Final Report: Intestinal Aluminum Absorption and Bioavailability from Representative Aluminum SpeciesEPA Grant Number: R829783
Title: Intestinal Aluminum Absorption and Bioavailability from Representative Aluminum Species
Investigators: Yokel, Robert A. , McNamara, Patrick J.
Institution: University of Kentucky
EPA Project Officer: Page, Angela
Project Period: July 1, 2002 through June 30, 2005 (Extended to June 30, 2006)
Project Amount: $515,720
RFA: Health Effects of Chemical Contaminants in Drinking Water (2001) RFA Text | Recipients Lists
Research Category: Health Effects , Drinking Water , Water
The application submitted to the EPA proposed two major studies. The first involved the use of Caco-2 cells as an in vitro model of the gastrointestinal tract. It was proposed to compare the flux of Al as the ion, citrate, maltolate, hydroxide, and fluoride across a monolayer of Caco-2 cells as well as their uptake by these cells. The second study determined the time-course of oral Al absorption and the time-course of absorption of its associated ligand, when administered as the citrate and maltolate. Additionally, Al bioavailability was determined from Al administered as the ion, citrate, maltolate and fluoride.
Study 1. Assessment of the impact of chemical species of aluminum on its paracellular flux across, and uptake into, Caco-2 cells.
To utilize Caco-2 cells in culture to determine the comparative rates and properties of apical to basolateral Al flux across this model of the intestinal tract when Al is introduced as the ion, citrate, fluoride, hydroxide, or maltolate.
MATERIALS AND METHODS:
Synthesis of Al citrate and Al maltolate for flux and uptake studies.
Al citrate was prepared from aqueous stock solutions of Al (1M), prepared from aluminum chloride hexahydrate, and citrate (1M), prepared from citric acid trisodium salt dehydrate in water. Al citrate was prepared by combining Al and citrate stock solutions in a ~1:1.1 Al to citrate molar ratio, and incubated 2 hours at room temperature. This was used in the flux experiments. Al citrate was believed to be successfully synthesized because no Al hydroxide precipitate was observed at neutral pH and previously conducted speciation calculations to predict the products of this synthesis suggest the primary product is Al citrates (Yokel et al., 2002). Al citrate was also purchased from City Chemical LLC and used in uptake experiments.
Al maltolate was synthesized following procedures developed in the lab of Dr. Chris Orvig (Finnegan et al., 1986). Briefly, a concentrated aqueous solution of maltol and aluminum nitrate at a molar ratio 3:1 (pH = 8.3) was heated for a few minutes. Al maltolate was obtained by evaporating the water at 70°C overnight. Verification of the product was obtained using proton NMR (Varian Model 958220 300 MHz), which showed M/z peaks corresponding to Al maltolate and no peaks corresponding to maltol, whereas an ~ 10-year-old product provided by Dr. Chris Orvig showed ~ 2% maltol. The results obtained with mass spectroscopy, using a Bruker Daltonics Autoflex time-of-flight mass spectrometer in reflector mode with alpha-cyano-4-hydroxycinnamic acid as the MALDI matrix, were nearly identical for our Al maltolate product and the Al maltolate provided by Dr. Orvig, except that the Orvig product had a peak at 126 (corresponding to maltol) that was 5-6 times larger than our product. This Al maltolate was used in the flux and some uptake experiments. Al maltolate was also purchased from Gelest Zac and used in uptake experiments.
Source of cells.
Caco-2 cells were used in these studies because they form a highly polarized monolayer and exhibit many of the features of the cells lining the small intestine. Caco-2 cells are widely used in the drug industry to study drug absorption and have been used to study the transport of zinc, iron, and calcium (Jovani et al., 2001). Caco-2 cells at passage 17 were purchased from American Type Culture Collection, Cat # HTB-37.
Growth conditions of Caco-2 cells.
Sub-cultures were grown in T-75 culture flasks and passaged with a trypsin-EDTA solution (Gibco) at 80% confluency. They were seeded, at passage 20-37, on Costar Snapwell polycarbonate 12 mm diameter, 0.4 μm pore size filters (1.13 cm2growth area) with a seeding density of 105/filter. They were grown in a Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids and 100 U/mL penicillin and 100 ug/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every other day and 24 hours before a flux experiment. These conditions result in growth of polarized cells as evidenced by different apical-to-basolateral (A-to-B) than B-to-A flux rates (results, below) and as reported (Delie and Rubas 1997).
Flux studies of aluminum across a Caco-2 monolayer.
Flux studies were conducted with cells 20-30 days post-plating. Flux studies were conducted using a Hanks' Balanced Salt Solution, modified by removing phosphate, containing: 25 mM HEPES, 138 mM NaCl, 4 mM KCl, 0.5 mM MgCl2·6H2O, 0.27 mM MgSO4·7H2O, 4.2 mM NaHCO3, 1.25 mM CaCl2, and 5.5 mM D-glucose at pH=7.4.
In initial flux experiments, 95% air/5% carbon dioxide was bubbled through the media of the diffusion chambers. In 2 hours this decreased the pH of the media from 7.4 to ~ 7.2. Subsequent flux and the uptake experiments are conducted in the absence of bubbled gas. The pH did not change under this condition.
Flux in the apical (A) to basolateral (B) and/or B-to-A direction was studied by removing 200 μL aliquots from both the donor and receiver chambers at 6 time points (15, 30, 45, 60, 90, 120 min). Flux was terminated by rinsing the cell monolayer with ice-cold flux medium (not containing test substance) 4 times. Cells were lysed with 0.1% Triton X 100.
a) Integrity of the Caco-2 monolayer
The tightness of the Caco-2 monolayer was measuring as trans-epithelial electrical resistance (TEER) across the Caco-2 cell layer using a Millicell-ERS voltohmmeter (Millipore Corp.; RMA3121-Millicell-ERS). The TEER at the beginning of these studies was ~ 350 ohms/cm2.
b) Aluminum quantification
Al concentration in media and cell lysates was measured by atomic absorption spectrometry (AAS) using a Perkin-Elmer 4100 ZL spectrophotometer.
c) Cell protein quantification
Cell protein was measured by the bicinchoninic acid method (Pierce, Rockford, IL).
d) Assessment of cytotoxicity - Lactate dehydrogenase (LDH) release Lactate dehydrogenase (LDH) release from the Caco-2 cells was measured, using a Sigma kit, as an indicator of cytotoxicity and lysis.
e) Trans-cellular diffusion rate through Caco-2 cells.
To determine the rate of trans-cellular diffusion through Caco-2 cells, DL-[4-3H-propranolol (as the hydrochloride, Sigma) was added to the medium on the apical side of the cells. [3H]-propranolol was quantified by liquid scintillation counting.
f) Paracellular flux rate between Caco-2 cells.
To determine the rate of flux through the paracellular pathway (through the tight junctions between Caco-2 cells), 100 μg/mL lucifer yellow (LY) (dipotassium salt, Sigma) was added to the medium on the donor side of the cells. It was quantified using a Shimadzu RF-5301PC Spectrofluorophotometer at excitation 485 nm and emission 535nm. Normal LY permeability has been reported by others to be 1-7 nm/sec (1-7 x 10-7 cm/sec) (Irvine et al., 1999).
g) Expression of a marker carrier protein in the Caco-2 cells at the time of their use for flux and uptake studies.
To assess the expression of a marker carrier, P-glycoprotein (P-gp), by the Caco-2 cells, their ability to take up and transport a P-gp substrate, rhodamine 123, introduced on the basolateral side of the Caco-2 cells, as previously conducted (Takano et al., 1998), was determined 20 to 30 days after plating, in the absence and presence of the P-gp inhibitor verapamil. Rhodamine 123 was introduced into the media facing the B side of the cells.
h) Assessment of necessity for calcium in the flux medium.
To address our concern that the citrate introduced as Al citrate might complex calcium, leaving free Al, some studies were conducted in the absence of calcium in the uptake media.
Uptake studies of aluminum into Caco-2 cells
Caco-2 cells were plated into six well plates containing 35 mm dishes. The above growth medium was used. Uptake studies were conducted with cells 5-10 days post-plating. Prior to the uptake study the growth medium was washed out with uptake medium containing no uptake substrates. The uptake medium was the same as the above flux medium. At the end of the uptake experiment the uptake medium was washed out with 4 rapid (few second) rinses of ice-cold uptake medium containing no substrates.
a) Time course of Al uptake into Caco-2 cells in the vertical diffusion chamber.
To characterize the time course of Al uptake in the vertical diffusion chamber, A-to-B flux studies were conducted for 15, 30, 45, 60, 90, and 120 min in the absence and presence of Al citrate (0.5, 2, 8, and 50 mM), Al maltolate (2 and 8 mM), Al hydroxide (8 mM) and the Al ion (8 mM). The Al concentration in the absence of added Al was considered to be contamination. This value was subtracted from the values obtained in the presence of added Al.
b) The time course of Al uptake into Caco-2 cells in cell culture dishes.
To determine the time-course of Al uptake in Caco-2 cells in cell culture dishes, studies were conducted with Al citrate and Al maltolate, followed by washes of the cells that contained 0 or 2 mM EDTA. EDTA was included to remove Al associated with labile storage sites such as that adsorbed to the cell surface and possibly intracellular sites.
c) Temperature dependence of Al uptake into Caco-2 cells.
To gain insight into the mechanism of Al uptake into Caco-2 cells, the temperature dependence of Al ion, citrate, fluoride and maltolate was determined after 30 minutes. Five temperatures, between 4 and 37°C were studied. The uptake rate constant was calculated from the Arrhenius equation (Lnk=lnA-Ea/R*T), where Ea is the temperature-dependent energy of activation, k is the uptake rate constant, R is the gas constant, and T is the absolute temperature (in Kelvin). A graph of Lnk versus the reciprocal of the absolute temperature, 1/T, yields a straight line with a slope of –Ea/R and an intercept lnA, enabling determination of Ea which is used to interpret the uptake process.
d) A concurrent flux and uptake study of Al ion, citrate, maltolate, and fluoride using 26Al with its quantification by accelerator mass spectrometry.
The objective was to determine if the above observations, obtained with exposure to 8 mM Al, generalize to much lower Al concentrations. Caco-2 cells were grown on Snapwell membranes and mounted in the vertical diffusion chamber, as above. 26Al (~0.12 nCi, ~ 5.75 ng; total Al ~ 200 ng), was added to 5 mL of medium in the chamber on the apical (A) cell surface, yielding ~ 55 mcg Al/l (2 μM), similar to the typical drinking water Al concentration. Al was added as the Al ion, citrate, maltolate or fluoride to one of the vertical diffusion chambers and compared to no added Al. The experiment was conducted a total of 3 times. After 120 minutes, a sample of the medium on the basolateral (B) side of the cells as well as the cells were taken and processed to convert the Al to Al oxide. This was sent to the Purdue Rare Isotope Measurement Laboratory for 26Al analysis by accelerator mass spectrometry to determine Al uptake into and flux across the Caco-2 cells.
e) Lumogallion staining and confocal microscopic imaging of Al localization.
Results of the above studies suggested concentrative uptake of Al ion and Al fluoride, yet the temperature dependence study suggested Al uptake was not carrier mediated. To ascertain if the Al associated with Caco-2 cells was simply adsorbed onto the outer cell surface or had distributed intracellularly, and its intracellular localization, Al localization was conducted using lumogallion, which quite specifically forms a fluorescent complex with Al (Silva et al., 2000; Uchiumi et al., 1998). Caco-2 cells were seeded at a density of 1x105 cells/cm2onto 35 mm diameter MatTeck glass bottom culture dishes and cultured for 5 to 7 days. The Al uptake procedure was used. After 30 min uptake at 37°C, cells were fixed in 1.5 mL 4% paraformaldehyde solution for 15 min at room temperature and then stained in 1 mL pH 4 acetate buffer containing 2.5 x 10-5M lumogallion in darkness for 1 h at 50° C. Caco-2 cells not exposed to Al but stained with lumogallion served as a negative control. The cells were rinsed twice with acetate buffer to remove excess stain. In the second study using this procedure the cells were incubated with the nuclear stain DAPI (50 μg/ml) for 10 min at room temperature. Caco-2 cells not exposed to Al but stained with lumogallion and DAPI were included. A Leica TCS SP inverted confocal microscope was used to visualize the Al-lumogallion complex and DAPI. The excitation wavelength was 488 nm and 364 nm to visualize the Al-lumogallion complex and DAPI, respectively. Emitted fluorescence was collected at wavelengths from 530 to 580 nm for the Al-lumogallion complex and 461 nm for DAPI. The voltage applied to the photomultiplier tube (PMT) was varied to generate images showing the Al distribution but not “washed out” by overexposure. In the first study the voltages were 1075, 1025, 755, and 451 for control cells and those exposed to Al citrate, ion and fluoride, respectively, reflecting a negative correlation between the Al concentration associated with the Caco-2 cells (see results) and the voltage used to image Al localization. In the second study the photomultiplier tube (PMT) voltage was selected to produce images of ~ equal fluorescence intensity to generate clear images showing the Al distribution. Differential interference contrast (DIC) images were concurrently collected. In the second study Al was determined in similarly treated cells by atomic absorption spectrometry.
Data Analysis, Calculations and Statistics:
Apparent permeability coeffients (Papp) were calculated using the equation: Papp=V*dC/A*Co*dt=cm·sec-1, where V = the volume of the solution in the recipient compartment, A = the membrane surface area, Co = the initial concentration in the donor compartment, and dC/dt = the change in drug concentration in the recipient solution over time (Irvine et al., 1999; Walgren et al., 1998). The results of flux and uptake over time experiments were fit to linear or non-linear functions using Graph Pad Prism. Differences in Papp were evaluated using student’s t-test. A P value < 0.05 was considered significant.
Flux studies of aluminum across a Caco-2 monolayer.
a) Assessment of cytotoxicity - Lactate dehydrogenase (LDH) release.
LDH was not detected in the media in the absence of added Al.
b) Expression of a marker carrier protein in the Caco-2 cells at the time of their use for flux and uptake studies.
The results (Figure 1) show greater uptake and reduced flux through the Caco-2 cells in the presence of the P-gp inhibitor. These results are consistent with reduced P-gp-mediated efflux in the presence of the P-gp inhibitor, showing expression of P-gp by these cells. Consultation with Dr. Martin Dowty revealed that he found maximal P-gp expression 21-25 days after plating Caco-2 cells, consistent with the time course of use of Caco-2 cells in the present studies.
Figure 1: Rhodamine 123 uptake into Caco-2 cells in the absence and presence of verapamil (left panel) and rhodamine 123 flux through Caco-2 cells, from the B-to-A direction, in the absence and presence of verapamil (right panel). Results are mean ± S.D. from single experiments conducted with three replicate observations.
c) Trans-cellular and paracellular diffusion rates through and between Caco-2 cells.
The fluxes of LY and propranolol were concurrently determined. They were found to not correlate (Figure 2, left panel) and would not be expected to correlate due to their different routes of flux across Caco-2 cells. Similarly, concurrent determination of propranolol and Al citrate showed that the fluxes did not correlate (Figure 2, center panel), suggesting Al citrate did not flux across Caco-2 cells by the same pathway as propranolol. In contrast, concurrent determination of Al citrate and LY flux showed a high correlation (r2 = 0.96) (Figure 2, right panel), suggesting a similar pathway of flux across Caco-2 cells. These results suggest Al citrate fluxes across Caco-2 cells through the paracellular pathway. Similar high correlations were seen when the flux of the Al ion, Al maltolate and Al hydroxide were compared to LY (Figure 3). The lower permeability of Al hydroxide than the other 3 Al species, when compared to LY, may be due to limited solubility of Al hydroxide or the presence of charged Al hydroxide species, resulting in a lower concentration of soluble uncharged Al species available to diffuse through the paracellular pathway.
Figure 2. Apparent A-to-B permeability of propranolol compared to LY at 2 hours (left panel), propranolol compared to Al citrate (center panel) and Al citrate compared to LY (right panel). Each point represents the results of an experiment. The lines represent linear best fit, and 95% confidence intervals in the left and center panels.
Figure 3. Apparent A-to-B permeability of Al ion, Al citrate, Al maltolate (Al(ma)3 and Al hydroxide (Al(OH)3 compared to LY at 2 hours. Each point represents the results of an experiment. The lines represent linear best fit of the 4 Al species.
d) Time course of Al flux across Caco-2 cells.
Al contamination in the flux medium averaged 7 ng/mL.
The time course of Al flux is shown in Figure 4 after introduction of the 4 Al species. Flux was nearly linear over 120 minutes and was greater for Al maltolate than Al citrate, which were greater than Al ion and Al hydroxide. There is no evidence of saturation. To the contrary, there is some indication of increased flux over time.
Figure 4. Time course of A-to-B Al flux. Al was introduced as 8 mM Al citrate and Al maltolate (Al(ma)3 at pH 7.4, Al ion at pH 4 or Al hydroxide (Al(OH)3 at pH 6.2. Results are mean ± S.D. of 4 experiments each with 2 replicates, 4 experiments each with 2 replicates, 1 experiment with 4 replicates and 1 experiment 5 replicates, respectively. The line shows a linear best fit of the results.
e) A-to-B and B-to-A Al flux across Caco-2 cells.
The results (Figure 5) show greater B-to-A than A-to-B Al citrate flux. This observation would not be expected if Al citrate is diffusing at equal rates in each direction across the Caco-2 cells.
Figure 5. Time course of 8 mM Al citrate A-to-B and B-to-A flux. Results are mean ± S.D. of 4 A-to-B experiments each with 2 replicates and 2 B-to-A experiments each with 2 replicates.
f) The influence of reduced calcium in the flux medium on Al permeability.
In the absence of calcium, TEER greatly decreased compared to its presence (e.g., 74 vs. 298 after 4 hours in the presence of 0.5 mM Al citrate) and the percentage of LY that moved to the recipient side greatly increased (3.02% vs. 0.19%). Therefore, calcium was included in the media in all subsequent studies except when decreased calcium was desired to disrupt the integrity of the tight junctions between the Caco-2 cells. Some studies were conducted with reduced calcium in the flux medium. Figure 6 shows all the data, including results obtained when the apical medium had reduced or no calcium. The very high correlation between Al and Lucifer yellow flux suggests Al, like Lucifer yellow, crosses Caco-2 cells through the paracellular pathway.
Figure 6. Apparent A to B permeability of the 5 Al species compared to Lucifer yellow at 2 hours in the presence of 1.25 mM calcium, unless noted otherwise as a fraction of 1.25 mM calcium. Each point represents the results of an experiment. The correlation coefficient is R2 = 0.9902 for all the data using Prism.
g) The influence of Al citrate on Caco-2 cell tight junction integrity.
The effect of various Al citrate concentrations, introduced on either the A or B side, on LY permeation was studied. Increasing concentrations of Al citrate increased the apparent permeability of LY (Figure 7). Increased paracellular permeability was greater when Al was introduced on the B side. These results suggest that the greater B-to-A Al permeability seen in Figure 5 might be due to Al-induced disruption of the paracellular pathway, permitting greater Al permeation through that pathway.
Figure 7. Apparent permeability of lucifer yellow after 2 hours in the presence of Al citrate at various concentrations introduced into the medium on the apical or basal side. Each symbol represents the results of an experiment.
Uptake studies of aluminum into Caco-2 cells.
a) The time course of Al uptake into Caco-2 cells in cell culture dishes.
The uptake rate of Al fluoride was greater than the other Al species (Figure 8). Al fluoride, 8 mM, caused cell toxicity after 90 min, as shown by increased LDH release. The uptake rates of Al citrate and maltolate were slower. These ligands may inhibit Al uptake, as seen in plant cells (Ma and Furukawa, 2003).
Figure 8. Time course of uptake of the Al species into Caco-2 cells. Results are mean of three replicates in one representative experiment, of the 4 to 5 experiments conducted, for each Al species.
The time course of Al maltolate uptake was linear, irrespective of the absence or presence of EDTA in the washout medium (Figure 9, left panel). However, inclusion of EDTA resulted in less Al associated with the Caco-2 cells after Al citrate exposure (Figure 9, right panel), suggesting Al uptake into at least two compartments from Al citrate, including one that is labile.
Figure 9. Time course of 8 mM Al citrate (left panel) and Al maltolate (right panel) uptake in Caco-2 cells in the absence and presence of an EDTA wash. Results are mean ± S.D. of 2 (no EDTA) or 3 (+ EDTA) experiments each with 3 replicates for Al citrate and 1 (no EDTA) or 4 (+ EDTA) experiments each with 3 replicates for Al maltolate.
Uptake experiments were conducted with 8 mM Al citrate and Al maltolate over longer durations. After 4 hours exposure to Al citrate and Al maltolate, the cell/media Al ratio was 0.7, based on three experiments with triplicate observations, and 1.9 (three experiments with triplicate observations), respectively. These results suggest concentrative uptake of Al maltolate. An experiment with 3 replicates showed non-linear uptake after 7 hour exposure to Al citrate but linear uptake after Al maltolate exposure. The cell/medium ratios were 1.0 for Al citrate and 4.0 for Al maltolate. Exposure of Caco-2 cells to Al citrate for 12 and 18 hours resulted much more cell detachment from the dish than seen with Al maltolate exposure, suggesting greater toxicity.
b) Temperature dependence of Al uptake into Caco-2 cells.
Creating Arrhenius plots of Al uptake into Caco-2 cells versus temperature (Figure 10), yielded an activation energy (Ea) for each of the Al species of 13.3-21.6 KJ/mol (3.2-5.2 Kcal/mol). These values were not statistically significantly different by t-test.
The Årrhenius plot can be used to interpret the mechanism of Al uptake. The low activation energies seen in the present study are generally below the range associated with active, energy-dependent transport (Ingermann and Bissonnette 1983; Makhey et al., 1998), suggesting Al enters Caco-2 cells through a channel rather than by carrier-mediated transport. Ea is generally 6 kcal/mol for water movement through an aqueous pore and 8–10 kcal/mol for water movement through a lipid bilayer (Finkelstein, 1987). The low activation energy indicates the uptake mechanism was close to water diffusion and was probably channel mediated.
Figure 10. Arrhenius plots for the 4 Al species tested in the temperature dependence study. Results are mean ± S.D. of 3 experiments each with 3 replicates, conducted at 8 mM Al. The results were best fit to a linear function
c) A concurrent flux and uptake study of Al ion, citrate, maltolate, and fluoride using 26Al with its quantification by accelerator mass spectrometry.
With exposure to 2 μM Al (containing 26Al) as the ion, hydroxide, citrate and fluoride, the apparent permeability (Papp) of Al was 1.5 to 16 × 10-8 cm/sec while the Papp of LY was 2.8 to 13 × 10-7 cm/sec. Al and lucifer yellow (LY) Papp were consistent with results obtained with 8 mM Al. There were no significant differences in the decrease of transendothelial cell electrical resistance or increased Papp of LY among the Al species and control. Al uptake and flux were not significantly different among the Al species (Figure 11). Approximately 0.015% of the Al in the uptake medium fluxed across the cell monolayer while ~ 0.75% was associated with the Caco-2 cells (Figure 11).
Figure 11. Flux across and uptake of Al (using 26Al as a tracer) by Caco-2 cells exposed to 2 μM Al. Flux and uptake of Al into Caco-2 cells are expressed as the % of Al introduced on the donor (apical) cell side. Left Y-axis scale is for flux results, right Y-axis scale is for uptake results. Note the difference in the Y-axis scales. Values shown are mean ± SD of three experiments, each conducted with a single replicate.
d) Lumogallion staining and confocal microscopic imaging of Al localization.
Confocal images of the lumogallion-Al complex (Figure 12) show a similar intracellular Al distribution after exposure to the three Al species. Al is not limited to the outside of the plasma membrane. The lumogallion-Al fluorescence signal is more intense in some intracellular regions. Overlays of the DIC and fluorescent images indicate a high Al concentration associated with cell nuclei.
To further test this conclusion that Al was concentrated in the cell nucleus, a second study was conducted using confocal microscopy to visualize the intracellular localization of Al. This study included a nuclear marker, DAPI, and Al hydroxide and Al maltolate exposure. The results were consistent with the conclusion of our previous study suggesting considerable accumulation of Al in the nucleus of Caco-2 cells exposed to soluble Al forms. The intracellular Al concentration was calculated based on cell volume. It was found to be 37, 392, 741, 8024, 9629, and 12390 μM for cells not exposed to added Al and cells exposed to Al as the citrate, maltolate, ion, hydroxide and fluoride, respectively. The intensity of Al-lumogallion fluorescence was proportional to the intracellular Al concentration. A significant negative correlation existed between the intracellular Al concentration and the PMT voltage used to generate the confocal images of comparable intensity. Images of Al-lumogallion (Figure 13) showed a similar intracellular Al distribution after exposure to the 5 Al species and control. As in the first of these two confocal studies, Al was not limited to the plasma membrane outer surface and overlays of the fluorescent images of lumogallion stain for Al and DAPI, with images using DIC to reveal cellular detail, indicate the Al was localized in the nucleus of Caco-2 cells.
Figure 13. Confocal microscopic images of Al localization in Caco-2 cells. Left panels: the fluorescent images of DAPI, a nuclear stain. Middle panels: the confocal laser images of lumogallion-stained cells (the voltage of the PMT used was 785, 642, 608, 727, 672, and 602 for control, Al as the ion, hydroxide, citrate, maltolate, and fluoride, respectively). Right panels: overlays of DAPI, Al-lumogallion and DIC images. Scale bar in right Al citrate panel = 20 μm.
SUMMARY OF STUDY 1:
The results suggest that Al, when introduced at 8 mM 27Al as the ion, citrate, maltolate, hydroxide or fluoride, fluxes across Caco-2 cells via diffusion through the paracellular pathway. Results of uptake studies show the association of Al with Caco-2 cells was considerably greater when introduced as the fluoride or ion than the citrate, maltolate or hydroxide. Uptake was concentrative for the Al ion and Al fluoride. Comparison of the uptake of Al citrate and Al maltolate to their A-to-B flux after 2 hours suggests uptake is ~ 25% and 40% of flux, based on comparable cell surface areas. The temperature dependence of Al uptake suggested diffusion-mediated uptake.
A limited flux and uptake study utilizing 26Al and 2 μM total Al showed no significant difference in the uptake into or flux across the cells when the Al was introduced as Al fluoride, ion, citrate and maltolate. However, ~ 50-fold more 26Al was taken up into than fluxed across the Caco-2 cells.
Al uptake was found to result in distribution into the cells, particularly into the nucleus.
Study 2: Oral bioavailability of Al as the ion, citrate, maltolate and fluoride in the rat, using the tracer 26Al.
Determine the absolute oral bioavailability of Al when introduced as the Al3+ion alone, or as a complex with citrate, maltolate, and fluoride at an Al concentration relevant to drinking water.
Test the null hypothesis that citrate, maltolate, and fluoride do not significantly influence oral Al bioavailability, maximum concentration (Cmax) or the time to reach maximum concentration (Tmax).
Test the null hypothesis that Al citrate and Al maltolate do not disassociate in the gastrointestinal (GI) tract, under the Al to ligand ratios studied, resulting in the intact absorption of these Al complexes.
MATERIALS AND METHODS:
26Al (15 Ci/mmol, 26Al:27Al ratio = 1:34) in 0.1 N HCl was obtained from the Purdue Rare Isotope Measurement Lab (PRIME Lab). 14C-citric acid (109 mCi/mmol) was purchased from Amersham Biosciences and 14C-maltol (50.9 mCi/mmol) was custom synthesized for this project by PerkinElmer.
Subjects. The subjects were 23 male Fisher 344 rats, weighing 270 ± 18 gm (mean ± SD). Al bioavailability of Al was using 26Al as a tracer and accelerator mass spectrometric analysis.
All rats were implanted with two femoral venous cannulae 1 day prior to oral dosing. This enabled i.v. infusion through one cannula and blood withdrawal from another, to avoid contamination of withdrawn blood by the administered Al. The oral absorption of Al was determined in the un-anesthetized rat. Blood was withdrawn 1 h prior to, and 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 8, 24 h after, oral dosing. The blood withdrawn, 0.4 mL in the first 9 samples, then 0.6 and 2.2 mL in the 8 and 24 h samples, was replaced by an equal
volume of injected saline. The rats had free drinking water access throughout the study except for the period from 14 h before to 4 h after oral dosing. Serum was obtained for quantification of 26Al, 27Al and 14C. Blood urea nitrogen (BUN) and creatinine were determined in the 24 h sample to monitor renal function.
The subjects were randomly assigned to receive oral 26Al by gastric administration in the absence of ligands or presence of citrate, maltol or fluoride. There were 5 rats per treatment group. The total Al:ligand ratio was 1:1 for citrate, 1:3 for maltolate and 1:4 for fluoride. Each rat received a 1 ml dose of solution containing ~50 ng (1 nCi) 26Al and 1700 ng 27Al, e.g., the Al concentration in the administered solution was ~ 65 μM. When the ligand was citrate or maltolate, it was administered with [14C]-citrate or [14C]-maltolate. The pH of the administered solution was adjusted to ~5 for Al in the absence of ligands, ~7.4 for Al in the presence of citrate or maltolate, and ~4 for Al in the presence of fluoride. Two control rats similarly received intragastric administration of water that did not contain 26Al. The absence of food in the stomach was produced by limiting food access to a 10% protein diet that was designed to minimize food retention in the stomach (Harlan Teklad 95215). This diet was available from 08:00 to 18:00 h daily for 5 days prior to the oral dosing. Food was removed 14 h before dosing and a fecal collection cup, modified from (Wang and Peters, 1963), installed to prevent fecal recycling. To maintain a serum 27Al concentration of ~700 ng/mL, all rats received iv infusion of 100 μg 27Al/kg/h as AlK(SO4)2 from 14 h prior to 24 h after oral dosing.
To assess if there was significant loss of Al due to adsorption to either the syringe or gastric feeding needle used to deliver the oral Al solution, the oral delivery procedure was simulated by delivery of identical Al solutions into a tube. The Al concentration in the solution delivered by this procedure was compared to the solution loaded into the syringe from which it was delivered through the feeding needle. For the Al ion, Al citrate, Al maltolate and Al fluoride, the delivered solution contained 104, 92, 97 and 107% of the solution for delivery, showing no significant loss due to adsorption to the syringe or feeding needle.
Aluminum speciation in the administered solutions from pH 2 to 8 was calculated using the computer modeling program SPECIES (Academic Software, Trimble, Otley, UK). Values for the aluminum hydrolysis constants were taken from Baes and Mesmer (1976), while the solubility constant for freshly prepared Al(OH)3 was taken from Öhman and Wågberg (1997). Aluminum citrate binding constants were taken from Harris et al. (2003). The Al maltolate binding constants were reported by Hedlund and Öhman (1988), and the Al fluoride constants were taken from Martin (1996). The presence of insoluble Al species, which would presumably be Al hydroxide, was determined by analysis of Al in dosing solutions prepared as described above (without 26Al addition) and including the condition where the Al ion is at pH 7 in the absence of ligand. Aliquots of the solutions were passed through a 0.22 μm filter. The unfiltered and filtered solutions, and concentrated Al solution from which they were prepared, were analyzed by ETAAS to determine their Al concentration.
Total aluminum quantification.
Before analysis, serum samples were diluted ten-fold with 0.2% HNO3 containing 2.5 mM Mg as a matrix modifier, and compared to Al aqueous standards in the same matrix (10% rat serum plus 0.2% HNO3 and 2.5 mM Mg). All serum samples were repeatedly analyzed until their Al concentration relative standard deviation was < 10%.
Analysis of 26Al by accelerator mass spectrometry.
Quality control serum samples containing 26Al were prepared by oral administration of 1 nCi 26Al to 2 rats. Blood was collected at 4 hours. Quality control serum samples containing 14C were prepared by oral administration of 30 nmoles of [14C]-citrate with 27Al. Blood was collect at 2 hours. For 26Al quantification, 4 mg 27Al (ICP/DCP standard, Aldrich) was added to an aliquot of each serum sample in a 7 ml Teflon screw cap container (Tuf-Tainer®). This represented > 1 × 1010 as much added 27Al as the 26Al in the sample, and >1 × 104 as much added 27Al as the 27Al in the sample. This enabled determination of the 26Al/27Al ratio by AMS and quantification of serum 26Al by its comparison to the known 27Al (from the 4 mg 27Al added). The sample was digested in a 70:30 HNO3:H2O2 v/v mixture. The liquid was evaporated, as described by Yokel and Melograna (1983), and the residue ashed at 1000°C. The radionuclide (26Al) to stable nuclide (27Al) ratio was determined by the PRIME Lab (Sharma et al., 2000) to a precision of ≤ 10% error.
Analysis of 14C by accelerator mass spectrometry.
Fifty μl serum samples frozen in microcentrifuge tubes were sent to the Prime Lab for 14C sample processing and analysis. The instrumentation for 14C analysis is similar to that used for 26Al; however, sample preparation is quite different. The procedures have been used by the PRIME Lab for 14C samples obtained in biological studies, e.g. (Hillegonds et al., 2001). The PRIME Lab prepared the samples for AMS analysis. Briefly, the samples are lyophilized in quartz tubes. The tubes are then flame-sealed and heated to 900° C for 3 hours to combust the sample to CO2 that is collected in another tube and reduced, with iron, to graphite by heating to 500° C for 3 hours and then 50° C for 2 hours. Every five samples were compared to 2 standards of known 14C/12C ratio and a blank.
Data Analysis, Calculations and Statistics:
Pharmacokinetic analysis of 26Al serum × time data was conducted using WinNonlin. One and two compartment models were used to fit the 26Al data of individual rats to the estimated area under the curve (AUC), maximum concentration (Cmax), and time to Cmax (Tmax). The mean 27Al serum concentration was calculated by the AUC of 27Al divided by the duration of the infusion. Oral 26Al bioavailability was calculated using the following equation:
(AUC 26Al × 27Al infusion rate)/(Mean 27Al serum concentration × 26Al dose)
A one-way ANOVA was used to test for significant treatment differences. A P value < 0.05 was considered significant. The results were expressed as mean ± SD.
Results of the Al species calculations for the freshly prepared administered solutions are shown in Figure 14. The dotted lines in Figure 14 denote solutions that would be supersaturated with respect to the precipitation of Al hydroxide ((OH)3) based on the solubility product of freshly prepared aluminum hydroxide (Öhman and Wågberg, 1997).
Figure 14. Results of Al speciation calculations at a total of 65 μM Al in the absence of ligands (panel A) and in the presence of citrate (cit) (65 μM) (panel B), maltolate (mal) (195 μM) (panel C), and fluoride (F) (260 μM) (panel D) in the pH range 2 to 8. The dotted lines indicate solutions that would be supersaturated with respect to freshly prepared Al(OH)3.
In the absence of ligands, a 65 μM solution of Al at pH 5 consists of comparable amounts of Al3+and Al(OH)2+, with a smaller concentration of Al(OH)2+. A neutral solution of Al citrate consists primarily of the trimer Al3(H-1cta)3(OH)4-, with about 20% of the Al(H-1cta)-monomer. The Al fluoride solution at pH 4 consists of approximately 55% AlF2+, 40% AlF3, 4% AlF2+, and 1.7% AlF4-. The speciation calculations indicate that the Al would be fully soluble in all three of these administered solutions. The speciation results for the administered Al maltol solution show a mixture of 64% Al(mal)3, 25% Al(OH)3, and approximately 5% each of Al(OH)4-and Al(mal)2+. Although the calculated concentration of Al(OH)3 exceeds the solubility of freshly prepared Al(OH)3, no visible precipitate was observed in this solution. It appears that for these dilute solutions, the formation of insoluble Al(OH)3 is too slow to be observed during the one hour incubation time of the preparation of the administered solutions. Öhman and Wågberg (1997) reported that neutralization of a 10 mM Al3+solution to pH 7 led to the formation of colloidal particles of Al(OH)3 with a diameter of ~ 400 nm, which resulted only in a faint opalescence, rather than an obvious precipitate. Batchelor et al. (1986) have shown that such colloids remain labile and reactive for at least 30 minutes following neutralization. True equilibration of Al solutions with the less soluble, crystalline form of Al(OH)3 (gibbsite) takes months (Smith, 1996).
The Al concentration in the unfiltered and filtered Al citrate (pH 7), Al maltolate (pH 7) and Al fluoride (pH 4) solutions at 20, 65, and 200 μM and 2 mM Al was not greatly different from that expected, based on the Al concentration in the solution from which these simulated dosing solutions were prepared. For the 65 μM Al condition, the Al concentration in the unfiltered solutions was 105, 86 and 96%; and for the filtered solutions it was 91, 86 and 92% of that expected, respectively. At pH 7, in the absence of ligand, the Al concentration in the 20, 65, and 200 μM and 2 mM Al conditions was 11, 13, 51 and 52% in the unfiltered and 10, 4, 1.5 and 1.5 % in the filtered solutions, as would be expected if there is significant Al hydroxide present, as predicted by the results shown in Figure 14, Panel A. At pH 5, the Al concentration in the absence of ligand was 59, 59, 68 and 69% in the unfiltered and 44, 38, 38, and 16% in the filtered solutions, consistent with the formation of some Al hydroxide, as predicted in Figure 14, Panel A. For the 65 μM Al at pH 5, only 50% of Al passed through the 0.22 μM filter, even though the speciation results do not predict a significant amount of precipitation at this pH. This discrepancy could reflect some error in the experimental KSP for freshly precipitated Al(OH)3 or the possible impact of polynuclear Al hydroxide complexes that were not included in the speciation calculations.
The BUN and serum creatinine values of the rats ranged from 3.9 to 17.8 mg/dl and 0.2 to 0.5 mg/dL, respectively, within normal limits. Therefore data from these subjects were used in the analysis.
There were seven 14C serum samples with an analytical error > 20%, which were not included in the data analysis. Since the excluded samples were from seven different rats, this did not greatly influence the data analysis. The average 26Al concentration in the serum samples obtained from all rats prior to 26Al dosing was 0.71 ± 0.76 fg/mL (mean ± SD, range = 0 to 3.27 fg/mL). The 26Al concentration in serum samples from non-26Al treated rats was 1.07 ± 1.16 fg/mL (range = 0 to 4.64 fg/mL). The peak serum 26Al concentration after oral 26Al dosing was ≥ 70 times the 26Al concentration in serum from rats that did not receive 26Al. The peak 14C concentration after the oral 14C dose was ≥ 30 times that seen in non-14C dosed rats. The serum total Al concentration in the rat that did not receive the 27Al infusion was ~ 50 ng/mL. The serum total Al concentration in the rats that did receive the 27Al infusion was ≥ 200 ng/mL. The time courses of serum 26Al following oral 26Al dosing are shown in Figure 15.
Figure 15. Time courses of serum 26Al concentrations when dosed as 52 ng 26Al in the absence of ligands or in the presence of citrate, maltolate and fluoride. Values are mean ± SD from the 5 rats of each Al treatment group.
Cmax, Tmax and absolute bioavailability values are shown in Table 1.
Table 1 The Cmax, Tmax and absolute bioavailability of 26Al in the absence of ligands or in the presence of citrate, maltolate and fluoride. Values shown are the mean ± SD.
Although the mean oral bioavailability and Cmax of Al as the citrate, maltolate and fluoride was 2.1, 1.7 and 1.2 and 1.6, 1.3 and 1.3 times higher than Al in the absence of ligands, respectively, no statistically significant differences were observed among these 4 Al species, for any of the 3 endpoints. The time courses of serum 26Al and 14C after Al citrate and Al maltolate administration are shown in Figure 16. The serum 14C, as the percentage of the 14C dosed, was ~100-fold that of serum 26Al. The shapes of the serum 14C and 26Al concentration versus time curves for individual rats were similar. Both 26Al and 14C concentrations returned close to the baseline by 24 h.
Figure 16. Comparison of the time courses of serum 14C and 26Al. Left axis is the percentage of the 26Al dose per mL serum. Right axis is the percentage of the 14C dose per mL serum. Each panel represents one rat.
SUMMARY OF STUDY 2:
Al bioavailability, Cmax and Tmax were not significantly different when Al when introduced as the ion, citrate, maltolate, and fluoride, although Al bioavailability was 2.1-, 1.7- and 1.2-fold greater after administration as the citrate, maltolate, and fluoride than the ion. These results suggest a non-significant enhancement of Al oral bioavailability and Cmax by some associated ligands at the dose studied. The serum 14C concentration was much higher after Al citrate and Al maltolate administration than 26Al, suggesting considerable dissociation of Al from ligands in the GI tract and much greater absorption of citrate and maltolate than Al.
Overall, the results suggest flux of these five Al species across Caco-2 cells is via the same route (paracellular pathway) and mechanism (diffusion), suggesting the chemical species of Al should not impact on its site and mechanism of absorption. However, the extent of Al flux across and uptake into Caco-2 cells is Al species dependent, suggesting oral Al bioavailability would be Al species dependent. These results suggest that at low concentrations of Al which model the Al concentration in drinking water, these soluble Al species would be similarly absorbed. However, at much higher Al concentrations which model the use of Al antacid/phosphate binders, the presence of ligands that complex Al, such as fluoride, citrate and maltolate, can have a significant effect on Al bioavailability.
The absorption of Al at drinking water Al concentrations would not be expected to be greatly influenced by the presence of similar molar concentrations of citrate, maltolate and fluoride.
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Journal Articles on this Report : 4 Displayed | Download in RIS Format
|Other project views:||All 8 publications||2 publications in selected types||All 2 journal articles|
||Zhou Y, Yokel RA. Aluminum transport and uptake in Caco-2 cells. Abstract presented at the 43rd Annual Meeting of the Society of Toxicology, March 21-25, 2004, Baltimore, MD, Abstract #392.||
||Zhou Y, Yokel RA. The rates of paracellular flux and uptake of aluminum (Al) in Caco-2 cells are influenced by the chemical species of Al. Abstract presented at the 2004 Ohio Valley Society of Toxicology Meeting, Lexington, KY, November 4-5, 2004.||
||Zhou Y, Yokel RA. The chemical species of aluminum influences its paracellular flux across and uptake into Caco-2 cells, a model of gastrointestinal absorption. Toxicological Sciences 2005;87(1):15-26.||
||Zhou Y, Harris WR, Yokel RA. The influence of citrate, maltolate and fluoride on the gastrointestinal absorption of aluminum at a drinking water-relevant concentration: a 26Al and 14C study. Journal of Inorganic Biochemistry 2008;102(4):798-808.||