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1. 4-Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25 "C.2. Samples of (3R)-and (35')-4'-hydro~yphenyl[3-~H~]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto -enol-isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium-labelled homogentisic acids, which were isolated as the crystalline 2',5'-diinethyl derivatives after treatment with dimethyl sulphate.3. 2',5'-Dimethoxy-(4-cinnamate was deuterated by enoate reductase from Clostridiurn sp. La 1 to afford (2S, 3R)-3-(2',5'-dimetho~yphenyI)-[2-~H~, propionate which was in turn chemically degraded to (S)-2,5-dimethoxyphenyl r2 Hl lacetic acid. 5. In the 500-MHz 'H-NMR spectrum the 2-methylene protons of unlabelled 2,5-dimethoxyphenylacetic acid (-)-menthy1 ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2-HR, atom could be correlated with the high-field half and the 2-HsI atom with the lowfield half of the AB system. 6. By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side-chain migration occurred with retention of configuration at the methylene C atom.4-Hydroxyphenylpyruvate dioxygenase is involved in the enzymic degradation of tyrosine and catalyses the following reaction [Eqn (l)] :The oxidative decarboxylation of the substrate is coupled with an ortho shift of the side chain reminiscent of the wellknown NIH shift of the para-hydrogen atom during the hydroxylation of phenylalanine [I, 21. Several mechanisms have been suggested for this remarkable transformation [2 -71, none of them has, however, been proved. By the use of l 8 0 2 it has been shown that both atoms of molecular oxygen are incorporated into the same homogentisic acid molecule thus justifying the name dioxygenase [8].The enzyme has been isolated from mammalian, avian and human liver [9-121 and recently from Pseudoinonas sp. P.J. 874 as well [13]. No specific prosthetic group has been yet detected. However, vitamin C and Fe2+ ions function as activators. In vim the best activator is 2,6-dichlorophenolindophenol in its reduced form [14]. (1 W) was applied over the deuterium lock circuit of the Proton magnetic 312 standard 'H-observe probe head. 'H-homodecoupling at the aromatic protons was performed simultaneously in the conventional fashion. Mass spectra were measured with a Hitachi-Perkin-Elmer RMU-6A spectrograph. The limit of error of the mass spectroscopic measurements was estimated to be 1 %. Optical rotatory dispersion (ORD) measurements were conducted with the polarimeter Jasco V. The circular dichroism (CD) curves were obtained with dichrograph Mark I11 of Jobin-Yvon. For ultraviolet spectroscopic measurements, a Unicam SP-1800 spectrophotometer was used.Dialyses were carried out with a hollow-fiber system model DC 2 of Amicon. MaterialsPhenylpyruvate ke...

The test principle and the optimization of the reactive ingredients are described for the one-step dip and-read immunochromatographic FRONTLINE rapid tests for drugs-of-abuse testing in urine samples. In a multicenter evaluation the rapid tests were compared with FPIA and EMIT immunoassays. Discrepant results were further analyzed by gas chromatography-mass spectrometry methods. In the comparison of the cannabinoids rapid tests versus both immunoassays using clinical and forensic urine samples (399 versus FPIA and 755 versus EMIT), sensitivities and specificities were 97% or better for both comparisons. For cocaine, a sensitivity of 100% versus both routine technologies was obtained, whereas the specificity was reduced somewhat to 91% because of some cross-reactivity with metabolites of methadone and of clozapine. Specificity was very high for the cocaine rapid tests (98-100%) when applied to urine samples of persons not in a methadone maintenance program. Sensitivities and specificities for the opiates rapid tests were 99% or better at all sites when compared with the routine methods. In the screening of about 1200 clinical urine samples for cannabinoids, cocaine or opiates misuse only six samples would have stayed undetected by rapid test analyzes. These results show the FRONTLINE assays allow a reliable and immediate screening for drugs of abuse.

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