Arsenosugar phospholipids and arsenic hydrocarbons in two species of brown macroalgae

Arsenic-containing organic compounds are abundant in marine ecosystems where they are thought to play a pivotal role in the cycling and detoxification of potentially toxic inorganic arsenic (arsenate) present in seawater.^[1] Although most of the arsenic compounds identified so far have been water-soluble species, the early work on arsenic marine chemistry focussed on lipid-soluble compounds, so called arsenolipids.^[2–4] Identification of these arsenolipids proved difficult, however, and it was not until 1988 that an arsenolipid was first rigorously characterised and identified as an arsenosugar-containing phospholipid^[5] (see Table 1, compound As-PL958).

Table 1.  Arsenosugar-phospholipids in algae identified by high resolution mass spectrometry (for spectra, see Supplementary material)
The positions of R₁ and R₂ relative to each other are arbitrary; the proposed position of the double bond is based on the natural occurrence of unsaturated fatty acid groups. Compounds are coded to designate that they are arsenosugar-phospholipids (As-PL) with the stated nominal molecular mass. Compounds are listed in order of increasing molecular mass; the order of high performance liquid chromatography elution differs at times from the molecular mass order because of the increased polarity resulting from the inclusion of a double bond. Δm is the mass difference between calculated and found molecular masses

Subsequently, the range of naturally occurring arsenolipids has been extended with the discovery of arsenic-containing fatty acids in fish oils,^[6] and arsenic-containing hydrocarbons in fish oils,^[7] fish liver,^[8] sashimi tuna^[9] and fish meal.^[10] The origin of these compounds was presumed to be algae. We report the arsenolipid profiles of two species of brown algae, determined mainly by high performance liquid chromatography–mass spectrometry (HPLC-MS), and we briefly discuss the possible biosynthetic origin of these unusual compounds.

Samples of Wakame (Undaria pinnatifida, 40 ± 3 µg As g^–1 dry mass) and Hijiki (Hizikia fusiformis, 113 ± 5 µg As g^–1 dry mass),^A obtained from a Japanese commercial source, were extracted with a mixture of chloroform and methanol using a modification^B of the classical procedure of Bligh and Dyer.^[11] The lipid fraction containing 6.7 % (Wakame) and 1.6 % (Hijiki) of the initial total arsenic was passed through a small plug of silica, which removed 90 % of the sample mass with little loss of arsenic. Both the pre- and post-silica fractions were then analysed by reversed-phase HPLC with simultaneous detection of arsenic and organo-arsenic compounds by using inductively coupled plasma mass spectrometry (ICPMS) and electrospray mass spectrometry (ESMS) respectively.^C The clean-up step with silica was essential to obtain good ESMS data. ICPMS, a more robust detection method, was capable of measuring the arsenolipids in both pre- and post-silica fractions, and provided data showing that the pattern of arsenolipids was essentially unchanged by the silica clean-up step.

HPLC-MS revealed the presence of at least 10 arsenic-containing peaks or bands (Fig. 1). The peaks eluted at ~23 to 25 min from the HPLC column were shown to contain two known arsenic-hydrocarbons (Fig. 2, As-HC332 and As-HC360) by accurate mass measurements and gas chromatography (GC)-MS analysis (see Supplementary material, Fig. S1).^[12] In addition, a new arsenic-hydrocarbon (retention time (RT) 26.5 min; Fig. 2, As-HC388) was identified in Wakame by accurate mass measurements and GC-MS. A major peak in the HPLC chromatogram (RT = 27.5 min) for both Wakame and Hijiki had a protonated molecular mass of 959, which matched that for the arsenosugar-phospholipid previously characterised by Morita and Shibata.^[5] In addition, there was a series of peaks before and after the peak with m/z 959 separated by 28, 26 or 24 mass units, which indicated the presence of a homologous series of saturated and unsaturated arsenosugar-phospholipids (Fig. 1). These assignments were strongly supported by accurate mass measurements^D performed on 11 of the compounds, which agreed in every case to within 2 ppm of the calculated molecular masses (Table 1). The possible presence in algae of a range of arsenosugar-phospholipids differing only in their fatty acid content had been predicted in 1988 by Morita and Shibata^[5]; our data confirm their astute early prediction.

Fig. 1.  High performance liquid chromatography (HPLC)–inductively coupled plasma mass spectrometry of lipid extracts (post-silica clean-up) from Wakame and Hijiki.^C A small peak (polar arsenic) was always present at the column void volume (~2 min) resulting from partial degradation of the arsenolipids during sample handling.

Fig. 2.  Arsenic-hydrocarbons in Wakame and Hijiki; As-HC332 & As-HC360, previously identified in fish oil^[7] and sashimi tuna,^[9] were present in both Wakame and Hijiki. The homologue As-HC388 is first reported here where it was shown to be present in Wakame (but not Hijiki). As-HC388 was identified by high resolution mass spectrometry {[M + H]⁺_calc. 389.2759, [M + H]⁺_found 389.2755, Δm 1.04} with supporting tandem mass spectrometry and gas chromatography–mass spectrometry data (see Supplementary material).

Quantification of the major arsenic compounds in the two algal species was achieved by HPLC-ICPMS using a modification of reported methods (Table 2).^[13,14] Although both species contained arsenosugar-phospholipid As-PL958 as a major compound, Wakame also contained significant quantities of other arsenosugar-phospholipids, whereas Hijiki contained a higher proportion of arsenic-hydrocarbons. The possible significance of these differences awaits studies with more samples of these two algae and other algal species from various locations. Such studies would be best carried out with samples collected directly from the natural environment, rather than with commercial products, thereby excluding the possibility of artefact formation.

Table 2.  Quantification of arsenolipids in two species of brown algae
Data represent the mean ± s.d. of three analyses each involving extraction, clean-up and high performance liquid chromatography (HPLC)–inductively coupled plasma mass spectrometry. The percentage lipid values are based on the total As applied to the HPLC column (post-silica fraction); column recoveries ranged from ~70 to 110 %

The presence of both arsenic-hydrocarbons and arsenosugar-phospholipids in algae raises questions as to how these unusual compounds are synthesised, and what their possible roles in algae might be. The early experiments of Benson and co-workers provide some information on the biochemical processes involved.^[3] By using radiolabelled arsenic and monitoring products by radiochromatography, they showed that unicellular algae take up arsenate from seawater and convert it into lipid- and water-soluble arsenic species. By selective use of enzymes (e.g. phospholipases A and D), they also demonstrated that the lipid-soluble arsenic comprised three types of lipids: phospholipids, lyso-phospholipids and an unknown lipid class. Although the water-soluble products from the enzyme treatments were incorrectly assigned at that time, they were later correctly ascribed to arsenosugars.^[1,15–17] Our definitive results on the structures of the major arsenolipids in algae are consistent with the earlier observations. Collectively, the data suggest that the arsenosugar-phospholipids might be the target arsenic species synthesised by algae, and that the arsenosugars are merely degradation products of compounds excess to the algae’s requirements.

The third, unknown, class of lipid reported by Benson and co-workers^[3] did not degrade with the enzymes they tested. Possibly, that group of compounds comprised the arsenic-hydrocarbons found in our study. These compounds are likely to have a biosynthetic pathway quite different from that of the arsenosugar-phospholipids, although their properties might be similar since they both contain polar head groups and long non-polar tails. We speculate that both these types of arsenolipids could be used by algae in membrane chemistry.

Possibly, arsenolipids impart a particular useful property to cell membranes not attainable from normal membrane lipids. In this regard, the recent work by Van Mooy et al.^[18] is relevant because they reported that algae can utilise S and N in place of P to form membrane lipids. In that case, however, the driving force was considered to be the need for algae to conserve phosphate for use in other essential cell biochemistry. Thus, the relative quantities of arsenosugar-phospholipids and arsenic-hydrocarbons in the algae might reflect phosphate levels in water or the relative phosphate requirements of algal species; i.e. when phosphate levels are limiting, arsenic-hydrocarbons are preferentially synthesised by algae for membrane chemistry. We are currently investigating these biological questions, building on the preliminary quantitative results reported here for different types of arsenolipids in algae.

The following information is available as Supplementary material:

• GC/MS of arsenic-hydrocarbons in Wakame and Hijiki.

• High resolution mass spectra of arsenic-hydrocarbons found in Wakame or Hijiki.

• High resolution mass spectra of arsenosugar-phospholipids found in Hijiki.