Binding of aluminium to plasma proteins: Comparative effect of desferrioxamine and deferiprone (L1)

ELSEVIER

Clinica Chimica Acta 230 (1994) 137-145

Binding of aluminium to plasma proteins: comparative effect of desferrioxamine and deferiprone (L 1) JosC L. Fernhdez-Martin, Pilar MenCndez-Fraga, M. Alejandra Canteros, J. Bernardino Diaz-Lbpez, Jorge B. Cannata-And~a* Unidad

de Investigacidn,

Metabolismo

Oseo y Mineral, (33080)

Oviedo.

Hospital

General de Asrurias.

Apdo 243,

Spain

Received 5 April 1994; revision received 15 July 1994; accepted 18 July 1994

Abstract Ultramicrofiltration techniques were used to study both the binding of aluminium to high molecular weight proteins in the presence of different concentrations of desferrioxamine and deferiprone (Ll) and the kinetics of aluminium release from human serum proteins. Human serum from healthy volunteers was used in all studies. The serum was spiked with aluminium (100 @g/l)and different concentrations of chelators. Ultramicrofiltration was performed with Amicon YMT membranes which had a nominal cut-off of 30 000 Da. Aluminium was measured by graphite furnace atomic absorption spectrometry in total serum and ultrafiltered fluid. Deferiprone shows a higher capability to displace aluminium from serum proteins (80%) than desferrioxamine (60%) at equivalent concentrations of the chelators. The kinetics of the release were also faster for deferiprone, taking 20 min to achieve its maximum effect, whereas, desferrioxamine achieved only 80% of its maximum effect after 2 h. Thus, deferiprone could be an attractive alternative to desferrioxamine, as an aluminium chelator agent. Keywords:

Aluminium;

Chelating agents; Deferiprone; Desferrioxamine;

Protein binding

1. Introduction Aluminium toxicity has been recognised as one of the most sitznificant moblems 1 patients with chronic renal failure who are undergoing haemodialysis ill.

in those

* Corresponding author, Tei.: 34-8-5106137;Fax; 34-8-5254509. 0009.8981/94/%07.000 1994 Elsevier Science B.V. All rights reserved SSDI

0009-8981(94)05918-I

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One way to remove aluminium from tissues is to administer desferrioxamine (DFO) to the patient [2]. Desferrioxamine is a trihydroxamide siderophore isolated from the bacterium Streptomycespilosus and is capable of chelating several metal ions to form complexes of varying stability. This drug was first used as a chelator in 1963 for the treatment of iron overload [3] and for aluminium intoxication in 1980 [4]. However its action mechanism is still unclear. Today it is known that serum aluminium is largely bound to high molecular weight proteins [5-71 and, even though most authors propose transferrin as the main carrier of the metal [8- 111,other proteins may be implicated [ 12- 141.Thus, aluminium and iron share common biochemical features, are carried by the same serum proteins and are chelated by the same drug (desferrioxamine) and ligands [ 151;some investigators have recently stressed this relationship [ 16- 171. When desferrioxamine is administered to patients, aluminium is removed from tissues and forms a tight complex (1: 1) of aluminium-desferrioxamine, low in molecular weight, easily ultrafiltrable and dialysable. Desferrioxamine is capable of displacing aluminium from transferrin also forming the same complex [ 181.This last aspect is also discussed by some authors who consider that the binding of aluminium to transferrin is very tight and cannot be dissociated by prolonged dialysis or desferrioxamine administration [9]. Others investigators, on the other hand, estimate that desferrioxamine treatment decreases aluminium bound by transferrin/albumin and increases that bound by an unknown protein termed albindin [14]. The use of desferrioxamine in dialysis patients is not free from side-effects [19]. In some patients, ocular toxicity, disturbances of colour vision and loss of hearing have been described. The most serious problems, however, are infections such as mucormycosis [20] even when a single dose is administered [21]. For this, and for other reasons (e.g. it is inactive orally), a great deal of interest has recently been shown in a new iron chelator, deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, Ll), as a possible alternative to desferrioxamine. Deferiprone is known to be effective in removing iron from both thalassemic patients [22] and iron-overloaded rats [23]. This drug form a neutral complex (1:3) with aluminium and is also effective in aluminium-overloaded rats with normal renal function and chronic renal failure [24]. The purpose of this study was, firstly, to evaluate in vitro the amount of ultrafiltrable aluminium in serum in the presence of different concentrations of desferrioxamine or deferiprone and, secondly, to evaluate the kinetics of aluminium release from human serum proteins. 2. Materials and methods 2.1. Samples Blood samples were obtained from healthy volunteers and placed in aluminiumfree plastic tubes. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983. The serum was stored at -20°C until required and then spiked with the desired amount of aluminium and chelators (desferrioxamine or deferiprone).

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2.2. Ultrafiltration Ultrafiltration studies were carried out with an Amicon micropartition system (MPS-1) (Amicon Div., Danvers MA) incorporating YMT membranes with a nominal cut-off value of 30 000 Da. Precautions were taken to avoid contamination of the samples with exogenous aluminium. All the plastic material of the micropartition system was immersed in 10% nitric acid for 24 h, and later rinsed in copious amounts of ultrapure water as indicated previously [251. YMT membranes contain large amounts of aluminium and exhaustive rinsing is therefore necessary. The washing process took place in the following order: ultratiltration of 1 mol/l sodium citrate solution several times, ultrapure water, 0.1 mol/l sodium hydroxide, ultrapure water again and finally 500 mg/l of desferrioxamine with copious amounts of ultrapure water. In order to check the membranes a blank serum sample from healthy volunteers (aluminium < 4 &l) spiked by 50 mg/l of desferrioxamine was ultrafiltered. The rinsing process was then repeated until the ultratiltered blank contained no detectable aluminium. For the ultrafiltration, 0.3-0.5 ml of serum was placed in the reservoir of the micropartition system and centrifuged for 15 min. Measurements were made for the presence of aluminium in total serum prior to centrifugation and then in the clear colourless fluid that passed through the membrane. The reproducibility of the ultrafiltration technique in these conditions is 2.8% for 60 fig/l of ultratiltrable aluminium (N = 8). Recovery of aluminium-desferrioxamine complex added to unspiked serum samples was 105.7 f 4.5% (N = 3). 2.3. Determination of aluminium Alumininm was measured by Electrothermal Atomic Absorption Spectrometry with the following Perkin Elmer equipment (Perkin Elmer Co., Uberlingen, Germany): a graphite furnace (Model HGA-600) coupled to an atomic absorption spectrophotometer (Model Z-3030) possessing Zeeman background correction, an autosampler (AS-60) and a PR-100 printer. The program for temperature maintenance and analytical conditions is shown in Table 1. The detection limit is 0.5 &l for aqueous samples and 1.0 &l for serum samples. Accuracy of the analysis of total serum aluminium was routinely monitored by the use of an internal and external quality control program. The external program was executed as part of an international Trace Element Quality Assessment Scheme organised by the University of Surrey, England. The coefficient of variation for analysis of total serum aluminium ranges typically between 2% and 5%. 2.4. Reagents An aluminium stock solution of 1 g/l (Standard solution, E. Merck, Darmstadt, Germany) was diluted ten times with 1:19 (v/v) nitric acid. Standard solutions were freshly prepared by direct dilution of the above solution with 1% nitric acid. High purity deionised water (specific resistivity 18 MWcm, NANOpure II, Barnstead Co., Boston, MA) was used to dilute serum samples (1: 1, v/v). This was checked daily for aluminium contamination.

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Table 1 Temperature program and analytical conditions for the measurement of aluminium in total serum and ultrafiltered fluid by graphite furnace atomic absorption spectrometry Step

Dry 1 Dry II Ash I Ash II Atomization Clean

Temperature (“C)

80 150 800 1300 2500 2700

Time (s) Ramp

Hold

5 15 IO 15 0

40 IO 10 10 4 3

I

Gas flow (mUmin)

Read

300 300 300 300 10 300

* -

Wavelength: 309.3 nm. Graphite tube: pyrocoated with L’Vov platform. Slit: 0.7 nm. *The lecture is performed during atomization.

Desferrioxamine (molecular weight 560) was obtained from Ciba-Geigy, Basle, Switzerland and deferiprone (molecular weight 139) was provided by Dr. George J. Kontoghiorghes of the Royal Free Hospital School of Medicine (London, UK). 2.5. Procedure The effect of different chelator concentrations was studied according to the following procedure: 5 ml of human serum obtained from healthy volunteers were spiked with 100 @I of aluminium and incubated for 24 h at 4OCto allow the binding of aluminium to plasma proteins. Serum was placed in eight plastic tubes (0.5 ml each) and different concentrations of chelators were then added. The concentrations of the chelators studied ranged between 0 and 40 pmol/l for desferrioxamine and 0 and 160 pmol/l for deferiprone. Taking into account the stoichiometry of the complexes, we used a range of equivalent concentrations (equivalent weight was consideFed as the weight of the chelator that combines with 1 g/mol of aluminium, 27 g). The samples were incubated at 37°C for 24 h and then ultrafiltered in a centrifuge at 2000 rev./min (= 750 x g). Measurements were then made of aluminium in total serum and ultrafiltration fluid. The kinetic study was also carried out in serum obtained from healthy volunteers and spiked with 100 &l of aluminium. Five millilitres of this serum were then spiked with 10 mgil of chelator (desferrioxamine or deferiprone) and the level of ultrafiltrable aluminium was measured at different times. All the experiments were performed at 37°C. 3. Results As shown in Fig. 1, deferiprone, in addition to desferrioxamine, was capable of displacing aluminium from high molecular weight proteins. Ultrafiltrable aluminium

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141

80

0

10

20

30

40

50

60

Chelator concentration@Et&) Fig. 1. Ultrafiltrable aluminium (%) versus chelator concentration (Eq = equivalents).

for both chelators were similar up to 10 pEq/l. However, when the concentration was greater than 10 PEqil (10 FM for desferrioxamine and 30 PM for deferiprone) the ultrafiltrable aluminium was higher for deferiprone. Both chelators followed the same model with a lower slope for high concentrations (> 10 pEq/l for desferrioxamine and > 20 pEq/l for deferiprone). Fig. 2 reveals that the mobilization of aluminium from high molecular weight proteins is very fast with deferiprone, the maximum effect being obtained after a few minutes. Desferrioxamine, on the other hand, has a slower response and reaches 80% of its maximum effect 2 h after its addition to the serum. 4. Discussion The transfer of aluminium during dialysis is limited as a result of its binding to plasma proteins of high molecular weight, e.g. transferrin. As a consequence it is not dialysable and the only dialysable fraction is the ultrafiltrable serum aluminium (5-10%) [26]. When desferrioxamine is administered, the removable aluminium increases because the chelator mobilises the aluminium from the tissues and from high molecular weight proteins, thus producing an increase in total serum aluminium at the expense of the desferrioxamine-aluminium complex [27] which is easily ultrafiltrable due to its low molecular weight. Our results demonstrate that deferiprone, in addition to desferrioxamine, was capable of displacing aluminium from high molecular weight proteins: this finding is in agreement with previous reports [28]. Taking into account the stoichiometry of the complex (1: 1 for desferrioxamine and 1:3 for deferiprone), in our comparison we have used equivalent concentrations for both chelators. The results demonstrate that

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40

olt-. .@ 0

1 46

60 120*24 Minutes

HOUfS Tii

100 1 1

60 $f

60-

8 &

40-

3

20:

0

4

l

(4

N=4

!.,_,v,.,_,.,_, 060120 Minutes

46

24 HoUfS

Time Fig. 2. Ultrafiltrable aluminium (X) versus time after the addition of the chelator (A, desferrioxamine; B, deferiprone).

three times higher molar concentrations of deferiprone, but equivalent and thus comparable with desferrioxamine, obtained a higher ultrafiltrable aluminium. The effkiencies for both chelators diminish at high concentrations. This finding reinforces the necessity of using lower doses. As the figure shows, there would not be any advantage in using concentrations greater than 20 pEq/l (60 pmol/l) for deferiprone and 10 FEqll (10 pmol/l) for desferrioxamine. In this way, some sideeffects of the chelators could be avoided without significant loss of ultrafiltrable aluminium. A second point of great interest is the speed of generation of the ultrafiltrable aluminium. Desferrioxamine has been currently administered 44-48 h before dialysis, thus having enough time to generate ultrafiltrable aluminium. Nevertheless, we have

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recently demonstrated [29,30] that by using desferrioxamine 1 h before dialysis we avoid unnecessary increments in serum aluminium between dialyses, with less risk of adverse effects [31,32] without losing efficiency in the removal of aluminium. Moreover, if the level of serum ferrioxamine is the growth determinant for murcomycosis [33], then, in order to keep this to a minimum during the inter-dialysis interval, desferrioxamine would be best administered at the beginning of dialysis at the smallest effective dose [34]. As this new strategy has been shown to be valid and it now can be used with desferrioxamine and probably, in the future, with deferiprone, we need to ensure that both chelators are able to displace aluminium from high molecular weight proteins rapidly. One of the most important findings in our study is related to the speed of complex formation. The kinetics of the release of aluminium from high molecular weight proteins was found to be faster with deferiprone than with desferrioxamine. This was possibly due to the small size of the former and consequently to lesser steric impediments. Thus, if in the future deferiprone becomes available for clinical use, its administration at the beginning of the dialysis will guarantee an early removal of aluminium from blood. However, the use of this drug is not free from side-effects and some cases of agranulocytosis, zinc deficiency and systemic lupus erythematosus have been observed in iron-overloaded patients under chelating therapy with doses greater than 100 mg/kg per day [35,36]. On the other hand, good tolerance of the drug was observed when it was used in renal dialysis patients at doses of 40 mg/kg [37]. In addition to previous studies [24] in which we showed the beneficial effect of deferiprone in removing aluminium from rats, the results presented in this paper demonstrate that deferiprone promotes a great increase in the ultrafiltrable aluminium and that it could be a valid alternative chelator for patients undergoing longterm haemodialysis. Acknowledgements This study has been supported by FIS 9110329 and by the IRSIN (Instituto Reina Sofia de Investigacibn Nefrolbgica) supported by the Fundaci6n Renal Iiiigo Alvarez de Toledo. Thanks are due to Dr. G.J. Kontoghiorghes for providing the deferiprone. References [I] Wills MR, Savory J. Aluminium poisoning: dialysis encephalopathy, osteomalacia, and anaemia. Lancet 1983;2:29-34. [2] Malluche HH, Smith AJ, Abreo K, Faugere MC. The use of deferoxamine in the management of aluminium accumulation in bone in patients with renal failure. N Engl J Med 1984;31I: 140-144. [3] Keberle H. The biochemistry of desferrioxamine and its relation to iron metabolism. Ann NY Acad Sci 1964;119:758-768. [4] Ackrill P, Ralston AJ, Day JP, Hodge KC. Successful removal of aluminium from patients with dialysis encephalopathy. Lancet 1980;2:692-693. [S] King SW, Wills MR, Savory J. Serum binding of aluminum. Res Commun Chem Pathol Pharmacol 1979;26:161-169.

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