The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.     

Identification of the major urinary metabolite of prostaglandin E3 in the rat

[11,12-3H2]Prostaglandin E3 was administered subcutaneously into male Sprague-Dawley rats in doses of 0.4 microgram-10 mg/kg body weight. 40-60% of the administered radioactivity was excreted in the urine. The major metabolite was isolated by solid phase extraction followed by three steps of high-performance liquid chromatography. The structure of the major metabolite (5-11% of the administered radioactivity) was 7 alpha,11 alpha-dihydroxy-5-ketotetranorprosta-9,13-dienoic acid as shown by gas-liquid chromatography-mass spectrometry and by its conversion into 11 alpha-hydroxy-5-ketotetranorprosta-4(8),9, 13-trienoic acid.     

Enterohepatic circulation of N-acetyl-leukotriene E4

N-Acetyl-leukotriene E4, the end product of leukotriene C4 metabolism in the mercapturic acid pathway, was rapidly eliminated from the blood circulation into the bile of rats. Part of the N-acetyl-leukotriene E4 secreted from bile into the intestine underwent enterohepatic circulation. Leukotriene absorption occurred from the small intestine and from the colon. Biliary and urinary excretion within 5.5 h amounted to 15 and 2%, respectively, of the intraduodenally administered N-acetyl- 3H leukotriene E4 in animals anesthetized with ketamine. HPLC analyses indicated that 35% of the biliary radioactivity corresponded to unchanged N-acetyl-3H leukotriene E4, while 65% in bile and 100% in urine were polar metabolites. Enterohepatic circulation extends the biological half-life of N-acetyl-leukotriene E4.     

In vivo metabolism of leukotriene C4 in germ-free and conventional rats. Fecal excretion of N-acetylleukotriene E4

[5,6,8,9,11,12,14,15-3H8]Leukotriene C4 was subcutaneously injected into rats. Substantial amounts of the administered radioactivity were excreted in feces of germ-free and conventional animals during a 72-h period (78 and 64%, respectively). Analyses of fecal extracts by high performance liquid chromatography showed eight radioactive components for each type of animal. One metabolite amounted to 4.6% of the injected radioactivity in germ-free and 0.6% in conventional rats. Its chemical structure, 5-hydoxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14-c is-eicosatetraenoi c acid (N-acetylleukotriene E4) was determined by ultraviolet spectroscopy, fast atom bombardment mass spectrometry, chemical and enzymatic transformations, and confirmed by chemical synthesis. Another metabolite (2.7% of the administered radioactivity in germ-free and 0.5% in conventional rats) was characterized as the 11-trans isomer of the former metabolite. The pathway of formation of these compounds appears to be analogous to the pathway of mercapturic acid biosynthesis.     

Inhibition of leukotriene D4 catabolism by D-penicillamine

Inhibition of the catabolism of the most biologically potent cysteinyl leukotriene, LTD4, was studied in rat hepatoma cells in vitro and in the rat in vivo. LTD4 dipeptidase, an ectoenzyme on the surface of AS-30D hepatoma cells, exhibited an apparent Km value of 6.6 microM for LTD4. D-Penicillamine and L-penicillamine inhibited this enzyme activity with apparent Ki values of 0.46 mM and 0.21 mM respectively. Bestatin, an inhibitor of the aminopeptidase activity of hepatoma cells, did not affect LTD4 hydrolysis at concentrations as high as 5 mM, indicating that the aminopeptidase did not contribute to LTD4 catabolism. In the rat in vivo, D-penicillamine also inhibited LTD4 catabolism. After intravenous injection of [3H]LTC4 an accumulation of [3H]LTD4 and a retarded formation of [3H]LTE4 were observed in the circulating blood after D-penicillamine pretreatment. Within 1 h after intravenous [3H]LTC4 injection, about 80% of the administered radioactivity was recovered in bile. After D-penicillamine pretreatment [3H]LTD4 was the major biliary leukotriene metabolite, whereas in untreated controls leukotriene metabolites more polar than LTC4 predominated in bile. After stimulation of endogenous leukotriene production in vivo by platelet-activating factor, N-acetyl-LTE4 was the major cysteinyl leukotriene detected in bile. D-Penicillamine treatment prior to platelet-activating factor resulted in the accumulation of LTD4, which under these circumstances was the major endogenous leukotriene metabolite detected in bile.     

Steroid metabolism in the rabbit: Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose a total of approx. 56-80% of the radioactivity was excreted in bile and urine. After infusion total recovery of radioactivity was approx. 63-75%. 3. The mean ratio of metabolites in urine to those in bile was 0.77+/-0.41 (range 0.3-1.5). 4. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. In both bile and urine neutral metabolites extracted by ethyl acetate-ether were found mainly after beta-glucuronidase hydrolysis, but a considerable proportion of the dose was converted into substances not extractable from alkaline aqueous solution after all forms of hydrolysis used.     

Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 by the conscious marine toad, Bufo marinus.

Tissue distribution, elimination, and metabolism of 3H-labelled leukotriene (LT) C4 were studied in ureter-catheterized conscious marine toads, Bufo marinus. Six and 24 h after injection, organs containing the highest percent of injected radioactivity were small intestine, liver, and kidney. Radioactivity declined in these organs at 24 h by approximately threefold. Peak elimination time for radioactivity in the urine was between 2 and 4 h after the injection. During the 24-h collection period, 55.2 +/- 0.2% of the injected radioactivity was eliminated in the urine. Polar metabolites represented 40.3 +/- 1.1, 57.3 +/- 5.6, and 62.8 +/- 1.6% of the radioactivity at 2, 4, and 6 h, respectively. The primary urinary polar metabolite was 20-carboxy-LTE4, with 18-carboxydinor-LTE4 and 20-hydroxy-LTE4 also present. [3H]LTE4 decreased from 37.2 +/- 1.8% at 2 h to 15.8 +/- 3.3 and 15.0 +/- 2.1% of the radioactivity at 4 and 6 h, respectively. Bile radioactivity was low. N-Acetyl-LTE4 was not detected in urine or bile samples. Radioactivity in the pan water was 14.3 +/- 2.4 and 15.8 +/- 2.5% of the injected radioactivity, at 6 and 24 h, respectively, suggesting that the skin was a route for excretion of leukotrienes. The marine toad is an interesting model demonstrating both similarities and differences from mammals in distribution, elimination, and metabolism of peptide leukotrienes.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]cortisone

1. [4-(14)C]Cortisone was administered to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. After the single dose a total of 69.6-89.6% of the radioactivity was excreted in bile, and 0.5-7.1% in urine. After infusion total recovery in bile was 73.4-92.1%, and 1.2-1.7% in urine. 3. When bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid, most of the radioactivity was converted into substances not extractable from neutral aqueous solution by ethyl acetate-ether. 4. In bile, metabolites hydrolysable by beta-glucuronidase were found in only small proportions (3-4%) of the dose.     

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered intravenously to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. Most of the administered radioactivity was excreted in the bile (70-80%); only 2.9-5.5% of the dose was excreted in the urine. 3. Bile and urine samples were hydrolysed successively to yield glucuronide, ;cold-acid-hydrolysed' and ;hot-acid-hydrolysed' fractions. 4. The proportion of glucuronides in bile decreased in successive samples, but cold-and hot-acid-hydrolysed metabolites showed no consistent change. 5. After hydrolysis most of the radioactivity in both bile and urine could not be extracted by ether from neutral aqueous solution.     

Direct photoaffinity labeling of leukotriene binding sites

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.     

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]progesterone

1. [4-(14)C]Progesterone was administered intravenously to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose about 60% of the radioactivity was recovered in 6hr., and twice as much radioactivity was present in bile as in urine. After infusion total recovery of radioactivity was only about 40% in 6hr., but the relative proportions of metabolites in bile and urine were about the same as after a single dose. 3. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. 4. In bile the major proportion of metabolites appeared in the glucuronide fraction; in urine beta-glucuronidase hydrolysis yielded the greatest amounts of ether-extractable radioactivity, but the greatest proportion of radioactivity could not be extracted by ether from an alkaline solution of the hydrolysed urine. 5. There was no apparent difference in the quantity or distribution of metabolites excreted by male and female animals.     

w-oxidation products of leukotriene E4 in bile and urine of the monkey

The intravenous administration of [3H]leukotriene C4 in the monkey Macaca fascicularis results in the biliary and urinary elimination of [3H]leukotriene D4 and [3H]leukotriene E4 in addition to more-polar metabolites. Separation of these polar metabolites and chromatographic comparison with synthetic w-oxidized leukotrienes indicated the in vivo formation of w-hydroxy-[3H]leukotriene E4 and w-carboxy-[3H]leukotriene E4. Time course studies of the [3H]leukotriene metabolite pattern in bile and urine showed that w-hydroxy-leukotriene E4 was decreasing as w-carboxy-leukotriene E4 and additional polar derivatives were increasing.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.     

The development of a sensitive and specific radioreceptor assay for leukotriene B4

To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.     

Quantification of leukotriene B4 glucuronide in human urine

We have developed a method for measuring leukotriene B4 glucuronide, a marker of systemic leukotriene B4 biosynthesis, in human urine. This method involves the separation of two positional isomers of leukotriene B4 glucuronide by high-performance liquid chromatography, followed by hydrolysis with beta-glucuronidase and then leukotriene B4 quantification by enzyme immunoassay after purification by high-performance liquid chromatography. One of two positional isomers of leukotriene B4 glucuronide was predominantly present in urine. The concentration of the isomer increased in urine from aspirin-intolerant asthma patients after aspirin challenge. Urinary leukotriene E4 and leukotriene B4 glucuronide concentrations in 13 normal healthy adults were 94.6 pg/mg-creatinine (median) and 22.3 pg/mg-creatinine, respectively. Urinary LTE4 concentration increased during the first 3h after allergen inhalation in atopic patients. However, allergen-induced bronchoconstriction was not associated with an increased concentration of LTB4 glucuronide in urine. The method enabled us to precisely determine urinary leukotriene B4 glucuronide concentration.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.     

The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.     

On the enterohepatic cycle of triiodothyronine in rats; importance of the intestinal microflora

Until 70 h after a single iv injection of 10 uCi [125I]triiodothyronine (T3), normal rats excreted 15.8 +/- 2.8% of the radioactivity with the feces and 17.5 +/- 2.7% with the urine, while in intestine-decontaminated rats fecal and urinary excretion over this period amounted to 25.1 +/- 7.2% and 23.6 +/- 4.0% of administered radioactivity, respectively (mean +/- SD, n = 4). In fecal extracts of decontaminated rats 11.5 +/- 6.8% of the excreted radioactivity consisted of T3 glucuronide (T3G) and 10.9 +/- 2.8% of T3 sulfate (T3S), whereas no conjugates were detected in feces from normal rats. Until 26 h after ig administration of 10 uCi [125I]T3, integrated radioactivity in blood of decontaminated rats was 1.5 times higher than that in normal rats. However, after ig administration of 10 uCi [125I]T3G or [125I]T3S, radioactivity in blood of decontaminated rats was 4.9- and 2.8-fold lower, respectively, than in normal rats. The radioactivity in the serum of control animals was composed of T3 and iodide in proportions independent of the tracer injected, while T3 conjugates represented less than 10% of serum radioactivity. These results suggest an important role of the intestinal microflora in the enterohepatic circulation of T3 in rats.     

Biliary and urinary excretion of peptide leukotrienes in the domestic pig

The metabolism of leukotriene (LT)C4 and its major routes of elimination in vivo have been studied in four anesthetized domestic pigs administered intravenous [3H]-LTC4 (0.5 microCi/kg). The kinetic profile of LTC4 in the blood was followed for 60 min after administration while the biliary and urinary excretion of LTC4 and its metabolites were determined over a 120 min interval. The total recovery of radioactivity in bile and urine was 45% +/- 1 (n = 3) and 18% (n = 2) respectively. Examination of the radioactive metabolites in bile showed LTD4 (44% of biliary content) and LTE4 (21% of biliary content) as the major identified lipoxygenase products at t 1/2 (27 min). The only identified cysteinyl leukotriene observed in the urine was LTE4 (13% of urinary content). In both bile and urine substantial amounts of radioactivity were detected at the solvent front of the reverse phase chromatographic system indicating the presence of additional unidentified metabolites. We suggest that measurement of metabolites using these sampling methods may be useful for the detection and measurement of peptide leukotriene production in vivo.