Destruction of liver haem by norethindrone. Conversion into green pigments

1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with ¹⁴C by using 5-amino[4-¹⁴C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major ¹⁴C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with ⁵⁹Fe by using ⁵⁹FeCl₃, norethindrone administration resulted in the detection of ⁵⁹Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H₂SO₄. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.     

Interaction of progestins with steroid receptors in human uterus

Norethindrone (17β-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) and norethindrone acetate (17β-acetoxy-19-nor-17α-pregn-4-en-20-yn-3-one) interfered to a varying degree, by competitive inhibition, with the binding of progesterone and oestradiol to respective cytoplasmic receptors in the human uterus. Progesterone binding to 4S macromolecule was saturable and co-specific for progestins. Competitors like norgestrel (17β-hydroxy-18-methyl-19-nor-17α-pregn-4-en-20-yn-3-one), 19-norprogesterone, medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione) and compound R₅₀₂₀ (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for the progestin receptor. The dissociation constant (K_d) for the progesterone–receptor interaction was 0.6–1.6nm and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptor with inhibition constants (K_i) of 6.8 and 72nm respectively. Gradient displacement and competitive-receptor assays indicated that norethindrone acetate-binding affinity for progestin receptor was approximately one-tenth that of norethindrone and progesterone. The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8–12%, involving interaction at the oestradiol-binding site with a calculated K_i value of 0.5–0.8μm. The competitive interaction of progestins with steroid receptors may be of putative importance in explaining the progestin action at the target site.     

Variations in the concentrations of androstenedione in peripheral plasma of women the day and during the menstrual cycle

The target tissues (e.g., hypothalamus, pituitary, uterus and vagina) of mature female ovariectomized rats show selective uptake of radioactivity in one hour after the injection of 6,7, ³H-estradiol-17β in a dose of 0.1 μg per 100 g body weight. Injection of 100 μg norethindrone or norgestrel per 100 g body weight 15 min before or 15 min after the administration of tritiated estradiol reduced the radioactivity in most target tissues, and also in the non-target tissues to a lesser extent. The uptake of radioactivity in the pituitary and uterus is reduced more by norethindrone than by norgestrel treatment when these Steroids were injected 15 min after estradiol-17β injection. It appears that there exists a competitive inhibition of estradiol-17β by these contraceptive Steroids in the rat. It is speculated that such competition with estradiol-17β may be an inherent property of the 17-substituted 19-nortestosterone group of Steroids.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

Inhibition of the Extraneuronal Uptake of Catecholamine in the Isolated Rat Heart by Cholesterol

EARLIER work has demonstrated that the removal of catecholamines from plasma involves several different mechanisms: uptake into sympathetic nerve terminals¹; extraneuronal uptake (“Uptake₂”) into cardiac and smooth muscle and other tissues², a process which is subsequently followed by metabolism of the amine^3,4 and active transport across kidney tubules followed by excretion⁵. Various steroids, including corticosterone, 17-β-oestradiol and deoxycorticosterone, can selectively inhibit the extraneuronal uptake of noradrenaline (Uptake₂) in the isolated perfused rat heart^6,7. The inhibition of Uptake₂ occurred at concentrations of steroid which exceeded those normally found in the plasma and is thus unlikely to be of physiological significance under normal conditions. We have now found, however, that cholesterol which is present in plasma at much higher concentrations than any other steroid^8,9 also acts as an inhibitor of the Uptake₂ mechanism.     

Method for quantitative analysis of PGA2 from plasma using deuterated carrier and gas-liquid chromatography-mass spectrometry

The preparation of [3,3,4,4,-²H₄, 17, 18-³H₂]prostaglandin A₂ (PGA₂) and [17, 18-³H₂]PGA₂ is described. These compounds have been prepared for use in quantitative determination of corresponding nondeuterated prostaglandin by gas-liquid chromatography-mass spectrometry. The method is based on addition of a known amount of carrier to the sample. After purification and derivatization, the ratio between the protium and deuterium form is measured in the mass spectrometer. Ions originating from deuterated and nondeuterated moleeules are focused intermittently on the electron multiplier using an accelerating voltage alternator. With this technique, 60 pg of PGA₂ can be determined with a precision of ±8.4%. The recoveries from plasma of added PGA₂ were about 97%. Basal levels of PGA₂ in human plasma were lower than 10 pg/ml.     

Metabolism of estrone sulfate in the pregnant sheep

The metabolism of ³H-estrone sulfate (³H-E₁S) in 4 pregnant sheep, two injected i.v. and two i.m., has been studied. Intravenously injected ³H-E₁S had a plasma half-life of approximately 8 min, and metabolic clearance rate of approximately 800 ml/min. Using this clearance rate and the previously published mean plasma concentration of E₁S, the estimated production rate of E₁S is between 8.8 nmol (3.3 μg) and 78.2 nmol (29.1 μg) per min from 2-day to 0-day before parturition.Intramuscularly injected ³H-E₁S disappeared from plasma linearly and was completely cleared well within 3 hours. In all cases, whether i.v. or i.m. injected, the main metabolite isolated was ³H-estradiol-17β-3-sulfate, with only a trace amount as ³H-estradiol-17β-3-sulfate.     

Spectroscopic measurements of the parameters of the helium plasma jets generated in the plasma focus discharge at the PF-3 facility

The spectroscopic technique used to measure the parameters of the plasma jets generated in the plasma focus discharge and those of the plasma of the immobile gas through which these jets propagate is described. The time evolution of the intensities and shapes of spectral lines in experiments carried out with helium at the PF-3 facility was studied by means of electron-optical streak cameras. The plasma electron temperature, T ≈ 4–5 eV, was determined from the intensity ratio of two spectral lines, one of which (λ₁ = 5876 Å) belongs to neutral helium, while the other (λ₂ = 4686 Å), to hydrogen-like helium ions. The plasma density at different time instants was determined from the Stark broadening of these lines in the electric fields of different nature. The plasma density is found to vary from 4 × 10¹⁴ to 2 × 10¹⁷ cm^?3.     

Failure of contraceptive steroids to modify human chorionic gonadotrophin secretion by hydatidiform mole tissue and choriocarcinoma cells in culture

The effect of steroids contained in oral contraceptives, namely ethinylestradiol:17α-ethinyl-1,3,5(10)-estratriene-3, 17-diol (E) and norethindrone acetate: 17β-acetoxy-17-ethinyl-4-estren-3-one (N), on cell replication and human chorionic gonadotropin (hCG) secretion by choriocarcinoma cells in monolayer culture and by hydatidiform mole tissue maintained in organ culture were studied. The steroids were added to the culture medium individually or in combination to achieve a range of concentrations (10^?10 to 10^?4), within and beyond the presumed concentration of these substances in the blood of women taking oral contraceptives. The effect of luteinizing hormone releasing hormone (LHRH) on hCG secretion by choriocarcinoma cells in monolayer culture also was investigated. The rate of hCG production by either choriocarcinoma cells in monolayer culture or by hydatidiform mole tissue maintained in organ culture was not affected by the hormones used in this study; indeed hCG secretion remained reasonably unchanged even with high concentrations of steroids (up to 10^?14 M) or LHRH (up to 10^?4 mg × ml^?1). Cell replication, as measured by increase in amount of cellular protein and DNA, was not stimulated by either of these compounds.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

We previously demonstrated that the ATP/PKA?dependent activation of the human intermediate conductance, Ca²⁺?activated K⁺ channel, hIK1, is dependent upon a C?terminal motif. The NH₂?terminus of hIK1 contains a multi?basic ¹³RRRKR¹⁷ motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (¹⁵RKR¹⁷/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole?cell patch?clamp. However, cell?surface -immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the ¹⁵RKR¹⁷/AAA mutation we performed inside?out patch clamp recordings where we observed no difference in Ca²⁺ affinity between the wild?type and mutated channels. However, in contrast to wild?type hIK1, channels expressing the ¹⁵RKR¹⁷/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the ¹⁵RKR¹⁷/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the ¹⁵RKR¹⁷/AAA mutation resulted in a four?fold lower channel open probability (P_o), in the presence of saturating Ca²⁺ and ATP, compared to wild?type hIK1. In conclusion, these results represent the first demonstration for a role of the NH₂?terminus in the second messenger?dependent regulation of hIK1 and, in -combination with our previous findings, suggest that this regulation is dependent upon a close NH₂/C?terminal association.     

Hepatotoxicity studies with primary cultures of rat liver cells

Summary A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes [arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10⁻⁶ to 1×10⁻³ m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10⁻⁴ m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10⁻⁴ m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures. Part of this investigation was presented orally at the 17th Annual Meeting of the Society of Toxicology, San Francisco, March 13, 1978.     

Single-dose Pharmacokinetics of Nestorone, a potential female-contraceptive

A synthetic progestin Nestorone^® is being developed for female-contraception. This study was conducted to determine the distribution, metabolism, and excretion of tritium-labeled Nestorone (³H Nestorone) in adult female rats. Rats were injected subcutaneously (S.C.) with a single dose of 400 μCi ³H Nestorone/kg BW. Its distribution and concentrations in blood, plasma and other tissues were determined at defined times. The excreta were examined for elimination of ³H Nestorone. Radioactivity in all samples was analyzed by liquid scintillation counter. Metabolite profiling was performed by HPLC and LC/MS analysis of the plasma, urine, and feces samples. Following subcutaneous injection of ³H Nestorone, the mean peak concentrations of radioactivity (C_max) in the blood and plasma were 58.1 and 95.5 ng equiv. ³H Nestorone/g, respectively, at 2-h postdose (T_max). Thereafter, the concentration of drug steadily declined through 96-h postdose with a terminal elimination half-life (t_1/2) of 15.6 h. ³H Nestorone-derived radioactivity was widely distributed in most tissues by 0.5 h and attained a mean maximal concentration by 2-h postdose. Approximately, 81.4% and 7.62% of the administered dose was excreted via feces and urine, respectively. In vivo metabolism of ³H Nestorone resulted into a total of 19 metabolites. Among them, two metabolites viz., 17α-deacetyl-Nestorone (M9) and 4,5-dihydro-17α-deacetyl-Nestorone (M19) were identified by HPLC and LC/MS analysis. Metabolite profiling of plasma samples showed that most of the circulating radioactivity was associated with unchanged parent drug, and M19. The M19 was a major metabolite in the profiled urine and feces samples. Presence of large proportion of drug/drug-related material in feces suggested that the biliary excretion is a main elimination route of ³H Nestorone. The distribution, metabolism, and excretion profiles of ³H Nestorone obtained in this study provide a fairly good insight about its fate in women.     

Influence of short-term oestradiol treatment on plasma thyroid hormone kinetics in rainbow trout, Salmo gairdneri

The effects of 17β-oestradiol (E₂) on plasma kinetics of thyroid hormones (T₄, l-thyroxine; T₃, 3,5,3′-triiodo-l-thyronine) were studied in immature rainbow trout. E₂-3-benzoate (0.5 mg/100 g) was injected intraperitoneally on days 0 and 3, and on the morning of day 4 each trout received an intracardiac injection of either [¹²⁵I]T₄ and Na ¹³¹I or [^I25I]T₃. Groups of trout were bled and killed from 5 min to 4 days post-injection of tracer. E₂ did not alter the plasma T₄ level but depressed the T₄ plasma clearance rate, plasma-to-total tissue flux of T₄ and thyroidal T₄ secretion rate. Monodeiodination of T₄ to T₃ was also depressed, as judged from plasma [^I25I]T₃ and ^I25I ^? levels in [¹²⁵I]T₄-injected trout. E₂ had no major effect on T₃ plasma clearance rate but depressed the plasma T₃ level, plasma-to-total tissue flux of T₃ and the T₃ plasma appearance rate. E₂ had no influence on biliary transport of [^I25I]T₄ or [¹²⁵I]T₃. The above results suggest that E₂, at the dose range employed, depresses extrathyroidal T₄ to T₃ conversion, which may in turn decrease plasma T₄ clearance and thyroidal T₄ secretion.     

Gonadal development and sex steroids in a female Arctic charr broodstock

Gonadal development and plasma levels of sex steroids were investigated in female Arctic charr at 3-week intervals over a 12-month period. Circulating levels of oestradiol-17β (E₂), testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were measured by radioimmunoassay, and gonadal status assessed through histological examination and measurement of gonadosomatic index (GSI) and frequency distribution of oocyte size-classes. Gonadal recrudescence during March-July was characterized by modest but insignificant increases in plasma levels of E₂ (2–4 ng ml^?1) and T (2–5 ng ml^?1) and recruitment of oocytes into yolk accumulation. Only a small and insignificant rise in GSI and no apparent increase in oocyte diameter occurred during this period, indicating that the rate of yolk formation and oocyte growth was low. Following transformation from stage V (peripheral yolk granule stage) to stage VI (yolk granule migration stage) in late July, the vitellogenic oocytes entered a phase of rapid growth which resulted in a marked rise in GSI until ovulation commenced in late September. Gonadal growth during this period was accompanied by increases in plasma levels of E₂ and T which peaked at 11 ± 1 (mid-August) and 71 ± 5ng ml^?1 (late September), respectively. The levels of both steroids dropped rapidly during final maturation and ovulation, followed by a surge in plasma levels of 17,20β-P which peaked at an average of 74 ± 17 ng ml^?1 in early October. All three steroids returned to basal levels within a month after ovulation, and all steroids, except E₂, remained low until March of the following year. A slight increase in E₂ detected in February and March during the second season may have been associated with recruitment into vitellogenesis of a new generation of oocytes. It is suggested that the abrupt increase in vitellogenesis in late July may reflect a condition-dependent decision to proceed with maturation, once the energy reserves have been repleted during spring-early summer.     

铕标记抗癌胚抗原单克隆抗体C17的研究和应用

用合成的N′-(对-异硫氰基苄基)-二乙三胺-N¹,N²,N³,N³-四乙酸铕钠为螫合剂.用Sephadex G-50凝胶柱层析分离标记C₁₇和过量试剂.分部收集的层析物的光密度图和荧光强度图完全一致.蛋白峰处两管标记C₁₇的比活性分别为5.2和8.9Eu³⁺离子每C₁₇分子,而且C₁₇的标记回收率为64%.标记C₁₇至少在6个月内是稳定的.用铕标记物得到的时间分辨免疫荧光分析的标准曲线具有良好线性和斜率,表明标记物免疫活性良好,而且已用于血清CEA分析.     

Increased testicular 5α-androstane-3α, 17β-diol formation induced by treatment with [D-Ser(TBU)6, des-Gly-NH210]LHRH ethylamide in the rat

While the intact male adult rats respond to LH with a predominant increase of testicular and plasma testosterone levels, the response to LH stimulation in animals treated with the LHRH agoriist, [D-Ser(TBU)⁶, des-Gly-NH₂10]LHRH ethylamide is characterized by a major production of 5α-androstane-3α, 17β-diol. The marked increase of 5α-androstane-3α, 17β-diol levels in the presence of a 90% decrease of testosterone concentration strongly suggests that 5α-reductase and 3α-hydroxysteroid oxidoreductase activities are increased during testicular desensitization induced by treatment with the LHRH agonist.     

Hardness does not affect the physiological responses of wild and domestic strains of diploid and triploid rainbow trout Oncorhynchus mykiss to short‐term exposure to pH 9.5

This study examined the effects of water hardness on the physiological responses associated with high pH exposure in multiple strains of diploid and triploid rainbow trout Oncorhynchus mykiss. To accomplish this, three wild strains and one domesticated strain of diploid and triploid O. mykiss were abruptly transferred from control soft water (City of Vancouver dechlorinated tap water; pH 6·7; [CaCO₃] < 17·9 mg l^?1) to control soft water (handling control), high pH soft water (pH 9·5; [CaCO₃] < 17·9 mg l^?1), or high pH hard water (pH 9·5; [CaCO₃] = 320 mg l^?1) followed by sampling at 24 h for physiological measurements. There was a significant effect of ploidy on loss of equilibrium (LOE) over the 24 h exposure, with only triploid O. mykiss losing equilibrium at high pH in both soft and hard water. Furthermore, exposure to pH 9·5 resulted in significant decreases in plasma sodium and chloride, and increases in plasma and brain ammonia with no differences between soft and hard water. There was no significant effect of strain on LOE, but there were significant differences between strains in brain ammonia and plasma cortisol. Overall, there were no clear protective effects of hardness on high pH exposure in these strains of O. mykiss.     

Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.