Gut 1994; 35: 1053-1057

Intestinal perfusion of dietary levels of aluminium:association with the mucosa

J J Powell, C C Ainley, R Evans, R P H Thompson

AbstractAn aluminium (93 ,uM) sulphate solutionfreshly adjusted to pH 7 0 was perfusedthrough the rat small bowel to mimic thereported physiological conditions thatfollow dietary aluminium ingestion. Onethird ofthis aluminium was taken up fromthe perfusate, but >90% of this wasthen recovered from the intestinalmucus/mucosa and most (>70%) from thedistal third of the small bowel. The freshperfusate was shown by ultrafiltration tocontain largely particulate/colloidalaluminium-hydroxide, and this probablyadhered to intestinal mucus which maybe an important barrier to the gastro-intestinal absorption ofaluminium.(Gut 1994; 35: 1053-1057)

uptake of both soluble and precipitatedaluminium by the bowel, but not its systemictransfer, has been consistently demon-strated,'3-15 showing that retention in themucosa, rather than luminal precipitation, isthe major limiting factor in absorption. Thiscould be due to either mucosal'3 or extra-mucosal factors, such as the mucus layer.7These studies'3-15 again used supraphysiologi-cal levels of aluminium.The aim of this study was to develop an in

situ rat gut perfusion technique to study thefate of dietary levels of aluminium, and to seewhether such physiological quantities ofthe metal also associate with the intestinalmucosa.

Methods

GastrointestinalLaboratory, The RayneInstitute, St Thomas'Hospital, LondonJ J PowellC C AinleyR P H Thompson

Division ofBiochemistry, UMDS,Guy's Hospital,LondonR Evans

Correspondence to:Dr J J Powell,Gastrointestinal Laboratory,The Rayne Institute, StThomas' Hospital, LondonSE1 7EH.

Accepted for publication23 November 1993

The toxicity of aluminium is now wellrecognised, and occurs at even low levels inplants,' fish,2 and human cells.3 Encephalo-pathy, bone disease, and anaemia have beenreported in patients with impaired renalexcretion of aluminium,4 particularly thosewho are exposed to the metal in dialysiswater. The efficiency of renal excretion andimpermeability of the intestine to aluminiumare sufficient to prevent acute toxicity innormal subjects, although not all absorbedaluminium is necessarily excreted, and longterm loading may occur even in the normalpopulation.5

It is therefore important to understandthe mechanisms of the intestinal absorptionof typical low dietary levels of aluminium.Previous studies have considered onlyunphysiologically high levels and the chemistryof aluminium is such that the results cannotbe extrapolated to much lower dietaryconditions.6The average UK daily intake of aluminium

is around 8 mg,7 diluted by food plus 1.5 litresfluid, all matched by a similar volume ofendogenous secretions during ingestion andtransit. Thus, the concentration of aluminiumin the intestinal lumen is about 8 mg in 4 litresor 75 pmol/l, although clearly this derivedestimate is highly variable. At such a concen-tration, aluminium is expected to precipitate atthe near neutral pH8 found in the small bowel,and Partridge et a19 showed that at muchhigher concentrations aluminium will indeedprecipitate in the intestinal lumen. This hastherefore been proposed as one limiting factorin the gastrointestinal absorption of the muchlower, physiological concentrations of dietaryaluminium.'1012 However, a large intestinal

PREPARATION OF PERFUSATEA solution of 50 mM 4-morpholinepropane-sulphonic acid (MOPS) buffered saline wasprepared at pH 7-0. Immediately before eachexperiment an aliquot of aluminium sulphate(pH 2X5, stock solution) was added to theMOPS buffer to yield an isotonic solution atpH 7*0 containing 93 ,M aluminium.

In preliminary experiments, this solutionwas thoroughly mixed by shaking, and precipi-tation was then allowed to ensue. The precipi-tate was not visible by inspection, but wasconfirmed under laser light (Malvem autosizer2C). The precipitate remained as a suspensionand did not significantly alter under the laserlight for at least 15 hours, both at roomtemperature and at 37°C.The fresh perfusate was maintained in vir-

gin polypropylene containers (Nalgene; BDHLtd) that had been previously acid washed(0.32M nitric acid/24 hours) and then soakedfor 24 hours, twice, in ultrapure water (ElgaUHP). The concentration of total aluminiumin aliquots of the fresh perfusate was con-firmed by analysis with inductively coupledplasma optical emission spectrometry(ICPOES) as below. The concentration ofaluminium remaining in solution in the freshlyprepared perfusate was assessed with pre-cleaned Centricon-10 (10 000 molecularweight cut-off, Amicon Ltd) ultrafiltrationdevices. Results were compared to similarsolutions containing 7X4 ,uM aluminium, alsoultrafiltered through precleaned devices.These were precleaned by centrifuging 2 mlsodium hydroxide (0 1M) and then 2 mlMOPS buffered saline (pH 7 0) through theultrafilters, according to the manufacturer'sinstructions.

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Powell, Ainley, Evans, Thompson

PERFUSION TECHNIQUETen male Wistar rats (300-350 g) were fastedwithout coprophagy overnight (food with-drawn for 12 hours) and then anaesthetised(five further experiments were discarded; see'Perfusion system'). Two cannulae wereinserted, one immediately distal to the liga-ment of Treitz and the other proximal to theileo-caecal junction. The gut was perfused withan isotonic aluminium-free MOPS/saline solu-tion (pH 7-0) for 10 minutes at 0-4 ml/min,and then with air at the same rate until thebowel was clear of all perfusate. The wholesmall intestine was then perfused in situ, usinga different and specially prepared single passperfusion system (see below), with aluminium(93 ,M total) in MOPS buffered saline at37°C and 0 4 ml/min for 40 minutes. Afterthis, residual luminal perfusate was removedby perfusing with air and the animal was killed.The whole small bowel was excised anddivided into three segments of equal length(proximal; middle; distal). Sections (3-4 mm)of bowel tissue were taken from the proximalend of each section of bowel for histologicalexamination and comparison with similarsections of bowel taken from non-perfusedrats. In the first six experiments the mucus andmucosa were scraped off each of the threesegments using an acid-washed perspex slideand were then weighed; for the last fourexperiments the mucus was removed bysqueezing the bowel evenly along its length,and again the collected mass was weighed.Sections of the squeezed and scraped bowelwere also taken for histological examinationand assessment of loss of the mucus/mucosa.Two animals were perfused as above, but

with aluminium-free MOPS saline buffer,and the scraped mucus and mucosa were

similarly collected, digested, and analysed foraluminium.

All sections of bowel were fixed in formalin,processed for wax sections, and stained withhaematoxylin and eosin for light microscopy.

PERFUSION SYSTEMWarmed perfusate (45°C) was maintained in aclosed polypropylene reservoir and drawnthrough polyethylene tubing (4 mm internaldiameter) with a constant pump delivery rate.This was passed through a heating coil (42°C),and then a plastic three way tap immediatelybefore delivery to the proximal bowel via a

glass cannula. The final perfusate temperaturewas 37°C and the flow rate was 0-4 ml/min.The perfusate from the distal bowel was col-lected through a glass cannula and teflon tubeinto an acid washed polypropylene container.The perfused bowel was kept moist with asaline (37°C) soaked tissue on the serosal sideand maintained at 37°C with an overheadheating lamp, thermostatically controlled by aprobe on the serosa of the perfused bowel.Two important features of this system tominimise contamination or adhesion of alu-minium were firstly, keeping the overall lengthof tubing (150 cm including the coil) to aminimum (which also reduces heat loss

from the perfusate) and secondly, the use ofpolyethylene tubing except for the glass cannu-lae and heating coil. In addition, the systemwas acid washed (1 6M HNO3 Aristar grade)for one hour by perfusion, and then for onehour with ultrapure water. The aluminiumcontaining perfusate was then passed throughthe system for two hours to allow aluminium toequilibrate with the tubing.One perfusion experiment was performed

each day, but the preparation was discarded ifluminal blockage could not be cleared or ifintestinal swelling rather than peristalsis wasnoted during perfusion; thus five preparationswere discarded before the 10 successfulexperiments were completed.

ANALYSESThe scraped mucus and mucosa were collectedinto acid washed polypropylene tubes,weighed, and digested with 1-5-3 ml concen-trated nitric acid (11 2 M: Aristar grade -BDH Ltd) for 72 hours at 40°C. This digestatewas then diluted with 3-6 ml ultrapure water.The perfusate from the bowel was collectedquantitatively into a similar container andacidified with 0-6 M nitric acid before analysisfor sodium, sulphur, and aluminium. Samplesof perfusate from the first six animals were alsocollected for analysis of aluminium just beforeentering the bowel from the three way tap atthe beginning and end of the perfusion period.

Analyses were performed in duplicate bysimultaneous ICPOES at 308-21 nm foraluminium, 588-99 nm for sodium, and180-67 for sulphur, using a Philips PV8050spectrometer. Spiking and recovery experi-ments showed a linear response and fullrecovery for all three elements.The squeezed mucus samples from the

proximal, middle, and distal bowel were frozenat -70°C, before being thawed and analysedfor protein content by sodium dodecylsulphate-polyacrylamide gel electrophoresis.Standards of myoglobin (MW 18 000), oval-bumin (MW 43 000), albumin (MW 67 000),transferrin (MW 77 000) and lactoferrin,which has a similar molecular weight to trans-ferrin but a slightly lower mobility, were alsorun on the gel.

Results

ULTRAFILTERABLE ALUMINIUMOnly 14-3 (1-3)% of aluminium (13-3 (1.2),uM, mean (SD)) from freshly prepared per-fusate solution (93 ,uM) was filterable throughthe Centricon-10 membrane ultrafilter (n=4);but at the lower concentration of 7-4 ,uM, 94-4(6&3)% aluminium was recovered through thesame filters and in the same solution at pH 7 0(n=4).

HISTOLOGYThe typical light microscopic histologicalappearance of the bowel mucosa immediatelyafter perfusion is shown in Figure 1.

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Intestinal perfusion of dietary levels of aluminium: association with the niucosa

Figure 1: Light microscopy section (original Figure 2: Light microscopy section (originalmagnification x 400) showing villi of the rat small bowel magnification x 100) showing the intestinal mucosa andfollowing successful perfusion; shedding of the tip of the lumen after removal of the luminal contents by squeezingmiddle villus is seen. the bowel. The mucosa remains intact but not all luminal

contents are removed; the residual is mainly mucus.

Compared with non-perfused control tissue,the mucosa was normal, except for someadditional sloughing of the villus tips (Fig 1).The mucosal scrape removed all mucosa, witha small amount of submucosa remaining onthe muscle layer. The squeezed mucosa wasintact (Fig 2), but occasionally some luminalmucus was still present indicating that squeez-ing did not completely remove all the mucus.

PERFUSATE CONCENTRATIONSOnce aluminium-containing perfusate hadbeen equilibrated with the perfusion appar-atus, there was no further loss of aluminiumfrom the perfusate by adhesion to the system(or sodium from the sodium chloride orsulphur from the MOPS buffer). In contrast,the percentage change in the perfusate concen-trations of aluminium, sodium, and sulphurafter perfusion through the small bowel areshown in Figure 3 (one sulphur atom is presentin one molecule of MOPS buffer).

ALUMINIUM RECOVERYFigure 4 shows the distribution of aluminiumafter perfusion. The total aluminium perfusedwas 1-48 p.mol/experiment, and of this 62-2(6- 1)% (mean (SD)) was recovered in theeffluent; 92-8 (15-4)% (mean (SD)) of theunrecovered (37-8%) perfused aluminium wasdetected in the mucus/mucosal scrape, ofwhich 11-2 (4-8)% was in the proximal, 17-5(4-8)% in the mid, and 71-3 (5.3)% in thedistal segments (p<0 00l, Student's t test;distal v others). The total aluminiumrecovered, from perfusate plus all sections of

the intestinal mucus/mucosa, was 97 5 (6-4)%.Aluminium recovery from the mucus/mucosaof the two control perfusions was 0-008 and0-010 p.mol respectively.

POLYACRYLAMIDE GELSAlthough in all 12 mucus specimens weakbands were seen for substances that ran withmobilities close to those of transferrin andlactoferrin, their intensities were the same inthe three areas of the gut. In contrast, therewere strong bands for a substance thatmigrated with the same mobility as albumin inall specimens of mucus from the proximal andmiddle gut, but not from any mucus of thedistal gut. There were no protein bands thatran with the same intensity of distribution as

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Sodium Sulphur AluminiumFigure 3: The concentrations of aluminium, sodium, andsulphur remaining in the perfusate after intestinal perfusion,as a percentage of their concentrations in the perfusatebefore intestinal perfusion (n=6); mean (SD)).

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Powell, Ainley, Evans, Thompson

0

C

Total Effluent Proximal Mid Distal

perfused small bowel small small

Recovered from

Figure 4: The total amount of aluminium (Al) perjfused(Total Al) and the amount of aluminium recovered in the

perfusate (Effluent) or the mucus/mucosa of the proximal,mid, and distal small bowel after perfusion (n =6; mean

(SD)). The individual total aluminium recoveries (fromeffluent+total mucus/mucosa) of each rat were: 90 7%,

109%, 96-1%, 99.90o, 94.99o~, and 94-3%.

aluminium along the bowel, namely pre-

dominately distally.

Discussion

Contamination is a major problem when

working with the low, physiological levels of

aluminium, but may be excluded by rigorousacid washing and rinsing with deionised water

or an appropriate buffer. We found that alu-minium adheres to the perfusion apparatus,perhaps to the glass parts, and so the perfusatewas allowed to pre-equilibrate with the system;

the results then showed no change in the

concentration of aluminium in the perfusate

entering the bowel at the beginning and end of

the experiment, and furthermore, did not differ

from the concentration in the closed reservoir.

Peristalsis is an important physiologicalresponse to distension of the bowel,t16 and whenabsent it indicates an ileus; such preparations

(five) were therefore discarded. This seemed to

be related to too high a perfusion rate or over

handling of the bowel, and so handling was

kept to a minimum and the perfusion rate to

0h4 mllmin. We detected some sloughing of thevillus tips even at this perfusion rate, and so it is

surprising that at 25 times this rate no histo-

logical damage was reported by Van der Voet

and de Woolf.17 Histological observations are

important after perfusion of the bowel; for

example aluminium could enter the tissuebetween cellst8 of the damaged areas.

Histological examination of squeezed bowel

consistently showed that most of the mucus

and no mucosa was collected; in addition, theabsence of albumin in all distal samples of

collected mucus showed that the samples were

not significantly contaminated by plasma,

interstitial, or cellular proteins. The mucus was

not collected quantitatively and so it was not

possible to assess its aluminium content.

Attempts to characterise experimentalsolutions containing aluminium have been

made.t9 20 A buffer that does not bindaluminium should be used, and so phosphatebuffers20 should be avoided; we thereforeused the non-interactive MOPS buffer.The aluminium sulphate dissociates when itdissolves yielding an acid pH, but then

precipitates as the pH is raised towardsneutral. The amount of aluminium remainingin solution will depend on a number of factors,such as the initial aluminium compound,temperature, pH, solution age, and electro-lyte composition.2" A chelator may be usedto prevent precipitation, if physiologicallyrelevant, although it has been suggested that aprecipitate may be physiological and occur inthe normal bowel.9-12 We therefore charac-terised this by ultrafiltration to separate solublefrom colloidal and particulate aluminium, andshowed that in freshly prepared physiologicalsolutions only about 14% of the aluminiumwas indeed 'in solution'.

After perfusion, the sodium concentration ofthe perfusate did not change, but sulphur,initially from the MOPS buffer, increased,probably because there is sulphur in bowelsecretions. In contrast to these controlelements, the concentration of aluminium inthe perfusate fell significantly after perfusion,and of the total aluminium perfused only62% was recovered in the collected perfusate.The retained aluminium was almost all (92.8(15A4)%) recovered either on or in themucus/mucosa, the apparent loss probablybeing partly due to errors in recovery at theselow levels. Less than 1% of the total perfusedaluminium would have been absorbed.7The overall mucus/mucosal uptake of

aluminium (38%) far exceeded the initialtotal ultrafilterable aluminium (14%) in theperfusate, showing that chiefly colloidal/particulate aluminium hydroxide was taken upby the mucus/mucosa. The intramucosaluptake of macromolecules from the lumen islow,22 and it is therefore more likely that thefreshly precipitated aluminium hydroxide,which is a fine positively charged floc,23 wasadsorbed to negatively charged species of thesurface mucosa or mucus.24 25 The distribu-tion of such perfused aluminium down thebowel (Fig 4) suggests adhesion to mucus,since distally the number of goblet cells andmucus increases,26 while the surface area of themucosa decreases. The aluminium may associ-ate with mucus glycoprotein,25 becausealthough other potential metal bindingproteins were present in the collected mucus,these proteins did not correlate in their distri-bution along the bowel to the uptake ofaluminium. Furthermore, such proteins couldonly specifically bind aluminium ions and notthe particulate aluminium that was mainlyperfused in these experiments.

In further work we shall therefore considerwhether other forms of ingested aluminium atthese dietary levels precipitate to form thehydroxide along the intestinal lumen, orwhether the interaction with endogenous com-ponents such as mucus prevents precipitation.7Nevertheless, we confirm that under these nearnormal conditions dietary levels of aluminiumstrongly associate with the intestinal mucus/mucosa.We thank Dr N Walsh, Royal Holloway and Bedford NewCollege, for access to ICPOES and the Water ResearchCouncil, The Jean Shanks Foundation, London UniversityCentral Research Fund and the Special Trustees for StThomas's Hospital for their support.

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