Measurement of prostaglandin F2α metabolites by radioimmunoassay

Antibodies against 15 keto PGF_2α and 13,14 dihydro 15 keto PGF_2α were produced in goats and rabbits using the appropriate prostaglandin protein conjugate. Tritium labeled 15-keto, and 13,14 dihydro 15-keto PGF_2α were prepared from ³H-PGF_2α. These antibodies and ³H-labeled compounds were used to develop radioimmunoassays for the respective F_2α metabolites. The antibodies had relatively little cross-reactivity (≤0.1%) with the parent F_2α molecule. Infusion of PGF_2α in monkeys increased 15-keto-h₂ levels 10–20 fold higher than PGF_2α in peripheral plasma. The levels of this metabolite were not altered detectably during clotting, indicating relatively slow rates of PGF_2α metabolism in vitro. These assays should be useful to follow release rates of exogenous prostaglandins from various formulations and delivery systems, and in vivo tissue synthesis of PGF_2α, where low levels preclude measuring the parent compound.     

草鱼(♀)×赤眼鳟(♂)杂交F1倍性分析和性腺发育特点

为探讨草鱼(Ctenopharyngodon idella♀)×赤眼鳟(Squaliobarbus curriculus♂)杂交F₁的倍性和生育性能, 对杂交F₁的染色体核型和红细胞核DNA含量进行分析和测定, 并运用组织学方法对繁殖期和非繁殖期共124尾杂交F₁的性腺发育特点进行研究。结果显示: (1)杂交F₁染色体2n=48, 核型公式2n=26 m+20 sm+2 st, NF=94, 未发现端部着丝点染色体、随体和次缢痕等结构; (2)杂交F₁相对DNA含量为对照组鸡血的1.29倍, 其绝对DNA含量为(3.21±0.61) pg/N, DNA指数为0.92; (3)经解剖观察和切片鉴定, 杂交F₁卵细胞分为两种类型: 一类卵细胞第Ⅰ—Ⅴ时相均发育正常, 可完成排卵并产生后代, 为正常发育型; 另一类卵细胞发育到第Ⅴ时相后, 卵黄颗粒减少, 油滴增加, 呈无卵黄空洞状, 为异常发育型。杂交F₁精巢包括初级精母细胞、次级精母细胞和精子细胞, 无成熟精子; 部分精小囊内精细胞量极少, 多为结缔组织或脂肪组织; 生产实践中暂未发现可以排精的杂交F₁个体。研究表明, 草鱼与赤眼鳟杂交获得的杂交F₁为二倍体, 部分雌性可育, 雄性可能为败育群体。     

翘嘴红鲌(♀)×团头鲂(♂)杂交种F1的形态特征及遗传分析

为优化太湖鲂鲌配合饲料配方, 设计3个饲料蛋白水平(36%、39%和42%)和3个脂肪水平(6%、8%和10%)的3×3因子实验, 配制9种实验饲料(P36L6、P36L8、P36L10、P39L6、P39L8、P39L10、P42L6、P42L8和P42L10), 分别饲喂9组3重复平均体重为8.59 g的太湖鲂鲌冬片鱼种60d, 以探讨饲料中不同含量的蛋白和脂肪对太湖鲂鲌冬片鱼种生长、饲料利用、体成分和肠道消化酶等的影响。结果表明: 实验鱼摄食蛋白水平为39%和42%的6种饲料(P39L6、P39L8、P39L10、P42L6、P42L8和P42L10) 后, 其鱼体增重率和饲料系数在组间无显著差异(P>0.05), 但鱼体增重率均比摄饲料蛋白水平为36%的3种饲料(P36L6、P36L8和P36L10)的实验组要高(P<0.05), 而饲料系数则较低; 在同一饲料蛋白水平时, 饲料脂肪水平变化对蛋白质沉积率无显著影响; 摄食蛋白质水平为39%饲料的3个实验组的蛋白质沉积率均高于饲料蛋白为36%的3个实验组。饲料同一脂肪水平时, 饲料蛋白水平从39%提高到42%对蛋白质沉积率有降低的趋势, 但差异不显著; 脏体指数(VSI)与饲料脂肪水平(L)呈正相关(VSI=0.223L+4.611, R=0.746, P=0.000); 全鱼粗脂肪随饲料蛋白的升高而降低, 但随饲料脂肪水平的升高而增加; 摄食3种低脂肪水平(6%)的饲料(P36L6、P39L6和P42L6)的太湖鲂鲌肌肉粗脂肪含量均显著低于摄食其他6种脂肪水平较高(8%和10%)饲料组; 摄食3种蛋白水平为36%的饲料(P36L6、P36L8和P36L10)后, 太湖鲂鲌肠道淀粉酶活性显著高于蛋白水平39%和42%的饲料组; 太湖鲂鲌肠道蛋白酶和脂肪酶活性均不受饲料蛋白和脂肪水平及其交互作用的影响; 因此, 能使实验鱼获得良好生长和饲料利用的适宜饲料蛋白和脂肪水平分别为39%和6%。过低的饲料蛋白水平和过高的饲料脂肪水平易导致鱼体脂肪积累过多。

     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37°C and incubated for five hours with ³H-choline and ¹⁴C-palmitate in the presence of various concentrations of prostaglandins (PGs) E₂ and F_2α. In the control (no PG) the level of % palmitate incorporation was 13.5 × as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 μM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 × better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 μM, PGE₂ was much more effective than PGF_2α in stimulating the incorporation of both precursors.     

The induction of labour by the intravenous infusion of prostaglandin F2α

labour was induced by the intravenous infusion of prostaglandin F_2α in 106 patients at 36–44 weeks of pregnancy. The induction was successful in 80% of the women. The total dose needed ranged from 0.1 to 14.2 mg of PGF_2α. The uterine activity and fetal heart rate were recorded by cardiotocography. The contraction pattern and induction-delivery time were the same as reported for induction with oxytocin. In one case uterine hyperactivity occurred after rupture of the membranes. No serious adverse effects were seen, but in a few cases local irritation was noted at the site of infusion. The condition of the infants was generally good.It might be concluded that PGF_2α seems valuable for the induction of labour, but due to the risk for overstimulation careful supervision is needed.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Induction of abortion by intra- and extra-amniotic prostaglandin F2α administration

Legal abortion was induced by intrauterine administration of prostaglandin F_2α in 115 patients during the 11th – 20th week of pregnancy. An intra-amniotic method was used in 61 of the cases, an extra-amniotic one in 54 cases. The average total dose administered was 35.1 mg (range 5 – 65 mg) in the intra-amniotic group, and 6358 μg (range 1500 – 14000 μg) in the extra-amniotic group. Abortion rate was 92 % in the intra-amniotic material and 72 % in the extra-amniotic material, and side-effects, mainly gastrointestinal irritation, were noted in 74 % of the intra-amniotic cases and 54 % of the extra-amniotic ones. If total doses of 4750 μg or more were administered in the extra-amniotic cases, abortion rate went up to 80 %, but the frequency of side-effects simultaneously increased to 64 %. No serious complications occurred. Intrauterine prostaglandin induction is well suited therapeutic abortions in the second trimester, and the intra-amniotic technique is more practicable than the extra-amniotic one. The latter is applicable in cases where the puncture of the amniotic cavity is difficult to achieve, e.g. in cases of fetus mortuus and hydatiform mole.     

Corpus luteum regression induced by ultra-low pulses of prostaglandin F2α

In view of the pulsatile nature of PGF_2α secretion from the ovine uterus at the time of luteolysis, experiments were designed to examine the effect of pulsed infusions of PGF_2α on luteal function and to re-examine the minimal effective levels of PGF_2α required to induce luteolysis. To mimic physiological conditions, hour-long infusions of PGF_2α in increasing concentrations were given either 4 times in 19 h or 5 times in 25 h into the arterial supply of the autotransplanted ovary in conscious sheep on day 12 of an induced cycle. Blood flow and progesterone secretion rate from the ovary were used to monitor directly the luteolytic effect of administered PGF_2α. The concentration of LH in peripheral plasma was measured throughout each infusion experiment and the presence of a preovulatory peak of LH was used as an indicator of the permanence of luteal regression. Four pulses of PGF_2α in 19 h caused complete corpus luteum regression in only 1 of 4 animals whereas the addition of a fifth pulse (5 pulses in 25 h) caused permanent regression in 4 out of 4 animals. Infusion of 5 hour-long pulses of saline or PGF_2α at a rate of <0.04 μg/h did not induce permanent suppression of progesterone secretion. The average total effective dose of PGF_2α required to induced luteal regression when given as 5 pulses was 1/40th of the amount currently regarded as the minimal effective one when given by constant infusion into the ovarian artery. In another series of experiments the luteolytic effect of a single hour-long pulse of 0.1 μg/h PGF_2α given daily for either 3 or 4 days was investigated. A significant fall (ANOVA, F_0.01) in progesterone secretion rate, which reached a nadir at $\text{5.3 ± 2.2}\text{h (}\text{x}\text{±}\text{S.D.}\text{, n=15)}$, was followed by a recovery of progesterone secretion rate. Permanent luteal regression did not occur with this protracted regimen, suggesting that a relatively short pulse frequency of PGF_2α over a minimal period of 24 h is a necessary condition for physiological regression of the corpus luteum in sheep.     

草鱼(♀)×赤眼鳟(♂)F1及其亲本CAST基因cDNA全长克隆与结构差异

钙蛋白酶抑制蛋白(Calpastatin, CAST)在肌肉生长和肉质特征形成中发挥重要作用。为探究草鱼(Ctenopharyngodon idellus)(♀)×赤眼鳟(Squaliobarbus curriculus)(♂)正交F1的肉质相关分子基础,通过RACE(Rapid-amplification of cDNA ends)技术分别克隆了草鱼(♀)×赤眼鳟(♂) F1及其亲本的CAST基因cDNA全长,并利用生物信息学方法分析比较了三种鱼的CAST结构差异。结果表明:草鱼(♀)、赤眼鳟(♂)及草鱼(♀)×赤眼鳟(♂) F1的CAST基因cDNA全长分别为3036、3165和3086 bp,编码901、893和904个氨基酸;预测蛋白质分子量分别为93.72、92.77和94.02 kD;推测的理论等电点分别为5.92、6.01和6.02。草鱼(♀)×赤眼鳟(♂) F1 CAST与草鱼(♀)和赤眼鳟(♂)核苷酸序列相似性分别为94.52%和90%。三种CAST蛋白均包括4个含有典型七肽的钙蛋白酶抑制结构域。草鱼(♀)、赤眼鳟(♂)和F1 CAST氨基酸残基中分别存在73、82和...     

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Analysis of prostaglandins by electron-capture gas chromatography. I. Thermal decomposition of heptafluorobutyrate methyl esters of F1α and F2α

Triheptafluorobutyrate methyl ester (THFB-Met) derivatives are easily prepared from prostaglandins F_1α and F_2α by successive methylation and heptafluorobutyrylation. The derivatives are reasonably stable during storage, are volatile, and can be detected in the picogram range by electron-capture gas chromatography. Both derivatives exhibit peak broadening or multiple peak formation during gas chromatography at 190°–210°C. Decomposition is independent of the nature of the stationary phase and can be increased by prior heating. Studies with other derivatives suggest that thermal decomposition of the THFB-Met derivatives occuring during gas chromatography involves loss of a heptafluorobutyrate group from the allylic position 15 of the prostaglandins.     

Radioimmunoassay for urinary metabolites of prostaglandin F2α

Antibodies against the main urinary metabolite of PGF_2α in the human, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the ω position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the ω carbon, and thus the assay could be used also for other metabolites of PGF_2α, e.g. the main urinary metabolite in the guinea pig, 5α,7α-dihydroxy-11-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either $\text{in vivo}$ by injection of |17,18-³H|-PGF_2α into humans after several days' treatment with indomethacin, or $\text{in vitro}$ by incubation of |17,18-³H|-15-keto-13,14-dihydro-PGF_2α with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively.The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF_2α; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment.The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.     

Determination of prostaglandin F2α and E2 contents in human cerebrospinal fluid by the radioisotope dilution method

Prostaglandin F_2α and E₂ contents in human cerebrospinal fluid were determined by the radioisotope dilution method. The mean values of PGF_2α and PGE₂ of men were 9.8±0.87 ng/ml and 6.5±1.39 ng/ml, respectively. Those of women were 8.3±1.4 ng/ml and 6.9±1.72 ng/ml, respectively. The correlation between age and PG was significantly with PGE₂ of men and with PGF_2α of women.     

Rapid block of gonadotropin uptake by corpora lutea in vivo induced by prostaglandin F2α

Intravenous administration of ¹²⁵I-hCG to 7–8 day pseudopregnant rats resulted in maximum uptake of radioactivity to corpora lutea 2 hours after treatment. At this time tissue/plasma radioactivity ratios on an equal weight basis were: corpora lutea, 70.2 ± 12.8; ovarian interstitium, 4.6 ± 0.2; kidney, 2.2 ± 0.1. No appreciable uptake was seen by adrenals or liver. Radioactivity in corpora lutea was associated primarily with membranes which sedimented at 2000g and when released by heat it was more than 63% bound to luteal LH receptor preparation in vitro. Radioactivity in renal tissue was associated primarily with the 100,000g supernatant fraction and was bound less than 1% to luteal LH receptors in vitro.PGF₂α significantly reduced uptake (p<.001) of ¹²⁵I-hCG by corpora lutea within 30 minutes (?63%) as well as at 1 (?64%), 2 (?75%), 4 (?68%) and 24 hours (?85%). No clear effect of PGF₂α on uptake of ¹²⁵I-hCG by ovarian interstitial tissue was seen. Plasma progesterone was significantly decreased (p<.001) within 30 minutes (?47%; p<.01) after PGF₂α treatment and also at 1 (?65%), 2 (?82%), 4 (?68%) and 24 hours (?92%). Two hours after PGF₂α treatment the content of progesterone in corpora lutea was depressed (?46%; p<.001). It is suggested that the rapid inhibition of luteal progesterone production induced by PGF₂α in vivo occurs through a block in gonadotropin uptake by corpora lutea.     

Central cardiovascular activity of prostaglandin E2, prostagladin F2∝ and prostacyclin

Prostacyclin (PGI₂), prostaglandin E₂ (PGE₂) and prostaglandin F_2∝ (PGF_2∝) were tested here in unanesthetized male Sprague-Dawley rats for their effects on the cardiovascular system as mediated by the Central nervous system. Cannulae were chronically implanted into the third cerebral ventricle, femoral arteries and femoral veins of rats. Both PGE₂ and PGF_2∝ induced increased arterial blood pressure and tachycardia by an action on the central nervous system. The changes seen with PGE₂ were larger than those observed with PGF_2∝. Only transient depressor effects were seen with PGI₂ and these changes appeared to be due to the leakage of the substance into the peripheral vascular system.     

Quantitative determination of prostaglandins E1 and F1α by multiple ion analysis

Quantitative assays for prostaglandins (PG) E₁ and PGF_1α are described using [3,3,4,4,5,6-²H₆]labeled prostaglandins as carriers and methyl ester-O-methyloxime-acetate (PGE₁) and methyl ester-acetate (PGF_1α) derivatives for gas - liquid chromatography/mass spectrometric analysis. Thin-layer argentation chromatography was used to separate PGE₁ from PGE₂ and 13, 14-dihydro-PGE₂. These latter compounds, which do not separate from PGE₁ using conventional thin-layer chromatography or under the gas - liquid chromatographic conditions used, can significantly interfere with the quantitative analysis of PGE₁. The method described prevents this interference and is therefore suitable for the accurate analysis of PGE₁ in biological samples containing a high concentration of PGE₂ and/or 13, 14-dihydro-PGE₂.