Detection of sodium channel toxins: directed cytotoxicity assays of purified ciguatoxins, brevetoxins, saxitoxins, and seafood extracts.

Neuroblastoma cells in culture were used to detect sodium channel-specific marine toxins based on an end-point determination of mitochondrial dehydrogenase activity. The assay responds in a dose-dependent manner to ciguatoxins, brevetoxins, and saxitoxins, and delineates the toxic activity as either sodium channel enhancing or sodium channel blocking. The assay responds rapidly to sodium channel activating toxins, allowing dose dependent detection in 4 to 6 h. Brevetoxins can be detected at 250 pg, and purified ciguatoxins are detected in the low picogram and subpicogram levels. The results obtained from cell bioassay of ciguatoxic finfish extracts correlates with those obtained from mouse bioassays. Sodium channel blocking toxins can also be detected with an approximate sensitivity of 20 pg in 24 to 48 h. This cell-based technique is simple, sensitive, demonstrates potential as an alternative to animal testing for sodium channel activating and blocking toxins, and can be automated.

Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins.

In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.

Determination of tri-n-butyltin in oysters by reaction-gas chromatography of hydride derivatives.

A reaction-gas chromatography method for determining tri-n-butyltin (TBT) as the hydride derivative has been adapted to allow determination of TBT in oysters. The extraction method has been modified to prevent fouling of the hydride formation reactor and the gas chromatography has been made faster by employing a different column and temperature program. The detection limit is 3-6 ng/g in oyster tissue. Apparent recoveries of TBT from oyster tissue at 25 and 125 ng/g levels are 107 and 97%, respectively.

Selective determination of histamine by flow injection analysis.

A flow injection analysis (FIA) method for the determination of histamine is described. Control of reaction timing allows exploitation of a transient, chemical-kinetic increase in selectivity that occurs when o-phthalaldehyde reacts with histamine. The molar fluorescence ratio (selectivity) of histamine/histidine reaches a maximum value of 800 in 32 s, precluding the need for separation of histamine from histidine, spermidine, and other potential interferences in biological samples. On-line dilution prevents matrix effects and affords a linear response up to approximately 4.45 mM histamine, or 500 mg of histamine free base/100 g. Under these conditions the detection limit (3 times peak-to-peak baseline noise) is 5.5 pg (corresponding to 0.60 mg of histamine free base/100 g of sample) and throughput is 60 injections per hour. The high sensitivity and high selectivity of the method allow the rapid determination of histamine in fish with minimal sample conditioning and will find application in the determination of endogenous histamine as well, such as in blood plasma and brain tissue.

Diffuse reflectance infrared Fourier transform spectroscopic characterization of a silica-immobilized N-hydroxysuccinimide active ester crosslinking agent and its precursors.

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.