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Drug Monitoring and Toxicology |
1
Laboratory for Analytical Chemistry, Institute for Nuclear Sciences, University of Gent, Proeftuinstr. 86, B-9000 Gent, Belgium.
2
Renal Division, Department of Medicine, University
Hospital, De Pintelaan 185, B-9000 Gent, Belgium.
a Author for correspondence. Fax 32 (0)9 2646699;
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As–protein binding occurs mainly in the cytosol, most notably in liver tissue (6). The binding of As to proteins in plasma and packed blood cells was also observed by several authors, although most of these studies were based on animal models and the mechanism is not well understood. Vahter and Marafante (7) observed a binding percentage up to 20% of As to plasma proteins after intravenous administration of [As]arsenite or [As]arsenate to mice and rabbits. Bertolero et al. (8) found that ~10% and 50% of As in plasma were bound to plasma proteins after 5- and 48-h intraperitoneal administration of 1 µg of As-(III)/kg to rabbits, respectively. De Kimpe et al. (9) observed that the percentage of protein-bound As reached a maximum of ~18% of total As at around 20 h and then slowly decreased to ~10% 120 h after intraperitoneal administration of an activity concentration of 4.44 MBq (120 µCi) per dose of As to rabbits. High binding percentages (~70%) of As to plasma proteins were observed in the nonmethylating marmoset monkeys (5), but dogs showed an extremely low binding percentage (10). These results indicate that binding of As to plasma proteins is a complex phenomenon and is dependent on the animal species.
De Kimpe et al. previously showed that As can be bound to serum transferrin in human serum after in vitro incubation of serum with inorganic As(V) (11). Whether the binding of As(V) to transferrin affects its elimination or toxicity is unknown . However, As is known to accumulate in renal failure patients. Samples from such patients could be helpful to elucidate the study of As–protein binding. The aim of the present investigations was to identify the proteins and the As species that are bound together in human serum. Patients with chronic renal insufficiency undergoing continuous ambulatory peritoneal dialysis (CAPD) were chosen for this study, as they showed highly increased As concentrations in their blood.1
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The patients gave their informed consent before blood
sampling. Patients were requested to abstain from eating seafood for 3
days before the blood sampling. Serum and packed cells were separated
by centrifugation at 1000g.
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apparatus and reagents
A UF system with 10-kDa molecular mass cutoff (Filtron Microsep,
Filtron Technology Corp.) was used for the separation of the
low-molecular-mass (LMM) As species from serum.
A fast-protein liquid chromatography (FPLC) system (Pharmacia) with three types of columns are used for the separation and identification of As–protein bindings: size-exclusion (SEC) (Superose HR 10/30 analytical column and Sephadex G-50 XK 50/30 preparative column), anion-exchange (MonoQ HR 16/10), and affinity (AFC) [N-hydroxysuccinimide (NHS)-activated HiTrap Superose® HR 10/2, coupled with goat anti-human transferrin].
A Perkin-Elmer 3030 atomic absorption spectrometer with an As electrodeless discharge lamp, operating at a power of 8W, was used throughout for the detection of the As signals by hydride generation. A flow injection atomic spectrometry (FIAS) 200 flow-injection system (Perkin-Elmer) was used to generate the hydride.
As species calibrators of monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) were provided by the Commission of the European Union, DGXII, Measurements and Testing Programme. Sodium arsenite and arsenate were obtained from Merck. Sodium tetrahydroborate solution of 5 g/L (Janssen Chimica) in 0.5 g/L NaOH (UCB) was freshly prepared every day and filtered before use. A HCl solution, 100 mL/L, was prepared from concentrated HCl solution (320 g/L) purified at subboiling conditions. The mobile phases of FPLC are 0.025 mol/L Tris-HCl (pH 7.4) for SEC and 0.025 mol/L Tris-HCl (pH 8.0) in a linear NaCl gradient (0–0.5 mol/L) for ion-exchange chromatography (IEC). The buffer solutions used for AFC are as follows: buffer A, 0.2 mol/L NaHCO3–0.5 mol/L NaCl, pH 8.3; buffer B, 1 mol/L ethanolamine in buffer A; buffer C, 75 mmol/L Tris-HCl at pH 8; and buffer D, 0.5 mol/L glycine-HCl solution at pH 2.0 (11).
procedure
The procedures for the separations are summarized in a flow
diagram (Fig. 1
).
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Procedure 1: separation procedure by UF.
LMM As species
were separated from the serum matrix by UF. The concentration of
high-molecular-mass (HMM) As species was estimated by calculating the
difference between the total As concentration and the As concentration
in LMM form.
Procedure 2: separation procedure by SEC.
A Superose-12
HR 10/30 analytical column was used for the identification of As
species based on a procedure of separation of HMM from LMM As species
in a serum matrix. This column is a type of commercial column
(Pharmacia) composed of highly cross-linked porous agarose beads (8–12
µm) with a fractionation range of 1000–300 000 Da. A 200-µL
aliquot of 11 diluted serum sample (with mobile phase) was introduced
into the column and eluted with 0.025 mol/L Tris-HCl buffer. The
fractions were collected into 30 carefully cleaned quartz tubes fit for
the subsequent digestion of the proteins. The collection procedure was
repeated 10 to 30 times according to the As concentrations in the serum
samples and all corresponding fractions were pooled to obtain a
sufficient amount of As for detection.
Procedure 3: separation procedure by IEC.
A 0.5-mL
lyophilized protein fraction obtained from the SEC preseparation step
was injected onto the MonoQ HR 16/10 anion-exchange column and eluted
with 0.025 mol/L Tris-HCl (pH 8) in a linear NaCl gradient (0–0.5
mol/L NaCl). This procedure was repeated two to five times (according
to the total As concentration in the sample) and the eluent was
collected into the quartz tubes for evaporation, digestion, and
detection.
The SEC preseparation step was carried out as follows: Undiluted serum sample (5 mL) was chromatographed by the Sephadex G-50 preparative column (XK 50/30). The fractions containing the HMM As species were lyophilized (~3 Pa). The residue was dissolved in 2 mL of water.
Procedure 4: AFC for transferrin.
The column preparation
was carried out as follows: The goat anti-human transferrin was
dissolved in 1.0 mL of buffer A and introduced slowly into the column
with a syringe. The column was then left at 4 °C overnight. NHS
groups that had not reacted with goat anti-transferrin were deactivated
by injecting 10 column volumes (0.5 mL/min) of buffer B through the
column. The column was then washed with buffer C until a stable
absorbance signal at 280 nm was obtained. Serum (1.0 mL) was put on the
conditioned affinity column with a flow rate of 0.05 mL/min. The
unbound components were eluted from the column with buffer C at a flow
rate of 0.5 mL/min. Then 10 mL of buffer D was introduced into the
column for desorption of the transferrin–antitransferrin binding.
Detection of proteins and As.
The chromatograms of
proteins were obtained on line with the UV detector at 280 nm. The
chromatograms of As were obtained off line by determination of the As
in the fractions by a previously described procedure (12).
Briefly, the As(V) calibrators and chromatographic eluents were
digested in high-purity quartz vials with 3.0 mL of digestion solution.
The solution was heated at about 200 °C for 1–2 h. After digestion
and evaporation until dryness (only H2SO4
left), 1.0 mL of HCl (32%) and 1.0 mL of KI (10%) ascorbic acid (5%)
solutions were added to the digestion tubes and diluted to 10.0 mL.
These solutions were left standing at room temperature for at least 45
min before measurement to ensure that all As(V) was reduced to As(III).
To detect the As signals, 500 µL of the digested solution was introduced into the FIAS 200 flow injection system, where the solution met the continuous flow of HCl and NaBH4 to generate arsine, which was then separated from the solution in a gas–liquid separator. Arsine was then introduced to the T-type quartz tube for atomic absorption measurement.
The accuracy of the method was tested by the analysis of As in three
certified reference materials including BCR-CRM-185 bovine liver,
NBS-SRM-1577a bovine liver, and a certified freeze-dried reference
serum of the University of Gent (13). Table 2
summarizes the As concentrations of the reference materials
obtained in this work and the certified values. No significant
differences were established by the Student t-test at the
95% confidence level for all three reference materials. The results
prove that the proposed method is accurate for the determination of the
As concentrations in serum.
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The precision (CV) of the method for replicate analyses of As species in a digested serum and in the digested fraction solutions after FPLC separation by flow injection hydride generation atomic absorption spectrometry (HGAAS) calculated on the peak height was always better than 5%. By using a sample volume of 500 µL, the detection limit of the method, defined as 3x the SD of the blank, is 0.031 µg/L As. The characteristic mass is 37 pg of As for 0.0044 absorbance. The response was linearly related to the As concentrations in the ranges 0.1–10 µg/L.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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speciation of as–protein binding in serum of patients by sec
Because the results obtained by UF were based on an indirect
calculation, which may have a large error, further identification of
the As–protein binding in serum of patients was carried out by SEC on
an analytical scale. Only three serum samples from patients 1, 2, and 3
(see Table 1
) with As–protein concentrations 1.04, 0.57, and 1.09
µg/L, respectively, were selected for this study.
After SEC separation, As was distributed into two peaks. The first peak contained HMM As species with a molecular mass of about 80 000 Da, whereas the second peak contained the LMM As species with a molecular mass of <1000 Da. The calibration was done with a set of compounds including blue Dextran 2000, transferrin, ovalbumin, myoglobin, ribonuclease A, aprotinin, and acetone with molecular masses of 2 000 000, 81 000, 43 000, 17 600, 13 700, 6500, and 58 Da, respectively. We assumed that the protein molecules and the As, detected in the same fraction, were associated with one another. The concentrations (SD) of As bound to proteins in serum for patients 1, 2, and 3 were 0.44 (0.12), 0.19 (0.09), and 0.59 (0.09) µg/L (n = 3), respectively. High SD values are due to insufficient sensitivity of the detection. The separation and collection procedure had to be repeated up to 30 times for a total of 3 mL of serum sample, the fractions pooled and lyophilized, and the As measured.
To identify which As species were bound to serum protein, 10 ng of each
of the five As species As(III), As(V), MMA, DMA, and AsB were incubated
in vitro with a "blank" serum (the concentration of total As in the
serum was only 0.3 µg/L and the concentration of As–protein was
lower than the detection limit) at 37 °C for 24 h. An excellent
resolution was obtained between the peaks of HMM and LMM As species by
the SEC column. The recoveries for all the As species incubated were in
the range of 100% ± 5% in the eluent. As species were detected in
the fractions containing proteins for As(III) and As(V), but not for
MMA, DMA, and AsB. These results imply that As–protein binding can
only take place between inorganic As and serum proteins. By incubating
10 ng of inorganic As species with 1 mL of serum at 37 °C for
24 h, the percentages of As–protein binding are 6.5% ± 0.51%
and 5.3% ± 4.9% (n = 3) for As(V) and As(III), respectively.
The typical UV chromatograms of serum proteins and the As elution
patterns for As(V) and AsB are shown in Fig. 2
A and B.
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speciation of the serum proteins bound to as species by iec
The SEC used in the present study is sufficient for the separation
of LMM As species from HMM As species, but this method does not allow
adequate separation of proteins with small differences in molecular
mass. The identification of the serum protein bound to As species was
therefore carried out on an anion-exchange column (MonoQ 16/10,
Pharmacia) because this column shows a high resolution for the
separation of serum proteins. Before the serum sample was submitted to
the column, the resolution for the separation of transferrin and
albumin was examined, since previous in vitro experiments have shown
that As may bind to transferrin (11). The chromatogram
shown in Fig. 3
demonstrates the excellent separation that was obtained between
transferrin and albumin.
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The chromatographic behavior of LMM As species on the column was also
studied because we assumed that the protein molecules and the As
species, measured in the same fraction, are indeed associated with one
another. This assumption is usually correct for the SEC if a suitable
column is selected for the separation. But the LMM As species, carrying
the same apparent charges as the macromolecules, may also elute
together with the protein molecules when an ion-exchange column is used
for the separation. Therefore, 100 ng of As(V) was incubated with 10 mL
of fresh serum at 37 °C for 24 h. The incubated sample was then
injected into the anion-exchange column (MonoQ 16/10, Pharmacia) for
the separation. Two peaks of As-containing compounds were detected by
HGAAS in the different fractions (Fig. 4
). One peak is the LMM As(V) species peak, representing 51.3%
of total As, which was identified by injecting an As(V) calibrator.
Another As peak (48.7% of total As incubated) was eluted in the same
fraction as transferrin. One of two possibilities could explain the
48.7% As in this fraction instead of the anticipated 5%. The first
could be a true As–transferrin binding, and the second could be the
presence of LMM As(III) species, due to reduction of As(V) by proteins
or by other LMM serum compounds.
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Therefore, two experiments were designed for the identification of this unknown peak: (a) injecting 10 ng of As(III) calibrator into the anion-exchange column; (b) after incubating As(V) with the serum, both HMM and LMM As species were separated by SEC on a preparative scale, and only the HMM As species were injected into the anion-exchange column. In the first experiment, we observed that As(III) species has the same retention time as the unknown peak. The second experiment showed that only a small amount of As bound to transferrin molecules exists. These results showed that LMM As(III) species elute in the same fractions as the HMM As species, interfering with the identification of As bound to proteins. Therefore, a preseparation of the LMM As species from HMM As species by, e.g., SEC, is needed before submitting the sample to anion-exchange chromatography. This separation removes the interference of As(III) with As–transferrin molecules.
The separation of proteins in the serum samples of patients 1 and 3 was
carried out according to the procedure suggested above. No significant
differences of UV absorption diagrams and As elution patterns were
observed for the two patients. The chromatograms showed nine distinct
protein regions (see Fig. 4
). Isoelectric focusing of the protein peaks
revealed the nature of the different proteins in each peak. Peaks 4 and
5 are transferrin, which may exist in two different forms: asialo and
sialo transferrin, with iron in different sites of the molecules.
Albumin is mainly situated in peaks 6 and 7, and to a lesser extent
also in peaks 8 and 9. The As distribution over the different fractions
containing proteins was measured by HGAAS. Almost all the As molecules
were distributed over the fractions containing transferrin (peaks 4 and
5). No As was detected in the fractions containing albumin.
identification of as–transferrin binding by afc
Considering the occurrence of As together with transferrin in the
same fractions, it was useful to look for further proof of the
statement that As is bound to serum transferrin. Therefore, two more
experiments were carried out: (a) in vitro incubation of
As(V) solely with human transferrin, and separation of the incubated
solution with SEC and anion-exchange chromatography; (b)
identification of the As–transferrin binding by means of AFC based on
the immunoassay of transferrin–antitransferrin. In the first
experiment, the As was detected exactly in the fraction containing
transferrin, confirming the statement that As can be bound to serum
transferrin. In the second experiment, the serum samples were applied
to the affinity columns and, as expected, only transferrin and
As–transferrin molecules were absorbed on the columns. After
desorption, the fractions obtained from five repetitive elutions were
collected in the quartz tubes, digested, and As was measured. The
results showed the As in the fraction containing transferrin for the
two patients, but with concentrations about 30% lower than the results
obtained by SEC (0.31 ± 0.10 and 0.40 ± 0.11 µg/L for
patients 1 and 2, respectively). Considering the very low As–protein
concentration in serum, as well as the systematic error resulting from
two different analytical methods, these values are acceptable. The
conclusion that As is bound to serum transferrin is valid.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The percentage of As–protein in serum of CAPD patients measured in the present work was lower than the percentages reported for animals, where 10–70% of As was bound to serum proteins (7)(8)(9)(10). However, the results obtained for humans and animals are not comparable because of differences in metabolism and protein binding. Also, most of the animal experiments were carried out by administration of As species. This protocol used in animal studies is impossible for human studies because a high dose of As cannot be administered to humans. Furthermore, As species ingested by humans can be inorganic as well as organic, depending on the nature of the foodstuffs; also, the estimation of the time interval between the intake of As and measurement is difficult. As is ubiquitously present in foodstuffs. As the As species are undefined and the time between intake and blood sampling is unknown, the influence of the major variables affecting the percent As–protein binding remains an open question.
To circumvent this problem, in vitro incubation of As species in human serum had to be used for the identification of As species bound to serum proteins. The detoxification of inorganic As in the human body involves several complicated biotransformation processes such as As–protein binding in tissues and blood, methylation of inorganic As species to MMA and DMA, and excretion of MMA and DMA into the urine (3)(4)(5). Equilibria and dynamic reactions that take place during in vivo metabolization may never occur in the process of in vitro incubation. The stability of protein–As complexes when carried through the procedures should also be an issue for further study.
Although UF has been used for the speciation of As–protein bindings in the literature (18), a complete separation of LMM molecules from HMM molecules by UF is difficult, especially when the concentration of LMM molecules is much higher than that of the HMM molecules (e.g., As in human serum). The method reported in the literature (18) for the speciation of As–protein binding in the residues on the UF membranes may be unreliable. During our in vitro experiment, >10% AsB species was present in the fraction containing HMM As species after 3 h of UF, although this species was known not to bind to proteins. This 10% residual AsB species in the fraction containing HMM species cannot be ignored because the As–protein concentration is even lower than this concentration. Washing residues and repeated UF could remove the small molecules from the residues more efficiently, but result in a loss of As–protein binding. Therefore, we used a more complicated procedure based on FPLC separation.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  D. Douer and M. S. Tallman Arsenic Trioxide: New Clinical Experience With an Old Medication in Hematologic Malignancies J. Clin. Oncol., April 1, 2005; 23(10): 2396 - 2410. [Abstract] [Full Text] [PDF]
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