RBC Flashcards
Composition of blood
RBC
Buffy coat (WBC, Platelets)
Plasma
Normal pH of blood?
7.35-7.45
Plasma vs. Serum
Plasma
- no clotting factors
- centrifuged then add anticoagulant
- used in physiologic
Serum
- no clotting factors
- no fibrinogen!!
- used in lab reference
Can it synthesize new membrane proteins?
No but it can freely exchange in circulating lipoprotein lipids.
How does an RBC generate energy?
Anaerobic glycolysis
Shape of an RBC? Importance?
Biconcave disc for better gas exchange and most importantly, allows it to be deformable (it’ll undergo a lot of pressure inside vessels). If not deformable, will easily lyse.
RBC Proteins?
Peripheral (5)
integral (2)
How do you form a red blood cell? Major site of production?
Pluripotent stem cell — IL3 —> Mixed myeloid progenitor (CFU-GEMM) — Erythropoietin from kidney —> erythroid progenitor —> Erythroblast —-loss of nucleus —> Reticulocyte —-leaves BM into blood —> Erythrocyte
Major site of production: BM
Characteristics of a mature erythrocyte?
- components missing?
- ATP generation?
- replication
- life span
>No intracellular organelles >Can generate ATP by glycolysis ONLY >Like a dead bag containing hemoglobin >Cannot replicate >Has a finite life span
How does a red blood cell metabolize glucose?
- Embden-Meyerhoff Pathway (Glycolysis)
- Accts for 90% of glucose metabolism in RBC
- Anaerobic so: Glucose —-oxidized—> Pyruvate
- END PRODUCT: Lactate
- ATP: 2 per glucose molecule - Hexose Monophosphate Shunt (Pentose Phosphate Pathway)
- Accts for 10% of glucose metabolism
- Aerobic
- Alternative pathway for producing a 5C sugar
- Product: NADPH; keeps glutathione reduced
- No enzyme -> Low glutamine levels -> Oxidation of RBC -> Hemolysis -> Hemolytic anemia - Rapaport-Luebering Shunt
- Converts 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate (2,3 BG) via BPG mutase
- 2,3 BG = negative allosteric effector of O2 affinity, decreases oxygen affinity of hemoglobin, promoting its release in peripheral tissues; impt in high altitudes & cases of anemia - Glutathione System (Anti-oxidative system of RBC)
- Major defense against free radicals
- Oxidized glutathione reduces protective effects
- GSH is reduced glutathione
- GSSG is oxidized glutathione
What is the Warburg Effect?
Cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol.
Myoglobin and hemoglobin structural similarities?
- Both contain heme
Structure of myoglobin?
What happens when to structure when O2 is bound vs unbound?
- CO binds 25k times better than O2 so why doesn’t it completely displace O2 from heme iron in both myoglobin and hemoglobin?
- Structure of hemoglobin?
- Similarities and differences of myoglobin and hemoglobin?
- Cooperative vs hindered binding?
- Dissociation curves
- Discuss
1. Myoglobin is rich in alpha helix. Its surface is rich in polar and charged amino acid side chains. Globular.
- Apoproteins of the globins create a hindered environment for their gaseous ligands. When C-O binds to free heme, all 3 atoms lie perpendicular to the plane of the heme maximizing overlap between the lone pair of electrons on the sp hybridized carbon of the CO molecule and Fe2+ iron. BUT DISTAL HISTIDINE sterically precludes this high affinity (= reduces binding strength) and permits O2 to attain most favorable orientation. Since O2 madami amt, it dominates.
- Hemoglobin is tetrameric composed of pairs of two diff polypeptide subunits (a2b2, a2y2, a2bs2, a2o2). Primary structures of greek letter chains of human Hemoglobin are highly conserved.
- Myoglobin and beta polypeptide of Hemo A share almost identical 2’ and 3’ structures - location of heme and helical regions, presence of amino acids specifically properties at comparable regions.
Diff: Hb has 8 rather than 7 helical regions - Hindered binding: #4;
COOPERATIVE BINDING: Hb can bind up to 4 molecules of O2/tetramer; 1 per heme. Hemoglobin will bind O2 if other O2 are already bound permitting Hb to maximize both qty of O2 loaded at PO2 of lungs and qty of O2 released at PO2 of peripheral tissues; exclusive property of certain multimeric proteins - Myoglobin good for O2 storage but not transport because the oxygen-binding curve for myoglobin is HYPERBOLIC (It loads O2 readily but inefficient release at PO2).
Hemoglobin behaves as if it were 2 proteins. High PO2, high affinity for O2 (this form is referred to as R for relaxed state Hb). At lower PO2, Hb has lower affinity for O2 (aka T or taut state) enabling release in large proportions. Dynamic change between states = sigmoidal O2-binding curve.
Role of CO and O2 in relation to heme structure?
> CO2
15% CO2 are carried by Hb as carbamates. Carbate formation changes CHARGE on amino terminals (+ —> -). Change in charge favors salt bridge formation between alpha and beta chains. Remaining CO2 carries as bicarbonate.
In venous blood CO2 —hydration —> H2CO3 —dissoc —> HCO3- + H+
T-state Hb binds 2H+/tetramer. Protein binding helps buffer against blood acidification. It enhances qty of CO2 absorbed by RBC by favoring conversion of CO2 to carbonic acid to bicarb.
CO2, in turn, enhances O2 delivery to respiring tissues by stabilizing T state by inducing carbamation and lowering pH.
In lungs, process reverses.
*Bohr effect
What is hemoglobinopathy?
Discuss:
1. Methemoglobin
- HbM
- HbS
Abnormalities resulting from mutations in genes encoding the alpha or beta Hb subunits affecting its biological function.
Examples:
- Methemoglobin
- heme iron: Fe2+ -> Fe3+
- result: methemoglobin unable to bind or transport O2
- normally, Fe3+ is returned to Fe2+ by methemoglobin reductase
- causes: agents, low methemoglobin reductase activity, inheritance of mutated gene for Hb called HbM - HbM
- His F8 replaced by Tyr
- Fe of HbM forms a tight ionic complex with phenolate anion of Tyr stabilizing Fe3+ form
- In alpha chain HbM, R-T equilib favors T state (low O2 affinity and bohr effect absent)
- In beta chain HbM, switching - HbS
- Nonpolar aa valine —> polar surface residue Glu6 os the beta subunit
- effect: generating hydrophobic “sticky patch” on surface of beta subunit of both oxyHbS and deoxyHbS
- char: deoxyHbS polyermizes to form long, insoluble fibers; binding of deoxyHbA terminates fiber polymerization; since HbA lacks 2’ sticky patch; twisted helical fibers DISTORT ERYTHROCYTE INTO SICKLE SHAPE rendering it vulnerable to lysis
Biomedical implications
- Myoglobinuria
- Anemia
- Thalassemias
- Glycated Hemoglobin (HbA1c)
- Myoglobin may appear in urine
Scenario: Ff massive crush injury in skeletal muscle then renal damage. Mb can be detected in plasma ff a MI; enzyme assays more sensitive index of myocardial injury - Reduction in no. of RBC or Hb in blood
Causes: Impaired synthesis of Hb (lack of Fe) or erythrocytes (lack of folic acid/Vit B12) - Genetic defect resulting from partial or total absence of 1 or more alpha or beta chains of Hb
Common: alpha thal, beta thal mutations
Nomenclature: (alpha^0) = absent alpha chain; (alpha^-) = alpha chain synthesis reduced
Remedies: marrow transplantation + treatment - Glycation: Blood glucose enters erythrocytes and forms a covalent adduct with e-amino groups of lysyl residues and the N-terminal val of Hb beta chains
- not enzyme-catalyzed unlike glycosylation
- fraction of Hb glycated is proportionate to blood glucose concentration
- Measurement provides info for diabetes mellitus management
- represents mean blood glucose conc over preceding 6-8 weeks
Heme biosynthesis. Which of the enzymes are mitochondrial? Cytosolic?
8 steps!
Mitochondrial: Enzyme 1, 6-8
Cytosolic: 2-5
Where does heme biosynthesis occur?
Almost all mammalian cells except mature erythrocytes (no mitochondria). 85% heme made in erythroid progenitor in bone marrow and the majority of the remaining in hepatocytes.
Porphyrinogen vs Porphyrin
Porphyrinogen
-colorless
Porphyrin
- colored due to conjugate double bonds in the pyrrole rings linking methylene groups
- has a Soret band (sharp absorption band near 400 nm)
ALAS1 vs ALAS2?
ALAS1
- throughout body tissues
- reaction catalyzed by ALAS1 is rate-limiting for biosynthesis of heme in liver
- has a short half-life
- heme acts as a negative regulator of ALAS1 synthesis & also affects translation and translocation from cytosolic site of synthesis to mitochondrion.
ALAS2
-expressed exclusively in erythrocyte precursor cells
What happens in lead poisoning?
Lead poisoning
Pb inactivates Ferrochelatase and ALA dehydratase by combining with essential thiol groups.
Signs: Elevated levels of protoporphyrin in erythrocytes and elevated urinary levels of ALA and coproporphyrin.
Heme catabolism
- Globin -> degraded to constituent amino acids
- Release Fe -> Enters the iron pool
- Iron-Free portion of heme -> degraded mainly in reticuloendothelial cells of liver, spleen, bone marrow
-Heme catabolism from all heme proteins:
Location: Microsomal fraction of cell
Enzyme: Heme oxygenase
Rxn: Hemin + 3 O2 + 7e- —-> Biliverdin + CO + Fe3+
Biliverdin +NADPH ——biliverdin reductase—-> Bilirubin + NADP+
Observed as the purple color of heme conversion to yellow bilirubin by reticuloendothelial cells
- Neonatal “Physiologic Jaundice”
Defects of Bilirubin UDP-Glucuronosyltransferase:
- Gilbert Syndrome
- Type I Crigler-Najjar Syndrome
- Type II Crigler-Najjar Syndrome
- Toxic Hyperbilirubinemia
- Most common cause of conjugated hyperbilirubinemia
* Dubin-Johnson Syndrome - Urobilinogen and Bilirubin as Clinical Indicators
- Unconjugated hyperbilirubinemia
- results from accelerated hemolysis and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin
- Effect: Bilirubin-glucuronosyltransferase activity reduced
- UDP-glucuronate synthesis reduced
- If left untreated, bilirubin enter blood-brain barrier = hyperbilirubinemic toxic encelopathy or kernicterus - mental retardation
- Treatment: Phototherapy (Blue Light) and Phenobarbital administration - Harmless syndrome if 30% of bilirubin UDP-glucuronosyltransferase activity is retained
- Severe congential jaundice ( X> 20mg bilirubin/dL serum).
Symptoms: w/ Brenda = complete absence of hepatic UDP-glucuronosyltransferase; Fatal w/in 1st 15 months of life
Treatment: Phototherapy reduces plasma bilirubin but phenobarbital no effect - Some bilirubin UDP-glucuronosyltransferase activity is retained. More benign than type 1.
- Unconjugated hyperbilirubinemia can result from toxin-induced liver dysfunction caused by Cf, CCl4, etc.
Cause: Hepatic parenchymal cell damage impairing bilirubin conjugation
Treatment: Large doses of phenobarbital. - Most common cause: Blockage of hepatic or common bile ducts due to gallstone or cancer of the head of pancreas
Cause: Bilirubin diglucuronide cannot be excreted into hepatic veins and lymphatics
Manifestations: Pale blood, urine, and stool (Choluric jaundice)
Dubin-Johnson syndrome
- autosomal recessive consists of conjugated hyperbilirubinemia in childhood or adult life
- caused by mutations in gene encoding protein involved in secretion of conjugated bilirubin into bile
7.
Complete obstruction of bile duct
= bilirubin no access to intestine to be converted to urobilinogen
= no urobilinogen present in urine
= thus, w/ conjugated bilirubin but w/o urobilinogen = intrahepatic or posthepatic obstructive jaundice
Jaundice secondary to hemolysis
*Table
Function of Platelets? Low platelet count causes? What is thrombocytopenia?
Platelets - help staunch the outflow of blood from damaged tissues
Low platelet = increase patient’s vulnerability to hemorrhage
Thrombocytopenia - low platelet count in the case of anemia
What regulates HSC dx?
Cytokines (secreted glycoproteins)
How is the extraordinaty Hb conc achieved by mature erythrocye?
Devoids internal organelles including nucleus. Thus, can’t reproduce.
RBC shape importance?
(1) Disc-like configuration possess a much higher ratio of SA to Vol than spherical shapes
(2) Enables RBC to fold over and squeeze through capillaries whose diameter is smaller than the erythrocyte’s
How does glucose enter RBC?
Via facilitated diffusion mediated by GLUT1 (glucose transporter 1 or glucose permease)
T or F. GLUT1 is specific to L-hexoses.
False!! Glucose and related D-hexoses only!!
2.3 BPG generation importance?
1,3-BPG converted —2,3-BPG mutase–> 2,3 BPG
2,3-BPG binds to and stabilizes T-state of Hb
*2nd enzyme: Multiple inositol polyphosphate phosphatase converts to 2-bisphosphoglycerate = keep lvls of 2,3-BPG appropriately for oxygen transport
Carbonic anhydrase importance in RBC?
Impt for CO2 transport. Carbonic Anhydrase enables to absorb water CO2 by catalyzing its rapid conversion to carbonic acid and reverse process to release in lungs.
How is methemoglobin reduced in RBC?
-By the NADH-Cytochrome b5 methemoglobin reductase
What is hereditary hemochromatosis?
Genetic condition wherein body absorbs excessive amt of iron
Sources of superoxide?
(1) Autooxidation of hemoglobin to methemoglobin
(2) NADPH - hemoprotein reductase
- reduces Fe3+ in methemoglobin to Fe2+
Deficiency in G-6-P Dehydrogenase
RBC hypersensitive to oxidative stress and ROS-induced formation of Heinz bodies;
Vulnerable to attacks of hemolytic anemia
Hemolytic anemia causes?
Causes:
Extrinsic:
1. Hypersplenism wherein enlargement of spleen sequesters RBC
2. Incompatible Ab present (e.g. transfusion)
3. Rh disease
4. AID
5. Hemolysins (lytic factor) from bacteria
6. Parasitic infection e.g. /Malaria/
Intrinsic (Root cause of many cases): 1. G6P Dehydrogenase deficiency 2. Hemoglobinopathies 3. Insufficient enzyme pyruvate kinase 4. Mutations affecting cytoskeletal proteins (membrane-specific causes) 5. HEREDITARY SPHEROCYTOSIS 6. HEREDITARY ELLIPTOCYTOSIS 5 and 6 arise from abnormalities in amt or structure of spectrin; GPI synthesis
Band 3
>Characteristic?
>Function?
> Multipass TM
COOH at external; NH3 intracellular
> Anion exchange protein to provide a channel through the membrane where Cl- and HCO3- can be exchanged
> Amino terminal end also anchors several other RBC proteins (band 4.1 and 4.2, ankyrin, Hb, several glycolytic enzymes)
Glycophorins A, B, C
>Single-pass TM >alpha helical config >Most predominant: A —basis of MN blood group system >Absence of glycophorin A = no adverse effects
Spectrin
> Alpha and beta chains dimer intertwine in antiparallel orientation
Ankyrin
- Pyramid-shaped protein that binds spectrin
- binds tightly to band 3, securing attachment of spectrin to membrane
- sensitive to proteolysis
Actin and protein 4.1
- short, double-helical filaments of F-actin
- tail end of spectrin dimers binds to actin
- also binds to protein 4.1, a globular protein that binds tightly to a site near actin-binding domain in the tail of spectrin to form protein 4.1-spectrin-actin ternary complex; protein 4.1 also binds to glycophorin A and C and certain PPL to anchor complex to membrane
Hereditary spherocytosis
- autosomal dominant
- presence of spherocytes in peripheral blood by hemolytic anemia and splenomegaly
- spherical RBC are less deformable and prone to destruction in the spleen, shortening their life in circulation
- caused by deficiency in amount or abnormalities in structure of spectrin (mostly) or ankyrin/band 3, band 4.1 or 4.2 proteins
- weakening of link = swelling of erthyrocyte = spherical shape
- relieved by surgical removal of patient’s spleen (splenectomy)
Hereditary elliptocytosis
- affected RBC are elliptical
- results from genetic abnormalities that affect spectrin (mostly) or band 4.1 protein or glycophorin C.
Glucose transporter independent of insulin?
Glut-1 found in erythrocyte, blood-tissue barriers
Is HbF less or more sensitive to the effects of 2,3-BPG?
LESS SENSITIVE!!
HMP shunt end products? What happens to the other products?
End product: Ribulose-5-Phosphate & NADPH
Other products return to glycolysis
Function of glutathione system in RBC?
Reduced form helps protect RBC (needs NADPH)
Heme catabolism
(1) Extravascular destruction
(2) Intravascular destruction
Sites of Heme Biosynthesis
- 85% RBC
- 15 % Liver
- Mitochondrion/cytosol
Mutations causing:
1. Accumulation of heme and precursors
- Accumulation of porphyrinogen in skin & tissues
- Neuropsychiatric disorders
2. Spont ox -> Photosensitivity
