The transport of aluminum and water across the rat small intestine

The Transport of Aluminum and Water Across the Rat Small Intestine K. A. Renton, K. L. Manchester,* and T. A. Kilroe-Smith National

for Occupational

Johannesburg, South

ABSTRACT Aluminum transport across the epithelium of the rat small intestine has been investigated to determine factors affecting its absorption and its effect on the transport of other substances across the membrane. The intestines were attached to a perfusion apparatus and perfused with Krebs-Ringer-bicarbonate buffer containing aluminum. The transport of aluminum and buffer ions across the small intestine were measured. Phosphate transport was found to be the most satisfactory marker for viability. It is impossible to accurately measure the aluminum transport across a biological membrane unless the aluminum concentration of the solution is stable over the period of measurement. Hence, the solutions were stabilized with citrate ions which made them stable over a period of at least two hours. The velocity of transport of aluminum across the epithelium increased steadily and only became constant after about one hour. The steady state value of 0.12 pg atom of Al/hr/mg dry tissue compares well with that reported in the literature for stable aluminum solutions. Aluminum inhibited the transport of water across the membrane, but the inhibition took about two hours to reach a steady state of about 50% of the control value. This indicates that aluminum-containing medications and foods are able to interfere with the absorption of nutrients from the gut. Aluminum salts may therefore be useful to prevent rapid dehydration in the treatment of certain diseases such as cholera.

INTRODUCTION Foods and many medicines, especially antacids, contain significant amounts of aluminum, an ion which has been reported exert profound effects [l-31. sulphate is extensively in food, beer, wine

Address requests and Health, P. Box 4788, * Department Biochemistry, University Journal of Inorganic Biochetihy, 0 1993Elsevier Science Publishing

(1993)

to: Mr. Renton, 2000, South the Witwatersrand,

National

for CkcupaSouth Africa. 21

Cc., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/93/$6.00

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industries [4]. A total of two milhon kilograms of aluminum was used as food additives in America during 1982. On average, each American consumed 21.5 mg (0.8 mgatom) of aluminum per day [5]. The aluminum cation self associates in aqueous solution to a maximum extent at around neutral pH, resulting in the formation of polymeric species, for example the six-membered ring Al,(OH),,6+ which coalesces into the extremely stable planar crystalline structure of Gibbsite [6]. Polymerization and subsequent precipitation effectively removes aluminum from solution and decreases the aluminum available for absorption into the body. The intestinal absorption of aluminum by rats has been studied using everted rat gut sacs 171and by perfusing cannulated intestine segments of anesthetised rats [8-141. The rate of aluminum transport reported in the literature varies considerably depending on the conditions used in the experiments. Some workers consider aluminum transport to be para-cellular and energy-independent [9, 151, while others postulate it to be an energy-dependent transcellular mechanism [7, 101. The aim of this work has been to measure the rate of aluminum transport across the rat small intestine from a stable aluminum solution and to observe whether aluminum interferes with the transport of water across the intestine.

Aluminum and phosphate standard solutions for inductively coupled plasma optical emission spectrophotometry (ICP) were obtained from Hopkin and Williams Ltd., England. All other chemicals were of analytical grade.

AlCl, (in excess of 12 mmoles) was dissolved in 100 ml distilled water. NaOH (2.2 M) was added slowly with stirring until the pH was just alkaline to litmus. The precipitate formed was collected by centrifugation in 4 x 50 ml tubes (700 x gl for 5 min and washed by resuspending in water and centrifuging 3 times. Citric acid (12 mmoles) was dissolved in 40 ml of distilled water and stirred with the freshly precipitated AI( for 30 min. The mixture was centrifuged as before and the aluminum citrate solution decanted, leaving behind unreacted AI(O Some of the supernatant was freeze-dried and kept in a glass-stoppered jar; the rest was used immediately. Aluminum-Krebs-Ringer-Bicarbonate Freeze-dried aluminum citrate powder, or fresh solution, was added to KrebsRinger-bicarbonate buffer, prepared according to Umbreit 1161,and the pH of the mixture adjusted to 7.4 using isotonic CO, saturated bicarbonate. Glucose and Chloride Measurement Glucose was measured by the o-toluidine method of Dubowski [17]. Chloride was assayed using the reaction of thiocyanate with ferric chloride and measuring the absorbance of the red ferric thiocyanate product [181.

ALUMINUM AND WATER TRANSPORT

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Ion Measurements Aluminum and phosphate (as phosphorus) were measured using an inductively coupled plasma optical emission spectrophotometer (ICP-ARL 34000) [191. Aliquots of serosal exudate (outside) and mucosal buffer (inside) were taken at 15 min ntervals. They were immediately diluted 15 times with 0.1 N nitric acid to prevent sample deterioration and then aspirated into the ICP. Argon gas in a cross flow nebulizer carried sample droplets into a plasma sustained by 1.25 kilowatts of 27 x lo6 Hertz radio frequency energy. The Isolated Rat Intestine Preparation Rat small intestine was perfused in vitro using apparatus similar to that of Smyth and Taylor [20]. The apparatus had a reservoir 4 cm in diameter by 7 cm high and the intestine sections were connected to 3 pairs of tubes 24 cm apart. White Sprague Dawley male rats (250-250 g>, starved overnight but with access to water, were killed by decapitation. The jejunum and ileum were excised from the ileo-caecal junction (site of the ileum joining the large bowel or caecum) for 90 cm towards the stomach. The gut was rinsed twice with 50 ml of buffer, using a syringe to force out gently the residual intestinal contents. The excised gut was attached to the perfusion apparatus and rinsed again. The gas lift pump delivered carbogen at 2 ml per minute which circulated the intestinal perfusion fluid and kept the intestinal tissue oxygenated. Droplets of exudate, formed on the serosal surface, ran down the segment of the intestine and were collected in weighed polystyrene test tubes. The volume in the reservoir was kept constant by the addition of fresh buffer equal to the volume lost in the exudate. Samples collected were diluted immediately in 0.1 M nitric acid and analyzed for aluminum and phosphate ions using the ICP. The serosal volume was calculated from the weight of the exudate collected every 15 min, assuming a specific gravity of 1.0. Each experiment was stopped after 2 h. The dry mass of each intestine was measured after drying the tissue in an oven overnight at 105°C. Analysis of the phosphate difference was used to validate the viability of the intestine used in each experiment.

Viability of the Intestine

d : The concentration of glucose in the serosal fluid was slightly lower than in the mucosal buffer during a control perfusion without aluminum, There was also a 20% decrease in chloride concentration across the intestinal membrane in the direction mucosa to serosa. The concentration of phosphate in the serosal exudate which varied from 1.5 to 2.5 times the concentration of the original mucosal buffer ,reservoir (Fig. 1) is indicative of concentrative uptake and was therefore chosen to monitor viability. ;“!. Aluminum Stability :

,‘,

Aluminum introduced as the chloride salt was unstable in Krebs bicarbonate buffer and its concentration steadily declined with time (Fig. ,2). This was also perceived by the formation of a fine white precipitate which settled on the bottom of the perfusion apparatus reservoir. The concentration of .aluminum

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K A. .Renton et al.

5,

A

A

A

A

A

0’

I

0

0.5

1

I

1.5

I

I

2

Time (h) FIGURE 1. The changes in concentration of phosphate (measured as phosphorus) during a typical perfusion experiment. Samples were taken from the reservoir of the perfusion apparatus at 15 min intervals. The buffer, kept at 37°C was circulated through the intestine by, means of a gas lift pump. The pH of the buffer was maintained at 7.4 by saturating it with carbogen (95% 02, 5% COs). The mucosal (A) and serosal (0) phosphate concentrations were measured at the indicated times after the start of the

$erfusion.

introduced as the,citrate, however, was stable for two h in the buffer and did not precipitate (Fig. 3). Aluminum citrate was therefore prepared for use in this study. Aluminum and Water Transport Aluminum passed through the intestinal epithelium and appeared in the serosal fluid (Fig. 3). The average total aluminum transport from the mucosa to the serosa ,increased gradually during the first hour of the experiment (Fig. 3). The average rate of aluminum transport calculated for these experiments was 0.12 f 0.04 ( f SEM for n = 4) pgatoms per gram dry intestine tissue per hour from 0.26 mgatoms aluminum per liter of mucosal fluid. - The serosal fluid derived from aluminum preparations kept in solution reached 50% f 11% (+ SEM for n = 5) of the mucosal aluminum concentration whereas that derived from aluminum preparations which were freeze-dried reached a maximum concentration of 15.8% f 3.5% (IIZSEM for n = 2) of the mucosal aluminum concentration; this discrepancy indicated a marked difference in the molecular composition of the solutions. The effect ,of aluminum citrate on the volume of serosal exudate is shown in Figure 4. Regression analysis of the data (Fig. 4) showed that the rate of fluid exudation was significantly correlated with time for both the high aluminum

ALUMINUM AND WATER TRANSPORT

0

0.5

1

1.5

2

25

2.5

Time (h) FIGURE 2. The stability of aluminum chloride in Krebs buffer. Aluminum chloride (37 mmoles/l) was added to Krebs buffer to an aluminum concentration of 0.12 mgatoms// and the pH adjusted to 7.4 using isotonic sodium bicarbonate. Samples were taken from the reservoir of the perfusion apparatus at 30 min intervals. The buffer, kept at 37°C was circulated through rubber tubing (in place of intestine) by means of a gas lift pump. The pH of the buffer was maintained at 7.4 by saturating it with carbogen (95% 0,, 5% CO,). The bar labeled “stirred” represents the aluminum concentration in a sample taken after stirring the buffer in the reservoir at the end of the experiment and includes the precipitate formed.

(p = 0.003) and the low aluminum concentration (p = 0.001). The deceleration of serosal fluid production is more for the high aluminum concentration in the mucosal fluid than that observed with the low aluminum concentration, indicating that aluminum citrate inhibits water transport.

concentration

DI!!EUSSlON Aluminum Chemistry When trace elements are analyzed by inductively coupled plasma optical emission spectrometry the high temperature of = 6OOO“Censures that all elements are in the atomic or ionic state, which largely prevents chemical associations from interfering with the analysis [19]. In contrast to other workers in the field who used atomic absorption spectrometry which may be subject to chemical interferences [23], the ICP method was chosen for our study;

Perfusion of the rat s&l intestine in vitro has been used by several workers to study ion and water transport [24, 25, 26, 201. Gihnan observed that in Krebs-

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K A. Renton et al.

0

0.5

1

1.5

2

Time (h)

Aluminum transport across the rat small intestine. Aluminum citrate (kept as a 37 mmoles/l solution) was added to Krebs buffer to give an aluminum concentration of 0.26 mgatoms Al/Z’ and the pH adjusted to 7.4. Fifteen mm after the start of the experiment the mucosal reservoir of Krebs buffer was emptied and replaced with aluminum-containing Krebs buffer. The aluminum concentration ( f SEM) in both the mucosal fluid (0, left hand y-axis) and the serosal fluid was monitored for two hours. The aluminum transport across the epithehum was calculated from the volume of exudate and its concentration (A, right hand y-axis) in pgatoms/lS mm. The results are the average of four experiments. FIGURE 3.

bicarbonate buffer “the function of the gut remains remarkably constant for a period of 2 hours” 1261. When everted rat intestinal sacs are used for studying the intestinal transport of substances, the intestinal epithelium may be damaged during the washing and everting procedure [27]. As a precaution, the gut sacs are usually monitored for viability by testing the ability of the epithelium to transport glucose against a concentration gradient [6]. To test the viability of the perfused intestine in the present study, the glucose, chloride, and phosphate concentrations were measured in the perfusing buffer and in the fluid appearing on the serosal surface of the intestine. Only the phosphate was transported from a low concentration to a higher concentration against the osmotic gradient, suggesting that phosphate transport requires energy. Phosphate transport was therefore used in our experiment to assess viability.

Water and Aluminum Transport The mechanism of inhibition of water transport by aluminum is not known. It has been reported that aluminum can affect both the fluidity of the membrane and enzymes requiring ATP [28-301. Either or both of these mechanisms may be responsible for the observed inhibition of water transport.

ALUMINUM AND WATER TRANSPORT

27

iii01, , , , 0

0.5

1

1.6

2

Time (h)

FIGURE 4. The average volumes of serosal fluid exuded during intestine perfusion experiments. The control without aluminum produced a steady flow of serosai fluid for 2 h (0, the average of four experiments). Addition of aluminum to a cbncentration of 0.26 mgatom/’ caused a decrease in the average volume of serosal fluid collected (0, the average of four experiments). Aluminum at a concentration of 1.20 mgatom/l, however, resulted in a more precipitous decrease of serosal fluid collected (A, the average of two experiments).

Aluminum transport at pH 7.0 measured by other workers was generally low except where tris buffer or citrate were used in the perfusion medium [7-12, 14, 311. A probable explanation for the low aluminum concentration in the serosal fluid found by others is that they used an “unstable” compound of aluminum in their work. Most workers in the field used aluminum chloride which hydrolyzes and polymerizes rapidly to form insoluble AKOH), polymers. The most important factor determining the rate of aluminum transport at pH 7 is likely to be the concentration of aluminum ion-complexes. In the absence of a complexing agent, the maximum concentration of AI(OH the predominant aluminum species in aqueous solution at pH 7.4, from its solubility product, is 8 PM [21]. The formation of a precipitate of an aluminum compound in Krebs buffer demonstrated in Figure 2 illustrates this point. This fact has also been recently reported on by Froment [22]. The freeze-dried aluminum preparation produced 32% of the aluminum transport seen with aluminum prepared and kept in solution. The aluminum citrate solution made into a freeze-dried powder may have polymerized during freeze-drying, an event which could have reduced the “transportable” aluminum and resulted in less aluminum appeamg in the serosal fluid. The results of our study are similar to those of Partridge and van der Voet [lo, 141 who measured aluminum transport in the presence of citrate at pH 7.4. Our results are higher than those of workers who studied aluminum absorption

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K A. Renton et al.

from aluminum &r>orjbe done. Tne rare 0E decrease of ;ilummum measured jn Krebs buffer depicted in Figure 2 was similar to that reported as duminum “uptake” in the paper of Provan. The particularly high apparent rates of absorption of Provan [9] and Adler [29] were, we believe, due to the mistaken assumption that the loss of aluminum from the mucosal fluid is an estimate of aluminum transport. Feinroth et al. [7] has demonstrated that aluminum “uptake” was about 1000 times greater than aluminum transport. The results presented here do not show any “uptake” Erom Ibe a!mmnmm &rare jn tie mucosal buffer which remains at a nearly constant concentration for 2 h in the case of the aluminum kept in solution. Aluminum transport across the mucosal membrane slowly increased over a period of about 1 h, but this was not due to a loss of viability of the tissue since the positive phosphate gsz&& ws ma&a&d f~ tti MS p&ed r& 2 B {F?g. 1). There may be an initial reaction of the aluminum with components of the membrane which prevents the aluminum from getting across the membrane until these components have been saturated after about 1 h; then the aluminum passes through & a &..a9 r&e a F z?IZ /.q@SITWX$Tl*,51qJ &y &X&?. TX% figure agrees with that of others who measured it in the presence of stable solutions of aluminum [lo, 141. The inhibition of water transport is shown by the steady decrease of volume of serosal fluid collected in 15 min. The rate of fluid transport reached a steady value of about SD% of fhe control value witbout &n&mm. That the cause js a combinatiorrof ahuninum with a membrane constituent is indicated by the fact that the rate of inhibition is related to the concentration of ahtminum (Fig. 4). This observation is of interest in regard to the use of aluminum-containing foods and medications. Although the aluminum transport across the gut slowly increases with time, the reduced transport of water could result in a reduced absorption of nutrients. On the other hand it may indicate a possible use for aluminum for the treatment of diseases where the transport of water across the gut leads to rapid dehydration, as in the case of cholera. Caution needs to be exercised in this regard since it has been shown that absorbed aluminum can accumulate in the brains of rabbits exposed to aluminum [32].

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ALUMINUM AND WATER TRANSPORT

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