- Email: [email protected]
Maechanism of diarrhea of villous adenomas
Mechanism GERALDO M.
of Diarrhea
G. DACRUZ,
M.D., JERRY D.
of Villous
Adenomas*
GARDNER, M.D., AND GERALD W.
PESKIN, M.D.,~.
Philadelphia, Pennsylvania
From the Harrison Deeartment of Surgical Research, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. This work was supported by U..!i.P.H.S. Grant HE-O-2757-1 1,
absorption by colonic or rectal mucosa. No firm experimental evidence has been presented to date to substantiate any of these propositions. Furthermore, an additional postulate can be advanced, that of the existence of a factor secreted by the tumor which would alter the intestinal mucosa by humoral means. In an attempt to define the mechanism of gastrointestinal losses associated with this tumor, we have performed the following experiments utilizing tumor extracts and measuring the net transfer of water and electrolytes (sodium, potassium, and chloride) and the bidirectional transport of sodium in vitro.
LTHOUGH the
original clinical description of a villous tumor was set forth by Holmes in 1861 [I], it was not until 1954 with the report of McKittrick and Wheelock [2] that attention was directed to the depletion syndrome associated with this adenoma. A profuse, watery diarrhea leading to excessive losses of fluid and electrolytes, dehydration, circulatory collapse, prerenal azotemia, and metabolic acidosis was produced by a villous adenoma. In recent years much information on the diagnosis and effects of villous adenomas has become available, and over seventy well documented cases of the depletion syndrome have been reported, of which fourteen resulted in death due to hydroelectrolytic disturbance ]31. .4 review of published information reveals an average daily rectal discharge of 1,420 ml. (range 285 to 3,400), with an average sodium content of 120 mEq./L. (range 40 to IGO), a potassium content of 44 mEq./L. (range 15 to 107), and a chloride content of 123 mEq./L. (range 80 to 163). Thus, a characteristic hyponatremia, hypokalemia, and hypochloremia are produced. .Many mechanisms have been suggested to account for the loss of water and electrolytes caused by this tumor. These include (1) simple transudation from the colon, (2) active secretion of fluid and electrolytes by the cells of the tumor, and (3) an inappropriate balance between secretion of the tumor and
A
METHODS Experiments were carried out in 164 adult male Sprague-Dawley rats weighing 250 to 350 g-m. The animals were maintained on a commercial diet with unrestricted access to food and water. Immediately after sacrifice, the terminal ileum was tied and sectioned at the level of the ileocecal junction After the mesocolon was peeled away the large intestine was divided in two segments (proximal and distal colon) of approximately equal size. Both segments were everted according to the method of Wilson and b’iseman [4]. Each sac was filled with 1.105 ml. of Krebs-Ringer’sbicarbonate-glucose solution and suspended vertically in 25 ml. of the same solution. The sacs were incubated for one hour at 37”~. while the mucosal solution was continuously gassed with 95 per cent oxygen and 5 per cent carbon dioxide. The composition (mM/L.) of the solution on both sides of the sac at the beginning of the incubation period was sodium, 140; potassium, 5.8; calcium, 1.3; magnesium, 1; chloride, 125; bicarbonate ion, 26; sulfate, 1; phosphorous, 3; glucose, 15. The pH was 7.4. The substances under study were colonic mucosa, adenomatous
*Presented at the Eighth Annual Meeting of the Society for Surgery of the Alimentary Tract, Atlantic City, New Jersey, June 17 and 18, 1967. ?PREsENT Vol.
ADDRESS:
115, February
Department 1968
of Surgery, Michael
Reese Hospital 203
and Medical Center, Chicago,
Illinois 90616.
DaCruz, Gardner, and Peskin
204
TABLE EFFECT
OF NORMAL IN
MUCOSA
VARIOUS
ON NET
PORTIONS
I FLUID
AND
OF EVERTED
ELECTROLYTE RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred
Proximal Colon
Control
Normal Mucosa
Control
f
3.68
II= 1.06 (6)
1.76 f
0.63 (6)
1.81 f
0.55 (6)
0.63 & 0.18 (6)
0.68
f
0.22 (6)
0.31 f
0.09 (6)
0.30
f
0.05 (6)
0.06 f
0.07 f
0.02 (6)
0.03 f
0.01 (6)
0.03 f
0.01 (6)
0.70 & 0.24 (6)
0.32 f
0.10 (6)
0.32 f
0.11 (6)
Normal Mucosa
Fluid (pl./mg. dry wt. sac/hr.) Sodium (FM./mg. dry wt. sac,Jhr.) Potassium (pM./mg. dry wt. sac/hr.) Chloride (pM./mg. dry wt. sac/hr.)
3.44
1.11 (6)t
0.02 (6)
0.65 z!z 0.17 (6)
* Values expressed as mean i t Number of experiments.
standard deviation.
The final result was computed in hl. of fluid per milligram of dry weight of sac per hour. The net electrolyte transfer was determined by taking the difference between the product of serosal fluid recovered and its electrolyte concentration and the product of the initial volume (1.105 ml.) and the starting electrolyte concentrations. These determinations were made in terms of PM of sodium, potassium, and chloride per milligram of dry weight of sac per hour. Bidirectional Sodium Movement. To explore the effect of villous adenoma on the bidirectional movement of sodium across the colonic wall, the same preparation was used as when net fluid and electrolyte movement was studied. Approximately 0.4 PC. of sodium 22 was added to the serosal solution, and the rate at which the isotope entered the mucosal solution was determined. Samples were obtained and counted for ten minutes each in a Packard AutoGamma liquid scintillation spectrometer. All readings were at least twenty times background. The
polyp, adenocarcinoma, and villous adenoma, all taken from the rectum and distal colon of human subjects at operation and immediately utilized for extraction. Extracts of normal mucosa and of the superficial layer of the three related tumors were made at a concentration of 100 mg./ml. in distilled water and kept frozen. One milliliter of the extract was added to the solution bathing the mucosal surface of the sac in any experiment. In no instance did the addition of any of the test substances alter the electrolyte composition or pH of the bath. Concentrations of sodium and potassium were measured simultaneously with an Instrumentation Laboratory flame photometer. Chloride concentration was measured with an Aminco-Cotlove chloride titrator, and glucose with the Worthington glucostat technic. The net fluid volume change was determined gravimetrically by taking the difference of the weight of the sac filled before and one hour after incubation. No gross leaks from the sacs could be detected. TABLE EFFECT
OF ADENOMATOUS Iii
VARIOUS
ON NET
PORTIONS
II FLUID
OF EVERTED
AND
ELECTROLYTE
RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred
Fluid (pl./mg. dry Sodium (pM/mg. dry Potassium (pM/mg. dry Chloride (pM/mg. dry
POLYP
Control
wt. sac/hr.)
4.24
wt. sac/hr.)
Polyp
Control
Adenomatous
Polyp
1.03 (8)t
4.40 f
1.50 (6)
2.01 f
0.45 (8)
2.08 f
0.51 (6)
0.88 f
0.20 (8)
0.90 f
0.15 (6)
0.29 f
0.09 (8)
0.30 f
0.06 (6)
wt. sac/hr.)
0.08 f
0.02 (8)
0.08 f
0.02 (6)
0.03 f
0.01 (8)
0.03
f
0.01 (6)
wt. sac/hr.)
0.81 zk 0.21 (8)
0.83 f
0.16 (6)
0.32 f
0.09 (8)
0.32
f
0.08 (6)
*Values expressed as mean f tNumber.of experiments.
f
Proximal Colon
Adenomatous
standard deviation.
American Journal
of Surgery
Diarrhea
of Villous
Adenoma
TABLE III EFFECT
OF ADENOCARCINOMA IN VARIOUS
Substance Transferred Fluid (pl./mg. dry wt. sac/hr.) Sodium (~.M./mg. dry wt. sac/hr.) Potassium (pM/mg. dry wt. sac/hr.) Chloride (pM/mg. dry wt. sac/hr.) *Values expressed as mean f tNumber of experiments.
ON NET FLUID
PORTIONS
1968
ELECTROLYTE RAT
Control
TRANSFER
COLON*
Distal Colon
Proximal Colon
Adenocarcinorna
Control
Adenocarcinoma
4.14 f
1.39 (8)t
4.26 z+=1.05(13)
1.71 =Iz0.50(S)
1.74 + 0.29 (13)
0.78 f
0.23 (8)
O.i3 zt 0.16 (13)
0.26 =t 0.08 (8)
0.29 f
0.05 (13)
0.06 f
0.04 (8)
0.06 f
0.02 (13)
0.03 i
0.01 (8)
0.03 f
0.00 (13)
O.i6f 0.20 (13)
0.29 f
0.09 (8)
0.31 zt 0.08 (13)
0.7% zt 0.24 (8) standard deviation.
serosal to mucosal sodium movement was determined from the rate at which Naz2 entered the mucosal solution and expressed as PM of sodium entering the mucosal solution per milligram of dry weight sac per hour. Mucosal to serosal sodium was taken as the serosal to mucosal movement plus the net movement. Intestinal Motility. Two technics were used to evaluate the effects of the various extracts on intestinal motility. To monitor the intrasac pressure, the sacs were prepared as they were when the net transfer was being studied. A polyethylene cannula was inserted into the upper end of the sac and connected to a Statham strain gauge transducer, and intrasac pressure was recorded under the same conditions as when fluid and electrolyte transfer was measured. An attempt wds made to demonstrate an effect on intestinal motility using the ileum of the guinea pig. Circular muscle strips of isolated ileum were suspended in a 10 ml. organ bath according to the method described by Harry [T]. The muscle bath containing Tyrode’s solution was stirred by continuous gassing with 95 per cent oxygen and 5 per cent carbon dioxide. The bath and all solutions were kept at 37”~. Contractions were measured using a strain gauge transducer. After adding the test substa.nce to the bath, contractions were recorded for three minutes or until the tracing returned to baseline value. The bath was changed and then after one minute, changed again. The procedure was repeated. To test for possible inhibition of guinea pig ileal contraction, the effect of these substances on histamine-induced or serotonin-induced stimulation was studied. Histamine, 5 mg. /L., was placed in the bath, and when the tracing returned to base line the bath was changed. After two minutes the bath was changed again and extract was placed in the bath. After three minutes or when the tracing returned to base line, histamine was added and the Vol. 115, February
AND
OF EVERTED
tracing allowed to return to base line. The bath was changed and after two minutes, changed again. Histamine and the substance being studied were placed in the bath simultaneously, the tracing allowed to return to base line, and the bath changed twice. The procedure was repeated using serotonin (17 mg./L.). RESULTS
Tables I, II, III, and IV present the data on the net mucosal to serosal transfer of fluid, sodium, potassium, and chloride by everted sacs from the proximal and distal colon of the rat influenced by extracts of normal mucosa, adenomatous polyp, adenocarcinoma, and villous adenoma, respectively. In sacs from the distal segment of colon, the addition of human villous adenoma was associated with a significant decrease (p
DaCruz,
Gardner, TABLE
EFFECT
OF VILLOUS IN
VARIOUS
ON NET
PORTIONS
IV FLUID
AND
OF EVERTED
ELECTROLYTE RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred Fluid (rl./mg. dry Sodium (pM/mg. dry Potassium (pM/mg. dry Chloride (rM/mg. dry
TUMOR
and Peskin
Control
Proximal
Villous Tumor
Control
Colon Villous Tumor
wt. sac/hr.)
5.49 zk 0.76 (1O)t
4.03
f
0.58$ (10)
2.58 zk 0.70 (8)
2.87 f
0.72 (8)
wt. sac/yr.)
0.97 f
0.13 (10)
0.70 f
0.122 (10)
0.44 f
0.12 (8)
0.47 f
0.11 (8)
wt. sac/hr.)
0.09
f
0.01 (10)
0.06 f
0.15$ (10)
0.04 f
0.01 (8)
0.03 f
0.01 (8)
wt. sac/hr.)
1.04 f
0.10 (10)
0.77 f
0.024 (10)
0.46 f
0.11 (8)
0.49 f
0.15 (8)
*Values expressed as mean i standard deviation. tNumber of experiments. $?+nnficantly less than control values (p < 0.01).
the mechanism by which this tumor diminishes fluid and electrolyte transfer. Studies using isolated ileal muscle of the guinea pig also failed to reveal an effect of this extract on intestinal smooth muscle contractility. In the in VZVO as well as in the in vitro distal colon of the rat, the normal pattern is that of absorption of water and sodium and secretion of potassium. The fluid transfer is thought to be a passive process, dependent on active solute transfer. Mechanisms by which this coupling between solute and fluid movement may occur have been suggested [6]. It appears likely, therefore, that the observed increase in serosal to mucosal movement of fluid and electrolytes produced by the villous adenoma extract is a reflection of an alteration of cellular function inducing increased permeability of limiting membranes. Although in the colon the net movement of fluid and electrolytes, except for potassium, occurs from mucosa to serosa, this has been demonstrated as the resultant of two simultaneous ion shifts in opposite directions, one
Tables v and VI present the data on the effects of villous adenoma on the bidirectional movement of sodium (Naz2) across sacs of distal and proximal colon in the rat. Villous adenoma significantly increased the serosal to mucosal movement of sodium (p
Data using everted sacs demonstrate the capacity of villous adenoma extracts to inhibit the net mucosal to serosal transfer of fluid, sodium, potassium, and chloride in the distal portion of the colon of the rat. Wilson and Wiseman [4] have reported that increasing the intrasac pressure can retard the transfer of fluid in this preparation; however, the results obtained by monitoring intrasac pressure exclude a rise in the intrasac pressure as TABLE EFFECT
OF VILLOUS
TUMOR
DISTAL
Substance
Control (7) Villous tumor (6)
HALF
OF COLON
Sodium Movement Mucosal
v
ON BIDIRECTIONAL
to Serosalt
1.22 f 1.12 f
0.13 0.21
MOVEMENT
OF THE
(pM/mg.
RAT
OF SODIUM
ACROSS
(NAza)*
dry wt. sac/hr.) Serosal to Mucosal 0.33 f 0.56 f
0.07* 0.12$
Net
0.85 f 0.06 f
0.09 0.071
*Values expressed as mean f standard deviation. tcalculated values. $Signifkantly different from control values (p < 0.01).
AmericanJournal
of Surgery
Diarrhea of Villous Adenoma
EPPECTOF
TABLE VI VILLOUSTUMORON BIDIRECTIONALMOVEMENTOF SODIUM PROXIMAL HALF OF LARGE INTESTINE OFTHE RAT (,Az2)* Sodium
Substance -__ Control (7) Villous tumor (6)
Movement
Mucosal to Serosal (MSt) 0.66 f 0.86 f
(NM/mg.
ACROSS
dry wt. sac/hr.)
Serosal to Mucosal (SM)
0.12 0.10
0.25 f 0.43 f
0.02 O.lO$
Net Movement (NM) -__ 0.37 f 0.39 f
0.02 0.10
*Values expressed as mean f standard deviation. tcalculated values. JSignificantly different from control values (p < 0.01). occurring from mucosa to serosa (insorption) and the other from serosa to mucosa (exsorption). The observed decrease in net sodium, chloride, and fluid movement produced by villous adenoma extracts could have resulted from an increased serosal to mucosal transfer, from a decreased mucosal to serosal transfer, or from a combination of the two. Studies of unidirectional sodium (Na22) indicated that tumor activity diminished net transfer by increasing serosal to mucosal sodium movement. Additional work will be necessary to establish the precise site of tumor activity, whether this be the mucosal or serosal cell membrane. From previous studies it is apparent that humoral agents can influence the permeability of’ membranes of cells to sodium and fluid without a direct effect on the active sodium transport system itself, assuming that this is located on the inner membrane of the cells [7,8]. Additionally, the incubation medium in our experiments always contains glucose (15 mM/ L.), and since glucose and other hexoses stimulate sodium movement [9], this tumor hormone may act by altering the hexose-induced stimulation of sodium transfer. It is important to bear in mind that none of the tumor extracts used in these studies was pure in the sense that we were dealing with a specific molecular entity. Our results, therefore, must be accepted with some reservation on this score. The fact that control tumor and normal mucosa were employed would lead one to believe that the results obtained were not due to a nonspecific protein effect. From our observations that villous tumors have the capacity to increase net fluid and electrolyte transfer into the distal colon, it is interesting to speculate on the mechanism of diarrhea in patients possessed of this tumor. Although one cannot exclude the secretion from
Vol. 115, February
1968
the huge surface area of the tumor itself, it would appear that of all the other alternate suggestions, humoral activity of the tumor influencing exsorption of fluid and electrolytes is of great significance. There is only one previously reported finding of rates of movement across isolated intestinal sacs with villous tumors in experimental work carried out by Duthie and Atwell in 1963 [IO]. They studied the transport of water and electrolytes in colonic loops of five patients undergoing surgical treatment for their villous adenomas of the rectum or sigmoid, and in addition, performed similar studies on control patients with various other tumors of this portion of the gastrointestinal tract. The segments of the intestine containing villous tumor secreted water, sodium, and potassium whereas the control loops absorbed water and sodium but secreted potassium. In studying bidirectional rates of movement, they found that in segments with villous tumor, exsorption of water, sodium, and potassium was increased, sodium being the most affected. Insorption differed only slightly from that in the control sacs. Accordingly, they concluded that the loss of water and electrolytes in the villous adenoma-containing loops was due to an increase in exsorption and suggested an intrinsic difference of the cells of the villous tumor itself compared with the normal colonic mucosal cells. In view of our studies, it is reasonable to suspect that the increase in exsorption may also have been related to the secretion of a humoral factor by the tumor which was affecting adjacent colonic mucosa, increasing its ability to secrete. SUMMARY
A study was carried out to determine the influence of villous adenoma extracts on in 1.
DaCruz,
Gardner,
colonic absorption of fluid and electrolytes. 2. Employing everted gut sacs, it was noted that villous tumor inhibited the net transfer of sodium, potassium, chloride, and fluid across the colon wall of the rat. 3. Furthermore, by measuring the bidirectional flux of sodium (Naz2) across the colonic wall, it was demonstrated that the diminution of net transfer of this ion resulted from an increase in the serosal to mucosal component. 4. These results are discussed in relation to the hydroelectrolytic depletion syndrome associated with some villous adenomas in man. vitro
REFERENCES
1. HOLMES, T. Villous tumor of the rectum. Tr. Path. Sot. London, 12: 120, 1861. 2. MCKITTRICK, L. S. and WHEELOCK, F. C., JR. Carcinoma of the Colon, p. 61. Springfield, Ill., 1954. Charles C Thomas. 3. DACRUZ, G. M. A. and PESKIN, G. W. Villous adenoma. To be published. 4. WILSON, T. H. and WISEMAN, G. Use of sacs of everted small intestine for the study of transference of substances from mucosal to the serosal surface. J. Physiol., 123: 116, 1954. 5. HARRY, J. The action of drugs on the circular muscle strip from the isolated guinea-pig ileum. Brit. J. Pharmacol., 20: 399, 1963. 6. CURRAN, P. F. Na, Cl and water transport by rat ileum in vitro. J. Gen. Physiol., 43: 1137, 1960. 7. GARDNER, J. D., PESKIN, G. W., CERDA, J. J., and BROOKS, F. P. Alterations in in vitro fluid and electrolyte absorption by gastrointestinal hormones. Am. J. Surg., 113: 57, 1967. 8. CURRAN, P. F., HERRERA, F. C., and FLANIGAN, W. J. Effect of calcium and antidiuretic hormone on Na transport across frog skin. J. Gen. Physiol., 46: 1011, 1963. 9. BARRY, R. J. C., SYMTH, D. H., and WRIGHT, E. M. Short-circuit current and solute transfer by rat jejunum. J. Physiol., 181: 410, 1965. 10. DUTHIE, H. L. and ATWELL, J. D. The absorption of water, sodium, and potassium in the large intestine with particular reference to the effects of villous papillomas. GUT, 4: 373, 1963. DISCUSSION
GERALD W. PESKIN (Philadelphia, Pa.): We have been interested in categorizing the effects of various tumors of the gastrointestinal tract with regard to their influence on absorption and the production of diarrhea. Numerous tumors have been thought to produce diarrhea by the fact that they are partially obstructive in nature or because they occupy a large surface area of the gastrointestinal tract. Our studies would indicate that in general tumors fall into three categories in relation to the production
and Peskin of diarrhea: (1) the one which is typified by the functioning carcinoid tumor in which motility is influenced primarily and diarrhea results on this basis; (2) the variant of the Zollinger-Ellison tumor which may produce diarrhea by inhibiting absorption from the lumen of the gastrointestinal tract into the blood stream; (3) the type mentioned by Dr. DaCruz, namely, the villous adenoma which apparently causes an active secretion of fluid from the blood stream into the intestinal lumen. These studies are continuing, and it is hoped that the limiting membrane and mechanism of action within the cell may be determined so that a better evaluation of the entire process will be afforded. HENRY N. HARKINS (Seattle, Wash.): I would like to ask two questions. First, I have been interested for about two years in the possible increase in magnesium in this diarrhea fluid. Unfortunately, in a recent case of villous adenoma, the staff destroyed the entire tumor and therefore I was unable to determine the magnesium content of the diarrhea fluid. I am reasonably certain, from armchair reasoning, that just as the potassium goes up to 80 mEq. or even more, the magnesium will be similarly elevated; however, since I cannot prove this hypothesis, I appeal to some of you who have such cases to look into it. I wonder if Dr. DaCruz has studied this. My second question is whether there might be a fourth or fifth explanation of the high electrolyte content of this diarrhea fluid, namely, that because of the conformation of the fronds in villous adenoma and of the well known fact that the intestinal mucosa is normally almost completely replaced every thirtysix hours (and the turnover in villous adenoma may be even greater), perhaps a large part of these electrolytes comes from degenerated and castoff cells rather than from merely a transudation of fluid. GERALD W. PESKIN (closing): Dr. Harkins, in answer to your first question on the magnesium problem, I do not know of any figures, in reviewing the seventy cases that appear in the literature, regarding magnesium turnover. In our particular study we had a fixed magnesium in the standard solution and did not measure it at the end; however, we will do so. In regard to your second question on the rapid turnover in a large surface area of tumor, I can understand your belief in this matter and I suspect it is a contributing factor. In view of the shortterm experiments performed by Duthie and Atwell in which for one hour, they were able to isolate a loop of colon and demonstrate a significant change in electrolytes, I would doubt very much that cell turnover of twenty-four or forty-eight hours in the colon was contributing that much to the over-all fluid and electrolyte loss.
American
Journal of Surgevy
of Diarrhea
G. DACRUZ,
M.D., JERRY D.
of Villous
Adenomas*
GARDNER, M.D., AND GERALD W.
PESKIN, M.D.,~.
Philadelphia, Pennsylvania
From the Harrison Deeartment of Surgical Research, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. This work was supported by U..!i.P.H.S. Grant HE-O-2757-1 1,
absorption by colonic or rectal mucosa. No firm experimental evidence has been presented to date to substantiate any of these propositions. Furthermore, an additional postulate can be advanced, that of the existence of a factor secreted by the tumor which would alter the intestinal mucosa by humoral means. In an attempt to define the mechanism of gastrointestinal losses associated with this tumor, we have performed the following experiments utilizing tumor extracts and measuring the net transfer of water and electrolytes (sodium, potassium, and chloride) and the bidirectional transport of sodium in vitro.
LTHOUGH the
original clinical description of a villous tumor was set forth by Holmes in 1861 [I], it was not until 1954 with the report of McKittrick and Wheelock [2] that attention was directed to the depletion syndrome associated with this adenoma. A profuse, watery diarrhea leading to excessive losses of fluid and electrolytes, dehydration, circulatory collapse, prerenal azotemia, and metabolic acidosis was produced by a villous adenoma. In recent years much information on the diagnosis and effects of villous adenomas has become available, and over seventy well documented cases of the depletion syndrome have been reported, of which fourteen resulted in death due to hydroelectrolytic disturbance ]31. .4 review of published information reveals an average daily rectal discharge of 1,420 ml. (range 285 to 3,400), with an average sodium content of 120 mEq./L. (range 40 to IGO), a potassium content of 44 mEq./L. (range 15 to 107), and a chloride content of 123 mEq./L. (range 80 to 163). Thus, a characteristic hyponatremia, hypokalemia, and hypochloremia are produced. .Many mechanisms have been suggested to account for the loss of water and electrolytes caused by this tumor. These include (1) simple transudation from the colon, (2) active secretion of fluid and electrolytes by the cells of the tumor, and (3) an inappropriate balance between secretion of the tumor and
A
METHODS Experiments were carried out in 164 adult male Sprague-Dawley rats weighing 250 to 350 g-m. The animals were maintained on a commercial diet with unrestricted access to food and water. Immediately after sacrifice, the terminal ileum was tied and sectioned at the level of the ileocecal junction After the mesocolon was peeled away the large intestine was divided in two segments (proximal and distal colon) of approximately equal size. Both segments were everted according to the method of Wilson and b’iseman [4]. Each sac was filled with 1.105 ml. of Krebs-Ringer’sbicarbonate-glucose solution and suspended vertically in 25 ml. of the same solution. The sacs were incubated for one hour at 37”~. while the mucosal solution was continuously gassed with 95 per cent oxygen and 5 per cent carbon dioxide. The composition (mM/L.) of the solution on both sides of the sac at the beginning of the incubation period was sodium, 140; potassium, 5.8; calcium, 1.3; magnesium, 1; chloride, 125; bicarbonate ion, 26; sulfate, 1; phosphorous, 3; glucose, 15. The pH was 7.4. The substances under study were colonic mucosa, adenomatous
*Presented at the Eighth Annual Meeting of the Society for Surgery of the Alimentary Tract, Atlantic City, New Jersey, June 17 and 18, 1967. ?PREsENT Vol.
ADDRESS:
115, February
Department 1968
of Surgery, Michael
Reese Hospital 203
and Medical Center, Chicago,
Illinois 90616.
DaCruz, Gardner, and Peskin
204
TABLE EFFECT
OF NORMAL IN
MUCOSA
VARIOUS
ON NET
PORTIONS
I FLUID
AND
OF EVERTED
ELECTROLYTE RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred
Proximal Colon
Control
Normal Mucosa
Control
f
3.68
II= 1.06 (6)
1.76 f
0.63 (6)
1.81 f
0.55 (6)
0.63 & 0.18 (6)
0.68
f
0.22 (6)
0.31 f
0.09 (6)
0.30
f
0.05 (6)
0.06 f
0.07 f
0.02 (6)
0.03 f
0.01 (6)
0.03 f
0.01 (6)
0.70 & 0.24 (6)
0.32 f
0.10 (6)
0.32 f
0.11 (6)
Normal Mucosa
Fluid (pl./mg. dry wt. sac/hr.) Sodium (FM./mg. dry wt. sac,Jhr.) Potassium (pM./mg. dry wt. sac/hr.) Chloride (pM./mg. dry wt. sac/hr.)
3.44
1.11 (6)t
0.02 (6)
0.65 z!z 0.17 (6)
* Values expressed as mean i t Number of experiments.
standard deviation.
The final result was computed in hl. of fluid per milligram of dry weight of sac per hour. The net electrolyte transfer was determined by taking the difference between the product of serosal fluid recovered and its electrolyte concentration and the product of the initial volume (1.105 ml.) and the starting electrolyte concentrations. These determinations were made in terms of PM of sodium, potassium, and chloride per milligram of dry weight of sac per hour. Bidirectional Sodium Movement. To explore the effect of villous adenoma on the bidirectional movement of sodium across the colonic wall, the same preparation was used as when net fluid and electrolyte movement was studied. Approximately 0.4 PC. of sodium 22 was added to the serosal solution, and the rate at which the isotope entered the mucosal solution was determined. Samples were obtained and counted for ten minutes each in a Packard AutoGamma liquid scintillation spectrometer. All readings were at least twenty times background. The
polyp, adenocarcinoma, and villous adenoma, all taken from the rectum and distal colon of human subjects at operation and immediately utilized for extraction. Extracts of normal mucosa and of the superficial layer of the three related tumors were made at a concentration of 100 mg./ml. in distilled water and kept frozen. One milliliter of the extract was added to the solution bathing the mucosal surface of the sac in any experiment. In no instance did the addition of any of the test substances alter the electrolyte composition or pH of the bath. Concentrations of sodium and potassium were measured simultaneously with an Instrumentation Laboratory flame photometer. Chloride concentration was measured with an Aminco-Cotlove chloride titrator, and glucose with the Worthington glucostat technic. The net fluid volume change was determined gravimetrically by taking the difference of the weight of the sac filled before and one hour after incubation. No gross leaks from the sacs could be detected. TABLE EFFECT
OF ADENOMATOUS Iii
VARIOUS
ON NET
PORTIONS
II FLUID
OF EVERTED
AND
ELECTROLYTE
RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred
Fluid (pl./mg. dry Sodium (pM/mg. dry Potassium (pM/mg. dry Chloride (pM/mg. dry
POLYP
Control
wt. sac/hr.)
4.24
wt. sac/hr.)
Polyp
Control
Adenomatous
Polyp
1.03 (8)t
4.40 f
1.50 (6)
2.01 f
0.45 (8)
2.08 f
0.51 (6)
0.88 f
0.20 (8)
0.90 f
0.15 (6)
0.29 f
0.09 (8)
0.30 f
0.06 (6)
wt. sac/hr.)
0.08 f
0.02 (8)
0.08 f
0.02 (6)
0.03 f
0.01 (8)
0.03
f
0.01 (6)
wt. sac/hr.)
0.81 zk 0.21 (8)
0.83 f
0.16 (6)
0.32 f
0.09 (8)
0.32
f
0.08 (6)
*Values expressed as mean f tNumber.of experiments.
f
Proximal Colon
Adenomatous
standard deviation.
American Journal
of Surgery
Diarrhea
of Villous
Adenoma
TABLE III EFFECT
OF ADENOCARCINOMA IN VARIOUS
Substance Transferred Fluid (pl./mg. dry wt. sac/hr.) Sodium (~.M./mg. dry wt. sac/hr.) Potassium (pM/mg. dry wt. sac/hr.) Chloride (pM/mg. dry wt. sac/hr.) *Values expressed as mean f tNumber of experiments.
ON NET FLUID
PORTIONS
1968
ELECTROLYTE RAT
Control
TRANSFER
COLON*
Distal Colon
Proximal Colon
Adenocarcinorna
Control
Adenocarcinoma
4.14 f
1.39 (8)t
4.26 z+=1.05(13)
1.71 =Iz0.50(S)
1.74 + 0.29 (13)
0.78 f
0.23 (8)
O.i3 zt 0.16 (13)
0.26 =t 0.08 (8)
0.29 f
0.05 (13)
0.06 f
0.04 (8)
0.06 f
0.02 (13)
0.03 i
0.01 (8)
0.03 f
0.00 (13)
O.i6f 0.20 (13)
0.29 f
0.09 (8)
0.31 zt 0.08 (13)
0.7% zt 0.24 (8) standard deviation.
serosal to mucosal sodium movement was determined from the rate at which Naz2 entered the mucosal solution and expressed as PM of sodium entering the mucosal solution per milligram of dry weight sac per hour. Mucosal to serosal sodium was taken as the serosal to mucosal movement plus the net movement. Intestinal Motility. Two technics were used to evaluate the effects of the various extracts on intestinal motility. To monitor the intrasac pressure, the sacs were prepared as they were when the net transfer was being studied. A polyethylene cannula was inserted into the upper end of the sac and connected to a Statham strain gauge transducer, and intrasac pressure was recorded under the same conditions as when fluid and electrolyte transfer was measured. An attempt wds made to demonstrate an effect on intestinal motility using the ileum of the guinea pig. Circular muscle strips of isolated ileum were suspended in a 10 ml. organ bath according to the method described by Harry [T]. The muscle bath containing Tyrode’s solution was stirred by continuous gassing with 95 per cent oxygen and 5 per cent carbon dioxide. The bath and all solutions were kept at 37”~. Contractions were measured using a strain gauge transducer. After adding the test substa.nce to the bath, contractions were recorded for three minutes or until the tracing returned to baseline value. The bath was changed and then after one minute, changed again. The procedure was repeated. To test for possible inhibition of guinea pig ileal contraction, the effect of these substances on histamine-induced or serotonin-induced stimulation was studied. Histamine, 5 mg. /L., was placed in the bath, and when the tracing returned to base line the bath was changed. After two minutes the bath was changed again and extract was placed in the bath. After three minutes or when the tracing returned to base line, histamine was added and the Vol. 115, February
AND
OF EVERTED
tracing allowed to return to base line. The bath was changed and after two minutes, changed again. Histamine and the substance being studied were placed in the bath simultaneously, the tracing allowed to return to base line, and the bath changed twice. The procedure was repeated using serotonin (17 mg./L.). RESULTS
Tables I, II, III, and IV present the data on the net mucosal to serosal transfer of fluid, sodium, potassium, and chloride by everted sacs from the proximal and distal colon of the rat influenced by extracts of normal mucosa, adenomatous polyp, adenocarcinoma, and villous adenoma, respectively. In sacs from the distal segment of colon, the addition of human villous adenoma was associated with a significant decrease (p
DaCruz,
Gardner, TABLE
EFFECT
OF VILLOUS IN
VARIOUS
ON NET
PORTIONS
IV FLUID
AND
OF EVERTED
ELECTROLYTE RAT
TRANSFER
COLON*
Distal Colon
Substance Transferred Fluid (rl./mg. dry Sodium (pM/mg. dry Potassium (pM/mg. dry Chloride (rM/mg. dry
TUMOR
and Peskin
Control
Proximal
Villous Tumor
Control
Colon Villous Tumor
wt. sac/hr.)
5.49 zk 0.76 (1O)t
4.03
f
0.58$ (10)
2.58 zk 0.70 (8)
2.87 f
0.72 (8)
wt. sac/yr.)
0.97 f
0.13 (10)
0.70 f
0.122 (10)
0.44 f
0.12 (8)
0.47 f
0.11 (8)
wt. sac/hr.)
0.09
f
0.01 (10)
0.06 f
0.15$ (10)
0.04 f
0.01 (8)
0.03 f
0.01 (8)
wt. sac/hr.)
1.04 f
0.10 (10)
0.77 f
0.024 (10)
0.46 f
0.11 (8)
0.49 f
0.15 (8)
*Values expressed as mean i standard deviation. tNumber of experiments. $?+nnficantly less than control values (p < 0.01).
the mechanism by which this tumor diminishes fluid and electrolyte transfer. Studies using isolated ileal muscle of the guinea pig also failed to reveal an effect of this extract on intestinal smooth muscle contractility. In the in VZVO as well as in the in vitro distal colon of the rat, the normal pattern is that of absorption of water and sodium and secretion of potassium. The fluid transfer is thought to be a passive process, dependent on active solute transfer. Mechanisms by which this coupling between solute and fluid movement may occur have been suggested [6]. It appears likely, therefore, that the observed increase in serosal to mucosal movement of fluid and electrolytes produced by the villous adenoma extract is a reflection of an alteration of cellular function inducing increased permeability of limiting membranes. Although in the colon the net movement of fluid and electrolytes, except for potassium, occurs from mucosa to serosa, this has been demonstrated as the resultant of two simultaneous ion shifts in opposite directions, one
Tables v and VI present the data on the effects of villous adenoma on the bidirectional movement of sodium (Naz2) across sacs of distal and proximal colon in the rat. Villous adenoma significantly increased the serosal to mucosal movement of sodium (p
Data using everted sacs demonstrate the capacity of villous adenoma extracts to inhibit the net mucosal to serosal transfer of fluid, sodium, potassium, and chloride in the distal portion of the colon of the rat. Wilson and Wiseman [4] have reported that increasing the intrasac pressure can retard the transfer of fluid in this preparation; however, the results obtained by monitoring intrasac pressure exclude a rise in the intrasac pressure as TABLE EFFECT
OF VILLOUS
TUMOR
DISTAL
Substance
Control (7) Villous tumor (6)
HALF
OF COLON
Sodium Movement Mucosal
v
ON BIDIRECTIONAL
to Serosalt
1.22 f 1.12 f
0.13 0.21
MOVEMENT
OF THE
(pM/mg.
RAT
OF SODIUM
ACROSS
(NAza)*
dry wt. sac/hr.) Serosal to Mucosal 0.33 f 0.56 f
0.07* 0.12$
Net
0.85 f 0.06 f
0.09 0.071
*Values expressed as mean f standard deviation. tcalculated values. $Signifkantly different from control values (p < 0.01).
AmericanJournal
of Surgery
Diarrhea of Villous Adenoma
EPPECTOF
TABLE VI VILLOUSTUMORON BIDIRECTIONALMOVEMENTOF SODIUM PROXIMAL HALF OF LARGE INTESTINE OFTHE RAT (,Az2)* Sodium
Substance -__ Control (7) Villous tumor (6)
Movement
Mucosal to Serosal (MSt) 0.66 f 0.86 f
(NM/mg.
ACROSS
dry wt. sac/hr.)
Serosal to Mucosal (SM)
0.12 0.10
0.25 f 0.43 f
0.02 O.lO$
Net Movement (NM) -__ 0.37 f 0.39 f
0.02 0.10
*Values expressed as mean f standard deviation. tcalculated values. JSignificantly different from control values (p < 0.01). occurring from mucosa to serosa (insorption) and the other from serosa to mucosa (exsorption). The observed decrease in net sodium, chloride, and fluid movement produced by villous adenoma extracts could have resulted from an increased serosal to mucosal transfer, from a decreased mucosal to serosal transfer, or from a combination of the two. Studies of unidirectional sodium (Na22) indicated that tumor activity diminished net transfer by increasing serosal to mucosal sodium movement. Additional work will be necessary to establish the precise site of tumor activity, whether this be the mucosal or serosal cell membrane. From previous studies it is apparent that humoral agents can influence the permeability of’ membranes of cells to sodium and fluid without a direct effect on the active sodium transport system itself, assuming that this is located on the inner membrane of the cells [7,8]. Additionally, the incubation medium in our experiments always contains glucose (15 mM/ L.), and since glucose and other hexoses stimulate sodium movement [9], this tumor hormone may act by altering the hexose-induced stimulation of sodium transfer. It is important to bear in mind that none of the tumor extracts used in these studies was pure in the sense that we were dealing with a specific molecular entity. Our results, therefore, must be accepted with some reservation on this score. The fact that control tumor and normal mucosa were employed would lead one to believe that the results obtained were not due to a nonspecific protein effect. From our observations that villous tumors have the capacity to increase net fluid and electrolyte transfer into the distal colon, it is interesting to speculate on the mechanism of diarrhea in patients possessed of this tumor. Although one cannot exclude the secretion from
Vol. 115, February
1968
the huge surface area of the tumor itself, it would appear that of all the other alternate suggestions, humoral activity of the tumor influencing exsorption of fluid and electrolytes is of great significance. There is only one previously reported finding of rates of movement across isolated intestinal sacs with villous tumors in experimental work carried out by Duthie and Atwell in 1963 [IO]. They studied the transport of water and electrolytes in colonic loops of five patients undergoing surgical treatment for their villous adenomas of the rectum or sigmoid, and in addition, performed similar studies on control patients with various other tumors of this portion of the gastrointestinal tract. The segments of the intestine containing villous tumor secreted water, sodium, and potassium whereas the control loops absorbed water and sodium but secreted potassium. In studying bidirectional rates of movement, they found that in segments with villous tumor, exsorption of water, sodium, and potassium was increased, sodium being the most affected. Insorption differed only slightly from that in the control sacs. Accordingly, they concluded that the loss of water and electrolytes in the villous adenoma-containing loops was due to an increase in exsorption and suggested an intrinsic difference of the cells of the villous tumor itself compared with the normal colonic mucosal cells. In view of our studies, it is reasonable to suspect that the increase in exsorption may also have been related to the secretion of a humoral factor by the tumor which was affecting adjacent colonic mucosa, increasing its ability to secrete. SUMMARY
A study was carried out to determine the influence of villous adenoma extracts on in 1.
DaCruz,
Gardner,
colonic absorption of fluid and electrolytes. 2. Employing everted gut sacs, it was noted that villous tumor inhibited the net transfer of sodium, potassium, chloride, and fluid across the colon wall of the rat. 3. Furthermore, by measuring the bidirectional flux of sodium (Naz2) across the colonic wall, it was demonstrated that the diminution of net transfer of this ion resulted from an increase in the serosal to mucosal component. 4. These results are discussed in relation to the hydroelectrolytic depletion syndrome associated with some villous adenomas in man. vitro
REFERENCES
1. HOLMES, T. Villous tumor of the rectum. Tr. Path. Sot. London, 12: 120, 1861. 2. MCKITTRICK, L. S. and WHEELOCK, F. C., JR. Carcinoma of the Colon, p. 61. Springfield, Ill., 1954. Charles C Thomas. 3. DACRUZ, G. M. A. and PESKIN, G. W. Villous adenoma. To be published. 4. WILSON, T. H. and WISEMAN, G. Use of sacs of everted small intestine for the study of transference of substances from mucosal to the serosal surface. J. Physiol., 123: 116, 1954. 5. HARRY, J. The action of drugs on the circular muscle strip from the isolated guinea-pig ileum. Brit. J. Pharmacol., 20: 399, 1963. 6. CURRAN, P. F. Na, Cl and water transport by rat ileum in vitro. J. Gen. Physiol., 43: 1137, 1960. 7. GARDNER, J. D., PESKIN, G. W., CERDA, J. J., and BROOKS, F. P. Alterations in in vitro fluid and electrolyte absorption by gastrointestinal hormones. Am. J. Surg., 113: 57, 1967. 8. CURRAN, P. F., HERRERA, F. C., and FLANIGAN, W. J. Effect of calcium and antidiuretic hormone on Na transport across frog skin. J. Gen. Physiol., 46: 1011, 1963. 9. BARRY, R. J. C., SYMTH, D. H., and WRIGHT, E. M. Short-circuit current and solute transfer by rat jejunum. J. Physiol., 181: 410, 1965. 10. DUTHIE, H. L. and ATWELL, J. D. The absorption of water, sodium, and potassium in the large intestine with particular reference to the effects of villous papillomas. GUT, 4: 373, 1963. DISCUSSION
GERALD W. PESKIN (Philadelphia, Pa.): We have been interested in categorizing the effects of various tumors of the gastrointestinal tract with regard to their influence on absorption and the production of diarrhea. Numerous tumors have been thought to produce diarrhea by the fact that they are partially obstructive in nature or because they occupy a large surface area of the gastrointestinal tract. Our studies would indicate that in general tumors fall into three categories in relation to the production
and Peskin of diarrhea: (1) the one which is typified by the functioning carcinoid tumor in which motility is influenced primarily and diarrhea results on this basis; (2) the variant of the Zollinger-Ellison tumor which may produce diarrhea by inhibiting absorption from the lumen of the gastrointestinal tract into the blood stream; (3) the type mentioned by Dr. DaCruz, namely, the villous adenoma which apparently causes an active secretion of fluid from the blood stream into the intestinal lumen. These studies are continuing, and it is hoped that the limiting membrane and mechanism of action within the cell may be determined so that a better evaluation of the entire process will be afforded. HENRY N. HARKINS (Seattle, Wash.): I would like to ask two questions. First, I have been interested for about two years in the possible increase in magnesium in this diarrhea fluid. Unfortunately, in a recent case of villous adenoma, the staff destroyed the entire tumor and therefore I was unable to determine the magnesium content of the diarrhea fluid. I am reasonably certain, from armchair reasoning, that just as the potassium goes up to 80 mEq. or even more, the magnesium will be similarly elevated; however, since I cannot prove this hypothesis, I appeal to some of you who have such cases to look into it. I wonder if Dr. DaCruz has studied this. My second question is whether there might be a fourth or fifth explanation of the high electrolyte content of this diarrhea fluid, namely, that because of the conformation of the fronds in villous adenoma and of the well known fact that the intestinal mucosa is normally almost completely replaced every thirtysix hours (and the turnover in villous adenoma may be even greater), perhaps a large part of these electrolytes comes from degenerated and castoff cells rather than from merely a transudation of fluid. GERALD W. PESKIN (closing): Dr. Harkins, in answer to your first question on the magnesium problem, I do not know of any figures, in reviewing the seventy cases that appear in the literature, regarding magnesium turnover. In our particular study we had a fixed magnesium in the standard solution and did not measure it at the end; however, we will do so. In regard to your second question on the rapid turnover in a large surface area of tumor, I can understand your belief in this matter and I suspect it is a contributing factor. In view of the shortterm experiments performed by Duthie and Atwell in which for one hour, they were able to isolate a loop of colon and demonstrate a significant change in electrolytes, I would doubt very much that cell turnover of twenty-four or forty-eight hours in the colon was contributing that much to the over-all fluid and electrolyte loss.
American
Journal of Surgevy