Toxin Concentration

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

By utilizing the HPLC method, it is possible to determine the level of each individual toxin in sample solutions. This provides a "toxin profile" that can be very useful in PSP toxin research studies. The ability to examine relative changes in toxin concentration and profile has greatly facilitated studies relating to toxin production by dinoflagellates, metabolism of toxins in shellfish, and movement of toxins up the food chain. Since the HPLC method is easily automated and requires only very small sample sizes (< 1 g tissue), it has clear advantages over other analytical procedures for the toxins in many research situations. Two examples of the utilization of HPLC for the study of the PSP toxins follow. 

K. = inhibition constant in units of toxin concentration which... 

Figure 7. Inhibition of tritiated PbTx-3 specific binding by unlabeled brevetox-ins, PbTx-2, PbTx-1, PbTx-3, PbTx-7. Specific binding was measured in intact synaptosomes at 4 C degrees in standard binding medium using four different tritiated toxin concentrations—5.0, 7.5, 10.0, and 15.0 nM (inverse [ H] PbTx-3 abscissa values)—in the presence of unlabeled toxins at 0 (o), 5.0 ( ), 7.5 (A), 10.0 (A), 25.0 ( ), 50.0 ( ), or 100.0 (data not shown) nM. Points are means of triplicate determinations at each concentration.

AufhammerW, Hermann W, KublerE, LauberU and SchollenbergerM (1999), Fusarium (F. graminearum) infection of ears and toxin concentration of grains of winter wheat, triticale and rye depending on cultivars and production intensity , Pflanzenbauwissenschaften, 3 (1), 32-39. 

Figure 4. Michaelis-Menten type plot of percent of contractile activity versus the histamine substrate concentration and the toxin concentration in the saline solution.

When the duration of exposure to GT-4 was limited to 10 minutes and the percent inhibition for an agonist concentration equivalent to the 50% control response was measured at 90 minutes as a function of toxin concentration, a linear response was obtained (Figure 5). Figure 5 shows that the exposure of the preparation to 2.8 ng/ml, 7.0 ng/ml and 70 ng/ml for 10 minutes resulted in inhibitions of 30, 36, and 66% of the contractile response from control levels, respectively. In addition, exposure of the preparation to 2.8, 7.0 and 70 ng/ml for 15 minutes resulted in 45, 55, and 98% inhibition of the contractile response, respectively. We can express the effects of GT-3 by stating both the dose and time to achieve a 50% inhibition from the control or a DCI50 value. 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

Acetylcholinesterase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. As the inhibition is irreversible and stoichiometric, it is possible to calculate the toxin concentration by the percentage of the enzyme that has not been inhibited and remains active. The use of several enzyme concentrations and the... 

R [ 3I]Lectin/Toxin Concentration 5% TCA wash 5% TCA + Chloroform-methanol (2 1) wash... 

The line shown in Figure 14.9 is in fact the line of best fit. It intercepts the vertical axis at a value of 10.6 pg/100 g and it has a gradient of+6.73 (i.e. an increase in rainfall of 1 cm/week is associated with an increase in toxin concentration of 6.73 pg/lOOg). Therefore, the line corresponds to the relationship ... 

The rainfall and toxin data will be entered into two appropriately labelled columns. You will then have to indicate the relevant columns. However, there is an important difference from correlation. With regression you must be careful to indicate correctly which is the dependent and which the independent variable. Unfortunately, statistical packages use a varied terminology. The toxin concentrations may be entered as the dependent variable or response and the rainfall may be the independent variable or the predictor . 

Table 14.4 Generic output for regression analysis of toxin concentration and rainfall...

The term b is referred to as a coefficient . There was a positive relationship between rainfall and toxin, so the value of coefficient b was positive. In that way the greater the rainfall, the greater the predicted toxin concentration. 

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