Gut, 1991, 32, 754-759

Impaired intestinal barrier function measured bydifferently sized polyethylene glycols in patientswith chronic renal failure

M Magnusson, K-E Magnusson, T Sundqvist, T Denneberg

AbstractThe intestinal mucosa plays a fundamental roleas the site for absorption ofnutrients, and as animportant barrier from potentially harmfulagents in the intestinal lumen. Little is knownof the permeability properties of the intestinalmucosa in uraemic patients. The intestinalpermeability to differently sized polyethyleneglycols (PEGs; range 326-1254 daltons) wasstudied in nine patients with chronic renalfailure (24 hour endogenous creatinine clear-ance 5-27 ml/minute). The maximum 24 hoururinary recovery ofPEGs was decreased in theuraemic patients but relatively more of thelarger than the smaller PEGs were found inthese patients. The results suggest a reducedurinary recovery of PEGs caused by renaldysfunction but also a relatively increasedintestinal permeability to larger PEGs in theuraemic patients.

Departments ofNephrology and MedicalMicrobiology, Universityof LinkBping, SwedenM MagnussonK-MagnussonT SundqvistT DennebergCorrespondence to:Dr M Magnusson,Department of Nephrology,Faculty of Health Sciences,University of Linkoping,S-581 85 Linkoping, Sweden.

Accepted for publicationAugust 1990

Although the intestinal membranes are the site ofabsorption of nutrients, they also provide a

barrier that prevents potentially harmful agentsfrom leaving the intestinal lumen.' However,infections such as rotavirus, and villous atrophycaused in disorders such as coeliac disease, can

lead to altered barrier properties.2` Arthritis inassociation with chronic inflammatory boweldiseases has been thought to depend on absorp-tion of immunogenic substances via the alteredgut mucosa.5 Furthermore, several potentiallytoxic substances are produced by the bacterialflora in the intestinal lumen and these mayaccumulate after absorption in the uraemicstate.i9

Histological changes, including reduction ofvillous height, increased crypt depth, and infil-tration of inflammatory cells and functionalchanges such as decreased activity of dipepti-dases and increased activities of disaccharidases,have been shown in the intestine in patients withchronic uraemia.'1-3Although proteinaceous macromolecules are

thought to be digested in the lumen and furtherbroken down during the absorption process,some material may enter the bloodstream.'4Several studies have indicated that even largermolecules and particles can be transmitted across

the intestinal membranes by persorption.'5Several inert or weakly metabolised molecules

have been used to assess intestinal permeability,including 5Cr-EDTA, urea, mannitol, rham-nose/lactulose, and polyethylene glycols.'"We have recently investigated the intestinal

permeability to differently sized polyethyleneglycols (PEGs) (range 326-1162 daltons) and

their recovery in urine after intravenous injectionin rats with chronic uraemia.'20 Firstly, wefound that the intestinal permeability to thelarger PEG molecules (range 546-1162 daltons)increased in the uraemic rats. Secondly, intesti-nal permeability decreased in normal anduraemic rats fed a low protein diet. The investi-gation also showed that intravenously adminis-trated PEGs were excreted without glomerularexclusion in the size range used (326-1162daltons) in both normal rats and those withchronic uraemia.This study aimed to investigate the intestinal

permeability to differently sized polyethyleneglycols (range 326-1162 daltons) in nine patientswith chronic renal failure.We found an over all reduction in intestinal

permeability of PEGs but a relatively increasedpermeability to larger PEG molecules (414-898daltons) in patients with chronic uraemia. Thepermeability profiles of the uraemic patients andnormal subjects were also compared with theresults of computer simulations of a multicom-partment model, focussing on the effects ofreduced renal excretion capacity.

Patients and methods

PATIENTSThe uraemic group consisted of nine patientswith stable chronic renal insufficiency withoutovert uraemic symptoms. None had undergonegastrointestinal surgery or had any history ofgastrointestinal disease. The patients were inhospital during the study and most were on anunrestricted protein intake diet. One patient hada terminal uraemia and was on a moderatelyprotein restricted diet of approximately 40 g perday (patient 9 in Table I). Daily energy intakevaried between 1800-2200 kcal. Phosphatebinders, vitamin B, and calcium carbonate weregiven and antihypertensives were added whenneeded. No patient was treated with glucocorti-coides or non-steroidal anti-inflammatory drugs(NSAIDs). Further patient data are given inTable I. The control group comprised six healthyvolunteers (four women and two men) with nohistory of renal or gastrointestinal disease (TableI). The study was approved by the local ethicalcommittee.

PEG TESTA mixture of PEG 400 and PEG 1000 (range326-1244 daltons) (100 mg PEG 400 and 2.5 gPEG 1000 dissolved in 10 ml of water, obtainedas Macrogolum from Apoteksbolaget, Stock-

754

Impaired intestinal barrierfunction measured by differently sized polyethylene glycols in patients with chronic renalfailure

TABLE I Details ofuraemic patients and control subjects

Serum 24 hr creatinineAge creatinine Serum urea clearance (mil

Patient Sex (yrs) Diagnosis (pMWl,l) (mmolll) min/1 73m2)

1 M 35 CGN 274 20 272 M 73 HN 355 23 183 M 55 CGN 451 27 164 F 75 NSC 413 41 125 M 54 CGN 796 31 116 M 82 NSC 446 33 107 F 51 PC 542 22 98 M 63 CGN 667 30 89 F 66 CPN 586 45 5Mean 62 503 30 13Range 35-82 274-7% 20-45 5-27Controls:Mean 31 83 5 99Range 23-41 63-99 3-7 69-124

CGN=chronic glomerulonephritis; HN=hydronephrosis; NSC=nephrosclerosis; PC=polycysticdisease; CPN=chronic pyelonephritis.

holm, Sweden) was given orally in 150 ml ofwater after overnight fasting. Ingestion of foodand drugs was not allowed during the first twohours. Urine was collected over six, 24, and 48hours. The urine was frozen at -20° for furtheranalysis of PEGs. When the urinary recovery isdiscussed below it refers to the 24 hour recovery

unless otherwise stated.

ANALYSES OF PEGSSix, 24, and 48 hour samples of urine were

analysed. Since the urine contained visibleflocculated material which seemed to interferewith the separation and analysis ofPEGs accord-ing to a previously published procedure,2' theextraction of PEG from the urine was modified.Thus, 6 ml samples and controls with knownamounts of PEGs in duplicates were mixed with15 ml trichloroacetic acid (TCA 50%) for 10minutes at room temperature and centrifugedfor 10 minutes at 3700 rpm (n1150 g) in aWifug Doctor Centrifuge (Wifug, Stockholm,Sweden). Then 7 ml of the combined super-natants of the duplicates were neutralised toaround pH 7 with 0 5 mol/l NaOH, as measuredwith pH indicator paper. Some 6 ml was thenmixed in a vortex mixer with 3-7 g AmberliteMB-3 mixed anionic and cationic resin (BDHLtd, England), inverted repeatedly for 30minutes at 37°C, mixed with new resin, andinverted for another 30 minutes at 37°C. A 2 mlsample was freeze dried overnight, dissolvedin methanol (42%) and water (58%), filteredthrough a 0.45 [im filter, and analysed with highperformance liquid chromatography (HPLC;HSRI 931, Tecator, Sweden, equipped withreverse phase C-8 column, Lichrosorb RP-8Fertigkolonnen, Merck AG, Darmstadt, WestGermany). The pressure was about 20 MPa andthe flow rate (waste mode) around 40 ml/hour.By comparing the peak height of each PEGmolecular weight species in the urine with theamount given orally, the percentage recovery ofeach PEG molecular size was calculated.

CHARACTERISTICS OF THE MUCOSAL BARRIER

Based on the recovery of different sized PEGs ineach patient, the barrier was characterised inthe following ways. Firstly by the breakpoint(dalton) - that is the PEG size where the recovery

1 2

Figure 1: Graphic representation ofthe multicompartmentmodel, where kij represent the rate constantfromcompartment i toj.

(R) (%)=(R max-R min)/2+R min. R max and Rmin are the maximum and minimum urinaryrecoveries of the molecules used; this parameterdescribes the PEG size where 50% of the intesti-nal filtering is expressed.22 Secondly by the slopeof the recovery values for increasing PEG size,since the intestinal exclusion of moleculesbecause of size was roughly linear and inverselyproportional to the molecular weight. Todescribe the correlation between urinary recov-ery and molecular weight, a linear correlationcoefficient was calculated by using the eight PEGsizes symmetrically surrounding the breakpoint.This was done because the urinary recoverypatterns, as a result of the relative low recovery,did not fit well with a previously describedmodel.23

COMPUTER SIMULATIONS OF URINARY RECOVERYIN RENAL FAILUREThe passage of molecules from the intestine tothe urine was approximated with a multicom-partment model (Fig 1), and simulated fordifferent critical parameters, using Stella soft-ware (High Performance System Inc, Lyme,New Hampshire, USA) and a Macintosh SEcomputer. The transit times between the intesti-nal segments were described by ramp functions.During the first 30 minutes, absorption wasassumed to occur from a segment correspondingto the jejunum, and from 30 to 60 minutes theintestinal contents were transported further intothe ileum. Absorption from the ileum took placeonly between 60 and 105 minutes, when thetransport to the colon started. Absorption fromthe colon alone occurred between 135 and 360minutes, when the simulation ended. The rate

TABLE II Mucosal barrier characteristics calculated fromthe urinary recovery ofpolyethelyne glycols (PEGs) in theuraemic and control groups. Values are mean (SEM)

Barier Significancecharacteristics Controls (n=6) Uraemic (n= 9) (p<)

Breakpoint(Da PEG)* 541 (60) 688 (25) 0.05

Regressioncoefficientt 3-92 (0.98) 1.18 (0.42) 0-01

*Breakpoint Da PEG, is the PEG molecular weight at which 50%of the intestinal filtering has occurred.tThe regression coefficient is based on a linear approximation ofthe recovery of molecules according to molecular weight for theeight molecules surrounding the breakpoint of each individual.

755

Magnusson, Magnusson, Sundqvist, Denneberg

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Molecular weight (daltons)Figure 2: Twenty four hour urinary recovery ofpolyethylene glycols (PEGs) in the controlgroup (open bars) and in the uraemic group (closed bars). The recovery is significantly higherfor all molecular weight species in the control group (p<005-O001).

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Molecular weight (daltons)Figure 3: (A) Six hour (open bars) and 24 hour (closed bars) urinary recovery ofpolyethyleneglycols (PEGs) in the control group. Only small quantities ofsmall molecules (range 326-546daltons) are excreted between 6 and 24 hours. (B) Six hour (open bars) and 24 hour (closedbars) urinary recovery ofPEGs in three uraemic patients (nos 7-9 in Table I). Smallquantities are excreted between 6 and 24 hours.

constants (k41, k42, k43) for the absorption ofdifferent sized PEG molecules from the jejunum,ileum, and colon, respectively, were calculatedfrom exponential functions applied to steadystate intestinal perfusion data in man.2 The rateconstants describing exchange with the extra-vascular space and renal clearance were takenfrom earlier results obtained in pigs.24 Withregard to the aims of the present investigation,the simulations were performed by changing therate constant describing the passage from bloodto urine between 20% to 150% of the basal value- the value of the rate constant k,4 for eachmolecular weight species of PEG was changedbetween 0.2*kM and 1.5*k,4

LABORATORY TESTS AND STATISTICSSerum and urine creatinine and serum ureaconcentrations were determined by routinemethods at the Department of Clinical Chemis-try, University Hospital, Linkoping, Sweden.The results are given as mean (SEM) and thestatistical calculations were performed byStudent's t test for unpaired data.

ResultsThe 24 hour urinary recovery of PEGs in theuraemic and control groups are shown in Figure2. The reduced recovery of PEGs in the uraemicgroup is statistically significant for all molecularweight species (p<005-0001). The six and 24hour urinary recovery in the control group and inthree uraemic patients (patients 7-9) are shownin Figures 3A and B. As the Figures indicate,only minor quantities of the smaller molecules(326-414 daltons PEG) are excreted between sixand 24 hours, particularly in the control group(Fig 3A). This pattern was not so striking in thethree uraemic patients (Fig 3B). Negligibleamounts ofPEGs were, however, detected in 24-48 hour urine samples in both the control and inthe uraemic groups.Table II shows the calculated permeability

characteristics ofthe two groups. The breakpointwas significantly shifted towards a larger value -that is to the right in Figure 1 - in the uraemicgroup (p<005). Moreover, the regressioncoefficient was lower in the uraemic group(p<Q01).The relative urinary recovery of the molecules,

compared with the recovery of 326 daltons isshown in Figure 4, suggesting a relative in-creased recovery of larger PEGs in the uraemicpatients. The simulated urinary recovery of PEG400 (range 282-590 daltons) with various renalfunction is displayed in Figure SA. The resultssuggest that the over all urinary recovery ofmolecules is decreased when the renal excretioncapacity is reduced. Furthermore, the simulationalso suggests that there would be a relativelyincreased recovery of the larger molecules whenthe renal clearance is reduced (Fig 5B). Thedifference was much less pronounced, however,

*than in Figure 4. The breakpoint did not changesignificantly solely by reducing the renal func-tion. It varied only between 397-408 daltons inFigure SA, when the renal excretion was alteredfrom 150 to 20%.

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DiscussionThe urinary recovery of PEGs given orallydepends on several factors, the absorptive sur-face area, the intestinal transit time, the perme-ability of the intestinal membranes, and renalexcretion - that is, renal function.16 18 The relativerecovery of molecules differing in size is thoughtto depend mainly on the permeability of theintestine, provided the transit time is not greatlyaffected.22 25The reduced over all recovery of the PEGs in

the uraemic group (Fig 2) was probably primarilythe result of the impaired renal function. Therelative recovery of the individual molecules,however, was not identical in the two groups.The breakpoint - that is the molecular size where50% of the intestinal filtering is expressed -shifted significantly towards a larger value, 698(23) daltons in the uraemic group compared with

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540 (60) daltons in the healthy subjects (TableII). This indicates that relatively more largermolecules were recovered in the uraemic patientsthan in the control subjects. The relative urinaryrecovery of the molecules, compared with that of326 dalton PEG, is shown in Figure 4. Forcomparison the recovery of 326 dalton has beenset to 1-0 in both groups.The urinary recovery of orally given PEGs was

inversely proportional to molecular weight (Fig2). The intestinal exclusion due to the size of theprobes probably does not show a truly lineardependence, but it seems reasonable to use alinear approximation to describe the data for thelimited size range of probes used. A gut mucosawith an increased selectivity will give a compara-tively low recovery oflarge molecules and hence asteeper slope of the curve, resulting in a largernegative regression coefficient. On the contrary,a less selective gut, that allows larger molecules topass more freely, would give a flatter curve andthen a comparatively smaller value of the regres-sion coefficient. Using this approach, we inter-pret the smaller regression coefficient in theuraemic group as a sign of a reduced intestinalselectivity towards the larger PEGs (Table II).An explanation for the increased breakpoint

and the smaller regression coefficient in theuraemic patients could be a higher relativeexcretion of the larger PEGs in the uraemicpatients compared with subjects with normalrenal function.The opposite was found, however, when PEGs

were given intravenously to uraemic rats. Theoverall 24 hour recovery was decreased for allmolecular weight species as could be expectedbecause of the impaired renal function.20 But theurinary recovery of the smaller molecules (326-590 daltons) was relatively higher than therecovery of the larger ones (634-1162 daltons).The opposite was found in the control rats, inwhich the urinary recovery of the smaller mole-cules was lower than the recovery of the largerPEGs.20 Furthermore, the urinary recovery ofthe larger PEGs (590-1162 daltons) increased indirect proportion to the molecular weight in both

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Magnusson, Magnusson, Sundqvist, Denneberg

normal and uraemic rats, which was probably theresult of a larger distribution volume of thesmaller tracers.20 This suggests that a simplecomparison of ratios between larger and smallermolecules might not completely compensate forthe reduced renal function in uraemic subjects.20Moreover, intravenous administration of PEGssuggested that there was no glomerular exclusionof PEGs up to 1162 daltons.2' Neither haveprevious studies shown any reduced renal excre-tion of PEG 400, 600, or 1000.2129

Although there was no sign ofaltered intestinaltransit time in the uraemic subjects, thesechanges could affect the urinary recovery ofPEGs.'6 However, a recent study has shown thatmoderate changes in gastric emptying time andintestinal transit time have a negligible effect onthe relative recoveries of di- and monosacchar-ides.25 In another study the authors were unableto show a correlation between the absorption ofpolyethylene glycols and the gut transit time.30These results should also have a bearing on therelative recovery and the breakpoint of differ-ently sized PEGs.

Differences in the distribution volume of themolecules in the two groups could cause disparityin the urinary recovery between uraemic andcontrol subjects. Previous studies have indeedshown an increased distribution volume ofthe smaller PEGs compared with the largermolecules.2429 However, this is an unlikelyexplanation of the difference, since there was anegligible increase in recovery between six and24 hours in the control group. Moreover, thebreakpoint - that is the molecular size where50% of the intestinal filtering is expressed - wasvery similar for the six hour and 24 hour urinaryrecovery (531 (53) and 539 (49) daltons PEG,respectively). Delayed urinary excretion of thesmaller molecules in the control subjects, asindicated in Fig 3A, compared with the uraemicpatients (Fig 3B) would be more likely to increasethe breakpoint in the control subjects.The mean age was higher in the uraemic than

in the control subjects (Table I), which may haveinfluenced the permeability tests. Earlier studiesusing sugars as markers have shown, however,that increased age does not change intestinalpermeability.3'32 In several independent studiesusing PEGs as probes, the permeability charac-teristics have been shown to be remarkablysimilar in different adult control groups, irre-spective of age and sex.333 Only in 6-8 yearold children was a slightly higher recoveryobserved.2' Furthermore, in the present study,there was no correlation between age and thebreakpoint value. The only correlation seen wasbetween creatinine clearance and the breakpoint- that is, the lower the clearance the larger thebreakpoint (r=0.55).The experimental data obtained from the two

groups have also been compared with a mathe-matical multicompartment model simulatingurinary recovery of PEG 400 (282-590 daltons)in subjects with normal and reduced renal excre-tion ofPEGs. The simulation shows that the overall recovery of the molecules should decreasewhen the renal function is reduced (Fig 5A).Furthermore, the relative recovery of the largermolecules should increase compared with the

smaller ones at the same time (Fig SB). Therecovery patterns of the PEGs described bymathematical simulation agree with the recoverypattern found in the uraemic patients and in thecontrol group (Figs 2, 4, and SA and B). Thebreakpoint, however, varies only between 397-408 daltons (data not shown), when the renalexcretion capacity is reduced from 150% to 20%in the mathematical model. In addition, thechanged relative recovery of the individualmolecules was much less pronounced in themathematical model (Fig SB) than in the datafrom the experimental groups (Fig 4). Whenlooking at the results obtained by the mathemati-cal model it must be remembered that the modelis based on data obtained form perfusion studiesin man2 and from results gained after intravenousadministration ofPEGs in pigs.24The proposal that there is increased intestinal

permeability (larger average pore size35) in theuraemic patients is a logical one supported byseveral observations: (1) increased breakpoint inthe uraemic group; (2) smaller regression coeffi-cient in the uraemic group; (3) relatively higherurinary recovery of the larger molecules in theuraemic group. The results from intravenousinjection of PEGs in rats with chronic uraemiaalso suggest that the mucosal membranes havedifferent permeability, which shows up in theurinary recovery of PEGs.20These findings suggesting an increased per-

meability to larger molecules, agree with recentobservations in rats, where increased intestinalpermeability was shown in acute uraemia36137 andin those with chronic renal failure.`' Moreover,increased permeability of the blood brain barriertowards inert molecules in chronic uraemic ratshas been shown by other investigators.38

Shortening of the villi, elongation of thecrypts, and infiltration of lamina propria withinflammatory cells have previously been seen inpatients with chronic renal failure."''3 Indeed,transmigration of polymorphonuclear cells hasbeen observed to increase the permeability oflarger molecules, probably via paracellular path-ways.39 Increased intestinal permeability has alsobeen shown in mucosal inflammation, as inCrohn's disease.44' Moreover, several studieshave suggested that the bacterial flora ofthe gut isaltered in chronic uraemia and that bacteriaassociated materials could be implicated in thepathogenesis of this disorder.'94243 In fact,enterotoxin from Escherichia coli and syntheticpeptides which mimic bacterial peptides areknown to increase the intestinal permeability tolarge molecules - for example dextran 3000.`'7These observations may explain the impaired gutbarrier in uraemic subjects.An alternative explanation is the accumulation

of harmful, endogenous low molecular weightsubstances in serum in the uraemic state.948These uraemic toxins could also affect the intesti-nal integrity and allow larger molecules to passmore freely over the mucosal barrier than undernormal conditions.In conclusion, the results suggest that despite

the reduced over all recovery there is an increasedleakage of the larger PEG molecules (414-898daltons PEG in the test mixture) in chronicuraemic patients. An explanation could be open-

758

Impaired intestinal barrier function measured by differently sized polyethylene glycols in patients with chronic renalfailure 759

ing of the paracellular pathways, particularly thetight junctions - for example by bacterialpeptides or oligopeptides accumulated inplasma, or both. A possible effect is an impairedbarrier function in the intestinal wall towardspotential toxic substances in the intestinallumen. The results encourage further efforts toidentify and isolate the causative agent(s) and todelineate the precise mechanisms of the alteredmucosal barrier properties in chronic uraemia.

The excellent assistance of Anita Sjo and Gunnel Karisson isgratefully acknowledged. This research was supported by grantsfrom the Swedish Medical Research Council (Project No 6251),the Swedish Society of Medicine, King Gustaf the Vth 80-YearFoundation, the Professor Nanna Svartz Foundation, the MedicalResearch Council of the Swedish Life Insurance Companies andOstergotlands Lans Landstings Forskningsfond.

1 Tomasi TB Jr. The immune system of secretions. EnglewoodCliffs, New Jersey: Prentice Hall, 1976: 93-108.

2 Chadwick VS, Phillips SF, Hoffman AF. Measurements ofintestinal permeability using low molecular weight poly-ethylene glycols (PEG 400). II. Application to normal andabnormal permeability states in man and animals. Gastro-enterology 1977; 73: 247-51.

3 Johansen K, Stintzing G, Magnusson KE, et al. Intestinalpermeability assessed with polyethylene glycols in childrenwith diarrhea due to rotavirus and common bacterial patho-gens in a developing community. J Pediatr Gastroenterol Nutr1989; 9: 307-13.

4 Stenhammar L, Falth-Magnusson K, Jansson G, MagnussonKE, Sundqvist T. Intestinal permeability to inert sugars anddifferent-sized polyethyleneglycols in children with celiacdisease.J Pediatr Gastroenterol Nutr 1989; 9: 281-9.

5 Katz KD, Hollander D. Intestinal mucosal permeability andrheumatical diseases. Baillieres Clin Rheumatol 1989; 3: 271-84.

6 SimenhoffML, Asatoor AM, Milne MD, Zilva JF. Retentionof aliphatic amines in uraemia. Clin Sci 1963; 25: 65-77.

7 Simenhoff ML. Metabolism and toxicity of aliphatic amines.Kidney Int 1975; 7: S-314-7.

8 Mitch WE. Effects ofintestinal flora on nitrogen metabolism inpatients with chronic renal failure. AmJ Clin Nutr 1978; 31:1594-600.

9 Bergstrom J, Furst P. Uremic toxins. In: Drukker W, ParsonsFM, Maher JF, eds. Replacement ofrenalfunction by dialysis.Boston: Nijhoff Publishing, 1983: 354-90.

10 Denneberg T, Lindberg T, Berg NO, Dahlqvist A. Morphol-ogy, dipeptidases and disacchardidases of small intestinalmucosa in chronic renal failure. Acta Med Scand 1974; 195:465-70.

11 Goldstein DA, Horowitz RE, Petit S, Haldimann B, Massry S.The duodenal mucosa in patients with renal failure:Response to 1,25(OH)2D3. Kidney Int 1981; 19: 324-31.

12 Vaziri ND, Dure-Smith B, Miller R, Mirahmadi MK. Pathol-ogy ofgastrointestinal tract in chronic hemodialysis patients:an autopsy study of 78 cases. Am J Gastroenterol 1985; 80:608-11.

13 Arvanitakis C, Nakos V, Kalekou-Greka H, Tourkantonis A.Small intestinal function and structure in patients withchronic renal failure. Clin Nephrol 1988; 29: 235-43.

14 Walker WA. Intestinal transport of macromolecules. In:Johnson R, ed. Physiology of the gastrointestinal tract. NewYork: Raven Press, 1981: 1271-90.

15 Volkheimer G. Passage of particles through the wall of thegastrointestinal tract. Environ Health Perspect 1974; 9: 215-25.

16 Lifschitz C. Intestinal permeability. J7 Pediatr GastroenterolNutr 1985; 4: 520-2.

17 Lifschitz C, Shulman R. Intestinal permeability tests: are theyclinically useful?7 Pediatr Gastroenterol Nutr 1990; 10: 283-7.

18 Ma T, Hollander D, Krugliak P, Katz K. PEG 400, ahydrophilic molecular probe for measuring intestinal perme-ability. Gastroenterology 1990; 98: 39-46.

19 Magnusson M, Magnusson K-E, Sundqvist T, Denneberg T.Increased intestinal permeability to differently sized poly-ethylene glycols in uremic rats: effects of low- and high-protein diets. Nephron 1990; 56: 306-11.

20 Magnusson M, Magnusson K-E, Sundqvist T, Denneberg T.Urinary excretion of differently sized polyethylene glycolsafter intravenous administration in uremic and control rats:effects oflow and high-protein diets. Nephron 1990; 56: 312-16.

21 Falth-Magnusson K, Kjellman M, Magnusson K-E, Sundq-vist T. Intestinal permeability in healthy and allergic sub-jects before and after sodium-cromoglycate treatmentassessed with different-sized polyethylene glycols (PEG 400and PEG 1000). Clin Allergy 1984; 14: 277-86.

22 Sundqvist T, Sjodahl R, Magnusson KE, Stjernstrom I,Tagesson C. Passage of molecules through the wall of the

intestinal tract. II. Application of low-molecular weightpolyethylene glycol and a deterministic mathematical filterfunction for determining the intestinal permeability in man.Gut 1980; 21: 208-14.

23 Sundqvist T, Tagesson C, Magnusson KE. Simulation of amulti-compartment model for the intestinal permeability tolow-molecular-weight probes (polyethyleneglycol 400). JMath Biosci 1981; 56: 287-309.

24 Sundqvist T, Magnusson KE, Tagesson C. Characterization ofsize-dependent exchange of PEG molecules between theblood and the extravascular space in pig. Comput Biomed Res1988;21: 110-6.

25 Brunetto AL, Pearson ADJ, Gibson R, Bateman DN, RashidMU, Laker MF. The effect of pharmacological modificationofgastric emptying and mouth-to-caecum transit time on theabsorption of sugar probe marker molecules of intestinalpermeability in normal man. EurJ Clin Invest 1990; 20: 279-84.

26 Shaffer CB, Critchfield FH, Carpenter CP. Renal excretionand volume distribution of some polyethylene glycols in thedog. AmJ Physiol 1948; 152: 93-9.

27 Berglund F. Renal clearance of inulin, polyfructosan-S and apolyethylene glycol (PEG 1000) in the rat. Acta PhysiolScand 1965; 64: 238-44.

28 Berglund F. Renal excretion and volume distribution ofpolyethylene glycol (PEG) in the rat and the dog. ActaPhysiol Scand 1968; 19: 20A-1.

29 Tagesson C, Sjodahl R. Passage of molecules through the wallof the gastrointestinal tract. Urinary recovery of different-sized polyethylene glycols after intravenous and intestinaldeposition. ScandJ Gastroenterol 1984; 19: 315-20.

30 Philipsen EK, Batsberg W, Christensen AB. Gastrointestinalpermeability to polyethylene glycol: an evaluation of urinaryrecovery ofan oral load of polyethylene glycol as a parameterof intestinal permeability in man. EurJ Clin Invest 1988; 18:139-45.

31 Saweirs WM, Andrews DJ, Low-Beer TS. The double sugartest of intestinal permeability in the elderly. Age Ageing1985; 14: 312-5.

32 Black DA. Intestinal permeability in the elderly [Letter]. DigDis Sci 1988; 33: 382.

33 Magnusson KE, Sundqvist T, Sjodahl R, Tagesson C. Alteredintestinal permeability to low-molecular-weight poly-ethyleneglycols (PEG 400) in patients with Crohn's disease.Acta Med Scand 1983; 149: 323-7.

34 Falth-Magnusson K, KielIman NIM, Sundqvist T, Magnus-son KE. Gastrointestinal permeability in atopic and non-atopic mothers, assessed with different-sized polyethyleneglycols (PEG 400 and PEG 1000). Clin Allergy 1985; 15:565-70.

35 Sundqvist T and Magnusson KE. Modelling of intestinalpermeability in man to polyethylene glycols (PEG 400 andPEG 1000). Acta Physiol Scand 1985; 125: 289-96.

36 Kimura T, Kobayashi A, Kobayashi M, et al. Intestinalabsorption of drugs in rats with glycerol-induced acute renalfailure. Chem Pharm Bull 1988; 5: 1847-56.

37 Yamashita S, Takahashi M, Masada M, et al. Effect ofexperimental acute renal failure of a polar drug in ratjejunum: An electrophysiological analysis. Chem Pharm Bull1988; 7:2538-43.

38 Fishman RA. Permeability changes in experimental uremicencephalopathy. Arch Intern Med 1970; 126: 835-7.

39 Nash S, Stafford J, Madara JL. The selective and superoxide-independent disruption of intestinal epithelial tight junc-tions during leukocyte transmigration. Lab Invest 1988; 59:531-7.

40 Olaison G, Leandersson P, Sjodahl R, Tagesson C. Intestinalpermeability to polyethyleneglycol 600 in Crohn's disease.Peroperative determination in a defined segment of the smallintestine. Gut 1988; 29: 196-9.

41 Hollander D. Crohn's disease - a permeability disorder of thetight junction? Gut 1989; 29: 1621-4.

42 Simenhoff ML, Burke JF, Saukkonen JJ, et al. Aminemetabolism and the small bowel in uraemia. Lancet 1976; ii:818-21.

43 Simenhoff ML, Saukkonen JJ, Burke JF, et al. Bacterialpopulation of the small intestine in uremia. Nephron 1978;22: 63-8.

44 Magnusson KE, Dahlgren C, Sjolander A. Effect of N-for-mulated methionyl-phenylalanine (FMP) and methionyl-leucyl-phenylalanine (FMLP) on gut permeability. Inflam-mation 1985; 4: 365-73.

45 Magnusson KE, Kihlstrom E, Sundqvist T. Effect of choleratoxin on rat intestinal permeability assessed with fluorescentdextran 3000. FEBS Microbiol Lett 1985; 29: 15-8.

46 Magnusson KE, Kihlstrom E, Sundqvist T. Elevation of ratintestinal permeability by enterotoxigenic Escherichia coli incomparison to non-toxigenic E coli and Salmonellatyphimurium. Application of fluorescent dextran 3000 aspermeability probe. FEBS Microbiol Lett 1986; 34: 199-204.

47 v Ritter C, Sekizuka E, Grisham MB, Granger DN. Thechemotactic peptide N-Formyl Methionyl-Leucyl-Phenylalanine increases mucosal permeability in the distalileum of the rat. Gastroenterology 1988; 95: 651-6.

48 Rergstrom J, Furst P, Zimmerman L. Uremic middlemolecules exist and are biologically active. Clin Nephrol1979; 11: 229-38.