Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Simple radioimmunological method for urinary 6-keto-PGF1 alpha measurement

A radioimmunoassay for the determination of 6-keto-PGF1 alpha (stable metabolite of Prostacyclin) in urine using a specific antiserum, is described. The antibody used, showed no significant cross-reactivity with other PGS. (3H)-6-keto-PGF1 alpha has been used as tracer and a 30% Hydroxyapatite suspension was utilized for the separation of bound and free fractions. Standard curve ranges between 2 and 1000 pg thus allowing measurements in urine. Aliquots of 0.2 to 1 ml of 24 hours urinary samples were submitted to a preextraction step with Hexane in order to eliminate neutral fats. After acidifying with 0.1 N HCl to pH = 3.5-4, an Ethyl Ether extraction step was performed showing a recovery of 66%. Repeated analysis of a rat urine pool, showed 7.7 intraassay and 9.8 interassay variation coefficients. Values obtained amounted to 38.3 +/- 4.6 ng/24 h in normal rats and 133 +/- 32 ng/24 h in 5% NaCl loaded rats (p less than 0.001). The values obtained in healthy men were 508 +/- 94 ng/24 h and in women 375 +/- 136 (p less than 0.05).     

Plasma 6-keto-PGF1 alpha, thromboxane B2 and PGE2 during diabetic ketoacidosis

In 10 patients admitted to hospital with diabetic ketoacidosis plasma prostanoids 6-keto-PGF alpha, thromboxane B2 and PGE2 were studied before treatment and following recovery. During ketoacidosis the median plasma 6-keto-PGF1 alpha and PGE2 were significantly increased compared to those of a normal reference group: 5.2 pg/ml and 3.9 pg/ml versus 1.7 pg/ml and 0.4 pg/ml (p less than 0.01 and p less than 0.05). In response to therapy both prostanoids decreased significantly towards a normal level, 6-keto-PGF1 alpha: 0.5 pg/ml p less than 0.01 and PGE2: 0.08 p less than 0.05 respectively. The changes in plasma 6-keto-PGF1 alpha were negatively correlated to changes in pH, rho: -0.7788 p = 0.0135, whereas the changes in PGE2 were positively correlated to serum creatinine at admittance, rho: 0.6976, p = 0.0368 and to the amount of intravenous fluid and insulin used during treatment, rho: 0.7500 p = 0.0126 and rho: 0.8424, p = 0.0023 respectively. Plasma thromboxane B2 concentrations were not elevated and did not change after treatment of the ketoacidosis.     

Measurement of 6-keto-PGF1 alpha and thromboxane B2 levels in critically ill surgical patients

Systemic arterial and mixed venous plasma concentrations of 6-keto-PGF1 alpha and TxB2 were measured by radioimmunoassay in 63 critically ill patients with major trauma (n = 20) or sepsis (n = 43). Patients undergoing elective catheterization procedures served as controls (n = 10). Arterial and mixed venous 6-keto-PGF1 alpha and TxB2 levels were significantly elevated in patients with recent major trauma or active sepsis. The 6-keto-PGF1 alpha levels were found to be significantly elevated in the non-survivors and in patients with hepatic failure. The presence of severe pulmonary failure was not associated with increased levels of either 6-keto-PGF1 alpha or TxB2. Comparison of arterial and mixed plasma samples did not demonstrate increased pulmonary release of either compound. Increased eicosanoid production may account, in part, for the local vascular and humoral responses to tissue injury or infection.     

Analysis of the eicosapentaenoic acid (EPA) metabolite delta 17-6-keto-PGF1 alpha in human plasma by GC/SIM using [18O] delta 17-6-keto-PGF1 alpha.

A method of the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI3, is described. An authentic delta 17-6-keto-PGF1 alpha (120 mg) was prepared from eicosapentaenoic acid (EPA) incubated with homogenate of bovine aortic intima. [18O]delta 17-6-Keto-PGF1 alpha was synthesized by repeating base-catalyzed hydrolysis of methyl ester derivatives in [18O]water, to obtain an internal standard in gas chromatography/selected ion monitoring (GC/SIM) of delta 17-6-keto-PGF1 alpha. Good linear response over the range of 10 pg-10ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. We were able to detect delta 17-6-keto-PGF1 alpha in the range from 6 to 26 pg/ml of the human plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma.     

Effect of cicletanine on reperfusion-induced arrhythmias and its relation to 6-keto-PGF1 alpha and thromboxane B2 release

Isolated hearts, excised from spontaneously hypertensive male rats treated orally with cicletanine, a new furopyridine anti-hypertensive drug, were subjected to 30 min of global ischemia followed by 10 min of reperfusion. The effect of cicletanine on reperfusion-induced arrhythmias in relation to 6-keto-PGF1 alpha and thromboxane (TXB2) release was studied. After 30 min of global ischemia, the incidence (total) of ventricular fibrillation (VF) and ventricular tachycardia (VT) was reduced by 2-week pretreatment of the rats with 30 and 100 mg/kg of cicletanine (VF, 33% at 30 mg/kg and 25% at 100 mg/kg vs. 91% in untreated rats; VT, 42% at 30 mg/kg and 42% at 100 mg/kg vs. 100% in untreated rats), while lower doses of cicletanine (3 and 10 mg/kg) failed to reduce the incidence of reperfusion-induced rhythm disturbances. Reperfusion of the ischemic myocardium resulted in a fivefold increase of 6-keto-PGF1 alpha and TXB2 release in the perfusion effluent of fibrillated hearts but not in the perfusion effluent of nonfibrillated hearts. Cicletanine failed to influence the reperfusion-stimulated release of 6-keto-PGF1 alpha and TXB2. These results indicate that the anti-arrhythmic effect of cicletanine in the reperfused myocardium is not related to PGI2 and thromboxane A2 release.     

The concentrations of 6-keto-PGF1 alpha and TXB2 in plasma samples from patients with benign and malignant tumours of the breast

Peripheral plasma concentrations of 6-keto-PGF1 alpha and TXB2 were measured in patients with benign and malignant tumours of the breast, in patients with non-gynecological diseases, and in healthy female controls. The values were significantly higher in female patients with malignant tumours of the breast than in healthy controls (146 +/- 28 vs 13 +/- 2.5 pg/ml for 6-keto-PGF1 alpha p less than 0.01 and 78 +/- 17 vs 11 +/- 2 pg/ml for TXB2, p less than 0.01). Benign tumours of the breast were also associated with significantly raised plasma levels of 6-keto-PGF1 alpha and TXB2 compared to normal controls (52 +/- 5 vs 13 +/- 2.5 pg/ml for 6-keto-PGF1 alpha, p less than 0.01 and 26 +/- 5 vs 11 +/- 2 pg/ml for TXB2, p less than 0.05). The high levels of 6-keto-PGF1 alpha 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 concentrations of 6-keto-PGF1 alpha and TXB2 over a follow-up period of 9 days after operation. The lack of alterations in the ratio of TXB2:6-keto-PGF1 alpha 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.     

Age-, cycle- and topographic dependency of human myometrial prostaglandin (6-keto-PGF1 alpha, PGF2 alpha)-synthesis in vitro

Specimens of human myometrium (isthmus and fundus) freshly obtained at hysterectomy were immediately transferred in ice cold Tyrode solution and placed in superfusion chambers. Spontaneous contractions were recorded, the effluent of the myometrium was analyzed for PGF2 alpha and 6-keto-PGF1 alpha by use of specific radioimmunoassay systems. Dating of the menstrual cycle was achieved by histological evaluation of the endometrium. The PG release rates expressed as ng/min/g wet weight were correlated to the patients age and to the phase of the menstrual cycle. The production rates of 6-keto-PGF1 alpha were negatively correlated to the age of the patients and declined in fundus specimens from 2.89 +/- 0.35 ng/min/g wet weight in 39-42 years old patients to 0.52 +/- 0.17 ng/min/g wet weight in 48-52 years old women during the secretory phase (p less than 0.001). Similar significant correlations were found in specimens obtained from the isthmus uteri. During the proliferative phase fundus specimens produced on average 1.61 +/- 0.67 ng/min/g wet weight in 39-42 years old patients and 0.49 +/- 0.12 ng/min/g wet weight 6-keto-PGF1 alpha in 48-52 years old women respectively (p les than 0.001). The PGF2 alpha synthesis in myometrial specimens of fundus or isthmus origin was significantly lower than 6-keto-PGF1 alpha and did not correlate to the age of the patients during the proliferative phase. However, PGF2 alpha release rates during the secretory phase were significantly (p less than 0.001) higher in younger women. These results suggest an age-, cycle- and topographic dependency of PGI2 synthesis in human myometrial tissue.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1 alpha on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF1 alpha were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 microgram PGI2, 10 micrograms PGI2, and 10 micrograms PGI2 plus 5 ng LH averaged 99 +/- 42, 353 +/- 70, 152 +/- 35, 252 +/- 45, and 287 +/- 66 ng/ml (n = 4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.     

Effects of 1 gram oral or intravenous aspirin on urinary excretion of thromboxane B2 and 6-keto-PGF1 alpha in healthy subjects

Aspirin inhibits cyclo-oxygenase, thus preventing prostanoids formation. After oral administration aspirin is hydrolysed to inactive salicylate partly within the gastrointestinal tract, partly during first pass in the liver, partly in the circulation by plasma esterases. Intravenous aspirin, in contrast, mainly undergoes plasma esterase-catalysed deacetylation. Six healthy male subjects were given 1 g aspirin orally and intravenously two weeks apart according to a cross-over randomized design. Whereas serum TxB2 generation reflecting platelet cyclo-oxygenase activity was suppressed by aspirin by both routes, urinary excretion of TxB2 and 6-keto-PGF1 alpha was not affected by oral aspirin, but was partially though significantly reduced by the i.v. drug. Drug disposition seems therefore to be essential in determining the "biochemical selectivity" of aspirin as related to platelet and renal prostanoids generation.     

心交感神经对缺血心肌区血液中TXB2和6—酮—PGF1α浓度变化的影响

本实验在54只麻醉开胸犬,分别观察了心交感神经和α、β受体阻断剂对心肌缺血早期血小板功能变化的影响。结果表明,阻断冠脉后,心肌缺血区血液中TXB_2和6-酮-PGF_(1α)含量明显升高,血小板计数减少,随缺血时间延长,变化程度也增大。缺血心肌局部外敷2％利多卡因湿沙条或切除双侧星状神经节,分别阻断心交感神经的传入和传出效应,发现阻断冠脉后各参数变化程度明显减轻,与单纯阻断冠脉后各参数变化相比,有显著差异,P<0.01。切除星状神经节并由静脉输注去甲肾上腺素后再阻断冠脉,可重新恢复单纯阻断冠脉后的各参数变化,但输注生理盐水无影响。α和β受体阻断剂对上述参数的影响途径不同。α_2受体阻断剂育亨宾和非选择性α受体阻断剂酚妥拉明,可明显减轻TXB_2和6-酮-PGF_(1α)升高及血小板计数降低的程度,与单纯阻断冠脉后的各参数变化程度相比,有显著差异,P<0.01。但α_1受体阻滞剂哌唑嗪无此作用。和α_2受体阻断剂一样,β受体阻断剂心得安对缺血后上述参数的变化也具有明显改善效应。这些结果提示,心交感神经在血小板功能变化中具有重要作用;育亨宾和酚妥拉明是通过阻断血小板膜α_2受体发挥作用的;在输注心得安而未阻断血小板膜α_2受体时所看到对缺血后血小板功能参数的改善效应,提示β受体阻断剂可能     

Differential effects of various vasoactive drugs on basal and stimulated levels of TXB2 and 6-keto-PGF1 alpha in rat brain

Thromboxane B2 and 6-keto-PGF1 alpha (6KPGF1 alpha), the major stable metabolites of thromboxane and prostacyclin, are present in the CNS, where they appear to be mainly produced within and/or acting upon the vascular district. Their concentrations are of few pg/mg protein in rat brain cortex of animals sacrificed by microwave (MW) radiation, procedure which inactivates tissue enzymes and allows the determination of endogenous "basal" levels of eicosanoids. Levels of 6KPGF1 alpha and especially those of TxB2 increase several fold over the basal values in brain cortex of animals sacrificed by decapitation followed by a few minute interval before analysis (post-decapitation ischemia, PDI). Pretreatment of animals with the vasoactive drug papaverine, resulted in elevation of brain basal levels of 6KPGF1 alpha and with the carbochromene derivative AD6 in reduction of basal levels of TxB2, whereas the calcium antagonist nifedipine and dipyridamole did not modify basal levels of the two eicosanoids. Treatments with papaverine and AD6 reduced the accumulation of TxB2 and enhanced that of 6KPGF1 alpha occurring after PDI, to different extents, both resulting, however, in reduction of the TxB2/6KPGF1 alpha ratio. Nifedipine instead, decreased the release of both eicosanoids and resulted in elevation of the TxB2/6KPGF1 alpha ratio, whereas dipyridamole had no effect. In conclusion, the evaluation of the overall effects of drug treatments on the TxB2/6KPGF1 alpha ratio in cerebral tissue, provided useful informations on the pharmacological modulation of vascular eicosanoids in this district.     

Metabolism of 2-mercaptobenzothiazole by Rhodococcus rhodochrous

2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.     

Metabolism of 2-Mercaptobenzothiazole by Rhodococcus rhodochrous

2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant. We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous. Three metabolites, among four detected, were identified.     

Studies of the mechanisms involved in the fate of prostacyclin (PGI2) and 6-keto-PGF1alpha in the pulmonary circulation.

We have investigated the metabolism of [3]H-prostaglandin (PG)I2 and its non-enzymatic breakdown product [3]H-6-keto-PGF1alpha by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of beta-NAD, [3]H-PGI2 was extensively degraded into at least one metabolite, while [3]H-6-keto-PGF1alpha was only minimally metabolized. However, on passage through isolated perfused rat lungs, neither [3]H-PGI2 nor [3]H-6-keto-PGF1alpha were removed from the circulation into the lung or degraded. This demonstration that PGI2 is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference ot the structure-activity requirements necessary for pulmonary transport and subsequent metabolism.     

Allosteric regulation of pyruvate kinase from Mycobacterium tuberculosis by metabolites

Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that poses multiple dangers. Mtb reprograms its metabolism in response to the host niche, and this adaptation contributes to its pathogenicity. Knowledge of the metabolic regulation mechanisms in Mtb is still limited. Pyruvate kinase, involved in the late stage of glycolysis, helps link various metabolic routes together. Here, we demonstrate that Mtb pyruvate kinase (Mtb PYK) predominantly catalyzes the reaction leading to the production of pyruvate, but its activity is influenced by multiple metabolites from closely interlinked pathways that act as allosteric regulators (activators and inhibitors). We identified allosteric activators and inhibitors of Mtb PYK originating from glycolysis, citrate cycle, nucleotide/nucleoside inter-conversion related pathways that had not been described so far. Enzyme was found to be activated by fructose-1,6-bisphosphate, ribose-5-phosphate, adenine, adenosine, hypoxanthine, inosine, L-2-phosphoglycerate, l-aspartate, glycerol-2-phosphate, glycerol-3-phosphate. On the other hand thiamine pyrophosphate, glyceraldehyde-3-phosphate and L-malate were identified as inhibitors of Mtb PYK. The detailed kinetic analysis indicated a morpheein model of Mtb PYK allosteric control which is strictly dependent on Mg²⁺ and substantially increased by the co-presence of Mg²⁺ and K⁺.     

Microbial degradation of 2 alpha, 3 alpha-dihydroxy-5 alpha-cholestan-6-one by Mycobacterium vaccae.

2 alpha,3 alpha-Dihydroxy-5 alpha-cholestan-6-one (3), which had the substitution pattern of brassinosteroids in the A/B-ring moiety, was transformed by Mycobacterium vaccae to give 2 alpha,3 alpha,6 alpha-trihydroxy-5 alpha-androstan-17-one (4) and 2 alpha-hydroxyandrost-4-ene-3,17-dione (5). The structures of these compounds were determined by spectroscopic methods, especially 1H nuclear magnetic resonance studies.