PH microclimate

Mucus layer (glycoprotein gel, 90% oligosaccharide, implicated in pH microclimate) Unstirred water layer... 

The absorption of short-chain weak acids in the rat intestine, as a function of pH, does not appear to conform to the pH partition hypothesis [44]. Similar anomalies were found with weak bases [77]. The apparent pKa values observed in the absorp-tion-pH curve were shifted to higher values for acids and to lower values for bases, compared with the true pKa values. Such deviations could be explained by the effect of an acid layer on the apical side of cells, the so-called acid pH microclimate [44,70,73,76-84],... 

YF Shiau, P Fernandez, MJ Jackson, S McMonagle. Mechanisms maintaining alow-pH microclimate in the intestine. Am J Physiol 248 G608-G617, 1985. 

Shiau et al. [73] directly measured the microclimate pH, pHm, to be 5.2-6.7 in different sections of the intestine (very reproducible values in a given segment) covered with the normal mucus layer, as the luminal (bulk) pH, pH/, was maintained at 7.2. Good controls ruled out pH electrode artifacts. With the mucus layer washed off, pHm rose from 5.4 to 7.2. Values of pHfo as low as 3 and as high as 10 remarkably did not affect values of pHm. Glucose did not affect pHm when the microclimate was established. However, when the mucus layer had been washed off and pHm was allowed to rise to pHfo, the addition of 28 mM glucose caused the original low pHm to be reestablished after 5 min. Shiau et al. [73] hypothesized that the mucus layer was an ampholyte (of considerable pH buffer capacity) that created the pH acid microclimate. 

The tips of villi have the lowest pHm values, whereas the crypt regions have pHm > 8 values [70]. Most remarkable was that an alkaline microclimate (pHm 8) was observed in the human stomach, whose bulk pHfo is generally about 1.7. In the stomach and duodenum, the near-neutral microclimate pH was attributed to the secretion of HCO3 from the epithelium [70]. 

Said et al. [78] directly measured the acid microclimate on the surface of gastrointestinal tract (GIT) epithelial cells (intact with mucus layer) in rats. The pH on the apical (donor) side of the cells varied from 6.0 to 8.0, while the pH on the basolateral (acceptor) side was 7.4. Furthermore, the pH gradient between... 

TABLE 7.2 Microclimate pH on the Apical Side of Epithelial Cells in the GIT in Rats... 

Permeability is a property closely tied to the environment of the epithelial cell surface. There is little point in measuring permeability at pH 1.7, if the microclimate barrier has pH >5 and <8, averaging 6. An in vitro permeability screen based on donor pH 5.0-7.4 and acceptor pH 7.4 seems about right. It will be useful to correct the data for the unstirred water layer effect, using computational methods. 

Precellular solute ionization dictates membrane permeability dependence on mucosal pH. Therefore, lumenal or cellular events that affect mucosal microclimate pH may alter the membrane transport of ionizable solutes. The mucosal microclimate pH is defined by a region in the neighborhood of the mucosal membrane in which pH is lower than in the lumenal fluid. This is the result of proton secretion by the enterocytes, for which outward diffusion is slowed by intestinal mucus. (In fact, mucosal secretion of any ion coupled with mucus-restricted diffusion will provide an ionic microclimate.) Important differences in solute transport between experimental systems may be due to differences in intestinal ions and mucus secretion. It might be anticipated that microclimate pH effects would be less pronounced in epithelial cell culture (devoid of goblet cells) transport studies than in whole intestinal tissue. 

Membrane uptake of nonionized solute is favored over that of ionized solute by the membrane/water partition coefficient (Kp). If Kp = 1 for a nonionized solute, membrane permeability should mirror the solute ionization curve (i.e., membrane permeability should be half the maximum value when mucosal pH equals solute pKa). When the Kp is high, membrane uptake of nonionized solute shifts the ionization equilibrium in the mucosal microclimate to replace nonionized solute removed by the membrane. As a result, solute membrane permeability (absorption rate) versus pH curves are shifted toward the right for weak acids and toward the left for weak bases (Fig. 7). 

Figure 11 Schematic of mucosal membrane sodium-proton exchanger and chloride-bicarbonate exchanger responsible for pH homeostasis in enterocyte cytosol. Microclimate pH is maintained by mucosal slowing of proton diffusion away from the lumenal membrane.

Figure 12 Schematic of generation of mucosal microclimate pH as a transmucosal proton-gradient driving force for di- and tripeptide carrier-mediated translocation across the mucosal membrane into the enterocyte.

The majority of evidence supporting the pH-partition hypothesis is from studies of gastrointestinal absorption, renal excretion, and gastric secretion of drugs [11]. While correlation between absorption rate and pKa was found to be consistent with the pH-partition hypothesis, deviations from this hypothesis were often reported [12]. Such deviations were explained by the existence of a mucosal unstirred layer [13,14] and/or a microclimate pH [15]. 

Tab. 3.2. Microclimate pH on the apical side of epithelial cells in the gastrointestinal (Gl) tract in rats [32].

Physiological Buffer Systems Recently, a lot of efforts have been made on how to increase the biorelevance of the Caco-2 model [63, 47, 64, 65,105], Historically, the media used for Caco-2 experiments were buffered at pH 7.4 on both sides of the monolayer. The pH in the cellular interstice and blood compartment is known to be 7.4. However, the pH in the upper GI tract under fasted conditions ranges from 5.0 to 6.5, with an acidic microclimate existing just above the epithelial cell layer estimated to be between 5.8 and 6.3 [90], The pH of the apical medium can have a critical effect on the transport of drugs, especially for drugs with a pKa close to 7, or when pH-dependent transporters are involved. 

Similar to the PAMPA and the Caco-2 models, the experimental pH of the buffer solution can be changed in the Ussing chambers model. However, it seems that the impact of changing the pH of the mucosal (= apical) buffer solution is lower than for the other two systems [82], This is probably due to the presence of the mucus layer retaining the microclimate pH regardless of the luminal pH using the Ussing chambers technique [82],... 

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