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1.
In an isotope dilution assay, prostaglandin (PG) E2, 6-keto-PGF, 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, 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.  相似文献   

2.
We report a convenient and efficient method for the preparation of prostaglandin 2,3-dinor-6-keto-F1α by incubating prostaglandin 6-keto-PGF1α (6-keto-PGF1α) with dispersed rat hepatocytes. Chromatographic separation revealed a single product from the hepatocyte metabolism of 6-keto-PGF1α whose structure was positively confirmed by mass spectrometry as 2,3 dinor-6-keto-PGF1α. This methods allowed for the preparation of high specific activity radioactive 2,3-dinor-6-keto-PGF1α which can be utilized to determine the recovery of urinary dinor-6-keto-PGF1α during extraction and separation of the compound for radioimmunoassay measurements, as well as deuterated 2,3-dinor-6-keto-PGF1α which can be used as an internal standard in the gas chromatography-mass spectrometric assay of this compound.  相似文献   

3.
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∝.  相似文献   

4.
A method for quantification of 6-keto-PGF, 2,3-dinor-6-keto-PGF, TXB2, 2,3-dinor TXB2, PGE2, PGD2 and PGF in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF and 55% for tritiated TXB2. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.  相似文献   

5.
This paper describes a method for selective two-step solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F 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 immunoreactivity consisted of interfering material coeluting with 6-ketoprostaglandin F and 2,3-dinor-6-ketoprostaglandin F. Among the alkyl silica sorbents studied (methyl, butyl, octyl, and octadecyl), an efficient separation of 2,3-dinor-6-ketoprostaglandin F from 6-ketoprostaglandin F 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 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 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.  相似文献   

6.
We microanalyzed 2,3-dinor-6-keto-prostaglandin F (2,3-dinor-6-keto-PGF 1) and 11-dehydrothromboxane B2 (11-dehydro-TXB2, 2) in human urine. Samples containing a [2H4]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.  相似文献   

7.
The pulmonary formation of prostacyclin (PGI2), as reflected by the difference in concentration of pulmonary and systematic arterial radioimmunoassayed 6-keto-PGF, was determined in six healthy waking subjects. The systematic arterial 6-keto-PGF levels were low (50 pg/ml), and no evidence of pulmonary formation and release of the compound was noted. In other experiments systemic arterial 6-keto-PGF levels were determined in patients prior to and during artificial ventilation, as well as during and after occlusion of the pulmonary circulation (extra-corporeal circulation, ECC). The arterial 6-keto-PGF concentration prior to artificial ventillation was 17±4 pg/ml, i.e. within the range observed in the healthy subjects. During artificial ventilation the arterial levels of 6-keto-PGF increased to 191±21 pg/ml, suggesting that pulmonary formation of PGI2 was stimulated. In the patients subjected to ECC with occluded pulmonary circulation the arterial content of 6-keto-PGF was stabilised at an elevated level (120−170 pg/ml). Following re-establishment of the pulmonary circulation the arterial concentrations of 6-keto-PGF increased markedly, to 284±50 pg/ml. It is suggested that the basal pulmonary formation of PGI2 in man is low or non-existent, and that enhanced formation of the compound in the lungs is a consequence of intervention with normal pulmonary ventilation or perfusion.  相似文献   

8.
Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF, PGE-M, 2,3-dinor-TxB2 and 11-dehydro-TxB2) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE2 rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF from 7.2 ± 1.3 to 51.5 ± 17.0, and txB2 from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m2 ( ) at the maximal urine flow. Except for 6-keto-PGF and TxB2, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB2 one can assume that this prostanoid represents renal as well as systemic TxA2 activity.  相似文献   

9.
Pulmonary arterial prostacyclin (as 6-keto-PGF) concentrations of near term, fetakl lambs and goats were determined following fetal surgery and 24, 48, and 72 hrs later. Blood gases, pH, and arterial pressure were determined also. At the end of 2.5 hrs of surgery including exteriorization of the uterus and fetal thorocotomy, pulmonary arterial concentrations of 6-keto-PGF was 948 ± 92 (SEM) pg/ml of blood. Twenty-four hrs later it had fallen to 435 ± 92 pg/ml and remained constant for the duration of monitoring. Maternal arterial 6-keto-PGF concentration was much lower (105 ± 20 pg/ml of blood). No significance changes in fetal PaO2, PaCO2, pH, or arterial pressure were observed, although PaCO2 appeared to be elevated and pH reduced following surgery. These values normalized within 24 hrs. We conclude that surgical perturbation increases fetal arterial prostacyclin concentration. Increased prostacyclin levels are transient, reaching stable values within 24 hrs following completion of extensive surgery.  相似文献   

10.
The transformation of 6-keto-PGF to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF (II) by Mycobacterium rhodochrous UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ13 reductase enzyme systems.  相似文献   

11.
The cross-reactivity of the PGI3 metabolite, Δ17-6-keto-PGF, with antibodies against 6-keto-PGF for radioimmunoassays (RIA) has been investigated. Δ17-6-keto-PGF 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 and Δ17-6keto-PGF produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified Δ17-6-keto-PGF with 6-keto-PGF 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. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF, the Δ17-6-keto-PGF formed being undetected at 90%. It is concluded that 6-keto-PGF antibodies generally used for RIA of 6-keto-PGF are highly specific since they can discriminate a metabolite bearing an additional double bond such as the PGI3 metabolite Δ17-6-keto-PGF.  相似文献   

12.
A radioimmunoassay (RIA) for the estimation of 6-keto-PGF 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 was found to be lower than that reported for PGE2 or PGF. However, during the first week of life, significantly more 6-keto-PGF was excreted. The very high levels of 6-keto-PGF 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.  相似文献   

13.
Spirals of human saphenous veins (HSV), mounted in a 5 ml organ bath containing Krebs-Henseleit solution (37°C), when kept in contact with defibrotide (100–200 ug/ml) for 15 min, enhance (2 and 3 fold) their own basal release of 6-keto-PGF (61 ± 1.3 pg/mg w.t. n = 12). The phenomenon was long lasting upon repeated washing and sensitive to indomethacin (1 ug/ml). Endothelin-1 (ET-1, 20–40 ng) induced a sustained contraction of HSV and concomitantly released from the venous tissue a proportional amount of 6-keto-PGF.Indomethacin (1 ug/ml), by inhibiting cyclo-oxygenase enzyme, potentiated the contractile activity of ET-1 in HSV whereas exogenous PGE2 (20 ng/ml) considerably reduced the tension developed by the peptide on this venous tissue.Defibrotide (200 ug/ml), by releasing 6-keto-PGF, and other vasoactive prostaglandins, antagonized the contractile effect ET-1 (20 ng) in HSV. This data indicates that the eicosanoid metabolism is involved in the modulation of the potent vasoconstrictor effect of ET-1 in HSV and that PGI2-releaser, such as defibrptide, may have therapeutical value against immoderate changes of venous tone.  相似文献   

14.
The production of vasodilatory, antiaggregatory prostacyclin (PGI2) and vasoconstrictory, proaggregatory thromboxane A2 (TxA2) by the placenta was studied in the cases of hypertensive pregnancy complications by superfusing pieces from maternal and fetal sides of placentae of 9 pre-eclamptic, 6 hypertensive and 11 healthy women and measuring the release of 6-keto-prostaglandin F (6-keto-PGF) and thromboxane B2 (TxB2), the breakdown products of PGI2 and TxA2 respectively, from the superfusate. Both sides of the placentae from the controls produced 6-keto-PGF (maternal side 0.5±0.1 ng/g/min dry weight of tissue, mean±SEM; fetal side 0.7±0.2 ng/g/min) and TxB2 (maternal side 2.5±0.4 ng/g/min; fetal side 2.7±0.5 ng/g/min with no correlation between the two. The 6-keto-PGF production was normal in hypertensive complications whereas the TxB2 production was increased on the fetal side of the placentae obtained from the pre-eclamptic (3.7±0.3 ng/g/min: p<0.05) and hypertensive women (4.1±0.4 ng/g/min; p<0.025). This may explain the occurrence of microthrombi and infarctions in placentae of hypertensive women.  相似文献   

15.
PGI2 and 6-keto-PGF were converted to 6-methoxime-PGF (6-MeON-PGF) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (3H-6-MeON-PGF, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE2, 1% with PGF and less than 0.2% with PGD2, PGF, PGF and TXB2. The radioimmunoassay was used to estimate release of PGI2 and 6-keto-PGF from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf 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 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.  相似文献   

16.
Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing 3H-labeled prostacyclin (PGI2), thromboxane (TxB2) or 6-keto-PGF and 14C-labeled sucrose or thiourea. Following 1 to 30 min incubation with 14C-sucrose, 3H-TxB2 or 3H-6-keto-PGF, 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 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.  相似文献   

17.
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 (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 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.  相似文献   

18.
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 (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 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, the stable metabolite of prostacyclin (PGI2), has the ability to influence cervical protein metabolism and that this effect is steroid hormone dependent.  相似文献   

19.
Endogenous thromboxane production is best assessed by the measurement of its excreted metabolites, of which 11-dehydrothromboxane B2 (11-dehydro-TxB2) is most abundant. Gas chromatographic—mass spectrometric assays have been developed for this compound but suffer from the presence of co-eluting impurities which make the measurement of 11-dehydro-TxB2 difficult. Furthermore, these assays are often time-consuming. We now report a modified assay for the measurement of this compound employing gas chromatography—mass spectrometry which alleviates the problem of co-eluting impurities primarily through modification of extraction and chromatographic methods. Furthermore, the time to complete the assay is significantly shortened. It is adaptable to both urine and plasma. Precision of the assay is ± 7% and accuracy is 90%. The lower limit of sensitivity in urine is approximately 20 pg/mg creatinine. Normal levels of urinary excretion of this metabolite were found to be 370 ± 137 pg/mg creatinine (mean ± 1 S.D.) and normal plasma levels were found to be 1.5 ± 0.4 pg/ml (mean ± 1 S.D.). Urinary excretion of 11-dehydro-TxB2 is markedly altered in situations associated with abnormalities in thromboxane generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous thromboxane production and to further explore the role of this compound in human disease.  相似文献   

20.
The TxA2 synthetase inhibitor, dazoxiben, and the TxA2 antagonist, ±SQ 29, 548, were examined for effects on release and vasoactivity of TxA2 and prostacyclin. Isolated perfused guinea pig lungs were used as the enzyme source from which TxA2 and prostacyclin were released in response to injections of arachidonic acid or bradykinin. Both dazoxiben and ±SQ 29, 548 inhibited contraction of the superfused rat aorta and bovine coronary artery after arachidonic acid injection through the lung. ±SQ 29, 548 abolished contractions of the rat aorta, but significant aorta contracting activity persisted during dazoxiben treatment. Dazoxiben significantly inhibited arachidonate-induced release of TxA2 (immunoreactive TxB2)iinto the superfusate, but TxA2 release was significantly potentiated by ±SQ 29, 548. Thus, in the presence of enhanced TxA2 concentrations, ±SQ 29, 548 effectively antagonized the vasospastic effect of TxA2. Dazoxiben diverted a significantly greater amount of arachidonic acid into prostacyclin synthesis (immunoreactive 6-keto-PGF), changing original coronary vasoconstriction into relaxation. ±SQ 29, 548 did not significantly modify lung prostacyclin synthesis. Moreover, with ±SQ 29, 548, the absence of TxA2-mediated coronary contraction unmasked active relaxation of the superfused bovine coronary artery, coincident with thromboxane and prostacyclin release. Dazoxiben consistently inhibited TxA2 synthesis and enhanced prostacyclin synthesis. ±SQ 29, 548 augmented TxB2 release in response to arachidonate, but not bradykinin, and did not significantly alter 6-keto-PGF release in response to either arachidonate or bradykinin. In terms of vasoactivity measured , ±SQ 29, 548 and dazoxiben produced similar anti-vasospastic effects, although this was accomplished by completely different mechanisms.  相似文献   

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