Biological chemistry of aluminium studied using 26Al and accelerator mass spectrometry

Nuclear Instruments and Methods in Physics Research B 92 (1994) 463-468 North-Holland

NION! B

Beam Interactions with Materials&Atoms

Biological chemistry of aluminium studied using 26A1and accelerator mass spectrometry J.P. Day a,*, J. Barker a, S.J. King a, R.V. Miller a, J. Templar a, J.S. Lilley b, P.V. Drumm b, G.W.A. Newton b, L.K. Fifield ‘, J.O.H. Stone ‘, G.L. Allan ‘, J.A. Edwardson d, P.B. Moore d, I.N. Ferrier d, N.D. Priest e, D. Newton e, R.J. Talbot e, J.H. Brock f, L. S5nchez f, C.B. Dobson g, R.F. Itzhaki g, A. RadunoviC h and M.W.B. Bradbury h a Department of Chemistry, University of Manchester, Manchester Ml3 9PL, UK b Nuclear Struct,ure Facility, SERC Daresbury Laboratory, Warrington, UK ‘Department of Nuclear Physics, Australian National Uniuersiiy, Canberra, Australia d MRC Neurochemical Pathology Unit, Newcastle General Hospital, Newcastle, UK e Biomedical Research Department, AEA Technology, Hanvell, UK f Department of Immunology, University of Glasgow, Glasgow, Scotland, UK g Department of Optometry and Vision Sciences, UMIST, Manchester, UK h Department of Physiology, King’s College, London, UK

Developments in sample preparation and presentation for accelerator mass spectrometry now permit the determination of 26Al/27Al ratios in biological materials with an accuracy of ca. 2%. We describe the use of 26Al as a tracer for aluminium, in a number of biological experiments which demonstrate the power of this technique to determine Al content and distribution within complex chemical and biological systems, and to investigate the kinetics and mechanisms of Al-transport pathways. Applications to human, animal and cellular systems are described. 1. Introduction

Although aluminium is toxic to humans, animals and plants, its biochemistry has been little studied and

is poorly understood [l]. However, the increasing availability of 26Al now opens the way for the study of the biochemical pathways and kinetics of aluminium distribution, both in vitro and in vivo, using either radiochemical techniques [2] or accelerator mass spectrometry CAMS) [3-61. The object of this paper is to summarise the general principles and scope for the application of 26Al as a tracer to investigate human or animal biochemistry, by reference particularly to our own programme of experiments using 26Al-AMS. The applications of AMS to biomedical research in general, including 26Al-AMS, are reviewed in a parallel paper 171.

2. ‘“Al determination

by

AhIS

Although accelerator mass spectrometry to determine 26Al has been applied for some years in the

* Corresponding author. Tel. + 44 61 275 4661, fax + 44 61 275 4598. 0168-583X/94/$07.00

geological sciences, the first applications to biological systems were reported only recently [3-51. For biological applications, three important technical requirements of the instrumentation are: (al good discrimination between 26Al and 26Mg, as magnesium is a common element in biology, and in practice 26Mg/26Al ratios in samples may well exceed 10”; (b) ability to measure *‘Al with an accuracy equivalent to that achieved for the tracer, as the stable isotope is invariably added to the sample as yield monitor; and (c) ability to produce stable beam currents for extended periods (e.g. up to 1 h) from aluminium oxide or similar sources of low mass (ca. 100 p,g Al or less). We have described the development and characteristics of two systems which achieve these objectives. In one, we used the 20 MV tandem accelerator (now closed) at Daresbury, UK [4,6]. The system accelerated the 26A10- ion, and relied for 26Al/26Mg differentiation on full stripping of the nuclides at > 150 MeV, and subsequent separation and detection of 26A113+ and 26Mg12+ by magnetic spectrometer. In the other, we have used the 14UD accelerator at ANU, Canberra [8,9]. In this system we also use Cs sputtering on Al,O,, but accelerate Al-, relying on the instability of the Mg- ion in the ion source to achieve the primary Al/Mg separation. Under optimum conditions, the 26Al/27Al atom ratio at the detection limit is ca. 10-14,

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VIII. BIOMEDICINE

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464

and the useful working range is from lo-l2 to lo-’ with a precision of measurement ca. 2%. More details of the methodology for the determination of 26Al in biological materials are given in a parallel paper [lo].

3. Application

of 26Al to biological

measurements

It should be appreciated that the types of compartment represented in Fig. 1 may be physically, chemically or biologically distinct, and range from the microscopic to the macroscopic. Examples of the use of 26Al as a tracer to investigate aluminium distribution and kinetics in human, animal and cellular systems, and between body organs, cellular compartments and chemical species, will now be described.

3.1. Generalised methodology

3.2. Measurement of AI uptake in humans An important application of 26Al as a tracer for aluminium is to overcome the analytical problems associated with the determination of the element by chemical analysis. Major problems arise in the measurement of aluminium distribution in biological or environmental systems from adventitious gain (from contamination) or loss (precipitation, adsorption, etc.) during the analytical process. In general, these difficulties are diminished by the use of an isotopic tracer [ll]. With reference to the general system shown in Fig. 1, equilibration of the 26Al isotope with the various chemical forms of the natural element (27Al) throughout the system, prior to the sampling of the various compartments, subsequent addition of an aliquot of 27Al to each sample to monitor chemical yield, and determination of the isotope ratios in each sample, allows unambiguous determination of the original Al contents of the system compartments. A distinct but equally useful application of an isotopic tracer is in the investigation of the dynamics of a system. An isotopic spike introduced at a specific point in a complex system in steady state (e.g. Fig. 1) acts as a time dependent marker for the redistribution of the element amongst the various compartments, and kinetic information can be obtained [ll]. An important precondition in this type of experiment is that the chemical speciation of the 26Al spike should match that of the natural Al in the compartment to which the spike is introduced.

INPUT

A

*___-_--_*

B

z

OUTPUT +

D

4

Fig. 1. Schematic diagram representing transfers of a given component of a complex system under steady state conditions. The compartments A, B, etc. may represent discrete phases of a physical system, chemical species in a chemical system, entities of a biological system, etc.

An essential parameter to quantify the effects of aluminium exposure by ingestion is the so-called gastrointestinal (GI) uptake factor (usually denoted F, [12]), defined as the ratio of the amount (of aluminium) absorbed into the body to the amount ingested: F, = (mass Al absorbed)/(mass

Al ingested).

However, it should be recognised that gastrointestinal absorption of any element is likely to vary widely between individuals, and will also be dependent on the chemical speciation of the element in the ingested material. Although it is possible to derive a value for the gut uptake of Al by direct chemical analysis of input and subsequent body load, the amounts (ca. 1 g [13]) which need to be ingested in order to produce accurately measurable increases in systemic concentrations are arguably large enough to affect the absorption process itself. Potentially, the use of 26Al tracer overcomes this problem, by allowing the measurement of F, under physiologically normal circumstances. In a continuing series of experiments designed to measure Fl for aluminium in humans under a variety of physiological and chemical conditions, a relatively simple approach has been adopted [14,15,20]. An aliquot of 26Al, carried by a relatively large but still physiologically insignificant amount of the stable element, is added to orange juice and ingested by the subject (e.g. 100 ng 26Al with 100 ug “Al in 100 ml juice). The presence of 27Al and citrate carriers (albeit at low concentrations) in the juice ensures that the aluminium is held in solution both before and during the ingestion process (this is a very important detail, and omission of suitable carriers probably accounts for the anomalously low values of gut absorption recently reported for 26A1uptake in rats [16]). Blood samples are then taken sequentially for several hours (in later experiments, the 1 h sample only was taken, as this appears to correspond approximately to the maximum plasma-Al level), and after suitable preparation the 26Al plasma concentrations are determined by AMS. As a simplifying approximation, it is assumed that multiplication of the measured plasma concentration

465

J.P. Day et al. /Nucl. Instr. and Meth. in Phys. Res. B 92 (1994) 463-468

by the plasma volume gives the total amount absorbed by the subject (errors implicit in this assumption are discussed later). Division by the amount ingested (also determined by AMS) leads directly to an estimate of FI. In most cases, we have studied subjects in groups of six, with an equal number of normal subjects in the control group. Values for F, obtained in a series of experiments are given in Table 1 [14,15]. The enhancement of gastrointestinal uptake of Al by large amounts of citrate, as previously reported [17,18], was confirmed. Aluminium uptake is considerably reduced in the presence of silicate [15], confirming a prior hypothesis by Birchall [19] based on the recognition that the formation of polymeric aluminosilicates occurs in solution, which could make the aluminium less available biologically. In another study [20], it was shown that Al uptake in a group of patients with Down’s syndrome (a disease which may be genetically linked to Alzheimer’s disease) is both much more variable than in the control group, and on average enhanced by a factor of about 5 (Table 1). The effects of other disease states and physiological factors, for example Alzheimer’s disease and iron status, are currently being investigated. Thus, whilst a value ca. 1O-4 for gastrointestinal absorption, given for a range of aluminium compounds [12,13], would indeed be representative of our control group, this quantity was shown to be enhanced or depressed by chemical speciation (e.g. citrate or silicate, respectively), and might also be affected by the physiology of the individual. It is implicit in our methodology that losses from the blood volume by excretion and absorption to other tissues would have been negligible over the 1 (or 6) h period from ingestion to sampling in these experiments. This, of course, cannot be the case as the plasma-Al levels rise sharply after ingestion of the Al compound, pass through a maximum value, and then more slowly decline [l&21]. Thus, although these losses are probably small over the first hour, we have previously pointed out that the value of Al uptake obtained

Table 1 Values of the gastrointestinal absorption factor (F,) determined for 26A1uptake in humans under various conditions Study

Group size

Uptake (104F,) mean

Al with excess citric acid Al inorange juice Control (no additives) Silicate added Control (for DS group) Down’s Syndrome patients

1

100

Ref.

range 1141

6 6 6

1.2 0.5 -i.s 0.18 0.05-0.3 0.91 0.04-1.3

1151 ll51 1191

6

4.7

[ZO)

1.1 -8.0

0.0001 0.01

0.1-

1

10

DAYS AFTER 1NJECTlON

100--T

IO

(Log scale)

Fig. 2. 26Al in blood plasma for 1000 days following the injection of aluminium citrate, determined by AMS [22]. Note that both scales are logarithmic, and that the ordinate spans 5 orders of magnitude in 26AIconcentration.

by this methodolo~ wiil necessarily be an underestimate [14]. A more rigorous but experimentally much more complex approach is to quantify the rates of excretion in a separate experiment, and then to use excretion measurements to determine uptake following aluminium ingestion. The first stages of an experiment using this methodology are described below.

In an experiment to characterise the retention and excretion of aluminium, independently of gut absorption, Priest and co-workers injected 26Al in citrate solution into a healthy subject [221. Sufficient isotope was used to permit blood, excreta and whole body measurements by gamma spectrometry; excretion could be followed for some 14 days, whilst whole body gamma measurements are still being made (after three years). Most of the aluminium was excreted rapidly in urine during the first few days after injection (80% of dose over 10 days). However, whifst whole body measurements matched the excretion data over the first 10 days, the whole body measurements also show that the aluminium remaining in the body is removed with a progressively increasing half-time, so that by 1000 days, 4% of the injected dose still remained and showed an instantaneous retention half-time of more than six years. Blood 26A1was also measured, for the first five days by gamma spectrometry, and then by AMS [6,221, so far up to 1000 days post-injection. From the plasma concentration almost immediately after injection (1.5 min; see Fig. 21, it appears that the 26Al-citrate dispersal volume was approximately 10 litres, i.e. approaching the extracellular fluid volume. The log-log plot (blood concentration vs. time; Fig. 21 is approximately VIII. BIOMEDICINE

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Table 2 26Alspeciation in blood Fraction

26Al(% of total)

Red cells Plasma-total Plasma-fractions Low mol. wt ( < 5 kDa) High mol. wt ( > 5 kDa) Protein association Transferrin Albumin Unassigned (5-50 kDa)

at 1 h [14]

at 880 days 1221

1 99

14 86

4 95 80 10 5

linear, and at 1000 days the fraction of injected dose still remaining in the blood plasma was approximately 1/106. These measurements have considerable clinical importance, as the cross calibration of tracer aluminium levels in blood with whole body retention provides a method for the estimation of aluminium body burdens from blood-Al measurements in Al-exposed individuals, for example patients in renal failure.

3.4. Aluminium blood

speciation and distribution in human

In the first experiment in this category, blood from the subject ingesting 26Al-citrate (Table 1) was fractionated down to protein level by membrane filtration and column chromatography, and the 26Al distribution determined by AMS (Table 2 [6,14]). These results confirmed the previous conclusion [23] that aluminium in plasma is bound to the iron-transport protein transferrin (Tf: molecular mass 72 kDalton). In a more recent experiment, blood taken from the 26Al-citrate injected subject (section 3.3 and Fig. 2) after 880 days was fractionated, and the plasma/red cell distribution determined by AMS (Table 2). In contrast to the situation with the earlier subject [14] where, after 1 h, no *“Al had entered the red cell, in the present case (after the much longer period) approximately 14% of the blood-26Al was contained in the red cell. This observation seems to confirm an earlier observation that aluminium in plasma does not equilibrate directly with aluminium contained in red cells, but more probably enters the red cell during erythropoiesis 1241. 3.5. Determination of aluminium distribution at the cellular level 3.5.1. Neuroblastoma cells

The analytical virtues of the use of an isotopic tracer are well demonstrated by the determination of

aluminium distribution in the various cellular compartments of neuroblastoma cells grown in culture, reported previously and in a parallel paper [10,25]. In those experiments, aluminium (with 26A1 spike) was introduced to the culture medium as the EDTA complex, which is soluble and reasonably stable, and which had previously been shown to enter the neuroblastoma cells under the experimental conditions. One result of the experiment was to show that, in this chemical form, the aluminium probably enters the neuroblastoma cells by a passive (i.e. diffusion controlled) mechanism. Additionally, it was demonstrated that the nuclear Al was not strongly bound to the DNA fraction. 3.5.2. Transferrin mechanism Aluminium is also thought to enter cells by active transport, using the transferrin receptor mechanism employed for iron incorporation [1,26]. Thus, in another experiment, designed to compare the active and passive transport mechanisms, aluminium bound to transferrin or to citrate, respectively, was used in a simple in vivo model system, the Bicameral Chamber [27]. In this system, a tight monolayer of cultured cells (human colon carcinoma), supported on a collagen membrane, was used to separate upper and lower solution compartments containing the culture medium. 2hAl-spiked aluminium-transferrin (Al-Tfl or aluminium-citrate (Al-tit) was introduced at non-toxic concentrations to the upper compartment, which was large enough for the aluminium concentration to remain unchanged for the duration of the experiment. Aliquots were withdrawn periodically from the lower compartment, and 26Al determined. The Al transfer factors across the membrane for the two chemical species are given in Table 3 [28]. Clearly, in the model system Al-Tf was transported more readily than Al-cit. It also appears that while transport of Al-Tf is nearly complete at 5 h, transport of Al-tit is still continuing even after 23 h, possibly suggesting an active, and specific, mechanism for the former species (which could presumably saturate), and a passive (i.e. diffusive) mechanism for the latter, which would be expected to continue until the concentrations on either side of the membrane were equalised.

Table 3 26Al transfer

factors

in the Bicameral

Chamber

Aluminium speciation (upper chamber)

Transfer 5h

23 h

[%I

Citrate Transferrin

0.15 1.35

0.26 1.66

73 23

a 26Al transfer factor in upper chamber).

factor [%I a

Increase

= 100 x ([26A11in lower chamber)/([26All

J.P. Day et al. / Nucl. Ins&. and Meth. 3.6. Kinetics and mechanism in the mouse

of aluminium

distribution

From the foregoing and other evidence, it can be plausibly suggested that tissue uptake of aluminium in animals mimics the iron pathway, in which Fe(III), transported round the body as the transferrin complex, Fe-Tf, undergoes endocytosis via the transferrin receptors on the cell surface [29]. In an experiment to test this hypothesis, three groups of mice were compared: one, a control group of normal animals, the second a group of similar mice treated with a monoclonal antibody against the transferrin receptor (which would in consequence largely cease to operate), and the third a group of homozygous hypotransferrinaemic mice (hpx /hpx), a strain without circulating transferrin. The mice in the three groups were infused with an %Alspiked aluminium citrate solution. After 2 h, the mice were killed by ~vipe~usion and tissues removed for analysis by AMS. Tissue uptake in all groups followed the order [30]: bone > kidney > spleen/liver/skeletal

muscle > brain.

However, levels of uptake were not consistently different in corresponding tissues across the three groups. Thus, under the conditions of this experiment (i.e. with aluminium introduced intravenously as citrate complex) it would appear that aluminium uptake can occur independently of a transfer&-mediated process, and this uptake is most marked in bone and renal cortex. However, the independence from a transferrinmediated mechanism might simply reflect the persistence of the Al-citrate complex within the experimental animal (over the short period of the experiment), and a resulting absence of Tf-bound Al in circulation. As would be expected [31], bone was the major target organ for aluminium deposition in these animals, whilst the brain appears to be relatively well protected compared to other soft tissues. The relative concentrations of 26Al found in bone and liver in this experiment (36: 1) are not greatly different to those observed by Fink et al. (8: 1) 1321following gut uptake of 2bAI in rats.

4. Summary and conclusions The application of 26Al as a tracer for aluminium has been exemplified for a diversity of biological systems. The tracer has been used to allow the determination of aluminium concentrations in cases where chemical analysis is impracticable, to investigate speciation and distribution within and between system compartments, and to measure the kinetics and mechanism of aluminium transport. The power of AMS to extend the range of concentration over which chemical species

in

Phys. Res. B 92 (1994) 463-468

may be studied demonstrated.

461

in a complex system has also been

We wish to thank the SERC (UK) for research studentships to J.B., L.J.A.E., R.V.M., S.J.K. and 3.T. Also, the following organisations for their generous financial support to various parts of this work: The Royal Society (London); Withington Hospital (Manchester) Renal Unit Endowment Fund; the Medical Research Council (UK); the Wellcome Trust; the Humane Research Trust; the International Primary Aluminium Institute (UK); the Aluminum Association (USA).

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1221 N.D. Priest, D. Newton, J.P. Day and R.J. Talbot, unpublished (currently submitted to Human Toxicology). [23] G.A. Trapp, Life Sci. 33 (1983) 311. [24] CD. Hewitt, P. Ackrill and J.P. Day, in: Aluminium and Other Trace Elements in Renal Disease, ed. A. Taylor (Balliere Tindall, London, 1986) p. 313. [25] C.B. Dobson, J. Templar, J.P. Day and R.F. Itzhaki, Biochem. Sot. Trans. 21 (1993) 321s. [26] S.J. McGregor, J.H. Brock and D. Halls, Biol. Metals 4 (1991) 173. [27] I.J. Hidalgo, T.J. Raub and R.T. Borchardt, Gastroenterology 96 (1989) 736. [28] L. Sanchez and J.H. Brock, unpublished. [29] K. Rao, J. van Renswoude and C. Kempf, FEBS Lett. 160 (1983) 213. [30] A. Radunovic, F. Ueda, K. Raja, J.P. Day, J. Templar and M.W.B. Bradbury, J. Physiol. (in press). [31] F. Van de Vyver and W.J. Visser, in: Trace Metals and Fluoride in Bones and Teeth, eds. N.D. Priest and F. Van de Vyver (CRC Press, Boca Raton, 1990) p. 83. [32] D. Fink, M.A.C. Hotchkis, G.E. Jacobsen, E.M. Lawson, A.M. Smith, C. Tuniz, J. Walton and D. Wilcox, unpublished.