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. 2004 Sep 28;101(39):14091-6.
doi: 10.1073/pnas.0405430101. Epub 2004 Sep 21.

Endogenous Formation of Morphine in Human Cells

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Free PMC article

Endogenous Formation of Morphine in Human Cells

Chotima Poeaknapo et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of (18)O(2) led to the [(18)O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule of morphine. Growth of DAN-G cells in an (18)O(2) atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-(13)C(6)]-tyramine, [1-(13)C, N-(13)CH(3)]-(S)-reticuline and [N-CD(3)]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of "endogenous morphine" in the neurosciences and immunosciences.

Figures

Fig. 1.
Fig. 1.
MS/MS product ion spectra of TMS-morphine. (A) The m/z 429 ion ([M]+ of reference TMS-morphine; plant-derived). (B) The m/z 429 ion ([M]+ of TMS-morphine isolated from the SH-SY5Y human neuroblastoma cell line).
Fig. 2.
Fig. 2.
MS/MS product ion spectra of morphine derivatives. (A) The m/z 477 ion ([M]+ of TFA-morphine isolated from SH-SY5Y cell line cultured in the presence of 16O2). (B) The m/z 481 ion ([M]+ of TFA-[18O2]-morphine labeled with two atoms of 18O per morphine molecule that was isolated from SH-SY5Y cell line cultured in the presence of 18O2). (C) The m/z 429 ion ([M]+ of TMS-morphine isolated from SH-SY5Y cell line). (D) The m/z 435 ion ([M]+ of TMS-[13C6]-morphine isolated from SH-SY5Y cell line cultured in the presence of 40 μM[ring-13C6]-tyramine).
Fig. 3.
Fig. 3.
Precursor studies in human neuroblastoma cell culture. (A) Labeling pattern in morphine molecules produced by SH-SY5Y cells after supplying 18O2 (asterisk denotes 18O), [ring-13C6]-tyramine (circle denotes 13C), [1-13C, N-13CH3]-(S)-reticuline (square denotes 13C), or [N-CD3]-thebaine (D denotes 2H) in the culture medium. (B) Predicted intermediates in the pathway from l-tyrosine to morphine in SH-SY5Y cells.
Fig. 4.
Fig. 4.
GC/MS chromatograms obtained from selected-ion monitoring (SIM) mode analysis. (A) The selected ion at m/z 234 represents the proposed fragment of reference TMS-morphine, which includes all five rings. (B) The selected ion at m/z 240, representing the proposed fragment of TMS-[13C6]-morphine, which includes all five rings. (C) The selected ion at m/z 236, which represents the proposed fragment of isoquinoline-derived rings of reference TMS-morphine. (D) The selected ion at m/z 242, which represents the proposed fragment of isoquinoline-derived rings of TMS-[13C6]-morphine.

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