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Alécio-Oliveira, Eduardo José Desenvolvimento de métodos para...

UNIVERSIDADE FEDERAL DE PERNAMBUCO

CENTRO DE CIÊNCIAS BIOLÓGICAS DOUTORADO EM CIÊNCIAS BIOLÓGICAS

DDEESSEENNVVOOLLVVIIMMEENNTTOO DDEE MMÉÉTTOODDOOSS PPAARRAA DDEETTEERRMMIINNAAÇÇÃÃOO DDEE MMIICCRROOCCIISSTTIINNAA--LLRR EEMM ÁÁGGUUAA

EDUARDO JOSÉ ALÉCIO DE OLIVEIRA

RECIFE – BRASIL

outubro de 2003

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FICHA CATALOGRÁFICA

ALÉCIO-OLIVEIRA, Eduardo José

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11,, 113399 pp..

TTeessee:: DDoouuttoorraaddoo eemm CCiiêênncciiaass BBiioollóóggiiccaass ((BBiiootteeccnnoollooggiiaa))

1. Cianobactérias

2. Microcistina-LR

3. Métodos de detecção

4. Dot-blot

5. Criptato

6. Európio

7. Térbio

i. Universidade Federal de Pernambuco – Teses

ii. Título




EDUARDO JOSÉ ALÉCIO DE OLIVEIRA

DDEESSEENNVVOOLLVVIIMMEENNTTOO DDEE MMÉÉTTOODDOOSS PPAARRAA DDEETTEERRMMIINNAAÇÇÃÃOO DDEE MMIICCRROOCCIISSTTIINNAA--LLRR EEMM ÁÁGGUUAA

Tese apresentada ao Curso de Doutorado do Centro de Ciências Biológicas da Universidade Federal de Pernambuco, visando a obtenção do título de:

Doutor em Ciências Biológicas Área de Biotecnologia

Orientador: Professor Dr. José Luiz de Lima Filho Departamento de Bioquímica, UFPE.

RECIFE–BRASIL Outubro de 2003




DDEESSEENNVVOOLLVVIIMMEENNTTOO DDEE MMÉÉTTOODDOOSS PPAARRAA DDEETTEERRMMIINNAAÇÇÃÃOO DDEE

MMIICCRROOCCIISSTTIINNAA--LLRR EEMM ÁÁGGUUAA EDUARDO JOSÉ ALÉCIO DE OLIVEIRA e-mail: [email protected]

Aprovada em: Comissão Examinadora: Aprovada por: _____________________________________________ Profº. Dr. Benildo Souza Cavada - UFC _____________________________________________ Profº. Dr. Maria do Carmo de Barros Pimentel- UFPE _____________________________________________ Profª. Dr. Rosa Amália Fireman Dutra - UPE _____________________________________________ Profª. Dr. Ana Lúcia Figueiredo Porto - UFRPE _____________________________________________ Profº. Dr. José Luiz de Lima Filho – UFPE (Orientador) Programa autorizado para oferecer o título de: ________________ Data: _____________


mailto:[email protected]



Dedicatória:

Aos meus Pais,

José Miguel de Oliveira e Ana Rosa Alécio de Oliveira pela educação que me

proporcionaram e os incentivos constantes.

À minha Esposa Vânia Soares de Carvalho pelo companheirismo, incentivo e

grande paciência.

Às minhas irmãs Ana Áurea, Rosa Valéria e Karina Maria, sempre envolvidas

em novos desafios profissionais.

A todos os amigos que comigo dividiram, discutiram e participaram deste

trabalho, crescendo com as novas experiências.




SUMÁRIO

AGRADECIMENTOS_____________________________________________i LISTA DE FIGURAS_____________________________________________iii LISTA DE TABELAS_____________________________________________vi

RESUMO______________________________________________________vii ABSTRACT____________________________________________________ix 1. INTRODUÇÃO__________________________________________________01 1.1. Cianotoxinas___________________________________________________01 1.2. Toxicidade_____________________________________________________04 1.3. Ocorrência_____________________________________________________05 1.4. Legislação ____________________________________________________08

1.5. Métodos de Análise de Microcistina _______________________________09 1.6. Complexos de Lantanídeos Fluorescentes: Criptatos de Eu3+ e Tb3+ ____15

1.7. Matrizes Poliméricas como Suporte para Complexos de Lantanídeos Fluorescentes __________________________________________________20 2. OBJETIVOS ____________________________________________________22

3. REFERÊNCIAS BIBLIOGRÁFICAS _________________________________23

CAPÍTULO 1: APPLICATION OF DOT BLOT IMMUNOASSAY WITH COMPUTERISED SCANNING TO ANALYSIS OF MICROCYSTIN-LR______29

Abstract_______________________________________________________30

Introduction ____________________________________________________31

Materials and Methods___________________________________________32 Results________________________________________________________38 Discussion_____________________________________________________46 Acknowledgements _____________________________________________49 References ____________________________________________________49




CAPÍTULO 2: A NEW FLUORESCENT-LABELED MICROCYSTIN-LR TERBIUM CRYPTATE____________________________________________53 Abstract_______________________________________________________54

Introduction____________________________________________________55 Materials and Methods___________________________________________57 Results________________________________________________________61 Discussion_____________________________________________________65

References_____________________________________________________67 CAPÍTULO 3: EUROPIUM CRYPTATE GEL IMMUNOSENSOR TO MICROCYSTIN-LR_______________________________________________72

Abstract_______________________________________________________73

Introduction____________________________________________________74 Materials and Methods___________________________________________75 Results and Discussion__________________________________________80 References_____________________________________________________85 4. CONCLUSÕES__________________________________________________87 5. CONSIDERAÇÕES FINAIS E PERSPECTIVAS CIENTÍFICAS ____________89 6. OUTROS TRABALHOS COM PARTICIPAÇÃO DO AUTOR______________92 6.1. NEUROTOXIC CYANOBACTERIA BLOOM IN A BRAZILIAN NORTHEAST TROPICAL RESERVOIR (Em preparação)___________________________92 Abstract_______________________________________________________93 Introduction____________________________________________________94 Materials and Methods___________________________________________95 Results________________________________________________________98 Discussion____________________________________________________104 Acknowledgements ____________________________________________108 References ___________________________________________________109




6.2. PUBLICAÇÕES REALIZADAS NO PERÍODO DA TESE _______________113 7. ANEXO I: PROTOCOLO DE VALIDAÇÃO DE METODOLOGIA DE ANÁLISE DE MICROCISTINA-LR EM ÁGUA ULTRAPURA POR CROMATOGRAFIA LÍQUIDA DE ALTA EFICIÊNCIA – CLAE____________114




AGRADECIMENTOS

Às Instituições de pesquisa e de ensino público LIKA-UFPE, DQF-UFPE, ITEP,

UnB, AGGEU MAGALHÃES-FIOCRUZ, CEFET-PE e CNPq que permitiram a

realização deste trabalho. Ao Professor José Luiz de Lima Filho pelo constante incentivo e confiança

demonstrada, além dele mesmo ser um exemplo de otimismo e perseverança na

obtenção dos objetivos de formação e desenvolvimento regional.

À Professora Elizabete Malageño do LIKA-UFPE cujo conhecimento e

confiança demonstrados permitiu que os trabalhos imunológicos fossem realizados. À Professora Célia Castro do LIKA pelas discussões sobre ensaios dot-blot.

Aos Professores do DQF-UFPE Petrus Santa Cruz e Severino Alves Junior, e

a Doutoranda Suzana Vilanova pela prática concreta do termo cooperação.

Ao Professor Antonio Teixeira do Laboratório Multidisciplinar de Pesquisa em

Doença de Chagas da UnB, pela colaboração efetiva, princípios científicos e de

parceria que foram imprescindíveis à conclusão desta tese.

Ao Profº Manoel Euzébio, ao aluno de Doutorado Pablo Viana e aos alunos de

I.C. do Departamento de Informática da UFPE, Atus Vianna e Leonardo Nascimento, pelo desenvolvimento do programa (software) para quantificação dot

blot.

Ao pesquisador e amigo do ITEP Renato Molica cujo profissionalismo,

conhecimento científico e senso de participação, foi fundamental para a realização

de meus trabalhos. À Professora Rosa Fireman da UPE por todo o apoio prestado.

Ao pesquisador Ian Drummond do ITEP cujas críticas e experiência analítica

foram essenciais aos trabalhos de HPLC. Ao Curso de Doutorado em Ciências Biológicas da Universidade Federal de

Pernambuco (UFPE), representado pela Professora Luana Cassandra B. Coelho,

sem o qual esta formação não seria possível, e as Sras. Adenilda e Jacilene

sempre presentes nesta Coordenação.




Aos colegas do CEFET-PE Silvana Mendonça, Dulce Lins, Marcos Maciel e Sofia Suely, pelo incentivo e compreensão durante as horas de trabalho.

Aos dirigentes e profissionais do ITEP, que sempre me acolheram com

cordialidade e confiança, representados aqui na pessoa das Sras. Fátima Bryner, Márcia Lyra, Conceição, Ana Arnaud, Adélia, Sônia Pereira, Ana Rita, Cláudia Neves, e dos Srs. Raimundo, Celerino, Gilberto Pompeu, Carlos e Manoel.

Aos colegas de turma do Curso de Doutorado cuja convivência foi bastante construtiva, em especial a Laura Bruno, Ana Catarina, Adriana Argolo, Jaqueline, Vânia, e Emerson Peter.

Aos colegas dos diversos laboratórios do LIKA, com referência especial a Cosme Martinez, Keila Moreira, Jorge Silva e Daniele Bruneska, pela amizade,

discussões e divisão da experiência desta formação; À Mônica Belian e demais estudantes do DQF-UFPE pelos momentos de

convívio participativo. Aos funcionários do LIKA, especialmente as Sras. Ilma, Conceição

Chimendes e Vera, e aos Srs. Otaviano Tavares, Oscar Brasil e Moisés pela

presteza sempre demonstrada na execução de seus trabalhos.

Aos profissionais e estudantes do Laboratório Multidisciplinar de Pesquisa em Doença de Chagas da UnB que me acolheram como um dos seus, durante as

atividades lá desenvolvidas.




LISTA DE FIGURAS

INTRODUÇÃO:

Figura 1: Estrutura química das Microcistinas e Nodularinas; (A) Estrutura geral das microcistinas: ciclo-(D-Ala1-X2-D-MeAsp3-Z4-Adda5-D-Glu6-Mdha7); (B) Estrutura da nodularina: ciclo-(D-MeAsp1-Z2-Adda3-D-Glu4-Mdhb5) (Sivonen & Jones, 1999)......................................................................................................04

Figura 2: Esquema do mecanismo de extravasamento sangüíneo hepático provocado por microcistinas e nodularinas. (A) Fígado normal; (B) Fígado após ação das toxinas (Traduzido de Carmichael, 1994).....05 Figura 3: Esquema do processo de desativação envolvida na conversão de luz absorvida pelos ligantes do complexo e emitida pelo Eu3+ (Setas azuis = Processos não radioativos; Setas vermelhas = Processos radiativos).................................................................................................................................17 Figura 4: Espectro de excitação (..........) e de emissão (____) de um criptato de Eu3+ em solução (Flipsen, 2000)...........................................................................................................................................................18 Figura 5: Criptato de bis-piridina-piridina [Bip.bip.pi(CO2Et)2]3+ lantanídeo sintetizado no DQF (Departamento de Química Fundamental da UFPE)................................................................................19 CAPÍTULO 1: Application of Dot Blot Immunoassay with computerised scanning

to analysis of microcystin-LR Figure 1: Image scanning for the dot’s edge detecting. Blue and black lines are the horizontal and vertical scanning procedure, respectively……………………………………………………………………………… 36 Figure 2: virtual Dot blot membrane model……………………………………………………………………...37 Figure 3: Anti-IgG-HRP dilutions dot blot (three replicate) membrane revealed with DAB-Imidazol. …….37 Figure 4: Bovine serum albumin – BSA dot blot membrane revealed with 0.1% amidoblack solution (Henkel & Bieger, 1994)…………………………………………………………………………………………...38 Figure 5: Linear regression of a digitlilizing hypotetical dot-blot embrane. Vertical error bars represent standard deviation (n = 5; r = 0.9620; t = 17.0; p < 0.01)…………………………………………………..….39 Figure 6: Semi-log graphic of Anti-IgG-HRP dilution curve quantified by dot blot software. Linear range between 500 and 25000 dilution factor (r = 0.9868). Vertical error bars represent standard deviation (n = 3; t = 19.3; p < 0.01)……………………………………………………………………………………….………39 Figure 7: Bovine serum albumin – BSA concentration curve quantified by dot blot software. Linear range between 0 and 78 µg/ml (r = 0.9735). Vertical error bars represent standard deviation (n = 3; t = 34.1; p < 0.01)………………………………………………………………………………………………………………….40 Figure 8: Purified anti-microcystin-LR-KLH IgG titres determined with a ELISA procedure. Vertical error bars represent standard deviation (n=3)…………...…………………………………………………………….40 Figure 9: Semi-log graphic of 0.16, 0.50 and 1.60 µg/l microcystin-LR analysed in an indirect competitive ELISA. Vertical bars represent standard deviation (n=3; t = 10.8; p < 0.01)…………………………………41




Figure 10: A triplicate dot blot membrane of purified water (a) and lake water (b) spiked with microcystin-LR and cell extract dilution of M. aeruginosa (c)………………………………………………………………..42 Figure 11: Indirect competitive ELISA of purified (a) and lake water (b) spiked with micrcocystin-LR with 0.16, 0.50 and 1.60 µg/l. Vertical bars represent standard deviation (n=3) The right graphics are the correspondent semi-log scale …………………………………………………………………………………….43 Figure 12: Purified water (a) and lake water (b) spiked with Microcystin-LR and quantified by dot blot software. Vertical error bars represent standard deviation (a minimum n = 2). The right graphics are the correspondent semi-log scale………………………………………………………….………………………….44 Figure 13: Purified water (a) and lake water (b) spiked with Microcystin-LR and quantified by dot blot software. Vertical error bars represent standard deviation (a minimum n = 2) The right graphics are the correspondent semi-log scale ……………………………............................................................................45 Figure 14: Microcystin in M. aeruginosa extract quantified by dot blot software. Vertical error bars represent standard deviation (n = 3) The right graphics are the correspondent semi-log scale ……………………………………………………………………………………………………………………….45

CAPÍTULO 2 : A new fluorescent-labeled microcystin-LR terbium cryptate

Figure 1: Schematic synthesis of terbium cryptate……………………………………………………………..58 Figure 2: Chromatogram in gradient reversed-phase HPLC of mixture of reaction product: (1) microcystin-LR-aminoethanethiol and (2) microcystin-LR standard…………………………………………..62

Figure 3: Chromatogram in gradient reversed-phase HPLC at start of conjugation time of microcystin-LR-aminoethanethiol and terbium cryptate (toh) at 238 nm and 310 nm: (1) Terbium cryptate and (2) Microcystin-LR-aminoethanethiol…………………………………………………………………………………63

Figure 4: Chromatogram in gradient reversed-phase HPLC at the final conjugation time (t24h) of microcystin-LR-aminoethanethiol and terbium cryptate at 310 nm: (1) Microcystin-LR-Terbium cryptate and (2) Terbium cryptate…………………………………………………………………………………………..64

Figure 5: Emission spectra of microcystin-LR-terbium cryptate (b) recouped in gradient reversed-phase HPLC Excitation wavelength 310 nm.……………………………………………………………………………64

Figure 6: Semi-log graphic of competitive indirect ELISA of standard microcystin-LR using anti-microcystin-LR-KHL IgG on a microplate coated with Microcystin-LR-chicken-ovoalbumin (n = 3)……....65

Figure 7: Microcystin-LR terbium cryptate conjugation reactions……………………………………………..66

CAPÍTULO 3: Europium cryptate gel immunosensor for microcystin-LR Figure 1: Absorption spectrum in infrared region of commercial sample of polyacrylamide/acrilic acid gel…………………………………………………………………………………………………………………….80 Figure 2: Absorption of 0.1M saline solution for SAPG in diferent values of pH. …………………………...80 Figure 3: Percentual retention of bovine serum albumin in the SAPG in function of time contact………...81




Figure 4: : Emission spectrum of SAPG with Europium cryptate, IgG antimicrocystin-LR-KLH (IgG) and Microcystin-LR (Myc). Excitation wagelength 310 nm………………………………………………………….82 Figure 5: Elution of IgG anti-microcystin-LR-KLH-Europium cryptate conjugate obtained by maleimide method. Elution with 20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA (pH 6.5) in a fast desalting column HR 10/10 Pharmacia. …………………………………………………………………………………….83 Figure 6: Anti-microcystin-LR-KLH-Europium cryptate titre using ELISA plate coated with microcystin-LR-chiken ovoalbumin . Initial dilution was a 50 µg protein / 50 µl antibodie solution. Vertical error bar represent standard deviation (n = 3) . ………………………………………..………………………………….84 Figure 7: Emissium spectrum of conjugated IgG antimicrocystin-LR-KLH-Europium cryptate. Excitation in 315 nm with deuterium lamp. …………………………………………………………………………………….84 OUTROS TRABALHOS COM PARTICIPAÇÃO DO AUTOR: Neurotoxic cyanobacteria bloom in a brazilian northeast tropical reservoir Figure 1: Environmental parameters in Tapacurá reservoir from 19 March to 30 May 2002. (A) pH; (B) Conductivity and (C) = Total phosphorus. ………………………………………………………………………98 Figure 2: Cyanobacteria composition and concentration (cell number mL-1) during a bloom in Tapacurá reservoir. Anabaena spiroides, Anabaena sp., Synechocystis sp., Planktothrix sp., Merismopedia sp., Microcystis aeruginosa, Gomphosphaeria sp., Pseudoanabaena sp. and Cylindrospermopsis raciborskii. ...………………………………………………………………………………100 Figure 3: HPLC-FLD chromatograms of (A) 8-May bloom sample, (B) standards (reference material) and (C) mixture of standards of unknown concentrations. ……………………………………………………..…103 ANEXO I: PROTOCOLO DE VALIDAÇÃO DE METODOLOGIA DE ANÁLISE DE MICROCISTINA-LR EM ÁGUA ULTRAPURA POR CROMATOGRAFIA LÍQUIDA DE ALTA EFICIÊNCIA - CLAE Figura 1:Extração de microcistina-LR em amostras de água Milli-Q propositalmente incriminadas (Adaptado de Fastner et al., 1998 e Krishnamurthy, (1986). ………………………………………………..118




LISTA DE TABELAS

INTRODUÇÃO: Tabela 1: Classificação geral das cianotoxinas (Roset et al., 2001; Sivonen & Jones, 1999; Carmichael, 1992). ………………………………....................………………………….....................................................02

Tabela 2: Resumo dos Métodos Físico-Químicos para Detecção de Microcistinas...................................12

Tabela 3: Relação de anticorpos anti-microcistinas e técnicas ELISA.......................................................14

CAPÍTULO 1: Application of Dot Blot Immunoassay with computerised scanning to analysis of microcystin-LR

Table 1: Average of somatory of intensity of pixels and paired Student test values of purified and lake water membrane spiked with microcystin-LR and quantified by dot blot software. …………………………………………….......................................................................................................42

OUTROS TRABALHOS COM PARTICIPAÇÃO DO AUTOR: Neurotoxic cyanobacteria bloom in a brazilian northeast tropical reservoir

Table 1: Morphological features of three strains and natural population of A. spiroides from Tapacurá reservoir. ………………………………………………………………………………………………………..…102 Table 2: Toxicity and achetylcholinesterase inhibition assay of bloom samples and A. spiroides strains102




RESUMO

Na busca por métodos de análise de microcystinas de fácil execução,

baixo custo e sensibilidade alta o suficiente para atender as recomendações da

Organização Mundial da Saúde – OMS e da legislação Brasileira para água potável,

foi desenvolvida uma técnica de imunoensaio dot blot para determinação de

microcistina-LR em água e extratos de cianobactérias. Com esta técnica foi possível

a determinação visual direta de concentrações de microcystina-LR menores que 1

µg/L, em amostras de água purificada e água de superfície (lago) incriminadas com

concentrações na faixa de 0,16 a 10,0 µg/L de microcystina-LR. Nas condições

testadas não foi necessário nenhum processo de concentração ou limpeza das

amostras. Foi desenvolvido um programa de computador especificamente para a

leitura das membranas de nitrocelulose, permitindo uma melhor caracterização do

sinal com a obtenção de curvas analíticas similares às obtidas nas análises com

método de imunoensaio ELISA. Com a técnica dot blot computadorizada utilizando

um “scanner” convencional foram obtidas curvas de calibração com microcystina-LR

em concentrações de 0,16 a 1,6 µg/L, cujo coeficiente de correlação foi de 0,9551

para água purificada e 0,8402 em água de superfície. Quando analisadas pelo

método ELISA, coeficientes de correlação de 0,9713 e 0,8316 foram observados,

respectivamente. Quantificação em dot blot na faixa de 0,16 a 10,0 µg/L foi

realizada, porém com menor correlação entre a concentração da toxina e a

intensidade dos pixels. A análise computadorizada permitiu observar uma boa

correlação entre concentração e a intensidade dos pixels em extrato da cepa

toxigênica Microcystis aeruginosa - NPJB-1, produtora de microcistina. O programa

desenvolvido permitiu ainda a aplicação na quantificação de proteína de soro bovina

- BSA na faixa de 0 a 78 µg/mL (r = 0,9868). Esta reação foi corada usando solução

de amidoblack. Com o objetivo de obter um novo marcador fluorescente aplicável às

análises de microcystina-LR em baixas concentrações, foi sintetizado um complexo

de microcystina-LR-Criptato de térbio. A formação do complexo foi acompanhada

por cromatografia líquida de alta eficiência (CLAE) e o complexo formado analisado

quanto à atividade imune (ELISA), positividade de reação protéica e espectro de

luminescência, que confirmaram a conjugação. Na busca por novos métodos para

análise de microcistina foi monitorada a transição 5D0 à 7F2 de um complexo IgG -




antimicrocistina-LR-KLH-criptato de európio. Como suporte foi utilizado gel

superabsorvente de ácido acrílico / poliacrilamida. Os espectros de emissão da IgG

livre, do criptato de európio e do complexo IgG-Criptato de európio foram

monitorados. Os espectros de emissão na presença de IgG livre apresentaram uma

mudança específica na transição.




ABSTRACT

In the search for methods for microcystins analysis that are easy to use, low cost

and with sufficient high sensitivity to following the recommendations of the World Health

Organization –WHO and the Brazilian legislation, was developed a specific dot blot

assay. This dot blot imnunoassy is for visual determination of microcystin-LR in purified

and sufarce water (lake). Concentration as 0.16 up to 10.0 µg/l of microcystin-LR in

water were tested. Samples concentration or clean-up was not necessary for the water

analysis. A specific software for membranes reading dot was developed, allowing a

much better characterization of the dot with the attainment of similar analytical curves

obtaining troughout the analyses by enzyme linked imunoassay - ELISA. With the

computerized dot blot technique using a conventional scanner, coefficient of correlation

of 0.9551 to purified water and 0.8402 to surface water were observed in the range of

0.16 at 1.6 µg/l. For ELISA, the value of “r” were 0.9713 and 0.8316, respectively.

Quantification in the range of 0.16 at 10.0 µg/l was carried through, however with lesser

correlation between toxin concentration and intensity of pixels. The computerized

analysis allowed to observe a good correlation between concentration and intensity of

pixels using extract of toxic strain Microcystis aeruginosa - NPJB-1. This software was

used for the determination of the bovine serum albumin - BSA in the range of 0.0 at

78.0 µg/mL (r = 0.9868) when stained with amidoblack solution. In order to get a new

fluorescent labeled compound to the analysis of microcystin-LR in low concentrations, a

complex of microcystin-LR-Terbium criptate was synthecized. The complex formation

was followed by high performance liquid chromatography - HPLC and the ELISA,

proteic reaction and luminescence spectrum utilized to confirme the conjugation. In

order to lookinkg for new methods for microcystin analysis the formatoin of the

transistion 5D0 à 7F2 of a IgG complex antimicrocystin-LR-KLH- Europium cryptate was

monitored. Superabsorbent acrylic acid / polyacrilamyde gel was used as support. The

emission spectrum of free IgG, europium cryptate and IgG-Europium cryptate complex

had been monitored. The emission spectrum in the presence of IgG had presented a

specific change in the transistion.




1. INTRODUÇÃO

As cianobactérias são um dos mais primitivos grupos de organismos da terra,

com origem estimada de cerca de 3.500 milhões de anos (Roset et al., 2001). São

microrganismos procariotos fotossintetizantes capazes de sintetizar clorofila-a e de

produzir O2 como resultado da fotossíntese. Devido a presença do pigmento ficocianina

estes microrganismos são também denominados de algas azuis (Whitton & Potts,

2000). O termo cianobactéria deve-se ao fato destas algas estarem mais relacionadas

às bactérias procarióticas do que às algas eucarióticas (Lee, 1999).

As cianobactérias apresentam uma grande diversidade morfológica, variando de

unicelular (p.ex. Chroococcus) a filamentosas (p.ex. Anabaena), ocorrendo de forma

isolada ou agrupada em colônias (p.ex. Microcystis) (Whitton & Potts, 2000).

As células vegetativas das cianobactérias unicelulares variam de cerca de 0,4

µm até mais de 40 µm de diâmetro, enquanto as formas filamentosas da família

Oscillatoriaceae podem apresentar comprimentos de até 100 µm, ou mais (Whitton &

Potts, 2000).

A estrutura da parede celular das cianobactérias é basicamente a mesma das

bactérias Gram negativas e apresenta coloração típica de Gram. O maior constituinte

da parede é o peptídeoglicano, constituindo até 50% do peso seco da célula (Lee,

1999).

1.1 Cianotoxinas

As cianobactérias produzem uma variedade de metabólitos cuja função natural não

está bem esclarecida, mas são capazes de exercer ações tóxicas em seres humanos e

animais. Em alguns casos as toxinas são metabólitos secundários que são gerados

durante a formação dos pigmentos (Roset et al., 2001).




Quanto ao grupo químico as toxinas podem ser classificadas como peptídeos

cíclicos, alcalóides ou lipopolissacarídeos - LPS (Tabela 1). As biotoxinas (neurotoxinas

e hepatotoxinas) são capazes de levar os vertebrados à morte, enquanto que as

citotoxinas têm ação sobre grupos celulares ou tecidos, mas não são letais

(Carmichael, 1992).

Tabela 1. Classificação geral das cianotoxinas *

Biotoxinas Grupo Químico Congêneres Alvo Gênero(s Produtor(es)

Peptídeos cíclicos Microcistinas Nodularinas

> 60

>8

Fígado

Fígado

Microcystis, Anabaena, Planktothrix(Oscillatoria), Nostoc, Hapalasiphon, Anabaenopsis, Aphanocapsa. Nodularia.

Organofosforado Anatoxina-a(S) Alcalóides Anatoxina-a Cilindroespermopsina Saxitoxinas, toxinas GTX e neosaxitoxina.

1

1

1

Mínimo de 8

Sinapse nervosa

Sinapse nervosa

Fígado

Axônio nervoso

Anabaena Anabaena, Aphanizomenon, Planktothrix(Oscillatoria), Cylindrospermopsis, Aphanizomenon, Umezakia Anabaena, Planktothrix, Aphanizomenon, Lyngbya, Cylindrospermopsis.

Citotoxinas Grupo Químico Congêneres Alvo Gênero(s Produtor(es)

Alcalóides Aplisiatoxinas Lingbiatoxina-a

Pele

Pele, Trato gastrintestinal

Lyngbya, Schizothrix, Planktothrix(Oscillatoria) Lyngbya

Lipopolissacarídeos (LPS)

> 3

tecidos Todos




(* Baseada em Roset et al., 2001; Sivonen & Jones, 1999; Carmichael, 1992)

a. Neurotoxinas:

As neurotoxinas são alcalóides ou éster de guanidina metil-fosfato capazes de

bloquear a transmissão dos impulsos nervosos entre neurônio-neurônio e neurônio-

músculo, nos animais e no homem. Os sintomas de exposição incluem tontura,

fasciculações, dificuldade para respirar e convulsões. Podem ser fatais em altas

concentrações devido a paralisação do músculo diafragma. As neurotoxinas produzidas

são anatoxina-a, anatoxina-a(S). As toxinas paralisantes, do inglês Paralytic Shellfish

Poisons – PSP, são saxitoxinas e análogos (Lee, 1999; Carmichael, 1997).

b. Hepatotoxinas:

As toxinas peptídicas cíclicas da família das microcistinas e nodularinas

compõem o grupo das toxinas de cianobactérias mais freqüentemente encontradas em

florescimentos de águas doce e salobra. Uma outra toxina hepática conhecida é o

alcalóide cilindrospermopsina (Sivonen & Jones, 1999).

Os peptídeos cíclicos apresentam massa molecular de cerca de 800 a 1.100

Daltons. As microcistinas são heptapeptídeos monocíclicos produzidos por cepas dos

gêneros Microcystis, Anabaena, Planktotrix (Oscillatoria), Nostoc, Hapalasiphon,

Anabaenopsis e Aphanocapsa (Carmichael, 1992; Sivonen & Jones, 1999; Domingos

et al., 1999). Nestes compostos os dois aminoácidos terminais encontram-se

conjugados formando um composto cíclico (Figura 1). Estas toxinas apresentam dois

aminoácidos variáveis, três D-aminoácidos e os aminoácidos incomuns chamados de

N-metildehidroalanina e o ácido 3-amino-9-metoxi-10-fenil-2,6,8-trimetil-deca-4(E)6(E)-

dienóico (Adda). A estrutura geral é de um ciclo (D-ala-L-X-D-eritro-β-metil-isoAsp-Z-

Adda-D-isoGlu-N-metildehidroAla), onde X e Z são os aminoácidos variáveis. A

microcistina-LR apresenta os L-aminoácidos leucina e arginina , e é a toxina mais

hepatotóxica produzida por cepas de cianobactérias (Honkanen, 1990). Todas as

microcistinas contém Adda, estrutura essencial à atividade biológica (An & Carmichael,

1994).




De estrutura e ação semelhantes às microcistinas, as nodularinas são

pentapeptídeos monocíclicos (Maude, 1997), produzidos por Nodularia, uma

cianobactéria de águas salobras (Rinehart, 1988). As nodularinas diferenciam-se das

microcistinas pela ausência de um dos L e D aminoácidos, mas também apresenta o

aminoácido Adda (An & Carmichael, 1994). Tem sido relatado mais de oito tipos de

nodularinas, de acordo com o grau de metilação, composição e isomerização dos

aminoácidos (Roset et al., 2001).

Figura 1: Estrutura química das microcistinas e nodularinas; (A) Estrutura geral das microcistinas: ciclo-(D-Ala1-X2-D-MeAsp3-Z4-Adda5-D-Glu6-Mdha7); (B) Estrutura da nodularina: ciclo-(D-MeAsp1-Z2-Adda3-D-Glu4-Mdhb5) (Sivonen & Jones, 1999)

1.2. Toxicidade

As microcistinas e nodularinas são potentes inibidores das enzimas proteínas

fosfatases 1 e 2A (Craig, 1996; Yoshizawa, 1990).

As hepatotoxinas penetram nos hepatócitos via mecanismo dos receptores dos

ácidos biliares, provocando a inibição das fosfatases. Como conseqüência ocorre a

quebra do processo regulatório de fosforilação/desfosforilação, influenciando na

estrutura e função dos filamentos intermediários e microfilamentos. Ocorre então uma

excessiva fosforilação dos filamentos, ou das proteínas que atuam sobre eles,

aumentando a velocidade de perda das subunidades e da dissociação celular. O

resultado final é um encolhimento das células dos hepatócitos que provoca uma

separação das células intimamente ligadas. Com a separação dos hepatócitos as

células que formam o capilar sinusoidal também se separam permitindo o

extravasamento do sangue dos vasos para o tecido hepático (Figura 2). O dano




provocado ao tecido leva à formação de edema (hepatomegalia), com conseqüente

estado de choque hemodinâmico (Carmichael, 1994), falha cardíaca e morte (Kuipe-

Goodman et al., 1999).

As microcistinas também são promotoras de tumor (Nishiwaki-Matsushima et al.,

1992); Juntamente com as nodularinas, são suspeitas de provocarem câncer primário

de fígado em humanos expostos ao consumo de água contaminada com baixas

concentrações por longos períodos (Carmichael, 1994; Ueno et al, 1996).

Algumas formas de microcistinas não são tóxicas. Quando a estrutura apresenta

o aminoácido Adda com a dupla ligação do carbono 6 na forma isomérica (Z), ao invés

da forma (E), não é capaz de inibir a enzima fosfatase 1 (PP1 ) (An & Carmichael,

1994).

Figura 2: Esquema do mecanismo de extravasamento sangüíneo hepático provocado por microcistinas e nodularinas. (A) Fígado normal; (B) Fígado após ação das toxinas (Traduzido de Carmichael, 1994).

A cilindrospermopsina atua inibindo a síntese protéica, provocando necrose

hepática, renal e em outros órgãos (Chorus & Bartram, 1999).

1.3 Ocorrência

As cianobactérias são capazes de crescer em todos os ecossistemas,

destacando-se as águas doces e os ambientes marinhos. A afinidade das

cianobactérias por nitrogênio e fósforo é muito maior que a de muitos organismos

fotossintéticos (Roset et al., 2001), e este é um meio de se sobressair sobre outros

A

Capilar Sinusoidal

Hepatócitos

Duto Biliar

B

Capilar Danificado

Extravazamento de sangue para o tecido hepático




organismos fitoplanctônicos em condições limitantes de fósforo ou nitrogênio (MUR et

al., 1999). No entanto, estas bactérias também são capazes de florescer (“Blooms”)

tanto em ambientes eutrofizados como oligotróficos (Roset et al., 2001).

Florações de cianobactérias tóxicas têm sido observadas em várias partes do

globo (Rapala, 1997; Sedmak & Kosi, 1997; Tsuji et al., 1996; Mcdermott, 1995; Costa

& Azevedo, 1994; Zhang et al, 1991; Chu et al., 1989;) e vem se tornando comum em

fontes de águas doces e estuários (Molica et al., 2002; Bouvy et al., 1999; Porfírio et

al., 1999; Tsuji et al., 1997; Yunes et al., 1996). Estima-se que mais de 50% das

florações de cianobactérias são tóxicas (Azevedo et al., 1994).

As toxinas de cianobactérias são produzidas no interior das células e ficam

retidas até que ocorra a lise celular. Esta característica, associada à estabilidade

química e boa solubilidade em água, fazem das cianotoxinas um importante grupo

químico de persistência ambiental, com risco considerável de intoxicação para animais

e seres humanos. A ação tóxica no homem tem efeitos graves, que pode culminar com

a morte.

Os primeiros casos de intoxicação de populações humanas pelo consumo de

água contaminada com cianobactérias tóxicas foram descritos na Austrália, Inglaterra,

China e África do Sul (Roset et al., 2001).

Azevedo et al. (1994) foram os pioneiros a relatar a presença da cepa de

Microcystis aeruginosa produtora de microcistina em mananciais brasileiros. A amostra

isolada foi nomeada de NPJB-1 e foi obtida da Lagoa das Garças - Jardim Botânico da

cidade de São Paulo. Em cultivo, a cultura produziu as microcistinas -LF e -LR, com

predominância desta última. A DL100 de uma suspensão liofilizada foi de

aproximadamente 31 mg/Kg de massa corporal de camundongos.

Em 1996 ocorreu uma intoxicação humana grave na cidade de Caruaru-PE,

Região Agreste do Estado de Pernambuco, devido ao uso de água contaminada com

cianotoxinas no tratamento de pacientes em hemodiálise. A clínica era abastecida pelo

reservatório de Tabocas e por carros pipas. Cento e vinte e seis pacientes renais em




tratamento apresentaram sintomas de neurotoxicidade aguda e hepatotoxicidade

subaguda. Destes, 51 faleceram num intervalo de cerca de dois meses após a primeira

morte relatada, culminando com um total de 76 mortes até agosto de 1997. O

tratamento inadequado da água (Jochimsen, 1998), associado à falta de

monitoramento dos níveis de toxinas permitiram que o acidente ocorresse. Foi

constatado através das análises de amostras de sangue, fígado e dos componentes

dos equipamentos de tratamento de água, que a intoxicação deveu-se à presença de

microcistina-LR, -YR, -AR e de cilindrospermopsina, sendo esta última encontrada

apenas em amostras do carvão. Os níveis de microcistinas foram estimados em 19,5

µg/L (Carmichael et al., 2001).

Análises da água do reservatório de Tabocas, realizadas após o acidente,

revelaram a presença de cianobactérias toxigênicas em uma proporção de 99% da

densidade do fitoplâncton, com um número médio de 24.500 células/mL (Carmichael et

al, 2001). Cianobactérias pictoplanctônicas produtoras de microcistinas (gênero

Aphanocapsa) também foram isoladas do mesmo reservatório (Domingos et al, 1999),

o que demonstra a potencialidade de florescimentos tóxicos e a necessidade de

constante monitoramento.

Há vários outros casos suspeitos de intoxicação de seres humanos pela ingestão

hídrica e de alimentos contendo cianotoxinas, mas cuja presença química dos agentes

não puderam ser confirmadas (Carmichael et al, 2001).

Um grande caso de intoxicação humana, anterior ao acidente de Caruaru,

aconteceu na região de Paulo Afonso – BA. O consumo de água do reservatório de

Itaparica levou à morte 88 pessoas, de um total de 2.000 com sintomas de gastrenterite

(Teixeira, et al, 1993). O caso foi atribuído à presença de toxinas, o que não foi

comprovado, embora na água do reservatório tenha sido verificada a presença de

células de cianobactérias.

Há diversos outros relatos de florescimentos de cianobactérias tóxicas em águas

Brasileiras, como descrito a seguir:




Yunes et al. (1996) citaram vários florescimentos de Microcystis aeruginosa na

lagoa dos patos, Estado do Rio Grande do Sul. Destas florações foram isoladas cepas

tóxicas com DL50 de 22 a 250 mg de peso seco/Kg de massa corporal de

camundongos.

Porfírio et al. (1999) encontraram Microcystis aeruginosa em 95% da biomassa

de amostras de fitoplâncton da lagoa Manguaba, no Estado de Alagoas. A DL50

observada foi de 154,28 mg/Kg de massa corporal de camundongos.

Nhoato et al. (2001) analisaram amostras de água da barragem de Jurumirim, na

cidade de Itaí – SP, durante florescimentos de cianobactérias. Foram encontradas

concentrações de microcistinas de 0,58 ng/mg e 5,02 mg/g de biomassa, em maio de

1988 e janeiro de 1999, respectivamente.

A importância de um constante monitoramento da qualidade da água está no

fato de que um grande número de florescimentos de cianobactérias, 50% na média

mundial, deve-se a cepas de espécies tóxicas, ainda que uma mesma espécie de

cianobatéria apresente diferentes toxicidades (Rivasseau, 1998).

1.4 Legislação Brasileira

Os padrões de qualidade da água para consumo humano, hemodiálise,

produção de medicamentos, e outros usos variam em cada país, mesmo com os

esforços da Organização Mundial da Saúde para uma padronização mínima da

qualidade da água potável. Com relação ao controle de cianobactérias e/ou suas

toxinas, mesmo países considerados desenvolvidos não possuem normas específicas,

a exemplo da Espanha, membro da Comunidade Européia (Roset et al., 2001).

Atenção especial deve ser dispensada às águas utilizadas na preparação de

medicamentos injetáveis e para tratamento dialítico, cujo contato com o indivíduo é

realizado de forma direta na corrente sangüínea, sem passagem pelas barreiras

naturais do organismo humano.




No Brasil houve um avanço considerável no monitoramento da qualidade da

água potável. A publicação da Portaria nº. 1469/2000 do Ministério da Saúde - MS

estabelece as “Normas de Qualidade da Água para Consumo Humano”. Seguindo a

orientação da Organização Mundial de Saúde (WHO, 1998), a portaria estabelece o

valor provisório de 1 µg/L de microcistinas, considerando a ingestão de 2 litros de água

/ dia por uma pessoal adulta de massa corporal média de 60 Kg. Também recomenda

a análise de cilindroespermopsina e de saxitoxinas, cujo limite máximo é de 15 µg/L e 3

µg de equivalentes de saxitoxina /L, respectivamente (Brasil, 2000).

1.5 Métodos de Análise de Microcistinas

A quantificação e a identificação de cianotoxinas em água pode ser realizada por

diversos métodos. Estes podem variar no grau de sofisticação e de informação gerada,

bem como na seletividade, sensibilidade, custo e qualificação técnica necessária à sua

realização.

Os métodos podem ser classificados como Físico-Químicos, Biológicos e Mistos.

O monitoramento do número de cianobactérias em água é considerado a ação

primária para avaliação de florações potencialmente tóxicas. O exame microscópico é

usado na identificação e contagem do número de células. É uma ferramenta para a

tomada de decisão para o bioensaio em camundongos, ou outra análise de

identificação e quantificação de toxinas que utiliza técnicas imunológicas, enzimáticas

ou físico-químicas (Harada et al., 1999). A identificação microscópica é um método que

pode ser lento e necessita de especialista experiente em classificação planctônica.

a. Métodos Físico-Químicos a.1 Cromatografia Líquida de Alta Eficiência (CLAE ou HPLC)

A cromatografia líquida de alta eficiência (Lawton et al., 1994) é um dos métodos

mais usados atualmente para a análise de microcistinas. O sistema utiliza detector de




varredura de diodos (Diode array) que torna a análise mais específica. O método tem

limitações para a identificação das variantes devido à dificuldade de resolução de

muitos picos congêneres e do número reduzido de padrões. A necessidade de

concentração e limpeza (clean-up) das amostras e a utilização de método em gradiente

aumentam a complexidade do ensaio. A CLAE não é um método específico e a

atividade biológica para determinação da toxicidade deve ser confirmada por bioensaio.

a.2 Cromatografia em Camada Delgada (CCD ou TLC)

Detecção visual de microcistinas por cromatografia em camada delgada após

derivativação com fenilenodiamonium (N,N-DPDD) foi proposto por Pelander et al.,

2000. Porém, para detecção em água é necessário a limpeza e concentração em fase

sólida, como usado para HPLC. O método descrito exige uso de aplicador automático

para garantir o limite de detecção apresentado. a.3 Espectrometria de Massa com Ionização Assistida por Laser (MS-MALDI-TOF)

O método de espectrometria de massa com tratamento da amostra por ionização

da matriz assistida por laser (Matrix Assisted Laser Desorption Ionization Time of Flight)

permite a identificação de microcistinas em pequenos volumes de amostra, com

determinação da massa molecular de todos os peptídeos. Fornece indicações das

variantes de microcistinas presentes na amostra. Tem aplicação qualitativa. O custo é

elevado e necessita de pessoal muito qualificado (Chorus et al., 1999).

a.4 Cromatografia Líquida com Espectrometria de Massa (LC/MS)

A associação da cromatografia líquida à espectrometria de massa é uma

metodologia mais avançada que permite a separação e confirmação da identidade das

microcistinas (Chorus et al., 1999). É um método de alto custo de investimento e que

necessita de pessoal muito qualificado para a interpretação dos espectros.




Uma variação do método LC/MS utiliza a ionização por bombardeamento rápido

de átomos (Fast atom bombardment ionization - Frit-FAB) e tem aplicação para a

determinação da fórmula e massa molecular de microcistinas purificadas. Este método

tem baixa sensibilidade, requerendo microgramas da toxina para a identificação

(Chorus et al., 1999).

a.5 Cromatografia Gasosa e Líquida por Derivatização (MMPB/GC/LC)

A obtenção do derivado oxidado de microcistinas ácido 2-metil-3-metoxi-4-

fenilbutirico - MMPB, seguido da detecção por cromatografia gasosa ou líquida com

detector de fluorescência, foi sugerida como um método de triagem. Este é um ensaio

longo, em que a amostra tem que ser concentrada, oxidada e limpa para a eliminação

dos reagentes usados (Chorus et al., 1999). a.6 Ressonância Magnética Nuclear (RMN)

Método dos mais caros e cujos resultados são de difícil interpretação, a RMN

permite a identificação estrutural das microcistinas. É necessário grande quantidade de

amostra com alta pureza (miligramas). Não se aplica como método de rotina e tem

elevado custo de implantação e manutenção (Chorus et al, 1999).

Resguardadas as diferenças econômicas entre os vários países e regiões, é

apresentada abaixo uma síntese dos diversos métodos físico-químicos para detecção

de microcistinas (Tabela 2).




Tabela 2. Resumo dos Métodos Físico-Químicos para Detecção de Microcistinas

Método Custo Comentários Referências

TLC Médio Poucos padrões, limpeza da amostra, aplicador automático, Falsos positivos, uso de solventes orgânicos.

Harada et al., 1996.

Pelander et al., 2000. HPLC-PDA Alto Poucos padrões, limpeza da amostra,

equipamentos caros, uso de solventes orgânicos.

Lawton et al., 1994.

LC/MS Muito Alto Pessoal muito qualificado (Especialista na interpretação)

Kondo et al., 2000.

Edwards et al., 1993.

MS- MALDI-

TOF

Muito Alto Qualitativo; Pessoal muito qualificado. (Especialista na interpretação)

Erhardt et al., 1997.

MMPB/GC/LC

Alto Detecta microcistina total; Método longo com oxidação e limpeza da amostra.

Sano et al., 1992.


NMR Muito Alto Pode caracterizar; Necessita mg da amostra; Especialista na interpretação.


TLC = Cromatografía em camada delgada; HPLC-PDA = Cromatografia líquida de alta eficiência com detector de varredura de diodo; LC-MS = Cromatografia líquida/Espectrometria de massa; MALDI = Ionização de matriz assistida por dessorção de laser; NMR = Ressonância Magnética Nuclear; MMPB = Ácido 2-metil-3-metoxi-4-fenilbutírico

b. Métodos Biológicos b.1 Bioensaio em Camundongos/Ratos

O bioensaio em camundongos ou ratos fornece uma medida total da toxicidade,

mas geralmente é pouco sensível ou específico. (Chorus et al., 1999). Para a análise

de rotina é necessário infra-estrutura de biotério, e tem como uma das principais

desvantagens o sacrifício constante de animais

b.2 Ensaio de Inibição da Fosfatase

O método bioquímico mais estudado é o da inibição das enzimas fosfatases tipo

1 e 2A (PP1 e PP2A), que resulta em menor conversão do p-nitrofenilfosfato à sua

forma cromófora (An & Carmichael, 1994; Rivasseau et al., 1999).




É um método muito sensível, detecta nanogramas de microcistinas, e tem

mostrado correlação linear (r = 0,7400) com o método de HPLC, quando utilizado a

PP2A (Harada et al., 1999).

Uma variação do método enzimático colorimétrico é a medida de fluorescência

gerada pelo 4-metilumbeleferilfosfato – MUP após metabolização, pelas fosfatases, do

6,8-difluoro-4-metilumbeleferil fosfato – DIFMUP. Esta modificação do método

colorimétrico resultou em um método muito sensível, com detecção de quantidades

muito baixas de microcistina tal como 0,08 pg/poço, correspondente a 0,0004 µg/L

(Fontal et al., 1999), enquanto o método colorimétrico apresenta faixas de 0,1 a 0,4

µg/L (Rivasseau et al., 1999).

As desvantagens desta metodologia são a inibição das fosfatases por outros

compostos ativos, e efeitos de matriz que leva a falsos positivos ou a uma super

estimação da concentração de toxinas. Este último efeito é reduzido com uma limpeza

prévia da amostra (Rivasseau et al., 1999).

b.3 Imunoensaios

Os ensaios baseados no uso de anticorpos podem ser utilizados tanto para

triagem como para análise quantitativa de cianotoxinas em amostras de água.

O ensaio com enzima ligada a imunoadsorvente - ELISA (Enzyme-Linked

Immunosorbent Assay), é uma das técnicas de triagem e quantificação de

microcistinas cuja rapidez, sensibilidade e especificidade tem justificado seu largo uso

(Harada et al., 1999). Diversos anticorpos anti-microcistinas e técnicas ELISA vêm

sendo desenvolvidos desde a década de 80 e alguns destes encontram-se listados na

Tabela 3.




Tabela 3. Relação de anticorpos anti-microcistinas e técnicas ELISA Tipo de Anticorpo Autores Técnica ELISA

Monoclonal anti-Microcistina-LA Kfir et al., 1986 ----

Policlonal de coelho anti-Microcistina-LR-BSA Chu et al., 1989 Chu et al., 1989

Chu et al., 1990

Monoclonal anti-Microcistina-LR Nagata et al., 1995 Ueno et al., 1996

Monoclonal anti-Imunocomplexo Microcistina-

Anticorpo Monoclonal anti-Microcistina

Nagata et al., 1999 Nagata et al., 1999

Policlonal de coelho anti-Microcistina-LR-KLH Metcalf et al., 2000 Metcalf et al., 2000

Policlonal de coelho e camundongo anti –

Microcistina – LR/RR e Nodularina

Baier et al., 2000 Baier et al., 2000

Policlonal de coelho anti-microcistina-LR-KLH Karnikowski, 2000. Karnikowiski , 2001

O ensaio ELISA é referido como fácil e de baixo custo, porém os “kits”

disponíveis no mercado não são fabricados no Brasil e o valor de uma microplaca com

96 poços é de cerca de US$ 400 (Envirologix®, 2003), não incluídos os custos

operacionais e de pessoal.

A técnica dot-blot é uma variação do ELISA que utiliza membranas de nylon,

nitrocelulose, ou outra adequada, como suporte para o imunógeno. O ensaio envolve

diversas etapas como bloqueio e lavagens até a fase final, onde o reagente marcado

com a enzima é utilizado (Tijssen, 1985).

Entre as variações de dot-blot possíveis há o ensaio indireto, onde o antígeno

imobilizado na fase sólida captura o anticorpo da amostra teste, o qual é revelado pela

enzima do segundo anticorpo marcado. Este método tem uma sensibilidade de cerca

de dez vezes maior que o método direto (Tijssen, 1985).

Não tem sido relatada a descrição de método dot-blot para a análise de

microcistina-LR.

Os métodos imunosenssores ainda são considerados promissores para a

detecção de microcistinas em água, o que subsidia o desenvolvimento de




imunobiossenssores, uma alternativa para o monitoramento ambiental da presença de

microcistinas em água.

C. Método Misto C.1 Eletroforese Capilar de Alta Eficiência (HPCE)

Consiste de um sistema de eletroforese capilar equipado com detector de

varredura de diodo na região U.V.-Visível. Neste método as microcistinas são extraídas

e purificadas em cartuchos de imunoafinidade contendo anticorpo policlonal para

microcistina-LR (Aguete et al., 2003).

1.6 Complexos de Lantanídeos Fluorescentes: Criptatos de Eu3+ e Tb3+

Os elementos Európio (Eu) e térbio (Tb) pertencem ao grupo dos metais

chamados de terras-raras (número atômico de 58 a 71) e caracterizam-se por

apresentar maior estabilidade quando os orbitais 4f estão preenchidos pela metade (7

elétrons) (Gameiro, 1998).

O íon Eu3+ tem configuração eletrônica [Xe] 4f7 6s2 e íon Tb3+ [Xe] 4f9 6s2 ; Os

elétrons 4f estão internalizados, protegidos da interação com o meio devido a

blindagem provocada pelos elétrons “5s” e “5p” externos. Assim, os elétrons dos níveis

“4f” são apenas ligeiramente afetados pelo campo ligante (Alves, 1998).

A luminescência dos íons lantanídeos está relacionada a características

peculiares, tais como o longo período e as linhas finas de emissão. Estas

características devem-se ao fato que, tanto no estado excitado de emissão como no

estado fundamental, a configuração eletrônica “f n” é a mesma, além do fato acima

citado de que os orbitais “f” estão protegidos do ambiente pelos elétrons externos dos

níveis “s” e “p”. As transições entre os estados de configuração “f n “ são consideradas

proibidas (Sabbatini et al, 1993), mas ocorrem normalmente em virtude da mistura de

estados de diferentes paridades nos estados “4f”; No entanto estas transições são de

baixa intensidade (ε ≈ 1) (Alves, 1998).




Os íons lantanídeos, isoladamente, apresentam um coeficiente de absorção

muito pequeno nas regiões U.V. e visível do espectro (Sabbatine et al., 1993).

Também não apresentam grande habilidade para coordenação devido a sua

configuração eletrônica. Assim, ligantes convencionais não são capazes de gerar

complexos inertes, especialmente em soluções aquosas onde as moléculas do

solvente competem eficientemente para ocupar os sítios de coordenação (Sabbatine et

al., 1993).

Estas deficiências, no entanto, podem ser superadas pelo planejamento e

construção de complexos de lantanídeos estáveis, capazes de desempenhar o papel

de um Dispositivo Molecular para Absorção Eficiente de Luz e Emissão de

Luminescência - “Light Conversion Molecular Devices” – LCMDs (Sabbatine et al.,

1993).

Entre os complexos, os criptatos caracterizam-se por serem uma classe de

compostos de íons lantanídeos cujos ligantes possuem cavidade esferoidal e sítios

ligantes de natureza “rígida”. Possuem alta estabilidade e capacidade para proteger o

íon encapsulado das interações com o meio circundante (Sabbatine et al., 1993). Os

complexos do tipo criptato são termodinamicamente mais estáveis quando o tamanho

do íon é comparável às dimensões da cavidade do complexo (Lehn & Sauvage, 1975).

A capacidade de blindagem dos criptatos é muito maior que a dos complexos

macrociclicos que, devido a uma incompleta coordenação com o íon, deixa sítios

disponíveis para a ação de agentes supressores de luminescência que se aproximam

do cátion (Flipsen, 2000).

Nos criptatos de lantanídeos uma intensa luminescência do íon pode ser obtida

pelo efeito “antena” dos ligantes. Este efeito é definido como um processo de

conversão de luz via uma seqüência de absorção de energia, transferência e emissão

,que envolve os componentes absorvedores (ligantes) e emissor (ion metálico). O

estado excitado 5D0 é o estado luminescente envolvido na transferência de energia do

ligante para o metal (Figura 3). Neste processo contribuem para a intensidade de

luminescência (Sabbatine et al., 1993):




a) A intensidade de absorção pelo ligante

b) A eficiência de transferência de energia do ligante para o metal.

c) A eficiência luminescente do metal.

Figura 3: Esquema do processo de desativação envolvido na conversão de luz absorvida pelos ligantes do complexo e emitido pelo Eu3+ (Setas azuis = Processos não radioativos; Setas vermelhas = Processos radiativos)

Compostos do tipo piridina-Eu3+ exibem forte fluorescência quando irradiado por

luz U.V. A intensidade desta fluorescência indica que a transferência de energia do

ligante excitado para o íon metálico está ocorrendo. Uma outra característica

importante em todos os complexos de Eu3+ é que o comprimento de onda máximo de

excitação ocorre em região diferente do espectro, não havendo sobreposição entre os

picos de absorção e de emissão (Figura 4) (Flipsen, 2000).

Ligante Tb3+

hv

3 ππ*

1 ππ*

5D2

5D1

5D0

Estado Fundamental

Singleto

Tripleto

Absorção pelo Ligantes

Estado Emissor do Metal




Figura 4: Espectro de excitação (..........) e de emissão (____) de um criptato de Eu3+ em solução (Flipsen, 2000).

O aumento do tempo de luminescência do criptato em solução aquosa deve-se,

em parte, a um menor acoplamento do metal com radicais –OH de alta energia

vibracional, que ocorre pelo fato do íon Eu3+ estar fortemente protegido das interações

com as moléculas do solvente (Sabbatine et al., 1993).

Criptatos de bipiridinas caracterizam-se por apresentar como “antenas” unidades

de 2,2’-bipiridina, que leva à formação de um complexo altamente absorvente que

aumenta a intensidade de luminescência do lantanídeo. O aumento da absorção no

U.V. deve-se as transições π → π* nas unidades de bipiridinas (Sabbatine et al., 1993).

Os criptatos podem ser do tipo tris-bipiridina (Bip.bip.bip) (Prat et al., 1991;

Mathis, 1993; Lopez et al., 1993; Alpha-Bazin, 2000) ou podem apresentar outros

arranjos, como por exemplo, o Bip.bip.pi(CO2Et)2, sintetizado pelo grupo do

Departamento de Química Fundamental –DQF, da UFPE (Figura 5).




Figura 5: Criptato de bis-piridina-piridina [Bip.bip.pi(CO2Et)2]3+ lantanídeo sintetizado no DQF (Departamento de Química Fundamental da UFPE)

Os criptatos de lantanídeos têm sido utilizados como marcadores luminescentes

para fluorimetria de tempo resolvido – TRF (Time-Resolved Fluorometry). Esta

metodologia baseia-se no uso de composto com tempo de decaimento longo. Quando

excitado com pulsos de luz U.V., em sua faixa de absorção, o composto passa a emitir

fótons fluorescentes de longa duração, porém em outro comprimento de onda diferente

do de absorção. Os fótons emitidos são então medidos com uma contadora de fótons

após um período de tempo adequado para o decaimento da fluorescência de curta

duração (“background”) das espécies interferentes (Prat et al., 1991). No método

TRACE (Time Resolved Amplification of Cryptate Emission) utiliza-se o criptato de

európio ligado a um anticorpo, e um segundo anticorpo ligado a uma ficobiliproteína.

Quando ocorre a interação entre os anticorpos e o antígeno o criptato transfere energia

para a ficobiliproteína que passa a emitir fluorescência em comprimento de onda

diferente.

O uso de criptato de lantanídeo como sonda de marcação em ensaios biológicos

tem a vantagem de não necessitar de nenhuma etapa adicional de revelação. Assim,

uma medida direta da fluorescência no meio, ou do material retido em suporte sólido,

pode ser facilmente realizada após o contato com a molécula marcada (Prat et al. ,

1991). Um método para detecção de DNA em suporte sólido utilizando criptato de

európio foi desenvolvido por Prat et al.(1991). A vantagem sobre os métodos

3CF3COO-

N N

NN NN

O

O

O

O

N

Tb+3




convencionais do tipo DNA ligado à enzima, complexo biotina-avidina ou

imunocomplexos, é o da obtenção de um ensaio quantitativo, além da superação das

dificuldades dos métodos colorimétricos onde a cor resultante da ação enzimática é

fraca.

Os criptatos de lantanídeos como ferramenta para fluoroimunensaios já era

preconizado por Sabbatine et al. (1993) devido as características de estabilidade,

solubilidade em água e forte luminescência destes compostos. Os criptatos de tris-

bipiridina - Eu3+ têm sido utilizados no desenvolvimento de fluoroimunoensaios (Prat et

al., 1991; Mathis, 1993; Lopez et al., 1993; Alpha-Bazin, 2000).

1.7 Matrizes Poliméricas como Suporte para Complexos de Lantanídeos Fluorescentes.

Polímeros com densas ligações cruzadas são matrizes hospedeiras para

complexos lantanídeos fluorescentes. Devido ao alto grau de ligações, a mobilidade

dos compostos na malha é reduzida e a supressão da fluorescência é diminuída devido

a uma minimização das colisões das cadeias com os complexos de terra rara. Quando

o complexo já está bem protegido, como nos criptatos, a influência das colisões com as

cadeias poliméricas é menos importante, mas continua sendo altamente favorável para

a estabilidade do complexo, principalmente para a estabilidade térmica a altas

temperaturas (Flipsen, 2000).

O uso de complexos macrociclicos ancorados numa rede polimérica nem

sempre é quimicamente possível. Flipsen (2000), na tentativa para obter fibra óptica

amplificadora, testou algumas matrizes como suporte para o complexo de európio

[Eu(hfa)3(TOPO)2] – tris(1,5-hexafluoro-2,4-pentanodionato)bis (óxido de trioctilfosfina).

O polímero hexametilenodiisocianato poliisocianurato apresentou instbilidade para o

complexo devido a uma reação do catalisador de polimerização com o complexo; Com

um polímero de polifenilmetilvinilhidro-siloxano a cura foi realizada a 150 oC, o que

levou a uma desestabilização do complexo.

Para a imobilização de uma espécie biológica em sua forma ativa, é necessário

que as reações envolvidas na etapa de formação do suporte sejam brandas. Com isto

evita-se a inativação do composto pela exposição a temperaturas elevadas ou a




condições de pH extremos. Géis superabsorventes apresentam a capacidade de

absorver grandes quantidades de água ou soluções iônicas de baixa osmolaridade.

Esta característica possibilita a sua utilização como suporte para substâncias biológicas

ativas. A rede polimérica dos géis absorventes tem a vantagem da biocompatibilidade,

sendo compatíveis com diversas aplicações especializadas farmacêuticas e biológicas

tais como: A preparação de formas farmacêuticas de liberação controlada de drogas, a

fabricação de cateteres e de pele artificial, a construção de biossenssores, além de

várias outras aplicações comerciais, como o uso em absorventes de higiene pessoal

como fraldas infantis e produtos de higiene feminina (Buchholz & Peppas, 1994).

Os géis absorventes são normalmente obtidos pela polimerização de

monômeros de acrilatos com pequenas quantidades de agentes de ligação cruzada

que apresentam duas ou mais ligações duplas polimerizáveis, a exemplo do N,N’-

metilenobisacrilamida, trialilamina, etilenoglicoldiacrilato, etc. (Buchholz & Peppas,

1994; Garner et al., 1997). Hidrogéis com elevada capacidade de intumescimento

podem ser produzidos pela polimerização de monômeros como o ácido acrílico e

metacrílico, ou seus sais de sódio. Nestes géis o número de ligações cruzadas é muito

pequeno, mas o suficiente para tornar o material insolúvel em água; As regiões internas

do gel com elevada força iônica explica a capacidade de alta absorção de água (Garner

et al., 1997).




2. OBJETIVOS

• Desenvolver técnica dot-blot para a determinação de microcistina-LR em

amostras de água purificada e de superfície.

• Produzir marcador fluorescente de microcistina-LR-criptato de térbio para

aplicações em métodos de fluorescência.

• Construção de gel imunossensor em matriz de poliacrilamida / ácido acrílico

utilizando criptato de európio para detecção de microcistina-LR.




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UENO, Y., NAGATA, S., TSUTSUMI, T., HASEGAWA, A., YOSHIDA, F., SUTTAJIT, M., MEBS, D., PÜTSCH, M., and VASCONCELOS, V. Survei of microcystins in environmental water by a highly sensitive immunoassay based on monoclonal antibody. Natural Toxins, v.4, p. 271-276, 1996. WHITTON, Brian A.; POTTS, Malcolm (Eds). The ocology of Cyanobacteria – Their Diversity in Time and space. London: Kluwer Acad. Pub., 2000.

WHO, 1998 Guidelines for Drinking-water Quality In: CHORUS, Ingrid Jamie. Toxic Cyanobateria in Water – A guide to their public health consequences, and management. London: E & FN Spon, /WHO, 1999. YOSHIZAWA, S.; MATSUSHIMA, R.; WATANABE, M.F.; HARADA, K-L. et al. J. Canc. Res. Clin. Oncol., v. 116, 1990. YUNES, J.S.; SALOMON, P.S.; MATTHIENSEN, A.; BEATTIE, K.A.; RAGGETT, S.L. CODD, G.A. Toxic blooms of cyanobacteria in the patos lagoon etuary, southern Brazil. J. Aquat. Ecosyst. Health, v.5, p.223-229, 1996.

ZHANG, Q.Z.; CARMICHAEL, W.W.; YU, M.J. Cyclic peptide hepatotoxins from freshwater cyanobacterial (blue-green algae) waterbloons collected in central China. Environ. Toxicol. Chem., v.10, p.313-321, 1991.




CAPÍTULO 1: Application of Dot Blot Immunoassay with computerised scanning to analysis of microcystin-LR

Eduardo José Alécio-Oliveira1*, Elizabet Malageño2, Antonio Teixeira3, Carla Nunes3,

Manoel Eusébio4, Pablo Viana4, Leonardo Tavares do Nascimento4, Hatus Vianna4,

Renato J.R. Molica5, José Luiz de Lima Filho2.

1Centro Federal de Educação Tecnológica de Pernambuco (CEFET-PE), 50740-540,

Recife-PE, Brazil. 2Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de

Pernambuco (UFPE), Recife-PE, Brazil. 3Laboratório Multidisciplinar de Pesquisa em Doença de Chagas da Universidade de

Brasília (UnB), Brazil. 4Departamento de Informática, Universidade Federal de Pernambuco (UFPE), Recife-

PE, Brazil. 5Instituto de Tecnológico do Estado de Pernambuco (ITEP), Recife-PE, Brazil.

*Corresponding author: Centro Federal de Educação Tecnológica de Pernambuco

(CEFET-PE), Av. Profº Luiz Freire, 500, Cidade Universitária, Recife-PE, 50740-540,

Brazil. e.mail: [email protected]

KEYWORDS: Dot blot, Cyanobacteria, Microcystin-LR, ELISA, Immunoassay, Image

Processing.





ABSTRACT The aim of this work was to use a dot blot immunoassay by visual inspection and

by not expensive computerized scanning software to screening microcystins-LR

cyanobacaterial toxin in surface or purified water and cell cyanobacteria extract.

Ornamental lake water from Centro Federal de Educação Tecnológica de Pernambuco

– CEFET-PE, City of Recife, Brazil and the celular extract of microcystins producer

strain Microcystis aeruginosa - NPJB-1, were investigated. Presence of microcystin-LR

was visually and quantitatively detected in the level of WHO guidelines (1 ppb) in

purified and surface water as well as in the cells extract. The methodology described for

quantitative analysis of microcystin-LR in water, reached similar results to them

obtained by the enzyme-linked immunosorbent assay - ELISA. To our knowledge this is

the first report describing a visual or computerized scanning dot blot for analysis of

microcystin-LR presence in water and in cyanobaterial cellular extract.




INTRODUCTION

The eutrophication of ponds, rivers and others freshwater ecosystem is favorable

to form cyanobacteria (blue-green algae) blooms with potential contamination of water

(Pouria et al., 1998; Kim et al., 2003). Water blooms of cyanobacteria are increasing the

risk of toxic event because many strains can be present in surface water as species of

genera Microcystis, Anabaena, Aphanizomenon, Planktotrix (Oscillatoria), and Nostoc,

frequently described as producers of microcystins (Watanabe et al., 1997; Carmichael,

1994). Cyanobacterial blooms are very frequent events in drinking water supplies in

Brazil (Yunes et al., 1996; Bouvy et al., 2000) and the principal cyanotoxins in the

Brazilian waterbodies are microcystins (Azevedo et al., 2002; Magalhães et al., 2001;

Hirooka et al., 1999; Yunes et al., 1996; Azevedo et al., 1994). The risk of utilization of

contaminated water is also exemplified by the case occurred in the city of Caruaru,

Northeast of the Brazil, when humans in treatment in a haemodialysis clinic received

water contaminated with microcystin with death of 76 patients (Jochimsen et al. 1998;

Carmichael et al. 2001; Azevedo et al., 2002). In consequence of this accident the

Brazilian government established the limit of 1 µg/l for microcystins in the drinking water

(Brasil, 2000).

The contaminated water is also hazardous to animals and the low levels in the

environmental and drink water must be monitored. Microcystins are potent inhibitors of

protein phosphatases 1 (PP1 ) and 2A (PP2A ) which are regulatory enzymes present in

the cytosol of the mammalian cells (Fontal et al.., 1999). More than 60 diferent analogue

of microcystins were described (Mehto et al., 2001) and the principal structure is

composed of cyclo-(D-Ala1-X2-D-MeAsp3-Z4-Adda5-D-Glu6-Mdha7) in which X and Z are

variable L-amino acids, D-MeAsp is D-erythro-β-methylaspartic acid, Mdha is N-

methyldehydroalanine, and Adda is an unusual amino acid 2S,3S,8S,9S-3-amino-9-

methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid (Carmichael et al., 1988).

The microcystins are also a tumor promote (Nishiwaki-Matsushima et al., 1992) and

suspicion of cause primary cancer of liver in human that they consume contamined low

concentrations water for long periods (Carmichael, 1994; Ueno et al., 1996).




The methods for detection of microcystins in water and cells extract can be labor-

intensive, time-consuming and expensive as the case of mass spectroscopy, liquid

chromatography with mass spectrometry , gas chromatography (Chorus et al., 1999) or

high performance liquid chromatography – HPLC (Lawton et al., 1994). Additionally

some assays require a previous extraction and clean-up procedure (Rivasseau &

Hennion, 1999).

Enzyme-Linked Immunosorbent Assay - ELISA utilizing polyclonal or monoclonal

antibodies has become the prevailing method for detecting microcystins (KIM et al.,

2003; Baier et al., 2000; Metcalf et al., 2000; Nagata et al., 1999; Ueno et al., 1996; Chu

et al., 1990; Chu et al., 1989) but indirect solid phase ELISA method (Dot blot) was not

utilized for microcystin analysis.

The dot blot immunoassay have been utilized for some biological assays as

bacteria Salmonella enterica (Yoshimasu & Zawistowski, 2001), hybridoma screening

(Piwowarczyk & Matsuda, 1990), analysis of DNA (Nexlin & Mozes, 1995), heparin

(Scheere et al., 1989), proteins (Morçöl & Subramanian, 1999; Henkel & Bieger, 1994)

and Bacillus thuringienesis toxins (Tapp & Stotzky, 1995) but not for microcystins

detection.

The aim of this work is to develop a not expensive and convenient semi-

quantitative dot-blot assay to screening microcystin-LR in surface water, pure water

(haemodialysis) and cellular extracts of cyanobacteria.

MATERIALS AND METHODS

The water used was purified by distillation or deionization followed by passage

through a Milli-Q® water purification system (Millipore). The chromatographic

acetonitrile and methanol was purchase from Merck and all other reagents were of

analytical or synthesis grade when not specified.




Surface water and cyanobacteria analysis

In order to asses the ability of the assay to detect microcystin-LR in surface

water, a sample of an ornamental lake of CEFET-PE was collected (2.5 l) and 1 l

portion filtered immediately through 47 mm glass fiber discs (Millipore) and conserved at

-18ºC. One liter was utilized to analyze by light microscopy for cyanobacterial cells

presence.

Culture of Microcystin-LR producer Microcystis aeruginosa NPJB-1

Non-axenic culture of Microcystis aeruginosa was kept in a culture tube

containing approximately 5 ml sterile ASM-1 medium (Gorham et al. 1964). The strain

was cultivated under 26oC + 2oC with a photon flux density of 40 µmol m-2s-1

(Biospherical Instruments QSL-100) from cool-white fluorescent tubes on a 12:12 light:

dark cycle. The strain for toxins extraction was cultivated in 2.0 l Erlenmeyer flask

containing 1.5 l ASM-1 medium, under 80 µmol photon flux m-2 s-1 with a 12:12 h light :

dark cycle and aeration. Culture was harvested at the late exponential phase by

centrifugation. The cell material was lyophilized (ModulyoD, ThermoSavant) and stored

at -18oC. The strain was kindly provided by Dr. Sandra Azevedo.

Microcystins extraction and high-performance liquid chromatography (HPLC) quantification

Lyophilized culture material of M. aeruginosa (0.1g) was disrupting by adding 7.5

ml volume of 75% v/v methanol and sonicated (High Intensity Ultrasonic Processor -

50W Model) during 1 min at low temperature and centrifuged at 8,000xg for 15 minutes

for sediment separation. This operation was repeated twice and the liquid phase pooled,

the volume reduced to 1/3 under vacuum in rout-vapor at 40º C (Büchi mod. R-200).

The culture extract was filtered in a glass fiber discs (Millipore) and applied in a 1.0 g

octadecylsilica cartridge (Bond Elute) conditioned with 10 ml of methanol followed by 20

ml of water at a flow not exceeding 10 ml min-1. The cartridge was washed with 10 ml of

water and 10 ml of 30% v/v aqueous methanol. The toxins were eluted with 5 ml of




90% v/v acidified methanol (0.1% v/v trifluoracetic acid), concentrated by evaporation

and water added for a final volume of 1,600 µl. To confirm the microcystin-LR

production this solution was analyzed by means of HPLC (Shimadzu (Kyoto, Japan)

with a LC-10AT pump (column 250 x 4.0 mm, 5 µm; Lichrospher 100 RP-18) coupled to

a diode-array U.V. detector (SPDM-10Avp). Samples of 50 µl were injected and

separation was accomplished under a reverse phase gradient condition using a mobile

phase of 0.05% (v/v) acetonitrile : 0.05 % (v/v) trifluoracetic acid - TFA at flow rate of 1.0

ml/min at 40ºC. The linear gradient starting at 15% (v/v) acetonitrile increasing to 100%

over 35 min and was kept up to 40 minutes. Then acetonitrile was reduced for 15% over

45 minutes and kept in this ratio up to 55 minutes. Detector resolution was set at 1.0 nm

and run monitored at 238 nm. The total microcystins concentration in the extract were

calculed based in the area under the curve of the peak in the same retention time of the

microcystin-LR (Sigma) curve obtained with solutions at levels of 0.16, 1.6, 5.0 and

10.0 µg/l in isocratic condition using 0.05% (v/v) acetonitrile-trifluoracetic acid- TFA and

aqueous 0.05% (v/v) TFA (45:55).

Antibody production and purification

Microcystin-LR and keyhole-limpet hemocyanin - KLH were purchased of Sigma.

Polyclonal antibodies were produced by conventional methods (Karnikowski, 2001) with

immunization of rabbit with a solution of 500 µg microcystin-LR-KLH conjugated in 500

µl sodium phosphate buffer (pH 7.4). The conjugated was mixed with 500 µl Freund’s

complete adjuvante (Sigma) and injected subcutaneously at three dorsal sites. Booster

injections consisting of 500µg antigen were administered intravenously into the marginal

ear vein at days 28 and 56, with collection of 20 ml of blood at days 35, 63 and 93. The

blood was clot at room temperature and antisera were colected and centrifuged at

2,000xg (Micronal centrifuge). The colected antisera were purified in an affinity column

of protein G Pharmacia (HiTrap , Amersham, London, UK) stabilized with 5 ml of 20

mM phosphate buffer (pH 7.0). Serum portions of 0.5 ml were applied in the column

washed with 5.0 ml of the same phosphate buffer and the fraction of interest was

extracted with 3.0 ml of 0.1M glicina-HLC buffer (pH 2.7) directly in a tube with 0.3 ml of

0.1M tris-HCl buffer (pH 9.0). The antibodie solution was reduced for a volume of

approximately 200 µl using a 30 Kda Centricon centrifugal filter unit (Millipore). The




final concentration of antibodies was determined by the total protein analysis (Bradford,

1976). Indirect ELISA and dot blot assay were utilized to determine the antimicrocystin-

LR-KLH IgG work dilution. Competitive Indirect Microcystin ELISA

Samples, blank or standards microcystin-LR solutions were analysed in triplicate.

This assay is an indirect competitive enzyme immunoassay (Karnikowski, 2001) in a 96

well microtiter plate (Maxisorp , Nunc, Roskilde, Denmark) coated with microcystin-LR-

chicken ovoalbumine. It was calibrated with microcystin-LR (Sigma) solutions at levels

of 0.16, 0.5 and 1.6 µg/L . In summary, 50 µl of sample or microcystin-LR standard and

the antibody that were incubated for 90 minutes at 37º C were introduced into the wells

and incubated for 120 minutes at ambient temperature. The wells were emptied and

washed three times with 200 µl of phosphate buffer 15 mM- 0.05% Tween (pH 7.2). A

50 µl aliquot of polyclonal anti-rabbit IgG Horseradish Peroxidase – HRP from goat

(Sigma) diluted of 1 : 2,000 was added and incubated for another 90 min at 37º C. The

wells were washed again and a 50 µl aliquot of substrate o-phenylenediamine - OPD

(Sigma) added to each well and incubated for 10 minutes in ambiente temperature

protected of the light. Finally 50 µl of a 3.0 M hydrochloric solution was added to stop

the reaction and the absorbance immediately recorded at 492 nm using the microplate

reader ( EIA/ELISA reader mod. 2550, Biolab).

Computational analysis of the images (Dot blot Software)

The experimental membranes were digitilized by means of a traditional scanner,

obtaining a digital image for each essay. Before any analysis, the digital image was

filtered in order to eliminate the noise added during the image digitilizing. Most of these

noises comprise shadows and dust, unavoidably present in such approach of image

acquiring. The image filtering, or “cleaning”, was made using a popular software for

image enhancing Corel Photo-Paint 10 (Corel). All digitilized membranes were treated

as grayscale images, following a given set of standards such as size, resolution,

contrast and brightness, as will be described bellow.




The images were scanned with a resolution of 75 horizontal and vertical dpi, with

million colors, using a HP DeskScan II software (Hewlett Packard). The selected area

was then inverted in order to eliminate the parts of the figure that were not of interest. In

this computational analysis, initially each line and column of the frame was covered,

being situated the beginning and the end of each dot (Fig. 1). Starting from the

horizontal lines, the top of the first pixel is identified by the contrast between the

membrane colour (near to white) and the dot image. Thus, the border of each dot can

be clearly identified. The same process is used to get the lateral limits of the dots. This

procedure was applied for all images in sequence, having identified rectangular regions

bordering for each dot that was analyzed and quantified according to the number and

intensity of the pixels inside. Figure 1: Image scanning for the dot’s edge detecting. Blue and black lines are

the horizontal and vertical scanning procedure, respectively. Computerized protein dot blot software validation

In order to evaluate the dot blot program three different types of images have

been used: The first one was a hypothetical membrane constructed in the Corel Photo

Paint 10 software and composed by a know pre-established sequence of browning color

dots. The dots are depicted in Figure 2.




Figure 2: virtual Dot blot membrane model.

For the two others membranes a Bio-Dot micro filtration apparatus (Bio-Rad

Laboratories, USA) with a water tube of vacuum has been used. The second membrane

as depicted in Figure 3 was prepared after the application of 40 µl of goat anti-IgG-HRP

(Sigma) diluted to 500, 1000, 5000, 25000, 125000 and 625000 and revealed (8

minutes) with diaminobenzidine – DAB (Sigma) substrate solution prepared in 0.5 ml

methanol, 10.0 ml 0.01 M PBS buffer, 5 µl 0.1% w/v imidazol (Sigma) and 5 µl peroxide

hydrogen. This reaction was stopped after the addition of two portion of 200 µl of water.

This procedure was the same revelation assay used for dot-blot screening methods.

Figure3: Anti-IgG-HRP dilutions dot blot (three replicate) membrane revealed with DAB-Imidazol.

The third membrane (Fig. 4), an albumen dot, was obtained by applying 40 µl of

bovine serum albumin – BSA (Sigma) with dilutions of 0, 5 ,10, 20, 39, 78, 156, 312,

624, 1248, 2496 and 4.992 µg/ml revealed with amidoblack solution (0.1% w/v

amidoblack in acetic acid 10% v/v and 10% methanol v/v). This membrane was then

washed in a methanol 90% v/v, acetic acid 2% v/v (Henkel & Bieger, 1994). The

membranes were dry at the ambient temperature. All images were scanned as

described above.

A B C D E

0.5 1 5 25 125 625 ( Dilution x 1000)

1 2 3




Figure 4: Bovine serum albumin – BSA dot blot membrane revealed with 0.1% amidoblack solution (Henkel & Bieger, 1994).

Computerized microcystin dot blot Assay

The assays were carried out in a Bio-Dot micro filtration apparatus (Bio-Rad

Laboratories, USA) using a water tube of vacuum. The nitrocellulose membrane was

prewetted with 0.025 M Tris 0.15 M NaCl buffer (pH 7.4) –TBS prior sensitizing

applications. Five microliters of microcystin-LR chicken eggs albumen (200 ng/µl) were

placed in each well and incubated at 37ºC / 5 minutes. The excess was removed and

the wells washed with 200 µl of TBS - 0.1% Tween buffer. The membranes were then

blocked with 100 µl of TBS- 2.5% (w/v) BSA for 90 minutes at ambient temperature and

washed with 200 µl of TBS buffer.

Forty microliters of a mixture of the sample, blank or standard toxin solutions with

anti-microcystin-LR-KLH polyclonal antibodie (6.25 ng/µl) were then added into the

wells and incubated for 90 minutes at 37ºC. These wells were washed 3 times with

0.1% (v/v) TBS-Tween. After that, 40 µl of an 1:2,000 dilution of anti-IgG-HRP was

added to the mixture and incubated for 60 min more at 37ºC, followed by a wash with

0.1% (v/v) TBS-Tween and pure TBS buffer. Finally, the reaction was developed for 8

minutes with DAB – Imidazol. DAB - nickel chloride and DAB – cobalt chloride also they

had been analyzed.

RESULTS

Dot blot Technique

The program developed for the dot blot membranes quantification showed a

good linear correlation between the intensity of spots of the hypothetical membrane

0 5 10 20 39 78 156 312 BSA (µg/µl)




(Fig. 2) before and after digitilizing and quantification, with a coefficient of correlation (r)

of 0.9620 (Fig. 5) . The average relative standard deviation – RSD after quantification

was 1.3 ± 0.5%.

Figure 5: Linear regression of a digitilizing hypotetical dot blot membrane. Vertical error bars represent standard deviation after scaning (n = 5, r = 0.9620, t = 17.0, p < 0.01)

The software analysis with real biological membranes were prepared throug a

simplified method either using only dilutions of anti-IgG-HRP (Fig. 3) or BSA solution

(Fig. 4) showed a good correlations coeficient in a range dilution between 500 – 25000

and 0 – 78 µg/ml BSA concentration, respectively (Fig. 6 and Fig. 7). For the anti-IgG-

HRP membrane the average of RSD and for BSA were 9.0 ± 4.6 % and 13.6 ± 6.5%,

respectively.

Figure 6: Semi-log graphic of Anti-IgG-HRP dilution curve quantified by dot blot software. Linear range between 500 and 25000 dilution factor (r = 0.9868). Vertical error bars represent standard deviation (n = 3, t = 19.3, p < 0.01)

3000

4000

5000

6000

7000

6000 8000 10000 12000 14000

Average of Σ intensity of pixels

Ave

rage

of

Σ o

f int

ensi

ty

of p

ixel

s

0

2000

4000

6000

8000

100 1000 10000 100000

log anti IgG-HRP dilutions

Aave

rage

of

Σ o

f int

ensi

ty

of p

ixel

s




Figure 7: Bovine serum albumin – BSA concentration curve quantified by the dot blot software. Linear range between 0 and 78 µg/ml (r = 0.9735). Vertical error bars represent standard deviation (n = 3, t = 34.1, p < 0.01)

Computerized microcystin dot blot Assay

During the experiment, when it was tested under an indirect ELISA procedure it

was verified a satisfactory activity of purified polyclonal rabbit anti-microcystin-LR-KLH

antibodies. In this experiment a dilution of 1:32 (-log10 = 1.5) of a 200 µg/ml solution was

applied (Fig. 8). With these antibodies in ELISA assay for microcystin-LR it was possible

to detected concentrations as low as 0.16 µg/l. A linear range (r = 0.9713 ) was

obtained between 0.16 µg/l at 1.6 µg/l as depicted in Figure 9.

Figure 8: Purified anti-microcystin-LR-KLH IgG titres determined with a ELISA procedure. Vertical error bars represent standard deviation (n=3).

0,0

0,1

0,2

0,3

0,4

0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50

- log10 IgG dilution

Abso

rban

ce (

492n

m)

0

1000

2000

3000

4000

0 20 40 60 80 100

BSA concentration (µg/ml)

Aver

age

of Σ

inte

nsity

of

pixe

ls/d

ot




Figure 9: Semi-log graphic of 0.16, 0.50 and 1.6 µg/L microcystin-LR analysed in an indirect competitive ELISA. Vertical bars represent standard deviation (n = 3, t = 10.8, p < 0.01).

After several experiments, the concentration of several substances, taking into

account the best condition for the dot blot signal (sensitivity) were established;

concentrations of anti-microcystin-LR-KLH antibody (6.25 ng/µl), membrane

sensibilizing microcystin-LR- chicken ovoalbumin (20 µg/ml), blocker solution (TBS- 2.5

% BSA) and DAB stained conditions with imidazol solution (5 µl of 10 mg/l). Stained test

with DAB, DAB-NiCl2 and DAB-CoCl2 solution had produced minor sensitivity than DAB-

Imidazol (data not show).

The HPLC analysis of cyanobcterial extract of M. aeruginosa - NPJB-1 showed a

microcystin-LR concentration of 37 µg/ml. Microcystin-LR presence was confirmed by

the same retention time of microcystin-LR standard and characteristic absorption

spectrum in the U.V. region in accordance with Lawton et al. (1994).

The dot blot membrane obtained with purified water and lake water spiked with

microcystin-LR and M. aeruginosa cells extract presented different color intensities

between the negative control – NC (maximum signal) and the correspondent 0.16, 0.50,

1.60 or 5.00 µg/l dots (Fig. 10)

0,00

0,10

0,20

0,30

0,1 1 10

Microcystin-LR (µg/l)

Abs

orba

nce

(492

nm)




c

Figure 10: A triplicate dot blot membrane of purified water (a) and lake water (b) spiked with microcystin-LR and cell extract dilution of M. aeruginosa (c).

The difference of colors between the NC and all microcystin-LR concentration in

the waters membrane (a and b) had been significantly different when calculeted using a

paired Student t-test of the average intensity of pixels obtained by dot blot software

(Table 1). In M. aeruginosa extract significant difference was observed for 1.6 µg/l and

5.0 µg/l (p < 0.05).

Table 1: Average of somatory of intensity of pixels and paired Student test values of purified and lake water membrane spiked with microcystin-LR and quantified by dot blot software. Microcystin-LR (µg/l) -------------------------------------------------------------------------- PURIFIED WATER NC 0.16 0.50 1.60 5.00 ------------------------------------------------------------------------------------------------------------------ Average of Σ of number of Pixels 2641 1610 1631 1696 1963 t --- 14.50 8.94 22.76 4.73 p --- 0.005 0.012 0.028 0.042

--------------------------------------------------------------------------------------------------------------------

LAKE WATER

------------------------------------------------------------------------------------------------------------------- Average of Σ of number of Pixels 2543 1543 1661 1779 2194 t --- 49.38 67.47 116.7 45.4 p --- 0.013 0.009 0.005 0.014

---------------------------------------------------------------------------------------------------------------------

a b

B NC 0.16 0.50 1.60 5.00




The ELISA analysis of microcystin-LR in purified water and lake water showed

valures of “r” of 0.9713 (n = 3, t = 10.8, p < 0.01) and 0.8316 (n = 3, t = 3.56, p < 0.01),

respectively. Curves are show in Figure 11.

Figure 11: Indirect competitive ELISA of purified (a) and lake water (b) spiked with micrcocystin-LR with 0.16, 0.50 and 1.60 µg/l. Vertical bars represent standard deviation (n=3). The right graphics are the correspondent semi-log scale.

The quantitative dot blot assay for purified water spiked with microcystin-LR in

concentration of 0.16, 0.5 and 1.60 µg/l presented an “r” value of 0.9551 (n = 2, t = 8.5,

p < 0.01) and an average RSD of 10.7% with a minimum of 5.8% and maximum of

17.6%, while the lake water presented a “r” of 0.8402 (n = 2, t = 3.8, p < 0.01) with an

average of RSD of 5.5% with a minimum of 2.6% and maximum of 9.0% (Fig. 12).

0,000,050,100,150,200,250,30

0 0,5 1 1,5 2


Abs

orba

nce

(492

nm)

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0 0,5 1 1,5 2


Abs

orba

nce

(492

nm)

0,00

0,10

0,20

0,30

0,1 1 10


Abs

orba

nce

(492

nm)

0,00

0,10

0,20

0,30

0,1 1 10


Abs

orba

nce

(492

nm)




Figure 12: Purified water (a) and lake water (b) spiked with microcystin-LR and quantified by dot blot software. Vertical error bars represent standard deviation (a minimum n = 2). The right graphics are the correspondent semi-log scale.

In the range of microcystin-LR of 0.16, 5.0 and 10.0 µg/l the dot blot assay for

purified water presented a r = 0.9999 (n = 3; t = 173 ; p < 0.01) and an average RSD of

11.2% with a minimum of 4.5% and maximum of 20.2%. The lake water tested with 1.0,

5.0 and 10.0 µg/l of microcystin-LR presented a r = 0.7197 (n = 2; t = 2.54; p < 0.05)

and an average of RSD of 8.3% with a minimum of 6.3% and maximum of 9.4% (Fig.

13).

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Figure 13: Purified water (a) and lake water (b) spiked with Microcystin-LR and quantified by dot blot software. Vertical error bars represent standard deviation (a minimum n = 2). The right graphics are the correspondent semi-log scale. The dilution of the M. aeruginosa extract containing microcystin-

LR quantified by HPLC, when analyzed by dot-blot assay, showed a coefficient

correlation of 0.9980 in the range of 0.16 at 1.6 µg/l with an average of RSD of 9.2%

with a minimum of 5.1% and maximum of 14.5% (Fig. 14).

Figure 14: Microcystin in M. aeruginosa extract quantified by dot blot software. Vertical error bars represent standard deviation. The right graphics are the correspondent semi-log scale (n = 3, t = 41.8, p < 0.01)

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DISCUSSION

Methods as high performance liquid chromatography and mass spectroscopy or

capillary electrophoresis require an previous extraction and clean-up procedure before

analysis the presence of microcystin-LR in water, while Immunological microplate

ELISA methods need the immediate reading after color reaction.

The use of quantitative dot blot is cited to protein analysis by using an expensive

densitometer to made the measures (Sportsman & Elder, 1984; Morçöl & Subramanian,

1999). Until where we know there is not description dot blot technique for the

microcystin analysis.

The dot blot assay is an alternative detection system to sophisticated and

complex methods of analysis. This methodology can be aplied to screening water

quality, with a very simplified and not expensive control laboratorie. This assay may be

performed next the field using a direct visual detection, that is can be improved by

scanning reading.

In this technique, utilizing nitrocellulose membrane stained with

diaminobenzidine – Imidazol, surface water sample, did not need concentration or

clean-up procedure. The reaction yields a browning precipitated that does not fade over

time. The dried membrane can be stored and read again if necessary.

Software was also developed specifically for this application. The main goal of

this tool is to help the technician to analysis the samples based on their colors. The

system is able to detect the different color degress, counting the number and intensity of

the pixels in each dot. In order to validade the tool a hypotetical dot blot membrane was

used as example, and the results were satisfactories. This software also presented

good results when analyzing biological membranes of BSA stained with amidoblack.

The linear range observed with BSA (0 – 78 µg/ml) was smaller than that

obtained by Sportsman & Elder (1984) using a He-Ne laser densitometer, but the




linearity in our analysis (0.9735) was the same of them (0.9710). This software allows a

reliable BSA analysis because sensitivity (5 µg/ml) and correlation coefficient observed

are very good in the range cited. This sensibility was that one verified by Sportsman &

Elder (1984) and Morçöl & Subramanian (1999) for BSA detection in a dot blot assay,

10 µg/ml and 4 µg/ml, respectively. With regard to analysis of the quantification

response front anti-IgG-HRP dilutions revealed with DAB – Imidazol solution in the

dilutions range of 500 at 25000, a good correlation coefficient (0.9868) and RSD (9.0%)

showed to be adjusted to apply in the microcystin-LR assay in large anti-IgG-HRP range

dilutions.

The best result for sensitivity had been gotten with the use of the DAB-Imidazol.

The use of DAB – NiCl2 or DAB - CoCl2 genereted a strong background interference

disabling its application.

This technique has provided visual and quantitative detections of microcystin-LR

in a level of 1.0 µg/l directly in surface and purified water, spiked with this toxin in

accordance with World Health Organization guidelines (WHO, 1998) and Brazilian law

(Brasil, 2000).

The detection can be made directly by visual detection or after software

quantitation without the necessity of enrichment or clean-up. The accuracy of the

measurements was enhanced by the use of not expensive computer scanning. Direct

measure can have been possible because the advantage of nitrocellulose use that’s

possiblity eliminate the unbound products (Henkel & Bieger, 1994).

The criteria of acceptance for relative standard deviation - RSD depends the type

of matrix and the concentration of the analyte. In levels of concentration of 1 ppb values

of RSD up to 30% has been accepted in food analysis (AOAC, 1993). In our

experiments the maximum RSD observed was of 20.2% placing itself in the acceptable

range for this level of concentration.

The minor microcystin-LR concentration tested here with purified and surface

water was 0.16 µg/l, but it was sufficient to generated a visual color discrimination in




relation to the negative control . This concentration is very next to the limit of detection

of the comercial microcystins plate ELISA kit Envirologix (0.147 µg/l). In our tests the

system showed conditions to evaluate concentrations in the range of 0.16 at 10 µg/l, but

with a linearity smaller (r = 0.7197) than in the range of 0.16 at 1.6 µg/l (r = 0.8402). It

was also clearly absorved an interference in the quantitative results, particularly in the

quantified microcystin in surface water than in purified water, observed by a minor

coefficient correlation. Same results are also observed in the ELISA assay.

Dot blot assay described here can be used in routine screening detections.

Membranes with antigen and blocking steps can be prepared beforehand reducing the

time of analysis in aproxamately 1h 40 minutes. However, tests involving the use of

other commercial microcystins is necessary to evaluate the cross reactivity of the

polyclonal antibody.

This metodology can help in the substitution of biological animal test in qualitative

or quantitative microcysti-LR screening. The animal test have the disadvantage of life

use and are less sensitive (ppm) while, the immunoassays, are more sensitives

(Carmichael, 2002) and increasingly attractives due to the need for monitoring

microcystin levels at lower concentrations as ppb (Metcalf et al., 2000).

The results observed with purified water in the dot blot assay demonstrate the

capacity for application of this not expensive method to control of haemodyalise waters.

If such control is applied in clinics situated in places whose quality of the water varies

constantly, p.e. example in the Northeast region of Brazil, accidents as the occurred in

the City of Caruaru - Pernambuco in 1996, with death of more than 70 patients of renal

treatment (Jochimsen et al. 1998; Carmichael et al. 2001; Azevedo et al., 2002) could

be prevented.

Anoter perspective for application of this assay is in development of a direct

competitive assay based in the microcystin labeled with horseradish peroxidase enzyme

or fluorescent labeling as fluorescein or lanthanide cryptates.




ACKNOWLEDGEMENTS

We are greatful to Sandra M.F.O. Azevedo of Universidade Federal do Rio de

Janeiro, Rio de Janeiro, Brazil for a producer microcystin strain of Microcystis

aeruginosa - NPJB-1 and Maristela C. C. Cunha of Instituto Tecnológico do Estado de

Pernambuco – ITEP –Laboratório de Ecofisiologia de Microalgas by light microscopy

water cyanobacterial analysis.

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Kim, Y.M., Oh, S.W., Jeong, S.Y., Pyo, D.J., and Choi, E.Y. (2003) Development of an ultrarapid one-step fluorescence immunochromatographic assay system for the quantification of microcystins. Environmental Science & Tehnology 37 (9):1899-1904. Lawton, L.A., Edwards, C., and Codd, G.A. (1994) Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst 119, 1525-1530. Magalhães, Valéria Freitas de, Soares, Raquel Moraes, Azevedo, Sandra, M.F.O. (2001) Microcystin contamination in fish from the Jacarepaguá lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk. Toxicon 39, 1077-1085. Metcalf, J.S.; Bell, S.G.; Codd, G.A. (2000) Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin. Water Researche 34 (10): 2761-2769. Mehto, P., Ankelo, M., Hinkkanen, A., Mikhailov, A., Eriksson, J.E., Spoof, L., Meriluoto, J. (2001) A time-resolved fluoroimmunometric assay for the detection of microcystins, cyanobacterial peptide hepatotoxinas. Toxicon 39, 831-836. Morçöl, Tülin, Subramanian, Anuradha (1999) A red-dot-blot protein assay technique in the low nanogram range. Analytical Biochemistry 270, 75-82. Nagata, Satoshi; Tsutsumi, Tomoaki; Yoshida, Fuyuko; Ueno, Yoshio. (1999) A new type sandwich immuassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microcistin and anti -microcystin monoclonal antibody. Natural Toxins 7, 49-55.

Pouria, Shideh, Andrade, A de, Barbosa, J., Cavalcanti, R.L., Barreto, V.T.S., Ward, C.J., Preiser, W., Poon, Grace K., Neild, G.H., Codd, G.A. (1998) Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil 352, 21-26.

Rivasseau C.; Martins, S.; Hennion M.C. (1998) Determination of some physicochemical parameters of microcystins (cyanobacterial toxins) and trace level analysis in environmental samples using liquid chromatography. Journal of Chromatography A 799, 155-169.

Rivasseau, Corinne; Hennion, Marie-Claire (1999) Potential of immunoextraction coupled to analytical and bioanalytical methods (liquid chromatography, ELISA kit and phosphatase inhibition test) for an improved environmental monitoring of cyanobacterial toxins. Analytica Chimica Acta 399, 75-87.

Sportsman, J. Richard, Elder, John H (1984) A microanalytical protein assay using laser densitometry. Analytical Biochemistry 139, 298-300. Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Yoshida, F., Suttajit, M., Mebs, D., Pütsch, M., and Vasconcelos, V. (1996) Survey of microcystins in environmental water




by a highly sensitive immunoassay based on monoclonal antibody. Natural Toxins 4, 271-276. Whitton, Brian A.; Potts, Malcolm (Eds). (2000) The ocology of Cyanobacteria – Their Diversity in Time and space. London: Kluwer Acad. Pub. WHO, Guidelines for Drinking-water Quality. 1998 In: Chorus, Ingrid, Jamie. Toxic Cyanobateria in Water – A guide to their public health consequences, and management. London: E & FN Spon, /WHO, 1999. Yunes, J.S., Salomon, P.S., Matthiensen, A., Beattie, K.A., Raggett, S.L., and Codd, G.A. (1996) Toxic blooms of cyanobacteria in the patos lagoon estuary, Southern Brazil. Journal of Aquatic Ecosystem Health 5, 223-229.




CAPÍTULO 2: A new fluorescent-labeled microcystin-LR terbium cryptate

Eduardo José Alécio-Oliveira1*, Suzana Vilanova2, Severino Alves Junior2, Petrus Santa

Cruz2, Mathis, G.3, Bazin, H.3, Renato J. R. Molica4, Carla Nunes5, Antonio Teixeira 5,

Elizabete Malageño6, José Luiz de Lima Filho6


Recife-PE, Brazil. 2Departamento de Química Fundamental (DQF) da Universidade Federal de

Pernambuco (UFPE), Recife-PE, Brazil. 3Cis-Bio International (DIVT) Research and New Technologies, BP 175, 30200,

Bagnds/Cèze-France 4Instituto de Tecnológico do Estado de Pernambuco (ITEP), Recife-PE, Brazil. 5Laboratório Multidisciplinar de Pesquisa em Doença de Chagas da Universidade de

Brasília (UnB), Brazil. 6Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de

Pernambuco (UFPE), Recife-PE, Brazil.

*Corresponding author: Centro Federal de Educação Tecnológica de Pernambuco

(CEFET-PE), Av. Profº Luiz Freire, 500, Cidade Universitária, Recife-PE, 50740-540,

Brazil. e.mail: [email protected]

KEYWORDS: Cyanobacteria, Microcystin-LR, Terbium Cryptate.





ABSTRACT

A new fluorescent labeled compound of microcystin-LR with terbium cryptate was

obtained by initial conjugation of microcystins-LR with aminoethanethiol followed by the

liaison with the ester group of terbium cryptate. The product formation was followed by

high performance liquid chromatography - HPLC in 238 nm and 310 nm. The presence

of microcystin-LR in the labeled molecule was confirmed by enzyme linked

immunosorbent assay ELISA and protein reaction with bicinhonic acid. Luminescence

spectrum of cryptate and the conjugated molecule was carried through.




INTRODUCTION

Microcystins are a group of stable heptapetide hepatotoxins produced by

freshwater cyanobacteria genera that lower the water quality leading to an increase in

the risk of intoxication for animals and humans. Species of genera Microcystis,

Anabaena, Aphanizomenon, Planktothrix (Oscillatoria), and Nostoc are frequently

described as producers of microcystins (Watanabe et al., 1997; Carmichael, 1994). The

eutrophication of ponds, rivers and other freshwater ecosystems allows cyanobacteria

to flourish, leading to contamination of the water (Pouria et al., 1998; Kim et al., 2003).

Microcystins are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A),

which are regulatory enzymes present in the cytosol of mammalian cells (Fontal et al.,

1999). The general structure of microcystins is composed of cyclo-(D-Ala-X-D-MeAsp-

Z-Adda-D-Glu-Mdha) in which X and Z are variable L-amino acids, D-MeAsp is D-

erythro-β-methylaspartic acid, Mdha is N-methyldehydroalanine, and Adda is an

unusual amino acid 2S,3S,8S,9S-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-

4E,6E-dienoic acid (Carmichael et al., 1988). The most extensively studied form is

microcystin-LR that contains L-leucine and L-arginine in the two main variant positions

(Fontal, et al., 1999). More than 60 different analogues of microcystins have been

described and their acute mouse i.p. toxicity (DL50) varies between 50-600 µg/Kg

(Mehto et al., 2001).

Contamination of surface drinking water by microcystin-producing cyanobacteria

is a suspected cause of primary liver cancer (Ueno et al., 1996). Chronic oral ingestion

of a crude extract of Microcystis suggests that microcystins can cause tumors after

long-term exposure (Falconer, 1991). Human mortalities resulting from oral

consumption of cyanotoxins have been suspected but have not been confirmed, due the

difficulty of obtaining reliable data regarding vectors and the circumstances that would

confirm the presence of cyanotoxins in human food or water supplies (Azevedo et al.,

2002). Increasing care is being taken in the consumption of products contaminated with

cyanobacteria toxins, because of the risks involved in consuming food that contains

blue-green algae or water contaminated by their toxins (Gilroy et al., 2000). Recently an

acute case of poisoning of humans undergoing haemodialysis with water contaminated




with microcystin killed 76 of a total of 131 patients in the city of Caruaru in Northeast

Brazil (Jochimsen et al., 1998; Carmichael et al., 2001; Azevedo et al., 2002). As a

consequence of this, a microcystin limit of 1µg/l was established for drinking water in

Brazil (Brasil, 2000).

Currently the aim is to develop a fast, inexpensive and convenient analytical

method for detecting microcystins. Immunological fluorescent methods using antigen,

monoclonal or polyclonal antibody have come to be preferred (Nagata et al., 1999;

Nagata et al., 1997; Mcdermott et al., 1995; An & Carmichael, 1994; Chu et al., 1990).

Fluorescent-labeled reagents have been developed for biological applications. Kim et.

al. (2003) employed a one-step fluorescence immunochromatographic assay to quantify

microcystins using Alexa Fluor 647. A fluorescent phosphatases enzyme inhibition

assay for microcystin-LR was developed by Fontal et al. (1999).

Complexes with a lanthanide ion, when excited by ultraviolet (U.V.) radiation,

show long-lasting fluorescence and are excellent light conversion molecular devices –

LCMD’s (de Sá et al., 2000) applicable to time-resolved microscopy (Mikola et al.,

1995), fluorescent lighting (Ronda, 1995), U.V. dosimeters (Gameiro et al., 1998),

antirefelection coatings for solar cells (de Sá et al., 1998) and the labeling of biological

assays (Hemmilä et al., 1997; Takalo et al., 1994).

In the search for more sensitive markers, some chelates of lanthanides had

already been obtained and tested in various models of biological assays (Dakubu et al.,

1988; Mukala et al., 1993; Saha et al., 1993). Time-resolved fluoroimmunometric assay

- TR-FIA is a method when the fluorescence emission is measured after a delay has

elapsed from a pulsed excitation, enabling the short-lived background fluorescence

which has a rapid decay time to be excluded (Mehto et al., 2001). Soini et. al., (1988)

labeled anti-rabbit IgG with europium chelate as a model to locate rabbit IgG for human

smooth muscle myosin in a histological section; Bonin et al. (1999) used a complex of

europium and samarium for the detection, using the ELISA method, of diphteria

antitoxins in serum; and Xu et al. (1991) labeled anti-β-LH and monoclonal anti- β-FSH

antibodies with europium and samarium, respectively. The use of fluorescent lanthanide

as a substitute for labeled enzyme (e.g. horseradish peroxidase - HRP) in the ELISA




method for microcystin detection has been described by Mehto et al., (2001). This

system uses microcystin-LR labeled with a chelate of europium.

One of the classes of complexes of lanthanides that is increasingly being used in

immunological assays is the cryptates. These composites are highly stable when

dissolved in water, and have a high coefficient of molar absorption, a high quantum

income in water solution and kinetic and thermodynamic stability (Sabbatine et al.,

1993). Europium cryptate also has been used for labeling DNA (Prat et al. 1991) and

antibodies (Lopez et al. 1993), and the Cis-Bio-Schering Company (France) now

markets a kit with trisbipyridine europium cryptate for labeling biomolecules. Terbium

complex has been utilized in films as very efficient light converters under U.V.

excitation, where the ligands act as antennas, absorbing U.V. radiation and efficiently

transferring energy to Tb3+ emitters. A emission green 5D4 à 7F5 transition is monitoring

as function of U.V. excitations (de Sá et al., 2000). The use of a terbium cryptate for

labeling cyanotoxins, however, has not been investigated.

There is obviously a need to monitor cyanotoxins in water samples and products

that may have previously been in contact with cyanobacteria.

This paper describes how a new fluorescent terbium cryptate conjugate with

microcystin-LR was obtained.

MATERIALS AND METHODS

The water used was purified by distillation or deionization followed by passage

through a Milli-Q water purification system (Millipore) giving the water a resistivity of

18.2 MΩcm. All other reagents, where not specified, were of analytical, synthesis or

liquid chromatographic grade.




Terbium cryptate synthesis

The labels (a), (b) and (c) refer to the intermediate compounds shown in Fig. 1.

The synthesis of the cryptates was achieved by bromination of diethyl 2,6-dimethyl-3,5-

pyridinedicarboxilate (99% Aldrich), via the initiator 1,1'-Azo (cyclohexanecarbonitrile)

(98% Aldrich) and N-bromosuccinimide (NBS, 99% Aldrich) under reflux and irradiation

by a 100W incandescent lamp to give 2,6-dibromomethyl-3,5-diethyl-dicarboxilate (a).

The bromination of 6,6'-dimethyl-2,2'-bipyridine used benzoyl peroxide associated with

NBS (Lehn and Regnouf de Vains, 1989; Rodriguez-Ubis et al., 1984). In this procedure

the product 6,6'-Bis (bromomethyl)-2,2'-bipyridine (b) is obtained in the pure state after

re-crystallization. The ring (c) was synthesized following the procedure described in the literature (Rodriguez-Ubis et al., 1984; Newkome et al., 1983). The subsequent step

was the formation of the cryptate [Li⊂bpy.bpy.py(CO2Et)2]CO3-. The product (c) was

added to CH3CN(aq) in an atmosphere of N2(g). The product (a) was dissolved in an

aqueous solution of Li2CO3 under reflux. The cryptate thus formed was purified using

HPLC. The inclusion of the terbium ion in the cryptate was achieved by the single ionic

change with the Li+ cation and the lanthanide. The lanthanide cryptate had been purified

using HPLC.

Figure 1: Schematic synthesis of terbium cryptate.

1. EtOH

2. H2SO4

N NBrH2C CH2Br

S NHNa

O

O

2

2

+

(b)

N

NH

N

N

HN

N

(c)

N

O

O

O

O

Br Br

(a)

CF3COO-

N N

NN NN

O

O

O

O

N

Li+




Microcystin-LR – Terbium Cryptate Conjugation First stage: Conjugation of microcystin-LR to aminoethanethiol

Microcystin-LR was dissolved in a mixture of 20 percent methanol and 80 percent

100 mM phosphate buffer (pH 7.0) to a final concentration of 5.0 mg/ml and was

maintained at –20oC. In the first stage of conjugation 13.9 mg of 2-aminoethanethiol

hydrochloride (Sigma ) was added to 60 µg of microcystin-LR in a molar excess of

3,000-fold. The reaction was carried out in a solution of 24 µl of dimethyl sulfoxide

(Merck ), 18 µl of purified water and 8 µl of 5.0 M NaOH. The mixture was incubated for

one hour at 50º C under nitrogen, followed by the addition of 62 µl of concentrated

acetic acid and 496 µl of Trifluoracetic acid 0.1% (pH 1.5) as suggested by Moorhead et

al. (1994). Before the purification of conjugated microcystin-LR-aminoethanethiol, a

mixture of equal parts of the reaction and microcystin-LR solution of the same

concentration had been injected to verify the retention times. The conjugate was purified

by HPLC, freeze-dried (ModulyoD, ThermoSavant) and suspended in 200 µl of 50 mM

NaHCO3, 0.15 M NaCl buffer (pH 8.5).

Second stage: Conjugation of microcystin-LR- aminoethanethiol to terbium

cryptate

The reaction was carried out in molar excess of 25:1 cryptate: microcystin-LR-

aminoethanethiol for a 24-hour period (toh – t24h). Purified terbium cryptate (1.7 mg) was

dissolved in 200 µl of 50 mM NaHCO3, 0.15 M NaCl buffer (pH 8.5) and mixed with 200

µl of the microcystin-LR-aminoethanethiol solution and incubated at ambient

temperature for 24 hours.

HPLC terbium cryptate analysis

Microcystin-LR, microcystin-LR-aminoethanethiol, terbium cryptate and all

reactions were analyzed using HPLC. The system consisted of a Shimadzu (Kyoto,

Japan) LC-10AT pump and a column C18 LichroCart with 5 µm particle size, 250x4 mm

(Merck) attached to a U.V. detector. Samples of 50 µl were injected and separation was




accomplished using a reverse phase gradient and a mobile phase of acetonitrile: 1.0 %

(v/v) TFA at rate of flow of 1.0 mL/min at 40ºC. The linear gradient starting at 15% (v/v)

acetonitrile and increasing to 100% over 35 min and was maintained for 40 min. The

acetonitrile was then reduced by 15% for 45 min. and kept at this ratio until 55 min. had

elapsed and a new injection was administered. Detector resolution was set at 1.0 nm

and the reaction monitored at 238 nm and 310 nm. The fractions of terbium-

microcystins-LR conjugation were collected, pooled, freeze-dried and suspended in 500

µl of purified water for use in the fluorescence assays (luminescence), ELISA and

protein reaction.

Terbium cryptate luminescence spectroscopy

The fraction corresponding to the terbium cryptate and the terbium-microcystin-

LR conjugated, obtained at the end of the HPLC purification, were analyzed using

emission spectroscopy. The luminescence spectra were measured at 300 K using a

Jobin Yvon double monochromator model U - 1000 Ramanor attached to a water-

cooled RCA C310340-02 photomultiplier. In this case, a 450 U.V. Xe lamp was used as

the source of excitation. The registering and processing of the signal were carried out

using a Spectralink interface to an IBM microcomputer.

Purification of anti-Microcystin-LR-KLH antibodies

The affinity column of protein G (HiTrap, Amersham, London, UK) was stabilized

with 5 mL of 20 mM phosphate buffer (pH 7.0). Nine milliliters of the rabbit serum anti-

microcystin-LR-KLH were applied in portions of 0.5 ml and the column washed with 5.0

ml of the same buffer. The fraction of interest was extracted with 3.0 ml of 0.1M glicina-

HCl buffer (pH 2.7) directly into a tube with 0.3 ml of 0.1M tris-HCl buffer (pH 9.0). The

solution of IgG was reduced to a volume of approximately 200 µl using a Centricon

tube of 30 KD. The final concentration of antibodies was determined by the total protein

analysis (Bradford, 1976).




Competitive Indirect ELISA

The assay utilized was developed by Karnikowski (2001) for analysis of

microcystins. The standard curve was achieved with microcystin-LR solutions of 0.16,

1.6, 5.0 and 10.0 µg/l. The substrate wased was o-phenilenediamine (OPD, Sigma) and

the absorbance was recorded at 492 nm using a microplate reader ( EIA/ELISA reader

mod. 2550, Biolab). Protein analysis

The aqueous fraction of terbium-microcystin-LR conjugation and terbium cryptate

obtained at the end of HPLC purification were tested for the protein reaction utilizing a

bicinchonic acid protein assay kit (Pierce art. 23255).

RESULTS

Microcystin-LR – Terbium Cryptate Conjugation

No peak was detected in th HPLC analysis at the wavelengths of 238 nm and

310 nm on injection of 50 µl of bicarbonate buffer and aminoethanethiol utilized for the

conjugation of microcistina-LR to terbium cryptate. The microcystin-LR standard, when

injected separately, showed a peak in 22.6 min. at 238 nm and no peak at 310 nm. The

chromatogram of a mixture of equal parts of microcystin-LR and microcystin-LR-

aminoethanethiol after reaction (1 hour/50ºC) showed two peaks when observed at 238

nm, one with 22.6 min. and another in 21.5 min. respectively, showing good separation

under analytical test conditions (Fig. 2). The injection of the reactional mixture

microcystin-LR-aminoethanethiol (t24h) alone confirmed the appearance of only one

peak with 21.5 min (data not show).




Figure 2: Chromatogram in gradient reversed-phase HPLC of mixture of reaction product: (1) microcystin-LR-aminoethanethiol and (2) microcystin-LR standard.

Purified terbium cryptate dissolved in a NaHCO3 buffer showed a main peak in

18.6 min. at 238 nm and 310 nm (data not show). The same retention times of the

isolated products, terbium cryptate and microcystin-LR-aminoethanethiol, were

observed at the beginning of the conjugation - toh (Fig. 3). A small peak in 15.9 min. was

also observed at the beginning of the conjugation at 238 nm and also at 310 nm.

Time (minutes)

mA

bsor

banc

e (2

38 n

m)

1 2




Figure 3: Chromatogram in gradient reversed-phase HPLC at start of conjugation time (toh) of microcystin-LR-aminoethanethiol and terbium cryptate at 238nm and 310 nm: (1) Terbium cryptate and (2) Microcystin-LR-aminoethanethiol.

After 24 h of reaction (t24h) a sharp peak was observed in 15.9 min and an slightly

less intense peak with 18.6 min (Fig. 4).

The luminescence spectra of the isolated fractions with terbium-microcystin-LR

and terbium cryptate proved to be identical as represented in the Figure 5.




Figure 4: Chromatogram in gradient reversed-phase HPLC at the final conjugation time (t24h) of microcystin-LR-aminoethanethiol and terbium cryptate at 310 nm: (1) Microcystin-LR-Terbium cryptate and (2) Terbium cryptate.

Figure 5: Emission spectra of microcystin-LR-terbium cryptate recouped in

gradient reversed-phase HPLC. Excitation wavelength 310 nm.

The standard ELISA curve used as standard showed a good correlation

coefficient (r = 0.9346; t = 8.3; p < 0.01) during the interval from 0.16 µg/l to 10.0 µg/l

(Fig. 6). The fraction of microcystin-LR-terbium cryptate conjugation diluted 1/10

450 475 500 525 550 575 600 625 650 675 700 725

0

10

20

30

40

50

cts x

103 (s

-1)

Wavelengh (nm)




showed a 43.3% inhibition of antibody response while that corresponding to the terbium

cryptate and the blank did not show inhibition.

Figure 6: Semi-log graphic of competitive indirect ELISA of standard microcystin-LR using anti-microcystin-LR-KHL IgG on a microplate coated with microcystin-LR- chiken ovoalbumin (n = 3) The aqueous fraction of terbium-microcystin-LR conjugation was tested positive

for protein using bicinchonic acid reaction, while the fraction of terbium cryptate did not.

DISCUSSION

There is an increasing demand for more sensitive and inexpensive analytical

methods to deal with lower detection limits and this has given rise to a need for more

sensitive labeling.

A fluorescent complex of microcystin-LR-terbium cryptate was produced with a

view to using it for microcystin analysis.

The Microcystin-LR was used because it is one of the most common microcystins

(Metho et al., 2001), is more hepatotoxic (Honkanen et al., 1990) and testing water for it

is recommended in various countries including Brazil (Brasil, 2000), Great Britain and

other European countries (Roset et al., 2001). The latter is also recommended by the

World Health Organization - WHO that has set a provisional standard level of 1.0 µg/l

for drinking water (WHO, 1998).

0,100

0,150

0,200

0,250

0,300

0,1 1 10


Abs

orba

nce

(492

nm)




The cryptate complex of terbium is very advantageous because terbium is not so

sensitive to aqueous quenching (Hemmilä et al., 1997) and the characteristic of

transference of energy from the long lifetime excited metallic state to reactive ligand

triplet state overcomes the difficulty with the use of terbium chelates in bioanalytical

applications. Terbium cryptate molecule is also of small dimension when compared

with chicken egg albumin (44,000 Da), horseradish peroxidase (44,000 Da) or IgG

antibodies (150,000 Da) - molecules normally used in immunoassays - thereby

minimizing the possibility of steric impediment. The cryptate complexes as biomolecule

labeling groups, in contrast of chelates, has the advantage of being directly conjugated

without a displacement step (Prat et al., 1991).

The microcystin-LR conjugation with aminoethanethiol was chosen because it

was known that the reaction occurs with the amino acid methyldehydroalanine (Mdha)

of the toxin (Metcalf et al., 2000; Moorhead et al., 1994), in a position that does not

interfere with the main antigenic region of the molecule, the Adda amino acid.

Based on the reaction presented by Metho et al., (2001) for linking microcystin-

LR with europium chelate, the following reaction is proposed for the conjugation of toxin

with terbium cryptate (Fig. 7):

Figure 7: Microcystin-LR terbium cryptate conjugation reactions.

NH

ONH

R2

CH3CH3

OCH3

O

NH

O

NH

O

HNN

O

NH

O

CH3

COOH

O

CH3

CH2H3C

R1

COOH

CH3

HSCH2CH2NH2+

3CF3COO-

N N

NN NN

O

O

O

O

N

Tb+3

NH

ONH

R2

CH3CH3

OCH3

O

NH

O

NH

O

HNN

O

NH

O

CH3

COOH

O

CH3

R1

COOH

CH3

H3C

3CF3COO-

Tb+3

N N

NN NN

HN

O

O

O

N

SCH2




Microcystin-LR reacted in its entirety with the low molecular weight

aminoethanethiol, as verified in HPLC by the presence of a single peak in 21.5 min.

(chromatogram not shown), and the successful separation of microcystin-LR standard

(Fig 2.) allowing it to be recovered for the subsequent reaction. According to Moorhead

et al. (1994), the reaction of aminoethanethiol with the carbonyl group of the Mdha

residue is stronger at a high pH value, producing more than 95% conversion to

products.

The link inhibition of antibody with microcystin-LR-chicken ovoalbumin, in the

ELISA plate, by the conjugate (peak 1, Fig. 4) demonstrate the immunoreagent capacity

and confirm the microcystin presence in the structure. In accordance with Lopez et al.

(1993) trisbipyridine diamine europium cryptate was conjugated to antibody by the

method utilized here withtout any loss of immunoreactivity. The structure of the

hydrophobic amino acid Adda, which has the (E) form at the C-6 double bond is

essential for determining antibody specificity (An & Carmichael, 1994) and was

preserved because the reaction occurred selectively in the amino acid

methyldehydroalanie (Metcalf et al., 2000; Moorhead et al., 1994).

The presence of the toxin in this molecule is strengthened by a positive protein

reaction in this fraction when using the bicinchonic acid method, whose sensitivity is for

structures with up to four amino acids. The conjugation of the microcystin-LR is also

confirmed by the luminescence spectra of isolated cryptate and the microcystin-LR-

terbium cryptate fraction.

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Rodriguez-Ubis, Juan-Carlos, Alpha, Béatrice, Plancherel, Dominique, Lehn, Jean-Marie (1984) Photoactive Cryptands – Synthesis of the Sodium Cryptates of Macrobicyclic Ligands Containing Bipyridine and Phenanthroline Groups Helv. Chim. Acta 67, 2264-2269.

Sabbatini, Navida; Guardigli, Massimo; Lehn, Jean-Marie(1993) Luminescent lanthanide complexes as photochemical supramolecular devices. Coordination Chemistry Reviews 123, 201-228. Saha, A.K., Kross, K., Kloszewski, E.D., Upsen, D.A., Toner, J.L., Snow, R.A., Black, C.D.V., Desais, V.C. (1993) Time-resolved fluorescence of a new europium chelate complex: Demonstration of highly sensitive detection of protein and DNA samples. Journal American Chemical Society 115, 11032-11033. Soini, erkki J., Pelliniemi, Lauri J., Hemmilä, Ilkka A., Mukkala, Veli-Matti, Kankare, Jouko J., Fröjdman, Kim. (1988) Lanthanide chelates as new fluorochrome labels for cytochemistry. The Journal of Histochemistry and Cytochemistry 36 (11): 1449-1451. Takalo, H., Mukkala, V., Mikola, H., Liitti, P., and Hemmilä, I. (1994) Synthesis of Europium(III) chelates sutable for labeling of bioactive molecules. Bioconjugate Chemistry 5 (3): 278-282. Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.F., Park, H., Chen, G., Chen, G., and Yu, S. (1996) Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in haimen and fusui, endemic areas of primary liver cancer in china, by highly sensitive immunoassay. Carcinogenesis 17(6):1317-1321. Watanabe, Mariyo F., Park, Ho-Dong, Kondo, Fumio, Harada, Ken-ichi, Hayashi, Hidetake and Okino, Tokio. (1997) Identification and estimation of microcystins in freshwater mussels. Natural Toxins 5, 31-35. WHO (1998) Guidelines for drinking-water quality, 2nd ed. Addendum to Volume 2, Health Criteria and Other Supporting Information. World Health Organization, Geneva. Xu, Y., Hemmilä, I., and Lövgren, T. (1991) Co-fluorescence of Europium and samarium in time-resolved fluorimetric immunoassays. Analyst 116, 155-1158.




CAPÍTULO 3: Europium cryptate gel immunosensor to microcystin-LR Eduardo José Alécio-Oliveira1*, Petrus Santa Cruz2, Severino Alves Junior2, Antonio

Teixeira4, Carla Nunes4, Elizabete Malageño5, Renato J. R. Molica3, José Luiz de Lima

Filho5


Recife-PE, Brazil. 2Departamento de Química Fundamental (DQF) da Universidade Federal de

Pernambuco (UFPE), Recife-PE, Brazil. 3Instituto de Tecnológico do Estado de Pernambuco (ITEP), Recife-PE, Brazil. 4Laboratório Multidisciplinar de Pesquisa em Doença de Chagas da Universidade de

Brasília (UnB), Brazil. 5Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de

Pernambuco (UFPE), Recife-PE, Brazil.

*Corresponding author: Centro Federal de Educação Tecnológica de Pernambuco (CEFET-PE), Av. Profº Luiz Freire, 500, Cidade Universitária, Recife-PE, 50740-540, Brazil. e.mail: [email protected] KEYWORDS: Cyanobacteria, Microcystin-LR, Europium cryptate, polyacrylamide gel





ABSTRACT

Lanthanide cryptates are usually stable complexes and their solubility in aqueous

solutions allows to biological applications. In the present work, the Eu3+ ion was used as

a probe locally with microcystin-LR antibody neighborhood in the following system [Eu ⊂

bpy.bpy.py(CONH2CH2CH2NH2)2]3+-Anti-Microcystin-LR-KLH IgG immobilized in a

super-absorbent polyacrylamide gel (SAPG). The 5D0→7Fn transitions were monitored

by electric dipole transitions highly dependent of the site symmetry and magnetic dipole

ones, they were used as reference to evaluate the interaction antigen-antibody through

hypersensitive transitions. The spectra for 10-5 M free antibody, 10-5 M cryptate and 2:1

toxin concentration showed a specific change in the 5D0 → 7F2 transition in the presence

of free IgG. The conjugated antibody-Europium cryptate was carried out and the 5D0 → 7F2 transition monitored but no differences can be observed yet.




INTRODUCTION

Lanthanide ion complexes could be used as light conversion molecular devices –

LCMD’s. The absorption and energy-transfer emission sequence involve distinct

absorbing (the ligand) and emitting (the lanthanide ion) components (de Sá et al.,

2000).

The lanthanides complexes has been an important research goal and different

class of ligand have been proposed. The Eu (III), Tb (III) and Sm (III) ions have

excellent luminescence properties when chelated with ligands that have broad intense

absorption bands. The chelating agents used with Eu (III) and Tb(III) ion include

cryptands, β-diketonates tridentate pyridine dicarbolylic acids, multidentate pyridine,

bipyridine, terpyridine and their structural analogues as polyaminopolycarboxylates,

cyclodextrins and crown-ether derivatives (Lemmetyinen et al., 2000; de Sá et al.,

2000).

Cryptates are coordination compounds with exeptional thermodynamic and

kinetic stability and are extremely stable in aqueous media (Gasow et al., 1977; Prat et

al., 1991) and have the advantage to the conventional europium chelates which are

subject to dissociation at low concentration and to exchange with other ionic species,

despite the europium ion that cannot be displaced from a cryptate by others metal ions

present in samples or reagents (Prat et al., 1991).

To suitable bioaffinity assays is desired for the chelates of Ln(III) a long excitation

wavelength to avoid background fluorescence, high chemical, photochemical and

kinetic stability, good solubility in water, long luminescence life-time, high luminescence

quantum yield and minor interference in the affinity of the biomolecules (Lemmetyinen

et al., 2000).

Because of its characteristics lanthanide compounds have been utilized in

fluorometric assay (Karsilayan et al., 1997; Prat et al., 1991; Mukkala et al., 1995) and

as probes in time-resolved fluorometry (Prat et al., 1991).




Absorbent gel presents the capacity to absorb great amounts of water and ionic

solutions of low osmolaridade, what it makes possible its use as support for active

biological substances. The polymer net of the absorbent gel has the advantage of its

biocompatibilidade, being compatible with diverse specialized pharmaceutical and

biological applications as: Preparation of pharmaceutical controlled release drugs,

manufacture of catheters and artificial skin, construction of biosensors, beyond several

other commercial applications as in absorbents of personal hygiene as infantile diapers

and products of feminine hygiene (Bucholz & Peppas, 1994).

We report here the use of Eu3+ ion to probe locally the microcystin-LR antibody

neighborhood in the system [Eu ⊂ bpy.bpy.py(CONH2CH2CH2NH2)2]3+-anti-microcystin-

LR-KLH IgG immobilized in a commercial super-absorbent polyacrylamide gel - SAPG. MATERIALS AND METHODS All reagents when not specified were analytical grade; Europium cryptate [Eu ⊂

bpy.bpy.py(CONH2CH2CH2NH2)2] was prepared according to Rodriguez-Ubis et al.

(1984) and Lehn & Roth (1991); The pyridine group was reacted with ethylenediamine

to forming the amide groups and purified by liquid reversed-phase chromatography;

Acrylic acid/Polyacrylamide hydrogel was a commercial product; Microcystin-LR toxin

was purchased from Sigma and sulfo-SMCC from Pierce.

Antibody production and purification

Polyclonal antibodies were produced by conventional methods as described for

Karnikowski (2001). Using Microcystin-LR (Sigma) and keyhole-limpet hemocyanin -

KLH (Sigma) rabbit was immunized with a solution of 500 µg of microcystin-LR-KLH

conjugated in 500 µl sodium phosphate buffer (pH 7.4). The conjugated was mixed with

500 µl Freund’s complete adjuvant (Sigma) and injected subcutaneously at three dorsal

sites. Booster injections consisting of 500 µg antigen were administered intravenously

into the marginal ear vein at days 28 and 56, with collection of 20 ml of blood at days

35, 63 and 93. The blood was clot at room temperature and antisera were colected and

centrifuged at 2,000xg (Micronal centrifuge). The colected antisera were purified in an




affinity column of protein G Pharmacia (HiTrap, Amersham, London, UK) stabilized with

5 ml of 20 mM phosphate buffer (pH 7.0). Portions of 0.5 ml were applied in the

column washed with 5.0 ml of the same phosphate buffer and the fraction of interest

was extracted with 3.0 ml of 0.1M glicina-HCl (pH 2.7) buffer directly in a tube with 0.3

ml of 0.1M tris-HCl buffer (pH 9.0). The antibody solution was reduced to a volume of

approximately 200 µl using a 30 KDa Centricon centrifugal filter unit (Millipore).

Antibodies Activity

Indirect enzyme immunoassay (Karnikowski, 2001) utilizing a 96 well microtiter

plate (Maxisorp Nunc, Roskilde, Denmark) coated with microcystin-LR-chicken

ovoalbumin was utilized to verify the activity of conjugated IgG antimicrocystin-LR-KLH-

Europium cryptate. Pure IgG antimicrocystin-LR-LKH was used as control. The Dilution

for determination of the titres had been carried through from solution with 50 µg of

protein / 50 µl.

Super-Absorbent Polyacrylamide gel Properties

The used polymer was a commercial gel granules with 2 at 5 mm diameter. The

infrared spectrum was carried out in KBr tablets pressed under vacuum and read into

the region of 4000 at 500 cm-1 with resolution of 2 cm-1 in a Bruker model IF66

spectometer.

Gel particle with about 20 mg had been left in contact with 10 ml of the solutions

of 0.1 M NaCl pH 2.9, 7.1 and 10.8 for a period of 2 hours. After this time the solution

was drained and the excess in the surface absorbed with filter paper. Gel masses were

weighed in analytical balance with sensitivity 0.0001g (Ohaus Corporation, USA). The

results were expressed in mg of absorbed solution/mg of gel.

To verify the capacity of SAPG to immobilize protein, were used bovine serum

albumin - BSA and glucose oxidase – GOD as a model. Granules with approximately 10

mg were deeping in 2 ml of bovine serum albumin solution 0.034 mg/ml prepared in

0.1M sodium phosphate buffer (pH 6.5). As control a triplicate of SAPG was tested in




ultrapure water (resistivity > 18,3 MOhm). To each 20 min. one SAPG granule were

removed of the solution and washed with 0.9% (w/v) NaCl solution. The supernatant

and washing waters were pooled and analyzed to the protein concentration utilizing the

method of Bradford (1976) and the immobilized percentual calculated.

With glucose oxidase an granule with approximately 10 mg were immersed in

0.045 mg/ml glucose oxidase - GOD solution prepared in 0.1 M sodium phosphate

buffer (pH 5.9) and left in contact for up to 180 minutes. To each 20 minutes three

SAPG had been removed of the solution washed with 0,9% (w/v) NaCl solution and

cooled at -80ºC. The granules were lyophilized and stored at -18ºC before activity

enzimatic test.

The enzymatic activity of GOD was determined using a glucose kit (Laborlab,

Brazil) with peroxidase in the concentration of 440 U/l. The SAPG were placed in acrylic

cuvets of 3.2 ml, added 2 ml of the color reagent, incubated under soft agitation at

37ºC. To each 20 min. the samples were read in an digital spectrophotometer (Micronal

mod. B442, Brazil) in the wavelength of 505 nm. The activity of the free enzyme was

determined using 100 mg/dl glucose standard solution. Super-Absorbent Polyacrylamide gel antibody neighborhood: Test 1

Two hundred and fifty milliliters of solutions a) Europium cryptate (1x10-5 M) b)

Europium cryptate (1x10-5 M) + IgG antimicrocystin-LR-KLH (1x10-5 M) and c) Europium

cryptate (1x10-5 M) + IgG antimicrocystin-LR-KLH (1x10-5 M) + Microcystin-LR (2x10-5

M) were placed in contact by 60 minutes at ambiente temperature and left in contact

with the SAPG granules (10 mg each).

Spectroscopic measurements After liquid absorption the SAPG granules were analyzed using emission

spectroscopy in the range of 5000 at 7200 Aº. The luminescence spectra were obtained

by scanning with a 1m double-grating Jobin-Yvon U-1000 monochromator. The




excitation wavelength (310 nm) was selected by a 0.25 m Jobin-Yvon H-10

monochromator, using a 150 W Xe-Hg lamp as the excitation source. The light

detection was performed by a water-cooled RCA C31034 photomultiplier tube, the

photocurrent signal being acquired through an EG & G discriminator model 1182 and

digitally stored by a Jobin-Yvon Spectralink interface and a personal computer. This set-

up allows for measurements at room temperature (300 K).

The 5D0→7Fn transitions (J = 0-4) were monitored - electric dipole transitions

highly dependent of the site symmetry and magnetic dipole ones (5D0→7F1) - used as

reference to evaluate the interaction antigen-antibody through hypersensitive

transitions.

Europium Cryptate - IgG Conjugation (Maleimide Method)

Conjugation of the anti-microcystin-LR-KLH IgG to the europium cryptate

understood three main stages: a) Linking of the cryptate to sulfo-SMCC (Sulfo-

succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), b) Reduction of

dissulfide groups of the IgG to form –SH group and c) Linking of the cryptate-SMCC to

activated IgG.

Firstly, 8 mg of the europium cryptate had been dissolved in 5 ml of 20 mM

sodium phosphate buffer (pH 7.4) and mixed with 0.5 ml of 30 mM sulfo-SMCC. The

mixture was kept in reaction for 30 minutes protected of the light. To eliminate the sulfo-

SMCC excess reagent the solution was purified in Fast Performance Liquid

Chromatography -FPLC using a strong anionic Mono-Q HR 5/5 Pharmacia column (Prat

et al., 1991). The column was equilibrated with 5.0 ml of 20 mM sodium phosphate

buffer (pH 7.0) in flow of 0.5 ml/min. The mixture in analysis was injected in volumes of

1.5 mL and the column eluted with 20 mM sodium phosphate, 1M NaCl, 10% (v/v)

dimethyl sulphoxide (pH 7.0). The fractions (0.5 mL) were collected and the absorbance

read at 307 nm.




In the second stage dissulfide groups of the IgG were reduced with dithiothreitol

– DTT. Using eppendorf tube 450 µl of a 6.53 mg/ml IgG solution was added of 5.0 µL

of a 1.0 M DTT solution and kept for 30 min. protected by the light without agitation.

The DTT excess was eliminated by FPLC in a fast desalting column HR 10/10

Pharmacia. The column was stabilized with 50 ml of 20 mM sodium phosphate and 150

mM NaCl (pH 7.0) in a flux of 2 ml/min. Volumes of 200 µL was injected and volumes of

0.5 ml recovered each 15 seconds. The samples were analyzed by protein presence

using the Bradford reaction. Positive protein fractions were pooled and concentrated

centrifugation in 30 kD Centricon centrifugal filter unit (Millipore). After, the volumes

were measured and the protein concentration quantitatively analyzed in a microplate

protein assay.

The third and last stage was the process of linking the europium cryptate

activated with carbodiimide group (SMCC) to the IgG whose sulfhydryl groups were

reduced with DTT. A volume of 2.2 ml of 0.21 mg/ml protein concentration antibodies

was mixed a 1 ml solution contend 553 µg of activated cryptate. The mixture was kept

in reaction for 14 hours at 4ºC under light protected and gently agitation. After that the

sample was purified in HR 10/10 desalt column and the fractions collected analyzed by

spectrophotometry in 238 nm and 307 nm. Super-Absorbent Polyacrylamide gel antibody-Europium cryptate neighborhood: Test 2

Two hundred and fifty milliliters of solutions with a) Europium cryptate-IgG

antimicrocystin-LR-KLH (1x10-5 M) and b) Europium cryptate-IgG antimicrocystin-LR-

KLH (1x10-5 M) + Microcystin-LR (2x10-5 M) were placed in contact and the

fluorescence analyzed as described above in the Test 1.




RESULTS AND DISCUSSION

Gel Properties

The aimed of infrared analysis of SAPG was to demonstrate the presence of

C=O groups characteristic of polyacrylates polymers, while the capacity of absorption in

function of differents pH solution evaluated by gravimetrical method was tested to

evaluate the capacity to absorbing solutions in neutral pH.

The absorption spectra in the infrared presented bands of carbonila groups in

1665 cm-1 as show in Figure 1.

Figure 1: Absorption spectrum in infrared region of commercial sample of polyacrylamide/acrilic acid gel.

The gel showed good capacity to absorb 0.1M saline solution in neutral pH as

show in figure 2. The capacity of the SAPG to absorb a bigger amount of solution in

neutral and basic pH is in accordance with polyacrylimide/acrilic acid gel type because

in acid pH it has a diminished capacity of absorption (Garner et al., 1997).

Figure 2: Absorption of 0.1M saline solution for SAPG in diferent values of pH.

0,4

0,5

0,6

0,7

0,8

0,9

5001000150020002500300035004000

Wave numbers (1/cm)

Tran

smita

nce

(%)

02468

101214161820

mg

NaC

l 0,1

M /

mg

SAPG

2.9 7.1 10.8pH




The SAPG showed to be capable to hold back bovine serum albumin in polymer

net. A maximum of 37,7% of protein retention was observed in 40 minutes, with

reduction of 20% of the restrained amount from 60 minutes as show in Figure 3. This

phenomenon probably occurs by net expansion and effect of concentration balance

between external and intern system. Accord to Bucholz & Peppas (1994) the amount of

pure water that can be absorbed is typically around 400x the wight of polymer.

Figure 3: Percentual retention of bovine serum albumin in the SAPG in function of time contact.

The capacity of SAPG in to keep the biological activity using the enzyme glucose

oxidase showed a constant activity of the enzyme in a period of 180 minutes (0.90

U/mg).

The commercial gel used here showed good compatibility with biological activity

as praised for Buchholz & Peppas (1994) with the advantage of a passive

immobilization. In the case of gel polyacrylamide synthesis it has the disadvantage of

the manipulation of acrylamide, a neurotoxicity and probably carcinogenic activity

product (IARC, 1994). Additionaly, immobilization during gel synthesis can submited the

biological material to high temperatures or extreme conditions of pH with inactivation

(Buchholz & Peppas, 1994)

Super-Absorbent Polyacrylamide gel Immunosensor

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

0 20 40 60 80 100 120 140 160 180 200

Time contact (minutes)

Imob

ilize

d Pr

otei

n (%

)




Test to identify the microcystin-LR presence with the antibody neighborhood in

the system bpy.bpy.py(CONH2CH2CH2NH2)2]3+ + Anti-Microcystin-LR-KLH IgG

immobilized in a super-absorbent polyacrylamide gel - SAPG showed a specific change

in the transistion 5D0 à 7F2 in the presence of IgG (Fig. 4) but no significative

differences was observed by the interaction of microcystin-LR with IgG.

In the spectrum of Figure 4 was expecteted a significative signal resultant of

interaction antigen-antibody with an modification in the sensitive transistion 5D0 à 7F2

that is highly dependent of the site symmetry and magnetic dipole ones. The absence of

a significative signal when of the immunological interaction can be occurred by a very

weak linking of IgG with europium cryptate. This situation can have been incapable to

provoke alteration of symmetry in the europium cryptate by IgG neighborhood, also

considering the low molecular weight of microcystin-LR (995 g/mol).

Figure 4: Emission spectrum of SAPG with europium cryptate alone, IgG - Antimicrocystin – LR - KLH + Europium cryptate and in the presence of microcystin-LR. Excitation wavelength 310 nm.

Wavelenght (A)

Wavelenght (A)




Anti-Microcystin-LR-KLH Europium Cryptate Conjugated Atibodies

In this stage was verified the effect of antigen-antibody interaction in the trasition 5D0 à 7F2 using the IgG-europium cryptate conjugate. The use of well know

heterobifunctional reagent allows the conjugation to antibodies withowt loss of

immunoreacitivity (Mathis, 1993). The fractions between 60 and 105 seconds resulting

of the purification stage (Fig. 5) were collected and concentrated in 30 KDa Centricon

centrifugal filter unit (Millipore). The cryptate presence in the conjugated compouds was

observed by the typical fluorescence when the concentrated solution was displayed to

the U.V. of long wavelength (360 nm).

Figure 5: Elution of IgG anti-microcystin-LR-KLH-Europium cryptate conjugate obtained by maleimide method. Elution with 20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA (pH 6.5) in a fast desalting column HR 10/10 Pharmacia The conjugated antibodie-cryptate showed similar activity to the one of the

antibodie before conjugation (control). Titre until 1/128 dilutions (-log10 = 2.1) was

observed as depicted in Figure 6.

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

15 45 75 105 135 165 195 225 255 285

Time (seconds)

UA

bs

0,00

0,02

0,04

0,06

0,08

0,10

0,12

UA

bs

280 nm

307nm




Figure 6: Anti-microcystin-LR-KLH-Europium cryptate titre using ELISA plate coated with microcystin-LR-chiken ovoalbumin. Initial dilution was a 50 µg protein / 50 µl antidobie solution. (Vertical error bar represent satnadar deviation, (n = 3)

The conjugate solution presented the emission spectrum between 6100 and

6200 Ao characteristic of the 5Do à 7F2 europium electric dipole transiction (Fig. 7), but

not difference in the signal was observed when 1x10-5 M solutions (calculed by protein

concentration) were analysed with microcystin-LR (2x10-5 M) in SAPG. The inensity of

observed fluorescence was very small probably because low proportion of cryptate in

the complex or insufficiente U.V. excitement. Is necessary verify the emission spectrum

with high intensity energy excitation as nitrogen laser pulser at 337 nm as cited in other

works (de Sá et al., 1998; Moutereau & Rymer, 1998; Mathis, 1993)

Figure 7: Emission spectrum of conjugated IgG antimicrocystin-LR-KLH-Europium cryptate. Excitation in 315 nm with deuterium lamp.

6100 6120 6140 6160 6180 6200

200

400

600

800

1000

1200

1400

1600

1800

I(cts

/s)

Wavelength (A)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0 1 2 3 4-log10 Igg dilution

Abs

orba

nce

(492

nm

) Conjugated

Control




REFERENCES Bradford, Marion M. (1976) A rapid and sensitive method for the quantitation microgram quantities of protein utilizing the principle of protein-Dye bindig. Analytical Biochemistry 72, 248-254. Buchholz, Frederic L.; Peppas, Nicholas A. (eds.) Superabsorbent Polymers, Washington: American Chemical Society, 1994. de Sá, G.F.; Malta, O.L.; Donegá, C. de Mello; Simas, A.M.; Longo, R.L.; Santa-Cruz, P.A.; da Silva Jr., E.F. (2000) Spectroscopic properties and design of highly luminescent lanthanide coordination complexes. Coordination Chemistry Reviews 196, 165-195. de Sá, Gilberto F.; Alves Jr., Severino; Silva, Blenio J.P. da; Silva Jr., Eronides F. da (1998) A novel fluorinated Eu(III) β-diketone complex as thin film for optical device applications, Optical Material 11, 23-28. Gasow, Otto A.; Kausar, A. Rashid; Kelly, M. (1977) Synthesis and Chemical Properties of Lanthanide Cryptates . J. Am. Chem. Soc. 99, 7087-7089. Garner, Charles M.; Nething, Matthew; Nguyen, Phuc. (1997) Synthesis of a superabsorbent polymer, Journal of Chemical Education 74 (1): 95-96.

IARC - International Agency for Research on Cancer - Summaries & Evaluations (1994) 60, p. 389 Disponible in: http://www. http://www.inchem.org/documents/iarc/vol60/m60-11.html , last updatee 08/26/1997. Access in: 10/20/2003. Karnikowski, M.G.O. Desenvolvimento e padronização de teste imunoenzimático para diagnóstico de microcistinas. Tese de Doutorado, Doutorado em Patologia Molecular – Faculdade de Medicina, Universidade de Brasília, Brasília – DF, 2001. Karsilayan, Huriye; Hemmilä, Ilkka; Takalo, Harri; Toivonen, Airi; Pettersson, Kim; Lövgren, Timo; Mukkala, Veli-Matti. (1997) Influence of coupling method on the luminescence properties, coupling efficiency, and binding affinity of antibodies labeled with europium(III) chelates. Bioconjugate Chemistry 8 (1): 71-75. Lehn, Jean-Marie; Roth, Christine O. (1991) Synthesis and Properties of Sodium and Europium (III) Cryptates Incorporating the 2,2´-Bipyridine 1,1´-Dioxide and 3,3´-Biisoquinoline 2,2´-Dioxide Units. Helvetica Chimica Acta 74, 572-578.

Lemmetyinen, H.; Vuorimaa, E.; Jutila, A.; Mukkala, Veli-Matti; Takalo Harri; Kankare, Jouko (2000) A time-resolved study of the mechanism of the energy transfer from a ligand to the lanthanide (III) ion in solutions and solid films. Luminescence 15, 341-350.


http://www.inchem.org/documents/iarc/vol60/m60



Moutereau, S.; Rymer, J.C. (1998) Kryptor – Évaluation finale en situation de routine. Immunoalanal. Biol. Spéc. 13, 307-316. Mukkala, V.-M.; Takalo, H.; Liitti, P.; Hemmilä, I. (1995) Synthesis and luminescence properties of some Eu(III) and Tb(III) chelate labels having 2,2’:6’,2”-terpyridine as an energy absorbing part. Journal of Alloys and Compounds 225, 507-510. Mathis, Gérard (1993) Rare earth cryptates and homogeneous fluoroimmunoassays with human sera. Clinical Chemistry 39/9, 1953-1959. Prat, Odette; Lopez, Evelyne; Mathis, Gérard. (1991) Europium (III) cryptate: A fluorescent label for the x detection of DNA Hybrids on solid support. Analytical Biochemistry 195, 283-289. Rodriguez-Ubis, Juan-Carlos ; Alpha, Béatrice; Plancherel, Dominique; Lehn, Jean-Marie (1984) Synthesis of the Sodium Cryptates of Macrobicyclic Ligands Containing Bipyridine and Phenanthroline Groups. Helvetica Chimica Acta 67, 2264-2269.




4. CONCLUSÕES

Capítulo 1:

• A técnica dot blot desenvolvida foi eficaz para a análise visual de amostras de

água purificada e água bruta (lago) incriminadas com microcistina-LR na

concentração de 0,16 a 5,0 µg/L; As amostras não necessitaram de

concentração ou limpeza (clean-up) antes da análise.

• A sensibilidade do ensaio dot blot visual permitiu a detecção de concentrações

de microcistina-LR inferiores a 1µg/L, valor limite para água potável especificado

na legislação brasileira (Portaria 1469/2000 – MS) e recomendada pela

Organização Mundial da Saúde.

• O programa (software) para quantificar os ensaios dot blot apresentou

concordância entre a intensidade dos pixels e a concentração protéica (BSA),

com faixa linear entre 0 e 78 µg/mL (r = 0,9735).

• As curvas padrões em água purificada e água bruta (lago) obtidas com os

ensaios dot blot informatizado, possibilitou a análise de microcistina-LR na faixa

de 0,16 a 10,0 µg/L, com melhores correlações entre 0,16 e 1,6 µg/L.. Também

foi possível determinar a concentração de microcistina em extrato da cepa

Microcystis aeruginosa – NPJB-1.




Capítulo 2:

• Foi sintetizado um novo marcador fluorescente microcistina-LR-Criptato de

térbio capaz de ser reconhecido pela IgG anti-microcistina-LR-KLH em

ensaio ELISA. Este marcador tem uso potencial para aplicação em ensaios

imunofluorescentes direto ou resolvido no tempo.

Capítulo 3:

• O gel superabsorvente de poliacrilaminda/ácido acrílico é um suporte

promissor para a construção de um sistema imunosenssor para microcistina-

LR em amostras de água.




5. CONSIDERAÇÕES FINAIS E PERSPECTIVAS CIENTÍFICAS A utilização de técnicas consideradas convencionais, para o desenvolvimento de

métodos em áreas ainda não exploradas, pode contribuir para a obtenção de

aplicações com menores custos de investimento e retorno mais rápido. Os ensaios

imunológicos para a detecção de microcistinas têm sido utilizado com sucesso; São

atualmente os mais adaptados aos ensaios em amostras naturais, a exemplo das

técnicas ELISA em microplaca e tubos de ensaio, colunas de imunoafinidade e

fluoroimunoensaios diretos ou resolvidos no tempo como TR-FIA (Time-resolved

fluoroimmunometric assay) e TRACE (Time Resolved Amplification of Cryptate

Emission).

O crescente aumento da presença de cianotoxinas em mananciais hídricos,

resultado de florações causadas pela eutrofização dos reservatórios, torna necessário

uma rápida difusão de sistemas de controle e monitoramento que permitam o manejo

seguro da água destinada ao consumo humano e animal.

Um de nossos trabalhos permitiu a adaptação da técnica dot blot à determinação

da concentração de microcistina-LR em amostras de água de superfície e água

purificada. O princípio da precipitação da diaminobenzindina – DAB pela ação da

peroxidase em meio contendo imidazol permitiu a observação visual da reação a olho

desarmado, sem a necessidade de equipamentos sofisticados.

O crescente acesso em massa aos produtos informatizados tem permitido a

construção de equipamentos com bom desempenho e custo acessível. Foi desta

observação que verificamos a possível aplicação de um “scanner” comercial em

substituição ao sofisticado densitômetro na quantificação dos dots. Assim, foi possível

quantificar a microcistina-LR em água e extratos celulares de Microcystis aeruginosa.

Os resultados mostraram-se promissores, permitindo a construção de curvas padrão

utilizando um programa (“software”) desenvolvido para este fim. É necessário

considerar que apenas uns poucos padrões de microcistinas (-LR, -RR, -YR) são

comercializados, e que testes de reações cruzadas para o anticorpo policlonal utilizado




nesta pesquisa devem ser realizados. Para a utilização como método oficial ou kit

comercial a validação através de ensaios intra e inter-laboratoriais é essencial; A

utilização de uma técnica ELISA ou HPLC já validada deve ser utilizada como

referência para um estudo comparativo.

Complexos luminescentes de lantanídeos têm sido cada vez mais aprimorados

para uso em técnicas de espectrofluorimetria, na forma de dispositivos moleculares

conversores de luz (Light Conversion Molecular Devices – LCMD). Técnicas de ELISA

(Enzyme-Linked Immunosorbent Assay) direta, onde o antígeno é marcado com a

molécula sinalizadora e o anticorpo específico imobilizado na superfície adsorvente,

são mais rápidas, porém o marcador fluorescente deve apresentar alta intensidade

luminescente. Os complexos de lantanídeos, e em particular os criptatos, apresentam

alta intensidade de emissão além de tempo de vida de emissão prolongado, o que

permite a utilização em técnicas de fluorescência direta e resolvidas no tempo (TR-FIA

, TRACE).

A obtenção do conjugado microcistina-LR-Criptato de térbio vem contribuir com

um marcador de microcistina-LR fluorescente para aplicação em técnicas imunológicas.

A síntese foi obtida de forma simples utilizando o espaçador etilenodiamina,

aprensentando ao final um composto com atividade imune verificada em teste ELISA.

As limitações no uso da técnica resolvida no tempo encontra-se, ainda, na sofisticação

das leitoras que utilizam fonte de laser de nitrogênio e fotomultiplicadoras de custo

elevado. Complexos com íon Tb3+ apresentam perda de rendimento quântico

luminescente provocado por processos não-radiativos de retorno de energia transferida

(energy back-transfer); De acordo com a posição dos níveis excitados do ligante e do

metal este fenômeno é mais ou menos intenso; A fenda de energia (energy gap) entre

o estado tripleto excitado do ligante doador e o nível emissor do íon Tb3+ deve ser larga

o suficiente para previnir o retorno de energia transferida, do íon excitado para o

ligante; Quanto maior esta diferença de energia, melhora a eficiência da emissão. Nos

criptatos o fenômeno de “energy back-transfer” é reduzido, porém as propriedades

luminescentes do conjugado microcistina-LR-Cripatato de térbio devem ser ensaiadas

para demonstrar a correlação entre o estado tripleto de energia e seus tempos de

decaimento e eficiência luminescente.




A hipótese da modificação da transição hipersensitiva (5D0 à 7F2) do criptato -

Eu3+ ancorado em gel superabsorvente, após a interação microcistina-LR – IgG, foi

verificada. Primeiro pela influência da interação com o anticorpo não ligado ao criptato

e, em seqüência, sob a influência da IgG covalentemente ligada ao criptato. Os

resultados ainda não foram conclusivos, mas esta linha de pesquisa mostra-se

bastante promissora e pode direcionar à obtenção de sensores simples e baratos para

a análise qualitativa, semi-quantitativa e quantitativa de contaminantes em água.




6. OUTROS TRABALHOS COM PARTICIPAÇÃO DO AUTOR 6.1. NEUROTOXIC CYANOBACTERIA BLOOM IN A BRAZILIAN NORTHEAST TROPICAL RESERVOIR (Em preparação) R. J. R. Molica1,4*, E. J. Alécio-Oliveira2, P. V. V. C. Carvalho3, A. N. S. F. Costa3, M. C.

C. Cunha1, G. L. Melo1, S. M. F. O. Azevedo4.

1Instituto de Tecnologia de Pernambuco (ITEP), Av. Prof. Luis Freire 700, Recife-PE, 50.740-540, Brazil 2Centro Federal de Educação Tecnológica de Pernambuco, (CEFET), Av. Prof. Luis Freire 500, Recife-PE, 50740-540, Brazil 3Companhia Pernambucana de Sanamento – COMPESA, Gerência de Controle de Qualidade, Largo Dois Irmãos 1012, Dois Irmãos, Recife-PE, 52.071-440, Brazil 4Instituto de Biofísica Carlos Chagas Filho, CCS, Blogo G, UFRJ, Ilha do Fundão, Rio de Janeiro, 21949-900, Brazil *Corresponding author: Instituto de Tecnologia de Pernambuco, Laboratório de Ecofisiologia de Microalgas, Av. Prof. Luis Freire 700, Cid. Universitária, Recife-PE, 50.740-540, Brasil. Fone: 55 81 3272-4264 Fax: 55 81 3272-4287 e.mail: [email protected]





ABSTRACT

The blooms of toxic cyanobacteria are very common in Brazilian waterbodies as

a consequence of eutrophication process. This situation is more critical in the Northeast

of Brazil, a region subject to recurrent periods of droughts and consequently with a

greater number of surface reservoirs to store water. In this work we have investigated

the presence of neurotoxins during a cyanobacterial bloom in Tapacurá reservoir, which

is used to supply water to Recife city. It is a eutrophic system with frequent blooms of

cyanobacteria. We also investigated the presence of neurotoxins in strains of Anabaena

spiroides isolated from this reservoir. Samples were collected from March to May 2002

at the surface close to the dam. Cyanobacteria density was analyzed. The samples

were assayed for toxicity by mouse bioassay, acetylcholinesterase inhibiting activity by

colorimetric method and saxitoxins (paralytic shellfish toxins) by HPLC-FLD postcolumn

derivatization method. The dominant species during the bloom were A. spiroides,

Pseudoanabaena sp. Cylindrospermopsis raciborskii and Microcystis aeruginosa. The

mouse bioassays have showed the presence of neurotoxins during A. spiroides and C.

raciborskii dominance but an antiacetylcholinesterase activity only during A. spiroides

dominance. The A. spiroides strains have also demonstrated posses an

acetylcholinesterase inhibitor. HPLC-FLD chromatograms reveled the presence of

saxitoxin, neosaxitoxin and dc-saxitoxin, probably produced by C. raciborskii. The

presence of saxitoxins and anatoxin-a(s)-like in a drinking water supply alerts the local

authorities for the possibility of the presence of these high toxic compound in potable

water.

KEYWORDS: Anabaena spiroides, Cylindrospermopsis raciborskii, acetylcholinesterase inhibitor, anatoxin-a(s), saxitoxin, cyanobacterial bloom, Tapacurá reservoir.




INTRODUCTION

Eutrophication favors occurrence of cyanobacterial blooms and many of which

are toxic (Bartram et al., 1999). Animal poisonings by toxic cyanobacteria are

extensively recorded in the literature (Carmichael, 1992). The most serious event

involving human being took place in Caruaru city, Northeast Brazil, when 76 people died

during hemodialysis treatment (Jochimsen et al., 1998; Carmichael et al., 2001). The

toxic compounds produced by cyanobacteria can be grouped in two classes according

to the targets of their toxic actions as hepatotoxins and neurotoxins (Sivonen, 1996).

The first one includes microcystins and nodularins, cyclic peptides that inhibit protein

phosphatases type 1 (PP1) and type 2 (PP2A) and cylindrospermopsin, an alkaloid

suppressor of protein synthesis (Sivonen and Jones, 1999). Neurotoxins include

anatoxin-a, a postsynaptic cholinergic nicotinic agonist, anatoxina-a(s), an inhibitor of

acetylcholinesterase activity and PSP toxins (saxitoxins), sodium channel blockers in

the nerves (Sivonen and Jones, 1999).

Cyanobacterial blooms are very frequent events in drinking water supplies in

Brazil (Yunes et al., 1996; Souza et al., 1998; Bouvy et al., 2000; Huszar et al., 2000)

and the cyanotoxins registered to occur in Brazilian waterbodies until today are

microcystins (Azevedo et al., 1994; Yunes et al., 1996; Matthiensen et al., 2000),

saxitoxins (Lagos et al., 1999; Molica et al., 2002) and anatoxin-a(s)-like (Monserat et

al., 2001). Cylindrospermopsin was only detected in a carbon from a hemodialysis clinic

in Caruaru (Carmichael et al., 2001) and anatoxin-a has never been reported.

In this report we present results of presence of saxitoxins and anatoxin-a(s)-like

during a cyanobacterial bloom in Tapacurá reservoir. This eutrophic waterbody is

located in Pernambuco state - Northeast of Brazil - and it is used to supply water to

Recife city (Braga, 2001; Bouvy et al., 2003). The local Water Company has been

realizing a water-monitoring program in Tapacurá reservoir since 2001 including

cyanobacteria cells count, toxicity measurements and some limnological parameters.

We also investigated the presence of neurotoxins in cultures of Anabaena spiroides

isolated from this reservoir.




MATERIALS AND METHODS Sampling site, analytical procedures and nutrients

Sampling was carried out weekly from 19 March to 10 April and from 8 May to 30

May at the surface close to the dam of Tapacurá reservoir. The interruption on sampling

between 10 April and 8 May was due a technical problem. The conductivity and pH

were recorded using specific electrodes. Water samples for nutrients (NH4-N, NO3-N,

NO2-N and total phosphorus) determinations were transported under refrigeration to the

laboratory. Analysis were performed according to APHA (1992).

Cyanobacteria identification and counts

Cyanobacteria species were identified by light microscopy and cells counts were

performed using a Sedgwick-Rafter Cell.

Isolation and culture

Non-axenic cultures of A. spiroides were initiated from a bloom sample by

transferring successively single trichomes with a Pasteur pipette to drops of culture

media and at last to a culture tube containing approximately 5 ml sterile ASM-1 medium.

The three strains (ITEP-024, ITEP-025 and ITEP-026) were maintained under 26oC +

2oC with a photon flux density of 40 µmol photon flux m-2 s-1 (Biospherical Instruments

QSL-100) from cool-white fluorescent tubes on a 12:12 light:dark cycle. The strains

were cultivated in 2 L Erlenmeyers flasks containing 1.5 L ASM-1 medium (Gorham et

al., 1964), under 80 µmol photon flux m-2 s-1 with a 12:12 h light:dark cycle and aeration.

Cultures were harvested at the late exponential phase by centrifugation. The cell

material was lyophilized and stored at -18oC.

The measurements of A. spiroides were made taking 50 vegetative cells,

heterocytes, akinets, spirals wide and distance between coils of each strain and natural

population.




Toxins extraction and mouse bioassays

The toxicity of bloom samples and A. spiroides strains were tested by i.p.

injection in at least three Swiss Webster mice weighting 15-22 g.

Bloom samples were collected in 5 liters plastic bottles and vacuum-filtered

through 47 mm fiberglass filters and the volumes were recorded. The toxins were

extracted disrupting cells by adding 4 mL of 0,9% NaCl pH 4.0 (adjusted with HCl 0.1

M) to the filters and submitted them to freeze-thawed process. The saline solution was

separated from the filters by centrifugation (8,014 g, 10 min). The doses injected in

three mice varied from 150 to 520 mg Kg-1 (mg of dry weight of seston per Kg of mice

body weight). The dry weights of bloom material (seston) were determined by filtering

know volumes through tared 25 mm fiberglass filters, which were then dried for 24 h at

100oC and weighted.

Lyophilized culture material was resuspended in 0.9% NaCl pH 4.0 (adjusted with

HCl 0.1 M) and sonicated (High Intensity Ultrasonic Processor - 50W Model) during 1

min at low temperature. Two or three doses in the range of 14 to 156 mg Kg-1 were

injected i.p. into mice.

Animals were observed for poisoning symptoms during the first 2 hours and

deaths were recorded until the first 24 hours. Control animals were injected with 1 mL of

0.9% NaCl (pH 4.0).

Acetylcholinesterase assay

All the chemicals products were of analytical grade purity. Eel

acetylcholinesterase (AChE), propionylthiocoline iodide, 5,5’-dithiobis- (2-nitro) benzoic

acid (DTNB) and eserine were purchased from the Sigma Co. (St. Louis, MO, USA).

The centrifuged saline aqueous extracts of bloom samples were used for the

assay. The lyophilized culture material of the three strains (25 mg) were extracted in

ethanol:1M acetic acid (20:80) solution for 4 hours and centrifuged as described by

Henriksen et al. (1997).




The in vitro inhibition of acetylcholinesterase (AChE) was determined by Ellman

method (Dietz et al., 1973) using propionylthiocoline iodide (10 mM) as a substrate.

The capacity of inhibition of eel AChE by extracts was determined by incubation of

enzyme in an end point assay. The strains extracts were diluted 1:10 in saline solution

and bloom extracts tested without dilution. All activity assays were made in triplicate.

Eel AChE solution (100 µL) and extracts (10 µL) were incubated 2 min at 37ºC.

Then 290 µL of PBS and 1200 µL of DTNB (0.423 M) were added and incubated for 5

min. Substrate (400 µL) were added and incubate more 3 min at 37ºC. The reaction

was stopped with 80 µL of eserine (2.4 mM) and absorbance read at 410 nm (Hitachi U-

2000). The positive control (PBS) and blank reaction were assayed during the period of

the test. For negative control eserine (80 µL) was added first and PBS instead extract

solution. The final absorbance at 410 nm was utilized as inhibition measurement.

PSP toxins analysis

The saline aqueous extracts of bloom samples and lyophilized culture material

utilized for mice bioassay were analyzed for the presence of the three classes of

saxitoxins (C toxins, gonyautoxins and saxitoxin) separately according to Oshima

(1995). The Shimadzu HPLC system used a silica-base reversed phase column (125 x

4.0 mm, 5µm; Lichrospher 100 RP-8e) and separations were carried out under specifics

mobile phases for each group of saxitoxins. Samples were filtered through 0.22 µm

Millex (Millipore) prior injection. Post column oxidation also followed the method of

Oshima (1995) excepted for the temperature of the oven (Shimadzu CTO-10Avp) that

was 80o C. Fluorescent saxitoxins derivatives were detected using a Shimadzu RF-

10Axl fluorometric detector with excitation at 330 nm and emission at 390 nm. Toxins

were identified and quantified by comparison of retention times and integrated areas

with standards, respectively. Standards (Reference Material) of saxitoxins (saxitoxin,

neosaxitoxin, GTX1/4 and GTX2/3) were purchased from Institute for Marine Bioscience

– National Research Council of Canada (Halifax, Canada). A mixture of saxitoxin,

neosaxitoxin and dc-saxitoxin of unknown concentration and C1/C2 toxins were kindly

provided by Dr. Nestor Lagos of Chile University (Chile).




RESULTS

Environmental parameters and nutrients

The pH was predominantly alkaline and as conductivity showed a tendency to

decrease, but progressively rises up from mid-April to the end of the survey (Fig.1).

Nitrite and ammonium were always below the detection limit (d.l.) of the method (25 and

40 µg L-1, respectively) except on 24 May when ammonium concentration was 40 µg L-1

and 15 May when nitrite concentration was 150 µg L-1. Nitrate (d.l. 50 µg L-1) was not

detected during the entire period and total phosphorus varied in the range of 20 to 228

µg L-1 (Fig. 1).

Figure 1: Environmental parameters in Tapacurá reservoir from 19 March to 30 May 2002. (A) pH; (B) Conductivity and (C) = Total phosphorus.

0

50

100

150

200

250

P-P

O43-

(mg

L-1)

7

7,4

7,8

8,2

8,6

9

pH

420

430

440

450

460

470

19/03

27/03

04/04

12/04

20/04

28/04

06/05

14/05

22/05

30/05

µS. c

m-1




Cyanobacteria species composition during the bloom

In general, a bloom is considered as a drastic development of one or two species

of microalgae. We observed a cyanobacterial bloom with a succession of different

species in Tapacurá reservoir. It was recorded from mid-March to the end of May and

during this period the average number of cyanobacterial cells was about 140,000 cells

mL-1 (Fig 2). The monitoring program realized at Tapacurá reservoir just managed

cyanobacteria species, thus the data about other phytoplankton group are not available.

On 19 March A. spiroides was the dominant species representing 72% (approximately

1.4 x 105 cells mL-1) of total cyanobacteria and their number gradually declined until 3

April when Pseudoanabaena sp. become the dominant species. Cylindrospermopsis

raciborskii started to increase at end of March and dominate until 10 April. The samples

between 10 April and 8 May could not be collected but C. raciborskii dominance

continued to be registered from 8 to 15 May. The last part of the bloom was dominated

by Microcystis aeruginosa constituting 66% to 82% (3.7 and 2.7 x 104 cells mL-1,

respectively) of cyanobacterial cells. Other cyanobacterial species were observed

during the bloom but their numbers were not expressive (Fig. 2). Raphidiopsis-like was

included in the enumeration of C. raciborskii because the difficult to separate them as

recommended by McGregor and Fabbro (2000).




Figure 2: Cyanobacteria composition and concentration (cell number mL-1) during a bloom in Tapacurá reservoir. Anabaena spiroides, Anabaena sp., Synechocystis sp., Planktothrix sp., Merismopedia sp., Microcystis aeruginosa,

Gomphosphaeria sp., Pseudoanabaena sp. and Cylindrospermopsis raciborskii. Characterization of isolated strains

Three clones of A. spiroides were isolated and cultivated. Under the culture

conditions the organism formed solitary trichomes, coiled, with many aerotopes, spirals

32-62 µm wide and distance between the coils 10-50 µm. In general all the taxonomic

characteristics of strains and natural population agreed well with those described for A.

spiroides. The only exception was the strain ITEP-026, which spirals wide and distance

between coils were smaller (Table I).

19/M

ar

27/M

ar

03/A

pr

10/A

pr

08/M

ay

15/M

ay

24/M

ay

30/M

ay

0

50000

100000

150000

200000C

ell n

umbe

r m

L-1




Toxicity of bloom samples and isolated strains

Mouse bioassay demonstrated a neurotoxic response of bloom material from 19

March to 24 May except the sample collected on 03 April that was not toxic. The time of

death varied from 3 to 12 min. Anatoxin-a(s)-like anticholinesterase symptoms,

including salivation and limbs fasciculation, were only observed for sample collected on

27 March (Table II).

The acetylcholinesterase assay reveled an AChE inhibitor activity in samples

collected on 19 and 27 March with 100 and 88% of inhibition, respectively. This period

coincided with A. spiroides dominance. All other samples did not show AChE inhibition

(Table II).

The three isolated strains were toxic in mouse bioassay and the observed

symptoms were typical for that caused by anatoxin-a(s). The AChE enzymatic assay

reveled a 100% of inhibition for strains extracts (Table II).

HPLC-FLD analysis

HPLC-FLD chromatograms reveled the presence of saxitoxins variants only in

sample collected on 8 May. The peaks had retention times identical to those of the

standards (Fig. 3). The variants identified were saxitoxin (Stx), neosaxitoxin (NeoStx)

and dc-saxitoxin (dc-Stx). The concentrations calculated by comparison of peak areas

with those of the standards were 52 and 51 ng L-1 for Stx and NeoStx, respectively.




Table 1. Morphological features of three strains and natural population of A. spiroides from Tapacurá reservoir. Cell lenght

(µm) Cell width

(µm) Heterocyst length (µm)

Heterocyst width (µm)

Akinetes length (µm)

Akinetes width (µm)

Spirals width (µm)

a Between coils (µm)

N.P 4.87 ± 0.70 5.47 ± 0.50 6.18 ± 0.7 6.55 ± 0.7 10.05 ± 1.0 10.01 ± 1.0 42.90 ± 7.10 32.60 ± 11.80 ITEP-024 5.30 ± 0.52 5.95 ± 0.57 7.22 ± 1.0 7.09 ± 0.8 9.04 ± 1.3 9.24 ± 1.0 42.80 ± 6.26 30.20 ± 11.02 ITEP-025 5.28 ± 0.50 6.04 ± 0.50 7.88 ± 0.6 7.80 ± 0.7 N.D N.D 39.85 ± 4.70 24.35 ± 8.59 ITEP-026 5.03 ± 0.40 5.60 ± 0.50 7.21 ± 0.6 7.33 ± 0.6 N.D N.D 14.30 ± 3.71 32.00 ± 3.81

b - 6 – 8 - 6 - 8 17 - 21 10 - 14 20 - 45 20 - 45

A. spiroides

c - 6.5 – 8 - 6 - 7 13 - 18 8 - 9 30 - 40 30 - 48 NP – natural population ND – not determined a Distance between coils b Komárková.-Legnerová & Eloranta (1992) c Sant’Anna & Azevedo (2000) Table 2. Toxicity and achetylcholinesterase inhibition assay of bloom samples and A. spiroides strains.

Date Injected Dose (mg Kg-1)

Toxicity by mouse bioassay

Acetylcholinesterase assay - % inhibition

Symptoms

19 March 519.4 + 100 Convulsions and respiratory arrest 27 March 495.2 + 88 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation 03 April 276.4 - 0 No observed symptoms 10 April 319.3 + 0 Trembling head, convulsions, jumping and respiratory arrest 08 May 493.0 + 0 Trembling head, convulsions, jumping and respiratory arrest 15 May 149.1 + 0 Trembling head, convulsions, jumping and respiratory arrest 24 May 444.4 + 0 Trembling head, convulsions, jumping and respiratory arrest 30 May 253.0 - 0 No observed symptoms ITEP-024 148.4 + 100 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation ITEP-025 150.4 + 100 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation ITEP-026 156.1 + 100 Salivation, tremors, convulsions, respiratory arrest and limbs fasciculation



Alécio-Oliveira, Eduardo José Desenvolvimento de métodos para... Figure 3: HPLC-FLD chromatograms of (A) 8-May bloom sample, (B) standards (reference material) and (C) mixture of standards of unknown concentrations.

It was not possible to calculate dc-Stx concentration because its concentration

on mixture standards was unknown. When analyzed replacing the oxidizing reagent

by water the peaks changed in the same manner as with the respective standards

toxins. NeoStx increased several fold while Stx and dc-Stx almost disappeared (data

not shown). No matched peaks were detected under the conditions for Gtx and C

toxins (data not shown). Moreover, no suspected peaks of these groups of saxitoxins

variants were observed in the chromatograms. The three A. spiroides strains were

also analyzed for the presence of saxitoxins but no suspicious peaks were observed.

Minutes

A

B

C




DISCUSSION

The Northeast Brazil is subjected for periods of drought especially in years of

El Niño phenomenon. Man-made reservoirs, most designed for multiple purposes,

including drinking water sources, are very common in this region as a consequence

of this meteorological characteristic (Bouvy et al., 1999). Many of these waterbodies

receive a high level of nutrients from human activities – mainly industry and urban

effluents – creating favorable conditions for cyanobacterial blooms (Bouvy et al.,

2000). Since the hemodialysis accident in Caruaru city (Azevedo et al., 2002) more

attention has been given for the presence of cyanobacteria in drinking water supplies

in Pernambuco state. Tapacurá reservoir supplies water for about 1.35 million

inhabitants in Recife city and as cyanobacterial blooms are recurrent in this

environment (Nascimento et al., 2000; Ferreira, 2002) the local Water Company

realizes a monitoring program due to the potential of human health consequences.

Bouvy et al. (2003) through a study during two years (May 1998 – May 2000)

showed that Tapacurá reservoir is a eutrophic environment. These authors observed

concentrations as high as 124 µg L-1 chlorophyll-a, 200 µg L-1 of particulate

phosphorus and 2 mg L-1 of ammonium. In our study the concentration of total

phosphorus was also high and ranged from 20 to 228 µg L-1 (Fig.1C). On the other

hand, the level of ammonium was nearly always below the detection limit. The lack of

dissolved nitrogen show a nitrogen limitation. According to Sas (1989) concentrations

lower than 100 µg L-1 would be considered limiting. The dominant species during the

bloom were the nitrogen-fixing A. spiroides and C. raciborskii. The exception was

Pseudoanabaena sp. which dominance was during a short period of time. Low

concentration of ammonium was detected in 24 May coinciding with M. aeruginosa

non-nitrogen fixing species dominance.

According to Reynolds et al. (2002) A. spiroides can be classified in a H1

assemblage – a separation of group H - that includes heterocytic cyanobacteria. C.

raciborskii was previously grouped in H, but a new one (Sn) was created for this




species due to its low light requirements rather than its N2 fixing capacity (Reynolds

1997). However, Ferreira (2002) reported a variation from 0 to 77% in the percentage

of thricomes of C. raciborskii carrying heterocytes in Tapacurá reservoir indicating

that the capacity of C. raciborskii to fix N2 could not be disregarded. Huszar et al.

(2000) observed a codominance of C. phillippinensis and A. spiroides during a strong

N-limitation in Juturnaíba reservoir. These authors observed that 30% of C.

phillippinensis carried heterocytes, indicating that at least part of the population was

fixing nitrogen. We did not count the number of heterocytes of C. raciborskii but

according to the data of those authors, it is reasonable to suppose that even though

C. raciborskii is included in Sn assemblage the lack or low concentration of dissolved

inorganic nitrogen observed in Tapacurá reservoir during our study would not be a

limitation factor for its development. Moreover, C. raciborskii has been reported as a

species with high affinity for ammonium, using this nutrient in such concentrations in

which the other heterocytic species already need to fix N (Padisák, 1997). Huszar et

al. (2000) pointed out Cylindrospermopsis as genus with alternative physiological

adaptations between S (Oscillatoriales – non-N2-fixing) and H (heterocytic

cyanobacteria) assemblages.

Neurotoxic cyanobacterial blooms have already been observed in Tapacurá

reservoir but the toxins identified were saxitoxins detected during C. raciborskii

dominance (Nascimento et al., 2000). The 19 March sample showed typical

symptoms of neurointoxication during mouse bioassay but no clinical signs of

anatoxin-a(s) toxicosis were observed (Tab II). These results suggests that saxitoxins

or even anatoxin-a could be the mainly toxins produced. However, anatoxin-a(s)-like

anticholinesterase symptoms were observed in sample collected on 27 March.

Henriksen et al. (1997) reported that mouse bioassays developed with high doses

generally could mask the characteristic sings of anatoxin-a(s)-like anticholinesterase

and only doses around LD50 would allow the visualization of typical symptoms. In fact,

the acetylcholinesterase assay revealed an inhibition in samples collected on 19 and

27 March of 100 and 88% (Tab II), respectively.




The percentages of inhibition were significantly different (t = 19.34; p <

0.00004), suggesting that AChE inhibitor was in a higher concentration in 19 March

sample. The period in which was observed an AChE inhibition coincided with a

dominance of A. spiroides (Tab II and Fig 2). Interesting to note that the presence of

this species was not reported during a two years survey (May 1998 – May 2000)

conducted in Tapacurá reservoir. In that time the dominant species of cyanobacteria

were C. raciborskii and Raphidiopsis cf. mediterranea (Ferreira, 2002). The

appearance and dominance of A. spiroides probably are related to a specific

limnological and nutrients conditions not present during that period. However, the

environmental parameters analyzed in this study are not enough to explain the

change in the environment that favored A. spiroides. Since March 2002, A. spiroides

neurotoxic blooms have been observed in Tapacurá reservoir (data not showed).

The production of anatoxin-a(s) was only described for species of Anabaena

genus including A. flos-aquae (Mahmood and Carmichael, 1986) and A. lemmermanii

(Henriksen et al., 1997; Onodera et al., 1997). Monserrat et al. (2001) reported an

AChE inhibition by an aqueous extract from A. spiroides bloom collected from an

ornamental lake in South of Brazil. In order to confirm whether A. spiroides observed

in Tapacurá reservoir synthesize anatoxin-a(s)-like anticholinesterase three strains

were isolated and cultivated. The mouse and anticholinesterase bioassay reveled

that the three strains are AChE inhibitor producer (Tab. II). It has been recently

verified by LC/MS that strain ITEP-024 produces anatoxin-a(s) (Dr. Wayne

Carmichael, Wright State University, personal communication, 2003). The others

strains and bloom material will also be analyzed to confirm the presence of this toxin.

Nevertheless, the mouse and anticholinesterase bioassay more the LC/MS analysis

suggest that the toxin present in Tapacurá reservoir during A. spiroides dominance

and the toxin produced by the other two A. spiroides strains is anatoxin-a(s).




During the Pseudoanabaena sp. dominance no toxicity was observed by the

mouse bioassay (Fig. 2; Tab II). It is reasonable, since there are no reports about

toxins production for this genus. From 10 April to 24 May the mouse bioassay

indicated again the presence of neurotoxins in bloom samples. The mice exhibited

typical neurotoxicosis symptoms but not caused by anatoxin-a(s) since the

acetylcholinesterase assay of these samples reveled 0% of activity inhibition (Tab II).

This period coincided with C. raciborskii dominance. Several strains of C. raciborskii

isolated from different parts of the world have been reported to be toxic. European

strains were toxic on mouse bioassays but no evident toxin was identified (Bernard et

al., 2003; Saker et al. 2003). The presence of cylindrospermopsin, a potent

hepatotoxic alkaloid, was only identified in Australian and Thailandian strains (Li et

al., 2001; Neilan et al., 2003; Griffiths and Saker 2003). On the other hand, only

Brazilian C. raciborskii strains have been reported to produce saxitoxins (Lagos et

al., 1999; Molica et al., 2002, Bernard et al., 2003; Pomati et al., 2003). Saxitoxins

were only identified in the sample 8 May (Fig. 3) and their profile (NeoStx, Stx and

dc-Stx) was the same with those reported by Bernard et al. (2003), which have

worked with a strain isolated from a reservoir close to Tapacurá. Unfortunately, we

were not successful to isolate C. raciborskii strains from bloom samples to confirm

saxitoxin production in culture conditions and to better investigate the saxitoxins

profile. The reasons for non-identification of saxitoxins in the other samples despite

the positive mouse bioassay could be due to the degradation of toxins during 7

months of storage at -18oC and successively freeze-thawed for mouse and

acetylcholinesterase bioassay. The detection limits in our HPLC method for Stx,

NeoStx, GTX1, GTX4, GTX2 and GTX3 are 0.5, 3.9, 4.7, 14.2, 0.81 and 1.2 µg L-1

respectively, enough to detect any trace of these toxins in the samples. Moreover, no

suspicious peaks of other saxitoxins variants were observed in the chromatograms.

Another possibility would be the presence of another neurotoxin, in this case

anatoxin-a since AChE inhibition assays were negative. Kiss et al (2002) suggested

that a Hungarian strain of C. raciborskii could has an anatoxin-a-like compound but

the structure of this molecule is unknown. For these reasons, the neurotoxical

symptoms exhibited for the mice injected with samples collected between 10 April

and 24 May were probably caused by saxitoxins produced by C. raciborskii.




On 30 May M. aeruginosa was the dominant species in Tapacurá reservoir

(Fig 2). This species is more related with microcystin production (Carmichael, 1996)

but no symptoms induced by this toxin were observed during the mouse bioassay

(Tab. II). Four strains of Microcystis sp. isolated from Tapacurá did not possess the

molecular marker mcyB involved in the biosynthesis of microcystins (Bittencourt-

Oliveira, 2003). Previous M. aeruginosa blooms in Tapacurá reservoir were also non-

toxic when analyzed by mouse bioassay or HPLC (unpublished results).

In conclusion, these results show the necessity of continuous monitoring in

Tapacurá reservoir since it is used for drinking water supply, fishing and recreation

activities and the implementation of management plan to control cyanobacterial

blooms. The Brazilian guideline for potable water established the maximum

concentration of 1 µg L-1, 3 µg L-1 and 15 µg L-1 for microcystin, saxitoxin equivalents

and cylindrospermopsin, respectively (FUNASA, 2001). Anatoxin-a(s) was not

included because it has never been reported its presence in Brazilian drinking water

supplies before. However, it could be detected indirectly because the same

Legislation recommends the realization of AChE inhibition assay for detection of

organophosphates and carbamates, with tolerable maximum inhibition of 15% and

20%, when the enzyme is from insect or mammalian, respectively. Thus, this first

study revealing the presence of anatoxin-a(s)-like in a Brazilian drinking water supply

alerts the local authorities for the possibility of the presence of this high toxic

compound in potable water.

ACKNOWLEDGEMENTS

We are greatful to Dr Marc Bouvy for critical review and ??? . Maristela

Cunha and Gustavo Melo were supported by scholarships from CNPq and FACEPE,

respectively.




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Carmichael WW, Azevedo SMFO, An JS, Molica RJR, Jochimsen EM, Lau S, Rinehart KL, Shaw GR, Eaglesham GK. 2001. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ Health Persp 109(7):663-668. Dietz AA, Rubinstein HM, Lubrano T. 1973. Colorimetric determination of serum cholinesterases and its genetic variants by the propionylthiocholine-dithiobis (nitrobenzoic acid) procedure. Clinical Chem 19(11):1309-1313. Ferreira ACS. 2002. Dinâmica do fitoplâncton de um reservatório hipereutrófico (reservatório Tapacurá, Recife, PE), com ênfase em Cylindrospermopsis raciborskii e seus morfotipos. Rio de Janeiro: Master thesis, Museu Nacional, Universidade Federal do Rio de Janeiro. 79 p. FUNASA 2001. Portaria no 1469/2000, de 29 de dezembro de 2000: aprova o controle e vigilância da qualidade da água para consumo humano e seu padrão de potabilidade. Brasília. 32 p. Gorham PR, McLachlan J, Hammer UT, Kim WK. 1964. Isolation and culture of toxic strains of Anabaena flos-aquae (Lyngb.) de Bréb. Verh.Internat.Verein.Limnol. 15:796-804. Griffiths DJ, Saker ML. 2003. The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin. Environ Toxicol 18:78-93. Henriksen P, Carmichael WW, An J, Mostrup Ø. 1997. Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds. Toxicon 35(6):901-913. Huszar VLM, Silva LHS, Marinho M, Domingos P, Sant'Anna CL. 2000. Cyanoprokaryote assemblages in eight productive tropical Brazilian waters. Hydrobiologia 424:67-77. Jochimsen EM, Carmichael WW, An J, Cardo DM, Cookson ST, Holmes CEM, Antunes BC, Melo Filho DA, Lyra TM, Barreto VST, Azevedo SMFO, Jarvis WR. 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. New Engl J Med 338(13):873-878. Kiss T, Vehovszky Á, Hiripi L, Kovács A, Vörös L. 2002. Membrane effects of toxins isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified molluscan neuorenes. Comp Biochem Phys C 131:167-176. Komárková.-Legnerová J, Eloranta P. 1992. Planktic blue-green algae (Cyanophyta) from central Finland (Jyväskylä region) with special reference to the genus Anabaena. Algolg Studies 67:103-133.




Lagos N, Onodera H, Zagatto PA, Andrinolo D, Azevedo SMFO, Oshima Y. 1999. The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. Toxicon 37:1359-1373. Li R, Carmichael WW, Brittain S, Eaglesham GK, Shaw GR, Mahakhant A, Noparatnaraporn N, Yongmanitchai W, Kaya K, Watanabe WW. Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria). Toxicon 39:973-980. Mahmood NA, Carmichael WW. 1986. The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17. Toxicon 24(5):425-434. Matthiensen A, Beattie KA, Yunes JS, Kaya K, Codd GA. 2000. [D-Leu1]-Microcystin-Lr, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil. Phytochemistry 55:383-387. McGregor GB, Fabbro LD. 2000. Dominance of Cylindrospermopsis raciborskii (Nostocales, Cyanoprokaryota) in Queensland tropical and subtropical reservoirs: implications for monitoring and management. Lakes Reserv: Res Manage 5:195-205. Molica R, Onodera H, García C, Rivas M, Andrinolo D, Nascimento S, Meguro H, Oshima Y, Azevedo S, Lagos N. 2002. Toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii (Cyanophyceae) isolated from Tabocas reservoir in Caruaru, Brazil, including demonstration of a new saxitoxin analogue. Phycologia 41(6):606-611. Monserat JM, Yunes JS, Bianchini A. 2001. Effects of Anabaena spiroides (Cyanobacteria) aqueous extract on the acetycholinesterase activity of aquatic species. Environ Toxicol Chem 20(6):1228-1235. Nascimento SM, Molica RJR, Bouvy M, Ferreira A, da Silva LHS, Huszar V, Azevedo SMFO. 2000. Toxic cyanobacterial blooms in the Tapacurá reservoir, northeast Brazil. In: Conference abstracts of the IX International Conference on Harmful Algal Bloom, Hobart, Australia, 7-11 February 2000. Neilan BA, Saker ML, Fastner J, Töröknés A, Burns BP. 2003. Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii. Mol Ecol 12:133-140. Onodera H, Oshima Y, Henriksen P, Yasumoto T. 1997. Confirmation of anatoxin-a(s), in the cyanobacterium Anabaena lemmermanannii, as the cause of bird kills in Danish lakes. Toxicon 35(11):1645-1648. Oshima Y. 1995. Postcolumn derivatization liquid chromatographic methods for paralytic shellfish toxins. J AOAC Int 78(2):528-532. Padisák J. 1997. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju, an expanding, highly adaptative cyanobacterium: worldwide distribution and review of its ecology. Arch hydrobiol. 107(suppl 4):563-593.




Pomati F, Neilan BA, Suzuki T, Manarolla G, Rossetti C. 2003. Enhancement of intracellular saxitoxin accumulation by lidocaine hydrochloride in the cyanobacterium Cylindrospermopsis raciborskii T3 (Nostocales). J Phycol 39:535-542. Reynolds CS. 1997. Vegetation process in the pelagic: a model for ecosystem theory. Germany: Ecology Institute. 371 p. Reynolds CS, Huszar V, Kruk C, Naselli-Flores L, Melo S. 2002. Towards a functional classification of the freshwater phytoplankton. J. Plankton Res 24(5):417-428. Saker ML, Nogueira ICG, Vasconcelos VM, Neilan BA, Eaglesham GK, Pereira P. 2003. First report and toxicological assessment of the cyanobacterium Cylindrospermopsis raciborskii from Portuguese freshwaters. Ecotoxicol Environ Saf 55:243-250. Sant’Anna CL, Azevedo MTP. 2000. Contribution to the knowledge of potentially toxic cyanobacteria from Brazil. Nova Hedwigia 71(3-4):359-385. Sas H. 1989. Lake restoration by reduction of nutrient loading: expectations, experiences, extrapolations.St. Augustin: Academia Verlag Richarz. Sivonen K. 1996. Cyanobacterial toxins and toxin production. Phycologia 35 (suppl 6):12-24. Sivonen K and Jones GJ. 1999. Cyanobacterial toxins. In: Chorus I, Bartram J, editors. Toxic cyanobacteria in water – a guide to their public health consequences, monitoring and management. London: E & FN Spon. p 41-111. Souza RCR, Carvalho MC, Truzzi AC. 1998. Cylindrospermopsis raciborskii (Wolosz.) Seenaya and Subba Raju (Cyanophyceae) dominance and a contribution to the knowledge of Rio Pequeno Arm, Billings Reservoir, Brazil. Environ Toxicol Water Qual 13:73-81. Yunes JS, Salomon PS, Matthiensen A, Beattle KA, Raggett SL., Codd GA. 1996. Toxic blooms of cyanobacteia in the Patos lagoon estuary, Southern Brazil. J Aquat Ecosys Health 5:223-229.




6.2. PUBLICAÇÕES REALIZADAS NO PERÍODO DA TESE ALÉCIO-OLIVEIRA, E.J.; AIRES, P.S.R.; SOUZA, I.A.; MENDONÇA, S.C.; BRANDÃO, S.S.F. Avaliação da toxicidade aguda oral do óelo essencial da Lippia gracillis Schau. 55ª SBPC, 2003, Recife. MOLICA, R.J.; ALÉCIO-OLIVEIRA, E.J.; CARVALHO, P.; COSTA, A; CASÉ, M.; MELO, G.; AZEVEDO, S. Floração de cianobactérias neurotóxicas em reservatórios de abastecimento. IX Congresso Brasileiro de Limnologia, 2003, Juiz de Fora. ALÉCIO-OLIVEIRA, E.J.; MOLICA, R.J.; PIMENTEL, F.; ALBUQUERQUE, E.C.J.; LIMA-FILHO, J.L. Validação de método de análise de microcistinas por CLAE. IX Reunião Brasileira de Ficologia, 2002, Espírito Santo. MELO, Janaína V. de; ALÉCIO-OLIVEIRA, E.J.; MOLICA, R.J. Validação de metodologia para análise de saxitoxinas dissolvidas em água por CLAE. IX Reunião Brasileira de Ficologia, 2002, Espírito Santo. PORFÍRIO, Z.; ALÉCIO-OLIVEIRA, E.J.; MOLICA, R.J. Atividade abortiva e teratogênica de cianobactérias (Anabaena spiroides) isolada da lagoa Manguaba – Alagoas. VII Encontro Nacional de Microbiologia Ambiental – ENAMA, 2000, Recife. ALÉCIO-OLIVEIRA, E.J.; MENDONÇA, S.C.; CRUZ, J.M.; SILVA, J.C.; MOTA, C.M.S. Avaliação preliminar da qualidade microbiológica do ar em ambiente escolar na cidade de Recife. VII Encontro Nacional de Microbiologia Ambiental – ENAMA, 2000, Recife. ALÉCIO-OLIVEIRA, E.J., OLIVEIRA, M.C.M.; ALMEIDA, M.C.L.; CRUZ, L.B.; M.C.R.; MARÇAL. Detecção de alfatoxinas em amendoim e derivados comercializados na cidade de Recife-PE durante o período de 1996 a 1999. Encontro Nacional de Analistas de Alimentos – ENAAL, 2000, Recife. ALÉCIO-OLIVEIRA, E.J.; ATAÍDE, E.J.; ASSIS, O.B.G.; LIMA FILHO, J.L. Inhibition of bytyrylcholinesterase by parathion-methyl using open-pore soda-lime glass membranes like as support. XXIX Reunião Annual da Sociedade Brasileira de Bioquímica e Biologia Molecular – SBBq, 2000, Caxanbu. GURGEL, I.G.D.; NETO, O.C.; FILHO, P.C.; ALÉCIO-OLIVEIRA, E.J.; BATALHA, V.G.; ALMEIDA, M.C.S. Uma outra face do programa de erradicação do Aedes aegypti em Pernambuco: A vigilância da saúde dos trabalhadores expostos a agrotóxicos, em Recife-PE. VI Congresso Brasileiro de Saúde Coletiva, 2000, Salvador. ALÉCIO-OLIVEIRA, E.J.; PIMENTEL, M.C.B.; SILVA FILHO, A.G.; OLIVEIRA, .I.P.; NASCIMENTO, P.S.B.; GONDIM, P.B.; CORREIA, T.; LEDINGHAM, W.; LIMA-FILHO, J.L. Development of a cholesterol biosensor using choleterol oxidase immobilized on sinanased glass beads. XXXVIII Reunião Anual da SBBq, 1999, Caxanbu.




7. ANEXO I: PROTOCOLO DE VALIDAÇÃO DE METODOLOGIA DE ANÁLISE DE MICROCISTINA-LR EM ÁGUA ULTRAPURA POR CROMATOGRAFIA LÍQUIDA DE ALTA EFICIÊNCIA - CLAE

Os ensaios cromatográficos foram baseados no trabalho de Lawton et al.

(1994). Para extração foi utilizado o procedimento sumarizado na Figura 1

adapatado de Fastner et al. (1998) e Krishnamurphy et al. (1986). Os trabalhos

foram desenvolvidos no Laboratório de Ecofisiologia de Microalgas – LEMI do

Instituto Tecnológico do Estado de Pernambuco –ITEP.

Antes do início da validação foram verificados variações e erros das pipetas

automáticas, bem como os parâmetros de qualidade do CLAE tais como o poder de

separação da coluna, energia da lâmpada, volume dispensado pela bomba, etc.

a) Robustez do Método

As amostras utilizadas são homogêneas, por serem preparadas em água, e as

variáveis que podem interferir no método estão controladas, a saber: A temperatura

da coluna é controlada por forno com precisão de ± 0,1oC (monitoramento contínuo),

o detector utilizado é do tipo U.V. (e não índice de refração onde a variação de

temperatura é crítica), o equipamento é dotado de degaseificador de solventes, e a

variação de concentração da curva está em faixa estreita de concentração (0,25 a

10,0 µg/mL => 12,5 a 500 ng de toxina injetada). Foram utilizados reagentes de

mesma marca e lote.

b) Amostragem

A microcistina-LR é uma substância solúvel em água, o que possibilita uma

distribuição homogênea no meio quando presente na forma livre. Assim, testes de

homogeneidade da amostra não são necessários visto que o método aplica-se à

determinação da toxina em água.




c) Parâmetros de Detectabilidade do Instrumento: Limite de Detecção – LDI, Limite de Determinação - LDmI e Limite de Quantificação– LQI

Trabalhando em quintiplicata foram preparadas soluções de concentração

decrescente de microcistina-LR em água ultrapura (Milli-Q ®). Volumes de 50 µL

foram injetados e a menor concentração (massa de toxina injetada) que produziu

uma relação sinal x ruído (s/r) de no mínimo 2 a 3 vezes a da linha de base foi

tomada como referência para cálculo dos limites.

Obtido os valores da média e do desvio padrão da área sob a curva, os

valores do LDI, do LDmI e do LQI foram calculados como segue:

LDI = 3,3 x DP

LDmI = 6 x DP

LQI = 10 x DP

d) Curva de Calibração / Precisão

A construção de uma curva de calibração permite avaliar a correlação entre a

concentração e o sinal (através da análise de regressão), o intervalo dinâmico da

curva (através do gráfico dos desvios da regressão) e a precisão nos diferentes

níveis de concentração (através do coeficiente de variação). Foram utilizados cinco

níveis de concentração injetados em quintiplicata.

Curva de Calibração

Faixa de 0,25 a 10,0 µg/mL (12,5 a 500 ng de massa de toxina injetada)

A linearidade da curva de calibração de microcistina-LR foi avaliada pela

injeção em quintiplicata de 50 µL de soluções de microcistina-LR nas concentrações

de 0,25 / 0,50 / 1,0 / 2,0 e 10,0 µg/mL (massa de toxina injetada de 12,5 / 25,0 /

50,0 / 100,0 e 500 ng, respectivamente).




Após a injeção de cada amostra (n=5) foi calculado a média (M), o Desvio

Padrão (DP) e o Coeficiente de Variação (CV = DP x 100/M) das áreas sob a curva,

dos picos correspondentes ao padrão.

Linearidade da curva de calibração

Com os dados obtidos para a curva de calibração foi construído o gráfico de

Concentração x área sob a curva, calculando-se o coeficiente de correlação (r),

através da análise de regressão (método dos mínimos quadrados).

A significância de “r” foi verificada utilizando o teste “t” de significância do

coeficiente de correlação descrito por Cochran (1974).

Faixa Dinâmica da Curva de calibração

Este procedimento permite verificar desvios na linearidade que muitas vezes

não se pode visualizar por uma simples inspeção visual da curva de regressão.

A faixa dinâmica foi avaliada verificando-se a região horizontal, com limites

superior e inferior de 5% da média, após grafar no eixo de “y” a resposta relativa de

cada concentração da curva e no eixo de “x” o valor da concentração em escala

logarítmica.

Precisão

Foi avaliada através do coeficiente de variação em cada concentração da

curva de calibração. O critério para avaliação da variação máxima aceitável foi o da

AOAC (1993) que considera variações de até 30% e 21% para concentrações de 1 e

10 ppb, respectivamente.




e) Parâmetros de Detectabilidade Do Método: Limite de Detecção (LDM), Limite de Determinação (LDMm) e Limite de Quantificação (LQM).

Considerando o limite da Portaria 1469/2000-MS de 1,0 µg/L para a presença

de microcistinas em água tratada, os testes para determinação dos limites de

detectabilidade foram realizados utilizando extratos de soluções de microcistina-LR

0,5 µg/L, preparadas em água Milli-Q ® e extraída conforme Figura 1. Para a

extração foi utilizado um único cartucho C18 de 1g, condicionado com metanol e

água Milli-Q antes de cada extração.

O critério para avaliação da recuperação foi o da AOAC (1993) que aceita

variações da média de 40 a 120%, para o nível de 1 ppb.

O cálculo dos limites de detectabilidade foi realizado utilizando equações

adotados para o cálculo dos limites do instrumento e, desde que o valor do LQM ou

do LDMm encontrados estejam abaixo da concentração de interesse legal (1 µg/L), o

valor de 0,5 µg/L deve ser assumido como a concentração de incriminação controle

para as análises diárias (“report limit”).

f) Precisão e Exatidão (recuperação) do Método

Volumes de 500 mL de amostras de água Milli-Q foram incriminadas em

quintiplicatas com solução de microcistina-LR nas concentrações de 0,8 / 1,6 / 3,2 /

2,0 e 10 µg/L, e submetidas à extração, purificação (“clean-up”) e concentração, de

acordo com a Figura 1. Para as réplicas de cada concentração foi utilizado um único

cartucho C18 condicionado com metanol e água Milli-Q antes de cada extração.

A concentração da toxina nos extratos foi expressa pela equação da reta de

curva padrão construída no dia da análise. (concentrações padrão de 0,5; 1,0; 2,0 e

10,0 µg/mL).




O critério para avaliação da exatidão (recuperação) foi o da AOAC (1993) que

aceita variações da média de 40 a 120% para o nível de 1 ppb e de 60 a 115% para

10 ppb. Para a precisão nos diferentes níveis foram aceitos variações de 21 a 30%

para concentrações de 10 e 1 ppb, respectivamente.

Incriminar 500 mL da amostra de Água Milli-Q com solução da toxina

Aplicar a amostra sobre cartucho com 1g de C18 ativado com 10 mL de Metanol 100% (v/v) e 10 mL de água Milli-Q (Fluxo < 10 mL/min)

Lavar o cartucho com 20 mL de água Milli-Q, 20 mL de metanol 20% Fluxo < 10 mL/min, Secar por sucção

Recolher o eluato com 3,5 mL de metanol 90% grau CLAE+TFA 0,1%

Evaporar em rota-vapor a 40ºC, re-dissolver o extrato em 500 µL de metanol 100% grau CLAE, filtrar em membrana 0,22 µm e injetar em CLAE .

Injetar 3 ou 4 concentrações do padrão de microcistina-LR para construção da curva de calibração do dia

Figura 1: Extração de microcistina-LR em amostras de água Milli-Q propositalmente incriminadas (Adaptado de Fastner et al., 1998 e Krishnamurthy, 1986).




REFERÊNCIAS BIBLIOGRÁFICAS AOAC Peer Verified Methods Program, Manual on Policies and Procedures, Arlington, VA, 1993. FASTNER, J., FLIEGER, I., and NEWMANN, U. Optimised extraction of microcystins from field samples - A comparison of different solvents and procedures. Wat. Res., v.32, n.10, p.3177-3181, 1998. KRISHNAMURTHY, T., CARMICHAEL, W.W., and SARVER, E.W. Toxic peptides from freshwater cyanobacteria (blue-green algae). I. isolation, purification and characterization of peptides from microcystis aeruginosa and anabaena flos-aquae. Toxicon, v. 24, n.9, p. 865-873, 1986. LAWTON, L.A.; EDWARDS, C.; CODD, G.A. Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters, Analyst, v.119, n.7, p.1525-1530, 1994.



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