Mammalian morphine: de novo formation of morphine in human cells.

BACKGROUND: Morphine, the major alkaloid of opium of Papaver somniferum, is one of the strongest analgesic compounds known. "Endogenous morphine" has been long isolated and authenticated by mass spectrometry in trace amounts from animal and human specific tissue or fluids. The most widely accepted explanation presently is that morphine detected in human and animal tissue is of exogenous sources (e.g. dietary origin). MATERIAL/METHODS: The biosynthetic experiments were performed with human neurobalstoma cells (SH-SY5Y) and human pancreas carcinoma (DAN-G) cells. The application experiments were done in the presence of isotopically labeled potential precursors such as (18)O(2) and [ring-(13)C(6)]-L-tyrosine. Benzylisoquinoline alkaloids present in mammalian cells were identified by GC-MS/MS. RESULTS: Growth of the SH-SY5Y cells in the presence of (18)O2 led to [(18)O(2)]-labeled morphine and [(18)O(2)]-labeled reticuline, each had a molecular mass four mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule. DAN-G cells cultured in an (18)O(2) atmosphere produced (S)-[ (18)O(2)]-norlaudanosoline and (S)-[ (18)O(2)]-reticuline, each labeled with (18)O atoms at only two of the four possible positions. Feeding of [ring-(13)C(6)]-tyramine, (S)-[1-(13)C, N-(13)CH(3)]-reticuline and [N-CD(3)]-thebaine to SH-SY5Y cells led each to the labeling of morphine, as established by GC-MS/MS. CONCLUSIONS: Taken together, morphine, reticuline and norlaudanosoline are unequivocally biosynthesized by cultured human cells. The precursors of morphine in the human cell lines were conclusively shown to be oxygen, tyramine, reticuline and thebaine.

High-performance liquid chromatographic determination of enalapril in human plasma by enzyme kinetic analytical method.

A high-performance liquid chromatographic method for indirect determination of enalapril in human plasma, was developed and validated. An exogenous angiotensin converting enzyme after drug inhibition was determined by reacting with hippuryl-histidyl-leucine to produce hippuric acid (HA) which was inversely proportional to the amount of enalaprilat in plasma. The HPLC was carried out on a Lichrosphere 60RP-select B, C18, 5 microm (125 mm x 4.0 mm i.d.) column at flow rate of 1.0 ml/min. The analysis time per injection was within 6.5 min. The lowest concentration of enalaprilat to be quantitated was 3.0 ng/ml with the acceptable accuracy and precision. This successfully developed method was practically and accurately used for pharmacokinetics and bioequivalent study of enalapril.

Endogenous formation of morphine in human cells.

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.

Evaluation of the mass spectrometric fragmentation of codeine and morphine after 13C-isotope biosynthetic labeling.

All major fragment ions of codeine and morphine were elucidated using LC-electrospray MS/MS and high resolution FT-ICR-MS combined with an IRMPD system. Nanogram quantities of labeled codeine were isolated and purified from Papaver somniferum seedlings, which were grown for up to 9 days in the presence of [ring-13C6]-l-tyrosine, [ring-13C6]-tyramine and [1,2-13C2], [6-O-methyl 13C]-(R,S)-coclaurine. The labeling degree of codeine up to 57% into morphinans was observed.

1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway.

A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.

Electrospray tandem mass spectrometric investigations of morphinans.

In this study positive ESI tandem mass spectra of the [M + H]+ ions of morphinan alkaloids obtained using an ion trap MS were compared with those from a triple quadrupole MS. This allows to assess the differences of the tandem-in-time versus the tandem-in-space principle, often hampering the development of ESI MS/MS libraries. Fragmentation pathways and possible fragment ion structures were discussed. In order to obtain elemental composition, accurate mass measurements were performed. According to the MS/MS fragmentation pathway, the investigated compounds can be grouped into 4 subsets: (1) morphine and codeine, (2) morphinone, codeinone, and neopinone, (3) thebaine and oripavine, (4) salutaridine and salutaridinol. Salutaridinol-7-O-acetate shows a different fragmentation behavior because of the favored loss of acetic acid. Although most fragment ions occur in both ion trap and triple quad tandem mass spectra, some are exclusively seen in either type. For triple quad, quadrupole time-of-flight and FT-ICR MS/MS, the base peak of morphine results from an ion at m/z 165 that contains neither nitrogen nor oxygen. This ion is not found in ion trap MS/MS, but in subsequential MS3 and MS4.