Total histamine content of mast cells was calculated by freezing and thawing suspensions for 3 cycles. Cell suspensions containing 500,000 cells/ml released 1.27μg/ml (0.21) histamine, corresponding to 2.53 pg (0.41) histamine per mast cell.

Mast cells stimulated with 0.25μg/ml calcium ionophore released 63% (7.2) of total histamine.

The effect of histamine release from mast cells treated with both clinically attained concentrations of morphine and concentrations above those seen clinically was determined.

To chemically defined reaction mixtures, morphine was added at varying concentrations (18 ng/ml, 450 ng/ml, 6.68μg/ml and 668μg/ml). Enzymes causing free radical activation were not present. The blank sample contained no morphine. Reaction mixtures were incubated in a water bath at 37°C for 60 minutes. Experiments were performed in triplicate. Histamine release from samples was determined following derivitization with o-phthaldialdehyde and mercaptoethanol using high-performance liquid chromatography as detailed below. Histamine release from reaction mixtures was expressed as a % of total histamine present in the cell pellet.

Histamine release from reaction mixtures containing morphine at all concentrations was not statistically different (p > 0.1) from histamine release from blank samples (Table 1).

To verify the time dependence of histamine release, a morphine concentration of 668 μg/ml was tested. Release was detectable as early as 5 minutes after the addition of drug and appeared to plateau between 40 and 60 minutes (Figure 1).

To determine whether the addition of sodium bisulfite causes increased histamine release from mast cells, sodium bisulfite at a concentration of 0.1% of morphine concentration (the % of sulfite present in morphine most formulations) was added to the reaction mixtures described previously. Blank samples contained neither morphine nor sulfite. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as above.

There was no statistical difference in histamine release at any concentration of morphine with sulfite compared with blank samples (Table 1).

Reaction mixtures containing 668μg/ml morphine with 0.1% sodium bisulfite were incubated for 5, 10, 20, 40 and 60 minutes yielded results identical to those observed in the absence of sodium bisulfite (results not shown).

Prostaglandin H synthestase may catalyze free radical formation [20]. The first experiment investigated its effect on mast cell histamine release in the absence of sulfite and morphine. This ensured that any increase in histamine release in subsequent experiments was not due to the effect of the enzyme only.

Prostaglandin H synthetase was added to reaction mixtures at a concentration of 25 mU. In reaction mixtures containing no drug, the enzyme was added to mast cell samples containing neither morphine nor sulfite. Controls contained no enzyme.

Reaction mixtures containing the enzyme alone (no sulfite or morphine) produced similar histamine release compared to blank samples (3.2% (0.7) of total and 2.9% (2.6) of total respectively). The presence of prostaglandin H synthetase had no effect.

The effect of clinically attained concentrations of morphine in the presence of prostaglandin H synthetase on mast cell histamine release was investigated. In vivo studies show histamine release at these concentrations of morphine [3-7]. Morphine sulfate was added to the mast cell solution at clinically attained concentrations of 450 ng/ml (a typical peak in vivo concentration following an intravenous morphine bolus) and 18 ng/ml (a typical steady-state concentration during a 20 μg/kg/hour morphine infusion) [27]. Mast cell histamine release from higher concentrations of morphine (668μg/ml and 6.68μg/ml) was also investigated.

Blank samples contained water instead of morphine. All samples contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Histamine release values are shown in Table 2. There was no statistical difference between histamine release from morphine samples at both clinically-attained concentrations (18 ng/ml (p = 0.87) and 450 ng/ml (p = 0.08)) and histamine release from blank samples. Histamine release from HMC-1 cells incubated with morphine 668μg/ml and 6.68μg/ml were significantly increased compared with blank samples (p = 0.03 at both concentrations). Results are shown in table 2. Maximum histamine release (16.1% of total (6.3)) occurred at the highest concentration of morphine (668μg/ml) (Figure 1).

The effect of sodium bisulfite 0.1% in varying concentrations of morphine in the presence of prostaglandin H synthetase was investigated. This preservative is present in many morphine preparations and its effect on histamine release from mast cells is determined here. Morphine was added to the mast cell samples at concentrations of 668μg/ml, 6.68μg/ml, 450 ng/ml and 18 ng/ml. Sodium bisulfite was added to samples at a concentration of 0.1% of morphine concentration. Controls contained morphine only. Each sample also contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Reaction mixtures containing morphine 668μg/ml with 0.1% sodium bisulfite showed a statistically significant increase in histamine release compared with blank samples (p = 0.03). There was no statistical difference in histamine release between morphine only samples at all concentrations and samples containing morphine and 0.1% sodium bisulfite (Table 2) (morphine 668μg/ml (p = 0.8), morphine 6.68μg/ml (p = 0.13), morphine 450 ng/ml (p = 0.89) and morphine 18 ng/ml (p = 0.56). Sulfite did not have any additive effect on histamine release from mast cells in the presence of morphine and prostaglandin H synthetase.

Studies looking at plasma histamine levels following morphine administration in humans have shown peak histamine levels within 5 minutes of administration [3,4,6]. This experiment investigated whether maximum histamine release occurred at a similar time in vitro in the presence of the enzyme prostaglandin H synthetase. HMC-1 cells were incubated at 37°C for 5, 10, 20, 30 and 60 minutes with morphine 668μg/ml, 0.1% sodium bisulfite and 25 mU prostaglandin H synthetase. The cells were centrifuged and the supernatant was examined for the presence of histamine and expressed as a % of total histamine.

Histamine release was time dependent with a maximum effect after 1 hour of incubation (Figure 2).

Interestingly, reaction mixtures containing a typical peak morphine concentration (450 ng/ml) showed a significant increase in histamine release in the presence of prostaglandin H synthetase compared with no enzyme. This was true for both sulfite-free (p = 0.003) and sulfite-containing (p = 0.009) morphine solutions. This suggests that even at clinically-attained morphine concentrations, it is not only possible to cause the metabolic activation of sulfite, but also that of morphine.

The effect of sulfite alone (without morphine) on histamine release from mast cells was investigated. To 4 mast cell reaction mixtures, varying concentrations of sulfite (0.018 ng/ml, 0.45 ng/ml, 0.007μg/ml and 0.67μg/ml) were added. These concentrations of sulfite correspond to the concentration of sulfite present in morphine solutions of 18 ng/ml, 450 ng/ml, 6.6μg/ml and 668μg/ml. To each sample 25 mU prostaglandin H synthetase was added. The highest concentration of sulfite (0.67μg/ml) was incubated for 10, 20, 40 and 60 minutes to assess time dependence. The reaction mixtures were incubated, centrifuged and analyzed as described below.

In the presence of prostaglandin H synthetase, sulfite resulted in histamine release from mast cells. There was a significant increase in histamine release (9.7% (0.65) of total) in the reaction mixture containing 0.67μg/ml sulfite (p = 0.01). There was no statistically significant difference in histamine release compared with blank samples at all other concentrations of sulfite (0.018 ng/ml sulfite (2.8% (0.3) of total, p = 0.91), 0.45 ng/ml sulfite (1.93% (0.35) of total, p = 0.53) and 0.007μg/ml sulfite (4.5% (0.7) of total, p = 0.36) (Table 3).

The time-dependent experiment showed histamine release of 8% of total (3.3) at 10 minutes incubation, 7.7% of total (2.2) at 20 minutes, 10.5% of total (6.1) at 40 minutes and 9.7% of total (0.65) at 60 minutes.

Human leukemic mast cells, HMC-1 [33] were kindly provided by J. Butterfield, Rochester, Minnesota. This cell line shows many characteristics of immature mast cells. They have been shown to release histamine following stimulation [28-30] Cells were carried in 75 cm² vented tissue culture flasks (BD Falcon, Franklin Lakes, NJ) containing Iscoves modified Dulbecco's medium (Sigma, St.Louis, MO) with 10% iron-supplemented calf serum (Hyclone, Logan, Utah) and monothioglycerol 1.2 mM (Sigma, St.Louis, MO). Incubations were conducted in a humidified atmosphere at 5% CO₂ and 37°C. Cells were passed weekly and fed only when necessary (evidence of color change in media).

To pass cells, the cell suspension was placed in a 50 ml polypropylene centrifuge tube (BD Falcon, Franklin Lakes, NJ) and centrifuged at 250 × g at 4°C for 10 minutes. The culture medium was discarded and the cells were re-suspended in fresh medium. Cell density was maintained at 500,000 cells/ml.

Reagent grade O-phthaldialdehyde (OPA), mercaptoethanol, dihydrogen sodium phosphate, disodium EDTA, sodium bisulfite, morphine sulfate, hydrogen peroxide, xanthine oxidase, boric acid and phosphate buffered saline (PBS) were purchased from Sigma (St.Louis, MO, USA). Methanol (HPLC grade), acetonitrile (HPLC grade) and sodium hydroxide were from Fisher Scientific (Hampton, NH). Phosphate buffered saline (PBS) pH 7.4, contained 120 mmol/L NaCl, 2.7 mmol/L KCl and 10 mmol/L phosphate. Horseradish peroxidase was purchased from Cooper Biomedical. Prostaglandin H synthetase was purchased from Calbiochem (Darmstadt, Germany).

Mast cell suspensions, containing approximately 500,000 mast cells were, aliquoted into 1 ml samples. These were rinsed 4 times with 4 ml of PBS, pH 7.4. Cells were then re-suspended in 1 ml PBS. Reaction mixtures were supplemented with morphine, sulfite and prostaglandin H synthase at this stage according to experimental design, unless otherwise specified. Reaction mixtures were incubated for 60 minutes in a water bath at 37°C. Reactions were stopped by centrifugation at 250 × g for 4 minutes at 4°C and placed on ice. Then 200μl of the supernatant was collected and dried under nitrogen at 40°C.

Histamine in the supernatant was measured using derivitization with OPA and mercaptoethanol (ME) as described [34]. Dried samples were re-dissolved in 100μl of derivitisation buffer (0.4 M boric acid adjusted to pH 9.5 with 1 M sodium hydroxide) and then derivitized with 20 μL of a freshly made 1:1 mixture of OPA (3.8 mM in methanol) and ME (2.5 ml/L in methanol) and analyzed within 30 minutes. The derivitized samples were used for chromatography.

The response of HMC-1 cells to the stimulant calcium ionophore was investigated to assess the releasability of HMC-1 cells. 0.25μg/ml calcium ionophore was added to mast cell suspensions. Reaction mixtures were incubated and analyzed as above.

To extract the remaining histamine from the original sample, the mast cell pellet in suspension was frozen and thawed 3 times. It was then centrifuged at 250 × g for 4 minutes and 200μl of the supernatant was dried under nitrogen as described previously. Samples were re-dissolved, derivitized and analyzed as above. Histamine release during morphine incubations was expressed as a percentage of total histamine release.

Histamine was measured by high-performance liquid chromatography using a Varian model 5500 chromatograph. The mobile phase was 0.1 M dihydrogen sodium phosphate and 1 mM disodium EDTA with 16% methanol and 14% acetonitrile adjusted to pH 6.4. The flow rate was 0.8 ml/minute.

Separations were performed using a 15 cm × 4.6 mm, Ace 5 C18 reversed-phase cartridge column (Advanced Chromatography technologies, UK). A Zorbax C8 guard column with a 5 micron, 4.6 × 12.5 mm analytical guard-column cartridge (Agilent technologies, Palo Alto, CA) was used. Fluorescence detection was used with the fluorimeter (Varian 9070) set at an excitation of 348 nm and an emission of 444 nm (determined by running individual excitation and emission spectra on the fluorometer).

Chromatographic conditions involved the use of a mobile phase gradient to optimize peak separation. A linear gradient starting at 90% mobile phase, 10% acetonitrile and ending at 80% mobile phase, 20% acetonitrile took place over 20 minutes. The column was washed by increasing the acetonitrile to 30% by 22 minutes and by 23 minutes the proportions were returned to the original values. The length of each run was 30 minutes.

Values are expressed as a mean +/- SD of 3 experiments. One-way analysis of variance was used to determine the significance of the difference between groups. Results were considered significantly different when p < 0.05.