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In this study, saxitoxin dihydrochloride in skim milk was reacted with sodium hydroxide and hydrogen peroxide to yield nontoxic 8-amino-6-hydroxymethyl-iminopurine-3(2H)-propionic acid (AHIPA), which was quantified by fluorescence spectroscopy using excitation and emission wavelengths of 330 and 425 nm, respectively. Samples of saxitoxin dihydrochloride (in 20% ethanol, vol/vol) were used as controls. The limits of detection of AHIPA, based on the concentration of saxitoxin prior to inactivation, were 5 and 10 μg/ml for the control and skim milk, respectively. These values are considerably below the concentration of saxitoxin that corresponds to the lethal dosage of 1 mg for an adult of average weight (70 kg). The inactivation of saxitoxin proceeded at a lower rate in skim milk than in the control, as its reaction rate constant was only 0.004 min(-1) compared with 0.011 min(-1) for the control. We were unable to detect AHIPA in 2% milk contaminated with saxitoxin because of possible interference from what we believed were products of secondary reactions involving milk fat and sodium hydroxide. Our results also indicated that the conversion of saxitoxin to AHIPA increased initially with temperature up to 40°C but decreased thereafter. We observed a decrease in the formation of AHIPA when the concentration of hydrogen peroxide was increased except at 22°C, where there was an initial increase in AHIPA formation between 1.2 and 2.4 mg/ml hydrogen peroxide but its formation decreased thereafter.

Ricin is a potential bioterrisiom agent. There is a critical need for a method that can rapidly and simply detect ricin and other bioterrisiom agents in complex food matrices such as milk. In this study, we demonstrated a rapid method that combined immunomagnetic separation (IMS) and surface-enhanced Raman spectroscopy (SERS) to detect ricin in whole milk. IMS was used to specifically capture the ricin out of the milk. Then, SERS was applied to analyze the IMS eluate mixed with silver dendrite nanosubstrates. This approach facilitated detection and quantification down to 4 μg/mL ricin in milk within 20 min, based on the results of principal component analysis and partial least squares analysis. The feasibility of using a portable Raman instrument shows great promise for on-site detection in a processing facility. Practical Application: The method described in this manuscript that combined IMS and SERS could be used for rapid detection of ricin and other protein toxins in complex food matrices such as milk within 20 min. The use of a portable Raman could facilitate the on-site detection in a processing facility.

The effect of lactose at the concentration typically found in milk (134 mM) on the ability of ricin to inhibit protein synthesis in HeLa cells was studied. Ricin (0.001 to 300 mg/ml) that was either not treated or treated with 134 mM lactose was added to test tubes containing 1 ml of HeLa cells (approximately 3 | 10 5 cells in a low-leucine medium). After 2 h of incubation at 37uC, 0.5 mCi of L-[U- 14 C]-leucine was added to each tube and incubated for another 60 min. The cells were harvested by centrifugation and lysed, and cellular proteins were separated. The amount of radioactivity incorporated into the proteins was determined by liquid scintillation. The biological activity of ricin, i.e., the amount of radioactivity in a sample relative to that of the control (cells not treated with ricin), was calculated for each treatment. The inhibitory effect of 134 mM lactose on the biological activity of ricin was only significant at concentrations of ricin below 1 mg/ml. At higher ricin concentrations, the effect of 134 mM lactose decreased as the concentration of ricin increased, resulting in an increase in the inhibition of proteins synthesis. Our results also indicated that bovine milk, when used in place of 134 mM lactose, was more effective for reducing the activity of ricin at concentrations below 1 mg/ml but was ineffective against ricin concentrations greater than 1 mg/ml. These results suggest that milk may not protect against ricin intoxication at the concentration (0.89 mg/ml) equivalent to the lowest limit of its 50% lethal dose for a 20-kg child consuming 225 ml (8 oz) of milk. Ricin is a globular protein that can be used as a warfare