Hemoglobin Flashcards
Both functions of Hb are dependent on _____ gradients of O2 and CO2
partial-pressure
Myoglobin is synthesized inside ___ cells
muscle
Rank myoglobin concentration in different skeletal muscle fiber types:
IA>IIA>IIB
Describe the structure of myoglobin:
Single polypeptide chain, with ~80% alpha-helical. Closely packed tertiary structure. Single heme molecule covalently bound. Binds one O2 molecule at heme.
Is oxygen hydrophilic or phobic?
Hydrophobic
Excessive strong _____ interactions between ___ and ___ subunits and between ___ and ____
Hydrophobic interaction between alpha-1 and beta-1, and between alpha-2 and beta-2
weaker ____ interactions between heterodimers ___ and ___
Weaker polar interactions between a1B1 and a2B2
Hemoglobin A (HbA):
alpha2beta2 (a2B2). 97 - 98.5% of total Hb in adult. Glycosylation is marker for chronically elevated blood sugar.
Hemoglobin A2 (HbA2):
alpha2delta2. 1.5 - 3% of total Hb in adult.
Hemoglobin F (HbF):
alpha2gamma2. Fetal Hb is major Hb from 1 month gestation until near birth. About 40% at birth.
Hemoglobin Epsilon (Hb E):
Can be alpha2epsilon2 = embryonic Hb. Have from about 1 week post-conception until birth.
Deletion of ___ genes lead to Thalassemia
globin genes
Deletion of 1 beta globin gene leads to:
B-thalassemia minor
Deletion of 2 beta globin genes leads to:
B-thalassemia major (excess alpha chains, doesn’t form tetramer)
Deletion of 1 alpha globin gene leads to:
usually no symptoms
Deletion of 2 alpha globin genes leads to:
alpha-thalassemia trait
Deletion of 3 alpha globin genes leads to:
HbH disease (named after B4 tetramer formed)
Deletion of 4 alpha globin genes leads to:
Hb Bart’s disease or hydrops fetalis (Hb Barts is gamma-4 tetramer)
____ rings form coordinate covalent bonds to Fe2+ in heme
pyrrole
The ____ chains from the pyrrole rings interact with the surrounding alpha/beta-globin chains to stabilize heme binding
hydrophobic
Heme belongs to the class of pigments known as:
porphyrins (cyclic tetrapyrroles)
What are two other common porphyrin rings?
Chlorophyll (green) and Cyanocolamain or Vitamin B12 (pink)
What contains 2 alpha and 2 beta chains?
Hemoglobin A
What contains 2 alpha and 2 delta chains?
Hemoglobin A2
What contains 2 alpha and 2 gamma chains?
Hemoglobin F
Describe the O2 binding curve for myoglobin:
hyperbolic
Describe the O2 binding curve for hemoglobin:
sigmoidal; indicates changing affinity for O2 over the binding curve
What does P50 show on a binding curve?
The partial pressure of O2 at which 50% of O2 binding sites are occupied
What is the P50 for oxygen for hemoglobin?
26
The ___ conformation of hemoglobin favors the deoxy form (O2 release)
T (taut)
The ___ conformation of hemoglobin favors the oxy form (O2 binding)
R (relaxed)
O2 is a ____ regulator of Hb O2 binding
positive allosteric regulator
The movements of the proximal and distal _____ cause their attached ___-helices to move. This causes a conformational change at the interface of each alpha-beta dimer in the Hb molecule.
proximal and distal histidines; attached alpha-helices.
Physiological modulators of O2 binding by Hb most often ____ the affinity of Hb for O2
decrease the affinity
2,3-DPG is produced in RBCs when _____ is abundant; it binds in the cleft between the ____-subunits and stabilizes the ______ state. Binding allows Hb-bound O2 to dissociate and to supply O2 to tissues operating at a high metabolic rate.
when glucose is abundant. beta-subunits; T (deoxy) state.
makes O2 release by Hb more responsive to hypoxia by allowing more O2 release at ____ pO2
low pO2
Haldane effect:
In the lungs, high pO2 drives binding of O2 to Hb, release of H+, and formation of H2CO3. carbonic anhydrase equilibrium favors the synthesis of CO2 and H2O. Haldane - High pO2 in lungs..
Bohr effect:
in RBCs, protons bind to Hb and favor the T state, favoring O2 release
CO2 produced in metabolism enters RBCs, forming ____ with Hb or is converted to ____ by carbonic anhydrase, then dissociates into ____
carbamate adducts. Converted to H2CO2 then HCO3- and H+
Increase in pH favors O2 ____
binding
Decrease in pH favors O2 ____
release
Fetal hemoglobin has a different ___ chain, and it has a ___ affinity for oxygen than adult hemoglobin
different beta chain. Higher affinity.
Hb F binds 2,3-DPG very ____. Why?
Binds very poorly, because the cavity is not as positively charged.
The most common Hb variant associated with significant pathology is the ____ variant, caused by a mutation at position __ of the ___-globin gene, changing a Glu to Val
The most common Hb variant associated with significant pathology is the Bs variant, caused by a mutation at position 6 of the BETA-globin gene, changing a Glu to Val
What compounds act as competitors with oxygen at the iron-binding site on heme?
Cyanide (CN-), Carbon monoxide (CO), Nitrogen dioxide (NO2), and hydrogen sulfide (H2S)
Carboxyhemoglobin:
A stable complex of carbon monoxide and heme in Hb. Forms more readily than oxyhemoglobin (HbO2)
Mutations that change any of the 3 amino acids on the distal side of heme result in _______. What are the physical symptoms of this?
hereditary methemoglobinemia. Characterized by cyanosis and brown color of blood
In addition to AA mutations on heme, what are other hereditary mutations resulting in Methemoglobinemia?
Mutations in PPP (especially G-6-P dehydrogenase deficiency) and mutations in cytochrome b5 reductase.
Glutathione normally ____ reactive oxygen species, thereby preventing the formation of _____
normally reduces ROS, preventing the formation of methemoglobin
Cytochrome B5 reductase:
(NADH-dependent methemoglobin reductase) - enzyme uses NADH generated through glyceraldehyde phosphate dehydrogenase (glycolysis) to reduce Fe3+ in methemoglobin to Fe2+ thereby reversing formation of Fe3+.
At high elevations, 2,3-DPG concentration _____ and hemoglobins affinity for oxygen _______
At high elevations, 2,3-DPG concentration INCREASES and hemoglobins affinity for oxygen DECREASES
Define Thalassemia:
- genetically heterogeneous conditions resulting from imbalance between the amounts of alpha and beta globin chains that are synthesized.
- These arise from mutations which partially or completely inactivate production of either alpha or beta globin chains.
In general, we tend to have full deletions of ____ genes and point mutation in the ____ genes
Full deletions of alpha
point mutation in beta
Broadly, what are different ways that Thalassemias are classified?
- Severity of clinical manifestations
- The major hemoglobin species formed
- The genotype
What regions are Beta Thalassemias most commonly found?
•most common in southern Europe. Also seen in SE Asia, Africa, Middle East
Bº Thalassemia
Mutation where there is absent beta globin synthesis
B+ Thalassemia
The B+ thalassemia mutations have decreased (but still present) beta globin synthesis
What is the genotype of B-Thalassemia Major?
Bº/Bº
In patients with homozygous Beta Thalassemias, when do symptoms arise?
Clinically, severe anemia develops b/w 2 and 12 mo.
Infants are well at birth, since HbF and HbA2 predominate, and less HbA is needed
How are Homozygous Beta Thalassemias diagnosed?
Diagnosis by hemoglobin electrophoresis showing a4 tetramers and absence (or relative deficiency) of normal hemoglobin A
What are common symptoms of B-thal major? Why?
Anemia - a4 tetramers are very unstable and cause RBC hemolysis in periphery and inside bone marrow
Splenomegaly - A lot of destruction takes place in spleen, and red cells are drawn to it as it gets bigger
Bony deformaties - marrow is told to make more RBCs and enlarges
Iron Overload symptoms (bronze skin, liver failure, endocrine failure) - over-absorpotion of iron from gut and transfusions
Describe the hemoglobin type/structure in Beta Thalassemia Major and the consequences of this:
- Deficiency of beta globin chain leads to relative excess of alpha, which precipitates out as a4 tetramers
- a4 tetramers are unstable and leads to ineffective erythropoiesis (aka intramedullary hemolysis—hemolysis of red cell precursors inside the marrow) and destruction of produced RBCs
What is the genotype of B-Thalassemia Minor (Trait)?
B/B+
What are the lab findings for a patient with B-Thalassemia Trait?
- Very microcytic (MCV usually around 70)
- May or may not be anemic, if anemia is present, it is very mild
- RBC count will typically be very elevated
- RDW is normal, since ALL of the cells are microcytic and hypochromic
How is B-Thalassemia Minor diagnosed?
- Diagnosis by hemoglobin electrophoresis in adults
- Synthesis of delta chains is upregulated, so proportion of Hgb A2 levels will rise
- Typically see Hb A2 levels 4-8%
- We do not see this increase in HbA2 in newborns, since newborn hemoglobin species are predominantly HbF
What are the characteristics of a patient with Alpha Thalassemia aa/a- (one alpha gene deletion)?
- Typically silent.
- May have minimal microcytosis.
- Anemia not present.
- Hg electrophoresis normal
What are the characteristics of Alpha Thalassemia patients with two alpha gene deletions, e.g. aa/– or a-/a- ?
- Mildly anemic (Hg 10-11),
- Microcytosis with MCV around 70.
- Hgb electrophoresis normal in adults
- Newborns will make Hemoglobin Barts (g4), so the newborn screen (electrophoresis) will be abnormal
- Make dx by molecular techniques in adults.
Hemoglobin H Disease:
Alpha Thalassemia with 3 alpha gene deletion (a-/–)
Form Hemoglobin H aka B4 tetramers
•Hg H is unstable and precipitates as the RBC ages, forming Heinz bodies, which causes bite cells and a hemolytic anemia.
Hg H is unstable and precipitates as the RBC ages, forming _____, which causes ____ cells and a _______ anemia.
Hg H is unstable and precipitates as the RBC ages, forming Heinz bodies, which causes bite cells and a hemolytic anemia.
What is the clinical presentation of patients with Hemoglobin H Disease?
- Variable presentation (Most are like b-thal intermedia; some are barely affected and some are severely affected)
- Most have splenomegaly
- Hgb b/w 7-11, but may be as low as 3-4
- Low MCV and MCH, high RDW
Hydrops fetalis:
An Alpha Thalassemia where NO alpha globin genes are formed (genotype –/–)
No alpha chains form
Predominant chain synthesized is gamma-4 tetramers (Hemoglobin Barts)
Intrauterine death, followed by stillbirth at 25-40 weeks
Can be treated in utero by exchange transfusions followed by immediate bone marrow transplant after birth
In patients with hemoglobin S, the _____ in the B chain is changed from ____ (charged) to ____ (hydrophobic)
In patients with hemoglobin S, the 6th AA in the B chain is changed from glutamate (charged) to valine (hydrophobic)
Deoxygenated HbS is 50x less ___ than deoxy HbA. Explain.
50x less soluble
- Polymerizes and forms long fibers
- Rate of polymerization is logarithmically related to intracellular concentration of HbS
- Heterozygous cells do not usually sickle in vivo
Hemoglobin C mutations arise at the _____ in the B chain, changing a _____ to ____, which leads to increased cellular _______.
Hemoglobin C mutations arise at the 6th AA in the B chain, changing a glutamate to lysine, which leads to increased cellular dehydration.
Hemoglobin C disease is typically seen in individuals of _____ descent
West African
What is shown in this image?
Red arrows point to target cells
Yellow arrows point to Hemoglobin C crystal
This is a slide from a patient with Hemoglobin C
HbS _______ and cells sickle in microvascular beds under conditions of _____ and _____. When blood returns to lungs, HbS _______, and cells unsickle
HbS polymerizes and cells sickle in microvascular beds under conditions of hypoxia and acidosis. When blood returns to lungs, HbS depolymerizes, and cells unsickle
Multiple cycles of sickling/unsickling lead to cellular _______, which reduces threshold for ________
Multiple cycles of sickling/unsickling lead to cellular dehydration, which reduces threshold for further sickling
Abnormal Hb/membrane interactions activate the ______ ion channel, leading to cellular _______
Abnormal Hb/membrane interactions activate the Gardos ion channel, leading to cellular dehydration
Both sickled and unsickled red cells are less deformable and suffer ________
Both sickled and unsickled red cells are less deformable and suffer hemolysis
Sickle cells have abnormal WHAT
adhesion to endothelial cells
Sickle Cell Disease encompasses 3 genotypes:
SS, SC, and SB-thalassemia
What is the genotype of Sickle Cells Anemia?
Homozygous SS
What is the disctinction between SS and SC sickle cell disease?
In SC disease, the presence of HbC leads to more intracellular dehydration, worsening sickling
What are the forms of SB-Thalassemia?
- S-Bº thalassemia – indistinguishable from SS
- S-B+ thalassemia – milder disease, since some normal beta chains are produced
What are the clinincal manifestations of Sickle Cell Disease?
- Hematologic
- Crises
- Pain
- Infection
- Complications affect multiple organ systems
- Special considerations (e.g. surgery and pregnancy)
Describe the hematologic findings in patients with Sickle Cell Disesase:
- Anemia - Hemolysis is chronic and ongoing, but reticulocyte count should be elevated as patient compensates, so if Hb falls below pt’s baseline, look for other causes:
- Parvovirus B19
- Splenic or hepatic sequestration
- Acute chest syndrome
- Leukocytosis - elevated WBC count because spleen infarcts and they have to go to blood instead
- Thrombocytosis - elevated platelets because spleen infarcts
By adulthood in SS patients, the spleen has _____ and thus the patients are functionally _____
By adulthood in SS patients, the spleen has infarcted and thus the patients are functionally asplenic
See Howell Jolly Bodies
Susceptible to infections
SS patients are at an increased risk of ______ (1/3 of patients will have one by the age of 40)
venous clots
What are the 3 important aspects of the Sickle Cell Crises in SS patients?
Splenic sequestration crisis
Aplastic Crisis
Painful (vaso-occlusive) crisis
Splenic sequestration crisis:
- In children, rapid and extensive trapping of RBCs in spleen
- Profound anemia, massive splenomegaly, hypovolemic shock—occurs quickly
Aplastic crisis:
Parvovirus B19 leads to marrow suppression, with rapid development of anemia (but not so fast as in sequestration crisis)
Painful Crisis:
- Periodic episodes of acute vascular occlusion
- Attacks of pain usually affecting bones and large joints
- Triggers include, but are not limited to: Exercise, Dehydration, Infection, Cold, Stress, Menstruation, Surgery/trauma, Pregnancy
How should a painful crisis in SS patients be managed?
- Treat underlying infection, if any
- Treat pain adequately
- Supplemental oxygen
- Folate replacement
- Replace fluid if dehydrated, but watch for pulmonary edema
- Evaluation:
- Check retic count, looking for aplastic crisis
- Are they on hydoxyurea?
- Are they having acute chest syndrome?
Explain the unique infection risks of patients with SS:
- Immunocompromise - “autosplenectomy” from splenic infarct – increased susceptibility to encapsulated organisms
- Overwhelming bacterial sepsis – (esp in children) from encapsulated organisms
- If undergoing iron chelation therapy, infection with Yersinia or Vibrio organisms is a problem
- Pneumonia – can worsen acute chest syndrome
- Osteomyelitis and septic arthritis (Salmonella osteomyelitis)
What is the most common cause of death in patients with sickle cell disease?
Acute Chest Syndrome
What respiratory complications arise in sickle cell disease patients?
Acute Chest Syndrome
Pulmonary hypertension
How is Acute Chest Syndrome classified?
- Hypoxemia (new)
- New infiltrate on chest x-ray
- New fever, chest pain, dyspnea, or cough
- Acute worsening of anemia
Explain the etiology of acute chest syndrome
- Infection – especially with “atypical” organisms—chlamydia and mycoplasma
- Fat embolism from necrotic bone marrow
- Sequestration of sickled red cells
- Pulmonary infarction
- Hypoventilation from rib infarct, narcotic administration
- Pulmonary edema from fluid overload
Treatment of acute chest syndrome:
Early detection is key
Antibiotics, oxygen
Transfusion to lower HbS concentration (simple or exchange)
______ is one of the most frequent causes of pulmonary hypertension
Sickle Cell Disease
(occurs in 1/3 of SCD adults)
What are the consequences of pulmonary hypertension in SCD patients?
- leads to substantial morbidity with dyspnea, chest pain, hypoxia
- Leads to death within 2 years (no good treatment)
SCD patients often suffer from a stroke by the age of ___
5
Why are SCD patients susceptible to stroke?
Not due to atherosclerosis—due to disordered blood vessels (vasculopathy)
What is the appropriate treatment for stroke in SCD patients?
- Acutely – exchange transfusion
- Chronically in children, chronic regular transfusions prevent further stroke
- If transcranial dopplers show narrowing in the circle of Willis, then chronic regular transfusions prevent the first instance of stroke
In SCD patients, if transcranial dopplers show narrowing in the circle of _____, then chronic regular transfusions prevent the first instance of _____
In SCD patients, if transcranial dopplers show narrowing in the circle of Willis, then chronic regular transfusions prevent the first instance of stroke
What type of transfusion is appropriate for SCD patients with stroke?
Exchange transfusion
What complications can arise with transfusions in SCD?
- Iron overload (treat with chelation)
- Alloimmunization (help by transfusing with blood negative for certain blood groups - C, E, and Kell)
What are other general complications that can arise in SCD patients?
- Renal (Isosthenuria = Inability to concentrate urine due to renal papillary necrosis, Hematuria, proteinuria, Renal failure and need for dialysis)
- Leg ulcers
•AVN = Avascular Necrosis (More common in SC or Sb-thal. Pain in shoulders or hips – not like a regular pain crises)
- Priapism (episodic or chronic, may lead to impotence. Treat with transfusion, local measures)
- Proliferative retinopathy (more common in SC or S b thal)
Hydroxyurea effects:
Increases amount of HbF, thus decreasing concentration of HbS
Increases baseline hemoglobin values
Reduces number of sickle crises
Reduces episodes of acute chest syndrome
Prevents (not treats) pulmonary hypertension
Reduces mortality
Side effects and contraindications of Hydroxyurea:
Side effects: bone marrow suppression
Contraindications: pregnancy, poor/erratic follow-up, allergy
In general, patients with Sickle Cell Trait should not have crises or splenic infarctions unless they are ______
severely hypoxic
Patients with Sickle Cell Trait may have renal manifestations, including ____ and ______
Isosthenuria - suggests kidneys aren’t filtering properly
Microscopic hematuria - urinary blood only visible under the microscope
Hemoglobin C Trait:
30-40% HbC
Not anemic
Hemoglobin C Disease:
Mild hemolysis
Microcytosis and target cells
Splenomegaly
