EFFECT OF LUMEN ACI'DITY AND OSMOLALITY 0N..DUODENAL BLOOD FLOW AND MOTILITY

Transcription

1 HHIIHIHIIHM > 1 ( 'THS' EFFECT OF LUMEN ACI'DITY AND OSMOLALITY 0N..DUODENAL BLOOD FLOW AND MOTILITY Thesis for the Degree of M. S. MICHIGAN STATE UNWERSITY CHlH-PENG HSIEH 1971

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4 ABSTRACT EFFECT OF LUMEN ACIDITY AND OSMOLALITY ON DUODENAL BLOOD FLOW AND MOTILITY By Chih-Peng-Hsieh Blood flow through the mesenteric artery increases after a meal. The contents of the stomach after a meal are usually acidic and hypertonic to plasma. Thus, the duodenal mucosa will be exposed to acid and hypertonic chyme when the stomach empties. The purpose of this present study was to determine if perfusion of the duodenum with acidic or hyperosmotic solutions would alter local blood flow and motility. The study was performed in in E122 duodenal segments of anesthetized dogs. Venous outflow from the segment and the luminal pressure was measured while perfusing the lumen with various acidic, alkaline and hyperosmotic solutions. Tyrode's solution was used as control and was perfused before and after test solutions. Venous blood ph, osmolality and glucose concentration, and the ph of luminal outflow were measured.

5 Chih-Peng Hsieh Luminal perfusion with ph 1.5 or 2.0 solution significantly raised duodenal venous outflow and decreased venous blood ph. Motility was regularly increased by ph 1.5 solution and occasionally by ph 2.0 solution. Luminal perfusion with ph 2.5 or 3.0 solution did not alter the venous outflow, motility or venous blood ph. Arterial blood ph was not altered during the perfusion of any of these acid solutions. There was a linear correlation between the degree of increase in duodenal venous outflow and the degree of decrease in duodenal venous blood ph. Luminal perfusion with alkaline solutions, ph 11.0, 9.0 and 8.0 did not alter blood flow, blood ph or motility. The ph of the acidic solutions increased while the ph of alkaline solutions decreased after the solution perfused through the lumen. A 50% glucose solution significantly increased duodenal venous outflow which was associated with an increase in venous osmolality. Other glucose solutions, 5%, 15% and 22% did not alter venous outflow and osmolality. A 18% glucose solution having an osmolality of 1500 m0sm/ Kg and ph of 2.5 also did not change venous outflow and osmolality. Both 5% and 50% glucose solutions increased the venous glucose concentration but did not change arterial blood glucose concentration. Motility was occasionally increased by 15%, 18%, 22% and 50% glucose solutions, but not by 5% glucose.

6 Chih-Peng Hsieh It is concluded that high luminal acidity or osmolality increases duodenal blood flow and osmolality. The threshold of the luminal ph for the increase in blood flow and motility is The threshold of luminal osmolality for the increase in blood flow is 5000 m0sm/ Kg, while the threshold for the increase in motility is 1000 m0sm/kg. In addition to the increased local hydrogen ion concentration and hyperosmolality, the rise in blood flow and motility may also be mediated by intestinal hormones and neural reflexes.

7 EFFECT OF LUMEN ACIDITY AND OSMOLALITY ON DUODENAL BLOOD FLOW AND MOTILITY By Chih-Peng Hsieh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1971

8 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. C.C. Chou and Dr. J.M. Dabney for their invaluable encouragement and guidance during the course of this study. Sincere appreciation is also extended to Dr. J.B. Scott for his service on the examination committee. The author also wishes to express his special thanks to Mrs. Jodi Johnston, Mr. T.D. Burns and Mrs. Yanee Thoongsuwan for their technical assistance in the present study. ii

9 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES iv LIST OF FIGURES V Chapter I. INTRODUCTION.. o. o. o o.. 1 II. METHODS AND MATERIALS Analysis of Results III. RESULTS o Perfusion with the Control Solution.. 10 Effect of Changing ph Perfusion with Hyperosmotic Solutions.. '20 Effect on the Motor Activity IV. DISCUSSION V. SUMMARY AND CONCLUSION BIBLIOGRAPHY iii

10 LIST OF TABLES Page ph and Osmolality of the Solutions Studied Average Venous Outflow During Perfusion with Control Solution Average Venous Outflow (gm/min) During Perfusion of the Lumen with Acidified Tyrode's Solutions Average ph of the Arterial and Venous Blood and Luminal Content During Perfusion of the Lumen with Acidified Tyrode's Solutions Average Venous Outflow (gm/min) During Perfusion of the Lumen with Alkaline Solutions Average ph of the Arterial and Venous Blood and Luminal Content During Perfusion of the Lumen with Alkaline Tyrode's Solutions Average Venous Outflow (gm/min) During Perfusion of the Lumen with Hyperosmotic Glucose Solutions Mean Plasma Osmolality (m0sm/kg) of the Venous Outflow During Perfusion of Hyperosmotic Glucose Solutions Mean Blood Glucose Concentration (mg%) During Perfusion of 5% and 50% Glucose Solutions iv

11 LIST OF FIGURES Figure Page 1. Diagram Showing the Preparation of the Segment of the Dog Duodenum. The Solutions Perfused the Lumen Via the Multiple Stopcock, then Drained into Beaker. The Venous Flow was Collected into the Beaker Back to the Animal Via the Femoral Vein. Two Small Tubes in the Lumen were Connected to the Transducer for Recording the Luminal Pressure Relationship Between Changes in Blood ph (zpo) and Changes in Blood Flow (gm/min) (sz.f.) During the Perfusion of Acid Solutions. The Equation for the Regression Line is:a B.F. = ApH I (P 0.001). g 0 o o o o o Duodenal Luminal Pressures and Venous Outflow (Bl. F1.) during the Perfusion of Control (Tyrode's), ph 1.5, ph 2.0 and 50% Glucose Solutions

12 CHAPTER I INTRODUCTION Blood flow through the mesenteric artery increases after a meal (6). The mechanism of the increased blood flow however is not clear. After a meal, the gastric acid secretion is increased and the gastric content will be hyperosmotic if the meal is hyperosmotic. Thus, the duodenal mucosa is usually exposed to acidic and hyperosmotic chyme when the stomach empties (5,21). It has been shown that introduction of hyperosmotic glucose solution into the jejunum increases local blood flow (15). Also, introduction of air containing 10% to 90% C02 into the jejunum or ileum loop increases blood flow through the superior mesenteric artery (26). These studies indicate that exposure of the jejunal mucosa to hyperosmotic fluid or the acid forming CO2 gas increases local blood flow. The same phenomena may also occur in the duodenum. The rise in splanchnic blood flow following a meal may be, in part, due to exposure of the duodenal mucosa to hyperosmotic and/or acidic chyme. In addition to its effect on blood flow, introduction of acid into the duodenum also alters motor activities of the duodenum (37). Motor

13 activity of the circular muscle of the jejunum is altered by hyperosmolality (36). The purpose of this present study was to (a) determine if perfusion of the duodenum with acidic or hyperosmotic solution would alter local blood flow and (b) provide more data on change in motility.

14 CHAPTER-II METHODS AND MATERIALS The response of duodenal blood flow and motility to perfusion of the lumen with acidic, alkaline and hyperosmotic solutions were studied in anesthetized dogs. Mongrel dogs (15-18 Kg) of both sexes were anesthetized with sodium pentobarbital (30 mg/kg) and a cuffed tube was inserted into the trachea and connected to a positive pressure respiration pump (Harvard, Model 607, Dover, Mass.). After the abdomen was opened through a midline incision, a segment of the duodenum about cm in length and 5-10 cm oral to the ligament of Treitz was exteriorized. Taking care not to disturb the nerves and arteries, the veins (usually two or three) that drain the segment were dissected free for cannulation. After the intravenous administration of heparin sodium (6mg/Kg), these veins were cannulated with polyethylene tubing (i.d mm, o.d mm). Venous blood was directed into a beaker initially containing 250 ml of 6% dextran in normal saline. This blood was continuously pumped back to the animal via a femoral vein at a rate equal to venous outflow (Sigmamotor pump, Model TmlO, Middleport, 3

15 N.Y.).., As shown in Figure l, a L-shaped plastic tubing (i.d. 1.1 cm, o.d. 1.5 cm) was inserted into the proximal end of the segment and connected to a multiple stopcock which in turn was connected to bottles containing the perfusate. Another plastic tubing was inserted into the distal end of the segment and connected to a tygon tube to drain the perfusate into a beaker. Both ends of the segment were tied around these plastic tubes to prevent bleeding. The cut ends of the duodenum proximal and distal to the experimental segment were tied and severed from the segment and the mesentery was cut to exclude collateral flow. Thus, a duodenal segment was made free from collateral flow with its total venous outflow draining into a beaker. Two polyethylene tubes (i.d mm, o.d mm) were inserted via the distal plastic tubing into the lumen of the segment to measure duodenal luminal pressure. A polyethylene catheter (i.d mm, o.d mm) was inserted into the abdominal aorta via the femoral artery for monitoring the systemic pressure. Both arterial and luminal pressures were monitored continuously with pressure transducers (Statham, Model P23 Gb, Hato Rey, Puerto Rico) and recorded on a direct writing oscillograph (Sanborn, Model 7714A, Waltham, Mass.). The duodenal lumen was perfused with various solutions (Table 1) at a constant pressure from the syphon bottle.

16 Diagram Showing the Preparation of the Segment of the Dog Duodenum. The Solutions Perfused the Lumen via the Multiple Stopcock, the Drained into Beaker. The Venous Flow was Collected into the Beaker and Pumped Back to the Animal via the Femoral Vein. Two Small Tubes in the Lumen Were Connected to the Transducer for Recording the Luminal Pressure. Figure l.

17 TABLE 1. ph and osmolality of the solutions studied A114 4.x g Solutions ph Osmolality (mosm/kg) Control solution (Tyrode's) Acid solutions* PH 1.5 (0.043 N HCl) ? ph 2.0 (0.022 N H01) ph 2.5 (0.014 N HCl) ph 3.0 (0.012 N HCl) Alkaline solutions* ph 8.0 ( N NaOH) ph 9.0 (0.003 N NaOH) ph 11.0 ( N NaOH) Glucose solutions 5% glucose % glucose; % glucose* % glucose; % glucose, *Tyrode's solution (ph 7.4, 300 mosm/liter, Na meq/liter, Cl me liter, K+ 2.7 meq/liter, H meq/liter, Ca++. meq/liter, HP04" 0.7 meq/liter, Mg meq/liter, Glucose 0.1%) was used as the control for all test solutions. All test solutions, therefore, were made by adding an appropriate amount of 1 N HCl, 1 N NaOH or glucose into Tyrode's solution. Both 5% and 50% glucose solutions were made by adding glucose to distilled water.

18 The rate of perfusion was set initially at 4 ml per min (50-56 drops) by adjusting a hosecock placed on the perfusing tubing. Each solution perfused the duodenum for 16 min during which venous outflow from the segment was collected continuously in 2 minutes sample. The blood was weighed on a top loading precision balance (Mettler, Model P1200, Hightown, N.Y.) and hence blood flow was expressed as grams per minute. Tyrode's solution was used as control for all test solutions. After perfusing the lumen with the control solution for 16 minutes, the perfusate was changed to a test solution. Following perfusion with a test solution for 16 minutes, the perfusate was switched back to the control solution. Thus, the lumen was perfused with Tyrode's solution alternating with a test solution. No more than four test solutions were studied in a single About 2 minutes was required to switch to a new solution and adjust the rate of perfusion. Blood flow was not measured during this period. Blood samples were obtained at 8 and 16 minutes after starting the perfusion for the measurement of ph, dog and the test solutions were perfused in random sequence. osmolality or glucose concentration. Samples of luminal outflow were also taken at the same time to measure its ph. The ph of the blood or luminal content was measured immediately after obtaining the sample (ph meter 22, Type PHA 621b,

19 Radiometer, Copenhagen). Osmolality was determined by freezing point depression (Advanced Instruments, Model LAS, Newton Highlands, Mass.) and glucose concentration by the Folin-Wu method. The 31 dogs used in this study were divided into two groups. In the first group (18 dogs), the effects of acid solutions, 5% and 50% glucose solutions were studied. In the second group (13 dogs), the effects of alkaline solutions, 13%, 18% and 22% glucose solutions were studied. Analysis of Results Venous outflow, blood ph, osmolality and glucose concentration usually became steady 14 to 16 minutes after perfusing the duodenal lumen with Tyrode's solution (control). Therefore, the data obtained at this time were used as the control for the data obtained during the perfusion of next test solution. The significance of changes from the control of blood flow, ph, osmolality or glucose concentration which occurred during the perfusion of the next solution were tested by Student's t-test modified for paired comparison between two sample means (33). Since venous outflow usually decreased with time, the effects of a test solution on venous outflow may be underestimated if the solution is a vasodilator or over- -estimated if the solution is a vasoconstrictor. Therefore, the change in blood flow during perfusion of a test solution was compared to the change in blood flow during

20 perfusion with the control solution. To obtain the latter, the duodenum was perfused with the control solution in two successive periods in 10 animals chosen randomly from the total of 31. The second period was considered as the test period and changes in blood flow were estimated as described in the previous paragraph (Table 2). Since the lumen was perfused with the same solution in both periods, the change in flow was considered to be a measure of the rate at which blood flow decreased in these experiments. The Student's tatest modified for unpaired comparison was used in these calculations (33).

21 CHAPTER III RESULTS Systemic arterial pressure was not significantly altered by any experimental procedures. The mean systemic arterial pressure of the 31 experimental dogs ranged from 95 to 135 mmhg with an average of 120 mmhg. Perfusion Control Solution In 10 dogs, the duodenal lumen was perfused with control solution (Tyrode's solution) in two successive periods. The purpose was to measure the degree of spontaneous changes in blood flow that may occur with time and the degree of changes in flow that may result from mechanical manipulations of changing perfusates. The results are shown in Table 2. Using the last blood flow value of the first period as the control, blood flow had on the average decreased in the second period by 0.20 and 0.66 gm/min at 2 and 16 minutes respectively. These amounted to 1.4 and 3.9% of the control respectively. The decreases in flow however are not statistically significant (p>0.1). The study thus indicated that venous outflow was not affected by mechanical manipulations of 10

22 Controla 0-2 Blood flow (gm/min)b solution perfused 11 TABLE 2. Average venous outflow during perfusion with control solution. (N-lO) Time after starting perfusion (in minutes) Changes from control (gm/min) C -o.49 : i i Percent changesc the preceding period during which control percent change from control are mean : S.E. flow value of 8Control a the last blood bmean the lumen. cthe values of changes from control and

23 12 changing perfusates and adjusting the rate of perfusion because venous outflow before and after manipulations were not significantly different. Effect of Changing ph The effects of perfusing the lumen with acidic solutions on venous outflow is shown in Table 3. Arterial and venous ph and the ph of the solution flowing out of the lumen are shown in Table 4. As shown in Table 3, perfusion of the duodenal lumen with ph 1.5 or 2.0 solution, caused a significant increase in duodenal venous outflow. The flow started to increase from the first 2 min collection period and remained high throughout the entire 16 min perfusion. The mean flow values measured at 8-10 min and min after the perfusion were not statistically different (p>0.3). The venous ph was decreased significantly during the perfusion with ph 1.5 or 2.0 solution (Table 4). The arterial ph, however, was not significantly altered. The solution became significantly less acidic after perfusing through the lumen, ph of 1.5 being increased to 1.79 and ph of 2.0 being increased to 2.33 (p40.01)(table 4). Perfusing a solution having a ph of 2.5 or 3.0 into the duodenal lumen did not significantly alter the venous outflow. The flow tended to fall over the 16 min perfusion period and the degree of fall in flow with ph 2.5 (-0.17 gm/min/l6 min) or ph 3.0 (-0.68 gm/min/l6 min)

24 0-2 Solution No. ph * * :_2.11* i i TABLE 3. Average venous outflow (gm/min) during perfusion of the lumen with acidified Tyrode's solutions. after starting perfusion (in minutes) Time Control ph * 2.56* * + + +l 2.59* ph i i 2.56* ph i i i *Denotes that the value is significantly different from the control value at p<0.05. Control - the last blood value of the preceding period during which control solution perfused the lumen. All values are mean : S.E.

25 Solution A 7.30* ph ph TABLE 4. Average ph of the arterial and venous blood and luminal content during perfusion of the lumen with acidified Tyrode's solutions. (N=7) ph Time after starting perfusion Control (Tyrode's) Control 16 min min (Tyrode's) A V L A V L A V L V L 1.79* 7.33* ph * * 2.50* * 2.33* * * * 4.71* *Denotes that the value is significantly different from the control value at p<0.05. A - arterial blood; V - venous blood; L = the perfusate after perfusing through the lumen.

26 15 solution however was not different from that occurring during the perfusion with control solution (-0.66 gm/min/ 16 min)(p>0.3)(table 2). Neither the venous nor arterial ph changed during the perfusion with ph 2.5 or 3.0 solution. However, the solution became less acidic after perfusing through the lumen, ph of 2.5 being increased to 3.33 and ph of 3.0 being increased to 4.90 (P<0.01) (Table 4).. Tables 5 and 6 show the effects of perfusing the alkaline solution on venous outflow, arterial and venous ph and the ph of the perfusate. Perfusion of the lumen with a ph 8.0 solution did not significantly alter venous outflow. Arterial or venous ph was not significantly altered, but the ph of the perfusate was significantly decreased from 8.0 to 7.78 during its perfusion through the lumen (p<0.05)(table 6). The effects of ph 9.0 were similar to that of ph 8.0 solution. Venous outflow as well as the arterial and venous ph were also not significantly altered during the perfusion with ph 9.0 solution. However, the ph of the perfusate was significantly decreased (p<0.05) (Table 6). Perfusing the lumen with ph 11.0 solution caused a transient rise in average venous outflow in the first 2 min after the perfusion (Table 5). This rise in flow, however, was not statistically significant (p60.2). A large increase in blood flow occurred in one of the five dogs studied and the remaining four dogs showed only a

27 16 very small increase in flow. Following this transient rise, the flow returned to slightly below control level. The blood flows measured at 8, 10, and 16 min after the perfusion were not different from control flow (Table 5). Neither arterial nor venous ph was altered during perfusion with ph 11.0 solution, but the ph of the perfusate significantly decreased (p<0.05)(table 6). In Figure 2 the changes in blood flow occurring min after the perfusion of various acidic solutions are plotted against the changes in bloode occurring at the corresponding time. Changes in blood flow were calculated by subtracting the control blood flow from the flow measured during the perfusion of the acidic solution. Changes in blood ph were calculated by subtracting the A-V difference obtained during perfusion with control solution from the A-V difference obtained during perfusion with acidic solution, i.e.,.aph = (Arterial ph - Venous ph)acid Figure 2, - (Arterial ph - Venous ph) As shown in control the greater the change in blood ph the greater was the rise in flow (r = 0.61, p<0.001). Since systemic arterial ph was not altered by changing perfusate and also since arterial and venous ph during the perfusion of control solution were essentially the same (Table 4), a change in ApH indicates a fall in venous ph during the perfusion of acidic solution. It can also be seen in Figure 2 that if venous ph is lowered to the same extent the blood flow is raised to the same extent whether the

28 Solution No PH TABLE 5. Average venous outflow (gm/min) during perfusion of the lumen with alkaline Tyrode's solutions. Time after starting perfusion (in minutes) Control i ph i " + ph _+_ i i i perfused Control - the last blood flow value of the preceding period during which control solution the lumen. All values are mean :_S.E.

29 8 min Solution A V L ph V L * TABLE 6. Average ph of the arterial and venous blood and luminal content during perfusion of the lumen with alkaline Tyrode's solutions. (N-S) Time after starting perfusion Control (Tyrode's) 16 min Control (Tyrode's) A V L * A V L * A * ph * * ph *Denotes that the value is significantly different from the control value at p<0.05. A - arterial blood; V - venous blood; L a the perfusate after perfusing through the lumen.

30 l9.9"ph105 X'9'PH 200 OoooPH 2.5 Aesop" 3.0 U A B.F..1 lit. 0. )K. ) A_:x (D I. x o l l I J I J V A0 A x Figure 2. aph Relationship Between Changes in Blood ph ( ph) and Changes in Blood Flow (gm/min)( B.F.) During the Perfusion of Acid Solutions. The Equation for the Regression Line is: B.F. = ph, r = 0.61 (p 0.001).

31 20 perfusate was ph 1.5, 2.0 or 2.5. Perfusion with Hyperosmotic Solutions The effects of perfusing the duodenal lumen with hyperosmotic solutions on the rate and osmolality of the venous outflow are shown in Tables 7 and 8. Perfusion with 5% (300 mosm/kg), 13% (1000 m0sm/kg) or 22% (1500 m0sm/kg) glucose solution did not significantly change the venous outflow or osmolality. During the perfusion with 5% or 22% glucose, blood flow tended to fall slightly and gradually but with 13% glucose flow tended to rise slightly and gradually. However, these slight changes in blood flow were not statistically significant (Table 7). Perfusion of 50% (3000 m0sm/kg) glucose solution caused a significant increase in venous outflow for the entire 16 minute perfusion period (p<0.05). The flow progressively increased upon switching perfusate from control to 50% glucose solution and reached its maximum in about 8 min. The flow then remained at the same level thereafter. The blood flow that occurred at 6-8 min after onset of the perfusion ( % of the control) was significantly greater than that occurring at 0-2 min ( % of the control)(p<0.02). But the blood flow occurring at min after starting the perfusion ( % of the control) was essentially the same as that occurring at 6-8 min after the perfusion. The venous osmolality was significantly increased during the

32 50% glucose TABLE 7. Average venous outflow (gm/min) during perfusion of the lumen with hyperosmotic glucose solutions. Time after starting_perfusion (in minutes) Control Solution No l i % glucose % glucose i i % glucose l l H 2.35* +l 2.81* * * % glucose *Denotes that the value is significantly different from the control value at p<0.05. Control = the last blood flow value of the preceding period during which control solution perfused the lumen. All values are mean i;s.e. The ph of 18% glucose solution was 2.5 but the ph of all other glucose solutions was 7.4.

33 22 TABLE 8. Mean plasma osmolality (m0sm/kg) of the venous outflow during perfusion of hyperosmotic glucose solutions. f v w Time after starting Igrfusion(in minutes) Solution Ng. Control 6:8 14:16 Control 5% glucose % glucose % glucose % glucose * 300* % glucose * Denotes that the Talus is significantly different from the control value at p<0.05. The ph of 18% glucose solution was 2.5 but the ph of all other solutions was 7.4. perfusion with 50% glucose solution (Table 8). A 18% glucose solution having a ph of 2.5 caused a slight increase in mean blood flow. Although the mean blood flow remained above control for the entire 16 min perfusion period, the rise in flow was not statistically significant (Table 7). The change in blood flow was also not statistically different from that occurring during the perfusion of the control solution.(table 2). Neither arterial nor venous ph was altered during perfusion with 18% glucose solution, but the ph of the perfusate (ph 2.5) significantly increased after perfusing through the lumen (ph 3.65 at 16 min)(p<0.05). Venous osmolality was not significantly altered (Table 8).

34 25 The blood glucose concentration during the perfusion with 5% or 50% glucose solution are shown in Table 9. Venous glucose concentration was significantly increased during the perfusion of both 5% and 50% glucose (p 0.02), but the arterial glucose concentration was not significantly altered. TABLE 9. Mean blood glucose concentration (mg%) during erfusion of 5% and 50% glucose solutions. N=3> Time after starting perfusion (in minutes) Solution Control A_ V Control H V % * 165* ~50% « * ~ 4251* < * Denotes that the value is significantly different from the control value at p<0.05. A = arterial blood; V = venous blood. Effect My; Activity The motor activity of the duodenum, as indicated by changes in luminal pressure, was consistently increased during the perfusion with ph 1.5 solution. Increased motor activity occurred in all the 17 dogs studied. Perfusion of a solution with a ph of 2.0 increased motility in 5 of the 15 animals studied; a ph of 11.0 increased motility in l of 5 animals; a 50% glucose solution increased motility in 4 of 13 animals; 13%, 22% glucose

35 24 solutions and 18% glucose solution having a 1500 mosm/kg with ph of 2.5 each increased motility in 2 of 7 animals. Solutions having a ph of 2.5, 3.0, 8.0 or 9.0 and the 5% glucose solution did not alter duodenal motility in any of the dogs studied. These studies indicate that very acidic (ph ), or very hyperosmotic ( mosm/ Kg) solutions increased motility of the duodenum. The increased motor activities were rhythmic contractions showing on the pressure tracing as rhythmic phasic oscillation of luminal pressure or without a rise in basal pressure. Figure 3. A typical presssure recording is shown in As soon as the perfusate was switched from control to ph 1.5, ph 2.0 or 50% glucose solution, the duodenum started to contract rhythmically. The contractions usually lasted as long as the duodenum was perfused with the acidic or hyperosmotic solution. Upon switching back to control solution, the contractions disappeared gradually. Figure 3 also shows that venous outflow was increased by all three test solutions. The increase in motility did not appear to substantially affect the venous outflow. As shown in Figure 3, the mean blood flow was greater at 8-12 min than at 0-4 min after the perfusion with ph 2.0 or 50% glucose solution but the magnitude of the increase in motility appeared the same. Also, the degree of rise in blood flow was about the same whether the motility was increased or not altered. The motility was increased by

36 mg ma 7.10 "a.nic-cxslixx..600 In.vote-v on on n. the Perfusion o f Control (Tyrode's), ph 1.5, ph 2.0 and 50% Glucose Solut lons. Duodenal Luminal Pressures and Venous Outflow (Bl. Fl.) Dur1ng 8065 we as Figure 3. a o l-2mln-i 7K3 D I. II. Fl. GMnlhflnn {inuhndn at

37 26 perfusing the ph 2.0 solution in 5 of the 15 dogs studied. Concurrent with this increase in motility venous outflow rose to % of the control at 6-8 min after starting the perfusion. 0f the remaining 10 dogs in which the motility was not altered, the mean venous outflow was raised by 8.62 i 0.87% of the control at 6 8 min after the perfusion. The rise in blood flow in these was not statistically different (p>0.5).

38 CHAPTER IV DISCUSSION The purpose of this study was to determine whether altering the ph or osmolality of the duodenal lumen content will affect local blood flow and motor activity. A The study was performed in an in'situ segment of the canine duodenum. Venous outflow fnom the segment and the luminal pressure were measured while perfusing the lumen with various acidic, alkaline and hyperosmotic solutions. The data obtained during the perfusion with the test solution were compared to those obtained during perfusion with the control solution, i.e., Tyrode's solution. Blood flow through an organ is determined by the pressure gradient across the vascular bed and the resistance to flow exerted by the vascular bed. Since pressure gradient, i.e., the systemic arterial pressure minus venous outflow pressure remained unchanged during these present experiments, changes in blood flow indicated changes in vascular resistance. A decrease in flow indicated an increase in vascular resistance and an increase in flow a decrease in resistance. 27

39 28 This present study shows that duodenal venous outflow was increased during the perfusion of Tyrode's solution with ph 1.5 or 2.0 solution through the duodenal lumen (Table 3). Accompaning with this increase in flow was a decrease in venous blood ph and an increase in duodenal motility. Acidic Tyrode's solution having a ph of 2.5 or greater, on the other hand, did not significantly alter venous outflow, motility or venous blood ph. The arterial ph was not altered during perfusion of any acidic solution. These studies thus indicate that increases in venous outflow and motility caused by ph 1.5 or 2.0 solution may be related to decreases in venous ph, i.e., increase in venous hydrogen ion concentration. This is further suggested by the fact that the magnitude of rise in venous outflow is related to the magnitude of fall in venous ph, i.e., the greater the decrease in venous ph the greater was the rise in venous outflow (Figure 2). It has been reported that injection of air containing 10% to 90% CO into an intestinal loop increased blood 2 flow in the superior mesenteric artery. The rise in blood flow was associated with a rise in pco2 and a fall in ph of the mesenteric venous blood (26). It has been also shown that decreases in arterial blood ph caused either by intra-arterial infusion of acid or by equibrating the perfusing blood with a gas mixture containing 20% CO2 produce a decrease in resistance in various

40 29 organs (17,20). These studies indicate that a decrease in arterial or venous ph regardless of how it is achieved decreases vascular resistance. The decreased vascular resistance appears to be due to a direct effect of the hydrogen ion on the blood vessels because the effect is not abolished by blocking the nerves (20). Studies done in preparations also suggest the direct action of the hydrogen ion is on the vascular smooth muscle. The rates of flow through an isolated arterial segment have been shown to be linearly related to the ph of the perfusate (Tyrode's solution). A decrease in ph of 0.05 units significantly increased the flow and a decrease in ph from 7.40 to 7.15 increased flow by 87% of the control (7). This present study showed a similar finding, i.e., the greater decrease in venous blood ph was associated with the greater increase in blood flow (Figure 2). A decrease in ph of 0.05 units increased the duodenal blood flow by 6% of the control. None of the alkaline solutions studied significantly change venous outflow (Table 5). Arterial and venous blood ph remained unchanged during perfusion of any alkaline solution (Table 6). It has been shown that intra-arterial infusion of ph 10.0 solution (diluted NaOH) caused vasodilation in the skeletal muscle vasculature (17). In the isolated segments of arteries perfused with alkaline Tyrode's solution, flow decreased when the ph of the perfusate was increased from 7.40 to 7.50, but

41 30 flow increased when the ph was further increased to 7.65 (7). In the present study, neither arterial nor venous blood ph were altered during the perfusion of any alkaline solution. The lack of changes in duodenal blood flow may be due to the lack of changes in blood ph. As shown in Tables 7 and 8, the flow and osmolality of the venous outflow was increased during the perfusion of 50% glucose solution. The other glucose solutions (5%-22%) did not significantly alter venous flow or osmolality. Venous glucose concentration however was increased by both 5% and 50% glcuose solutions (Table 9). These studies indicate that the increase in flow was associated with a rise in venous osmolality, but not with the rise in blood glucose concentration. In the jejunum, Chou 23 a1. (11) have found a similar finding with luminal placement of glucose solutions. In addition to the luminal placement, they also studied the effect of intra-arterial infusion of glucose solutions and concluded that hyperglycemia pg; s2 does not affect local vascular resistance. They found that intra-arterial infusions of 5.4% glucose solution did not alter blood flow and osmolality but raised plasma glucose concentration. Intra-arterial infusions of a hypertonic glucose solution (16.4%) increased blood glucose concentration, osmolality and local blood flow. Luminal placement of the other hyperosmotic solutions such as NaCl, KCl, MgCl2 and Ca012 (1500 mosm/kg) into

42 31 the ileum also raises local blood flow with increased venous osmolality (8). Hyperosmolality caused by intraarterial infusion of various hyperosmotic solution also lowers the vascular resistance of various peripheral vascular beds in dog (22,25). This evidence thus suggests that plasma hyperosmolality can lower vascular resistance. The mechanisms by which plasma hyperosmolality decreases vascular resistance is not entirely clear. However, at least three mechanisms are involved, namely changes in blood viscosity, in vessel wall hydration and in active vasomotion. It has been shown that placement of the hypertonic Tyrode's solution into the lumen of a jejunal segment increases the osmolality (+8 to +10 m0sm/kg) and decreases the mean corpuscular volume of the red cells of the jejunal venous blood. The decrease in the mean corpuscular volume, i.e., the size of red cells, results from an increase in blood osmolality which remove water from the cells (18). The decrease in the size of the red cells will lead to decrease in blood viscosity which in turn decreases the vascular resistance. In this present study, venous osmolality increased by about 8 mosm/kg during perfusion of 50% glucose (Table 8). Thus, the increase in blood flow during 50% glucose (Table 7) may, in part, result from a decrease in plasma viscosity. The decrease in vascular resistance caused by plasma

43 32 hyperosmolality can also result from a passive increase in vessel radius. Hyperosmolality can dehydrate the vessel wall and increase its radius. The third possibility is the active vasomotion. It has been proposed that increases in plasma osmolality induce active vasomotion by altering intracellular ionic concentrations in vascular smooth musble cells subsequent to osmotic movement of water (22). Placement of 20% glucose solution into the jejunum has been shown to increase both the local blood flow and venous osmolality (11). However, in this present study perfusion of 22% glucose solution through the duodenum did not significantly alter venous outflow or osmolality (Tables 7 and 8). The difference in the result is most likely due to the difference in technique. In the present study, the test solution was continuously perfused at a constant rate (4 ml/min) through the lumen of the duodenum. In the jejunum the glucose solution remained in the closed lumen for 15 minutes. Although the osmolalities of the experimental solution perfused into the lumen of the duodenum and jejunum was similar, the area of the mucosa exposed to glucose solution may differ. The area exposed to glucose solution was probably greater in the closed jejunal segment. Since perfusion of the lumen either with a ph 2.5 or a 1500 m0sm/kg solution did not change the venous outflow (Tables 3 and 7), it was interesting to see if perfusion of a glucose solution having an osmolality of 1500 m0sm/kg

44 33 and ph of 2.5 would increase the venous outflow. As shown in Table 7, the solution (18% glucose) did not significantly increase the venous outflow. This indicates that the effect of acid and hyperosmolality (ph 2.5 and 1500 mosm/kg) was not additive. However, it is still possible that other combinations of ph and hyperosmolality would show potentiation. The presence of acid of hypertonic solution in the duodenum can cause a variety of changes in digestive function which are mediated by humoral (24,32) and neural mechanisms (23). The gastrointestinal hormones, such as secretin, pancreozymin and cholecystokinin and enterogastrones are released and neural reflexes are activated to alter the functions of the stomach, pancreas and gallbladder (24,34). Thus, in addition to the direct effect of the hydrogen ion or hyperosmolality on the vessel, the increased duodenal blood flow following the perfusion with acidic or hypertonic solution may in part mediated by humoral and neural mechanisms. Intravenous injection of secretin has been shown to increase small intestinal blood flow. The dilator effect appears to result from a direct effect on the vessel since the dilation occurs within a few seconds after the injection and persists after the denervation (28). Cholecystokininapancreozymin (CCK-PZ) also causes vasodilation in the superior mesenteric vasculature (13,19). Thus,

45 54 the increased blood flow observed in the present study following the perfusion of ph 1.5, 2.0 or 50% glucose solution may be, in part, mediated by secretin and CCK-PZ. The lack of blood flow change following the perfusion of ph 2.5, 3.0, or other glucose solutions (5%-22%) may be due to insufficient release of secretin or CCK-PZ. It has been shown that no measurable release of secretin occurred when the duodenal lumen ph is above 4.5 (5). It has also been shown that the degree of vasodilation caused by intra-vascular infusion of secretin or CCK-PZ depends on the dosages, i.e., depends on the plasma concentration of the hormones (13). In the present study, the duodenal lumen ph, as indicated by the ph of the perfusate drained out from the lumen, was 3.0 to 3.3 during the perfusion of ph 2.5 solution and was above 4.5 (4.71 to 4.90) during the perfusion of ph 3.0 solution (Table 4). The possible effect of nerves on the blood flow changes observed in this present study is not clear. However, it is known that presence of acid in the duodenum initiates neural reflexes to inhibit gastric secretion. The reflex is mediated by the vagi and the threshold to initiate this reflex is intraduodenal ph of 2.5 or less (16). Electrophysiological studies also show that perfusion of the intestinal lumen with 4-10% glucose, amino acids (leucine, cystine, or tyrosine), or % HCl

46 35 solution increases the afferent impulses in the intestinal nerves. Perfusion of HCl solution caused more afferent impulses (frequency and amplitude) from the duodenum than from the other segments of the small intestine (38). Furthermore, perfusion with N HCl (ph 2.0) produced slight activity but perfusion with 0.05 N HCl produced more intense and longer responses (30). In the present study, the threshold of the ph value of the perfusate that increased blood flow is 2.0 (0.022 N HCl) or less. Thus, the increase blood flow caused by ph 1.5 or 2.0 solution may be in part mediated by the neural mechanisms. Two studies support the thesis that hypertonic solutions in the lumen may affect mucosal nerves to increase local blood flow. The increased local blood flow caused by intraluminal placement of 50% glucose solution in the jejunum was abolished by anesthetizing the lumen with a local anesthetic (dibucaine)(l4). Similarly, the in- - creased local blood flow caused by intraluminal placement of the hyperosmotic solution of CaCl2 or NaCl was also abolished by dibucaine (9). Since the venous glucose or cation concentration and osmolalities were increased to the same extent before and after dibucaine, the increased local blood flow might not be completely dependent on the plasma ion concentration or osmolality. Since the local anesthetics anesthetized the mucosal nerves, the increased flow appears to be, in part, mediated by the mucosal nerves.

47 36 The mucosal nerve receptors which are sensitive to the alkaline, however, have not been identified in the duodenum (29). Perfusion of the intestinal loop with N NaOH solution did not affect the afferent discharge in the mesenteric nerves (30). Thus, it is possible that perfusion of the alkaline solution in this present study did not initiate neural reflexes that may alter duodenal blood flow. Intestinal blood flow can be influenced by the pressure in the lumen of the gut and the tension in its wall (12). Tonic contractions of the intestinal wall will reduce the inflow of blood and the pumping actions produced by rhythmic contractions of the intestinal smooth muscle will increase the intestinal blood flow (31). The results obtained in this study showed that a ph of 1.5, 2.0 or 50% glucose solution in the duodenal lumen caused an increase in intestinal wall activity, i.e., increases in lumen pressure and rhythmic contractions (Figure 3). However, the increased blood flow observed was not consistently correlated with the increased intestinal motor activity. The degree of rise in blood flow caused by a ph 2.0 solution in the duodenum was the same whether the motility was increased or not changed (p. 24). Therefore, it appears that the rhythmic contractions of the duodenum caused by ph 1.5, 2.0 or 50% glucose solution was not strong enough to substantially affect the duodenal blood flow.

48 37 This present study shows that duodenal motor activity was consistently increased during perfusion of ph 1.5 or 2.0 solution through the lumen. Perfusion of the lumen with 13%, 18%, 22% or 50% glucose solution also increased motility. motility. The alkaline solution, however, did not alter Other investigators also showed that presence of acid in the duodenum increases duodenal motility in dogs and men (4,37). The mechanisms by which acid or hyperosmolality in the duodenal lumen increases duodenal motility may be similar to the mechanisms that increase duodenal blood flow. This is supported by the fact that those solutions, e.g., ph 1.5 or 50% glucose that increased blood flow also increased motility and the solutions, e.g., ph 3.0 or 5% glucose that did not alter flow did not alter motility. The possible mechanisms under the consideration are the effect of the hydrogen ion, hyperosmolality, secretin, CCK-PZ and local nerves on the intestinal smooth muscle. Since a fall in venous ph was associated with increased motility while no change in venous ph was associated with no change in motility, it is reasonable to speculate that the increased motility results from a direct action of the hydrogen ion on the duodenal smooth muscle. The effect of acid on the intestinal smooth muscle have not been studied in 13,11339 preparations nor with intra-arterial infusion. Such studies may give an answer to whether the hydrogen ion will directly act on

49 38 the smooth muscle. Whether the increased duodenal motility caused by perfusion of hyperosmotic solution results from a direct action of hypertonicity on the visceral muscle is not clear. Since 13%, 18% and 22% glucose solutions increased duodenal motility but did not alter venous osmolality, an indicator of the tissue fluid osmolality, the increased motility appears not to be a direct osmotic effect on the muscle. In an study, Tomita has shown that hypertonic solution (sucrose added to Krebs solution) hyperpolarizes and stops the spontaneous electrical and mechanical activity of the guinea pig taenia coli (35). Although Vogt (36) has found that hypertonicity increases the motor activity of the circular muscle of the rabbit jejunum, he attributed this effect secondary to the stimulation of the intramural nerve plexuses. The stimulating effect of the hypertonicity was abolished by nicotine given in the amount that blocks the ganglia. The effect, however, was not affected by atropine given in the amount that blocks the action of muscarine. From these studies Vogt suggested that the stimulating effect of hypertonicity on the rabbit jejunal motility is mediated by the Auerbach's plexus in the jejunal wall. Thus, the increased duodenal motility observed in this present study may be secondary to the action of hypertonicity on the local nerves.

50 39 In addition to the neural mechanism, presence of acidic or hypertonic solution in the duodenal lumen releases the gastrointestinal hormones. These hormones have been shown to affect intestinal motility. Adlercreutz 33'21. (l) have demonstrated that intravenous injection of cholecystokinin cause a tonic contraction and markedly accelerated the peristalsis of the second part of the human duodenum. Ramirez _a1. (27) also reported that intravenous infusion of CCK-PZ in conscious dogs caused a prompt increase in the amplitude and frequency of the pressure waves recorded in the jejunum. Since the increased motility occurred within 30 seconds after the infusion, they suggested that the increased motility result from a direct action of CCK-PZ on the intestinal smooth muscle. In contrast to excitatory action of CCK-PZ, secretin has been shown to inhibit the intestinal motility (10,27). Intravenous injection of secretin caused a prompt and almost complete inhibition of intestinal activity. However, Andersson showed that injection of secretin caused a rhythmic motor activity of the human duodenum (2). This evidence thus suggests that release of cholecystokinin-pancreozymin and secretin may play a role in the increased duodenal motility observed in this present study during the perfusion of acidic and hyperosmotic solution. This present study shows that the ph of all acidic solutions increased after they perfused through the lumen.

51 40 This indicates that all the acidic perfusate underwent neutralization while they were in the lumen. Since no bile or pancreatic secretion drained into the duodenal segment used in this present study, the neutralizing substances appear to come from the duodenal mucosa.. It has been shown in human subjects that when the mean ph of the antrum is 1.4 (ranged 1.0 to 1.9), the mean ph of duodenal bulb is 2.4 (ranged 1.8 to 4.0). Also, when the mean ph of the antrum is 2.3 (ranged 2.0 to 2.7), the mean ph of the duodenal bulb is 4.4 (ranged 2.5 to 6.7) (3). These findings thus indicate that in addition to the hepatic and pancreatic secretions, the secretion from the duodenal mucosa and the absorption of the hydrogen ion contribute in the dissipation of gastric acid emptying into the duodenum. This present study also shows that the ph of all alkaline solutions decreased after the solution perfusing through the lumen. This indicates that the duodenum is not only capable of neutralizing acid but also capable of neutralizing alkali.

52 CHAPTER V SUMMARY AND CONCLUSION The responses of duodenal blood flow and motility to perfusion of the lumen with acidic, alkaline and hyperosmotic solutions were studied in anesthetized dogs. Venous outflow and luminal pressure of the 12'situ duodenal segments were measured while perfusing the lumen of the segments with control and various test solutions. The results show that: l. Perfusion of the lumen with the control solution (Tyrode's) in two successive experimental periods did not significantly alter the venous outflow or motility. This indicates that there was no significant spontaneous change in flow and motility during two experimental periods. This also indicates that flow and motility was not significantly changed by mechanical manipulation of changing perfusates. Luminal perfusion with ph 1.5 or 2.0 solution significantly raised duodenal venous outflow and decreased venous blood ph. Motility was regularly.41