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A reappraisal of the magnitude and implications of the intestinal unstirred layer
GASTROENTEROLOGY
SPECIAL
REPORTS
1991;101:843-847
AND REVIEWS
A Reappraisal of the Magnitude and Implications of the Intestinal Unstirred Layer ALESSANDRA
STROCCHI
and MICHAEL
D. LEVITT
Research Service, Veterans Affairs Medical Center, Minneapolis,
Until recently, a variety of studies had suggested that luminal stirring in the jejunum is relatively poor, with unstirred layers of about 600 pm reported for humans and 300-900 pm for animals. Unstirred layers of this magnitude would markedly retard the absorption of all solutes, and diffusion through this layer would be the rate-limiting step in the uptake of all rapidly absorbed compounds. As a result, luminal stirring, rather than epithelial transport, would be the major variable influencing absorption rate. However, recent studies in dogs and humans have shown that the unstirred layer has a maximal apparent thickness of only about 40 pm. This layer is far thinner than what can be achieved in vitro with vigorous stirring with a magnetic bar, suggesting that some unique stirring mechanism, perhaps villous contractions, is responsible for this extraordinarily efficient mixing. A 40-Frn unstirred layer would produce only about l/15 the resistance of the previously reported 600 pm value; with this thinner layer, alterations in either luminal stirring or epithelial function could readily influence the absorption rate of rapidly transported compounds.
It
has been known for 100 years that, despite vigorous mixing, water adjacent to a solid surface remains unstirred. However, it was not until the studies of Dietschy et al. (1,2)in the 1970s that gastroenterologists focused their attention on the influence of unstirred layers on intestinal absorption. Most previous discussions of this topic have been relatively complex and have used a good deal of mathematics. Thus, whereas gastroenterologists recognize that an unstirred layer exists in the intestine, there is limited understanding of how it has been measured and of the quantitative implications of this layer on the absorptive process, In this brief review we attempt to provide a simple, intuitive, and largely nonmathematical discussion of this topic, with particular emphasis on recent studies indicating that lumi-
Minnesota
nal stirring is far more efficient than previously proposed. If radial stirring of luminal fluid is perfect, the concentration of solutes would be constant throughout a cross-section of luminal contents. However, with imperfect luminal stirring, the concentration of rapidly absorbed compounds in the fluid adjacent to the mucosa decreases relative to the concentration in the center of the lumen. This peripheral, solutedepleted fluid then creates an aqueous diffusion barrier separating the brush border from the higher concentrations of solute present in more centrally located fluid. Whereas the diffusion barrier created by imperfect luminal stirring probably takes the form of a gradient of progressively poorer stirring of fluid as the mucosa is approached, this complex situation is commonly modeled as a uniform layer of totally unstirred fluid separating perfectly mixed bulk luminal contents from the brush border. Until recently, all measurements of the unstirred layer in the human intestine were obtained with the osmotic transient technique (3-5). These studies suggested that a 600~km unstirred layer separated the brush border from bulk luminal contents. Whereas this value seems to have been widely accepted in the scientific literature, little attention has been paid to the seemingly impossible physiological implications of a 600~km unstirred layer. The resistance to absorption created by such an unstirred layer is inversely proportional to its area. The maximal area would be that of the entire villous surface, whereas the minimal value would be the area of a “cylinder” of fluid over the villous tips. Because the intervillous space is only about XI Frn wide, 25 Km is the maximal thickness of unstirred water that could separate solute from epithelium in this space. Thus, a 600~km unstirred layer This is a U.S. government work. There are no restrictions use.
on its
844
STROCCHI AND LEVITT
GASTROENTEROLOGY
must be located over the villous tips and have a surface area roughly equal to that of the villous tips. Absorption is extremely slow when solutes have to diffuse across an unstirred layer of 600 pm to reach the villi. For example, the maximal rate that glucose could be absorbed through such a layer would be equal to about 1% of the luminal content per minute (see Appendix for calculation). This slow absorption is seemingly at odds with studies showing that 30% 50% of ingested glucose is absorbed during the few minutes that elapse as glucose passes from the pylorus to the ligament of Treitz (6,7). In the absence of bile salts, long-chain fatty acid absorption should be dependent on the rate that the poorly water-soluble monomers can diffuse through the unstirred layer to reach the brush border. An unstirred layer of 600 km should result in malabsorption of least 90% of the unsaturated, long-chain fatty acids present in the usual meal (see Appendix for calculation). However, fecal fat measurements in patients with biliary tract obstruction indicate malabsorption of only about 30% of ingested fat (8). A second implication of a 600-pm unstirred layer is
600pm
Vol. 101, No. 3
that diffusion through this layer becomes the ratelimiting step in absorption of all rapidly absorbed substances and variations in epithelial function play little role in the absorptive process. Figure 1 schematically portrays relative resistances of the unstirred layer and the epithelial cell to the passage of compounds that are rapidly transported across the epithelial cell membrane, i.e., lipid-soluble compounds or water-soluble compounds that are transported by an efficient carrier system. Whereas the precise resistance of the epithelial cell to such rapidly transported compounds is unknown, for the purpose of this discussion, membrane resistance is considered to be negligible and the epithelial resistance is assumed to equal an aqueous diffusion barrier with a thickness of the epithelial cell (about 30 km). The unstirred layer creates an aqueous barrier that is 600 t.t.rnthick. As the two barriers are in series, their resistances are additive, and the unstirred layer provides X1/21 of the total resistance. In this situation, enhanced epithelial transport cannot appreciably increase absorption rate because infinite epithelial permeability (zero resistance) only decreases the total resistance by about 5%.
Relative Resistance A 20
B 20
C 20 Relative Resistance
30pm
1 ---
0
22
2tal21
20
42
40pm 30pm
{ 4
A 1.3 1 ---
B 1.3 0
C 1.3 3.3
Total2.3 1.3 4.3
Figure 1. Schematic representation of the relative resistances to absorption of the unstirred layer and the epithelial cell for solutes with high membrane permeability. On the left, unstirred layer has the resistance of a 600-pm layer of water over the villous tips. The membrane of the epithelial cell is assumed to have infinite permeability to the solute (resistance = 0), and the resistance of the epithelium equals the thickness (30 pm) of the aqueous barrier of the cell. The unstirred layer represents 20/H of the total resistance, and an infinite increase in epithelial function (resistance = 0) has a minimal (5%) effect on total resistance, and, hence, absorption. Epithelial resistance would have to increase by Z&fold to double the total resistance and halve the absorption rate. The diagram on the right shows a similar analysis for an unstirred layer of 40 pm, the maximal value calculated to exist in the human jejunum (see text). In this situation, modest increases or decreases in epithelial resistance have appreciable effects on total resistance.
September
INTESTINAL
1991
Whereas defective epithelial function could reduce absorption, an enormous (22-fold) decline in the efficiency of epithelial transport would be required to double total resistance and halve the absorption rate (Figure 1).If this scenario is correct, the malabsorption of rapidly absorbed compounds observed with diseases of the intestine must be largely a result of decreased luminal stirring, rather than diminished mucosal transport. Similarly, the adaptation that improves absorption of such compounds after intestinal resection must produce better luminal stirring rather than enhancement of epithelial cell transport. For some compounds (i.e., hydrophylic solutes lacking an efficient carrier system), epithelial resistance could markedly exceed that of the unstirred layer and, thus, become rate limiting. However, such compounds would be only partially absorbed, even by the normal small intestine. For example, a compound with an epithelial resistance three times that of a 600-km unstirred layer would have a fractional absorption rate of only about 0.25%/min, yielding only about 50% absorption during a J-hour transit in the small bowel. Because most nutrients are nearly completely absorbed in the small bowel, it seemingly follows that epithelial transport cannot be the limiting step in their absorption if the unstirred layer truly were 600 pm thick. The 600-t.Lm value for the unstirred layer in the human intestine was derived from studies using the osmotic transient technique, a method initially used to measure unstirred layers in the gallbladder (9). The potential difference (PD) across the gallbladder (or intestinal) mucosa is a function of the osmolality of the solution bathing the mucosal surface. When a solution was emptied from the gallbladder and replaced with a solution of different osmolality, a gradual rather than instantaneous change in PD was observed. This finding reflected the presence of a residual layer of the initial solution adjacent to the mucosa that acted as a diffusion barrier to the solute present in the second solution; the thickness of this residual layer could be calculated from the half-time required to obtain a new steady-state PD. Whereas this measurement reflects the thickness of the layer of fluid left behind when the gallbladder is manually emptied, it need bear little resemblance to the unstirred layer that develops when solutes are absorbed under physiological conditions. For example, if the gallbladder lumen were entirely unmixed, the unstirred layer would be many times thicker than the residual layer left behind after emptying. This technique was then applied to the intestine by moving a sheet of rabbit jejunal mucosa between beakers containing solutions of different osmolality (10). The thickness of the unstirred layer was about 300 Frn with no stirring of the solution and 115 pm
UNSTIRRED
LAYER
845
with very vigorous mixing with a magnetic bar. Once again, these measurements presumably reflected the fluid layer that adhered to the mucosa as it was moved between the solutions rather than the unstirred layer that might be present, in vivo. In an attempt to obtain in vivo measurements of intestinal unstirred layers in humans, several investigators recorded the PD of constantly perfused jejunal segments after a sudden alteration of the osmolality of the infusate (3-5). Whereas the unstirred layer was reported to be about 600 km thick, the relatively slow alteration of the osmolality of the bulk luminal fluid in these studies appears to render the conclusions uninterpretable. For example, in one study (3) a 25-cm segment was perfused at a rate of 10 mL/min. Because the volume of luminal fluid in this segment would be about 75 mL (1 I), 3.5 minutes would have been required to merely washout half of the initial perfusion solution from the lumen. Thus, a very thick unstirred layer would have been measured with this technique even if no unstirred layer existed in the gut. A second means of determining the unstirred layer thickness is based on measurements of absorption rate of rapidly transported probes. Because net secretion or absorption of fluid, respectively, increases or decreases the resistance of a given thickness of unstirred layer, the unstirred layer measurement obtained with absorption technique represents an apparent or effective thickness. The thickness of the unstirred layer t is calculated from the formula that determines the rate of diffusion of a solute (i.e., the absorption rate Q across an unstirred layer) :
t=
VNANG> - GA
Q
,
(Equation
1)
where D is the aqueous diffusion constant of the solute; A is the surface area of the unstirred layer; and C, - C, is the concentration gradient driving diffusion across the unstirred layer, where C, and C, are the concentrations of the probe in bulk luminal and brush border fluid, respectively. The value of D for many solutes is available in the literature; C, is known, and Q is experimentally measured. As discussed above, the unstirred layer appears to reside over the villous tips and thus has an area equal to that of the villous tips which is calculated from the length and volume of the gut segment. The only unknown in Equation 1, CM, is a function of the epithelial resistance to the probe. The direct determination of C, requires measurement of the uptake of probes in the absence of the unstirred layer. To this end, the infusion of gas bubbles (I 2) or a gaseous infusate (13) have been used to minimize the unstirred layer effect in animal studies. Because these studies showed that
846
STROCCHI AND LEVITT
the unstirred layer created the bulk of the resistance to the absorption of rapidly transported probes, a second, simple approach has been to assume that epithelial resistance is negligible (C, = 0),an approach that yields a maximal value for the unstirred layer (see Equation 1). Nearly all in vivo determinations of the unstirred layer via measurement of the uptake of probes have been performed in anesthetized, laparotomized rats. Whereas unstirred layers of 300-900 km have been it should be emphasized that the observed (12-15), jejunum is virtually’amotile in this situation. Perfused fluid moves through the lumen with nearly perfect laminar flow, indicating that the gut segment is acting like an inert tube (16). When perfusion studies were performed via chronically implanted cannulas in awake, nonlaparotomized rats, maximal apparent unstirred layers (assuming C, to be negligible) were found to be only about 100 pm (17).Thus, the previously reported values of 300-900 km in rats apparently were an artifact of the amotile gut resulting from laparotomy. Recently, we perfused the jejunum of conscious dogs via a Thomas cannula and calculated the maximal apparent unstirred layer thickness that would have permitted the observed absorption rate of two rapidly absorbed probes (glucose and [14C]warfarin) (18). At perfusion rates of 5 and 25 mL/min, maximal apparent unstirred layers were about 50 and 35 pm, respectively. In addition, the maximal unstirred layer in the human jejunum was calculated from the rates of absorption of glucose reported in nine different constant perfusion studies (18).This calculation showed that the observed absorption rate of glucose could not have occurred if the apparent thickness of the unstirred layer were greater than 40 Frn, and, to the extent that the epithelial cell offers appreciable resistance to glucose absorption, unstirred layer thickness would have been even less than 40 pm. Surprisingly, the normally functioning intestine maintains a much thinner unstirred layer than apparently can be achieved with vigorous stirring with a magnetic bar, in vitro (10). Presumably, the intermittent contractions of the longitudinal and circular muscle could not induce this excellent mixing. Although speculative, it seems possible that the thin, in vivo unstirred layer results from contractions of the villi, a unique stirring mechanism that functions at the absorptive surface rather than in the center of the lumen. An unstirred layer of 40 pm would permit absorption to occur up to 15 times more rapidly than the previously reported 600~pm value; this faster transport is more compatible with clinical and experimental measurements of the absorption of glucose or fatty
GASTROENTEROLOGY
Vol. 101, No. 3
acids. from nonmicellar solutions. In addition, this thinner barrier would markedly reduce the disparity between unstirred layer and mucosal resistances to rapidly absorbed compounds. As shown in Figure 1, if the unstirred layer is 40 pm thick, conditions that enhance or mildly impair mucosal transport could have an appreciable influence on absorption rate of rapidly absorbed compounds. Thus, our present concepts concerning the relative roles of the unstirred layer and epithelial transport in nutrient absorption need to be reassessed in light of a jejunal unstirred layer that has a maximal thickness of only 40 urn. Appendix Estimation of maximal fractional absorption rate of glucose from the human intestine if an unstirred layer of 600 pm existed over the villous tips:
Fraction Absorbedlmin Absorption
Rate
= Quantity in Lumen Absorption
Rate =
(D)(A)(C, - C,) t ’
(Equation A)
(Equation B)
where D, the aqueous diffusion coefficient of glucose, is 3.8 x 10m4cm’/min; A is the area/cm of the unstirred layer over the villous tips (A = 2rrr); C, is the concentration of glucose in bulk luminal contents; CM, the concentration of glucose at the mucosa, is assumed to be zero, yielding a maximal absorption rate; and t, unstirred layer thickness, is 0.06 cm. Quantity in Lumen/cm = (C,) (luminal volume/cm), where luminal volumelcm ing in Equation A:
= &.
(Equation
Therefore,
C)
substitut-
Fraction Absorbedlmin = (3.8x 10-4)(2~r)(CL) WWC,)(~f?
= 0.012/r.
(Equation D)
Because the gut volume is roughly 3 mI_Jcm (ll), r is roughly 1 cm and the fractional absorption rate of luminal glucose would be about l%/min. Fractional absorption rate decreases inversely with increasing unstirred layer or epithelial resistance. Thus, if epithelial resistance were three times that of a 600+&m unstirred layer, the total resistance would be equivalent to an unstirred layer of 0.24cm in Equation D, and the fractional absorption rate would decrease to 0.25%/min.
INTESTINAL
1991
September
Estimation of maximal absorption rate (QI of long-chain fatty acid monomers in the absence of bile salts if an unstirred layer (t) of 600 pm existed over the villous tips:
(9) =
PJb‘W, - Cd (Equation D) @I ’
where D is 4 x 10m4cm’/min; A is 1500 cm* calculated from 250 cm of small bowel and an area over the villous tips of 6 cm’/cm (11); CL = 0.68 mmol/L [the maximal aqueous solubility reported for laurate (19), the most soluble of the longer chain fatty acids]; C, = 0;and t = 0.06 cm. Substituting in Equation D: (~ = (4 x lo-‘)(1500)(0.68/1000) (0.06) = 0.0068 mmol/min.
(Equation E)
Assuming a residence time of the meal in the small bowel of 480 minutes (8 hours), a maximum of 3.3 mmol (660 mg) of laurate would be absorbed. If I/3 of the the 20-g fat content of a meal is laurate, a maximum of 10% of the laurate could be absorbed. The maximal aqueous solubilities of longer chain fatty acids such as palmitate and stearate are appreciably less than that of laurate (19), and their maximal absorption rates would be proportionately reduced relative to that calculated above for laurate. References 1. Dietschy
2.
3.
4.
5.
JM, Sallee VL, Wilson FA. Unstirred water layer and absorption across the intestinal mucosa. Gastroenterology 1971; 61:932-934. Wilson FA, Dietschy JM. Characterization of bile acid absorption across the unstirred water layer and brush border of the rat jejunum. J Clin Invest 1972;51:3015-3025, Read NW, Barker DC, Levin RJ, Holdsworth CD. Unstirred layer and kinetics of electrogenic glucose absorption in the human jejunum in situ. Gut 1977;18:865-876. Frase LL, Strickland AD, Kachel GW, Krejs GJ. Enhanced glucose absorption in the jejunum of patients with cystic fibrosis. Gastroenterology 1985;88:478-484. Sparso BH, Luke M, Wium E. Electrogenic transport of glucose in the normal upper duodenum. II. Unstirred water layer and estimation of real transport constants. Stand J Gastroenterol 1984;19:568-574.
6. Borgstrom
B, Dahlqvist
A, Lundh
UNSTIRRED
G, Sjovall
LAYER
847
J. Studies
of
intestinal digestion and absorption in the human. J Clin Invest 1957;36:1521-1536. 7. Shay H, Gershon-Cohen J, Fels SS, Munro FL, Siplet H. The absorption and dilution of glucose solutions in the human stomach and duodenum. Am J Dig Dis 1939-1940;6:535-544. 8. Wilson
clinical
FA, Dietschy JM. Differential diagnostic approach to problems of malabsorption. Gastroenterology 1971;61:
911-931. 9. Diamond
concentration
JM.
A rapid method for determining voltagerelations across membranes. J Physiol (Lond)
1966;183:83-100. H, Dietschy
10. Westergaard
JM. Delineation
of the dimensions
and permeability characteristics of the two major diffusion barriers to passive mucosal uptake in the rabbit intestine. J Clin Invest 54:718-732, 1974. 11. Dillard RL, Eastman H, Fordtran JS. Volume-flow relationship during the transport of fluid through the small intestine. Gastroenterology 1965;49:58-66. 12. Winne D. Unstirred layer thickness in perfused rat jejunum in vivo. Experientia (Basel) 1976;32:1278-1279. 13. Levitt MD, Aufderheide T, Fetzer CA, Bond JH, Levitt DG. Use of carbon monoxide to measure luminal stirring in the rat gut. J Clin Invest 1984;74:2056-2064, H, Miyamoto Y, Iga T, Hanano M. Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer. Biochim Biophys Acta
14. Yuasa
1986;856:219-230, H, Holtermuller KH, Dietschy JM. Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am J Physiol 1986;250:G7274735. 16. Levitt MD, Knelp JM, Levitt DG. Use of laminar flow and unstirred layer models to predict intestinal absorption in the rat. J Clin Invest 1988;81:1365-1369. 17. Anderson BW, Levine AS, Levitt DG, Kneip JM, Levitt MD. 15. Westergaard
Physiological measurement of luminal stirring in perfused rat jejunum. Am J Physiol 1988;254:G843-G848. 18. Levitt MD, Furne JK, Strocchi A, Anderson BW, Levitt DG. Physiological measurements of luminal stirring in the dog and human small bowel. J Clin Invest 1990;86:1540-1547. 19. Westergaard H, Dietschy JM. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. J Clin Invest 1976;58:97108.
Received December 28,199O. Accepted March 18,199l. This study was supported in part by Veteran’s Affairs Merit Review Funds and NIDDK no. 2 ROlDK133309-22. Address requests for reprints to: Michael D. Levitt, M.D., Veterans Affairs Medical Center (151), One Veteran’s Drive, Minneapolis, Minnesota 55417.
SPECIAL
REPORTS
1991;101:843-847
AND REVIEWS
A Reappraisal of the Magnitude and Implications of the Intestinal Unstirred Layer ALESSANDRA
STROCCHI
and MICHAEL
D. LEVITT
Research Service, Veterans Affairs Medical Center, Minneapolis,
Until recently, a variety of studies had suggested that luminal stirring in the jejunum is relatively poor, with unstirred layers of about 600 pm reported for humans and 300-900 pm for animals. Unstirred layers of this magnitude would markedly retard the absorption of all solutes, and diffusion through this layer would be the rate-limiting step in the uptake of all rapidly absorbed compounds. As a result, luminal stirring, rather than epithelial transport, would be the major variable influencing absorption rate. However, recent studies in dogs and humans have shown that the unstirred layer has a maximal apparent thickness of only about 40 pm. This layer is far thinner than what can be achieved in vitro with vigorous stirring with a magnetic bar, suggesting that some unique stirring mechanism, perhaps villous contractions, is responsible for this extraordinarily efficient mixing. A 40-Frn unstirred layer would produce only about l/15 the resistance of the previously reported 600 pm value; with this thinner layer, alterations in either luminal stirring or epithelial function could readily influence the absorption rate of rapidly transported compounds.
It
has been known for 100 years that, despite vigorous mixing, water adjacent to a solid surface remains unstirred. However, it was not until the studies of Dietschy et al. (1,2)in the 1970s that gastroenterologists focused their attention on the influence of unstirred layers on intestinal absorption. Most previous discussions of this topic have been relatively complex and have used a good deal of mathematics. Thus, whereas gastroenterologists recognize that an unstirred layer exists in the intestine, there is limited understanding of how it has been measured and of the quantitative implications of this layer on the absorptive process, In this brief review we attempt to provide a simple, intuitive, and largely nonmathematical discussion of this topic, with particular emphasis on recent studies indicating that lumi-
Minnesota
nal stirring is far more efficient than previously proposed. If radial stirring of luminal fluid is perfect, the concentration of solutes would be constant throughout a cross-section of luminal contents. However, with imperfect luminal stirring, the concentration of rapidly absorbed compounds in the fluid adjacent to the mucosa decreases relative to the concentration in the center of the lumen. This peripheral, solutedepleted fluid then creates an aqueous diffusion barrier separating the brush border from the higher concentrations of solute present in more centrally located fluid. Whereas the diffusion barrier created by imperfect luminal stirring probably takes the form of a gradient of progressively poorer stirring of fluid as the mucosa is approached, this complex situation is commonly modeled as a uniform layer of totally unstirred fluid separating perfectly mixed bulk luminal contents from the brush border. Until recently, all measurements of the unstirred layer in the human intestine were obtained with the osmotic transient technique (3-5). These studies suggested that a 600~km unstirred layer separated the brush border from bulk luminal contents. Whereas this value seems to have been widely accepted in the scientific literature, little attention has been paid to the seemingly impossible physiological implications of a 600~km unstirred layer. The resistance to absorption created by such an unstirred layer is inversely proportional to its area. The maximal area would be that of the entire villous surface, whereas the minimal value would be the area of a “cylinder” of fluid over the villous tips. Because the intervillous space is only about XI Frn wide, 25 Km is the maximal thickness of unstirred water that could separate solute from epithelium in this space. Thus, a 600~km unstirred layer This is a U.S. government work. There are no restrictions use.
on its
844
STROCCHI AND LEVITT
GASTROENTEROLOGY
must be located over the villous tips and have a surface area roughly equal to that of the villous tips. Absorption is extremely slow when solutes have to diffuse across an unstirred layer of 600 pm to reach the villi. For example, the maximal rate that glucose could be absorbed through such a layer would be equal to about 1% of the luminal content per minute (see Appendix for calculation). This slow absorption is seemingly at odds with studies showing that 30% 50% of ingested glucose is absorbed during the few minutes that elapse as glucose passes from the pylorus to the ligament of Treitz (6,7). In the absence of bile salts, long-chain fatty acid absorption should be dependent on the rate that the poorly water-soluble monomers can diffuse through the unstirred layer to reach the brush border. An unstirred layer of 600 km should result in malabsorption of least 90% of the unsaturated, long-chain fatty acids present in the usual meal (see Appendix for calculation). However, fecal fat measurements in patients with biliary tract obstruction indicate malabsorption of only about 30% of ingested fat (8). A second implication of a 600-pm unstirred layer is
600pm
Vol. 101, No. 3
that diffusion through this layer becomes the ratelimiting step in absorption of all rapidly absorbed substances and variations in epithelial function play little role in the absorptive process. Figure 1 schematically portrays relative resistances of the unstirred layer and the epithelial cell to the passage of compounds that are rapidly transported across the epithelial cell membrane, i.e., lipid-soluble compounds or water-soluble compounds that are transported by an efficient carrier system. Whereas the precise resistance of the epithelial cell to such rapidly transported compounds is unknown, for the purpose of this discussion, membrane resistance is considered to be negligible and the epithelial resistance is assumed to equal an aqueous diffusion barrier with a thickness of the epithelial cell (about 30 km). The unstirred layer creates an aqueous barrier that is 600 t.t.rnthick. As the two barriers are in series, their resistances are additive, and the unstirred layer provides X1/21 of the total resistance. In this situation, enhanced epithelial transport cannot appreciably increase absorption rate because infinite epithelial permeability (zero resistance) only decreases the total resistance by about 5%.
Relative Resistance A 20
B 20
C 20 Relative Resistance
30pm
1 ---
0
22
2tal21
20
42
40pm 30pm
{ 4
A 1.3 1 ---
B 1.3 0
C 1.3 3.3
Total2.3 1.3 4.3
Figure 1. Schematic representation of the relative resistances to absorption of the unstirred layer and the epithelial cell for solutes with high membrane permeability. On the left, unstirred layer has the resistance of a 600-pm layer of water over the villous tips. The membrane of the epithelial cell is assumed to have infinite permeability to the solute (resistance = 0), and the resistance of the epithelium equals the thickness (30 pm) of the aqueous barrier of the cell. The unstirred layer represents 20/H of the total resistance, and an infinite increase in epithelial function (resistance = 0) has a minimal (5%) effect on total resistance, and, hence, absorption. Epithelial resistance would have to increase by Z&fold to double the total resistance and halve the absorption rate. The diagram on the right shows a similar analysis for an unstirred layer of 40 pm, the maximal value calculated to exist in the human jejunum (see text). In this situation, modest increases or decreases in epithelial resistance have appreciable effects on total resistance.
September
INTESTINAL
1991
Whereas defective epithelial function could reduce absorption, an enormous (22-fold) decline in the efficiency of epithelial transport would be required to double total resistance and halve the absorption rate (Figure 1).If this scenario is correct, the malabsorption of rapidly absorbed compounds observed with diseases of the intestine must be largely a result of decreased luminal stirring, rather than diminished mucosal transport. Similarly, the adaptation that improves absorption of such compounds after intestinal resection must produce better luminal stirring rather than enhancement of epithelial cell transport. For some compounds (i.e., hydrophylic solutes lacking an efficient carrier system), epithelial resistance could markedly exceed that of the unstirred layer and, thus, become rate limiting. However, such compounds would be only partially absorbed, even by the normal small intestine. For example, a compound with an epithelial resistance three times that of a 600-km unstirred layer would have a fractional absorption rate of only about 0.25%/min, yielding only about 50% absorption during a J-hour transit in the small bowel. Because most nutrients are nearly completely absorbed in the small bowel, it seemingly follows that epithelial transport cannot be the limiting step in their absorption if the unstirred layer truly were 600 pm thick. The 600-t.Lm value for the unstirred layer in the human intestine was derived from studies using the osmotic transient technique, a method initially used to measure unstirred layers in the gallbladder (9). The potential difference (PD) across the gallbladder (or intestinal) mucosa is a function of the osmolality of the solution bathing the mucosal surface. When a solution was emptied from the gallbladder and replaced with a solution of different osmolality, a gradual rather than instantaneous change in PD was observed. This finding reflected the presence of a residual layer of the initial solution adjacent to the mucosa that acted as a diffusion barrier to the solute present in the second solution; the thickness of this residual layer could be calculated from the half-time required to obtain a new steady-state PD. Whereas this measurement reflects the thickness of the layer of fluid left behind when the gallbladder is manually emptied, it need bear little resemblance to the unstirred layer that develops when solutes are absorbed under physiological conditions. For example, if the gallbladder lumen were entirely unmixed, the unstirred layer would be many times thicker than the residual layer left behind after emptying. This technique was then applied to the intestine by moving a sheet of rabbit jejunal mucosa between beakers containing solutions of different osmolality (10). The thickness of the unstirred layer was about 300 Frn with no stirring of the solution and 115 pm
UNSTIRRED
LAYER
845
with very vigorous mixing with a magnetic bar. Once again, these measurements presumably reflected the fluid layer that adhered to the mucosa as it was moved between the solutions rather than the unstirred layer that might be present, in vivo. In an attempt to obtain in vivo measurements of intestinal unstirred layers in humans, several investigators recorded the PD of constantly perfused jejunal segments after a sudden alteration of the osmolality of the infusate (3-5). Whereas the unstirred layer was reported to be about 600 km thick, the relatively slow alteration of the osmolality of the bulk luminal fluid in these studies appears to render the conclusions uninterpretable. For example, in one study (3) a 25-cm segment was perfused at a rate of 10 mL/min. Because the volume of luminal fluid in this segment would be about 75 mL (1 I), 3.5 minutes would have been required to merely washout half of the initial perfusion solution from the lumen. Thus, a very thick unstirred layer would have been measured with this technique even if no unstirred layer existed in the gut. A second means of determining the unstirred layer thickness is based on measurements of absorption rate of rapidly transported probes. Because net secretion or absorption of fluid, respectively, increases or decreases the resistance of a given thickness of unstirred layer, the unstirred layer measurement obtained with absorption technique represents an apparent or effective thickness. The thickness of the unstirred layer t is calculated from the formula that determines the rate of diffusion of a solute (i.e., the absorption rate Q across an unstirred layer) :
t=
VNANG> - GA
Q
,
(Equation
1)
where D is the aqueous diffusion constant of the solute; A is the surface area of the unstirred layer; and C, - C, is the concentration gradient driving diffusion across the unstirred layer, where C, and C, are the concentrations of the probe in bulk luminal and brush border fluid, respectively. The value of D for many solutes is available in the literature; C, is known, and Q is experimentally measured. As discussed above, the unstirred layer appears to reside over the villous tips and thus has an area equal to that of the villous tips which is calculated from the length and volume of the gut segment. The only unknown in Equation 1, CM, is a function of the epithelial resistance to the probe. The direct determination of C, requires measurement of the uptake of probes in the absence of the unstirred layer. To this end, the infusion of gas bubbles (I 2) or a gaseous infusate (13) have been used to minimize the unstirred layer effect in animal studies. Because these studies showed that
846
STROCCHI AND LEVITT
the unstirred layer created the bulk of the resistance to the absorption of rapidly transported probes, a second, simple approach has been to assume that epithelial resistance is negligible (C, = 0),an approach that yields a maximal value for the unstirred layer (see Equation 1). Nearly all in vivo determinations of the unstirred layer via measurement of the uptake of probes have been performed in anesthetized, laparotomized rats. Whereas unstirred layers of 300-900 km have been it should be emphasized that the observed (12-15), jejunum is virtually’amotile in this situation. Perfused fluid moves through the lumen with nearly perfect laminar flow, indicating that the gut segment is acting like an inert tube (16). When perfusion studies were performed via chronically implanted cannulas in awake, nonlaparotomized rats, maximal apparent unstirred layers (assuming C, to be negligible) were found to be only about 100 pm (17).Thus, the previously reported values of 300-900 km in rats apparently were an artifact of the amotile gut resulting from laparotomy. Recently, we perfused the jejunum of conscious dogs via a Thomas cannula and calculated the maximal apparent unstirred layer thickness that would have permitted the observed absorption rate of two rapidly absorbed probes (glucose and [14C]warfarin) (18). At perfusion rates of 5 and 25 mL/min, maximal apparent unstirred layers were about 50 and 35 pm, respectively. In addition, the maximal unstirred layer in the human jejunum was calculated from the rates of absorption of glucose reported in nine different constant perfusion studies (18).This calculation showed that the observed absorption rate of glucose could not have occurred if the apparent thickness of the unstirred layer were greater than 40 Frn, and, to the extent that the epithelial cell offers appreciable resistance to glucose absorption, unstirred layer thickness would have been even less than 40 pm. Surprisingly, the normally functioning intestine maintains a much thinner unstirred layer than apparently can be achieved with vigorous stirring with a magnetic bar, in vitro (10). Presumably, the intermittent contractions of the longitudinal and circular muscle could not induce this excellent mixing. Although speculative, it seems possible that the thin, in vivo unstirred layer results from contractions of the villi, a unique stirring mechanism that functions at the absorptive surface rather than in the center of the lumen. An unstirred layer of 40 pm would permit absorption to occur up to 15 times more rapidly than the previously reported 600~pm value; this faster transport is more compatible with clinical and experimental measurements of the absorption of glucose or fatty
GASTROENTEROLOGY
Vol. 101, No. 3
acids. from nonmicellar solutions. In addition, this thinner barrier would markedly reduce the disparity between unstirred layer and mucosal resistances to rapidly absorbed compounds. As shown in Figure 1, if the unstirred layer is 40 pm thick, conditions that enhance or mildly impair mucosal transport could have an appreciable influence on absorption rate of rapidly absorbed compounds. Thus, our present concepts concerning the relative roles of the unstirred layer and epithelial transport in nutrient absorption need to be reassessed in light of a jejunal unstirred layer that has a maximal thickness of only 40 urn. Appendix Estimation of maximal fractional absorption rate of glucose from the human intestine if an unstirred layer of 600 pm existed over the villous tips:
Fraction Absorbedlmin Absorption
Rate
= Quantity in Lumen Absorption
Rate =
(D)(A)(C, - C,) t ’
(Equation A)
(Equation B)
where D, the aqueous diffusion coefficient of glucose, is 3.8 x 10m4cm’/min; A is the area/cm of the unstirred layer over the villous tips (A = 2rrr); C, is the concentration of glucose in bulk luminal contents; CM, the concentration of glucose at the mucosa, is assumed to be zero, yielding a maximal absorption rate; and t, unstirred layer thickness, is 0.06 cm. Quantity in Lumen/cm = (C,) (luminal volume/cm), where luminal volumelcm ing in Equation A:
= &.
(Equation
Therefore,
C)
substitut-
Fraction Absorbedlmin = (3.8x 10-4)(2~r)(CL) WWC,)(~f?
= 0.012/r.
(Equation D)
Because the gut volume is roughly 3 mI_Jcm (ll), r is roughly 1 cm and the fractional absorption rate of luminal glucose would be about l%/min. Fractional absorption rate decreases inversely with increasing unstirred layer or epithelial resistance. Thus, if epithelial resistance were three times that of a 600+&m unstirred layer, the total resistance would be equivalent to an unstirred layer of 0.24cm in Equation D, and the fractional absorption rate would decrease to 0.25%/min.
INTESTINAL
1991
September
Estimation of maximal absorption rate (QI of long-chain fatty acid monomers in the absence of bile salts if an unstirred layer (t) of 600 pm existed over the villous tips:
(9) =
PJb‘W, - Cd (Equation D) @I ’
where D is 4 x 10m4cm’/min; A is 1500 cm* calculated from 250 cm of small bowel and an area over the villous tips of 6 cm’/cm (11); CL = 0.68 mmol/L [the maximal aqueous solubility reported for laurate (19), the most soluble of the longer chain fatty acids]; C, = 0;and t = 0.06 cm. Substituting in Equation D: (~ = (4 x lo-‘)(1500)(0.68/1000) (0.06) = 0.0068 mmol/min.
(Equation E)
Assuming a residence time of the meal in the small bowel of 480 minutes (8 hours), a maximum of 3.3 mmol (660 mg) of laurate would be absorbed. If I/3 of the the 20-g fat content of a meal is laurate, a maximum of 10% of the laurate could be absorbed. The maximal aqueous solubilities of longer chain fatty acids such as palmitate and stearate are appreciably less than that of laurate (19), and their maximal absorption rates would be proportionately reduced relative to that calculated above for laurate. References 1. Dietschy
2.
3.
4.
5.
JM, Sallee VL, Wilson FA. Unstirred water layer and absorption across the intestinal mucosa. Gastroenterology 1971; 61:932-934. Wilson FA, Dietschy JM. Characterization of bile acid absorption across the unstirred water layer and brush border of the rat jejunum. J Clin Invest 1972;51:3015-3025, Read NW, Barker DC, Levin RJ, Holdsworth CD. Unstirred layer and kinetics of electrogenic glucose absorption in the human jejunum in situ. Gut 1977;18:865-876. Frase LL, Strickland AD, Kachel GW, Krejs GJ. Enhanced glucose absorption in the jejunum of patients with cystic fibrosis. Gastroenterology 1985;88:478-484. Sparso BH, Luke M, Wium E. Electrogenic transport of glucose in the normal upper duodenum. II. Unstirred water layer and estimation of real transport constants. Stand J Gastroenterol 1984;19:568-574.
6. Borgstrom
B, Dahlqvist
A, Lundh
UNSTIRRED
G, Sjovall
LAYER
847
J. Studies
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intestinal digestion and absorption in the human. J Clin Invest 1957;36:1521-1536. 7. Shay H, Gershon-Cohen J, Fels SS, Munro FL, Siplet H. The absorption and dilution of glucose solutions in the human stomach and duodenum. Am J Dig Dis 1939-1940;6:535-544. 8. Wilson
clinical
FA, Dietschy JM. Differential diagnostic approach to problems of malabsorption. Gastroenterology 1971;61:
911-931. 9. Diamond
concentration
JM.
A rapid method for determining voltagerelations across membranes. J Physiol (Lond)
1966;183:83-100. H, Dietschy
10. Westergaard
JM. Delineation
of the dimensions
and permeability characteristics of the two major diffusion barriers to passive mucosal uptake in the rabbit intestine. J Clin Invest 54:718-732, 1974. 11. Dillard RL, Eastman H, Fordtran JS. Volume-flow relationship during the transport of fluid through the small intestine. Gastroenterology 1965;49:58-66. 12. Winne D. Unstirred layer thickness in perfused rat jejunum in vivo. Experientia (Basel) 1976;32:1278-1279. 13. Levitt MD, Aufderheide T, Fetzer CA, Bond JH, Levitt DG. Use of carbon monoxide to measure luminal stirring in the rat gut. J Clin Invest 1984;74:2056-2064, H, Miyamoto Y, Iga T, Hanano M. Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer. Biochim Biophys Acta
14. Yuasa
1986;856:219-230, H, Holtermuller KH, Dietschy JM. Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am J Physiol 1986;250:G7274735. 16. Levitt MD, Knelp JM, Levitt DG. Use of laminar flow and unstirred layer models to predict intestinal absorption in the rat. J Clin Invest 1988;81:1365-1369. 17. Anderson BW, Levine AS, Levitt DG, Kneip JM, Levitt MD. 15. Westergaard
Physiological measurement of luminal stirring in perfused rat jejunum. Am J Physiol 1988;254:G843-G848. 18. Levitt MD, Furne JK, Strocchi A, Anderson BW, Levitt DG. Physiological measurements of luminal stirring in the dog and human small bowel. J Clin Invest 1990;86:1540-1547. 19. Westergaard H, Dietschy JM. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. J Clin Invest 1976;58:97108.
Received December 28,199O. Accepted March 18,199l. This study was supported in part by Veteran’s Affairs Merit Review Funds and NIDDK no. 2 ROlDK133309-22. Address requests for reprints to: Michael D. Levitt, M.D., Veterans Affairs Medical Center (151), One Veteran’s Drive, Minneapolis, Minnesota 55417.