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Xiao-Chun Le, William R. Cullen,' and Kenneth J. Reimer. We studied ... arsenic varies a great deal with its chemical species. (3-5), the ...... Irvin TR, Irgolic KJ.
CLIN.CHEM.40/4,

617-624

(1994)

#{149} Drug

Monitoring

and

Toxicology

Human Urinary Arsenic Excretion After One-Time Ingestion of Seaweed, Crab, and Shrimp Xiao-Chun Le, William R. Cullen,’ and Kenneth J. Reimer We studied chemical speciation of arsenic compounds in urine samples by using HPLC with inductively coupled plasma mass spectrometry detection. We examined urinary arsenic excretion patterns and the arsenic species excreted from nine human subjects who ingested seaweed products and crab (or shrimp). Fast urinary excretion of unchanged arsenobetaine was seen after ingestion of crab and shrimp, which contain arsenobetaine as the major arsenic species. In contrast, the arsenosugars, which comprise the major arsenic species in seaweed, are metabolized and have a longer retention time in the human body. When nine volunteers ingested the commercial seaweed product non, both the urinary arsenic excretion pattern and the excreted arsenic species varied from individual to individual, and as many as six metabolites could be detected. It seems that arsenosugars are not decomposed by stomach acid and that reactions invoMng enzymatic and (or) microbial activity in the human body may be responsible for the metabolism of arsenosugars. Indexing

Terms:

a,senobetaine/arsenosugars/toxicology/metabolism

wood preservatives; exposure to MMAA and DMAA is due to their use as selective herbicides. Urinary excretion of these compounds from humans (10-16), monkeys (17), dogs (18), and hamsters (19) has been studied. Workers who are exposed to airborne arsenic compounds, particularly smelter workers who inhale As203, seem to eliminate this arsenic in urine, principally in the thmethylated form (12). Reports of the biotransformation of inorganic arsenic to DMAA and MMAA are numerous (9-24). Consumption of seafood can result in significant intake of arsenic because seafood contains high (usually ,ig/g) arsenic concentrations, generally in the form of organoarsenicals such as arsenobetaine and arsenosugars (3, 4, 6, 7). Many studies have dealt with the consumption of arsenobetaine-rich seafood (24-28). Arsenobetaine shrimp,

upon consumption of crab, lobster, is excreted rapidly into urine unchanged (7, 23, 24, 28, 29), and no toxic effect has been observed (5, 30). However, little is known about the effect of consumption of seaweed (31, 32), which con-

tains

ingested and fish

arsenosugars

as the

major

arsenic

species

(33).

Arsenic has had the reputation of being a poison for centuries (1, 2). Although certain arsenic compounds are toxic to humans, others are not. The toxicity of arsenic varies a great deal with its chemical species (3-5), the 50% lethal dose (LD50) values in rats for some arsenic species being (in mg/kg): arsine 3, arsenite 14, arsenate 20, monomethylarsonic acid (MMAA) 7001800, dimethylarsinic acid (DMAA) 700-2600, arsenobetaine >10 000, and arsenocholine 6500.2 The limit for arsenic in drinking water in the US and Canada (50 .gfL) is largely based on inorganic arsenite and arsenate. If this limit were applied to seafood as 50 ng/g, all seafood would be unfit for consumption, given contents often 1000 times this concentration (6, 7). Therefore, one must differentiate arsenic forms during biological monitoring to assess health risks involving arsenic exposure and intake. Industrial and agricultural uses of arsenic compounds (8, 9) can result in excess human exposure, especially in occupations such as mining, smelting, glass making, and pesticide manufacture (10). Exposure to arsenate arises from its use in insecticides, cotton desiccants, and

product processed from red algae (Porphyra tenera and other Porphyra species), for example, contains 17-28 g/g of arsenic (dry weight), almost all of

Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z1. ‘Author for correspondence. Fax 604-822-2847. 2NonsdI abbreviations: MMAA, monomethylarsonic acid; DMAA, dimethylarsiic acid; AA, atomic absorption; FLA/ HGAAS, flow injection analysis with hydride generation atomic absorption spectrometry; ICPMS, inductively coupled plasma mass spectrometer (-try); and SRM, Standard Reference Material. Received November 8, 1993; accepted December 30, 1993.

Materials and Methods

Nori,

a seaweed

which is present as arsenosugars (34). Average daily consumption of seaweed by the Japanese is reported to be 20-30 g with a high of 120 g (wet weight) (35). Although Shibata et al. suggested that arsenosugars have no cytotoxicity or mutagenecity (34), it is not known whether arsenosugars are metabolized in the body after ingestion of seaweed and whether the metabolites are toxic. Such information is vital to evaluate any toxicological implications and health risks accompanying seaweed consumption. Urinary excretion is the major pathway for the elimination of arsenic from the body (14-16, 24-26, 28). Chemical analysis of urine samples is a convenient approach to the study of metabolism of arsenic compounds. Therefore, we decided to study the speciation of arsenic compounds in the urine of human subjects who had ingested arsenosugar-containing seaweed products and arsenobetaine-contaiing crab and shrimp. Here we report our findings.

Apparatus We performed

atomic absorption (AA) measurements (Victoria, Australia) Model AA 1275 atomic absorption spectrophotometer equipped with a standard Varian air-acetylene flame atomizer, as described previously (28, 36, 37). A conventional openwith

a Varian

CLINICAL CHEMISTRY, Vol. 40, No. 4,

1994

617

ended T-shaped quartz absorption tube [11.5 x 0.8 cm (i.d.)] was mounted in the air-acetylene flame of the burner. Hydrides were introduced to the quartz tube by way of its side arm for atomization and AA measurements. We used a system coupling flow injection analysis with hydride generation atomic absorption spectrometry (FIA/HGAAS) as described previously (36). A 500-W domestic microwave oven (Toshiba, Japan) was used to digest the human urine samples. The HPLC system consists of a Waters (Milford, MA) Model 510 solvent delivery pump, a Waters U6K injector, and an appropriate column. The columns were two new reversed-phase C18 columns (10j.Bondclone C15, 300 x 3.9 mm, Phenomenex, Torrance, CA; and Inertsil ODS-2, 250 x 4.6 mm, GL Sciences, Tokyo, Japan). A guard column was packed with the same material as that in the analytical column. We used an inductively coupled plasma mass spectrometer (ICPMS) for HPLC detection as described elsewhere (38). We used a VG PlasmaQuad 2 Turbo Plus ICPMS (VG Elemental, Winsford, Cheshire, UK) equipped with a SX300 quadrupole mass analyzer. We operated

the quadrupole

mass

analyzer

in the single-ion

monitoring mode (mlz 75 for arsenic). Polytetrafluoroethylene tubing [20 cm x 0.4 mm (i.d.)] with appropriate fittings connected the outlet of the HPLC analytical column directly to the inlet of the ICP nebulizer. Reagents Arsenic compounds used are listed in Table 1. We prepared standard solutions in deionized water. We prepared the HPLC eluents, 10 mmoIIL tetraethylammonium hydroxide and 4.5 mmol/L malonic acid, pH 6.8 (eluent 1), and 10 mmol/L sodium heptanesulfonate and 4 mmol!L tetramethylammonium hydroxide, pH 3.5 (eluent 2), in distilled deionized water and filtered them through a 0.45-nm (pore size) membrane ifiter. We adjusted the pH of all HPLC eluents with dilute nitric

acid

and

sodium

hydroxide.

We

added

methanol

(1

mIlL) (HPLC grade; Fisher Scientific, Fair Lawn, NJ) to both eluents before the pH adjustment and the ifitration. We obtained sodium heptanesulfonate from Aldrich Chemical Co., Milwaukee, WI; tetramethylammonium hydroxide and tetraethylammonium hydroxide from Eastman Kodak, Rochester, NY; and creatinine calibrators (10, 30, and 100 mgfL) from Sigma Chemical Co., St. Louis, MO. Sodium borohydride (Aldrich) solutions were made fresh in 0.1 molIL sodium hydroxide (BDH, Toronto, Canada) and filtered before use. We added L-cysteine (BDH) to the sample solution (10 g/L) when needed. Samples We purchased the commercial seaweed products non and powdered kelp from a local food store (Vancouver, Canada) and fresh crab and shrimp from a local fish market. We purchased a Standard Reference Material (SRM), oyster tissue 1566a, from the National Institute of Standards and Technology, Gaithersburg, MD.

We extracted nori, kelp, or oyster tissue SRM (0.5-1 g dry weight), and crab or shrimp (5-10 g wet weight) by a procedure similar to that described by Shibata and Morita (39). We weighed each sample into a test tube (15 mL). To each tube we added 5-10 mL of a methanol! water mixture (1:1, by vol), sonicated this for 10 min, and after centrifiigation, removed the extract and placed it in a round-bottom flask. We repeated the extraction process with the aid of sonication a further four times for each sample. We combined the extracts in the flask, evaporated them to dryness, and dissolved the residue in 10 mL of deionized water. The sample was analyzed by both FLA/HGAAS and HPLC/ICPMS. We obtained urine samples from nine adult volunteers from various professional occupations. All volunteers refrained from eating any seafood for at least 72 h prior to commencing the seafood ingestion experiment.

Table 1. Arsenic compounds used In the present studles.a Compound

Arsenite Arsenate Monomethylarsonate Dimethylarsinic acid

III IV V

Trimethylarsine oxide

VI

TetramethylarsoniumIodide

VII

Asenocholine Arsenobetaine

VIII Ix Arsenosugars XI XII

Dimethylarsinylethanol

Formula

Source

NaAsO2 Na2HAsO4 7H20

Aldrich

CH3AsO(ONa)2 (CHAsO(OH) (CH3)3AsO

Ma

.6HO

(CH3)4Asl (CH3)3AsCH2CH2OH

(CH3)AsCH2COO (CH3)2As(O)CH2CH2OH

Aldrich

Sigma Synthesized Synthesized Synthesized Synthesized Synthesized

Y

0 =As-

CM2\O/ocH2cHoHcH2Y

-OH -0P03--C H2CH(OH)CH2OH -0S03-

for

In SRM In SAM Not available

The first 10 arsenic compounds are available in our laboratory and are used as standards HPLC identification. Arsenic concentration in solutions prepared from these compounds were standardized against an arsenicatomicabsorption standard solution (Aldrich).

618

CLINICAL CHEMISTRY, Vol. 40, No. 4, 1994

Each volunteer was instructed to collect 2-3 urine samples during the 12 h before the consumption of seafood. We used these samples to determine the background concentration of arsenic. We

discussed

experimental

details

and

possible

health effects concerning seafood ingestion in this experiment with volunteers before conducting the experiment. All procedures followed were in accordance with the ethical standards of the University of British Columbia Clinical Screening Committee for Research and Other Studies Involving Human Subjects. Procedures Ingestion of non. After not eating any seafood for 3 days, each volunteer then consumed -9.5 g (dry weight) of nori in one meal (time zero). Following the one-time consumption of nori, urine samples (usually midstream) were collected at -3-5-h intervals for the next 4 days or longer. No other seafood was eaten during the experiment. Ingestion of powdered kelp. In another experiment, two volunteers ingested kelp, and urine samples were collected as described above for nori. Ingestion of crab and shrimp. In another set of experiments, volunteers 3 and 4 ingested crabmeat, and volunteers 1,2,5, and 6 ingested shrimp. Midstream urine samples from volunteers 3-fl were collected for 3 days. Volunteers 1 and 2, however, were required to eat a known amount of stir-fried shrimp including the juice from the cooking and to collect all urine samples for 3 days after consumption of the shrimp. We measured the volume of each urine sample. This procedure enabled the determination of the total amount of all arsenic species excreted by volunteers 1 and 2. All urine samples were stored at 4#{176}C and analyzed within 48 h. No preservative was added. FIA/HGAAS. Method 1: We directly analyzed urine samples for arsenic by FJ.AJHGAAS (28, 36). We dissolved -0.2 g of cysteine in each 10-mL urine sample, left the solution for 10-30 mm at room temperature, and then injected a 100-L sample into a deionized water carrier stream by using a sample injection valve. The injected sample met with continuously introduced streams of hydrochloric acid (0.5 mol/L) and sodium borohydride (30 g/L) at two T-joints. Arsines were generated and separated from liquid waste in a locally made gas/liquid separator as described previously (36). The gaseous arsines were then swept by a continuous flow of nitrogen carrier gas into a quartz absorption tube mounted in the air-acetylene flame for AA measurement. We used arsenate solutions of known concentration for calibration. Method 2 (28): We first subjected urine samples to batch-type microwave digestion as follows: We combined the urine sample or calibrator (40 mL), potassium persulfate (4.5 g), and sodium hydroxide (2.7 g) in a 125-mL Erlenmeyer flask and placed six of these sample-containing flasks in the microwave oven at one time. We then operated the microwave oven at full power for 3 mm followed by a 3-mm cooling period, and repeated

this heating and cooling cycle four more times. After these samples were cooled to room temperature, they were each diluted to 50 mL with deionized water. We also determined arsenic in the digested samples by FIA/ HGAAS as described in method 1, except that the concentrations of hydrochloric acid and sodium borohydride were 3 mol/L and 30 g/L, respectively, and no cysteine was needed. HPLCIICPMS. We used the ODS-2 column with eluent 1 at a flow rate of 0.7 or 0.8 mL/min for the separation of arsenosugar derivatives and metabolites. We used the Phenomenex C18 column when running eluent 2 at a flow rate of 1 mLlmin for the separation of arsenobetaine. We centrifuged all samples and filtered them through a 0.45-gm membrane filter before injecting 5-10 aL of the sample onto the HPLC column for chromatographic analysis. We identified arsenic compounds in the samples by matching the retention times of the chromatographic peaks of the sample with those of standards sometimes added to the sample. [We obtained the chromatograms shown later in Figs. 2 and 3 under slightly different flow rates (0.7 mtdmin) and void volumes from that shown in Fig. 5 (0.8 mL/min and a larger void volume)]. Determination of creatinine in urine samples. We determined creatinine in the urine samples by using HPLC with ultraviolet absorption spectrophotometric detection, essentially as described by Achari et al. (40). We diluted urine samples 50-fold with deionized water and injected a 5-10-tL aliquot onto the Phenomenex C18 column. The eluent was 50 mmoJJL sodium acetate (pH 6.5) in 98:2 (by vol) water:acetonitrile. The flow rate was 1.0 mldmin. We used an ultraviolet/visible spectrophotometer (Lambda-Max Model 481, Waters) at 254 nm as the HPLC detector.

Results and Discussion Urinary arsenic excretion after ingestion of kelp. The arsenic concentration normalized against the concentration of creatinine in urine samples collected from a volunteer (30-year-old man) before and after the ingestion of kelp is shown in Fig. 1. We analyzed both microwavedigested and undigested urine samples for arsenic species by HGAAS, and compared the results. As described previously (28, 36), only hydride-forming arsenic species are determined by HGAAS analysis on samples that have not been subjected to microwave-assisted digestion. Arsenosugars, arsenobetaine, arsenocholine, and the tetramethylarsonium ion are “hidden” and are not detected. When urine samples are decomposed in the presence of potassium persulfate and sodium hydroxide, all arsenic compounds are converted to arsenate, which is readily determined by HGAAS. Under these conditions, we measured the total arsenic concentration. Figure 1 shows that a considerable concentration of direct hydride-forming arsenic compounds is present in urine, particularly 17-37 h after the ingestion of kelp. The small difference in the arsenic concentrations measured before and after the microwave-assisted decomposition indicates that only small amounts of “hidden” CLINICAL CHEMISTRY, Vol. 40, No. 4,

1994

619

retention times of arsenic species previously reported for some related macroalgae (33, 41-45). We also studied the arsenic species in urine samples with HPLC/ICPMS. Chromatograms obtained from urine samples collected 12, 23, and 46 h after the ingestion of kelp are shown in Fig. 3(A-C). Only arsenate and DMAA are the major arsenic species in urine samples, each at -5-7 tg/L, before ingestion of kelp. After eating kelp, a small peak, Ui, with the same retention time as that of arsenosugar XI (-5.7 mm), appears in the chromatograin of the 12-h urine sample (Fig. 3A). In addition to this compound, two other peaks appear (at retention times -9.1 and 10.8 mm) in the 23-h urine samples -10 -7 -5 -3 2 Time

3 12 17 19 23 24 25 28 33 37 43 46 w.r.t.

kelp consumption,

h DMAA

Fig. 1. Relative concentration of arsenic in urine samplesnormalized against the concentration of creatinine collected from volunteer 1 before and after consumption of kelp. Determination wascarried outby FINHGAAS () without digestion and () with microwave-assisted digestion of the sample.w.r.t., with respect to.

8 0,

arsenic compounds are present in the urine samples. The urinary excretion of arsenic after ingestion of kelp shown in Fig. 1 is much slower than the excretion of arsenic after ingestion of crabmeat (28). Arsenobetaine is the major arsenic compound present in crab (7, 33, 41), and the ingestion of crab resulted in a fast excretion of this arsenical, unchanged (23,24,28,29). In contrast, kelp, prepared from brown algae, contains arsenosugars as the major arsenic compounds (33, 42, 43). Analysis of a kelp sample extract by HGAAS gave an arsenic concentration of 1.9 gfg; after microwave-assisted digestion, the arsenic content was 19.6 g/g. The difference between the two measurements is attributed to the presence of arsenosugars. As shown in Fig. 2, an analysis of the extract of the powdered kelp with HPLC/ICPMS shows the presence of three major arsenic species, two of which are identified as arsenosugars X and XI (structures shown in Table 1) on the basis of a match of their retention

times

with those of standards. The other major compound that appears at a longer retention

arsenic time (-7.5 mm) is not identified; it is probably another arsenosugar, on the basis of the occurrence and relative

3.2

0

1.5

3.0

4.5 6 7.5 9.0 Retention time, mm

10.5

12

10.5

12

10.5

12

DMAA

8

I 6

4

2

0

1.5

4.5

3.0

6

Retention

7.5

9.0

time, mm

DMAA

I 0

0

1.5

3.0

4.5

6

Retention

7.5

9.0

10.5

time, mm

Fig. 2. HPLC/ICPMS trace of an extract from powdered kelp. Flow rate, 0.8 mL/min.

620 CLINICAL CHEMISTRY, Vol. 40, No. 4,

1994

12

1.5

3.0

4.5 6 Retention

7.5

9.0

time,mm

Fig. 3. HPLC/ICPMS traces of urine samples collected (B), and 46 h (C) after volunteer 1 ingested kelp. Flowrate, 0.8 mLImln. h

12 h (A), 23

(Fig. 3B). The retention times of these two peaks do not match those of any of the standard arsenic compounds currently available to us (Table 1). These two peaks may indicate two new arsenic species that are metabolic products of the arsenosugars. These new compounds are not detected in the 46-h urine sample (Fig. 3C). Analysis on a 25-h urine sample from volunteer 2 shows three unknown arsenic compounds (retention times 9.1, 10.8, and 14.7 min). We did not observe compound Ui in the urine samples from volunteer 2; however, we observed another arsenic compound at retention time 14.7 mm in the urine sample from the latter volunteer. The difference in arsenic species found in the urine samples from these two volunteers after eating kelp suggests that metabolism of arsenosugars may vary from person to person. Therefore, we decided to repeat the experiment with a few more volunteers. We chose a small group of nine volunteers so that the number of samples could be handled easily in our laboratory. For simplicity we chose nori because it contains a single arsenosugar as the major arsenic compound and is readily available from local food markets. Analysis of a nori sample extract by HGAAS before and after microwave decomposition gave arsenic concentrations of 0.7 and 21 pg/g, respectively. Urinary arsenic excretion after ingestion of non. We asked each of the nine volunteers to eat -9.5 g of nori (containing -193 pg of arsenic as arsenosugar X); urine samples were collected before and after the time of ingestion. We determined the arsenic concentration in the urine samples by HGAAS with microwave decomposition of the sample, and results are shown in Fig. 4. We saw an increase in the arsenic concentration in urine samples from volunteers 1-3 and 6-9; the highest concentration was found 10-GO h after eating nori, with a return to background concentrations after -80 h. In contrast, we saw very little change in arsenic concentrations in urine samples from volunteers 4 and 5, even though they had ingested the same amount of nori as the others. Only background concentrations of arsenic species were found in these urine samples. Both volunteers had no apparent problem in digesting the seaweed and no abnormal activities or feelings. Volunteers 1-4 are in the same family, have a very similar diet, and have similar activities, yet their urinary arsenic excretion patterns are significantly different. Similarly, two female volunteers (5 and 6) of the same age also demonstrate very different urinary arsenic excretion patterns. These results strongly suggest that different individuals metabolize arsenosugars in different ways. To further study the excreted arsenic species, we subjected selected urine samples from each volunteer to HPLC/ICPMS analysis. Fig. 5 shows a typical chromatogram obtained on analysis of the 26-h urine sample from volunteer 3, revealing three metabolites (Ui, U3, and U4). Only DMAA and arsenate were detected in their urine samples collected before the ingestion of nori. We obtained similar chromatograms by analyzing urine samples from the other volunteers. We also determined the creatinine concentration in the urine samples

I VU

A

80 60

40 0 V V

20

a

0 -20

0

20 Time

40

60

w.r.t.

non

80

100 120 140 160 180 h

consumption,

:1. 0

8 C 0 V

V C

a

-20

0

20 40 60 80 100 Time w.r.t. non consumption,

Fig. 4. Urinary arsenic concentrations

120

140

h

of nine volunteers

before and

after consumption of non. (A) Volunteers 1-4; (B) 5-9. Volunteers: 1, 0, male, age 32 years; 2, #{149}, female,30; 3, X, male,59; 4, 0, female, 53; 5, A, female,20; 6, & female,20; 7, 0, male, 60; 8, #{149}, female, 48; 9, +, male, 24. w.r.t.,with respectto.

and used this to correct for variations in urine volume. Concentrations of creatiine and HPLC/ICPMS peak intensities from some urine samples are summarized in Table 2. Up to six arsenic species (U1-U6) at retention times 5.7, 6.6, 8.2, 9.9, 13.0, and 14.7 min, in addition to DMAA 0

Retention

time, mm

16

1: 0

2

4

6 8 10 Retention time, mm

12

14

16

Fig. 5. Typical chromatogram obtained on analysis of the urine sample from volunteer 3 collected 26 h after non ingestion. Inset trace

wasobtained

at higher sensitivity setting.

CLINICAL CHEMISTRY, Vol. 40, No. 4, 1994

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Table 2. HPLC/ICPMS peak Intensity of arsenic CompoUnds and creatinine concentration In selected urine samples. Peak Intensity

Time after Volunteer no.

Ingestion, h

2

18

DMAA

x

iO counts/s Creatinine conc, g/L

As(V)

Ui

U2

U3

U4

U5

U6

3.5 8

1 X

ND

26

12

6

X

19 26 16

8 8

8

7 8 10

1 0.5 0.5

1 X 1.6 X X X

X X X X X

X 0.5 X X X

1.11

4

1.6 0.4 2 X X X

0.2

3

X X X X X X

X

27

5 12

29.5

7

10

X

X

X

X

X

X

ND 2.1 1.4

5

8

ND 0.94

-8

16

8

X

X

X

X

X

X

15 29 -13 13.5 19 34.5

43 40

10 12

0.2 0.4

0.2 0.4

2.4 6.4

1.4 2

X 0.4

0.8

ND

0.6

1.0

3.5 10

2 2

X

X

0.6

X 4

X X

X 0.3

8

1.5

X

0.4

X

3.5

3.5 0.5

X X

0.5

8

U U

X 7 9.5

0.5

0.8

-14

1 38 7

3.5 10 7

X 1.2 4

X 0.8 X

34

22

0.2 5.7

0.5 6.6

X X X 0.1

X 1.2 0.5 0.8

13.0

14.7

6

7

0.96

15 23.5 19.5

9 Peak retention tIm, mis

X 25 14 12

X 8 2 8

8.2

9.9

0.73 1.6 ND 1.0 1.0 ND 1.2

0.2

Ps(V), arsenate;X, undetected,U, unresolvedfrom DMAA peak; ND, not determined.

arsenate and DMAA, were present in urine samples from these volunteers. Two members of one family, volunteers 7 and 8, each excreted five metabolites in their urine samples following ingestion of nori. However, volunteers i-4 excreted three, four, three, and one metabolite(s) (Fig. 3B and Table 2), respectively, although these four volunteers are all from the same family and have a similar diet. Furthermore, volunteers 5 and 6, both 20-year-old women, also showed different metabolic patterns for arsenosugars. We detected none of the arsenic metabolites in the 29.5-h urine sample of volunteer 5, whereas six metabolites of arsenic species were present in the 29-h urine from volunteer 6. These results are consistent with those obtained from the timecourse studies and support the contention that the ability to metabolize arsenosugars varies from individual to individual. In addition to the appearance of various unknown metabolites Ui-U6, the DMAA concentration was also significantly increased in urine samples collected after the ingestion of seaweed. Detailed results on the spedation of arsenic in urine samples from volunteer 1 who ingested kelp are summarized in Fig. 6. We obtained the relative intensity shown in the Figure by normalizing the intensity of each chromatographic peak from a urine sample against the concentration of creatiine in the corresponding urine sample. Clearly, the concentration of DMAA in the 23-h and 33-h urine sample following the ingestion of kelp was double the background concentration (in the -7-h urine sample). These results indicate that arsenosugars are metabolized to arsenic species such as DMAA, which is more toxic. Metabolism and the nature of metabolites should be taken into consideration when assessing the overall toxicological effect of seaweed ingestion. 622

CLINICAL

CHEMISTRY,

Vol. 40, No. 4, 1994

I -7

12

23

33

46

Time w.r.t. kelp consumption, h Fig. 6. Relative intensity of chromatographic peaks of arsenic species in urine samples from volunteer 1 before and after consumption of kelp. w.r.t.,with respect to.

Urinary arsenic excretion after ingestion of crab and shrimp. We decided to establish if this difference in metabolic patterns applied to arsenobetaine as well. We chose six volunteers for this study; of particular interest was a comparison between volunteers i-3 and 4 of the same family, and between 5 and 6 of the same age and sex. Figure 7 shows urinary arsenic excretion patterns from volunteer 4 after the ingestion of some crabmeat. We saw a fast excretion of arsenic compounds. Volunteer 3 shows a similar arsenic excretion pattern following ingestion of crab. HPLC/ICPMS analysis clearly shows that arsenobetame is the major arsenic compound in urine samples from both volunteers 3 and 4, indicating that arsenobe-

:1. 600

40O

8 0 C., C.,

200

a)

IJJj

-1 3

lJ

....

8 1216182023252835404345495258 Time

w.r.t.

crab consumption,

h

Fig. 7. Concentrationof arsenic in urine samples collected from volunteer 4 before and after consumption of crab. () without digestion; (U) with microwave-assisteddigestion. w.r.t.,with respect to.

tame is excreted unchanged following the ingestion of crab. Arsenobetaine was also excreted unchanged after volunteers 1, 2, 5, and 6 ingested shrimp. We saw similar arsenic excretion patterns to that shown in Fig. 7. These patterns are in clear contrast to those obtained after the ingestion of nori by the same volunteers. Percentage of excreted arsenobetaine compared with total intake. To carry out a quantitative study, we required volunteers 1 and 2 to eat a known amount of shrimp in one meal. All their urine samples were collected for 3 days following the meal, and the volume of each sample was measured. We determined the arsenobetaine concentration in both shrimp and urine samples, and determined amounts of intake and excretion of arsenobetaine. For volunteer 1, who ingested 163 g of arsenobetaine from the shrimp, the excreted arsenobetame amounted to 120 g (73% of the total intake) within 37 h after ingestion. Of the 415 g of arsenobetame ingested by volunteer 2, 274 .tg (66%) was excreted as arsenobetaine within 37 h. These results confirm that urinary excretion is the major pathway for the elimination of arsenobetaine. Excretion of arsenobetaine and metabolism of arsenosugars. Arsenobetaine is a relatively stable compound. Its decomposition often requires either vigorous chemical and physical conditions, such as strong alkaline digestion with the aid of microwave energy (28, 36) and photon energy (46), or enzymatic reactions involving microbial activity (47). In contrast, arsenosugars are more labile (41-43). Thus, one should be cautious when assessing the impact of seafood ingestion on urinary arsenic excretion. The type of seafood consumed, i.e., whether it contains arsenobetaine (crustaceans) or arsenosugars (seaweed), should be specified. Otherwise, false conclusions may result, such as the one reached by Tamaki and Frankenberger (48), who claimed that the ingestion of sea-

food (crustaceans, fish, and seaweed) resulted in arsenic excretion without any change in the chemical species. Urinary arsenic excretion pattern and excretion rate vary with the different arsenic compounds ingested. Buchet et al. (23) calculated that the half-life of arsenic following the ingestion of arsenobetaine and sodium arsenite was 18 and 30 h, respectively. Similar fast excretion of arsenobetaine and relatively slower elimination of inorganic arsenic by the kidney were observed by others (10-16, 24, 25, 28). Freeman et al. (25) and Charbonneau et al. (17) reported that most of the arsenic ingested through eating arsenic-containing fish is excreted in the urine from both man (25) and monkey (17) within 2 days. Following the ingestion of 500 pg of arsenic in the form of arsenite, 45% of the dose was excreted within 4 days, of which 24% was as DMAA, 10% as MMAA, and 11% as inorganic arsenic (15). The urinary arsenic excretion pattern (and rate) after ingestion of seaweed obtained in this study is similar to those obtained after ingestion of inorganic arsenic (11-15, 23, 24). We made comparisons by extracting nori and SRM oyster tissue with 0.01 mol/L hydrochloric acid vs deionized water, and noted no significant differences. These results suggest that arsenosugars are not simply decomposed by the acid present in the stomach, and that reactions involving enzymatic and (or) microbial activity may be responsible

for their

metabolism.

Therefore,

any

difference in urinary arsenic excretion patterns of arsenosugars may be attributed to a difference in individual metabolic functions. A larger number of subjects is required to study any possible correlations between urinary excretion and a particular group of subjects. We thank B. Mueller for technical assistance on ICPMS andY. Shibata for helpful technical advice. We also acknowledge the Natural Sciences and Engineering Research Council of Canada, Canada’s Department of Fisheries and Oceans, and the University of British Columbia for financial support. X.-C.L. thanks Canada’s Killam Trust and its Scholarship Committee for a Killam Predoctoral Fellowship. References 1. PoIson CJ, Tattersall

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