Phosphate

• Important intracellular and urinary buffer.
• Phosphate exists in four forms, these are:

H₃PO₄     H₂PO₄^-      HPO₄^-2     PO₄^-3

• They are converted through this series by titration, with three pK values of 2.1, 6.8, and 12.7, as shown:

Thus, when a solution of phosphoric acid (H₃PO₄) is titrated completely, there will exist three buffering regions. However, physiologically, we only need to deal with the pH range between pH 4.5 (acidic urine) and 8.0 (alkaline urine). For this reason we only have to concern ourselves with two forms of phosphate (H₂PO₄^- and HPO₄^-2) and one pK value (6.8). H₂PO₄^- is the acid form, and HPO₄^-2 is the salt form. At a pH of 6.8, the concentration of these two forms in a given compartment are equal.

Bicarbonate
Probably the most important physiological buffer. Other buffers, such as protein, quantitatively buffer more acid. It’s important because:

• it’s the bicarbonate buffer system that is regulated,
• the acid component of the system is volatile (CO₂), and can be excreted rapidly by pulmonary respiration, and
• it’s in high concentration in plasma (HCO₃^- = 24 meq/l).

Concentrations and sum reaction indicates that we don't need to consider carbonic acid.

Carbonic anhydrase

• Located in cytosol of cells (renal cells and RBC’s).
• Along the brush border of the proximal tubule. Membrane-bound enzyme with the active site facing the nephron lumen.
• Not found in plasma.

In the Henderson - Hasselbalch (HH) equation, HCO₃^- is the salt and CO₂ is the acid, as shown:

pH = pK + log ([HCO₃^-] / [CO₂])

where pK = 6.1 and both [HCO₃^-] and [CO₂] is in units of mmol/l.  But, since 0.03 mmoles/l CO₂ = 1 mmHg CO₂, then:

pH = pK + log ([HCO₃^-] / 0.03 pCO₂)

Normal values:

pCO₂ = 40 mmHg [HCO₃^-] = 24 mmol/l

[HCO₃^-] / 0.03 pCO₂ = 20

Hemoglobin:

• Located in RBC.
• Very important buffer of plasma pH.
• pK of hemoglobin is dependent on oxygenation.

pK_oxy = 6.7   pK_deoxy = 7.9

pH = pK + log ([Hb^-] / [Hb-H])

What effect does the change in pK have?

Keq = [Hb^-] [H⁺] / [Hb-H]

With a drop in the oxygenation, the reaction will be shifted toward Hb-H formation.   You can calculate that when

• pK = 6.7,  Keq = 0.2 x 10^-6
• pK = 7.9,  Keq = 0.013 x 10^-6

Renal proton secretion (H⁺)

Normally, there is no bicarbonate in the urine. 100% is reabsorbed.

In this figure, note the source of protons that get into the lumen in each case.

• In reabsorption of bicarbonate, the protons are just cycled across the luminal membrane. They are secreted into the lumen, but return as CO₂. In this case, we have no net secretion of acid.
• In the second example, we have net acid excretion. The proton that was used was derived from CO₂ from the peritubular fluid. It was pumped into the lumen and buffered by luminal phosphate. The phosphate formed is a titratable acid. No titratable acid is formed during bicarbonate reabsorption.
• Both of these process occur simultaneously and are under control by the same mechanism (renal acidification). What neutralizes a given proton after it enters the lumen depends on what it encounters. Most of the bicarbonate is reabsorbed early in the nephron because it is there to buffer this acid being excreted. Only after the urinary buffers are titrated does the luminal pH begin to fall significantly. This occurs primarily in the collecting duct.

This figure also illustrates the role of carbonic anhydrase in bicarbonate reabsorption. 

In each case, note the source of acid.

The cells of the nephron can also directly secrete HCO3- into the lumen of the distal tubule under conditions of a metabolic alkalosis.  However, the significance of this pathway is probably minor.  Bicarbonate can appear in the urine during a metabolic alkalosis just by a decrease in its reabsorption.

What is titratable acid anyway?
It is the quantity of acid excreted into the urine. It can be measured by determining the amount of base (OH^-) needed to bring the pH back to that of blood (7.4).

The ratio [HPO₄^-2] / [H₂PO₄^-]

• at pH 7.4 is ~4
• at pH 5.5 is 0.05

Thus, during the formation of acidic urine, almost the entire quantity of phosphate is titrated from HPO₄^-2 to H₂PO₄^-. As protons are secreted into the nephron lumen, the pH only decreases significantly in the late distal tubule and in the collecting duct.  After this occurs, the pH of the tubular fluid drops to the lower limit quickly.  The titration of the urinary buffers during the transit of filtrate through the nephron can be seen in the following figure.

Although ammonia/ammonium is synthesized exclusively in the proximal tubule, it is concentrated in the collecting duct (as ammonium) by the process of diffusion trapping. The pH in the late portion of the nephron are low, for reasons discussed above, and this causes concentration of ammonium in this part of the nephron.

Some text books consider ammonium a titratable acid, but this not is uniformly believed.   Since the pK of ammonia is 9.3, it is essentially always all in the protonated form.