Hemoglobin Flashcards
1
Q
Hemoglobin structure?
A
- consists of 4 polypeptides (proteins), called globins
- two types of globins, alpha and beta
- each molecule has 2 alphabeta dimers (tetramer) in hemoglobin (held together by ionic bonds for flexibility between the dimers)
- has 4 heme prosthetic groups, prosthetic group is not a polypeptide, but forms part of a functional part of protein
- each globin chain binds on heme, and each heme can bind one molecule of O2
- globin chains within dimers held together by hydrophobic bonds
2
Q
Globin chains?
A
- heme binds in crevice between two helices of each globin
- 2 alpha types (zeta, alpha)
- 4 beta types (epsilon, gamma, delta, beta)
- developmental regulation controlled by differentiation specific transcription factors
- genes transcribed only in a narrow stage of development in 5-7 days from pro erythroblast to enucleation
- mRNAs very stable, translation continues after enucleation
3
Q
globin genes? (6)
A
- alpha genes on chromosome 16
- beta genes on chromosome 11
- combining them, different forms of hemoglobin
- Hb gower expressed in embryonic yolk sac
- Hb F expressed in fetus from first trimester until 6 months after birth
- Hb A2 found throughout life
- HbA normal adult form
4
Q
developmental regulation of globin chains? (7)
A
- alpha globin begins with zeta, then drops
- alpha picks up and is throughout life
- 6 months after birth, entirely beta chains
- different properties in each type
- F has higher affinity for O2 than A
5
Q
Hemoglobin F (Hb F, alpha2gamma2)?
A
- gamma globin not expressed after 6 months of age
- less than 1% total Hb in adults
- found in small number of RBC called F cells
- HbF has higher affinity for O2 than HbA
- therapy goals for sickle cell anemia when there is a problem with beta globin chain, is to increase number of F cells
6
Q
Hemoglobin A1c?
A
- under physiological conditions, Hb is slowly non enzymatically glycated (added glucose)
- extent of glycation depends on plasma concentration of glucose
- measured in diabetics (information about blood glucose levels over the 120 day lifespan of RBC)
7
Q
How many different types of globin chains are there?
A
-6 (2 alpha, 4 beta), each encoded by a different gene
8
Q
What is the major adult form of hemoglobin?
A
- has 2 alpha globin chains and 2 beta chains
- it is called HbA or HbA1 (alpha2beta2)
9
Q
Sickle cell disease as it relates to globin chains? therapy?
A
- there is a mutation in the beta globin chain
- goal of therapy is to increase number of F cells, which still express some HbF, because they have higher affinity for O2
10
Q
Heme structure?
A
- heme is a prosthetic group also found in other proteins including cytochromes (ETC, cytochrome p450), catalase, peroxidases
- largely planar molecule, consisting of tetrapyrrole ring (protoporyphyrin IX) with one ferrous ion (Fe 2+) in its center
- Fe 2+ does not fit perfectly in the plane, but puckers out to one side as it binds to the 4 N atoms of 4 pyrrole groups
11
Q
What is heme if not associated with protein?
A
- lipophilic pro oxidant
- tends to damage membranes, don’t want it to build in tissues
- pathways for synthesis are highly regulated so as to not have too much heme
12
Q
What are the conjugated double bonds responsible for on heme?
A
- color
- oxygenated is red
- deoxygenated is blue (cyanosis)
13
Q
Oxidation state of iron in heme?
A
- Fe 2+ can only bind O2 (protoporyphrin IX)
- if oxidized to 3+ form, cannot bind O2 (met-hemoglobin)
14
Q
How much heme binds each globin chain?
A
- one heme per globin chain
- 4 heme total
15
Q
What does iron bind to in heme?
A
- 4 N in porphyrin rings
- a histidine AA on globin protein
- free to also bind O2
16
Q
What happens under normal conditions to the ferrous ion?
A
- iron becomes oxygenated, it binds O2
- it is not oxidized
17
Q
Proximal histidine? (12)
A
-a bond is formed between the Fe2+ of heme and a histidine AA in the F helix of the globin chain
18
Q
Distal histidine? (12)
A
- histidine on the E helix is in close proximity helps to stabilize the interaction with O2
- also prevents oxidation of Fe2+ to Fe3+
- reduces Hb affinity for CO
19
Q
Sources of iron?
A
- Fe absorbed in small intestine as heme or free Fe
- absorption of free Fe is less efficient and affected by diet
- increased by Vit C, acidic pH
- decreased by tannates (tea), carbonates, phosphates - transfer of Fe from mucosal cell to capillary is regulated by Fe requirement
- Hepcidin produced by liver when Fe levels are high, inhibits transport from mucosal cells - Fe binds apotransferrin (transferrin after binding) in capillary, carried to Fe consuming tissues, especially bone marrow, liver
- no physiological path for excretion of Fe
- lost by bleeding, sloughed off cells, urine, feces
20
Q
Transferrin?
A
- two binding sites for Fe3+
- carries Fe in blood, delivers to cells by receptor mediated endocytosis
- presence of unbound sites protects against infection by Fe dependent pathogens
- level of saturation changes with Fe availability
- usually only 33% saturated
- TIBC (transferring iron binding capacity) increases in Fe deficient states
21
Q
Ferritin?
A
- 24 subunits (apoferritin) surround a core of 3-4000 Fe3+
- stores iron
- mostly in tissues, but small amount in blood, indicator of Fe reserves
- hemosiderin- microscopic aggregates of partially degraded ferritin (and iron) in lysosomes, increases in iron overload
22
Q
Regulation of iron absorption?(16)
A
- absorb iron in diet in form of heme iron or free Fe
- heme is transported into cell and heme oxygenase breaks it apart
- free iron is reduced to get through DMT1 by ferrireductase, it is then broken down
- there can be competition for DMT1 with other metals - iron leaves cell through ferroportin 1 where it is oxidized to Fe3+ by ferrioxidase
- some extra iron is stored in ferritin, can be mobilized if needed, or is shed with epithelial cells - it is picked up by transferrin and transported to bone marrow and liver
- regulation:
- increased by ascorbic acid, citric acid
- decreased by tannates (tea), carbonates, phosphates
- hepcidin is increased by Fe or IL-6 (inflammatory) to block ferroportin (anemia of chronic disease) so that iron does not leave cell into blood stream
- transfer of iron from mucosal cell to capillary bed is regulated by iron requirement and synthesis of storage protein (apoferritin)
23
Q
How are synthesis of ferritin and transferrin receptor regulated by Fe levels? (17)
A
- iron low: don’t need to make ferritin to store Fe, but need to make receptor to bring Fe into cell
- iron high: express more ferritin to store Fe, limit expression of receptor
- ferritin is regulated at translational level by iron response element (IRE) in the 5’ untranslated region, translation is inhibited when iron levels are low
- transferrin receptor mRNA has an IRE in 3’ untranslated region, when iron is high the mRNA is degraded quickly
- when iron is low, IRE is bound by IRE-BP and mRNA is stabilized so more receptor is made
24
Q
Where is the major site of absorption of iron?
A
in mucosal cells of the proximal duodenum
25
Q
What does ferrireductase do?
A
- reduces free Fe3+ to Fe2+ in intestinal mucosal cells so that it can enter DMT1
- reductase activity is increased by acidic pH and by reducing substances such as Vit C
- it is inhibited by tannates (tea), carbonates, phosphates
26
Q
Why can Fe have a difficult time entering the cells by DMT1 (divalent metal transporter)
A
-there is competition from other metals such as calcium
27
Q
Function of ferriportin?
A
- transports Fe2+ out of the mucosal cell
- since apotransferrin binds only Fe3+, ferriportin is coupled ferroxidase, hephaestin, ceruloplasmin
- ceruloplasmin is also a transport protein for copper in blood
28
Q
Function of hepcidin? what increases its synthesis?
A
- small circulating peptide, synthesized and released from the liver in response to increased intrahepatic levels of Fe
- inhibits transfer of Fe from the enterocyte to plasma by binding ferriportin and causing it to endocytosed and degraded
- hepcidin synthesis is increased by IL-6 (inflammatory), contributes to anemia of chronic diseases
29
Q
hematochromatosis?
A
- condition of iron overload in which there is abnormally increased iron absorption
- it is associated with decreased hepcidin activity
- iron accumulates in various organs and can lead to cirrhosis, liver tumors, diabetes, cardiac failure
30
Q
What is the most common cause of iron deficiency anemia in children?
A
- excess consumption of cows milk
- causes inflammation and damage to intestinal lining, leading to blood loss and decreased iron absorption
31
Q
Where does heme synthesis take place?
A
- most heme is made in bone marrow and liver, but some is made in all cells because all cells have cytochromes
- first reaction and last 3 take place in mitochondria
32
Q
substrates for heme synthesis?
A
- glycine AA
- succinyl CoA from TCA
- organic portion of heme is derived entirely from eight molecules each of the above
33
Q
Heme synthesis process? (20)
A
- succinyl CoA and glycine turn into ALA by ALA synthase in the mitochondria
- this is the rate limiting step - ALA converts to porphobilinogen (first of 4 rings) by ALA dehydratase
- inhibited by lead - goes on to make other intermediates to get protoporyphyrin IX
- Fe2+ is added to make heme by ferrochetolase
- ferrochetolase is inhibited by lead
- underlined reactions are in mitochondria, the rest is in cytosol
- the first reaction is inhibited by the product, heme (feedback inhibitor)
34
Q
ALA synthase reaction? (21)
A
- occurs in mitochondrial matrix
- first step, rate limiting
- substrates: glycine and succinyl CoA
- cofactor: pyridoxal phosphate (Vit B6)
- product: alpha aminolevulinic acid (ALA)
- inhibited by heme
35
Q
ALA dehydrates reaction? (21)
A
- occurs in cytosol
- second step
- product: porphobilinogen (PBG)
- inhibited by heavy metals such as lead
36
Q
Ferrochetolase reaction? (22)
A
- final reaction
- occurs in mitochondria
- catalyzes insertion of Fe2+ into protoporyphryin IX
- inhibited by heavy metals such as lead
38
Q
ALA synthase 1 (ALAS 1)? (24)
A
- one of two isoforms of ALA synthase
- found in liver and other tissues
- short half-life and more regulation
- major regulation by heme (negative feedback):
- inhibits transcription
- increases mRNA degradation (decreases stability of mRNA)
- blocks post transcriptional translocation of ALAS 1 to mitochondria
- induced by steroid hormones (oral contraceptives)
- inhibited by glucose or proximal glycolytic intermediate
- activity increased by certain drugs, especially those that are metabolized by the cytochrome p450 monooxygenase system
39
Q
ALA synthase 2 (ALAS 2)? (25)
A
- isoform of ALA synthase
- erythroid, bone marrow, makes 85% of daily heme
- makes heme primarily for hemoglobin, which has a long half life, so its response to heme levels is less regulated
- regulated in response to iron levels:
- IRE in 5’ UTR regulate translation, inhibited when iron levels are low
- heme (more leisurely) regulates synthesis indirectly by regulating acquisition of Fe from its transport protein transferrin
- may also inhibit translocation to mitochondria
- transcription regulated by erythroid specific promoter and same transcription factors that regulate globin synthesis
41
Q
Process of inhibition of translation of globins in erythrocytes by a kinase? (27)
A
- when heme is high, it inactivates a protein kinase, no phosphorylation of eIF-2-GDP
- translation is allowed to continue to synthesize globin chains - when heme is low, the protein kinase is active to phosphorylate eIF-2-GDP
- translation is inhibited, no need for globin chains
42
Q
Lead poisoning? (31)
A
- affects heme synthesis path
- lead inhibits ALA dehydratase and ferrochetolase
- anemia due to decreased Hb
- lead competes with Calcium:
- lead accumulates in bone and developing teeth
- neurotoxic effects primarily due to inhibition of neurotransmitters due to disruption of calcium homeostasis
- symptoms are age dependent (greater effect in children) and dose dependent
43
Q
Porphyrias? (31)
A
- genetic defects in heme synthesis
- rare but possibly under diagnosed
- symptoms are often intermittent and non specific
- classified as erythropoietic or hepatic
- most are dominantly inherited, but with low penetrance:
- 90% of people with genetic trait for acute intermittent porphyria never show symptoms
- precipitating factors may include:
- certain drugs (cytochrome p450)
- alcohol
- fasting/severe diet
- hormones
- stress
45
Q
porphyria cutanea tarda? (31)
A
- most common of porphyria
- dominant inheritance
- expressed mainly in patients with alcoholism or liver damage
- no neurologic or abdominal symptoms
- photosensitivity or pigmented urine
- treatment:
- abstain from alcohol
- reduce extra iron
- avoid sunlight
46
Q
Acute intermittent porphyria? (31)
A
- intermittent attacks:
- acute abdominal pain, constipation, muscle weakness, cardio abnormalities
- neurological dysfunction: agitation, seizures, psychosis
- colored urine and stool
- episodes last few days to several months
- often misdiagnosed
- inheritance is dominant, but low penetrance
- many have no symptoms except with precipitating factors:
- certain drugs
- fasting/dieting
- stress/infection
- anything that increases glucagon and decreases insulin
47
Q
conformational changes in Hb due to O2 binding? process? (36, 37, 38)
A
- oxygen binds to Fe2+ from opposite side of poryphryin plane
- iron atom, which had been puckered out from poryphryin plane, is pulled into the plane of heme on oxygenation
- proximal histidine residue of globin chain is pulled along with the iron overcoming steric hindrance and resulting in conformational change of hemoglobin molecule
- process:
1. deoxy state (T form): - Fe is puckered out from center of heme
- heme plane has slight dome shape
- F helix of globin chain is not perpendicular, but at slight angle
2. transition: - O2 binds Fe2+ from opposite plane from globin
- pulls Fe2+ down into center of plane, creating strain
3. oxygenated state (R form): - movement of globin chain relieves strain
- these changes are transmitted to the rest of the Hb molecule
- becomes perpendicular
49
Q
What happens to ionic bonds as Hb transitions from T to R form? (40)
A
-some ionic bonds are broken in the oxygenated state (R)
50
Q
T form of Hb? (41)
A
- tight, taut
- deoxygenated
- low affinity for O2
- lots of ionic bonds and H bonds between dimers that constrain the movement of globin chains
51
Q
R form of Hb? (41)
A
- relaxed
- oxygenated
- high affinity for O2
- fewer bonds between dimers
- binding O2 ruptures some ionic bonds between two dimers, allowing more freedom of movement
- transition from T to R form increases in likelihood as each heme group becomes oxygenated
- less steric hindrance
- affinity of Hb for O2 increases over 300x in R form
- O2 is allosteric activator of Hb binding of O2
52
Q
O2 dissociation curve? (42)
A
- myoglobin:
- one globin chain and one heme
- not allosterically regulated, not sigmoidal
- in muscle cells, holds iron until needed my exercising muscles
- fully saturated, normal curve
- hemoglobin:
- sigmoidal curve suggests allosteric regulation
- O2 is an allosteric activator
- allosteric inhibitors decreases Hb affinity for O2 in tissues:
1. 2, 3 BPG
2. H+
3. CO2 - none of the allosteric regulators has much effect in lungs, where O2 is really high to overcome inhibitors
- association of globin chains in tetrameric structure in Hb allows for greater O2 delivery than is possible with only one chain, as in myoglobin
54
Q
Why HbF has higher affinity for O2 than HbA? (44)
A
- HbF has alpha2gamma2 chains
- gamma globin chains have fewer positive charges in center pocket (no beta chains)
- HbF has very low affinity for 2, 3 BPG
- has to pick up O2 from mothers HbA, so must have higher affinity in tissues
- HbF curve more to the left, less sigmoidal
55
Q
2, 3 BPG and adaptation to altitude and hypoxia? (45)
A
- adaptation to high altitude is complex and takes several weeks to complete
- increased number of RBCs
- increased Hb concentration in RBC - within a day there is increased synthesis of 2, 3 BPG
- curve shifts to right
- helps Hb drop off O2 in tissues even when not fully saturated - 2, 3 BPG levels are all increased in conditions of hypoxia
- anemia
- cardiopulmonary insufficiency (COPD)
57
Q
Role of H+ in releasing O2? (47)
A
- as pH decreases (H+ increases), O2 affinity decreases
- H+ stabilizes the T form
- shift dissociation curve right
- little effect at high O2
- more lactate means more H+
58
Q
Role of CO2 in releasing O2? (48,49)
A
- 70-80% CO2 produced in aerobic metabolism is converted to bicarbonate in RBC by carbonic anhydrase
- carbonic acid spontaneously dissociates to bicarbonate and H+
- bicarbonate diffuses out of RBCs and helps buffer blood
- H+ helps to release O2 in tissues
- reverse reaction happens in lungs, CO2 is exhaled
CO2 + H2O H2CO3 HCO3- + H+
- 15-20% of CO2 is transported to the lungs on Hb as carbamino-Hb
- CO2 binds N terminal AA of globin chains
- this reaction produces H+ so binding of CO2 promotes release of O2 from Hb in tissues
- smaller amount of CO2 is transported to lungs as Carbamino-Hb
59
Q
RBC in capillaries of tissues vs lungs? (50)
A
- RBC:
- as CO2 binds N terminal AA of globin, releases H+ which help to release O2 to tissues
- lungs:
- O2 comes in and release H+, which releases CO2 exhalation
63
Q
Alpha thalassemia? (56)
A
- usually cause by deletion of 1 or more of the 4 alpha globin genes, severity depends on how many
- lack of alpha globins cause gamma or beta tetramers to form that don’t work as well
- one deleted = silent carrier
- two deleted= mild symptoms
1. Hb Barts syndrome - all 4 genes deleted
- Hydrops fetalis: abnormal accumulation of interstitial fluid in a fetus or newborn
- Hb Barts- only gamma chains in fetus (gamma 4), forms tetramer
- increased O2 affinity makes it a poor transporter
- usually fatal at or before birth
2. Hb H disease - 3 alpha genes deleted
- HbH is formed (4 beta globes)
- unstable tetramer found in patients with 3 alpha chains deleted
- moderate to severe disease
64
Q
Beta thalassemia? (57)
A
- 170 different mutations
- excess alpha chains precipitate, causes destruction of RBC (hemolysis) anemia
1. beta thalassemia minor - heterozygous
- usually asymptomatic except for mild anemia
2. beta thalassemia major (cooley anemia) - homozygous
- transfusions for severe anemia
- often suffer from iron overload
65
Q
beta thalassemia leading to iron overload? (58)
A
- reduced beta globin, excess alpha
- insoluble alpha globes make abnormal erythroblast
- most die in bone marrow (ineffective erythropoiesis)
- anemia increases iron absorption and iron overload
- few abnormal RBC leave in circulation, and destroyed by spleen
- blood transfusion helps anemia but increases iron overload
- tissue anoxia, marrow expansion, skeletal deformities
68
Q
CO poisoning? (61)
A
- acquired hemoglobinopathies
- most common fatal poisoning in US
- CO competes with O2 for binding to Hb
- CO increases O2 affinity for remaining sites and makes it impossible for Hb to drop off O2 in tissues
- levels of carboxyhemoglobin:
- normal less than 5%
- smoker up to 9%
- toxic over 25%
- fatal over 95%
- treatment: hyperbaric O2 chamber
71
Q
How heme is converted to bilirubin in macrophages? (65)
A
- begins in macrophages of reticuloendothelial (RES) system, with removal of globin chains and conversion of heme to bilirubin
- usually for RBC at end of lifespan
1. heme oxygenase - cleaves pyrrole ring to produce biliverdin (linear)
- requires O2, NADPH
- releases CO and Fe3+
- rate limiting step
2. Biliverdin reductase - requires NADPH
- reduced to bilirubin
3. bilirubin leaves macrophage into blood stream to be carried by albumin to liver cells - bilirubin not soluble in blood and must be carried
73
Q
Conjugation of Bilirubin (Br)? (67)
A
- unconjugated Br is carried on albumin to liver
- in hepatocytes, Br is bound by glutathione S transferase and is conjugated to 2 molecules of UDP-glucuronate by bilirubin glucuronyl transferase
- conjugated Br (Br diglucuronide) is actively transported by multi drug resistance protein 2 (MRP2) into bile
- now soluble in blood
75
Q
What happens to conjugated bilirubin? (68)
A
- excreted in bile into intestine
- it is then deconjugated by bacterial hydrolases and is reduced to urobilinogens
- most are oxidized to urobilins and excreted in feces - small amount is reabsorbed to the blood where it combines with albumin and is carried to the liver by enterohepatic circulation
- small amount can be reabsorbed by the terminal ileum and large intestine to blood and excreted in urine
76
Q
summary of bilirubin metabolism? (69)
A
pic
80
Q
Pre hepatic jaundice (hemolytic)? (73)
A
- indirect hyperbilirubinemia
- increased production of Br, exceeding capacity of liver to conjugate
- excess indirect bilirubin in serum
- increased urobilinogen in blood and urine due to increased conjugated Br reaching intestine
- rarely severe unless problem with liver because capacity of liver is high
- mainly unconjugated
81
Q
Intrahepatic (hepatocellular) jaundice? (74)
A
- impaired hepatic uptake, conjugation, secretion or Br
- direct or indirect hyperbilirubinemia
- indirect- getting bilirubin into hepatocyte, conjugation
- direct- problem getting git out of liver into bile
- conjugated bilirubin in urine
- liver enzymes often increased
83
Q
Neonatal jaundice? (75)
A
- common, usually self limited
- more common in premature infants
- Br glucuronyl transferase (conjugates Br) is developmentally expressed (adult levels reached 4 weeks after birth)
- heme doesnt need to be conjugated in utero due to using mothers O2
- the enzyme increases close to birth, Br increases at birth and then decreases
- premature infants enzyme increases slower leading to increases Br in blood at birth, could be toxic
- if Br levels exceed binding capacity of albumin, Br can accumulate in basal ganglia and cause toxic encephalopathy called kernicterus
84
Q
Other factors that contribute to neonatal jaundice? (76)
A
- ineffective conjugation
- increased RBC lysis immediately after birth, increased unconjugated Br
- albumin synthesis is low in premature infants
- if not feeding properly, albumin can breakdown into AA for energy - blood brain barrier more permeable in newborns
- kernicterus
87
Q
Post hepatic jaundice (obstructive)? (79)
A
- bile duct obstruction that prevents excretion of conjugated Br
- direct hyperbilirubinemia
- bile acids may also accumulate in plasma
- stool pale
- conjugated Br in urine, dark
- no urobilinogens in stool
- prolonged obstruction can lead to liver damage and increased unconjugated (indirect) Br
88
Q
Haptoglobin? (82)
A
- bind alphabeta dimers in blood, produces a complex too large to be filtered by renal glomerulus
- delivers dimers to macrophages via specific receptor
- serum haptoglobin concentration reflects degree of intravascular hemolysis
- levels decline with sustained hemolysis
- haptoglobin is an acute phase reactant, so levels may increase during inflammation or infection
- basically removes complexes in normal path to not clog kidney and lose iron in urine
89
Q
hemopexin? (82)
A
- free Hb not bound by haptoglobin is oxidized to metHb, which dissociates to globin and met-heme
- hemopexin binds met-heme, delivers it to the liver
- delivery of heme to hepatocytes induces heme oxygenase
- hemopexin and its receptor are recycled, but may be depleted with prolonged hemolysis
- basically removes complexes in normal path to not clog kidney and lose iron in urine
