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Comment on "Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation"

Biocenter University Halle-Wittenberg Halle (Saale), Germany

Stefano et al. (1) observed that human polymorphonuclear (PMN) cells contained and biosynthesized morphine endogenously. We confirm here, by gas chromatography-tandem mass spectrometry (2, 3), the presence of morphine in human PMN cells (10.4 ± 1.6 pg/million cells) but furthermore also in mononuclear (MN) cells (8.5 ± 1.0 pg/million cells) and the erythrocyte fraction (81.3 ± 7.4 pg/ml packed volume) (morphine contamination excluded). It was also suggested (1) that morphine is biosynthesized in PMN from tyramine via conversion to dopamine by action of CYP2D6. Applications of unlabeled and [ring-¹³C₆]tyramine (10^–6 M) to PMN or MN as published (1) showed no significant increase in morphine level (<0.5%) (Fig. 1), and absolutely no labeled morphine (neither ¹³C₆- nor ¹³C₁₂-morphine) was detected. Furthermore, the incubation of PMN or MN cells with [ring-3,5-[³H]₂]tyramine showed no significant release of ³HOH from [ring-3,5-[³H]₂]tyramine (4), indicating that 3-hydroxylation of tyramine did not occur, and thus, dopamine was not formed by PMN or MN cells. In addition, we found that [1-¹³C,3'-¹⁸OH]dopamine was transformed by PMN or MN to its metabolites, [1-¹³C,3'-¹⁸OH]-3,4-dihydroxyphenylacetic acid (DOPAC) (identified as trimethylsilyl (TMS) derivative: m/z 387 [M]^·+, 16.4 ± 1.0 ng/million cells) and [1-¹³C,3'-¹⁸OH]homovanillic acid (HVA) (as TMS derivative: m/z 329 [M]^·+, 18.8 ± 3.1 ng/million cells) (Fig. 2). Neither DOPAC nor HVA was formed in PMN or MN supplied with either unlabeled or [ring-¹³C₆]tyramine, again showing that tyramine was not transformed to dopamine. [6-²H]Codeine applied to PMN or MN was demethylated to morphine (as TMS derivative: m/z 430 [M]^·+, 2.6 ± 0.7 ng/million cells), indicating the presence of functionally active CYP2D6 in PMN or MN (Fig. 1). Three types of experiments presented here show clearly that isolated human white blood cells (PMN and MN) do not transform tyramine by action of CYP2D6 to dopamine and fail to synthesize morphine.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Morphine (as TMS derivative) detection and enhancement following precursor exposure. Human white blood cells (WBC) isolated from heparinized blood were incubated with unlabeled [ring-13C6]tyramine and [3-2H]codeine at 10–6 M for 1 h. WBC morphine significantly increased when incubated with codeine (2600 ± 700 pg/106 cells), but neither with unlabeled tyramine (10.5 ± 0.7 pg/106 cells) nor [ring-13C6]tyramine (11 ± 3.4 pg/106 cells), compared with a control value of (10.4 ± 1.6 pg/106 cells). Each experiment was repeated four times, and the mean ± SEM was graphed.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Dopamine-derived acids (as TMS derivative) detection and enhancement following precursor exposure. Human WBC isolated from heparinized blood were incubated with unlabeled [ring-13C6]tyramine and [1-13C,3'-18OH]dopamine at 10–6 M for 1 h. WBC DOPAC and HVA significantly increased when incubated with labeled dopamine (DOPAC: 16,400 ± 1,000 pg/106 cells, HVA: 18,800 ± 3,100 pg/106 cells), but neither with unlabeled tyramine (76 ± 8.5 pg/106 cells nor with [ring-13C6]tyramine (67 ± 9.2 pg/106 cells), compared with a control value of (56 ± 10.2 pg/106 cells). Each experiment was repeated four times and the mean ± SEM was graphed.

Footnotes

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References

1. Zhu, W., P. Cadet, G. Baggerman, K. J. Mantione, G. B. Stefano. 2005. Human white blood cells synthesize morphine: CYP2D6 modulation. J. Immunol. 175: 7357-7362. [Abstract/Free Full Text]
2. Poeaknapo, C., J. Schmidt, M. Brandsch, B. Dräger, M. H. Zenk. 2004. Endogenous formation of morphine in human cells. Proc. Natl. Acad. Sci. USA 101: 14091-14096. [Abstract/Free Full Text]
3. Boettcher, C., M. Fellermeier, C. Boettcher, B. Dräger, M. H. Zenk. 2005. How human neuroblastoma cells make morphine. Proc. Natl. Acad. Sci. USA 102: 8495-8500. [Abstract/Free Full Text]
4. Reinhard, J. F., G. K. Smith, C. A. Nichol. 1986. A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of ³H₂O and adsorption of [³H]-tyrosine by charcoal. Life Sci. 39: 2185-2189. [Medline]

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