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.     

Dihydrotestosterone induces a sexual dimorphism in estrogen uptake by specific anterior pituitary cell types in vivo

Summary Castrated male and female rats pretreated with dihydrotestosterone (DHT) were injected i.v. with ³H-estradiol (E₂). Nuclear uptake and retention of ³H-E₂ was measured in each of five cell types of the anterior pituitary gland using a combined quantitative autoradiographic and immunocytochemical procedure. In non-pretreated groups, each cell type bound a characteristic amount of ligand but no sex differences were apparent. DHT pretreatment, however, caused a significant decrease in ³H-E₂ retention by gonadotrophs in both males and females. The treatment also caused a decrease in binding by lactotrophs and somatotrophs, but only in the females. No other cell types were altered. Thus, androgen appears to modulate E₂ binding and retention by pituitary cells in both a cell-type and sexdependent manner. Our results also indicate that the inhibitory effects of androgens on E₂ binding by the pituitary gland is more complex than can be explained by simple competition for the estrogen receptor.     

Estriol metabolism in the baboon: analysis of urinary and biliary metabolites

The metabolism of i.v. estriol was investigated in two intact baboons and four with biliary fistulas. Urine and bile samples were collected periodically and the radioactivity extracted by Amberlite-XAD resin. Metabolites were separated and purified by a combination of DEAE-Sephadex chromatography, celite partition, specific enzyme hydrolysis of the conjugates and identification of the aglycones. The excretion and metabolism of estriol in the animals closely resembled those of the human. Intact animals excreted an average of 50% of the radioactivity in the urine during 12 hours and two animals with biliary drainage excreted an average of 40% in the urine and 49% in the bile. When the steroid was injected into the portal vein an average of 11.5% and 84% were excreted in the urine and bile, respectively.In the urine of intact animals, approximately 65.8% of the radioactivity was in the form of E₃-16G; 14.2% as E₃-3G; 13.4% as E₃-3S and 5.1% as E₃-3S-16G. Over 73% of biliary radioactivity from the peripheral injections was made up of E₃-3S-16G and 3.6% as E₃-16G and 8.3% as 3-sulfate. In the urine,however, 57% of the label was made up of E₃-16G. No radioactive E₃-3G was detected in the bile of any of the animals. Following simultaneous injection of ³H-E₃ peripherally and ¹⁴C-E₃ intraportally, the 3-glucosiduronate excreted in the urine was derived exclusively from the ³H-label. Based on the results obtained, the baboon has been shown to metabolize estriol in the same fashion as the human, with E³-3S-16G as the predominant biliary metabolite and E³-16G as the major urinary metabolite. As in the human, evidence was also found for an enterohepatic circulation of e³ in the baboon, 16-glucuronidation in the kidney, and extrahepatic (enteric?) formation of E³-3G. $\text{In vitro}$ incubation of the baboon liver yielded 94% of the total conjugate as E³-16G without any trace of E³-3G.     

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.     

Solubilization of histamine H-1, GABA and benzodiazepine receptors.

Dopamine 3-0-sulfate is present in considerable amounts in mammalian plasma and peripheral tissues. Incubation of dopamine 3-0-sulfate (0.1 μmole) with purified bovine dopamine-β-hydroxylase resulted in the formation of free norepinephrine (7.3 × 10^?3 μmole). The conversion to norepinephrine was inhibited by 0.6 mM of fusaric acid, an inhibitor of dopamine-β-hydroxylase. The reaction of dopamine 3-0-sulfate with dopamine-β-hydroxylase followed Michaelis-Menten kinetics. The calculated K_m was 17 mM, different from the K_m for free dopamine (0.1 mM). The incubation medium does not contain any sulfatase activity.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

Estriol in pregnancy. III. Development, comparison and use of specific antisera for rapid radioimmunoassay of unconjugated estriol in pregnancy plasma

Estriol-6-(0-carboxymethyl) oxime (E₃-CMO) and estriol-4-azobenzoic acid (E₃-4-ABA) were linked to bovine serum albumin (BSA). Twelve rabbits were immunized, six with each E₃-BSA conjugate. All six E₃-6-CMO-BSA rabbits, but only one E₃-4-ABA-BSA animal, responded with useful antibody titers. All antisera exhibited good Ring D specificity. E₃-6-CMO-BSA (type 1) antisera cross-reacted up to 220 percent with 6-oxoestriol while the E₃-4-ABA-BSA (type 2) antiserum cross-reacted only 3.8 percent with this steroid. Neither type of antiserum cross-reacted with neutral steroids nor with estriol-16-glucosiduronate and estriol-3-sulfate-16-glucosiduronate, but both cross-reacted with estriol-3-sulfate and estriol-3-glucosiduronate.Both types of antisera could be utilized for a rapid and specific radioimmunoassay (RIA) of unconjugated E₃ in third trimester pregnancy plasma without need for further purification of the plasma extract. Blanks were negligible, sensitivity was sufficient, recovery was virtually complete by using ³H-E₃ as an internal standard, and precision was satisfactory. The measurements of unconjugated plasma E₃ concentrations in ninety apparently normal women between 29 and 40 weeks or gestation obtained by this RIA averaged 7.6, 10.2 and 16.7 ng/ml at 29 to 32, 33 to 36 and 37 to 40 weeks of gestation, respectively.The results obtained in this study indicate that antisera against E₃-6-CMO-BSA, despite their appreciable cross-reaction with 6-oxoestriol, are as useful for a rapid RIA of plasma unconjugated E₃ as antisera against E₃-4-ABA-BSA because very little, if any, 6-oxoestriol is present in late pregnancy plasma. As anti-E₃ titers were much higher and much more readily obtained in response to immunization with E₃-6-CMO-BSA than with E₃-4-ABA-BSA, E₃-6-CMO-BSA appears to be the preferable antigen to develop antisera for a rapid, yet specific, E₃ RIA.     

In vitro liver clearance of tritiated estradiol-17β in the female rat after retrochiasmatic transection and ovariectomy

It was reported in a previous study that serum estradiol-17β (E₂) was elevated in rats after retrochiasmatic transection (FC). Serum E₂ was also higher in estradiol cypionate treated, ovariectomized (ovx) rats that had been subjected to FC than in those that had not. This suggested that increased secretion of E₂ was not the only factor responsible for elevated serum E₂ after FC. To ascertain the contribution of decreased metabolism of E₂ to this response, liver tissue slices were incubated with 3H-estradiol-17β, and the rates of ³h uptake and conversion to water-soluble conjugates were measured.The rate of uptake of ³H by the tissue was not indicative of the rate of conjugate formation. Livers of rats with high serum E₂ exhibited lower rates of ³h uptake than those of rats with low serum E₂. Even so, the formation of ³H conjugates was greater in liver of rats with high serum E₂. As hypothesized, livers of rats with FC formed conjugates at a significantly lower rate than those of similarly treated rats without FC. Thus, FC of an intact rat leads to an increase in serum E₂ by increasing the secretion of E₂, and apparently also by decreasing the rate of E₂ metabolism by the liver.     

Estramustine binding in rat,baboon and human prostate measured by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) was used to determine ³H-estramustine (estradiol-17β3N-bis-[2-chlorethyl] carbamate), ³H-17β-hydroxy-5α-androstan-3-one (³H-dihydrotestosterone or ³H-DHT), ³H-estradiol-17β (³H-E₂) and ³H-3β-hydroxy-5-pregnen-20-one (³H-pregnenolone) binding in 50μ1 of cytosol utilizing a column which separates proteins in the molecular weight range of 2,000 to 70,000 daltons. The rat prostate contains a protein in considerable concentration and with the highest affinity for estramustine (375,000dpm ³H-estramustine per mg. cytosol protein) among the substances tested. Operationally, we have named this protein “estramustine binding protein” (EBP), though it is very likely similar to other previously described prostatic proteins (e.g., α-protein, prostatein, prostatic binding protein). The sensitivity of the HPLC method disclosed EBP-like proteins, but in much lesser concentrations, in some of the other tissues tested. The concentration of these proteins in the human and baboon prostates was much lower (average for the baboon cranial lobe 4800dpm/mg cytosol protein, with a somewhat higher value for the caudal lobe) than that in the rat gland. The amount of the EBP-like protein was higher in prostatic cancer than in that of benign prostatic hypertrophy (BPH) (range 9350 – 25,900 vs. 2200 – 18,900 dpm/mg cytosol protein). In the human, the highest value was found in one normal prostate tested (106,000dpm/mg cytosol protein).     

Human serum albumin (HSA) decreases prostaglandin A1 (PGA1) metabolism.

PGA₁ was incubated with rabbit renal cortical homogenates containing HSA (0–4.5%). The ability of this tissue to readily metabolize PGA₁ progressively decreased with increasing HSA levels in the incubates The rate of disappearance of ³H-PGA₁ was twice as rapid in rats treated with salicylic acid (S. A.) in comparison to control animals; since only 30% of the injected radioactivity was bound to the plasma of the S.A. treated rats, as compared to 90% bound to control plasma, an association may exist between the degree of binding of ³H-PGA₁ and its rate of clearance. The studies indicate that PGA₁ interaction with HSA decreases its metabolism $\text{in vitro}$, and slows down its clearance $\text{in vivo}$, implicating HSA as a possible factor in prostaglandins metabolism $\text{in vivo}$.     

Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals

We have prepared monoclonal antibodies (MoAbs) with the specific ability to bind metal chelates such as ¹¹¹In benzyl EDTA. One, 10, 50 and 100 μg MoAb CHA255 K_b = 4 × 10E9 was complexed with ¹¹¹In BLEDTA II, BLEDTA IV, and benzyl EDTA and injected i.v. in Balb/c mice with KHJJ tumor. The biological half-life by whole body counting was profoundly altered for all three compounds; from minutes to hours with 10 μg; to days with 100 μg. Tumor uptake increased 50 fold at 24 h with increasing MoAb but satisfactory tumor concentrations (3% per g) and tumor/blood ratios (1.8:1) were obtained with an amount equivalent to 7 mg for a human. Blood level and whole body activity were decreased 30–50% within 3 h or i.v. injection of a “flushing” dose of unlabeled indium benzyl EDTA, increasing tumor/blood ratios to 50:1.     

Pyrogenic effects of cytokines (IL-1β, IL- 6, TNF-α) and their mode of action on thermoregulatory centers and functions

The objective of this study was to compare the thermoregulatory responses of rabbits to fever-inducing doses of various cytokines (IL-1β, IL-6, TNF-α) injected peripherally (i.v.), or centrally (i.h.).TNF-α (1 μg kg⁻¹), when applied i.v. at thermoneutral conditions, induced a long lasting increase in hypothalamic temperature, which reached maximal values within 50 min and then persisted for at least 3 h. This monophasic fever-like response was due to extensive and long lasting attenuation of panting and due to transient vasoconstriction. Metabolic rate tended to increase during the early phase of the fever, only. On the other hand, IL-6 when applied i.v. in the same dose (1 μg kg⁻¹) and IL-1β, in a much lower dose (60 ng kg⁻¹), induced a short lasting monophasic hyperthermia due to transient attenuation of panting and due to transient vasoconstriction. No significant increase in metabolic rate was observed. The immediate attenuation of panting and induction of vasoconstriction rather resembled reflex (shock) responses than specific thermoregulatory reactions. Thirty minutes after i.v. administration of TNF-α, temperature thresholds for induction of cold thermogenesis, panting and vasomotion were shifted to higher body temperatures and remained elevated for at least 3 h. In case of IL-1β the increased temperature thresholds were observed 30 min after i.v. administration, only. I.v. applied IL-6 did not influence the thresholds neither 30 or 180 min after injection. Thus, peripheral IL-6 rather induced hyperthermia than fever.When injected i.h. all cytokines studied induced a long lasting increase in body temperature similar to that after i.v. injection of LPS. Temperature thresholds for induction of all thermoregulatory outputs were increased and remained increased for at least 3 h. No changes in hypothalamic thermosensitivity were observed. Data indicate a nonspecific effect of central cytokines on body temperature control.IL-1β appeared to be the most potent fever inducer. Nanogram doses of IL-1β injected i.v. induced a similar febrile response as microgram doses of other cytokines. On the other hand, TNF-α induced a longer lasting fever than other cytokines.     

Characterization by high performance liquid chromatography of nuclear metabolites of testosterone in the brains of male rhesus monkeys

Testosterone (T) restores the potency of castrated male rhesus monkeys, and our autoradiographic data have demonstrated that ³H-T or its metabolites concentrate in cell nuclei in the corticomedial amygdala, bed nucleus of stria terminalis, preoptic area, and hypothalamus. In rat, ³H-estradiol (³H-E₂) is a major nuclear metabolite of ³H-T in areas of the limbic system, but comparable data are lacking for the primate. We have therefore developed an improved technique using high performance liquid chromatography for investigating metabolites of ³H-T that accumulate in cell nuclei in small amounts of tissue obtained from the brain of the rhesus monkey. Two castrated male rhesus monkeys were injected with 5 mCi of ³H-T and were killed 30 min later. In amygdala, preoptic area-bed nucleus of stria terminalis, and hypothalamus, 48–70% of the nuclear radioactivity was in the form of ³H-E₂ (Type I tissues). In six other brain areas and in pituitary, 35–85% of the nuclear radioactivity was in the form of ³H-T (Type II tissues), whereas in genital tract tissues, 86–99% of the nuclear radioactivity was in the form of ³H-dihydrotestosterone (³H-DHT) (Type III tissues). In plasma and in supernatants from both Type I and Type II tissues, the proportions of ³H-T were high, and ³H-E₂ did not exceed 10% of the total extractable radioactivity. These data suggest that, as in rodents, some of the central actions of T in primates may be mediated by estrogen target neurons.     

Use of antisense oligodeoxynucleotide to determine δ-opioid receptor involvement in [d-Ala2]deltorphin II-induced locomotor hyperactivity

Intracerebroventricularly (i.c.v.)-administered [d-Ala²]deltorphin II (20 μg) produced a marked locomotor hyperactivity in male ICR mice. The locomotor hyperactivity induced in response to i.c.v. [d-Ala²]deltorphin II (20 μg) was suppressed by pretreatment with naltriben (NTB, 10 μg) but not 7-benzylidene naltrexone (BNTX, 1 μg) and d-Phe-Cys-Tyr-d-Try-Orn-Thr-Phe-Thr-NH₂ (CTOP, 100 ng). The influence of antisense oligodeoxynucleotide to δ-opioid receptor mRNA (δ-AS oligo) or a mismatch oligodeoxynucleotide (MM oligo) on the locomotor hyperactivity induced by [d-Ala²]deltorphin II was determined. Groups of mice pretreated i.c.v. with δ-AS oligo (1 μg), MM oligo (1 μg) or saline (4 μl) once a day for 3 days, were injected i.c.v. [d-Ala²]deltorphin II (10 or 20 μg) and the locomotor response to [d-Ala²]deltorphin II was measured. The locomotor hyperactivity of i.c.v. [d-Ala²]deltorphin II (10 or 20 μg) were significantly suppressed by i.c.v. pretreatment with δ-AS oligo but not MM oligo. The present results indicate that pretreatment with δ-AS oligo suppresses mouse locomotor hyperactivity produced by stimulation of δ₂-opioid receptors in the brain.     

The Diplomonad Fish Parasite Spironucleus vortens Produces Hydrogen

ABSTRACT. The diplomonad fish parasite Spironucleus vortens causes major problems in aquaculture of ornamental fish, resulting in severe economic losses in the fish farming industry. The strain of S. vortens studied here was isolated from an angelfish and grown in Keister's modified TY‐I‐S33 medium. A membrane‐inlet mass spectrometer was employed to monitor, in a closed system, O₂, CO₂, and H₂. When introduced into air‐saturated buffer, S. vortens rapidly consumed O₂ at the average rate of 62±4 nmol/min/10⁷ cells and CO₂ was produced at 75±11 nmol/min/10⁷ cells. Hydrogen production began under microaerophilic conditions ([O₂]=33.±15 μM) at a rate of 77±7 nmol/min/10⁷ cells. Hydrogen production was inhibited by 62% immediately after adding 150 μM KCN to the reaction vessel, and by 50% at 0.24 μM CO, suggesting that an Fe‐only hydrogenase is responsible for H₂ production. Metronidazole (1 mM) inhibited H₂ production by 50%, while CO₂ production was not affected. This suggests that metronidazole may be reduced by an enzyme of the H₂ pathway, thus competing for electrons with H⁺.     

Optimization of the therapeutic efficacy of human umbilical cord blood–mesenchymal stromal cells in an NSG mouse xenograft model of graft-versus-host disease

Background aimsAlthough in vitro studies have demonstrated the immunosuppressive capacity of mesenchymal stromal cells (MSCs), most in vivo studies on graft-versus-host disease (GVHD) have focused on prevention, and the therapeutic effect of MSCs is controversial. Moreover, optimal time intervals for infusing MSCs have not been established.MethodsWe attempted to evaluate whether human umbilical cord blood–MSCs (hUCB-MSCs) could either prevent or treat GVHD in an NSG mouse xenograft model by injection of MSCs before or after in vivo clearance. Mice were infused with either a single dose or multiple doses of 5 × 10⁵ hUCB-MSCs (3- or 7-day intervals) before or after GVHD onset.ResultsBefore onset, hUCB-MSCs significantly improved the survival rate only when repeatedly injected at 3-day intervals. In contrast, single or repeated injections after GVHD onset significantly increased the survival rate and effectively attenuated tissue damage and inflammation. Furthermore, the levels of prostaglandin E₂ and transforming growth factor-β1 increased significantly, whereas the level of interferon-γ decreased significantly in all MSC treatment groups.ConclusionsThese data establish the optimal time intervals for preventing GVHD and show that hUCB-MSCs effectively attenuated symptoms and improved survival rate when administered after the onset of GVDH.     

Estrogen induced changes in uterine sensitivity for the decidual cell reaction: Interactions between prostaglandin E2 and histamine or bradykinin

In rats receiving high doses of estrogen along with progesterone, the uterus is desensitized and does not respond to artificial stimuli with increased endometrial vascular permeability or decidualization. In addition, prostaglandin E₂ (PGE₂), the putative mediator of endometrial vascular permeability changes in sensitized uteri, is ineffective when given into the uterine lumen. The possibility that this inability of PGE₂ to increase endometrial vascular permeability may be related to the unavailability of hitamine of bradykinin was investigated. Rats were differentially sensitized for the decidual cell reaction by the daily injection of 2 mg progesterone with either 0.5 of 10 μg estrone for the 3 days preceding the unilateral intra-uterine injection of 50 μl phosphate buffered saline containing gelatin with or without 10 μg PGE₂ and with or without 1 mg histamine or 1 μg bradykinin. Prior to the intrauterine injection, all rats were treated with indomethacin to inhibit endogenous prostaglandin production. Endometrial vascular permeability changes were determined 8 h later by determining radioactivity levels in injected and non-injected uterine horns 15 min after the i.v. injection of ¹²⁵I-labelled bovine serum albumin. PGE₂ increased endometrial vascular permeability in rats receiving 0.5 μg estrone, but not in those receiving 10 μg. Histamine or bradykinin, alone or with PGE₂, did not affect endometrial vascular permeability in rats receiving either estrogen dose. The data suggest that the unresponsiveness of uteri from rats treated with high doses of estrogen is not simply due to the unavailability of bradykinin or histamine.     

An acridinium sulphonylamide as a new chemiluminescent label for the determination of carboxylic acids in liquid chromatography

The synthesis of a new acridinium sulphonylamide label for the liquid chromatographic determination of carboxylic acids is described. The label 10-methyl-N-(p-tolyl)-N-(p-iodoacetamidobenzenesulphonyl)-9-acridinium carboxamide iodide is synthesized from 9-acridinecarboxylic acid by a seven-step reaction. Ibuprofen, used as test compound, is coupled to the reactive iodoacetamide group of the label by means of an alkylation reaction in dry acetonitrile for 20 min at 50°C in the presence of 18-crown-6 and potassium carbonate as base catalyst. The reaction mixture is injected into a liquid chromatographic system with chemiluminescence detection. Separation is performed on a Zorbax C₁₈ column with acetonitrile-water-tetrahydrofuran (39:57:4, v/v/v) containing 10 mmol/L TBABr and 0.035% H₂O₂ as the mobile phase at a flow rate of 1.0 ml/min. Chemiluminescence detection is achieved by the post-column addition of 200 mmol/L potassium hydroxide dissolved in methanol–water (1:1, v/v) at a flow rate of 20 μL/min. The detection limit (S/N = 3) of derivatized ibuprofen is 60 pg (3 pg injected). © 1998 John Wiley & Sons, Ltd.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Effect of PGD2, PGE2, PGF2α and PGI2 on blood pressure,heart rate and plasma catecholamine responses to spinal cord stimulation in the rat

The following experiments were designed in order to examine the inter-relationships of various prostaglandins (PG's) and the adrenergic nervous system, in conjunction with blood pressure and heart rate responses, $\text{in vivo}$. Stimulation of the entire spinal cord (50v, 0.3–3 Hz, 1.0 msec) of the pithed rat increased blood pressure, heart rate and plasma epinephrine (EPI) and norepinephrine (NE) concentration (radioenzymatic-thin layer chromatographic assay). Infusion of PGE₂(10–30 μg/kg. min, i.v.) suppressed blood pressure and heart rate responses to spinal cord stimulation while plasma EPI (but not NE) was augmented over levels found in control animals. PGI₂ (0.03–3.0 μg/kg. min, i.v.) suppressed the blood pressure response to spinal cord stimulation without any effect on heart rate or the plasma catecholamine levels. PGE₂ and PGF_2α(10–30 μg/kg. min, i.v.) did not change the blood pressure, heart rate or plasma EPI and NE responses to the spinal cord stimulation although PGF_2α disclosed an overall vasopressor effect during the pre-stimulation period. At the pre-stimulation period it was also observed that PGE₂, PGF_2α and PGI₂, had a positive chronotropic effect on the heart rate, the cardiac accelerating effect of PGE₂ was not abolished by propanolol. These $\text{in vivo}$ studies suggest that in the rat, PGE₂ and PGI₂ modulate sympathetic responses, primarily by interaction with the post-synaptic elements — PGE₂ on both blood vessels and the heart and PGI₂ by acting principally on blood vessels.