Intestinal Perfusion Studies in Tropical Sprue

Vol. 65, No.2 Printed in U.S.A .

GASTHOENTEROLOGY 65: 192- 198, 1973 Copyright© 1973 by The Williams & Wilkins Co.

INTESTINAL PERFUSION STUDIES IN TROPICAL SPRUE I. Transport of water, electrolytes, and d-xylose Josf J . CORCINO, M.D. , MILAGROS MALDONADO, AND FREDERICK A. KLIPSTEIN, M.D.

Tropical Malabsorption Unit of the Universities of Rochester and Puerto Rico, University (District) Hospital, San J uan, Puerto Rico

Segmental jejunal perfusion studies were performed to assess water, sodium , chloride, and d-xylose transport in 10 carefully selected patients with untreated tropical sprue who had diarrhea at the time of perfusion, and in 8 control subjects. All of the patients with tropical sprue were found to be in a net secretory state in regards to water, sodium, and chloride as compared with control subjects . d-Xylose absorption was impaired in patients with tropical sprue as compared with control subjects. A significant correlation was demonstrated between the absorption of xylose as measured by the segmental perfusion technique and the 5-hr urinary xylose excretion as well as the peak serum xylose level obtained with a standard xylose absorption test utilizing a 25-g dose of xylose when combined data from control and tropical sprue subjects are analyzed. The net secretory state for water, sodium, and chloride observed in patients with tropical sprue may be of relevance in explaining, at least partially, the pathogenesis of the diarrhea seen in this syndrome. Contamination with coliform bacteria has been reported in tropical sprue, and, more recently, certain of these organisms have been shown to produce enterotoxins. Whether these enterotoxins account fully for the derangements in water and electrolyte transport found present in patients with tropical sprue remains undetermined. Intestinal malabsorption is a well recognized feature of the syndrome known as tropical sprue. 1 Although much emphasis has been placed on the pathophysiology of Received May 12 , 1972. Accepted March 9, 1973. Address requests for reprints to: Dr. J . J. Corcino, Tropical Malabsorption Unit , University (District) Hospital, Puerto Rico Medical Center, San Juan , Puerto Ri co 00935. This work was supported by grants from the Research Corporation, ew York City: the Wellcome Trust, London , England; Training Grant No . 5-T0-1 AMO 5177 (Gastroenterology) from the United States Public Health Service, Bethesda, Maryland; Grant 445 from the Nutrition Foundation, Inc ., New York City; an d Grant RR-63-09 from the General Clini cal Research Center, Program of the Division of Research 192

impaired absorption of nutrients such as fat, vitamins, and carbohydrate in this disease , only limited attention has been given to fluid and electrolyte tran sport. Apart from isolated reports 2 • 3 only the Resources , National Institutes of H ealth, Bethesda, M a ryland. The enco uragement and s upport of Drs . N. Maldonado and A. A. Cint r6n -Rivera is gratefu lly ackn owledged. The authors t ha nk Dr. J. T. Tomasini for evaluating t he jejunal biopsies, Dr. R. Santini. Jr .. a nd Mrs. S. Muriel for performing t he serum folate determinations, an d Mrs. A. Pagan, Miss F. Rodriguez, Mr. W. Roma n , Mrs. I. Fiol. Miss H. Monagas, and Mr. J . Roditguez for superb technical ass istance .

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PERFUSION STUDIES IN TROPICAL SPRUE

193

int estinal contents were collected by siphonage at the two distal openings at a rate of 1 ml per mm . A 60-min period was allowed fo r equilibration after which three 20-min collections were obtained; staggering of collections was not performed. Chemical analyses. All deter minations were performed in duplicate. Immediately after collection, the perfusion solutions were deproteinized and then frozen. Sodium and chloride were measured by standard tec hniques ·using a fla me photometer with internal lithium standards and a Cotlove chloridometer, respectively . d -Xylose concentrations were measured in protein-free fil trates by the met hod of Roe and Rice• and polyethylene glycol values were determined by a modification 10 of the method of Hyden." Net transport of water, sodium , chloride, and d-xylose was determined by standard formulas for the 30-cm study segment utilizing polyethylene glycol corrections. • Results were exMaterials and Methods pressed as milliliters of water per hour per Subjects. Eighteen Puerto Rican subjects ce ntimeter, microequi valents of electrolytes were investigated; 10 patients with overt, un- transported per hour per centimeter, and millitreated tropical sprue , aged 15 to 67 years, with grams per hour per centimeter of d-xylose a history of diarrhea for at least 1 month prior to absorbed. The results regarding crystalline pterevaluation, a nd active diarrhea at the time of oylglutamic acid absorption will be the subject perfusion ; and 8 asy mptomatic control subjects, of a subsequent communication. The statistical significance of the data was randomly selected from a large group undergo ing screening studies for subclinical malabso rp- evaluated with Student's t-test. tion, 6 aged 20 to 60 years. Informed oral and written consent was obtained in all instances. Results Techniques utilized for the laboratory evalu Biochemical and Absorption Studies ation of intestinal function and structure in these individua ls were similar to those deAll but 1 of the 8 control subjects had scribed in previous communications from this normal absorption of xylose, fat , and vita6 8 Ia bora tory. min B,2 . Observations concerning intestiT echnique. The small bowel was intubated nal function and perfusion findings in this after an overnight fast with three fused polyvinyl tubes which were weighted by a mercury 1 subject will be discussed. All had normal bag and positioned under fluoroscopic control so hemoglobin , folate , and B, 2 levels. Impaired intestinal absorption and morthat the infusion a perture lay adjacent to t he liga men t of Treitz. Openings of the distal tubes phological abnormalities of the mucosa we re located 15 an d 45 em from t he infusion were present in all subjects with tropical point. sprue (table 1) . All 10 patients presented A warmed (37 C) isoos motic solution (osmo - with diarrhea of at least 1-month duration; lality 280 milliosmoles per kg, pH 6.5) contain- all 10 had megaloblastic bone marrows, ing 124 mEq per liter of sodium chloride, 5 g per and 8 were severely anemic . All had low liter of d-xylose , 25 11g per liter of crystalline se rum folate levels and all but 2 also had pteroylgluta mic ac id , and 10 g per liter of polyethylene glycol was delivered into the jeju- subnormal serum B, 2 levels . The 2 patients num with a peristaltic infusion pump (Model with normal serum B, 2 levels had had 1023, Ha rva rd Instrument Co ., Dover, Mass.) at diarrhea for only 1 to 2 months before a constant rate of 9.2 ml per min. Samples of evaluation . All ha d ma labsorption of vita-

group of Ban well and colleagues, • workin g in Calcutta , have addressed themselves to this problem. These investigators found net secretion of water into the jejunum of 6, and into the ileum of 7, of 13 patients with tropical sprue. The variable findings described in their report prompted us to study a group of Puerto Ricans with tropical sprue to determine whether, by selecting a more homogeneous group of patients, none of whom had received previous therapy, a more uniform pattern concerning abnormal water and electrolyte transport could be obtained. In this paper, our observations regarding water, electrolyte, and d-xylose transport by utilizing a triple lumen tube perfusion technique 5 in a group of 10 patients with untreated tropical sprue and in 8 normal control subjects are reported .

TABLE

Patient

A.M.V. M.D .M. A. C. D. M.M.O. J.D. F . F.N.G . R.A. A. T. G.R. B.Q. S. R.M . M. a b

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CORCINO ET AL.

194

1. Laboratory in vestigations in subjects with tropical spruea

Duration Hemoglobin of diarrhea

Xylose

Serum Folate

Vitamin Albumin B"

Urin e

Serum

Jejunal

"CoB 12 absorption

Fecal fat

mar-

phology

mo

g/JOOml

ng/ml

pg/ml

g/5 hr

mg/100 ml

g/24 hr

%

(0)

( > 13)

(>5)

(> 150)

(>3 .5)

( >5)

(>30)

( < 6)

(> 8)

(0)

4 48 4 1 2 6 6 3 4 4

13.6 7.1 5.7 5.6 13.0 4.8 8.4 3.6 8.6 7.9

2.5 2.5 3.0 4.5 1.5 1.5 3.5 2.0 2.5 2.0

150 70 70 175 160 25 8 5 75 100

3.8 3.2 4.2 3 .3 4.0 3 .4 3 .2 2.9 3. 1 3.4

1.7 1.3 2.3 1.9 1. 2 0 .7 1.4 1.6 1.9 2.3

14

7.4 3 .1 5.0 4.5 10.3 12.6 14 .8 21.8 5.1 7.8

2.0 3.5 4.3 1.5 1.3 1.2 3.6 1.1 5.4 10.2b

2+ 4+ 3+ 3+ 2+ 3+ 4+ 3+ 3+ 3+

g/100 ml

25 35 21 13 14 6 28 22

Normal values are in parentheses at tops of columns. Performed with whole body counter technique (normal, 35 to 75% retention in 7 days).

min B 12 and d-xylose; 6 had steatorrhea and 5 had a low serum albumin . All patients were receiving a diet containing at least 80 g of fat at the time the fat balance studies were performed.

CONTROLS TROPICAL SPRUE

D f~~~~l

300

3

Water and Electrolyte Transport (Fig. 1) Normal subjects. Perfusion studies with the isoosmotic solution described were per2 200 formed in 8 asymptomatic subjects. Absorption of water was 1.41 ± 0.23 (mean ± SE) ml per hr per em of jejunum. Absorp100 tion of sodium and chloride were 141.7 ± 29.3 ~Eq, and 129 .9 ± 24.9 ~Eq per hr per 8 em; the mean geometric concentrations of sodium and chloride in the study segment }-Orf-------+-v--~-t-------i- 0 --.... •• were 124.2 and 122 .9 mEq per liter, respeca w • tively. ~ ,,,,, Tropical sprue subjects. Studies performed in 10 subjects with tropical sprue ,,,, ' ' ' " 100 revealed a net secretion of water of 0.9 ± 0.2 (mean ± SE) ml per hr per em of • jejunum. Secretion of sodium and chloride 200 2 were 74.1 ± 22 .1 ~Eq and 110.6 ± 23 .1 ~Eq per hr per em; the mean geometric concen trations for sodium and chloride in the study segment were 124.5 and 121.3 mEq "00 3 per liter, respectively. No Cl Differences concerning water, sodium, FIG. 1. Transport of water, sodium , and chloride and chloride transport in tropical sprue as in healthy (open circles) and tropical sprue (closed compared with control subjects are highly circles) subjects . Bars represent mean ± SE. 0

0

0

00

....

..

0

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PERFUSION STUDIES IN TROPICAL SPR UE

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tion in the study segment being 3.9 mg per ml. The difference between control and tropical sprue subjects is significant to the P < 0.001 level. 20

Correlation of d-Xy lose Transport as Measured by the Tripl e Lumen Tub e Absorption Techniqu e as Compared with the 5-hr Urinary Excretion of Xylose and Peak Serum Level after the Oral Administration of a 25 -g Dose of X y lose

0

oo 15

e

I)

'.c:..

'e

8 0

There was an excellent correlation (r = 0.78, P < 0.001) between the triple lumen tube assessment of xylose absorption and its 5-hr urinary excretion (fig. 3), as well as

-

Q

10

0 0

10

\

••

• =·•

0._--~----------r---------

CONTROLS

TROPICAL SPRUE

FIG. 2. Tra n s p ort of d-xy lose by jejunum in healthy (o p en circles) a nd tropica l s prue (c losed circles ) s ubject s. Bar and dotted lines represent m ea n

...

~

0

>. ><

5

'

± SE.

"0

~

-~

4

significant to the P < 0.001 levels in all instances. The mean geometric osmolalities for the study segment were 270.1 and 271.6 milliosmoles per kg for the control and tropical sprue subjects, respectively . y,(038l X+ 0.42

d-Xy lose Transport (Fig. 2) Normal subjects. Healthy volunteers absorbed 14.1 ± 1.2 (mean ± SE) mg per hr per em when perfused with an isoosmotic solution containing 5 mg per ml of dxylose. The geometric mean concentration of d-xylose in the study segment was 3.5 mg per ml. Tropical sprue subjects. The mean absorption of d -xylose observed in 10 perfusion studies was 4.8 ± 0.8 (mean ± SE) mg per hr per em ; geometric mean concentra-

4

6

B

10

12

14

16

18

20

mg/ hr I em d- Xylose Absorbed

FIG. 3. Correla ti on o f d- xy lose absorption as m easured by the tripl e lum e n tube technique and tha t determined by th e st a nda rd t est meas uring th e 5 -hr urin a ry excretion of d -xylose a fter th e oral a dmini stra ti on of a 25 -g d ose in hea lthy (op en circl es ) a nd tropi ca l s prue (closed circles) s ubject s.

196

CORCINO ET AL.

with its peakserum level (fig. 4, r = 0.84, P < 0.001), when the data obtained from tropical sprue as well as control subjects are. combined. It became obvious from examining figures 3 and 4 that there was a clear cut clustering between the observations made in control subjects when compared with those obtained in patients with tropical sprue. When the data obtained in control subjects are analyzed separately, the correlation is not as significant; r = 0.097, P > 0.8, and r = 0.59, P > 0.1 for urinary xylose versus milligrams per hour per centimeter absorbed and peak serum xylose level attained versus milligrams per hour per centimeter absorbed, respectively. Similarly, when the tropical sprue patients are analyzed as a separate group, the correlation coefficient obtained is not significant. Thus, for this group the correlation between the urinary xylose and the milligrams per hour per centimeter of the pentose absorbed is not statistically significant (r = 0.16, P > 0.6) , and the correla80

70 .

60

~50 2 . -"" 0

0

"'~ ..40 0. 0

-;;::. X

~ 30 E

<=

"'

(/)

20

Y= ( 314 l X +8.66 10

2

4

6

8

10

12

14

16

18

20

mg/ hr I em d- Xylose Absorbed FIG . 4. Correlation of d-xylose absorption as measured by the triple lumen tube technique and the serum d-xylose peak le\·el after the oral ad ministra tion of 25 g of xylose in hea lthy (open circles) and tropical sprue (closed circles) su bjects.

Vol . 65, No.2

tion between the peak serum level and the milligram per hour per centimeter of dxylose absorbed is poor (r = 0.28, P > 0.4).

Discussion In this study, net jejunal secretion of water and electrolytes was observed in all the patients with tropical sprue evaluated, who were from a group of carefully selected patients who had active diarrhea at the time of perfusion , and who were not previously treated. As shown in table 1, all the patients investigated had diarrhea for at least 1 month before eva luation; they all had megaloblastic bone marrows and folate deficiency and all but 2 were also B, 2 -deficient. In the study of Ban well et al.,< no clinical data concerning the presence of diarrhea or its duration prior to the perfusion studies are given; only 4 of9 patients who had bone marrow aspirates performed had megaloblastic erythropoiesis, even though 5 of these 9 patients had subnormal serum folate levels. Of even more significance may be the fact that all the patients with tropical sprue in the study from Calcutta had normal serum B, 2 levels in spite of a uniformly subnormal vitamin B, 2 absorption. The latter findings suggest that these patients had been treated with vitamin B, 2 prior to the perfusion studies, most likely during the performance of a Schilling test. This may account for the differences concerning abnormal jejunal water and electrolyte transport in patients with tropical sprue observed in both studies, since the effect of treatment with pharmacological doses of vitamin B, 2 on the deranged water and electrolyte transport observed in such patients has, to our knowledge, not been evaluated . It is, nevertheless, well known that symptomatic as well as gastrointestinal improvement may be seen in patients with tropical sprue treated with this vitamin . 8 It is of interest that in the report from Calcutta, 4 treatment with folic acid (15 mg daily for 10 days to 4 weeks) resulted in improvement of the abnormal transport of water (at the ileal level) in 1 of 2 patients with tropical sprue who presented with net secretion when originally

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PERFUSION STUDIES IN TROPICAL SPRUE

evaluated. No significant change was observed at the jejunal level in the same subject. The finding of a net secretory state for water and electrolytes in patients with tropical sprue may be of relevance in explaining the pathogenesis of the diarrhea seen in this syndrome. A similar secretory pattern has been reported in a variety of diarrheal disorders resulting from intestinal colonization with organisms that produce enterotoxins such as Vibrio cholerae 12 and certain strains of Escherichia coli. 13 Bacterial contamination 14 (and F. A. Klipstein, L. V. Holdeman, J. J. Corcino, et al., unpublished observations) of the upper small bowel by coliform bacteria has been found in subjects with tropical sprue, and certain of these organisms have been shown to elaborate an enterotoxin capable of inducing a secretory state when injected into rabbit ileal loops (Klipstein et al. , unpublished observations). Whether such metabolic products of bacteria present within the proximal small intestine of subjects with tropical sprue account fully for the pathogenesis of the secretory state seen in this syndrome is uncertain since such a condition is also present in nontropical sprue where no contamination of the upper small bowel is present. 15 A borderline value for sodium and chloride transport with net water absorption was observed in 1 of the 8 control subjects (fig. 1). This was the only subject found to have an abnormal determination of intestinal function (urinary excretion of xylose of 3.5 g per 5 hr). Jejunal biopsy showed morphological changes of mild (1 + ) severity such as is found in 48% of the asymptomatic Puerto Rican population . 6 The finding of a net secretory state for sodium and chloride in a subject with a moderately reduced xylose absorption suggests that some persons with the so-called "nonspecific jejunitis" seen in the tropics may have more extensively impaired intestinal function than has hitherto been recognized using the standard clinical determinations, since such impairment may be detectable only by the application of more sensitive techniques.

197

Fordtran et al. 16 have observed a high degree of correlation between the urinary excretion of d-xylose and the intestinal absorption of this pentose in young, healthy male subjects. Figures 3 and 4 summarize our data concerning the relationship between xylose absorption as measured by the triple lumen tube technique and that determined by the 5-hr urinary excretion test (fig. 3) as well as by peak serum xylose concentrations (fig. 4). Our results confirm Fordtran's findings and extend his observations to include subjects with intestinal malabsorption. Although such a finding may seem, to some investigators, logical, in view of a morphologically abnormal intestinal mucosa, there is considerable controversy concerning the correlation between xylose malabsorption and morphological mucosal abnormalities. 1 The role that intraluminal (i.e., bacterial utilization 1 '), as well as mucosal factors, plays in the malabsorption of d-xylose observed in tropical sprue remains undetermined. REFERENCES 1. Klipstein FA: Tropical sprue. Gastroenterology 54:275-293, 1968 2. Fordtran JS, Rector FC, Locklear TW, et al: Water and solute movement in the small intest ine of patients with sprue. J Clin Invest 46:287-298, 1967 3. Gerson CD: Glucose and intest inal absorption in man. Am J Clin Nutr 24:1393-1398, 1971 4. Banwell JG, Gorbach SL, Mitra R, et al: Tropical sprue and malnutrition in West Bengal. II. Fluid and electrolyte transport in the small intest ine. Am J Clin N utr 23:1559-1568, 1970 5. Cooper H, Levitan R, Fordtran JS , et al: A method for studying absorption of water and solute from the human small intestine. Gastroenterology 50:1-7, 1966 6. Klipste in FA, Beauchamp I, Corcino JJ , et al: Nutritional status and intest inal function among rural populations of the West Indies. II . Barrio Nuevo , Puerto Rico. Gastroenterology 63:758- 767' 1972 7. Klipstein FA, Falaiye JM: Tropical s prue in expatriates from the tropics li ving in the continental United States. Medicine (Baltimore) 48:475-491, 1969 8. Rickles FR, Klipstein FA, Tom asi ni J , et al: Long-term follow up of antibiotic-treated tropical sprue. Ann Intern Med 76:203-210, 1972

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9. Roe JH, Rice EW: Photometric meth od for determination of free pentoses in anima l tissues. J Bioi Chern 173:507-5 12, 1948 10. Malawer SJ, Powell DW: An improved turbidometric analysis of polyethylene glycol utilizing an emu ls ifier. Gastroenterology 53:250- 256, 1967 11. H y den S : Turbidometric method for determination of higher polyet hylene glycols in biologic materials. Ann R Agric Co li Sweden 22:139- 145, 1955 12. Burrows W, Mustei kis GM: Cholera infection and toxin in the rabb it ilea l loo p. J Infect Dis 116:183- 190, 1966 13. Banwell JG , Gorbach SL, Mitra RC, et al: Acute undifferentiated diarrhea in the trop ics. II . Alterations in in testina l f1 uid and electrolyte move-

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ment. J Clin Invest 50: 890- 900, 1971 14. Gorbach SL, Mitra R , Jacobs B, et al: Bacterial contamination of the upper sma ll bowel in tropical s prue. Lancet 1:74-77, 1969 15 . Anderson CM, Langford RF: Bacte ri a l content of small intest in e in children in health in coe li ac disease and in fibrocystic di sease of pancreas. Br Med J 1: 803-806, 1958 16. Fordtra n JS , Soergel KH , lngelfinger FJ: Intest ina l absorption of d-xylose in m a n. N Eng! J Med 267:274-279, 1962 17. Goldstein F , Karacadag S , Wirts CW , et a!: Intraluminal s mall-intesti nal utili zat ion of dxylose by bacteria. Gastroenterology 59:380-386, 1970