Arsenic‐Containing Phosphatidylcholines: A New Group of Arsenolipids Discovered in Herring Caviar

Abstract A new group of arsenolipids based on cell‐membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic‐containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic‐containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non‐arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids.

synthetic origin different from that of the arsenosugar lipids. Thew idespread distribution of these arsenolipids among marine organisms,i ncluding those used as human food, encouraged the chemical synthesis of the compounds [10] and investigations of their toxicological properties. [11,12] Am ore fundamental biological role for the compounds,f or example in membrane chemistry,has also been suggested, [6] but so far there is no evidence supporting this speculation.
Ar ecent study of af ish oil showed the presence of an unidentified group of arsenolipids thought to be conjugates of arsenic fatty acids because they could be base-hydrolyzed to known arsenic fatty acids. [13] We wondered if this group of arsenolipids might be related to phosphatidyl compounds integral to membrane chemistry.B ut in the fish oil samples, the fatty acid conjugates were present at such trace levels that they could not be identified. Seeking ar icher source of cell membrane compounds,w et urned to fish eggs (roe). [14] Herein, we report the identification of am ajor new group of arsenolipids from three samples of herring roe (herring caviar) and ap reliminary investigation of arsenolipids in salmon caviar.
Extracting freeze-dried herring caviar (Clupea harengus; % 0.8 mgAsg À1 dry mass,originating from the Norwegian Sea) with DCM/MeOH and washing the extract with water yielded an organic layer containing approximately 80 %ofthe initial arsenic.A fter all traces of DCM were removed, the residue was re-dissolved in absolute ethanol and analyzed by reversed-phase HPLC coupled to an elemental mass spectrometer (inductively coupled plasma mass spectrometry, ICPMS). TheH PLC/ICPMS measurements performed in selected-ion monitoring mode with m/z 75 (As + )indicated the presence of several known arsenic fatty acids and arsenic hydrocarbons,i na ddition to ac luster of later-eluting arsenolipids (RT25-28 min;F igure 2a). Silica column chromatography,p reviously used to purify known arsenolipids, [15] was found to be ineffective for this group of arsenolipids,possibly because it degraded the native compounds.Thus,further mass spectrometric analyses were performed on the crude extracts by using HPLC coupled via electrospray ionization (positive mode) to ah igh-accuracym ass spectrometer with collisioninduced-dissociation capability (Figure 2b). During the elution, alarge number (approximately 700) of distinct ions were isolated and fragmented, yielding more than 18 000 MS/MS spectra in each run (Supporting Information).
Almost all of the arsenolipids identified so far contain the dimethylarsinoyl group À As(O)Me 2 ,w hich gives rise to two characteristic fragment ions,o ne with m/z = 105 (C 2 H 6 As + ) and one with m/z = 123 (C 2 H 8 OAs + ), when subjected to collision-induced-dissociation. Owing to their mass defects, these fragment ions can be uniquely identified by ah igh- resolution mass spectrometer even from ac omplex matrix. Searching the fragmentation spectra for these two fragment ions exposed am ultitude of precursor ions,f rom which six known arsenic fatty acids (AsFA334, AsFA362, AsFA388, AsFA390, AsFA436, AsFA448) and five known arsenic hydrocarbons (AsHC 330, AsHC 332, AsHC 358, AsHC 360, AsHC 404) could be expressly identified (Supporting Information, Table S2 and Figure S2). This procedure similarly implied an ion with [M+ +H] + at m/z = 529. Theaccurate mass and fragmentation data of this ion were consistent with ah itherto unknown arsenic fatty acid with eight ethylene groups (AsFA528;F igures 3a nd S3).
In the later part of the HPLC/ESIMS chromatogram (RT 20.5-24 min;F igure 2b), weak signals for one or the other of the two indicator fragments were observed. Thep recursor ions were singly or doubly charged, and had masses revealing the presence of an odd number of nitrogen atoms in their formulae.A lso present were fragment ions attributable to apreviously observed arsenic-containing (free) fatty acid and its ketene derivative.F ive such examples were discovered.
Theo bserved masses were all consistent with the general formula C x H y O 9 NAsP,a nd inspection of the MS/MS (fragmentation) spectra of these ions confirmed similarity as well as as et of prominent fragment ions characteristically observed in the MS/MS spectra of phosphatidylcholines. [16] An example is given in Figure 4. These observations indicated the presence of phosphatidylcholine compounds where one of the fatty acids is an arsenic fatty acid. In phosphatidylcholines, fragment ions relating to the fatty acid moieties are normally of minor intensity. [16] In contrast, for the arsenic-containing phosphatidylcholines (AsPCs), two significant ions representing the protonated free arsenic fatty acid and the protonated ketene can be seen. This difference is an expected consequence of the proton affinity of the arsinoyl group of the As fatty acids.B ased on these data, five AsPCs were identified ( Figure 5a nd Table 1). It should be noted that, while the phosphatidylcholine-revealingfragment ions are conspicuous, the small arsenic-containing fragments that led to the discovery of these ions are not.
Thec ollision energy used was originally optimized to obtain intense arsinoyl-group fragments,a nd therefore was too high to partly preserve the phosphatidylcholine precursor   Small differences in the HPLC measurement conditionsresulted in au niform shift in retention times between the two measurements. AsHC 332, for which we had an authenticstandard, [10] could be used to normalize the two HPLC data sets (RT = 22.5 min by HPLC/ICPMS and RT = 18.9 min by HPLC/ESIMS. See Figure 1and   Communications ions when fragmenting these.C onsequently,i nF igure 4, the precursor ion at m/z = 986.5250 is absent. In contrast, ap rominent fragment ion at m/z = 803 (corresponding to are-arranged diacylglycerol-type fragment resulting from the loss of the neutral phosphorylcholine zwitterion [16] )a nd protonated phosphorylcholine at m/z = 184, along with the smaller assigned fragments at m/z = 86, 104, and 125, are present together with the two above-mentioned fragment ions of the arsenic fatty acid ester.A na nalogous fragmentation pattern was observed for the other four AsPCs (Table 2). Phosphatidylcholines form am ajor lipid class comprising many compounds varying only in their fatty acid composition. It is thus likely that the cluster of unresolved arsenic compounds with HPLC/ICPMS retention times 25-28 (Figure 2a), which collectively constitute roughly 50 %o ft he total arsenic,b elong predominantly to the new group of arsenic phosphatidylcholines described here.T his study identified five of those compounds,a nd opens the door to discovering many more of these complex arsenic natural products.
Examination of two other sources of herring caviar purchased in Sweden produced very similar patterns of arsenolipids (Table S2), and both contained the five AsPCs described above.T he same approach applied to an extract of salmon caviar (Oncorhynchus tshawytscha; % 0.7 mgAsg À1 dry mass,o riginating from Alaska) did not reveal the presence of AsPCs,b ut again AsFA528 ([M + H] + exp.: 529.2642; j Dm/m j [ppm]: 2.9) and also an arsenic-containing phosphatidylethanolamine (AsPE) incorporating AsFA528 were identified (Figure 6a nd Table 1). Arsenic-containing phosphatidylethanolamines were not found in herring caviar, but as for the AsPCs in salmon caviar,wecannot exclude their presence at low levels (which indeed appears likely).
Thefragmentation behavior of the protonated AsPE 1035 ( Figure S19) parallels the behavior observed for the protonated AsPCs with the exception that protonated phosphorylethanolamine at m/z = 142 is not present. Such am assspectrometric difference has also been noted between usual (non-arsenic-containing) PEs and PCs. [17] Thef ragment ions of the arsenic fatty acid ester as well as the re-arranged diacylglycerol-type fragment (loss of the neutral phosphorylethanolamine zwitterion) were observed in the MS/MS spectrum of the singly charged species.I na ddition, the MS/ MS spectrum of the doubly charged species revealed af ragment ion at m/z = 585.2919, corresponding to the arsenic fatty acid with part of the glycerol molecule after loss of phosphorylethanolamine and docosahexaenoic acid as the corresponding ketene.   Theo rigin of the arsenic phosphatidylcholines in the membrane-rich fish eggs remains an open question. Phosphatidylcholines are key components of biological membranes.Itispossible that infidelity in their biosynthesis results in the arsenic analogues,a lthough am ore fundamental role for the arsenic compounds cannot be dismissed. These compounds are likely to be widespread in fatty fish, based on the ubiquity of free arsenic fatty acids and the recent report of unidentified arsenic fatty acid conjugates in fish oil, [13] and thus they have relevance to human health. Preliminary toxicity testing with human cells has shown that one group of arsenolipids,t he arsenic hydrocarbons,a re highly cytotoxic, and that their enhanced toxicity relative to arsenic fatty acids might be related to their less-polar nature. [11,12] Thea rsenic phosphatidylcholines and the phosphatidylethanolamine reported here are,b ased on reversedphase HPLC retention times,l ess polar than the arsenic hydrocarbons,a nd thus might also be expected to be cytotoxic. In future work, we will synthesize representative members of this new group of arsenolipids to enable af ull assessment of their chemical and toxicological properties.