Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae)

Abstract

Viscera (48.3 kg) from moray eels (Lycodontis javanicus) collected in a ciguatera endemic area were extracted and the ciguatoxins characterized. Three major ciguatoxins, CTX-1, CTX-2 and CTX-3, were isolated and purified to homogeneity on reverse phase high performance liquid chromatography. Several minor toxins were also detected. CTX-1 (490 micrograms) was comparable by both 1H nuclear magnetic resonance (1H NMR) and mass spectroscopy (MH+ m/z = 1111) to ciguatoxin isolated previously from moray eels. CTX-2 (280 micrograms) and CTX-3 (100 micrograms) were less polar ciguatoxins not previously characterized. CTX-2 and CTX-3 differed from CTX-1 by 16 mass units, suggesting that they were less oxygenated analogues. 1H NMR revealed that the hydroxyl at C54 in CTX-1 was absent in CTX-2 and CTX-3. An additional change in the chemistry of CTX-2 compared to CTX-1 and CTX-3 was also suggested on the basis of 1H NMR, indicating that CTX-2 may arise from a different precursor to CTX-1. CTX-3 is likely to be an intermediate in the oxidation of a gambiertoxin (sodium channel toxins from Gambierdiscus toxicus) to CTX-1. The i.p. LD50 values for CTX-1, CTX-2 and CTX-3 were 0.25, 2.3 and 0.9 micrograms/kg, respectively. The signs induced in mice by the ciguatoxins were similar, except that CTX-2 and CTX-3 induced hind-limb paralysis that was absent with CTX-1. Each ciguatoxin was potent orally. CTX-1, CTX-2 and CTX-3 competitively inhibited the binding of [3H]brevetoxin-3 to voltage-dependent sodium channels with relative potencies qualitatively (but not quantitatively) comparable to mouse lethality. This study reveals that the relatively small chemical differences between CTX-1, CTX-2 and CTX-3 give rise to significant structure-activity and pharmacokinetic differences.