Defective gallium-transferrin binding in Alzheimer disease and Down syndrome: possible mechanism for accumulation of aluminium in brain

Gillian Farrar, Paul Altmann, Simon Welch, Oryst Wychrij, Bharati Ghose, Jerome Lejeune, John Corbett, Vee Prasher, John A. Blair

Lancet 1990; 335:747-750


Résumé :

The plasma distribution of gallium (as an analogue of aluminium) was investigated in patients with Alzheimer disease. Down syndrome, or stroke dementia, in subjects on haemodialysis for chronic renal failure, and in healthy controls. Gallium-transferrin binding was significantly lower in the Alzheimer (mean [SEM] 7.9 [1.1]%) and Down syndrome groups (6.9 [0.7]%) than in the controls (17.1 [1.6]%), whereas stroke dementia and haemodialysis patients had normal binding. There were no differences among the groups in plasma citrate concentration. The plasma transferrin concentration was slightly lower in the Alzheimer and Down syndrome groups than in the controls, but even lower in stroke dementia patients (1.74 [0.14] g/l vs 2.98 [0.18] g/l in controls). Transferrin iron saturation was higher in the Alzheimer (58.9%) and Down syndrome groups (81.6%) than in the controls (39.0%) or stroke dementia patients (33.4%). This deficiency of gallium/aluminium binding would leave more unbound aluminium which could move readily into the brain, where it has neurotoxic effects.

Sommaire

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Introduction

Alzheimer disease is an insidious and ultimately fatal form of dementia affecting as many as 1% of the population. The diagnosis can be made only by exclusion of other causes of encephalopathy or at necropsy, when characteristic neuropathological changes are found. Its cause is not yet known, although both genetic and environmental factors are thought to be involved. Aluminium has been implicated in the pathogenesis of the disorder: animal studies as early as 1897 showed behavioural changes similar to human dementia [1], and many studies have reported increased levels of aluminium in the affected parts of the brain in Alzheimer disease patients [2,3] even when serum aluminium levels are within normal limits. Aluminium-induced encephalopathy has been reported after heavy industrial exposure in subjects with normal renal function [4,5] and in patients undergoing maintenance haemodialysis for chronic renal failure, who are exposed to much higher concentrations of aluminium than normal [6]. Dialysis patients (even if not encephalopathic) show some similarities in neurochemistry, neuropathology, and cerebral function to patients with Alzheimer disease [3,7-9].

Epidemiological studies have shown a link between the incidence of Alzheimer disease and the concentration of aluminium in drinking water [10].

Patients with Down syndrome have similar neurochemical features to those with Alzheimer disease [11,12] and a predisposition to the disease itself [13]. In necropsy studies more than 90% of Down syndrome patients aged over 30 years had similar neuropathology to Alzheimer disease patients [14]. These patients also have higher than normal concentrations of aluminium in their brains [3].

Although we have shown objective psychomotor impairment in dialysis patients possibly related to aluminium, the incidence of Alzheimer disease in these patients is no higher than in the general population -an unexpected finding if aluminium has a causal effect in the pathogenesis of Alzheimer disease. In an attempt to resolve this discrepancy we have postulated that the factor protecting the majority of people from the adverse neurotoxic effects of aluminium is also protective in dialysis patients (except when they are exposed to overwhelming amounts).

Experiments on the intestinal absorption of aluminium showed the importance of its chemical species for bioavailability [15]. The species distribution may also be important in vivo, affecting the transport of aluminium to susceptible organs such as the brain. Aluminium binds almost exclusively to the plasma glycoprotein transferrin [16] a large transport protein of approximately 70 kD, which normally transports iron and certain other metals. The release of metals from transferrin depends on the presence of transferrin receptors on the cell surface. Once bound to the receptor, transferrin becomes incorporated into an endosomal compartment within the cell which has a sufficiently low pH to cause dissociation of the metal from transferrin. Under normal conditions there is very little non-transferrin-bound aluminium in the circulation and there are few transferrin receptors in the brain, so the normal uptake into the brain may be negligible. The transferrin molecule has spare metal-binding capacity and is found in very large quantities in the blood. It binds aluminium avidly and because of the excess of transferrin over aluminium it is difficult under normal circumstances to saturate the total blood binding capacity. However, any free aluminium in the circulation - most likely as aluminium citrate - could enter the central nervous system without hindrance [17]. Thus, any impairment of aluminium binding to transferrin could give rise to far greater deposition of aluminium in the brain, thereby increasing the risk of neutotoxic effects.

We have attempted to determine the distribution of aluminium in plasma from subjects with Alzheimer disease, Down syndrome, stroke dementia, end-stage renal disease on haemodialysis, and normal subjects. Because there are no biologically useful radionuclides of aluminium, we used the chemically similar radioisotope gallium-67 (half-life 78 h) as an analogue for aluminium [18]. Both aluminium and gallium bind to the same sites on transferrin [19] and aluminium does not necessarily compete with iron in binding to transferrin [20].

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Patients and methods

Blood (10 ml) was drawn into heparinised containers for plasma separation from five groups of subjects (table I). The control subjects were laboratory staff and non-demented, depressed inpatients. In the patients with Alzheimer disease, the diagnosis was confirmed by standard neuropsychiatric techniques, as well as (in 5 patients) by the presence of delayed flash-stimulated visual evoked potentials [21]. All plasma samples were stored at 4°C for immediate assay of gallium-transferrin binding and subsequent assay of citrate, iron, ferritin, and transferrin and transferrin subtyping.

To measure the binding of gallium to transferrin, gallium nitrate (Ga[NO3]3, Aldrich Chemical Company) and 1-5 µCi 67Ga (Amersham International) were added to 1800 µl plasma to a final concentration of 10 µg/l. This concentration is similar to that of aluminium in the serum of normal individuals [22] and those with Alzheimer disease [23]. The plasma was incubated at 37°C for 1 h in a shaking water bath (60 oscillations/mn), then applied to a Sephadex G75 column (60 x 1.5 cm; Amicon Wright) at a flow rate of 1 ml/min. The column was eluted upwards with 25 mmol/l "tris"-HCl, 100 mmol/1 sodium chloride buffer at pH 7.4, and 50 fractions of 6 ml were collected. Recovery of 85-90 % was achieved and after each run any remaining gallium was eluted by means of 5 ml 0.1 % EDTA solution. Each fraction and standards of a known volume were counted for 67Ga activity by measuring gamma emissions over the energy range 50-420 keV (LKB Compugamma Counter; Pharmacia-LKB). The percentage binding of gallium was calculated from the recovered radioactivity. No difference in gallium distribution was seen when a smaller quantity of gallium (0.15 µg/l) was added. We examined the possible effect of sample storage on gallium binding and found no difference between samples stored at 4°C or - 20°C, nor any effect of duration of storage over 1 week in either set of samples. Since we were concerned with relative differences in binding, absolute levels of gallium binding are not necessarily comparable with other human studies because the assay conditions, such as incubation times, differ.

Plasma citrate levels (normal range below 140 µmol/l) were measured by an enzymic method (Boehringer Mannheim) [24] by means of the unextracted protocol of Borland et al [25] modified for the 'Multistat III' microcentrifugal analyser (Instrumentation Laboratories Ltd), plasma iron (normal range 500-1680 µg/l) by the guanidine/'FerroZine' method (JT Baker BV), and plasma ferritin (normal range 4-330 µg/l) by radioimmunoassay (Becton Dickinson).

Plasma transferrin levels were measured by immunonephelometry on the Technicon Instruments DPA1. The normal range is 1.96-4.58 g/l. Transferrin iron saturation is the ratio between plasma iron and total iron-binding capacity (itself derived from the transferrin concentration, 1 mole being able to bind 2 moles of iron), and is normally between 20 and 40 %. Subtyping was carried out by a modification of an isoelectric focusing procedure [26]. Before electrophoresis, one volume of plasma was mixed with four volumes of rivanol (3 mg/ml). The mixture was saturated with ferric iron by the addition of iron nitrilotriacetate. The precipitated proteins were removed by centrifugation. 5 µl transferrin-containing supernatant was used for isoelectric focusing on polyacrylamide gels containing 3 % (by volume) LKB ampholine pH 4-6 and 2 % LKB ampholine pH 5-7. The gels (250 mm x 120 mm x 1.5 mm) were run for 3 h at 25 mA and a constant power of 25 W. The transferrin bands were detected by staining with coomassie blue. The genotype frequency in the general population is transferring-(1) 60.8 %, transferrin(2-1) 24.9 %, transferrin-(2) 2.6%, transferrin-(3-1) 9.4%, transferrin-(3-2) 1.9%, and transferrin-(3) 0.4%.

Group data are given as mean and SEM, with ranges where appropriate. Differences between grouped data were assessed by the Mann-Whitney U test, and correlations between variables by linear regression analysis [27]. A two-tailed significance value of p < 0.05 was taken as significant.

TABLE I. - COMPOSITION OF STUDY GROUPS
GroupnSex (M/F)Mean (SEM) age (yr)
Control 2211/1127.5 (4.0)
Alzheimer disease 133/1075.9 (3.0)
Down syndrome* 125/738.4 (7.0)
Stroke dementia 109/176.6 (2.0)
Haemodialysis**53/252.6 (6.0)
*Non-demented, all trisomy 21, **No evidence of cerebral impairment.

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Results

A bimodal distribution of gallium was observed in all test and control subjects, the two peaks eluting in similar fractions in all experiments. The first peak, a high-molecular-weight gallium-protein species (fractions measured for protein by the Lowry assay), corresponded to that of transferrin; the low-molecular-weight species (less than 4 kD) is as yet unidentified, although we presume it is citrate since that is the major species of non-transferrin-bound aluminium in man [20].

The mean (SEM) percentage binding of gallium to transferrin was similar in control (17.1 [1.58]%) and haemodialysis subjects (17.6 [2.38]%) and patients with stroke dementia (19.8 [1.45]%). The Alzheimer disease and Down syndrome groups showed significantly lower binding (7.92 [1.11]% and 6.92 [0.70]%, respectively) than the control group and there was no significant difference in binding between these two groups (see accompanying figure). There was no significant difference in age between the Alzheimer and stroke dementia groups, and there was no correlation between age and gallium-transferrin binding, either overall or in any of the groups. All citrate concentrations were within the normal range, and there were no significant differences among the groups.

The transferrin concentration was lower in the Alzheimer group than in the controls (table II), but the difference did not reach significance (p<0.07), whereas the difference between the Down syndrome and control groups was significant. However, the stroke dementia and haemodialysis groups had much lower transferrin concentrations (below the normal range), despite having normal gallium binding. Iron and ferritin levels (table II) were generally higher in patients with Alzheimer disease or Down syndrome than in control subjects, but because of the wide normal range this difference was not significant. Patients with stroke dementia had significantly lower iron and higher ferritin levels than controls, but the ferritin level was not significantly different from that of me Alzheimer disease group. There were no correlations between levels of transferrin, citrate, iron, or ferritin and gallium-transferrin binding.

Transferrin iron saturation (table II) was higher in the Alzheimer disease group and much higher in the Down syndrome group than in the control group, whereas the stroke dementia group had normal saturation. The results of transferrin subtyping showed a slightly different distribution of genotype from the general population in the combined group of Alzheimer disease and Down syndrome patients: 50% were transferrin-(1) homozygotes and 50% transferrin-(2-1) heterozygotes. However, this difference did not reach significance, possibly owing to the small numbers.

Table II. - Plasma transferrin, iron and ferritin concentrations and transferrin iron saturation
GroupnTransferrin (g/l)Iron (µg/l)Ferritin (µg/l)Transferrin saturation (%)
Controls152.98 (0.18)1540 (273)63 (11)39.0 (7.4)
Alzheimer disease 102.45 (0.20)2056 (770)133 (35)58.9 (20.2)
Down syndrome 142.44 (0.13)*2604 (509)124 (40)81.6 (14.2)**
Alzheimer + Down 242.44 (0.11)*2358 (437)127 (27)71.4 (11.9)*
Stroke dementia 101.74 (0.14)***865 (230)*164 (46)*33.4 (8.9)
Haemodialysis51.90 (0.10)ND120 (32)ND
*p < 0.05; **p < 0.01; ***p < O.OOO1 for difference from control group. ND = not done.


Percentile plots (percentiles 10, 25, 50, 75, 90) of gallium-transferrin binding in Alzheimer disease. Down syndrome, stroke dementia, and control groups.p values refer to differences between patients and controls.

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Discussion

Our results show that significantly lower amounts of gallium are bound to plasma transferrin in patients with Alzheimer disease and Down syndrome than in normal subjects or patients with stroke dementia. Consequently, greater amounts of gallium are present as a low-molecular-weight complex. De Wolffet al [28] observed a similar component in rat plasma and identified it as a citrate complex. In comparative experiments the two gallium species eluted in similar positions in both rat and human plasma [17]. We did not find any differences in plasma concentrations, of citrate. Although transferrin levels were slightly lower in the Alzheimer disease and Down syndrome groups than in the controls, this difference should not affect the capacity to bind gallium, both the stroke dementia and dialysis groups had a greater reduction in transferrin concentrations (often found in chronically ill people) but had normal gallium binding. This result is corroborated by the finding that transferrin saturation by iron was greater in the Alzheimer disease and Down syndrome groups than in the controls or stroke dementia patients, suggesting separately that there is an abnormality of transferrin function in both groups. All subjects had normal iron status: none had low iron stores, as measured by ferritin, or ferritin levels in the haemochromatosis range (several thousand (µg/l). The transferrin genotype distribution was slightly different from normal in the Alzheimer disease and Down syndrome group. The reduced binding of gallium, and by implication aluminium, to transferrin must be due to a functional defect of transferrin, to a very high-affinity, low-molecular-weight substance (much less likely in view of the transferrin iron saturation results), or to a third factor which changes the equilibrium between these two species. The high transferrin iron saturation may be secondary to the greater number of available binding sites owing to reduced occupation by gallium, aluminium, or other metals, or a primary factor leading to a reduction in the capacity of transferrin to bind gallium, aluminium, and other metals. In either case the result is consistent with a substantial change in transferrin function.

Previous studies with gallium and citrate have shown that a lipid-soluble species is formed between the two, and that there is enhanced transport across the intestinal membrane [15], as seen with aluminium [29]. It seems likely that a low-molecular-weight aluminium citrate species in plasma would move across the blood-brain barrier. Transferrin-bound aluminium transport is limited, since it is mediated by brain transferrin receptors [30] which are not increased in number in Alzheimer disease compared with age-matched controls [31]. Similarly, levels of transferrin in both grey and white matter are not abnormal in Alzheimer disease. Since Alzheimer disease and Down syndrome patients show similar neuropathology, it is possible that both have a predisposition to aluminium accumulation. Patients with stroke dementia, whose dementia is due to repeated episodes of cerebrovascular occlusion and who have very different neuropathological changes in the brain, had normal binding characteristics, as would be expected.

The finding that the few haemodialysis patients studied did not have the gallium-transferrin binding defect, despite slightly low total transferrin levels, may solve the question of why such patients do not show a high incidence of Alzheimer disease. Because transferrin has such a large capacity to bind aluminium normally, these patients remain reasonably well detoxified, and acute aluminium toxicity develops only when levels of exposure rise above the saturation point for transferrin or other binding proteins. In addition, we know little of the interaction between aluminium and silicon, which is present in the plasma of dialysis patients in much greater amounts than normal [32]. It is possible that the insoluble aluminium silicate complex which may form in the presence of non-transferrin-bound aluminium also helps to prevent neurotoxicity. This possibility is also pertinent to healthy people whose diet and drinking water contains substantial amounts of silicon [32].

In conclusion, we have found a substantial difference in the plasma distribution of gallium, and aluminium by implication, between patients with Alzheimer disease or Down syndrome and control subjects or those with stroke dementia. The study indicates a mechanism by which aluminium may accumulate in the brains of individuals with defective transferrin, thereby leading to neurotoxic changes seen in Alzheimer disease and Down syndrome. We suggest that people without this defect would be protected from the neurotoxic effects of aluminium. The presence of the defect could be used as a diagnostic test for Alzheimer disease and may prove to be useful in screening for susceptibility to the disorder, although we are investigating other, simpler, techniques to show these abnormalities.

The exact nature of the functional defect of transferrin remains unclear and we are undertaking studies to elucidate it further. Once the molecular defect is identified, it should be possible to explore its genetic basis, which would open the door to future preventive and therapeutic possibilities.


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