Comparative Studies of Enzymatic and Non-enzymatic oxidation of 3. 4-Dihydroxyphenylalanine (DOPA) In Vitro
by Ngo Manh Tran and Marcel Laplante
Department of Nuclear Medicine and Radiobiology, Centre Hospitalier Universitaire, Sherbrooke, Quebec, Canada

So sánh hiện tượng oxýt-hoá chất 3, 4-dihydroxypheneylalanie (DOPA).
L-DOPA là thuốc được dùng điều trị bệnh run chân tay người già Parkinson.

The decarboxylation of 3, 4-dihydroxyphenylalanine (DOPA) to dopamine by aromatic L-decarboxylase, a pyridoxal-phosphate-(PLP-) dependent enzyme, has been described in animal tissues and human post-mortem specimens. A so-called spontaneous decarboxylation of DOPA was reported recently when [carboxyl-14C]DOPA was incubated in phosphate buffer without any tissues. It was suggested later that such a “spontaneous decarboxylation” of DOPA in vitro might contribute significantly to a rapid decarboxylation in vivo, resulting in low plasma levels in patients with Parkinson’s disease after administration of large doses of this drug. However, we demonstrate here that this reaction is due to a well known non-enzymatic oxidation of DOPA to CO2 and melanin rather than to the spontaneous decarboxylation of DOPA to CO2 and dopamine. A continous-flow ionization chamber method was used for measurements of an enzymatic decarboxylation and a non-enzymatic oxidation of DOPA under various experimental conditions in vitro.

Details of the ionization chamber method for an instantaneous and continuous measurement of 14CO2 production from [14C] biochemical have been published previously. Approximately 0.12µC. [carboxyl-14C]DOPA was added to 0.1 M phosphate buffer, pH 7.0 at 37ºC., and incubated with and without enzymes or rat liver homogenates. The livers of male rats were homogenized with a “Stir” in 30ml. of cold phosphate buffer, pH 7.0, and centrifuged at 3000 r.p.m. for 10 minutes. Compressed gases with 95% 02 and 5% CO2, 100% N2 or compressed air with approximately 20% O2 and 80% N2 were passed continuously through the incubation chamber at a constant flow-rate (100ml per minute). The gas flow then passed through an ionization chamber connected to a vibrating-reed electrometer which, in turn, was connected to a chart recorder.
For the purpose of comparing various curves obtained from either the non-enzymatic oxidation or the decarboxylation of DOPA, we have utilized T max and the total fraction of the incubated of [carboxyl-14C]DOPA.

Spectral measurement: Visible and ultra-violet absorption measurements were made with an Acta ÌI Beckman recording spectrometer equipped with a 0-3-0-absorbance expanded scale. Non-radioactive DOPA was added to 0.1 M phosphate buffer, pH 7.0, incubated at 37ºC., and gassed with 95% O2 and 5% CO2. The spectra were scanned repeatedly. Similarly, a cuvette of non-radioactive DOPA solution was scanned repeatedly when exposed to air only.

Discussion: In previous studies measurements of liberated 14CO2 from [carboxyl-14C]DOPA suggested that DOPA was easily “decarboxylated in phosphate buffer without the addition of tissue homogenates (Vogel). We observe here that such a very large amount of 14CO2 can be detected in the presence of O2 and air atmospheres, or H2O2 but not in the presence of N2 atmospheres. The result indicated, therefore, that there is no important spontaneous decarboxylation of DOPA in 0.1 M phosphate buffer, pH 7.0, and in N2, whereas there is a considerable non-enzymatic oxidation of DOPA to melanin occurring in O2 or in H2O2.
This non-enzymatic oxidation of DOPA by H2O2 was probably due to its ions, HO-2 or O2(-2) or O3(-2), formed in the phosphate buffer. The formation of polyphenols from DOPA in air or O2 can be seen in our study by changes in the absorption spectrum of this substrate at typical peaks at 210 nm. and 280 nm. This is supported further by the observed formation of black DOPA melanin from 1-10x10-3M non-radioactive DOPA incubated with O2 for 10 hours, or H2O2 in the phosphate buffer. In addition, such a non-enzymatic oxidation of DOPA was inhibited significantly by a relatively large dose of LPL, possibly due to the formation of DOPA-PLP complex.
Interestingly, we observed that both the non-enzymatic oxidation of DOPA in O2 and the spontaneous decarboxylation of DOPA in N2 were completely inhibited by human plasma and erythrocytes. Such a non-enzymatic oxidation of DOPA was easily reversed by Cu2+ in O2, but not in N2. We recently found a two-fold increase in 14CO2 production from [carboxyl-14C]DOPA in 14CO2 production incubated with rat liver homogenates, in 0.1 M phosphate buffer, pH 7.0 and in an O2 atmosphere, as compared to values obtained in N2. Similar results were noted with this 14C-labelled DOPA and L-histidine decarboxylase in the presence of PLP in O2 and N2 respectively. These observations suggest the occurrence of an enzymatic decarboxylation of DOPA in H2 and, on the other hand, the occurrence of both an enzymatic decarboxylation and a non-enzymatic oxidation of DOPA in O2. This suggests also that measurement of DOPA decarboxylase activity in isolated tissues with [carboxyl-14C]DOPA, using either a modified standard Warburg technique or a continuous-flow ionization chamber, should be performed in N2 only.
Our overall results demonstrate that under the conditions employed, DOPA can undergo an enzymatic or a non-enzymatic reaction.

References:
1) Laplante, M., Tran Ngo, and LeBel, E.: ‘Influence du Cu++ sur l’oxydation non-enzymatique de DOPA dans le foie de rat”, Proc. Can. Fedn Biol. Socs, 14: 22, 1971.
2) Tran, Ngo: Interaction of aromatic L-amino-acid decarboxylases with pyridoxal phosphate. Int. J. Biochem., 2: 700-704, 1971.
3) Tran, Ngo: Complex-formation between hydrogen peroxide and L-phenylalanine decarboxylase. Int. J. Biochem. 3: 61-65, 1972.
4) Tran, Ngo and Laplante, M. , unpublished data, 1972.
5) Tran, Ngo, Laplante M., and LeBel, E.: “ Altered catabolism of 14C-formate by erythrocytes of folic acid-deficient rats: a possible in vitro means for differential diagnosis of megaloblastic anemias in man? J. Nucl. Med., 12: 222-226, 1971.

Acknowledgements: This work was supported by the Sherbrooke Medical School Fund. Marcel Laplante was a Ph.D. candidate and a recipient of a studentship from the Medical Research Council of Canada.

Copyright, 2009. Muốn phổ biến bài viết này, cần xin phép tác giả và xin ghi rõ nguồn Y Dược Ngày Nay, www.yduocngaynay.com

>>>back>>>