Metabolism and mode of action 



The first experiment on absorption revealed a most unexpected result. 

 At the end of the first four hours, over all five concentrations there were 

 marked gains in the amounts of C^'* present in the tissues, but between 4 and 

 24 hours the trend for absorption was dependent on the concentration. At 

 4 and 8 p. p.m. the amount present remained unchanged, but at 16 p. p.m. 

 and more, particularly at 32 and 44 p. p.m., there was a sharp fall in the 

 activity between 4 and 8 hours followed by a further but less steep decline 

 between 8 and 24 hours. Thus, although at the end of 24 hours the 

 amount of C^^ in the sample was roughly proportional to the external 

 concentration, these amounts bore little relation to the amounts present 

 after 4 hours since the proportion 'lost' in the interval ranged from 

 nought to two thirds. 



The experiment was then repeated with observations at 1, 2, 3, and 4 

 hours and in this instance maximal values for C^"* were recorded at either 1 

 (8 p. p.m.) or 2 hours (16 and 32 p. p.m.) followed by progressive declines up 

 to 4 hours. In another experiment the time intervals were shortened to 20, 

 40, and 60 minutes and under these conditions, after 20 minutes the rate of 

 uptake was roughly proportional to time and concentration. It was noted 

 that when for each concentration the line was extrapolated back to zero time 

 it did not pass through the origin. 



It seemed that there were two possible explanations for the loss of C^* from 

 the tissues. Firstly, that the phenoxyacetic acid was rapidly broken down and 

 the C^* respired as carbon dioxide. Secondly, that either the compound 

 itself or some breakdown product was transferred back to the solution. The 

 first explanation appeared unlikely on account of the speed and the amounts 

 which would have to be respired since where such respiratory losses have been 

 reported the percentage lost has been small (e.g. Weintraub et al., 1952). It 

 was decided, therefore, to test the second hypothesis by placing Lemna minor 

 in labelled 2:4-dichlorophenoxyacetic acid (16 and 56 p. p.m.) for a short 

 period (0-75 hours) and then to transfer samples to {a) double distilled water, 

 {b) culture solution at the same pH, and [c) culture solution containing the 

 same concentration of the unlabelled growth regulator. It was found that 

 at the end of 0-75 hours more than half of the original C^* was no longer 

 present in the tissues and that at 3 hours the loss was ca. 90 per cent. This 

 hyperbolic relationship between loss and time was the same irrespective of 

 the composition of the external solution. 



When a balance sheet was constructed between {a) the original C^* in the 

 sample and {b) the amounts present either in the tissue or in the solution at 

 the end of 3 hours, then over 99 per cent could be accounted for, thus demon- 

 strating that any respiratory loss of C^'* as carbon dioxide was negligible. To 

 discover whether it was the compound itself which was transferred to the 

 solution an aliquot of the final solution was concentrated, the residue 

 spotted on to chromatogram paper, run in a mixture of uopropanol, 

 ammonia, and water, and the paper placed in an electronic scanning device. 

 Only one radioactive spot was recorded and this had the same Rf value as 

 the original labelled 2:4-dichlorophenoxyacetic acid. 



From these experiments it can be concluded that the absorption of 

 2:4-dichlorophenoxyacetic acid is characterized by a very rapid rate of 

 entry during the first few minutes followed by a steady rate of accumulation 



256 



