Study on the effects of nitrilotriproprionic acid and 4,5-dihydroxy-1,3-benzene disulphonate on the fractionation of beryllium in human serum using graphite furnace atomic absorption spectrometry
© Stephan et al 2008
Received: 13 February 2008
Accepted: 14 May 2008
Published: 14 May 2008
Occupational exposure to beryllium may cause Chronic Beryllium Disease (CBD), a lung disorder initiated by an electrostatic interaction with the MHC class II human leukocyte antigen (HLA). Molecular studies have found a significant correlation between the electrostatic potential at the HLA-DP surface and disease susceptibility. CBD can therefore be treated by chelation therapy. In this work, we studied the effect of two complexing agents, nitrilotriproprionic acid (NTP) and 4,5-dihydroxy-1,3-benzene disulphonate (Tiron), on the fractionation of beryllium in human serum analysed by graphite furnace atomic absorption spectrometry (GFAAS).
We found the average serum beryllium concentration of fourteen non-exposed individuals to be 0.53 (± 0.14) μg l-1, with 21 (± 3)% of the beryllium mass bound to the low molecular weight fraction (LMW), and 79 (± 3)% bound to the high molecular weight fraction (HMW). The addition of Tiron increased the beryllium mass in the HMW fraction, while NTP was not seen to have any influence on the fractionation of beryllium between the two fractions. NTP was, however, shown to complex 94.5% of the Be mass in the LMW fraction. The beryllium GFAAS detection limit, calculated as three times the standard deviation of 10 replicates of the lowest standard (0.05 μg L-1), was 6.0 (± 0.2) ng L-1.
The concentration of beryllium or its fractionation in human serum was not affected by sex or smoking habit. On average, three quarters of the beryllium in serum were found in the HMW fraction. Of the two ligands tested, only Tiron was effective in mobilising beryllium under physiological conditions, thus increasing the Be content in the HMW fraction.
Beryllium, the first of the alkaline earth metals, is naturally found in mineral rocks, coal, soil, and volcanic dust . Beryllium ore is mined and purified for its use in nuclear reactors, weapons, aircraft and space vehicle structures, x-ray machines, as well as telecommunication and high-tech devices . Occupational exposure to beryllium may cause Chronic Beryllium Disease (CBD), a lung disorder characterised by a granulomatous inflammation initiated by an electrostatic interaction with the MHC class II human leukocyte antigen (HLA) [3, 4]. Molecular epidemiological studies have shown that interaction between beryllium and specific HLA-DP alleles is a factor in disease susceptibility . Furthermore, molecular modelling has been used to investigate a potential mechanistic basis for these observations. A significant correlation has been found between the risk of chronic beryllium disease associated with specific alleles, and the predicted electrostatic surface potential, suggesting that the alleles associated with the most negatively charged proteins carry the greatest risk of beryllium sensitisation and disease .
Nitrilotripropionic acid was selected because of its physico-chemical properties: it complexes beryllium in a tetrahedral complex [Be(NTP)]- where the beryllium cation lies at the centre of a slightly distorted tetrahedron of C3υ symmetry, with a longer Be-N bond and three equal Be-O bonds . Tiron is a hydrophilic chelator and was selected because of its reported efficiency in mobilising beryllium [15, 16], restoring the altered biochemical parameters  and improving the altered hepatorenal biochemistry and ultramorphology in different rat tissues and organs .
Beryllium, like any other metal ions in serum, can be fractionated into four distinct groups: rigidly bound to metalloproteins; loosely bound to other types of proteins (labile equilibrium); complexed by the so-called low-molecular-weight fraction (LMW); and occurring as free (or hydrated) metal ions .
In this study, we looked first at the natural distribution of beryllium in serum and the influence of NTP and Tiron on this distribution. We focused on two operationally-defined fractions; the first fraction combines the Be rigidly and loosely bound to proteins, and is called the high molecular weight fraction (HMW); whilst the second fraction, that is the low molecular weight fraction (LMW), combines the Be complexed to the low molecular-weight fraction or that which occurs in its free ionic form. We separated the HMW and LMW fractions by ultrafiltration with Centricon centrifugal filter devices at a 10,000 nominal molecular weight limit (NMWL) cut-off. Beryllium quantification in serum and serum fractions was carried out using an optimised graphite furnace atomic absorption spectrometer (GF-AAS). The accuracy of the analytical method was tested using Seronorm samples (Seronorm trace elements whole blood level 2, STEWB; Ref # 201605; Lot # 0503109).
Results and discussion
Beryllium in serum
The average beryllium concentration in fourteen non-exposed individuals (9 females and 5 males; 9 non smokers and 5 smokers) found to be 0.53 (± 0.14) μg L-1. Neither sex nor smoking habit was shown to have a significant influence on the concentration of beryllium in serum (independent sample T-test, p > 0.05). The method detection limit, calculated as three times the standard deviation of 10 replicates of the lowest standard (0.05 μg L-1), was 6.0 (± 0.2) ng L-1. The accuracy of the method varied from 99 to 104% and was verified by analysing a control sample (STEWB level 2 certified blood material with a concentration of 5.9 (± 0.5) μg Be L-1).
Beryllium distribution in serum
Concentration and natural distribution of beryllium in the human serum of non-exposed individuals.
Serum (μg L-1)
In an attempt to investigate the chelation effect of Tiron and NTP, as well as study their effect on the repartition of beryllium in human serum, dose-response experiments were conducted showing the influence of both complexing agents on the repartition of beryllium between the two serum fractions. We tested different equilibration times (2, 4 and 6 h), but found no differences for both ligands, suggesting that the exchange reactions rates are almost instantaneous and that equilibrium is reached within the 2 hour equilibration timeframe employed. This is similar to the Al-transferrin exchange reaction rate with desferrioxamine B (DFO) where the addition of DFO increased the amount of ultrafiltrable Al sharply up to 90% of the total plasma Al content [28, 29]. Due to the similarity between Be and Al "hard ions", and DFO, NTP and Tiron "complexing agents with soft oxygen donors", we should expect almost instant exchange reaction rates for the Be-Tiron-HMW and Be-NTP-HMW systems, thus confirming that the 2-h equilibration time is more than enough for the exchange to occur.
Neither sex nor smoking habit was shown to influence beryllium concentration or its fractionation in human serum. On average, three quarters of the beryllium in serum were found to be bind to the HMW fraction. Tiron, unlike NTP, showed a significant interaction with beryllium under physiological conditions by increasing the Be content in the HMW fraction – in contrast to that which is usually observed for standard complexing agents used in chelation therapy. The addition of NTP did not affect the MW distribution between the two fractions but was shown to complex most of the Be in the LMW fraction. Further work could employ anion exchange fast protein liquid chromatography (FPLC) coupled with electrospray tandem mass spectrometry (ES-MS-MS) or inductively coupled plasma mass spectrometry to confirm the nature of the ligands or proteins that complex Be in both serum fractions [20–22, 27].
All of the reagents used were of analytical grade or better. Antifoam B silicone emulsion (J.T. Baker, NJ, USA), ammonium hydroxide (certified A.C.S. Plus, Fisher scientific, NJ, USA), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Fluka chemika, Switzerland), Triton X-100 (Acros, NJ, USA), nitric acid (trace metal grade, Fisher Scientific, Ontario, Canada), beryllium plasma standard solution (Specpure, Alfa Aesar, MA, USA), nitrilotripropionic acid (NTP) (MP Biomedicals, Ohio, USA) and 4,5-dihydroxy-1,3-benzene disulphonate (Tiron) (Acros Organics, New Jersey, USA).
Serum samples were diluted with a Nash reagent (NR) solution prepared weekly and containing 5% (v/v) nitric acid, 5% (v/v) of ammonium hydroxide, 0.2% (v/v) Triton X-100, 0.2% (v/v) antifoam B and 0.5% (w/v) of EDTA in distilled-deionised water. Solutions of NTP and Tiron were prepared at 10-1, 10-2, 10-3, 10-4 and 10-5 M by dissolving the appropriate mass in 2% (v/v) HNO3 aqueous solution. A 50 μg L-1 working Be (II) solution was prepared by dilution of the beryllium stock solution (1000 μg L-1) in 2% (v/v) HNO3. The Be standard solution was prepared daily by dilution of the working Be (II) solution with the Nash reagent to give a final concentration of 0.5 μg L-1.
Blood from unexposed individuals was collected in BD vacutaine SST tubes (BD Franklin lakes, New Jersey, USA). Tubes were left at room temperature for 30 minutes to allow clot formation and then centrifuged at 500 g for 10 minutes. Supernatants of each individual were collected and recombined to ensure sample homogenisation. Each sample was divided into twelve 2.5 mL sub-samples in borosilicate disposable culture tubes, two served as controls, five were spiked with NTP and five were spiked with Tiron at concentrations ranging from 10-7 to 10-3 M. Sub-samples were left to equilibrate for two hours. A 2 mL aliquot of each sub-sample was transferred to a Centricon centrifugal device with ultracel YM-10 (10,000 MWCO) (Millipore Corporation, Ireland) and centrifuged at 4500 g for 90 minutes. The LMW and HMW fractions obtained were analysed for beryllium by graphite furnace atomic absorption spectrometry (GFAAS) after respective 2-fold and 5-fold dilutions with NR. The same experimental procedure was repeated on serum samples that were spiked with 1 μg L-1 of beryllium. We also measured the beryllium concentration in fourteen individuals and looked at the natural distribution of beryllium between the HMW and LMW fractions. Serum was diluted 5-fold with NR before GFAAS analysis.
Graphite furnace atomic absorption spectrometer
The furnace programme for the determination of beryllium in serum.
Argon flow rate (L min-1)
For the chemical speciation calculations, we used MINEQL+ (version 4.5 for Windows – Environmental Research Software, Hallowell, ME), a chemical equilibrium modelling system that can be used to perform calculations at low temperatures (0–50°C) and low to moderate ionic strength (< 0.5 M). MINEQL+ operates over three steps: creation of a system by selecting chemical components from a menu with the possibility of adding new ligands, then scanning the thermodynamic database and finally running the calculations with actual measured concentrations included. The output data module yields the activity for each species of each component. Our chemical speciation calculations were made assuming the following parameters (unless specified otherwise): T = 37°C (fixed), ionic strength: I = 0.01 M (fixed), Log pCO2 = -3.5 (open atmosphere). Different chemical equilibrium software should yield similar results when the same stability constants are used. The following input data were added to the MINEQL+ thermodynamic database; Log KBe-Ox = 3.47, Log KBe-(Ox)2 = 5.24, Log KBe-NTP = 13.94, Log KH3-NTP = 11.54, Log KH2-NTP -1 = 10.31 and Log KH-NTP -2 = 7.59.
Statistical analysis was performed with SPSS (version 13 for Windows, SPSS Inc, Chicago, IL) using independent sample T-tests to monitor significant differences among groups. We also used One-Way ANOVA tests with Fisher's LSD (least significant difference) to make pair wise comparisons as a way of monitoring significant changes within a group.
We thank the Natural Sciences and Engineering Research Council of Canada and the Institut de Recherche Robert-Sauvé en Santé et en Sécurité du Travail for financial support.
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