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Effect of changing intestinal flow rate on a measurement of intestinal permeability
GASTROENTEROLOGY1995;108:983-989
Effect of Changing Intestinal Flow Rate on a Measurement of Intestinal Permeability KENNETH D. FINE, CAROL A. SANTA ANA, JACK L. PORTER, and JOHN S. FORDTRAN Department of Internal Medicine, Baylor UniversityMedical Center, Dallas, Texas
BackEround/Aims: The flow rate of fluid through the proximal small intestine varies widely under normal physiological conditions. The aim of this study was to assess the effect of changes in flow rate on the passive permeability of the aqueous paracellular pathway of the human jejunum. Methods: Normal subjects were studied in vivo during constant perfusion of 30-cm loops of jejunum at flow rates of 5, 10, or 20 mL/min. The permeability ratio of L-xylose/urea was used to assess apparent permeability of the mucosa and to calculate the average pore radius of the aqueous pathway for passive diffusion. Results: Increasing jejunal flow rate from 5 to 20 mL/min significantly decreased the L-xylose/urea permeability ratio from 0.35 to 0.23 and decreased average calculated pore radius of the diffusion pathway from 13 A to 8 A. Conclusions: Increases in flow rate in the normal physiological range decrease the estimated pore size of normal healthy jejunal mucosa. Because increasing flow rate is known to increase exposure of luminal fluid to the intervillus space, the results of this study are best explained by postulating that cells lining the sides of villi are less permeable than cells lining the villus tips.
he flow rate of fluid through the proximal small intestine varies widely from an average of 2.5 mL/ min in fasting subjects* to as high as 20 mL/min after meals. 2'3 The purpose of the research presented here was to explore the effect of such changes in flow rate on the permeability of the intestine to three passively absorbed solutes: urea (molecular radius, 2.4 A), L-xylose (molecular radius, 3.4 A), and mannitol (molecular radius, 4.0 ~).4,5 These solutes permeate the intestinal mucosa by passive diffusion via an aqueous pathway believed to be paracellular, presumably through tight junctions. <7 This is the same route by which sodium and chloride diffuse passively across the mucosa, 8'9 lending physiological importance to this absorptive pathway. In this study, solute absorption rate from 30-cm segments of human jejunum was measured in vivo by the constant perfusion, nonabsorbable marker technique. The experiments were performed at perfusion rates of 5, 10, or 20 mL/min, which are within the normal physiological
T
range. Absorption rate divided by the mean concentration gradient across the mucosa was used as a measure of the permeability of the 30-cm segment of intestine to each solute. From the ratio of permeabilities of L-xylose and urea, the average pore radius of the pathway for passive diffusion could be calculated using an equation relating the permeability ratio to their molecular radii.
Materials and Methods Subjects and Solutions The study consisted of two experiments. The first experiment involved 5 normal subjects (3 women and 2 men; age range, 2 3 - 5 0 years). One test solution was infused at two rates: 5 mL/min and 20 mL/min. The composition of this solution is shown in Table 1. Mannitol was added to the solution mainly to keep net water movement near zero. In this way, solvent drag of the probe molecules would be absent, absorption would occur only by passive diffusion, and absorption rate (J) divided by mean lumen to serum concentration gradient (AC) would represent the permeability of the 30-cm test segment. In the first experiment, it was observed that varying the flow rate of the test solution altered mean test segment concentrations of the probe molecules. Therefore, a second experiment involving 9 normal subjects (5 women and 4 men) was designed to evaluate the effect of three flow rates: 5, 10, and 20 mL/min on J/dlC in the absence of flow rate-induced differences in AC. AC of L-xylose and urea was controlled by varying their concentrations in the infused test solutions as shown in Table 1.
Choice of Solutes L-Xylose and urea were the primary probe molecules chosen for study because their absorption rates can be measured with a high degree of accuracy during in vivo intestinal perfusion of the human jejunum and because they possess the characteristics necessary for assessing the passive permeability of the aqueous pathway of the jejunum through which small hydrophilic solutes and ions diffuse. First, I_-xylose and urea are hydrophilic, water-soluble solutes; they are highly restricted from directly permeating the lipid bilayer of cells and must © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
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Table 1, Composition of Test Solutions NaCI
(mmo//L) Experiment 19 Experiment 2 b Solution 1 Solution 2 Solution 3
Urea
L-Xylose
Mannitol
(mmo//L) (mmo//L) (mmo//L)
110
50
20
60
90 90 90
64 39 27
26 18 15
49 48 60
aSingle solution infused at 5 and 20 mL/min. bSolutions 1, 2, and 3 infused at 5, 10, and 20 mL/min, respectively.
therefore permeate an aqueous pathway for passive diffusion. Second, urea and L-xylose are uncharged molecules, so that permeation is determined only by the average pore radius of the diffusion pathway. For charged molecules, the charge state within and on either side of the pores, in addition to pore radius, influences permeability. Third, in the absence of water absorption, these molecules are absorbed only by passive diffusion. This latter statement is based on the following lines of reasoning. For pentoses (of which xylose is one), there are no known specific membrane carriers in the human intestine. 1° Although there has been debated speculation that D-xylose has some cross-affinity for the D-hexose carrier, 1°'11 L-enantiomers of all monosaccharides are structurally excluded from these transport proteins. 1° In support of these points, absorption of L-xylose (and D-xylose) in the human intestine has been shown to be linearly related to its luminal concentration, 12'13a characteristic of passively absorbed solutes. The absorption of urea across both animal 14 and human jejunum (personal observation, January 1993) is also linearly related to its luminal concentration; in general, urea behaves as a passively absorbed small hydrophilic solute and lacks absorption characteristics of carrier-mediated transport. 6'15-17 Furthermore, estimations of pore size in human small intestine by osmotic activity of urea yielded the same value as other solutes absorbed only by passive diffusion18; this could occur only if urea was absorbed exclusively by passive diffusion. As mentioned above, mannitol was added to the test solutions mainly to keep net water movement near zero. Because mannitol is a widely used marker of paracellular absorption, its presence gave us a third probe to assess jejunal permeability. Mannitol is a hexitol that possesses all of the characteristics of passively absorbed molecules mentioned above for z-xylose and urea (i.e., it is water soluble and uncharged, absorption in the human jejunum is related linearly to luminal concentration, 19 and there are no known intestinal carriers for sugar alcohols in mammalian intestine). W i t h a molecular radius of 4.0 ~l, mannitol is larger than L-xylose but is still capable of permeating the paracellular pathway, albeit to a limited extent. However, because its concentration in the infused solutions and/or the test segment varied with each perfusion, mannitol was not selected as one of the primary paracellular probes in these studies.
Measurement of Absorption The steady-state, in vivo perfusion method was used to measure absorption of water and solutes in the jejunum of
normal human subjects. 1<2° A triple lumen tube was used; it contained a 10-cm mixing segment between the infusion aperture and proximal aspiration port and a 30-cm test segment between the proximal and distal aspiration ports. Test solutions were infused in random order for a 50-minute equilibration period and then for a 1-hour experimental period. Absorption was assessed using the nonabsorbable marker polyethylene glycol 3350.19'21 Polyethylene glycol concentration was measured turbidimetrically,22 L-xylose and mannitol concentrations were measured colorimetrically,23#4 and urea concentration was measured enzymatically by the Urease/Berthelot enzymatic kit (Sigma Diagnostics, St. Louis, MO). To determine the mean concentration gradient of solute across the mucosa (AC), the serum concentrations of L-xylose and urea were measured before and after each l l 0 - m i n u t e perfusion period, and the arithmetic mean of these measurements was subtracted from mean luminal concentration. Mean luminal concentration of solute was calculated as the geometric mean of the concentration entering and leaving the test segment.
Assessment of Permeability and Average Pore Radius of the Aqueous Pathway for Passive Diffusion The permeability of a 30-cm test segment of jejunum to L-xylose and urea was assessed as absorption rate divided by mean serum to lumen concentration gradient (J/AC) when net water flow was nil. Average pore radius of the aqueous pathway for passive diffusion was calculated from the ratio of J/AC of L-xylose/urea (hereafter referred to as permeability ratio) according to the following mathematical reasoning. Assuming a spherical molecule traverses a cylindrical pore, we begin with a published equation 25 that relates pore area to two factors numerically accounting for the chance that the molecule enters the pore opening untouched and the friction created as the molecule permeates the pore. The magnitude of these two factors is determined by the ratio of molecular radius(mr) to pore radius (pr) as follows:
_-(1_mq2 Ao
pr /
\pr/
'
\pr/
_1
(Equation 1). In this equation, A is the effective diffusion area of the pore available to the diffusing molecule and Ao is the actual crosssectional area of the pore. A is different for every molecule depending on the ratio of mr to pr, whereas Ao is the same for different molecules and is therefore a constant. Because numerical values for mr for most solutes can be obtained by one of a variety of methods, pr could be calculated if A/Ao was known. However, A/A0 is an unknown and unmeasurable variable. But, because Ao is a constant, dividing equation 1
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FLOW RATE AND INTESTINAL PERMEABILITY 985
for one molecule (of radius mr0 by that for a second molecule (of radius mr2) causes A0 to cancel from the equation as follows:
(1 7,)2[1 2 04( t A1
A2
+ 2.09(mr1/3 \pr/
__
0.95(mr1/5 ] \pr/ A
(1--p:2)2[1--2.104(~2r2 ) + 2.09(~2r2)3- 0.95(~2r2)5 ]
(Equation 2). AJA2 is related to the measured permeability ratio by the following expression derived from Fick's first law of diffusion: A1 _ Jl~C2D2 A2
(Equation 3).
J2~CID1
In this equation, J1ACJJ2AC1 is the permeability ratio for solutes 1 and 2 and D2/D1 is the ratio of aqueous diffusion coefficients. By substitution from equation 2 into equation 3, we obtain (1-
~r~)211 - 2.104(~rl )
+ 2 . 0 9 ( ~ r l ) 3 - 0.95(~rl)5 ]
(1_ r2)2[1 - 2.104(r2) + 2.09(2r2)3- 0.95(r2)51
-- J1AC2D2 J2AC1D1
Table 2. E f f e c t o f Flow R a t e on P e r m e a b i l i t y o f t h e H u m a n J e j u n u m t o L-Xylose and U r e a U s i n g a S i n g l e T e s t Solution Flow rate (mL/min)
Infusion Entering segment Leaving segment Mean test segment L-Xylose Luminal concentration (mmol/L) Infused Proximal Distal Mean Mean serum concentration (mmol/L) AC a Load entering test segment (mmol/h)b J (mmol, h 1 . 3 0 cm-1) c Fractional absorption (%)d J/AC e Urea Luminal concentration (mmol/L) Infused Proximal Distal Mean Mean serum concentration (mmol/L) AC a Load entering test segment (mmol/hY' J (mmol. h 1.30 cm-1) ° Fractional absorption (%)~ J/AC ~ L-Xylose/urea permeability ratio s Average pore radius (A)g
5 5.2 _+ 0.4 5.8 + 0.4 5.5 +_ 0.4
20 19.3 ± 1.3 20.2 ± 1.3 19.8 ± 1.3
12.8 8.4 10.4 0.4 9.9 4.0 1.1 27,1 0.10
20 + 0.7 _+ 0.5 + 0.5 + 0.1 _+ 0.5 -- 0.3 ± 0.1 ± 3.4 ± 0.01
16.8 14.9 15.8 0.4 15.4 19.4 1.4 7.0 0.08
20 ~ 0.5 h _ 0.4 h ± 0.5 h ± 0.09 ± 0.5 ~ £ 1,2" _ 0.2 h ~ 0.7" ± 0.01 h
22.9 11.7 16.3 6.2 10.1 7.1 3.0 47.8 0.30 0.35 12,1
50 +_ 1.4 _+ 1.1 _+ 1.1 _+ 0.5 ± 0.7 ± 0.5 ± 0.2 _+ 5.9 + 0.02 + 0.03 ± 1.3
32.9 23.8 28.0 6.2 22.8 38.3 9.0 24.6 0.41 0.24 9.3
50 ± 1.0 h + 2.1 h + 1.6 h + 0.6 _+ 1.3 h +_ 3.W +_ 1.2 h ± 4.5 h ~ 0,08 _ 0,06 _ 3.2
NOTE. All values are expressed as mean _+ SEM (n = 5). aMean luminal concentration - mean serum concentration. bProximal concentration x proximal flow rate. CAbsorption rate. d(Absorption rate + load entering segment) × 100. ePermeability, calculated as absorption rate + AC. ~J/ACL_xy,os.+ J / A C ..... gCalculated from permeability ratio (see text). hStatistically significantly different from that measured at the 5 mL/min infusion rate (P < 0.05).
and/or paired, two-tailed t tests with a value of P ~ 0.05 representing statistical significance. 27 (Equation 4).
From equation 4, pr can be calculated because J and AC are experimentally measured values, and values for D and mr are known. For urea/L-xylose, D2/D 1 is 1.726 assuming aqueous diffusion in vitro is similar to diffusion through water-filled pores in vivo and the diffusion coefficient of xylose is numerically similar to that of its stereoisomer arabinose; mr for Lxylose and urea are 3.4 and 2.4 A, respectively. 4'5 A computer program 27 was used to solve this equation for pr (expressed in angstroms) for the different infusion rates tested.
Statistical Analysis The presence of significant differences between measured values for permeability of L-xylose and urea, L-xylose/ urea permeability ratio, and average pore radius for the different flow rates studied were looked for by analysis of variance
Institutional Approval The Institutional Review Board for Protection of Human Subjects of Baylor University Medical Center, Dallas, approved this protocol, and informed consent was obtained from each subject.
Results Experiment 1 Experiment 1 involved perfusion of a single solution at two different flow rates. As shown in Table 2, at both rates of infusion, the flow rates of solution entering and leaving the test segment were very similar, indicating that there was little if any net water absorption or secretion. As infusion flow rate was increased from 5 to 20 mL/min, mean lumen to serum concentration gradient
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(dlC) of both L-xylose and urea increased (Table 2). dlC increased because at the higher flow rate, solute load to the test segment (calculated by multiplying flow rate by solute concentration entering the test segment) increased, and fractional absorption of solute decreased. The combination of these factors at high flow rates results in a greater number of molecules remaining in the test segment per liter of fluid, i.e., a higher test segment concentration. The absorption rate, J, of both L-xylose and urea also increased significantly as flow rate increased. However, J/dlC for L-xylose decreased slightly, whereas J/dlC for urea increased by 37%. The permeability ratio of t-xylose/urea (i.e., J/die for L-xylose divided by J/dlC for urea) consequently decreased from 0.35 to 0.24 as flow rate increased from 5 to 20 mL/min (Table 2). As the result of the decrease in permeability ratio, average pore radius of the aqueous passive diffusion pathway decreased from a mean of 12.1 A at the low flow rate to 9.3 A at the higher flow rate. Neither the decrease in permeability ratio nor the decrease in average pore radius reached statistical significance (P < 0.14 and P < 0.44, respectively). Because a decrease in permeability ratio and calculated pore radius was observed in 4 of the 5 subjects (data not shown), more studies were necessary to help rule out a type II error in the lack of statistical significance. However, to insure that the changes in permeability ratio were not in some way attributable to the discordant changes in dlC of L-xylose and urea with increasing flow rate (Table 2), a second experiment was designed involving more subjects, more tested flow rates, and a constant die of the solutes.
Experiment 2
J.
Experiment 2 involved perfusion of three test solutions at three different flow rates while dlC of L-xylose and urea were held constant. As shown in Table 3, net water movement with each solution was again nil because the flow rates of solution entering and leaving the test segment were the same or similar. J/dlC of L-xylose did not change as flow rate was increased from 5 to 10 and 10 to 20 mL/min, whereas J/dlC of urea increased significantly as infusion rate increased. As shown in Table 3, this resulted in statistically significant decreases in the L-xylose/urea permeability ratio as flow rate was increased (P = 0.0007). Mean values for average pore radius also decreased significantly from 13.3 A to 9.2 A to 7.6 ~l as infusion rate was increased from 5 to 10 and 10 to 20 mL/min (P < 0.006). Mannitol
Permeability
As mentioned in Materials and Methods, mannitol was added to the test solutions mainly to keep net
Table 3. Effect of Flow Rate on Permeability of the Human Jejunum to L-Xylose and Urea When Luminal Solute Concentrations Are Constant Flow rate (mL/min) Infusion Entering segment Leaving segment Mean test segment L-Xylose Concentration Mean luminal (mmol/L) Mean serum (mmol/L)
AC (mmol/L)a J ( m m o l - h - l ' 3 0 cm-1) b J/AC e Fractional absorption (%)d Urea Concentration Mean luminal (mmol/L) Mean serum (mmol/L)
AC (mmol/L)a J (mmol " h - 1 - 3 0 cm-1) b J/ZxC ~ Fractional absorption (%)o L-Xylose/urea permeability ratio ° Average pore radius (~,)r
5 4.8 _+ 0.4 5.0 ± 0.4 4.9 _+ 0.3
10 10.1 ± 0.5 10.1 _+ 0.4 10.1 _+ 0.4
20 19.6 + 0.3 19.6 _+ 0.4 19.6 _+ 0.3
12.8 0.3 12.5 1.4 0.12 32.0
± 0.7 ± 0.05 + 0.7 + 0.1 -+ 0.01 ± 2,5
12.6 0.3 12,3 1.3 0.11 16.1
± 0.4 _+ 0.05 + 0.4 ± 0.1 ± 0.01 ± 1.2
12.4 0.3 12.1 1.3 0.11 8.3
_+ 0.3 ± 0.05 ± 0,3 ± 0.2 ± 0.01 --+ 1.0
18.3 5.6 12.7 4.2 0,34 54.2
+ 1,0 ± 0.4 + 0.6 _+ 0.2 ± 0.02 ± 2.6
18.2 5.5 12.6 5.0 0,40 36.3
± ± ± + ± +
18.1 5,7 12.4 5.8 0.49 24.0
± 0.8 ± 0.4 ± 0.9 ± 0,7 g _+ 0.06 g ± 2.8
0.35 ± 0.03 13.3 ± 1.9
0.6 0.3 0.8 0,3 0.02 g 1.3
0.28 ± 0.02 g 9.2 ± 0.5 g
0.22 +_ 0.01 g 7.6 _+ 0,2 g
NOTE. All values are expressed as mean _+ SE (n = 9). aMean luminal concentration - mean serum concentration. °Absorption rate. CPermeability calculated as absorption rate + AC. °(Absorption rate + load entering segment) x 100. ea/ACL.~los e + J/ACurea. eCalculated from permeability ratio (see text]. gStatistically significantly different from 5 mL/min flow rate (P --< 0.05).
water absorption near zero, thus eliminating solvent drag as an absorptive force. The mannitol concentrations in the test segment were subject to change by the effects of flow rate per se (as described above for the other probes in experiment 1) or changes in the perfused concentration of mannitol (as required because of varying concentrations of L-xylose and urea in experiment 2). Nevertheless, the permeability of the 30-cm test segments to mannitol could be calculated by measuring J/AC. Combining the data from experiments 1 and 2, there was no significant difference in J/AC of mannitol at the three flow rates tested: J/dlC at 5 mL/min was 0.038 + 0.004 (n = 14), at 10 mL/min was 0.030 + 0.005 (n = 9), and at 20 mL/min was 0.029 + 0.006 (n = 14) (P > 0.42).
Discussion In the present experiments, triple-lumen in vivo perfusion was used to assess intestinal permeability at different flow rates within the physiological range. Solute absorption rate (J) divided by lumen to serum concentration gradient (AC) was used to measure the permeability of a 30-cm segment of jejunum to small hydrophilic solutes. As jejunal flow rate was increased from 5 to 20 mL/min, J/dlC for urea increased by 37% and 44% in two separate experiments. In contrast, J/AC for L-xylose and mannitol did not change. As pointed out in Materials and Methods, the perme-
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ability ratio of L-xylose/urea (J/AC for L-xylose divided by J/AC for urea) can be used to calculate the average pore radius of the aqueous pathway through which these solutes diffuse passively. When the jejunum was perfused at 5 mL/min, the average calculated pore radius of the diffusion pathway was about 13 /i, whereas when the perfusion rate was 20 mL/min, pore radius averaged about 8 A. This 38% reduction in average pore size can have a large impact on permeability because according to the assumptions and principles regarding diffusion through aqueous pores set out in Materials and Methods, a spherical molecule cannot permeate a cylindrical pore to any significant extent unless its molecular radius is less than half that of the pore radius. 1s'25 Therefore, a pore radius of 8 A would restrict the permeation of many more molecules (including most organic molecules) than would a pore radius of 13 A. We considered four possible explanations for the resuits of this study. The first postulates that high flow rates cause a perfused test solution to be exposed to a larger surface area than is available to the test solution when it is perfused slowly and that the newly recruited surface area is permeable to small molecules the size of urea but not to somewhat larger molecules like L-xylose and mannitol. Two lines of evidence in the literature support the notion that an increasing flow rate through a defined length of intestine causes an increase in the mucosal surface area exposed to fluid within the intestinal lumen. First, studies in rats have shown that the absorption rate of tritiated water and the absorption rate of D-glucose under maximum velocity conditions increase when perfusion rate is increased. 28 These changes could not be explained by changes in unstirred water layer thickness and were therefore attributed to an increased surface area exposed to the perfused test solutions by high flow. The second line of evidence also comes from studies in rats in which increasing the volume of a loop of small intestine perfused at a constant rate caused the intervillus space to open widely. 29 This was presumed to enhance exposure of the perfused test solution to cells deeper in the intervillus space. Perfusion studies in humans using dye dilution methodology showed that the average volume of a constantly perfused 30-cm jejunal segment would increase from 70 to 130 mL as flow rate was increased from 5 to 20 mL/min. 3° It seems reasonable to suggest that a flow rate-induced increase in luminal volume in humans, like in rats, would allow increased exposure of a perfused test solution to the intervillus space. Previous studies therefore suggest that increasing flow rates cause the exposure of luminal fluid to a greater surface area of mucosal cells and that this newly recruited
FLOW RATE AND INTESTINAL PERMEABILITY
987
surface area consists of mucosal cells lining the sides of villi. Therefore, the results of our present research, when interpreted in light of these previous observations, are consistent with the hypothesis that cells at the villus tips (exposed to luminal fluid at low rates of perfusion) are permeable to urea, L-xylose, and mannitol but that cells along the sides of the villi (exposed by higher flow rates) are permeable to urea but not to t-xylose or mannitol and therefore possess a smaller pore radius. The decrease in average pore size at higher flow rates is entirely consistent with our conclusion that fast flow rates cause luminal fluid to be exposed to mucosal cells of lower permeability than the more permeable mucosal ceils exposed when perfusion rate is slow. In addition, it should be pointed out that the values for pore radius calculated for the various flow rates tested reflect the average pore radius of all of the pores exposed at the respective flow rate. Therefore, our finding of a smaller average pore radius at higher flow rates is not attributed to a reduction in the size of a single population of pores but instead represents the average size of the larger pores exposed at slow perfusion rates and smaller pores exposed when perfusion rate is higher. Because the relative amount of additional surface area exposed by increasing flow rate is unknown, the pore radius of the newly exposed area in the intervillus space cannot be measured. However, the fact that J/AC for L-xylose did not increase at all suggests that the average pore radius of this newly exposed surface is less than twice the radius of L-xylose (i.e., < 6 . 8 A) because, as mentioned above, a pore radius/ molecular radius ratio of at least two is necessary for significant pore permeation. The second possible explanation we explored is that preferential stimulation of urea permeation, in the setting of a flow rate-induced deepening of the intervillus space, was the result of urea's ability to diffuse from the bulk contents of the lumen into the intervillus space more rapidly than L-xylose or mannitol because of its smaller molecular size. Two consequences of a smaller molecular size could theoretically favor greater delivery of urea into the newly opened intervillus space. One is more rapid diffusibility of urea in water per se (i.e., a larger aqueous diffusion coefficient). The second is more rapid permeation of the mucosa (i.e., a larger membrane permeability coefficient) as it relates to the development of a greater concentration gradient down the intervillus space. A concentration gradient of solute might develop in the intervillus space assuming the bulk of the test solution was excluded from the space while solute diffused perpendicularly into the space. The concentration of the solute would be greatest at the space entrance (and equal to that in luminal contents) and would progressively de-
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crease along its path down the intervillus space as solute was absorbed. Because of greater permeability to the smaller urea compared with L-xylose or mannitol (even if the overall permeability of the mucosa was constant along the entire villus), a greater concentration gradient would develop for urea. To the extent that a concentration gradient down the intervillus space could enhance diffusion of solute from bulk luminal contents, proportionally more urea would diffuse into the intervillus space, possibly facilitating a more rapid absorption rate. 31 Assuming that the decrease in concentration of urea down the intervillus space was slow enough to allow urea absorption to continue well into this space, this explanation could account for a greater flow rate-induced increase in permeability to urea relative to L-xylose or mannitol, but it could not explain an increase in permeability to urea with no increase whatsoever in the permeability to L-xylose or mannitol. Moreover, if the urea concentration decreased so rapidly that very little urea was left part way into the intervillus space, further deepening of the intervillus space would provide little if any increase in functional absorbing surface and consequently no further increase in its permeation. 32 Under these circumstances, a deepening intervillus space could result in a proportionally greater increase in functional surface area and absorption of mannitol and t-xylose relative to urea because a greater number of these larger molecules would be present over a longer path down the intervillus space. 32 For these reasons, we do not believe that our results can be satisfactorily explained by urea's greater aqueous diffusability or membrane permeability as it relates to a steeper concentration gradient down the intervillus space. The third mechanism we explored to explain our findings was reduction in unstirred water layer thickness at higher flow rates. 33'34 According to this theory, the bulk contents of the intestinal lumen are stirred completely, whereas a layer of pre-epithelial fluid of fixed thickness is unstirred and therefore acts as a diffusion barrier to solute absorption. The unstirred water layer as a diffusion barrier can be expressed mathematically as follows35: D J = -- AC, t in w h i c h J is solute flux across the unstirred water layer, D is the aqueous diffusion coefficient, t is the thickness of the unstirred water layer, and AC is the concentration gradient across the unstirred water layer. If the value for t decreases, J for a molecule with a larger D (like urea) would increase to a larger numerical extent than would J for a molecule with a smaller D (like L-xylose); however,
the fractional change in J for each molecule would be the same because it is changing by the same reduction in t. Looked at in another way, expressing JL-~ylose/J.... as a ratio results in a cancellation of t from the mathematical expression. Therefore, if in our experiments increased flow rate decreased unstirred water layer thickness, JL-xylose and J . . . . would have increased proportionally and the ratio of JI._xylose/J.... would have been unaffected. Thus, we cannot attribute our finding a decreasing L-xylose/ urea permeability ratio at higher flow rates to a thinning unstirred water layer. The fourth possibility we considered was whether changes in the laminar flow properties of the perfused fluid could account for a flow rate-induced increase in urea permeability with no change in L-xylose or mannitol permeability. According to the principles of laminar flow, the flow rate of fluid is most rapid (and most perfectly stirred) in the center of the lumen with progressively slower flow rates (and poorer stirring) in fluid near the intestinal epithelial cells. 36-38 According to this theory, for a given solute, fractional absorption is inversely proportional to flow rate if mucosal permeability and absorptive surface area remain constant. 38 Therefore, in our study, if there were no changes in mucosal permeability or absorptive surface area, sequentially doubling the flow rate should have sequentially halved the fractional absorption rate of all three solutes. Whereas this was observed for z-xylose (mean fractional absorption rates of 32%, 16%, and 8% at flow rates of 5, 10, and 20 mL/ rain, respectively) and mannitol (respective mean fractional absorption rates of 11%, 5%, and 2.4%), the fractional absorption of urea decreased by a lesser factor each time flow rate was doubled (fractional absorption rates of 54%, 36%, and 24% at flow rates of 5, 10, and 20 mL/min, respectively). Therefore, a change in laminar flow properties of fluid cannot explain the higher permeation rate of urea at faster flow rates unless a concomitant selective increase in urea permeability and/or absorptive surface occurred. In our opinion, the best explanation of the findings of this study is that increasing intestinal flow rate from 5 to 20 mL/min enhances exposure of a perfused test solution to mucosal cells lining the intervillus space and that these cells are less permeable (i.e., have "tighter" tight junctions) than ceils lining the tips of villi. Regardless of the explanation, these studies show that changes in flow rate within the physiological range can alter the apparent permeability of normal healthy jejunal mucosa. This fact needs to be considered when interpreting measures of tight junction permeability in experimental situations in which there exist significant differences in intestinal flow rates and/or intestinal volume.
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References 1. Soergel KH. Flow measurements of test meals and fasting contents in the human small intestine. Gastrointestinal motility. International symposium on motility of the GI tract. Erlangen, West Germany, July 15 and 16, 1969. New York: Academic, 1969. 2. Fordtran JS, Locklear TW. Ionic constituents and osmolality of gastric and small-intestinal fluids after eating. Am J Dig Dis 1966;11:503-521. 3. Fordtran JS, Ingelfinger FJ. Absorption of water, electrolytes, and sugars from the human gut. Handbook of physiology--alimentary canal. Washington, DC: American Physiological Society, 1967: 1457. 4. Schultz SG, Solomon AK. Determination of the effective hydrodynamic radii of small molecules by viscometry. J Gen Physiol 1961;44:1189-1199. 5. Soil AH. A new approach to molecular configuration applied to aqueous pore transport. J Gen Physiol 1 9 6 7 ; 5 0 : 2 5 6 5 - 2 5 7 8 . 6. Mandel LJ, Curran PE. Response of the frog skin to steady-state voltage clamping. J Gen Physiol 1 9 7 2 ; 5 9 : 5 0 3 - 5 1 8 . 7. Powell DW. Barrier function of epithelia. Am J Physiol 1981; 241:G275-G288. 8. Ussing HH, Windhager EE. Nature of shunt path and active sodium transport path through frog skin epithelium. Acta Physiol Scand 1 9 6 4 ; 6 1 : 4 8 4 - 5 0 4 . 9. Frizzell RA, Schuitz SG. Ionic conductances of extracellular shunt pathway in rabbit ileum. J Gen Physiol 1 9 7 2 ; 5 9 : 3 1 8 - 3 4 6 . 10. Crane RK. Absorption of sugars. Handbook of physiology. Section 6: alimentary canal. Volume III. Intestinal Absorption. Washington, DC: American Physiological Society, 1 9 6 8 : 1 3 2 3 - 1 3 5 1 . 11. Cook GC. Rates and mechanisms of glucose, galactose, and xylose absorption in man in vivo. Scand J Gastroenterol 1977; 1 2 : 7 3 3 - 7 3 7 . 12. Ohkohchi N, Himukai M, Igarashi Y, Kasai M. Mechanism of Dxylose transport in human small intestine. J Pediatric Gastroenterol Nutr 1 9 8 6 ; 5 : 3 7 2 - 3 7 8 . 13. Fine KD, Santa Ana CA, Porter JL, Fordtran JS. Mechanism by which glucose stimulates the passive absorption of small solutes by the human jejunum in vivo. Gastroenterology 1994; 1 0 7 : 3 8 9 395. 14. Wilson TH. Intestinal absorption. Chapter 3. Mechanisms of absorption. Philadelphia: Saunders, 1962. 15. Lippe C, Bianchi A, Cremaschi D, Capraro V. Different types of asymmetric distribution of hydrosoluble and liposoluble substances at the two sides of a mucosal intestinal preparation. Arch Int Physiol Biochim Biophys 1 9 6 5 ; 7 3 : 4 3 - 5 4 . 16. Lifson N, Hakim AA. Simple diffusive-convective model for intestinal absorption of a nonelectrolyte (urea). Am J Physiol 1966; 2 1 1 : 1 1 3 7 - 1 1 4 6 . 17. Ochsenfahrt H, Winne D. The contribution of solvent drag to the intestinal absorption of tritiated water and urea from the jejunum of the rat. Naunyn Schmiedebergs Arch Pharmacol 1973;279: 133-152. 18. Fordtran JS, Rector FC, Ewton MF, Soter N, Kinney J. Permeability characteristics of the human small intestine. J Clin Invest 1965; 4 4 : 1 9 3 5 - 1 9 4 4 . 19. Fine KD, Santa Ana CA, Porter JL, Fordtran JS. Effect of D-glucose on intestinal permeability and its passive absorption in human small intestine in vivo. Gastroenterology 1 9 9 3 ; 1 0 5 : 1 1 1 7 1125. 20. Cooper H, Levitan R, Fordtran JS, Ingelfinger FJ. A method for studying absorption of water and solute from the human small intestine. Gastroenterology 1966; 5 0 : 1 - 7 .
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21. Jacobsen ED, Bondy DC, Broitman SA, Fordtran JS. Validity of polyethylene glycol in estimating intestinal water volume. Gastroenterology 1 9 6 3 ; 4 4 : 7 6 1 - 7 6 7 . 22. Hyden S. A turbidimetdc method for determination of higher polyethylene glycols in biological materials. Lantbrukshoegsk Ann 1955;22:139-145. 23. Roe JH, Rice EW. A photometric method for the determination of free pentoses in animal tissues. J Biol Chem 1948; 1 7 3 : 5 0 7 512. 24. Corcoran AC, Page IH. A method for the determination of mannitol in plasma and urine. J Biol Chem 1947; 1 7 0 : 1 6 5 - 1 7 1 . 25. Renkin EM. Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physio11954; 3 8 : 2 2 5 - 2 4 3 . 26. International critical tables of numerical data, physics, chemistry, and technology. U.S. National Research Council. New York; McGraw Hill, 1 9 2 9 : 6 9 - 7 0 . 27. SAS/STAT user's guide, version 6 . 4 t h ed. Volume 2. Cary, NC: SAS Instutue Inc., 1989. 28. Lewis LD, Ferdtran JS. Effect of perfusion rate on absorption, surface area, unstirred water layer thickness, permeability, and intraluminal pressure in the rat ileum in vivo. Gastroenterolog~ 1975; 6 8 : 1 5 0 9 - 1 5 1 6 . 29. Harris SM, Kennedy JG, Siegesmund KA, Yorde DE. Relationship between distention and absorption in rat intestine. I. Effect of luminal volume on the morphology of the absorbing surface. Gastroenterology 1 9 8 8 ; 9 4 : 1 1 6 4 - 1 1 7 1 . 30. Diilard RL, Eastman H, Fordtran JS. Volume-flow relationship during the transport of fluid through the human small intestine. Gastroenterology 1 9 6 5 ; 4 9 : 5 8 - 6 6 . 31. Harris MS, Kennedy JG. Relationship between distention and absorption in rat intestine. I1. Effects of volume and flow rate on transport. Gastroenterology 1988; 9 4 : 1 1 7 2 - 1 1 7 9 . 32. Winne D. The permeability coefficient of the wall of a villous membrane. J Math Biol 1 9 7 8 ; 6 : 9 5 - 1 0 8 . 33. Winne D. Rat jejunum perfused in situ: effect of perfusion rate on intraluminal radius on absorption rate and effective unstirred layer thickness. Arch Pharmacol 1 9 7 9 ; 3 0 7 : 2 6 5 - 2 7 4 . 34. Westergaard H, Holtermuller H, Dietschy JM. Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am J Physiol 1986;250:G727-G735. 35. Dietschy JM, Sallee VL, Wilson FA. Unstirred water layers and absorption across the intestinal mucosa. Gastroenterology 1971;61:932-934. 36. Amidon GL, Kou J, Elliott RL, Lightfoot EN. Analysis of models for determining intestinal wall permeabilities. J Pharm Sci 1980; 6 9 : 1 3 6 9 - 1 3 7 3 . 37. Levitt MD, Fetzer CA, Kneip JM, Bond JH, Levitt DG. Quantitative assessment of luminal stirring in the perfused small intestine of the rat. Am J Physiol 1987;252:G325-G332. 38. Levitt MD, Kneip JM, Levitt DG. Use of laminar flow and unstirred layer models to predict intestinal absorption in rat. J Clin Invest 1988; 8 1 : 1 3 6 5 - 1 3 6 9 . Received June 9, 1994. Accepted December 13, 1994. Address requests for reprints to: Kenneth D. Fine, M.D., Department of Internal Medicine, Baylor University Medical Center, 3500 Gaston Avenue, Dallas, Texas 75246. Fax: (214) 820-4837. Supported by U.S. Public Health Service grant 5-RO1-DK37172-05 from the National Institute of Diabetes and Digestive and Kidney Diseases and the Southwest Digestive Disease Foundation. The authors thank Diana Santa Ana for her help with preparation of the manuscript and Dr. Donald Mclntire, University of TexasSouthwestern Medical Center at Dallas, for his help with mathematical and statistical computation.
Effect of Changing Intestinal Flow Rate on a Measurement of Intestinal Permeability KENNETH D. FINE, CAROL A. SANTA ANA, JACK L. PORTER, and JOHN S. FORDTRAN Department of Internal Medicine, Baylor UniversityMedical Center, Dallas, Texas
BackEround/Aims: The flow rate of fluid through the proximal small intestine varies widely under normal physiological conditions. The aim of this study was to assess the effect of changes in flow rate on the passive permeability of the aqueous paracellular pathway of the human jejunum. Methods: Normal subjects were studied in vivo during constant perfusion of 30-cm loops of jejunum at flow rates of 5, 10, or 20 mL/min. The permeability ratio of L-xylose/urea was used to assess apparent permeability of the mucosa and to calculate the average pore radius of the aqueous pathway for passive diffusion. Results: Increasing jejunal flow rate from 5 to 20 mL/min significantly decreased the L-xylose/urea permeability ratio from 0.35 to 0.23 and decreased average calculated pore radius of the diffusion pathway from 13 A to 8 A. Conclusions: Increases in flow rate in the normal physiological range decrease the estimated pore size of normal healthy jejunal mucosa. Because increasing flow rate is known to increase exposure of luminal fluid to the intervillus space, the results of this study are best explained by postulating that cells lining the sides of villi are less permeable than cells lining the villus tips.
he flow rate of fluid through the proximal small intestine varies widely from an average of 2.5 mL/ min in fasting subjects* to as high as 20 mL/min after meals. 2'3 The purpose of the research presented here was to explore the effect of such changes in flow rate on the permeability of the intestine to three passively absorbed solutes: urea (molecular radius, 2.4 A), L-xylose (molecular radius, 3.4 A), and mannitol (molecular radius, 4.0 ~).4,5 These solutes permeate the intestinal mucosa by passive diffusion via an aqueous pathway believed to be paracellular, presumably through tight junctions. <7 This is the same route by which sodium and chloride diffuse passively across the mucosa, 8'9 lending physiological importance to this absorptive pathway. In this study, solute absorption rate from 30-cm segments of human jejunum was measured in vivo by the constant perfusion, nonabsorbable marker technique. The experiments were performed at perfusion rates of 5, 10, or 20 mL/min, which are within the normal physiological
T
range. Absorption rate divided by the mean concentration gradient across the mucosa was used as a measure of the permeability of the 30-cm segment of intestine to each solute. From the ratio of permeabilities of L-xylose and urea, the average pore radius of the pathway for passive diffusion could be calculated using an equation relating the permeability ratio to their molecular radii.
Materials and Methods Subjects and Solutions The study consisted of two experiments. The first experiment involved 5 normal subjects (3 women and 2 men; age range, 2 3 - 5 0 years). One test solution was infused at two rates: 5 mL/min and 20 mL/min. The composition of this solution is shown in Table 1. Mannitol was added to the solution mainly to keep net water movement near zero. In this way, solvent drag of the probe molecules would be absent, absorption would occur only by passive diffusion, and absorption rate (J) divided by mean lumen to serum concentration gradient (AC) would represent the permeability of the 30-cm test segment. In the first experiment, it was observed that varying the flow rate of the test solution altered mean test segment concentrations of the probe molecules. Therefore, a second experiment involving 9 normal subjects (5 women and 4 men) was designed to evaluate the effect of three flow rates: 5, 10, and 20 mL/min on J/dlC in the absence of flow rate-induced differences in AC. AC of L-xylose and urea was controlled by varying their concentrations in the infused test solutions as shown in Table 1.
Choice of Solutes L-Xylose and urea were the primary probe molecules chosen for study because their absorption rates can be measured with a high degree of accuracy during in vivo intestinal perfusion of the human jejunum and because they possess the characteristics necessary for assessing the passive permeability of the aqueous pathway of the jejunum through which small hydrophilic solutes and ions diffuse. First, I_-xylose and urea are hydrophilic, water-soluble solutes; they are highly restricted from directly permeating the lipid bilayer of cells and must © 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
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Table 1, Composition of Test Solutions NaCI
(mmo//L) Experiment 19 Experiment 2 b Solution 1 Solution 2 Solution 3
Urea
L-Xylose
Mannitol
(mmo//L) (mmo//L) (mmo//L)
110
50
20
60
90 90 90
64 39 27
26 18 15
49 48 60
aSingle solution infused at 5 and 20 mL/min. bSolutions 1, 2, and 3 infused at 5, 10, and 20 mL/min, respectively.
therefore permeate an aqueous pathway for passive diffusion. Second, urea and L-xylose are uncharged molecules, so that permeation is determined only by the average pore radius of the diffusion pathway. For charged molecules, the charge state within and on either side of the pores, in addition to pore radius, influences permeability. Third, in the absence of water absorption, these molecules are absorbed only by passive diffusion. This latter statement is based on the following lines of reasoning. For pentoses (of which xylose is one), there are no known specific membrane carriers in the human intestine. 1° Although there has been debated speculation that D-xylose has some cross-affinity for the D-hexose carrier, 1°'11 L-enantiomers of all monosaccharides are structurally excluded from these transport proteins. 1° In support of these points, absorption of L-xylose (and D-xylose) in the human intestine has been shown to be linearly related to its luminal concentration, 12'13a characteristic of passively absorbed solutes. The absorption of urea across both animal 14 and human jejunum (personal observation, January 1993) is also linearly related to its luminal concentration; in general, urea behaves as a passively absorbed small hydrophilic solute and lacks absorption characteristics of carrier-mediated transport. 6'15-17 Furthermore, estimations of pore size in human small intestine by osmotic activity of urea yielded the same value as other solutes absorbed only by passive diffusion18; this could occur only if urea was absorbed exclusively by passive diffusion. As mentioned above, mannitol was added to the test solutions mainly to keep net water movement near zero. Because mannitol is a widely used marker of paracellular absorption, its presence gave us a third probe to assess jejunal permeability. Mannitol is a hexitol that possesses all of the characteristics of passively absorbed molecules mentioned above for z-xylose and urea (i.e., it is water soluble and uncharged, absorption in the human jejunum is related linearly to luminal concentration, 19 and there are no known intestinal carriers for sugar alcohols in mammalian intestine). W i t h a molecular radius of 4.0 ~l, mannitol is larger than L-xylose but is still capable of permeating the paracellular pathway, albeit to a limited extent. However, because its concentration in the infused solutions and/or the test segment varied with each perfusion, mannitol was not selected as one of the primary paracellular probes in these studies.
Measurement of Absorption The steady-state, in vivo perfusion method was used to measure absorption of water and solutes in the jejunum of
normal human subjects. 1<2° A triple lumen tube was used; it contained a 10-cm mixing segment between the infusion aperture and proximal aspiration port and a 30-cm test segment between the proximal and distal aspiration ports. Test solutions were infused in random order for a 50-minute equilibration period and then for a 1-hour experimental period. Absorption was assessed using the nonabsorbable marker polyethylene glycol 3350.19'21 Polyethylene glycol concentration was measured turbidimetrically,22 L-xylose and mannitol concentrations were measured colorimetrically,23#4 and urea concentration was measured enzymatically by the Urease/Berthelot enzymatic kit (Sigma Diagnostics, St. Louis, MO). To determine the mean concentration gradient of solute across the mucosa (AC), the serum concentrations of L-xylose and urea were measured before and after each l l 0 - m i n u t e perfusion period, and the arithmetic mean of these measurements was subtracted from mean luminal concentration. Mean luminal concentration of solute was calculated as the geometric mean of the concentration entering and leaving the test segment.
Assessment of Permeability and Average Pore Radius of the Aqueous Pathway for Passive Diffusion The permeability of a 30-cm test segment of jejunum to L-xylose and urea was assessed as absorption rate divided by mean serum to lumen concentration gradient (J/AC) when net water flow was nil. Average pore radius of the aqueous pathway for passive diffusion was calculated from the ratio of J/AC of L-xylose/urea (hereafter referred to as permeability ratio) according to the following mathematical reasoning. Assuming a spherical molecule traverses a cylindrical pore, we begin with a published equation 25 that relates pore area to two factors numerically accounting for the chance that the molecule enters the pore opening untouched and the friction created as the molecule permeates the pore. The magnitude of these two factors is determined by the ratio of molecular radius(mr) to pore radius (pr) as follows:
_-(1_mq2 Ao
pr /
\pr/
'
\pr/
_1
(Equation 1). In this equation, A is the effective diffusion area of the pore available to the diffusing molecule and Ao is the actual crosssectional area of the pore. A is different for every molecule depending on the ratio of mr to pr, whereas Ao is the same for different molecules and is therefore a constant. Because numerical values for mr for most solutes can be obtained by one of a variety of methods, pr could be calculated if A/Ao was known. However, A/A0 is an unknown and unmeasurable variable. But, because Ao is a constant, dividing equation 1
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FLOW RATE AND INTESTINAL PERMEABILITY 985
for one molecule (of radius mr0 by that for a second molecule (of radius mr2) causes A0 to cancel from the equation as follows:
(1 7,)2[1 2 04( t A1
A2
+ 2.09(mr1/3 \pr/
__
0.95(mr1/5 ] \pr/ A
(1--p:2)2[1--2.104(~2r2 ) + 2.09(~2r2)3- 0.95(~2r2)5 ]
(Equation 2). AJA2 is related to the measured permeability ratio by the following expression derived from Fick's first law of diffusion: A1 _ Jl~C2D2 A2
(Equation 3).
J2~CID1
In this equation, J1ACJJ2AC1 is the permeability ratio for solutes 1 and 2 and D2/D1 is the ratio of aqueous diffusion coefficients. By substitution from equation 2 into equation 3, we obtain (1-
~r~)211 - 2.104(~rl )
+ 2 . 0 9 ( ~ r l ) 3 - 0.95(~rl)5 ]
(1_ r2)2[1 - 2.104(r2) + 2.09(2r2)3- 0.95(r2)51
-- J1AC2D2 J2AC1D1
Table 2. E f f e c t o f Flow R a t e on P e r m e a b i l i t y o f t h e H u m a n J e j u n u m t o L-Xylose and U r e a U s i n g a S i n g l e T e s t Solution Flow rate (mL/min)
Infusion Entering segment Leaving segment Mean test segment L-Xylose Luminal concentration (mmol/L) Infused Proximal Distal Mean Mean serum concentration (mmol/L) AC a Load entering test segment (mmol/h)b J (mmol, h 1 . 3 0 cm-1) c Fractional absorption (%)d J/AC e Urea Luminal concentration (mmol/L) Infused Proximal Distal Mean Mean serum concentration (mmol/L) AC a Load entering test segment (mmol/hY' J (mmol. h 1.30 cm-1) ° Fractional absorption (%)~ J/AC ~ L-Xylose/urea permeability ratio s Average pore radius (A)g
5 5.2 _+ 0.4 5.8 + 0.4 5.5 +_ 0.4
20 19.3 ± 1.3 20.2 ± 1.3 19.8 ± 1.3
12.8 8.4 10.4 0.4 9.9 4.0 1.1 27,1 0.10
20 + 0.7 _+ 0.5 + 0.5 + 0.1 _+ 0.5 -- 0.3 ± 0.1 ± 3.4 ± 0.01
16.8 14.9 15.8 0.4 15.4 19.4 1.4 7.0 0.08
20 ~ 0.5 h _ 0.4 h ± 0.5 h ± 0.09 ± 0.5 ~ £ 1,2" _ 0.2 h ~ 0.7" ± 0.01 h
22.9 11.7 16.3 6.2 10.1 7.1 3.0 47.8 0.30 0.35 12,1
50 +_ 1.4 _+ 1.1 _+ 1.1 _+ 0.5 ± 0.7 ± 0.5 ± 0.2 _+ 5.9 + 0.02 + 0.03 ± 1.3
32.9 23.8 28.0 6.2 22.8 38.3 9.0 24.6 0.41 0.24 9.3
50 ± 1.0 h + 2.1 h + 1.6 h + 0.6 _+ 1.3 h +_ 3.W +_ 1.2 h ± 4.5 h ~ 0,08 _ 0,06 _ 3.2
NOTE. All values are expressed as mean _+ SEM (n = 5). aMean luminal concentration - mean serum concentration. bProximal concentration x proximal flow rate. CAbsorption rate. d(Absorption rate + load entering segment) × 100. ePermeability, calculated as absorption rate + AC. ~J/ACL_xy,os.+ J / A C ..... gCalculated from permeability ratio (see text). hStatistically significantly different from that measured at the 5 mL/min infusion rate (P < 0.05).
and/or paired, two-tailed t tests with a value of P ~ 0.05 representing statistical significance. 27 (Equation 4).
From equation 4, pr can be calculated because J and AC are experimentally measured values, and values for D and mr are known. For urea/L-xylose, D2/D 1 is 1.726 assuming aqueous diffusion in vitro is similar to diffusion through water-filled pores in vivo and the diffusion coefficient of xylose is numerically similar to that of its stereoisomer arabinose; mr for Lxylose and urea are 3.4 and 2.4 A, respectively. 4'5 A computer program 27 was used to solve this equation for pr (expressed in angstroms) for the different infusion rates tested.
Statistical Analysis The presence of significant differences between measured values for permeability of L-xylose and urea, L-xylose/ urea permeability ratio, and average pore radius for the different flow rates studied were looked for by analysis of variance
Institutional Approval The Institutional Review Board for Protection of Human Subjects of Baylor University Medical Center, Dallas, approved this protocol, and informed consent was obtained from each subject.
Results Experiment 1 Experiment 1 involved perfusion of a single solution at two different flow rates. As shown in Table 2, at both rates of infusion, the flow rates of solution entering and leaving the test segment were very similar, indicating that there was little if any net water absorption or secretion. As infusion flow rate was increased from 5 to 20 mL/min, mean lumen to serum concentration gradient
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(dlC) of both L-xylose and urea increased (Table 2). dlC increased because at the higher flow rate, solute load to the test segment (calculated by multiplying flow rate by solute concentration entering the test segment) increased, and fractional absorption of solute decreased. The combination of these factors at high flow rates results in a greater number of molecules remaining in the test segment per liter of fluid, i.e., a higher test segment concentration. The absorption rate, J, of both L-xylose and urea also increased significantly as flow rate increased. However, J/dlC for L-xylose decreased slightly, whereas J/dlC for urea increased by 37%. The permeability ratio of t-xylose/urea (i.e., J/die for L-xylose divided by J/dlC for urea) consequently decreased from 0.35 to 0.24 as flow rate increased from 5 to 20 mL/min (Table 2). As the result of the decrease in permeability ratio, average pore radius of the aqueous passive diffusion pathway decreased from a mean of 12.1 A at the low flow rate to 9.3 A at the higher flow rate. Neither the decrease in permeability ratio nor the decrease in average pore radius reached statistical significance (P < 0.14 and P < 0.44, respectively). Because a decrease in permeability ratio and calculated pore radius was observed in 4 of the 5 subjects (data not shown), more studies were necessary to help rule out a type II error in the lack of statistical significance. However, to insure that the changes in permeability ratio were not in some way attributable to the discordant changes in dlC of L-xylose and urea with increasing flow rate (Table 2), a second experiment was designed involving more subjects, more tested flow rates, and a constant die of the solutes.
Experiment 2
J.
Experiment 2 involved perfusion of three test solutions at three different flow rates while dlC of L-xylose and urea were held constant. As shown in Table 3, net water movement with each solution was again nil because the flow rates of solution entering and leaving the test segment were the same or similar. J/dlC of L-xylose did not change as flow rate was increased from 5 to 10 and 10 to 20 mL/min, whereas J/dlC of urea increased significantly as infusion rate increased. As shown in Table 3, this resulted in statistically significant decreases in the L-xylose/urea permeability ratio as flow rate was increased (P = 0.0007). Mean values for average pore radius also decreased significantly from 13.3 A to 9.2 A to 7.6 ~l as infusion rate was increased from 5 to 10 and 10 to 20 mL/min (P < 0.006). Mannitol
Permeability
As mentioned in Materials and Methods, mannitol was added to the test solutions mainly to keep net
Table 3. Effect of Flow Rate on Permeability of the Human Jejunum to L-Xylose and Urea When Luminal Solute Concentrations Are Constant Flow rate (mL/min) Infusion Entering segment Leaving segment Mean test segment L-Xylose Concentration Mean luminal (mmol/L) Mean serum (mmol/L)
AC (mmol/L)a J ( m m o l - h - l ' 3 0 cm-1) b J/AC e Fractional absorption (%)d Urea Concentration Mean luminal (mmol/L) Mean serum (mmol/L)
AC (mmol/L)a J (mmol " h - 1 - 3 0 cm-1) b J/ZxC ~ Fractional absorption (%)o L-Xylose/urea permeability ratio ° Average pore radius (~,)r
5 4.8 _+ 0.4 5.0 ± 0.4 4.9 _+ 0.3
10 10.1 ± 0.5 10.1 _+ 0.4 10.1 _+ 0.4
20 19.6 + 0.3 19.6 _+ 0.4 19.6 _+ 0.3
12.8 0.3 12.5 1.4 0.12 32.0
± 0.7 ± 0.05 + 0.7 + 0.1 -+ 0.01 ± 2,5
12.6 0.3 12,3 1.3 0.11 16.1
± 0.4 _+ 0.05 + 0.4 ± 0.1 ± 0.01 ± 1.2
12.4 0.3 12.1 1.3 0.11 8.3
_+ 0.3 ± 0.05 ± 0,3 ± 0.2 ± 0.01 --+ 1.0
18.3 5.6 12.7 4.2 0,34 54.2
+ 1,0 ± 0.4 + 0.6 _+ 0.2 ± 0.02 ± 2.6
18.2 5.5 12.6 5.0 0,40 36.3
± ± ± + ± +
18.1 5,7 12.4 5.8 0.49 24.0
± 0.8 ± 0.4 ± 0.9 ± 0,7 g _+ 0.06 g ± 2.8
0.35 ± 0.03 13.3 ± 1.9
0.6 0.3 0.8 0,3 0.02 g 1.3
0.28 ± 0.02 g 9.2 ± 0.5 g
0.22 +_ 0.01 g 7.6 _+ 0,2 g
NOTE. All values are expressed as mean _+ SE (n = 9). aMean luminal concentration - mean serum concentration. °Absorption rate. CPermeability calculated as absorption rate + AC. °(Absorption rate + load entering segment) x 100. ea/ACL.~los e + J/ACurea. eCalculated from permeability ratio (see text]. gStatistically significantly different from 5 mL/min flow rate (P --< 0.05).
water absorption near zero, thus eliminating solvent drag as an absorptive force. The mannitol concentrations in the test segment were subject to change by the effects of flow rate per se (as described above for the other probes in experiment 1) or changes in the perfused concentration of mannitol (as required because of varying concentrations of L-xylose and urea in experiment 2). Nevertheless, the permeability of the 30-cm test segments to mannitol could be calculated by measuring J/AC. Combining the data from experiments 1 and 2, there was no significant difference in J/AC of mannitol at the three flow rates tested: J/dlC at 5 mL/min was 0.038 + 0.004 (n = 14), at 10 mL/min was 0.030 + 0.005 (n = 9), and at 20 mL/min was 0.029 + 0.006 (n = 14) (P > 0.42).
Discussion In the present experiments, triple-lumen in vivo perfusion was used to assess intestinal permeability at different flow rates within the physiological range. Solute absorption rate (J) divided by lumen to serum concentration gradient (AC) was used to measure the permeability of a 30-cm segment of jejunum to small hydrophilic solutes. As jejunal flow rate was increased from 5 to 20 mL/min, J/dlC for urea increased by 37% and 44% in two separate experiments. In contrast, J/AC for L-xylose and mannitol did not change. As pointed out in Materials and Methods, the perme-
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ability ratio of L-xylose/urea (J/AC for L-xylose divided by J/AC for urea) can be used to calculate the average pore radius of the aqueous pathway through which these solutes diffuse passively. When the jejunum was perfused at 5 mL/min, the average calculated pore radius of the diffusion pathway was about 13 /i, whereas when the perfusion rate was 20 mL/min, pore radius averaged about 8 A. This 38% reduction in average pore size can have a large impact on permeability because according to the assumptions and principles regarding diffusion through aqueous pores set out in Materials and Methods, a spherical molecule cannot permeate a cylindrical pore to any significant extent unless its molecular radius is less than half that of the pore radius. 1s'25 Therefore, a pore radius of 8 A would restrict the permeation of many more molecules (including most organic molecules) than would a pore radius of 13 A. We considered four possible explanations for the resuits of this study. The first postulates that high flow rates cause a perfused test solution to be exposed to a larger surface area than is available to the test solution when it is perfused slowly and that the newly recruited surface area is permeable to small molecules the size of urea but not to somewhat larger molecules like L-xylose and mannitol. Two lines of evidence in the literature support the notion that an increasing flow rate through a defined length of intestine causes an increase in the mucosal surface area exposed to fluid within the intestinal lumen. First, studies in rats have shown that the absorption rate of tritiated water and the absorption rate of D-glucose under maximum velocity conditions increase when perfusion rate is increased. 28 These changes could not be explained by changes in unstirred water layer thickness and were therefore attributed to an increased surface area exposed to the perfused test solutions by high flow. The second line of evidence also comes from studies in rats in which increasing the volume of a loop of small intestine perfused at a constant rate caused the intervillus space to open widely. 29 This was presumed to enhance exposure of the perfused test solution to cells deeper in the intervillus space. Perfusion studies in humans using dye dilution methodology showed that the average volume of a constantly perfused 30-cm jejunal segment would increase from 70 to 130 mL as flow rate was increased from 5 to 20 mL/min. 3° It seems reasonable to suggest that a flow rate-induced increase in luminal volume in humans, like in rats, would allow increased exposure of a perfused test solution to the intervillus space. Previous studies therefore suggest that increasing flow rates cause the exposure of luminal fluid to a greater surface area of mucosal cells and that this newly recruited
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surface area consists of mucosal cells lining the sides of villi. Therefore, the results of our present research, when interpreted in light of these previous observations, are consistent with the hypothesis that cells at the villus tips (exposed to luminal fluid at low rates of perfusion) are permeable to urea, L-xylose, and mannitol but that cells along the sides of the villi (exposed by higher flow rates) are permeable to urea but not to t-xylose or mannitol and therefore possess a smaller pore radius. The decrease in average pore size at higher flow rates is entirely consistent with our conclusion that fast flow rates cause luminal fluid to be exposed to mucosal cells of lower permeability than the more permeable mucosal ceils exposed when perfusion rate is slow. In addition, it should be pointed out that the values for pore radius calculated for the various flow rates tested reflect the average pore radius of all of the pores exposed at the respective flow rate. Therefore, our finding of a smaller average pore radius at higher flow rates is not attributed to a reduction in the size of a single population of pores but instead represents the average size of the larger pores exposed at slow perfusion rates and smaller pores exposed when perfusion rate is higher. Because the relative amount of additional surface area exposed by increasing flow rate is unknown, the pore radius of the newly exposed area in the intervillus space cannot be measured. However, the fact that J/AC for L-xylose did not increase at all suggests that the average pore radius of this newly exposed surface is less than twice the radius of L-xylose (i.e., < 6 . 8 A) because, as mentioned above, a pore radius/ molecular radius ratio of at least two is necessary for significant pore permeation. The second possible explanation we explored is that preferential stimulation of urea permeation, in the setting of a flow rate-induced deepening of the intervillus space, was the result of urea's ability to diffuse from the bulk contents of the lumen into the intervillus space more rapidly than L-xylose or mannitol because of its smaller molecular size. Two consequences of a smaller molecular size could theoretically favor greater delivery of urea into the newly opened intervillus space. One is more rapid diffusibility of urea in water per se (i.e., a larger aqueous diffusion coefficient). The second is more rapid permeation of the mucosa (i.e., a larger membrane permeability coefficient) as it relates to the development of a greater concentration gradient down the intervillus space. A concentration gradient of solute might develop in the intervillus space assuming the bulk of the test solution was excluded from the space while solute diffused perpendicularly into the space. The concentration of the solute would be greatest at the space entrance (and equal to that in luminal contents) and would progressively de-
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crease along its path down the intervillus space as solute was absorbed. Because of greater permeability to the smaller urea compared with L-xylose or mannitol (even if the overall permeability of the mucosa was constant along the entire villus), a greater concentration gradient would develop for urea. To the extent that a concentration gradient down the intervillus space could enhance diffusion of solute from bulk luminal contents, proportionally more urea would diffuse into the intervillus space, possibly facilitating a more rapid absorption rate. 31 Assuming that the decrease in concentration of urea down the intervillus space was slow enough to allow urea absorption to continue well into this space, this explanation could account for a greater flow rate-induced increase in permeability to urea relative to L-xylose or mannitol, but it could not explain an increase in permeability to urea with no increase whatsoever in the permeability to L-xylose or mannitol. Moreover, if the urea concentration decreased so rapidly that very little urea was left part way into the intervillus space, further deepening of the intervillus space would provide little if any increase in functional absorbing surface and consequently no further increase in its permeation. 32 Under these circumstances, a deepening intervillus space could result in a proportionally greater increase in functional surface area and absorption of mannitol and t-xylose relative to urea because a greater number of these larger molecules would be present over a longer path down the intervillus space. 32 For these reasons, we do not believe that our results can be satisfactorily explained by urea's greater aqueous diffusability or membrane permeability as it relates to a steeper concentration gradient down the intervillus space. The third mechanism we explored to explain our findings was reduction in unstirred water layer thickness at higher flow rates. 33'34 According to this theory, the bulk contents of the intestinal lumen are stirred completely, whereas a layer of pre-epithelial fluid of fixed thickness is unstirred and therefore acts as a diffusion barrier to solute absorption. The unstirred water layer as a diffusion barrier can be expressed mathematically as follows35: D J = -- AC, t in w h i c h J is solute flux across the unstirred water layer, D is the aqueous diffusion coefficient, t is the thickness of the unstirred water layer, and AC is the concentration gradient across the unstirred water layer. If the value for t decreases, J for a molecule with a larger D (like urea) would increase to a larger numerical extent than would J for a molecule with a smaller D (like L-xylose); however,
the fractional change in J for each molecule would be the same because it is changing by the same reduction in t. Looked at in another way, expressing JL-~ylose/J.... as a ratio results in a cancellation of t from the mathematical expression. Therefore, if in our experiments increased flow rate decreased unstirred water layer thickness, JL-xylose and J . . . . would have increased proportionally and the ratio of JI._xylose/J.... would have been unaffected. Thus, we cannot attribute our finding a decreasing L-xylose/ urea permeability ratio at higher flow rates to a thinning unstirred water layer. The fourth possibility we considered was whether changes in the laminar flow properties of the perfused fluid could account for a flow rate-induced increase in urea permeability with no change in L-xylose or mannitol permeability. According to the principles of laminar flow, the flow rate of fluid is most rapid (and most perfectly stirred) in the center of the lumen with progressively slower flow rates (and poorer stirring) in fluid near the intestinal epithelial cells. 36-38 According to this theory, for a given solute, fractional absorption is inversely proportional to flow rate if mucosal permeability and absorptive surface area remain constant. 38 Therefore, in our study, if there were no changes in mucosal permeability or absorptive surface area, sequentially doubling the flow rate should have sequentially halved the fractional absorption rate of all three solutes. Whereas this was observed for z-xylose (mean fractional absorption rates of 32%, 16%, and 8% at flow rates of 5, 10, and 20 mL/ rain, respectively) and mannitol (respective mean fractional absorption rates of 11%, 5%, and 2.4%), the fractional absorption of urea decreased by a lesser factor each time flow rate was doubled (fractional absorption rates of 54%, 36%, and 24% at flow rates of 5, 10, and 20 mL/min, respectively). Therefore, a change in laminar flow properties of fluid cannot explain the higher permeation rate of urea at faster flow rates unless a concomitant selective increase in urea permeability and/or absorptive surface occurred. In our opinion, the best explanation of the findings of this study is that increasing intestinal flow rate from 5 to 20 mL/min enhances exposure of a perfused test solution to mucosal cells lining the intervillus space and that these cells are less permeable (i.e., have "tighter" tight junctions) than ceils lining the tips of villi. Regardless of the explanation, these studies show that changes in flow rate within the physiological range can alter the apparent permeability of normal healthy jejunal mucosa. This fact needs to be considered when interpreting measures of tight junction permeability in experimental situations in which there exist significant differences in intestinal flow rates and/or intestinal volume.
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21. Jacobsen ED, Bondy DC, Broitman SA, Fordtran JS. Validity of polyethylene glycol in estimating intestinal water volume. Gastroenterology 1 9 6 3 ; 4 4 : 7 6 1 - 7 6 7 . 22. Hyden S. A turbidimetdc method for determination of higher polyethylene glycols in biological materials. Lantbrukshoegsk Ann 1955;22:139-145. 23. Roe JH, Rice EW. A photometric method for the determination of free pentoses in animal tissues. J Biol Chem 1948; 1 7 3 : 5 0 7 512. 24. Corcoran AC, Page IH. A method for the determination of mannitol in plasma and urine. J Biol Chem 1947; 1 7 0 : 1 6 5 - 1 7 1 . 25. Renkin EM. Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physio11954; 3 8 : 2 2 5 - 2 4 3 . 26. International critical tables of numerical data, physics, chemistry, and technology. U.S. National Research Council. New York; McGraw Hill, 1 9 2 9 : 6 9 - 7 0 . 27. SAS/STAT user's guide, version 6 . 4 t h ed. Volume 2. Cary, NC: SAS Instutue Inc., 1989. 28. Lewis LD, Ferdtran JS. Effect of perfusion rate on absorption, surface area, unstirred water layer thickness, permeability, and intraluminal pressure in the rat ileum in vivo. Gastroenterolog~ 1975; 6 8 : 1 5 0 9 - 1 5 1 6 . 29. Harris SM, Kennedy JG, Siegesmund KA, Yorde DE. Relationship between distention and absorption in rat intestine. I. Effect of luminal volume on the morphology of the absorbing surface. Gastroenterology 1 9 8 8 ; 9 4 : 1 1 6 4 - 1 1 7 1 . 30. Diilard RL, Eastman H, Fordtran JS. Volume-flow relationship during the transport of fluid through the human small intestine. Gastroenterology 1 9 6 5 ; 4 9 : 5 8 - 6 6 . 31. Harris MS, Kennedy JG. Relationship between distention and absorption in rat intestine. I1. Effects of volume and flow rate on transport. Gastroenterology 1988; 9 4 : 1 1 7 2 - 1 1 7 9 . 32. Winne D. The permeability coefficient of the wall of a villous membrane. J Math Biol 1 9 7 8 ; 6 : 9 5 - 1 0 8 . 33. Winne D. Rat jejunum perfused in situ: effect of perfusion rate on intraluminal radius on absorption rate and effective unstirred layer thickness. Arch Pharmacol 1 9 7 9 ; 3 0 7 : 2 6 5 - 2 7 4 . 34. Westergaard H, Holtermuller H, Dietschy JM. Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am J Physiol 1986;250:G727-G735. 35. Dietschy JM, Sallee VL, Wilson FA. Unstirred water layers and absorption across the intestinal mucosa. Gastroenterology 1971;61:932-934. 36. Amidon GL, Kou J, Elliott RL, Lightfoot EN. Analysis of models for determining intestinal wall permeabilities. J Pharm Sci 1980; 6 9 : 1 3 6 9 - 1 3 7 3 . 37. Levitt MD, Fetzer CA, Kneip JM, Bond JH, Levitt DG. Quantitative assessment of luminal stirring in the perfused small intestine of the rat. Am J Physiol 1987;252:G325-G332. 38. Levitt MD, Kneip JM, Levitt DG. Use of laminar flow and unstirred layer models to predict intestinal absorption in rat. J Clin Invest 1988; 8 1 : 1 3 6 5 - 1 3 6 9 . Received June 9, 1994. Accepted December 13, 1994. Address requests for reprints to: Kenneth D. Fine, M.D., Department of Internal Medicine, Baylor University Medical Center, 3500 Gaston Avenue, Dallas, Texas 75246. Fax: (214) 820-4837. Supported by U.S. Public Health Service grant 5-RO1-DK37172-05 from the National Institute of Diabetes and Digestive and Kidney Diseases and the Southwest Digestive Disease Foundation. The authors thank Diana Santa Ana for her help with preparation of the manuscript and Dr. Donald Mclntire, University of TexasSouthwestern Medical Center at Dallas, for his help with mathematical and statistical computation.