P50 results in increased affinity (shift to the left)

Question 22

Allosteric effectors of hemoglobin affinity to Oxygen

Answer 22

PO2 (increased levels: higher affinity)

PCO2 (increased levels: lower affinity)

PH changes (Lower pH: lower affinity/ Higher pH: high affinity)

2,3-BPG (increased levels: lower affinity)

Question 23

Bohr effect

Answer 23

Decreases in pH results in decreased oxygen affinity (shift to the right) for hemoglobin since hemoglobin will bind H+ ions (protonates the heme groups) that are produced via bicarbonate

• this causes stabilizing (taut) affects on the deoxygenated hemoglobin, reducing the overall binding sites

PH in lungs is higher than tissues, so oxygen affinity is higher in lungs than tissues

Question 24

2,3 BPG and hemoglobin affinity

Answer 24

2,3 BPG is an allosteric regulator of O2 and ONLY binds to deoxy-Hb; NOT to oxy-Hb

2,3 BPG binding promotes taut structure and release of oxygen from heme groups, produces a chronic hypoxia effect, but also allows RBCs to release oxygen to the target tissue effectively

Causes right shift in the oxygen saturation curve

Question 25

Oxygen saturation mechanisms

Answer 25

A measure of the number of occupied O2-binding on a hemoglobin molecule * independent on the amount of hemoglobin present* - increases in hemoglobin levels only increase the total amount of O2 content possibly bound

Question 26

Carbon monoxide saturation mechanisms

Answer 26

Hemoglobin affinity for carbon monoxide is 220x greater than oxygen - binds tightly (but reversible) to hemoglobin at the same site as oxygen Presence of CO both reduces o2 carrying capacity and decreases unloading of oxygen from hemoglobin -It’s binding causes heme sites to very tightly bind to oxygen, decreasing releases as long as CO is present Cuts the curve peak in half and results in a slight shift towards the left *CO poisoning produces bright cherry red skin since the hemoglobin bound to carbon monoxide turns bright cherry red*

Question 27

Types of hemoglobin in adults

Answer 27

HbA (normal): 90% HbA2 (2 alpha chains, 2 delta chains): 2-3% HbF (2 alpha chains, 2 gamma chains): <2% HbA1c (heme bound to glucose) 4-6%

Question 28

What is special about HbA1c?

Answer 28

Hemoglobin bound to glucose, high levels (>5.7) suggest prediabetes or diabetes. Prediabetic levels = 5.7 - 6.4% Diabetic levels = >6.5%

Question 29

Expression of hemoglobin genes

Answer 29

Both alpha and beta genes: - alpha gene only produces alpha chains (zeta only when 0-6 weeks of prenatal age) * begins at 2 weeks of prenatal age through life* -beta gene produces gamma, delta and beta chains (epsilon only when 0-6 weeks of prenatal age)

Question 30

Time frames for gamma, beta and delta chain transcription in the beta chains for hemoglobin

Answer 30

Gamma: 2 weeks prenatal -> 30 years *peaks at 18 weeks prenatal* Beta: 7 weeks prenatal -> life * peaks at 18 years* Delta: 30 weeks -> 40 years * very low levels throughout though*

Question 31

Why are gamma chains present at such Huge levels when prenatal vs postnatal?

Answer 31

HbF (2 alpha and 2 gamma chains) has a higher affinity to oxygen and doesn’t bind as well to 2,3 BPG - 2,3 BPG is used to help facilitate transfer of oxygen from maternal circulation to the fetal circulation HbF is preferred over HbA (normal) at this stage

Question 32

Groups of hemoglobinopathies

Answer 32

Qualitative: structural defects Quantitative: defects in total synthesis

Question 33

What is the most common structural defects in hemoglobin?

Answer 33

Point mutations in the B-globin gene Sickle cell= glu -> Val HbC = glu -> lys

Question 34

Treatment of sickle cell disorder

Answer 34

Hydration, analgesics, antibody therapy - hydroxyurea (increases circulating levels of HbF and reduces RBC sickling) - stem cell transplantation is possible in severe cases

Question 35

Treatment of Methemoglobinemia

Answer 35

Methylene blue dye (acts as electron acceptor and helps reduce iron beck to Fe2+/normal state) Vitamin c is also used in conjunction with methylene blue to speed up this process

Question 36

Selective advantage against malaria

Answer 36

Homozygous individuals for malaria are much more likely to die than heterozygote individuals in fact, heterozygous individuals have increases resistance to some strains (such as plasmodium falciparum malaria). - because of this, the trait of sickle cell anemia is actually selected for in cultures with high rates of malaria (such as Africa) Results in disease maintaining a high frequency in populations that are often exposed to malaria, but not in populations with low exposure to malaria

Question 37

Glutathione (GSH) structure

Answer 37

Glycine, cysteine and glutamate residues bound together and have a sulfide binding site

Question 38

Types of G6PD deficiencies

Answer 38

Class 1: very severe (<10% activity) Class 2: severe (<10% enzyme activity) - CGPD Mediterranean Class 3: moderate (10-60% enzyme activity) - G6PD A- Class 4: asymptomatic (60% enzyme activity) Mutations can affect the stability active site or allosteric site of G6PD. - most common is overall stability

Question 39

Nutritional anemia types

Answer 39

``` Microcytic anemia (MCV <80) - often signals deficency in iron, Vit. C or pyridoxine ``` Normocytic (MCV = 80-100 - protein malnutrition Macrocytic (MCV >100) - often signals deficency in vitamin B12 and/or folic acid