Stimulation of human T-lymphocyte chemokinesis by arachidonic acid.

The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 ± 34% enhancement (mean ± SD) at 8 μM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cyclo-oxygenase inhibitors indomethacin (ID₅₀ = 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID₅₀ = 20 μM) suppressed the stimulation of migration by arachidonic acid. The cyclo-oxygenase product 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) as well as PGE₂, PGF_2α, and thromboxane B₂ had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.     

The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolities produced by thrombin-treated human platelets: I. The validation of the technique

We have developed a technique for the rapid separation and quantitative collection of thromboxane B₂ (TXB₂), PGE₂, PGD₂, PGF_2α, 12-hydroxy-5,8,10 heptadecatrienoic acid (HHT), 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE), and arachidonic acid released from thrombin treated human platelets. Platelets were pre-labeled with ³H-arachidonic acid and then isolated by gel filtration. They were then exposed to thrombin for various intervals and separated by centrifugation. Aliquots of the cell-free medium were applied directly to a high pressure liquid chromatograph containing a fatty acid column as the stationary phase. A quarternary solvent system containing tetrahydrofuran (THF), acetonitrile (CH₃CN), water and acetic acid (HOAC) resolved and eluted the arachidonic acid metabolites within 30 minutes. Since no sample preparation is required and since the solvent system does not quench the counting efficiency of a standard liquid scintillation fluor the technique permits rapid separation and quantitation of radiolabeled arachidonic acid and its metabolites.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Prostaglandin Biosynthesis from Endogenous Precursors in Rabbit Kidney

THIS report describes the biosynthesis of the naturally occurring renal prostaglandins E₂ (PGE₂) and F_2α (PGF_2α)^1,2 by homogenates and slices of rabbit renal medulla, from endogenous precursors. I have confirmed that rabbit renal cortex contains little prostaglandin and cannot synthesize them from endogenous lipids³. Hamberg has reported that arachidonic acid, which is converted to PGE₂ and PGF_2α by enzymes present in ram seminal vesicles⁴, can be efficiently converted to PGE₂ and PGF_2α by homogenates of rabbit renal medulla³. I have now confirmed that arachidonic acid, added to such medullary homogenates, can increase the quantities of prostaglandins synthesized. There was no evidence that the major prostaglandin biosynthesized, PGE2, was further metabolized to inactive products.     

Corpus luteum function in the guinea pig: effect of pgf2 alpha and inhibitors of prostaglandin and progesterone biosynthesis.

Exogenous PGF_2α failed to consistently alter estrous cycle length of the guinea pig. A wide range of dose levels were administered with varying frequency, at different stages of the estrous cycle, in different vehicles and by various routes. Massive doses of PGF_2α (5.0–10.0 mg) produced a significant (p<.05), although transient, lowering of plasma progesterone levels. Smaller doses were ineffectual. An i.p. injection of 25 mg of PGF_2α was toxic in four of five treated animals. It would appear that the intact guinea pig is extremely resistant to the luteolytic effects of parenterally administered PGF_2α.Estradiol-17β, administered s.c. on days 3 to 10 of the estrous cycle, significantly (p<.05) reduced corpus luteum diameter and plasma progesterone levels. Estrous cycle length was unaffected. Clomiphene, in the same experiment, caused premature vaginal opening in some treated animals, but corpus luteum size and plasma progesterone levels were unaffected and no ovulations occurred.The prolactin secretion inhibitor, CB-154, administered early in the estrous cycle, did not have any effect on estrous cycle length of the guinea pig alone or in combination with PGF_2α. The prostaglandin precursor, arachidonic acid, also failed to influence estrous cycle length when administered on days 8 and 9 of the cycle. Plasma progesterone levels remained unaltered.Oral administration of a prostaglandin synthetase inhibitor, (MK-715), caused a small, but non-significant (p>.05) prolongation of the estrous cycle. The progesterone biosynthesis inhibitors, aminoglutethimide and 6β-hydroxy-3α, 5α-cyclo-androstane-17-one did not effect estrous cycle length or plasma progesterone levels of the guinea pig.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Production of prostaglandins by cells in vitro: radioimmunoassay measurement of the conversion of arachidonic acid to PGE2 and PGF2 alpha

A specific radioimmunoassay has been applied to the measurement of the conversion of arachidonic acid to PGE₂ and PGF_2α. PGE₂ and PGF_2α biosynthesis was linearly related to the amount of arachidonic acid added and was significantly inhibited by indomethacin in concentrations as low as 10^?10 M. Sonicated Hela, L, and HEp-2 cells synthesized 244.0, 42.3, and 22.6 ng PGE₂ per mg of protein, but made substantially less PGF_2α.     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ ¹⁴C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F_1α, PGE₂, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF_1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF_1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Effect of polyamines on prostaglandin synthesis in various cell-free systems

Synthesis of PGF_2α by bovine uterus and guinea pig lung microsomes and that of TXB₂ by human platelet and rat spleen microsomes were stimulated by spermine. PGE₂ synthesis by bovine seminal vesicle and porcine lung microsomes, and 6-keto-PGF_1α synthesis by bovine seminal vesicle and uterus microsomes were inhibited by spermine. When phospholipid-free prostaglandin synthetase from bovine seminal vesicle was used instead of microsomes, the inhibition of PGE₂ synthesis by spermine disappeared. The inhibition of PGE₂ synthesis by spermine gradually appeared with an increase of phospholipid added. Among phospholipids tested, phosphatidylcholine was the most effective for the inhibition of PGE₂ synthesis by spermine.     

Separation of icosenoic acids,monohydroxyicosenoic acids,and prostaglandins by high-pressure liquid chromatography on a silver ion-loaded cation-exchange column

Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E₁(PGE₁), PGF_1α, PGD₂, PGE₂, PGF_2α, 6-oxoPGF_1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid.     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

PGE2 and PGF2α biosynthesis in stimulated and nonstimulated peritoneal preparations containing macrophages

The biosynthesis of PGE₂ and PGF_2α was measured in intact peritoneal exudate preparations obtained fom $\text{C. parvum}$-treated and control C₃H mice. Although both the control and stimulated preparations biosynthesized PGF_2α and PGE₂ from [1–14C] arachidonic acid, the stimulated preparations generated more of both prostaglandins than did nonstimulated preparations, probably as a result of increased synthesis within macrophages. Increased transformation of PGE₂ into PGF_2α 9-ketoreductase was noted in stimulated preparations when compared to that in control cells. The data suggest that stimulated macrophages are capable of generating increased quantities of PGF_2α and therefore might function as one source of this substance in resolving inflammatory reactions.     

Prostaglandin metabolism during circulatory shock

The rates of metabolic degradation and the patterns of metabolite formation of tritium-labeled prostaglandins E₂ and F_2α were assessed in vitro in tissues obtained from normal rabbits and from rabbits subjected to hemorrhagic or endotoxic shock. Normal rabbit tissues metabolized prostaglandin E₂ at the following rates: renal cortex 479 ± 34, liver 389 ± 95, and lung 881 ± 93 pmol of PGE₂ metabolized/mg soluble protein per min at 37°C (mean ± S.E.). Prostaglandin F_2α metabolism proceeded in normal animal tissues at rates of 477 ± 39, 324 ± 95, and 633 ± 69 pmol of PGF_2α metabolized/mg soluble protein per min for renal cortex, liver and lung, respectively. There were no significant differences between these rates of PGE₂ and PGF_2α metabolism when compared to rates in tissues obtained from animals subjected to either hemorrhagic or endotoxic shock. In addition, no significant differences were observed between the rate of PGE₂ metabolism and that of PGF_2α metabolism for any tissue. However, the lung was able to metabolize PGE₂ and PGF_2α significantly more rapidly than the liver, and to degrade PGE₂ at a significantly greater rate than the renal cortex. Although slightly different patterns of metabolite production were observed between lung and kidney homogenates, only the liver metabolized prostaglandins almost exclusively to more polar metabolites. While hemorrhagic or endotoxic shock induced slight changes in the patterns of PGE₂ metabolite formation in all three tissues studied, PGF_2α metabolite formation patterns were not significantly altered by circulatory shock. Thus, prostaglandin metabolism is not significantly impaired during the first 2 h of hemorrhagic or endotoxic shock in rabbit tissues. Therefore, impairment of prostaglandin metabolism is not the major factor responsible for the early increase in circulating prostaglandin concentrations in these forms of shock.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Lipoxygenase conversion of arachidonic acid in males and inseminated females of the firebrat,Thermobia domestica (Thysanura)

This is the first investigation concerning prostaglandin-like compounds in the primitive insect, Thermobia domestica. The incubation of homogenates of reproductive tissues in the presence of [U-¹⁴C]arachidonic acid yielded several compounds which have been characterized by their chromatographic mobilities as well as by the enzyme systems involved in their formation. The three major compounds (I to III) had R_f values very different from those of several prostaglandin standards (PGE₂, PGF_2α and 6-keto PGF_1α). As the addition of aspirin or indomethacin had no effect on the conversion of arachidonic acid, a cyclo-oxygenase pathway leading to prostaglandins seems to be excluded. However, another compound (noted V), present in very small quantities, could be a prostaglandin, owing to its chromatographic mobility near that of the PGE₂ standard. By contrast, compounds I and II co-migrated with 8- and 5-hydroxyeicosatetraenoic acid standards, respectively, and the addition of 4,7,10,13-eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) showed a pronounced and dose-dependent inhibition of arachidonic acid conversion. These data demonstrate lipoxygenase activity. Such a pathway in the metabolism of arachidonic acid had not, as yet, been reported in insects. This enzyme system can be demonstrated in the genital tract of the male and also in the seminal receptacle of the female, especially after insemination. So the enzyme system is probably transferred from male to female during mating.     

A radioimmunoassay for 6-keto- prostaglandin F1α

A radioimmunoassay for 6-keto-prostaglandin F_1α has been developed. The assay is accurate and sensitive but since the antiserum cross-reacts 5–10% with prostaglandins (PGs) of the E and F series, solvent extraction and thin layer chromatography are required fo absolute specifity. The assay has been validated by comparison with a radiochemical assay and by the use of an inhibitor of 6-keto PGF_1α formation, 15-hydroperoxy arachidonic acid. 6-Keto PGF_1α was found to have a low cross reaction with antisera directed against PGE₂, PGF_2α and thromboxane B₂.     

Studies on the oxidation of ω-hydroxyprostaglandins by an NAD-dependent dehydrogenase from mammalian liver cytosol

The oxidation of 12-hydroxylauric acid methyl ester (12-OH-L-Me) and of ω-hydroxy-prostaglandins (ω-OH-PGs) such as 20-OH-PGB₁ and 20-OH-PGE₁, was demonstrated with liver cytosol from rat, rabbit, and guinea pig in the presence of NAD; however, NADP did not support this oxidation. (ω-1)-Hydroxy-compounds (11-OH-laurate and 19-OH-PGB₁) and PGE₁, PGF_1α, and PGB₁, all lacking the terminal (ω)-hydroxyl, did not reduce NAD. However, at pH 10, PGE₁ slightly enhanced NAD reduction, suggesting that at this pH PGE₁, could be a substrate for 15-hydroxy-PG dehydrogenase (PGDH). The oxidation products from incubations of 12-OH-L-Me, 20-OH-PGB₁-Me, and 20-OH-PGE₁ with guinea pig liver cytosol were isolated and identified by gas chromatography/mass fragmentation spectrometry as being the corresponding dicarboxylic acids. In contrast to the liver cytosol, guinea pig kidney cytosol had only a minimal effect on NAD reduction by 12-OH-L-Me but nevertheless did support the stimulation of NAD reduction by PGE₁, and PGF_1α, but not by PGB₁, indicating the participation of kidney cytosolic PGDH in PGE₁ and PGF_1α oxidation and demonstrating that the oxidation of ω-OH to the carboxylic acid is not mediated by PGDH. Though the in vivo rate of oxidation of ω-OH-PGs has not been established, these results suggest that the urinary dicarboxylic-PG metabolites involve a multiple sequentialstep oxidation of PGs involving ω-hydroxylation by an NADPH-cytochrome P-450 system in the endoplasmic reticulum and the subsequent oxidation of the ω-OH by an NAD-dependent dehydrogenase in the cytosol.