In this article we will discuss about Buffers:- 1. Definition of Buffers 2. Principles of Buffers 3. Determining the pH 4. Buffer Mixture 5. Buffer Pairs in the Blood 6. Uses 7. Tissue Fluids and Tissues 8. Role in pH Regulation 9. Acidosis and Alkalosis Acidosis 10. Role of Lungs and Kidneys in pH Regulation 11. Elimination of Free Acids 12. Renal Correction of Acidosis 13. Renal Correction of Alkalosis.
- Definition of Buffers
- Principles of Buffers
- Determining the pH of Buffers
- Buffer Mixture
- Buffer Pairs in the Blood
- Uses of Buffers
- Buffers of Tissue Fluids and Tissues
- Role of Buffers in pH Regulation
- Acidosis and Alkalosis Acidosis
- Role of Lungs and Kidneys in pH Regulation by Means of Buffers
- Elimination of Free Acids
- Renal Correction of Acidosis
- Renal Correction of Alkalosis
1. Definition of Buffers:
Buffers are the mixtures of weak acids and their salts of strong bases (or strong acids and their salts of weak bases).
Acetic acid (CH3COOH) + Sodium acetate (CH3COONa).
2. Principles of Buffers:
HAC + NaAC → Na+ + H+ + 2AC−
where, HAC=Acetic acid; NaAC = Sodium acetate.
If alkali (NaOH) is added to this system, it will form salt and no free H+ or OH− will be available.
HAC + NaAC + NaOH → 2NaAC + H2O
If acid (HCl) is added to this system, it will also form salt and no free H+ or OH− will be available.
HAC + NaAC + HCl NaCl + 2HAC
In either cases there is no change in hydrogen ion concentration. The buffer acts almost as if it were “absorbing” the added free hydrogen or hydroxyl ions.
3. Determining the pH of Buffers:
The pH of buffers can be determined by the Henderson-Hasselbalch equation:
In case of blood, the ratio between [BHCO3]: [H2CO3] can be found out by applying the above equation to maintain average pH of blood 7.4:
The pH of human blood is 7.4. In normal health, it lies between 7.3 and 7.5 although CO2 (i.e. carbonic acid) is always added. If the pH of human blood becomes 7.0 and 7.6, it alarms danger; if not fatal.
The difference in pH between arterial and venous blood is rarely more than 0.04. A marked decrease in pH of blood has been observed during severe muscular exercise when the blood lactic acid content rises over 100 mg per 100 ml.
4. Buffer Mixture:
(a) Glycine and HCl.
(b) Acid potassium phthalate and HCl.
(c) Acid potassium phosphate and NaOH.
(d) Sodium bicarbonate and Sodium carbonate.
5. Buffer Pairs in the Blood:
Actually in blood the buffering of carbonic acid is complicated by the presence of the red cells:
6. Uses of Buffers:
i. Buffers are used for preparing standard solutions in which it is always desired to maintain a constant pH. This is required for the colorimetric determination of the pH of unknown solutions.
ii. These are used to maintain H+ concentration which is necessary for optimal activity of enzymes.
iii. These are practically important in all physiological systems.
7. Buffers of Tissue Fluids and Tissues:
i. The buffering system of lymph, cerebrospinal fluid etc., are similar to that of blood although the quantity is much less.
ii. The chief buffering system in these fluids is BHCO3 and H2CO3.
iii. The buffering system in tissues is mainly BHCO, and H2CO3; protein buffers and organic acid salt.
8. Role of Buffers in pH Regulation:
(i) Bicarbonate Buffer:
a. It is the main buffer in blood plasma and consists of bicarbonate (HCO−3) and carbonic acid (H2CO3).
b. The bicarbonate buffer neutralizes stronger dietary and metabolic acids (HA) converting them into weak bases (A–) with the increase in H2CO3. Stronger bases (B) are also changed into weak acids (BH+) with the rise in HCO−3.
c. The pH of blood is maintained 7.4 when the buffer ratio becomes 20. If the bicarbonate buffer neutralizes any acid or base, there may be the change of buffer ratio and the blood pH value. But the buffer ratio remains unchanged by the respiratory elimination of H2CO3 as CO2 or the urinary elimination of HCO−3.
d. Since cells contain much lower amounts of HCO−3 the importance of bicarbonate buffer inside the cell is negligible.
(ii) Phosphate Buffer:
a. Since the concentration of phosphate buffer in the blood plasma is about 8 per cent of that of the bicarbonate buffer, its buffering capacity is much lower than bicarbonate in the plasma.
b. The phosphate buffer consists of dibasic phosphate (HPO−−4) and monobasic phosphate (H2PO−4). Its PKa value is about 6.8. It is more effective in the pH range 5.8 to 7.8. Plasma has a [HP0—4]:
c. The concentration of phosphate buffer is much higher in intra-cellular fluid than in extracellular fluids. The pH of intracellular fluids (6.0 – 6.9) is nearer to the PKa of the phosphate buffer. Therefore, the buffering capacity of the phosphate buffer is highly elevated inside the cells and the phosphate buffer is also effective in the urine inside the renal distal tubules and collecting ducts.
d. In case the ratio of [HPO−−4]: [H2PO−4] tends to be changed by the formation of more H2 PO−4, there occurs the renal elimination of H2PO4 for which the ratio ultimately remains unaltered.
(iii) Protein Buffers:
a. The protein buffers are very important in the plasma and the intracellular fluids but their concentration is very low in C.S.F., lymph, and interstitial fluids.
b. They exist as anions serving as conjugate bases (Pr) at the blood pH 7.4 and form conjugate acids (HPr) accepting H+.
3=c. They have the capacity to buffer some H2CO3 in the blood:
H2CO3 + Pr ⇋ HCO−3 + HPr
(iv) Hemoglobin Buffers:
a. They are involved in buffering CO2 inside erythrocytes. The buffering capacity of hemoglobin depends on its oxygenation and de-oxygenation. Inside the erythrocytes CO2 combines with H2O to form H2CO3 under the action of carbonic anhydrase.
At the blood pH 7.4, H2CO3 dissociates into H+ and H2CO3 and needs immediate buffering. Oxy-hemoglobin (HBO−2) on the other side loses O2 to form de-oxy-hemoglobin (Hb−) which remains un-dissociated (HHb) by accepting H+ from the ionization of H2CO3.
Thus, Hb buffers H2CO3 in erythrocytes:
Some of the HCO−3 diffuse out into the plasma to maintain the balance between intracellular and plasma bicarbonates. This causes influx of some CI− into erythrocytes along the electrical gradient produced by the HCO−3 outflow (chloride shift).
b. HHbo2, produced in lungs by oxygenation of HHb, immediately ionizes into H+ and HbO−2. The released hydrogen ions (H+) are buffered by HCO–3 inside erythrocyte to form H2CO3 which is dissociated into H2O and CO2, by carbonic anhydrase. CO2 diffuses out of erythrocytes and escapes in the alveolar air. Some HCO−3 return from the plasma to erythrocytes in exchange of Cl− and are changed to CO2.
9. Acidosis and Alkalosis Acidosis:
a. Accumulation of acid or loss of alkali is called acidosis.
b. It occurs due to the loss or fall of [HCO–3] : [H2CO3] of blood below 20.
c. There are two types of acidosis: (a) Metabolic; (b) Respiratory.
(i) The concentration of plasma bicarbonate is decreased in excessive loss of bases.
(ii) It happens in renal failure, diabetic ketosis, severe diarrhoea.
(i) The retention of CO2 is caused in hypoventilation resulting in the rise of H2CO3. This lowers [HCO−3]: [H2CO3] ratio.
(ii) It happens in chronic obstructive airway diseases (asthma, respiratory paralysis), prolonged anesthesia, and unconsciousness due to any cause.
a. Accumulation of alkali and loss of acid is called alkalosis.
b. There is increase in the ratio of [HCO−3]: [H2CO3] of blood above 20, resulting the rise in blood pH.
3. There are two types:
(i) High intake of alkaline substances like NAHCO3 may elevate the plasma HCO−3.
(ii) It happens in severe vomiting due to any cause, indiscriminate use of antacid.
(i) The excess CO2 is removed from the blood due to hyperventilation and causes decrease of H2CO3.
10. Role of Lungs and Kidneys in pH Regulation by Means of Buffers:
a. Lungs maintain the normal ratio of [HCO–3]: [H2CO3] and the pH of blood by altering the rate of respiratory elimination of CO2 from the blood. This lowers the alveolar PCO2 and increases the diffusion of CO2 to the alveolar air. Since the concentration H2CO3 is in equilibrium with that of dissolved CO2 in the blood, hyperventilation increases the ratio of [HCO−3]. : [H2CO3] with the fall in the CO2 concentration. The doubling of ventilation may raise the blood pH by 0.4.
b. Hypoventilation, on the other hand, raises the blood concentration of dissolved CO2 and consequently lowers the buffer ratio. A fall in alveolar ventilation to one-fourth of the normal value may lower the blood pH by 0.46. The pulmonary ventilation is adjusted according to the blood pH. Hypoventilation not only retains CO2 decrease the ratio of [HCO−3] : [H2CO3] and the blood pH but also curtails the O2 supply—an undesirable effect.
c. Lungs also have a role in the functioning of hemoglobin buffers through oxygenation and de-oxygenation which has been discussed earlier.
a. The pH of the glomerular filtrate is about 7.4. But the pH falls to about 6.9 in the proximal tubule, then to about 6-6.5 in the distal tubule and ultimately to about 4.5-4.7 in the collecting duct.
b. The urinary pH is maintained by a cooperation between the urinary buffers and the renal ion-exchange mechanism. The major urinary buffers are bicarbonate and phosphate buffers. As the filtrate proceeds along the tubules, the ratio between base member and the acid member of each urinary buffer falls progressively with a consequent fall in the urinary pH.
c. In the renal ion exchange mechanism, some urinary Na+ are actively reabsorbed in the exchange of HH secreted in the tubular filtrate. 85 percent of H+ are secreted by the proximal tubule and 15 per cent from the distal tubules and collecting ducts. The hydrogen ions (H+) are mainly formed from the ionization of H2CO3 formed from CO2 and H2O by carbonic anhydrase in the tubule cells.
The bicarbonate formed is returned to blood along with the reabsorption of Na+.
d. Most of the secreted H+ are immediately buffered by HCO–3 filtered from the plasma into the glomerular filtrate.
e. H2CO3 produced by such buffering in the proximal tubules is immediately removed through its cleavage to CO2 and cannot play any role in lowering the pH there.
f. The buffering of the secreted H+ is essential for continuing their secretion in the urine. This buffering is held in the tubular filtrate in several ways.
(a) Buffering by Bicarbonate:
(i) Normally about 3.50 mM of H+ are secreted in the tubules per minute.
(ii) The major portion of the secreted H+ can be buffered by HCO−3 in the tubular filtrate to form H2CO3, except a small amount of free H+ to pass into the urine.
(iii) H2CO3 is then immediately cleaved into H2O and CO2 in the proximal tubular lumen by carbonic anhydrase.
(iv) CO2 then diffuses very readily into the proximal tubule cell and therefrom to the blood.
(v) In erythrocytes, carbonic anhydrase converts this CO2 into H2CO3 which dissociates to form fresh HCO–3. This HCO–3 is restored in the plasma.
(vi) When H+ are secreted in excess due to the fall in blood pH, almost all the filtered HCO−3 changes into H2CO−3 and is returned to the plasma. So the urinary HCO−3 is negligible so long as the urinary pH does not exceed 6. But whenever the blood pH tends to rise, much more HCO−3 is filtered than the amount of H+ secreted. So, some of the filtered HCO3 fails to get H+ to combine and fails to return to the plasma. This causes the urinary elimination of bicarbonate.
(vii) Buffering of the secreted H+ by the filtered HCO−3 serves two purposes:
1. It does not allow the pH to fall below 6.9 in the proximal tubule and allows more H+ to be eliminated by tubule cells into the urine.
2. It helps to reabsorb the filtered HCO–3 and to restore it in the blood.
(b) Buffering by Phosphate Buffer:
(i) In the distal tubules, some secreted H+ is buffered by the phosphate buffer. HPO−−4, filtered into the glomerular filtrate, receives the secreted H+ to form H2PO−4. This changes the ratio of [HPO−−4]: [H2PO−4] from 4 of Bowman’s capsule to 0.02-0.05 in the final urine. The lower this ratio, the more acidic is the urine.
(ii) H2PO–4 is eliminated in urine carrying some Na+ with it resulting in the urinary loss of Na+.
(iii) The total buffering capacity of urinary phosphate is much less than that of bicarbonate.
(iv) As the urine gets more concentrated in the distal tubules and collecting ducts, the rise in tubular concentration of HPO−−4 enhances the buffing capacity of the phosphate buffer.
(c) Buffering by Ammonia:
(i) The base NH3, synthesized and secreted by the tubule cells, can buffer some H+ in the distal tubule.
(ii) In the tubular lumen, NH3 combines with H+ to form NH+4 which is excreted in the urine in association with CI– and SO−−4 left behind by the reabsorbed Na+.
(iii) NH+4 behaves like a weak acid and does not dissociate much. Its formation lowers the tubular concentration of free H+, enabling further secretion of H+. The tubular membrane is not permeable to NH+4 which is retained in the tubular filtrate instead of diffusing back into the tubule cells (diffusion trapping).
(iv) The secretion of NH3 rises whenever the free H+ concentration is high enough to lower the urinary pH below 6; the more acidic the urine, the higher is the urinary ammonia.
(v) An alkaline urine contains little or no ammonia, kidneys normally excrete about 40 mEq of NH+4 in 24 hours. The elimination of highly acidic urine may enhance more ammonia secretion.
11. Elimination of Free Acids:
Strong conjugate bases such as lactate, acetoacetate, urate and oxalate anions accept some H+ replacing the Na+ reabsorbed from their salts. As a result, the free acids such as lactic acid, acetoacetic acid, uric acid are excreted. Their elimination changes the urinary pH a little only.
12. Renal Correction of Acidosis:
In acidosis, the blood carries a high amount of dissolved CO2 compared to that of HCO−3 and the tubule cells secrete far more H+ than the HCO−3 filtered from glomeruli. As a result, all the filtered H2CO−3 combines with H+ to form H2CO3.
Thus, the urine does not contain HCO−3 while the reabsorbed HCO−3 is retained in blood increasing the buffer ratio. In addition, tubular secretion of NH3 increases for buffering the H+ left in the tubules after all the HCO3 has been reabsorbed.
13. Renal Correction of Alkalosis:
In alkalosis, the blood carries a high amount of HCO−3, and the glomerular filtrate contains far more HCO−3, than H+ secreted in the tubules. The urinary elimination of unabsorbed HCO−3, causes a loss of HCO−3, from blood and finally lowers the buffer ratio and the pH of blood. Alkalosis also reduces the tubular secretion of NH3.