Tosicron Vol. 28, No . 2, pp. 129-132, 1990 . Printed in Great Britain.

0041-0101/90 53 .00+ .00 CC9 1990 Pergamon Press pk

TETRODOTOXIN IN "ZOMBIE POWDER" (Accepted jor publication 6 July 1989)

WE wEr.cohtE the publication by BENEDEK and RIVER (1989) of their analyses for

tetrodotoxin in one sample of "zombie powder". Until this publication, the only data in this regard were those of YASUMOTO and Kno (1986), and those in a private and unpublished report provided to RNIER by A. LOMBET and M. LAZDUNSKI (quoted in part in BENEDEK and RNIER; copy available to Kno and permission to quote granted by LAZDUNSKI, 4 September 1987). All of these analyses were performed at the direct or indirect behest of WADE DnVS on samples which he collected during two field trips in Haiti in 1982 and 1984 . It might be recalled that based on what he thought to be similarities between the symptomatology of Haitian zombies and victims of fugu poisoning, and his observation of the incorporation of some dried puffer fish in samples of zombie powder, Dnvls had disseminated a claim, virtually exclusively via the lay press, that Haitian zombies were made through the use of tetrodotoxin present in the puffer fish . In all, eight samples were collected (DnVS, W., The Ethnobiology of the Haitian Zombie, Ph .D. thesis, Harvard University, 1986; DnVS, 1988). YASUMOTO and Kno examined two samples. RrvIER received six samples (personal communication to Kno even though only five were acknowledged in BENEDEK and RIVrER). After failing to detect any tetrodotoxin in one or more samples, RNIER sent portions of his six samples to LnznuNSx[. In their report to RNIER, LOMBET and LAZDUNSKI WI'Ote that five of the samples, with pHs ranging between 6.76 and 7.70, contained no tetrodotoxin that could be detected by the displacement of 3H-TTX from a sodium~hannel preparation (DnVO and FONTELLA, 1984). The sixth sample, which RNIER identified as sample D had a pH of 11 .75, and contained the equivalent of 69 ng tetrodotoxin/g of powder (quoted in BENEDEx and RIVrER, 1989). There is now some confusion over sample D. The two samples which YASUMOTO and Kno examined were both marked as having been collected in the Artibonite region, one in 1982 and the other in 1984 . Both were extremely alkaline, as the acetate-buffer extracts of both had pHs exceeding 10 . Sample D was collected in the Artibonite region in 1984 (BENEUEx arid RMER, 1989). RIVER informed Dnvls (letter dated 11 March 1987), and DnVS acknowledged the information (footnote, p. 194, Dnvls, 1988), that sample D was the same as one of the two samples examined by YnsuMO~ro and Kno. RrvIER also wrote that he was focussing his attention on sample D because YASUMOTO and Kno had found a trace of tetrodotoxin (20 ng/g of powder) in it. Further support for that identity is found in the observations of LOMBET and Lnznuxsxl that only sample D had the extreme alkalinity . In their published report, however, BENEDEK and RNIER (1989) did not make this important identification, but instead implied that the samples analyzed by YnsuNIOTo and Kno were different. Because we are in possession of a copy of the March 1987 letter, we will proceed with the following comments on the basis that sample D has indeed been 129

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analyzed independently by YASUMOTO and Kno (1985), LOMBET and LAZDUNSKi (private report to RIVIER, 1987), and now by BEIVEDEK and RrnER (1989) . From their GC-MS analyses, BENEDEK and RIVIER concluded that sample D contained 20 hg tetrodotoxin/g of powder (three orders of magnitude higher than the finding of either YASUMOTO arid KAO Of Of LOMBET and LAZDUNSKI) . The GC-MS analyses were based on determining the amount of an alkaline degradation product of tetrodotoxin, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline, the so-called Cv-base. From the early days of studies on the chemistry of tetrodotoxin (e .g . Tsunn et al ., 1964; see also summary by MosxEx, 1986), it has been known that this product can be derived not only from the pharmacologically potent tetrodotoxin, but also from much weaker analogues, as well as from the inactive tetrodonic acid . BENEDEK arid RIVIER, who were aware of the nonspecificity of the test, nevertheless concluded that all of their C9-base was due to tetrodotoxin . Ignoring the private report of LOMBET and Lnznuxsxl, they suggested that YASUMOTO and Kno had inadvertently brought about the destruction of the high content of tetrodotoxin through careless extraction of the powder sample. In fact, there is no basis for their conclusion that all the C9-base was due to tetrodotoxin, because even the inactive tetrodonic acid could be derived from a host of sources other than the uniquely potent tetrodotoxin . The enormous difference between their results and those of LOMBET and Lnznuxsxl and of YASUMOTO and Kno suggests that most of the C9-base did not originate in tetrodotoxin . Most importantly, at 20 pg tetrodotoxin/g of powder, sample D would have readily produced the characteristic paralysis of tetrodotoxin in a mouse-lethality bioassay . Such a simple and standard test would have readily clarified the plausibility of their GC-MS results. YnsuMO~ro and Kno (1986) reported that the sample had no noticeable effects in mice . Inexplicably, and in spite of Kno's advice (letter to RIVIER, 9 June 1988), no such test results were included in their paper. Therefore, it is not the competence of YASUMOTO and Kno that is open to question, but the objectivity of BETVEDEK and RIVIER . The LC-MS analyses of BENEDEK and RIVIER were equally flawed . The difficulty of accurate quantitation in thermospray LC-MS analyses is well known. The rate of ionization is dependent on the amount of the sample and on the amount and nature of the contaminants . Responsible analyses of unknown samples require comparisons with standards of authentic compounds, sprayed at precisely controlled rates. BENEDEK and RIVIER acknowledged that, aside from tetrodotoxin, they did not use any standards at all . The (M+H)+ ion at m/z 320 they recorded can be derived not only from tetrodotoxin but also from 4-epi-tetrodotoxin and tetrodonic acid . Their claim as to having separated isomers of tetrodotoxin by LC-MS has no foundation, because they did not prove the separation by comparing with reference compounds. Separation of 4-epi-tetrodotoxin or anhydrotetrodotoxin from tetrodotoxin requires ion-pairing reagent in the solvent and very careful examination of the conditions (YASiJMOTO arid MICHISHITA, 1985). There is no evidence in BENEDEK and RIVIER that these steps were employed . The two peaks in their chromatogram are most likely to be tetrodonic acid and a mixture of tetrodotoxin isomers, which are relatively easy to separate, but which are indistinguishable by the molecular ion. Having addressed the specific deficiencies in the analyses of BENEDEK and RIVIER, we wish now to address the issue of WnnE Dnvls's claim that tetrodotoxin was responsible for the initial zombification process, because the exp11C1t pllrpOSe Of BENEDEx and RIVIER was to provide some support for Dnvls's claim. That BENEDEK and RIVIER (1989), with all its serious deficiencies, was published is a tribute to the editorial sagacity of Toxicon. It is

Letters to the Editor

13 1

infinitely better to have bad data and wrong conclusions published in a scientific journal where rebuttals can be properly joined than to have such information selectively disseminated through the lay press where sensationalism is often held higher than truth. A general report on this affair based on impartial investigations has been published by Boo~rx (1988) . We will confine ourselves to three specific points which have not been raised in adequate detail before : (1) In a letter received by Dnv~s on 3 April 1987, Rtv1Ex wrote of Lnznurrsxl's report to him, citing Lnznuxsxl as saying that at the concentration of 69 ng tetrodotoxin/g zombie powder, 10 kg of the powder would be necessary to reach an LD P for a 70 kg man. In spite of RrvlEtt's expressions of confidence in his own ability to demonstrate the presence of tetrodotoxin in the zombie powder, there was not a shred of evidence up to that time to support the occurrence of tetrodotoxin in any of the samples in concentrations sufficient to produce the paralysis called for in Dnvls's scenario of zombification. Yet from 1983 when he made his first claim until May 1987 when he defended his thesis at Harvard University, Dnvls consistently maintained a position that was contrary to the extant factual evidence . When Davis wrote in his thesis, ". . . laboratory tests have shown both the presence of tetrodotoxin in the samples, and have indicated that the powders when applied topically to rats and monkeys are biologically active (Dnvls, 1983a) ." (p. 276, Dnvls, thesis), Rivier was many months away from demonstrating (erroneously) the presence of 20 hg of tetrodotoxin/g of zombie powder . The reference cited by Dnv~s (1983) contained no supportive information whatsoever for the simple reason that it predated any analytical endeavors on his samples. It might be further noted that in an advanced copy of the thesis submitted for approval to his thesis committee, the citation in place of "(1983)" was "(Appendix A)", and that no "Appendix A" existed in that copy . Furthermore, Dnvts himself, working with a very alkaline sample of zombie powder (sample D?) in November 1984 in collaboration with Joxty HARTUNG, already knew that even very large amounts of that sample, applied parenterally or topically, did not produce any biological effects in mice and rats (see BooTx, 1988). (2) A central flaw of Dnvls's research on the question of zombies is his unquestioning acceptance of the authenticity of Clairvius Narcisse, the individual who claimed to be a "returned" zombie . Narcisse was supposed to have died in 1962 of heart failure in the Albert Schweitzer Hospital . Terminally, he was .described by attending physicians as being conscious, immobile, cyanotic, and with a blood pressure of 26/15. After his death, the body was kept in cold storage for 20 hr before being removed for burial . In the Dnvls scenario, however, Narcisse was only profoundly paralyzed by tetrodotoxin, and was later exhumed from his grave and subjugated into slavery as a zombie . An objective reading of Narcisse's health record (in Dnvts, Ph.D. thesis, Harvard University) revealed that Narcisse had actually been ill for about a year before his terminal episode. His symptoms were consistent with chronic congestive heart failure associated with hypertensive cardiovascular diseases (see also independent view of $KRABANEK, 1988). Dnvts who is untrained in medical diagnosis, however, chose to focus only on Narcisse's terminal immobility and consciousness as indications of parallelism with the main symptoms in fugu poisoning. It is a common occurrence in people with severely impaired cardiac functions to be cyanotic, immobile and conscious. Few of those people would ever be properly diagnosed as having been poisoned by tetrodotoxin . Moreover, the very low systemic arterial blood pressure and the long cold-storage after death would hardly be compatible with a recovery of brain functions, especially those involved in higher intelligence. Yet, in 1982 the `returned' Narcisse showed so little

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permanent brain damage for his earlier ordeal as to be capable, apparently, of giving DAMS lengthy and persuasive interviews . (3) On the inclusion of some dried puffer fish in zombie powders, DAMS attributed some pharmacological knowledge to the bokors, the evil voodoo practitioners, without considering the possible alternative of sheer chance . Because of the poverty in Haiti, the sea around Haiti has been depleted of edible fish for some years. What remains to be caught in a cast seine net (as DAVIS'S bokor did, DAMS, 1985) are mainly fish too small to be consumed or puffer fish (COUSTEAU, 1985) . Whatever pharmacological sense DAVIS'S learned bokors might possess, their taxonomical ability concerning toxic puffer fish was wanting. Of the specimens of fish DAMS provided us as being representative of those included in the zombie powders, half were the non-toxic porcupine fish (YASUMOTO and KAO, 1986) .

We cannot conclude this rebuttal without commenting on an important point which has not received proper attention. This issue concerns DAVIS'S role in the illegal exhumation of a freshly buried child whose remains were incorporated into a sample of zombie powder . If WADE DAMS stumbled onto such a scene, or if the remains had been obtained before his amval, the moral issues would be moot. In this case, however, the bokor involved prepared a sample of zombie powder solely at DAVIS'S instigation, and on the lure of DAVIS'S cash payment; he dug into the child's grave under the light provided by DAVIS'S torch (DAMS, 1985), and displayed the remains on the end of a stick for DAMS to photograph (p. 415 in PRESS, 1987 ; p. 116 in DAMS, 1988) . The monstrosity of this moral transgression is in itself overwhelming, but if the action was committed in the guise of scientific investigations, then it surely must be the scientist's responsibility to confront the fundamental moral issues squarely and honestly . We hold firmly that science done without a moral and ethical foundation can never be more than a mockery of science. And that, in our view, is what DAMS (thesis; 1988) and BENEDEK and RtvIER (1989) really are. REFERENCES BENEDEK, C. and RtVtER, L. (1989) Evidence for the presence of tetrodotoxin in a powder used in Haiti for zombification. Toxicon 27, 47380. Boo~rti, W. (1988) Voodoo science. Science 240, 274-277. Cous~trau, J. (1985) Haiti, Waters of Sorrow . Video tape. DAVtO, S. R. and Fotv'tErrt.o, P. A. (1984) A competitive displacement assay to detect saxitoxin and tetrodotoxin . Analyt. Biochem. 141, 199-204. DAVt3, E. W. (1983) The ethnobiology of the Haitian zombie. J. Ethnopharmac . 9, 85-104 . DAMS, E. W. (1985) The Serpent and the Rainbow. New York : Simon and Schuster . DAMS, E. W. (1988) Passage ojDarkness . Chapel Hill : The University of North Carolina Press. Moste=tt, H. S. (1986) The chemistry of tetrodotoxin. In: Tetrodotoxin, Saxitoxin and the Molecular Biology of the Sodium Channel (Kao, C. Y. and Levinson, S. R., Eds.) Ann. New York Acad. Sci. 479, 323. PRtss, M. (1987) American Scientist interviews. Am . Scientist 75, 412-417. SKRAHANEK, P. (1988) Book review on Passage of Darkness . Lancet II, 826. TSUDA, K., IKUMA, S., KAWAMURA, M ., TACHIKAWA, R., SAKAt, K., TAMURA, C. and AMAKASU, O. (1964) Tetrodotoxin VII. On the structure of tetrodotoxin and its derivatives. Chem . Pharmac. Bull . 12, 1357-1374. YASUMOTO, T. and KAO, C. Y. (1986) Tetrodotoxin and the Haitian zombie . Toxicon 24, 747-749. YASUMOi'O, T. and MtctttstnTA, T. (1985) Flurometric determination of tetrodotoxin by high performance liquid chromatography. Agric. Biol. Chem . 49, 3077-3080. C. Y. KAO Department of Pharmacology State University of New York Downstate Medical Center Brooklyn, NY 11203, U.S .A .

T. YASUMOTO Faculty of Agriculture Tohoku University Sendai 980, Japan

Tetrodotoxin in "zombie powder".

Tosicron Vol. 28, No . 2, pp. 129-132, 1990 . Printed in Great Britain. 0041-0101/90 53 .00+ .00 CC9 1990 Pergamon Press pk TETRODOTOXIN IN "ZOMBIE...
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