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LETTERS TO THE EDITOR |
* Neuroscience Research Institute State University of New York at Old Westbury Old Westbury, NY 11567
Department of Biochemistry State University of New York Downstate Medical Center Brooklyn, NY 11203
We are very pleased that our article has engendered such interest in the biologically important realm of endogenous morphine expression in eukaryotes by the group noted for its work in opiate alkaloid biosynthesis in plants. We now provide a point-by-point explication of potential confounds in the analyses of Boettcher et al. as follows:
1) Boettcher et al. corroborate, via gas chromatography-tandem mass spectrometry, that morphine is found in white blood cells (WBC) without commenting on the remarkable concurrence of cellular concentration values (
10 pg morphine/million cells) with our values validated by quadrupole-time of flight-mass spectrometry.
2) They conclude that WBC do not synthesize morphine originating from CYP2D6-catalyzed conversion of tyramine into dopamine without addressing our results, supporting a parallel pathway of morphine biosynthesis that derives from tyrosine hydroxylase-catalyzed conversion of tyrosine to 3,4-dihydroxy-L-phenylalanine.
3) They used our published incubation conditions, without success, to metabolically label the pool of cellular morphine from tyramine without addressing the requisite sensitivity of our RIA/HPLC detection system capable of quantification of fmol concentrations of morphine. The low sensitivity of their detection system is indicated by the 7-day incubation period that was required to monitor 13C isotope enrichment into 13C-labeled morphine from precursors in neuroblastoma cells (1, 2). The lack of consideration in determining detection limits in the labeling of various intracellular substrate pools in SHY5Y (3) may have also weakened their conclusions pertaining to the metabolic and biosynthetic pathway for morphine in human cells (1, 2).
4) The inability to detect significant levels of 3H2O arising from CYP2D6-catalyzed ring hydroxylation of [3H]tyramine is predictable based on the low specific activity of commercially available [3H]tyrosine or [3H]tyramine (typically 50 Ci/mmol for 3H3,3H5, ring-labeled tyrosine) and the inappropriate adaptation of an in vitro assay originally developed to monitor tyrosine hydroxylase activity (2, 4). The feasibility of the [3H]tyramine/3H2O release assay as an accurate and sensitive method for monitoring CYP2D6 activity is not established in vitro or in viable cell cultures. Boettcher et al. selectively present data indicating conversion of isotopically labeled dopamine to [1-13C, 3'-18OH]-3,4-dihydroxyphenylacetic acid and [1-13C, 3'-18OH]homovanillic acid without addressing the basic issue of whether isotopically labeled tyramine is directly converted to dopamine by CYP2D6, which is consistent with our published work (5) and that of others (6, 7, 8, 9).
5) They demonstrate the presence of both monoamine oxidase and catechol-O-methyltransferase enzymes previously identified as critical for morphine biosynthesis (1, 2). Additionally, the authors demonstrate conversion of radiolabeled codeine to morphine in WBC, although they have not used appropriate CYP2D6 inhibitors. This is not surprising based on in vitro studies demonstrating that CYP2D6 demethylation occurs faster than ring hydroxylation, i.e., rapid conversion of codeine to morphine (8, 9). In light of the above, we do not understand why the authors conclude that WBC do not synthesize morphine.
6) In conclusion, our results demonstrating a relatively slow conversion of tyramine, via dopamine, into morphine are supported by our enzymology studies of CYP2D6 showing a 10-fold lower binding affinity of tyramine to the active site of the enzyme as compared with the 3-OH-analog (8, 9).
References
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