Carriage of Oxygen in blood

Question 1

Important concepts-oxygen

Answer 1

• Vast majority of oxygen in blood carried on haemoglobin (Hb: 98% of total oxygen)
• Need carrier molecule (oxygen poorly soluble in water and blood is mostly water)
• Hb can carry up to 4 oxygen molecules
• Haemglobin saturation: percentage of total Hb binding sites available for oxygen binding that are occupied by oxygen
• If alveolar oxygen is increased (i.e. person given supplemental oxygen) once all Hb molecules fully saturated (100% saturation) - only dissolved oxygen levelsl can increase
• There is small amount oxygen dissolved in plasma (1-2% of total oxygen)- part of blood with no cells
• Amount oxygen arterial blood determined by amount oxygen in lung alveoli

Question 2

Important concepts-carbon dioxide

Answer 2

• Contrasted with oxygen, carbon dioxide (CO2) is soluble in water- so soluble in blood plasma
• CO2 in blood exists in 3 forms:
  a. Dissolved CO2: 10%
  b. CO2 reacted with water to form bicarbonate (HCO3-): 60%
  c. CO2 bound to haemoglobin (at different site from oxygen): 30%
• Amount dissolved CO2 arterial blood plasma determined by amount CO2 in lung alveoli-equilibrium
• The majority of CO2 in blood is not transported by a carrier. This means that the more CO2 produced by tissues the more can be delivered to lungs to be exhaled
• CO2 is not just a waste product but is also used in the buffer system

Question 3

What is the function of the cardiovascular system?

Answer 3

1. Supply oxygen and metabolic fuel (e.g glucose) to tissues and remove product of metabolism (e.g CO2)
2. Maintain defenses against invading micro-organisms
3. Deliver hormones&nutrients
   - Carriage of oxygen (O2) is problem because oxygen is powerful oxidising agent
   - Most organic molecules are damaged by too high concentration of O2
   - Erythrocytes are specially designed to carry this dangerous cargo

Question 4

What is oxdiation?

Answer 4

Oxidation = loss electrons
Oxidising agents combine with other atoms or molecules and remove electrons leaving oxidised molecule
This process releases energy

Question 5

What is reduction?

Answer 5

Reduction=gain electrons
Reducing agents protect against electron loss by donating electrons back
This process requires energy

Question 6

Describe the haemoglobin

Answer 6

1. Oxygen carrier must be able to bind to oxygen reversibly
2. Haemoglobin is unique molecule-can combine rapidly and reversibly with oxygen without becoming permanently oxidised
3. RBCs biconcave disks-diameter: 7um, thickness: 2um, volume of 90femtolitres
4. RBC contains 270 million molecules, weighing 30 picograms (mean corpuscular haemoglobin or MCH)
5. Sizes RBCs can vary in several common illness: smaller-microcytic anaemia, larger-macrocytic anaemia

Question 7

Haemoglobin equation

Answer 7

Low level haemoglobin: anaemia
High level of haemoglobin: polycythaemia

MCHC=MCH/volume
= mean corpuscular haemoglobin concentration/volume of RBC

Haemoglobin concentration=measure amount of haemoglobin per litre x whole blood

Question 8

Haematocrit-the blood test

Answer 8

1. Ratio of the volume of RBCs to the total volume of blood- can be presented as ratio or percent
2. The normal ranges depend on age and beginning in adolescence, sex of person
3. Normal range for adults:
   Adult cis-males: 0.40-0.52
   Adult cis-women: 0.37-0.45

Question 9

What are reticulocytes?

Answer 9

1. Reticulocytes: immature RBCs just before and after leaving bone marrow
2. 1-2% of circulating red blood cells in healthy people (but larger proprotion after haemorrhage)
3. Reticulocytes change into mature RBCs ~1 day after entering circulation. Able to carry oxygen but not as efficiently as mature RBCs
4. Called reticulocytes because of reticular (mesh-like) network of ribosomal RNA visible with methylene blue stain. Ribsomes enable reticulocytes to complete production of haemoglobin

Question 10

Describe RBC metabolism&lifespan

Answer 10

1. Mature RBCs have no nuclei or mitochondria
2. Because of lack of nuclei and organelles, RBCs cannot divide or repair themselves- survive about 120 days
3. Because no mitochondria RBCs cannot get energy by oxidative metabolism. But require ATP to maintain sodium pumps in cell membranes and for other ion pumping operations
4. RBCs produce ATP by glycolysis: (conversion of glucose to pyruvate followed by conversion of pyruvate to lactic aci-less efficient than aerobic metabolism)
5. RBC glucose uptake is mediated by Glut1 transporters. Glut 1 works by facilitated diffusion and not regulated by insulin

Question 11

Describe the anti-oxidants of RBC

Answer 11

1. Due to high pO2 in RBCs, NAD+ spontaneously formed from NADH (O2 took electron-oxidation)
2. RBCs then use enzyme convert NAD+ to NADPH
3. Formation of NADPH counteracts oxidative stress in RBCs. NADPH required for enzyme glutathione reductase which is required to maintain adequate cellular levels glutathione-key anti-oxidants
4. RBCs also have Vitamin C-another anti-oxidant

Question 12

Describe the role of G6PD (glucose 6 phosphate dehydrogenase)

Answer 12

Glutathione reductase requires NADPH, not NADP+ to work
- In cells throughout body there are multiplie pathways to regenerate NADPH
- But, in RBCs enzyme G6PD is key enzyme that regenerates NADPH from NADP+
- G6PD deficincy most common enzyme deficiency in world

Question 13

Describe the strucutre of human haemoglobin.

Answer 13

1. Human haemoglobin made up of four polypeptide subunits (globin chains), each with haem prosthetic group attached
2. Four subunits bound to each other by salt bridges, hydrogen bond, and hydrophobic interactions
3. Amino acid sequence of each subunit, and exact way subunits fit together, vital for porper haemoglobin function

Question 14

Describe the structure of Haem.

Answer 14

1. Porphyrin ring: large ring molecule consisting of 4 pyrroles (smaller rings made from 4 carbons and 1 nitrogen)
2. Haem: porphyrin ring with iron atom bound
3. Haem proteins: several enzymes contain haem
4. Involved in chemical reactions requiring transfer of electron-oxidation or reduction

Question 15

How does haem enable oxidation-reduction reactions?

Answer 15

1. Histidine group underneath porphyrin ring binds one iron electron of a pair
2. This leaves other one iron electron sticking up out of plane which can react with other molecules
3. Electron-hungry oxygen molecule sees single unpaired electron sticking out haem and forms weak reversible bond
4. Bond weak as oxygen molecule cannot get close enough to iron to fully remove electron due to steric hindrance from other parts of haemoglobin molecule

Question 16

What is sterin hindrance in Hb?
How does it affect in high and low partial pressure of O2?

Answer 16

1. Hard for first O2 molecule to bind to haemoglobin so it needs high partial pressure O2 in lungs, forming oxyhaemoglobin
2. Binding of the first O2 to Hb alters teric hindrance-> easier for other O2 molecules to bind until all 4 haem irons occupied
3. Low partial pressure O2 environment (e.g tissue): O2 diffuses off haemoglobin and as each O2 molecule dissociates, it makes it harder for the remaining O2 to remain on the haemoglobin or new O2 to attach -> leaving with deoxyhaemglobin
4. High partial pressure O2 environment (with steric hindrance not quite right)=oxygen DOES oxidse iron to ferric state Fe+3

Question 17

Methaemoglobin

Definition
How it forms
Why its bad
How it can be solved
Condition

Answer 17

Meaning: haemoglobin with ferric iron
How it forms: steric hindrance allowing reversible bonding oxygen to haem iron but not oxidation iron finely balanced
1. No longer has spare one electron to attract O2 -> so cannot pick up or transport O2
2. Newly released RBCs methaemoglobin converted back to haemoglobin by methaemoglobin reductase (RBC NADH- dependent enzyme)
3. Cells age -> enzyme level decreases -> methaemoglobin increases
Condition: Methaemoglobinemia
1. Caused by G6PD deficiency when there is increased oxidative stress, exposure to various chemicals
2. High percentage genetic deficiency methaemoglobin reductase (rare)
3. Healthy amount: 1-2% in the body

Question 18

RBC damage & destruction

Answer 18

1. RBCs progressively damaged by oxygen
2. RBC senescence: rise RBC methaemoblogin -> causes marker to change on RBC membrane -> change detected by cells in liver and spleen which remove RBCs
3. As aging RBCS undergo changes (e.g becomes more spherical) in plasma membrane -> suscpetible to recognition by phagocytes and subsequent phagocytosis in spleen, liver and bone marrow

Question 19

Haemoglobin saturation -SaO2

What is SaO2: definition, units
How it measured and what instrument is u

Answer 19

SaO2 (%Hb saturation): percentage of haemoglobin that is carrying oxygen
- basically how many RBCs carry oxygen
1. Arterial SaO2 can be measured with pulse oximeter
2. Pulse oximeter detects difference in absorption of light between oxygenated and deoxygenated Hb
Advantages
1. Non-invasive
2. Portable
3. Relatively inexpensive
4. Relatively accurate
5. Widely used
Disadvantages
1. Only detects pulsatile arterial blood levels
2. Can’t detect non-pulsatile venous blood or tissue oxygen levels
3. Less accurate in darker skin

Question 20

What is the normal and abnormal range of haemoglobin saturation?

Answer 20

Healthy individuals at sea level: haemoglobin oxygen saturation 96-99%, >94%
hypoxaemia (<90%): low partial pressure oxygen in arterial blood
- when there is not enough in tissue

Question 21

Describe the oxygen/haemoglobin (Hb) dissociation curve

Definition
Saturation
What does it tell us (4 marks)
Reasons for S shape (3 marks)
Describe the graph

Answer 21

Definition: graph showing how changes in partial pressure of oxygen change Hb saturation
Saturation: & of Hb that has bound oxygen against 100% saturation, independent of Hb concentration
O2 content: mmol/L
e.g if 1 Hb molecule in your body but it was carrying 4 oxygen molecules, it would be 100% saturated
Haemoglobin satruation itself therefore doesn’t tell us how much haemoglobin is in blood

What does it tells us?
1. How easy/difficult it is to saturate/desaturate haemoglobin depending on part of curve
2. How much O2 will be bound or given up when moving from one partial pressure to another
3. Work out difference in percentage saturations between two pO2 values
4. Work out the effects of changed conditios on how easily haemoglobin binds or releases oxygen

Reason for S shape:
1. Harder for first oxygen to get on
2. Easier for the next oxygen one wards
3. Steep line: all 4 haem groups are occupied so increase in pressure doesn’t affect the saturation anymore

Describe the graph:
1. Steep middle part of curve: Hb releases large amounts of oxygen for a small decrease in pO2 in respiring tissues
2. straight line at the end of the curve: saturation maintained high even as pO2 falls
- wide range of safe partial pressure oxygen: especially at rest

Question 22

Molecular basis of haemoglobin cooperativity

Answer 22

Molecular re-arrrangement of haem group so that ironir more accessible to oxygen
Decreased steric hindrance as more O2 bound to haemoglobin (high pO2 environment (e.g lungs)
Incrased steric hindrance as less O2 bound to haemoglobin (low pO2 environment (e.g tissues)

Question 23

When does the oxygen/haemoglobin (Hb) dissociation curve can be shifted?

Factors that shift the curve to left (4 marks) and right (4 marks)
Explanation (3 points)

Answer 23

Left shift:
1. Decreased temperature
2. Decreased 2,3-DPG
3. Decreased H+
4. CO
Right shift:
1. reduced affinity
2. Increased temperature
3. Increased 2,3-DPG
4. Increased H+ (lower pH)
5. Higher oxygen partial pressure needed for 50% saturation

Explanations:
1. Bohr effect: Acid conditions (right) facilitates O2 unloading in tissues. metabolically active tissues-> lower pH
2. Temperature effect: higher temperature (right) facillitates O2 unloading in tissues. metabolically active tissue-> higher temperature
3. 2,3 DPG effect: 2,3-DPG intermediate of RBC glycolysis normally rapidly consumed but in hypoxaemia (low oxygen) RBC production of 2,3 DPG increases (right) facilitates O2 unloading tissues

Question 24

What is the myoglobin (Mb)?

Definition
Affinity compared to Hb
Where is O2 transferred from
Special function
Where is it usually not found?

Answer 24

Definition: Mb form of haemoglobin found in muscle
- single subunit protein with higher affinity for oxygen than haemoglobin (Hb)
- O2 is transferred to MbO2 from Hb as blood passses through muscle capillaries
- Mb forms a buffer store of oxygen in muscle (important in start of exercise)
- It isn’t usually found in smooth muscle

Question 25

Describe myoglobin/oxymyoglobin dissociation curve

Describe Mb dissociation durve
What happens when myoglobin oxidised and how reduction happens
Why is high concentrations of myoglobin sometimes helpful
What is rhabdomyolysis?
What is ATN?

Answer 25

1. Mb dissociation curve is hyperbolic. As myoglobin is a single subunit, steric hindrance is fixed
2. Myoglobin slowly becomes oxidised to metmyoglobin (iron gets oxidsed to Fe3+) but metmyoglobin reductase enzymes in muscle reduce it back to myoglobin
3. High concentrations of myoglobin muscle cells allow organisms to hold their breath for longer. (e.g diving mammals (whales and seals) have muscles with particularly high abundance of myoglobin)

Clincial application
Rhabdomyolysis: Myoglobin is released from damaged muscle tissue process
Acute Tubular Necrosis: Released myoglobin filtered by kidneys but toxic to renal tubular epithelium and so may cause acute renal failure

Question 26

What is erythropoetin?

Deifition
Where it is found
Function
What happens in erthropoiesis
What factors affect tissue oxygen levels
Relationship between Epo and Hb concentration
Why the renal cortex?

Answer 26

Definition: hormone that regulates the production of RBC
- primarily found in kidneys
- promotes production of mature RBCs in the bone marrow
In hypoxia (low tissue oxygen relative to need)
1. Kidney renal cortex interstitial peri-tubular cells produce Epo
2. More RBCs in the circulation leads to incrased oxygenation
3. Lower levels of HIF (degrated under conditions of normal oxygen tension, promotes gene transcription of EPO in anemia or hypoxia)
4. Suppresses Epo production

Tissue oxygen levels depend on:
1. Hb concentration
2. Arterial pO2
3. Hb-O2 affinity
4. Blood circulation

Concentration of circulating Epo increases exponentially with decreasing Hb concentration in uncomplicated anaemia (absence of renal disease or inflammation)

Why the renal cortex?
pO2 in the renal cortex is little affected by blood flow changes, as renal O2 consumption decreases in proportion with blood flow.

Question 27

What are the 3 forms of carbon dioxide in blood?

Factors, percentage of blood, how its formed

Answer 27

1. Dissolved CO2 (CO2 is more soluble than oxygen): 10%
2. Carbaminohaemoglobin (CO2 also reacts with Hb, different site from O2): 30%
3. HCO3- (CO2 reacts chemically with water to form bicarbonate, primarily happens inside RBCs): 60%

Question 28

RBCs and carbon dioxide: carbaminohaemoglobin

Answer 28

-CO2 binds to Hb
- Hb carrying less O2 can carry more CO2 (vice versa)
- Favours picking up CO2 from tissues (low oxygen) and delivering Co2 to lungs (high oxygen)