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Articles |
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Centre de Spectrométrie Nucléaire et de Spectrométrie de Masse (CSNSM), CNRS-IN2P3, Bat 108, 91405 Orsay, France.
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INSERM U 90, Hôpital Necker, Paris, France.
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Département de Physiologie, Faculté de
Pharmacie, Châtenay-Malabry, France.
a Author for correspondence. Fax 33 1 69 15 52 68; e-mail raisbeck{at}csnsm.in2p3.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: mass spectrometry • rats • citrate • variation, source of • nutritional status • bone • brain • liver • toxicology
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At rates of ordinary dietary intake, the low intestinal absorption of Al, its preexisting concentrations in all tissues, and ubiquitous laboratory contamination, all make kinetic studies with 27Al, the only stable isotope, very difficult, if not impossible (1). The obvious solution, namely, the use of a radioactive isotope, was until recently not practical because the half-lives of the available isotopes were either too short (<7 min) or too long (716 000 years). The development of accelerator mass spectrometry (AMS) has now made feasible the use of the latter isotope, 26Al (7)(8)(9)(10), although at present in only a few highly specialized centers.
In a previous study (11)(12), we used AMS to determine the fraction of 26Al ingested by rats that was transiently present in plasma, excreted in urine, or retained in bone 8, 24, and 48 h after oral intake. Somewhat surprisingly, we found that the fraction retained in bone (~0.02%) was about the same as that excreted in urine after the administration of a single dose. Another surprising result was that, taking into account sample-to-sample variability, and the limited number of rats studied, we were unable to confirm a statistically significant enhancing effect of concomitant ingestion of citrate on Al absorption, in contrast to reports by other workers (13)(14)(15)(16)(17)(18)(19).
In the present work, our previous study has been expanded in two directions: (a) We have extended the period considered from 0.5 h to 30 days after ingestion; and (b) we have added brain and liver to the organs studied (in the previous study we gave only upper limits for 26Al accumulation in the liver).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal handling and experimentation conformed to guidelines issued by the European Economic Community, as published in Journal Officiel des Communautés Européennes (Déc 18) 1986; L358.
Samples consisted of 1 mL of blood plasma, total brain, a fraction of the liver, one femur cleaned of adhering matter, and a fraction of total urinary volume; all were treated according to the experimental protocol described elsewhere (11). The 26Al/27Al ratios of the resulting Al2O3 were measured by using the "Tandetron" AMS facility at Gif/Yvette, France (8).
All results are expressed as the 26Al present in total plasma, bone, cumulative urine, brain, and liver divided by the ingested 26Al for each rat. The fractions are sometimes expressed as %. Measurement uncertainties were generally ~10%, except for the brain samples, for which the statistical error increased to 15–30% or, in three cases, permitted us to report only an upper limit. To convert the 26Al present in measured samples to the 26Al present in the total volume of blood (or plasma) or in the total skeleton, we used the following estimations.
Total plasma volume (Vp), in milliliters, was calculated as (21) Vp = (0.0276 x W) + 3.38, where W is rat body weight in grams.
Total blood volume was calculated by assuming a hematocrit of 50% (22) for rat blood.
Skeleton was assumed to represent 8% of rat body weight (23), and the 26Al concentration observed in the femur was assumed to be representative of the content in the whole skeleton.
We used the nonparametric Mann–Whitney U-test to examine the statistical significance of differences between rats treated either with or without citrate. Because of the relatively small number of animals at each time point, results have been expressed throughout both as median values (with range) and as mean ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. For the rats treated without citrate, the 26Al
present in plasma appeared to reach a maximum (near 0.01%) between 1
and 2 h after ingestion, and decreased by more than three orders
of magnitude (10-3) after 1 month. For rats treated with
citrate, the 26Al concentration in plasma apparently peaked
before the first time point of 30 min (with a value of ~0.1% at 30
min). From this maximum value to the value observed at 1 month was a
decrease of 10-4.
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To investigate the fraction of 26Al in blood associated with components other than plasma, we also measured whole-blood aliquots from the rats killed at 1 h after gavage. The plasma results accounted for 70% of the 26Al in blood after gavage without citrate, and ~85% after gavage with citrate.
urinary 26AL ELIMINATION
Figure 2
shows the distribution of urinary 26Al
excretion as a function of time for rats killed after 5, 15, or 30
days. In the last-named group, ~94% of cumulative 26Al
was excreted after 5 days, ~6% in the next 24 days, and ~0.1%
during the final (30th) day. Results were similar for the rats killed
on day 15, again with ~95% of the 26Al being excreted
during the first 5 days. For the rats killed on day 5, ~90% of
cumulative 26Al excretion took place within 24 h, and
~95% within 48 h. Together, these data show that ~90% of
cumulative urinary excretion occurs during the first 48 h after
ingestion, with or without the concomitant ingestion of citrate. Thus,
a cumulative 48-h urine sample can be used to make a convenient and
reliable estimate of total urinary excretion after the administration
of a single oral dose.
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Figure 3
c summarizes the cumulative urine data. The values found in rats
treated with citrate were both greater and more variable than those in
rats without citrate. From the time profiles seen in plasma, one might
argue that at least part of the difference at 8 h could result
from a more rapid absorption and excretion of 26Al in the
rats treated with citrate. Therefore, for purposes of comparison, we
considered only the results at 5, 15, and 30 days (n = 6). The
resulting median values were 16.4 x 10-4
(range, 9.4–91 x 10-4; mean ± SD, 28 ±
32 x 10-4) for rats with citrate and 5.6 x
10-4 (range, 2.5–6.8 x 10-4; mean
± SD, 5.2 ± 1.8 x 10-4) for rats without
citrate. Thus, an enhancement effect of about threefold (P
<0.005) was associated with the citrate ingestion.
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bone 26AL ACCUMULATION
Figure 3d
shows an estimate of the fraction of ingested
26Al retained in the whole skeleton, for each of
the 40 rats studied, according to measurement of femur
26Al. There is no strong correlation of amounts of Al
ingested—either with citrate (r2 = 0.01) or
without citrate (r2 = 0.05)—as a function of
time. This indicates that 26Al uptake into bone took place
very rapidly (~1 h) and irreversibly (at least within the 30 days
studied). The median value of the 20 determinations in rats without
citrate was 4.2 x 10-4 (range, 1.7–20 x
10-4; mean ± SD, 6.7 ± 6.7 x
10-4) compared with 23 x 10-4 (range,
6.8–73 x 10-4; mean ± SD, 29 ± 20
x 10-4) in rats with citrate. Thus, the concomitant
intake of 26Al with citrate increased the accumulation of
26Al in bone by a factor of ~5 (P <0.0002,
n = 20 for each group).
brain and liver 26AL ACCUMULATION
The fractions of 26Al observed in brain and
liver at 
8 h are presented in Fig. 3
(a, b). We also measured some
samples from animals killed at earlier time points. However, because we
made no special effort to remove blood from the blood vessels of these
organs, we cannot neglect a contribution from this source at the
earlier times. In fact, on the basis of our measured plasma
26Al concentrations, we calculate that a vascular blood
component of ~20% and ~5% for liver and brain, respectively,
would be sufficient to explain the total 26Al observed in
these organs <8 h after gavage—plausible fractions under our
experimental conditions. We thus restricted ourselves to using only the
results from 5, 15, and 30 days after ingestion. The median fraction of
26Al retained in the brain was 3.8 x
10-8 (range, 0.8–6.5 x 10-8; mean
± SD, 3.7 ± 1.1 x 10-8) (n = 6, using
upper limits for 3 of these values) in rats that received no citrate,
and 5.8 x 10-8 (range, 3.2–46 x
10-8; mean ± SD, 13.1 ± 18.5 x
10-8) (n = 5) in rats that had received citrate
treatment.
Using the same reasoning, we determined that the respective fractions of 26Al retained in liver were 2.2 x 10-6 (range, 0.9–11.4 x 10-6; mean ± SD, 4.1 ± 4.0 x 10-6) and 4.8 x 10-6 (range, 2.6–19.4 x 10-6; mean ± SD, 8 ± 6.6 x 10-6), respectively (n = 6 each group).
Thus for these two tissues, at the times when blood 26Al contribution was negligible, the concomitant intake of citrate led to a moderate median increase (twofold) in aluminum retention in the liver and brain. Given the limited number of data, and the fact that only upper limits were available for 3 brain samples, this increase was not statistically significant (P >0.1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study confirms our earlier observation that the amount of ingested Al retained by bones in young rats is as great as that excreted in urine. The accumulation in the skeleton also appears to be relatively permanent. For example, by comparing the amount of 26Al excreted in urine after 30 days with that accumulated in bone in the same rats, one can calculate a lower limit (because not all excreted 26Al is necessarily from bone) of 500 days for the residence time in bone. It will be important to investigate whether similar results are observed for older rats, where bone growth is considerably slower.
The only other experiments using 26Al and rats that, to our knowledge, can be directly compared with ours, are those of Schönholzer et al. (16)(17) and Ittel et al. (24). The former investigators found urinary excretion for 26Al ingested as Al(OH)3 to be 0.07–0.11%, only slightly more than our value of 0.05% for gavage without citrate. Their results for Al ingested as Al citrate (0.7%) or Al citrate plus 76 mg of citrate (5%) are considerably larger than our median value of 0.16% for Al plus 20 mg of citric acid. The difference may be due to the different conditions of gavage (volume, pH, nature of citrate) or to the fact that the animals of Schönholzer et al. were studied after a period of fast (private communication). Indeed, we have obtained results indicating that prior fasting substantially increases Al absorption (25). Ittel et al. (24) found urinary excretion of 26Al (administered with substantial quantities of 27Al as AlCl3) to be 0.032% and 0.018% in two rats. These are slightly lower than our values, even though their animals were in a fasted state. This is in contrast to what would be expected if, as suggested by Ittel et al., absorption was correlated positively with Al dose.
Fink et al. (26), measuring the accumulation of orally ingested 26Al in the brains and livers of two 6-month-old Wistar rats (~550 g), found results for brain (10 and 28 x 10-8) somewhat larger than our maximum value of 6.5 x 10-8. Because Fink et al. do not give the total weight of the livers for their animals, an exact calculation of the fraction accumulated is not possible. However, estimating these weights as 4% of the rat body weight (i.e., 22 g) yields accumulation values of 8.8 x 10-6 and 33 x 10-6, also somewhat larger than our median value of 2.2 x 10-6. Although these estimates are based on only two measurements in their study, one reason for the differences could be the difference in age of the rats.
Walton et al. (27) also recently reported brain absorption
factors for single doses of 26Al administered orally to
eight mature rats after a 30-h fast. Adopting the results from column 3
of their Table 1
[using the experimental results of their column 1,
and the quantity of 27Al carrier indicated in the paper (4
mg), we in fact calculate different values], we calculated that six of
the rats had absorption factors for total brains of 0.5 x
10-8 to 4.5 x 10-8—i.e., within the
range found by us. Two other rats had considerably larger factors of
35 x 10-8 and 53 x 10-8. Once
again, the prolonged period of fasting may account for the enhanced
absorption in these cases.
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Several authors (18)(19)(28) have used serum concentrations of 26Al to estimate aluminum absorption in humans. Our results in rats, however, show that the instantaneous amount of 26Al present in the circulation at any time was never >10% of the total amount effectively absorbed. In studies of two human subjects, Hohl et al. (29) also found that the maximum 26Al in circulation was never >20% of that excreted in urine. Therefore, in addition to requiring measurement of a complete time profile to ensure observation of the peak, estimates based on serum concentrations are likely to seriously underestimate total Al absorption.
One striking feature shown in Fig. 2
is that individual rats showing
the largest urinary 26Al excretion (e.g., rats 33 and
40) also tend to have the largest 26Al retention in the
organs studied. To examine this quantitatively, we calculated in Table 1
the ratio of retention in bone, brain, and liver to excretion
in the urine. Interestingly, for the concomitant intake of citrate,
these ratios are less variable than are the absolute retention factors
themselves. Consequently, urinary excretion can be used to make
reasonably reliable estimates of Al retention in other organs under
physiological conditions. Similarly, although the concomitant ingestion
of citrate increases Al absorption, Table 1
shows that this did not
appear to modify the relative distribution of 26Al in bone,
brain, and liver in comparison with ingestion without citrate. This
suggests that the critical step in Al retention is its initial passage
through the gastrointestinal barrier.
From these data we draw the following conclusions. From the retention of Al in bone, liver, and brain and its excretion in urine, the gastrointestinal absorption of normal dietary quantities of Al by rats can be estimated as near 10-3 (0.1%); however, this estimation does not take into account a possible distribution of Al into muscle. Because the amount of Al in the circulatory system never exceeds ~10% of that absorbed, blood samples are a poor indicator of gastrointestinal absorption of Al. About 50% of absorbed Al is rapidly (<2 h) and permanently (within the 30-day scale of our experiment) accumulated in the skeleton of young rats. About 50% of absorbed Al is excreted in urine, with 90% of this excretion occurring during the first 48 h after ingestion; this makes a 48-h sample of urine a relatively good indicator of total gastrointestinal absorption of Al. About 2 x 10-6 and 4 x 10-8 of ingested Al is permanently (within the 30 days of our experiment) deposited in the liver and brain, respectively. The concomitant intake of citrate leads to a more rapid, larger, and more variable absorption, with an average enhancement factor under our experimental conditions ranging from 2 to 5. Finally, the retention of Al in bone, brain, and liver, relative to that excreted in urine, is statistically indistinguishable between rats fed Al with citrate and those fed no citrate supplement (P >0.1); this suggests that the principal effect of citrate is to enhance gastrointestinal absorption, with little influence on subsequent distribution in various organs.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  R. A. Yokel, S. S. Rhineheimer, P. Sharma, D. Elmore, and P. J. McNamara Entry, Half-Life, and Desferrioxamine-Accelerated Clearance of Brain Aluminum after a Single 26Al Exposure Toxicol. Sci., November 1, 2001; 64(1): 77 - 82. [Abstract] [Full Text] [PDF]
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) or with citrate (
).
