Synthesis of thromboxane B2 and 6-keto-prostaglandin F1α by bovine gastric mucosal and muscle microsomes

Bovine gastric mucosal and muscle microsomes synthesize prostaglandins and thromboxane B₂ (TXB₂) from arachidonic acid (AA). TXB₂ and 6-keto-prostaglandin F₁α (6-keto-PGF₁α) were the major products synthesized by pylorus, body, and cardiac region of the gastric mucosa. Gastric muscle mainly synthesized 6-keto-PGF₁α. TXB₂ and 6-keto-PGF₁α synthesis occurs at an appreciable rate from endogenous precursors but more rapidly with added arachidonate. Prostaglandins E₂, F₂α and D₂ were synthesized in smaller amounts under the conditions studied.     

Facile method for preparation of 2,3-dinor-6-keto PGF1α, the major urinary metabolite of prostacyclin

We report a convenient and efficient method for the preparation of prostaglandin 2,3-dinor-6-keto-F_1α by incubating prostaglandin 6-keto-PGF_1α (6-keto-PGF_1α) with dispersed rat hepatocytes. Chromatographic separation revealed a single product from the hepatocyte metabolism of 6-keto-PGF_1α whose structure was positively confirmed by mass spectrometry as 2,3 dinor-6-keto-PGF_1α. This methods allowed for the preparation of high specific activity radioactive 2,3-dinor-6-keto-PGF_1α which can be utilized to determine the recovery of urinary dinor-6-keto-PGF_1α during extraction and separation of the compound for radioimmunoassay measurements, as well as deuterated 2,3-dinor-6-keto-PGF_1α which can be used as an internal standard in the gas chromatography-mass spectrometric assay of this compound.     

Simplified assay for the quantification of 2,3-dinor-6-ketoprostaglandin F1α by gas chromatography—mass spectrometry

Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F_1α (2,3-dinor-6-keto-PGF_1α). Gas chromatographic—mass spectrometric (GC—MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF_1α employing GC—MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is ± 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 ± 54 pg/mg creatinine (mean ± S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF_1α is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.     

Cross-Reactivity of Δ17-6-Keto-PGF1α with 6-Keto-PGF1α antibodies

The cross-reactivity of the PGI₃ metabolite, Δ17-6-keto-PGF_1α, with antibodies against 6-keto-PGF_1α for radioimmunoassays (RIA) has been investigated. Δ17-6-keto-PGF_1α was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37°C with 10 μM eicosapentaenoic acid (EPA) in the presence of 2 μM 13-hydroperoxy-octadecadienoic acid, an activator of the EPA cyclooxygenation, and the 6-keto-PGF_1α and Δ17-6keto-PGF_1α produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified Δ17-6-keto-PGF_1α with 6-keto-PGF_1α antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF_1α. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF_1α, the Δ17-6-keto-PGF_1α formed being undetected at 90%. It is concluded that 6-keto-PGF_1α antibodies generally used for RIA of 6-keto-PGF_1α are highly specific since they can discriminate a metabolite bearing an additional double bond such as the PGI₃ metabolite Δ17-6-keto-PGF_1α.     

Analysis of 6-keto-prostaglandin F1α in human urine: Age-specific differences

A radioimmunoassay (RIA) for the estimation of 6-keto-PGF_1α in human urine is described in detail. The RIA method was validated by direct comparison to gas chromatography-mass spectrometry. In adults and in one year old children basal excretion of 6-keto-PGF_1α was found to be lower than that reported for PGE₂ or PGF_2α. However, during the first week of life, significantly more 6-keto-PGF_1α was excreted. The very high levels of 6-keto-PGF_1α in urine seen on the third day of life seemed already to decrease during the first week of life. It is concluded that prostacyclin may have a major role for kidney function in the newborn, possibly by protecting the immature kidney from high levels of angiotensin II.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

Production rates of prostaglandin F, 6-keto-PGF1α and thromboxane B2 by perifused human endometrium

Human endometrium obtained from fresh hysterectomy specimens was perifused for 7 hr in 95% O₂/5% CO₂ at 37°C. The phase of the menstrual cycle was determined by histological examination. The concentrations of PGF, 6-keto-PGF_1α and TxB₂ in 20 min fractions of the perifusion medium were measured by radioimmunoassay and production rates were calculated in terms of dry weight of tissue. Biphasic patterns of production were observed; high initial values fell to about 20% at 2 hr and then increased to relatively stable values at about 4 hr which were maintained for the next 2 hr. During this latter period, production rates in endometria taken at different phases of the cycle differed markedly from each other; the production rates of PGF in secretory and early proliferative endometria were low (15.8 ± 2.6, mean ± SEM and 67.2 ± 8.3 ng/min/g respectively) whereas they were high in late proliferative and premenstrual endometria (188.0 ± 16.7 and 196.4 ± 16.9 ng/min/g respectively). The patterns of production of 6-keto-PGF_1α and TxB₂ were similar to those of PGF but the absolute values were much lower (<10%). We conclude that the observed rates of production of prostaglandins by perifused human endometrium are consistent with synthesis being stimulated either by estrogen or withdrawal of hormonal support and being inhibited by progesterone.     

Changes in urinary 6-keto-prostaglandin F1α excretion during pregnancy and labor

Urinary excretion of 6-keto-PGF_1α was measured by high pressure liquid chromatography and radioimmunoassay at various stages of pregnancy and labor. In the first trimester of pregnancy, urinary 6-keto-PGF_1α concentrations were nor different from those measured before pregnancy, but they showed a significant increase in the second trimester of pregnancy (p <0.001). The levels rose further in the third trimester, although this increase was not statistically significant when compared to levels obtained in the second trimester. There was no evidence for a significance change in 6-keto-PGF_1α excretion with the onset of labor. During well-established, progressive labor mean values of 6-keto-PGF_1α excretion were about twice as high as before the onset of labor, but the range of values during labor was so wide that there was no statistical difference with values obtained in the second half of pregnancy.It is concluded that the increase in the urinary excretion of 6-keto-PGF_1α occurs later in pregnancy than the increase in TXB₂ excretion and that labor at term is not associated with marked changes in 6-keto-PGF_1α excretion.     

Effects of indomethacin, prostaglandin E2, prostaglandin F2α and 6-keto-prostaglandin F1α on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) or 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were added to the culture media with indomethacin. (1) The hatching was inhibited by indomethacin yet the inhibition was reversible. (2) In the groups with indomethacin and PGE₂, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. (3) In the groups with indomethacin and PGF_2α, inhibition of hatching was improved in comparison with the group with indomethacin. (4) In the groups with indomethacin and 6-keto-PGF_1α, no improvement was seen. The above results indicated that PGF_2α possibly had an accelerating effect on hatching and a high concentration of PGE₂ would exert cytotoxic effect on blastocysts.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater importance.     

The impermeability of the basic cell membrane to thromboxane-B2, prostacyclin and 6-keto-PGF1α

Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing ³H-labeled prostacyclin (PGI₂), thromboxane (TxB₂) or 6-keto-PGF_1α and ¹⁴C-labeled sucrose or thiourea. Following 1 to 30 min incubation with ¹⁴C-sucrose, ³H-TxB₂ or ³H-6-keto-PGF_1α, the ¹⁴C or ³H space of packed RBCs remained essentially constant, yielding mean values (±S.E.) for all time periods of 6.1 ± 0.3, 9.5 ± 0.5 and 6.5 ± 0.4%, respectively. After 1 min of incubation at 4° or 23°C at a pH of 7.4 or 8.5 with trace amounts (10⁻⁹M) of ³H-PGI₂ or in the presence of added PGI₂ (10⁻⁵M) or ethacrynic acid (1.6 × 10⁻⁴M), the apparent PGI₂ space of packed RBCs ranged from 16 to 27%, decreasing to about 7% by 30 min. When RBCs were resuspended in fresh ³H-PGI₂ every 5 min, their ³H content increased very slowly (apparent PGI₂ space <40% at 30 min) as compared to thiourea (distribution space > 80% within 5 min). Over 90% of this ³H activity was lost from the RBCs in less than 2 min during elution at 4° or 23°C. It is concluded that RBC membranes and thus, presumably, the basic cell membrane in general, is not fundamentally permeable to PGI₂, 6-keto-PGF_1α or TxB₂. Hence, the effective entry of these cyclooxygenase products into some cells or their passage across tight-junctional capillaries or epithelial membranes must require facilitated or active transport processes as was shown to be the case for E, F and A PGs. This implies that the distribution, pharmacological action and metabolism of these and presumably all related cyclooxygenase products are selective rather than unrestricted.     

Release of 6-keto-prostaglandin F1α and thromboxane B2 from mouse peritoneal macrophages during their adhesion and spreading on a glass surface

The release of 6-keto-prostaglandin F_1α (6KF_1α)_and of thromboxane B₂ (TXB₂) from cells were investigated using mouse peritoneal exudate cells (PECs) and non-cultured peritoneal macrophages. They were prepared by adhesion to glass dishes and incubated for 1 hr at 37°C in 5% Co₂ in air. Both the percentage of spreading macrophages and the release of 6KF_1α and TXb₂ increased in proportion to the incubation time. 6KF_1α and TXB₂ were released from the macrophages, not from the non-adherent cells. When PECs were incubated in silicon-coated glass dishes, the spreading of macrophages was hardly detected and lower amounts of 6KF_1α and TXB₂ were released from these cells compared with cells incubated in non-treated glass dishes. These findings suggest that adhesion with the correlated spreading of macrophages on glass dishes serve as a considerable physical factor for the release of 6KF_1α and TXB₂.     

Effects of nitrogen dioxide exposure on the contents of prostaglandins and thromboxane B2 in broncho alveolar lavage

Effects of 10 ppm nitrogen dioxide (NO₂) exposure on the contents of prostaglandins (PGs) and thromboxane (TX) B₂ in broncho-alveolar lavage (BAL) of rats were studied. In the BAL of normal rats, the amounts of PGs and TXB₂ in the whole lavage were 6-keto-PGF_1α (38.0 ± 6.4 ng) > TXB₂ (11.8 ± 4.0 ng) > PGF_2α (5.7 ± 1.6 ng) PGE (0.5 ± 0.3 ng). Rats were exposed to NO₂ for 1, 3, 5, 7 and 14 days. The NO₂ exposure decreased in the level of 6-keto-PGF_1α by about 35% throughout the exposure. The level of TXB₂ was higher in the day 5 exposure group (155%). The contents of PGF_2α and PGE first, decreased and then transiently increased on days 3 and 5. PG 15-hydroxy-dehydrogenase activity of lung homogenate decreased correspondingly on day 3 and 5. Then the contents PGF_2α and PGE decreased on day 7 and 14.6-keto-PGF_1α and TXB₂ are stable metabolites of PGI₂, a strong bronchorelaxant and TXA₂, a strong bronchoconstrictor respectively. Therefore the results suggested that the decrease in 6-keto-PGF_1α, a major prostanoid in the BAL and the increase in TXB₂ may correlate with broncho constriction by NO₂ exposure.     

The concentration of 6-keto-PGF1α and TXB2 in plasma samples from patients with benign and malignant tumours of the breast

Peripheral plasma concentrations of 6-keto-PGF_1α and TXB₂ were measured in patients with benign and malignant tumours of the breast, in patients with nongynecological disease,a nd in healthy female controls. The values were significantly higher in female patients with maligants tumours of the breast than in healthy controls (146 ± 28 vs 13 ± 2.5 pg/ml for 6-keto-PGF_1α p<0.01 and 78 ± 17 vs 11 ± 2 pg/ml for TXB₂, p<0.01). Benign tumours of the breast were also associated with significantly raised plasma levels of 6-keto-PGF_1α and TXB₂ compared to normal controls (52 ± 5 vs 13 ± 2.5 pg/ml for 6-keto-PGF_1α, p < 0.01 and 26 ± 5 vs 11 ± 2 pg/ml for TXB₂, p < 0.05). The high levels of 6-keto-PGF_1α and TXB₂ were not found to be correlated with clinical and histopathological data. The surgical removal of the primary tumour has apparently no effect on the plasma concentration of 6-keto-PGF_1α and TXB₂ over a follow-up period of 9 days after operation. The lack of alterations in the ratio of TXB₂: 6-keto-PGF_1α in the cancer patients and other subjects studied before and after surgery is indicative of the regulatory power of metabolic systems to preserve the homeostatic balance.     

Development of a GC-MS method for quantitation of 2,3-dinor-6-keto-PGF1∝ and determination of the urinary excretion rates in healthy humans under normal conditions and following drugs

Tetradeuterated 2,3-dinor-6-keto-PGF₁∝ was used as internal standard in the development of a method for quantitation of 2,3-dinor-6-keto-PGF₁∝ in human urine based on gas chromatography - mass spectrometry. The urinary excretion rates of 2,3-dinor-6-keto-PGF₁∝ in twenty normal healthy males and females were 9.7 ± 4.6 and 8.8 ± 8.5 (mean ± SD) ng/h respectively. A considerable inter- and intra-individual variation was found under normal conditions. It was also found that the urinary excretion of 2,3-dinor-6-keto-PGF₁∝ was increased about fivefold during and shortly after 30 min of strenuous jogging. Any data about the effect of nonsteroidal antiinflammatory drugs on the excretion rate of 2,3-dinor-6-keto-PGF₁∝ are difficult to interpret when considering the above findings. However, oral administration of 500 mg of aspirin did not seem to reduce the excretion rate of 2,3-dinor-6-keto-PGF₁∝.     

Formation of 6-keto-PGF1α by collecting tubule cells isolated from rabbit renal papillae

Homogeneous populations of collecting tubule epithelial cells have been isolated from rabbit renal papillae by a sequence of procedures involving: (a) dissociation of the tissue by mincing and treatment with trypsin; (b) destruction of contaminating non-collecting tubule cells by differential lysis in hypotonic media and (c) collection and washing by repeated centrifugation. The isolated cells have been characterized as being derived from the collecting tubules on the basis of anatomical source, size and histological staining for both NADH diaphorase activity and cyclooxygenase antigenicity. The cells are judged to be viable by several criteria including their ability to exclude both trypsin and vital dyes, their capacity to metabolize glucose and leucine and their ability to retain distinctive morphology following 10–14 days in culture media. Homogenates of freshly isolated collecting tubule cells when incubated with [³H]-arachidonic acid yielded radioactive products identified by thin-layer chromatographic behavior in multiple solvent systems as 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂ and a monohydroxy acid, probably HHT. No lipoxygenase-like activity was detected. At arachidonate concentrations of 2 or less, the major product was 6-keto-PGF_1α; while at substrate concentrations of greater than 10 , PGE₂ was the major radioactive prostaglandin formed. Similar distributions of products were observed when homogenates of dissociated renal papillae enriched in medullary interstitial cells were incubated with arachidonic acid. Our results indicate that collecting tubule cells do contain significant prostacyclin synthetase activity and suggest that PGI₂ plays a role in the function of mammalian collecting tubules.     

Selective Solid-Phase Extraction of Urinary 2,3-Dinor-6-ketoprostaglandin F1α for Determination with Radioimmunoassay

This paper describes a method for selective two-step solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F_1α for reliable determination with radioimmunoassay. In the immunoreactivity profile of nonselectively extracted urine after HPLC separation, over 90% of the total 2,3-dinor-6-ketoprostaglandin F_1α immunoreactivity consisted of interfering material coeluting with 6-ketoprostaglandin F_1α and 2,3-dinor-6-ketoprostaglandin F_1α. Among the alkyl silica sorbents studied (methyl, butyl, octyl, and octadecyl), an efficient separation of 2,3-dinor-6-ketoprostaglandin F_1α from 6-ketoprostaglandin F_1α and the lowest immunoreactive concentration of analyte were achieved in extraction on the methyl silica sorbent by elution of 2,3-dinor-6-ketoprostaglandin F_1α with chloroform:hexane (85:15, v/v) from the cartridge. The proportion of specific immunoreactivity could be further increased by two-step extraction of sample on methyl silica cartridges, first at pH 3 and then at pH 10 using diethyl ether:hexane (85:15, v/v) and chloroform as eluent, respectively. After this, a high correlation was found with concentrations of samples determined by radioimmunoassay using three different antisera. A significant correlation of values was also observed between samples measured by radioimmunoassay and those measured by GC-MS. The values of 12-h excretion of 2,3-dinor-6-ketoprostaglandin F_1α in eight volunteers (268 ± 204 ng/g creatinine, mean ± SD) as well as the inhibitory effect of acetylsalicylic acid (74 ± 12%) are in accordance with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring subsequent TLC or HPLC purification.     

Influence of 6-keto-PGF1α on collagen synthesis in the human cervix during various phases of the menstrual cycle

Uterine cervix tissue, obtained from nonpregnant fertile women undergoing hysterectomy, was mechanically chopped into 1 mm thick slices and incubated in Krebs-Ringer bicarbonate buffer containing 6-keto-PGF_1α (0.03–10 μg/ml) and ³H-proline. After incubation of 30–120 min the incorporated radioactivity was determined and related to the protein content of each slice. 6-keto-PGF_1α had specific and significant effects on the incorporation of ³H-proline into cervical tissue. In the follicular phase of the cycle a decreased incorporation was registered, indicating a reduced net synthesis of protein. However, increased radiolabelling was observed in the luteal phase, reflecting an augmented protein synthesis. It is suggested that 6-keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), has the ability to influence cervical protein metabolism and that this effect is steroid hormone dependent.     

Fish oil supplementation reduces excretion of 2,3-dinor-6-oxo-PGF1α and the 11-dehydrothromboxane B2/2,3-dinor-6-oxo-PGF1α excretion ratio in adult men

Dietary supplementation with a fish oil concentrate (FOC) reduced the endogenous synthesis of prostacyclin (PGI₂), as measured by the excretion of its major urinary catabolite, 2,3-dinor-6-oxo-PGF_1α (PGI₂-M). Thirty-four healthy men (24–57 years old) were given controlled diets and supplements that provided 40% of the energy from fat and a minimum of 22 mg/d of α-tocopherol for two consecutive experimental periods of 10 weeks each. During the experimental periods, the men received capsules containing 15 g/d of a placebo oil (PO) (period 1) or 15 g/d of the FOC (period 2). In addition to the PO or FOC, capsules contained 1 mg of α-tocopherol per g of fat as an antioxidant. The average daily excretion of PGI₂-M during the last week of FOC supplementation (period 2) was 22% less (P = 0.0001) than at the end of the first period. These results are at variance with those reported in comparable human studies conducted by other investigators during the middle and late 1980s. A 20% reduction (P = 0.003) in the 11-dehydrothromboxane B₂ to 2,3-dinor-6-oxo-PGF_1α excretion ratio at the end of period 2 in this study demonstrates that a shift of the n-6 to n-3 polyunsaturated fatty acid ratio from 12.5 to 2.3 brings about a substantial modulation of the eicosanoid system.