The effect of dietary sulfur on opiate analgesia, conjugations and binding

Female Sprague Dawley rats weighing between 80 and 120 grams were individually housed and fed diets with a basal formulation of 15 percent casein with inorganic sulfur supplementation at 0.0002 percent, 0.02 percent, and 0.42 percent. Three variations in organic sulfur were achieved by no supplement, supplemental methionine (0.625 percent), and supplemental cysteine (0.505 percent).

Five animals on each of the nine diets (45 tests) were investigated for each phase of the study. The tests were made following a period of ad libitum feeding lasting 17 days.

The animals received a subcutaneous injection of ³H-etorphine (2.6 x 10⁶ CPM) in saline solution. Counts per minute in blood and brain homogenates were determined. The animals were sacrificed and cerebroside sulfates were isolated from the brain homogenates by florisil column chromatography.

Ratios of sulfated:glucuronidated ³H-etorphine in 24-hour urine collections from injected animals were calculated from differences in radiation levels following hydrolysis with type IX glucuronidase and type V sulfatase. The freed etorphine was extracted by dichloromethane.

Incubation of brain homogenate in varying concentrations of ³H-etorphine and ³H-naloxone followed by displacement of the unbound drug by centrifugation determined the levels of drug specifically bound to receptors. Number of binding sites was determined by Scatchard analysis.

No differences were detected in analgesic response with the opiate tested due to the fractional occupancy of etorphine receptors necessary for eliciting the effect. Differences in conjugation ratios were significant for level of dietary inorganic sulfate and form of organic sulfur supplementation. Increased levels of inorganic sulfate increased sulfated:glucuronidated ratios (S:G) and supplementation with cysteine resulted in higher S:G than methionine supplementation.

Dietary sulfur raised the number of binding sites for the agonist and antagonist tested. There was an increase in effect on apparent number of binding sites for the agonist etorphine with methionine supplementation. Conversely, inorganic sulfate at 0.42 percent supplementation increased the apparent number of binding sites for the antagonist naloxone.

This study provides evidence of a requirement for sulfate for the development of the opiate receptor. Sulfur status of the rat was shown to affect the number of receptors for opiates in brain homogenates.