Haemoglobin Flashcards
What is the role of red blood cells?
carry oxygen from lungs to tissues
transfer carbon dioxide from tissues to lungs
How many RBCs are there in a litre of blood?
3.5~5 x 10^12 RBCs/L
What key component do RBCs contain?
Haemoglobin (Hb)
How many molecules of Hb are in each red cell?
~640 million molecules
What is the normal concentration of Hb in adults?
120-165g/L
How much Hb is produced each day?
~90mg/kg produced and destroyed in the body every day
How much Fe is in each gram of Hb?
3.4mg
Where can haem groups be found?
bound within pockets of the globin protein
and other proteins
e.g. myoglobin, cytochromes peroxidases, catalases
What does the haem group contain?
protoporphyrin ring with central iron atom
iron is usually in ferrous form (Fe2+)
What is Fe2+ in the haem group able to do?
reversibly bind with oxygen
What are globins coded by?
several genes in a couple of clusters
alpha globin cluster + beta globin cluster
(see page 7 of Haematology class notes)
various types of products combine with haem to form different haemoglobin molecules
How many functional globin chains are produced?
8
arranged in two clusters
What are the two clusters?
alpha cluster: alpha + zeta genes
beta cluster: beta, gamma, delta + epilson genes
What do two alpha globins and two beta globins combine to form?
tetramer
(4 subunit protein)
(see page 7 of Haematology class notes)
What are the proportions of haemoglobin within RBCs in a healthy adult?
HbA (a2b2): 96-98%
HbA2 (a2d2): 1.5-3.2%
HbF (a2g2): 0.5-0.8%
What proportion of the secondary structure of haemoglobin are made up of a-helices?
75%
Describe the quaternary structure of haemoglobin.
- approximate sphere
- hydrophilic surface (charged polar side chains)
- hydrophobic core
- haem groups found within pockets
- 4 haem groups pe
What is electrophoresis looking for?
mobilities of haemoglobin variants
(cellular acetate electrophoresis in a Tris-glycine buffer at pH 9.5)
a method of analysing molecules by measuring their migration in an electric field.
(see insendi page for electrophoresis protocol)
Define absorbance in spectrophotometry.
is the fraction of incident light absorbed by a solution, which is typically measured by apparatus known as a spectrophotometer.
The absorbance, A (sometimes refereed to as Optical Density or OD) can be defined as follows:
A = -log10T = -logI/I0 (light transmitted through blank solution/light transmitted through test solution)
(see insendi page for spectrophotometry protocol)
What is the Beer-Lambert Law?
A = Ecℓ
A is the absorbance of the solution
c is the concentration of the absorbing substance (typically in mol/L)
ℓ is the path length (in cm)
E is the extinction coefficient, a constant for the substance being measured at that particular wavelength.
What does cooperative binding mean?
that binding of one oxygen molecule to a haem group results in a change in the conformation of the other subunits, affecting their ability to bind oxygen
What kind of cooperative binding effect occurs in haemoglobin?
positive cooperative effect
This is exemplified by the sigmoidal nature of the oxygen association curve for haemoglobin
the positive cooperativity displayed by the binding of oxygen to haemoglobin enables it to deliver almost twice as much oxygen than would be possible in the absence of cooperativity
What proportion of the potential oxygen-binding sites contribute to oxygen transport?
66%
Where is myoglobin found and what is it?
found in muscle
oxygen binding protein
has one haem group her molecule
Which one has a greater affinity of oxygen between myoglobin and haemoglobin?
myoglobin
but the lack of copperativity means that it is poor at releasing oxygen under the same conditions
What is a method used when checking the respiratory status of newborn infants?
spectrophotometry
What is pulse oximetry used for?
non-invasive way of measuring oxygen saturation levels
What does pulse oximetry rely on?
the difference in absorbance of oxyhaemoglobin and deoxyhaemoglobin
How does pulse oximetry work?
Oxygenated haemoglobin absorbs more infrared light but less red light than deoxygenated haemoglobin. The transmitted light at each wavelength is corrected for the pulse of arterial blood by subtraction of the minimum absorbance.
The ratio of absorbances at each wavelength can be converted to SpO2 by reference to the Beer-Lambert law.
When is carboxyhaemoglobin (CoHb) generated?
by the binding of carbon monoxide to ferrous iron (Fe2+) in haemoglobin
By how much does haemoglobin have a greater affinity for carbon monoxide than oxygen?
haemoglobin has a 200-fold greater affinity for carbon monoxide than
oxygen
Levels of 0.2% carbon monoxide can lead to death within an hour or two
When is methaemoglobin (MetHb) generated?
When the Fe2+ ion is oxidised to the Fe3+ (ferric) state which results in greatly impaired oxygen binding.
What colour does MetHb give blood if present at high levels?
bluish/chocolate colour
What can low levels of MetHb do to the oxygen dissociation curves?
shift leftwards
result in tissue anoxia as oxygen is not readily released by MetHb
Which enzyme reduced methaemoglobin to haemoglobin?
methemoglobin reductase
What kind of disorder is methaemaglobinaemia?
can be hereditary
(for example as a deficiency in methemoglobin reductase or production of a mutant form of haemoglobin known as haemoglobin M, which is resistant to reduction)
can also be acquired following exposure to chemicals including aniline dues such as p-chloroaniline, nitrates, and local anaesthetics such as benzocaine
Why does normal haemoglobin A travel further towards the positive electrode that sickle haemoglobin (S) in the electrophoresis of HbA and HbS?
due to a point mutation occurring in one amino acid of the beta chain.
n this mutation, the amino acid glutamate in the normal protein (hydrophilic, negatively charged) is replaced by valine (hydrophobic, uncharged).
When was sickle cell anaemia first described?
James B Herrick (1861-1954)
it was the first molecular disease to be described in humans
How does sickle haemoglobin (HbS) occur?
from a missense mutation at codon 6 of the b globin chain (βS) - superscript S
Glu replaced by Val (Glu is polar and soluble, Val is non-polar and insoluble)
Deoxyhaemoglobin S is insoluble
How does HbS result in a sickled shape of RBCs?
HbS polymerises to form tactoids that distort the RBC
What kind of inheritance pattern is HbS?
autosomal recessive
What disease does HbSS result in?
sickle cell anaemia
sickle trait (usually asymptomatic)
Describe the global distribution of the βS gene.
- Distribution matches that of endemic Plasmodium falciparum malaria
- Up to 25% Africans (sub-Saharan) and 10% Caribbeans carry HbS gene
- Around 300,000 affected births annually worldwide
What does Sickle cell disease (SCD) incorporate?
Sickle cell anaemia (HbSS)
a number of compound heterozygous states that lead to a disease syndrome due to sickling e.g. HbSC, HbS β thalassaemia
What is the epidemiology of SCD in the UK?
- prevalence 12000-15000
- 70% reside in Greater London
- 350 new births per annum. Most common monogenic disorder
- National Haemoglobinopathy Registry (NHR) established in 2013. Currently 11000 SCD patients registered
- ~600 patients at the Imperial College Healthcare NHS trust
What two processes occur with SCD?
Haemolysis (breakdown) of sickled RBCs
Blockage to microvascular circulation (vaso-occlusion)
What can haemolysis of sickled RBCs lead to?
(shortened red cell lifespan: 20 days)
anaemia: baseline Hb concentration in HbSS is ~60-80b/L (cf 125-160 for HbA)
gall stones (increased red cell breakdown products)
Aplastic Crisis (Parvovirus B19 where bone marrow erythropoiesis has been shut down)
(see diagram 11/28)
What can blockage to microvascular circulation (vaso-occlusion) lead to?
tissue damage and necrosis (infarction)
therefore: pain + dysfunction
(see diagram 11/28)
What are some of the early presentations of sickle cell disorders?
(symptoms rare before 4-6 months of age)
onset coincides with switch from fetal to adult Hb synthesis
early manifestations:
- dactylitis (inflammation of digits)
- pooling of RBCs in spleen (splenic sequestration)
- infection
splenic sequestration is an unpredictable and life-threatening complication in young children
What is spectrophotometry?
a way of analysing molecules on the basis of their spectral properties
What are the roles of the spleen?
immune defence
breakdown and removal of old, malformed or damaged RBCs (quality control)
What is functional hyposplenism?
repeated splenic vaso-occlusion (usually with no symptoms) in HbSS leads to the spleen no longer working by the age of 5
What does hyposplenism increase susceptibility to?
encapsulated bacterial infection
e.g. pneumococcus or meningococcus
Why is it important for individuals with Sickle cells disease to receive immunisations?
to reduce the risk of them contracting these infections.
they also take lifelong prophylactic or preventative antibiotics on a daily basis because they no longer have a functioning spleen
What can vaso-occlusion in the chest lead to?
acute chest syndrome
How can you diagnose Acute chest syndrome in SCD?
New pulmonary infiltrate on chest X-ray
with:
fever, cough, chest pain, tachypnoea (rapid breathing)
(see diagram 14/28)
What proportion of individuals who develop acute chest syndrome require ventilation?
15%
What is the mortality in adults who develop acute chest syndrome?
10%
How common is stroke in sickle cell disease?
affects 8% of patients with HbSS
Most common in childhood
involves major cerebral vessels
(see diagram 15/28)
What are other complications you can have in sickle cell disease?
avascular necrosis of the femoral head
osteomyelitis due to Salmonella infection
(see diagram 16~17/28)
What is the prevalence of gallstones in HbSS individuals by the age of 25?
50%
due to increase in the bilirubin as a result of the breakdown of the red blood cells due to their enhanced turnover
What are the laboratory features of sickle cell disease?
Hb low (typically 60-80 g/L)
reticulocytes usually high
blood film:
- sickled cells
- boat cells
- target cells (feature of hyposplenism)
- Howell Jolly bodies (feature of hyposplenism - DNA remnants)
(see diagram 20~23/28)
What is the sickle solubility test?
In the presence of a reducing agent oxyHb converted to deoxyHb
solubility decreases, solution becomes turbid
does not differentiate AS from SS
(see diagram 24/28)
What does definite diagnosis require?
Electrophoresis or High Performance Liquid Chromatography (HPLC) to separate proteins according to charge
Does sickle cells anaemia include both HbSS and HbSC?
No
Is sickling due to a change in the alpha globin chain?
no
Is the molecular alteration a deletion which protect against malaria?
no
Does sickle Hb makes red cells less deformable?
yes
Can clinical manifestations start in utero because beta-globin is part of fetal Hb?
No
Is osteomyelitis the name given to inflammation of a digit?
No
Can chest crises be fatal?
Yes
If a lady with HbAS has a partner with HbSS, should she be offered genetic counselling?
Yes
