9 1994 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/94/4101-2-0129 $03.00

Determination of Aluminum Levels in the Kidney, Liver, and Brain of Mice Treated with Aluminum Hydroxide GOr~hL ~AHIN, *'1 I. VAROL,2 A. TEMIZER, 2 K. BENLI, 3 R . DEMIRDAMAR, 4 AND S .

DURU 1

'Department of Pharm. Toxicology; 2Department of Analytical Chemistry; 3Department of Neurosurgery; and 4Department of Pharmacology, Hacettepe University, 06100 Ankara, Turkey Received August 3, 1993, Accepted September 21, 1993

ABSTRACT In the present study, aluminum (A1) accumulation has been examined after aluminum loading in mice. The kidney, liver, and brain aluminum levels for mice that had been treated orally with aluminum hydroxide for 105 d and for the control group were determined using graphite furnace atomic absorption spectrophotometry (GFAAS) following an acid digestion. Matrix modifier consisted of 2% Triton X-100 and 2% Mg (NO3)2. A1 loaded mice showed a significant increase in tissue aluminum levels, relative to the control group. Index Entries: Accumulation of aluminum; graphite furnace atomic absorption; acid digestion bomb; matrix modifier.

INTRODUCTION A l u m i n u m is the third most a b u n d a n t e l e m e n t in the o u t e r crust of the earth, occurring as various aluminosilicates. Because its w i d e use in i n d u s t r y a n d medicine, this is an e l e m e n t to w h i c h h u m a n s are highly e x p o s e d . A l u m i n u m is also i m p o r t a n t b e c a u s e of its role in the etiology of s o m e disorders, such as dialysis e n c e p h a l o p a t h y , o s t e o m a l a c i a , and A l z h e i m e r ' s disease (1-4). *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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The clinical syndrome of a l u m i n u m toxicity was first recognized in 1972 as the dialysis encephalopathy s y n d r o m e (DES) in patients undergoing hemodialysis. In 1976, brain tissue of adult patients who died from a progressive neurological disorder while on hemodialysis was reported to contain large quantities of aluminum. A progressive encephalopathy similar to the one observed in adults was d e v e l o p e d in five children w h o were not in dialysis in 1977. Each child had a congenital type of renal disease, and developed renal failure within a few months of birth, after receiving a large a m o u n t of a l u m i n u m - c o n t a i n i n g p h o s p h a t e binders for months to years. Plasma At levels were found to be elevated in two of these children (4). The a m o u n t of a l u m i n u m absorbed through the gastrointestinal tract is still open to discussion, but it is generally accepted that the amount absorbed is about 10% of the total A1 ingested (1,2). Although the biochemical basis of A1 intoxication is not completely understood, A1 is known to interact with a n u m b e r of proteins and with cofactors that are involved in intermediary metabolism (4). A1 combines with a d e n o s i n e triphosphate (ATP) to form A1-ATP, a competitive inhibitor of hexokinase; inhibits catechol-O-methyl transferase, ceruloplasmin, cholinesterase, choline acetyl-transferase, glycerokinase, Mga d e n o s i n e triphosphatase, calmodulin, and activates adenylate cylase and D-aminolevulinic dehydratase (4). In this study, we have examined whether oral administration causes changes in the amounts of a l u m i n u m in various tissues of mice or not. A l u m i n u m levels of the liver, kidney, and brain in mice treated with alum i n u m hydroxide for 105 d and in the control group were determined using GFAAS after acid digestion.

METHODS Instrumentation Determination of aluminum was performed on a Perkin-Elmer 3030 Spectrometer (Perkin-Elmer [Norwalk, CT]), an HGA 400 electrothermal furnace, and a Perkin-Elmer pr-100 recorder. A Perkin-Elmer a l u m i n u m hollow cathode lamp was operated at 25 mA. The atomic absorption of a l u m i n u m was monitored at 309.3 nm, the spectral b a n d w i d t h was 0.7 nm, and background correction was performed using a d e u t e r i u m arc. The electrothermal furnace was p u r g e d with prepurified argon d u r i n g operation. The external argon flowrate was 900 m L / m i n and the internal argon flowrate was 310 m L / m i n . Gas "stop condition" was used only d u r i n g the atomization cycle. A Perkin-Elmer AS-40 autosampler was used to deliver 20 ~tL aliquots of solution. Pyrolytically-coated graphite tubes (id 5.8 mm-od 8.00 m m and length 28.0 mm) were used.

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Reagents and Solutions T h r o u g h o u t the study, triply distilled water was used for w a s h i n g and for the preparation of standards. S u p r a p u r e grade nitric acid (Merck [Darmstadt, Germany]) was u s e d to acid-leach containers a n d for the pre-treatment procedure. A dilute solution of triton X-100 (Sigma) was u s e d t o g e t h e r w i t h m a g n e s i u m nitrate (extra pure, Merck) as matrix modifier. In order to prepare the matrix modifier, 2.0 g m a g n e s i u m nitrate (Mg (NO3)2.6H20) was mixed with 2 mL concentrated Triton-X 100 a n d the v o l u m e was adjusted to 1000 mL w i t h water. It w a s then transferred to polypropylene bottles. A 1000 p p m certified a l u m i n u m atomic absorption reference solution (Fisher Scientific [Pittsburgh, PA]) was u s e d to prepare s e c o n d a r y standards. Dilute a l u m i n u m solutions were prepared daily.

Experimental Design In this study, male Swiss albino mice (20-23.38 g) were used and fed a commercial standard diet. The mice were divided into 2 groups: Group 1 (control group, n = 9) received a commercial standard diet a n d drank distilled, deionized water. Group 2 (n = 9) also received a standard diet a n d d r a n k distilled, d e i o n i z e d w a t e r s u p p l e m e n t e d w i t h a l u m i n u m hydroxide gel (provided from Wyeth Co.). The water s u p p l e m e n t e d with the gel contained 4.0 ___1.7 gg A1/mL. Each animal had 5.0 + 1.8 g commercial standard diet and drank 5.0 _+ 1.2 mL water daily. For statistical analysis, the Mann-Whitney U Test was used.

Sample Preparation For a l u m i n u m analysis of the brain, liver, a n d kidney, a 50-mg aliquot of tissue was d i s s o l v e d at 150~ for 45 rain in acid d i g e s t i o n b o m b (Parry-4547) with 2 mL of c o n c e n t r a t e d u l t r a p u r e nitric acid (Merck). In 6 separate p o l y p r o p y l e n e tubes, 0.050 mL of each digested tissue s a m p l e was mixed with 0.100 mL of matrix modifier. After this, starting with second tube 0.025, 0.050, 0.075, 0.100, 0.150 mL of 200 ppb s e c o n d a r y stock A1 solution were a d d e d , in an order. All tubes were filled u p to 0.500 mL with triply distilled water. The mixtures were transferred to the automatic sampler of the graphite furnace and the results were taken from the printer after starting the furnace program.

RESULTS Table 1 shows the furnace p r o g r a m that was obtained by optimization studies for m e a s u r e m e n t s of A1. The control g r o u p had lower tissue A1 levels than those of the group treated with A1 (p < 0.05). Table 2 indi-

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132 Table 1 The Furnace Setting for Aluminum Measurement Phases

Values

Drying temp. 1, ~ Time, ramp/hold, s Drying temp. 2, ~ Time, ramp/hold, s Ashing temp, ~ Time, ramp/hold, s Atomization temp., ~ Time, ramp/hold, s Cleaning temp., ~ Time, ramp/hold, s

90 15/10 120 15/10 1500 25/15 2650 0/5 2700 1/3

Table 2 Avarage Aluminum Contents of Various Tissues for the Control Group and for the Mice Aluminum Treated with A1 Hydroxide for 105 d Biological material Liver Kidney Brain

Control group, n = 9 ~tmol/g

A1 treated group, n = 9 ~tmol/g

17.69 _+4.51 14.28 _+5.41 0.32 + 0.16

28.63 _+6.37 18.13 _+4.75 1.41 -+ 0.40

cares the average A1 contents of three tissues for the control group and for the mice treated with A1 for 105 d. The a l u m i n u m measurements in A1 loaded mice showed increases in all tissues; approx 30% in the kidney, 60% in the liver, 340% in the brain.

DISCUSSION The present s t u d y shows that oral administration of a l u m i n u m is effective in altering the amounts of a l u m i n u m contents of various tissues of mice (Table-2H) We have found especially that the mice treated with a l u m i n u m hydroxide had significantly higher A1 levels in the brain samples than those of the control group (Table-2H). Since AI3+ is neurotoxic, brain A1 metabolism is most important. The normal and lethal toxic brain levels of A13+ are well documented. The normal brain uptake of A13+ is very slow. A13+ cannot be eliminated by the brain and is therefore accumulated (3). The d e t e r m i n a t i o n of a l u m i n u m in biological materials is complicated by the low concentration of the element, the limited a m o u n t s of Biological Trace Element Research

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sample available, and the matrix interferences. Several methods are used to determine the a l u m i n u m in biological fluids, including neutron activation (5), spectroflourimetry (6), inductively coupled atomic emission spectroscopy (7), graphite furnace atomic emission spectroscopy (8), and graphite furnace atomic absorption spectroscopy (8-10). Neutron activation analysis is hindered by the severe nuclear interference reaction 31p (n, c~) 28AI, and the short half-life of 28A1 isotope (11). Spectroflourimetry is sufficiently sensitive. Nevertheless, such methods are time-consuming and increase the risk of sample contamination. Graphite furnace atomic absorption spectrometry (GFAAS) is sufficiently sensitive to detect the ultralow levels of metals found in biological samples (12). In most cases, GFAAS is the method of choice because of its high sensitivity, specificity, scope, speed of measurement, comparatively low cost of the apparatus, and low cost of analysis with respect to neutron activation analysis, and the potential of direct analysis. In this study, the temperature and duration of the sample drying are crucial factors in eliminating the negative interference associated with chloride in aqueous solutions and the tendency for enhanced recovery of a l u m i n u m in biological samples. The temperature program used by various workers show wide variations (13). It was found that both of the abovementioned factors can be controlled by using 2-step drying at 90 and 120~ together with the r a m p and hold times of 5/10 s. Table 1 shows the furnace program that was obtained by careful optimization studies. Drying temperature over 120~ led to rapid and uneven boiling of the sample and consequently poor precision. Some workers offered two ashing steps at 800 and 1500~ (14), 450 and 1500~ (12), 400 and 1400~ (15,16), 400 and 1500~ (8), 700 and 1500~ (17), or omission of the ashing stage (18). We have found that for the case of the tissues A1 determination one ashing step at 1500~ is the best choice. GFAAS sensitivity for A1 increase with increasing atomization temperature. The high atomization temperature, however, lowered tube life and caused loss can be m i n i m i z e d by using a temperature b e t w e e n 2500-2700~ and by the use of pyrocoated graphite tubes. The automatic sampler, m a d e of Teflon| material enhances precision and obviates the contamination that can occur w h e n manual injection performed. Contamination has been minimized by the use of high-purity chemicals, alum i n u m - f r e e water, and working in the laminar flow. Acid digestion procedure increased reproducibility and accuracy. There is little agreement concerning the extent of sample pretreatment and the choice of instrumental parameters required for the analysis of the tissues samples. Mg (NO3)2 (19,20), Triton X-100 (16), nitric acid (21), Triton X-100 plus Mg (NO3)2 (22), and other less c o m m o n matrix modifiers such as EDTA, NH3, and H2SO4, and different surfactants (14) can be used. In order to overcome or minimize the matrix effect, all the possibilities were tried, and Triton X-100 plus Mg (NO3)2 were found the best for our purposes. Matrix modifier mixture convert Biological Trace Element Research

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the sample to less volatile c o m p o u n d s that are not lost in the thermal pretreatment step and that are atomized only after the furnace tube has reaches a steady temperature. The analytical recovery of a d d e d alum i n u m in the samples have been found as 100.91 + 2.61%, variation coefficient of 5.62 g m o l / L A1 has been found as 1.64%. The results were compared with those by use of the nonpyrolytically covered graphite tubes. It has been found that the use of pyrolytically coated tube has at least three a d v a n t a g e s over conventional uncoated tubes for the determination of a l u m i n u m in biological materials. Pyrolytically coated tubes attain higher temperatures and longer lifetimes, and, most importantly, the injected samples do not permeate deep into the pores of the tube. Pyrolytic coating of the graphite tube provides m i n i m i z i n g of surface interaction of the a l u m i n u m with the porous graphite surface. In conclusion, the kidney, liver, and brain a l u m i n u m levels in mice that had been treated orally with a l u m i n u m hydroxide for 105 d control mice and in were determined using graphite furnace atomic absorption spectrophotometry. The a l u m i n u m m e a s u r e m e n t s in A1 loaded mice s h o w e d increases in all tissues; approx 30% in the kidney, 60% in the liver, 340% in the brain. Our previous study also indicated that the bone a l u m i n u m levels of the mice fed the a l u m i n u m c o m p o u n d s were significantly greater than the control group (23). It is possible that A13+ may cause or contribute to some specific diseases.

SUMMARY In the present study, A1 levels in the tissue samples obtained from Al-loaded mice and from control group were examined by the GFAAS. The tissues utilized in this study were the liver, kidney, and brain of mice. The technique employed is free from major matrix interferences and provides high precision and accuracy. It was also observed that alum i n u m might accumulate in the liver, brain, and kidney in mice after oral A1 administration.

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