Sex-dependence of the metabolism of aldosterone in adrenalectomized and intact rats

The metabolism of aldosterone at physiological levels was shown to be significantly different in male and female rats: more aldosterone was metabolized in the male rats leading to significantly higher quantities of non-extractable polar derivatives of aldosterone in all subcellular fractions of kidney, particularly in the cytosol fractions. These results may be correlated with our findings in which it was shown that the physiological responses to aldosterone in both adrenalectomized and intact rats were significantly greater in the males than the females. These sex differences support the concept that the metabolism of aidosterone may be essential for the components of the physiological response to aldosterone to occur. Furthermore, the sex-dependence of the metabolism of aldosterone appears to be independent of the presence of the previously identified protein receptor-hormone complexes. At all dosages within the physiological range, no significant differences were observed between the extent of the ³H-aldosterone labeling of these binding proteins in the kidney cytosol fractions of male and female adrenalectomized rats, even at dosages where no physiological response was demonstrable in the females.     

Influence of glucocorticoid on the metabolism of aldosterone in the isolated perfused rat liver and kidney.

The effect of glucocorticoid deficiency and excess on the extraadrenal metabolism of D-[4-14C]aldosterone (at 4 nM) was studied by radioimmunoassay and by high-performance liquid chromatography in the isolated perfused liver and kidney of adult Wistar rats. Bilateral adrenalectomy was performed 3 weeks before experiments. In nonadrenalectomized rats, 0.3 mg/kg/day dexamethasone was continuously infused subcutaneously for 1 week before experiments. Adrenalectomy did not affect hepatic or renal metabolism of aldosterone. Dexamethasone treatment did not change the renal handling of aldosterone. However, the hepatic clearance of aldosterone was 19% lower (P less than 0.05) in livers of dexamethasone treated rats than in livers of normal rats. After 5 minutes, perfusate [4-14C]aldosterone metabolites were lower in livers of dexamethasone-treated than in livers of normal rats (P less than 0.05). Similar perfusate levels were then obtained. Radiometabolite peaks with similar relative retention times were found in the hepatic perfusate of all groups. However, the ratio between circulating polar metabolites of aldosterone and the metabolites less polar than tetrahydroaldosterone, after 5 and 15 minutes, was highest in livers of dexamethasone-treated rats. Biliary elimination of 14C was similar in all groups. Significant amounts of conjugated tetrahydroaldosterone were only excreted in the bile of dexamethasone-treated rats. In conclusion, glucocorticoid excess reduced the hepatic clearance of aldosterone and changed the pattern of the hepatic metabolites of aldosterone both in circulation and in bile.     

Effects of adrenalectomy and spironolactone on urinary metabolites of aldosterone in rats

The quantities and temporal sequences of appearance of aldosterone metabolites in the urine of adrenalectomized rats, and adrenalectomized rats treated with spironolactone, were compared following subcutaneous administration of a physiological dosage (0.05 microgram) of [1,2,-3H]aldosterone. Large amounts of radiometabolites were rapidly excreted during 0-1 and 1-3 h and only small quantities by 3-4 h in urine of both groups of rats. The majority of the urinary radiometabolites (70-85%) were identified by Sephadex DEAP-LH-20 chromatography as neutral metabolites of aldosterone (NMA), together with lesser quantities of acidic, sulfate, and glucuronide conjugates. Further characterization by high pressure liquid chromatography (HPLC) showed that 90% of the NMA excreted by adrenalectomized rats were polar metabolites which could be separated into at least 15 peaks eluting in regions of increasing polarity (designated A, B, C, and D). Only small quantities of unaltered [3H]aldosterone and no ring-A-reduced metabolites were excreted by the adrenalectomized rats. Spironolactone treatment caused large changes in the excretion of acidic and sulfate derivatives of aldosterone, as well as discrete alterations in the HPLC patterns of the polar NMA (particularly those metabolites in regions A and B). Such discrete changes in these metabolic pathways which occur at the same time as the hormonal actions of aldosterone in the kidney may provide further insight into understanding the biological role of aldosterone metabolism.     

Effect of spironolactone on the renin-aldosterone system in rats

PRA, PRC and the plasma concentration of aldosterone (Aldo) were measured in rats (Sp-rats) receiving a daily sc injection of Spironolactone, (Sp, 20 mg in olive oil) and in control rats (C-rats) receiving olive oil only. Animals were studied one day after starting treatment, 5 days on treatment or after 5 weeks on the study. PRA, PRC and Aldo were significantly increased in Sp-rats as compared to C-rats throughout all the study. In additional Sp-rats and C-rats, the urine volume, serum Na+ and K+ concentration, Na+ and K+ intake and the urinary excretion of Na+, K+ and aldosterone-18-glucuronide (UAldV) were serially measured during 5 weeks. The total radioactivity plasma clearance after an i.v. bolus injection of 3H-aldosterone was subsequently measured in (5 Sp-rats and 5 C-rats). No significant differences in serum Na+ and K+ concentration and in Na+ and K+ balance were observed between Sp-rats and C-rats. UAldV was significantly higher in Sp-rats than in C-rats during all the study. After 5 weeks on treatment the total radioactivity plasma clearance was significantly higher in Sp-rats than in C-rats. These results indicate that Sp, at high dosage, stimulates renin release and aldosterone secretion by a mechanism unrelated to alterations in Na+ and K+ balance.     

Formation and enterohepatic circulation of metabolites of retinol and retinoic acid in bile duct-cannulated rats

Four hours after intraportal injection of retinoic acid-(14)C into bile duct-cannulated rats, less than 10% of the radioactivity was recovered in the liver, intestine, and kidneys. Within 6 hr, 40% of the radioactivity had appeared in bile. When suspensions of retinol-(14)C or retinal were similarly injected, 25-35% of the dose was excreted in bile within 24 hr and equivalent amounts were deposited in the liver as retinol ester. The isolated perfused liver also produced these bile metabolites and is probably the major site of their formation in vivo. The intestine may metabolize retinoic acid, however, since some metabolites were found in the intestinal wall and lumen, even in bile duct-cannulated rats. The bile metabolites of retinol-(14)C and retinoic acid-(14)C undergo extensive enterohepatic circulation. The bile radioactivity was not volatilized on boiling at acid pH, was not present in digitonin-precipitated sterols, and did not migrate with bile salts on reversed-phase paper chromatography. Anion-exchange chromatography resolved the metabolites of bile into three fractions containing nonionic compounds, acidic substances like retinoic acid, and more polar acidic derivatives.     

Metabolism of aldosterone in the colostomized duck (Anas platyrhynchos): partial characterization of urinary metabolites

1. Chronically colostomized ducks were injected with [4-14C]-aldosterone to study the metabolism of aldosterone and the pattern of metabolite excretion via the kidney. 2. Nearly half of the injected dose was excreted as radiometabolites during the first 24 hr; the largest amounts being excreted during the first 3 hr after injection. 3. Ion-exchange chromatography showed that monosulfate, disulfate, glucuronide, acidic, and neutral metabolites were excreted during each collection period, and that their relative proportions changed with time after injection of [4-14C]-aldosterone. 4. HPLC analysis of the neutral radiometabolites revealed 15 major peaks with retention times corresponding to both polar and reduced derivatives of aldosterone. 5. Only small quantities of unaltered labelled aldosterone were excreted. 6. Treatment of the birds with SKF 525-A caused a decrease in the total quantity of radiometabolite excreted and a change in the proportions of neutral and acidic metabolites in the cloacal fluid. 7. The decreases that occurred in the absolute amounts of some of the polar metabolites excreted by the birds treated with SKF-525A suggests that they may be hydroxylated and at least part of the aldosterone metabolizing system in the duck is cytochrome P450 dependent.     

Inhibitors of sterol synthesis. Metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one after intravenous administration to bile duct-cannulated rats

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one has been studied after intravenous administration to bile duct-cannulated rats. Very rapid and substantial conversion of the 15-ketosterol to polar biliary metabolites was observed in both male and female rats. For example, upon intravenous injection of [4-14C]5 alpha-cholest-8(14)-en-3 beta-ol-15-one to male bile duct-cannulated rats, approximately 86% of the administered 14C was recovered in bile in the first 38 h. Of the total amount of 14C recovered in bile in 38 h, approximately 50% was excreted in bile in the first 70 min and approximately 90% was excreted within 8 h after the injection of the 15-ketosterol. A substantial fraction of the polar biliary metabolites was shown to undergo enterohepatic circulation. Of the radioactivity derived from the labeled 15-ketosterol which was not recovered in bile or other excreta at 48 h after the intravenous administration of the 15-ketosterol, most (approximately 79%) was recovered in the form of cholesterol and cholesteryl esters of blood and the various tissues. The very substantial and rapid biliary excretion of polar metabolites of the 15-ketosterol (or of cholesterol derived from the 15-ketosterol), coupled with inhibition of the intestinal absorption of cholesterol by the 15-ketosterol, may contribute to the overall hypocholesterolemic action of the 15-ketosterol which has been observed in rodents and in nonhuman primates by providing a metabolic pathway(s) wherein a substantial fraction of the absorbed 15-ketosterol is rapidly removed from the body by biliary excretion in the form of polar metabolites.     

Complex formation by 3 H-aldosterone in rat kidney and liver

Low molecular weight polar complexes were shown to be formed $\text{in vivo}$ from ³H-aldosterone in both kidney and liver subcellular fractions, the majority being present in the cytosol fractions. Significant differences were observed between the quantities of polar complexes present in kidney subcellular fractions from intact and adrenalectomized male rats and also between the quantities of these kidney polar complexes from spironolactone treated male rats. ³H-aldosterone macro-molecule complexes were shown to exist in appreciable quantities only in the kidney cytosol fractions of adrenalectomized male rats. These gel filtration studies also showed the ³H-aldosterone labeled macromolecule complexes to consist of two protein peaks; one of high molecular weight and the other of lower molecular weight (～50,000 mol. wt.). The amount of ³H-aldosterone labeled protein complexes in kidney cytosol was greatly reduced when adrenalectomized rats were pretreated $\text{in vivo}$ with spironolactone.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

The excretion of metabolites of cortisol and aldosterone in male patients on haemodialysis treatment

A single intravenous injection of ¹⁴C-cortisol and ³H-aldosterone was given to four male uraemic patients on haemodialysis (HD) treatment. The excretion of radioactivity was measured during two weeks in urine, HD fluid and faeces. In two patients, who were injected just before dialysis, large amounts of radioactivity were eliminated in the HD fluid (38 % and 56 % for ³H, 45 % and 57 % for ¹⁴C) and minor amounts were found in the urine (< 5 %); in the faeces respectively 32 % and 30 % of ³H and 18 % and 26 % of ¹⁴C were excreted. Two patients who were injected immediately after dialysis (and who also had a somewhat better kidney function) excreted larger amounts of radioactivity in the urine (10 % and 24 % for ³H, 13 % and 41 % for ¹⁴C) and in the faeces (44 % and 62 % for ³H, 29 % and 37 % for¹⁴C), while in the HD fluid respectively 18 % and 4 % of ³H and 30 % and 12 % of ¹⁴C was eliminated. The plasma radioactivity just before and just after dialysis showed a very good correlation (r = 0.96 to 0.99, p < 0.001) with the radioactivity eliminated in the first and last hour of HD treatment. Between HD treatments, the radioactivity in plasma did not change or decreased only very little. This finding suggests that metabolites of Cortisol and aldosterone to be excreted in the faeces, are very quickly removed from the circulation.     

Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Disposition of 1-[14C]phenyl-1-(3,4-dimethyl) phenylethane, a component of some PCB replacement materials, following intravenous administration to the thorny skate, Raja radiata

1-[14C]Phenyl-1-(3,4-dimethyl)phenylethane ([14C]PXE), a labelled analogue of a component of some commercial PCB replacement materials, was cleared rapidly from plasma of the thorny skate, Raja radiata, after i.v. injection. A polyexponential fit to the clearance data yielded three linear components with half-lives of 7, 128 and 924 min. The metabolic clearance rate for the mean animal was approximately 41 X kg-1 X d-1. The main storage site for [14C]PXE was the liver, which contained about 40% of the injected dose. Approximately 25% of the injected radioactivity was recovered from the bile within 36 hr of injection in the form of polar metabolites, indicating rapid degradation of [14C]PXE.     

Effect of infusion of "tris-galactosyl-cholesterol" on plasma cholesterol, clearance of lipoprotein cholesteryl esters, and biliary secretion in the rat

As shown by us previously (van Berkel et al. 1985. J. Biol. Chem. 260: 2694-2699 and van Berkel et al. 1985. J. Biol. Chem. 260: 12203-12207) the clearance of both low density lipoproteins (LDL) and high density lipoproteins (HDL) from the blood can be greatly enhanced by pretreatment of these lipoproteins with a tris-galactosylated cholesterol derivative, which makes these particles recognizable by hepatic galactosyl-receptors. Here we report that intravenous infusion of the (water-soluble) tris-galactosyl-cholesterol in rats caused a dose-dependent decrease of the plasma cholesterol level. This fall was sustained long after termination of the infusion. It was not observed upon infusion of tris-glucosyl-cholesterol. The fall in plasma cholesterol was accompanied by an increase in hepatic cholesterol. Upon injection of rat HDL and LDL labeled in their cholesteryl ester moieties, plasma clearance of label in both lipoproteins was enhanced in rats infused with tris-galactosyl-cholesterol, the stimulation being more pronounced when the label was in HDL. The appearance of label in bile was also enhanced in the rats receiving the compound, again more markedly when the label was given as HDL. Ninety four percent or more of the radioactivity excreted in the bile was in the form of bile salts, with conjugated cholate being the major species in both control and treated rats; 6% or less of the radioactivity in the bile was as free cholesterol. Infusion of tris-galactosyl-cholesterol constitutes a new and defined method of lowering plasma lipoprotein levels by enhancing their uptake in the liver.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.     

Investigations on metabolites of vitamin D in rat bile. Separation and partial identification of a major metabolite

1. Young rats with cannulated bile ducts were given 0.34mg. of [1alpha-(3)H]cholecalciferol or 0.54mg. of [(14)C]ergocalciferol by intravenous infusion. Of the radioactivity in the dose of [1alpha-(3)H]cholecalciferol 31% was recovered in bile within 24hr. 2. The metabolites in bile were separated by gradient-elution column chromatography on silicic acid into five components, all more polar than cholecalciferol or 25-hydroxycholecalciferol. [(14)C]Ergocalciferol gave a similar pattern of metabolites in bile. 3. The three most polar metabolites were shown to be ionic. The major component has been identified as a glucuronide conjugate, which was not identical with synthetic cholecalciferyl glucuronide.     

Mutagenic activity of biliary metabolites of 6-hydroxymethylbenzo[a]pyrene

The biliary excretion of the carcinogen 6-hydroxy-methylbenzo[a]pyrene was investigated in rats after i.p. administration. Mutagenicity of the parent compound and its biliary metabolites was tested in Ames Salmonella/microsome mutagenicity assay. Approximately 40% of the dose administered (0.25-0.5 mg/kg) to the rats was excreted in the bile within 6 h. 6-Hydroxymethylbenzo[a]pyrene was excreted primarily as water-soluble metabolites, including glucuronide and sulfate conjugates. Negligible quantities of unchanged 6-hydroxymethylbenzo[a]pyrene were excreted in the bile. In the presence of Aroclor-induced S9, 6-hydroxymethylbenzo[a]pyrene was a potent mutagen. The mutagenicity of bile from rats treated with 6-hydroxymethylbenzo[a]pyrene was variable in the absence of an activation system. However, the same bile samples were mutagenic in the presence of beta-glucuronidase and/or S9. These results indicate that biliary metabolites of 6-hydroxymethylbenzo[a]pyrene can be metabolically activated to mutagenic species.     

Identification of aldosterone metabolites formed by A6 cells

The A6 cell line of the toad kidney is well known to form an Na+ transporting tight epithelium in culture and is often used as an experimental model for Na+ transport systems. Although it has been shown that A6 cells can convert aldosterone to polar metabolites, these metabolites have not been identified. Therefore, in this study, we tried to identify the metabolites of aldosterone formed by A6 cells in culture. A6 cells at confluence were incubated with serum-free culture media containing [3H]aldosterone. When radioactive compounds in incubation media were separated by reversed phase high-pressure liquid chromatography (HPLC), four fractions (fractions A-D) were obtained. Fraction A, a mixture of two components, comprised the majority of metabolites formed. The more polar material (fraction A-1) and the less polar material (fraction A-2) of fraction A contained 47-71 and 9-19% of total radioactivity, respectively. When incubated in cell-free media, fraction A-2 was found to be unstable and partially converted to fraction A-1. Fraction B, 0.7-1.5% of total radioactivity, and fraction C, 8-21% of total radioactivity, cochromatographed with iso-aldosterone and D-aldosterone, respectively. Fraction D, 4-8% of total radioactivity, was a mixture of two components, which cochromatographed with 3 beta,5 beta-tetrahydroaldosterone and 5 alpha-dihydroaldosterone, respectively. In order to identify fraction A-2 material, large-scale cultures were performed and fraction A-2 was separated and purified by reversed phase HPLC. The purified material was analyzed by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. These two procedures unambiguously revealed that this material was 6 beta-hydroxyaldosterone. These results demonstrate that aldosterone can be converted to at least four metabolites by the incubation with A6 cells, and that major metabolites are polar compounds, a portion of which is 6 beta-hydroxyaldosterone.     

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.