Ascaris suum, an Intestinal Parasite, Produces Morphine

Extraction

The extraction experiments using internal or external morphine standards were performed in different rooms to avoid morphine contamination of Ascaris samples. Single-use siliconized tubes also were used to prevent the loss of morphine as well as contamination between assays (14). Tissues were extensively washed with PBS buffer (three times, 1 min) to avoid an additional potential source of morphine contamination. Tissues were weighed, homogenized in 1 N HCl (1 ml/0.1 g), and then extracted with 5 ml chloroform/isopropanol 9:1 (24). After 5 min, homogenates were centrifuged (3,000 rpm, 15 min). The supernatant was centrifuged twice (15 min, 12,000 rpm, 4°C), dried, and dissolved in 0.05% trifluoroacetic acid (25). Samples were loaded on a Sep-Pak Plus cartridge (Waters, Milford, MA) and eluted in water/acetonitrile/trifluoroacetic acid (89.95%:10%:0.05%, v/v/v). Samples were dissolved in buffer A before HPLC analysis (see below).

HPLC and electrochemical detection

HPLC separations were performed with two Acuflow Series VI and a C18 Unijet microbore column (Bioanalytical Systems, West Lafayette, IN). Morphine detection was performed with an amperometric detector LC-4C (Bioanalytical Systems) using a Unijet glassy carbon working electrode (3 mm) and a 0.02 Hz filter (500 mV; range, 5 nA). The mobile phases were buffer A (10 mM ammonium bicarbonate, 10 mM ammonium chloride, 10 mM sodium chloride, 0.2 mM EDTA (pH 5.0)) and buffer B (10 mM ammonium bicarbonate, 10 mM ammonium chloride, 10 mM sodium chloride, 0.2 mM EDTA, 50% acetonitile (pH 5.0) (24)). The injection volume was 5 μl, and the flow rate was 70 μl/min. Conditions were: t = 0, 0% buffer B; t = 10 min, 10% buffer B; t = 20 min, 30% buffer B; and t = 25 min, 100% buffer B. Several HPLC purifications were performed between each sample to prevent residual morphine contamination remaining on the column. Furthermore, the fraction of blank chromatography corresponding to the elution time of the morphine (flat line) was determined by gas chromatography/mass spectrometry (GC/MS)³ analysis, confirming that no morphine was remaining. Five nanograms of internal morphine standard was first chromatographed (Fig. 1⇓A) along with Ascaris morphine (Fig. 1⇓B), and then the same sample with 4 ng of morphine external standard was added after the extraction (Fig. 1⇓C).

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FIGURE 1.

Purification of morphine from Ascaris. Morphine was isolated by HPLC. Running conditions: range, 5 nA; filter, 0.02 Hz; potential, 500 mV; flow rate, 500 μl/min; A buffer: 1 mM ammonium bicarbonate, 10 mM ammonium chloride, 10 mM sodium chloride, 0.2 mM EDTA (pH 5.0); B buffer: same as A buffer but with 50% acetonitrile. Gradient: t = 0, 0% buffer B; t = 10 min, 10% buffer B; t = 20 min, 30% buffer B; t = 25 min, 100% buffer B. A, Five nanograms of morphine internal standard. B, Standard curve of low morphine concentrations. C, Ascaris extract. D, Ascaris extract + 4 ng morphine external standard. E, Ascaris incubation medium.

GC/MS

The identity of the morphine detected by HPLC was further confirmed by GC/MS using a modification of a method reported by Allen et al. (26). A fraction of the eluent corresponding to the HPLC morphine peak was collected in a 0.5-ml siliconized plastic vial, evaporated to dryness, and stored at −70°C. The sample was dissolved in 50 μl of 2% ammonia (in methanol) and transferred to 0.1-ml conical glass vials (Kimax, Alphuretta, GA) fitted with Teflon-lined septum liners. The sample was evaporated under nitrogen gas and then mixed with N,O-bis (trimethylsilyl) trifluoroacetimide (20–50 μl) catalyzed with 1% trimethylchlorosilane (90°C, 30 min). This reaction yields a ditrimethylsilyl derivative of morphine (m.w. = 429). The reaction conditions chosen resulted in higher yields than did reactions conducted at lower temperature (70°C) or for shorter times. The derivatized morphine was analyzed on a Varian Saturn III GC/MS equipped with a 30-m (0.25 mm internal diameter and 0.25 μm) DB-5 capillary column (J & W Scientific, Folsom, CA). Two-microliter injections were made in splitless mode at an initial column temperature of 180°C, and after 2 min the column temperature was heated at a rate of 15°C/min up to 300°C. Morphine was detected at 10 min; injection port temperature was 275°C. Mass spectrum indicated major ions (base peak depended on instrument tune conditions) at 429 (M+) and 414 (M-CH₃+). Analyses of samples were conducted using a selected ion storage method, in which mass windows (± 2 atomic mass units) around ions 429 and 414 were collected. Morphine identity was confirmed by the retention time, peak shape, and comparison of parasite-derived morphine to authentic morphine standards injected.

Bioassay of Ascaris morphine: NO release

Human peripheral monocytes (Long Island Blood Services, Melville, NY) were isolated using the Accurate Scientific (Westbury, NY) monocyte kit and were washed as previously described (27, 28, 29). We used monocytes to measure the biological activity of Ascaris-derived morphine because we have demonstrated that they contain stereospecific opiate alkaloid-selective receptors that are coupled to morphine and NO release that is naloxone reversible (27, 28).

NO release from the incubated monocytes (10⁷ cells/chamber) was measured directly using an NO-specific amperometric probe (World Precision Instruments, Sarasota, FL) (30, 31). Briefly, the cells were placed in a superfusion chamber in 1 ml PBS. The probe was positioned 15 μm above the cell surface by a micromanipulator (Zeiss-Eppendorff, Suwanee, GA) attached to the stage of an inverted microscope (Diaphot; Nikon, Melville, NY). The system was calibrated daily by adding potassium nitrite to a solution of potassium iodide, resulting in the liberation of a known quantity of NO (World Precision Instruments). Baseline levels of NO release were determined by evaluation of NO release in PBS. Cells were stimulated with the respective purified morphine-like material, and the concentration of NO gas in solution was measured in real time with the DUO 18 computer data acquisition system (World Precision Instruments). The amperometric probe was equilibrated for at least 12 h in PBS before being transferred to the superfusion chamber containing the cells, and manipulation of the cells was performed only with glass instruments. To evaluate NO release, the cells were exposed to morphine as indicated.

Morphine immunohistochemistry

Adult specimens of A. suum were obtained from Italcarni (Carpi, Italy) immediatly after the slaughter of parasitized pigs. The worms were fixed either in phosphate-buffered formalin (4% paraformaldehyde in 0.1 M (pH 7.4)) for 8 h at 4°C or in a mixture of glutaraldehyde/picric acid/acetic acid (GPA; 1:3 + 1%) overnight and then for 48 h at 4°C. Specimens were rinsed in 70% ethanol, dehydrated, embedded in paraffin, and sectioned (8 μm). Morphine was localized by indirect immunofluorescence using a polyclonal Ab with minimal cross-reactivity to codeine (Biogenesis, Bournemouth, U.K.). Briefly, rehydrated sections were washed in PBS (0.9% NaCl, 0.01 M sodium phosphate buffer (pH 7.4)), preincubated in 5% normal serum for 30 min, and then incubated overnight at 4°C in a moist chamber with the primary Ab. Sections were then washed with PBS and incubated with anti-sheep rabbit IgG-FITC (Dako, Aarhus, Denmark; 1:40; 1 h; room temperature).

Slides were rinsed in PBS and then dipped in 0.005% propidium iodide for nuclei staining (1 min). After a final rapid rinse with PBS, sections were mounted in buffered glycerol (87.7% glycerol, 2.3% 1,4-diazalbicyclo[2.2.2]octane, 10% Tris 20 mM (pH 8)), cover-slipped, and examined on a Zeiss microscope equipped for fluorescence with FITC excitation and barrier filter combination.

For comparison, we tested two other antisera, anti-adrenocorticotrophic hormone and anti-β-endorphin (Biogenesis). Reaction specificity for each technique was controlled by: 1) replacing the Ab with a nonimmune serum; 2) omitting the first antiserum; and 3) preincubating the primary Ab at 4°C for 24 h in the presence of excess Ag (morphine HCl 10⁻³ M).

μ Opiate receptor expression: isolation of total RNA

Tissue samples, excised pedal ganglia of Mytilus as a positive control (32), and all body sections of Ascaris were homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH) containing 1-bromo-3-chloropropane (0.1 ml/ml Tri-Reagent) using a Polytron homogenizer. The homogenates were stored at room temperature for 5 min to allow complete dissociation of nucleoprotein. The samples were vortexed vigorously (15 s), kept at room temperature for 7 min, and then centrifuged (15 min, 12,000 × g). The aqueous phase was transferred to a fresh tube, and the RNA was precipitated with isopropanol, washed with 75% ethanol, air-dried, and resuspended in water. RNA was analyzed on a 1% denaturing agarose gel, and purity was determined spectrophotometrically.

RT-PCR of total RNA

First-strand cDNA synthesis was performed using random hexamers (Life Technologies, Gaithersburg, MD). Three micrograms of total RNA isolated from pedal ganglia as positive control or Ascaris tissue was denatured at 95°C and reverse transcribed at 42°C for 1 h using Superscript II RNase H-RT (Life Technologies). Seven microliters of the reverse transcription product was added to the PCR mix containing specific primers for the μ opioid receptor gene and Taq DNA polymerase (Life Technologies). The PCR reaction was denatured at 95°C for 5 min before 30 cycles at 95°C for 1 min, 57°C for 1 min, 72°C for 1 min, and then an extension step cycle at 72°C for 10 min. PCR products were analyzed on a 2% agarose gel (Sigma, St. Louis, MO) and visualized by ethidium bromide staining.

The μ-specific primers used in the PCR reactions amplified a 441-bp fragment starting at map position 896 (primer M1, 5′-GGTACTGGGAAAACCTGCTGAAGATCTGTG-3′) and at map position 1336 (primer M4, 5′-GGTCTCTAGTGTTCTGACGAATTCGAGTGG-3′). This segment of the gene encodes the third extracellular loop of the receptor that is important for μ agonist selectivity. In addition, primers for the internal control gene G3PDH (forward primer, 5′-ACCACAGTCCATGCCATCAC; reverse primer, 5′-TCCACCACCCTGTTGCTGGTA) were used to amplify a 451-bp fragment by RT-PCR from Ascaris total RNA.