Buffers Flashcards
Define buffer
a buffer is a solution containing a weak acid and its conjugate base which has the ability to minimise changes in [H+] when a stronger acid or base is added to it
Buffer efficacy influence by
◦ pKa of the buffer - Buffering capacity maximal at the pKa of the weak acid and majority (80%) of buffering +/- 1 of pKa
◦ pH of the solution
◦ Amount of buffer
◦ Open or closed system - open system can have the amount of chemical at one or bodth ends adjusted by physiological means altering concentration
* Effectiveness can be compared by measuring how much base/acid required to change pH by 1
What is the dominant buffer in the ECF
Bicarbonate
Where does bicarbonate come from
Erythrocyte production by carbonic anhydrase and limited spotnaneous CO2/H20 reaction in plasma
Bicarbonate distirbution
Evenly in ECF
pKa of bicarbonate buffer system
- pKa 6.1 and physiological extracellular pH is 7.4
◦ 2nd dissociation reaction pKa 9.3
Why is the bicarbonate buffer so effective if its pKa is so far removed from the body
- Works very effectively as pCO2 can be altered by the lungs - as an open buffer system minimising changes
◦ i.e. adding a strong acid pushes the equation to the left forming more CO2 and H20 which is then exhaled - Additionally HCO3 is regulated in the kidneys again adding to the open system buffer in maintaining balance
- This allows it to be an effective buffer system despite its low pKa
What is bicarbonate a poor buffer of
Respiratory acid base disturbances as relies heavily on pulmonary ventilation to be effective
What is the bicarbonate buffer equation
Equation: CO2 + H20 –> H2CO3 –> HCO3- + H+ –> 2H+ + CO3 2-
What are the buffers of the ECF and what is their relative contribution
Bicarbonate 75%
Haemoglobin buffering system 20%
Plasma proteins 5%
Haemoglobin buffering occurs where?
Major buffer for H+ in the red cell when HCO3 formed form CO2 and water
Is haemoglobin a weak acid or base?
Weak acid
Why is Hb such a prominent buffer
◦ High concentration compared to plasma proteins
◦ Each molecule contains 38 histidine residues (3x the buffering capacity per g compared with plasma proteins)
Mechanism of Hb as a buffer
◦ Hb is a weak acid: HHb –> H+ + KHb
◦ Additional H+ ions bound to Hb, HCO3 diffuses down its concentration gradient into plasma with electroneutrality maintained through inward movement of Cl (Hamburger shift) by the Band 3 transport protein. This basically means you never reach a situation where CO2 encounters a concentration gradient entering cells
◦ Imidazole group of histidine amino acid residue can accept H+ (negatively charged nitrogen group)
Where on proteins is the buffering done
Imidazole group of histidine amino acid residue can accept H+ (negatively charged nitrogen group)
Why is Hb a variable buffer?
- Variable pKa due to imidazole group structural alteration between deoxy and oxyhaemoglobin
◦ Deoxyhaemoglobin pKa of 8.2 and therefore readily accepts H+ and is more effective at buffering acidic solutions
◦ Oxyhaemoglobin pKa of 6.6
◦ per mmol of oxyhaemoglobin reduced
‣ 0.8 mmol of H+ can be buffered
‣ 0.7mmol of CO2 can enter blood without a change in pH (mechanism behind Haldene effect and why venous blood only slightly more acidic than arterial)
Which is a more powerful buffer oxyhaemoglobin or deoxyhaemoglobin?
- Deoxyhaemoglobin is a more powerful buffer than oxyhaemoglobin - so oxygen unloading increases the capacity for CO2 carriage (Haldene effect);
Does CO2 binding have any relationship to Hb’s role as a buffer?
- Deoxyhaemoglobin is also more effective (3.5x) at forming carbamino compounds - but this is not responsible for buffering capacity
◦ Carbamino compounds come from CO2 reaction with terminal amine groups of Hb –> carbamic acid with low pKa so it is dissociated increasing H+ ions
◦ The formation of carbamino compounds does not change the buffering capacity of H
◦ Thus both CO2 carriage as carbamino compounds and HCO3 produce H+
How can you manipulate the role of Hb as buffer in blood
- Buffering capacity affected by
◦ pH (effective within pH of PkA +/- 1)
◦ State of oxygenation of Hb - deoxyHb better
◦ Hb concentration - higher the more buffering capacity per unit of blood
Plasma proteins act as a buffer how? pKa? Why less effective than Hb
(20% of non bicarbonate buffering)
* Involves the buffering effects of imidazole groups (ring) of histidine molecule an amino acid residue providing buffering on proteins containing it
* pKa 6.8 - so most of the imidazole groups have a nitrogen atom with a negative charge and can accept proton binding as pH reduces/H+ rises
* As there is a reduced concentration of proteins compared to Hb and reduced residues per protein it is a less significant buffer - 6x less important as Hb has 2x the concentration of plasma proteins, and each Hb molecule has 3x more histidine residues (with imidazole group) than the average plasma protein
Proteins also are less important in CO2 carriage as carbamino compounds because
* Reduced concentration compared with Hb
* 1/4 of the amount of terminal amine groups compared to Hb on the main other protein albumin
* Hb also becomes better at carrying CO2 as a carbamino group when deoxygenated at the same time this ability is required
Phosphate buffer system located where?
Urine and intracellular
Phosphate buffer system reaction
- H2PO4- ⇔ H+ + HPO4-
pKa of phosphate buffer system
- Tribasic and can therefore potentially donate three hydrogen ions
- However, only one of these reactions is relevant at physiological pH, with a pKa of 6.8:
What state does most phosphate reside at physiological pH
HPO4
Can you get extra phosphate from anywhere to act as a buffer?
Bones in prolonged acidossi
Proteins as intracellular buffers
- All proteins are potentially buffers - however, the useful one at physiological pH is the imidazole groups of the histidine residues.
- Extracellularly, proteins have a small contribution which is entirely due to their low pKa
- Intracellularly proteins have a much greater contribution because:
◦ Intracellular protein concentration is much greater than extracellular concentration
◦ Intracellular pH is much lower (~6.8) and closer to their pKa
The urine has a limit on its aciditiy however once it reaches maximum acidity what buffers start to play a part?
Creatrinine is not usually a uirinary buffer as its pKa is 5 however if the pH is very low it can be responsible for up to 1/4 of titratable aciditiy. As can betahydroxybutyrate pKa 4.8 in DKA
Ammonium as a buffer in the urine comes from? What state does it exist primarily? pKa?
- Glutamine and glutamate are the major source –> catalysed by glutaminase in proximal tubular cells
- Ammonia is present almost completely as ammonium as pKa 9.2
- Ammonium mostly removed in LOH and if the pH is low in the distal tubule this augments transfer of ammonium from medually intersitiam back into tubular fluid
What is the Henderson equation
H+ = K x PCO2/HCO3
Rearranged 24 = K x sPCO2
K is the dissociation constant
S is the solubility coefficient = 0.03 mmol/L/mmHg at 37 degrees
How do we calculate pH using the Henderson Hasselbach equation
Draw the equations and provide the pKa of the phosphate buffer system
