Hematology 1 - Exam 2 Flashcards
Describe the component parts of a basic hemoglobin molecule.
One hemoglobin molecule contains:
- 2 pairs of 2 different polypeptide chains (dimers). This means 4 globin chains are arranged as a tetramer.
- 2 molecules of protoporphyrin IX ().
- 4 iron (Fe2+) atoms (combine with protoporphyrin IX to form 4 heme rings.
- (Optional) one 2,3 BPG molecule may or may not occupy the center of the entire Hgb molecule.
Construct the heme synthesis pathway.
- Transferrin (plasma protein) transports ferric (Fe3+) iron to the developing RBCs.
- Iron (ferro-chelatase - is another word for iron) is actively carried across the RBC membrane to the mitochondria.
- In the mitochondria, iron is matched with protoporphyrin IX to make heme.
- Heme leaves the mitochondria and travels to the cytosol (cytoplasm) to join the globin chains.
Evaluate normal hemoglobin, their polypeptide chain composition and relative concentrations in human blood (adult and fetal).
- 3 months after conception, the embryo produces 3 embryonic Hgbs: Portland (GZ), Gower I (EZ), Gower II (AE), where each is comprised of 2 different pairs of globin chains.
-2nd trimester (hepatic phase) is when true fetal Hgb (Hgb-F (AG)) begins to form.
> At birth, Hgb-F (AG) = 80% total hemoglobin. Hgb-A1 (AB)= 20%.
> At 1 year, all Hgb is in adult forms Hgb-A1 (AB) = 97-98%, and Hgb-A2 (AD) = 2-3%.
> About 1% in adults remains as Hgb-F (AG).
Describe the following structural levels of a normal Hgb molecule: primary, secondary, tertiary, and quaternary.
Primary - linear amino acid structure. Begins with 1 at N-terminal and ends at C-terminal.
Secondary - arrangement in helices and non-helices. Each chain is divided into 8 helices (separate and structurally rigid, designated by A-H) and 7 nonhelical (flexible and lie between helical segments) segments.
Tertiary - roughly globular shape that secondary folding assumes spontaneously to support non-hydrogen bonds, such as sulfhydryl bridges formed between neighboring amino acid side chains.
*Note: at this stage, 1 heme group is inserted inside each of the 4 globin chains.
Quaternary - refers to tetramer formed by 2 pairs of polypeptide chains. Complete hemoglobin molecule is spherical and has 4 heme groups attached to 4 polypeptide chains. It can carry 4 molecules of oxygen.
Evaluate the complete pathway involved in Hgb synthesis, including locations.
- A ferric (Fe3+) iron molecule (originally obtained from ferritin, but being transported by transferrin) is chemically reduced to the ferrous (Fe2+) form in the nucleated RBCs cytoplasm.
- Fe2+ (ferrous) is transported into mitochondria & inserted into the center of a protoporphyrin IX molecule. Now the molecule can be called heme. Protoporphyrin IX + Fe = heme.
- Finished heme is released from the mitochondria back into the cytoplasm.
- In the cytosol, finished alpha & beta globin polypeptide chains are released from nearby ribosomes. (Heme is transported in tertiary form into the globin chains).
- One heme molecule is inserted into each globin polypeptide.
- Alpha & beta chains quickly form dimers, then dimers to tetramers (quaternary structure).
- (Optional) One 2,3 BPG/DPG molecule is inserted into the central cavity of each finished Hgb molecule as needed. (This step isn’t always present because if it was, we wouldn’t be able to bind oxygen as 2,3 BPG encourages oxygen off-loading.)
Examine the concept of Hgb-oxygen affinity in detail, including:
- The basics of heme-heme interaction
- The normal oxygen dissociation curve and the causes of both left and right shifts.
- The “R” and “T” configurations of normal Hgb
- Heme-heme interactions: the binding of 1 molecule of O2 causes slight shift in Hgb molecule structure, which triggers an increased affinity of other nearby heme groups to bind more O2 molecules.
- Shift to right - decreased O2 affinity (tends to off-load O2). Hypoxia occurs in tissues, so this shift occurs in tissues. Is caused by increased CO2, body temp, and 2,3 BPG. Is caused by decreasing pH.
Shift to left - increased O2 affinity (tends to bind O2). Occurs in lungs. Caused by decreased CO2, body temp, and 2,3 BPG. Caused by increased pH. - “R” (relaxed) form = no 2,3 BPG present = high O2 affinity = increased O2 uptake in lungs.
“T” (tense) form = 2,3 BPG present = lower O2 affinity = increased O2 off-loading in tissues.
Distinguish between extravascular and intravascular hemolysis.
Extravascular hemolysis - occurs more often.
- Intramacrophage RBC breakdown occurs following phagocytosis when RBC is attacked by lysosomal enzymes. Hgb is broken down into heme, iron, and globin.
-Heme iron is stored as ferritin or hemosiderin within macrophage.
- Globin is broken down and returns to the amino acid pool.
- Protoporphyrin component of heme converts to bilirubin (released into the plasma and excreted by the liver in bile). Conjugated bilirubin is excreted from the liver into the small intestine via the bile duct where it is converted by bacterial flora to urobilinogen.
- Most urobilinogen is excreted in the stool as urobilin, 10-20% is reabsorbed by gut.
- With liver disease, the enterohepatic cycle is impaired and increased amount of urobilinogen is excreted in urine.
Intravascular hemolysis - cell components are released into the plasma. Haptoglobin and hemopexin work to salvage the released Hgb, so iron is not lost.
- they carry Hgb to the liver, where it is broken down into bilirubin.
- A decrease in serum haptoglobin may be used to indicate intravascular hemolysis.
- If haptoglobin is depleted, free Hgb is filtered by the renal glomerulus.
Differentiate oxygen tension and oxygen affinity.
Oxygen tension - occurs in the tissues and is regulated, partially, by oxygen affinity of Hgb.
Oxygen affinity - occurs in the RBCs and is modulated by the concentration of phosphates in the cell.
*Note: in areas of hypoxic tissue, as O2 moves from Hgb to tissue, the amount of reduced Hgb decreases, resulting in reduced O2 affinity. If tissue hypoxia persists , the depletion of 2,3 BPG leads to increased glycolysis and production of more 2,3 BPG, which will further lower O2 affinity.
Predict the effects of the following factors on Hgb’s affinity for oxygen:
- Temperature
- pH (Bohr effect)
- 2,3 BPG
- Fetal Hgb
- Abnormal Hgb variants
- CO2
Temperature - increased temp = decreased O2 affinity
pH (Bohr effect) - decreased pH = decreased O2 affinity
2,3 BPG - increased concentration = decreases affinity
Fetal Hgb - fetal Hgb binds O2 easier than Hgb-A1. Increased Hgb-F = increased affinity.
Abnormal Hgb variants - can shift the oxygen dissociation curve either way.
CO2 - Haldane effect is when increasing CO2 causes right shift (decreasing O2 affinity).
Contrast carboxyhemoglobin, methemoglobin, and sulfhemoglobin in terms of their:
- Pigment
- Causes of formation
- Effect
- Carboxyhemoglobin - abnormal Hgb that is a bright cherry color (hallmark). Results from the binding of CO to Hgb. CO binds to Hgb at rate 200X higher than O2, so asphyxiation results in a matter of minutes. This would be a left shift. Is most associated with auto exhaust, burning coal, charcoal, and smoking. Death results when carboxyHgb reaches 50-70% total Hgb, when normal levels are < 1%.
- Methemoglobin - abnormal hemoglobin that is a brownish to bluish color. Results from Hgb that contains iron in the ferric state (Fe3+). Accumulation produces a shift to the left resulting in O2 not being delivered to tissues. This is seen in the presence of nitrites and genetic disorders.
- Sulfhemoglobin - abnormal hemoglobin that is a green color. Results from oxidation of Hgb by drugs or chemicals (usually sulfa drugs). Displays a 100X less affinity for O2 than unmodified Hgb. Conversion is permanent for the life of the cell.
Where do 2,3 BPG molecules in Hgb molecules come from?
RLP (Rapaport-Luebering Pathway) Pathway
What RBC stage does hemoglobin synthesis begin in, and what stage is it actually visibly seen in?
What percentage of hemoglobin synthesis occurs during which stages of maturation?
Hemoglobin synthesis begins in the Pronormoblast stage, but isn’t seen until the polychromatic normoblast stage.
- 65% of hemoglobin synthesis occurs during nucleated stages of maturation.
- 35% of hemoglobin synthesis occurs during the reticulocyte stage.
What is hemoglobin and how much of the RBC does it take up?
Hemoglobin is a conjugated globular protein which constitutes 33% of RBC weight by volume.
What processes is normal hemoglobin production dependent on?
- Adequate iron delivery and supply
- Adequate synthesis of protoporphyrin
- Adequate globin synthesis
Where do sources of iron come from?
Iron has very little bioavailability in food (meaning very little is actually absorbed in our diets, only about 10% of what we eat).
Therefore, the body works to recycle the iron it already has by reusing what was in degraded RBCs (which have been stored in splenic macrophages as there is no mechanism for excretion).
About 1/4 of iron is in storage form [ferritin (apoferritin + Fe = ferritin, where apoferritin is a cylindrical protein sac that many iron molecules can be stuffed inside) or hemosiderin] in macrophages. A very small amount is in transferrin (plasma protein).
Where are sources of iron stored?
- Stored in splenic macrophages (no mechanism for excretion).
> 1/4 of iron is in storage form in macrophages.
> Ferritin (apoferritin + Fe = ferritin, where apoferritin is a cylindrical protein sac that many iron molecules can be stuffed inside)
> Hemosiderin - A very small amount is in transferrin (plasma protein).
Describe iron transport.
When needed, Fe3+ (ferric form) is released from ferritin in the gut’s mucosal cells.
This Fe3+ attaches to the iron transport protein: Transferrin.
Transferrin can transport up to 2 atoms of Fe3+ through the plasma and delivers it into the RBCs.
Afterwards, transferrin is returned to the cell surface for recycling, and Fe3+ is reduced to Fe2+ (ferrous form) in the cytoplasm of RBCs.
What chromosome in an immature (still nucleated) RBC contains the alpha and zeta globin genes? Beta globin genes?
Chromosome 16 - Alpha and zeta
Chromosome 11 - Beta and all others
Describe protoporphyrin synthesis pathway.
- Starts with Succinyl Coenzyme A (CoA) produced by the tricarboxylic acid cycle.
> succinyl coenzyme A + glycine + [delta ALA synthase (enzyme)] = aminolaevulinic acid (ALA). Occurs in the mitochondria of pronormoblast and requires vitamin B6 (pyridoxal phosphate). - Aminolaevulinic acid (ALA) combines with ALA dehydrase (enzyme) to form porphobilinogen (PGB).
- Porphobilinogen + porphobilinogen deaminase = hydroxymethylbilane.
- Hydroxymethylbilane > uroporphyrinogen III.
- Uroporphyrinogen III + uroporphyrinogen decarboxylase = coproporphyrinogen III.
- Coproporphyrinogen III > protoporphyrin IX
- Protoporphyrin IX + (Fe2+) + ferro-chelatase (heme synthase) = heme.
Recall general locations as DNA goes to proteins.
DNA is transcribed to RNA in the nucleus.
RNA is processed and spliced to form mRNA transcript, which exits the nucleus through nuclear pores and goes to the cytoplasm.
In the cytoplasm, mRNA is translated to a ribosomal polypeptide chain.
This polypeptide chain immediately begins to assume primary through quaternary folded forms.
What globin chains are found in the following hemoglobin molecules:
- Portland
- Gower I
- Gower II
- Fetal (F)
- A1
- A2
Portland - gamma + zeta (embryonic)
Gower I - epsilon + zeta (embryonic)
Gower II - alpha + epsilon (embryonic)
Fetal (F) - alpha + gamma (newborn and adult)
A1 - alpha + beta (newborn and adult)
A2 - alpha + delta (newborn and adult)
What are the 4 primary globin chains that can be produced?
Alpha
Beta
Gamma
Delta
What is glycosylated hemoglobin?
What is the most common glycosylated hemoglobin? Where does the sugar bind?
An indicator of how well managed a patient’s diabetes is.
- hemoglobin can be modified by nonenzymatic binding of various sugars with the globin chains. The most common being Hgb-A1c (glucose attaches to the N-terminal valine of the beta chain).
> Older cells typically contain more sugars due to more prolonged exposure where 4-6% is normal. This means 4-6% of Hgb-A1 circulates as Hgb-A1c, but this percentage increases with diabetes (proportional to the mean glucose level over 2-3 months).
What is the RBC mission?
Describe the hydrophobic/hydrophilic qualities of the RBC.
Describe the function of the proximal and distal histidine of the globin chain.
- Contain, transport, and protect hemoglobin molecules, so that oxygen can be carried.
- Each Hgb has 4 globin chains [has hydrophobic pocket to contain heme group, which protects Fe2+ (ferrous form) from oxidation to Fe3+ (ferric form)] and 4 heme groups (with a center iron molecule).
- Iron from each heme group is bonded to 2 histidines of the globin chain. Proximal histidine functions to increase oxygen affinity (the ability to bind oxygen) of the heme ring. Distal histidine functions to protect the iron in the Fe2+ (ferrous) state (hydrophobic pocket), which diminishes the binding of carbon monoxide (CO).
- The exterior of the hemoglobin chain is hydrophilic, which makes the molecules soluble.
What is FEP?
Free Erythrocyte Protoporphyrin = excess protoporphyrin left over in the mitochondria when Fe supply is diminished (iron deficiency anemia).
> It becomes complexed with Zn2+, then shipped into the cytoplasm. It can be measured as ZPP (zinc protoporphyrin).
> When Fe is low, FEP goes up.
What are 3 major functions of hemoglobin?
- Transport of oxygen from the lungs to the tissues.
- Transport of carbon dioxide from the tissues to the lungs.
- Buffering the blood to prevent major pH changes.
Describe the allosteric effects of hemoglobin.
- Cooperative binding of oxygen (where the 1st oxygen molecule is the hardest to bind, but as soon as it is bound, the following ones are easier).
- Regulation of oxygen affinity by 2,3 BPG
- Bohr effect (caused by a drop in pH or high CO2 concentration in blood. This reduces Hgb affinity for oxygen and encourages O2 off-loading to meet oxygen demand in tissues).
What are the hemoglobin saturation forms?
Oxyhemoglobin - Hgb saturated with oxygen (O2)
Deoxyhemoglobin - Hgb without oxygen (–)
Carbaminohemoglobin - Hgb with carbon dioxide (CO2)
Carboxyhemoglobin - Hgb with carbon monoxide (CO)
What are heme-heme interactions?
When the binding of 1 molecule of O2 causes an increased affinity of other heme groups to bind more O2 molecules.
This happens because at any time a Hgb may be carrying 1-4 O2 molecules, and as O2 binds to a heme group, that heme group shifts slightly & changes the overall shape of the Hgb, which then encourages another O2 to bind to another nearby heme.
Describe how 2,3 BPG affects oxygen affinity.
> Decreases O2 affinity.
- It binds between beta chains of Hgb-A1 via salt bridges when Hgb is in it’s more unoxygenated state (“T”/”tense” form). This binding process moves beta chains slightly further apart & results in a shape change causing decreased O2 affinity, such that any remaining bound O2 is off-loaded into the tissues.
- Once this deoxyHgb reaches the lungs, the binding of O2 to heme is a greater force than the weak bonds holding the 2,3 BPG inside the Hgb. This means Hgb-A1 becomes oxygenated (“R”/”relaxed” form) and the resulting heme-heme interactions cause 2,3 BPG to be expelled.
