Hemoglobin Metabolism

Question 1

When hemoglobin is denatured and free from

RBCs, how is removed from the body ?

Answer 1

• it is excreted by the kidneys
• outside of the RBC hemoglobin has a short half life
  
  • when released it is salvaged for its iron and amino acid components
  • if those mechanisms are saturated then the rest is excreted via the kidneys

Question 2

Aside from O2 and CO2 transport, what other

role does hemoglobin have in the body ?

Answer 2

• contributes to the acid base balance of the body by binding and releasing hydrogen ions
• transport nitric oxide
  
  • this is a main regulator of vascular tone

Question 3

What is the general structure of

hemoglobin ?

Answer 3

• globular protein
• composed of 2 different pairs of polypeptide chains
  
  • 4 globin chains
• four heme groups
  
  • one heme group is embedded in each of the four polypeptide chains

Question 4

What is the structure of the

heme group ?

Answer 4

• One heme group
  
  • ring of carbon, nitrogen, hydrogen atoms (called Protoporphyrin IX)
  • there is a central atom of divalen ferrous iron (Fe 2+)
    
    • can reversibly combine with one oxygen molecule
    • oxidized to Ferric (Fe 3+) –> cant bind oxygen anymore

p. 92 great pictures (review)

Question 5

What is Methemoglobin ?

Answer 5

• oxidized hemoglobin

Question 6

What is the structure of each globin

chain ?

Answer 6

• each hemoglobin molecule has 2 identical pairs of unlike polypeptide chains
• variations in amino acid sequences give rise to different types of chains
  
  • each one has a different greek letter designation

Question 7

What is the primary structure of hemoglobin ?

Answer 7

• primary structure is the amino acid sequnces of the polypeptide chains

Question 8

What is the secondary structure of hemoglobin ?

Answer 8

• refers to chain arrangements in helices and nonhelices

Question 9

What is the tertiary structure of hemoglobin ?

Answer 9

• arrangement of the helices into a pretzel like configuration
• globin chains loop to form a cleft pocket for heme
  
  • the chains are hydrophic in the pocket and hydrophilic on the outside rendering hemoglobin water soluble
  • this allows iron to stay in the divalent ferrous form

Question 10

What is the quaternary structure of hemoglobin ?

Answer 10

• it is a tetramer
• describes the complete hemoglobin molecule
  
  • four heme groups attached to 4 polypeptides
  • can carry up to 4 molecules of oxygen

Question 11

What is the main adult hemoglobin ?

Answer 11

• Hemoglobin A
• composed of 2 alpha globin chains and 2 beta globin chains

Question 12

What type of hemoglobin is Hemoglobin A1C ?

Answer 12

• it is a glycated hemoglobin A molecule
• glycation is a posttranslational modification that is non-enzymatic allowing the binding of various sugars to hemoglobin
• HgA1C
  
  • glucose attaches to the N-terminal of valine of the beta chain
  • normally have about 4-6% of circulating HgA1C
  • increases in diabetes
  • reflective of the sugar levels over the preceding 2-3 months

Question 13

Where does Heme biosynthesis occur ?

Answer 13

• biosynthesis occurs in the mitochondria and cytoplasm of bone marrow erythroid precursors
  
  • begins with the pronormoblast through the circulating polychromatophilic erythrocyte
  • as the erythroid precursors lose their ribosomes and mitochondria they can no longer make hemoglobin

mechanism described on p. 95

Question 14

What is the final step of heme production

and where does it occur ?

Answer 14

• Final step
  
  • occurs in mitochondria
  • Fe 2+ combines with protoporphyrin IX in the presence of ferrochelatase (aka heme synthetase)

Question 15

What is the role of transferrin in

Heme synthesis ?

Answer 15

• Transferrin
  
  • plasma protein that binds Fe 3+ and delivers it to developing erythroids
  • binds to transferrin receptors on erythroid precursor cell membranes
  • then endosomes bring the transferrin and iron into the erythroid precursor cytoplasm
  • iron is transported from the endosome into the mitochondria
    
    • there it gets reduced to Fe 2+
    • united to protoporhyrin to make Heme
    • Heme leaves mitchondria and in the cytoplasmic ribosomes is joined to the produced globins

Question 16

How many copies of globin genes are present in the human genome ?

Answer 16

• one copy of each globin gene per chromatid , so two per diploid cell with the exception of:
  
  • alpha and gamma globins
    
    • these have 2 per chromatid or 4 per diploid cell
• globins are only produced in immature erythroid precursors
• although more alpha globin mRNA is produced there is less efficient translation of the mRNA so there are equal amounts of alpha and beta globin chains produced

Question 17

How do the hemoglobin molecules assemble ?

Answer 17

• non-alpha chains have a charge difference that determines their affinity to bind alpha chains
• alpha chains are positively charged
  
  • alpha chains have the highest affinity for beta globin chains because they are negatively charged
  • next is gamma globin
  • then delta globin chain
• two heterodimers combine to form a tetramer = hemoglobin

Question 18

What is the major hemoglobin in the body ?

Answer 18

• Hemoglobin A
  
  • 2 alpha and 2 beta chains
  • present from 6 months to adulthood
• Hemoglobin A2 (second hemoglobin in adults)
  
  • 2 alpha and 2 delta chains
  • production of delta chains is low (< 3.5% in body)

Question 19

What is the composition of

Hemoglobin F ?

Answer 19

• usually comprises only 1-2% of adult hemoglobins
• present in an uneven distribution in the RBCs
• composed of 2 alpha and 2 gamma chains
• these RBCs are called F/A cells

Question 20

How are hemoglobins tested for ?

Answer 20

• the different amino acids on hemoglobins have different net charges
• electrophoresis or HPLC are used for fractionation and presumptive identification
• molecular genetic testing globin gene DNA provides definitive identification of variant hemoglobins

Question 21

Which hemoglobin genes are on

chromosome 16 ?

Answer 21

• zeta (first 3 months of embryonic development)
• alpha

Question 22

Which hemoglobin genes are on

chromosome 11 ?

Answer 22

• epsilon (first 3 months of embryonic development)
• gamma
• delta
• beta

Question 23

What hemoglobin predominates

from 4 months of embryonic development

through the first 6 months of life ?

Answer 23

• Hemoglobin F
  
  • composed of 2 alpha and 2 gamma chains

Question 24

When does the adult hemoglobin pattern

start to emerge?

Answer 24

• emerges by 6 months of age
  
  • alpha2, beta2 (HgA) major form
  • alpha2, delta2 (HgA2) minor form
  • alpha2, gamma2 (HgF) extremely small amounts if any

Question 25

What are the three hemoglobins

present in early embryonic development

(products of yolk sac erythroblasts) ?

Answer 25

• Gower 1 (zeta2, epsilon2)
• Gower 2 (alpha2, epsilon 2)
• Portland (zeta2, gamma 2)

Question 26

What is the main regulatory step in

Heme production ?

Answer 26

• key rate limiting step:
  
  • initial reaction of glycine with succinyl CoA
  • this forms Aminolevulinic acid (ALA)
  • this is catalyzed by ALA synthetase
• Heme acts as a negative feedback molecule
  
  • if lots of heme (also maybe portoporphyrin) then the pathway is blocked
• Heme and portoporphyrin also likely inhibit
  
  • Ferrochelatase enzyme

Question 27

Where in the body is hypoxia detected ?

Answer 27

• detected by the peritubular cells of the kidney
• the kidney responds by increasing EPO
  
  • this increases erythrocyte production and release into the peripheral tissues
  • in addition it also accelerates hemoglobin production

Question 28

How does hemoglobin concentration

vary based on altitude ?

Answer 28

• individuals living at higher altitudes have higher hemoglobin concentration levels

Question 29

What regulates oxygen affinity for hemoglobin ?

Answer 29

• the parital pressure of oxygen determines the affinity for hemoglobin
  
  • high partial pressure = high affinity
• the curve is sigmoidal
  
  • low hemoglobin affinity at low oxygen tension
• the hemoglobin molecules work together
  
  • whne there is no oxygen bound all have a low affinity for O2
  • but as soon as one binds it changes it’s conformation and the others bind to oxygen even more quickly

Question 30

What happens to oxygen affinity if the

O2 dissociation curve shifts to the left ?

Answer 30

• there is higher affinity for Oxygen with higher oxygen saturation
• a shift to the right of the curve lowers the oxygen affinity (greater release of O2)

Question 31

What causes a left shift of the O2 saturation curve

of Hemoglobin ?

Answer 31

• Higher affinity for oxygen
• causes of left shift
  
  • 2,3 BPG
  • increased pH levels (increase H+ ions)
  • lower partial pressure of CO2
  • lower temperature
  • Hemoglobin F
    
    • other variants of hemoglobin that have a higher affinity for O2
  • Myoglobin (predominantly in muscle)

Question 32

What causes a right shift of

the O2 dissociation curve ?

Answer 32

• Right shift has lower O2 affinity (causes release of O2)
• Causes of right shift
  
  • increased 2,3 BPG (release O2 molecules)
  • lowered pH (increased H+ ions)
  • increased pCO2
  • increased temperature
  • hemoglobin variants with decreased O2 affinity
  • hypoxic conditions
    
    • high altitude, pulmonary insufficiency
    • CHF and severe anemia

Question 33

What hemoglobins are incapable of O2 transport ?

(dyshemoglobins)

Answer 33

• methemoglobin
• sulfhemoglobin
• carboxyhemoglobin

most of these are acquired from drug and toxin exposure. A small percentage of methemoglobin is inherited

Question 34

How is Methemoglobin formed ?

Answer 34

• reversible oxidation of heme iron to the ferric state (Fe 3+)
  
  • normally a small amount of methemoglobin is continuously formed
    
    • limited by NADH cytochrom b5 reductase to ~1% of total hemoglobin
• can t carry O2 because ferric iron cant bind it

Question 35

At what levels are symptoms of

Methemoglobinemia felt and what are the symptoms ?

Answer 35

• essentially get decreased delivery of O2 to tissues
• < 25% no symptoms generally
• ~30% symptoms start to manifest
  
  • cyanosis
  • symptoms of hypoxia: dyspnea, headache, vertigo, change in mental status
• > 50%
  
  • leads to coma and death

Question 36

What can cause the acquired form or toxic Methemoglobinemia ?

Answer 36

• exogenous oxidants
  
  • nitrites
  • primaquine
  • dapson
  • benzocaine
• the oxidant overwhelms the reduction system

Question 37

What is the treatment for Methemoglobinemia

caused by an exogenous oxidant ?

Answer 37

• IV Methylene blue
  
  • reduces ferric iron to ferrous iron
  • goes through NADPH-Methemoglobin reductase
• in life threatening cases
  
  • exchange transfusion

Question 38

How is Methemoglobin detected in the lab?

Answer 38

• spectral absorption such as a CO-oximeter
  
  • absorbs at 630 nm
  • blood takes on a chocolate brown color and does not revert back to normal red after exposure to oxygen

Question 39

How is Sulfahemoglobin formed ?

Answer 39

• formed by irreversible oxidation of hemoglobin by drugs
• or exposure to sulfur chemicals in the industrial or environmental settings
• formed by addition of a sulfur molecule to the pyrole ring
  
  • takes on a greenish color
• has a similar peak to methemoglobin on spectrophotometry

Question 40

How are Sulfahemoglobin and Methoemoglobin differentiated ?

Answer 40

• since they have similar peaks on spectral absorption
• use cyanide
  
  • sulfahemoglobin spectral curves does NOT shift with the addition of cyanide
  • methemoglobin does

Question 41

What does Carboxyhemoglobin form from ?

Answer 41

• combination of Carbon monoxide and iron
• CO has 240 times the affinity for hemoglobin compared to oxygen
• it shifts the oxygene dissociation curve to the left
  
  • less release of O2 to tissues
  • carbon monoxide has been termed the silent killer

Question 42

What group of people tend to have

high carboxyhemoglobin levels ?

Answer 42

• smokers can have levels up to 15% (due to cigarettes)
• normal is <2%
• they may compensate by increasing their RBC amounts (polycythemia)

Question 43

How is carboxyhemoglobin detected ?

Answer 43

• detected by spectral absorption instruments (540 nm)
• makes the blood and sometimes skin look cherry red
• CO poisoning is diagnosed when:
  
  • CO is > 3% in nonsmokers
  • CO > 10% in smokers

Question 44

How is carboxyhemoglobin treated ?

Answer 44

• administration of 100% oxygen