Human metabolism of arsenolipids present in cod liver

Transcription

1 Anal Bioanal hem (26) 385: DI 1.17/s x RIGINAL PAPER Ernst Schmeisser. Walter Goessler. Kevin A. Francesconi Human metabolism of arsenolipids present in cod liver Received: 2 November 25 / Revised: 27 February 26 / Accepted: 28 February 26 / Published online: 28 March 26 # Springer-Verlag 26 Abstract We report results from the first investigation of the human metabolism of arsenic-containing lipids (arsenolipids), significant arsenic constituents of some seafood products. Two male volunteers ingested canned cod liver and the arsenic metabolites in their urine were monitored by high-performance liquid chromatography inductively coupled plasma mass spectrometry over a 66-h period. Volunteer A consumed 85 g (wet mass) of cod liver containing a total of approximately 12 μg arsenic, 77% of which was present as arsenolipids, and volunteer B consumed 85 g (wet mass) of cod liver, 25% of which was present as arsenolipids, together with 2 g of cod liver oil, containing a total of about 18 μg arsenic. The structures of the arsenolipids are currently unknown, whereas the majority of the non-lipid arsenic in the cod liver was identified as arsenobetaine, which was excreted unchanged. The arsenolipids were rapidly metabolised to water-soluble compounds and excreted in the urine; peak arsenic concentrations were recorded between 7 and 15 h (volunteer A) and between 6.5 and 15 h (volunteer B), and by the end of the experiment about 9% of the ingested arsenic had been accounted for in the urine for both volunteers. The major arsenolipid metabolite was dimethylarsinate (), constituting 73% (volunteer A) or 41% (volunteer B) of the total urinary arsenic, and most of the remaining arsenolipid-derived arsenic, constituting about 1% (volunteer A) and 5% (volunteer B), comprised four novel arsenic-containing fatty acids, namely oxo-dimethylarsenopropanoic acid, thio-dimethylarsenopropanoic acid, oxo-dimethylarsenobutanoic acid, and thio-dimethylarsenobutanoic acid. Unchanged arsenobetaine (15% for volunteer A and 51% for volunteer B) made up the remaining E. Schmeisser. W. Goessler. K. A. Francesconi (*) Institute of hemistry Analytical hemistry, Karl Franzens University Graz, Universitaetsplatz 1, 81 Graz, Austria kevin.francesconi@uni-graz.at Tel.: Fax: urinary arsenic together with trace quantities of other, mostly unknown, arsenicals. In a second experiment (volunteer A only), performed with pure cod liver oil, which contains only arsenolipids, and the same four arsenic fatty acids were excreted in the urine. The study shows that arsenolipids in cod liver are bioavailable, and that they are quickly biotransformed to several watersoluble arsenicals, the structures of which suggest that the native arsenolipids contain a dimethylarsine oxide moiety. Keywords Arsenolipids. Biotransformation. Arsenic speciation. Urine metabolites Introduction Arsenic is an element well known for the high toxicity of many of its compounds [1]. hronic exposure to inorganic arsenic species in drinking water has serious health effects, in many cases resulting in skin cancers and internal cancers, and is a major worldwide health problem [2]. Another significant source of arsenic to humans is food, particularly seafood, where concentrations can be as high as 3 μgg 1 (dry mass). In most seafood samples, however, arsenic is present primarily as organoarsenic compounds. The organoarsenicals found in food samples can be categorised generally as water-soluble and lipid-soluble arsenic species (arsenolipids). Most work on identification of arsenicals has focussed on the water-soluble compounds, and more than 4 species have been reported. The most widespread compounds are arsenobetaine (), which predominates in fish and crustaceans, and arsenosugars, which are major arsenic constituents of algae and molluscs [3]. Even though lipid-soluble arsenicals in some food samples can be in excess of 9% of the total arsenic [4], their structures remain largely unknown, with only one lipidsoluble arsenical, extracted from a brown alga, rigorously identified so far [5]. Previous studies have chemically or enzymatically hydrolysed the arsenolipids to water-soluble products which were then identified, and thereby provided some information on the original structure [6 11].

2 368 A risk assessment of arsenic in water and foods must be based not only on the types and quantities of arsenicals present, but also on the metabolism of these compounds [1]; thus, many studies have been undertaken and have shown that ingested inorganic arsenic is largely methylated in the body and excreted in urine, primarily as methylarsonate (MA) and dimethylarsinate () [1]. There have been, however, relatively few human metabolic studies on the organoarsenic species found in seafood, and they have produced contrasting results. Whereas is rapidly excreted unchanged in the urine [12], arsenosugars are mostly biotransformed and excreted in urine as a multitude of arsenic metabolites [13 17]. There have been no metabolic studies of arsenolipids. Despite the fact that the structures of arsenolipids are not known, valuable information on their potential toxicity and/or chemical nature may be obtained by investigating their metabolism in humans. With that goal, we have investigated the arsenic species excreted in urine after consumption of arsenolipids contained in commercially available canned cod liver products. In the first part of that work, we reported the structural elucidation of four novel urinary metabolites resulting from the arsenolipids [18]. Here, we report quantitative data using high-performance liquid chromatography (HPL)-inductively coupled plasma mass spectrometry (IPMS) on the intake and excretion of the arsenic species. Materials and methods Standards and reagents Standard solutions containing 1, mg dm 3 of each of the following compounds were prepared in Milli-Q water (18.2 MΩ cm): arsenite [(III)] and arsenate [(V)] prepared from Na 2 and Na 2 H 4 7H 2, respectively (Merck, Darmstadt, Germany); (see Fig. 1 for structures of organoarsenicals relevant to this study) prepared from sodium dimethylarsinate trihydrate (Fluka, Buchs, Switzerland); MA prepared from methylarsonic acid synthesised in-house from 2 3 and I in NaH (Meyer reaction); trimethylarsine oxide was synthesised according to Merijanian and Zingaro [19]; and the tetramethylarsonium iodide were prepared according to McShane [2] by reaction of trimethylarsine with bromoacetic acid or methyl iodide, respectively. Arsenocholine (A), as the bromide salt, was synthesised according to Irgolic et al. [21]. xo-dimethylarsenobutanoic acid (; Fig. 1) was synthesised according to Francesconi et al. [22]. xo-dimethylarsenopropanoic acid (oxo-p; Fig. 1) was prepared from 3-bromopropanoic acid and bis(dimethylarsenic) oxide (obtained from dimethyliodoarsine and NaH). Thio analogues (thio-p, thio-dm; Fig. 1) of the oxo-arsenic compounds oxo-p and were prepared according to Schmeisser et al. [23]. Electrospray mass spectra were obtained for the four standard arsenic-containing fatty acids: thio-dm m/z 225 and 227 [M+H] +, 17 and 19 [S] +, 87 [H 2 H 2 - H 2 H] + ; thio-p m/z 211 and 213 [M+H] +, 17 and 19 [S] + ; m/z 29 [M+H] +, 121 [( ) 2] +, 87 [H 2 H 2 H 2 H] + ; oxo-p m/z 195 [M+H] +, 121 [( ) 2 ] +. All four arsenic-containing fatty acids showed a signal at m/z 91 [] + formed from traces of 2 in the N 2 drying gas [24]. 1 H-NMR oxo-p (4 MHz, D 2, δ): (4H, m, 2H 2 ), 2.1 (6H, s, Me 2 ). 13 -NMR oxo-p (1 MHz, D 2, δ) (), 31.5 (H 2 ), 3.4 (H 2 ), 19.2 (Me 2 ). Ammonium dihydrogen phosphate (pro analysi, p.a.), aqueous ammonia solution (25%, Suprapur), hydrochloric acid (p.a., 32%); sodium borohydride, sodium hydroxide, ammonium hydrogen carbonate (p.a.), pyridine (p.a.), hydrogen peroxide (p.a., 3%), and acetic acid (p.a., 96%) were purchased from Merck (Darmstadt, Germany); hexane (p.a.) and chloroform (p.a.) were purchased from Acros rganics (Geel, Belgium); methanol (p.a.) was purchased from Fisher Scientific (Loughborough, UK); and nitric acid (p.a., concentrated) was purchased from Roth (Karlsruhe, Germany) and was further purified in a quartz subboiling distillation unit H Arsenobetaine () Dimethylarsinate () Methylarsonate (MA) Arsenocholine (A) H H R H R H H H xo-arsenosugar glycerol (Gly) Fig. 1 Structures and abbreviations of arsenic compounds R = xo-dimethylarsenopropanoic acid (xo-p) R = S Thio-dimethylarsenopropanoic acid (Thio-P) R = xo-dimethylarsenobutanoic acid (xo-dm) R = S Thio-dimethylarsenobutanoic acid (Thio-DM)

3 369 The certified reference material TRT-1, (hepatopancreas from lobster, Homarus americanus) was purchased from the National Research ouncil of anada (ttawa, anada), and NIES certified reference material (RM) no. 18 (human urine) was purchased from the National Institute for Environmental Studies (Ibaraki, Japan). Analysis of cod liver and cod liver oil shown in a recent study [4], cod liver products are a rich source of lipid-soluble arsenicals and therefore we used a commercially available canned smoked cod liver in its own oil (Larsen Danish Seafood). Before the ingestion experiments were started, we determined the arsenic species in several cans of this product. The oil was drained from the liver tissue, and the two parts were separately freeze-dried to yield dry (water-free) residues. Water-soluble and lipidsoluble fractions of all samples were then prepared as follows. Water-extractable arsenic Individual portions (approximately 3 mg weighed to.1 mg) of freeze-dried cod liver and cod liver oil were transferred together with water (1 cm 3 ) to 15-cm 3 polypropylene tubes and the mixtures were shaken overnight at room temperature. The water phase was filtered (.22 μm) prior to total arsenic determination and arsenic speciation analysis. Lipid extractable arsenic Portions (approximately 3 mg weighed to.1 mg) of freeze-dried cod liver were transferred with hexane or a mixture of Hl 3 /hexane/ MeH (4:5:1, v/v) to 12-cm 3 glass tubes (Pyrex) and the mixtures were shaken overnight at room temperature. Portions (approximately 1 mg weighed to.1 mg) of cod liver oil were completely soluble in hexane or Hl 3 / hexane/meh (4:5:1, v/v). The organic phase was filtered and evaporated prior to total arsenic analysis. These procedures were also performed on subsamples of the actual cod liver/cod liver oil products consumed by the volunteers in the experiments described next. Ingestion of cod liver/cod liver oil and collection of urine samples Two separate experiments were performed: the first experiment involved two volunteers (volunteer A, male, 51 years of age; volunteer B, male, 39 years of age), and the second experiment used volunteer A only. In both experiments, the volunteers refrained from eating food known to contain significant concentrations of arsenic (e.g. seafood or mushrooms) for 3 days before and during the experiment. n the morning of the first day of the experiments, three urine samples from the volunteer (s) were collected to give the background arsenic concentration. In experiment 1, volunteer A ingested 85 g of cod liver (wet mass) containing about 12 μg, and volunteer B ingested 85 g of cod liver (wet mass) together with 2 g of cod liver oil containing a total of approximately 18 μg. In the second experiment, volunteer A ingested pure cod liver oil (2 g) containing approximately 2 μg. All urine samples were individually collected in 5-ml polyethylene bottles for a 66-h (experiment 1) or a 15-h (experiment 2) period. The masses of the urine samples were recorded and the specific gravity was measured with a Leica TS 4 total solid refractometer (Leica Microsystems, Buffalo, USA); urine volumes were then calculated from the masses of the samples. The samples were stored at 4 and then analysed during the following 1 days. Total arsenic and arsenic species concentrations were normalised to the mean specific gravity using the equation ð norm ¼ spec:grav: mean 1 spec:grav: sample 1 Total arsenic analysis Þ sample : (1) Arsenic concentrations in the samples were determined with IPMS after mineralisation of the samples with microwave-assisted acid digestion. Portions (approximately 1 mg weighted to.1 mg) of freeze-dried cod liver or cod liver oil together with nitric acid (4 cm 3 ) and water (1 cm 3 ) were transferred to 12-cm 3 quartz vials of an autoclave digestion system (ultralave 2, EMLS, Leukirch, Germany). For digestion of the water extracts and urine samples, 2-cm 3 aliquots of the sample solutions, 2 cm 3 nitricacidand1cm 3 water were used. The TFM/PTFE vessel inside the high-grade steel pot was filled with 3 cm 3 1% H 2 2 (w/w) absorption liquid. The autoclave was pressurised with argon (4 1 6 Pa), and the samples were heated according to the following temperature program: step 1, 3 min to 25, step 2, 3 min at 25. The digested sample solutions were quantitatively transferred to 5-cm 3 polypropylene tubes and diluted to 2 cm 3 with Milli-Q water. The arsenic concentrations in these digest solutions were determined by IPMS (Agilent 75ce, Waldbronn, Germany). 72 Ge was used as the internal standard. The accuracy of the measurement was tested by the analysis of two reference materials: for TRT-1 (approximately 1 mg weighed to.1 mg, certified value 24.6±2.2 mg kg 1 dry mass), we obtained 24.5±.1 mg kg 1 dry mass (n=3), and for NIES RM no. 18 (human urine, 2 ml weighed to.1 mg, certified value.137±.11 mg kg 1 ), we obtained.145±.1 mg kg 1 (n=3). HPL-IPMS of water extracts and urine samples HPL was performed with a Hewlett-Packard 11 Series system (Hewlett-Packard, Waldbronn, Germany) equipped with a quaternary pump, a vacuum degasser, a column oven, and a thermostatted autosampler with a variable 1-mm 3 injection loop. Separation of the arsenic species was carried out mostly under anion- and cation-exchange

4 37 conditions. For anion-exchange chromatography, PRP- X1 columns (25 mm 4.1-mm inner diameter, or 1 mm 4.1-mm inner diameter; Hamilton ompany, Reno, NV, USA) at 4 were used with mobile phases of 2 mmol dm 3 NH 4 H 2 P 4 at ph 5.6 (adjusted with 25% aqueous N ), or 2 mmol dm 3 NH 4 with 3% MeH at ph 1.3 (adjusted with 25% aqueous N ) and a flow rate of 1.5 cm 3 min 1. For cation-exchange chromatography, a ZRBAX 3-SX column (15 mm 4.6-mm inner diameter; Hewlett-Packard, Waldbronn, Germany) at 3 was used with a mobile phase of 2 mmol dm 3 aqueous pyridine solution at ph 2.6 (adjusted with formic acid) and a flow rate of 1.5 cm 3 min 1. For the determination of oxo-p we used reversed-phase chromatography as previously reported [18]. The injection volume for all sets of chromatographic conditions was 2 mm 3. The outlet of the HPL column was directly connected to the IPMS instrument (equipped with a Babington-type nebuliser) with PEEK (polyetheretherketone) capillary tubing (.125-mm inner diameter). The ion intensities at m/z 75 and 77 were monitored. The IPMS signal was optimised with the mobile phase containing 1 μg dm 3 to give the maximum response on the signal (m/z 75). Arsenic compounds were quantified with external calibration against standard solutions of the relevant species. The accuracy of the measurements was tested by the analysis of the reference material NIES RM no. 18 (human urine: certified concentration.36±.7 mg dm 3 ; certified concentration.69±.12 mg dm 3 ). We obtained a concentration of.41±.5 mg dm 3 and an concentration of.69±.6 mg dm 3 (n=3 injections). Vapour (hydride) generation IPMS of the arsenic compounds The generation of volatile arsenic analytes was performed with the hydride generation accessory from Agilent Technologies as described in a recent study [25]. The HPL system was connected directly to a continuous-flow hydride generation IPMS instrument (Hewlett-Packard 45, Waldbronn, Germany) with an integrated sample introduction system (Agilent, Waldbronn, Germany). The hydride generation system was equipped with a concentric nebuliser, a modified cyclonic spray chamber, and a membrane filter. Vapour generation was performed with.7% NaBH 4 in.1 M NaH (flow rate.5 cm 3 min 1 )as reducing agent and 3 M Hl (flow rate.3 cm 3 min 1 ), and the volatile analytes were transported via a.5-m sample tube (4-mm inner diameter) directly to the torch of the IPMS instrument. Results and discussion Arsenic content and arsenic species in cod liver and its oil Before ingestion of the cod liver in its own oil, representative subsamples were analysed for total arsenic concentration and for water-extractable arsenic species. The cod liver sample for volunteer A had a total arsenic concentration of 2.6 mg kg 1 (dry mass) and the cod liver oil sample had a concentration of 1. mg kg 1 (dry mass), whereas for volunteer B the cod liver had 3.3 mg kg 1 (dry mass) and the cod liver oil 1.3 mg kg 1 (dry mass) (Table 1). These results are in good agreement with those in works by Lunde [6] and Sloth et al. [26]. The amount of water-extractable arsenic in the case of the cod liver sample ingested by volunteer B was about 3 times higher than that obtained for the cod liver sample which Table 1 Water-extractable species from cod liver and cod liver oil. Values are reported on a dry mass basis as the mean (mg kg 1 ) and in parentheses as the relative standard deviation, n=4 Sample Total Water-extractable arsenic [%] a GLY A U1 olumn recovery [%] Volunteer A cod liver 2.62 (3.4%) cod liver oil 1. (1.%) Volunteer B cod liver 3.33 (4.5%) cod liver oil 1.3 (5.4%) ±7. (11.%) (9.6%) (13.1%) (3.4%) (13.5%) < ±1.8 (2.5%) (9.3%) (3.9%) (4.2%) (4.3%) < arsenobetaine, dimethylarsinate, GLY oxoarsenosugar glycerol, A arsenocholine, U1 unknown arsenical a Determined with high-performnace liquid chromatography hydride generation inductively coupled plasma mass spectrometry

5 371 was used by volunteer A (Table 1, Fig. 2). This was unexpected because preliminary investigations had indicated little between-can variability for this food product (data not shown), giving results comparable with those obtained for the cod liver sample ingested by volunteer A. Extraction of the cod liver (ingested by volunteer A) with hexane removed more than 4% of the arsenic. Although the arsenic quantities were different, the patterns of the arsenic species in the two water-soluble fractions were similar (Table 1, Fig. 2). Both samples contained as the dominant species, with smaller quantities of, oxo-arsenosugar-glycerol, A, and an unknown arsenical. The unknown arsenic compound was eluted shortly before on anion-exchange chromatography, but a peak could not be assigned to it in the cation-exchange chromatogram. Using HPL hydride generation IPMS, no signal was obtained, which suggests that the unknown compound may contain a quaternary arsonium group. ur recent experience with this method has shown that all oxo- and thio-arsenicals tested so far give signals, whereas arsonium compounds (e.g. and A) do not. Aqueous extraction of the cod liver oil removed negligible quantities (less than.5%) of the total arsenic and this amount was below the detection limit for speciation. The cod liver oils were completely soluble in hexane. In summary, although both volunteers consumed lipidsoluble arsenic present in the cod liver/cod liver oil products, the sample consumed by volunteer B had a higher water-soluble arsenic content comprising mainly. These data impacted on the pattern of urine metabolites observed for the two volunteers, as described later. Excretion of total arsenic For convenience, we refer here to the total arsenic concentrations normalised by the specific gravity (Eq. 1) because this reduces confounding effects of variable fluid intake/excretion throughout the day. After the ingestion of the cod liver products (experiment 1), the two volunteers quickly excreted arsenic in their urine (Table 2). Increased urinary arsenic concentration was already evident in the first sample collected after ingestion of the cod liver (7 h a /Gly/(III) Water extract of cod liver ingested by Volunteer A at ph b Water extract of cod liver ingested by Volunteer A ation-exchange at ph arsenic cations U arsenic anions Gly A c /Gly/(III) Water extract of cod liver ingested by Volunteer B at ph d Water extract of cod liver ingested by Volunteer B ation-exchange at ph arsenic cations U Gly A arsenic anions Fig. 2 Anion- and cation exchange chromatograms of water extracts of cod liver ingested by volunteers A and B. For chromatographic conditions see Material and methods. a, b Water extracts of cod liver ingested by volunteer A. c, d Water extracts of cod liver ingested by volunteer B

6 372 for volunteer A and 2.5 h for volunteer B), and the peak arsenic concentration was recorded shortly thereafter (between 7 and 15 h for volunteer A, and between 6.5 and 15 h for volunteer B). The concentrations then steadily decreased and essentially reached background levels after about 2 days, by which time about 85% of the ingested arsenic was accounted for in the urine samples. Hence, the ingested arsenolipids were efficiently absorbed from the intestines. Ingestion of cod liver oil alone (experiment 2, volunteer A) also resulted in rapid excretion of arsenic, peaking at about 6 h. Table 2 Urinary excretion of total arsenic after ingestion of cod liver and cod liver oil Urine sample (h) a Volume (cm 3 ) Arsenic concentration (μg dm 3 ) Arsenic concentration (μg dm 3 ) normalised b Sum of arsenic species (μg dm 3 ) Total arsenic (μg) umulative excretion in μg c (%) Experiment 1, volunteer A ingestion of cod liver (12±5 μg ) (24) (41) (58) (66) (73) (78) (83) (84) (85) (87) (89) Experiment 1, volunteer B ingestion of cod liver and cod liver oil (19±1 μg ) (9) (27) (45) (58) (66) (71) (75) (77) (8) (82) (84) (86) (86) (87) (88) (88) (9) Experiment 2, volunteer A ingestion of pure cod liver oil (2±3 μg ) (51) (66) (76) a Values represent time (hours) after ingestion of cod liver and cod liver oil b alculated from Eq. 1 c Small background contribution (mean 8 μg dm 3 ) has been subtracted

7 373 Although there are no other reported excretion data after ingestion of arsenolipids, it is interesting to compare our results with those obtained when arsenosugars were ingested, either as a pure compound (oxo-arsenosugarglycerol) [15, 17] or as a mixture of species naturally present in algae [13, 14, 16]. In those experiments, arsenic was generally excreted more slowly compared with the results we obtained, with the peak fraction recorded between 1 and 3 h. Arsenic metabolites in urine Experiment 1, volunteer A Speciation analysis showed that the urine samples had qualitatively similar patterns of arsenic species; the following discussion deals with the urine sample collected at 7 h, which had the highest concentration of arsenic. HPL-IPMS at ph 5.6 revealed the presence of at least five arsenic species (Fig. 3a, panel i). With and without the incorporation of the hydride generation step, we were able to identify three arsenic species, namely, MA and. Quantitative comparison with the speciation data for background urine samples showed that and were clearly a Experiment 1: volunteer A: ingestion of cod liver meat (~23% water-soluble ) 7 6 i ph ii ph iii thio-p/thio-dm/ arsenic anions ation-exchange ph void /Gly/(III) MA i /Gly/(III) void ph thio-p thio-dm Arl Gly b Experiment 1: volunteer B: ingestion of cod liver (~75% water-soluble ) and cod liver oil ii thio-p thio-dm ph c Experiment 2: volunteer A: ingestion of pure cod liver oil iii Arl thio-p/thio-dm/ arsenic anions A ation-exchange ph 2.6 A 25 2 i ph ii ph iii thio-p/thio-dm/ arsenic anions ation-exchange ph void /Gly/(III) MA thio-p thio-dm Arl Gly Fig. 3 a High-performance liquid chromatography (HPL) inductively coupled plasma mass spectrometry (IPMS) chromatograms of urine of volunteer A (experiment 1) after ingestion of cod liver (23% water-soluble ). b HPL-IPMS chromatograms of urine of volunteer B (experiment 1) after ingestion of cod liver (75% water-soluble ) and cod liver oil. c HPL-IPMS chromatograms of urine of volunteer A (experiment 2) after ingestion of cod liver oil (1% lipid-soluble ). Panels i anion-exchange chromatography ph 5.6 (see Material and methods ), panels ii) anion-exchange chromatography ph 1.3, panels iii cation-exchange chromatography ph 2.6 (see Material and methods )

8 374 metabolites resulting from the ingested cod liver. MA, however, was present at a concentration comparable with that in the background samples, and hence was probably not derived from the cod liver. xo-p was determined with reversed-phase chromatography (data not shown), and, oxo-arsenosugar-glycerol, and A were also shown to be present in the urine samples by cationexchange HPL-IPMS (Fig. 3a, panel iii). Because was present in the ingested cod liver, and is known to be excreted unchanged in urine [12], its presence in this urine sample does not suggest that it is a metabolite from the ingested arsenolipids; indeed the relative quantities of ingested/excreted indicated that there was no significant contribution of from the arsenolipids, and a second experiment with pure cod liver oil confirmed this view (see later). Similarly, the presence of small amounts of A in Fig. 4 Time profile of the excreted arsenic species for a volunteer A after ingestion of cod liver containing approximately2% water-soluble and b volunteer B after ingestion of cod liver containing approximately75% water soluble and cod liver oil Normalized concentration [µg dm -3 ] a Normalized concentration [µg dm -3 ] Time [h] thio-p thio-dm Time [h] Normalized concentration [µg dm -3 ] b Normalized concentration [µg dm -3 ] Time [h] thio-p thio-dm Time [h]

9 375 urine could be explained by the small quantities contained in the ingested cod liver. Recent work on arsenic species in urine of sheep [27] and humans [17] has shown the presence of significant concentrations of thio-arsenicals. Accordingly, we specifically looked for thio-arsenicals in our samples by employing anion-exchange chromatography at ph 1.3 as described in a recent study [23]. With this system, the two novel arsenic-containing fatty acids, thio-p and thio-dm, were shown to be present (Fig. 3a, panel ii); a full description of the identification of these new arsenic compounds has recently been reported [18]. The likely source of these arsenicals are arsenolipids present in the cod liver because they were not detectable in the aqueous extracts of the liver. Experiment 1, volunteer B The urine samples from volunteer B also had qualitatively similar patterns of arsenic species up to about 25 h, after which several species were no longer detectable; the following discussion deals with the urine sample collected at 6.5 h, which had the highest concentration of arsenic. The arsenic species were the same as those found for volunteer A, namely,, oxo-p,, thio-p, thio-dm and A (Fig. 3b). The relative quantities, however, were markedly different: for volunteer B, the major species was, reflecting the much higher content of this arsenical in the cod liver sample ingested by this volunteer. was again a significant species, occurring at concentrations much higher than could be explained by the small quantities initially present in the liver. Similar to the results for volunteer A, the likely source of, oxo-p,, thio-p and thio-dm in the urine samples of volunteer B is arsenolipid in the cod liver. Experiment 2, volunteer A Although the data from experiment 1 provided strong evidence for the formation of and the four novel arsenicals oxo-p,, thio-p and thio-dm from the ingested arsenolipids, we sought further support by undertaking a second experiment whereby volunteer A consumed only cod liver oil. In contrast to cod liver, the oil contains no detectable, or other water-soluble arsenicals which might confound the metabolic pattern of arsenicals found in the urine. n this occasion, the results closely matched those found in experiment 1 by showing significant increases in, oxo-p,, thio-p and thio- DM (Fig. 3c); the concentrations of, however, did not increase above background levels, indicating that a lipid-soluble precursor to was not present in the cod liver oil. Time profile of excretion of arsenic metabolites The time profile of the excreted arsenic compounds, normalised to the mean specific gravity, for volunteer A, is shown in Fig. 4a. Except for, all the arsenicals identified had peak concentrations at 7 h, after which time their concentrations fell quite rapidly. reached its peak concentration at 11.5 h and it was detectable until the end of the experiment. These data suggest that arsenolipids are possibly hydrolysed/biotransformed in the human body, in the first step, to arsenic containing fatty acids, and then further to. was excreted unchanged in the urine and reached background levels after about 4 h. The arsenic-containing fatty acids (, thio-dm and thio-p) reached background level after 24 h (volunteer A) and 19 h (volunteer B). xo-p was only present in the urine sample collected 6.5 h after ingestion. The major metabolite excreted over the complete course (66 h) of the experiment was (73%). The relative quantities of the other arsenicals excreted during the 66 h, were 15% for, 5.9% for thio-dm, 2.6% for thio-p, 1.3% for and less than 2% for all the other arsenicals. Similar results (not shown) were obtained when volunteer A ingested only the cod liver oil. (69%) was again the major excreted arsenic metabolite, followed by thio-dm (7.9%), (8.%, background level), thio-p (4.2%) and (2.3%). The situation was different when volunteer B ingested the cod liver/cod liver oil (Fig. 4b). From 2.5 h, arsenic metabolites began to appear in the urine at concentrations considerably above background concentration, peaking between 7.5 and 15 h. was the major arsenic species (51%) because it was the major arsenical in the cod liver and is not metabolised by humans [12]. The relative quantities of the other arsenicals were 41% for, 2.4% for thio-dm, 1.9% for thio-p, 1.3% for, and less than 2% for unknown arsenicals. Similar to the situation with volunteer A, the excretion rate for was clearly slower than that for the other metabolites. onclusions This study with two human volunteers has provided data showing that arsenolipids present in cod liver are metabolised and excreted almost quantitatively in urine chiefly as and four arsenic-containing fatty acids. The structures of these metabolites and their relative excretion rates suggest that the native arsenolipids contain a dimethylarsine oxide [Me 2 () ] moiety, and that a considerable portion of the arsenic is bound in fatty acids.

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