Rationale for Methods 



Hreveiniim Tc maieriils which becnae eoaceatraied in nmae titniet throngfa annwl feeAng trtjuit^t 

 jf PP«f fwn ittr<< rrfmniant Thc tonu, lynthrtTTT/l by thc marine faofUgellate ft>cfcadbaa b»rw «rc aorm*l)> 

 fcKf^^n^wn uUte/i b fiher-feedtog orguisnu, but recent evidence hxfirnci that biomagnificatioc through the food 

 T*i«iti by ahenaie roates oiay ako occur. 



The »<^»*«f« and parificatoB procedure and iafividna] protocols for brevetoiis from dolphio bvcrs is 

 both jr^**^^ and laborious. Liver, bung the prindpa] drinrififatinn organ of «"»"""■>■■" i pcoc s (and more 

 ao in dolphins which lack a gaD bladder), is the organ we nprrt to find breveiams in their largest conccntratiom 

 ■hould they east. Liver by its nature contains high coBceatratioBS of the eazynes necessary to detoxify organic 

 ppH^ ftwn isduding toodns. Also by its funetional nature is the ttor^ge depot for er»n«wim«tm to which the bviag 

 doti^iin has been exposed. 



I ffitial aeps in the pnrificatioo seek to first denature any enzymatic machinery which may exist in th; 

 frazen qiedsens using a modified acetOBe predpitatioo stq>. TUs ttep also tends to dehydrate the specimen 

 and make e&raetioo of Iqiid soluble materiids easier (tlep 3 of protocols). Following dehydratioo, a noo-polar 

 solvent (chloroform) is used to extract the brevetoxins from the tissue samfde. Brevetooons are freely sofa^ in 

 the chloroform (step 4). Steps 5-16 are steps utthzed routinely and daily by Chira) Corp or ati o n in our protoctds 

 for extradioo and purificatioo of brevetoiins from laboratory cultures of the **^***^^g'"**^. and as sudi are the 

 results of 16 years of optimizatioa and progressioo from anal3^tical separation to large scale quantitative recovery. 



Flash chramatogra|diy (steps 6-9) is a routine and powerful way to separate toodc materials from 

 fP fffftnmatmg oOs and pigments, the former of which is a massive probefan in marine mammal tissues. These 

 initial steps are utlized in the order — and on the scale that thqr art ' t o isolate miotigram quantities of toadn 

 from gram quantities of interfering snlnianrrt 



Three sfqumtial thin-layer chromatograpfay oqs are utiEzed to isolate brevetoxins from one another, 

 and are the exact trfhniqnrt that were used in our laboratory to first determine the individual nature of PbTi- 

 1, PbTx-2, and PbTz-3. When compared to the known migration of standardized brevetoaons, preliminary identity 

 (tf suspect toxins can be made. 



The final step (tf high perf or m ance Equid riiTnmatogra|Ay is an analytical technique, «4iidi when ^ipiied 

 in the parallel presence of standard brevetoasas, can ^ve a further jmjjiigj basis for ooofirming or denying 

 brevetoxio identity. 



The Cambusia ajfinis fish bioassay is the most sensitive bioassay kimown for the brevetoxins, and 

 individual brevetoiins exhibit lethal dose ranges in the nanooolar ooBcentratioB ranges (Le. aanomoles of toxio 

 per liter of test water, fish are placed in 20 mL water «4iich yields a calculated sensitivity of 17.9 ^assay 

 volume. We bebeve it is accessary to emphasize that '*' bioassay means the fish dies witUn the time period 

 of observation. For most sensitive applications, observatioos are oiade over 48 hr, but positives fi«quently are 

 exhibiied within hours of initial laqxKure. Those fraaions which test *•', Le. do death, are not pursued futher. 

 Thus, a positive value does ajt necjsiarily iadi^tte br. noapn, bat a amative by these criteria does not contain 

 brevet axis. 



Eadi step of the purification and analysis which tests positive lends increasiag supp o r t to the indications 

 that brevetoxis are present in the sample. By back<alculatioa (knowing the amounts ot ataterial tested in a 

 destructive manner at each step) one can arrive at a value for toxin concentration in the origiaa] sample. The 

 value is calculated based on liplc traces against known concentrations of standards. 



For those samf^ which test positive throughout the entire assay protocol to step (16), we would suggest 

 further analysis to further confirm identity. The method requiring the least amount of material is brevetoada 

 radioimmunoassay, a technique pioneered at the University of Miami, and whi^ is part of the product line of 

 Chiral Corporation. Recent work confirms the polyether Vevetooon-Iike* sensitivity of the assays and a complete 

 insensitjvity to okadaic acid-type polyether toxins. Non-destructive tesu which could be employed included 

 Fourier transform infrared qiectrometry, or mass spectnnetry or FT nuclear magn^tir resonance qwctrometry 

 (the laner two being sub-contracted out). Short of X-ray crystallography, ao sia^e technique wiU unequivocally 

 identify brevetoxins. 



In summary, the progression of the steps m the protocol (i) ^'^™»»» potentiaOy imerferiiv f^Kr^ftuf^ ; 

 (a) have bees optimized over many years of hands-on fjjgiic D ce ; (m) are »>^4»««M2ii>f ased routinefy by the 

 Corporation to purify brevetoaias; and (W) progressively affirm or deay the ideatity of toobc «i««tiTi«K as 

 wevetoiins 



E)q>erience, Expertise, and Proficiency of Personnel 



The cnnicnhun vitae for Daniel G. Baden. President of Qiirs] Corporation, and Lloyd S. Schuhnaa, pan- 



121 



