The simultaneous assay of progesterone, 17-hydroxyprogesterone, and deoxycorticosterone in human plasma by competitive protein binding

Testosterone oenanthate was administered intramuscularly in six infertile men with oligozoospermia and its effects on serum gonadotropins and some constituents in the seminal plasma were studied. One week after injection the mean serum FSH level was decreased to about 50%. Serum LH levels did not change. The mean ornithine decarboxylase activity in human semen was increased by 100% after the testosterone administration. The androgen dependent nature of ODC, fructose and sialic acid have been demonstrated.     

Experimental studies on the positive feedback effect of progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone on the pituitary release of LH and FSH in the human female. The estrogen priming of the progesterone feedback on pituitary gonadotropins in the eugonadal woman

Administration of progesterone eugonadal women during the midfollicular phase of the menstrual cycle failed to induce a positive feedback effect on the serum concentrations of LH and FSH. The levels of estradiol in serum decreased following the injection of progesterone without a parallel change in LH and FSH concentrations indicating a direct ovarian effect of the exogenous progesterone. In the late follicular phase of the cycle, when preovulatory levels of estradiol were present in serum, or under a ethinyl estradiol treatment progesterone was able to induce an LH discharge indicating the requirement of an estradiol priming of the positive feedback of progesterone in eugonadal women. In order to establish the time required for a sufficient estrogen priming with preovulatory levels of estradiol in serum 3 mg of estradiol-benzoate were administered i.m. 1, 12 and 24 h prior to the administration of 30 mg of microcristalline progesterone in the midfollicular phase of the menstrual cycle, when progesterone alone did not cause an LH surge. Only when estradiol-benzoate was injected 24 h prior to the progesterone administration an LH surge reproducible in time course and magnitude occurred. Administration of estradiol-benzoate alone under these conditions did not cause an LH surge within the elapse of time after the injection when the progesterone induced LH surge occurred. Thus, these experiments demonstrate that a defined estrogen priming is required for the positive feedback effect of progesterone on the gonadotropin release in eugonadal women. Furthermore, progesterone levels in serum of about only 1--2 ng/ml were required for the induction of an LH surge indicating that under physiological conditions progesterone may have an supplementory effect on the primarily estradiol induced LH midcycle peak. 17-hydroxyprogesterone administered during the mid follicular phase of the menstrual cycle and under pretreatment with ethinyl estradiol failed to induce a positive feedback effect on the serum concentrations of LH and FSH, indicating that this steroid does not play a regulatory role on the midcycle LH release in women. 20alpha-dihydroprogesterone administered under the same experimental conditions as 17-hydroxyprogesterone seems to be able to induce an LH surge in serum provided there is an adequate estrogen priming.     

Interactions of 16alpha,17alpha-cyclohexane derivatives of progesterone with the progesterone receptor from rat uterus

Pregna-D'-pentaranes, 16alpha,17alpha-cyclohexanoprogesterone and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, were found to specifically interact with the progesterone receptor of soluble fraction from rat uterus. The formation of complexes between 3H-labeled derivatives of these steroids and the protein was complete within 1 to 3 h at 0-4 degreesC. The dissociation of these complexes was a two-phase process, the contribution of the fast dissociating complexes decreasing with increasing preincubation time. The dissociation constant (k-1) values for progesterone, 16alpha, 17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone complexes with the protein after 1 h preincubation were 6.5 +/- 0.8, 8.8 +/- 5.5, and (16.6 +/- 5.6).10(-4) sec(-1) for the fast phase and 5.1 +/- 0.5, 3.5 +/- 0.8, and (2.8 +/- 0.6).10(-5) sec(-1) for slow phase, respectively. The equilibrium Kd values were 11.7 +/- 2.1, 19.0 +/- 2.0, and 66.1 +/- 14.6 nM for progesterone, 16alpha,17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, respectively. The steroids mutually inhibited the binding of their 3H-labeled derivatives to the protein, the inhibition being of competitive type. In the case of [3H]6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone, the inhibitory efficacy of progesterone declined with an increase of its concentration; this points to possible heterogeneity of binding sites for the 3H-labeled ligand. The comparison of the results with those obtained by us earlier (Biochemistry (Moscow), 1996, 61, 1034-1041) suggests the existence of significant species differences in progesterone receptor structure within or near the region that interacts with the D-ring of a hormone molecule.     

Dual UV-absorbing background electrolytes for simultaneous separation and detection of small cations and anions by capillary zone electrophoresis

The simultaneous separation and detection of small cations and anions by capillary zone electrophoresis (CZE) with indirect ultraviolet (UV) detection was successfully demonstrated in a background electrolyte (BGE) containing two UV-absorbing components. Benzylamine, imidazole, benzenesulfonic acid, sulfosalicylic acid, and pyromellitic acid were tested as the components of the BGE. The success of the simultaneous separation of the cations and anions is dependent upon the proper selection of the electrolyte components and control of the migration of the ions towards the detector. High pH is beneficial to the detection of anionic analytes but not to the separation of cationic analytes because of large electroosmotic flow produced under this condition. The upper pH limit of the working pH range is confined by the pKa value of the cationic component of the BGE. The influence of pH and total electrolyte concentration on the electroosmatic flow (EOF) counteracted each other. This counteraction effect imposes an upper limit on the change of total electrolyte concentration at certain pH. It was found that the EOF should be larger by at least 10 x 10(-5) cm2V(-1)s(-1) than the electrophoretic mobilities of the anions so that the anions could be detected on the cathodic side within reasonable times and with good peak shapes. In the imidazole-sulfosalicylic acid BGE, the detection limits (signal to noise, S/N = 3) for the cations and anions ranged from 100 to 900 ppb. In the benzylamine-pyromellitic acid BGE, K+, Na+, Li+, CH3 COO-, HPO4(2-), F-, ClO3-, ClO4-, NO3-, NO2-, Cl- and SO4(2-) were separated within twelve minutes. The strategies for selection of the electrolyte components of the binary BGE were also discussed.     

Automated sample preparation for the determination of budesonide in plasma samples by liquid chromatography and tandem mass spectrometry

An automated bioanalytical method for the determination of the glucocorticosteroid drug budesonide in plasma samples at pM levels was investigated. The method was built using three separate automated analytical steps with manual transfer of samples between them. In the first step, a Tecan RSP150 (Genesis) pipetting robot was used to transfer 1 ml of centrifuged plasma samples and deuterated budesonide internal standard solutions into tubes and to homogenise the resulting admixture. In the second step, a solid-phase extraction was performed using an ASPEC Xli (Gilson) with 100 mg Isolute C18 columns. In order to avoid conventional time-consuming evaporation and reconstitution steps, the solid-phase extraction was coupled on-line to a trace enrichment system for further purification and concentration of the sample extracts. The concentrated samples were eluted in 300 microliters ethanol into injection vials, which were capped and transferred to the autosampler in the detection system. In the third step, the pre-treated samples were chromatographed in a gradient LC system and detected using a tandem MS system (Finnigan TSQ 7000), with an atmospheric pressure chemical ionisation interface. The described Analytical System consisting of one Tecan robot, two ASPEC systems and one LC-MS-MS system may analyse up to about 800 samples a week with less routine work for the analyst. The concentration range studied was 15 to 2500 pM in 1 ml spiked plasma samples and the limit of quantitation for the described method was determined as 15 pM, as defined by accuracy and precision better than 20%.     

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone

The information available concerning the effects of chemotherapy administered during pregnancy is limited and consists of case reports and small series. A registry has been established at the National Cancer Institute, but there are currently only several hundred cases of neonates exposed to chemotherapy registered. All clinicians who care for women receiving chemotherapy during pregnancy should report those experiences to the National Cancer Institute to increase the data base. When chemotherapy is used during the embryogenesis period in the first trimester there is an increased rate of spontaneous abortion and major birth defects. The most toxic chemotherapeutic agents administered during pregnancy are methotrexate and aminopterin and should be avoided when possible, particularly during the first trimester. Pregnancy-related physiologic changes should be kept in mind when dosing and administering cytotoxic chemotherapy. The risk of fetal malformation when chemotherapy is administered during the second and third trimesters is probably not greater than background rate, but there may be a greater risk of stillbirth, fetal growth restriction, premature birth, and maternal and fetal myelosuppression. Breastfeeding should be avoided in women receiving chemotherapy.     

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.     

Effect of 17 alpha hydroxy-20 beta dihydroprogesterone and progesterone on maturation and ovulation in vivo and on the plasma level of gonadotropic hormone t-GtH in rainbow trout Salmo gairdneri

Ovulation occurred 5 to 9 days after injection of 2 mg/kg 17 alpha-hydroxy-20 beta dihydroprogesterone (17 alpha-20 beta Pg) in rainbow trout with oocytes in which the germinal vesicle was subperipheral. Whereas oocyte maturation was initiated by 17 alpha-20 beta Pg without an immediate increase in the concentration of t-GtH, in controls an increased level was found to coincide with initiation of the process and the concentration of t-GtH was higher at all stages. Progesterone treatment was less effective.     

共沉淀富集分离-ICP-MS法测定地球化学勘探样品中的超痕量金、铂、钯

建立了碲作为共沉淀剂分离富集,采用ICP—MS测定地球化学勘探样品中超痕量金、铂、钯的方法,并研究了共沉淀时各种因素的影响;确定了采用盐酸和氯酸钾分解试样,以2mg碲对金、铂、钯进行共沉淀分离富集,抽滤,残渣用王水溶解,采用电感耦合等离子体质谱法测定的条件。该方法检出限分别为：Au0．062ng／g、Pt0．041ng／g、Pd0．043ng／g,相对标准偏差（n=12）：Au4．81％、R5．46％、Pd3．50％。采用该方法测定了国家一级地球化学标准物质中的痕量Au,Pt,Pd,测定值与标准值相符合。     

A comparative study of plasma 17beta-oestradiol, progesterone, placental lactogen and chorionic gonadotrophin in abortion induced with intra-amniotic prostaglandin F2alpha

Serial estimations were made in plasma of 17beta-oestradiol (E2), progesterone and human placental lactogen (HPL) in 43 patients and of human chorionic gonadotrophin (HCG) in 34 patients during mid-trimester abortions induced with intra-amniotic prostaglandin F2alpha (PGF2alpha. Mean plasma concentrations of all the hormones showed a progressive fall after PGF2alpha. There was no relationship between the fall in levels of progesterone, HPL and HCG and the induction-abortion interval, signs of fetal distress or of intrauterine fetal death. Both the control level and the rate of fall of E2 were related to the induction-abortion interval and a rapid decline preceded intrauterine fetal death. The relationships of the progesterone/E2 ratio and the amniotic fluid volume/progesterone ratio to the induction-abortion interval were examined. The variation in the time at which significant falls in the concentration of individual hormones occurred was probably related to their respective half-lives in plasma.     

Testosterone and testosterone precursors in the spermatic vein and in the testicular tissue of old men. Reduced oxygen supply may explain the relative increase of testicular progesterone and 17 alpha-hydroxyprogesterone content and production in old age

Testosterone and the testosterone precursors pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, androstenedione, androstenediol and dehydroepiandrosterone were measured in the spermatic vein plasma and in the testicular tissue of young and old men. Testosterone and its precursors decreased in the testicular tissue of old men. However, progesterone and 17 alpha-hydroxyprogesterone increased in relation to testosterone in the testicular tissue and in the spermatic vein of old men. It is assumed that these age-dependent changes are caused by an impaired oxygen supply of the ageing testes. This hypothesis is supported by the observation that the same changes in steroid pattern seen in old age can be observed under reduced oxygen supply in in vitro incubation experiments with testicular tissue.     

Influence of serum, estrogen, and gonadotropins upon growth and progesterone secretion by cultures of granulosa cells from small porcine follicles

Granulosa cells from small (less than 2mm) antral porcine follicles were grown in culture to study the effects of various hormones on growth, morphology and progesterone secretion. Culture medium 199D + 4% serum was found to be most suitable since it maintained a fairly constant cell population. Estradiol (1mug/ml) and human FSH stimulated cell growth. LH and FSH stimulated progesterone secretion and induced morphological changes associated with luteinization. Estradiol (0.1 mug/ml) inhibited progesterone secretion by granulosa cells.     

Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography

Biological samples have a high dielectric constant that can shorten RF wavelengths by a factor of 8 relative to the vacuum. At high field strengths, finite wavelength effects within larger samples are the dominant cause of RF field nonuniformity. A coil design is presented that can reduce and even eliminate this inhomogeneity; 4-T images in phantoms and in the head of a normal volunteer are presented, which demonstrate improved homogeneity relative to a standard coil. This coil design should aid in realizing the potential advantages of imaging large samples at high field strengths.     

The use of sequential affinity chromatography for separation of human neutrophil elastase, cathepsin G and azurocidin

Elastase, cathepsin G and azurocidin from human neutrophils are key components of body inflammatory defense. Perturbations in regulation of their activities lead to many serious pathological states. The paper describes a simple, fast and efficient method of joint purification of these proteins with the use of sequential affinity chromatography on squash trypsin inhibitor (CMTI I) and bovine pancreatic trypsin inhibitor (BPTI).