Prostacyclin metabolism in adults and neonates. Urinary profiles of 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha studied by gas chromatography-mass spectrometry

Metabolism of endogenous prostacyclin was studied in adults and neonates by measuring urinary levels of 6-ketoprostaglandin F1 alpha (spontaneous hydrolysis product) and 2,3-dinor-6-ketoprostaglandin F1 alpha (enzymatically formed by beta-oxidation). Quantification of prostanoids was achieved by capillary gas chromatography-mass spectrometry using the stable isotope dilution technique. Purification of the urinary lipid extract included silicic acid column chromatography and reverse- and straight-phase high-pressure liquid chromatographies. Accuracy of the method was proven by recovery experiments for both metabolites. Partial mass spectra of endogenous 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha were obtained from urine samples. In neonates (third day of life, n - 5 pooled urines) levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.28 +/- 0.18 ng/ml) were much lower than those of 6-ketoprostaglandin F1 alpha (2.13 +/- 1.10 ng/ml), indicating low beta-oxidation activity at high prostacyclin formation. In adults (n = 7), levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.27 +/- 0.21 ng/ml) and levels of 6-ketoprostaglandin F1 alpha (0.20 +/- 0.11 ng/ml) were about the same, indicating relatively high beta-oxidation at low prostacyclin formation. Values are expressed as mean +/- S.D.     

Production of 6-oxo-prostaglandin F1 alpha and prostaglandin E2 by isolated glomeruli from normal and diabetic rats.

Production of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) was measured by radioimmunoassay in supernatants of isolated glomeruli from rats with streptozocin-induced diabetes and non-diabetic rats. Production of 6-oxo-PGF1 alpha by discs of aortas from these rats was measured at the same time. As shown before, aortic discs from diabetic rats produced significantly less 6-oxo-PGF1 alpha than aortic discs from non-diabetic rats (diabetic 1.99 +/- SEM 0.27 ng v non-diabetic 2.92 +/- 0.46 ng/mg net weight aorta; p less than 0.05). In contrast production of 6-oxo-PGF1 alpha by isolated glomeruli was not reduced in the diabetic rats (diabetic 77 +/- 7 pg v non-diabetic 70 +/- 8 pg/micrograms glomerular DNA). Similarly production of PGE2 was not diminished in the diabetic glomeruli (diabetic 1.20 +/- 0.15 ng v non-diabetic 0.91 +/- 0.12 ng/microgram glomerular DNA). It is concluded that regional differences in production of prostacyclin and 6-oxo-PGF1 alpha occur in experimental diabetes. Diminished prostacyclin production may contribute to the increased susceptibility of diabetic patients to atherosclerosis but is less likely to have a role in the pathogenesis of microangiopathy.     

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

Tetradeuterated 2,3-dinor-6-keto-PGF1 alpha was used as internal standard in the development of a method for quantitation of 2,3-dinor-6-keto-PGF1 alpha in human urine based on gas chromatography - mass spectrometry. The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha 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 alpha 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 alpha 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 alpha.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1 alpha by Mycobacterium rhodochrous

The transformation of 6-keto-PGF1 alpha to two prostacyctin metabolites, 2,3-dinor-6-keto-PGF1 alpha (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF1 alpha (II) by Mycobacterium rhodochrous UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF1 alpha to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and delta 13 reductase enzyme systems.     

Urinary 8-epi-PGF2alpha and its endogenous beta-oxidation products (2,3-dinor and 2,3-dinor-5,6-dihydro) as biomarkers of total body oxidative stress

Although measurements of plasma F2-isoprostanes are established markers of oxidative stress, their quantification only reflects acute non-enzymatic lipid peroxidation. In this study, a new approach is described for the rapid isolation and measurement of urinary 8-epi-PGF2alpha and its endogenous beta-oxidation metabolites (2,3-dinor-8-epi-PGF2alpha and 2,3-dinor-5,6-dihydro-PGF2alpha) for use as index of total body oxidative stress. Isoprostanes were partitioned with ethyl acetate and subsequently purified by chromatography on an aminopropyl (NH2) and silica (Si) cartridge. Final analysis of F2-isoprostanes as trimethylsilyl-ester/pentafluorobenzyl ester derivatives was carried out by stable isotope dilution mass spectrometry. Overall recovery of F2-isoprostanes was 80+/-4%. Inter- and intra-assay coefficients of variation were 5% and 7%, respectively. In a group of healthy humans, the mean excretion rates expressed as nmol/mmol creatinine for 2,3-dinor-8-epi-PGF2alpha, 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha, and 8-epi-PGF2alpha were 5.43+/-1.93, 2.16+/-0.71, and 0.36+/-0.16, respectively. Correlations were obtained between 8-epi-PGF2alpha and 2,3-dinor-8-epi-PGF2alpha or 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.998 and r=0.937, respectively). A strong relationship was also seen between 2,3-dinor-8-epi-PGF2 and 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.949). The new technique allows for high sample throughput and avoids the need for HPLC and/or other expensive equipment required for the initial sample preparation. Simultaneous analysis of urinary 8-epi-PGF2alpha and its metabolites should provide unique tool in clinical trials exploring the role of oxidant injury in human disease.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Cigarette smoking: profiles of thromboxane- and prostacyclin-derived products in human urine

Thromboxane (TX) B2, 2,3-dinor-TXB2, 11-dehydro-TXB2, 6-oxoprostaglandin (PG)F1 alpha and 2,3-dinor-6-oxo-PGF1 alpha were measured in 24 h urine samples obtained from 30 apparently healthy chronic cigarette smokers and 37 closely matched non-smoking control subjects. Samples were analysed using a newly developed assay based on immunoaffinity chromatography and capillary column gas chromatography/electron capture negative ion chemical ionisation mass spectrometry. There were significant and comparable increases in the excretion rates of both 2,3-dinor-TXB2 and 11-dehydro-TXB2 in the smoking compared with the non-smoking group (2P less than 0.001). Excretion rates of 2,3-dinor-TXB2 were 418 +/- 35 and 265 +/- 26 pg/mg creatinine in the two groups, respectively. 11-Dehydro-TXB2 excretion rates were 440 +/- 54 and 221 +/- 18 pg/mg creatinine, respectively (mean +/- S.E.). There were significant (2P less than 0.05) positive correlations between average reported cigarette consumption and excretion of both thromboxane metabolites. There were small but significant (2P less than 0.02) increases in the excretion rates of both 6-oxo-PGF1 alpha and 2,3-dinor-6-oxo-PGF1 alpha in the smoking compared with the non-smoking group. There was no significant difference in the rates of excretion of TXB2 in the two groups. The effects of acute cigarette smoke exposure (five cigarettes in 2 h) was also studied in four normally non-smoking healthy volunteers. There was no significant change in the excretion rate of any of the eicosanoids measured during control and smoking periods (at least 2 weeks apart), indicating that increased TXA2 biosynthesis in chronic smokers is unlikely to be a consequence of acute platelet activation.     

Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.     

In vivo formation of metabolites of prostaglandins I2 and I3 in the marmoset monkey (Callithrix jacchus) following dietary supplementation with tuna fish oil

Recent studies have shown that ingestion of eicosapentaenoic acid (EPA) in man results in the formation of 'trienoic' prostanoids which amy partly explain the potent antithrombotic/antiatherogenic properties of long-chain polyunsaturated n-3 fatty acids (PUFAs). However, endogenous formation of cyclooxygenase metabolites of EPA has not been demonstrated in an animal model, and in vitro studies indicate a clear species difference in the conversion of EPA to PGI3. Thus, in the present study, the in vivo formation of PGI3 following long-term dietary tuna fish oil supplementation was investigated in a small non-human primate - the marmoset monkey (Callithrix jacchus). The excretion of major urinary metabolites 2,3-dinor-6-keto-PGF1 alpha (PGI2-M) and delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) was estimated as an index of total body synthesis of PGI2 and PGI3, respectively. Following extraction, dinor prostanoid metabolites were separated by capillary gas chromatography and identified by negative ion chemical ionization mass spectrometry. Supplementation of the standard (reference) diet with either sheep fat or sunflower seed oil did not alter the body production of PGI2-M. However, following the tuna fish oil-enriched diet, there occurred not only an increase in urinary PGI2-M (reference 70.7 +/- 9.0; tuna fish oil 115.5 +/- 12.1 ng/g creatinine, P less than 0.05), but also a considerable formation of PGI3-M (62.9 +/- 5.3 ng/g creatinine), which was not seen in any other dietary group; in addition, the urinary level of immmunoreactive 2,3-dinor-thromboxane B2/3 was reduced after ingestion of tuna fish oil. These urinary changes were accompanied by a rise in plasma phospholipid-bound EPA and docosahexaenoic acid (DHA). In addition, tuna fish oil supplementation resulted in a significant reduction in plasma cholesterol (53%) and triacylglycerols (44%). The present study provides for the first time experimental evidence for the in vivo formation of PGI3 in an animal model and also confirms the earlier observations in man following dietary fish oil supplementation.     

Thromboxane (TX)A3 and prostaglandin (PG)I3 are formed in man after dietary eicosapentaenoic acid: identification and quantification by capillary gas chromatography-electron impact mass spectrometry

The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3 and weakly proaggregatory TXA3 has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivo with collagen was traced by capillary GC/EIMS. Via external standard, TXB3 formation in platelets was estimated to be 5-15% of TXB2 formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) as an index of total body production of PGI3 in analogy to 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3-M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83 +/- 25 ng/24 h (SD) after ingestion of cod liver oil and 134 +/- 38 ng/24 h after mackerel ingestion, while excretion of PGI2-M was 162 +/- 52 ng/24 h and 236 +/- 32 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivo the spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Determination of prostaglandin F2 alpha, E2, D2 and 6-keto-F1 alpha in human cerebrospinal fluid.

Prostaglandin (PG)F2 alpha, E2, D2 and 6-keto-F1 ALha were determined in human cerebrospinal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6-keto-F1 alpha (0.12--15 ng/ml). PGF2 alpha and PGE2 were present in lower concentrations PGD2 was below the level of detection (0.05 ng/ml) except in one patient with extremely high total levels of PGs.     

Changes in the concentrations of plasma steroid hormone and plasma 13, 14-dihydro-15-oxo-prostaglandin F2 alpha in late pregnancy rabbits treated with an inhibitor of 3 beta-hydroxysteroid dehydrogenase

Changes in the concentrations of progesterone, 17 beta-estradiol and 13, 14-dihydro-15-oxo-prostaglandin F2 alpha (PGFM) were evaluated in the peripheral plasma of rabbits during late pregnancy by treating trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, in an attempt to obtain further insight into the involvement of progesterone and prostaglandin (PG) in the initiation of parturition. The concentrations of progesterone were 18.8 +/- 2.2 ng/ml (mean +/- SE, n = 6) before administration of the inhibitor, significantly (p less than 0.05) fell to 7.6 +/- 1.0 ng/ml (n = 6) at 30 min, and remained low until 10 h after the drug administration. The concentrations of progesterone were still low (5.4 +/- 0.5 ng/ml, n = 6) at 20-24 h after administration of the inhibitor, and were also low (4.9 +/- 2.2 ng/ml, n = 6) at delivery. Premature deliveries occurred at 28.8 +/- 2.0 h after injection of trilostane (on days 29 of gestation). Increased concentrations of PGFM were observed at delivery. However, administration of trilostane induced no discernible changes in the concentration of estradiol. These findings suggest that delivery is induced by progesterone withdrawal and that synthesis prostaglandin F2 alpha is remarkably increased at delivery in the rabbit.     

Monitoring of the thromboxane A2/prostacyclin ratio in the urine of patients with retinal vascular occlusion through the low-dose-aspirin therapy using the gas chromatography/selected ion monitoring method

We determined the levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) in patients with retinal vascular occlusion (RVO) to elucidate the change of the thromboxane A2/prostacyclin (TX/PGI) ratio with this disease and the effect of low-dose-aspirin therapy. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were converted to 1-methyl ester-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15-tris-dimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The average level of 11-dehydro-TXB2 in 30 patients with RVO was 1038 +/- 958 pg/mg creatinine. It was significantly higher than that of 27 healthy volunteers, which was 616 +/- 294 pg/mg creatinine (p < 0.05 with unpaired t-test). However, 2,3-dinor-6-keto-PGF1alpha levels were not significantly different between these two groups. The average ratio of TX/PGI in the RVO patients was 32 +/- 26 and it was significantly higher than that of healthy volunteers, 17 +/- 10 (p < 0.01). Patients with central retinal artery occlusion or branch retinal artery occlusion showed greatly high 11-dehydro-TXB2 levels and TX/PGI ratios, although the number of patients was limited in the current study. After the administration of low-dose aspirin (40 mg/day) for about 1 month, the TX/PGI ratio decreased to around the normal level. Following the levels for up to 10 months, they also remained at the normal level. These observations suggested that the 11-dehydro-TXB2 levels and the TX/PGI ratio reflect the pathological conditions of RVO and are useful markers of the treatment.     

Urinary levels of 2,3-dinor-6-oxo-PGF1 alpha: a reliable index of the production of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M), a major metabolite of PGI2, are determined by the balance between the amount of PGI2 synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI2-M can be used as a reliable index of the in vivo production of PGI2 in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI2-M during paired intravenous infusions of 6-oxo-PGF1 alpha (the stable product of the spontaneous hydrolysis of PGI2) in conscious, unrestrained SHR and WKY rats aged 12-15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI2 was infused instead of 6-oxo-PGF1 alpha. The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF1 alpha and PGI2 into PGI2-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF1 alpha infused and the amount of PGI2-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha as an index of PGI2 production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI2 than WKY rats in vivo, contrary to the situation prevailing in vitro.     

Urinary thromboxane A2/prostacyclin balance reflects the pathological state of a diabetic

Levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) were measured in diabetics to elucidate the relation between the thromboxane A2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.     

Biosynthesis of 5,6-dihydroxyprostaglandin E1 and F1 alpha from 5,6-dihydroxyeicosatrienoic acid by ram seminal vesicles

cis-5(6)Epoxy-cis-8,11,14-eicosatrienoic acid was recently found to be metabolized by ram seminal vesicles to 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta, 5(6)epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1. The epoxide can be hydrolyzed by epoxide hydrolases to 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The latter was incubated with microsomes of ram seminal vesicles for 2 min at 37 degrees C and the polar metabolites were purified by reversed phase HPLC and analyzed by capillary column gas chromatography-mass spectrometry. The major metabolite was identified as 5,6-dihydroxyprostaglandin F 1 alpha. In the presence of glutathione (1 mM), 5,6-dihydroxyprostaglandin E1 was also formed. The 3H-labelled vicinal diol and the 3H-labelled epoxide were metabolized to polar products to a similar extent, but the formation of prostaglandin E compounds in the presence of glutathione was lower from the diol than from the epoxide or from arachidonic acid. The likely prostaglandin endoperoxide intermediates in the metabolism of the diol (5,6-dihydroxyprostaglandin G1 and 5,6-dihydroxyprostaglandin H1) thus appear to be less prone to be isomerized to prostaglandin E compounds than prostaglandins G2 and H2 and their 5(6)epoxy counterparts. 5(6)Epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1 can be chemically transformed into 5,6-dihydroxyprostaglandin B1. The latter can be analyzed by HPLC or by mass fragmentography, and a simple chemical synthesis of 5,6-dihydroxyprostaglandin B1 from prostaglandin E2 is described.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

Determination of 9alpha, 11beta prostaglandin F2 in human urine. combination of solid-phase extraction and radioimmunoassay

This paper describes a new iodine-125 radioimmunoassay of 9alpha ,11beta-PGF2, and its use for the determination of urinary 9alpha,11beta-prostaglandin F2 after a selective one-step solid-phase extraction. The newly reported immunoassay is based on the use of 125I-tyrosyl methyl ester derivative of 9alpha,11beta-PGF2 and specific polyclonal antibody raised in rabbits.The assay detected as lowas 0.85 pg/tube 9alpha,11beta-PGF2, and the antibodyshowed lessthan 0.01 cross-reaction with PGF-ring metabolites (e.g., 8-iso-PGF2alpha, PGF2alpha 2,3-dinor-6-keto-PGF1alpha, and 5 more PGF-ring compounds). Both the intra-assay, and inter-assay CVs were lessthan 20% for internal controls containing low, medium and high concentrations of 9alpha,11beta-PGF2. Immuno-HPLC analysis showed a very low ratio of specific immunoreactivity in both non-extracted urine (6.5%), and in urine extracted on C18-silicacartridge (14.8%). By contrast, approximately 80% specific immunoreactivity could be achieved by using C2-silicaas the sorbent, acetonitrile: water (15:85, v/v) as wash solvent, and ethyl acetate as eluent of 9alpha,11beta-PGF2.This extraction procedure enabled a reasonably high extraction efficiency of 80.4 +/- 0.855 (mean +/- SEM, n=82), as determined by 3H-9alpha,11beta-PGF2. The new SPE/RIA method was applied for the determination of urinary 9alpha,11beta-PGF2 values in 50 healthy human volunteers. For the concentration and for the excretion rate 37.52 +/- 4.61 pg/ml (mean +/- SEM), and 3.50 + 0.35 ng/mmol creatinine (mean +/- SEM), respectively, was measured.The specificity of the SPE/RIA method was supported by the observed 69% decrease in 9alpha, 11beta-PGF2 excretion rate after acetylsalicylic acid treatment. The effect of nicotinic acid, a PGD2-stimulatory agent, was monitored by the urinary excretion of 9alpha ,11beta-PGF2 in 6 patients, by using the new SPE/RIA method. In patients responding with flushing symptoms nicotinic acid induced an increase of the urinary excretion of 9alpha,11beta-PGF2 in the range between 11% and 187%. In summary, the combination of the newly developed specific [125I] radioimmunoassay with solid-phase extraction on C2-silica cartridges enables the specific, sensitive, and reliable determination of 9alpha,11beta-PGF2 in human urine without the need for further laborious chromatographic purification before radioimmunoassay.     

Effects of age and resistance exercise on skeletal muscle interstitial prostaglandin F(2alpha)

Prostaglandin (PG) F2alpha has been shown to contribute to the anabolic events in skeletal muscle. We measured the skeletal muscle interstitial concentration of PGF2alpha at rest and following a standard bout of resistance exercise in eight young (27+/-2 year) and eight old (75+/-4 year) men. Interstitial PGF2alpha concentration was determined from microdialysate samples obtained from two microdialysis probes placed in the vastus lateralis. Microdialysates were collected 1h pre- and 5-6, 8-9, and 24-25 h postexercise. The exercise bout consisted of 4 exercises (3 sets of 8 replications at 80% 1 RM per exercise) emphasizing the quadriceps. Interstitial PGF2alpha levels were not different (P>0.05) between young and old at rest (1.50+/-0.35 vs. 1.52+/-0.30 ng ml-1) or at any time point following the resistance exercise bout. For the young and old combined there was a change (P<0.05) in PGF2alpha levels at 5-6 h (93%) and 8-9 h (95%), which had returned to preexercise levels by 24-25 h. These results show that PGF2alpha is increased in skeletal muscle following a standard bout of resistance exercise and aging does not alter interstitial levels of this PG at rest or after exercise. These data, coupled with previous findings, suggest that the anabolic factor PGF2alpha should be considered when discussing the complex processes that regulate muscle mass in young and old individuals.