LYMPHATIC AND VENOUS TRANSPORT OF WATER FROM RAT JEJUNUM: A VASCULAR PERFUSION STUDY

GASTROENTEROLOGY Copyright 1968 by The Williams & Wilkins Co. Vol. 54, No.4, Part 1 of 2 Parts Printed in U.S.A. LYMPHATIC AND VENOUS TRANSPORT OF WATER FROM RAT JEJUNUM: A VASCULAR PERFUSION STUDY J. S. LEE, PH.D., AND KATHRYN M. DUNCAN, B.S. Department of Physiology, University of Minnesota, Minneapolis, Minnesota Procedures for vascular perfusion of intestine have been described for a variety of studies l - 9 but by none of them was it possible to partition water transport by the lymphatic and venous systems. This report describes a procedure for perfusing rat upper jejunum with heparinized homologous blood showing net water transport into the venous and lymphatic systems. The rates of water transport via these pathways were studied under various conditions. Methods Male rats (350 to 450 g) were fasted for 18 to 24 hr with free access to water. A short upper jejunal segment (15 to 25 cm in length) was employed and its upper end began about 7 cm from the ligament of Treitz. The surgical procedure for isolating a segment with intact mesentery for in vitro study was that described previously.'o Before the segment was deprived of blood supply, its lumen was rinsed with bicarbonate Ringer solution containing glucose (27 mm per liter), which was saturated with 5% CO 2 in O2 and which was of ionic composition similar to that of Krebs and Henseleit solution." After food residue and secretions had been removed, 100 ml of the same solution were circulated through the lumen at a rate of about 20 ml per min to provide nutrients for the intestine. The reser- Received O ctober 14, 1967. Accepted November 22, 1967. Address requests for reprints to: Dr. J. S. Lee, Department of Physiology, University of Minnesota, School of Medicine, Minneapolis, Minnesota. 55455. This investigation was supported by Public Health Service Grant AM05073 from the National Institute of Arthritis and Metabolic Diseases and research funds of the Graduate School of the University of Minnesota. A preliminary report of this investigation has appeared in Physiologist 10 : 228, 1967. 559 voir-pumping system for circulating the mucosal fluid has been described in detail previously.12 After a few minutes, the segment with intact mesentery was removed from the body and placed in a warm oil (liquid petrolatum) chamber (38 C) as shown in figure 1. The tubings (as indicated by arrows) connected to the intestinal cannulas with stopcocks 1 and 2 were attached to the reservoirpumping system (not shown in fig. 1). The circulation of mucosal fluid was continued until vascular perfusion with blood via the mesenteric artery was begun. The volume and composition of the mucosal fluid are described with each type of experiment. The oil chamber was a double jacketed glass with warm water circulating through it. A stainless steel wire screen was used to support the segment, but it was found to be not essential. The mesenteric artery was cannulated with a lo-cm PE 50 (polyethylene) tubing (OD, 0.097 mm; inside diameter, 0.059 mm), the other end of which was attached to a 100-cm PE 240 tubing (T2' OD, 2.41 mm; inside diameter, 1.68 mm), which was in turn connected to the arterial perfusion blood reservoir (AP. reservoir) via stopcock 5 (three way) and 6 (one way). The three way stopcock was used to sample the a.rterial perfusion blood. The reservoir was a 25-ml graduated pipette fitted with stopcock -4 at its upper end leading to a gas source. In most experiments, the gas used was room ai r and the use of 5% CO 2-95% O 2 yielded the same results. The pressure of the gas (above the perfusion blood) plus the hydrostatic pressure of the perfusion blood in the reservoir constituted the perfusion pressure. The arterial pressure was determined from the junction between arterial cannula and tubing T2 with pressure transducer and electronic recording system. The pressure drop along the cannula to the superior mesenteric artery was about 5 mm Hg. The perfusion blood in the reservoir was at room temperature but it was raised to body temperature (38 C) after passing through T2 which was immersed in a warm water bath (not shown in

560 LEE. AND DUNCAN Vol. 54, No.4, Part 1 Mucosal fluid out in A.P. reservoir liquid in Warm waler FIG. 1. The setting up of the perfusion system. M, mesentery; A.P. reservoir, arterial perfusate reservoir; D.P., intraluminal distention pressure; 1-6, stopcocks; T" T 2, connecting tubes. fig. 1). A bubble trap was placed between the arterial perfusion blood reservoir and T2 (not shown in fig. 1). The portal vein was cannulated with 5-cm PE 240 tubing. The cannula was then attached to a T-tube connector and the latter was connected to PE 240 tubing of about 40 cm in length. The side branch of the T-tube was connected to the pressure transducer and electronic recording system. The level of the open end of the long tubing with reference to the pressure base line of the segment determined the venous pressure. In experiments with zero venous pressure, a lo-cm PE 90 tubing (OD, 1 mm ; inside diameter, 0.86 mm) was used as the venous cannula and its open end was held at a level of about 6 cm below the segment. The lymphatic duct was cannulated with PE 50 tubing by a procedure described previously:o In some cases, when lymph vessel cannulation failed, lymph was allowed to drip to the bottom of the oil chamber. The fluid transported across the serosal surface as "serosal fluid" or "serosal sweat" was practically zero in the segments perfused with blood. Determination of net water transport rate. For the determination of net water transport rate, a small volume of mucosal fluid (10 ml) was used. After vascular perfusion was begun, the circulation of the large volume of mucosal fluid was stopped by turning stopcocks 1 and 2 in a position as shown in figure 1. The lumen of the segment was then emptied of fluid by repeatedly injecting air through it. Then, exactly 10 ml of mucosal fluid were introduced from stopcock 1; it passed through the lumen of the segment, stopcocks 2 and 3, and the connecting tube (T 1, 20 cm in length, inside diameter, 2 mm) and entered a graduated mucosal fluid reservoir. The fluid level in the reservoir above the pressure base line of the segment determined the intraluminal distention pressure, which was 3 mm Hg in all experiments. About 1 to 1.5 ml of fluid were in the lumen of the segment of 15 to 25

Apj'ill968 LYMPHATIC AND VENOUS TRANSPORT OF WATER 561 cm in length, and the rest was in T, and the reservoir. At the end of a perfusion period, the mucosal fluid in the lumen and T, was emptied into the reservoir, and the final volume was measured. A decrease in volume resulted from net water transport from lumen to the blood and lymph and an increase in volume resulted from a secretory flux. The error of volume measurement was ±0.05 ml. Preparation of blood. Blood was bled through a cannula in the abdominal aorta of unfas ted rats under sodium pentobarbital anesthesia (50 mg per kg, intraperitoneally) with artifi cial respiration of 100% 0.. About 1 min before bleeding heparin sodium, 2 mg per 100 g body wt, was injected intravenously. Glucose was added to the blood to increase its concentration in plasma to about 12 mm per liter. Just before use, the blood was equilibrated with 5% CO 2-95% 0. The blood already contained a certain amount of sodium pentobarbital but an additional amount of 5 mg per 100 rnl was always added. A higher concentration of this drug was used to prevent the occurrence of a spasmodic type of motility. The segments perfused with blood exhibited feeble rhythmical contractions, which were similar to those occurring in situ under sodium pentobarbital anesthesia. For one experiment, the pooled blood from 3 rats (about 50 ml) was sufficient. Fresh blood was always used and no recirculation was adopted. After perfusion with blood under the specific conditions of an experiment for 10 min, determinations of various flow rates were begun. The arterial inflow rate was measured from the decrease in blood volume in the arterial perfusion blood reservoir. The venous outflow was collected into a graduated cylinder. Lymph was collected into a graduated tube. The V-A difference is the difference between the venous outflow rate and arterial inflow rate. A positive V -A difference indicates fluid transport into the venous system and a negative V-A difference indicates the loss of fluid from the venous system. All these rates are expressed as microliters per centimeters (of intestinal l ength) per hour. The mean wet weight per centimeter length of rat upper jejunum (350 to 450 g body wt) was 0.055 g ± 0.008 (mean of 69 rats ± S D) and the mean dry weight was 10.9 mg ± 1.1 (mean of 35 rats ± SD). These figures can be used fo r calculating the flow rates on either the wet or dry weight basis, if desired. Statistical analysis of data was done by the t-test. Results Comparison of V-A Difference, Lymph Flow, and Net Water Transport R ate In this series of experiments, the segments were perfused for 30 min with blood at an arterial pressure of 70 mm Hg with the venous pressure varying from 0 to 20 mm Hg. The net water transport rate was determined as the change in mucosal fluid volume. In one group of experiments, the mucosal fluid was 10 ml of bicarbonate Ringer solution with glucose (27 mm per liter) and, in another group, it was 10 ml of isotonic MgS0 4 solution without glucose. The results are presented in table 1. TABLE 1. Comparison of various flow rates and water transport rate a Mucosal fluid Flow rates Ipercentages of Water transport rate b Water transport rate Venous Lymph [ V-A dif- Lym- Venous outflow [V-A difference[ flow ference + phatic lymph flow system [ system Venous pressure Arterial inflow[ tnmhg )'liters/ em/ hr )'liters/ em/!lt % Bicarbonate o (4)C 1782 ± 326 1889 ± 333 107 ± 14 13 ± 0 120 ± 14 120 ± 14 10 ± 1 90 ± 1 Ringer solution 10 (5) 1982 ± 250 2075 ± 250 93 ± 18 27 ± 6 120 ± 6 133 ± 42 22 ± 5 78 ± 5 20 (6) 1503 ± 109 1503 ± 114 o ± 16 d 53 ± 5 53 ± 11 67 ± 5 100 0 Isotonic MgSO. o (4) 1596 ± 20 1556 ± 20-40 ± 7 0-40 ± 7 0 solution 10 (4 ) 1968 ± 47 1902 ± 53-66 ± 7 0-66 ± 7-27 ± 71 a P erfusion period, 30 min; arterial pressure, 70 mm Hg; venous pressure, as shown in t able. Values are means ± SEM. b Sum of V-A difference and lymph flow was taken as water transport rate. C Number in parentheses indicates number of experiments. d In two cases, V -A difference was negative. Negative V-A difference indicates loss of fluid from the venous system. f Negative water transport rate indicates net water transport in the secretory direction. ----

562 LEE AND DUNCAN Vol. 54, No.4, Part 1 It can be seen that, when mucosal fluid was bicarbonate Ringer solution, net water transport from lumen to blood and lymph occurred. The water transport rate was practically the same at venous pressures from 0 to 10 mm Hg, but it was significantly reduced (P < 0.01) when the venous pressure was raised to 20 mm Hg. At all venous pressures tested, the swn of V-A difference and lymph flow was about the same as the net water transport rate. This demonstrates that the positive V -A difference is a valid measure of net water transport into the venous system and lymph flow is that of net water transport into the lymphatic system. The percentages of water transport into these systems are listed in the last two columns of table 1. The percentage transport by the lymphatic system increased with increasing venous pressure and the reverse was the case for that by the venous system. At zero venous pressure, lymph accounted for only 10% of the total net transport but, at 20 mm Hg venous pressure, water transport was entirely by way of the lymphatic system. In two of six experiments with high venous pressure (20 mm Hg), the V -A difference was slightly negative, indicating loss of fluid from the venous system. When venous pressure was increased from 0 to 10 mm Hg, the V -A difference did not change significantly (P > 0.50). At high venous pressure (20 mm Hg), the V -A difference fell to zero. Lymph flow was 13 ± 0, 27 ± 6, and 53 ± 5 ftliters per cm per hi' at venous pressure of 0, 10, and 20 mm Hg, respectively. The difference between lymph flow rate at 0 and 10 mm Hg was not statistically significant (P > 0.05), but the increase in rate at 20 mm Hg was significant (P < 0.01). When the mucosal fluid was an isotonic MgS0 4 solution, net water transport was nil at zero venous pressure. At a higher venous pressure (10 mm Hg), the volume of mucosal fluid was increased, indicating net water transport into the lumen of intestine. This was in agreement with the finding that the V -A difference was slightly negative at zero venous pressure and more so at higher venous pressure. At both venous pressures, lymph flow was nil. These observations demonstrated that when an unabsorbable electrolyte solution was placed in the lumen of intestine, no positive V-A difference or lymph flow could be obtained. The arterial inflow and venous outflow rates as listed in table 1 are discussed together with the subsequent section. Effects of Arterial and Venous Pressures on Various Flow Rates In this series of experiment, the mucosal fluid was 100 ml of bicarbonate Ringer solution containing glucose (27 mm per liter), and it was circulated at a rate of about 20 ml per min. The perfusion period was 20 min. The intraluminal distention pressure was 3 mm Hg. The rate of fluid disappearance from the mucosal side was not determined. Based on the result of the preceding series of experiments, the sum of V -A difference and lymph flow was taken as an approximate measure of total net water transport rate. When an unabsorbable electrolyte solution (isotonic MgS0 4 solution without glucose) was circulated through the lumen of the perfused segments at an arterial pressure of 70 or 100 mm Hg with venous pressure from 0 to 20 mm Hg, it was repeatedly found that lymph flow was zero and V-A difference was either zero or negative. The results of the effects of arterial and venous pressures on various flow rates are presented in table 2. V -A difference. At a constant arterial pressure (70 or 100 rum Hg), the V -A difference decreased with increasing venous pressure, but the effect of arterial pressure varied with the range of venous pressure. At zero venous pressure, the increase of arterial pressure from 50 to 70 mm Hg caused no change in V -A difference (120 ftliters per em per hi') but, when arterial pressure was increased to 100 mm Hg, the V-A difference (200 ftliters per em per hi') increased significantly (P < 0.01). At higher venous pressures, the increase of arterial pressure caused either no change or decrease of V-A difference. For instance, when the arterial pressure was 70 mm Hg

Apl'il 1968 LYMPHATIC AND VENOUS TRANSPORT OF WATER 563 TABLE 2. Effect of arterial and venous pressures on vario1ts flow rates Mean flow rates Water transport Arterial pressure Venous 1- - --,------ ----;-----,-- - ----,-- - -- pressure Arterial inflowl Venous!V-A difference I Lymph flow IV-A difference Lymphatic I outflow + lymph flow system Venous system 1- - ----'---- mm H g "liters/em/ltr % 50 (4) b 0 1370 ± 206 1490 ± 213 120 ± 0 40 ± 14 160 ± 40 25 ± 4 75 ± 4 70 (6) 0 2128 ± 239 2248 ± 239 120 ± 11 27 ± 5 147 ± 16 18 ± 3 82 ± 3 70 (4) 5 2421 ± 499 2567 ± 532 146 ± 40 40 ± 14 186 ± 14 21 ± 4 79 ± 4 70 (5) 10 2647 ± 368 2753 ± 358 106 ± 12 93 ± 18 199 ± 12 47 ± 7 53 ± 7 70 (6) 15 2248 ± 222 2328 ± 228 80 ± 11 106 ± 16 186 ± 11 57 ± 6 43 ± 6 70 (7) 20 1636 ± 212 1676 ± 222 40 ± 15 8~ ± 15 120 ± 30 75 ± 9 25 ± 9 70 (3) 25 1157 ± 161 1117 ± 169-40 ± 8 e 93 ± 31 53 ± 23 100 0 100 (4) 0 5050 ± 353 5250 ± 347 200 ± 13 26 ± 0 226 ± 13 12 ± 1 88 ± 1 100 (3) 5 5453 ± 116 5599 ± 92 146 ± 46 40 ± 0 186 ± 46 21 ± 7 79 ± 7 100 (6) 10 3485 ± 385 3511 ± 385 26 ± 5 53 ± 11 79 ± 22 67 ± 7 33 ± 7 100 (5) 15 2008 ± 328 1982 ± 334-26 ± 6 e 80 ± 18 54 ± 18 100 0 a. Perfusion period, 20 min; mucosal fluid, 100 ml of bicarbonate Ringer solution containing 27 mm glucose. Values are means ± SEM. b N umber in parentheses indicates number of experiments. C Nega tive V-A difference indicates loss of fluid from the venous system. and venous pressure was 10 mm Hg, the V-A difference was 106 j.tliters per cm per hr, but when the arterial pressure was raised to 100 mm Hg and the venous pressure was 10 mm Hg, the V-A difference (26 j.tliters per cm per hr) decreased significantly (P < 0.01). Lymph flow. At zero venous pressure, lymph flow did not change significantly when arterial pressure was increased from 50 to 70 or to 100 mm Hg (P > 0.30). At a constant arterial pressure of 70 mm Hg the difference between lymph flow rate at zero and 5 mm Hg of venous pressure was not significant (P > 0.30). The increase of venous pressure from zero to 10 mm Hg caused a significant increase in lymph flow (P < 0.01). Further increase of venous pressure resulted in no further significant change in lymph flow. When arterial pressure was 100 mm Hg, the lymph flow rate was similar to that when arterial pressure was 70 mm Hg. Net water transport rate (sum of V-A difference and lymph flow). At zero venous pressure, an increase of arterial pressure from 50 to 70 or to 100 mm Hg caused no significant change in water transport rate (P > 0.80 or > 0.10, respectively). At higher venous pressures (5 rum Hg or above), the increase of arterial pressure from 70 to 100 mm Hg resulted in either no change or decrease of water transport rate. For instance, at a venous pressure of 10 mm H g, the water transport rate was 199 j.tliters per cm per hr when arterial pressure was 70 mm Hg and it was 79 j.tliters per cm per hr when arterial pressure was 100 mm Hg. This difference was s:gnificant (P < 0.01). At a constant arterial pressure, the increase of venous pressure above 10 mm Hg (arterial pressure, 100 mm Hg) or above 20 mm Hg (arterial pressure, 70 mm Hg) always resulted in a decrease of water transport rate (P < 0.01). Percentage water transport via the venous and lymphatic systems. At low venous pressure (0 to 5 mm Hg), water transport by the lymphatic system was 12 to 25% of the total absorbed water a t arterial pressures from 50 to 100 mm Hg. With t he increase of venous pressure, more water was transported by the lymphatic system. At venous pressure of 15 mm Hg with arterial pressure of 100 mm Hg or at venous pressure of 25 mm Hg with arterial pressure of 70 mm Hg, water transport was entirely via the lymphatic system. Arterial inflow rates and venous outflow rates. As shown in table 2, at zero venous pressure, an increase of arterial pressure

564 LEE AND DUNCAN Vol. 54, No. 4, Part 1 ~ Arterial inflow 3400 T 3OO0 J i I I I T 1 2 6 0 0 I 17 ~ T 4 I I!7 "00] 1800 r-------:----=----------i j J. E '- 100 Lymph flow E u '- 80 :;. 60 0 ~ 40 0::!< 0 220 G: 180 140 100 60 V- A difference o 10 20 30 40 50 Time (min) 1 I FIG. 2. Time course of arterial inflow, lymph flow, and V-A difference. Zero time is the interval between 0 and 10 min; 10 min is the interval between 10 and 20 min, and so forth. Arterial pressure, 70 mm Hg; venous pressure, 0; intraluminal distention pressure, 3 mm Hg. Values are means :±: SEM of three to seven experiments as indicated. The differences in V -A difference between 0 and 10 and 10 and 20, between 0 and 10 and 20 and 30, and between 10 and 20 and 20 and 30 time intervals are not statistically significant (P > 0.50, P > 0.30, and P > 0.20, respectively). from 50 to 70 mm Hg resulted in no significant change (P > 0.05) in arterial inflow rate as well as venous outflow rate, but at 100 mm Hg arterial pressure the arterial inflow rate (5050,.,.liters per cm per hr) was significantly greater (P < 0.01) than t hat at 50 mm Hg (1370 flliters per cm per hr) or at 70 mm Hg (2128 flliters per em per hr). At 5 mm Hg of venous pressure, arterial inflow rate was also significantly increased (P < 0.01) when arterial pressure was increased from 70 to 100 mm Hg, but at higher venous pressure (15 mm Hg) there was no significant change in arterial inflow rate (P > 0.70) when arterial pressure was increased from 70 to 100 mm Hg. I I As shown in both table 1 and 2, at an arterial pressure of 70 mm Hg, the arterial inflow rate or venous outflow rate did not change significantly with increasing venous pressure in the range of 0 to 20 mm Hg (P > 0.10 to 0.70). At very high venous pressure (25 mm Hg, table 2), arterial inflow rate was significantly reduced as compared with that at zero venous pressure (P < 0.05). When arterial pressure was 100 mm H g, no significant difference in arterial inflow rate was noted by the increase of venous pressure from 0 to 5 mm Hg, but the arterial inflow rate was significantly reduced when venous pressure was increased to 10 mm Hg (P < 0.05) or to 15 mm Hg (P < 0.01). Time Course of Arterial Inflow, Lymph Flow, and l' -A Difference The segments were perfused with an arterial pressure of 70 mm H g and venous pressure of 0 mm Hg. One hundred milliliters of bicarbonate Ringer solution containing glucose (27 mm per liter) were circulated through the lumen of the segments. Various flow rates were determined at lo-min intervals. As shown in figure 2, both V-A difference and lymph flow maintained for the first three successive lo-min intervals and then declined steadily with time. After 90 min, both V -A difference and lymph flow were negligible (not shown in fig. 2). Arterial inflow rate was well maintained for at least 60 min in all cases so far observed. After 60 min, it also declined..il! iscellaneous Observations The mean ph of the arterial blood was 7.41 ± 0.03 (mean of seven determinations ± SEM) and mean venous ph was 7.17 ± 0.Q1. This difference was significant (P < 0.01). The color of the venous blood was deep blue, indicating low O 2 concentration. When mucosal fluid was an isotonic MgS04 solution without glucose, the glucose concentration of the venous blood (9.2 mm per liter ± 0.5 mean of five determinations ± SEM) was signific antly (P < 0.02) lower than that of the arterial

April 1968 LYMPHATIC AND VENOUS TRANSPORT OF WATER 565 " blood (12.2 ± 0.8 mm per liter), demonstrating glucose utilization by the intestine. When mucosal fluid was bicarbonate Ringer solution containing glucose (27 mm per liter), the venous glucose concentration (16.3 mm per liter ± 1.0, mean of eight determinations ± SEM) was significantly higher (P < 0.01) than the arterial glucose concentration (12.6 mm per liter ± 0.3). This shows the transport of glucose from the lumen into the venous system. The water content of the segments perfused for 10 min or more at various arterial and venous pressures varied from 76.3 to 78.8%; this was higher than the water content of the nonperfused segments (75.4 ± 0.2%, mean of 38 determinations ± SEM). Discussion The results of this study demonstrate that an isolated and vascular perfused intestine can transport fluid into both the venous and lymphatic systems. The total water transport rate at an arterial pressure of 70 to 100 mm Hg with venous pressure in the range of 0 to 15 mm Hg (table 2) was 147 to 226 p.liters per cm per hr. This is similar to the water transport rate (215 p.liters per cm per hi', mean of five experiments, range 175 to 226 p.liters per cm per hi') of the similar segments in situ (under sodium pentobarbital anesthesia). The perfused segments consumed glucose. Glucose transport into the venous system was also indicated by the increase of glucose concentration in the venous blood. The perfused segments showed feeble rhythmical segmental contractions. Therefore, the behavior of the perfused segments was similar to that of the similar segments in situ in many respects. Although the maximal fluid transport rate lasted about 30 min, it appears to be suitable for many types of investigation. According to Barrowman and Roberts,13 in unanesthetized rats, the intestinal lymph flow accounted for 11% (range 7.0 to 16.0%) of the oral intake of 0.9% NaCI solution. Assuming complete gastric emptying and complete fluid absorption in their experiments, the lymphatic system apparently transported 11 % of the absorbed fluid. This is fairly close to the results obtained in the present perfused segments at low venous pressure. As shown in tables 1 and 2, 10 to 25% of water was transported by the lymphatic system with venous pressure from 0 to 5 mm Hg, but at high venous pressure (above 20 mm Hg) water transport depended entirely on the lymphatic system. In an in vitro rat upper jejunal preparation without vascular perfusion, 85% of the absorbed water is transported by the lymphatic duct and only 11% by the mesenteric vein 1o ; thus, it is postulated that water may be primarily absorbed into the lymphatic system but transferred into the venous system within the intestine in situ or under vascular perfused condition at low venous pressure. The close contact between blood capillary and lymphatic plexus in the submucosa would seem to permit net water transport between blood and lymph. From the effects of arterial and venous pressures on the increase of percentage transport by the lymphatic system (tables 1 and 2), the fraction of fluid transport by this route in situ is probably not constant, but varies with the existing blood pressure, especially the portal pressure. Intestinal motility, distension pressure, and lymphatic contractility have been shown to be essential factors influencing intestinal lymph flow,14 and these might also affect the fluid transport via the lymphatic system. When D 20 was placed in the lumen of rat intestine, Benson et aj.15 found that 1 % of the absorbed D 2 0 molecules appeared in the lymph and that the concentration of D 2 0 in the lymph was the same as that in the arterial plasma and lower than that in the venous plasma. They concluded that 1 % of water was transported by the lymphatic system. Based on the amount of tritiated water (T 2 0) recovered from the intestinal lymph and total body water during lavage of rat intestine with salt solution containing T 2 0, Noyan 16 reached a similar conclusion that the lymphatic system transported 3% of the absorbed water. We (Visscher, Grim, and Lee, in Vis-

566 LEE AND DUNCAN Vol. 54, No.4, Part 1 scher 17 ) have obtained a result similar to that reported by Benson et a l,15 When dog ileum was lavaged with salt solution containing D 2 0, the lymph D 2 0 concentration was lower than the venous but close to the arterial D 2 0 concentration. However, when the lumen was lavaged with hypotonic solution containing K42 or Na 22, lymph contained more tracer than did venous plasma. Thus, there is no reason to believe that D 2 0 does not enter lymph at a concentration higher than that in the venous plasma; the low lymph D 2 0 concentration was most likely due to re-equilibration with arterial plasma during its passage through the intestinal wall. Therefore, the amount of D 2 0 or TzO in the lymph is not a valid measure of net water transport under these conditions; this is apparently the cause for the low estimate of the lymphatic transport of water by these investigators. 15 16 The decrease of water absorption rate at high venous pressure has been observed in dogs in situ by Wells 18 and Shields and Code. 19 A similar result was obtained from the present perfused segments. The decrease of water absorption rate was due to the decrease of water transport into the venous system and the transport of water by the lymphatic system was increased at high venous pressure. When an isotonic MgS0 4 solution was placed in the lumen of intestine, the V-A difference was slightly negative, indicating the loss of fluid from the vascular system. Since lymph flow was nil (table 1), the lost fluid must appear either in the lumen or remain in the intestinal tissue. At zero venous pressure, no net water transport into the lumen was observed. At 10 mm Hg venous pressure water was indeed transported into the lumen but it was at a rate (-27,...liters per cm per hr) less than the V -A difference (-66,...liters per cm per hr). However, the estimate of the rate of water transport into the lumen could have been low because in these segments mucus secretion was much increased. The volume of water held by the mucus on the mucosal surface was not estimated. As shown in table 1, when venous pressure was varied between 0 and 20 mm Hg, there was no significant change in arterial inflow rate but significant increases in lymph flow (P < 0.01), and decreases in V-A difference (P < 0.01) and water absorption rate (P < 0.01) were obtained. This indicates that these changes are not related to arterial inflow rate. The data in table 2 also show no consistent correlation between arterial inflow rate and other p arameters in most instances. However, the decreases of V -A difference and water absorption rate at high venous pressure could be partly related to the decrease in arterial inflow rate. When the segments were perfused at an arterial pressure of 70 mm Hg with venous pressure of 0 mm Hg, the mean arterial inflow rate was 2128,...liters per cm per hi', or 65 ml per 100 g wet wt per min. At higher arterial presure (100 mm Hg), the arterial inflow rate increased by 134% or to 5050,...liters per cm per hr (152 ml per 100 g per min). But the water absorption rate was increased only by 54% from 147 to 226,...liters per cm per hr. At higher venous pressure (10 mm Hg), the water absorption rate (79,...liters per cm per hr) was significantly smaller (P < 0.01) at 100 mm Hg of arterial pressure than that (199,...liters per cm per hi') at 70 mm Hg. Therefore, it is felt that the lower arterial pressure IS more practicable in perfusion studies. Summary A procedure for vascular perfusion of rat upper jejunum with heparinized homologous blood is described. When bicarbonate Ringer solution was placed in the lumen of the intestine, net water absorption was demonstrated by a positive V-A difference (venous outflow rate > arterial inflow rate) and lymph flow. The sum of V-A difference and lymph flow was found to be equal to the rate of water disappearance from the lumen of intestine. When an unabsorbable salt solution (MgS0 4 ) was placed in the lumen, the V -A difference was either zero or negative and lymph flow was nil. Lymph flow was small at low venous pressure but increased at high venous pres-

April 1968 LYMPHA1'IC AND VENOUS TRANSPORT OF WATER 667 sure. The change of arterial pressure between 50 and 100 mm Hg exerted no significant effect on lymph flow. The rate of water transport into the venous system as well as that of the total net water transport were reduced at high venous pressure. The percentage transport of water by the lymphatic system increased with the increase of venous pressure and the reverse was the case for the percentage transport by the venous system. At very low venous pressure (0 to 5 mm Hg), lymph flow accounted for 10 to 25% of net water transport but, at high venous pressure (above 20 mm Hg), net water transport was entirely by way of the lymphatic system. REFERENCES 1. Ohnell, R. 1939. The artificially perfused mammalian intestine as a useful preparation for studying intestinal absorption. J. Cell. Camp. Physioi. 13: 155-159. 2. Embley, E. H., and C. J. Martin. 1905. The action of anesthetic quantities of chloroform upon the blood vessels of the bowel and kidney; with an account of an artificial circulation apparatus. J. Physioi. (London) 32: 147-158. 3. Jacobs, P., T. H. Bothwell, and R. W. Charlton. 1966. Intestinal iron transport: studies using a loop of gut with an artificial circulation. Amer. J. Physioi. 210: 694-700. 4. Salvioli, G. 1880. Ein neue Methode fur die Untersuchung der Functionen des Diinndarms. Arch. Physioi. (Leipzig), suppi., 95-112. 5. Roese, H. F. 1930. Methoden zum Studium der Physiologie und Pharmakologie des kunstlich durchblutetin Siiugotierdarmes. Pflueger. Arch. Ges. Physioi. 226: 171-183. 6. Gellhorn, S., and D. Northup. 1933. Quantitative investigations on the influence of hormones on absorption. Amer. J. Physioi. 103: 382-391. 7. Parsons, D. S., and J. S. Prichard. 1966. A preparation for the vascular perfusion of the small intestine of amphibia. J. Physiol. (London) 186: 1-2. 8. Kavin, H., N. W. Levin, and M. M. Stanley. 1967. Isolated perfused rat small boweltechnique, studies of viability, glucose absorption. J. Appi. Physioi. 22: 604-611. 9. Hohenleitner, F. J., and J. R. Senior. 1967. Metabolism of the excised, vascularly perfused whole small intestine. Physiologist 10: 204. 10. Lee, J. S. 1961. Flows and pressures in lymphatic and blood vessels of intestine in water absorption. Amer. J. Physioi. 200: 979-983. 11. Krebs, H. A., and K. Henseleit. 1932. Untersuchungen iiber die Harnsoffbildung im Tierkorper. Z. Physioi. Chern. 210: 33-66. 12. Lee, J. S. 1963. Role of mesenteric lymphatic system in water absorption from rat intestine in vitro. Amer. J. Physioi. 204: 92-96. 13. Barrowman, J., and K. B. Roberts. 1967. The role of the lymphatic system in the absorption of water from the intestine of the rat. Quart. J. Exp. Physioi. 52: 19-30. 14. Lee, J. S. 1965. Motility, lymphatic contractility and distention pressure in intestinal absorption. Amer. J. Physioi. 208: 621-627. 15. Benson, J. A., Jr., P. R. Lee, J. F. Scholer, K. S. Kim, and J. L. Bollman. 1956. Water absorption from the intestine via portal and lymphatic pathways. Amer. J. Physioi. 184: 441-444. 16. Noyan, A. 1964. Water absorption from the intestine via portal and lymphatic pathways in rats. Proc. Soc. Exp. BioI. Med. 117: 317-320. 17. Visscher, M. B. 1957. Transport of water and electrolytes across intestinal epithelia, p. 57-71. In Q. R. Murphy [ed.], Metabolic aspects of transport across cell membranes. University of Wisconsin Press, Madison. 18. Wells, H. S. 1940. The balance of physical forces which determines the rate and direction of flow of fluid through the intestinal mucosa. Amer. J. Physioi. 130: 410-419. 19. Shields, R., and C. F. Code. 1961. Effect of increased portal pressure on sorption of water and sodium from the ileum of dog. Amer. J. Physioi. 200: 775-780.