The metabolic pathway in the degradation of glyceryl trinitrate.

NEEDLEMAN, PHILIP, DELORES J. BLEHM, ANNE B. HARKEY, EUGENE M. JOHNSON. JR. AND STANLEY LANG : The metabolic pathway in the degradation of glyceryl trinitrate. J. Pharmacol. Exp. Ther. 179: 347-353, 1971. Glyceryl trinitrate (GTN) is degraded primarily in the liver by a partial denitration process catalyzed by organic nitrate reducta.se. The supporting evidence is as follows : 1) incubation of GTN with liver homogenates leads to the appearance of glyceryl dinitrate and glyceryl mononitrate (which also represents the primary urinary metabolite), 2) in vitro incubation of GTN with liver homogenates does not lead to CO2 formation even though denitration to glyceryl dinitrate and glyceryl mononitrate proceeded rapidly, 3) glycerol cannot be detected in liver homogenates after incubation with GTN. 4) evisceration of rats inhibits ‘4C02 formation after tile administration of GTN-14C and 5) on the other hand, glycerol-14C was readily oxidized to 14C02 in both eviscerated animals and tissue homogenates. There does not appear to be an active enzyme system in liver which is capable of catalyzing the complete denitration of GTN metabolites to glycerol. The site and mechanism of CO2 formation from GTN is unknown. The interruption in the GTN degradation pathway of rats after evisceration must lie somewhere in the denitration sequence. The liver enzyme glutathione-organic nitrate reductase previously has been partially characterized (Needleman and Krantz, 1965 ; Needleman and Hunter, 1965) . It can rapidly denitrate glyceryl trinitrate (GTN, nitroglycerin) to 1 ,3and 1 ,2-glyceryl denitrate (GDN) . Denitration of the GDNs proceeds at only 2 to 5% of the rate for GTN and glyceryl mononit.rate (GMN) is practically unaffected by the liver enzyme (Needleman and Hunter, 1965) . Di Carlo et al. (1968b) have briefly reported on the in vivo conversion of GTN to CO . Tile current study investigates the influence of the liver and other organs on the oxidation of GTN to CO2 and considers some factors which might influence Received for publication April 8, 1971. 1 This work was done during the tenure of an Established Investigatorship and a grant-in-aid from the American Heart Association (AHA-68115. AHA-69-722) and was also supported by the National Institutes of Health (HE-11771, GM00096). this process. In addition, we are interested in determining whether carbon dioxide represents the ultimate end product of metabolism and, if so, its time of appearance relative to GTN degradation. METHODS. ‘4C02 formation in vivo. Radioactive GTN or glycerol was administered through a silicon-treated glass syringe to unanesthetized rats (Holtzman, 150 to 200 g). Each labeled compound was given in the amount of 0.5 ftc/rat. Glycerol-214C and glycerol-i ,3_14t were administered s.c. in 0.05 ml of water. The GTN-’4C was synthesized as previously reported ( Needleman and Harkey, 1971) ; tile material was chromatographically and radiochemically pure and the alcoholic stock solution had a specific activity of 1 zc/ mol. GTN-2‘(C and GTN-1 ,3-14C were injected s.c. in 0.05 ml of 95% ethanol with 1 mg of added unlabeled GTN (5 mg/kg). Water (02 ml) was drawn into the syringe and injected into the animal s.c. and this process was repeated three times. Additional syringe washes were transferred to a scintillation vial and counted. The residual of radioactivity was subtracted from the amount calculated to have been injected. Room air was drawn by vacuum 348 NEEDLEMAN ET AL. Vol. 179 through a carbon dioxide trap (Mallcasorb, Mallinckrodt Chemical Works. St. Louis, Mo.) and a water vapor trap (Aquasorb. Mallinckrodt Chemical Works) and then into the metabolism cage housing the injected rat. A ground glass sealed thin-layer chromatography tank (Brinkmann Instruments, Inc., Westburv. N.J.) fitted with a gas flow valve was employed as the metabolism cage for each rat. A wire screen was fitted in the bottorn to elevate the animal above the cage floor. Urine samples were aspirated easily from the silicon-treated glass cage bottom. The expired air was drawn through a water vapor trap and the 14C02 was removed quantitatively in a gas washing bottie containing 100 ml of monoethanolamine (Fisher Scientific Company. Pittsburgh. Pa.). One-milliliter aliquots were taken from the gas bottle and added to 10 ml of scintillation fluid. The scintillation solution was comprised of 4-g of Omnifluor (New England Nuclear Corporation, Boston, Mass.), 350 ml of 2-ethoxyethanol and 650 ml of toluene. Pooled 24-hour urine was collected from the bottom of the metabolism cage and assayed to characterize and quantitate the GTN nietabolites. The various treatments employed to influence CO2 formation were as follows : phenobarbital sodium (50 mg/kg i.p.) twice daily for four days; SKF 525A (50 mg/kg i.p.) 30 minutes after the labeled compound ; tolerance was produced by injecting s.c. the rats three times daily with 100 mg/kg of GTN (Needleman, 1970) for four days; and bromobenzene pretreatment consisted of 1.5 g/kg dissolved in cottonseed oil administered i.m. once daily for two successive days. Urine extraction. The pooled urine was thoroughly extracted with diethyl ether. The ether fraction was air evaporated and spotted with added cold carrier on silica gel thin-layer plates (Brinkmann Instruments) . The nitrate separations were achieved with benzene-ethvl acetate (4:1) and the color was developed by ultraviolet light after spraying with a 10% solution of diphenylamme in methanol. The counts in each nitrate metabolite were quantitated either by scrapping the spot into a scintillation vial or by scanning the thin-layer chromatography strip. An aliquot of the urine was counted after ether extraction to determine residual counts. Evisceration and nephrectomy procedure. A two-part surgical procedure was performed. In the first stage the inferior vena cava was partially occluded by tying tightly down on a piece of PE 10 (outside diameter. 024 mm) tubing which was placed over the inferior vena cava above the renal vessels, and then the tubing was removed. After four weeks complete evisceration and nephrectomy were carried out as previously described by Farris and Griffith (1949). The animals were injected Witil labeled compound immediately after surgery. In order to prevent hypoglycemia the animals were injected with 200 mg/kg of glucose s.c. every hour. 14c02 collection in vitro. Rats were sacrificed by decapitation and tissue samples were removed rapidly and placed in ice-cold 0.1 M phosphate buffer (pH 7.4) or in 0.33 M sucrose (pH adjusted to 7.4). The tissues were blotted, weighed and then homogenized (1 : 10) in the above media with a VirTis homogenizer. Two milliliters of each homogenate were added to a 25-mi Erlenmeyer flask (on ice) which already contained 1 ml of medium with the isotope present. When minces were tested, 02 to 0.25 g of tissue was weighed. placed in 2 ml of medium and minced with fine iris scissors. The flasks were closed with a rubber diaphragm (Kontes Glass Company, Vineland, N.J.) fitted with a polyethylene center well which contamed a rolled piece of Whatman no. 1 filter paper (0.5 x 8 cm). The flasks were incubated in a reciprocating shaker at 37#{176}Cfor two hours. Then 0.30 ml of hyamine hydroxide (Packard Instrument Company, Downers Grove. Ill.) was added to the center well and 030 ml of 10 N H SO4 was added to the tissue. The flasks were shaken for an additional hour. The filter paper was transferred to 10 ml of scintillation solution and counted. Glycerol assay. Glycerol was assayed by measuring the appearance of reduced nicotinamide adenine dinucleotide (NADH2) fluorescence produced in the presence of glycero-P-dehydrogenase and glycerokinase as described by Burch et al. (1970). The rat liver homogenate (1:10 in 0.1 M phosphate buffer, pH 7.4, and centriguged 10 minutes at 1000 X g) was found to be contaminated with some endogenous glycerol. The glycerol was removed readily from the liver homogenate by means of a Sephadex G-25 column with no loss of organic nitrate reductase activity (see text). Organic nitrate reductase activity. The measurement of organic nitrate reductase was based on the erythrityl tetranitrate (ETN)-induced disappearanee of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of gbt,athione reductase and tissue extracts as was described previously (Needleman and Hunter, 1965). ETN was used as the nitrate substrate (instead of GTN) sinci it has a low rate of reaction with glutathione (GSH) alone. The reaction mixture contained 1 &mol of ETN. 4 mol of GSH, glut.athione reductase (OR), 1 mol of NADPH2(finai concentration after preincubation with GSH and Gil). 1 Mmol of ethylene diamine tetraacetate (EDTA) in 1 ml of 0.1 M phosphate buffer (pH 7.4). HOURS 1971 PATHWAY OF GTN METABOLISM 349