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Blood, and its constituents
Colloid containing [cells: erythrocytes, wbc, platelets] [free proteins: albumin, ferritins, clotting enzymes, hormones], and electrolytes
- flexible, biconcave structure
- 95% of cellular protein is HB
- variety of transporters on membrane
- 35-50% hematocrit [ volume of RBC in blood]
- no cell organelles, thus no repair/translation of proteins
- only participates in glycolysis [no oxidative phosphorylation]
You find a higher than normal number of reticulocytes in the blood, how does this affect your hematocrit and your hemoglobin levels?
Hematocrit stays the same (as volume of RBC should remain the same), but hemoglobin levels should drop (as hemoglobin accumulation phase is rushed)
Compare/contrast biochemical properties of myoglobin/hemoglobin
- Myoglobin+Hemoglobin: both made up of high % of a-helices.
- Myoglobin: 1 amino acid chain, 1 heme group, hyperbolic saturation curve
- Hemoglobin: 4 a.a. chains, 4 heme groups, sigmoidal saturation curve (thus, co-operativity)
Biochemical properties of Heme
- aka Fe(II)-protoporphyrin IX
- cyclic tetrapyrole (the 4 nitrogens co-ordinating in the plane of heme are from these tetrapyrole groups)
- makes 6 co-ordinating bonds (4 to in plane nitrogens, 1 to HisF8, and 1 to O2)
Catabolism of Heme
- Fe-> reused (transferritins)
- Globin-> Broken down and reused by peptidases
- Heme-> Bilirubin
Lung, Muscle and Tissue pO2
p50 of Myoglobin
- Lung: 100 torr
- Tissues: 20 torr
- Working Muscle: 5 torr
- p50 Myoglobin: 2.8 torr
Binding of ligand at one site alters binding of ligands at a distal site
Hill Plot (axis and what they mean)
- Measure of co-operativity
- y vs. x= log (y/y-1) vs log pO2
- where y= (number of binding sites occupied/ total number of binding sites)
- Slope of 1= no co-operativity
- Slope>1= co-operativity
- 0<Slope<1= negative co-operativity.
- If the slope= the number of total binding sites, it insinuates instant co-operativity.
- Proposed the T-state(deoxy) and R-state(oxy) nature of hemoglobin.
- Iron binding O2 is pulled into the plane by 0.4 Angstroms, this shift is transmitted to distal co-ordinating bond of HisF8 (which in turn shifts 0.6A), causing a change in formation.
- As a result, HisE7 crowds around O2, stabilizing it
- In t-state, D94(aspartic acid) and H146 are together in space, thus this interaction needs to be overcome for the first O2 to bind (subsequent O2 doesn't need to break this interaction)
What are the roles of His146, HisE7, D94, HisF8 in the Perutz mechanism?
- His146 and D94 are together in space in T-state, which creates an interaction; needs to be overcome for O2 to bind
- HisF8 is the distal histidine which shifts 0.6A as a result of Iron being pulled into the plane 0.4 A when O2 binds
- HisE7 stabilizes bound oxygen as a result of the HisF8 conformational change.
- Hemoglobin's oxygen binding affinity is inversely correlated to both Carbon dioxide and acidity (i.e. lower pH= less O2 binding).
- Thus at the same pressure, more oxygen will be released with increasing acidity,or increasing [CO2]
Isohydric Transport vs Chloride Shift
Isohydric transport: CO2 converted to bicarbonate in order to more efficiently expel CO2 [80% of total CO2]
Chloride Shift: bicarbonate ion exchanged with chloride ions to maintain electrical neutrality across RBC membrane.
Would you expect more Chloride ions in venous or arterial red blood cells?
Venous, due to chloride shift.
BPG-general reaction from which it is synthesized and its effect on Hb
- Synthesized as a side rxn of glycolysis
- Decreases O2 affinity by stabilizing T-state(remember that T-state has a larger BPG binding pocket in the middle of Hb) through cross linking of B-chains (salt bridges)
- BPG is the first response to drastic changes in elevation (RBC counts will change over weeks, BPG can be synthesized to a saturation altering level in 2 days)
- Found in lungs and RBCs
- Catalyzes the rxn:
- H2O + CO2 <-> HCO3- + H+
A drug, known to alter glycolysis, is altering a patient's respiration. Why might this be?
2,3-BPG is a side reaction from glycolysis.
How does 2,3-BPG stabilize the T-state?
- BPG is an overall very negative molecule with 5 -ve charges; these charges bind electrostatically to the B-chains (as opposed to the A-chains)in HbA T-state.
- Electrostatic interactions take place at Lys82, His2, and His143
- One molecule of BPG binds per hemoglobin tetramer.
- Fetal HbF doesn't bind BPG as well (as it's composed of 2A and 2delta chains)
Products of Glycolysis
2 Pyruvate, 2NADH, 2ATP
Why use glycolysis in RBC
- 1. Supplies ATP
- -RBC have 30-40 mins of energy reserve of ATP; majority used for Na/K transport, and Ca/ATPase, and some used to maintain glycolysis
- 2. Supplies NADH for methemoglobin reductase (converts Hb3+ -> Hb2+)
Na+/K+ ATPase, what does it do, and inhibitors of it
- Pumps sodium out of cell, pumps potassium in to cell
- Can be inhibited by digitalis and ouabain
- Constantly uses energy to pump Calcium out of cell
- Increased intracellular calcium is indicative of cell membrane damage and aging of the cell (this aged cell is called an echinocyte)
- Aged RBC (loss of cell membrane integrity=increased intracellular calcium levels)
- Described as crenated, dehydrated cell which are no longer biconcave
- They are removed by the spleen (by the reticulo-endothelial cells/macrophages)
Uses of NADPH in RBCs
- GSH reductase
- GSH peroxidase
- Catalase (requiers 4 bound NADPHs to be active)
Arsenate (and fluoride)
Arsenate Inhibits net gain of ATP in glycolysis. (fluoride just inhibits glycolysis, not sure how)
Ribulose-5-phosphate: it's predecessor, and how it reforms compounds to re-enter glycolysis
6-phosphogluconate->ribulose-5-phosphate (produces a NADPH, and catalyzed by 6phosphogluconate dehydrogenase).
Can isomerize into xylulose-5-phosphate (ribulose 5 phosphate epimerase) and ribose-5-phosphate (ribose-5-phosphate isomerase); both of these products are precursors to fructose-6-phosphate and G3P
3 R-5-P make 2-F-6-P and 1 G3P
Draw a schematic of RBC development
Lecture 1 slide 6
Draw a schematic of Hemoglobin production during gestation time (label axes)
Lecture 1, slide 22
Draw the saturation curve of myoglobin, indicate p50, and p(lung, tissue, and working muscle)
Lecture 1, Slide 11
Explain the bohr effect using only diagrams, label axes
Lecture 1, slide 21
Draw saturation curve of hemoglobin, indicate p50.
Lecture 1, slide 13
Draw a diagram explaining isohydric transport
Lecture 1, slide 23
Draw the steps required to synthesize bpg
Lecture 1, slide 26
Using a diagram, explain how CO2, BPG, and CO2+BPG affect hemoglobin saturation curves.
Lecture 1, slide 31
Write the first 3 steps in glycolysis; indicate where a side reaction might take place leading to production of NADPH
Lecture 1, slide 39
Write out the steps involved in the monophosphate shunt starting from Glucose-6-phosphate and ending with Ribulose-5-phosphate
Explain (i.e. show the mechanism) how ribulose 5 phosphate can regenerate products that will feed back into glycolysis
Lecture 1, Slide 40
Drugs that induce hemolytic anemia in G6PD individuals
- Aspirin (not sure how)
- Dapsone (prototypical MetHb inducing drug)
- Methylene Blue (not sure how, but NADPH required to convert to leucomethylene blue)
- Primaquine (anti-malarial)
- Salicylates (not sure how)
Carbon monoxide, its mechanism of action, a clear-cut symptom of CO poisoning, its affinity for Hb vs its affinity for heme and why
- Outcompetes O2 for heme,
- victims turn bright red
- 25000X affinity for heme, 200X greater affinity for Hb (distal histidine HisE7 makes it harder for monoxide to bind)
Colouring of hemoglobins
- deoxy HB2+ : purple
- oxy HB2+: red
- metHB: brown(rusting)
How does HbFe3+ cuz hypoxia?
Oxygen doesn't bind to Fe3+, but causes a conformational change that binds the other 3 oxygens (to the remaining three Fe2+) in a irreversible fashion. Causes a leftward shift of the hemoglobin saturation curve.
What is an antidote for cyanide poisoning, and how does it work?
- Oxidation of Fe2+ to Fe3+ in hemoglobin (usually by Amyl+ Sodium nitrite).
- This causes cyanide to bind to the Fe3+ reversibly; as a result, cytochrome C in mitochondria are saved. Really a cost-benefit approach to therapy.
How is peroxide produced and detoxified in RBCs?
Produced by oxidation of Oxyhemoglobin to Methemoglobin
- Catalase (H2O2-> O2 + 2H2O)
- GSH Peroxidase (H2O2-> H20 by GSH-> GSSG)
- both of these rely on sufficient amounts of NADPH being available. If not hemolysis will occur.
Draw how peroxide is produced and detoxified in RBC
Unit 2, slide 4
Show how methylene blue could confer protection against methemoglobinemia
Lecture 2, Slide 4
Hemolysis, how it presents clinically, and in which population is it most apparent in?
Name some drugs that would induce hemolytic anemia
- Destruction of RBCs or their progenitors.
- Presents as black urine (due to hemolysis) and/or hypoxia (due to large numbers of methemoglobins)
- Frequently seen in G6PD defeciency.
- Basically any drugs that would create large amounts of ROS ex:
- aromatic amines, nitro compounds, hydrazines, antimalarial drugs, FAVA beans.
Fast Twitch vs Slow Twitch Muscle
Fast: rapid contraction for short duration; primarily anaerobic glycolysis, large glycogen content, small mitochondrial content, little myoglobin
Slow: slow contraction for long duration, primarily oxidative respiration, large mitochondrial content+ blood supply, large number of myoglobin (red meat)
- pretty rare (1/12000 children, 1/45000 adults)
- chromosome 19, ryanodine receptor
- anesthetic produces excessive Ca2+ release causing excessive heat+lactic acid leading to death by acidosis.
- Reversible if caught in the first couple minutes, administer dantrolene.
Write out oxidative free radical equations
Lecture 2 Slide 9
What's the role of hypochlorous acid?
Why is peroxynitrate dangerous?
Hypochlorous acid is found in WBC to create hydroxyl radicals to destroy pathogens in a generic way
Peroxynitrate has been implicated in inflammaation as well as atherosclerosis.
Show the path of transformations of oxygen radicals from most oxidized to least oxidized (i.e. most reduced)
Lecture 2, Slide 10
Glutathione (and how it protects from oxidative stress)
- Principal RBC anti-oxidant (concentrations in the millimolar)
- tripeptide of glutamate, cysteine, glycine (cysteine disulfide is the driving force)
- co-factor for many cellular enzymes
- most located in cytosol, some in the mitochondria (obviously not the case in RBCs)
- Detoxifies reactive drug metabolites
- Scavenges free radicals
- maintains cysteine in reduced form(thus allowing for a reservoir of free cysteines)
What is the role of Gamma-Glutamyl-Transpeptidase
Specifically cleaves G-glutamyl linkages, for example glutathione (G-glutamyl-cysteine-glycine)
What does y-glutamyl cysteine synthase do?
What inhibits y-glutamyl cysteine synthase?
links glutamate with cysteine via a g-glutamyl linkage, using ATP
buthionine sulfoxamine (used in chemotherapy)
Why purpose does the acetylation of a glutathione conjugate serve
It serves to make the final product more water soluble.
Give an overview of the role of glutathione in erythrocytes
Lecture 2, slide 16
Explain how acetaminophen is metabolized, and how the toxicity seen in overdose can be minimized by glutathione.
Also, what drug can be administered to promote glutathione conjugation of acetaminophen
- Lecture 2, slide 17
G6PD defeciency, how it's inherited, its prevalence, the different degrees of defeciency, and the cutoff for symptoms
Also, what reaction does G6PD catalyze?
- x-linked (females=heterozygotes[wild type+affected rbcs], males=affected)
- very common (~400 milli world wide)
- Type 1: <2% enzyme count
- Type 2: <10% enzyme count
- Type 3: 10-50% enzyme count
- Type 4: normal enzyme count
- 20% is the cutoff for symptoms (thus some type 3 won't exhibit symptoms)
Glucose-6-phosphate -> 6-phosphogluconate
What are the two amino acid substitutions we learned for G6PD defeciency?
- a) N126D (asparagine to aspartate)
- b) V68M (valine to methionine)
- these mutations occur 8Angstroms apart.
- If you just have a), you will get a reduction in the G6PDa to 85% [not clinically significant], if you have a) and b) then you 12% G6PD activity.
b) mutation increases protein rigidity, and affects Lys205, which is the active site of G-6-P binding.
What is the most serious and prevalent parasite implicated in malaria? How does malarial infection take place? What are the Hb aggregates called?
How does having G6PD defeciency increase your chances of malarial survival?
- Plasmodium Falciparum
- attacks RBC by using specialized food vacuole
- can consume 60-80% of Hb (using its own proteases) in RBC for its own protein synthesis(which will be seen as aggregates called Hemazoin)
- Normal RBC have low glucose utilization, and low lactate formation, but malaria parasite will instill its own hexokinase, and make large amounts of atp + lactate(30X more glycolysis than average RBC) (using up glucose) to create its own RNA/DNA/Protein
G6PD protects against oxidative stress by creating NADPH. In deficiency, increased oxidative stress due to large amounts of free iron & lack of reduced GSH will lead to hydroxyl radicals that will attack the malarial infection.
Show in a schematic how malarial infection can lead to lactic acidosis. Also explain how malarial parasites can use glycolysis to produce their own dna/rna/proteins
Lecture 2, slide 27.
Show why drugs that produce MetHb can induce hemolytic anemia in G6PD defecient individuals (especially drugs with anilline as a base)
Lecture 2, slide 29.
In a diagram, show how FAVISM can be caused by ingestion of Fava beans
Lecture 3, slide 1
Favism occurs because _____ produces supraoxide anions by _____ cycling. To detoxify the supraoxide anions an increase in ____ stores is required, which requires an increase in the production of ______
In what populations is sickle Cell disease found, and what is the cause (on a structural level) of sickle cell disease.
- Found mostly in blacks (10-25%), and some brown people (1%)
- Glutamate to valine substitution at position 6 of B-chain of Hb.
Why do HbS create fibrils?
Are HbS always sickled?
What is the quarternary structure of HbS
The valine substitution wants to avoid water, attracts other Hb molecules. These stacks keep growing until they hit the wall of the RBC, creating a sickled look.
Sickling is not apparant until times of oxygen debt (T-state)
14 stranded braid stabilized by multiple interactions
Why does Sickle-cell cause anemia
- Aggregation of the hemoglobin causes sickling. When passing through capillaries, sickled cells will lose K+.
- These sickled cells are removed from circulation more rapidly by the spleen.
How does sickle-cell disease confer an evolutionary advantage during malarial infection
Heterozygotes for sickle-cell disease have altered potassium content in their RBC
When RBCs pass through capillaries, they sickle and lose potassium, however malaria needs high potassium conditions to survive.
Sickling is triggered by _____. What is the implication of this (i.e. how can this be prevented).
How should sickle-cell be treated?
Sickling is triggered by conditions that prolong capillary transit (i.e. abnormal adherence to endothelium).
This implies that if capillary transit time, when HbS is in deoxygenated state, is less than fibril formation lag time, then sickling will not occur.
- No adequate treatment for primary disease.
- Can administer hydroxyurea, which promotes the production of HbF (promotes Gammaglobin synthesis), which doesnt stack as readily as HbA
- However secondary diseases can be prevented
- -vaso-occulusive crises/stroke-shown as cloudyness or decreased vascularity in the white of the eye
- -manage chronic pain symptoms
- -manage chronic anemia
- -prevention and treatment of primary infections [say diabetes+sickle will need more aggressive treatment]
Thallasemia, what it is, and what it can cause (very generally)
What are the two types of thallasemias
- Group of diseases resulting from genetic defects in synthesizing one type of Hb chain (either A or B)
- -can form A4 tetramers and B4 tetramers which have weird dissociation curves
- Can lead to ineffective erythropoiesis, hemolysis, and possibly anemia
- Alpha thallasemia- can't produce A-chains, thus excessive B and Gamma chains seen, causing a variety of abnormal HB (very similar to hydroxyurea + sickle cell Hb)
- Beta thallasemia- Can't produce B-chains, excessive formation of A4 tetramers, and HbF
Treatment for thallasemias
Can use splenectomy (reduces the number of RBC that will be destroyed) or allogeneic hematopoietic stem cell transplantation
Do not use transfusion therapy, as these rbc will eventually die too, leading to even more iron and free radical production (if it has been done, use chelation therapy to reduce the damage)
Major sources of iron in the body
- Blood (32 ug), Liver (13 ug), Bone marrow (4 ug)
- Note how approximately 60% of body iron is bound to heme.
What are Heinz bodies?
Inclusions within RBCs composed of denatured Hb. Can be seen in hemolytic anemia, or in RBCs subject to large amounts of oxidative stress
What does ferroreductase do?
Allows iron to be brought in past the brush border in Fe2+ state (without being oxidized/turned into something else). This reaction requires vitamin C. thus the role of vitamin C in facilitating Fe absorption.
What does heme oxygenase do?
Breaks down Heme. Found in proximal duodenum cells.
Heme -> Carbon + billiverdin + free iron
What does DcytB do? How does this differ from hephaestin?
Catalyzez the conversion of Fe3+ -> Fe2+ in proximal duodenum cells.
Hephaestin does the reverse, i.e. Fe2+ -> Fe3+
Explain in a schematic the process of Fe absorption. Then explain your schematic in words
- Lecture 3, slide 13
- 1. Iron/heme released from food, chelated by compounds that keep them soluble (Fe2+ state by ferroreductase)
- 2. Uptake occurs in proximal duodenum (but only about 10% of dietary Fe is absorbed)
- 3. Iron enters as either inorganic ion (Fe2+) or heme (which is broken down by heme oxygenase)
- 4. Iron either stored in ferritin (hephaestin converts to Fe3+) or exits cell via ferroportin transporter (after conversion by Hephaestin) and gets picked up by transferrin to distribute to tissues.
What is hepcidin and what does it do?
25 a.a. peptide hormone produced in liver that inhibits ferroportin transporter.
- Responds to acute phase infections.
- Trapped iron inside cells is liberated when cells are sloughed (remember that intestinal cells have a quick turnover rate)
Name instances where iron absorption will increase
Low dietary iron (this is the only one that will not decrease hepcidin levels)
- Low body iron
- increased RBC production (anemia)
- low hemoglobin
- low blood oxygen content.
How can you reduce iron absorption
Systemic inflammation(infection)-> increased hepcidin
Ferritin, general things about
- Very large protein (450,000MW; 24X175 a.a. chains)
- Holds 4500 irons/protein (FeOH3- note how the iron is in Fe3+ form)
- synthesized and secreted by the liver
- protects body from free iron toxicity
- 60% glycosylated(helps to maintain host-protein status and provides stability)
- Normal saturation is about 20%, iron overload~35%
- Iron is released by reduction, and chelation, and taken into cell by endocytosis
Alcoholism and its effects on iron transport
- alcoholism causes decreased transferrin-bound iron uptake.
- as a result, get an increase in ferritin receptors, and increased hepatocyte iron overload.
Transferrin, general things about
- MW 76000 (still big, but not as big as ferritin)
- 679 a.a. (2 mols Fe3+/mol Tf)
- 6% carbohydrate sugars decorate surface to increase longevity/decrease immunity (much like ferritin)
- Transports ~25mg/day (95% of blood plasma iron from catabolized RBCs)
- Transfer ~22mg/day to Hb
- Typical saturation is 20-25% (overload is ~33%)
- Taken up by hepatocytes, bone marrow, placenta via transferrin receptor.
Explain the workings of the transferrin receptor
- 1) Iron loaded transferrin binds to transferrin receptor (the carboxy side with carbohydrate moieties) causing conformational change due to energy of binding
- 2) Conformational change causes internal proteins(clathrin) (at amino-terminus ends) to coat inside of cell surface
- 3) Endocytosis occurs via invagination and a vesicle coated with clathrin is created
- 4) Coated vesicle proteins disassemble, creating endosome
- 5) Endosome pumps in protons(using ATP), which changes the nature of electrostatic interactions between transferrin and Iron, which releases the iron.
- 6) Free iron either gets picked up by proteins (ex. ferritin) and the receptors concentrate in an arm-like extension of the endosome and are returned to the cell surface.
The principal control of the transferrin receptor occurs at the level of ______
- Low cellular Fe: TfR mrna increases stability which leads to increase receptor proteins.
What is the biochemical composition of iron responsive elements?
and how does this control expression of proteins?
- Stem-loop structure in non-coding region of mRNA
- Unpaired C(in the stem) and C-A-G-U-G-C (is the entirety of the loop) sections are conserved.
Binding of these elements will alter translational efficiency (of ferritin and transferrin receptor-> controls level of iron stores and iron transporters)
Explain how Ferritin is regulated by iron responsive elements.
Low cellular iron causes IRP(rotein) to bind ferritin mRNA at the 5' end at the site of the IRE, preventing translation
High cellular iron causes release of IRP from stem-loop IRE because iron will bind the IRP instead (thus iron is directly involved in controlling its own levels)-> as a result, translation can proceed .
Important to note that transcription of genes is still the same, but only the translation of RNA is affected. Thus translational efficiency is affected.
Explain how translational efficiency is altered in transferrin receptor synthesis
For TfR mRNA, IRE is located on 3' end.
Increased free iron causes iron to bind IRP, exposing IRE+poly-a-tail to degradation, thereby decreasing translational efficiency
Decreased free iron causes IRP to bind the IRE at the 3' end, protecting the poly-a-tail from degradation, increasing mRNA half-life, thus increasing translational efficiency.
What are some types of causes for Iron deficiency?
- Direct blood loss (chronic/acute)
- RBC destruction
- -extrinsic: dietary/environmental/drug induced
- -intrinsic: genetic, HbS, thallasemia
Bone marrow abnormalities (hypochromic/mycrocytic anemia)
What are some causes of iron dietary deficiency?
Eating phytates (cereals, nuts, legumes, soybeans), polyphenols (tea, vegetables), Tannates (tea, coffee, veg)
Low meat intake
Lack of vitamin C
What would be the best way to diagnose an iron deficiency?
(Best way) If you find a low serum ferritin count (which is usually seen in acute/chronic inflammation), and high amounts of transferrin+transferrin receptor.
- (Additionally) Hb will be low, and there will be an increase in protoporphyrin levels in RBC (due to not finishing Hb molecules in the protein accumulation phase)
- There will also be decreased serum iron (but this is the least indicative of everything, as this can be low for a variety of reasons)
How does your body react in regards to iron regulation when faced with acute inflammation?
Ferritin levels increase (to lock up available iron), and transferrin levels decrease (to not allow iron into cells)
How does your body react in regards to iron regulation when faced with pure iron deficiency?
Ferritin levels are low (just not enough iron), and transferrin levels are high (trying to mobilize available iron)
How can one increase their dietary iron absorption?
Vitamin c(increase absorption of non-heme iron), Lactoferrin (whey proteins in milk/cheese, complex to non-heme Fe)
Why might you want to give parenteral iron preparations?
What are some problems with parenteral administrations?
Basically if you want to avoid the mucosal block of the gut. (IBD, ulcers, etc. )
May lead to iron overload, may cause allergic reaction
Name 4 drugs that complex with iron, thereby decreasing availability
- tetracycline, penicillamine, levodopa, quinoline
- (pretty sure this would happen in the gut, thereby decreasing absorption)
What are some causes of iron overload?
- Heriditary hemochromatosis (Recognizable as a bronz-y tan, along with a bronze glow in the eye)
- Induced hemochromatosis (giving transfusions when you see decreased RBCs, but they may have thallasemia or HbS so more RBC get destroyed, releasing free iron)
- Alcoholic cirrhosis, oral iron therapy,
Why is iron overload more common in children?
What are some symptoms?
How can it be treated?
Do not have sufficiently developed mucosal lining, thus cannot limit free iron absorption.
Victims will exhibit black stools, acidosis, convulsions, comas, and renal failure
Treated with chelation therapy (desferoxamine)
How does iron overload occur (in regards to saturation), and why does it have adverse effects?
- Occurs due to saturation of transferrin stores
- Free serum iron will accumulate in liver Kupffer cells, bone marrow, myocardium, pancreas (due to high levels of transferrin receptors) (this is why a lot of haemachromatosis patients will have diabetes/ liver cirrhosis/cardiac dysfunction)
What are some anti-iron therapies
- Desferoxamine (chelating agent) + ascorbic acid (vitamin c)
genetic disorder (Mutation in HFE gene, which codes for transferritin receptor -> increases absorption of iron)
More than 95% of genetically caused iron overloads are caused by this
There are also others which affect hepcidin, transferrin receptor, ferroportin (but again, the main cause will probably HFE gene-> transferrin receptor)
Explain how iron overload can cause cirrhosis/fibrosis
Lecture 3, page 39
Explain what the values will be when diagnosing various iron related ailments (Long-term iron deficiency; Hemochromatosis; Chronic illness; Hemolytic anemia; Acute Iron Poisoning)
Lecture 3, slide 40
Folic Acid Deficiency May Manifest as:
- Neural tube defects
- Premature atherosclerosis/inflammation
- Anemia, weight loss, weakness (in elderly)
- G.I., mucosa, and bone marrow complications
Some drugs can promote folic acid deficiency. Examples include:
- Oral contraceptives
- Alcohol (inhibits methionine adenosyl transferase)
- Methotrexate (inhibits dihydrofolate reductase)
- Nitrous oxide (inhibits methionine synthase)
Some genetic defects are associated with folic acid deficiency. These include:
- Hyperhomocysteinemia patients
- Ulcerative cholitis (Increased colon cancer)
What are the main components of a molecule of folic acid?
Pteridine (5N) moiety, PABA (10N), L-glutamic acid, Poly-glutamate side chain
The folyl/pterolyl moiety is the workhorse of the molecule (Pteridine+PABA)
What components of folic acid are not able to be synthesized by humans?
Pteridine, PABA, gamma-glutamyl linkage between PABA and L-glutamate
Explain in a schematic the absorption of THF
Lecture 4, page 7
Explain how methanol can be metabolized
Methanol is metabolized to either formalin (causes blindness) or formate (can be fed into THF derivative synthesis to create 10N-formyl-THF)
Draw a schematic of the creation of folic acid derivatives, include the denovo pathway
Lecture 4, slide 29
Why can folinic acid be used effectively as an antidote for methotrexate toxicity?
It can recreate folic acid precursors without the need for dihydrofolate reductase.
Why can alcoholism cause a decrease in methylation as well as GSH defeciency?
Alcoholism inhibits methionine adenosyl transferase, causing those downstream effects via remethylation cycle and homocysteine deficiency.
Also, some oxygen radicals can inhibit methionine adenosyl transferase as well (hypoxia, liver cirrhosis, septic shock)
Draw a schematic of the remethylation cycle, and show how alteration of this cycle can cause glutathione deficiency
lecture 4, page 16
Folate trap occurs as a result of ______
vitamin b12 deficiency, not allowing methionine synthase to convert homocysteine-> methionine.
What is a consequence of vitamin B6(pyridoxine) deficiency
How does increasing dna synthesis demand protect from methanol poisoning?
Increasing DNA synthesis requires an increase in Guanine and Adenosine. These are created when 5/10 formyl THF and 5,10-methenyl THF donate their 1 carbon groups. Since formyl THF requires formate for its creation, the equilibrium for methanol metabolism will favour its production (as opposed to toxic formalin).
How do the inhibition mechanisms differ between methotrexate/aminopterin and fluorouracil?
aminopterin/Methotrexate is a competitive inhibitor for DHFR.
fluorouracil is a suicide inhibitor for thymidylate synthase due to its electron withdrawing effects (fluorine)
What are some side-effects of methotrexate/aminopterin?
What can be given to reduce side-effects in methotrexate chemotherapy?
Myelosuppression (decreased marrow activity) and mucositis (inflammation of digestive tract)
How can methionine synthase be inhibited?
- Lack of folate
- Lack of homocysteine
- Lack of B12
- Nitrous oxide anesthetic
- Can be due to deficiency in B6, B12, B9
- Genetic deficiency (MTHFR,methionine synthase, cystathione b-synthase[autosomally recessive disorder with the hallmark of lens dislocation, cns and skeletal dysfunction])
How is CBS overactivity related to Down's syndrome?
CBS is located on chromosome 21, trisomied in Down's syndrome-> thus overexpression.
Increased levels of homocysteine, elevated cystathione and hypermethylation
General properties of Vitamin B12
Cobalt-containing compounds possessing a corrin ring (4 co-ordinating bonds- 4 to ring nitrogens, 1 to a non-ring nitrogen, and 1 to a variable group [-OH, -CN, -CH3, adenosine])
Required for 3 enzymatic reactions: methinone synthase, methylmalonyl CoA mutase, leucine aminomutase
what are they used for, and what is their parent molecule?
- Cyanocobalamin (B12 supplement)
- Hydroxocobalamin (cyanide+sulfide poisoning)
- Deoxyadenosylcobalamin (amino acid metabolism)
- methylcobalamin (for methylation [homocysteine-> methionine] )
Explain how vitamin B12 is absorbed
Where is the cause for most cases of B12 deficiency
Cobalophillin (secreted from salivary glands) binds available B12 in saliva, protects from stomach acid hydrolysis
This complex gets hydrolyzed in duodenum, and binds to intrinsic factor (a glycoprotein)
Only IF/B12 complex can be absorbed (neither can be absorbed individually) in distal third of ileum
Cause for deficiency is usually impaired complexing of B12/IF (either autoimmune or genetic-> both due to duodenal damage)
How is B12 transported?
- Transporter by transcobalamin 2 in blood
- Stored bound to transcobalamin 1 and 3
What does Vitamin B12 deficiency cause?
- Decreased carboxylate carrying capacity of mitochondria-> severe growth retardation
- Decreases THF levels -> decreased DNA synthesis-> pernicious anemia (pale look)
Pale, shiny tongue (lack of DNA synthesis effecting g.i. tract [glositis] )
this deficiency common in gastectomy patients (removal of duodenum/ileum)
Nitrous oxide (N2O) causes oxidation of cobalt(I) to cobalt (II) which will inhibit this enzyme.
Arises under conditions of B12 or IF defeciency.
Decreased B12 causes decreases folic acid (secondary effect), leading to impaired erythropoiesis, leading to premature release of erythrocytes (megaloblastic anemia-> can see nuclear material in RBCs)
Can be treated readily (within 72 hours) with B12 supplementation.
Draw a schematic of Methionine synthase and how it may need reactivation occasionally
Lecture 4, slide 25.
Draw the favoured pathway of homocystinuria toxicity
Lecture 4, slide 18
Show how Vitamin B12 deficiency can lead to growth retardation
Lecture 4, slide 24
Show how folate affects purine synthesis
Lecture 4, slide 16.
ABO blood type
Polysaccharide cell surface makers on RBCs
everyone has antigen H, modification to it will be A or B, unmodified results in O.
O=universal donor, can't except unless from O
Ex. Bloodtype A has antibodies to B; bloodtype O has antibodies to both A and B; bloodtype A/B doesnt have antibodies to A or B
- lack/lowering of granulocytes (especially neutrophils, and also eosinophils and basophils) in blood.
- 75% of cases are drug induced.
- ex. of drugs: Anti-inflammatory, Anti-thyroid/Thionamides, Cardiovascular drugs, Psychotropic drugs (TCA), Antibiotics, Dermatologic drugs (dapsone)
Signs and symptoms: often asymptomatic, may present with sudden fever, sore throat, septic shock. Higher chances of infection
- Direct toxicity: drug's reactive metabolite directly binds to neutrophils/their reserves in bone marrow
- Immune mediated: 4 different mechanisms
Treatment: Neutrophil recovery, like hematopoietic growth factors like G-CSF (granulocyte colony stimlating factor) and GMCSF (granulocyte macrophage colony stimulating factor)
- symptoms: headache, drowsiness, vomiting, bloody urine, changes in fingernail pigmentation
- can be inhaled/ingested and mainly distributes to the liver
- carcinogenic due to oxidative stress (inhibition of glutathione), inhibition of p53, genotoxicity, altered DNA repair and tumour promotion
arsenite, trivalent (As3+) reacts with thiols, and blocks GSH reductase-> increased free radicals; Inhibits pyruvate deoxygenase (decreased citric acid cycle activity)-> Decreased ATP and gluconeogenesis
Arsenate (pentavalent As5+): replaces phosphate groups (thus ADP-arsenate, rather than ATP; glucose-6-arsenate rather than G6P; g3-arsenate rather than G3P)
Can be treated with DMSA
Coagulation is the process of blood clot formation (aka thrombogenesis)
Warfarin: vitamin K antagonist; interferes with hepatic synthesis of coagulation factors
Heparin: indirect thrombin inhibitor; increases endogenous antithrombin activity
- Apixaban: direct factor Xa inhibitor
- Dabigatran: direct thrombin inhibitor
Glycogen storage diseases
Glycogen synthase extends glycogen chains (forms α -1,4 glycosidic bonds) (glycogenesis); glycogen phosphorylase breaks those bonds(glycogenolysis).
- type 0: deficiency in glycogen synthase in muscle/liver
- Type 1 (most common): glucose-6-phosphate or g-6-p transporter deficiency.
- Type 2: deficiency of the enzyme alpha-1,4-glucosidase in lysosomes
- type 3: inherited: buildup of abnormal glycogen due to deficiency in debranching enzyme
- Type4: inherited: buildup of abnormal glycogen due to deficiency in branching enzyme
- Type 5: inherited: inability to break down glycogen in muscle cells (deficiency in myophosphorylase enzyme)
- Type 6: deficiency in liver glycogen phosphorylase, causes liver enlargement due to buildup of glycogen
- Type 7: deficiency in phosphofructokinase specific to muscles.
Categorization and Actions of Leukocytes:
- Basophils = granulocytes that release chemicals to promote inflammation
- Eosinophils = granulocytes, cytotoxic killers that primarily target parasites
- Neutrophils = phagocytic granulocytes that ingest and kill bacteria
- Monocytes/macrophages = phagocytic antigen-presenting cells that ingest pathogens and activate helper T cells
- B-lymphocytes = antigen specific cells that have NOT come in contact with their antigen
- Plasma cells = activated B lymphocytes that have been in contact with their antigen and produces specific antibodies
- Memory B cells = inactive B lymphocytes that have been in contact with their antigen and remains in body in the event of secondary exposure to same antigen
- Helper T cells = lymphocytes that release cytokines to help activate B lymphocytes
- Cytotoxic T cells = lymphocytes that kill self cells carrying foreign antigens by inducing apoptosis and secreting digestive enzymes and perforin
- Natural killer cells = lymphocytes that target abnormal self cells and induces apoptosis
- -helps to reduce kidney rejection
- -Sirolimus acts on T and B cells of the immune system; Sirolimus binds with FKBP (FK506 binding protein) to form an immunosuppressive complex that inhibits the activity of the protein, mTOR (mammalian target of rapamycin)
By inhibiting mTOR, Sirolimus prevents protein synthesis, synthesis of cell cycle proteins, and translation of mRNAs required for cell growth and proliferation
Warburg effect describes the preferred metabolic pathway exhibited by cancerous cells oxidative glycolysis
Cells generate ATP through amplified rates of glycolysis and do not go through oxidative phosphorylation as primary pathway of generating ATP, leading to high lactate levels
Show in a schematic how glutathione is synthesized, then show how it can conjugate electrophiles
Lecture 2 slide 15