Site and Characteristics of Electrolyte Loss and Effect of Intraluminal

Site and Characteristics of Electrolyte

Loss and Effect of Intraluminal Glucose

in Experimental Canine Cholera

CHARLES C. J. CARFENTER, R. BRADLEY SACK, JOHN C. FEELEY, andRicHARD W. STEENBERG

From the Departments of Medicine and Surgery, Johns Hopkins University School ofMedicine, Baltimore, Maryland and the Division of Biologics Standards,National Institutes of Health, Bethesda, Maryland 21205

A B S T R A C T The site and characteristics of gas-trointestinal electrolyte loss were investigated ineight dogs with experimental cholera induced byorogastric administration of 6-hr broth cultures ofVibrio cholerae, strain Ogawa 395. In these ani-mals, all electrolyte losses originated in the smallbowel, predominantly from the jejunum and ileum.The bicarbonate concentration of the small bowelfluid showed a progressive increase from duode-num, where it was less than that of plasma, to theterminal ileum, where it was significantly greaterthan that of simultaneously obtained plasma.

Studies of the responses of chronic Thiry-Vella jejunal loops (five dogs) and chronic Thiry-Vella ileal loops (five dogs) to intraluminal chal-lenge by cholera exotoxin demonstrated that allloops exhibited isotonic electrolyte loss for a 14-18 hr period after challenge. The bicarbonate con-centration of fluid produced by exotoxin-challengedjejunal loops was not significantly different fromthat of plasma, whereas the ileal loops producedfluid with a bicarbonate concentration approxi-mately three times that of plasma.The effect of intraluminal glucose on the re-

sponse of canine gut to cholera exotoxin was in-vestigated by perfusion studies in 12 dogs withchronic Thiry-Vella fistulae. Intraluminal glu-cose significantly enhanced isotonic fluid absorp-tion in both jejunal and ileal loops. The net effectsof glucose on isotonic fluid absorption were equal

Received for publication 13 November 1967 and in re-vised form 22 December 1967.

before and after intraluminal administration ofcrude cholera exotoxin. These data suggest thatcholera exotoxin causes gut electrolyte loss by amechanism independent of that by which glucoseenhances sodium absorption.

INTRODUCTION

The characteristics of the massive gastrointestinalelectrolyte losses in cholera have been describedduring the past decade (1, 2). The site of elec-trolyte loss has, however, not been defined, norhas the mechanism been delineated. The difficul-ties inherent in carrying out definitive studies inthe cholera patient and the absence of a suitablelarge animal model have been major obstacles toanswering these questions. During the past 2 yr, acanine model has been developed in which the clini-cal signs and biochemical changes of cholera canpredictably be produced by challenge with brothcultures of Vibrio cholerae (3). The characteristicgastrointestinal electrolyte losses can also con-sistently be produced in the dog by intraduodenalchallenge with bacteria-free filtrates of broth cul-tures of V. cholerae.1 The canine model has beenemployed in three experiments that have beendesigned to determine the site of electrolyte loss inexperimental cholera and to investigate the effect

1 Craig, J. P., C. C. J. Carpenter, and R. W. Steenberg.Response of the canine gut to intraluminal challenge witha bacteria-free filtrate of broth culture of V. cholerae.Manuscript in preparation.

1210 The Journal of Clinical Investigation Volume 47 1968

of cholera exotoxin on glucose-associated sodiumtransport in the small intestine.

METHODS

All studies were performed on adult mongrel dogs, weigh-ing 14-24 kg. Animals were fasted for 18 hr before eachstudy. Sodium and potassium determinations were per-formed on a Patwin flame photometer with lithium in-ternal standards (National Instrument Laboratories, Inc.,Rockville, Md.). Chlorides were determined on a Cotlovechloridometer (Buchler Instruments, Inc., Fort Lee,N. J.). Carbon dioxide content in experiment III wasdetermined by a Natelson microgasometer (ScientificIndustries, Inc., Springfield, Mass.). pH measurementswere made on a Beckman expanded scale pH meter,Beckman Instruments, Inc., Palo Alto, Calif. Reducingsubstances were determined by the technique of Hoffman(4) with the Technicon AutoAnalyzer, Technicon Corpo-ration, Ardsley, N. Y. Total proteins were determined bythe Folin-Ciocalteu method (5). PSP was determinedphotometrically by the technique of Schedl and Clifton(6).The crude cholera exotoxin employed in experiments

II and III consisted of a 0.22 1A Millipore filtrate (Milli-pore Filter Corp., Bedford, Mass.) of a 24 hr shakenculture of V. cholerae, strain Inaba 569B, grown in modi-fied Syncase broth; the broth was prepared by a formulaof Finkelstein, Atthasampunna, Chulasamaya, and Char-unmethee (7) but with sucrose content reduced from 0.5to 0.1%o. In each case, the challenge dose was 40-50 ml offiltrate containing 3-4 million blueing doses of choleraexotoxin as determined by the rabbit skin assay of Craig(8). (Although present evidence suggests that the "per-meability factor" measured by the rabbit skin assay isnot the toxic moiety responsible for gastrointestinal elec-trolyte loss in the mammalian gut, the titer of "perme-ability factor" has correlated well with the electrolyteloss produced in the canine gut after challenge with thecrude filtrate employed in these studies. Preliminarystudies indicated that crude filtrate containing 1 millionrabbit skin blueing doses consistently caused maximalelectrolyte loss by the canine small bowel loops.)Experiment I was designed to determine the origin and

solute composition of the fluid produced by the canine gutin experimental cholera. Eight dogs with clinically se-vere cholera were studied. The challenge technique wasas follows. An orogastric tube was placed in the normal,postabsorptive, unanesthetized animal, and the followingseries of solutions were administered into the stomachover a 5 min period: 50 ml of 6%o sodium bicarbonate,100 ml of a 6 hr Syncase broth (7) culture of V. chol-erae Ogawa 395, and 100 ml of fresh uninoculated Syn-case broth. The viable vibrio count in the 6 hr broth cul-ture ranged from 1 X 10' to 5 X 10' organisms per ml.With this challenge technique, approximately 40%o of theanimals developed clinical cholera, which was lethal un-less treated with intravenous fluids, within 12 hr afterchallenge.

After challenge, each dog was placed in a metabolic

cage, and fluid output was observed over the following16 hr period. After the initial 16 hr period, eight dogswith clinically severe cholera, manifest by profuse rice-watery diarrhea, poor skin turgor, weak femoral pulse,and cyanosis of the tongue, were studied in detail. Eachanimal was anesthetized with Nembutal, laparotomy wasperformed, and the bowel was divided into four segmentsby tight double ligatures at the pylorus, the ligament ofTreitz, and the ileocecal junction. Foley catheters wereinserted into the stomach, the terminal duodenum, theterminal ileum, and the rectum and were allowed to drainexternally into graduated cylinders during the remainderof the study. Each animal was then rapidly given intra-venous fluids (2 parts isotonic sodium chloride to 1 part1/6 M sodium lactate) in quantities sufficient to restorethe hematocrit to the base line level, and the abdominalincision was closed by a series of towel clamps. Duringthe following 14 hr period, each animal was kept underlight Nembutal anesthesia and was given the above in-travenous fluids in quantities sufficient to maintain astable hematocrit, as gaged by microhematocrit deter-minations performed in duplicate at hourly intervals.During this time, the hourly output from each of thesegments of bowel was measured, and sodium, chloride,potassium, bicarbonate, and total protein concentrationsof the gastrointestinal fluid were determined. In six ofthe eight dogs, 5 ml samples of jejunal fluid, obtained froma segment approximately 50 cm distal to the ligament ofTreitz, were also collected at hourly intervals for elec-trolyte analysis. The antemesenteric wall of this segmentof jejunum was loosely sutured to the abdominal walladjacent to the laparotomy incision, and the hourly sam-ples were obtained by releasing one of the towel clampsand directly puncturing the jejunal segment with an 18gauge needle.

In three of the eight animals, the thoracic duct wasdirectly cannulated with polyethylene tubing (I.D. = 0.047inches) immediately after the abdominal incision wasclosed; cannulation was performed immediately caudalto the junction of the thoracic duct and the externaljugular vein. Hourly thoracic duct lymph flow rates, aswell as lymph electrolyte and protein concentrations,were determined throughout the remainder of the study.These observations were made to ascertain whether therewas increased leakage of protein from the capillaries tothe interstitial fluid of the lamina propria during ex-perimental cholera.Each animal was sacrificed with Nembutal 30 hr after

the initial challenge, and sections of small bowel mucosawere fixed in Hartman's solution and in glutaraldehydefor light and electron microscopic study.Experiment II was designed to study the response of

upper and lower small bowel segments to challenge withcrude cholera exotoxin. Studies were performed in. 10dogs with chronic Thiry-Vella small bowel loops. Infive dogs, the loops included the 60-70 cm of small bowelimmediately distal to the ligament of Treitz, and in fivethe loops consisted of the 60-70 cm immediately proximalto the ileocecal valve. The loops represented from 20 to30% of the entire jejunoileal length in these animals.

Gut Electrolyte Loss in Experimental Cholera 1211

The isolated small bowel loop was initially challengedwith crude cholera exotoxin 2 wk after preparation ofthe loop. Four of the animals, two with jejunal and twowith ileal loops, were then repeatedly challenged by exo-toxin at weekly intervals over the following 3 wk andthen at monthly intervals over the following 6 months.The challenge technique was as follows. On the morn-

ing of the study, each animal was lightly anesthetized withNembutal, and Foley catheters (American CystoscopeMakers, Inc., Pelham Manor, N. Y.) were placed, withballoons inflated, in the proximal and distal orifices of theThiry-Vella loops. The crude exotoxin was then placedin the loop via the proximal Foley catheter. Foley cath-eters were then clamped and the exotoxin was allowedto remain in the loop for the following 60 min period.After 60 min, both proximal and distal catheters wereopened and allowed to drain into graduated cylinders.Volume of fluid output was recorded at 2-hourly inter-vals over the following 12 hr, and electrolytes were de-termined on the fluid collected. Hematocrits and plasmaelectrolyte concentrations were determined at 2-hourlyintervals, and intravenous fluids (2 parts isotonic so-dium chloride to 1 part 1/6 M sodium lactate) were givenat a rate sufficient to maintain a stable hematocrit.Experiment III was designed to determine the effect of

intraluminal glucose on electrolyte movement in isolatedsmall bowel loops before and after administration of chol-era exotoxin. Studies were performed in six animals withchronic 60-70 cm jejunal loops and in six animals withchronic 60-70 cm ileal loops. Each experiment consistedof two studies, each consisting of three 4 hr periods.During the initial 4 hr period of the first study, the bowelloop was perfused at a rate of 15 ml/kg per hr with anisotonic electrolyte solution containing the following sol-utes: sodium, 140 mEq/liter; potassium, 10 mEq/liter;chloride, 100 mEq/liter; and bicarbonate, 50 mEq/liter.At the end of this period the loop was flushed with 200ml of air, and the rate of absorption of the perfusion fluidwas determined. Crude cholera exotoxin, 40-50 ml ofbacteria-free filtrate as described above, was then placedin the loop via Foley catheter, and both proximal anddistal catheters were clamped for the next 2 hr period.The catheters were then allowed to drain freely over thefollowing 2 hr period, after which the loop was againflushed with 200 ml of air, and fluid output was recorded.The loop was then perfused with the same isotonic elec-trolyte solution for an additional 4 hr period, at the endof which time the loop was again flushed with 200 ml ofair. 1 wk later the second study was done in the samedog, this time with a perfusion fluid containing: glucose,60 mOsm/liter; Na, 110 mEq/liter; K, 10 mEq/liter; Cl,80 mEq/liter; and bicarbonate, 40 mEq/liter. In six ani-mals (three with jejunal and three with ileal loops), theperfusion fluid containing no glucose was administeredduring the first study, and the glucose-containing perfu-sion fluid during the second study; in the remaining sixanimals, the reverse sequence was employed.

In each study net electrolyte absorption or productionby the isolated gut loop, before and after toxin adminis-tration, was determined. In four of the studies, phenol-

sulfonphthalein (PSP) was added to the perfusion fluid ina concentration of 15 mg/100 ml, and recovery of PSPfrom the effluent was used to estimate the amount ofresidual fluid retained in the loops at the end of each4 hr study period.

RESULTS

Experiment I. In the eight animals with clini-cally severe cholera secondary to V. cholerae in-fection, the mean gastrointestinal fluid loss duringthe 30 hr after vibrio challenge was 34.9% of bodyweight (Table I). The maximum observed hourlyrate of gastrointestinal fluid loss was 50 ml/kgper hr. In general, the fluid output was maintainedat a relatively constant level from the 20th to the30th hour of observation, i.e., from the time thecatheters had been placed and the animals ade-quately rehydrated until termination of the study.In each animal, the greatest proportion of electro-lyte loss occurred from the jejunoileal segment ofthe small bowel, with a mean of 91% of fluid lossoccurring from this segment (Table I). From 3to 16%o of the fluid loss occurred from the du-odenum, and virtually no fluid (less than 1%o)was produced proximal to the pylorus. In no ani-mal was any fluid produced by the colon after seg-mentation of the bowel.

Table II presents the electrolyte composition offluid produced by the three segments of smallbowel during experimental cholera. The duodenaland ileal values represent mean electrolyte con-centrations of hourly samples obtained from the20th to 30th hour after infection in all animals

TABLE IGastrointestinal Fluid Losses in Eight Dogs with

Experimental Cholera Secondary to OrogastricAdministration of V. Cholerae

Per centMaxi- output Per cent

Gastro- mum proximal outputintestinal hourly to liga- by jeju-

fluid fluid ment of noilealDog No. output output Treitz segment

ml/kg mi/kg1 358 28 15 852 510 45 6 943 331 50 3 974 240 31 9 915 223 30 5 956 345 31 12 887 269 22 16 848 512 28 6 94

Mean values+SD 3494111 3319 9±5 91±5

1212 C. C. J. Carpenter, R. B. Sack, J. C. Feeley, and R. W. Steenberg

TABLE I IElectrolyte Composition of Fluid Produced by the ThreePortions of Canine Small Intestine during the 20th-30th

Hr after Infection witk V. Cholerae*

Sodium Chloride Potassium Bicarbonate

Duodenum, eight dogs 143 i13 119±7 7.1±2.1 144 8Jejunum, six dogs 153± 4 112±6 6.4±2.0 30± 8Ileum, eight dogs 143+ 5 87±8 9.6±4.1 66±11

* Mean values 4 SD.

studied; the jejunal values represent mean con-centrations of all hourly samples in-the six dogsfrom which jejunal specimens were obtained. Thefluid produced by each of the segments of thesmall bowel was approximately isotonic withplasma, and in each animal the electrolyte concen-trations of fluid produced by a given segment ofbowel remained relatively constant throughout thestudy. Mean bicarbonate concentration of fluidproduced by the ileum was significantly greaterthan that of fluid produced by the jejunum (t =6.78, P < 0.001) and duodenum (t= 10.1, P <0.001), respectively. (t is used hereafter to denotevalues derived from Student's t test.) Conversely,mean chloride concentration of fluid produced bythe ileum was significantly less than that producedby the jejunum (t = 6.62, P < 0.001), and du-odenum (t = 7.69, P < 0.001), respectively (Ta-ble II). There were no significant differences inthe mean potassium concentrations observed atdifferent levels of the bowel, although a widerrange of K concentrations were observed in theileum than in more proximal segments of smallbowel. Mean sodium concentration of jejunal fluidwas significantly greater than that of ileal fluid(t = 3.90, P < 0.01).The protein content of fluid obtained at all levels

of the small bowel was consistently less than 500mg/100 ml, with a mean total protein value of 251±67 (SD) mg/100 ml for jejunoileal fluid ob-tained from all animals studied.

Total protein concentrations of thoracic ductlymph varied inversely with the lymph flow ratebut never exceeded the normal range of values ofcanine thoracic duct lymph (9). At the time ofinitial cannulation of the thoracic duct, before sa-line repletion had been carried out, the mean tho-racic duct lymph flow was 3.5 ml/kg per hr, witha mean total protein concentration of 4.0 g/100 ml.The thoracic duct lymph flow rate invariably in-

creased with saline repletion to a mean value of10.8 ml/kg per hr 4 hr after initiation of intrave-nous fluid therapy; at this time, the mean totalprotein concentration was 1.6 g/100 ml. Electrolyteand reducing substance concentrations in thoracicduct lymph were essentially the same as those insimultaneous plasma samples. Table III presentsmean values for electrolytes, total protein, and re-ducing substances in lymph, plasma, jejunum, andileum, respectively during the period of maximalgastrointestinal fluid output in the three dogs inwhich thoracic duct lymph studies were performed.

Histologic sections of jejunal and ileal mucosashowed moderate edema of the lamina propria butdemonstrated no morphologic changes in epithelialor capillary endothelial cells by either light orelectronmicroscopy. These data are described in aseparate report (10).Experiment II. Fluid was produced by all je-

junal and ileal loops challenged by the exotoxinpreparation. In all instances, a lag period occurredbefore fluid loss was observed. The filtrate con-taining exotoxin was completely absorbed by allileal loops, and largely (75-100%o) absorbed byall jejunal loops, within 60 min after challenge.Output of an approximately isotonic fluid was al-ways observed by the end of the 2nd hr after chal-lenge in jejunal loops and during the 3rd hr afterchallenge in ileal loops. Rate of fluid productionusually reached maximum values by the 4th or5th hr after challenge in both jejunal and ilealloops and then continued at maximum levels foran additional 4-6 hr (Table IV). The rate of fluid

TABLE II ICorrelation of Electrolyte, Protein and Reducing SubstanceValues in Thoracic Duct Lymph with Simultaneous Plasmaand Gastrointestinal Fluid Values during Experimental

Cholera*

TotalSo- Chlo- Potas- Bicar- Pro- Reducingdium ride sium bonate tein- Substances

mEq/ mEq/ mEq/ mEql g/ mg/100 mlliter liter liter liter 100 ml

Lympht 147 114 2.4 19 1.59 89Plasmat 154 119 2.2 19 5.20 87Jejunum 155 119 4.9 26 0.22 7Ileum 150 90 3.4 56 0.17 3

* Mean values of determinations in three animals at 22, 24, 26, and 28hr after vibrio challenge.t All values are corrected to give electrolyte concentrations per milli-liter of plasma water (11).

Gut- Electrolyte Loss in Experimental Cholera 1213

TABLE IVMean Hourly Rates of Fluid Output by Jejunal and IlealLoops during 12 Hr Period after Intraluminal Challenge

with Cholera Exotoxin

Time afterexotoxinchallenge Jejunal loops* Ileal loops*

hr ml/hr ml/hr

0-2 34±31 03-4 94+17 42±t12T5-6 98±11 48±1917-8 91±+12 46±151T9-10 74+20 36±12§11-12 61±20 33±16

* Mean values ± SD, five dogs.1 Differences between mean rates of fluid ouput by jejunaland ileal loops significant at 1% level by t test.§ Differences between mean rates of fluid output by jejunaland ileal loops significant at 2% level by t test.

output then decreased gradually over the following6-8 hr period. Fluid output generally ceased en-tirely by 14 hr after challenge in ileal loops and by18 hr after challenge in jejunal loops. Mean ratesof fluid production by jejunal and ileal loops dur-ing the 12 hr after exotoxin challenge are pre-sented in Table IV. The mean total fluid output bythe jejunal loops during the 12 hr after exotoxinchallenge was 901 + 134 ml and that by the ilealloops was 421 + 133 ml (Table V); the differ-ences between mean 12 hr outputs by jejunal andileal loops are significant (t = 5.14, P < 0.001).

In the four animals (two with jejunal loops,two with ileal loops) in which exotoxin challengeswere administered at weekly intervals during thefirst 3 wk of the study, the volume responses

TABLE VTotal Fluid Output by Chronic Jejunal and Ileal Loops during

the 12 Hr Period after Intraluminal Challenge withCholera Exotoxin

Jejunal loops Ileal loops

Dog No. Output Dog No. Output

ml ml

1 1016 2 4723 860 4 3395 835 6 5727 735 8 2359 1060 10 485

MeanESD 901 ± 134 421 i133

TABLE VIVolume Responses of Chronic Jejunal and Deal Loops to

Repeated Exotoxin Challenge at W7eekly Intervals*

Dog Jej unal Dog HealNo. Week loops No. Week loops

1 1 1016 2 1 4722 1055 2 5473 990 3 412

5 1 835 6 1 5722 760 2 6303 883 3 473

* Fluid output during 12 hr after challenge, in milliliters.

showed little variation on the successive challenges(Table VI). Repeated challenges of these ani-mals at 6 months after initial challenge showed de-creased fluid production in all animals. Both je-junal and ileal loops showed decreases in re-sponse to 30-70% of initial levels. All four dogs,however, showed gross atrophy of the small bowelloops, with decrease in both length and diameter,at the time of the 6 month challenge.

In all animals challenged, the fluid output fromthe loops was essentially isotonic, but electrolytecomposition of fluid produced by jejunal loopsvaried significantly from that produced by ilealloops in regard to bicarbonate (t = 15.8, P <0.001) and chloride (t = 7.26, P <0.001) con-centrations (Table VII). The mean sodium con-centration of jejunal fluid was greater than that ofileal fluid, but the difference was of borderlinesignificance (t = 2.31, P = 0.05). The mean po-tassium concentration of fluid produced by jejunalloops was not significantly different from that offluid produced by ileal loops (t = 1.12, P > 0.5).Mean plasma electrolyte concentrations during

the studies, corrected for plasma proteins to giveconcentrations per liter of plasma water (11), arepresented in Table VII. There were no significantdifferences between mean electrolyte concentrationsin plasma and in jejunal effluent during the'exo-toxin challenge studies. There were, however,highly significant differences between mean elec-trolyte concentrations in ileal effluent and in plasmain regard to bicarbonate (t = 20.8, P < 0.001)and chloride (t = 5.12, P < 0.001). Mean potas-sium concentration in ileal effluent was also greaterthan that of plasma (t = 2.32, P < 0.05).The electrolyte concentrations of intestinal fluid


TABLE VI IElectrolyte Concentrations of Fluid Produced by Jejunal and hleal Loops in Response to

Challenge with Cholera Exotoxin

Jejunai* Ileal*

Plasma$ Jejunal effluent Plasmat Heal effluent

Sodium, mEqiliter 153 ±8 159 i5 153 ±4 145 ±11Chloride, mEqlliter 116 ±5 122 ±6 113 ±4 68 ±17Potassium, mEqiliter 5.140.9 6.9±3.2 5.0±0.6 9.6±3.5Bicarbonate, mEqiliter 24 ±2 26 ±4 21 ±2 76 + 5

* Mean values ± SD, five dogs.Plasma electrolytes are corrected to give concentrations in milliequivalents per liter of plasma water (11).

obtained at 2-hourly intervals after challenge re-mained essentially constant throughout each studyand bore no relationship to the rate of fluid pro-duction by the challenged loop. Likewise the elec-trolyte concentrations of jejunal and ileal fluid re-mained unchanged in response to repeated weeklychallenges over a 3 wk period. The mean total pro-tein concentration of the loop fluid was 242 + 116(SD) mg/100 ml for jejunal, and 181 + 101 (SD)mg/100 ml for ileal loops. Protein concentration ofloop fluid never exceeded 500 mg/100 ml.None of the animals developed diarrhea or

showed any clinical evidence of malfunction of theunchallenged intact portion of the gastrointestinaltract during the studies.

Experiment III. All 12 animals absorbed iso-tonic perfusion fluid containing no glucose duringthe control period. Mean absorption by jejunalloops was 43 + 22 ml/hr, and mean absorption byileal loops was 71 + 20 ml/hr (Table VIII). Thedifference between mean control absorption ratesby jejunal and ileal loops is significant (t = 2.23,P < 0.05). Within 2 hr after toxin administration,output of isotonic fluid was observed in all jejunalloops, and within 3 hr isotonic fluid output oc-curred in all ileal loops. During the perfusion pe-riod after toxin administration, all animals ex-hibited continued fluid and electrolyte loss. Themean rate of production of isotonic fluid by jejunalloops was 68 + 46 ml/hr and that by ileal loopswas 78 ± 19 ml/hr; the differences between je-junal and ileal loops are not significant.When the perfusion fluid contained glucose, 60

mOsm/liter, an increase in isotonic fluid absorp-tion during the control period was observed inevery animal. The mean rate of absorption of theperfusion fluid by the jejunal loops was 100 ± 61

ml/hr and that by ileal loops was 148 ± 63 ml/hr(Table VIII). During the posttoxin glucose per-fusion period, the mean rate of gut fluid productionwas significantly less than that observed when theperfusion fluid contained no glucose in both jejunal(t = 2.90, P < 0.02) and ileal (t = 3.35, P <0.01) loops. The mean rate of fluid production byjejunal loops was 12 + 17 ml/hr and that by ilealloops was 31 ± 25 ml/hr. Net absorption of iso-tonic fluid was observed during this period in twoof the jejunal and one of the ileal loops.The net effect of toxin on isotonic fluid move-

ment was not significantly different during per-fusion studies with glucose from that observedduring perfusion with isotonic fluid containing noglucose. In jejunal loops the mean net effect oftoxin was to decrease absorption (or increase pro-duction) by 111 ml/hr during perfusion studieswithout glucose and by 112 ml/hr during studieswith glucose. In ileal loops, the mean net effect oftoxin was to decrease absorption (or increase pro-

TABLE VIIINet Absorption or Production of Isotonic Fluid during Per-

fusion of Small Bowel Loops before and afterIntraluminal Administration of Cholera Exotoxin

Jejunal loops* Heal loops*

ml/hr mi/hrPerfusion fluid with no glucose

Control +43±t22 +71±-20After toxin -68±46 -78± 19

Perfusion fluid with glucose, 60 mOsm/literControl +100i61 +148i63After toxin -12±4 17 -31 i25

+ indicates net absorption, - indicates production.* Mean values, ± SD, six dogs.


duction) by 149 ml/hr during studies without glu-cose and by 179 ml/hr during studies with glucose.

Conversely, the effect of glucose in increasingabsorption (or decreasing fluid production) afteradministration of exotoxin was not significantlydifferent from that observed during control periodsin either jejunal or ileal loops. In jejunal loops,the mean net effect of glucose was to increase ab-sorption (or decrease fluid production) by 57 ml/hr in control loops and by 56 ml/hr in toxin-treatedloops. In ileal loops, the mean net effect of glucosewas to increase absorption (or decrease produc-tion) by 77 ml/hr in control loops and by 47ml/hr in toxin-treated loops.

In the four animals in which PSP, 15 mg/100ml, was added to the perfusate as a nonabsorbablemarker, from 88 to 97% of administered PSPwas recovered from the loop effluent at the end ofeach perfusion period.

Glucose was absorbed from perfusate by bothjejunal and ileal loops (Table IX). Administrationof cholera exotoxin caused no significant changein rate of glucose absorption by either jejunal orileal loops.

During perfusion studies of jejunal loops with-out glucose, the isotonic perfusion fluid showed nosignificant alteration in electrolyte concentrations.During perfusion with glucose-containing solution,glucose concentration of the perfusate consistentlydecreased and sodium concentration increased dur-ing passage through the loop. Plasma electrolyteswere not significantly altered during these studies.

During perfusion studies of ileal loops, the per-fusion fluid containing no glucose showed signifi-cant increases in bicarbonate concentration dur-ing passage through the loop both before (t =2.80, P < 0.05) and after (t = 3.17, P < 0.02)

TABLE IXGlucose Absorption before and after Administration of Cholera

Exotoxin by Jejunal and Ieal Loops Perfused by anIsotonic Solution Containing Glucose,

60 mOsm/liter

Absorption Absorptionof glucose of glucose

by jejunal loops* by ileal loops*

g/hr g/hr

Control 1.70±0.39 1.53 ±0.58After toxin 1.4440.30 1.3540.44

* Mean values ± SD, six dogs.

TABLE XPlasma Bicarbonate Concentration (mEq/liter) before and

after Cholera Exotoxin Administration in Dogs withJejunal and Real Loops Perfused by Isotonic

Electrolyte Solutions

Jej unal* Ileal*

Perfusion Perfusion Perfusion Perfusionby solu- by solu- by solu- by solu-tion with tion with tion with tion withno glucose glucose no glucose glucose

Control 22 43 23±+2 22 ±3 22 ±4

After 4 hr perfusionwith isotonic 2342 25 +4 22 ±2 21 ±2solution

4 hr after toxinadministration 22 3 24±-5 18 ±3 19 +3

After 4 hr perfusionwith isotonic 24±3 22+3 15±2 17+3solution

* Mean values ± SD, six dogs.

toxin administration. During perfusion with glu-cose-containing solution a significant increase inperfusate bicarbonate concentration occurred onlyafter toxin administration (t = 6.00, P < 0.001).A significant decrease in perfusate glucose con-centration and increase in perfusate sodium con-centration occurred during passage through theileal loop both before and after toxin administra-tion. Plasma bicarbonate concentration consistentlydecreased after toxin administration during theileal studies (Table X), whereas other electrolytesshowed no significant changes. The mean plasmabicarbonate concentration at the end of the ilealperfusion studies was significantly less than thatat the beginning of the studies (t = 5.53, P < 0.001for nonglucose perfusion; t = 3.10, P < 0.02 forperfusion with glucose-containing solution).

DISCUSSION

The studies of animals with clinical cholera afterchallenge with viable V. cholerae clearly indicatethat virtually all the electrolyte loss originates inthe small bowel. Although all segments of the smallbowel contribute to the electrolyte loss, the du-odenum, because of its relatively short length, ac-counts for only a small proportion of the total elec-trolyte output.The electrolyte concentrations of the fluid pro-

duced by the jejunal and ileal segments in dogswith V. cholerae infection were virtually identical


to those in spontaneous secretions from these seg-ments of canine small bowel (12). The observedelectrolyte concentrations also closely approxi-mated those which occur in jejunal and ileal seg-ments of small bowel during clinical cholera.2 Thusin experimental canine as well as in human cholera,the electrolyte composition of the fluid producedby a given segment of small bowel approximatesthat which is characteristic of secretions normallyproduced by that segment of bowel.The protein concentration of the thoracic duct

lymph in the current studies was within the nor-mal range observed by other investigators in tho-racic duct lymph of dogs anesthetized with Nem-butal (13, 14). The absence of an abnormally highthoracic duct lymph protein concentration, to-gether with the consistently low protein concentra-tions of gastrointestinal fluid produced in responseto live vibrio or exotoxin challenge, correlate wellwith the absence of any observed histologic dam-age to either gut epithelial cells or capillary endo-thelial cells in the lamina propria. These data areconsistent with the earlier observations of Cohn-heim (15) and Goodpasture (16) that histologi-cally demonstrable gut epithelial cell damage is notessential to the pathogenesis of cholera. They arealso in agreement with the more recent demonstra-tions by Gangarosa, Beisel, Benyajati, Sprinz, andPiyaratn (17) that no loss of mucosal integrity oc-curs in the small bowel of the cholera patient, andby Gordon (18) that intravenously injected poly-vinylpyrrolidone does not leak into the gut duringclinical cholera. The observations are also con-sonant with the demonstration by Elliott, Carpen-ter, Sack, and Yardley (10) that no electromi-croscopically detectable lesions occur in the capil-laries of the lamina propria of the small bowelduring experimental canine cholera, and withthe recent demonstration by Norris and Majno(19) that intravenously injected saccharated ironoxide fails to leak through capillaries of the laminapropria of exotoxin-challenged rabbit ileum.

In the current studies, the thoracic duct lumphflow rates were abnormally high at the time ofinitial thoracic duct cannulation and increased, af-ter intravenous saline repletion, to levels of two to

2 Banwell, J. A., N. F. Pierce, R. C. Mitra, G. J. Cara-nasos, R. I. Keimowitz, A. Mondal, and P. M. Manji.1968. Preliminary results of a study of small intestinalwater and solute movement in acute and convalescenthuman cholera. Indian J. Med. Res. 56: In press.

four times the basal rate observed in barbiturate-anesthetized dogs in which thoracic duct cannula-tion had been performed at the same site (9). Itappears likely that the increased thoracic ductlymph flow is related to isotonic fluid absorptionduring experimental cholera. Previous studieshave clearly demonstrated that thoracic ductlymph flow is increased during the absorption ofisotonic saline by the mammalian gut (20); andavaliable data suggest that gut sodium absorptionis not significantly impaired during clinical 2 orexperimental 3 (21) cholera. Absorption of iso-tonic intraluminal fluid would therefore appear toprovide an adequate explanation for the increasedthoracic duct lymph flow observed in experi-mental cholera.The studies of the response of the isolated small

bowel loops to exotoxin challenge demonstrateconsistent differences between jejunal and ileal re-sponses in regard to volume and electrolyte con-tent of the fluid produced. The mean rate of fluidproduction by the jejunal loops is significantlygreater than that of ileal loops. As in the dogswith experimental vibrio infection, the bicarbonateconcentration is significantly higher, and- the chlo-ride concentration significantly lower, in fluid pro-duced by the ileum than in that produced by thejejunum. The electrolyte concentrations of thefluid produced by the canine ileal loops in re-sponse to crude exotoxin are essentially the sameas those of fluid produced by ligated rabbit ilealloops challenged by sonic lysates of V. cholerae(22).The time sequence of the response to exotoxin

was also different in the proximal and distal smallbowel loops, as fluid production by jejunal loopsbegan by the end of the 2nd hr after toxin chal-lenge, whereas that by ileal loops did not occuruntil the 3rd hr after challenge. In both cases, therate of fluid production continued at maximal lev-els through the 8th hr after challenge, but fluidoutput ceased approximately 4 hr earlier in ilealthan in jejunal loops. The "latent period" betweenexotoxin administration and onset of fluid pro-duction was less than the 4 hr as observed byLeitch, Iwert, and Burrows in ligated rabbit ileal

3 Iber, F. L., T. J. McGonagle, H. A. Serebro, E. H.Luebbers, T. M. Baylass, and T. R. Hendrix. 1968. Mecha-nism of fluid production in experimental cholera toxin-induced diarrhea. J. Clin Invest. 47. (Abstr.) In press.


loops challenged with sonic lysates of V. cholerae(22); this finding may represent a species differ-ence in response to cholera exotoxin.The similar quantitative and qualitative re-

sponses to repeated exotoxin challenges over a 3wk period indicate that neither effective immunityagainst the responsible exotoxin nor tachyphylaxisdevelops during this period of time. Subsequentstudies have indicated that responses of smallbowel loops are essentially unchanged after weeklyexotoxin challenges over a 9 wk period. These ob-servations were believed to justify the use of eachanimal as its own control in repeated studies overa 3 wk period during the subsequent glucose per-fusion studies.The small bowel perfusion studies were per-

formed to determine the effect of cholera exotoxinon glucose-associated sodium transport in thesmall intestine. Several studies have clearly dem-onstrated that intraluminal glucose enhances smallbowel sodium absorption in both the experimentalanimal (23) and in man (24). On the basis ofthese data, Phillips and Wallace (25) performeda series of studies in 1962 that demonstrated thatoral administration of hypertonic, glucose-con-taining electrolyte solutions greatly decreased thenegative electrolyte balance in cholera patients.More recently, Pierce, Banwell, Mitra, Keimowitz,Caranasos, Mondal, and Manji 4 and Hirschhorn,Kinzie, Sachar Taylor, Northrup, and Phillips5have independently shown that continuous oro-gastric infusion of isotonic, glucose-containing elec-trolyte solutions can prevent negative electrolytebalance in the majority of acute cholera patients.These clinical studies did not indicate whether themechanism of action of intraluminal glucose incholera patients was to prevent the exotoxin-in-duced electrolyte secretion or to enhance isotonicfluid absorption to such an extent as to counter-balance the toxin-induced secretion.The current studies suggest that intraluminal

glucose does not alter the effect of cholera exo-

4 Pierce, N. F., J. G. Banwell, R. C. Mitra, R. I.Keimowitz, G. J. Caranasos, A. Mondal, and P. M. Manji.1968. Effect of intragastric glucose-electrolyte solution onwater and electrolyte enterosorption in Asiatic cholera.Gastroenterology. 55. In press.

5 Hirschhorn, N., J. L. Kinzie, D. B. Sachar, J. 0. Tay-lor, R. S. Northrup, and R. A. Phillips. Reduction ofstool output in cholera by glucose and electrolyte lavage.Manuscript submitted for publication.

toxin in the canine gut. The net effect of exotoxinin increasing isotonic fluid production (or decreas-ing isotonic fluid absorption) was equally great inthe presence or absence of intraluminal glucose.Conversely, the effect of glucose in enhancing iso-tonic fluid absorption (or decreasing isotonicfluid production) was equally great in control andtoxin-treated loops. These data indicate that oneof the characteristic gut epithelial cell functions,enhanced sodium absorption in the presence ofintraluminal glucose, is well maintained in thepresence of cholera exotoxin. The data also sug-gest that the mechanism by which cholera exo-toxin produces electrolyte loss in cholera is inde-pendent of the mechanism of glucose-enhancedsodium absorption.The mean rate of isotonic fluid production by

toxin-treated ileal loops did not differ significantlyfrom that by toxin-treated jejunal loops duringthe perfusion studies. These findings appear toconflict with those of experiment II, in which exo-toxin was administered to jejunal and ileal loopsin the absence of intraluminal perfusion fluid; un-der those circumstances, fluid production by je-junal loops was significantly greater than that byileal loops. The following working hypothesis,which is consistent with current data, provides apossible explanation for this apparent paradox.The mean rate of absorption of the isotonic per-fusing fluid by the ileal loops during the controlperiod is significantly greater than that by jejunalloops. Several recent studies2 8 (21), as well asthe current data, suggest that absorptive activityof the small bowel is not affected by the choleraexotoxin. When, therefore, the cholera exotoxincauses increased secretion of isotonic fluid by agiven segment of gut, net fluid production occursonly when the rate of secretion exceeds the ab-sorptive capacity for isotonic fluid by that segmentof gut (Fig. 1). If we assume that the rates ofexotoxin-induced secretion were approximatelyequal in jejunal and ileal loops, fluid production bynonperfused jejunal loops would be consistentlygreater than that by nonperfused ileal loops, sincethe absorptive capacity of jejunal loops is signifi-cantly less than that of the ileal loops. During thestudies of perfused loops, on the other hand, the ab-sorptive capacities of both jejunal and ileal seg-ments are exceeded by the rate of perfusion of iso-tonic fluid. All exotoxin-induced secretion, there-


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:IDeOl Fluid Output

E Jejunal Fluid Output

---- --- I----r l2 4 6 8 10 12 14 16

TIME AFTER TOXIN ADMINISTRATION (Hours)

FIGURE 1 Postulated effect of cholera exotoxin on iso-tonic fluid movement in the canine small bowel. Theordinate represents isotonic fluid secretion induced bycholera exotoxin. The line J represents the rate of secre-tion (43 ml/hr) at which the absorptive capacity of thejejunal loop is equalled (experiment III). Whenevertoxin-induced secretion exceeds 43 ml/hr, fluid is pro-duced by the jejunal loop. The line I represents the rateof secretion (71 ml/hr) which equals the absorptive ca-pacity of the ileal lop (experiment III). Whenever toxin-induced secretion exceeds 71 ml/hr, fluid is produced bythe ileal loop. The points indicated by open circles repre-sent the levels of fluid production by jejunal loops ob-served in experiment II, with line J as base line. Thepoints indicated by closed circles represent the levels offluid production by ileal loops observed in experiment II,with line I as base line. The line representing exotoxin-induced secretion is hand-fitted to the experimental points,on the assumption that the rates of exotoxin-induced se-cretion are not significantly different in jejunal and ilealloops.

fore, appears as net fluid production because thesmall bowel mucosa does not absorb any of theadditional toxin-induced intraluminal fluid. Thus,during the perfusion studies with nonglucose solu-tions, the net exotoxin-induced isotonic fluid losswas approximately equal in ileal and jejunal loops.

This working hypothesis would also account forthe greater time lag between administration oftoxin and onset of fluid production by ileal thanby jejunal loops, and also for the shorter durationof toxin-induced fluid loss in ileal than in jejunalloops. If we assume that the time of onset and therate of increase of toxin-induced secretion wereapproximately the same in jejunal and ileal loops,the absorptive capacity of jejunal loops would beexceeded sooner and fluid output would be ob-served earlier, as delineated in Fig. 1. Likewise, asthe rate of toxin-induced secretion began to de-crease, fluid output would cease sooner in ilealthan in jejunal loops. This hypothesis would alsoaccount for the recent observation by Banwell,

Pierce, and Mitra that, during recovery fromclinical cholera, absorption of isotonic fluid fromthe ileum is observed at a time when isotonic fluidloss is still occurring from the jejunum.2 It shouldbe emphasized that current data, although con-sistent with this hypothesis, do not prove that thishypothesis is correct. Critical testing of the hy-pothesis should be provided by studies of bidi-rectional fluxes of isotopically labeled sodium dur-ing experimental cholera. Such studies are cur-rently underway."

ACKNOWLEDGMENTSDr. Carpenter is a recipient of National Institute ofAllergy and Infectious Disease Research Career Devel-opment Award AI-28861.This work was supported by research grant AI-07625

from the National Institute of Allergy and InfectiousDiseases, under the auspices of the United States-JapanCooperative Medical Science Program.

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Vanikiati, and R. A. Phillips. 1959. Water and elec-trolyte studies in cholera. J. Clin. Invest. 38: 1879.

2. Carpenter, C. C. J., A. Mondal, R. B. Sack, P. P.Mitra, P. E. Dans, S. A. Wells, E. J. Hinman, andR. N. Chaudhuri. 1966. Clinical studies in asiaticcholera. II. Development of 2: 1 saline: lactate regi-men. Comparison of this regimen with traditionalmethods of treatment, April and May, 1963. Bull.Johns Hopkins Hosp. 118: 174.

3. Sack, R. B., C. C. J. Carpenter, R. W. Steenberg,and N. F. Pierce. 1966. Experimental cholera: acanine model. Lancet. 2: 206.

4. Hoffman, W. S. 1937. A rapid photoelectric methodfor the determination of glucose in blood and urine.J. Biol. Chem. 120: 51.

5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193: 265.

6. Schedl, H. P., and J. A. Clifton. 1961. Small intes-tinal absorption of steroids. Gastroenterology. 41: 491.

7. Finkelstein, R. A., P. Atthasampunna, M. Chulasa-maya, and P. Charunmethee. 1966. Pathogenesis ofexperimental cholera: biologic activities of purifiedprocholeragen A. J. Immunol. 96: 440.

8. Craig, J. P. 1965. A permeability factor (toxin) foundin cholera stools and culture filtrates and its neutrali-zation by convalescent cholera sera. Nature. 207: 614.

9. Yoffey, J. M., and F. C. Courtice. 1956. Lymphatics,Lymph and Lymphoid Tissue. Harvard UniversityPress, Cambridge, Mass. 2nd edition. 87.

10. Elliott, H., C. C. J. Carpenter, R. B. Sack, and J. H.Yardley. 1968. Small bowel morphology in experi-mental canine cholera. A light and electron micro-scopic study. Am. J. Pathol. 52: 15a. (Abstr.)


11. Eisenman, A. J., L. B. Mackenzie, and J. P. Peters.1936. Protein and water of serum and cells of humanblood, with a note on the measurement of red bloodcell volume. J. Biol. Chem. 116: 33.

12. de Beer, E. J., C. G. Johnston, and D. W. Wilson.1935. The composition of intestinal secretions. J. Biol.Chem. 108: 113.

13. Field, M. E., 0. C. Leigh, Jr., J. W. Heim, andC. K. Drinker. 1934. The protein content and osmoticpressure of blood serum and lymph from varioussources in the dog. Am. J. Physiol. 110: 174.

14. Courtice, J. C., and B. Morris. 1955. The exchange oflipids between plasma and lymph of animals. Quart.J. Exptl. Physiol. 40: 138.

15. Cohnheim, J. 1890. Lectures on General Pathology.A handbook for practitioners and students. Trans-lated from the 2nd German edition by Alexander B.McKee. The New Sydenham Society, London. 3: 941.

16. Goodpasture, E. W. 1923. Histopathology of the intes-tine in cholera. Philippine J. Sci. 22: 413.

17. Gangarosa, E. J., W. R. Beisel, C. Benyajati, H.Sprinz, and P. Piyaratn. 1960. The nature of thegastrointestinal lesion in asiatic cholera and itsrelation to pathogenesis: a biopsy study. Am. J. Trop.Med. Hyg. 9: 125.

18. Gordon, R. S. 1961. The failure of asiatic cholera togive rise to "Exudative Enteropathy." In SEATO

Conference on Cholera. Post Publishing Co., Ltd.,Bangkok. 54.

19. Norris, H. T., and G. Majno. 1967. Observations onthe pathogenesis of experimental human cholera inthe rabbit. Am. J. Pathol. 50: 32a. (Abstr.)

20. Barrowman, J., and K. B. Roberts. 1967. The role ofthe lymphatic system in the absorption of water fromthe intestine of the rat. Quart. J. Exptl. Physiol. 52:19.

21. Love, A. H. G. 1965. The effect of glucose on cationtransport. Proceedings of the Cholera Research Sym-posium. U. S. Government Printing Office, Washing-ton, D. C. 144.

22. Leitch, G. J., M. E. Iwert, and W. Burrows. 1966.Experimental cholera in the rabbit ligated ileal loop:toxin-induced water and ion movement. J. Infect.Diseases. 116: 303.

23. Fisher, R. B. 1955. The absorption of water and ofsome small solute molecules from the isolated smallintestine of the rat. J. Physiol. (London) 130: 655.

24. Malawer, S. J., M. Ewton, J. S. Fordtran, and F. J.Ingelfinger. 1965. Interrelation between jejunal ab-sorption of sodium, glucose and water in man. J.Clin. Invest. 44: 1072. (Abstr.)

25. Phillips, R. A. 1966. Cholera in the perspective of1966. Ann. Internal Med. 65: 922.


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Site and Characteristics of Electrolyte Loss and Effect of Intraluminal