- Email: [email protected]
Measurement of regional gastric mucosal blood flow by hydrogen gas clearance
Measurementof RegionalGastric Mucosal Blood Flow by HydrogenGas Clearance Laurence Y. Cheung, MD, St. Louis, Missouri Leonard A. Sonnenschein, BS, St. Louis, Missouri
Measurement of gastric muscosal blood flow has stimulated considerable interest among investigators, not only because muscosal blood flow plays a central role in the secretory process [I $1, but also because it is an essential factor in the ability of the gastric mucosa to protect itself against injury [3-51. Of the several techniques employed to measure mucosal blood flow in experimental models, aminopyrine clearance [I] and gamma-labeled microspheres [6] have been most widely used. However, none of these methods are capable of measuring mucosal blood flow at a focal area. Aminopyrine clearance only reflects the gastric mucosal blood flow of the entire stomach. Although the radioactive microsphere technique determines regional blood flow, it is inaccurate to measure mucosal blood flow at a small foci because a single sample must contain at least 400 microspheres in order to avoid significant sampling error. In addition, the microsphere technique offers only a limited number of measurements and requires the use of a significant amount of radioactive material. Recently, Murakami et al [7] developed a contact electrode method in hydrogen gas clearance technique that determines focal gastric mucosal blood flow in animals and human subjects. The method is noninvasive and safe. It also offers repeated measurement of mucosal blood flow at the site of the gastric mucosa in contact with the electrode. To further evaluate the accuracy of hydrogen gas clearance as a measure of gastric blood flow, we compared hydrogen clearance-determined flow with total gastric blood flow as measured by the venous outflow technique in an isolated canine gastric segment. In addition, we simultaneously measured the Frcm the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. Supported In part by the Medical Research Service of the Veterans Administration, Washington. DC and the National Institutes of Health Grant AM25998-04, Bethesda, Maryland. Requests for reprints should be addressed to Laurence Y. Cheung. MD, Department of Surgery, Washington University School of Medicine, 4989 Barnes Hospital Plaza, Suite 5108, St. Louis, Missouri 63110. Presented at the 24th Annual Meeting of the Society for Surgery of the Alimentary Tract, Washington, DC, May 24-25, 1983.
32
mucosal blood flow of the antrum and the corpus in the intact stomachs of rats and dogs, two commonly used species for experimental acute ulcers. The difference in regional mucosal blood flow to the antrum and the corpus would be of interest since acute stress-induced gastric erosion occurs predominantly in the corpus of the stomach [a]. Material and Methods Comparison of hydrogen gas clearance and total gastric blood flow as determined by venous outflow were studied in an ex vivo isolated segment of stomach in five dogs. Mongrel dogs that weighed 20 to 25 kg were anesthetized with sodium pentobarbitol(25 mg/kg) and maintained on a Harvard respirator throughout each experiment. A polyvinyl catheter (internal diameter 0.058 inch) was placed in the femoral vein for infusion of fluid and replacement of blood lost. Arterial pressure was monitored through a catheter in the left femoral artery (Statham transducer P23AA, a Hewlett Packard direct writing recorder). A chambered segment of gastric corpus with an isolated vascular pedicle was prepared as described previously by Moody and Durbin [9]. This chamber provides a mucosal surface of approximately 36 cm2. In all the experiments, isotonic hydrochloric acid solution (0.15 N) was placed into and recovered from the chamber at 15 minute intervals. Venous flow through the gastric segment was measured by occluding the gastrosplenic vein proximal to a Silastic@ catheter (internal diameter 0.56 inch) placed in the superior splenic vein. Venous pressure was continuously monitored by a polyvinyl catheter (internal diameter 0.058 inch) placed in a terminal splenic vein (Statham transducer P23BB). For each 15 minute period, timed venous samples were collected at baseline pressure in preweighed tubes containing EDTA. Volume was calculated on the basis of 1 g/ml. In each of the five dogs, three measurements of venous outflow were determined before intravenous infusion of histamine at 1 pg/kg/min. Thirty minutes after the beginning of histamine infusion, three additional measurements of total venous outflow at 15 minute intervals were made. Mucosal blood flow of the entire segment was estimated simultaneously during each measurement of total venous outflow. Details of the technique of measuring mucosal blood flow with the hydrogen gas clearance method have
The American
Journal of Surgery
Regional Gastric Mucosal Blood Flow
.
0.
TABLE I
/ Rats
1
Period 2
3
Mean
1
77 115.5 99 81.5 99 86.6 72.9 138.6
66 106.6 106.6 81.5 106.6 86.6 69.3 138.6
69.3 106.6 99 77 92.4 86.6 69.3 126
70.8 109.6 101.5 80 99.3 86.6 70.5 134.4
96.3 7.8
95.2 8.5
90.8 6.9
94.1 7.7
3
4 5 6 7 8
Mean SEM
ov
’
5
I
I
I
I
I
I
,
I
I
IO
I5
20
25
30
35
40
45
50
Consdc~lvo Measurements of Regional Gastric Mucosal Blood Flow of the Antrum In Rats by the Hydro$en Gas Ctearance Method (ml/min/lOO g of tissue)
SEM = standard error of the mean.
H2 Clearance (ml/min) Ftgut~ l.ConWtbn~etdlb&odttowweathnated byhykoglP~c~~~~b~~w#&termtmd by orrlllbw tn tlvs &#R Tin Ilnear -ton rewaki a h@& +Mcani tx~~tatlon (n = 30, I = 0.87, p CO.o07), a#qo = 1.21. l?10 upon ckchs h&ate the mttng etmaci~ md the sNu$ &&IJ butkate the stamch aMt stlml&ton wtth h&tanMM.
been reported previously by Murakami et al [7]. The contact electrodes were made from a 0.3 mm platinum wire as described by Murakami et al [7]. The spring portion of the electrode was placed nearly vertically against the gastric mucosa in the midportion of the chamber. At this angle, the tipped portion of the electrode lay horizontally against the mucosa with sufficient contact. A calomel reference
electrode was introduced through the abdominal incision and WBB placed in contact with the fluid or blood within the abdominal cavity. The terminals of the platinum and calomel electrodes were connected to an amplifier-chart recorder (linear S-channel chart recorder, Linear Instrument Corp., Reno, NV). The electrode current in the absence of hydrogen was adjusted to zero. Tissue saturation with hydrogen gas was achieved by allowing the animal to breathe under an inverted funnel (4
cm diameter) positioned about 2 cm above the nose or the endotracheal tube. Pure hydrogen gas was supplied to the funnel at a rate of 1 to 2 liters/min. Inhalation was terminated by removing the funnel within 30 seconds when a sharp increase in current was observed in the chart recorder. The current kept increasing for a short period of time, attained a peak, and then declined. The declining portion of the curve was plotted semilogarithmically at appropriate intervals. Gastric mucosal blood flow was calculated from the slope and expressed in milliliters per minute per 100 g of tissue. At completion of the experiment, the mucosa was bluntly separated from the gastric segment and weighed. Total mucosal blood flow to the gastric segment was calculated by multiplying the hydrogen gas clearance measurement by the weight of the entire mucosa. The measurement of mucosal blood flow could be repeated by reapplication of hydrogen gas to the same animal as long as the previous recording returned to zero.
Vokmo 147, Jimwry 1984
Regional mucosal blood flow of the antrum was compared with that of the fundus in the intact stomach of anesthetized rata or dogs. Eight male Wistar rata weighing 200 to 250 g were deprived of food for 12 hours but were allowed to have free access to water. The rats were anesthetized with 1.8 g/kg of urethane subcutaneously. Five mongrel dogs that weighed 20 to 25 kg were anesthetized with sodium pentobarbitol. A midline laparotomy incision was made in these animals. Through a gastrotomy incision along the greater curvature of the anterior wall of the stomach, it was possible to introduce two platinum electrodes at the same time and simultaneously record the current at the corpus and the antrum. Three measurements were made at 15 minute intervals in each animal. After three resting periods, histamine was infused intravenously at 1 pg/kg/min in the dogs. Thirty minutes after the initiation of continuous intravenous infusion of histamine, three additional measurements of blood flow to the antrum and the corpus were made. The average of the three measurements in each animal was used for calculation. All data were expressed as the mean f the standard error of the mean. The data were analyzed for statistical significance with the paired Student’s t test. Results
The relationship between mucosal blood flow as estimated by hydrogen gas clearance and total gastric blood flow as determined by venous outflow is illustrated in Figure 1. Correlation analysis revealed a slope of 1.12 and a correlation coefficient of 0.97. A highly significant (p CO.001) linear relationship was found between total gastric blood flow and mucosal blood flow as measured by hydrogen gas clearance. The slope of 1.21 indicates that the ratio of mucosal blood flow to total gastric blood flow is approximately 82 percent. This finding agrees with the fractional distribution of gastric blood flow previously demonstrated by radioactive microspheres [6,10,11]. Table I shows the consec&ve measurements of regional gastric mucosal blood flow of the antrum in rats by hydrogen gas clearance methods. Although there was a significant difference in mucosal blood
33
Cheung and Sonnenschein
z j .t 0
1 5 8 E ; 0 I: 0 I”
200ISO160-
IOO80 6040
-
20 OC
I
I
Corpus
Antrum (Dogs)
Antrum
Corpus
(Rats)
(Rats)
nlWsWmantsofregkmdtmuscosaibkod [email protected]#[email protected](&)ckarance meihod In etght rats. Each sotfd clmk rapresents the average of three consecutive measurements in ens rat. lRe open ctrcles mucosalbkodfkw (mean f standardenvr hldkatethetqlknat of tfw mean) of the anbum or the corpus In these etght rats.
flow between the rats, the variation of three consecutive measurements within the same animal was small. This finding is in agreement with that reported by Murakami et al [7] who also found that betweenrat variation was significantly greater than within-rat variation for consecutive recordings. The mean antral mucosal blood flow as estimated by the hydrogen gas clearance method from our laboratory (94.1 f 7.7 ml/min/lOO g of tissue) is very comparable to that reported by Murakami et al [ 71. Figure 2 illustrates the stimultaneous measurement of mucosal blood flow of the antrum and the corpus in eight rats. In each animal, antral mucosal blood flow was higher than carpal flow. The mean antral mucosal blood flow in these eight rats was significantly higher than the carpal flow (p
34
(Dogs)
Flgure 3. Simultaneousmeawemeni of tha regional mucosal bkod ftow of the a&urn and the corpus by the hydmgen (H2) clearance method In five dogs. Each solht cbck represants the average of tfuee conseu#tve-hon8wl?mqpm circles hdkaie the regknal mucosal bkod fkw (mean f standard error of the mean) of the anhum or the corpus In these five dogs.
mucosal blood flow was significantly greater than that of the corpus. As a result, the mean antral mucosal blood flow was almost 100 percent greater than the carpal flow (p
The American Journal ol Surgery
CheungandSonnenschein
advantages over radioactive microspheres also include unlimited and repeated measurement and that it can be safely used in human subjects. Hydrogen gas clearance has been shown to closely correlate with aminopyrine clearance when acid secretion is stimulated [7] and has also been shown to demonstrate a significant increase in mucosal blood flow during isoproterenol infusion, whereas aminopyrine clearance reflects a much smaller fraction of mucosal blood flow under such experimental conditions [ 71. This finding is consistent with the data and conclusion reported by Archibald et al [IS] when they compared microsphere-measured mucosaI blood flow with that estimated by aminopyrine clearance. These findings suggest that hydrogen gas clearance method is suitable for measuring mucosal blood flow in the nonsecreting, as well as the secreting, stomach. To further evaluate the accuracy of hydrogen gas clearance as a measurement of mucosal blood flow, we compared the gastric blood flow as estimated by this method with total venous outflow in the resting stomach and after stimulation with histamine. The close correlation found between venous outflow and hydrogen clearance measured flow lends additional validation to the use of hydrogen clearance as an estimate of mucosal blood flow. The ratio of hydrogen gas clearance to total gastric blood flow in this study was approximately 82 percent which is in agreement with previous flow distribution studies using radioactive microspheres. Archibald et al [6] reported that the ratio of mucosal to total gastric blood flow in dogs as calculated by microsphere distribution was 68 percent in the resting stomach and 87 percent during histamine stimulation. A statistically significant difference was found in the regional mucosal blood flow across the antralcarpal boundary in rats and dogs. The mucosal blood flow of the antrum was significantly greater than that of the corpus. Murakami et al [7] and Semb [15] made similar observations in experiments with anesthetized rats and cats, respectively. It is not known whether the difference in mucosal blood flow between the antrum and the corpus of the stomach has any physiologic or pathophysiologic significance. It is conceivable that the lower mucosal blood flow of the gastric corpus may contribute to the observation that stress ulcers are located predominantly in this portion of the stomach [8]. Recently, a number of factors have been found to play an important role in the self-defense mechanism of the stomach against acid injury. They include gastric mucosal blood flow, intrinsic bicarbonate release during active secretion, systemic acid base balance, gastric bicarbonate secretion into the lumen, the permeability barrier, and so on [8]. Of these factors, there is a uniform agreement among investigators that mucosal blood flow plays an essential role in protecting the stomach against acid injury. Therefore, the comparatively lower mucosal blood flow of the corpus observed in Vdumo 147, January
1984
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this and other studies may be an important factor in the propensity of the corpus rather than the antrum to develop acute erosion. Other factors, such as the difference in bioenergy metabolism between the antrum and the corpus during stress, have also been suggested to render the fundus more susceptible to acute injury [17,18]. Smith et al [19] reported that the histaminestimulated gastric mucosa of rabbits does not ulcerate when exposed to 120 mM of hydrochloric acid whereas in the resting, unstimulated stomach severe ulceration occurs under the same circumstances. Inhibition of acid secretion by metiamide negates the protective effect of histamine against ulceration. Since the amount of acid secretion into the gastric lumen corresponds exactly to the amount of bicarbonate given off within the tissue [20], they concluded that intramural alkalinixation during acid secretion exerts a protective effect on mucosal tolerance to acid. In this study, we demonstrated a selective increase in the mucosal blood flow of the corpus during histamine stimulation. It is possible that the protection provided by histamine stimulation is not only due to bicarbonate release but also contributed to by a selective increase in mucosal blood flow of the corpus. It is of interest to note that the increase in the mucosal blood flow of the corpus during histamine stimulation is not at the expense of antral mucosal blood flow. In this study, we found that histamine infusion significantly increased the mucosal blood flow of the corpus, whereas antral mucosal blood flow remained relatively unchanged. As a result, the mucosal blood flow of the corpus was not significantly different from that of the antrum during histamine 35
Cheung and Sonnenschein
infusion. corpus is secretion in gastric
The increase in mucosal blood flow of the a result of active secretion since active acid is known to induce a corresponding increase blood flow [1,2]. Summary
Gastric mucosal blood flow as measured by the hydrogen gas clearance method was compared with total gastric blood flow as determined by venous outflow in an isolated segment of canine stomach. During rest and histamine stimulation, hydrogen gas clearance correlated lineally with total gastric blood flow (p
36
12. Kauffman GL Jr, Grossman Ml. Use of aminopyrine clearance as a measure of gastric mucosal blood flow. In: Granger DN, Bulkley GB, eds. Measurement of blood flow: applications to the splanchnlc circulation. Baltimore: Willlams 8 Wilkins, 1981:203-17. 13. Cheung LY. Comparison of aminopyrlne clearance and mlcrosphere techniques for the measurement of gastric mucosal blood flow. In: eanger DN, Bulkley GB, eds. Measurement of blood flow: applications to the splanchnic clrculatton. Baltimore: Williams & Wilkins, 1981:221-6. 14. Aukland K, Bower BF, BerlinerRW.Measurement of local blood flow with hydrogen gas. Circ Res 1964;14:164-87. 15. Semb BKH. Gastric flow measured with hydrogen clearance technique. Stand J Gastroenterol 1979; 14:641-6. 16. Archibald LH, Moody FG, Simons MA. Comparlson of gastric mucosal blood flow as determlned by amMopyrine cleamnce and y labeled mfcrospheres Gastroenterology 1975;69: 630-5. 17. Menguy Ft. Role of gastric mucosal energy metabolism in the etiology of stress ulceration. World J Surg 1981;5:17580. 18. Martin LF, Dean WL, Ratcliffe RI, Suarez CP, Fry DE. Bloenergy metabolism of gastric mucosa during stress. Surgery 1983;92:337-47. 19. Smith p, O’Brien P, Fromm D, Sllen W. Secretory state of gastric mucosa and resistance to injury tiy exogenous acid. Am J Surg 1977;133:81. 20. Kivilaakso E, Barzilai A, Schiessel R, Crass R, Silen W. UIceration of isolated amphibian gastric mucosa. Gastroenterology 1979;77:31-7.
Discussion Frank G. Moody (Houston, TX): It is quite clear that there is a stoichiometric relationship between mucosal blood flow, back diffusion of hydrogen ions, and lesion formation in experimental situations. The problem is that we have not been able to measure flow at the point where ulcers occur. Dr. Cheung, you have pointed out that this will provide a technique for looking at focal flow. I hope you will tell us a little about the application of this technology to experimental erosive gastritis. Also, an opportunity to measure blood flow through the endoscope in a gastric remnant may show that the alkaline gastritis we see is related to a decrement in blood flow to the fundus of the stomach. Dr. Cheung, would you comment on the human applications of this technology? Thomas A. Miller (Houston, TX): As you are probably aware, Barry Levine and his associates have published a fair amount of information on mucosal blood flow using the miniature swine model. They used radioactive microspheres to measure the flow. If I recall their data correctly, they have consistently demonstrated that either fundic flow is the same or slightly higher than antral flow in the unstimulated stomach using microspheres. Dr. Cheung, do you have any explanation as to why this difference exists between their findings and your results? Is it a species difference or is it perhaps a methodologic difference? The second question relates to the gold standard by which we measure mucosal blood flow. I think 10 or 15 years ago, the aminopyrine clearance technique was the method of choice, and then the microsphere method came into vogue. Until recently, most of us have believed this was the gold standard. Dr. Cheung, are you suggesting now that hydrogen gas clearance is the gold standard by which mucosal blood flow is to be measured and that in the future this method should be used rather than the microsphere technique?
The AmericanJournalof Surgery
Regional Gastric Mucoaal Blood Flow
Jantea C, ‘Bmnpsen (Galveston, TX): Dr. Cheung, will you tell us what the specific advantages and disadvantages of this method are compared with the other available methods of measuring mucosal blood flow? Laurence Y. Cheung (closing): At this time the use of microspheres offers a very accurate measurement in animals, particularly in acute experiments. However, the microsphere method requires the removal of the organ, therefore, it has no clinical potential. Furthermore, when Archibald et al [S] validated the method it required 400 or more spheres in a sample in order to avoid sampling errors; so it has not provided a means to measure the mucosal blood flow at small foci. I think the advantage of hydrogen gas clearance is really twofold: It has significant clinical potential for measuring mucosal blood flow in patients. The other advantage is related to Dr. Moody’s question that this is the only method that can measure blood flow in one focal area. As Dr. Moody knows, when we were studying the effect of aspirin on mucosal blood flow we found a significant
Vohimo 147, Jenualy 1984
increa%e in flow with the microspheres, but we also saw little pale spots within the stomach; therefore, we could not exclude the possibility of focal ischemia in those areas. That is the area I am working on now. Concerning human application, we have not used the method in patients yet because the electrode we made was too thick to pass through the endoscope. I think we will validate measurements through the endoscope with the animals first before we go on to patients. Regarding Dr. Miller’s question, we are not quite sure that antral blood flow is greater than carpal flow when measured by other methods. As he said, some other studies using microspheres have shown that the two are very comparable. There are two explanations for that: One is that the animal’s secretory state may have a lot to do. When we stimulated acid secretion with histamine we found that the blood flow of the fundus was similar to that of the antrum. In piglets there probably is a significant amount of basal secretion in the corpus. That could explain why blood flow was very comparable. The other possibility is the difference in methods.
37
Measurement of gastric muscosal blood flow has stimulated considerable interest among investigators, not only because muscosal blood flow plays a central role in the secretory process [I $1, but also because it is an essential factor in the ability of the gastric mucosa to protect itself against injury [3-51. Of the several techniques employed to measure mucosal blood flow in experimental models, aminopyrine clearance [I] and gamma-labeled microspheres [6] have been most widely used. However, none of these methods are capable of measuring mucosal blood flow at a focal area. Aminopyrine clearance only reflects the gastric mucosal blood flow of the entire stomach. Although the radioactive microsphere technique determines regional blood flow, it is inaccurate to measure mucosal blood flow at a small foci because a single sample must contain at least 400 microspheres in order to avoid significant sampling error. In addition, the microsphere technique offers only a limited number of measurements and requires the use of a significant amount of radioactive material. Recently, Murakami et al [7] developed a contact electrode method in hydrogen gas clearance technique that determines focal gastric mucosal blood flow in animals and human subjects. The method is noninvasive and safe. It also offers repeated measurement of mucosal blood flow at the site of the gastric mucosa in contact with the electrode. To further evaluate the accuracy of hydrogen gas clearance as a measure of gastric blood flow, we compared hydrogen clearance-determined flow with total gastric blood flow as measured by the venous outflow technique in an isolated canine gastric segment. In addition, we simultaneously measured the Frcm the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. Supported In part by the Medical Research Service of the Veterans Administration, Washington. DC and the National Institutes of Health Grant AM25998-04, Bethesda, Maryland. Requests for reprints should be addressed to Laurence Y. Cheung. MD, Department of Surgery, Washington University School of Medicine, 4989 Barnes Hospital Plaza, Suite 5108, St. Louis, Missouri 63110. Presented at the 24th Annual Meeting of the Society for Surgery of the Alimentary Tract, Washington, DC, May 24-25, 1983.
32
mucosal blood flow of the antrum and the corpus in the intact stomachs of rats and dogs, two commonly used species for experimental acute ulcers. The difference in regional mucosal blood flow to the antrum and the corpus would be of interest since acute stress-induced gastric erosion occurs predominantly in the corpus of the stomach [a]. Material and Methods Comparison of hydrogen gas clearance and total gastric blood flow as determined by venous outflow were studied in an ex vivo isolated segment of stomach in five dogs. Mongrel dogs that weighed 20 to 25 kg were anesthetized with sodium pentobarbitol(25 mg/kg) and maintained on a Harvard respirator throughout each experiment. A polyvinyl catheter (internal diameter 0.058 inch) was placed in the femoral vein for infusion of fluid and replacement of blood lost. Arterial pressure was monitored through a catheter in the left femoral artery (Statham transducer P23AA, a Hewlett Packard direct writing recorder). A chambered segment of gastric corpus with an isolated vascular pedicle was prepared as described previously by Moody and Durbin [9]. This chamber provides a mucosal surface of approximately 36 cm2. In all the experiments, isotonic hydrochloric acid solution (0.15 N) was placed into and recovered from the chamber at 15 minute intervals. Venous flow through the gastric segment was measured by occluding the gastrosplenic vein proximal to a Silastic@ catheter (internal diameter 0.56 inch) placed in the superior splenic vein. Venous pressure was continuously monitored by a polyvinyl catheter (internal diameter 0.058 inch) placed in a terminal splenic vein (Statham transducer P23BB). For each 15 minute period, timed venous samples were collected at baseline pressure in preweighed tubes containing EDTA. Volume was calculated on the basis of 1 g/ml. In each of the five dogs, three measurements of venous outflow were determined before intravenous infusion of histamine at 1 pg/kg/min. Thirty minutes after the beginning of histamine infusion, three additional measurements of total venous outflow at 15 minute intervals were made. Mucosal blood flow of the entire segment was estimated simultaneously during each measurement of total venous outflow. Details of the technique of measuring mucosal blood flow with the hydrogen gas clearance method have
The American
Journal of Surgery
Regional Gastric Mucosal Blood Flow
.
0.
TABLE I
/ Rats
1
Period 2
3
Mean
1
77 115.5 99 81.5 99 86.6 72.9 138.6
66 106.6 106.6 81.5 106.6 86.6 69.3 138.6
69.3 106.6 99 77 92.4 86.6 69.3 126
70.8 109.6 101.5 80 99.3 86.6 70.5 134.4
96.3 7.8
95.2 8.5
90.8 6.9
94.1 7.7
3
4 5 6 7 8
Mean SEM
ov
’
5
I
I
I
I
I
I
,
I
I
IO
I5
20
25
30
35
40
45
50
Consdc~lvo Measurements of Regional Gastric Mucosal Blood Flow of the Antrum In Rats by the Hydro$en Gas Ctearance Method (ml/min/lOO g of tissue)
SEM = standard error of the mean.
H2 Clearance (ml/min) Ftgut~ l.ConWtbn~etdlb&odttowweathnated byhykoglP~c~~~~b~~w#&termtmd by orrlllbw tn tlvs &#R Tin Ilnear -ton rewaki a h@& +Mcani tx~~tatlon (n = 30, I = 0.87, p CO.o07), a#qo = 1.21. l?10 upon ckchs h&ate the mttng etmaci~ md the sNu$ &&IJ butkate the stamch aMt stlml&ton wtth h&tanMM.
been reported previously by Murakami et al [7]. The contact electrodes were made from a 0.3 mm platinum wire as described by Murakami et al [7]. The spring portion of the electrode was placed nearly vertically against the gastric mucosa in the midportion of the chamber. At this angle, the tipped portion of the electrode lay horizontally against the mucosa with sufficient contact. A calomel reference
electrode was introduced through the abdominal incision and WBB placed in contact with the fluid or blood within the abdominal cavity. The terminals of the platinum and calomel electrodes were connected to an amplifier-chart recorder (linear S-channel chart recorder, Linear Instrument Corp., Reno, NV). The electrode current in the absence of hydrogen was adjusted to zero. Tissue saturation with hydrogen gas was achieved by allowing the animal to breathe under an inverted funnel (4
cm diameter) positioned about 2 cm above the nose or the endotracheal tube. Pure hydrogen gas was supplied to the funnel at a rate of 1 to 2 liters/min. Inhalation was terminated by removing the funnel within 30 seconds when a sharp increase in current was observed in the chart recorder. The current kept increasing for a short period of time, attained a peak, and then declined. The declining portion of the curve was plotted semilogarithmically at appropriate intervals. Gastric mucosal blood flow was calculated from the slope and expressed in milliliters per minute per 100 g of tissue. At completion of the experiment, the mucosa was bluntly separated from the gastric segment and weighed. Total mucosal blood flow to the gastric segment was calculated by multiplying the hydrogen gas clearance measurement by the weight of the entire mucosa. The measurement of mucosal blood flow could be repeated by reapplication of hydrogen gas to the same animal as long as the previous recording returned to zero.
Vokmo 147, Jimwry 1984
Regional mucosal blood flow of the antrum was compared with that of the fundus in the intact stomach of anesthetized rata or dogs. Eight male Wistar rata weighing 200 to 250 g were deprived of food for 12 hours but were allowed to have free access to water. The rats were anesthetized with 1.8 g/kg of urethane subcutaneously. Five mongrel dogs that weighed 20 to 25 kg were anesthetized with sodium pentobarbitol. A midline laparotomy incision was made in these animals. Through a gastrotomy incision along the greater curvature of the anterior wall of the stomach, it was possible to introduce two platinum electrodes at the same time and simultaneously record the current at the corpus and the antrum. Three measurements were made at 15 minute intervals in each animal. After three resting periods, histamine was infused intravenously at 1 pg/kg/min in the dogs. Thirty minutes after the initiation of continuous intravenous infusion of histamine, three additional measurements of blood flow to the antrum and the corpus were made. The average of the three measurements in each animal was used for calculation. All data were expressed as the mean f the standard error of the mean. The data were analyzed for statistical significance with the paired Student’s t test. Results
The relationship between mucosal blood flow as estimated by hydrogen gas clearance and total gastric blood flow as determined by venous outflow is illustrated in Figure 1. Correlation analysis revealed a slope of 1.12 and a correlation coefficient of 0.97. A highly significant (p CO.001) linear relationship was found between total gastric blood flow and mucosal blood flow as measured by hydrogen gas clearance. The slope of 1.21 indicates that the ratio of mucosal blood flow to total gastric blood flow is approximately 82 percent. This finding agrees with the fractional distribution of gastric blood flow previously demonstrated by radioactive microspheres [6,10,11]. Table I shows the consec&ve measurements of regional gastric mucosal blood flow of the antrum in rats by hydrogen gas clearance methods. Although there was a significant difference in mucosal blood
33
Cheung and Sonnenschein
z j .t 0
1 5 8 E ; 0 I: 0 I”
200ISO160-
IOO80 6040
-
20 OC
I
I
Corpus
Antrum (Dogs)
Antrum
Corpus
(Rats)
(Rats)
nlWsWmantsofregkmdtmuscosaibkod [email protected]#[email protected](&)ckarance meihod In etght rats. Each sotfd clmk rapresents the average of three consecutive measurements in ens rat. lRe open ctrcles mucosalbkodfkw (mean f standardenvr hldkatethetqlknat of tfw mean) of the anbum or the corpus In these etght rats.
flow between the rats, the variation of three consecutive measurements within the same animal was small. This finding is in agreement with that reported by Murakami et al [7] who also found that betweenrat variation was significantly greater than within-rat variation for consecutive recordings. The mean antral mucosal blood flow as estimated by the hydrogen gas clearance method from our laboratory (94.1 f 7.7 ml/min/lOO g of tissue) is very comparable to that reported by Murakami et al [ 71. Figure 2 illustrates the stimultaneous measurement of mucosal blood flow of the antrum and the corpus in eight rats. In each animal, antral mucosal blood flow was higher than carpal flow. The mean antral mucosal blood flow in these eight rats was significantly higher than the carpal flow (p
34
(Dogs)
Flgure 3. Simultaneousmeawemeni of tha regional mucosal bkod ftow of the a&urn and the corpus by the hydmgen (H2) clearance method In five dogs. Each solht cbck represants the average of tfuee conseu#tve-hon8wl?mqpm circles hdkaie the regknal mucosal bkod fkw (mean f standard error of the mean) of the anhum or the corpus In these five dogs.
mucosal blood flow was significantly greater than that of the corpus. As a result, the mean antral mucosal blood flow was almost 100 percent greater than the carpal flow (p
The American Journal ol Surgery
CheungandSonnenschein
advantages over radioactive microspheres also include unlimited and repeated measurement and that it can be safely used in human subjects. Hydrogen gas clearance has been shown to closely correlate with aminopyrine clearance when acid secretion is stimulated [7] and has also been shown to demonstrate a significant increase in mucosal blood flow during isoproterenol infusion, whereas aminopyrine clearance reflects a much smaller fraction of mucosal blood flow under such experimental conditions [ 71. This finding is consistent with the data and conclusion reported by Archibald et al [IS] when they compared microsphere-measured mucosaI blood flow with that estimated by aminopyrine clearance. These findings suggest that hydrogen gas clearance method is suitable for measuring mucosal blood flow in the nonsecreting, as well as the secreting, stomach. To further evaluate the accuracy of hydrogen gas clearance as a measurement of mucosal blood flow, we compared the gastric blood flow as estimated by this method with total venous outflow in the resting stomach and after stimulation with histamine. The close correlation found between venous outflow and hydrogen clearance measured flow lends additional validation to the use of hydrogen clearance as an estimate of mucosal blood flow. The ratio of hydrogen gas clearance to total gastric blood flow in this study was approximately 82 percent which is in agreement with previous flow distribution studies using radioactive microspheres. Archibald et al [6] reported that the ratio of mucosal to total gastric blood flow in dogs as calculated by microsphere distribution was 68 percent in the resting stomach and 87 percent during histamine stimulation. A statistically significant difference was found in the regional mucosal blood flow across the antralcarpal boundary in rats and dogs. The mucosal blood flow of the antrum was significantly greater than that of the corpus. Murakami et al [7] and Semb [15] made similar observations in experiments with anesthetized rats and cats, respectively. It is not known whether the difference in mucosal blood flow between the antrum and the corpus of the stomach has any physiologic or pathophysiologic significance. It is conceivable that the lower mucosal blood flow of the gastric corpus may contribute to the observation that stress ulcers are located predominantly in this portion of the stomach [8]. Recently, a number of factors have been found to play an important role in the self-defense mechanism of the stomach against acid injury. They include gastric mucosal blood flow, intrinsic bicarbonate release during active secretion, systemic acid base balance, gastric bicarbonate secretion into the lumen, the permeability barrier, and so on [8]. Of these factors, there is a uniform agreement among investigators that mucosal blood flow plays an essential role in protecting the stomach against acid injury. Therefore, the comparatively lower mucosal blood flow of the corpus observed in Vdumo 147, January
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this and other studies may be an important factor in the propensity of the corpus rather than the antrum to develop acute erosion. Other factors, such as the difference in bioenergy metabolism between the antrum and the corpus during stress, have also been suggested to render the fundus more susceptible to acute injury [17,18]. Smith et al [19] reported that the histaminestimulated gastric mucosa of rabbits does not ulcerate when exposed to 120 mM of hydrochloric acid whereas in the resting, unstimulated stomach severe ulceration occurs under the same circumstances. Inhibition of acid secretion by metiamide negates the protective effect of histamine against ulceration. Since the amount of acid secretion into the gastric lumen corresponds exactly to the amount of bicarbonate given off within the tissue [20], they concluded that intramural alkalinixation during acid secretion exerts a protective effect on mucosal tolerance to acid. In this study, we demonstrated a selective increase in the mucosal blood flow of the corpus during histamine stimulation. It is possible that the protection provided by histamine stimulation is not only due to bicarbonate release but also contributed to by a selective increase in mucosal blood flow of the corpus. It is of interest to note that the increase in the mucosal blood flow of the corpus during histamine stimulation is not at the expense of antral mucosal blood flow. In this study, we found that histamine infusion significantly increased the mucosal blood flow of the corpus, whereas antral mucosal blood flow remained relatively unchanged. As a result, the mucosal blood flow of the corpus was not significantly different from that of the antrum during histamine 35
Cheung and Sonnenschein
infusion. corpus is secretion in gastric
The increase in mucosal blood flow of the a result of active secretion since active acid is known to induce a corresponding increase blood flow [1,2]. Summary
Gastric mucosal blood flow as measured by the hydrogen gas clearance method was compared with total gastric blood flow as determined by venous outflow in an isolated segment of canine stomach. During rest and histamine stimulation, hydrogen gas clearance correlated lineally with total gastric blood flow (p
36
12. Kauffman GL Jr, Grossman Ml. Use of aminopyrine clearance as a measure of gastric mucosal blood flow. In: Granger DN, Bulkley GB, eds. Measurement of blood flow: applications to the splanchnlc circulation. Baltimore: Willlams 8 Wilkins, 1981:203-17. 13. Cheung LY. Comparison of aminopyrlne clearance and mlcrosphere techniques for the measurement of gastric mucosal blood flow. In: eanger DN, Bulkley GB, eds. Measurement of blood flow: applications to the splanchnic clrculatton. Baltimore: Williams & Wilkins, 1981:221-6. 14. Aukland K, Bower BF, BerlinerRW.Measurement of local blood flow with hydrogen gas. Circ Res 1964;14:164-87. 15. Semb BKH. Gastric flow measured with hydrogen clearance technique. Stand J Gastroenterol 1979; 14:641-6. 16. Archibald LH, Moody FG, Simons MA. Comparlson of gastric mucosal blood flow as determlned by amMopyrine cleamnce and y labeled mfcrospheres Gastroenterology 1975;69: 630-5. 17. Menguy Ft. Role of gastric mucosal energy metabolism in the etiology of stress ulceration. World J Surg 1981;5:17580. 18. Martin LF, Dean WL, Ratcliffe RI, Suarez CP, Fry DE. Bloenergy metabolism of gastric mucosa during stress. Surgery 1983;92:337-47. 19. Smith p, O’Brien P, Fromm D, Sllen W. Secretory state of gastric mucosa and resistance to injury tiy exogenous acid. Am J Surg 1977;133:81. 20. Kivilaakso E, Barzilai A, Schiessel R, Crass R, Silen W. UIceration of isolated amphibian gastric mucosa. Gastroenterology 1979;77:31-7.
Discussion Frank G. Moody (Houston, TX): It is quite clear that there is a stoichiometric relationship between mucosal blood flow, back diffusion of hydrogen ions, and lesion formation in experimental situations. The problem is that we have not been able to measure flow at the point where ulcers occur. Dr. Cheung, you have pointed out that this will provide a technique for looking at focal flow. I hope you will tell us a little about the application of this technology to experimental erosive gastritis. Also, an opportunity to measure blood flow through the endoscope in a gastric remnant may show that the alkaline gastritis we see is related to a decrement in blood flow to the fundus of the stomach. Dr. Cheung, would you comment on the human applications of this technology? Thomas A. Miller (Houston, TX): As you are probably aware, Barry Levine and his associates have published a fair amount of information on mucosal blood flow using the miniature swine model. They used radioactive microspheres to measure the flow. If I recall their data correctly, they have consistently demonstrated that either fundic flow is the same or slightly higher than antral flow in the unstimulated stomach using microspheres. Dr. Cheung, do you have any explanation as to why this difference exists between their findings and your results? Is it a species difference or is it perhaps a methodologic difference? The second question relates to the gold standard by which we measure mucosal blood flow. I think 10 or 15 years ago, the aminopyrine clearance technique was the method of choice, and then the microsphere method came into vogue. Until recently, most of us have believed this was the gold standard. Dr. Cheung, are you suggesting now that hydrogen gas clearance is the gold standard by which mucosal blood flow is to be measured and that in the future this method should be used rather than the microsphere technique?
The AmericanJournalof Surgery
Regional Gastric Mucoaal Blood Flow
Jantea C, ‘Bmnpsen (Galveston, TX): Dr. Cheung, will you tell us what the specific advantages and disadvantages of this method are compared with the other available methods of measuring mucosal blood flow? Laurence Y. Cheung (closing): At this time the use of microspheres offers a very accurate measurement in animals, particularly in acute experiments. However, the microsphere method requires the removal of the organ, therefore, it has no clinical potential. Furthermore, when Archibald et al [S] validated the method it required 400 or more spheres in a sample in order to avoid sampling errors; so it has not provided a means to measure the mucosal blood flow at small foci. I think the advantage of hydrogen gas clearance is really twofold: It has significant clinical potential for measuring mucosal blood flow in patients. The other advantage is related to Dr. Moody’s question that this is the only method that can measure blood flow in one focal area. As Dr. Moody knows, when we were studying the effect of aspirin on mucosal blood flow we found a significant
Vohimo 147, Jenualy 1984
increa%e in flow with the microspheres, but we also saw little pale spots within the stomach; therefore, we could not exclude the possibility of focal ischemia in those areas. That is the area I am working on now. Concerning human application, we have not used the method in patients yet because the electrode we made was too thick to pass through the endoscope. I think we will validate measurements through the endoscope with the animals first before we go on to patients. Regarding Dr. Miller’s question, we are not quite sure that antral blood flow is greater than carpal flow when measured by other methods. As he said, some other studies using microspheres have shown that the two are very comparable. There are two explanations for that: One is that the animal’s secretory state may have a lot to do. When we stimulated acid secretion with histamine we found that the blood flow of the fundus was similar to that of the antrum. In piglets there probably is a significant amount of basal secretion in the corpus. That could explain why blood flow was very comparable. The other possibility is the difference in methods.
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