A rapid microplate fluorescence method to detect yessotoxins based on their capacity to activate phosphodiesterases.
This paper describes an easy and fast assay with enough sensitivity to detect yessotoxin (YTX) in shellfish samples. YTX decreases intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by increasing the activity of phosphodiesterases (PDEs). Looking for new methods to detect YTXs, we developed a technique based on this effect. We use the fluorescent derivative of cAMP, anthranyloyl-cAMP, whose fluorescence decreases in time by hydrolysis effect of PDEs. The fluorescence fall is quantified in a plate reader. PDEs induce an anthranyloyl-cAMP hydrolysis rate that is increased in the presence of YTX. This effect is dose dependent, and the representation of YTX concentration versus rate of hydrolysis follows a lineal regression. The measurable range of YTX in this assay is 0.1 to 10microM, while by mouse bioassay, the official method to detect YTXs, the detection limit is 2microM. We determined by this method the concentration of YTX from alcoholic extracts whose concentrations were first determined by high performance liquid chromatography and the variation of concentration was from 5.26microM by fluorescence to 6microM by high performance liquid chromatography and from 3.16 by fluorescence to 3microM by HPLC.
Alfonso
Amparo
A
Departamento de Farmacología, Facultad de Veterinaria, Univesidad de Santiago de Compostela, 27002 Lugo, Spain.
Vieytes
Mercedes R
MR
Yasumoto
Takeshi
T
Botana
Luis M
LM
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004988:Ethers, Cyclic / Q000032:analysis* / Q000494:pharmacology*; D005453:Fluorescence; D006868:Hydrolysis; D015394:Molecular Structure; D008978:Mollusk Venoms; D035964:Oxocins / Q000032:analysis* / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012758:Shellfish; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D004988:Ethers, Cyclic; D008978:Mollusk Venoms; D035964:Oxocins; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; C066632:yessotoxin