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
What is the Hasselbach equation
pH = pK + log (HCO3 / sPCO2)
Which is a weaker acid the hydrogen bicarbonate ion or Hb
Hb is a weaker acid pKa 6.8
Which is the area of haemoglobin that acts as a buffer
Imidazole group of histidine residue amino acid
pKa 6.8
What is oxyhaemoglobin best at buffering? What is deoxyhaemoglobin better at buffering?
Oxyhaemoglobin better at resisting acidic changes –> pKa 6.6
Deoxyhaemoglobin better at resisting basic changes –> pKa 8.2
How much H+ can be added to the system without a change in pH due to Hb
0.7mmol CO2 and 0.7mmol of H+ per mmol of Hb
Isohydric buffering
Describe the 3 stage buffer of phosphate
H3PO4 –> pKa 2
H2PO4 –> pKa 6.8
HPO4 –> pKa 11.7
What % of non carbonic acids in the body are buffered by plasma bicarbonate
70%
25% Hb and proteins
<5% by inorganic and organic phosphates
What is the principle buffer of carbonic acid in the body
Hb –> 90%
Interstitial fluid HCO3- concentration?
27mmol/L
How much protein and how much phosphate is in cells to act as buffers?
6mmol/L of each
Both with same pKa 6.8
What is the Hb concentration in the ECF
5g/100mL
Buffering capacicty 1/3 of that of blood
What is a volatile acid
Respiratory acids
CO2 is produced by [O] metabolism of carbohydrates and TG with the majority hydrated in plasma
15000mmol H+ /day produced
Do not contribute to net acid balance in the body
How much lactate is made per day
1500 mmol
Does lactate contribute to body acid non volatile
No
Lactate is oxidised in the liver to regenerate HCO2 in the Cori cycle
The exception being in excessive production
What are the main non volatile acids made each day?
Sulphuric - 45mmol/day
From metabolism of sulphur containing amino acids
Phosphoric acid from hydrolysis of phosphorite is —>13 mmol/day
HCl 12mmol/day
1-1.5mmol/kg/day of H+
70mmol/day
How are acids eliminated from the body
H+ excretion from the body:
o (1) Lungs o (2) Liver o (3) Kidney
Eliminate all “volatile acids” (H2CO3) → 15000 mmol H+/day Eliminates “fixed acids” (mainly lactate) → 1500 mmol H+/day Eliminates “fixed acids” (mainly phosphoric acid, sulphuric acid, other
acids) as NH4+ and “titratable acids” Accounts for at least 70 mmol H+/day → 30 mmol/day as “titratable
acids” and 40 mmol/day as NH4+
How does alteration in body acid base effect systems
Derangements of [H+] and pH can result in systemic effects (Eg. altered CNS reflexes, CVS
depression, Etc.; See below) as a result of direct intracellular disturbances:
- (i) Altered protein function (Eg. enzyme activity, transporter activity, Etc.)
- (ii) Altered membrane excitability
- (iii) Disruption in metabolic pathways (esp energy production)
- (iv) Ion trapping of biological molecules in compartments, Etc.
What 3 factors determine buffer effectiveness
- AMount of buffer
- Buffers pKa and pH of solution - 80% of buffering activity occurring within +/- 1 pH of buffers pKa with maximal effect AT its pKa
- Opens vs closed
Blood buffering is from?
- RBC
- primarily haemoglobin - 35% of buffer capacity of the blood, buffers 90% of H2CO3
- HCO3 18%
- Proteins and PO4 negligible - Plasma
- Primarily HCO3 buffering 35%, 70% of metabolic events dealt with by HCO3
- Protein - 7%
- PO4 2%
How is the interstitial fluid buffered?
HCO3
How is intracellular buffering performed
- Primarily protein and PO4- —> high intracellular amounts, pKa close to intracellular pH
- Fixed acid extrusion in exchange for strong electrolytes via antiporter
- Organelles buffering - sequestering in IC organelles
- Metabolic - alter production of acidic metabolites
How does the bicarbonate buffer function when exposed to a strong acid or base
o In presence of strong acid – H+ from acid buffered by HCO3- → shifts to
production of CO2 production (via H2CO3) → CO2 excreted from lungs o In presence of strong base – Buffered by H2CO3 → Forms HCO3- which is
excreted from kidneys
What is the pKa of H2CO3
6.1
Describe the factors that make H2CO3 a good buffer system, and factors which impair its effectiveness
o It’s low pKa (6.1) relative to physiological pH and relatively small amounts in
ECF ↓ its effectiveness as a buffer system in ECF o BUT this is offset by the fact that the system is “open” system → Can be
controlled independently by the:
(i) Lungs – Excretion of CO2 (or H2CO3) regulated rapidly by changes in
minute ventilation (ii) Kidneys – Excretion of HCO3- and H+ regulated more slowly
What % of total ECG buffering does HCO3 make up?
80%
How do we calculate pH using the Henderson Hasselbach equation
Where on the Hb molecule is a. Buffer
Histidine residues (38) with imidazole side chains pKa 6.8 on the globin chain of Hb
Anionic site binding H+
Is Hb a acid or base?
Weak acid
H-Hb and its salt K-Hb with a pKa of 6.8
Why is deoxyhaemoglobin a better buffer than oxyhaemoglobin?
It isn’t a better buffer, it is a better buffer of ACIDIC SUBSTANCES
Deoxyhaemoglobin has a pKa 8.2 which is a better buffer than Oxyhaemoglobin with a pKa of 6.6 as it is a weaker acid
What physiological processes enable the Haldane effect
Uptake and buffering of extracellular H+ (as reduced Hb; HHb)
produced from hydration of tissue CO2 and dissociation of H2CO3
→ 30% Haldane effect
Uptake of tissue CO2 (as carbamino compounds) → 70% Haldane
effect
For each mmol of Oxyhaemoglobin that is reduced how much H+ can be buffered?
0.7mmol of H+and o.7mmol of CO2 can enter the veinous system without significantly changing the pH
WHy is Hb a more important buffer than proteins?
Note: Despite being a protein, Hb has 6x ↑ buffering capacity than protein proteins because
- (i) [Hb] is 2x of plasma protein
- (ii) Hb has 3x buffering capacity gram per gram that of plasma protein → due to 3x ↑
content of imidazole-containing histidine residues
What is the pKa of amino and Carboxyl groups
Most amino groups (pKa 9) and carboxyl groups (pKa 2) have pKa very far from
physiological pH → Contributes little to buffering
Draw the equations and provide the pKa of the phosphate buffer system
What is bone buffering
Important in “bone buffering” → CaPO4 acts as “Alkali reserve” → During prolonged
acidosis, it solubilises in plasma to ↑ [PO43-]
What is the the relationship of pH to PCO2
Remember: pH = 6.1 + log ([HCO3-]/0.03xPCO2)
How is PCO2 related to minute ventilation
Remember: PaCO2 = 0.83 x VCO2/Va
Sodium bicarbondate Properties and pharmaceutics
Glass vial - to avoid CO2 exchange with atmospheric air
8.4% solution
1mol/L
1000mmol/L of HCO3 and Na ions
Osmolality 2000mosm/kg
Side effects of bicarbonate administration
Hypokalaemia
Hypernatraemia
Hypocalcaemia
Fluid overload
Hypercapnoea
Phlebitis
Intracellular acidosis - CO2 diffusion
When is bicarbonate contraindicated
Hypokalaemia
Lactic acidosis - increased lactate as it removes acidic inhibition of glycolysis by increased activity of PFK + shifts Hb-O2 dissociation curve, increased oxygen affinity of haemoglobin and thereby decreases oxygen delivery to tissues
DKA
Increases mortality
When might sodium bicarbonate be indicated?
Metabolic acidosis with pH <7.10 and hemodynamic instability
Normal anion gap metabolic acidosis (esp. RTA)
Alkalinisation of urine to enhance the elimination of acidic drugs
What happens to a bottle of sodium bicarbonate after it is infused?
Addition of
- 100ml of fluid
- 100mmol of Na
- 100mmol HCO3
Bicarb: If niether of the below happens bicarb would increased from 24 –> 31 but its Vd depends on bicarbonate concentration/pH –> Vd at baseline is similar to total body water (0.4L/kg) , at low pH it has high Vd as it is used up, at high bicarbonate values it remains in fluid and has a smaller Vd/
1. Some bicarbonate rapidly excreted by the kidney
2. 50% of bicarbonate metabolised to CO2 and water and disappears from fluid shift calculations
It actually prolduced a bicarb of 27 in a normal person from baseline –> but this is diluted by giving a hypertonic solution causing water to dilute it to 26.
Rise in CO2 during the infusion that rapidly returns to baseline over 30 minutes(100mmol caused a temporary 6mmHg increase)
Addition of 200mOsm to IV compartment and 100ml of fluid
Sodium remains in ECF
Water equilibrates between compartments - +83ml IV, +250 in interstitial, -235ml in intracellular
When might bicarbonate be useful?
Hyperkalaemia
Treatment of sodium channel blocker overdose with ECG features (e.g. tricyclic overdose)
Urinary alkalinisation (salicylate poisoning)
Normal anion gap metabolic acidosis
