Buffers: Definition, Principles and Uses

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.

Contents:

1. Definition of Buffers
2. Principles of Buffers
3. Determining the pH of Buffers
4. Buffer Mixture
5. Buffer Pairs in the Blood
6. Uses of Buffers
7. Buffers of Tissue Fluids and Tissues
8. Role of Buffers in pH Regulation
9. Acidosis and Alkalosis Acidosis
10. Role of Lungs and Kidneys in pH Regulation by Means of Buffers
11. Elimination of Free Acids
12. Renal Correction of Acidosis
13. Renal Correction of Alkalosis

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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).

Example:

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 PK_a 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.

(a) Meta­bolic:

(i) The concentration of plasma bicarbonate is decreased in excessive loss of bases.

(ii) It happens in renal failure, diabetic ketosis, severe diarrhoea.

(b) Respiratory:

(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.

Alkalosis:

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:

(a) Metabolic;

(b) Respiratory.

(a) Metabolic:

(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.

(b) Respiratory:

(i) The excess CO₂ is removed from the blood due to hyperventilation and causes de­crease of H₂CO₃.

(ii) It happens in high altitude (hyper-ventilation syndrome), hysteria.

10. Role of Lungs and Kidneys in pH Regulation by Means of Buffers:

Lungs:

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.

Kidney:

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 H^H 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.