AXIT DOMOIC CUA VI TAO VA DONG V A T CO VO d CUM DAO SONG TlT vA JACKSON ATOLL,
BIEN DONG
Pham Xuan Ky, Dao Viet Ha, Nguyen Thu Hong, Nguyen Tien Dung Vien Hai duong hgc, Nha Trang, Viet Nam
Ulysses M. Montojo
Phdng Nghien cim Ca bien, Vien Nghien cim va Phat trien Thuy san Qudc gia, Quezon, Philippin
TOM T A T
Axit doinoic, mot loai doc to bien gay ra hien tugng mat tri nho do ngo doc dong vat CO vo i^^C^) duoc san sinh boi cac loai \'i tao va tich luy d sinh vat bien. Trong nghien ciru nay, chiing toi quan tain den a.\it domoic trong vi tao va DVCV thu d Jackson Atoll va cum dao Song Tu, vung khoi Bien Dong suot chuyen khao sat JOMSRE IV trong thang 4 va 5, 2007. Su hien dien cua axit domoic da duoc xac dinh bang phuong phap ELISA va HPLC.
Cac ket qua da cho thay rang ham luong axit domoic trong tk ca cac mau vi tao deu o dudi gidi han c6 the phat hien duo'c, trong khi do ham lugng doc td nay cua DVCV la thap. 6 vung cum dao Song Tu, ham lugng axit domoic cija 3 loai DVCV, gom Tridacna croscea, Tectus pyramis va Turbo sp lan lugt la 0,98, 0,20, and 0,11 ()ag/g).
Ham lugng doc to nay cua Tridacna crosce. Spondilus sp va Turbo sp d vung Jackson Atoll lan luolla 1,01,0,71 va 0,10 ()ig/g).
I.GIOflTHIEU
Sinh vat bien la ngudn thuc pham quan trgng ddi vdi ddi sdng con ngudi. Tuy nhien, nhidu loai sinh vat lai chira cac loai ddc td nhu ciguatoxin (CTX), tetrodotoxin (TTX). saxitoxin (STX), dynophysistoxin (DTX)..., gay ddc doi vdi ngtrdi sir dung, tham chi thinh thoang gay chet. Cac loai ddc td nay hoac dugc tu san sinh bdi ban than vat chii hoac dugc tich luy tir mdi trudng-ngoai qua chudi thirc an.
Trong so cac loai ddc td da dugc biet, axit domoic (Hinh 1) la mot trong nhimg loai doc td nguy hi§m dugc tim thay cV mdt sd loai sinh vat biin.
t Axit domoic \hn dau tien dugc chiet tach tir mdt loai Tao dd Chondria armata vaten ciia nd dugc dat theo ten dia phuong ciia ti§ng Nhat Chondria armata domoi
• ^ 0 9 Chu-ong frinh Hop tac Khoo hpc & Cong nghe Viet Nam - Philippin
hoac hanayanagi (Takemoto \ a Diago. 1958; Daigo, 1959). Sau dd, no cung dugc tim thiy d moi loai Tao dd khac la Alsidium coralinum tai viing bien Hdng Hai (Todd. 1993). Day la mdt loai ddc td thin kinh, nguyen nhan gay ra hdi chung mat tri nhd do ngd ddc ddng vat cd \'d nhiem loai dgc td nay (Amnesic Shellfish Poisoning. ASP). Hien tugng ngd ddc axit domoic dau tien do an Vem xanh Mytilus edulis 6 dao Prince Edward, Canada dugc ghi nhan vao nam 1987. Trong vu ngd ddc nay. 108 nan nhan phai nhap vien vai cac trieu hirng budn ndn, di mira va tieu chay (Wright va cs. 1989). Sau dd, ngudi ta da xac djnh dugc mot loai Tao silic Pseudo-nitzschia multiseries trong tuyen tieu hda loai Vem nay cd kha nang san sinh axit domoic. Loai Vem xanh ndi tren da tich luy ddc td dugc san sinh tir loai tao nay vao co the trong qua trinh an loc (Bates va cs. 1989).
Sau trudng hgp nay, hang Ioat cai chet ciia cac loai sinh vat bien nhu Bd ndng nau Pelecanus occidentalis, Cdc Phalacrocorax penicillatus (Work va cs, 1993) va Su tir bien Zalophus californianus (Scholin va cs, 2000) cd lien quan den axit domoic da dugc ghi nhan tai nliieu noi thudc Canada, My va Chau Au. Axit domoic cung da dugc phat hien trong Trai Mya arenaria (Martin va cs, 1990) hay trong ca Trdng Engraulis mordax (McGinness \ a cs. 1995) d vinh Monterey, California, Sd bien Placopecten magellanicus (Douglas va cs, 1997) tai vinh Fundy (Canada), Trai Siliqua patula (Wekell va cs, 1994) va Cua Cancer magister (Van Apeldoom va cs,
1999) danh bat d bang Washington \'a Oregon, My.
Axit domoic dugc san sinh chii yeu bdi mdt sd loai vi tao thudc gieng Pseudo- nitzschia. Cho den nay 11 loai thudc gidng Pseudo-nitzschia, bao gdm P. multiseries (Bates va cs, 1989), P. calliantha (Martin va cs, 1990), P. australis (Garrison va cs,
1992), P. seriata (Lundholm va cs, 1994), P. pungens, P. turgidula, P. fraudulenta (Rhodes va cs, 1996, 1997, 1998). P. delicatissima (Smith va cs, 2000), P.
multistriata (Sarno va Dahlmann, 2000), P. galaxiae (Cerino va cs, 2004), P.
pseudodelicatissima (Bill va cs, 2005), va 3 loai thudc cac gidng khac gdm Amphora coffaeiformis (Maranda va cs, 1990). Nitzschia actydrophila (Worms va cs, 1991) va N. navis-varingica (Kotaki va cs, 2000) dugc chung minh la cd kha nang san sinh axit domoic. Nhirng loai tao nay phan bd o nhieu viing khac nhau, chu yeu tap trung d nhimg viing bien ndng. Loai Nitzschia navis-varingica chi dugc tim thay d viing nude Ig.
Axit domoic la mpt ddng phan ciia axit amin tucrng tu axit kainic, chira chu true ciia axit glutamic. Nd cd tinh chat dac trung ciia cac axit amin, tan trong nude, tan nhe trong methanol, ben nhiet nhung khdng ben trong di^u kien pH thay ddi. Axit domoic cd nhieu ddng phan gdm cac axit isodomoic tir A-F., Trong dd, axit domoic
(thanh phan ehinh), axit isodomoic D, E, F lien quan din hien tugng ASP. .j^
Axit domoic gay doc ddi vdi he than kinh d ngudi va ddng vat bac cao do nd cd <
ai luc gan ket manh vdi thu the glutamat va thuc dhy hoat ddng cua cac thu thi nay.
Sir hoat ddng manh ciia cac thu the glutamat da van chuyen manh ddng ion Na"" vao ' mang sau sinap, gay ra su khir cue. Dilu nay lam gia tang su thhm thhu ciia ddng ion Ca"" vao mang trong te bao, din din sir mit can bing dien thi mang va cudi ciing
(y yeu. HOI NGHI TONG KET CAC CHUYEN KHAO SAT NCKH BIEN PHOI HOP VIET NAM - PHIUPPIN TREN BIEN DONG 3 Q 3
pha vd te bao. Axit domoic tac ddng chii ylu vao trung khu dieu khien tri nhd va dinh vi khdng gian ggi la hoi hai ma (hippocampus) gay nen hien tugng mat tri nhd tam thdi (Nijjar va Nijjar, 2000).
Axit domoic cd the gay ra cac trieu chirng ngd doc nhu ndn mira, dau co that viing bung, tieu chay, mat tri nhd va kha nang dieu khien. Lieu gay chet 50% d chudt thi nghiem ddi vdi axit domoic qua dudng tiem vao bung la 3,6mg/lkg trgng lugng (Grimmelt va cs, 1990). Ddi vdi nhuyen the chira tii 20|ig axit domoic/g, chiing dugc de nghi cam sir dung (Hallegraeff, 1995).
Do axit domoic dugc tich luy d sinh vat bien, nhat la ddng vat cd vd qua chudi thirc an nen mdt sd loai trong nhdm ddng vat nay cd the dugc diing nhu cac sinh vat chi thi mirc nhiem ddc td nay trong mdi trudng. Viec giam sat ngudn san sinh ddc td nay la can thiet de ngan chan sir nhiem ddc va bao ve he sinh thai bien.
Khu vuc quan dao Trudng sa, Bien Ddng la mot ngu trudng tiem nang. Sir cd mat ciia mot sd loai tao san sinh axit domoic dugc tich luy d cac sinh vat viing bien gan bd ciia Philippin (Kotaki va cs, 2006; Bajarias va cs, 2006) va Viet Nam (Kotaki va cs, 2000) cung nhu sir tich liiy axit domoic trong Ngheu Meretrix meretrix va Vem xanh tai viing bien Cat Ba, Dd Son (Huyen va cs, 2006) va trong Hau huong Spondylus cruentus b Khanh hda (Ha va cs, 2006) da dugc xac nhan. Tuy nhien ehua cd tai lieu chinh thirc ve cac ngudn san sinh axit domoic va mirc do nhiem doc td nay d cac loai sinh vat d viing khai.
Cac ket qlia khao sat ciia JOMSRE IV giira thang 4 va thang 5 nam 2007 cung cap du lieu ve axit domoic trong vi tao san sinh va ddng vat cd vd viing khoi Bien Ddng.
IL VAT LIEU VA PHUONG PHAP 1. Dja diem
Cae mhu tao dugc thu tai mot sd tram ca d ben trong va ngoai viing ran thudc cum dao Song Tir (Hinh 2) va Jackson Atoll (Llinh 3) thudc quin dao Trudng Sa.
Trong khi dd, ddng vat cd vd chi dugc thu ben trong ran thudc cac viing nay d do sau nhd han 20m.
2. Phuong thirc thu mau
30 mlu tao dugc thu bang ludi thu sinh vat phii du vdi kich thudc mk ludi la 20|im va dudng kinh mieng ludi la 20 cm. Ludi dugc keo tir do sau 10-20m, 5 din
10 lin, tiiy theo do sau cac tram. Khoang 250ml nude biin chira tao dugc dung trong cac binh nhua dung tich 500mK giir lanh bang da lanh ngoai hien trudng va d 4°C trong tii lanh din khi ly tam. Miu tao dugc ly tam trong vdng 12 gid sau khi thu. Trudc khi ly tam, phin can ba hu-u ca dugc gan bd nhd lgc qua ludi cd kich cd mat ludi 200(.im. Phan nude chira tao dugc lgc tiep qua ludi cd kich thudc mit ludi ,20^m. Phin tao giir lai tren ludi dugc riia bang nude cit va cho vao dng nghiem.
204 chu-ong trinh Hpp tac Khoa hoc & Cong nghe Viet N a m - Philippin
Mlu tao dugc can va chia thanh 2 phan. Mdt phan sau khi ly tam a 10000 vdng/phut trong 15 phut, lugng nude cat dugc dieu chinh tir l-2ml de chiet axit domoic diing xac dinh ndng do bing phuong phap thir nghiem hap phu mien dich lien kit enzim (enzym-linked immunosorbent assay, ELISA). Phan mau tao cdn lai dugc chiet bing methanol 50% dl kilm tra nong do bang ELISA hay phuang phap sic ky Idng hieu nang cao (high performance liquid chromatography, HPLC).
4 loai ddng vat cd vd gdm 2 loai hai manh vd, Spondilus sp • a Tridacna croscea va 2 loai dc gom Turbo sp va Tectus pyramis (Hinh 4) cung da dugc thu bang viec lan vdi binh khi. Phan md cua loai nay da dugc tach khdi vd va ddng lanh a -20° C din khi tach chiet axit domoic. Mau md ciing dugc chiet bang nude cat (g : ml = 1 : 1) hoac methanol 50% (g : ml = 1 : 4) diing cho ELISA hoac HPLC.
3. Phuong phap chiet va do ham lucmg axit domoic
Mau tao va md ddng vat cd vd da dugc lam nhuyen trong nude cat dugc dun sdi trong 10 phut, de ngudi, ly tam, 10000 vdng/phut trong 5 phut. Phan djch chiet dugc giii- a -20°C din khi tiln hanh ELISA.
Mau tao va md ddng vat cd vd ngam trong methanol 50%) dugc giir khoang 24h d 4°C. Phan djch chiet thu dugc sau khi ly tam dirge cd tren may cd chan khdng de lam bay hoi methanol. Phan mau sau khi cd dirge hda tan trong PBS (lOg md : 10ml PBS) de xac djnh ndng do axit domoic bang HPLC. Chi cd nhiing mau duang tinh bdi ELISA mdi duac kiem tra bang HPLC.
'to4. Nguyen tac cua ELISA
Phuong phap ELISA dua tren phan img dac hieu giira cac khang the va khang nguyen da dugc md ta bdi Takata, 2007b. Khang nguyen (axit domoic, AD) dugc nhan biet bang mdt khang the dac hieu thur 2 (khang hiiyet thanh de) da gan ket vdi mdt enzyme (horse raddish peroxidase) thdng qua sir cdng hgp vdi khang the thu 1 la khang huyet thanh thd da ket hgp vdi Disuceinimidyl Suberate (DSS) trong Bovin Serum Albumin (BSA) (AD-DSS-BSA-No.2). Hdn hgp se hien mau khi tac dung vai CO chat Ortho-phenylenediamin. Cudng do mau ty le nghich vdi ham lugng axit domoic cd trong mau.
5. Cac bu-tfc tien hanh
Cac gieng ciia ban nhua 96 gieng dugc phii bdi 50|il antigen (axit domoic) va cd
dinh bang 0,3%) gelatin (Bio-Rad, EIA grade) cimg vdi 50)il ciia khang thi thu 1 d
ndng do pha loang 1: 3000 trong dem phosphate ehua mudi (Phosphate Buffered
Saline, PBS). Sau khi ii d 37°C trong vdng 1 gid, cae gilng dugc rira bing dung dich
dem PBS cd chira 0,1% Tween 20, 3 lan. Sau dd, cho vao moi gilng ciia ban nhua
nay lOOjil khang thi thir 2 (Dako, P0399, x 1000 lin vdi dem PBS) va u a nhiet dd
37 C trong 1 gid. Khang the sau khi gan ket vai axit domoic cd mat trong miu dugc
hien mau bang Pastimo-phenylediamines dihydrochloride (OPD, peroxidase
Ky yeu. HQI NGHI TONG KET CAC CHUYEN KHAO SAT NCKH BIEN PHOI HOP VIET NAM - PHIUPPIN TREN BIEN DONG 3 0 5
substrate, Sigma). Cudng do mau dugc do tren may Microplate Reader (Bio-Rad, model 680) d budc sdng 490nm. Ham lugng axit domoic trong mau dich chiet dugc tinh toan dira vao dudng axit domoic chuan (Bio Vectra).
III. KET QUA L Doc to ff vi tao
Ham luong axit domoic ciia cac miu tao deu nam dudi gidi han cd the phat hien dugc (Bang 1-2).
2. Doc to 6' dong vat co vo
Tat ca cac mau ddng vat cd vd duang tinh vdi axit domoic, nhung d ham lugng thap (Bang 3). Hinh 5 cho thay ket qua ciia mot mau Spondilus sp dugc phan tich bing HPLC.
IV. THAO LUAN
Cac kit qua da ggi y rang cd sir hien dien ciia cac tac nhan san sinh axit domoic trong mdi trudng viing ran nghien cim. Mot sd loai tao thugc gidng Pseudo-nitzschia da dugc tim thay d viing bien nay (Nguyen Ngoc Lam va cs, 2008, tai lieu ehua cong bo). Tuy nhien, mot cau hdi xuat hien la vi sao khdng phat hien dugc axit domoic trong cac mau tao trong nghien cim hien tai. Cd 2 kha nang: Mdt la, cd the mat do cac loai tao cd kha nang san sinh axit domoic thap vao thdi diem thu mau.
Khac vdi viing biin ven bd, cac ran nghien ciru la viing ngheo dinh dudng, nai ma ham lugng eac mudi dinh dudng can thiet cho sir phat trien cac loai tao ddc hai tai viing biin nay thap. Kha nang khac la kich thudc ciia mat ludi thu mau tao ehua thich hgp. Cac nghien cim trudc day da cho thay rang cae loai vi tao trong mdi tnrcmg biin cd kha nang san sinh axit domoic thudng cd kich cd tir 10-20|im trd len.
Tuy nhien, cac nghien cim gan day da phat hien trong cac phan doan tao dugc lgc tir nude biin viing dim Nha Phu, Viet Nam, phan doan 0,6|im cd ndng do axit domoic cao hon phan doan \Qpm, trong khi dd phan doan lOO^im cho ket qua am tinh vdi phuang phap ELISA (Takata va cs, 2007a). Nhu vay, cac loai tao cd kich cd < lOjim bao gom cac loai cd kha nang san sinh axit domoic da bi Igt ludi vdi kich ed mit ludi 20^im trong nghien curu hien tai. Them vao dd, tao ciing cd the da thoat ra khdi mieng ludi trong qua trinh keo. Mot khao sat ve thanh phan, mat do va kich cd cac loai tao thu tai cac tram se lam sang td nhan dinh nay. Mat khac, mdt nghien ciai vl axit domoic trong cac mau tao trong nude thu d vung bien nay loc qua mang cd kich CO 0,6(.im nen dugc tiln hanh.
Ddi vdi cac loai ddng vat cd vd thu dugc d viing bien nghien cim, cd thi xem la an toan vl axit domoic trong viec sir dung d thdi dilm hien tai. Ham lugng axit domoic trong cac loai nay la thip, phii hgp vdi cac kit qua ciia Huyin va es (2006);
' Bajarias va cs (2006) vl axit domoic trong mdt sd loai cd vd khac d viing biin Viet
3Q(^ chu-ong trinh Hop tac Khoa hoc & Cong nghe Viet Nam - Philippin
Nam va Philippin. Tuy nhien vdi loai Spondylus sp trong nghien cuu nay, ham lugng axit domoic dugc tim thiy la nhd hon rat nhieu so vdi cac loai thudc gidng Spondylus khac trong nghien cim ciia Takata va cs (2007b); Ha va cs (2006). Mdt dilu dang chu y d day la axit domoic trong 2 loai de Turbo sp va Tectus pyramis, nhtrng loai khdng an lgc, da dugc phat hien. Cd the la axit domoic a 2 loai dc nay dugc tich liiy tir ngudn khac ma khdng phai la vi tao trong thdi gian dai.
Cac kit qua ciing cho thay sir lien quan giti'a ham lugng axit ^.Dmoic trong cae ddi tugng hai manh vd va tao trong nghien ciru nay la khdng rd. Mdt ket qua tuong tu dugc Huyen va cs (2006) tim thay trong nghien cim ve ham lugng axit domoic trong Mytilus edulis va Meretrix meretrix; khdng cd sir lien quan giu:a ham lugng axit domoic trong cac loai nay vdi mat do tao san sinh trong mdi trudng nude. Kotaki va cs (1996) ciing chi ra rang viec nhiem axit domoic d' ddng vat cd vd d vinh Ofunato, Nhat ban khdng chi dan gian dugc giai thich bang sir xuat hien ciia cac loai tao Pseudo-nitzchia. Ciing nhu vay, neu su tich luy axit domoic d ddng vat hai manh vd tir vi tao qua qua trinh an lgc thi rat khd ly giai tai sao axit domoic ludn dugc phat hien trong cac loai hai manh vd thudc gidng Spondylus thu d nhieu dia diem khac nhau viing nhiet ddi, trong khi ddc td nay khdng dugc tim thay d cac loai khac tai ciing dia diem (Ha va cs, 2006; Takata va cs, 2007b). Nhu vay, nghien cim nay cimg vdi eac nghien cim khac dugc trinh bay a day khdng the giai thich ngudn gdc axit domoic.
Ve ket luan, nhung ket qua hien tai cho thay rang axit domoic khdng dugc phat hien d mdt sd vi tao nhung hien dien d cac loai dgng vat cd vd viing Ichai Bien Ddng vai ham lugng thap. Ddng vat cd vd d thdi diem khao sat la an toan cho ngudi sir dung.
LOfI CAM OIV
Cac tac gia xin cam. on nhdm nghien cim da dang sinh hgc da giup thu mau cae loai ddng vat cd vd va Lanh dao ciia JOMSRE IV da hd trg nghien ciai nay.
TAI LIEU THAM KHAO
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Ky yeu. HOI NGHI TONG KET CAC CHUYEN KHAO S.AT NCKH BIEN PHOI HOP VIET NAM - PHIUPPIN TREN BIcN DONG 3 0 7
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3 0 Q Chuong trinh Hop tac Khoa hoc & Cong nghe Viet N a m - Philippin
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3 1 0 chuong trinh Hop fac Khoa hoc & Cong nghe Viet N a m - Philippin
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DANH MUC HINH
ETmh 1: Cau triic hda hgc ciia (a) axit domoic, (b) axit glutamic va (c) axit kainic Hinh 2: Ban dd vi tri thu mau vimg cum dao Song Tir
Hinh 3: Ban dd vi tri thu mau viing Jackson Atoll
Hinh 4: Cac loai ddng vat cd vd (a) Spondylus sp, (b) Tridacna croscea, (e) Tiubo sp and (d) Tectus pyramis
Hinh 5: Sac ky dd HPLC ciia mdt mau Spondilus sp. AD: axit domoic DANH MUC BANG
Bang 1: Flam lugng axit domoic (i-ig/g) ciia mau vi tao d cum dao Song Tir Bang 2: Ham lugng axit domoic (|.ig/g) ciia miu vi tao d Jackson Atoll
Bang 3: Flam lugng axit domoic ()ig/g) ciia miu ddng vat cd vd d cum dao Song Tir va Jackson Atoll
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(a)
(c)
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Hinh 4: Cac loai dong vat co vo (a) Spondylus sp. (b) Tridacna croscea, (c) Turbo sp and (d) Tectus pyramis
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Bang 1: Ham luong axit domoic (ug/g) cua miu vi tao a cum dao Song TIP
Tram
• 1
2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19
Ham lygng axit domoic (i^g/g) Am tinh
Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh
314 Chuong trinh Hop tac Khoa hpc & Cong nghe Viet N o m - Philippin
Bang 2: Ham luang axit domoic (fig/g) cua m i u vi tao a Jackson Atoll
Tram 1 3 4 5 6 9 13 14 15 16 17 18
Ham lugng axit domoic (ug/g) Am tinh
Am tinh Am tinh Am tinh Am tinh Am tinh Am tinh
•f Am tinh Am tinh Am tinh Am tinh Am tinh
Bang 3: HSm luong axit domoic (.ug/g) cua m i u ddng vSt co vd a cum dao Song Ti> va Jackson Atoll
Loai Tridacna croscea Tectus pyramis Turbo sp
Tridacna croscea Spondilus sp Turbo sp
Dia diem
Cum dao Song Tir
Jackson Atoll
Ham lirgng axit domoic (ug/g mo) 0,98
0,20 0,11 1,01 0,71 0,10
