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Plasma levels of aluminium after tea ingestion in healthy volunteers
Fd Chem. Toxic. Vol. 31, No. 1, pp. 19-23, 1993
0278-6915/93 $6.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
PLASMA LEVELS OF ALUMINIUM AFTER TEA INGESTION IN HEALTHY VOLUNTEERS P. N. DREWITT, K. R. BUTTERWORTH, C. D. SPRINGALLand S. R. MOORHOUSE BIBRA Toxicology International, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK (Accepted 21 May 1992) Abstract--12 healthy volunteers on a controlled aluminium (A1) diet each consumed a tea infusion
(500 ml/70 kg body weight), with either milk or lemon juice as additives, or mineral water, following a three-way crossover design. The concentrations of AI were determined in the diet, mineral water and tea infusions, and in plasma samples collected before and up to 24 hr after consumption of tea or water, using graphite-furnace atomic absorption spectrophotometry or inductively coupled plasma emission spectrometry. Consumption of up to 1.60 mg A! from tea with milk or lemon juice did not increase plasma A1 levels compared with consumption of approximately 0.001 mg AI from mineral water. The results suggest that, in the short-term, drinking tea does not contribute significantly to the total body burden of A1.
INTRODUCTION
MATERIALS AND METHODS
Although aluminium (A1) has been shown to possess a potential for systemic toxic/ty (Alfrey et al., 1976; Wills and Savory, 1983), its low absorption from the gastro-intestinal tract (Ganrot, 1986) and its rapid urinary excretion (Alfrey, 1989; Lote and Saunders, 1991) suggest that in normal healthy individuals the bioavailability of A1 is too low to present a risk to health. However, the demonstration of an association of A1 with neuritic plaques (Candy et al., 1986) and neurofibrillary tangle-bearing neurones (Perl and Brody, 1980), the brain lesions that characterize Alzheimer-type dementia, has raised public anxiety about a possible insidious role for dietary A1 in the aetiology of this neurodegenerative disease. This concern has prompted studies of all sources of intake of the metal, including that from food and beverages (Delves et al., 1989). Tea leaves contain high natural levels of AI, since the tea bush accumulates large quantities of A1 from acid soil (Prescott, 1989). Black tea, as sold, contains 500-1500mg A1/kg, and the prepared beverage has been found to contain 2-6 mg Al/litre (Fairweather-Tait et aL, 1987; Flaten and Odegard, 1988). Koch et aL, (1988) have reported that urinary excretion of AI increases when tea is consumed, suggesting that some of the A1 present in tea is absorbed. Since tea could be an important dietary source of AI in the UK, a human study was undertaken to investigate the absorption of AI from black tea infusions with added milk or lemon juice. The latter was examined since citrus juices and citric acid have been shown to enhance dietary AI absorption (Slanina et al., 1984 and 1986; Weberg and Berstad, 1986).
Reagents. AristaR grade nitric acid (70%, w/w) and magnesium nitrate were purchased from BDH (Poole, Dorset, UK), together with AnalaR grade distilled deionized water, SpectrosoL grade aluminium nitrate standard (1000 /~g/ml) and hydrochloric acid (36%, w/w). Triton X-100 was obtained from Sigma Chemical Co. (Poole, Dorset, UK). Serum samples from the Robens Institute Trace Element Quality Assessment Scheme (University of Surrey, Guild(oral, UK) were used as the quality control specimens for determination of A1 in plasma. Diets and toiletries. Volunteers were given meals selected from the Birds Eye 'Menu Master' range. These were stored at - 2 0 ° C prior to cooking. On each leg of the study all volunteers had the same meals at designated times. Fresh tea infusions were prepared on each of the three study periods by adding 31.25 g tea (10 tea-bags of a typical blend used in the UK to 3000 ml boiling still Malvern mineral water (Schweppes International Ltd, UK). After stirring for 5 rain with a plastic spatula, each tea infusion was decanted and either 75 ml fresh semi-skimmed milk or 30 ml PLJ lemon juice (Beecham Products, Brentwood, UK) and 45 ml Malvern water were added. The control solution consisted of boiled, still Malvern mineral water. All dosing solutions were kept hot in vacuum flasks prior to use. A 5-mi aliquot of each test solution was kept at - 2 0 ° C until analysed for AI content. Toileteries prepared from low Al-containing compounds were used during the study [Impulse and Lynx deodorants, and Signal toothpaste (Elida Gibbs, London, UK)]. Subjects. 12 volunteers (six male and six female) were judged to be healthy from a detailed family and medical history and a comprehensive medical
Abbreviation: A1 = aluminium. 19
20
P.N. Dgswirr et al.
examination, which included serum chemistry, haematology and urinalysis profiles. The volunteers, aged between 25 and 54yr, were within 15% of the desirable weight range for their height and were further approved by an informed clinical judgement within 14 days before study commencement. Each female volunteer gave details of her recent menstrual history and was shown not to be pregnant using a Pregnosticon Planotest urinary pregnancy kit (Organon Teknika BV, Oss, The Netherlands). Experimental Procedure. Prior to study commencement, approval to proceed was obtained from the BIBRA Ethics Committee. All clinical procedures were in full compliance with Good Clinical Practice (non-clinical procedures conforming to the internationally accepted standards of Good Laboratory Practice and audited by the independent BIBRA Quality Assurance Unit). 1 wk before and during each of the three study periods, volunteers used only toiletries with a low A1 content. Identical diets were consumed during the 24-hr period prior to study commencement and during each study period. Samples of the diets were retained for analysis of A1 content. Mineral water was provided (150 ml at hourly intervals) during the first 7 hr after dosing. Thereafter, decaffeinated coffee or mineral water was available ad lib. The study followed an open threeway crossover design. Volunteers received either mineral water, tea with milk or tea with lemon juice (500 ml/70 kg body weight) on each of the three study periods, which were separated by 2 wk to prevent carry-over. The order of dosing was in accordance with a randomization schedule. Blood samples were collected by repeated venepuncture, using 5-ml B-D Plastipak disposable syringes (Becton Dickinson, Dublin, Ireland), immediately before dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hr post-dose. Blood samples were transferred to 5-ml lithium-heparin tubes (Teklab, Co. Durham, UK) prior to mixing on a roller mixer for 10min. Plasma was obtained following centrifugation at 3000 rpm at 4°C for 5 min in a refrigerated centrifuge and then transferred in duplicate to 5-ml plain storage tubes (Teklab) using a Gilson pipette with acid-washed disposable tips. All plasma samples were stored at - 2 0 ° C until A1 analysis. Aluminium contamination. Sample contamination is a potentially serious problem because of the ubiquitous distribution of A1 compounds in the environment. A careful and meticulous regime of sample collection and storage was adopted to minimize extraneous AI contamination. Before venepuncture, the forearm of each volunteer was cleaned with an alcohol Medi-Swab (Smith and Nephew Ltd, Hull, UK), and care was taken not to touch the skin during sampling. Preliminary work included verification that the disposable syringes, lithium-heparin tubes and plain storage tubes were not sources of AI contamination (Hall et al., 1988). Before use, disposable plastic pipette tips were washed three times with nitric
acid (10%,v/v) and then rinsed three times with deionized filtered water. Aluminium determination. Concentrations of AI in plasma, tea and mineral water were determined using an atomic absorption spectrometer (model 2380) equipped with a graphite furnace (HGA-400) and an automatic sampler (AS 40; Perkin-Elmer, Beaconsfield, Bucks., UK). All steps of sample preparation and measurement were performed in an isolated dust-free environment. Polypropylene analyser cups (0.5 ml; BCL, Lewes, Sussex, UK) and caps, disposable pipette tips and other plasticware were washed with nitric acid (10%, v/v) and rinsed with distilled deionized water before use to minimize AI contamination. Working standards were prepared daily by serial dilution of a substock (10#gA1/ml) of the certified AI standard with AnalaR grade distilled deionized water. Aliquots of 100#1 working standards were added to 300#1 diluent solution in analyser cups. The diluent consisted of AnalaR grade distilled deionized water containing magnesium nitrate (1 or 2 g/litre) and nitric acid (70%, 2 ml/litre). Test samples and quality control specimens were similarly diluted (1:3, v/v) with the diluent solution. All solutions were capped and mixed by inversion. The A1 content of the samples was determined using the instrument conditions shown in Table 1. Determinations were made in duplicate or triplicate, with two quality-control samples analysed in duplicate at the start of a sample run, and after every five or six test samples. Between samples the autosampler probe was flushed with a solution of distilled deionized water containing Triton X-100 (1 ml/litre) and nitric acid (5 ml/litre). Analyses were accepted if valid results were obtained for the concurrently analysed quality control samples. An inductively coupled plasma emission spectrometer (Perkin-Elmer Plasma 40) was used for the determination of A1 in food samples. Working standards were prepared by adding 50 or 100 #1 A1 standard to 100 ml HC1 (10%, v/v). HC1 (10%, v/v)
Table 1. Conditions used for the determination of AI in plasma, tea infusions and mineral water by atomic absorption spectrometry Step number
Temperature (°C)
Ramp time (see)
Hold time (see)
1 2 3 4 5 6 7 8
100 120 40O 650 1600 2650 2700 20
5 20 10 10 10 0 I 1
45 or 65 10 30 10 25 or 35 4 or 5 3 10
The instrument parameters used were: wavelength, 309.3; lamp current, 22mA; spectral slit width, 0.7 nm; signal, concentration; mode, peak area; and background correction, Deuterium Arc System. • An aluminium hollow cathode lamp and pyrolytically coated graphite tubes were used, with 10- or 20-/zl sample aliquots. Argon was used as the purge gas with internal flow interrupted during atomization (step 6).
Plasma levels of aluminium after tea ingestion was used as the blank solution. The food samples were first homogenized, and 20-g aliquots were then heated gradually to charring before being placed in a muffle furnace at 550°C for 16 hr. After cooling, sufficient HC1 (50%, v/v) was added to wet the ash, followed by reheating to dryness. When cool, 10 ml HCI (25%, v/v) was added, and the solution was brought to the boil before being filtered through a Whatman 541 filter paper. The latter procedure was repeated several times before the filter paper and funnel were washed with deionized filtered water. The final volume was made up to 50 ml and then analysed for AI content using the following instrument parameters: argon flow, 3.5 litres/min; read delay, 70sec; replicates, 3; wavelength, 308.215 nm; photo-mnitiplier tube (PMT), 900 v; element time, 500msec; standard 1, 0.500/zg/ml; and standard 2, 1.000/tg/ml. Standards were analysed at the start of a sample run, and approximately after every six samples. Statistical analyses. Plasma AI levels were statistically analysed at all time points using a one-sided paired t-test, with the mineral water group v. the two tea groups. For pre-dose results a two-sided paired t-test was performed. RESULTS
Quality control Serum samples supplied monthly by the Robens Institute Trace Element Quality Assessment Programme (University of Surrey, Guildford, UK) were analysed to check the accuracy and precision of the method for AI determination in plasma and serum and to assess laboratory performance on an ongoing basis. These samples were also used for internal quality control. Performance indices are based on the relative deviations of laboratory results from consensus target values, with a highest possible score of 2 points (i.e. a difference of < 10% of the target value at 4.0/~mol/litre, and < 20% of the target value at 1.0/zmol/litre on a sliding scale). The six-monthly report from the Robens Institute, which covered the present study, showed that all performance indices had a value of 2 and ranked our laboratory first of the 111 participating laboratories, indicating that the analytical methodology was satisfactory. The detection limit for AI in serum and plasma was found to be 0.03 #mol/litre (0.9/zg/litre) and the linear range was up to at least 100/tg/litre.
Aluminium input from diet and dosing solutions Table 2 shows the mean AI content of the meals consumed by the volunteers. During each of the three study periods, the volunteers were on the same diets and therefore ingested similar quantities of AI from food. The mean AI intake per individual serving was between 0.380 and 0.883 mg. The concentrations of AI in the two tea infusions and mineral water at each leg of the study are shown in Table 3. The mean total
21
Table 2. Mealscheduleand AI content(mg)per serving used in the study on plasma levelsof aluminiumafter tea i n g e s t i o n in healthyvolunteers Mean AI content Time per serving (br)
24 19.30 14 - 10
-
0 +4 + 10
Meal
(mg)
Breakfast Lunch Dinner Supper Tea or mineral water ingested Lunch Dinner
0.782 0.883 0.620 0.380 Total 2.665 0.782 0.450 Total 1.232
amount of AI ingested by volunteers between 0 and 24 hr were: tea with milk, 2.329 mg; tea with lemon, 2.808 rag; and mineral water, 1.233 mg.
Plasma aluminium levels The concentration of AI in plasma sampled before treatment was 4.21 _ 0.58 gg/litre (mean _ SD, n = 36). This value is comparable with recent published estimations (Delves et al., 1989; Slavin, 1986; Wang et al., 1991]. Figures 1 shows mean plasma A1 concentrations at various times after consumption of either mineral water or tea (with milk or lemon), At no time did the concentration of AI found in the plasma of the two tea-drinking groups differ significantly from that in the mineral water (control) group. DISCUSSION An average of 0.001, 1.097 or 1.576 mg A1 was ingested by the volunteers when they drank mineral water, tea with added milk or tea with lemon juice, respectively. In addition, the volunteers received A1 from the standardized diets. Although the A1 content of the selected meals ranged between 0.380 and 0.833 nag, the intake of A1 from the diet was the same on each occasion for all the participants. Thus, in this study the consumption of 500 ml (about two cups) tea/70kg body weight almost doubled the dietary intake of AI by the volunteers. However, this did not increase plasma AI levels monitored over a 24-hr period. This finding suggests that the bioavailability of AI in tea is low, and that in the short term drinking tea with added milk or lemon juice does not contribute significantly to the total Table 3. AI concentrationsin dosing solutions used duringthe studyon plasmalevelsof aluminiumaftertea ingestionin healthyvolunteers AI concentration(mg/litre) during study periods Dosing
solution I 2 3 Mineralwater 0.0029 0.0026 0.0027 Tea with milk 2.0050 2.1060 2.4290 Tea with lemon 2.9850 3.1110 3.2830 Volunteers consumedthe dosing solutionsat 500ml/ 70 kg body weightduringstudy periods 1, 2 and 3.
22
P. N. DREVCITT et al.
! r - MIMrll
oilllll i I ! I- Tea with milk
I
I
In conclusion, drinking 500ml tea (containing 1-1.6 mg natural AI) per 70 kg body weight does not increase plasma AI concentrations, suggesting that AI ingested from short-term tea drinking does not contribute significantly to the total body burden of this metal. Acknowledgements--We gratefully acknowledge the advice
i i
0 IIIIII I I 6 ,-- To,, with lemon
I
I
,
oilllll,, 01334
I !
I 8
I 13 Time (hi')
I 34
Fig. 1. Mean plasma AI concentration (#g/litre) before and after drinking tea with milk, tea with lemonjuice or Malvern mineral water (500 mi/70 kg body weight). Each point is the mean of 10 to 12 estimations + SEM. Volunteers were given tea or mineral water at 0 hr, immediately after giving a blood sample. When time points were compared, there were no statistically significant differences (P < 0.05) in plasma levels between the three groups.
plasma AI concentration. Support for this observation is provided by recent animal work (Fairweather-Tait et al., 1991), which showed that there was no increase in blood and liver A1 levels in rats that had consumed tea as the only source of fluid intake for 28 days. As Flaten and Odegard (1988) have pointed out, it may be that most of the extractable A1 in brewed teas is strongly bound to organic species (e.g. theaflavins, thearubigins and other polyphenolic 'fermentation' products) and that these high molecular weight complexes are not readily absorbed from the gastrointestinal tract. Recent work (French et al., 1989) has suggested that complexation plays an important part in A1 speciation in tea, even under simulated gastric conditions. Other investigators have reported that tea 'tannins' reduced the absorption of gallium-67, which is a marker for A1, in both nourisbed and starved rats (Farrar and Blair, 1989), suggesting that components in tea may have an inhibitory effect on A1 uptake from other dietary sources. The adverse effect of tea on iron bioavailability is well known (Disler et al., 1975; Fairweather-Tait et al., 1991). The apparent failure of citrate to augment A1 absorption and increase plasma levels in the present study may also be due to effective sequestration of the metal by components in tea. It is perhaps worth noting that in most studies where the chelating effect of citrate has been observed, relatively large doses of Al-containing compounds and citrate had been used (Slanina et al., 1986; Weberg and Berstad, 1986), often for extended periods, before observations were made (Slanina et al., 1984 and 1986).
and comments of Dr P. Collier and Dr R. Wilson (Unilever Research), and the Analytical Section of Unilever Research (UK) for their expert analysis of AI in the diets. The efforts of Miss L. Norris in the typing of this manuscript are very much appreciated, and we also acknowledge the valuable assistance of the BIBRA Statistical Department.
REFERENCES
Alfrey A. C. (1989) The physiology of alurninium in man. In Aluminium andHealth. Edited by H. J. Gitelman. pp. 101-124. Marcel Dekker, New York. Alfrey A. C., Le Gendre G. R. and Kaehny W. D. (1976) The dialysis encephalopathy syndrome. Possible aluminium intoxication. New England Journal o f Medicine 294, 184-188. Candy J. M., Oakley A. E., Klinowski J., Carpenter T. A., Perry R. H., Atack J. R., Perry E. K., Blessed G., Falrbairn A. and Edwardson J. A. (1986) Aluminosillcates and senile plaque formation in Alzheimer's disease. Lancet i, 354-357. Delves H. T., Suchak B. and Fellows C. S. (1989) The determination of aluminium in foods and biological materials. In Aluminium in Food and the Environment. Edited by R. Massey and D. Taylor. Royal Society of Chemistry. Disler P. B., Lynch S. R., Charlton R. W., Torrance J. D., Bothwell T. H., Walker R. B. and Mayet F. (1975) The effect of tea on iron absorption. Gut 16, 193-200. Fairweather-Tait S. J., Moore G. R. and Fatemi S. E. J. (1987) Low levels of aluminium in tea. Nature, London 330, 213. Fairweather-Tait S. J., Piper Z., Jemil S., Fatemi A. and Moore G. R. (1991) The effect of tea on iron and aluminium metabolism in the rat. British Journal of Nutrition 65, 61-68. Farrar G. and Blair J. A. (1989) Aluminium and Alzheimer's disease. Lancet i, 269. Flaten T. P. and Odegard M. (1988) Tea, aluminium and Alzheimer's disease. Food and Chemical Toxicology 26, 959-960. French P., Gardner M. J. and Gunn A. M. (1989) Dietary aluminium and Alzheimer's disease. Food and Chemical Toxicology 27, 495-496. Ganret P. O. (1986) Metabolism and possible health effects of aluminium. Environmental Health Perspectives 65, 363-441. Hall M., Loscombe S. and Taylor A. (1988) Trace element contamination from blood specimen containers. Trace Elements in Medicine 5, 126-129. Koch K. R., Pougnet M. A. B., de Villiers S. and Monteagudo F. (1988) Increased urinary excretion of aluminium after drinking tea. Nature, London 333, 122. Lote C. J. and Saunders H. (1991) Aluminium: gastrointestinal absorption and renal excretion. Clinical Science 81, 289-295. Perl D. P. and Brody A. R. (1980) Alzheimer's disease: X-ray spectrometric evidenceof aluminium accumulation in neurofibrillary tangle-bearing neurones. Science 208, 297-299. Prescott A. (1989) What's the harm in aluminium? New Scientist 1648, 58-62.
Plasma levels of aluminium after tea ingestion Slanina P., Falkeborn Y., Frech W. and Cedergren A. (1984) Aluminium concentrations in the brain and bone of rats fed on citric acid, aluminium citrate or aluminium hydroxide. Food and Chemical Toxicology 22, 391-397. Slanina P., Frech W., Ekstrom L.-G., Loof L., Slorach S. and Cedergren A. (1986) Dietary citric acid enhances absorption of aluminium in antacids. Clinical Chemistry 32, 539-541. Slavin W. (1986) An overview of recent developments in the determination of aluminium in serum by furnace atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry l, 281-285.
FCT 31/1---~
23
Wang S. T., Pizzolato S. and Demshar H. P. (1991) Aluminium levels in normal human serum and urine as determined by zceman atomic absorption spectrometry. Journal of Analytical Toxicology 15, 66-70. Weberg R. and Berstad A. (1986) Gastrointestinal absorption of aluminium from single doses of aluminium containing antacids in man. European Journal of Clinical Investigation 16, 428-432. Wills M. R. and Savory J. (1983) Aluminium poisoning: dialysis encephalopathy, osteomalacia and anaemia. Lancet ii, 29-34.
0278-6915/93 $6.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
PLASMA LEVELS OF ALUMINIUM AFTER TEA INGESTION IN HEALTHY VOLUNTEERS P. N. DREWITT, K. R. BUTTERWORTH, C. D. SPRINGALLand S. R. MOORHOUSE BIBRA Toxicology International, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK (Accepted 21 May 1992) Abstract--12 healthy volunteers on a controlled aluminium (A1) diet each consumed a tea infusion
(500 ml/70 kg body weight), with either milk or lemon juice as additives, or mineral water, following a three-way crossover design. The concentrations of AI were determined in the diet, mineral water and tea infusions, and in plasma samples collected before and up to 24 hr after consumption of tea or water, using graphite-furnace atomic absorption spectrophotometry or inductively coupled plasma emission spectrometry. Consumption of up to 1.60 mg A! from tea with milk or lemon juice did not increase plasma A1 levels compared with consumption of approximately 0.001 mg AI from mineral water. The results suggest that, in the short-term, drinking tea does not contribute significantly to the total body burden of A1.
INTRODUCTION
MATERIALS AND METHODS
Although aluminium (A1) has been shown to possess a potential for systemic toxic/ty (Alfrey et al., 1976; Wills and Savory, 1983), its low absorption from the gastro-intestinal tract (Ganrot, 1986) and its rapid urinary excretion (Alfrey, 1989; Lote and Saunders, 1991) suggest that in normal healthy individuals the bioavailability of A1 is too low to present a risk to health. However, the demonstration of an association of A1 with neuritic plaques (Candy et al., 1986) and neurofibrillary tangle-bearing neurones (Perl and Brody, 1980), the brain lesions that characterize Alzheimer-type dementia, has raised public anxiety about a possible insidious role for dietary A1 in the aetiology of this neurodegenerative disease. This concern has prompted studies of all sources of intake of the metal, including that from food and beverages (Delves et al., 1989). Tea leaves contain high natural levels of AI, since the tea bush accumulates large quantities of A1 from acid soil (Prescott, 1989). Black tea, as sold, contains 500-1500mg A1/kg, and the prepared beverage has been found to contain 2-6 mg Al/litre (Fairweather-Tait et aL, 1987; Flaten and Odegard, 1988). Koch et aL, (1988) have reported that urinary excretion of AI increases when tea is consumed, suggesting that some of the A1 present in tea is absorbed. Since tea could be an important dietary source of AI in the UK, a human study was undertaken to investigate the absorption of AI from black tea infusions with added milk or lemon juice. The latter was examined since citrus juices and citric acid have been shown to enhance dietary AI absorption (Slanina et al., 1984 and 1986; Weberg and Berstad, 1986).
Reagents. AristaR grade nitric acid (70%, w/w) and magnesium nitrate were purchased from BDH (Poole, Dorset, UK), together with AnalaR grade distilled deionized water, SpectrosoL grade aluminium nitrate standard (1000 /~g/ml) and hydrochloric acid (36%, w/w). Triton X-100 was obtained from Sigma Chemical Co. (Poole, Dorset, UK). Serum samples from the Robens Institute Trace Element Quality Assessment Scheme (University of Surrey, Guild(oral, UK) were used as the quality control specimens for determination of A1 in plasma. Diets and toiletries. Volunteers were given meals selected from the Birds Eye 'Menu Master' range. These were stored at - 2 0 ° C prior to cooking. On each leg of the study all volunteers had the same meals at designated times. Fresh tea infusions were prepared on each of the three study periods by adding 31.25 g tea (10 tea-bags of a typical blend used in the UK to 3000 ml boiling still Malvern mineral water (Schweppes International Ltd, UK). After stirring for 5 rain with a plastic spatula, each tea infusion was decanted and either 75 ml fresh semi-skimmed milk or 30 ml PLJ lemon juice (Beecham Products, Brentwood, UK) and 45 ml Malvern water were added. The control solution consisted of boiled, still Malvern mineral water. All dosing solutions were kept hot in vacuum flasks prior to use. A 5-mi aliquot of each test solution was kept at - 2 0 ° C until analysed for AI content. Toileteries prepared from low Al-containing compounds were used during the study [Impulse and Lynx deodorants, and Signal toothpaste (Elida Gibbs, London, UK)]. Subjects. 12 volunteers (six male and six female) were judged to be healthy from a detailed family and medical history and a comprehensive medical
Abbreviation: A1 = aluminium. 19
20
P.N. Dgswirr et al.
examination, which included serum chemistry, haematology and urinalysis profiles. The volunteers, aged between 25 and 54yr, were within 15% of the desirable weight range for their height and were further approved by an informed clinical judgement within 14 days before study commencement. Each female volunteer gave details of her recent menstrual history and was shown not to be pregnant using a Pregnosticon Planotest urinary pregnancy kit (Organon Teknika BV, Oss, The Netherlands). Experimental Procedure. Prior to study commencement, approval to proceed was obtained from the BIBRA Ethics Committee. All clinical procedures were in full compliance with Good Clinical Practice (non-clinical procedures conforming to the internationally accepted standards of Good Laboratory Practice and audited by the independent BIBRA Quality Assurance Unit). 1 wk before and during each of the three study periods, volunteers used only toiletries with a low A1 content. Identical diets were consumed during the 24-hr period prior to study commencement and during each study period. Samples of the diets were retained for analysis of A1 content. Mineral water was provided (150 ml at hourly intervals) during the first 7 hr after dosing. Thereafter, decaffeinated coffee or mineral water was available ad lib. The study followed an open threeway crossover design. Volunteers received either mineral water, tea with milk or tea with lemon juice (500 ml/70 kg body weight) on each of the three study periods, which were separated by 2 wk to prevent carry-over. The order of dosing was in accordance with a randomization schedule. Blood samples were collected by repeated venepuncture, using 5-ml B-D Plastipak disposable syringes (Becton Dickinson, Dublin, Ireland), immediately before dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hr post-dose. Blood samples were transferred to 5-ml lithium-heparin tubes (Teklab, Co. Durham, UK) prior to mixing on a roller mixer for 10min. Plasma was obtained following centrifugation at 3000 rpm at 4°C for 5 min in a refrigerated centrifuge and then transferred in duplicate to 5-ml plain storage tubes (Teklab) using a Gilson pipette with acid-washed disposable tips. All plasma samples were stored at - 2 0 ° C until A1 analysis. Aluminium contamination. Sample contamination is a potentially serious problem because of the ubiquitous distribution of A1 compounds in the environment. A careful and meticulous regime of sample collection and storage was adopted to minimize extraneous AI contamination. Before venepuncture, the forearm of each volunteer was cleaned with an alcohol Medi-Swab (Smith and Nephew Ltd, Hull, UK), and care was taken not to touch the skin during sampling. Preliminary work included verification that the disposable syringes, lithium-heparin tubes and plain storage tubes were not sources of AI contamination (Hall et al., 1988). Before use, disposable plastic pipette tips were washed three times with nitric
acid (10%,v/v) and then rinsed three times with deionized filtered water. Aluminium determination. Concentrations of AI in plasma, tea and mineral water were determined using an atomic absorption spectrometer (model 2380) equipped with a graphite furnace (HGA-400) and an automatic sampler (AS 40; Perkin-Elmer, Beaconsfield, Bucks., UK). All steps of sample preparation and measurement were performed in an isolated dust-free environment. Polypropylene analyser cups (0.5 ml; BCL, Lewes, Sussex, UK) and caps, disposable pipette tips and other plasticware were washed with nitric acid (10%, v/v) and rinsed with distilled deionized water before use to minimize AI contamination. Working standards were prepared daily by serial dilution of a substock (10#gA1/ml) of the certified AI standard with AnalaR grade distilled deionized water. Aliquots of 100#1 working standards were added to 300#1 diluent solution in analyser cups. The diluent consisted of AnalaR grade distilled deionized water containing magnesium nitrate (1 or 2 g/litre) and nitric acid (70%, 2 ml/litre). Test samples and quality control specimens were similarly diluted (1:3, v/v) with the diluent solution. All solutions were capped and mixed by inversion. The A1 content of the samples was determined using the instrument conditions shown in Table 1. Determinations were made in duplicate or triplicate, with two quality-control samples analysed in duplicate at the start of a sample run, and after every five or six test samples. Between samples the autosampler probe was flushed with a solution of distilled deionized water containing Triton X-100 (1 ml/litre) and nitric acid (5 ml/litre). Analyses were accepted if valid results were obtained for the concurrently analysed quality control samples. An inductively coupled plasma emission spectrometer (Perkin-Elmer Plasma 40) was used for the determination of A1 in food samples. Working standards were prepared by adding 50 or 100 #1 A1 standard to 100 ml HC1 (10%, v/v). HC1 (10%, v/v)
Table 1. Conditions used for the determination of AI in plasma, tea infusions and mineral water by atomic absorption spectrometry Step number
Temperature (°C)
Ramp time (see)
Hold time (see)
1 2 3 4 5 6 7 8
100 120 40O 650 1600 2650 2700 20
5 20 10 10 10 0 I 1
45 or 65 10 30 10 25 or 35 4 or 5 3 10
The instrument parameters used were: wavelength, 309.3; lamp current, 22mA; spectral slit width, 0.7 nm; signal, concentration; mode, peak area; and background correction, Deuterium Arc System. • An aluminium hollow cathode lamp and pyrolytically coated graphite tubes were used, with 10- or 20-/zl sample aliquots. Argon was used as the purge gas with internal flow interrupted during atomization (step 6).
Plasma levels of aluminium after tea ingestion was used as the blank solution. The food samples were first homogenized, and 20-g aliquots were then heated gradually to charring before being placed in a muffle furnace at 550°C for 16 hr. After cooling, sufficient HC1 (50%, v/v) was added to wet the ash, followed by reheating to dryness. When cool, 10 ml HCI (25%, v/v) was added, and the solution was brought to the boil before being filtered through a Whatman 541 filter paper. The latter procedure was repeated several times before the filter paper and funnel were washed with deionized filtered water. The final volume was made up to 50 ml and then analysed for AI content using the following instrument parameters: argon flow, 3.5 litres/min; read delay, 70sec; replicates, 3; wavelength, 308.215 nm; photo-mnitiplier tube (PMT), 900 v; element time, 500msec; standard 1, 0.500/zg/ml; and standard 2, 1.000/tg/ml. Standards were analysed at the start of a sample run, and approximately after every six samples. Statistical analyses. Plasma AI levels were statistically analysed at all time points using a one-sided paired t-test, with the mineral water group v. the two tea groups. For pre-dose results a two-sided paired t-test was performed. RESULTS
Quality control Serum samples supplied monthly by the Robens Institute Trace Element Quality Assessment Programme (University of Surrey, Guildford, UK) were analysed to check the accuracy and precision of the method for AI determination in plasma and serum and to assess laboratory performance on an ongoing basis. These samples were also used for internal quality control. Performance indices are based on the relative deviations of laboratory results from consensus target values, with a highest possible score of 2 points (i.e. a difference of < 10% of the target value at 4.0/~mol/litre, and < 20% of the target value at 1.0/zmol/litre on a sliding scale). The six-monthly report from the Robens Institute, which covered the present study, showed that all performance indices had a value of 2 and ranked our laboratory first of the 111 participating laboratories, indicating that the analytical methodology was satisfactory. The detection limit for AI in serum and plasma was found to be 0.03 #mol/litre (0.9/zg/litre) and the linear range was up to at least 100/tg/litre.
Aluminium input from diet and dosing solutions Table 2 shows the mean AI content of the meals consumed by the volunteers. During each of the three study periods, the volunteers were on the same diets and therefore ingested similar quantities of AI from food. The mean AI intake per individual serving was between 0.380 and 0.883 mg. The concentrations of AI in the two tea infusions and mineral water at each leg of the study are shown in Table 3. The mean total
21
Table 2. Mealscheduleand AI content(mg)per serving used in the study on plasma levelsof aluminiumafter tea i n g e s t i o n in healthyvolunteers Mean AI content Time per serving (br)
24 19.30 14 - 10
-
0 +4 + 10
Meal
(mg)
Breakfast Lunch Dinner Supper Tea or mineral water ingested Lunch Dinner
0.782 0.883 0.620 0.380 Total 2.665 0.782 0.450 Total 1.232
amount of AI ingested by volunteers between 0 and 24 hr were: tea with milk, 2.329 mg; tea with lemon, 2.808 rag; and mineral water, 1.233 mg.
Plasma aluminium levels The concentration of AI in plasma sampled before treatment was 4.21 _ 0.58 gg/litre (mean _ SD, n = 36). This value is comparable with recent published estimations (Delves et al., 1989; Slavin, 1986; Wang et al., 1991]. Figures 1 shows mean plasma A1 concentrations at various times after consumption of either mineral water or tea (with milk or lemon), At no time did the concentration of AI found in the plasma of the two tea-drinking groups differ significantly from that in the mineral water (control) group. DISCUSSION An average of 0.001, 1.097 or 1.576 mg A1 was ingested by the volunteers when they drank mineral water, tea with added milk or tea with lemon juice, respectively. In addition, the volunteers received A1 from the standardized diets. Although the A1 content of the selected meals ranged between 0.380 and 0.833 nag, the intake of A1 from the diet was the same on each occasion for all the participants. Thus, in this study the consumption of 500 ml (about two cups) tea/70kg body weight almost doubled the dietary intake of AI by the volunteers. However, this did not increase plasma AI levels monitored over a 24-hr period. This finding suggests that the bioavailability of AI in tea is low, and that in the short term drinking tea with added milk or lemon juice does not contribute significantly to the total Table 3. AI concentrationsin dosing solutions used duringthe studyon plasmalevelsof aluminiumaftertea ingestionin healthyvolunteers AI concentration(mg/litre) during study periods Dosing
solution I 2 3 Mineralwater 0.0029 0.0026 0.0027 Tea with milk 2.0050 2.1060 2.4290 Tea with lemon 2.9850 3.1110 3.2830 Volunteers consumedthe dosing solutionsat 500ml/ 70 kg body weightduringstudy periods 1, 2 and 3.
22
P. N. DREVCITT et al.
! r - MIMrll
oilllll i I ! I- Tea with milk
I
I
In conclusion, drinking 500ml tea (containing 1-1.6 mg natural AI) per 70 kg body weight does not increase plasma AI concentrations, suggesting that AI ingested from short-term tea drinking does not contribute significantly to the total body burden of this metal. Acknowledgements--We gratefully acknowledge the advice
i i
0 IIIIII I I 6 ,-- To,, with lemon
I
I
,
oilllll,, 01334
I !
I 8
I 13 Time (hi')
I 34
Fig. 1. Mean plasma AI concentration (#g/litre) before and after drinking tea with milk, tea with lemonjuice or Malvern mineral water (500 mi/70 kg body weight). Each point is the mean of 10 to 12 estimations + SEM. Volunteers were given tea or mineral water at 0 hr, immediately after giving a blood sample. When time points were compared, there were no statistically significant differences (P < 0.05) in plasma levels between the three groups.
plasma AI concentration. Support for this observation is provided by recent animal work (Fairweather-Tait et al., 1991), which showed that there was no increase in blood and liver A1 levels in rats that had consumed tea as the only source of fluid intake for 28 days. As Flaten and Odegard (1988) have pointed out, it may be that most of the extractable A1 in brewed teas is strongly bound to organic species (e.g. theaflavins, thearubigins and other polyphenolic 'fermentation' products) and that these high molecular weight complexes are not readily absorbed from the gastrointestinal tract. Recent work (French et al., 1989) has suggested that complexation plays an important part in A1 speciation in tea, even under simulated gastric conditions. Other investigators have reported that tea 'tannins' reduced the absorption of gallium-67, which is a marker for A1, in both nourisbed and starved rats (Farrar and Blair, 1989), suggesting that components in tea may have an inhibitory effect on A1 uptake from other dietary sources. The adverse effect of tea on iron bioavailability is well known (Disler et al., 1975; Fairweather-Tait et al., 1991). The apparent failure of citrate to augment A1 absorption and increase plasma levels in the present study may also be due to effective sequestration of the metal by components in tea. It is perhaps worth noting that in most studies where the chelating effect of citrate has been observed, relatively large doses of Al-containing compounds and citrate had been used (Slanina et al., 1986; Weberg and Berstad, 1986), often for extended periods, before observations were made (Slanina et al., 1984 and 1986).
and comments of Dr P. Collier and Dr R. Wilson (Unilever Research), and the Analytical Section of Unilever Research (UK) for their expert analysis of AI in the diets. The efforts of Miss L. Norris in the typing of this manuscript are very much appreciated, and we also acknowledge the valuable assistance of the BIBRA Statistical Department.
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FCT 31/1---~
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