Study 2
Comparison of assessments at two wavelengths
Glutathione
S-Transferase as a Biological Marker of Aquatic Contamination Return to the Index
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Introduction
Methods
Results
Discussion
Conclusions
This study was initiated when the cause of the double absorbance peaks had not been identified. Nevertheless it is included since it demonstrates the potential of utilising absorbances other than that at 340 nm in the test system. This is potentially useful to avoid backgound absorbance at 340 nm. The opportunity was also taken to determine any loss of activity caused by storage of samples.
An investigation was made of a series of GST homogenates from Mytilus edulis previously deployed at three sites on the river Mersey (Tranmere, Wallasey, and Pier Head) for 30 days. Absorbance was measured at 340 nm and 390 nm, and the results compared.
Cytosolic extracts from 10 mussels at each site, plus 10 from a control site were previously obtained using the method described in the introduction, and stored for 31 months at -18°C to -30°C (Johnson et al., 1992).
The assay technique was essentially that given in the introduction. Absorbance at 340 nm was recorded at 60 and 90 seconds, and the difference calculated. The absorbance at 390 nm was recorded at 100 and 130 seconds. In addition, the absorbance of a extract free blank was recorded on several occasions over the same time period. The experiment was conducted over two days using a randomised block design.
The results were calculated as dABS/min. The average value of the appropriate daily blanks was subtracted and the results are presented in Appendix B. The results were analysed using Minitab, and are presented in the table below and Figure 2.1 as mean + S.E.M. The results of a 2 way analysis of variance (ANOVA) are also given in the table, the value given being the p-value calculated by Minitab for the effects due to sample and treatment with each analytical technique.
| Treatment | dABS 340 | dABS 390 | Ratio 390/340 | Original Data dABS 340 |
| Wallasey | 0.101 + 0.012 | 0.038 + 0.005 | 2.631 + 0.089 | 0.257 + 0.021 |
| Pier Head | 0.150 + 0.019 | 0.050 + 0.006 | 3.003 + 0.040 | 0.234 + 0.015 |
| Tranmere | 0.199 + 0.026 | 0.067 + 0.009 | 3.060 + 0.174 | 0.321 + 0.037 |
| Control | 0.114 + 0.020 | 0.039 + 0.007 | 2.998 + 0.161 | 0.137 + 0.022 |
| 2-Way ANOVA | ||||
| Sample | 0.041 | 0.032 | 0.208 | 0.005 |
| Treatment | 0.002 | 0.005 | 0.067 | <0.001 |
The significance of the difference of each treatment from the control was analysed using a two-tailed Dunnet's test.
| ABS 340 | ABS 390 | Ratio | Original Data | |
| Wallasey | N.S | N.S. | 5% | 1% |
| Pier Head | N.S. | N.S. | N.S. | 1% |
| Tranmere | 1% | 1% | N.S. | 1% |
1. Comparison of new data with original.
There has evidently been loss of a great deal of activity since the samples were last tested. Although the mean value for Pier Head, Tranmere, and the controls show the same general trend, the significance of the results is much reduced (F-Ratio of original data = 15.4 ; F-Ratio of new data = 6.4). The Wallasey samples have degraded to a greater extent, so that the mean is almost identical to the control.
This loss of activity is probably principally due to gradual decay over time. However Garrood et al. (1990) noted that the freezing process itself causes loss of activity, presumably a physical effect with denaturation occuring at the ice/water interface. It could be that re-freezing after the previous analysis caused part of the loss in activity.
Furthermore although the results of the two way anova shows effects due to sample number in both tests, in the original data there is a clear increase in sample mean with sample number. This gradation of means (due to the original samples being made in order of body weight) has disappeared in the new test. Finally, there is little correlation between the old and the new results for each individual sample (r2 = 20.3%, p<0.01).
2. Comparison of the ratio between the two absorbances.
The ratio for all samples is approximately 3:1 for 340:390. The only significant difference being that for Wallasey which, given that fact that the sample absorbance has degraded faster than the other samples (see above), is probably an effect due to storage.
3. Comparison of absorbance at 340 nm with absorbance at 390 nm.
In general, an absorbance at 340 nm gives exactly the same results as that at 390 nm. A scatterplot across all test samples gives a very high correlation (r2 = 96.4%). The degradation found in activity in the Wallasey sample is reflected at both wavelengths. The discriminatory power of the test is somewhat lower at 390 nm (F-ratio at 340 nm = 6.42 ; F-Ratio at 390 = 5.28). This is probably due to the lower activity at 390 nm. An absorbance change of around 0.05 units is on the limits of detection and is therefore more susceptible to fluctuation.
Firstly, the samples analysed have lost a great deal of their activity. On the basis of this it was decided to use a different set of samples in further experiments.
Secondly, both absorbances are facets of the same reaction. The ratio between the two provides no further information, and has no potential for use as a discriminatory test. However measurement of the change of absorbance at 390 nm does therefore provide an alternative to the measurement at 340 nm, and further investigations should be carried out into the background absorbance at this wavelength of a less purified sample, with an aim to reducing the centrifugation required by the test procedure.