6 - Erythrocyte & Heme Biochemistry Flashcards
Majority of Hb synthesized before extrusion of nucleus from the normoblast to become a…
Reticulocyte
***Small amount made in reticulocyte
Describe the structure of adult Hb.
2 alpha-globin chains
2 beta-globin chains
1 Heme per subunit (chain)
Heme has an ________ atom and it carries O2. It is hydrophobic.
Iron (Ferrous: Fe2+)
There is a conformational change in Hb upon oxygen binding. The iron ion lies slightly outside the plane of porphyrin in heme. Upon oxygen binding, it moves into the plane of the heme. This 0.4 A change pulls down the proximal _________ of Hb and changes interaction with associated globin chain.
Histidine
What are the two types of curves for myoglobin and hemoglobin?
Hyperbolic curve = Myoglobin
Sigmoidal curve = Hemoglobin
Hb binds O2 in a __________ manner. The binding of one molecule of O2 to one heme facilitates the binding of an O2 to another heme.
Cooperative
The pH of actively respiring tissues is lower, it drops from 7.4 to 7.2. As pH decreases, binding affinity of Hb for O2 decreases. _________ picks up H+ from tissue. This changes conformation of Hb in a way that favors release of O2.
Histidine
This shifts the Oxygen Dissociation Curve to the right because it reduces the O2 affinity so Hb gives up more O2 to tissues. It provides a signal to Hb to let go of O2.
2,3-BPG
The drop in pO2 from _______ to ______ torr in exercising tissues corresponds to the steepest part of the Oxygen-binding curve. Consequently, Hb is very effective in providing Oxygen to exercising tissues.
40
20
The fetus needs Hb that has higher affinity for O2 than maternal Hb. O2 flows from mother to fetus. HbF does not bind well to _________, therefore it has a higher affinity for O2.
2,3-BPG
What is fetal and maternal Hb?
Fetal = HbF (2 alpha, 2 gamma) Maternal = HbA (2 alpha, 2 beta)
***The HbF has a left shift on the ODC because it has a higher affinity for oxygen!
Sickle cell anemia is due to a mutation in the Hemoglobin (HbS). There is a mutation at amino acid position 6 in Beta-globin. ________ ________ (negatively charged) changes to _________ (hydrophobic). This causes polymerization of Hemoglobin.
Glutamic Acid
Valine
Sickle shaped RBCs will impede circulation and cause hemolytic anemia. It can cause pain, organ damage, stroke, increased infections, etc. Research is ongoing to induce expression of HbF as a cure. Currently using __________ to induce HbF. It works but causes inflammation and is a toxic chemotherapeutic agent.
Hydroxyurea
Iron readily exchanges electrons, which makes it an ideal catalyst for oxidation-reduction reactions. Exists in Fe2+ (ferrous) or Fe3+ (ferric) state. Plays a role in ________ transport (is a component of Hemoglobin and Myoglobin).
Oxygen
Total body iron is about 3-5 g, most of it is in _______ (about 2.7 g). The average American male ingests 10-20 mg iron daily. We need _______ each day to support Hb production.
RBCs
25 mg
Too much _______ can promote cellular damage if not regulated by protein binding.
Iron
What is the distribution of iron in humans?
Hemoglobin = 67%
Myoglobin = 5%
Fe-Containing Proteins = 1%
Stored Iron = 27%
What are the Fe-Containing proteins in humans?
Cytochromes
Iron-sulfur Clusters
Enzymes using non-heme iron
Stored iron is in cells that line the intestines, liver, spleen, and bone marrow. The storage proteins are…
Ferritin – A protein that binds to ferric iron
Hemosiderin – Product of Ferritin breakdown
Heme iron (Fe2+ - from animal products) is easily absorbable. It enters the enterocyte, oxidized to Fe3+ by ___________, aka ___________.
Ferroxidase
Cerruloplasmin
The Fe3+ (from animal products) is stored in the form of ________ and its degradation product ________.
Ferritin
Hemosiderin
Non-Heme iron (Fe3+ - from plant products) is difficult to absorb. It is converted to Fe2+ by Ferric Reductase aka ________ in the presence of Vitamin C.
Dcytb (Duodenal Cyctochrome-Like B protein)
Fe2+ enters the enterocyte (from plant products) via _________ _________. After this, it is either converted to Fe3+ by __________ for storage or exported out of enterocyte by __________.
DMT1 (Divalent Transporter-1)
Ferroxidase (aka Cerruloplasmin)
Ferroportin
Ferroportin (exports Fe2+ out of enterocyte) requires ________ for its function. Ferroportin levels are regulated by ________.
Hephaestin
Hepcidin
Once in blood, Fe2+ is converted to Fe3+ by _________. Fe3+ gets bound to _________ for transport to target tissues.
Ferroxidase
Transferrin
Uptake of Transferrin occurs by receptor-mediated endocytosis via ________ ________. Once bound, it is internalized via clathrin-coated pits into endosomes. The low ______ of endosome releases Transferrin from its receptor.
Transferrin Receptor (TfR) pH
The uptake of iron in the __________ (where Heme is made) is very efficient. The endosome transiently docks on it and transfers iron directly via ________.
Mitochondria
DMT1
Iron content in body is regulated by modulating its absorption by the peptide hormone _________.
Hepcidin
Hepcidin is a 25 amino acid peptide made by the liver. It exerts its regulatory effect by binding to _________. The binding of Hepcidin to _________ causes its internalization and its subsequent degradation in lysosomes.
Ferroportin
Ferroportin
Levels of Hepcidin are controlled by a complex signaling pathway involving the protein Transferrin, its receptor and a protein called _______.
HFE (Human Homeostatic Iron Regulator Protein)
When iron is (HIGH/LOW) – Hepcidin expression is up, Ferroportin levels are down, and Iron absorption is low.
High
When iron is (HIGH/LOW) – Hepcidin expression is down, Ferroportin levels are up, and Iron absorption is high.
Low
If iron absorption is low, then there is Fe…
Deficiency
If iron absorption is high, then there is Fe…
Overloading
Iron deficiency can cause _________ _________ _________, and the treatment is dietary iron supplementation.
Hypochromic Microcytic Anemia
This is the increased absorption of iron which then accumulates in the heart, liver, and pancreas. It causes liver cirrhosis, hepatocellular carcinoma, diabetes, arthritis, and heart failure.
Hereditary Hemochromatosis (HH or Iron Overload)
Classical HH is autosomal recessive and is caused by mutations in the ________ protein. It causes a dysregulation of iron uptake and export by enterocytes (increased absorption). Treatment is blood letting and iron chelators.
HFE
***Remember HFE is important for Hepcidin, which is what causes the internalization of Ferroportin. Ferroportin is the transporter of iron out to the tissues.
What are the iron levels for HH?
15 g
***Normal is 3 to 5 g!
RBC production is dependent on ________ and ________. Deficiency of these can cause a severe anemia called Megaloblastic Anemia. This occurs due to diminished synthesis of _______ in developing RBC in bone marrow. Continual _______ synthesis is required for production of erythroblasts.
Folate (Folic Acid)
Vitamin B12 (Cobalamin)
DNA
DNA
This is caused by deficiency of Folate (Folic Acid) and Vitamin B12 (Cobalamin). It is characterized by large erythrocytes. There is normal Hb content in relation to size.
Megaloblastic Macrocytic Anemia
For Megaloblastic Macrocytic Anemia, the bone marrow shows large erythroblasts (called _________). There are also hyper-segmented ________ (more than 5 lobes – normally 3 to 4).
Megaloblasts
Neutrophils
Folic acid exists in a number of derivatives collectively known as folates. Folic acid has 3 parts, which are…
1) Pteridine – nitrogen containing ring
2) PABA (p-amino-benzoic acid ring)
3) Glutamate residue
Folate can be reduced to DHF by dihydrofolate reductase. It is then further reduced to ________ by dihydrofolate reductase. This is the active form.
THF (Tetrahydrofolate)
This is important for the synthesis of purines and the pyrimidine thymine. It serves a vital role in DNA synthesis.
THF
Folic acid is absorbed in the small intestine (jejunum). The liver stores 5 to 10 mg folate which can last 3 to 6 months. Most dietary sources have folate present in the ______ form. Once absorbed in the intestine, it’s reduced to ________. This is the primary circulating for in the bloodstream.
DHF
N5-Methyl-THF
This is an antineoplastic agent and inhibitor of DNA synthesis. It is a strong inhibitor of Dihydrofolate Reductase.
Methotrexate
If _______ is not available, then folate is stuck as N5-Methyl-THF. This is called the folate trap.
Vitamin B12
Vitamin B12 removes the ________ group from N5-Methyl-THF to make Methyl-Cobalamin (B12-CH3) and release THF.
Methyl
B12 is found in ________ products, and it can cause Megaloblastic Macrocytic Anemia because of its impact on DNA synthesis. 85% of B12 deficiency is from the lack of a protein called ________ ________.
Animal
Intrinsic Factor
Dietary B12 binds to ________ proteins. Proteases from the pancreas degrade R-binder proteins in the duodenum releasing B12. ________ ________ then carries B12 to the ileum from where it is released into the blood stream.
R-binder
Intrinsic Factor
Intrinsic Factor-Cobalamin complex is carried by ___________ in blood. The complex is taken up by cells via receptor-mediated endocytosis.
Transcobalamin II
Vitamin B12 deficiency can occur due to lack of Intrinsic Factor (necessary for B12 absorption). This is called __________ __________, which is a type of Megaloblastic Macrocytic Anemia. Condition results from failure to absorb Vitamin B12 resulting from unavailability of Intrinsic Factor (IF).
Pernicious Anemia
Pernicious Anemia can be tested via routine CBC. We test serum B12 and folate (folate deficiency vs. B12 deficiency). In a B12 deficiency, it is important to know how the deficiency occurred. We need to know if it’s lack of B12 in diet or a difficulty in absorption. What is the “gold standard” for testing this?
Schilling Test
Describe part one of the Schilling Test.
0.5-1.0 ug oral dose of 57Co-labeled B12. Also give injection of 1,000 ug unlabeled B12 cobalamin. This saturates all B12 cobalamin receptors in the liver to prevent radioactive B12 binding. If absorbed from the GI, it will pass into urine. Urine is collected for 24 hours, looking for presence of radioactive B12.
If present = Normal absorption of B12 and radioactivity shows in urine. B12 deficient diet is answer.
If absent = B12 not absorbed – Pernicious Anemia – Continue to Part 2
Describe part two of the Schilling Test.
0.5-1.0 ug oral dose of 57Co-labeled B12 plus Intrinsic Factor. Also give injection of 1,000 ug unlabeled B12 cobalamin. This saturates all B12 cobalamin receptors. Urine collected after 24 hours, looking for presence of radioactive B12.
If present = Pernicious Anemia due to lack of Intrinsic Factor
Briefly describe the composition of Hb and Heme.
Hb composed of 4 globular protein sub-units, each bound to an iron containing heme.
Heme has a heterocyclic porphyrin ring with iron present in the center. Porphyrin rings are four 5-membered rings.
In Heme, iron is present in the _________ state and oxidation to ________ states will inactivate Hemoglobin.
Ferrous
Ferric
Where does biosynthesis of Heme occur?
Primarily in liver and erythroid cells of bone marrow
What are the phases of Heme and where do they occur?
Phase I – in Mitochondria
Phase II – in Cytosol
Phase III – in Mitochondria
This phase of Heme synthesis occurs in the Mitochondria and is the synthesis of ALA from Glycine and Succinyl CoA.
Phase I
This phase of Heme synthesis occurs in the Cytosol and is the condensation of two delta-ALAs to form PB. Condensation of four PBs to assemble the tetrapyrrole ring system of coproporphyrinogen III.
Phase II
This phase of Heme synthesis occurs in the Mitochondria and consists of two oxidation reactions of coproporphyrinogen III to install the side-chain vinyl groups in protoporphyrinogen IX and generate the fully conjugated ring system of protoporhyrin IX. Insertion of Fe2+ by ferrochelatase gives heme.
Phase III
Defects in one or more stages of heme synthesis causes…
Porphyrias
For Phase I of Heme synthesis, ALA Synthase needs ______. A deficiency in this can cause diminished Heme synthesis, making RBCs pale. Iron stores are usually elevated (anemia).
B6 (Pyridoxal Phosphate)
In lead poisoning, lead inactivates 2 important enzymes in the heme synthesis pathway. These enzymes are…
ALA Dehydratase (contains Zn) Ferrochelatase (contains Fe)
In lead poisoning, both _______ and _________ accumulate.
ALA
Protoporphyrin IX
In lead poisoning, ALA is neurotoxic, which continues to neurological symptoms of lead poisoning. ALA resembles the neurotransmitter ________. Heme production is lowered. This causes anemia (microcytic and hypochromic). It also impacts _______ synthesis and energy metabolism because cytochromes (heme containing compounds) are not synthesized.
GABA
ATP
Porphyrias are inherited metabolic disorders. They are caused by defects in Heme synthesis. There are different types depending on the enzyme defect, which are…
Acute Hepatic – neurological symptoms
Erythropoietic – affect skin, photosensitivity
This type of porphyria is a defect in the enzyme PBG Deaminase (in liver). It is hepatic and has the following characteristics:
- Autosomal dominant
- Deficiency leads to excessive production of ALA and PBG
- Periodic attacks of abdominal pain and neurologic dysfunction
Acute Intermittent Porphyria
This type of porphyria is a defect in the enzyme Uroporphyrinogen III Synthase (in erythrocytes). It is erythropoietic and has the following characteristics:
- Autosomal recessive
- Deficiency leads to accumulation of Uroporphyrinogen I and its red-colored, air oxidation product Uroporphyrin I
- Photosensitivity; red color in urine and teeth; hemolytic anemia
Congenital Erythropoietic Porphyria
This type of porphyria is a defect in the enzyme Uroporphyrinogen Decarboxylase. It is hepatoerythropoietic and has the following characteristics:
- Autosomal dominant
- Deficiency leads to accumulation of Uroporphyrinogen III, which converts to Uropporphyrinogen I and its Uroporphyrin oxidation products.
- It is the most common porphyria in the United States; photosensitivity resulting in vesicles and bullae on skin of exposed area; wine red-colored urine
Porphyria Cutanea Tarda (PCT)
This type of porphyria is a defect in the enzyme Protoporphyrinogen IX Oxidase. It is hepatic and has the following characteristics:
- Autosomal dominant
- Photosensitivity and neurologic symptoms and developmental delay in children
Variegate Porphyria
This celebrity has Variegate Porphyria. He had intermittent episodes of abdominal pain, delirium, hallucinations, and convulsions. Medicines were given to him that contained arsenic which likely triggered these episodes. Became insane towards the last years of his life.
King George III
What system handles the degradation of Hemoglobin into Globin and Heme?
Reticulo-Endothelial System
Briefly describe the degradation of Heme into Bilirubin.
Heme Oxygenase removes bridge between pyrrole rings of Heme (requires oxygen) —
Carbon Monoxide released. Taken up by Hemoglobin —
Iron oxidized from ferrous to ferric —
Biliverdin (green pigment) synthesized and reduced to Bilirubin (red-orange) by Biliverdin Reductase (needs NADPH)
Bilirubin is released into the bloodstream. Free/unconjugated/indirect Bilirubin is insoluble. Therefore it is bound to ________. This complex is transported to the liver.
Albumin
Hepatic uptake of Bilirubin is mediated by a protein carrier. In hepatocyte microsomes, Bilirubin is conjugated with _______ _______, making it soluble. This is called conjugated/direct Bilirubin.
Glucuronic Acid
Briefly describe the conjugation of Bilirubin.
Glucose is activated by the enzymatic conversion to UDP-glucose —
Then converted to UDP-glucuronic acid —
UDP glucuronyl transferase enzymes in liver conjugate free Bilirubin with UDP-glucuronic acid —
This makes Bilirubin-Monoglucuronide and then Diglucuronide
What is the rate-limiting step of Bilirubin conjugation?
UDP glucuronyl transferase
***Remember, this takes free Bilirubin with UDP-glucuronic acid and makes Bilirubin-Monoglucuronide and then Diglucuronide
Conjugated Bilirubin is secreted by hepatocyte to biliary canaliculi which connect to form bile duct, which empties into the…
Gall Bladder
Bilirubin-Diglucuronide is released into the gall bladder as ________. This is then secreted into the small intestine in response to food. In the intestines it undergoes microbial reduction to __________.
Bile
Urobilinogen
Some Urobilinogen is reabsorbed and processed by the kidneys to produce the yellow pigment _______, found in urine. Some Urobilinogen undergoes further microbial reduction to the red-brown pigment _________, found in the feces.
Urobilin
Stercobilin
This is caused by elevated levels of Bilirubin in blood stream. There is an imbalance between production and excretion of Bilirubin.
Jaundice
What are the types of jaundice?
Pre-hepatic
Intra-hepatic
Post-hepatic
This type of jaundice is due to increased production of unconjugated Bilirubin. There are elevated blood levels of unconjugated or indirect Bilirubin (normal blood levels of conjugated Bilirubin).
Pre-hepatic
This type of jaundice is due to impaired hepatic uptake, conjugation, or secretion of conjugated Bilirubin. There is generalized hepatic dysfunction.
Intra-hepatic
Examples of this type of jaundice are due to liver cirrhosis (alcoholism), viral hepatitis, Criggler-Najjar Syndrome, and Gilbert Syndrome.
Intra-hepatic
T/F. For Intra-hepatic jaundice there are variable increases in unconjugated and conjugated Bilirubin depending on the cause (pre- or post-conjugation). There is an increase in serum ALT and AST.
True
This type of jaundice is due to problems with Bilirubin excretion. There is decreased bile flow. There can be dark urine or pale stool.
Post-hepatic
In this type of jaundice there is elevated blood levels of conjugated Bilirubin. The conjugated Bilirubin is present in urine (dark).
Post-hepatic
Many newborns develop jaundice due to elevation of unconjugated Bilirubin. This is called physiological jaundice. The immature hepatic metabolic pathways are unable to conjugate and excrete Bilirubin. There is a deficiency of the enzyme __________. There is a breakdown of fetal Hb as it is replaced with adult Hb. Accumulation of excess Bilirubin in blood leads to the symptoms of jaundice. Prematurity aggravates this.
UDP-GT
In neonatal jaundice, Bilirubin may diffuse into basal ganglia and cause encephalopathy. This is called…
Kernicturus
What are treatments for neonatal jaundice?
– Phototherapy with blue fluorescent light
– Intramuscular injection of tin-mesoporphyrin (strong inhibitor of heme oxygenate) to prevent heme breakdown of Bilirubin
This type of UDP-GT disorder results from a deficiency of the enzyme. This is Type I, meaning there is complete absence of the gene to produce it. Results in severe hyperbilirubinemia. Causes encephalopathy (kernicturus) and brain damage.
Criggler-Najjar Syndrome
This is the benign form, which results from a mutation in the UDP-GT gene. The enzyme has less activity at about 10% (not completely absent).
Criggler-Najjar Syndrome Type II
This UDP-GT disorder is relatively common. It is a benign disorder affecting 2-10% of population. Results from reduced activity of UDP-GT at about 25%. Serum Bilirubin is <6 mg/dL but may increase with fasting, stress, or alcohol consumption.
Gilbert Syndrome
This is inflammation of the liver and leads to liver dysfunction. Its causes are from viral infections, alcoholic cirrhosis, and liver cancer. It causes increased levels of unconjugated and conjugated Bilirubin in the blood.
Hepatits
Hepatitis causes accumulation of Bilirubin in the _______ and ________ of the eyes, causing yellow discoloration. There is dark, tea colored urine.
Skin
Sclera
Describe the colors of bruises based on Hemoglobin breakdown.
Heme – Red
Biliverdin – Green
Bilirubin – Orange/Yellow
Hemosiderin – Reddish-Brown
