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Individual Variability in the Human Metabolism of an Arsenic-Containing Carbohydrate, 2′,3′-Dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-β-D-riboside, a Naturally Occurring Arsenical in Seafood Reingard Raml,† Georg Raber,† Alice Rumpler,† Thomas Bauernhofer,‡ Walter Goessler,† and Kevin A. Francesconi*,† Institute of Chemistry, Analytical Chemistry, Karl-Franzens UniVersity Graz, UniVersitaetsplatz 1, 8010 Graz, Austria, and Department of Internal Medicine, DiVision of Oncology, Medical UniVersity Graz, 8010 Graz, Austria ReceiVed May 2, 2009
We report studies on the variability in human metabolism of an oxo-arsenosugar involving the ingestion of a chemically synthesized arsenosugar and quantitative determination of the arsenic metabolites in urine and serum by HPLC coupled with arsenic-selective mass spectrometric detection (ICPMS, inductively coupled plasma mass spectrometry). The total, four-day, urinary excretion of arsenic for six volunteers ranged widely from ca. 4-95%. The arsenic metabolites present in the urine also showed great variability: high arsenic excretion was accompanied by almost complete biotransformation of the ingested oxoarsenosugar into a multitude of metabolites (>10), whereas the subjects that excreted low amounts of arsenic produced low quantities of metabolites relative to unchanged oxo-arsenosugar and its thio-analogue. Major arsenic urinary metabolites were dimethylarsinate (DMA) and possible intermediates in the degradation of arsenosugar to DMA, namely, dimethylarsinoylethanol (DMAE) and dimethylarsinoylacetate (DMAA) present both as their oxo- and thio-analogues. Thio-DMAE and thio-DMAA were also found in blood serum indicating that these species were formed in the liver rather than on storage of the urine in the bladder. The large variability in the way individuals metabolize arsenosugars has implications for risk assessment of arsenic intake from seafood. Introduction Human health effects resulting from chronic exposure to inorganic arsenic include skin and internal cancers, cardiovascular disease, and possibly diabetes (1, 2). A major source of arsenic to humans is drinking water derived from underground water supplies where inorganic arsenic of natural geological origin can accumulate (3). As a safeguard to human health, the World Health Organization has recommended an acceptable maximum level of arsenic in drinking water of 10 µg As L-1; however, drinking water supplies from many parts of the world, including many regions of the USA, can easily exceed this value (4). The mode of toxic action of inorganic arsenic is still to be elucidated, but it is thought to be strongly related to metabolism. Arsenic ingested as inorganic arsenic (either arsenate or arsenite) is methylated and excreted via the kidney mainly as dimethylarsinate (DMA; see Table 1 for names, abbreviations, and structures of arsenicals relevant to this study) and methylarsonate (MA). The formation of these methylated metabolites and intermediates along the pathway has been implicated in arsenic’s toxic action (5). Hence, individual differences in metabolism may greatly influence susceptibility to arsenic toxicosis. Seafood also contains high concentrations of arsenic, present primarily as organoarsenic compounds biosynthesized by marine organisms from the inorganic arsenic naturally present in their * Corresponding author. E-mail:
[email protected]. † Karl-Franzens University Graz. ‡ Medical University Graz.
ambient seawater (6). A major form of seafood arsenic is arsenobetaine [(CH3)3As+CH2COO-], a harmless arsenical that is not biotransformed by humans (7). Other major seafood arsenicals are arsenosugars, which are found mainly in algae and algal-consuming animals such as oysters and mussels. Human metabolic studies with arsenosugars have produced results more complex than those with arsenobetaine. The initial studies (8, 9), performed with algae and humans, indicated differences in the excretion of arsenic metabolites, although a subsequent study, also with humans and algae, reported that all of the five subjects showed similar excretion patterns (10). A study with human volunteers who consumed mussels, which naturally contain arsenosugars in addition to arsenobetaine, also showed differences in arsenic metabolites in the urine of the volunteers (11). All of these studies provided valuable information on the human metabolism of arsenosugars, but interpretation of the results was compromised by the mixture of arsenicals present in the food items. Recently, considerable progress has been made in determining arsenic metabolites of an arsenosugar by using a pure chemically synthesized compound. Structures of the major metabolites have now been elucidated, standards have been synthesized, and HPLC systems have been optimized to determine these newly discovered species (12, 13). These advances have been built on in the following study comparing the metabolism of a chemically synthesized arsenosugar by six human volunteers. Furthermore, the methods are applied for the first time to investigate the arsenic metabolites present in human serum in these volunteers.
10.1021/tx900158h CCC: $40.75 2009 American Chemical Society Published on Web 07/23/2009
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Table 1. Arsenic Species Relevant to This Study: Names, Abbreviations, and Structures
Materials and Methods Reagents and Standards. The following commercial products were used: formic acid (p.a.), ammonium dihydrogen phosphate (p.a.), and ortho-phosphoric acid (p.a.) from Fluka (Buchs, Switzerland); methanol (p.a.) from Fisher Scientific (Leicestershire, UK); hydrogen sulfide from Messer Griesheim (Krefeld, Germany); and pyridine (p.a.), hydrogen peroxide 30% (p.a.), aqueous ammonia 25% (suprapure), and nitric acid (p.a.) from Merck (Darmstadt, Germany). Chemicals were used without further purification except for nitric acid, which was distilled in a quartz sub-boiling distillation unit. Standard solutions of dimethylarsinate (DMA) were prepared from sodium dimethylarsinate trihydrate purchased from Fluka (Buchs, Switzerland); trimethylarsine oxide (TMAO) (14), oxodimethylarsenoacetate (oxo-DMAA) (15), oxo-dimethylarsenoethanol (oxo-DMAE) (16), thio-DMAE and thio-DMAA (12), and thio-DMA (17) were prepared as previously reported. The oxo-
arsenosugar (2′,3′-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-βD-riboside) and its thio-analogue were synthesized as described by Traar and Francesconi (18). Trimethylarsine sulfide was synthesized on an analytical scale by addition of 0.1% (v/v) of a saturated aqueous H2S solution to a 100 µg As L-1 standard solution of TMAO; after 5 min, the excess H2S was removed by purging the solution with argon. The identity of trimethylarsine sulfide (TMAS) was established by electrospray mass spectrometry with a single quadrupole mass spectrometer of the SL type (Agilent Technologies, Waldbronn, Germany). Standard ESMS conditions used here and elsewhere (see below) were as follows: drying gas temperature, 350 °C; drying gas flow, 12 L min-1; capillary voltage, 4000 V. For TMAS, the spectrum was obtained with a fragmentor voltage of 150 V: m/z 153 [M + H]+, 107 [AsS]+. Synthesis of the Model Compound, (R,S)-4-Dimethylarsinothioyl-butane-1,2-diol. The full synthetic details for this compound are provided as Supporting Information. Briefly, com-
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mercially available (R,S)-4-(2-hydroxy-ethyl)-2,2-dimethyl-[1,3]dioxolane was converted into the bromide, which was then reacted with sodium dimethylarsenide followed by treatment with H2O2 to give (R,S)-4-(2-dimethylarsinoyl-ethyl)-2,2-dimethyl-[1,3]-dioxolane. This arsine oxide was converted to the sulfide by treatment with H2S; deprotection with aqueous CF3COOH gave the desired product (R,S)-4-dimethylarsinothioyl-butane-1,2-diol as a colorless syrup (50% overall yield). Electrospray MS m/z 227 [M + H]+. The oxo-analogue of the thio compound was prepared by treating it with aqueous H2O2, while the trimethylated analogue was prepared by reacting the oxo analogue with dithiothreitol and methyl iodide as previously reported for a related compound (19). The identities of the two products were supported by electrospray MS data showing [M + H]+ at m/z 211 and M+ at m/z 209 for the oxoand trimethylated analogues, respectively. Administration of Oxo-Arsenosugar to the Volunteers. A stock solution of the oxo-arsenosugar was prepared in water and the precise arsenic concentration determined by inductively coupled plasma mass spectrometry (ICPMS; see below). An appropriate volume of this stock solution equivalent to about 1 mg of As (850 µg to 1100 µg of As depending on the volunteer) was then ingested in ca. 200 mL of water by each of six volunteers. Collection of Urine Samples. The experiments were conducted with the approval (12-203 ex 01/02) of the Ethics Commission of the University of Medicine Graz. Six volunteers participated: volunteer 1, female 28 years; volunteer 2, male 27 years; volunteer 3, female 31 years; volunteer 4, male 39 years; volunteer 5, female 27 years; volunteer 6, male 40 years. The volunteers refrained from eating food known to contain significant concentrations of arsenic (e.g., seafood) for three days before and during the experiment. Four background urine samples were taken: three on the day before the ingestion and the morning urine on the day of ingestion. The general sampling protocol has been previously described (12). Briefly, the arsenosugar was ingested as an aqueous solution between 9:00 and 10:00 on the morning of the first day of the experiment. All urine samples were individually collected, in 500 or 1000 mL polyethylene bottles, during the following four days. All consumed foods and drinks in this period were recorded. The samples were stored at 4 °C (domestic refrigerator) for a maximum of 10 h before the mass of each sample was recorded and the specific gravity measured with a Leica TS 400 total solids refractometer (Leica Microsystems, Buffalo, United States). Two subsamples (40 mL each in polypropylene tubes) were then taken and stored separately at -80 and 4 °C. Six months after the experiment with six volunteers, another experiment was performed with volunteers 1 and 5 following the same ingestion and sampling procedures. Collection of Whole Blood and Serum Samples. From volunteers 2, 3, and 5, whole blood, approximately 6 mL (8 mL vacuette heparin tubes, Greiner bio-one, Frickenhausen, Germany), and serum, approximately 4 mL (8 mL vacuette serum separator tubes Greiner bio-one, Frickenhausen, Germany), were taken before ingestion and 2, 4, 6, 8, and 24 h after ingestion. For the remaining three volunteers (1, 4, and 6), blood and serum samples were taken only in the first hour: from volunteer 1, every 15 min; from volunteer 4, every 20 min; and from volunteer 6, every 10 min. Serum samples were obtained after centrifugation. The blood samples were stored at 4 °C (2 days) and the serum samples at -80 °C (up to 18 months) until the analysis. Determination of Total Arsenic in Urine, Blood, and Serum. Arsenic concentrations in samples of urine, blood, and serum were determined by ICPMS following microwave-assisted acid mineralization. In addition, urine samples were prepared for analysis by simple dilution without prior mineralization. In all cases, determinations were performed with an Agilent ICPMS 7500ce equipped with an ISIS (Integrated Sample Introduction System) (Agilent Technologies, Waldbronn, Germany) and a Microflow PFA-100 nebulizer. Arsenic standards for external calibration were prepared from a 1000 mg As L-1 standard solution in the same acid and/or methanol concentration as the samples. Each sample was analyzed in triplicate.
Raml et al. Microwave-assisted acid mineralization of samples of urine, blood, and serum was performed with an Ultraclave III (MLS, Leutkirch Germany). Portions of sample (usually 1.00 mL), nitric acid (2.00 mL), and water (2.00 mL) were transferred to 12 mL quartz tubes; the tubes were placed in a quartz rack, covered with Teflon caps, and the rack was mounted onto the microwave system. The holding vessel was filled with 270 mL of water and 30 mL of H2O2; the system was closed, loaded with argon to 4 × 106 Pa, and the mixture was heated for 30 min at 250 °C. After mineralization, the samples were diluted to 10.0 mL (blood and serum samples) or 50.0 mL (urine samples) with water in polypropylene tubes (Greiner, Bio-one, Frickenhausen, Germany) before analysis. The method was validated by analysis of NIES CRM 18-Human Urine (National Institute for Environmental Studies, Ibaraki, Japan) (certified [As] ) 137 ( 11 µg L-1; found [As] ) 146 ( 14 µg L-1, n ) 5; values represent mean ( 2 SD). For sample preparation without mineralization, urine samples (200 µL) were mixed with a solution (9.8 mL) of water containing conc. HNO3 (100 µL) and methanol (500 µL). The stability of the measurements recorded on these solutions was improved when methanol was also added at a level of 5% v/v to the wash solutions of the ICPMS. In the early part of the study, both sample preparation methods were applied to 54 urine samples with arsenic concentrations from ca. 5-1200 µg L-1; the data were compared and found to agree closely (r2 ) 0.997, slope ) 0.994). Consequently, the dilution method, which was simpler and faster to perform, was used in preference to the mineralization method for the remaining urine samples. All total arsenic data for urine samples reported here refer to data obtained using the dilution method. Determination of Arsenic Species in Urine. The urine samples were analyzed by HPLC/ICPMS using one or more of the following four sets of chromatographic conditions: (i) anion-exchange performed with a Hamilton PRP-X100 column (4.1 × 100 mm) and mobile phase of 20 mM NH4H2PO4 at pH 6.0, adjusted with aqueous NH3, containing 3% methanol (40 °C, 1.5 mL min-1); (ii) cation-exchange performed with a Chrompack Ionospher C column (3 × 100 mm) and a mobile phase of 10 mM pyridine at pH 3.0, adjusted with HCO2H, containing 3% methanol (40 °C, 1.5 mL min-1); (iii) reversed-phase with a Waters Atlantis dC18 column (4.6 × 150 mm) and a mobile phase of 20 mM NH4H2PO4 at pH 3.0, adjusted with aqueous H3PO4 (30 °C, 1 mL min-1); or (iv) reversed-phase with a Waters Atlantis dC18 column (4.6 × 150 mm) and a mobile phase of 20 mM NH4HCO2 at pH 3.0, adjusted with HCO2H (30 °C, 1 mL min-1). The different conditions were necessary to effect separation of all the various arsenic metabolites to obtain quantitative data. Determination of Arsenic Species in Serum. A 500 µL portion of the serum sample was transferred to a microcentrifuge tube (1.5 mL, Eppendorf AG, Hamburg, Germany), and 500 µL of acetonitrile was added. The solution was centrifuged at 7,000g for 15 min in a microcentrifuge, and the supernatant removed and dried in a centrifugal lyophilizer (Maxi-Dry Plus, Heto Holton, Allerød, Denmark). The resultant residue was dissolved in 100 µL of buffer (20 mM NH4HCO2 at pH 3.0) and the arsenic species determined by HPLC/ICPMS under reversed-phase conditions (Waters Atlantis dC18 column, 4.6 × 150 mm) (30 °C, 1 mL/min) and 20 mM NH4HCO2 at pH 3.0, adjusted with HCO2H. Triplicates of the t ) 2 h serum sample (no thio-DMAA and thio-DMAE detectable) were spiked with thio-DMAA and thio-DMAE, and the spiked samples were subjected to the complete cleanup procedure to investigate the recovery of these compounds during sample preparation. Purification of Unknown Urine Metabolite (Oxo-U/Thio-U) and Analysis by HPLC/Electrospray-MS. An unknown arsenic metabolite, termed thio-U, was purified from urine sample at t ) 23 h of volunteer 5 (first experiment) by preparative HPLC (Atlantis dC18 column, 19 × 150 mm, with water as mobile phase at 10 mL min-1; 5 × 800 µL injections). The effluent of the preparative column was split so that about 10% of the flow was directed to the ICPMS to determine the time interval of elution of thio-U. The eluent from the preparative column was collected during the
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Table 2. Cumulative Excreted Total Arsenic Expressed as a Percentage of the Ingested Arsenic after Ingestion of Oxo-Arsenosugara Cumulative excreted arsenic (%) hours after volunteer volunteer volunteer volunteer volunteer volunteer ingestionb 1 2 3 4 5 6
5 10 20 30 40 50 60 70 80 90
0.7 1.5 2.6 3.0 3.2 3.5 3.6 3.7 3.8 3.9
2.1 2.9 4.6 6.2 7.0 10 11 13 15 15
6.1 13 56 68 76 82 85 87 87 88
1.2 2.0 23 48 55 73 79 83 87 93
1.9 16 64 74 85 89 93 94 95
0.7 7.9 58 71 76 82 83 85 85
a See Supporting Information for complete data sets for all volunteers. Actual sampling times varied between individuals; to simplify the presentation of the data, time periods were divided into 5 or 10 h intervals. b
appropriate time interval and evaporated under reduced pressure in a centrifugal lyophilizer, and the resulting residue was dissolved in 200 µL of water. To one-half (100 µL) of this solution, 5 µL of H2O2 was added to convert the isolated thio-U to oxo-U. Portions of the two solutions (each ∼2 mg As L-1) were then each diluted in water (1 + 9 v/v) to give working solutions that contained thio-U or oxo-U at ∼200 µg As L-1. Analysis by HPLC/ICPMS showed that the solutions were arsenic-pure, i.e., no other arsenic peak (m/z 75) was detected (LOD ∼0.5 µg As L-1). Oxo-U was quaternized by treating 10 µL of the ∼2 mg As L-1 solution with 200 µL of dithiothreitol (10 mM) and 10 µL of methyl iodide, as previously described for dimethylarsinoyl compounds (19).
Results and Discussion Excretion of Total Arsenic. The administered amount of arsenic differed slightly between the volunteers (850-1100 µg As); therefore, to enable easier comparison of the six volunteers, the accumulative amount of arsenic excreted was calculated as a percentage of the ingested amount. After oral ingestion of the oxo-arsenosugar, the excretion of total arsenic over the subsequent 90 h varied significantly between the volunteers, with two distinct excretion patterns being observed (Table 2; see Supporting Information for full data set). Volunteers 3-6 excreted almost all (85-95%) of their ingested arsenic during the 90 h. The majority of the arsenic was excreted within the first day, except for volunteer 4 who showed slightly delayed excretion. Volunteers 1 and 2, however, were low-excretors: after 90 h, only ca. 4% and 15% of the ingested arsenic, respectively, were found in their urine. The experiment was repeated after six months with volunteers 1 and 5, low- and high-excretors, respectively, with a very similar outcome (see Supporting Information, Tables 2 and 7): volunteer 1 again excreted only a small amount of the ingested arsenic (6.3%), whereas volunteer 5 excreted >82% (this was a minimum value; a more precise value for volunteer 5 could not be assigned because the 20-h urine sample was inadvertently not collected). These data suggest that the intraindividual variability in arsenic excretion is minimal. The individual variability observed here agrees qualitatively with that reported by Le et al. (8) who investigated arsenic excretion after ingestion of edible algae. Although these authors did not report quantitative data, they observed increased urinary excretion of arsenic in seven of their nine subjects who ingested algae, whereas the remaining two showed only background levels of arsenic indicating that they did not excrete their ingested arsenic in the urine. Van Hulle et al., however, reported
only minor differences in the way six volunteers metabolized the arsenic compounds present in the alga Laminaria sp. (10). Seven of nine volunteers ingesting arsenic compounds contained in mussels produced generally similar arsenic urinary excretion patterns (11). The low excretion of arsenic found for two volunteers in our study was a surprise result since urinary excretion is considered to be the main pathway for the elimination of arsenic, both inorganic and organic arsenic species (4). There are several possible explanations for the fate of arsenic in the two lowexcretors. Possibly, the gastrointestinal absorption of the compound was low in volunteers 1 and 2 (i.e., the compound was not bioaccessible), and the arsenic was excreted in the feces. For inorganic arsenic, excretion is known to occur to some degree via the feces depending on the solubility of the compound and on the matrix in which the arsenical is ingested (4). Arsenic Metabolites in Urine. The major arsenic species in human urine following ingestion of an arsenosugar were identified in a previous study (12) as DMA, oxo-DMAE, thioDMAE, oxo-DMAA, thio-DMAA, oxo-arsenosugar, thio-arsenosugar, and TMAO. These species were all observed in the current study together with traces of thio-DMA and TMAS, both of which were identified by cochromatography with standards on the Atlantis dC18 column with 20 mM NH4HCO2 at pH 3.0 as the mobile phase. In addition, an unknown pair of species (oxo-U/thio-U; see below) was present in the samples in variable but significant quantities (up to about 120 µg As L-1 equivalent to about 10% of the total arsenic in one urine sample). The species patterns for the four volunteers termed highexcretors were similar to each other; Figure 1a shows the patterns for volunteer 3 (see Supporting Information for full data sets and some representative HPLC/ICPMS chromatograms). Volunteer 4 differed slightly from the other three by showing a delayed excretion profile and having a larger quantity of the unknown arsenical oxo-U and/or oxo-DMAE. In order to accommodate the slight differences in excretion rate between the four individuals, the total amount of the excreted arsenic metabolites were calculated as percentages of the total excreted arsenic at the time point when 80% of the ingested arsenic had been excreted. In this way, the main metabolites for the four high-excretors over the course of the experiment were determined as DMA (40-46% of total), thio-DMAA (15-19%), thio-DMAE (5-9%), and thio-U (3-8%). The arsenic species profiles for the two low-excretors were similar to each other, but quite different from those recorded for volunteers 3-6. Thus, for volunteers 1 and 2, only three arsenic species were detected: unchanged oxo-arsenosugar, thioarsenosugar, and DMA (Figure 1b). The arsenic species were also determined in the repeat experiment with volunteers 1 and 5 (Supporting Information, Tables 10 and 15); the results matched those obtained in the first experiment. There are several possible explanations for the observed differences between the low-excretors and high-excretors. The differences might lie with biotransformation enzymes in the liver; perhaps the oxo-arsenosugar is not significantly degraded by the low-excretors, at least not to products that are excreted in the urine. Conversely, differences in gut flora may be a factor; the arsenosugar might not readily cross the gut membrane, and biotransformations in the gut are perhaps necessary to facilitate the uptake of arsenic. The high-excretors, however, might be able to metabolize the arsenosugar to a readily bioaccessible form, which then undergoes further transformation in the liver. For the low-excretors, this first step in the gut might not occur
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Figure 1. Time series excretion of arsenic metabolites in urine for (a) volunteer 3, a high excretor, and (b) volunteer 2, a low excretor. See Supporting Information for tables of data sets for all volunteers.
Table 3. Total Arsenic Concentrations in Serum and Whole Blood of Volunteers 3 and 5 (Mean ( SD, n ) 3 Determinations) volunteer 3
volunteer 5 -1
concentration (µg As L )
concentration (µg As L-1)
time (h)
whole blood
serum
whole blood
serum
0 2 4 6 8 24
