Excretion patterns of arsenic and its metabolites in human saliva and urine after ingestion of Chinese seaweed

There are no reports in scientific literature on arsenic species in human saliva after seaweed exposure. The present article reports for the first time the regular excretion patterns of arsenic in the saliva of volunteers with one-time ingestion of Chinese seaweed. Total arsenic and speciation analyses were carried out by high-performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC-ICP-MS). Results show that the excretion time of total arsenic in saliva is a trifle earlier than that in urine, total arsenic in human saliva also shows a regular excretion pattern like that in urine within 72 h after exposure to seaweed. For speciation analysis, four species, including the major dimethylarsinic acid (DMA) species, were detected in urine prior to seaweed intake. Six species were detected in urine after seaweed ingestion, including DMA, methylarsonic acid (MMA), oxo-dimethylarsinoylethanol (oxo-DMAE), thio-dimethlyarsenoacetate (thio-DMAA), arsenite (As^III) and arsenate (As^V). In saliva samples, three species were found before seaweed ingestion, with the major peak identified as As^III. After consumption, the kinds of arsenic metabolites in saliva were less than those in urine. The major species was inorganic arsenic (iAs As^III+As^V), followed by DMA, MMA and a trace amount of oxo-DMAE. Taken together, the present study suggests that saliva assay can be used as a potential tool for understanding the regular excretion pattern of total arsenic after seaweed ingestion. Whether or not it’s an efficient tool for assessing arsenic metabolites in humans exposed to seaweed requires further investigation.     

Arsenic speciation in Chinese brake fern by ion-pair high-performance liquid chromatography-inductively coupled plasma mass spectroscopy

Ion-pair reverse-phase HPLC-inductively coupled plasma (ICP) MS was employed to determine arsenite [As(III)], dimethyl arsenic acid (DMA), monomethyl arsenic (MMA) and arsenate [As(V)] in Chinese brake fern (Pteris vittata L.). The separation was performed on a reverse-phase C18 column (Haisil 100) by using a mobile phase containing 10 mM hexadecyltrimethyl ammonium bromide (CTAB) as ion-pairing reagent, 20 mM ammonium phosphate buffer and 2% methanol at pH 6.0. The detection limits of arsenic species with HPLC-ICP-MS were 0.5, 0.4, 0.3 and 1.8 ppb of arsenic for As(III), DMA, MMA, and As(V), respectively. MMA has been shown for the first time to experimentally convert to DMA in the Chinese brake fern, indicating that Chinese brake fern can convert MMA to DMA by methylation.     

Transformation of arsenic(V) by the fungus Fusarium oxysporum melonis isolated from the alga Fucus gardneri

A fungus isolated from the macroalga Fucus gardneri was identified by using 28S rDNA sequence analysis, 99% similarity match, as Fusarium oxysporum meloni. The fungus was exposed to arsenic(V) (500 ppb) in artificial seawater to investigate the possibility that the fungus is the source of the metabolic activity that results in the presence of arsenosugars in the macroalga. High‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry was used to identify the arsenic species in the fungus, and in the growth medium. The fungus was able to accumulate arsenic(V) and an increase in arsenite and dimethylarsinate was also observed. Some reduction of arsenate led to a small increase of arsenite in the growth medium. The fungus does not seem to be involved with the accumulation of arsenosugars by the Fucus. Copyright © 2002 John Wiley & Sons, Ltd.     

High-performance liquid chromatography with hydride generation/atomic absorption spectrometry for the determination of arsenic species with application to some water samples

High-performance liquid chromatography (h.p.l.c.) is used for separation of arsenite, arsenate, monomethylarsinate (MMA) and dimethylarsonate (DMA) followed by continuous sodium tetrahydroborate reduction and atomic absorption spectrometric detection. Sample preconcentration, offering improved detection limits for the individual species and the removal of matrix interferences, is achieved with a pellicular anion-exchange column. The arsenic species are then separated on a strong anion-exchange column placed in series with the preconcentration column. Detection limits of 2 ng (as arsenic) for arsenite, arsenate and MMA, and 1 ng for DMA. Results for arsenic species in soil waters and commercial bottle waters are given.     

Evaluation of atomic fluorescence spectrometry as a sensitive detection technique for arsenic speciation

The potential of coupling anion-exchange high-performance liquid chromatography, hydride generation and atomic fluorescence spectrometry (HPLC–HG–AFS) for arsenic speciation is considered. The effects of hydrochloric acid and sodium tetrahydroborate concentrations on signal-to-background ratio, as well as argon and hydrogen flow rates, were investigated. Detection limits for arsenite, dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate were 0.17, 0.45, 0.30 and 0.38 μg l⁻¹, respectively, using a 20-μl loop. Linearity ranges were 0.1–500 ng for As(III) and MMA (as arsenic), and 0.1–800 ng for DMA and As(V) (as arsenic). Arsenobetaine (AsB) was also determined by introducing an on-line photo-oxidation step after the chromatographic separation. In this case the limits of detection and linear ranges for the different species studied were similar to the values obtained previously for As(V). The technique was tested with a human urine reference material and a volunteer's sample. © 1998 John Wiley & Sons, Ltd.     

Simultaneous separation of 17 inorganic and organic arsenic compounds in marine biota by means of high-performance liquid chromatography/inductively coupled plasma mass spectrometry

A method using high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) has been developed to determine inorganic arsenic (arsenite, arsenate) along with organic arsenic compounds (monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, tetramethylarsonium ion and several arsenosugars) in fish, mussel, oyster and marine algae samples. The species were extracted by means of a methanol/water mixture and a dispersion unit in 2 min, with extraction efficiencies ranging from 83 to 107% in the different organisms. Up to 17 different species were determined within 15 min on an anion-exchange column, using a nitric acid gradient and an ion-pairing reagent. As all species are shown in one chromatogram, a clear overview of arsenic distribution patterns in different marine organisms is given. Arsenobetaine is the major compound in marine animals whereas arsenosugars and arsenate are dominant in marine algae. The method was validated with CRM DORM-2 (dogfish muscle). Concentrations were within the certified limits and low detection limits of 8 ng g(-1) (arsenite) to 50 ng g(-1) (arsenate) were obtained.     

Arsenic Speciation Analysis in Solutions Treated with Zeolites

Experiments have been carried out to study the behaviour of organoarsenicals treated with zeolites by means of speciation analysis. IC-ICP-MS was applied to identify and quantify arsenite, arsenate and the following organoarsenicals: monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), tetramethylarsonium bromide (TMA⁺), arsenobetaine (AsB) and arsenocholine (AsC). Zeolites loaded with ferrous ions did not significantly increase the retention of inorganic arsenic species compared to the native zeolites, while there was a ten-fold removal of arsenate relating to arsenite. The formation of As(V) and DMA in the leachates containing clinoptilolites and mordenites was confirmed in the presence of natural and synthetic zeolites. Arsenobetaine and arsenocholine yielded higher levels of arsenate than the methylated species.     

Arsenic accumulation and speciation analysis in wool from sheep exposed to arsenosugars

Wool or hair fibre is a metabolically dead material after it has left the epidermis. During growth the fibre in the root is a metabolically very active organ, which is highly influenced by the health status of the living being. Arsenic is one of the elements that is easily taken up by the cells of the root and stored in the fibre afterwards. Here we show that arsenic can quantitatively be extracted by boiling the wool fibre or hair in water. The high intake of arsenic species by the sheep of North Ronaldsay (the seaweed-eating sheep) leads to a high arsenic concentration in wool (mean 5.2+/-2.3 mug g(-1)). The wool of lambs of these sheep, which are not exposed to seaweed, contains about 10 times less arsenic, which is still elevated compared to uncontaminated wool. The arsenic species identified in wool extract are arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)) and monomethylarsonious acid (MMA(III)) as minor species. The major species is dimethylated arsenic DMA in its tri- and pentavalent form (dimethylarsinous acid (DMA(III)) and dimethylarsinic acid (DMA(V))) accounting for 85% of the specified arsenic in the wool which reflects the amount of dimethylated species (i.e. the arsenoribofuranosides) taken up by seaweed being the main food source of the sheep. However, there are unknown arsenic species in the extract, which are not eluting from a strong anion exchange column. In vitro incubation experiments with this kind of wool showed that it has reducing properties but no demethylation was recorded. The absorption ability of the wool for methylated arsenic species is negligible, while inorganic arsenic is easier to be absorbed in the fibre (11-17%). This means that the species integrity is only guaranteed in terms of the degree of methylation but not in terms of their redox status.     

微波辅助提取-液相色谱-氢化物发生-原子荧光光谱法分析沉积物中砷的形态

采用微波辅助提取-液相色谱-氢化物发生-原子荧光光谱法(LC-HG-AFS)联用技术分析了太湖沉积物中砷的形态[亚砷酸(As(III))、二甲基砷酸钠(DMA)、一甲基砷酸二钠(MMA)和砷酸As(V)]。测得沉积物中以无机砷为主,且以As(V)居多。选定以1mol/L的磷酸和0.1mol/L抗坏血酸为提取液,在微波辅助萃取(功率为60W,时间12min)下,萃取率达79.84%～91.57%,回收率在94.78%～107.6%之间。4种砷的形态在0～160μg/L之间时线性良好,检测限为0.6～2.3μg/L,相对标准偏差RSD为1.62%～2.20%。方法具有简便、快速、灵敏的特点。     

A new hydride generation system applied in determination of arsenic species with ion chromatography-hydride generation-atomic fluorescence spectrometry (IC-HG-AFS)

A novel procedure was developed for the determination of arsenite (As(III)), arsenate (As(V)), monomethylarsonic (MMA) and dimethylarsinic acid (DMA) with ion chromatography-hydride generation-atomic fluorescence spectrometry (IC-HG-AFS) by employing a new gas-liquid separator (GLS). The effective separation of the four arsenic species was achieved in about 12 min. With a sample loading volume of 20 μl, the measurable minimum for As(III), DMA, MMA and As(V) were 0.02, 0.045, 0.043 and 0.166 ng, respectively, along with relative standard deviations of 1.1, 1.1, 1.7 and 2.2% at the 100 μg l⁻¹ level (n = 6) for As(III), DMA, MMA and As(V), respectively. The present procedure was applied for the speciation of arsenic in underground water and in urine samples, and the sum of the four arsenic species by IC-HG-AFS was in good agreement with the total value by HG-AFS.     

Urinary arsenic speciation by high-performance liquid chromatography/atomic absorption spectrometry for monitoring occupational exposure to inorganic arsenic

Total urinary arsenic determinations are often used to assess occupational exposure to inorganic arsenic. Ingestion of sea food can increase the normal background levels of total arsenic in urine by up to an order of magnitude, but this arsenic has relatively little toxicity; it is tightly bound as arsenobetaine. The excretion of inorganic arsenic and its metabolites dimethylarsenic acid (DMA) and monomethylarsonic acid (MMA) is not influenced by the consumption of arsenic from sea food. Specific measurements of DMA, MMA and inorganic arsenic provide a more reliable indicator or exposure than total urinary arsenic levels. An automated atomic absorption method involving high-performance liquid chromatographic separation of the arsenic species and continuous hydride generation is described for the determination of arsenite, arsenate, DMA and MMA at μg As l^?1 levels. The method is used to study normal urinary arsenic levels in laboratory staff and arsenic excretion by exposed workers.     

Arsenic speciation in xylem sap of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Flow injection analysis (FIA) and high-performance liquid chromatography double-focusing sector field inductively coupled plasma mass spectrometry (HPLC-DF-ICP-MS) were used for total arsenic determination and arsenic speciation of xylem sap of cucumber plants (Cucumis sativus L.) grown in hydroponics containing 2 μmol dm⁻³ arsenate or arsenite, respectively. Arsenite [As(III)], arsenate [As(V)] and dimethylarsinic acid (DMA) were identified in the sap of the plants. Arsenite was the predominant arsenic species in the xylem saps regardless of the type of arsenic treatment, and the following concentration order was determined: As(III) > As(V) > DMA. The amount of total As, calculated taking into consideration the mass of xylem sap collected, was almost equal for both treatments. Arsenite was taken up more easily by cucumber than arsenate. Partial oxidation of arsenite to arsenate (<10% in 48 h) was observed in the case of arsenite-containing nutrient solutions, which may explain the detection of arsenate in the saps of plants treated with arsenite.     

Demethylation of methylarsenic species by Mycobacterium neoaurum

Mycobacterium neoaurum demethylates both methylarsonic acid and methylarsonous acid to mixtures of arsenate and arsenite. After 28 days of incubation, the yields of inorganic arsenic were 27% from arsenate and 43% from arsenite. A time study of the demethylation of methylarsonic acid by M. neoaurum showed that demethylation occurs rapidly during the growth and stationary phases of the bacterium, and indicates that MMA(V) is reductively demethylated to arsenite. Copyright © 2003 John Wiley & Sons, Ltd.     

Speciation of arsenic in body fluids

Inorganic arsenic is metabolized by consecutive reduction and methylation reactions to dimethylated arsenic (DMA), and then excreted into the urine mostly in the form of DMA. Therefore, arsenic metabolites in the body fluids and organs/tissues are present in the form of inorganic (arsenite and arsenate) and methylated arsenics (MMA and DMA). Although pentavalent arsenics can be present mostly in the form of free ions, trivalent ones may be present more in the forms conjugated with thiol groups of glutathione (GSH) or proteins. Arsenic in the body fluids (plasma, bile and urine) is present in the soluble forms and can be speciated on ion exchange columns by HPLC with on-line detection by an inductively coupled argon plasma-mass spectrometer (ICP-MS). Free forms of arsenite, arsenate, and monomethylarsonous, monomethylarsonic, dimethylarsinous and dimethylarsinic acids in the body fluids have been demonstrated to be speciated simultaneously within 10 min or so on both anion and cation exchange columns together with arsenobetaine (AsB) and arsenocholine (AsC). Trivalent arsenics conjugated with GSH were eluted in intact forms on an anion exchange column but were liberated into free forms on a cation exchange column. Thus, free and GSH-conjugated arsenic metabolites in the bile and urine have been speciated simultaneously on ion exchange columns by HPLC-ICP-MS.     

Biotransformation of arsenate to arsenosugars by Chlorella vulgaris

Chlorella vulgaris was cultivated in a growth medium containing arsenate concentration of <0.01, 10, 100 and 1000 mg l^?1. Illumination was carried out in 12 h cycles for 5 days. The health status of the culture was monitored by continuous pH and dissolved oxygen (DO) readings. Destructive sampling was used for the determination of biomass, chlorophyll, total arsenic and arsenic species. The chlorophyll a content, the DO and pH cycles were not significantly different for the different arsenate concentrations in the culture. In contrast, biomass production was significantly (p < 0.05) increased for the arsenic(V) treatment at 1000 mg l^?1 compared with 100 mg l^?1. The arsenic concentration in the algae increased with the arsenate concentration in the culture. However, the bioconcentration factor decreased a hundred‐fold with increase of arsenate from the background level to 1000 mg l^?1. The arsenic species were identified by using strong anion‐exchange high‐performance liquid chromatography–inductively coupled plasma mass spectrometry analysis after methanol/water (1 : 1) extraction. The majority (87–100%) of the extractable arsenic was still arsenate; arsenite was found to be between 1 and 6% of total extractable arsenic in the algae. In addition to dimethylarsinic acid, one unknown arsenical (almost co‐eluting with methylarsonic acid) and three different arsenosugars have been identified for the first time in C. vulgaris growing in a culture containing a mixture of antibiotics and believed to be axenic. The transformation to arsenosugars in the algae is not dependent on the arsenate concentration in the culture and varies between 0.2 and 5% of total accumulated arsenic. Although no microbiological tests for bacterial contamination were made, this study supports the hypothesis that algae, and not associated bacteria, produce the arsenosugars. Copyright © 2003 John Wiley & Sons, Ltd.     

Determination of urinary arsenic and impact of dietary arsenic intake

An analytical method based on microwave decomposition and flow injection analysis (FIA) coupled to hydride generation atomic absorption spectrometry (HGAAS) is described. This is used to differentiate arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) from organoarsenic compounds usually present in seafood. Without microwave digestion, direct analysis of urine by HGAAS gives the total concentration of As(III), As(V), MMA and DMA because organoarsenic compounds such as arsenobetaine, usually found in most seafood, are not reducible upon treatment with borohydride and therefore cannot be determined by using the hydride generation technique. The microwave oven digestion procedure with potassium persulfate and sodium hydroxide as decomposition reagents completely decomposes all arsenicals to arsenate and this can be measured by HGASS. Microwave decomposition parameters were studied to achieve efficient decomposition and quantitative recovery of arsenobetaine spiked into urine samples. The method is applied to the determination of urinary arsenic and is useful for the assessment of occupational exposure to arsenic without intereference from excess organoarsenicals due to the consumption of seafood. Analysis of urine samples collected from an individual who ingested some seafood revealed that organoarsenicals were rapidly excreted in urine. After the ingestion of a 500-g crab, a 10-fold increase of total urinary arsenic was observed, due to the excretion of organoarsenicals. The maximum arsenic concentration was found in the urine samples collected approximately between 4 to 17 hr after eating seafood. However, the ingestion of organoarsenic-containing seafoods such as crab, shrimp and salmon showed no effect on the urinary excretion of inorganic arsenic, MMA and DMA.     

Urinary arsenic species in an arsenic‐affected area of West Bengal,India (part III)

Arsenic contamination of groundwater has long been reported in the Mushidabad district of West Bengal, India. We visited 13 arsenic‐affected families in the Makrampur village of the Beldanga block in Mushidabad during 18–21 December 2001 and collected five shallow tubewell‐water samples used general household purposes, four deep tubewell‐water samples used for drinking and cooking purposes, and 44 urine samples from those families. The arsenic concentrations in the five shallow tubewell‐water samples ranged from 18.0 to 408.4 ppb and those in the four deep tubewell‐water samples were from 5.2 to 9.6 ppb. The average arsenite (arsenic(III)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate (arsenic(V)) in urine were 28.7 ng mg^?1, 168.6 ng mg^?1, 25.0 ng mg^?1 and 4.6 ng mg^?1 creatinine respectively. The average total arsenic was 227.0 ng mg^?1 creatinine. On comparison of the ratio of (MMA + DMA) to total arsenic, the average proportion was 86.7 ± 9.2% (mean plus/minus to residual standard deviation, n = 43). The exception was data for one boy, whose proportion was 8.0%. One woman excreted the highest total arsenic, at 2890.0 ng mg^?1 creatinine. When using 43 of the urine samples (the exception being the one sample obtained from the boy) there were significantly positive correlations (p < 0.01) between arsenic(III) and MMA, between arsenic(III) and DMA and between MMA and DMA. Copyright © 2005 John Wiley & Sons, Ltd.     

Determination of arsenic species by capillary zone electrophoresis with large-volume field-amplified stacking injection.

Determination of arsenic species by large-volume field amplified stacking injection-capillary zone electrophoresis (LV-FASI-CZE) is reported in this paper. Whole column injection was employed. The optimum buffer pH for the separation of weak acids was discussed. It was found that the optimum buffer to analyze the stacked arsenate (As(V)), monomethylarsonate (MMA), and dimethylarsinate (DMA) was 25 mM phosphate at pH 6.5. However, the optimum buffer to analyze the concentrated arsenite (As(III)) was 20 mM phosphate - 10 mM borate at pH 9.28. The limits of detection of the method developed were 0.026 mg/L for As(III), 0.023 mg/L for As(V), 0.043 mg/L for MMA, and 0.018 mg/L for DMA. An enrichment factor of 34-100 for several arsenic species was obtained. In the end, this method was applied to determine the arsenic concentration in the environmental reference materials to show the usefulness of the method developed.     

Comparison of extraction procedures for arsenic speciation in environmental solid reference materials by high‐performance liquid chromatography–hydride generation‐atomic fluorescence spectroscopy

Water and ‘soft’ extractions (hydroxylammonium hydrochloride, ammonium oxalate and orthophosphoric acid) have been studied and applied to the determination of arsenic species (arsenite, arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in three environmental solid reference materials (river sediment, agricultural soil, sewage sludge) certified for their total arsenic content. The analytical method used was ion exchange liquid chromatography coupled on‐line to atomic fluorescence spectroscopy through hydride generation. Very low detection limits for arsenic were obtained, ranging from 0.02 to 0.04 mg kg^?1 for all species in all matrices studied. Orthophosphoric acid is the best extractant for sediment (mixed origin) and sludge samples (recent origin) but not for the old formation soil sample, from which arsenic is extracted well only by oxalate. Both inorganic forms (As(III) and As(V)) are significant in all samples, As(V) species being predominant. Moreover, organic forms are found in water extracts of all samples and are more important in the sludge sample. These organic forms are also present in the ‘soft’ extracts of sludge. Microwave‐assisted extraction appears to minimize the risk of a redox interconversion of inorganic arsenic forms. This study points out the necessity of combining direct and sequential extraction procedures to allow for initial arsenic speciation and to elucidate the different mineralogical phases–species associations. Copyright © 2002 John Wiley & Sons, Ltd.     

Stability study of As(III), As(V), MMA and DMA by anion exchange chromatography and HG-AFS in wastewater samples

The stability of arsenic species (arsenate [As(V)], monomethylarsonate [MMA], dimethylarsinate [DMA] and arsenite [As(III)]) in two types of urban wastewater samples (raw and treated) was evaluated. Water samples containing a mixture of the different arsenic species were stored in the absence of light at three different temperatures: +4 degrees C, +20 degrees C and +40 degrees C. At regular time intervals, arsenic species were determined by high performance liquid chromatography (HPLC)-hydride generation (HG)-atomic fluorescence spectrometry (AFS). The experimental conditions for the separation of arsenic species by HPLC and their determination by AFS were directly optimised from wastewater samples. As(III), As(V), MMA and DMA were separated on an anion exchange column using phosphate buffer (pH 6.0) as the mobile phase. Under these conditions the four arsenic species were separated in less than 10 min. The detection limits were 0.6, 0.9, 0.9 and 1.8 micro g L(-1) for As(III), DMA, MMA and As(V), respectively. As(V), MMA and DMA were found stable in the two types of urban wastewater samples over the 4-month period at the three different temperatures tested, while the concentration of As(III) in raw wastewater sample decreased after 2 weeks of storage. A greater stability of As(III) was found in the treated urban wastewater sample. As(III) remained unaltered in this matrix at pH 7.27 over the period studied, while at lower pH (1.6) losses of As(III) were detected after 1 month of storage. The results show that the decrease in As(III) concentration with time was accompanied by an increase in As(V) concentration.